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Oxoid Ltd part of
Thermo Fisher Scientific
Wade Rd Basingstoke
Hampshire RG24 8PW (UK)
NF VALIDATION
Validation of alternative analytical methods
Application in food microbiology
Summary report
EN ISO validation study of the
Listeria PrecisTM method for the
enumeration of Listeria monocytogenes in food products and environmental samples
Quantitative method
This report includes 38 pages with 4 appendixes
Only copies including the totality of this report are authorised
Version 1 November 19 2014
Cancel and replace the previous version
which must be returned to ADRIA Deacuteveloppement or destroyed internally
Thermo Fisher Scientific
ADRIA Deacuteveloppement 238 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
1 INTRODUCTION 4
11 Dates of the validation studies 4
12 Protocol and principle of the alternative method 4
13 Standard method 4
2 VALIDATION STUDY RESULTS 5
21 Initial validation (realised by ASEPT 2006) 5
211 Method Comparison Study 5
2111 Linearity 5
2112 Relative accuracy 9
2113 Detection limit (LOD) and quantification limit (LOQ) 17
2114 Relative Sensitivity 18
2115 SpecificitySelectivity 19
212 Practicability 20
213 Inter-laboratory study 21
2131 Organisation 21
2132 Verification of experimental parameters 21
2133 Results 23
214 Conclusion 29
22 Extension study (realised by ASEPT 2007) 30
Appendix 1 ndash Flow diagram of the Listeria PrecisTM method __________________________________ 31
Appendix 2 ndash ISO 11290-2 (1998) amp ISO 11290-2A1 (2004) Horizontal method for the detection and
enumeration of Listeria monocytogenes - Part 2 enumeration of Listeria monocytogenes in foods ____ 32
Appendix 3 ndash Mandelrsquos graphics _______________________________________________________ 33
Appendix 4 ndash Statistical results ________________________________________________________ 35
Thermo Fisher Scientific
ADRIA Deacuteveloppement 338 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
The modifications are highlighted
Quality assurance documents related to this study can be consulted upon
request from Oxoid part of Thermo Fisher Scientific
The technical protocol and the result interpretation were realised according to
the EN ISO 16140 and the AFNOR technical rules
o Company Oxoid Ltd
part of THERMO FISHER SCIENTIFIC
Wade Road Basingstoke
Hampshire RG24 8PW UK
o Expert Laboratory ADRIA Deacuteveloppement
ZA Creacrsquoh Gwen
F-29196 QUIMPER Cedex
o Studied method Listeria PrecisTM method for enumeration of
Listeria monocytogenes
o Validation standard NF EN ISO 16140 (October 2003) Food
microbiology ndash Protocol for the validation of
alternative methods
o Standard method ISO 11290-2 (1999) amp ISO 11290-2A1 (2004)
Horizontal method for the detection and
enumeration of Listeria monocytogenes - Part 2
enumeration of Listeria monocytogenes in foods
o Scope All human food products and environmental
samples
o Certification organism AFNOR Certification
Thermo Fisher Scientific
ADRIA Deacuteveloppement 438 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
1 INTRODUCTION
11 Dates of the validation studies
The Listeria PrecisTM method for enumeration of Listeria monocytogenes was
validated on September 15 2006 (certificate number UNI 0305 ndash 0906) for
food products and environmental samples
In 2007 an extension study was obtained for a new confirmation test OBIS
MONO
The alternative method was renewed in 2010 and 2014
12 Protocol and principle of the alternative method
The Listeria PrecisTM method is based on a specific chromogenic media for
Listeria monocytogenes enumeration
The protocol is described in Appendix 1
13 Standard method
The reference method is the ISO 11290-2 (1998) amp ISO 11290-2A1 (2004)
Horizontal method for the detection and enumeration of Listeria
monocytogenes - Part 2 enumeration of Listeria monocytogenes in foods
(See Appendix 2)
Analysis performed according to the COFRAC accreditation
Thermo Fisher Scientific
ADRIA Deacuteveloppement 538 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
2 VALIDATION STUDY RESULTS
21 Initial validation (realised by ASEPT 2006)
211 Method Comparison Study
2111 Linearity
Linearity is the ability of the method when used with a given matrix to give results that are in proportion
to the amount of analyte present in the sample that is an increase in analyte corresponds to a linear or
proportional increase in results
Food matrices and protocols
Six matrixstrain pairs were analysed with five contamination levels and two
replicates At least 50 analyses were performed by the alternative and the
standard methods
The contamination levels the samples and the inoculated strains are
presented in the table below
Table 1
Samples Strains Contamination levels
(CFUg)
Rillettes Listeria monocytogenes 4e 10 ndash 50
50 ndash 100
100 ndash 500
500 ndash 1 000
1 000 ndash 10 000
Raw milk Listeria monocytogenes 12a
Lettuce Listeria monocytogenes 12a
Smoked salmon Listeria monocytogenes 12b
Raw egg product Listeria monocytogenes 12b
Process water Listeria monocytogenes 12a
The samples were individually inoculated and were analysed in duplicate by
the alternative and the reference methods
Results
The bi-dimensional graphs are shown figure 1
Thermo Fisher Scientific
ADRIA Deacuteveloppement 638 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Figure 1 ndash Bi-dimensional graphs
Rillettes Raw milk
Smoked salmon Raw egg product
Lettuce Process water
Thermo Fisher Scientific
ADRIA Deacuteveloppement 738 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Statistical interpretations
Statistical interpretation results are provided in Table 2
Table 2 ndash Statistical interpretations
Matrix R Selected
regression
RobF Critical
value
P Correlation
coefficient
Regression equation
Rillettes 180 GMFR 609 541 4 09960 log (Alt) = 09887 log (Ref) + 01141
Lettuce 108 GMFR 000 541 100 09992 Log (Alt) = 10081 log (Ref) - 00257
Raw milk 102 GMFR 000 541 100 09997 Log (Alt) = 10188 log (Ref) - 00737
Smoked salmon 1048 OLS1 1806 541 263 09984 Log (Alt) = 10406 log (Ref) - 01418
Raw egg product 130 GMFR 548 541 49 09958 Log (Alt) = 09775 log (Ref) - 00161
Process water 184 GMFR 0079 541 969 09985 Log (Alt) = 09680 log (Ref) + 01317
GMFR = orthogonal linear regression
OLS1 = ordinary least-square linear regression
x ndash axis = reference method
y ndash axis = alternative method
OLS1 = ordinary least-square linear regression
x axis = alternative method
y axis = reference method
Statistical Interpretation
P gt 5 not significant 1 lt P lt 5 significant
P lt 1 highly significant
The regressions straight lines are shown figure 2
Thermo Fisher Scientific
ADRIA Deacuteveloppement 838 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Figure 2 ndash Regression straight lines
Lineacuteariteacute - Œuf
Process water Process water (2) Process water
Smoked salmon (GMFR) Smoked salmon (OLS 1)
Raw milk Raw egg product
Rillettes Lettuce
Thermo Fisher Scientific
ADRIA Deacuteveloppement 938 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Discussion
The linearity is accepted in all cases with
P values 5 and correlation coefficients superior to 099
For the matrix ldquoRillettesldquo the linearity test is significant (P = 4) with a high
correlation coefficient value (0996)
The Listeria PrecisTM method shows satisfying linearity
2112 Relative accuracy
The accuracy is the closeness of agreement between a test result and the accepted reference value
The bias is the difference between the expectation of the test results and an accepted reference value
Number and nature of the samples
The repartition per category and types is provided in table 3
Table 3 ndash Number and nature of the samples
Categories Types Number of samples
Analysed Exploited (1)
Meat products
Raw meat products 6 4
Ready to cook products 4 2
Ready to eat delicatessen 10 4
Total 20 10
Dairy products
Raw milk cheeses 7 4
Raw milks 4 3
Other dairy products 5 5
Total 16 12
Fishery products
Raw fishes 5 4
Processed fishes 7 2
Ready-to-eat products 6 4
Total 18 10
Vegetables
Raw vegetables 5 4
Frozen vegetables 3 3
Lettuce raw vegetables 5 5
Total 13 12
Egg products
Raw egg 5 5
Pastries 3 3
Pasteurised egg products 4 3
Total 12 11
Environmental samples
Wipes swabs 8 4
Process water 3 3
Dusts 3 3
Total 14 10
TOTAL 93 65
(1) Some data cannot be exploited because enumeration was under the detection level for at least one of the
method
Thermo Fisher Scientific
ADRIA Deacuteveloppement 1038 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Artificial contamination of the samples
Artificial contaminations were realised by spiking after performing injury
protocols on pure cultures or by cross-contaminations
56 samples were artificially contaminated 53 gave exploitable results among
a total of 65 samples 8150 of the samples were artificially contaminated
The injury protocol and the strains inoculated are provided in Table 4
Table 4
Stress Ndeg Injury protocol Strain Origin
A 45 min at 50 degC
48
LM -H123
LM-H170
Smoked salmon
Lettuce
Lettuce
B 30 min at 50 degC
and 2 h at ndash20degC
18
43
LM-O1
Raw milk (environment)
Environment
Egg product
C 30 min at 55degC
18
48
LM-H123
Raw milk
Smoked salmon
Lettuce
D 30 min at 50degC
et 2 h at ndash80degC
34
LM-H171
LM-O1
Rillettes
Lettuce
Egg product
E 30 min at 60 degC LM-O1 Egg product
Confirmatory tests
For the reference and the alternative methods confirmations were realised
by the tests described in the ISO 12290-2A1 standard For the alternative
method only one colony was confirmed while 5 colonies were tested for the
reference method
Results
Samples were analysed in duplicate by the reference and the alternative
methods
The contamination range is provided in table 5 per tested category
Thermo Fisher Scientific
ADRIA Deacuteveloppement 1138 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Table 5 ndash Contamination range
Food category Contamination level (log CFUg)
Meat products 100 ndash 404
Dairy products 148 ndash 408
Fishery products 100 ndash 384
Vegetables 170 ndash 511
Egg products 130 ndash 499
Environmental samples 170 ndash 511
All categories 100 ndash 511
Bi-dimensional graphs are given figure 3
Figure 3 ndash Bi-dimensional graphs
Graphe bi-dimensionnel Produits carneacutes
Fishery products
Vegetables
Egg products
Meat products
Thermo Fisher Scientific
ADRIA Deacuteveloppement 1238 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Statistical interpretation according to EN ISO16140 method
The results of the statistical interpretation are given in Table 6
Table 6 ndash Statistical interpretation results
Category n R Regression
used a t(a) b t(b) Critical T
P
Ordinate
at 0
Slope
at 1
Meat products 10 158 GMFR -0455 3321 115 3123 2306 001 001
Dairy products 12 065 GMFR -0027 0385 1002 0108 2228 092 071
Fishery products 10 130 GMFR -0305 1024 1034 0312 2306 076 034
Vegetables 12 086 GMFR -0015 0128 0993 0191 2228 085 090
Egg products 11 158 GMFR -0038 0866 1001 0090 2262 093 041
Environmental
samples 10 176 GMFR -0139 265 1034 231 2306 005 003
All products 65 118 GMFR -0182 3042 1040 2145 2000 004 000
All products
(estimated number) 65 118 GMFR -0182 3042 1040 2145 2000 004 000
All products (non
estimated results) 20 115 GMFR -0367 151 1138 1141 2101 027 015
Repeatability limits obtained for the alternative and reference methods as
well as bias obtained are presented in Table 7
Dairy products Environmental samples
Log (Ref) Log (Ref)
All categories
Thermo Fisher Scientific
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Listeria Precis Enumeration
Table 7
Categories Bias
Repeatability limits
Rob r
Ref Alt
Meat products 0008 0314 0343
Dairy products -0031 0136 0130
Fishery products -0133 0220 0214
Vegetables -0041 0180 0319
Egg products -0026 0219 0206
Environmental samples -0007 0198 0119
All products -0032 0196 0215
Regression straight lines (graph and equation representations) for each food
category and for all products are presented figure 4
Thermo Fisher Scientific
ADRIA Deacuteveloppement 1438 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Figure 4 ndash Regression straight lines
Meat products
Fishery products
Dairy products
Vegetables
Thermo Fisher Scientific
ADRIA Deacuteveloppement 1538 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Environmental samples
Egg products
All products (estimated number)
Thermo Fisher Scientific
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Summary Report (Version 1)
Listeria Precis Enumeration
The hypotheses that the ordinates are close to 0 and the slopes close to 1
are approved in all cases except for
- Meat products (slope P = 1 ordinate P = 1)
- Environmental samples (ordinate P = 3)
- All products (ordinate P = 0)
The bias between the two methods vary from ndash 0133 to 0008 log CFU For
all categories the bias is ndash 0032 log CFU The repeatability limits of the
alternative and the reference method are similar For all products the values
are respectively 0215 and 0196 log CFU For all products these
interpretations are provided taking into account data with low contamination
level or not (estimated colonies number)
For all food products the regression straight line is the following
log (Alt) = 1040 log Ref ndash 0182
The Listeria PrecisTM method shows satisfying relative accuracy
All products
All products (Level 2)
Thermo Fisher Scientific
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Summary Report (Version 1)
Listeria Precis Enumeration
2113 Detection limit (LOD) and quantification limit (LOQ)
The critical level is the defined as the smallest amount which can be detected (not null) but not
quantified as an exact value Below this value it cannot be sure that the true value is not null
The detection limit is defined as being higher than the critical level because it involves a power the
probability 1- which has to be well over 50 for example 95
The quantification limit is defined as the smallest amount of analyte (that is the lowest actual number of
organisms) which can be measured and quantified with defined precision and accuracy under the
experimental conditions by the method under validation
Protocol
The detection limit of the alternative method was realised with pure cultures
Three different inoculation levels were tested with six replicates per level ie
18 analyses were done by the alternative method
Listeria monocytogenes 18 was used for the detection limit of the alternative
method
Quantification limit was calculated for six independent blank samples
determinations
Results
These data are intrinsic to the method used and are presented in the
following tables
Table 8 ndash Inoculation 1 ml on 3 plates
Level (CFUml) Positive samples number Standard deviation Bias
0 06
05 26 1211 0
1 36 0816 05
5 66 2483 85
Thermo Fisher Scientific
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Summary Report (Version 1)
Listeria Precis Enumeration
Table 9 ndash Inoculation 01 ml on 1 plate
Level (CFUml) Positive samples number Standard deviation Bias
0 06
5 36 0548 05
10 46 1414 2
50 66 2258 55
Table 10
Formulas Obtained values
LC 165 S0 + X0 18
LOD 33 S0 + X0 32
LOQ 10 S0 + X0 87
2114 Relative Sensitivity
The relative sensitivity is defined as the ability of the alternative method to detect two different amounts
of analyte measured by the reference method within a given matrix at a specified average value or
over the whole measurement range that is it is the minimal quantity variation (increase of the analyte
concentration x) which gives a significant variation of the measured signal (response y)
These data are intrinsic to the used method and are obtained from the results
of the linearity study
Sensitivity patterns obtained for tested (matrixstrain) pairs are presented in
figure 5
Thermo Fisher Scientific
ADRIA Deacuteveloppement 1938 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Figure 5 ndash Sensitivity patterns for the tested (matrixstrain) pairs
2115 SpecificitySelectivity
The specificity is defined as the degree to which a method is affected (or not) by the other components
present in a multi-component sample That is the ability of a method to measure exactly a given
analyte or its amount within the sample without interference from non-target components such as a
matrix effect or background noise
The selectivity is defined as a measure of the degree of non-interference in the presence of non-target
analytes A method is selective if it can be used to detect the analyte under examination and that a
guarantee can be provided that the detected signal can only be a product by that specific analyte
The specificity and selectivity study was performed for the Listeria PrecisTM
detection method no additional test was realised for the enumeration
method The results were the following
- Among the Listeria monocytogenes strains tested 2 strains (Listeria
monocytogenes CIP 7834 and Listeria monocytogenes CIP 105459) gave
negative results These 2 strains were tested twice by running 48 h
incubation time of the compared agars Positive results were then
observed by the alternative method Pale characteristic colonies were
observed on the ISO agar by streaking the Half Fraser broth only
- No cross reaction was observed with the non-target strains except with
Listeria ivanovii which gave characteristic colonies on Brilliancetrade Listeria
Agar but the confirmatory tests provide a clear discrimination between
Listeria monocytogenes and Listeria ivanovi species
Rillettes
Lettuce
Raw milk
Smoked salmon
Raw egg
Process water
x (CFUg)
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2038 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
212 Practicability
1 Packaging Ready to use Brilliance Listeria agar plates are available
(x 10 plates)
2 Reagent volume
3 Storage conditionsndash
Shelf life of products
not opened
The plates are stored at 6 - 12degC in darkness conditions The
expiration date is mentioned on the package and on the plates It
is around 3 months
4 Modalities of use
after first use
5 Required
equipments No specific equipment is required
6 Reagents Ready to use plates
7 Training 05 day
8 Real time handling
Reference method
9 min for one sample
55 min for 10 samples
Alternative method
7 min for one sample
35 minutes for 10 samples
9 Time to result
Negative results
Reference method Day 2
Alternative method Day 2
Negative results after confirmation or positive results
Reference method Day 4 ndash Day 7
Alternative method Day 4 ndash Day 7
Positive results
Reference method Day 4 ndash Day 7
Alternative method Day 2 ndash Day 3 (with OBIS Mono Test)
or Day 4 ndash Day 7 (by the classical tests described in the
standardised method)
10 Technician skill Identical to that required for the reference method
11 Steps common to
the reference method Mother suspension revivification step and decimal dilutions
12 Traceability
13 Maintenance
Thermo Fisher Scientific
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Summary Report (Version 1)
Listeria Precis Enumeration
213 Inter-laboratory study
2131 Organisation
12 laboratories participated to this study Pasteurised milk was inoculated by
Listeria monocytogenes 12a isolated from raw milk The targeted
inoculation levels were
- 0 CFUml
- 50 CFU ml
- 500 CFU ml
- 5 000 CFU ml
Each laboratory received eight flasks of 10 ml sample ie two flasks per
inoculation level Furthermore one non-inoculated sample has been added
to the package for total viable count microflora (NF ISO 4833 method)
Detailed instructions were transmitted to the collaborators by the expert
laboratory
Collaborative study laboratories and the expert laboratory carried out the
analyses with the alternative and reference methods
2132 Verification of experimental parameters
Strain stability during transport
In order to evaluate the Listeria monocytogenes strain stability during
transport bacterial count of all inoculated flasks were checked at different
time ie inoculation time after 24 h of conservation at 2deg - 8degC Results are
reported in table 11
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2238 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Table 11 ndash Listeria monocytogenes count
by ISO 11290-2A1 method (log CFUml)
Day of the analyses Level contamination Reference method
Day 0
(29052006)
1 45
2 440
3 4 500
Day 1
(30052006)
1 60
2 430
3 4 900
No evolution of the strain was observed after storage for 24 h at 2degC ndash 8degC
Contamination level
The real contamination levels are provided in table 12
Table 12 ndash Real contamination level (CFUml)
Target inoculation
(CFU ml) Samples Ndeg
Real contamination levels
(CFUml)
0 1 - 8 0
50 5 - 6 45
500 3 - 7 440
5000 2 - 4 4 500
Sample temperature at receipt
Measured temperatures at receipt are listed in table 13
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2338 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Table 13 - Sample temperature on receipt
Laboratories Receipt
day
Hour
receipt
Measured temperature
at receipt (degC)
Measured temperature by the
temperature probe (degC)
A
B
C
D
E
F
G
H
I
J
K
L
M
30052006
10h45
10h15
11h15
10h00
09h30
8h30
11h50
10h00
08h30
14h45
08h30
09h45
08h45
82
55
45
63
60
62
81
38
48
-
14
67
76
03
03
-13
03
04
-14
05
04
01
05
-13
04
Conclusion
The temperatures during the transport were correct for all the labs except for
Lab J which didnrsquot provide the information
All the labs received their packages with several broken flasks
Three labs (B D and L) received some damaged samples but proceeded to
analyses
All the labs started the analyses at Day 1 except Lab J
2133 Results
Aerobic mesophilic flora
The count of aerobic mesophilic flora varied from 94 102 CFUml to
33 103 CFUml
Thermo Fisher Scientific
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Listeria Precis Enumeration
Expert lab results
The results obtained by the expert lab are the following
Table 14
Target inoculation level (CFUml)
Reference method ISO 11290-2
Alternative method Listeria Precis Enumeration
Rep 1 Rep 2 Rep 1 Rep 2
lt 10 lt 10 lt 10 lt 10 lt10
50 50 (Ne) 60 (Ne) 50 (Ne) 30 (Ne)
500 470 390 490 500
5 000 4 700 5 100 5 900 3 400
Ne estimated number
The obtained results are in agreement with those expected
Collaborator Labs results
A synthesis of the results obtained by the collaborator labs is provided
Table 15 (CFUml) and Table 16 (log CFUml)
Table 15 ndash Synthesis of results obtained by the alternative
and the reference methods (CFUml)
Labs Level 1 Level 2 Level 2 bis Level 3
Reference method
Alternative method
Reference method
Alternative method
Reference method
Alternative method
Reference method
Alternative method
A 40 40 70 40 480 410 210 340 1000 300 400 300 4900 4600 4900 5100
B 40 15 40 20 350 300 360 450 350 650 800 600 4100 4700 5100 3700
C 50 20 20 70 440 430 480 450 300 650 300 500 4600 5400 4600 5000
D 40 60 40 40 490 460 600 540 250 450 600 1000 4900 4900 4400 4600
E 50 40 50 20 490 520 530 560 750 550 400 200 5400 4500 4100 5400
F 60 20 60 50 370 250 300 130 300 350 600 300 4400 4700 4900 5900
G 70 35 120 50 440 390 570 370 450 550 700 700 5000 4500 4000 4400
H 110 40 100 90 520 440 450 540 150 650 300 300 4200 4700 5600 4800
L 35 60 20 40 390 390 170 380 650 350 800 200 5300 4600 5600 5900
M 45 45 30 40 450 490 550 410 400 550 800 180 4900 4900 6400 4800
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2538 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Table 16 ndash Synthesis of results obtained by the alternative
and the reference methods (log CFUml)
Labs Level 1 Level 2 Level 2 bis Level 3
Reference method
Alternative method
Reference method
Alternative method
Reference method
Alternative method
Reference method
Alternative method
A 1602 1602 1845 1602 2681 2613 2322 2531 3000 2477 2602 2477 3690 3663 3690 3708
B 1602 1176 1602 1301 2544 2477 2556 2653 2544 2813 2903 2778 3613 3672 3708 3568
C 1699 1301 1301 1845 2643 2633 2681 2653 2477 2813 2477 2699 3663 3732 3663 3699
D 1602 1778 1602 1602 2690 2663 2778 2732 2398 2653 2778 3000 3690 3690 3643 3663
E 1699 1602 1699 1301 2690 2716 2724 2748 2875 2740 2602 2301 3732 3653 3613 3732
F 1778 1301 1778 1699 2568 2398 2477 2114 2477 2544 2778 2477 3643 3672 3690 3771
G 1845 1544 2079 1699 2643 2591 2756 2568 2653 2740 2845 2845 3699 3653 3602 3643
H 2041 1602 2000 1954 2716 2643 2653 2732 2176 2813 2477 2477 3623 3672 3748 3681
L 1544 1778 1301 1602 2591 2591 2230 2580 2813 2544 2903 2301 3724 3663 3748 3771
M 1653 1653 1477 1602 2653 2690 2740 2613 2602 2740 2903 2255 3690 3690 3806 3681
Level 2 results obtained taking account the 3 plates inoculated with the mother-suspension
Level 2 bis results obtained taking account the plates inoculated with 01 ml of the mother-suspension
Scrutiny of the measurement results for consistency
In order to identify the measurement results or laboratories that could be
inconsistent two graphical consistency techniques were realised Mandelrsquos
h-ank-statistics Mandel indicators h and k at 5 significance highlighted
some possible inconsistent data (See tables and graphics in Appendix 3 and
Appendix 4)
Mandelrsquos values Number of values observed over the threshold
Reference method Alternative method
h gt 1 Lab F Level 2 Lab F Level 2
h gt 5 Lab F Level 2
Lab B Levels 1 and 2
Lab A Level 2
Lab D Level 3
Lab F Level 2
Lab L Level 2
kgt 1 Lab F Level 3
k gt 5 Lab F Level 2 Lab C Level 2
Lab F Level 3
Lab L Levels 1 and 3
Lab M Level 1
Thermo Fisher Scientific
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Listeria Precis Enumeration
The emphasized results appear (i) coherent with the targeted contamination
levels (ii) concordant between both methods
All the results were thus kept for the statistical analysis
Comparison of the trueness and precision characteristics of
the reference method and alternative methods
The statistical values are summarised hereafter
Level p
Reference method Alternative method
Repeatability ratio
Reproducibility ratio Median
Repeatability standard deviation
Reproducibility standard deviation
Median Repeatability
standard deviation
Reproducibility standard deviation
1 10 16519 02415 02425 16021 02045 02433 0847 1007
2 10 26551 00543 00659 26719 00914 01325 1682 2011
2 bis 10 26581 02607 02607 25951 01900 01953 0729 0749
3 10 18671 00378 00378 18671 00478 00611 1264 1617
Bias of the alternative method
In order to estimate the bias of the alternative method with regard to the reference method for every
level Dij and t are calculated according to the following equations
RefAlt ijYijYDij
Diffp
Dijit
)2(
)(median
If the t value is superior to 2 the alternative method is significantly biased
with regard to the reference method
The values are given in table 17
Table 17 ndash t (d) values obtained by level
Level Bias t (d) Conclusion
1 0068 114 Non significant bias
2 0021 082 Non significant bias
2 bis - 0037 035 Non significant bias
3 - 0037 043 Non significant bias
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2738 November 19 2014
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Listeria Precis Enumeration
The statistical test concludes to a non significant bias for all the inoculation
levels The bias varies from ndash 0037 to 0068 log CFUml
Comparison of the repeatability standard deviations
If the ratio Srj Alt Srj Ref of the repeatability standard deviations of the
alternative method and the reference method is larger than 2 the precision
under repeatability conditions of the alternative method is considered to be
lower than that of the reference method If this ratio is smaller than 05 the
precision under repeatability conditions of the alternative method is
considered to be greater than that of the reference method
The ratio values are given in table 18
Table 18
Level
Repeatability standard deviation
Reference method Alternative method Alternative method
reference method ratio
1 0242 0205 0847
2 0054 0091 1682
2 bis 0261 0190 0729
3 0038 0048 1264
Level Repeatability limit
Reference method Alternative method
1 0676 0573
2 0152 0256
2 bis 0730 0532
3 0106 0134
The ratios of the repeatability standard deviations are below 2 in all cases
The precision under repeatability conditions of the alternative method is
equivalent to that of the reference method
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2838 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Comparison of the reproducibility standard deviations
If the ratio Srj Alt Srj Ref of the reproducibility standard deviations of the alternative method and the
reference method is larger than 2 the precision under reproducibility conditions of the alternative
method is considered to be lower than that of the reference method If this ratio is smaller than 05 the
precision under reproducibility conditions of the alternative method is considered to be greater than
that of the reference method
The ratio values are given in table 19
Table 19
Level
Reproducibility standard deviation
Reference method Alternative method Alternative method
reference method ratio
1 0242 0243 1007
2 0066 0133 2011
2 bis 0261 0195 0749
3 0038 0061 1617
Level Reproducibility limit
Reference method Alternative method
1 0676 0681
2 0185 0371
2 bis 0730 0547
3 0106 0171
The ratios of the reproducibility standard deviations are below or close to 2
The precision under reproducibility conditions of the alternative method is
equivalent to that of the reference method
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2938 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
214 Conclusion
Method comparison study
The Listeria PrecisTM enumeration method shows satisfying linearity and
relative accuracy
The alternative method shows satisfying inclusivity and exclusivity results
Two Listeria monocytogenes strains gave negative results with the
alternative method These strains grew weakly on the reference method
media (OampA Palcam and Oxford)
The limits of repeatability of the alternative method are similar to those of the
reference method
The bias between both methods are low between - 0133 Log CFUg and
- 0008 Log CFUg
The Listeria PrecisTM enumeration method offers the saving for confirmatory
tests
Positive results are obtained with the alternative method in 2-3 days (with
OBIS Mono test) or in 4-7 days (by the classical tests described in the
standardized method) against 4-7 days for the reference method
Inter-laboratory study
The bias between the reference and the alternative methods shows satisfying
and correct values
- level 1 (100 - 1 000 CFUg) bias = + 007 log CFUg
- level 2 (1 000 - 10 000 CFUg) bias = 002 log CFUg
- level 2 bis (1 000 - 10 000 CFUg) bias = - 004 log CFUg
- level 3 (10 000 - 100 000 CFUg) bias = - 004 log CFUg
The repeatability and the reproducibility of the alternative method look similar
to those of the reference method
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3038 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
22 Extension study (realised by ASEPT 2007)
An extension study was performed in 2007 for a new confirmatory test OBIS
MONO
150 Listeria monocytogenes strains and 100 non-target strains were isolated
onto TSAYE and BrillianceTM Listeria Agar plates All the Listeria
monocytogenes and the non-target strains gave the expected results
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3138 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Appendix 1 ndash Flow diagram of the Listeria PrecisTM method
x g sample + 9 x ml buffered peptone water
Revivification step for 1 h plusmn 5 min at 20degC plusmn 1degC
Streaking 1 ml onto 3 Brilliance Listeria Agar plates (mother suspension)
or 01 ml onto 1 Brilliance Listeria Agar plates (mother suspension and decimal dilutions)
Incubation for 48 h plusmn 3 h at 37degC plusmn 1degC
Confirmatory tests on at least one typical colony
- by the tests described in the ISO method
- by applying the OBIS mono test on an isolated colony
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3238 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Appendix 2 ndash ISO 11290-2 (1998) amp ISO 11290-2A1 (2004) Horizontal
method for the detection and enumeration of Listeria monocytogenes -
Part 2 enumeration of Listeria monocytogenes in foods
10 g cheese + 90 ml buffered peptone water or Half Fraser Broth (without supplements) (110 dilution)
Stomach 1 min 30 seconds
Keep at 20degC plusmn 2degC for 1 h plusmn 5 min
Decimal dilutions in 9 ml peptone salt tubes
Dilution 110
Dilution 1100
Dilution 11000
1 ml on 3 Chromogenic Listeria medium (ISO) plates (x 2) (-1)
01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-2)
01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-3)
01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-4)
Incubation at 37degC plusmn 1degC
24 h - 48 h
Numerate typical colonies at 24 h
(bluegreen surrounded by an opaque halo)
Numerate at 48 h
Streak typical colonies on TSYEA
(5 coloniesplate 2 successive dilutions)
Confirmation tests
Catalase
Gram stain
For confirmation of L monocytogenes
Haemolysis
Carbohydrate (rhamnose and xylose) utilisation
CAMP Test
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3338 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Appendix 3 ndash Mandelrsquos graphics
Laboratoire h Ref
Niveau 1 h Ref
Niveau 2 h Ref
Niveau 3 h Alt
Niveau 1 h Alt
Niveau 2 h Alt
Niveau 3 h5 h1 h5 h1
A -050 -015 083 062 -212 -039 198 299 -198 -299
B -261 -270 021 -077 -058 173 198 299 -198 -299
C -151 -031 -014 -015 -004 -005 198 299 -198 -299
D 038 040 -137 000 072 207 198 299 -198 -299
E -001 090 155 -052 056 -101 198 299 -198 -299
F -112 -321 -153 070 -325 023 198 299 -198 -299
G 042 -071 040 147 -009 176 198 299 -198 -299
H 169 046 -169 192 018 -083 198 299 -198 -299
L 009 -120 021 -077 -231 005 198 299 -198 -299
M 001 031 014 -032 004 -011 198 299 -198 -299
-300
-200
-100
000
100
200
300
A B C D E F G H L M
Vale
urs
h d
e M
an
del(
rob
uste
s)
Laboratoires
Meacutethode de reacutefeacuterence analyse robuste
h Ref Niveau 1
h Ref Niveau 2
h Ref Niveau 3
h 5=198
h 1=299
-300
-200
-100
000
100
200
300
A B C D E F G H L M
Vale
urs
de h
de M
an
del(
rob
uste
s)
Laboratoires
Meacutethode alternative analyse robuste
h Alt Niveau 1
h Alt Niveau 2
h Alt Niveau 2
h 1=299
h 5=198
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3438 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Laboratoire k Ref
Niveau 1 k Ref
Niveau 2 k Ref
Niveau 3 k Alt
Niveau 1 k Alt
Niveau 2 k Alt
Niveau 3 k1 k5
A 000 089 142 084 162 046 259 181
B 125 087 073 104 075 046 259 181
C 117 013 091 188 022 083 259 181
D 052 036 069 000 035 083 259 181
E 028 034 037 138 019 112 259 181
F 140 222 018 027 281 112 259 181
G 088 068 024 131 145 000 259 181
H 129 094 173 016 061 000 259 181
L 069 000 073 104 270 224 259 181
M 000 048 038 043 099 241 259 181
000
050
100
150
200
250
300
350
400
A B C D E F G H L M
Vale
urs
k d
e M
an
del(
rob
uste
s)
Laboratoires
Meacutethode de reacutefeacuterence analyse robuste
k Ref Niveau 1k Ref Niveau 2k Ref Niveau 3
k 1=259
k 5=181
000
050
100
150
200
250
300
A B C D E F G H L M
Vale
urs
k d
e M
an
del
(ro
bu
ste
s)
Laboratoires
Meacutethode alternative analyse robuste
k Alt Niveau 3
k Alt Niveau 1
k Alt Niveau 2
k 1=259
k 5=181
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3538 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Appendix 4 ndash Statistical results
Level 1
Nombre de laboratoires (p) 10
A 1602 1602 1602 000 0000 0000 -0495 0000 A 1845 1602 1724 017 0122 -0122 0621 0840 0122
B 1602 1176 1389 030 0213 -0213 -2613 1247 B 1602 1301 1452 021 0151 -0151 -0769 1041 0062
C 1699 1301 1500 028 0199 -0199 -1510 1165 C 1301 1845 1573 038 -0272 0272 -0148 1881 0073
D 1602 1778 1690 012 -0088 0088 0380 0516 D 1602 1602 1602 000 0000 0000 0000 0000 -0088
E 1699 1602 1651 007 0048 -0048 -0013 0284 E 1699 1301 1500 028 0199 -0199 -0522 1376 -0151
F 1778 1301 1540 034 0239 -0239 -1116 1397 F 1778 1699 1739 006 0040 -0040 0698 0274 0199
G 1845 1544 1695 021 0151 -0151 0425 0881 G 2079 1699 1889 027 0190 -0190 1467 1315 0194
H 2041 1602 1822 031 0220 -0220 1689 1286 H 2000 1954 1977 003 0023 -0023 1917 0158 0155
L 1544 1778 1661 017 -0117 0117 0092 0685 L 1301 1602 1452 021 -0151 0151 -0769 1041 -0210
M 1653 1653 1653 000 0000 0000 0013 0000 M 1477 1602 1540 009 -0062 0062 -0319 0432 -0114
m 165186 m 160206 mediane D 00678
Cn(2p) 1867 Cn(2p) 1867 Cp 16101
Cn(p) 16101 Cn(p) 16101 Qn(D) 0092926
n=2p n 20 n=2p n 20 Qdiff 01496169
f 11 f 11 t 114
l 55 l 55
n=p n 10 n=p n 10
f 6 f 6
l 15 l 15
Qn(2p) 0091465 Qn(2p) 007745
Qn(p) 0062469 Qn(p) 0121519
Qintra 017078 Qintra 014461
Qinter 010058 Qinter 019565
Sr(eacutecart-type de reacutepeacutetabiliteacute) 02415 Sr 02045
RSDr(coefficient de variation de la reacutepeacutetabiliteacute)1462 RSDr 1277
r(repeatability limit)) 0676 r 0573
SL(eacutecart-type interlaboratoire) -001905 SLlt0 SL 001737
SR(eacutecart-type de reproductibiliteacute) 02415 SR 02433
RSDR(coefficient de variation de la reproductibiliteacute)0146 RSDR 0152
R(limite de reproductibiliteacute)) 06762 R(limite de reproductibiliteacute))068123
h k h MoyenneLaboratoires
Niveau 1
si LaboratoiresDeviationMoyenne Dk DeviationsiAlt
Niveau 1
Ref
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3638 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Level 2
Nombre de laboratoires (p) 10
A 2681 2613 2647 005 0034 -0034 -0151 0891 A 2322 2531 2427 015 -0105 0105 -2119 1620 -0220
B 2544 2477 2511 005 0033 -0033 -2699 0872 B 2556 2653 2605 007 -0048 0048 -0581 0750 0094
C 2643 2633 2638 001 0005 -0005 -0311 0130 C 2681 2653 2667 002 0014 -0014 -0040 0217 0029
D 2690 2663 2676 002 0014 -0014 0400 0357 D 2778 2732 2755 003 0023 -0023 0721 0354 0079
E 2690 2716 2703 002 -0013 0013 0897 0336 E 2724 2748 2736 002 -0012 0012 0556 0185 0033
F 2568 2398 2483 012 0085 -0085 -3214 2217 F 2477 2114 2296 026 0182 -0182 -3255 2811 -0188
G 2643 2591 2617 004 0026 -0026 -0707 0682 G 2756 2568 2662 013 0094 -0094 -0085 1453 0045
H 2716 2643 2680 005 0036 -0036 0460 0945 H 2653 2732 2693 006 -0040 0040 0181 0613 0013
L 2591 2591 2591 000 0000 0000 -1196 0000 L 2230 2580 2405 025 -0175 0175 -2307 2704 -0186
M 2653 2690 2672 003 -0018 0018 0311 0481 M 2740 2613 2677 009 0064 -0064 0040 0987 0005
m 2655082 m 26719001 mediane D 00209
Cn(2p) 1867 Cn(2p) 1867 Cp 16101
Cn(p) 16101 Cn(p) 16101 Qn(D) 0039911
n=2p n 20 n=2p n 20 Qdiff 00642591
f 11 f 11 t 082
l 55 l 55
n=p n 10 n=p n 10
f 6 f 6
l 15 l 15
Qn(2p) 0020570 Qn(2p) 00345985
Qn(p) 00332437 Qn(p) 00718158
Qintra 003841 Qintra 006460
Qinter 005352 Qinter 011563
Sr(eacutecart-type de reacutepeacutetabiliteacute) 00543 Sr 00914
RSDr(coefficient de variation de la reacutepeacutetabiliteacute)205 RSDr 342
r(repeatability limit)) 0152 r 0256
SL(eacutecart-type interlaboratoire) 000139 SL 000920
SR(eacutecart-type de reproductibiliteacute) 00659 SR 01325
RSDR(coefficient de variation de la reproductibiliteacute)0025 RSDR 0050
R(limite de reproductibiliteacute)) 018446 R(limite de reproductibiliteacute))03708618
Laboratoires
Niveau 2
Moyenne si Laboratoires
Niveau 2
DRef Alt Moyenne si Deviation h Deviation h k k
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3738 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Level 2 bis
Nombre de laboratoires (p) 10
A 3000 2477 2739 037 0261 -0261 0832 1418 A 2602 2477 2540 009 0062 -0062 -0391 0465 -0199
B 2544 2813 2678 019 -0134 0134 0211 0729 B 2903 2778 2841 009 0062 -0062 1732 0465 0162
C 2477 2813 2645 024 -0168 0168 -0135 0911 C 2477 2699 2588 016 -0111 0111 -0049 0826 -0057
D 2398 2653 2526 018 -0128 0128 -1370 0692 D 2778 3000 2889 016 -0111 0111 2074 0826 0363
E 2875 2740 2808 010 0067 -0067 1547 0365 E 2602 2301 2452 021 0151 -0151 -1012 1120 -0356
F 2477 2544 2511 005 -0033 0033 -1525 0182 F 2778 2477 2628 021 0151 -0151 0230 1120 0117
G 2653 2740 2697 006 -0044 0044 0400 0236 G 2845 2845 2845 000 0000 0000 1764 0000 0148
H 2176 2813 2495 045 -0318 0318 -1692 1727 H 2477 2477 2477 000 0000 0000 -0832 0000 -0017
L 2813 2544 2678 019 0134 -0134 0211 0729 L 2903 2301 2602 043 0301 -0301 0049 2241 -0076
M 2602 2740 2671 010 -0069 0069 0135 0375 M 2903 2255 2579 046 0324 -0324 -0112 2411 -0092
m 2658114 m 259505 mediane D -00372
Cn(2p) 1867 Cn(2p) 1867 Cp 16101
Cn(p) 16101 Cn(p) 16101 Qn(D) 01656915
n=2p n 20 n=2p n 20 Qdiff 02667753
f 11 f 11 t 035
l 55 l 55
n=p n 10 n=p n 10
f 6 f 6
l 15 l 15
Qn(2p) 0098745 Qn(2p) 007195
Qn(p) 006007 Qn(p) 008805
Qintra 018437 Qintra 013435
Qinter 009672 Qinter 014176
Sr(eacutecart-type de reacutepeacutetabiliteacute) 02607 Sr 01900
RSDr(coefficient de variation de la reacutepeacutetabiliteacute)981 RSDr 732
r(repeatability limit)) 0730 r 0532
SL(eacutecart-type interlaboratoire) -002464 SLlt0 SL 000205
SR(eacutecart-type de reproductibiliteacute) 02607 SR 01953
RSDR(coefficient de variation de la reproductibiliteacute)0098 RSDR 0075
R(limite de reproductibiliteacute)) 072996 R(limite de reproductibiliteacute))054686
Laboratoires
Niveau 2 bis
Moyenne si Laboratoires
Niveau 2 bis
DRef Alt Moyenne si Deviation h Deviation h k k
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3838 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Level 3
Nombre de laboratoires (p) 10
A 3690 3663 3676 002 0014 -0014 -0311 0513 A 3690 3708 3699 001 -0009 0009 0354 0257 0022
B 3613 3672 3642 004 -0030 0030 -1851 1109 B 3708 3568 3638 010 0070 -0070 -0844 2061 -0005
C 3663 3732 3698 005 -0035 0035 0645 1302 C 3663 3699 3681 003 -0018 0018 0000 0536 -0017
D 3690 3690 3690 000 0000 0000 0311 0000 D 3643 3663 3653 001 -0010 0010 -0545 0286 -0037
E 3732 3653 3693 006 0040 -0040 0429 1480 E 3613 3732 3673 008 -0060 0060 -0162 1769 -0020
F 3643 3672 3658 002 -0014 0014 -1157 0536 F 3690 3771 3731 006 -0040 0040 0975 1193 0073
G 3699 3653 3676 003 0023 -0023 -0328 0856 G 3602 3643 3623 003 -0021 0021 -1141 0612 -0053
H 3623 3672 3648 003 -0024 0024 -1615 0913 H 3748 3681 3715 005 0033 -0033 0664 0990 0067
L 3724 3663 3694 004 0031 -0031 0461 1150 L 3748 3771 3760 002 -0011 0011 1544 0335 0066
M 3690 3690 3690 000 0000 0000 0311 0000 M 3806 3681 3744 009 0062 -0062 1234 1848 0054
m 368334 m 368086 mediane D 00089
Cn(2p) 1867 Cn(2p) 1867 Cp 16101
Cn(p) 16101 Cn(p) 16101 Qn(D) 00325359
n=2p n 20 n=2p n 20 Qdiff 00523852
f 11 f 11 t 043
l 55 l 55
n=p n 10 n=p n 10
f 6 f 6
l 15 l 15
Qn(2p) 0014323 Qn(2p) 001811
Qn(p) 0013719 Qn(p) 003164
Qintra 002674 Qintra 003381
Qinter 002209 Qinter 005094
Sr(eacutecart-type de reacutepeacutetabiliteacute) 00378 Sr 00478
RSDr(coefficient de variation de la reacutepeacutetabiliteacute)103 RSDr 130
r(repeatability limit)) 0106 r 0134
SL(eacutecart-type interlaboratoire) -000023 SLlt0 SL 000145
SR(eacutecart-type de reproductibiliteacute) 00378 SR 00611
RSDR(coefficient de variation de la reproductibiliteacute)0010 RSDR 0017
R(limite de reproductibiliteacute)) 0105894 R(limite de reproductibiliteacute))017119
Ref Alt DLaboratoires
Niveau 3
Moyenne si Deviation h k Laboratoires
Niveau 3
Moyenne si Deviation h k
Thermo Fisher Scientific
ADRIA Deacuteveloppement 238 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
1 INTRODUCTION 4
11 Dates of the validation studies 4
12 Protocol and principle of the alternative method 4
13 Standard method 4
2 VALIDATION STUDY RESULTS 5
21 Initial validation (realised by ASEPT 2006) 5
211 Method Comparison Study 5
2111 Linearity 5
2112 Relative accuracy 9
2113 Detection limit (LOD) and quantification limit (LOQ) 17
2114 Relative Sensitivity 18
2115 SpecificitySelectivity 19
212 Practicability 20
213 Inter-laboratory study 21
2131 Organisation 21
2132 Verification of experimental parameters 21
2133 Results 23
214 Conclusion 29
22 Extension study (realised by ASEPT 2007) 30
Appendix 1 ndash Flow diagram of the Listeria PrecisTM method __________________________________ 31
Appendix 2 ndash ISO 11290-2 (1998) amp ISO 11290-2A1 (2004) Horizontal method for the detection and
enumeration of Listeria monocytogenes - Part 2 enumeration of Listeria monocytogenes in foods ____ 32
Appendix 3 ndash Mandelrsquos graphics _______________________________________________________ 33
Appendix 4 ndash Statistical results ________________________________________________________ 35
Thermo Fisher Scientific
ADRIA Deacuteveloppement 338 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
The modifications are highlighted
Quality assurance documents related to this study can be consulted upon
request from Oxoid part of Thermo Fisher Scientific
The technical protocol and the result interpretation were realised according to
the EN ISO 16140 and the AFNOR technical rules
o Company Oxoid Ltd
part of THERMO FISHER SCIENTIFIC
Wade Road Basingstoke
Hampshire RG24 8PW UK
o Expert Laboratory ADRIA Deacuteveloppement
ZA Creacrsquoh Gwen
F-29196 QUIMPER Cedex
o Studied method Listeria PrecisTM method for enumeration of
Listeria monocytogenes
o Validation standard NF EN ISO 16140 (October 2003) Food
microbiology ndash Protocol for the validation of
alternative methods
o Standard method ISO 11290-2 (1999) amp ISO 11290-2A1 (2004)
Horizontal method for the detection and
enumeration of Listeria monocytogenes - Part 2
enumeration of Listeria monocytogenes in foods
o Scope All human food products and environmental
samples
o Certification organism AFNOR Certification
Thermo Fisher Scientific
ADRIA Deacuteveloppement 438 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
1 INTRODUCTION
11 Dates of the validation studies
The Listeria PrecisTM method for enumeration of Listeria monocytogenes was
validated on September 15 2006 (certificate number UNI 0305 ndash 0906) for
food products and environmental samples
In 2007 an extension study was obtained for a new confirmation test OBIS
MONO
The alternative method was renewed in 2010 and 2014
12 Protocol and principle of the alternative method
The Listeria PrecisTM method is based on a specific chromogenic media for
Listeria monocytogenes enumeration
The protocol is described in Appendix 1
13 Standard method
The reference method is the ISO 11290-2 (1998) amp ISO 11290-2A1 (2004)
Horizontal method for the detection and enumeration of Listeria
monocytogenes - Part 2 enumeration of Listeria monocytogenes in foods
(See Appendix 2)
Analysis performed according to the COFRAC accreditation
Thermo Fisher Scientific
ADRIA Deacuteveloppement 538 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
2 VALIDATION STUDY RESULTS
21 Initial validation (realised by ASEPT 2006)
211 Method Comparison Study
2111 Linearity
Linearity is the ability of the method when used with a given matrix to give results that are in proportion
to the amount of analyte present in the sample that is an increase in analyte corresponds to a linear or
proportional increase in results
Food matrices and protocols
Six matrixstrain pairs were analysed with five contamination levels and two
replicates At least 50 analyses were performed by the alternative and the
standard methods
The contamination levels the samples and the inoculated strains are
presented in the table below
Table 1
Samples Strains Contamination levels
(CFUg)
Rillettes Listeria monocytogenes 4e 10 ndash 50
50 ndash 100
100 ndash 500
500 ndash 1 000
1 000 ndash 10 000
Raw milk Listeria monocytogenes 12a
Lettuce Listeria monocytogenes 12a
Smoked salmon Listeria monocytogenes 12b
Raw egg product Listeria monocytogenes 12b
Process water Listeria monocytogenes 12a
The samples were individually inoculated and were analysed in duplicate by
the alternative and the reference methods
Results
The bi-dimensional graphs are shown figure 1
Thermo Fisher Scientific
ADRIA Deacuteveloppement 638 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Figure 1 ndash Bi-dimensional graphs
Rillettes Raw milk
Smoked salmon Raw egg product
Lettuce Process water
Thermo Fisher Scientific
ADRIA Deacuteveloppement 738 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Statistical interpretations
Statistical interpretation results are provided in Table 2
Table 2 ndash Statistical interpretations
Matrix R Selected
regression
RobF Critical
value
P Correlation
coefficient
Regression equation
Rillettes 180 GMFR 609 541 4 09960 log (Alt) = 09887 log (Ref) + 01141
Lettuce 108 GMFR 000 541 100 09992 Log (Alt) = 10081 log (Ref) - 00257
Raw milk 102 GMFR 000 541 100 09997 Log (Alt) = 10188 log (Ref) - 00737
Smoked salmon 1048 OLS1 1806 541 263 09984 Log (Alt) = 10406 log (Ref) - 01418
Raw egg product 130 GMFR 548 541 49 09958 Log (Alt) = 09775 log (Ref) - 00161
Process water 184 GMFR 0079 541 969 09985 Log (Alt) = 09680 log (Ref) + 01317
GMFR = orthogonal linear regression
OLS1 = ordinary least-square linear regression
x ndash axis = reference method
y ndash axis = alternative method
OLS1 = ordinary least-square linear regression
x axis = alternative method
y axis = reference method
Statistical Interpretation
P gt 5 not significant 1 lt P lt 5 significant
P lt 1 highly significant
The regressions straight lines are shown figure 2
Thermo Fisher Scientific
ADRIA Deacuteveloppement 838 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Figure 2 ndash Regression straight lines
Lineacuteariteacute - Œuf
Process water Process water (2) Process water
Smoked salmon (GMFR) Smoked salmon (OLS 1)
Raw milk Raw egg product
Rillettes Lettuce
Thermo Fisher Scientific
ADRIA Deacuteveloppement 938 November 19 2014
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Listeria Precis Enumeration
Discussion
The linearity is accepted in all cases with
P values 5 and correlation coefficients superior to 099
For the matrix ldquoRillettesldquo the linearity test is significant (P = 4) with a high
correlation coefficient value (0996)
The Listeria PrecisTM method shows satisfying linearity
2112 Relative accuracy
The accuracy is the closeness of agreement between a test result and the accepted reference value
The bias is the difference between the expectation of the test results and an accepted reference value
Number and nature of the samples
The repartition per category and types is provided in table 3
Table 3 ndash Number and nature of the samples
Categories Types Number of samples
Analysed Exploited (1)
Meat products
Raw meat products 6 4
Ready to cook products 4 2
Ready to eat delicatessen 10 4
Total 20 10
Dairy products
Raw milk cheeses 7 4
Raw milks 4 3
Other dairy products 5 5
Total 16 12
Fishery products
Raw fishes 5 4
Processed fishes 7 2
Ready-to-eat products 6 4
Total 18 10
Vegetables
Raw vegetables 5 4
Frozen vegetables 3 3
Lettuce raw vegetables 5 5
Total 13 12
Egg products
Raw egg 5 5
Pastries 3 3
Pasteurised egg products 4 3
Total 12 11
Environmental samples
Wipes swabs 8 4
Process water 3 3
Dusts 3 3
Total 14 10
TOTAL 93 65
(1) Some data cannot be exploited because enumeration was under the detection level for at least one of the
method
Thermo Fisher Scientific
ADRIA Deacuteveloppement 1038 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Artificial contamination of the samples
Artificial contaminations were realised by spiking after performing injury
protocols on pure cultures or by cross-contaminations
56 samples were artificially contaminated 53 gave exploitable results among
a total of 65 samples 8150 of the samples were artificially contaminated
The injury protocol and the strains inoculated are provided in Table 4
Table 4
Stress Ndeg Injury protocol Strain Origin
A 45 min at 50 degC
48
LM -H123
LM-H170
Smoked salmon
Lettuce
Lettuce
B 30 min at 50 degC
and 2 h at ndash20degC
18
43
LM-O1
Raw milk (environment)
Environment
Egg product
C 30 min at 55degC
18
48
LM-H123
Raw milk
Smoked salmon
Lettuce
D 30 min at 50degC
et 2 h at ndash80degC
34
LM-H171
LM-O1
Rillettes
Lettuce
Egg product
E 30 min at 60 degC LM-O1 Egg product
Confirmatory tests
For the reference and the alternative methods confirmations were realised
by the tests described in the ISO 12290-2A1 standard For the alternative
method only one colony was confirmed while 5 colonies were tested for the
reference method
Results
Samples were analysed in duplicate by the reference and the alternative
methods
The contamination range is provided in table 5 per tested category
Thermo Fisher Scientific
ADRIA Deacuteveloppement 1138 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Table 5 ndash Contamination range
Food category Contamination level (log CFUg)
Meat products 100 ndash 404
Dairy products 148 ndash 408
Fishery products 100 ndash 384
Vegetables 170 ndash 511
Egg products 130 ndash 499
Environmental samples 170 ndash 511
All categories 100 ndash 511
Bi-dimensional graphs are given figure 3
Figure 3 ndash Bi-dimensional graphs
Graphe bi-dimensionnel Produits carneacutes
Fishery products
Vegetables
Egg products
Meat products
Thermo Fisher Scientific
ADRIA Deacuteveloppement 1238 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Statistical interpretation according to EN ISO16140 method
The results of the statistical interpretation are given in Table 6
Table 6 ndash Statistical interpretation results
Category n R Regression
used a t(a) b t(b) Critical T
P
Ordinate
at 0
Slope
at 1
Meat products 10 158 GMFR -0455 3321 115 3123 2306 001 001
Dairy products 12 065 GMFR -0027 0385 1002 0108 2228 092 071
Fishery products 10 130 GMFR -0305 1024 1034 0312 2306 076 034
Vegetables 12 086 GMFR -0015 0128 0993 0191 2228 085 090
Egg products 11 158 GMFR -0038 0866 1001 0090 2262 093 041
Environmental
samples 10 176 GMFR -0139 265 1034 231 2306 005 003
All products 65 118 GMFR -0182 3042 1040 2145 2000 004 000
All products
(estimated number) 65 118 GMFR -0182 3042 1040 2145 2000 004 000
All products (non
estimated results) 20 115 GMFR -0367 151 1138 1141 2101 027 015
Repeatability limits obtained for the alternative and reference methods as
well as bias obtained are presented in Table 7
Dairy products Environmental samples
Log (Ref) Log (Ref)
All categories
Thermo Fisher Scientific
ADRIA Deacuteveloppement 1338 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Table 7
Categories Bias
Repeatability limits
Rob r
Ref Alt
Meat products 0008 0314 0343
Dairy products -0031 0136 0130
Fishery products -0133 0220 0214
Vegetables -0041 0180 0319
Egg products -0026 0219 0206
Environmental samples -0007 0198 0119
All products -0032 0196 0215
Regression straight lines (graph and equation representations) for each food
category and for all products are presented figure 4
Thermo Fisher Scientific
ADRIA Deacuteveloppement 1438 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Figure 4 ndash Regression straight lines
Meat products
Fishery products
Dairy products
Vegetables
Thermo Fisher Scientific
ADRIA Deacuteveloppement 1538 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Environmental samples
Egg products
All products (estimated number)
Thermo Fisher Scientific
ADRIA Deacuteveloppement 1638 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
The hypotheses that the ordinates are close to 0 and the slopes close to 1
are approved in all cases except for
- Meat products (slope P = 1 ordinate P = 1)
- Environmental samples (ordinate P = 3)
- All products (ordinate P = 0)
The bias between the two methods vary from ndash 0133 to 0008 log CFU For
all categories the bias is ndash 0032 log CFU The repeatability limits of the
alternative and the reference method are similar For all products the values
are respectively 0215 and 0196 log CFU For all products these
interpretations are provided taking into account data with low contamination
level or not (estimated colonies number)
For all food products the regression straight line is the following
log (Alt) = 1040 log Ref ndash 0182
The Listeria PrecisTM method shows satisfying relative accuracy
All products
All products (Level 2)
Thermo Fisher Scientific
ADRIA Deacuteveloppement 1738 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
2113 Detection limit (LOD) and quantification limit (LOQ)
The critical level is the defined as the smallest amount which can be detected (not null) but not
quantified as an exact value Below this value it cannot be sure that the true value is not null
The detection limit is defined as being higher than the critical level because it involves a power the
probability 1- which has to be well over 50 for example 95
The quantification limit is defined as the smallest amount of analyte (that is the lowest actual number of
organisms) which can be measured and quantified with defined precision and accuracy under the
experimental conditions by the method under validation
Protocol
The detection limit of the alternative method was realised with pure cultures
Three different inoculation levels were tested with six replicates per level ie
18 analyses were done by the alternative method
Listeria monocytogenes 18 was used for the detection limit of the alternative
method
Quantification limit was calculated for six independent blank samples
determinations
Results
These data are intrinsic to the method used and are presented in the
following tables
Table 8 ndash Inoculation 1 ml on 3 plates
Level (CFUml) Positive samples number Standard deviation Bias
0 06
05 26 1211 0
1 36 0816 05
5 66 2483 85
Thermo Fisher Scientific
ADRIA Deacuteveloppement 1838 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Table 9 ndash Inoculation 01 ml on 1 plate
Level (CFUml) Positive samples number Standard deviation Bias
0 06
5 36 0548 05
10 46 1414 2
50 66 2258 55
Table 10
Formulas Obtained values
LC 165 S0 + X0 18
LOD 33 S0 + X0 32
LOQ 10 S0 + X0 87
2114 Relative Sensitivity
The relative sensitivity is defined as the ability of the alternative method to detect two different amounts
of analyte measured by the reference method within a given matrix at a specified average value or
over the whole measurement range that is it is the minimal quantity variation (increase of the analyte
concentration x) which gives a significant variation of the measured signal (response y)
These data are intrinsic to the used method and are obtained from the results
of the linearity study
Sensitivity patterns obtained for tested (matrixstrain) pairs are presented in
figure 5
Thermo Fisher Scientific
ADRIA Deacuteveloppement 1938 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Figure 5 ndash Sensitivity patterns for the tested (matrixstrain) pairs
2115 SpecificitySelectivity
The specificity is defined as the degree to which a method is affected (or not) by the other components
present in a multi-component sample That is the ability of a method to measure exactly a given
analyte or its amount within the sample without interference from non-target components such as a
matrix effect or background noise
The selectivity is defined as a measure of the degree of non-interference in the presence of non-target
analytes A method is selective if it can be used to detect the analyte under examination and that a
guarantee can be provided that the detected signal can only be a product by that specific analyte
The specificity and selectivity study was performed for the Listeria PrecisTM
detection method no additional test was realised for the enumeration
method The results were the following
- Among the Listeria monocytogenes strains tested 2 strains (Listeria
monocytogenes CIP 7834 and Listeria monocytogenes CIP 105459) gave
negative results These 2 strains were tested twice by running 48 h
incubation time of the compared agars Positive results were then
observed by the alternative method Pale characteristic colonies were
observed on the ISO agar by streaking the Half Fraser broth only
- No cross reaction was observed with the non-target strains except with
Listeria ivanovii which gave characteristic colonies on Brilliancetrade Listeria
Agar but the confirmatory tests provide a clear discrimination between
Listeria monocytogenes and Listeria ivanovi species
Rillettes
Lettuce
Raw milk
Smoked salmon
Raw egg
Process water
x (CFUg)
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2038 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
212 Practicability
1 Packaging Ready to use Brilliance Listeria agar plates are available
(x 10 plates)
2 Reagent volume
3 Storage conditionsndash
Shelf life of products
not opened
The plates are stored at 6 - 12degC in darkness conditions The
expiration date is mentioned on the package and on the plates It
is around 3 months
4 Modalities of use
after first use
5 Required
equipments No specific equipment is required
6 Reagents Ready to use plates
7 Training 05 day
8 Real time handling
Reference method
9 min for one sample
55 min for 10 samples
Alternative method
7 min for one sample
35 minutes for 10 samples
9 Time to result
Negative results
Reference method Day 2
Alternative method Day 2
Negative results after confirmation or positive results
Reference method Day 4 ndash Day 7
Alternative method Day 4 ndash Day 7
Positive results
Reference method Day 4 ndash Day 7
Alternative method Day 2 ndash Day 3 (with OBIS Mono Test)
or Day 4 ndash Day 7 (by the classical tests described in the
standardised method)
10 Technician skill Identical to that required for the reference method
11 Steps common to
the reference method Mother suspension revivification step and decimal dilutions
12 Traceability
13 Maintenance
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2138 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
213 Inter-laboratory study
2131 Organisation
12 laboratories participated to this study Pasteurised milk was inoculated by
Listeria monocytogenes 12a isolated from raw milk The targeted
inoculation levels were
- 0 CFUml
- 50 CFU ml
- 500 CFU ml
- 5 000 CFU ml
Each laboratory received eight flasks of 10 ml sample ie two flasks per
inoculation level Furthermore one non-inoculated sample has been added
to the package for total viable count microflora (NF ISO 4833 method)
Detailed instructions were transmitted to the collaborators by the expert
laboratory
Collaborative study laboratories and the expert laboratory carried out the
analyses with the alternative and reference methods
2132 Verification of experimental parameters
Strain stability during transport
In order to evaluate the Listeria monocytogenes strain stability during
transport bacterial count of all inoculated flasks were checked at different
time ie inoculation time after 24 h of conservation at 2deg - 8degC Results are
reported in table 11
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2238 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Table 11 ndash Listeria monocytogenes count
by ISO 11290-2A1 method (log CFUml)
Day of the analyses Level contamination Reference method
Day 0
(29052006)
1 45
2 440
3 4 500
Day 1
(30052006)
1 60
2 430
3 4 900
No evolution of the strain was observed after storage for 24 h at 2degC ndash 8degC
Contamination level
The real contamination levels are provided in table 12
Table 12 ndash Real contamination level (CFUml)
Target inoculation
(CFU ml) Samples Ndeg
Real contamination levels
(CFUml)
0 1 - 8 0
50 5 - 6 45
500 3 - 7 440
5000 2 - 4 4 500
Sample temperature at receipt
Measured temperatures at receipt are listed in table 13
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2338 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Table 13 - Sample temperature on receipt
Laboratories Receipt
day
Hour
receipt
Measured temperature
at receipt (degC)
Measured temperature by the
temperature probe (degC)
A
B
C
D
E
F
G
H
I
J
K
L
M
30052006
10h45
10h15
11h15
10h00
09h30
8h30
11h50
10h00
08h30
14h45
08h30
09h45
08h45
82
55
45
63
60
62
81
38
48
-
14
67
76
03
03
-13
03
04
-14
05
04
01
05
-13
04
Conclusion
The temperatures during the transport were correct for all the labs except for
Lab J which didnrsquot provide the information
All the labs received their packages with several broken flasks
Three labs (B D and L) received some damaged samples but proceeded to
analyses
All the labs started the analyses at Day 1 except Lab J
2133 Results
Aerobic mesophilic flora
The count of aerobic mesophilic flora varied from 94 102 CFUml to
33 103 CFUml
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2438 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Expert lab results
The results obtained by the expert lab are the following
Table 14
Target inoculation level (CFUml)
Reference method ISO 11290-2
Alternative method Listeria Precis Enumeration
Rep 1 Rep 2 Rep 1 Rep 2
lt 10 lt 10 lt 10 lt 10 lt10
50 50 (Ne) 60 (Ne) 50 (Ne) 30 (Ne)
500 470 390 490 500
5 000 4 700 5 100 5 900 3 400
Ne estimated number
The obtained results are in agreement with those expected
Collaborator Labs results
A synthesis of the results obtained by the collaborator labs is provided
Table 15 (CFUml) and Table 16 (log CFUml)
Table 15 ndash Synthesis of results obtained by the alternative
and the reference methods (CFUml)
Labs Level 1 Level 2 Level 2 bis Level 3
Reference method
Alternative method
Reference method
Alternative method
Reference method
Alternative method
Reference method
Alternative method
A 40 40 70 40 480 410 210 340 1000 300 400 300 4900 4600 4900 5100
B 40 15 40 20 350 300 360 450 350 650 800 600 4100 4700 5100 3700
C 50 20 20 70 440 430 480 450 300 650 300 500 4600 5400 4600 5000
D 40 60 40 40 490 460 600 540 250 450 600 1000 4900 4900 4400 4600
E 50 40 50 20 490 520 530 560 750 550 400 200 5400 4500 4100 5400
F 60 20 60 50 370 250 300 130 300 350 600 300 4400 4700 4900 5900
G 70 35 120 50 440 390 570 370 450 550 700 700 5000 4500 4000 4400
H 110 40 100 90 520 440 450 540 150 650 300 300 4200 4700 5600 4800
L 35 60 20 40 390 390 170 380 650 350 800 200 5300 4600 5600 5900
M 45 45 30 40 450 490 550 410 400 550 800 180 4900 4900 6400 4800
Thermo Fisher Scientific
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Summary Report (Version 1)
Listeria Precis Enumeration
Table 16 ndash Synthesis of results obtained by the alternative
and the reference methods (log CFUml)
Labs Level 1 Level 2 Level 2 bis Level 3
Reference method
Alternative method
Reference method
Alternative method
Reference method
Alternative method
Reference method
Alternative method
A 1602 1602 1845 1602 2681 2613 2322 2531 3000 2477 2602 2477 3690 3663 3690 3708
B 1602 1176 1602 1301 2544 2477 2556 2653 2544 2813 2903 2778 3613 3672 3708 3568
C 1699 1301 1301 1845 2643 2633 2681 2653 2477 2813 2477 2699 3663 3732 3663 3699
D 1602 1778 1602 1602 2690 2663 2778 2732 2398 2653 2778 3000 3690 3690 3643 3663
E 1699 1602 1699 1301 2690 2716 2724 2748 2875 2740 2602 2301 3732 3653 3613 3732
F 1778 1301 1778 1699 2568 2398 2477 2114 2477 2544 2778 2477 3643 3672 3690 3771
G 1845 1544 2079 1699 2643 2591 2756 2568 2653 2740 2845 2845 3699 3653 3602 3643
H 2041 1602 2000 1954 2716 2643 2653 2732 2176 2813 2477 2477 3623 3672 3748 3681
L 1544 1778 1301 1602 2591 2591 2230 2580 2813 2544 2903 2301 3724 3663 3748 3771
M 1653 1653 1477 1602 2653 2690 2740 2613 2602 2740 2903 2255 3690 3690 3806 3681
Level 2 results obtained taking account the 3 plates inoculated with the mother-suspension
Level 2 bis results obtained taking account the plates inoculated with 01 ml of the mother-suspension
Scrutiny of the measurement results for consistency
In order to identify the measurement results or laboratories that could be
inconsistent two graphical consistency techniques were realised Mandelrsquos
h-ank-statistics Mandel indicators h and k at 5 significance highlighted
some possible inconsistent data (See tables and graphics in Appendix 3 and
Appendix 4)
Mandelrsquos values Number of values observed over the threshold
Reference method Alternative method
h gt 1 Lab F Level 2 Lab F Level 2
h gt 5 Lab F Level 2
Lab B Levels 1 and 2
Lab A Level 2
Lab D Level 3
Lab F Level 2
Lab L Level 2
kgt 1 Lab F Level 3
k gt 5 Lab F Level 2 Lab C Level 2
Lab F Level 3
Lab L Levels 1 and 3
Lab M Level 1
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2638 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
The emphasized results appear (i) coherent with the targeted contamination
levels (ii) concordant between both methods
All the results were thus kept for the statistical analysis
Comparison of the trueness and precision characteristics of
the reference method and alternative methods
The statistical values are summarised hereafter
Level p
Reference method Alternative method
Repeatability ratio
Reproducibility ratio Median
Repeatability standard deviation
Reproducibility standard deviation
Median Repeatability
standard deviation
Reproducibility standard deviation
1 10 16519 02415 02425 16021 02045 02433 0847 1007
2 10 26551 00543 00659 26719 00914 01325 1682 2011
2 bis 10 26581 02607 02607 25951 01900 01953 0729 0749
3 10 18671 00378 00378 18671 00478 00611 1264 1617
Bias of the alternative method
In order to estimate the bias of the alternative method with regard to the reference method for every
level Dij and t are calculated according to the following equations
RefAlt ijYijYDij
Diffp
Dijit
)2(
)(median
If the t value is superior to 2 the alternative method is significantly biased
with regard to the reference method
The values are given in table 17
Table 17 ndash t (d) values obtained by level
Level Bias t (d) Conclusion
1 0068 114 Non significant bias
2 0021 082 Non significant bias
2 bis - 0037 035 Non significant bias
3 - 0037 043 Non significant bias
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2738 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
The statistical test concludes to a non significant bias for all the inoculation
levels The bias varies from ndash 0037 to 0068 log CFUml
Comparison of the repeatability standard deviations
If the ratio Srj Alt Srj Ref of the repeatability standard deviations of the
alternative method and the reference method is larger than 2 the precision
under repeatability conditions of the alternative method is considered to be
lower than that of the reference method If this ratio is smaller than 05 the
precision under repeatability conditions of the alternative method is
considered to be greater than that of the reference method
The ratio values are given in table 18
Table 18
Level
Repeatability standard deviation
Reference method Alternative method Alternative method
reference method ratio
1 0242 0205 0847
2 0054 0091 1682
2 bis 0261 0190 0729
3 0038 0048 1264
Level Repeatability limit
Reference method Alternative method
1 0676 0573
2 0152 0256
2 bis 0730 0532
3 0106 0134
The ratios of the repeatability standard deviations are below 2 in all cases
The precision under repeatability conditions of the alternative method is
equivalent to that of the reference method
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2838 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Comparison of the reproducibility standard deviations
If the ratio Srj Alt Srj Ref of the reproducibility standard deviations of the alternative method and the
reference method is larger than 2 the precision under reproducibility conditions of the alternative
method is considered to be lower than that of the reference method If this ratio is smaller than 05 the
precision under reproducibility conditions of the alternative method is considered to be greater than
that of the reference method
The ratio values are given in table 19
Table 19
Level
Reproducibility standard deviation
Reference method Alternative method Alternative method
reference method ratio
1 0242 0243 1007
2 0066 0133 2011
2 bis 0261 0195 0749
3 0038 0061 1617
Level Reproducibility limit
Reference method Alternative method
1 0676 0681
2 0185 0371
2 bis 0730 0547
3 0106 0171
The ratios of the reproducibility standard deviations are below or close to 2
The precision under reproducibility conditions of the alternative method is
equivalent to that of the reference method
Thermo Fisher Scientific
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Summary Report (Version 1)
Listeria Precis Enumeration
214 Conclusion
Method comparison study
The Listeria PrecisTM enumeration method shows satisfying linearity and
relative accuracy
The alternative method shows satisfying inclusivity and exclusivity results
Two Listeria monocytogenes strains gave negative results with the
alternative method These strains grew weakly on the reference method
media (OampA Palcam and Oxford)
The limits of repeatability of the alternative method are similar to those of the
reference method
The bias between both methods are low between - 0133 Log CFUg and
- 0008 Log CFUg
The Listeria PrecisTM enumeration method offers the saving for confirmatory
tests
Positive results are obtained with the alternative method in 2-3 days (with
OBIS Mono test) or in 4-7 days (by the classical tests described in the
standardized method) against 4-7 days for the reference method
Inter-laboratory study
The bias between the reference and the alternative methods shows satisfying
and correct values
- level 1 (100 - 1 000 CFUg) bias = + 007 log CFUg
- level 2 (1 000 - 10 000 CFUg) bias = 002 log CFUg
- level 2 bis (1 000 - 10 000 CFUg) bias = - 004 log CFUg
- level 3 (10 000 - 100 000 CFUg) bias = - 004 log CFUg
The repeatability and the reproducibility of the alternative method look similar
to those of the reference method
Thermo Fisher Scientific
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Summary Report (Version 1)
Listeria Precis Enumeration
22 Extension study (realised by ASEPT 2007)
An extension study was performed in 2007 for a new confirmatory test OBIS
MONO
150 Listeria monocytogenes strains and 100 non-target strains were isolated
onto TSAYE and BrillianceTM Listeria Agar plates All the Listeria
monocytogenes and the non-target strains gave the expected results
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3138 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Appendix 1 ndash Flow diagram of the Listeria PrecisTM method
x g sample + 9 x ml buffered peptone water
Revivification step for 1 h plusmn 5 min at 20degC plusmn 1degC
Streaking 1 ml onto 3 Brilliance Listeria Agar plates (mother suspension)
or 01 ml onto 1 Brilliance Listeria Agar plates (mother suspension and decimal dilutions)
Incubation for 48 h plusmn 3 h at 37degC plusmn 1degC
Confirmatory tests on at least one typical colony
- by the tests described in the ISO method
- by applying the OBIS mono test on an isolated colony
Thermo Fisher Scientific
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Summary Report (Version 1)
Listeria Precis Enumeration
Appendix 2 ndash ISO 11290-2 (1998) amp ISO 11290-2A1 (2004) Horizontal
method for the detection and enumeration of Listeria monocytogenes -
Part 2 enumeration of Listeria monocytogenes in foods
10 g cheese + 90 ml buffered peptone water or Half Fraser Broth (without supplements) (110 dilution)
Stomach 1 min 30 seconds
Keep at 20degC plusmn 2degC for 1 h plusmn 5 min
Decimal dilutions in 9 ml peptone salt tubes
Dilution 110
Dilution 1100
Dilution 11000
1 ml on 3 Chromogenic Listeria medium (ISO) plates (x 2) (-1)
01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-2)
01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-3)
01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-4)
Incubation at 37degC plusmn 1degC
24 h - 48 h
Numerate typical colonies at 24 h
(bluegreen surrounded by an opaque halo)
Numerate at 48 h
Streak typical colonies on TSYEA
(5 coloniesplate 2 successive dilutions)
Confirmation tests
Catalase
Gram stain
For confirmation of L monocytogenes
Haemolysis
Carbohydrate (rhamnose and xylose) utilisation
CAMP Test
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3338 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Appendix 3 ndash Mandelrsquos graphics
Laboratoire h Ref
Niveau 1 h Ref
Niveau 2 h Ref
Niveau 3 h Alt
Niveau 1 h Alt
Niveau 2 h Alt
Niveau 3 h5 h1 h5 h1
A -050 -015 083 062 -212 -039 198 299 -198 -299
B -261 -270 021 -077 -058 173 198 299 -198 -299
C -151 -031 -014 -015 -004 -005 198 299 -198 -299
D 038 040 -137 000 072 207 198 299 -198 -299
E -001 090 155 -052 056 -101 198 299 -198 -299
F -112 -321 -153 070 -325 023 198 299 -198 -299
G 042 -071 040 147 -009 176 198 299 -198 -299
H 169 046 -169 192 018 -083 198 299 -198 -299
L 009 -120 021 -077 -231 005 198 299 -198 -299
M 001 031 014 -032 004 -011 198 299 -198 -299
-300
-200
-100
000
100
200
300
A B C D E F G H L M
Vale
urs
h d
e M
an
del(
rob
uste
s)
Laboratoires
Meacutethode de reacutefeacuterence analyse robuste
h Ref Niveau 1
h Ref Niveau 2
h Ref Niveau 3
h 5=198
h 1=299
-300
-200
-100
000
100
200
300
A B C D E F G H L M
Vale
urs
de h
de M
an
del(
rob
uste
s)
Laboratoires
Meacutethode alternative analyse robuste
h Alt Niveau 1
h Alt Niveau 2
h Alt Niveau 2
h 1=299
h 5=198
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3438 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Laboratoire k Ref
Niveau 1 k Ref
Niveau 2 k Ref
Niveau 3 k Alt
Niveau 1 k Alt
Niveau 2 k Alt
Niveau 3 k1 k5
A 000 089 142 084 162 046 259 181
B 125 087 073 104 075 046 259 181
C 117 013 091 188 022 083 259 181
D 052 036 069 000 035 083 259 181
E 028 034 037 138 019 112 259 181
F 140 222 018 027 281 112 259 181
G 088 068 024 131 145 000 259 181
H 129 094 173 016 061 000 259 181
L 069 000 073 104 270 224 259 181
M 000 048 038 043 099 241 259 181
000
050
100
150
200
250
300
350
400
A B C D E F G H L M
Vale
urs
k d
e M
an
del(
rob
uste
s)
Laboratoires
Meacutethode de reacutefeacuterence analyse robuste
k Ref Niveau 1k Ref Niveau 2k Ref Niveau 3
k 1=259
k 5=181
000
050
100
150
200
250
300
A B C D E F G H L M
Vale
urs
k d
e M
an
del
(ro
bu
ste
s)
Laboratoires
Meacutethode alternative analyse robuste
k Alt Niveau 3
k Alt Niveau 1
k Alt Niveau 2
k 1=259
k 5=181
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3538 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Appendix 4 ndash Statistical results
Level 1
Nombre de laboratoires (p) 10
A 1602 1602 1602 000 0000 0000 -0495 0000 A 1845 1602 1724 017 0122 -0122 0621 0840 0122
B 1602 1176 1389 030 0213 -0213 -2613 1247 B 1602 1301 1452 021 0151 -0151 -0769 1041 0062
C 1699 1301 1500 028 0199 -0199 -1510 1165 C 1301 1845 1573 038 -0272 0272 -0148 1881 0073
D 1602 1778 1690 012 -0088 0088 0380 0516 D 1602 1602 1602 000 0000 0000 0000 0000 -0088
E 1699 1602 1651 007 0048 -0048 -0013 0284 E 1699 1301 1500 028 0199 -0199 -0522 1376 -0151
F 1778 1301 1540 034 0239 -0239 -1116 1397 F 1778 1699 1739 006 0040 -0040 0698 0274 0199
G 1845 1544 1695 021 0151 -0151 0425 0881 G 2079 1699 1889 027 0190 -0190 1467 1315 0194
H 2041 1602 1822 031 0220 -0220 1689 1286 H 2000 1954 1977 003 0023 -0023 1917 0158 0155
L 1544 1778 1661 017 -0117 0117 0092 0685 L 1301 1602 1452 021 -0151 0151 -0769 1041 -0210
M 1653 1653 1653 000 0000 0000 0013 0000 M 1477 1602 1540 009 -0062 0062 -0319 0432 -0114
m 165186 m 160206 mediane D 00678
Cn(2p) 1867 Cn(2p) 1867 Cp 16101
Cn(p) 16101 Cn(p) 16101 Qn(D) 0092926
n=2p n 20 n=2p n 20 Qdiff 01496169
f 11 f 11 t 114
l 55 l 55
n=p n 10 n=p n 10
f 6 f 6
l 15 l 15
Qn(2p) 0091465 Qn(2p) 007745
Qn(p) 0062469 Qn(p) 0121519
Qintra 017078 Qintra 014461
Qinter 010058 Qinter 019565
Sr(eacutecart-type de reacutepeacutetabiliteacute) 02415 Sr 02045
RSDr(coefficient de variation de la reacutepeacutetabiliteacute)1462 RSDr 1277
r(repeatability limit)) 0676 r 0573
SL(eacutecart-type interlaboratoire) -001905 SLlt0 SL 001737
SR(eacutecart-type de reproductibiliteacute) 02415 SR 02433
RSDR(coefficient de variation de la reproductibiliteacute)0146 RSDR 0152
R(limite de reproductibiliteacute)) 06762 R(limite de reproductibiliteacute))068123
h k h MoyenneLaboratoires
Niveau 1
si LaboratoiresDeviationMoyenne Dk DeviationsiAlt
Niveau 1
Ref
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3638 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Level 2
Nombre de laboratoires (p) 10
A 2681 2613 2647 005 0034 -0034 -0151 0891 A 2322 2531 2427 015 -0105 0105 -2119 1620 -0220
B 2544 2477 2511 005 0033 -0033 -2699 0872 B 2556 2653 2605 007 -0048 0048 -0581 0750 0094
C 2643 2633 2638 001 0005 -0005 -0311 0130 C 2681 2653 2667 002 0014 -0014 -0040 0217 0029
D 2690 2663 2676 002 0014 -0014 0400 0357 D 2778 2732 2755 003 0023 -0023 0721 0354 0079
E 2690 2716 2703 002 -0013 0013 0897 0336 E 2724 2748 2736 002 -0012 0012 0556 0185 0033
F 2568 2398 2483 012 0085 -0085 -3214 2217 F 2477 2114 2296 026 0182 -0182 -3255 2811 -0188
G 2643 2591 2617 004 0026 -0026 -0707 0682 G 2756 2568 2662 013 0094 -0094 -0085 1453 0045
H 2716 2643 2680 005 0036 -0036 0460 0945 H 2653 2732 2693 006 -0040 0040 0181 0613 0013
L 2591 2591 2591 000 0000 0000 -1196 0000 L 2230 2580 2405 025 -0175 0175 -2307 2704 -0186
M 2653 2690 2672 003 -0018 0018 0311 0481 M 2740 2613 2677 009 0064 -0064 0040 0987 0005
m 2655082 m 26719001 mediane D 00209
Cn(2p) 1867 Cn(2p) 1867 Cp 16101
Cn(p) 16101 Cn(p) 16101 Qn(D) 0039911
n=2p n 20 n=2p n 20 Qdiff 00642591
f 11 f 11 t 082
l 55 l 55
n=p n 10 n=p n 10
f 6 f 6
l 15 l 15
Qn(2p) 0020570 Qn(2p) 00345985
Qn(p) 00332437 Qn(p) 00718158
Qintra 003841 Qintra 006460
Qinter 005352 Qinter 011563
Sr(eacutecart-type de reacutepeacutetabiliteacute) 00543 Sr 00914
RSDr(coefficient de variation de la reacutepeacutetabiliteacute)205 RSDr 342
r(repeatability limit)) 0152 r 0256
SL(eacutecart-type interlaboratoire) 000139 SL 000920
SR(eacutecart-type de reproductibiliteacute) 00659 SR 01325
RSDR(coefficient de variation de la reproductibiliteacute)0025 RSDR 0050
R(limite de reproductibiliteacute)) 018446 R(limite de reproductibiliteacute))03708618
Laboratoires
Niveau 2
Moyenne si Laboratoires
Niveau 2
DRef Alt Moyenne si Deviation h Deviation h k k
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3738 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Level 2 bis
Nombre de laboratoires (p) 10
A 3000 2477 2739 037 0261 -0261 0832 1418 A 2602 2477 2540 009 0062 -0062 -0391 0465 -0199
B 2544 2813 2678 019 -0134 0134 0211 0729 B 2903 2778 2841 009 0062 -0062 1732 0465 0162
C 2477 2813 2645 024 -0168 0168 -0135 0911 C 2477 2699 2588 016 -0111 0111 -0049 0826 -0057
D 2398 2653 2526 018 -0128 0128 -1370 0692 D 2778 3000 2889 016 -0111 0111 2074 0826 0363
E 2875 2740 2808 010 0067 -0067 1547 0365 E 2602 2301 2452 021 0151 -0151 -1012 1120 -0356
F 2477 2544 2511 005 -0033 0033 -1525 0182 F 2778 2477 2628 021 0151 -0151 0230 1120 0117
G 2653 2740 2697 006 -0044 0044 0400 0236 G 2845 2845 2845 000 0000 0000 1764 0000 0148
H 2176 2813 2495 045 -0318 0318 -1692 1727 H 2477 2477 2477 000 0000 0000 -0832 0000 -0017
L 2813 2544 2678 019 0134 -0134 0211 0729 L 2903 2301 2602 043 0301 -0301 0049 2241 -0076
M 2602 2740 2671 010 -0069 0069 0135 0375 M 2903 2255 2579 046 0324 -0324 -0112 2411 -0092
m 2658114 m 259505 mediane D -00372
Cn(2p) 1867 Cn(2p) 1867 Cp 16101
Cn(p) 16101 Cn(p) 16101 Qn(D) 01656915
n=2p n 20 n=2p n 20 Qdiff 02667753
f 11 f 11 t 035
l 55 l 55
n=p n 10 n=p n 10
f 6 f 6
l 15 l 15
Qn(2p) 0098745 Qn(2p) 007195
Qn(p) 006007 Qn(p) 008805
Qintra 018437 Qintra 013435
Qinter 009672 Qinter 014176
Sr(eacutecart-type de reacutepeacutetabiliteacute) 02607 Sr 01900
RSDr(coefficient de variation de la reacutepeacutetabiliteacute)981 RSDr 732
r(repeatability limit)) 0730 r 0532
SL(eacutecart-type interlaboratoire) -002464 SLlt0 SL 000205
SR(eacutecart-type de reproductibiliteacute) 02607 SR 01953
RSDR(coefficient de variation de la reproductibiliteacute)0098 RSDR 0075
R(limite de reproductibiliteacute)) 072996 R(limite de reproductibiliteacute))054686
Laboratoires
Niveau 2 bis
Moyenne si Laboratoires
Niveau 2 bis
DRef Alt Moyenne si Deviation h Deviation h k k
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3838 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Level 3
Nombre de laboratoires (p) 10
A 3690 3663 3676 002 0014 -0014 -0311 0513 A 3690 3708 3699 001 -0009 0009 0354 0257 0022
B 3613 3672 3642 004 -0030 0030 -1851 1109 B 3708 3568 3638 010 0070 -0070 -0844 2061 -0005
C 3663 3732 3698 005 -0035 0035 0645 1302 C 3663 3699 3681 003 -0018 0018 0000 0536 -0017
D 3690 3690 3690 000 0000 0000 0311 0000 D 3643 3663 3653 001 -0010 0010 -0545 0286 -0037
E 3732 3653 3693 006 0040 -0040 0429 1480 E 3613 3732 3673 008 -0060 0060 -0162 1769 -0020
F 3643 3672 3658 002 -0014 0014 -1157 0536 F 3690 3771 3731 006 -0040 0040 0975 1193 0073
G 3699 3653 3676 003 0023 -0023 -0328 0856 G 3602 3643 3623 003 -0021 0021 -1141 0612 -0053
H 3623 3672 3648 003 -0024 0024 -1615 0913 H 3748 3681 3715 005 0033 -0033 0664 0990 0067
L 3724 3663 3694 004 0031 -0031 0461 1150 L 3748 3771 3760 002 -0011 0011 1544 0335 0066
M 3690 3690 3690 000 0000 0000 0311 0000 M 3806 3681 3744 009 0062 -0062 1234 1848 0054
m 368334 m 368086 mediane D 00089
Cn(2p) 1867 Cn(2p) 1867 Cp 16101
Cn(p) 16101 Cn(p) 16101 Qn(D) 00325359
n=2p n 20 n=2p n 20 Qdiff 00523852
f 11 f 11 t 043
l 55 l 55
n=p n 10 n=p n 10
f 6 f 6
l 15 l 15
Qn(2p) 0014323 Qn(2p) 001811
Qn(p) 0013719 Qn(p) 003164
Qintra 002674 Qintra 003381
Qinter 002209 Qinter 005094
Sr(eacutecart-type de reacutepeacutetabiliteacute) 00378 Sr 00478
RSDr(coefficient de variation de la reacutepeacutetabiliteacute)103 RSDr 130
r(repeatability limit)) 0106 r 0134
SL(eacutecart-type interlaboratoire) -000023 SLlt0 SL 000145
SR(eacutecart-type de reproductibiliteacute) 00378 SR 00611
RSDR(coefficient de variation de la reproductibiliteacute)0010 RSDR 0017
R(limite de reproductibiliteacute)) 0105894 R(limite de reproductibiliteacute))017119
Ref Alt DLaboratoires
Niveau 3
Moyenne si Deviation h k Laboratoires
Niveau 3
Moyenne si Deviation h k
Thermo Fisher Scientific
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Listeria Precis Enumeration
The modifications are highlighted
Quality assurance documents related to this study can be consulted upon
request from Oxoid part of Thermo Fisher Scientific
The technical protocol and the result interpretation were realised according to
the EN ISO 16140 and the AFNOR technical rules
o Company Oxoid Ltd
part of THERMO FISHER SCIENTIFIC
Wade Road Basingstoke
Hampshire RG24 8PW UK
o Expert Laboratory ADRIA Deacuteveloppement
ZA Creacrsquoh Gwen
F-29196 QUIMPER Cedex
o Studied method Listeria PrecisTM method for enumeration of
Listeria monocytogenes
o Validation standard NF EN ISO 16140 (October 2003) Food
microbiology ndash Protocol for the validation of
alternative methods
o Standard method ISO 11290-2 (1999) amp ISO 11290-2A1 (2004)
Horizontal method for the detection and
enumeration of Listeria monocytogenes - Part 2
enumeration of Listeria monocytogenes in foods
o Scope All human food products and environmental
samples
o Certification organism AFNOR Certification
Thermo Fisher Scientific
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Summary Report (Version 1)
Listeria Precis Enumeration
1 INTRODUCTION
11 Dates of the validation studies
The Listeria PrecisTM method for enumeration of Listeria monocytogenes was
validated on September 15 2006 (certificate number UNI 0305 ndash 0906) for
food products and environmental samples
In 2007 an extension study was obtained for a new confirmation test OBIS
MONO
The alternative method was renewed in 2010 and 2014
12 Protocol and principle of the alternative method
The Listeria PrecisTM method is based on a specific chromogenic media for
Listeria monocytogenes enumeration
The protocol is described in Appendix 1
13 Standard method
The reference method is the ISO 11290-2 (1998) amp ISO 11290-2A1 (2004)
Horizontal method for the detection and enumeration of Listeria
monocytogenes - Part 2 enumeration of Listeria monocytogenes in foods
(See Appendix 2)
Analysis performed according to the COFRAC accreditation
Thermo Fisher Scientific
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Summary Report (Version 1)
Listeria Precis Enumeration
2 VALIDATION STUDY RESULTS
21 Initial validation (realised by ASEPT 2006)
211 Method Comparison Study
2111 Linearity
Linearity is the ability of the method when used with a given matrix to give results that are in proportion
to the amount of analyte present in the sample that is an increase in analyte corresponds to a linear or
proportional increase in results
Food matrices and protocols
Six matrixstrain pairs were analysed with five contamination levels and two
replicates At least 50 analyses were performed by the alternative and the
standard methods
The contamination levels the samples and the inoculated strains are
presented in the table below
Table 1
Samples Strains Contamination levels
(CFUg)
Rillettes Listeria monocytogenes 4e 10 ndash 50
50 ndash 100
100 ndash 500
500 ndash 1 000
1 000 ndash 10 000
Raw milk Listeria monocytogenes 12a
Lettuce Listeria monocytogenes 12a
Smoked salmon Listeria monocytogenes 12b
Raw egg product Listeria monocytogenes 12b
Process water Listeria monocytogenes 12a
The samples were individually inoculated and were analysed in duplicate by
the alternative and the reference methods
Results
The bi-dimensional graphs are shown figure 1
Thermo Fisher Scientific
ADRIA Deacuteveloppement 638 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Figure 1 ndash Bi-dimensional graphs
Rillettes Raw milk
Smoked salmon Raw egg product
Lettuce Process water
Thermo Fisher Scientific
ADRIA Deacuteveloppement 738 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Statistical interpretations
Statistical interpretation results are provided in Table 2
Table 2 ndash Statistical interpretations
Matrix R Selected
regression
RobF Critical
value
P Correlation
coefficient
Regression equation
Rillettes 180 GMFR 609 541 4 09960 log (Alt) = 09887 log (Ref) + 01141
Lettuce 108 GMFR 000 541 100 09992 Log (Alt) = 10081 log (Ref) - 00257
Raw milk 102 GMFR 000 541 100 09997 Log (Alt) = 10188 log (Ref) - 00737
Smoked salmon 1048 OLS1 1806 541 263 09984 Log (Alt) = 10406 log (Ref) - 01418
Raw egg product 130 GMFR 548 541 49 09958 Log (Alt) = 09775 log (Ref) - 00161
Process water 184 GMFR 0079 541 969 09985 Log (Alt) = 09680 log (Ref) + 01317
GMFR = orthogonal linear regression
OLS1 = ordinary least-square linear regression
x ndash axis = reference method
y ndash axis = alternative method
OLS1 = ordinary least-square linear regression
x axis = alternative method
y axis = reference method
Statistical Interpretation
P gt 5 not significant 1 lt P lt 5 significant
P lt 1 highly significant
The regressions straight lines are shown figure 2
Thermo Fisher Scientific
ADRIA Deacuteveloppement 838 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Figure 2 ndash Regression straight lines
Lineacuteariteacute - Œuf
Process water Process water (2) Process water
Smoked salmon (GMFR) Smoked salmon (OLS 1)
Raw milk Raw egg product
Rillettes Lettuce
Thermo Fisher Scientific
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Listeria Precis Enumeration
Discussion
The linearity is accepted in all cases with
P values 5 and correlation coefficients superior to 099
For the matrix ldquoRillettesldquo the linearity test is significant (P = 4) with a high
correlation coefficient value (0996)
The Listeria PrecisTM method shows satisfying linearity
2112 Relative accuracy
The accuracy is the closeness of agreement between a test result and the accepted reference value
The bias is the difference between the expectation of the test results and an accepted reference value
Number and nature of the samples
The repartition per category and types is provided in table 3
Table 3 ndash Number and nature of the samples
Categories Types Number of samples
Analysed Exploited (1)
Meat products
Raw meat products 6 4
Ready to cook products 4 2
Ready to eat delicatessen 10 4
Total 20 10
Dairy products
Raw milk cheeses 7 4
Raw milks 4 3
Other dairy products 5 5
Total 16 12
Fishery products
Raw fishes 5 4
Processed fishes 7 2
Ready-to-eat products 6 4
Total 18 10
Vegetables
Raw vegetables 5 4
Frozen vegetables 3 3
Lettuce raw vegetables 5 5
Total 13 12
Egg products
Raw egg 5 5
Pastries 3 3
Pasteurised egg products 4 3
Total 12 11
Environmental samples
Wipes swabs 8 4
Process water 3 3
Dusts 3 3
Total 14 10
TOTAL 93 65
(1) Some data cannot be exploited because enumeration was under the detection level for at least one of the
method
Thermo Fisher Scientific
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Listeria Precis Enumeration
Artificial contamination of the samples
Artificial contaminations were realised by spiking after performing injury
protocols on pure cultures or by cross-contaminations
56 samples were artificially contaminated 53 gave exploitable results among
a total of 65 samples 8150 of the samples were artificially contaminated
The injury protocol and the strains inoculated are provided in Table 4
Table 4
Stress Ndeg Injury protocol Strain Origin
A 45 min at 50 degC
48
LM -H123
LM-H170
Smoked salmon
Lettuce
Lettuce
B 30 min at 50 degC
and 2 h at ndash20degC
18
43
LM-O1
Raw milk (environment)
Environment
Egg product
C 30 min at 55degC
18
48
LM-H123
Raw milk
Smoked salmon
Lettuce
D 30 min at 50degC
et 2 h at ndash80degC
34
LM-H171
LM-O1
Rillettes
Lettuce
Egg product
E 30 min at 60 degC LM-O1 Egg product
Confirmatory tests
For the reference and the alternative methods confirmations were realised
by the tests described in the ISO 12290-2A1 standard For the alternative
method only one colony was confirmed while 5 colonies were tested for the
reference method
Results
Samples were analysed in duplicate by the reference and the alternative
methods
The contamination range is provided in table 5 per tested category
Thermo Fisher Scientific
ADRIA Deacuteveloppement 1138 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Table 5 ndash Contamination range
Food category Contamination level (log CFUg)
Meat products 100 ndash 404
Dairy products 148 ndash 408
Fishery products 100 ndash 384
Vegetables 170 ndash 511
Egg products 130 ndash 499
Environmental samples 170 ndash 511
All categories 100 ndash 511
Bi-dimensional graphs are given figure 3
Figure 3 ndash Bi-dimensional graphs
Graphe bi-dimensionnel Produits carneacutes
Fishery products
Vegetables
Egg products
Meat products
Thermo Fisher Scientific
ADRIA Deacuteveloppement 1238 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Statistical interpretation according to EN ISO16140 method
The results of the statistical interpretation are given in Table 6
Table 6 ndash Statistical interpretation results
Category n R Regression
used a t(a) b t(b) Critical T
P
Ordinate
at 0
Slope
at 1
Meat products 10 158 GMFR -0455 3321 115 3123 2306 001 001
Dairy products 12 065 GMFR -0027 0385 1002 0108 2228 092 071
Fishery products 10 130 GMFR -0305 1024 1034 0312 2306 076 034
Vegetables 12 086 GMFR -0015 0128 0993 0191 2228 085 090
Egg products 11 158 GMFR -0038 0866 1001 0090 2262 093 041
Environmental
samples 10 176 GMFR -0139 265 1034 231 2306 005 003
All products 65 118 GMFR -0182 3042 1040 2145 2000 004 000
All products
(estimated number) 65 118 GMFR -0182 3042 1040 2145 2000 004 000
All products (non
estimated results) 20 115 GMFR -0367 151 1138 1141 2101 027 015
Repeatability limits obtained for the alternative and reference methods as
well as bias obtained are presented in Table 7
Dairy products Environmental samples
Log (Ref) Log (Ref)
All categories
Thermo Fisher Scientific
ADRIA Deacuteveloppement 1338 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Table 7
Categories Bias
Repeatability limits
Rob r
Ref Alt
Meat products 0008 0314 0343
Dairy products -0031 0136 0130
Fishery products -0133 0220 0214
Vegetables -0041 0180 0319
Egg products -0026 0219 0206
Environmental samples -0007 0198 0119
All products -0032 0196 0215
Regression straight lines (graph and equation representations) for each food
category and for all products are presented figure 4
Thermo Fisher Scientific
ADRIA Deacuteveloppement 1438 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Figure 4 ndash Regression straight lines
Meat products
Fishery products
Dairy products
Vegetables
Thermo Fisher Scientific
ADRIA Deacuteveloppement 1538 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Environmental samples
Egg products
All products (estimated number)
Thermo Fisher Scientific
ADRIA Deacuteveloppement 1638 November 19 2014
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Listeria Precis Enumeration
The hypotheses that the ordinates are close to 0 and the slopes close to 1
are approved in all cases except for
- Meat products (slope P = 1 ordinate P = 1)
- Environmental samples (ordinate P = 3)
- All products (ordinate P = 0)
The bias between the two methods vary from ndash 0133 to 0008 log CFU For
all categories the bias is ndash 0032 log CFU The repeatability limits of the
alternative and the reference method are similar For all products the values
are respectively 0215 and 0196 log CFU For all products these
interpretations are provided taking into account data with low contamination
level or not (estimated colonies number)
For all food products the regression straight line is the following
log (Alt) = 1040 log Ref ndash 0182
The Listeria PrecisTM method shows satisfying relative accuracy
All products
All products (Level 2)
Thermo Fisher Scientific
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Listeria Precis Enumeration
2113 Detection limit (LOD) and quantification limit (LOQ)
The critical level is the defined as the smallest amount which can be detected (not null) but not
quantified as an exact value Below this value it cannot be sure that the true value is not null
The detection limit is defined as being higher than the critical level because it involves a power the
probability 1- which has to be well over 50 for example 95
The quantification limit is defined as the smallest amount of analyte (that is the lowest actual number of
organisms) which can be measured and quantified with defined precision and accuracy under the
experimental conditions by the method under validation
Protocol
The detection limit of the alternative method was realised with pure cultures
Three different inoculation levels were tested with six replicates per level ie
18 analyses were done by the alternative method
Listeria monocytogenes 18 was used for the detection limit of the alternative
method
Quantification limit was calculated for six independent blank samples
determinations
Results
These data are intrinsic to the method used and are presented in the
following tables
Table 8 ndash Inoculation 1 ml on 3 plates
Level (CFUml) Positive samples number Standard deviation Bias
0 06
05 26 1211 0
1 36 0816 05
5 66 2483 85
Thermo Fisher Scientific
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Summary Report (Version 1)
Listeria Precis Enumeration
Table 9 ndash Inoculation 01 ml on 1 plate
Level (CFUml) Positive samples number Standard deviation Bias
0 06
5 36 0548 05
10 46 1414 2
50 66 2258 55
Table 10
Formulas Obtained values
LC 165 S0 + X0 18
LOD 33 S0 + X0 32
LOQ 10 S0 + X0 87
2114 Relative Sensitivity
The relative sensitivity is defined as the ability of the alternative method to detect two different amounts
of analyte measured by the reference method within a given matrix at a specified average value or
over the whole measurement range that is it is the minimal quantity variation (increase of the analyte
concentration x) which gives a significant variation of the measured signal (response y)
These data are intrinsic to the used method and are obtained from the results
of the linearity study
Sensitivity patterns obtained for tested (matrixstrain) pairs are presented in
figure 5
Thermo Fisher Scientific
ADRIA Deacuteveloppement 1938 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Figure 5 ndash Sensitivity patterns for the tested (matrixstrain) pairs
2115 SpecificitySelectivity
The specificity is defined as the degree to which a method is affected (or not) by the other components
present in a multi-component sample That is the ability of a method to measure exactly a given
analyte or its amount within the sample without interference from non-target components such as a
matrix effect or background noise
The selectivity is defined as a measure of the degree of non-interference in the presence of non-target
analytes A method is selective if it can be used to detect the analyte under examination and that a
guarantee can be provided that the detected signal can only be a product by that specific analyte
The specificity and selectivity study was performed for the Listeria PrecisTM
detection method no additional test was realised for the enumeration
method The results were the following
- Among the Listeria monocytogenes strains tested 2 strains (Listeria
monocytogenes CIP 7834 and Listeria monocytogenes CIP 105459) gave
negative results These 2 strains were tested twice by running 48 h
incubation time of the compared agars Positive results were then
observed by the alternative method Pale characteristic colonies were
observed on the ISO agar by streaking the Half Fraser broth only
- No cross reaction was observed with the non-target strains except with
Listeria ivanovii which gave characteristic colonies on Brilliancetrade Listeria
Agar but the confirmatory tests provide a clear discrimination between
Listeria monocytogenes and Listeria ivanovi species
Rillettes
Lettuce
Raw milk
Smoked salmon
Raw egg
Process water
x (CFUg)
Thermo Fisher Scientific
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Listeria Precis Enumeration
212 Practicability
1 Packaging Ready to use Brilliance Listeria agar plates are available
(x 10 plates)
2 Reagent volume
3 Storage conditionsndash
Shelf life of products
not opened
The plates are stored at 6 - 12degC in darkness conditions The
expiration date is mentioned on the package and on the plates It
is around 3 months
4 Modalities of use
after first use
5 Required
equipments No specific equipment is required
6 Reagents Ready to use plates
7 Training 05 day
8 Real time handling
Reference method
9 min for one sample
55 min for 10 samples
Alternative method
7 min for one sample
35 minutes for 10 samples
9 Time to result
Negative results
Reference method Day 2
Alternative method Day 2
Negative results after confirmation or positive results
Reference method Day 4 ndash Day 7
Alternative method Day 4 ndash Day 7
Positive results
Reference method Day 4 ndash Day 7
Alternative method Day 2 ndash Day 3 (with OBIS Mono Test)
or Day 4 ndash Day 7 (by the classical tests described in the
standardised method)
10 Technician skill Identical to that required for the reference method
11 Steps common to
the reference method Mother suspension revivification step and decimal dilutions
12 Traceability
13 Maintenance
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Listeria Precis Enumeration
213 Inter-laboratory study
2131 Organisation
12 laboratories participated to this study Pasteurised milk was inoculated by
Listeria monocytogenes 12a isolated from raw milk The targeted
inoculation levels were
- 0 CFUml
- 50 CFU ml
- 500 CFU ml
- 5 000 CFU ml
Each laboratory received eight flasks of 10 ml sample ie two flasks per
inoculation level Furthermore one non-inoculated sample has been added
to the package for total viable count microflora (NF ISO 4833 method)
Detailed instructions were transmitted to the collaborators by the expert
laboratory
Collaborative study laboratories and the expert laboratory carried out the
analyses with the alternative and reference methods
2132 Verification of experimental parameters
Strain stability during transport
In order to evaluate the Listeria monocytogenes strain stability during
transport bacterial count of all inoculated flasks were checked at different
time ie inoculation time after 24 h of conservation at 2deg - 8degC Results are
reported in table 11
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2238 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Table 11 ndash Listeria monocytogenes count
by ISO 11290-2A1 method (log CFUml)
Day of the analyses Level contamination Reference method
Day 0
(29052006)
1 45
2 440
3 4 500
Day 1
(30052006)
1 60
2 430
3 4 900
No evolution of the strain was observed after storage for 24 h at 2degC ndash 8degC
Contamination level
The real contamination levels are provided in table 12
Table 12 ndash Real contamination level (CFUml)
Target inoculation
(CFU ml) Samples Ndeg
Real contamination levels
(CFUml)
0 1 - 8 0
50 5 - 6 45
500 3 - 7 440
5000 2 - 4 4 500
Sample temperature at receipt
Measured temperatures at receipt are listed in table 13
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2338 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Table 13 - Sample temperature on receipt
Laboratories Receipt
day
Hour
receipt
Measured temperature
at receipt (degC)
Measured temperature by the
temperature probe (degC)
A
B
C
D
E
F
G
H
I
J
K
L
M
30052006
10h45
10h15
11h15
10h00
09h30
8h30
11h50
10h00
08h30
14h45
08h30
09h45
08h45
82
55
45
63
60
62
81
38
48
-
14
67
76
03
03
-13
03
04
-14
05
04
01
05
-13
04
Conclusion
The temperatures during the transport were correct for all the labs except for
Lab J which didnrsquot provide the information
All the labs received their packages with several broken flasks
Three labs (B D and L) received some damaged samples but proceeded to
analyses
All the labs started the analyses at Day 1 except Lab J
2133 Results
Aerobic mesophilic flora
The count of aerobic mesophilic flora varied from 94 102 CFUml to
33 103 CFUml
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2438 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Expert lab results
The results obtained by the expert lab are the following
Table 14
Target inoculation level (CFUml)
Reference method ISO 11290-2
Alternative method Listeria Precis Enumeration
Rep 1 Rep 2 Rep 1 Rep 2
lt 10 lt 10 lt 10 lt 10 lt10
50 50 (Ne) 60 (Ne) 50 (Ne) 30 (Ne)
500 470 390 490 500
5 000 4 700 5 100 5 900 3 400
Ne estimated number
The obtained results are in agreement with those expected
Collaborator Labs results
A synthesis of the results obtained by the collaborator labs is provided
Table 15 (CFUml) and Table 16 (log CFUml)
Table 15 ndash Synthesis of results obtained by the alternative
and the reference methods (CFUml)
Labs Level 1 Level 2 Level 2 bis Level 3
Reference method
Alternative method
Reference method
Alternative method
Reference method
Alternative method
Reference method
Alternative method
A 40 40 70 40 480 410 210 340 1000 300 400 300 4900 4600 4900 5100
B 40 15 40 20 350 300 360 450 350 650 800 600 4100 4700 5100 3700
C 50 20 20 70 440 430 480 450 300 650 300 500 4600 5400 4600 5000
D 40 60 40 40 490 460 600 540 250 450 600 1000 4900 4900 4400 4600
E 50 40 50 20 490 520 530 560 750 550 400 200 5400 4500 4100 5400
F 60 20 60 50 370 250 300 130 300 350 600 300 4400 4700 4900 5900
G 70 35 120 50 440 390 570 370 450 550 700 700 5000 4500 4000 4400
H 110 40 100 90 520 440 450 540 150 650 300 300 4200 4700 5600 4800
L 35 60 20 40 390 390 170 380 650 350 800 200 5300 4600 5600 5900
M 45 45 30 40 450 490 550 410 400 550 800 180 4900 4900 6400 4800
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2538 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Table 16 ndash Synthesis of results obtained by the alternative
and the reference methods (log CFUml)
Labs Level 1 Level 2 Level 2 bis Level 3
Reference method
Alternative method
Reference method
Alternative method
Reference method
Alternative method
Reference method
Alternative method
A 1602 1602 1845 1602 2681 2613 2322 2531 3000 2477 2602 2477 3690 3663 3690 3708
B 1602 1176 1602 1301 2544 2477 2556 2653 2544 2813 2903 2778 3613 3672 3708 3568
C 1699 1301 1301 1845 2643 2633 2681 2653 2477 2813 2477 2699 3663 3732 3663 3699
D 1602 1778 1602 1602 2690 2663 2778 2732 2398 2653 2778 3000 3690 3690 3643 3663
E 1699 1602 1699 1301 2690 2716 2724 2748 2875 2740 2602 2301 3732 3653 3613 3732
F 1778 1301 1778 1699 2568 2398 2477 2114 2477 2544 2778 2477 3643 3672 3690 3771
G 1845 1544 2079 1699 2643 2591 2756 2568 2653 2740 2845 2845 3699 3653 3602 3643
H 2041 1602 2000 1954 2716 2643 2653 2732 2176 2813 2477 2477 3623 3672 3748 3681
L 1544 1778 1301 1602 2591 2591 2230 2580 2813 2544 2903 2301 3724 3663 3748 3771
M 1653 1653 1477 1602 2653 2690 2740 2613 2602 2740 2903 2255 3690 3690 3806 3681
Level 2 results obtained taking account the 3 plates inoculated with the mother-suspension
Level 2 bis results obtained taking account the plates inoculated with 01 ml of the mother-suspension
Scrutiny of the measurement results for consistency
In order to identify the measurement results or laboratories that could be
inconsistent two graphical consistency techniques were realised Mandelrsquos
h-ank-statistics Mandel indicators h and k at 5 significance highlighted
some possible inconsistent data (See tables and graphics in Appendix 3 and
Appendix 4)
Mandelrsquos values Number of values observed over the threshold
Reference method Alternative method
h gt 1 Lab F Level 2 Lab F Level 2
h gt 5 Lab F Level 2
Lab B Levels 1 and 2
Lab A Level 2
Lab D Level 3
Lab F Level 2
Lab L Level 2
kgt 1 Lab F Level 3
k gt 5 Lab F Level 2 Lab C Level 2
Lab F Level 3
Lab L Levels 1 and 3
Lab M Level 1
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2638 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
The emphasized results appear (i) coherent with the targeted contamination
levels (ii) concordant between both methods
All the results were thus kept for the statistical analysis
Comparison of the trueness and precision characteristics of
the reference method and alternative methods
The statistical values are summarised hereafter
Level p
Reference method Alternative method
Repeatability ratio
Reproducibility ratio Median
Repeatability standard deviation
Reproducibility standard deviation
Median Repeatability
standard deviation
Reproducibility standard deviation
1 10 16519 02415 02425 16021 02045 02433 0847 1007
2 10 26551 00543 00659 26719 00914 01325 1682 2011
2 bis 10 26581 02607 02607 25951 01900 01953 0729 0749
3 10 18671 00378 00378 18671 00478 00611 1264 1617
Bias of the alternative method
In order to estimate the bias of the alternative method with regard to the reference method for every
level Dij and t are calculated according to the following equations
RefAlt ijYijYDij
Diffp
Dijit
)2(
)(median
If the t value is superior to 2 the alternative method is significantly biased
with regard to the reference method
The values are given in table 17
Table 17 ndash t (d) values obtained by level
Level Bias t (d) Conclusion
1 0068 114 Non significant bias
2 0021 082 Non significant bias
2 bis - 0037 035 Non significant bias
3 - 0037 043 Non significant bias
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2738 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
The statistical test concludes to a non significant bias for all the inoculation
levels The bias varies from ndash 0037 to 0068 log CFUml
Comparison of the repeatability standard deviations
If the ratio Srj Alt Srj Ref of the repeatability standard deviations of the
alternative method and the reference method is larger than 2 the precision
under repeatability conditions of the alternative method is considered to be
lower than that of the reference method If this ratio is smaller than 05 the
precision under repeatability conditions of the alternative method is
considered to be greater than that of the reference method
The ratio values are given in table 18
Table 18
Level
Repeatability standard deviation
Reference method Alternative method Alternative method
reference method ratio
1 0242 0205 0847
2 0054 0091 1682
2 bis 0261 0190 0729
3 0038 0048 1264
Level Repeatability limit
Reference method Alternative method
1 0676 0573
2 0152 0256
2 bis 0730 0532
3 0106 0134
The ratios of the repeatability standard deviations are below 2 in all cases
The precision under repeatability conditions of the alternative method is
equivalent to that of the reference method
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2838 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Comparison of the reproducibility standard deviations
If the ratio Srj Alt Srj Ref of the reproducibility standard deviations of the alternative method and the
reference method is larger than 2 the precision under reproducibility conditions of the alternative
method is considered to be lower than that of the reference method If this ratio is smaller than 05 the
precision under reproducibility conditions of the alternative method is considered to be greater than
that of the reference method
The ratio values are given in table 19
Table 19
Level
Reproducibility standard deviation
Reference method Alternative method Alternative method
reference method ratio
1 0242 0243 1007
2 0066 0133 2011
2 bis 0261 0195 0749
3 0038 0061 1617
Level Reproducibility limit
Reference method Alternative method
1 0676 0681
2 0185 0371
2 bis 0730 0547
3 0106 0171
The ratios of the reproducibility standard deviations are below or close to 2
The precision under reproducibility conditions of the alternative method is
equivalent to that of the reference method
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2938 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
214 Conclusion
Method comparison study
The Listeria PrecisTM enumeration method shows satisfying linearity and
relative accuracy
The alternative method shows satisfying inclusivity and exclusivity results
Two Listeria monocytogenes strains gave negative results with the
alternative method These strains grew weakly on the reference method
media (OampA Palcam and Oxford)
The limits of repeatability of the alternative method are similar to those of the
reference method
The bias between both methods are low between - 0133 Log CFUg and
- 0008 Log CFUg
The Listeria PrecisTM enumeration method offers the saving for confirmatory
tests
Positive results are obtained with the alternative method in 2-3 days (with
OBIS Mono test) or in 4-7 days (by the classical tests described in the
standardized method) against 4-7 days for the reference method
Inter-laboratory study
The bias between the reference and the alternative methods shows satisfying
and correct values
- level 1 (100 - 1 000 CFUg) bias = + 007 log CFUg
- level 2 (1 000 - 10 000 CFUg) bias = 002 log CFUg
- level 2 bis (1 000 - 10 000 CFUg) bias = - 004 log CFUg
- level 3 (10 000 - 100 000 CFUg) bias = - 004 log CFUg
The repeatability and the reproducibility of the alternative method look similar
to those of the reference method
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3038 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
22 Extension study (realised by ASEPT 2007)
An extension study was performed in 2007 for a new confirmatory test OBIS
MONO
150 Listeria monocytogenes strains and 100 non-target strains were isolated
onto TSAYE and BrillianceTM Listeria Agar plates All the Listeria
monocytogenes and the non-target strains gave the expected results
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3138 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Appendix 1 ndash Flow diagram of the Listeria PrecisTM method
x g sample + 9 x ml buffered peptone water
Revivification step for 1 h plusmn 5 min at 20degC plusmn 1degC
Streaking 1 ml onto 3 Brilliance Listeria Agar plates (mother suspension)
or 01 ml onto 1 Brilliance Listeria Agar plates (mother suspension and decimal dilutions)
Incubation for 48 h plusmn 3 h at 37degC plusmn 1degC
Confirmatory tests on at least one typical colony
- by the tests described in the ISO method
- by applying the OBIS mono test on an isolated colony
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3238 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Appendix 2 ndash ISO 11290-2 (1998) amp ISO 11290-2A1 (2004) Horizontal
method for the detection and enumeration of Listeria monocytogenes -
Part 2 enumeration of Listeria monocytogenes in foods
10 g cheese + 90 ml buffered peptone water or Half Fraser Broth (without supplements) (110 dilution)
Stomach 1 min 30 seconds
Keep at 20degC plusmn 2degC for 1 h plusmn 5 min
Decimal dilutions in 9 ml peptone salt tubes
Dilution 110
Dilution 1100
Dilution 11000
1 ml on 3 Chromogenic Listeria medium (ISO) plates (x 2) (-1)
01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-2)
01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-3)
01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-4)
Incubation at 37degC plusmn 1degC
24 h - 48 h
Numerate typical colonies at 24 h
(bluegreen surrounded by an opaque halo)
Numerate at 48 h
Streak typical colonies on TSYEA
(5 coloniesplate 2 successive dilutions)
Confirmation tests
Catalase
Gram stain
For confirmation of L monocytogenes
Haemolysis
Carbohydrate (rhamnose and xylose) utilisation
CAMP Test
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3338 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Appendix 3 ndash Mandelrsquos graphics
Laboratoire h Ref
Niveau 1 h Ref
Niveau 2 h Ref
Niveau 3 h Alt
Niveau 1 h Alt
Niveau 2 h Alt
Niveau 3 h5 h1 h5 h1
A -050 -015 083 062 -212 -039 198 299 -198 -299
B -261 -270 021 -077 -058 173 198 299 -198 -299
C -151 -031 -014 -015 -004 -005 198 299 -198 -299
D 038 040 -137 000 072 207 198 299 -198 -299
E -001 090 155 -052 056 -101 198 299 -198 -299
F -112 -321 -153 070 -325 023 198 299 -198 -299
G 042 -071 040 147 -009 176 198 299 -198 -299
H 169 046 -169 192 018 -083 198 299 -198 -299
L 009 -120 021 -077 -231 005 198 299 -198 -299
M 001 031 014 -032 004 -011 198 299 -198 -299
-300
-200
-100
000
100
200
300
A B C D E F G H L M
Vale
urs
h d
e M
an
del(
rob
uste
s)
Laboratoires
Meacutethode de reacutefeacuterence analyse robuste
h Ref Niveau 1
h Ref Niveau 2
h Ref Niveau 3
h 5=198
h 1=299
-300
-200
-100
000
100
200
300
A B C D E F G H L M
Vale
urs
de h
de M
an
del(
rob
uste
s)
Laboratoires
Meacutethode alternative analyse robuste
h Alt Niveau 1
h Alt Niveau 2
h Alt Niveau 2
h 1=299
h 5=198
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3438 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Laboratoire k Ref
Niveau 1 k Ref
Niveau 2 k Ref
Niveau 3 k Alt
Niveau 1 k Alt
Niveau 2 k Alt
Niveau 3 k1 k5
A 000 089 142 084 162 046 259 181
B 125 087 073 104 075 046 259 181
C 117 013 091 188 022 083 259 181
D 052 036 069 000 035 083 259 181
E 028 034 037 138 019 112 259 181
F 140 222 018 027 281 112 259 181
G 088 068 024 131 145 000 259 181
H 129 094 173 016 061 000 259 181
L 069 000 073 104 270 224 259 181
M 000 048 038 043 099 241 259 181
000
050
100
150
200
250
300
350
400
A B C D E F G H L M
Vale
urs
k d
e M
an
del(
rob
uste
s)
Laboratoires
Meacutethode de reacutefeacuterence analyse robuste
k Ref Niveau 1k Ref Niveau 2k Ref Niveau 3
k 1=259
k 5=181
000
050
100
150
200
250
300
A B C D E F G H L M
Vale
urs
k d
e M
an
del
(ro
bu
ste
s)
Laboratoires
Meacutethode alternative analyse robuste
k Alt Niveau 3
k Alt Niveau 1
k Alt Niveau 2
k 1=259
k 5=181
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3538 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Appendix 4 ndash Statistical results
Level 1
Nombre de laboratoires (p) 10
A 1602 1602 1602 000 0000 0000 -0495 0000 A 1845 1602 1724 017 0122 -0122 0621 0840 0122
B 1602 1176 1389 030 0213 -0213 -2613 1247 B 1602 1301 1452 021 0151 -0151 -0769 1041 0062
C 1699 1301 1500 028 0199 -0199 -1510 1165 C 1301 1845 1573 038 -0272 0272 -0148 1881 0073
D 1602 1778 1690 012 -0088 0088 0380 0516 D 1602 1602 1602 000 0000 0000 0000 0000 -0088
E 1699 1602 1651 007 0048 -0048 -0013 0284 E 1699 1301 1500 028 0199 -0199 -0522 1376 -0151
F 1778 1301 1540 034 0239 -0239 -1116 1397 F 1778 1699 1739 006 0040 -0040 0698 0274 0199
G 1845 1544 1695 021 0151 -0151 0425 0881 G 2079 1699 1889 027 0190 -0190 1467 1315 0194
H 2041 1602 1822 031 0220 -0220 1689 1286 H 2000 1954 1977 003 0023 -0023 1917 0158 0155
L 1544 1778 1661 017 -0117 0117 0092 0685 L 1301 1602 1452 021 -0151 0151 -0769 1041 -0210
M 1653 1653 1653 000 0000 0000 0013 0000 M 1477 1602 1540 009 -0062 0062 -0319 0432 -0114
m 165186 m 160206 mediane D 00678
Cn(2p) 1867 Cn(2p) 1867 Cp 16101
Cn(p) 16101 Cn(p) 16101 Qn(D) 0092926
n=2p n 20 n=2p n 20 Qdiff 01496169
f 11 f 11 t 114
l 55 l 55
n=p n 10 n=p n 10
f 6 f 6
l 15 l 15
Qn(2p) 0091465 Qn(2p) 007745
Qn(p) 0062469 Qn(p) 0121519
Qintra 017078 Qintra 014461
Qinter 010058 Qinter 019565
Sr(eacutecart-type de reacutepeacutetabiliteacute) 02415 Sr 02045
RSDr(coefficient de variation de la reacutepeacutetabiliteacute)1462 RSDr 1277
r(repeatability limit)) 0676 r 0573
SL(eacutecart-type interlaboratoire) -001905 SLlt0 SL 001737
SR(eacutecart-type de reproductibiliteacute) 02415 SR 02433
RSDR(coefficient de variation de la reproductibiliteacute)0146 RSDR 0152
R(limite de reproductibiliteacute)) 06762 R(limite de reproductibiliteacute))068123
h k h MoyenneLaboratoires
Niveau 1
si LaboratoiresDeviationMoyenne Dk DeviationsiAlt
Niveau 1
Ref
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3638 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Level 2
Nombre de laboratoires (p) 10
A 2681 2613 2647 005 0034 -0034 -0151 0891 A 2322 2531 2427 015 -0105 0105 -2119 1620 -0220
B 2544 2477 2511 005 0033 -0033 -2699 0872 B 2556 2653 2605 007 -0048 0048 -0581 0750 0094
C 2643 2633 2638 001 0005 -0005 -0311 0130 C 2681 2653 2667 002 0014 -0014 -0040 0217 0029
D 2690 2663 2676 002 0014 -0014 0400 0357 D 2778 2732 2755 003 0023 -0023 0721 0354 0079
E 2690 2716 2703 002 -0013 0013 0897 0336 E 2724 2748 2736 002 -0012 0012 0556 0185 0033
F 2568 2398 2483 012 0085 -0085 -3214 2217 F 2477 2114 2296 026 0182 -0182 -3255 2811 -0188
G 2643 2591 2617 004 0026 -0026 -0707 0682 G 2756 2568 2662 013 0094 -0094 -0085 1453 0045
H 2716 2643 2680 005 0036 -0036 0460 0945 H 2653 2732 2693 006 -0040 0040 0181 0613 0013
L 2591 2591 2591 000 0000 0000 -1196 0000 L 2230 2580 2405 025 -0175 0175 -2307 2704 -0186
M 2653 2690 2672 003 -0018 0018 0311 0481 M 2740 2613 2677 009 0064 -0064 0040 0987 0005
m 2655082 m 26719001 mediane D 00209
Cn(2p) 1867 Cn(2p) 1867 Cp 16101
Cn(p) 16101 Cn(p) 16101 Qn(D) 0039911
n=2p n 20 n=2p n 20 Qdiff 00642591
f 11 f 11 t 082
l 55 l 55
n=p n 10 n=p n 10
f 6 f 6
l 15 l 15
Qn(2p) 0020570 Qn(2p) 00345985
Qn(p) 00332437 Qn(p) 00718158
Qintra 003841 Qintra 006460
Qinter 005352 Qinter 011563
Sr(eacutecart-type de reacutepeacutetabiliteacute) 00543 Sr 00914
RSDr(coefficient de variation de la reacutepeacutetabiliteacute)205 RSDr 342
r(repeatability limit)) 0152 r 0256
SL(eacutecart-type interlaboratoire) 000139 SL 000920
SR(eacutecart-type de reproductibiliteacute) 00659 SR 01325
RSDR(coefficient de variation de la reproductibiliteacute)0025 RSDR 0050
R(limite de reproductibiliteacute)) 018446 R(limite de reproductibiliteacute))03708618
Laboratoires
Niveau 2
Moyenne si Laboratoires
Niveau 2
DRef Alt Moyenne si Deviation h Deviation h k k
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3738 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Level 2 bis
Nombre de laboratoires (p) 10
A 3000 2477 2739 037 0261 -0261 0832 1418 A 2602 2477 2540 009 0062 -0062 -0391 0465 -0199
B 2544 2813 2678 019 -0134 0134 0211 0729 B 2903 2778 2841 009 0062 -0062 1732 0465 0162
C 2477 2813 2645 024 -0168 0168 -0135 0911 C 2477 2699 2588 016 -0111 0111 -0049 0826 -0057
D 2398 2653 2526 018 -0128 0128 -1370 0692 D 2778 3000 2889 016 -0111 0111 2074 0826 0363
E 2875 2740 2808 010 0067 -0067 1547 0365 E 2602 2301 2452 021 0151 -0151 -1012 1120 -0356
F 2477 2544 2511 005 -0033 0033 -1525 0182 F 2778 2477 2628 021 0151 -0151 0230 1120 0117
G 2653 2740 2697 006 -0044 0044 0400 0236 G 2845 2845 2845 000 0000 0000 1764 0000 0148
H 2176 2813 2495 045 -0318 0318 -1692 1727 H 2477 2477 2477 000 0000 0000 -0832 0000 -0017
L 2813 2544 2678 019 0134 -0134 0211 0729 L 2903 2301 2602 043 0301 -0301 0049 2241 -0076
M 2602 2740 2671 010 -0069 0069 0135 0375 M 2903 2255 2579 046 0324 -0324 -0112 2411 -0092
m 2658114 m 259505 mediane D -00372
Cn(2p) 1867 Cn(2p) 1867 Cp 16101
Cn(p) 16101 Cn(p) 16101 Qn(D) 01656915
n=2p n 20 n=2p n 20 Qdiff 02667753
f 11 f 11 t 035
l 55 l 55
n=p n 10 n=p n 10
f 6 f 6
l 15 l 15
Qn(2p) 0098745 Qn(2p) 007195
Qn(p) 006007 Qn(p) 008805
Qintra 018437 Qintra 013435
Qinter 009672 Qinter 014176
Sr(eacutecart-type de reacutepeacutetabiliteacute) 02607 Sr 01900
RSDr(coefficient de variation de la reacutepeacutetabiliteacute)981 RSDr 732
r(repeatability limit)) 0730 r 0532
SL(eacutecart-type interlaboratoire) -002464 SLlt0 SL 000205
SR(eacutecart-type de reproductibiliteacute) 02607 SR 01953
RSDR(coefficient de variation de la reproductibiliteacute)0098 RSDR 0075
R(limite de reproductibiliteacute)) 072996 R(limite de reproductibiliteacute))054686
Laboratoires
Niveau 2 bis
Moyenne si Laboratoires
Niveau 2 bis
DRef Alt Moyenne si Deviation h Deviation h k k
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3838 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Level 3
Nombre de laboratoires (p) 10
A 3690 3663 3676 002 0014 -0014 -0311 0513 A 3690 3708 3699 001 -0009 0009 0354 0257 0022
B 3613 3672 3642 004 -0030 0030 -1851 1109 B 3708 3568 3638 010 0070 -0070 -0844 2061 -0005
C 3663 3732 3698 005 -0035 0035 0645 1302 C 3663 3699 3681 003 -0018 0018 0000 0536 -0017
D 3690 3690 3690 000 0000 0000 0311 0000 D 3643 3663 3653 001 -0010 0010 -0545 0286 -0037
E 3732 3653 3693 006 0040 -0040 0429 1480 E 3613 3732 3673 008 -0060 0060 -0162 1769 -0020
F 3643 3672 3658 002 -0014 0014 -1157 0536 F 3690 3771 3731 006 -0040 0040 0975 1193 0073
G 3699 3653 3676 003 0023 -0023 -0328 0856 G 3602 3643 3623 003 -0021 0021 -1141 0612 -0053
H 3623 3672 3648 003 -0024 0024 -1615 0913 H 3748 3681 3715 005 0033 -0033 0664 0990 0067
L 3724 3663 3694 004 0031 -0031 0461 1150 L 3748 3771 3760 002 -0011 0011 1544 0335 0066
M 3690 3690 3690 000 0000 0000 0311 0000 M 3806 3681 3744 009 0062 -0062 1234 1848 0054
m 368334 m 368086 mediane D 00089
Cn(2p) 1867 Cn(2p) 1867 Cp 16101
Cn(p) 16101 Cn(p) 16101 Qn(D) 00325359
n=2p n 20 n=2p n 20 Qdiff 00523852
f 11 f 11 t 043
l 55 l 55
n=p n 10 n=p n 10
f 6 f 6
l 15 l 15
Qn(2p) 0014323 Qn(2p) 001811
Qn(p) 0013719 Qn(p) 003164
Qintra 002674 Qintra 003381
Qinter 002209 Qinter 005094
Sr(eacutecart-type de reacutepeacutetabiliteacute) 00378 Sr 00478
RSDr(coefficient de variation de la reacutepeacutetabiliteacute)103 RSDr 130
r(repeatability limit)) 0106 r 0134
SL(eacutecart-type interlaboratoire) -000023 SLlt0 SL 000145
SR(eacutecart-type de reproductibiliteacute) 00378 SR 00611
RSDR(coefficient de variation de la reproductibiliteacute)0010 RSDR 0017
R(limite de reproductibiliteacute)) 0105894 R(limite de reproductibiliteacute))017119
Ref Alt DLaboratoires
Niveau 3
Moyenne si Deviation h k Laboratoires
Niveau 3
Moyenne si Deviation h k
Thermo Fisher Scientific
ADRIA Deacuteveloppement 438 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
1 INTRODUCTION
11 Dates of the validation studies
The Listeria PrecisTM method for enumeration of Listeria monocytogenes was
validated on September 15 2006 (certificate number UNI 0305 ndash 0906) for
food products and environmental samples
In 2007 an extension study was obtained for a new confirmation test OBIS
MONO
The alternative method was renewed in 2010 and 2014
12 Protocol and principle of the alternative method
The Listeria PrecisTM method is based on a specific chromogenic media for
Listeria monocytogenes enumeration
The protocol is described in Appendix 1
13 Standard method
The reference method is the ISO 11290-2 (1998) amp ISO 11290-2A1 (2004)
Horizontal method for the detection and enumeration of Listeria
monocytogenes - Part 2 enumeration of Listeria monocytogenes in foods
(See Appendix 2)
Analysis performed according to the COFRAC accreditation
Thermo Fisher Scientific
ADRIA Deacuteveloppement 538 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
2 VALIDATION STUDY RESULTS
21 Initial validation (realised by ASEPT 2006)
211 Method Comparison Study
2111 Linearity
Linearity is the ability of the method when used with a given matrix to give results that are in proportion
to the amount of analyte present in the sample that is an increase in analyte corresponds to a linear or
proportional increase in results
Food matrices and protocols
Six matrixstrain pairs were analysed with five contamination levels and two
replicates At least 50 analyses were performed by the alternative and the
standard methods
The contamination levels the samples and the inoculated strains are
presented in the table below
Table 1
Samples Strains Contamination levels
(CFUg)
Rillettes Listeria monocytogenes 4e 10 ndash 50
50 ndash 100
100 ndash 500
500 ndash 1 000
1 000 ndash 10 000
Raw milk Listeria monocytogenes 12a
Lettuce Listeria monocytogenes 12a
Smoked salmon Listeria monocytogenes 12b
Raw egg product Listeria monocytogenes 12b
Process water Listeria monocytogenes 12a
The samples were individually inoculated and were analysed in duplicate by
the alternative and the reference methods
Results
The bi-dimensional graphs are shown figure 1
Thermo Fisher Scientific
ADRIA Deacuteveloppement 638 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Figure 1 ndash Bi-dimensional graphs
Rillettes Raw milk
Smoked salmon Raw egg product
Lettuce Process water
Thermo Fisher Scientific
ADRIA Deacuteveloppement 738 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Statistical interpretations
Statistical interpretation results are provided in Table 2
Table 2 ndash Statistical interpretations
Matrix R Selected
regression
RobF Critical
value
P Correlation
coefficient
Regression equation
Rillettes 180 GMFR 609 541 4 09960 log (Alt) = 09887 log (Ref) + 01141
Lettuce 108 GMFR 000 541 100 09992 Log (Alt) = 10081 log (Ref) - 00257
Raw milk 102 GMFR 000 541 100 09997 Log (Alt) = 10188 log (Ref) - 00737
Smoked salmon 1048 OLS1 1806 541 263 09984 Log (Alt) = 10406 log (Ref) - 01418
Raw egg product 130 GMFR 548 541 49 09958 Log (Alt) = 09775 log (Ref) - 00161
Process water 184 GMFR 0079 541 969 09985 Log (Alt) = 09680 log (Ref) + 01317
GMFR = orthogonal linear regression
OLS1 = ordinary least-square linear regression
x ndash axis = reference method
y ndash axis = alternative method
OLS1 = ordinary least-square linear regression
x axis = alternative method
y axis = reference method
Statistical Interpretation
P gt 5 not significant 1 lt P lt 5 significant
P lt 1 highly significant
The regressions straight lines are shown figure 2
Thermo Fisher Scientific
ADRIA Deacuteveloppement 838 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Figure 2 ndash Regression straight lines
Lineacuteariteacute - Œuf
Process water Process water (2) Process water
Smoked salmon (GMFR) Smoked salmon (OLS 1)
Raw milk Raw egg product
Rillettes Lettuce
Thermo Fisher Scientific
ADRIA Deacuteveloppement 938 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Discussion
The linearity is accepted in all cases with
P values 5 and correlation coefficients superior to 099
For the matrix ldquoRillettesldquo the linearity test is significant (P = 4) with a high
correlation coefficient value (0996)
The Listeria PrecisTM method shows satisfying linearity
2112 Relative accuracy
The accuracy is the closeness of agreement between a test result and the accepted reference value
The bias is the difference between the expectation of the test results and an accepted reference value
Number and nature of the samples
The repartition per category and types is provided in table 3
Table 3 ndash Number and nature of the samples
Categories Types Number of samples
Analysed Exploited (1)
Meat products
Raw meat products 6 4
Ready to cook products 4 2
Ready to eat delicatessen 10 4
Total 20 10
Dairy products
Raw milk cheeses 7 4
Raw milks 4 3
Other dairy products 5 5
Total 16 12
Fishery products
Raw fishes 5 4
Processed fishes 7 2
Ready-to-eat products 6 4
Total 18 10
Vegetables
Raw vegetables 5 4
Frozen vegetables 3 3
Lettuce raw vegetables 5 5
Total 13 12
Egg products
Raw egg 5 5
Pastries 3 3
Pasteurised egg products 4 3
Total 12 11
Environmental samples
Wipes swabs 8 4
Process water 3 3
Dusts 3 3
Total 14 10
TOTAL 93 65
(1) Some data cannot be exploited because enumeration was under the detection level for at least one of the
method
Thermo Fisher Scientific
ADRIA Deacuteveloppement 1038 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Artificial contamination of the samples
Artificial contaminations were realised by spiking after performing injury
protocols on pure cultures or by cross-contaminations
56 samples were artificially contaminated 53 gave exploitable results among
a total of 65 samples 8150 of the samples were artificially contaminated
The injury protocol and the strains inoculated are provided in Table 4
Table 4
Stress Ndeg Injury protocol Strain Origin
A 45 min at 50 degC
48
LM -H123
LM-H170
Smoked salmon
Lettuce
Lettuce
B 30 min at 50 degC
and 2 h at ndash20degC
18
43
LM-O1
Raw milk (environment)
Environment
Egg product
C 30 min at 55degC
18
48
LM-H123
Raw milk
Smoked salmon
Lettuce
D 30 min at 50degC
et 2 h at ndash80degC
34
LM-H171
LM-O1
Rillettes
Lettuce
Egg product
E 30 min at 60 degC LM-O1 Egg product
Confirmatory tests
For the reference and the alternative methods confirmations were realised
by the tests described in the ISO 12290-2A1 standard For the alternative
method only one colony was confirmed while 5 colonies were tested for the
reference method
Results
Samples were analysed in duplicate by the reference and the alternative
methods
The contamination range is provided in table 5 per tested category
Thermo Fisher Scientific
ADRIA Deacuteveloppement 1138 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Table 5 ndash Contamination range
Food category Contamination level (log CFUg)
Meat products 100 ndash 404
Dairy products 148 ndash 408
Fishery products 100 ndash 384
Vegetables 170 ndash 511
Egg products 130 ndash 499
Environmental samples 170 ndash 511
All categories 100 ndash 511
Bi-dimensional graphs are given figure 3
Figure 3 ndash Bi-dimensional graphs
Graphe bi-dimensionnel Produits carneacutes
Fishery products
Vegetables
Egg products
Meat products
Thermo Fisher Scientific
ADRIA Deacuteveloppement 1238 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Statistical interpretation according to EN ISO16140 method
The results of the statistical interpretation are given in Table 6
Table 6 ndash Statistical interpretation results
Category n R Regression
used a t(a) b t(b) Critical T
P
Ordinate
at 0
Slope
at 1
Meat products 10 158 GMFR -0455 3321 115 3123 2306 001 001
Dairy products 12 065 GMFR -0027 0385 1002 0108 2228 092 071
Fishery products 10 130 GMFR -0305 1024 1034 0312 2306 076 034
Vegetables 12 086 GMFR -0015 0128 0993 0191 2228 085 090
Egg products 11 158 GMFR -0038 0866 1001 0090 2262 093 041
Environmental
samples 10 176 GMFR -0139 265 1034 231 2306 005 003
All products 65 118 GMFR -0182 3042 1040 2145 2000 004 000
All products
(estimated number) 65 118 GMFR -0182 3042 1040 2145 2000 004 000
All products (non
estimated results) 20 115 GMFR -0367 151 1138 1141 2101 027 015
Repeatability limits obtained for the alternative and reference methods as
well as bias obtained are presented in Table 7
Dairy products Environmental samples
Log (Ref) Log (Ref)
All categories
Thermo Fisher Scientific
ADRIA Deacuteveloppement 1338 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Table 7
Categories Bias
Repeatability limits
Rob r
Ref Alt
Meat products 0008 0314 0343
Dairy products -0031 0136 0130
Fishery products -0133 0220 0214
Vegetables -0041 0180 0319
Egg products -0026 0219 0206
Environmental samples -0007 0198 0119
All products -0032 0196 0215
Regression straight lines (graph and equation representations) for each food
category and for all products are presented figure 4
Thermo Fisher Scientific
ADRIA Deacuteveloppement 1438 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Figure 4 ndash Regression straight lines
Meat products
Fishery products
Dairy products
Vegetables
Thermo Fisher Scientific
ADRIA Deacuteveloppement 1538 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Environmental samples
Egg products
All products (estimated number)
Thermo Fisher Scientific
ADRIA Deacuteveloppement 1638 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
The hypotheses that the ordinates are close to 0 and the slopes close to 1
are approved in all cases except for
- Meat products (slope P = 1 ordinate P = 1)
- Environmental samples (ordinate P = 3)
- All products (ordinate P = 0)
The bias between the two methods vary from ndash 0133 to 0008 log CFU For
all categories the bias is ndash 0032 log CFU The repeatability limits of the
alternative and the reference method are similar For all products the values
are respectively 0215 and 0196 log CFU For all products these
interpretations are provided taking into account data with low contamination
level or not (estimated colonies number)
For all food products the regression straight line is the following
log (Alt) = 1040 log Ref ndash 0182
The Listeria PrecisTM method shows satisfying relative accuracy
All products
All products (Level 2)
Thermo Fisher Scientific
ADRIA Deacuteveloppement 1738 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
2113 Detection limit (LOD) and quantification limit (LOQ)
The critical level is the defined as the smallest amount which can be detected (not null) but not
quantified as an exact value Below this value it cannot be sure that the true value is not null
The detection limit is defined as being higher than the critical level because it involves a power the
probability 1- which has to be well over 50 for example 95
The quantification limit is defined as the smallest amount of analyte (that is the lowest actual number of
organisms) which can be measured and quantified with defined precision and accuracy under the
experimental conditions by the method under validation
Protocol
The detection limit of the alternative method was realised with pure cultures
Three different inoculation levels were tested with six replicates per level ie
18 analyses were done by the alternative method
Listeria monocytogenes 18 was used for the detection limit of the alternative
method
Quantification limit was calculated for six independent blank samples
determinations
Results
These data are intrinsic to the method used and are presented in the
following tables
Table 8 ndash Inoculation 1 ml on 3 plates
Level (CFUml) Positive samples number Standard deviation Bias
0 06
05 26 1211 0
1 36 0816 05
5 66 2483 85
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Summary Report (Version 1)
Listeria Precis Enumeration
Table 9 ndash Inoculation 01 ml on 1 plate
Level (CFUml) Positive samples number Standard deviation Bias
0 06
5 36 0548 05
10 46 1414 2
50 66 2258 55
Table 10
Formulas Obtained values
LC 165 S0 + X0 18
LOD 33 S0 + X0 32
LOQ 10 S0 + X0 87
2114 Relative Sensitivity
The relative sensitivity is defined as the ability of the alternative method to detect two different amounts
of analyte measured by the reference method within a given matrix at a specified average value or
over the whole measurement range that is it is the minimal quantity variation (increase of the analyte
concentration x) which gives a significant variation of the measured signal (response y)
These data are intrinsic to the used method and are obtained from the results
of the linearity study
Sensitivity patterns obtained for tested (matrixstrain) pairs are presented in
figure 5
Thermo Fisher Scientific
ADRIA Deacuteveloppement 1938 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Figure 5 ndash Sensitivity patterns for the tested (matrixstrain) pairs
2115 SpecificitySelectivity
The specificity is defined as the degree to which a method is affected (or not) by the other components
present in a multi-component sample That is the ability of a method to measure exactly a given
analyte or its amount within the sample without interference from non-target components such as a
matrix effect or background noise
The selectivity is defined as a measure of the degree of non-interference in the presence of non-target
analytes A method is selective if it can be used to detect the analyte under examination and that a
guarantee can be provided that the detected signal can only be a product by that specific analyte
The specificity and selectivity study was performed for the Listeria PrecisTM
detection method no additional test was realised for the enumeration
method The results were the following
- Among the Listeria monocytogenes strains tested 2 strains (Listeria
monocytogenes CIP 7834 and Listeria monocytogenes CIP 105459) gave
negative results These 2 strains were tested twice by running 48 h
incubation time of the compared agars Positive results were then
observed by the alternative method Pale characteristic colonies were
observed on the ISO agar by streaking the Half Fraser broth only
- No cross reaction was observed with the non-target strains except with
Listeria ivanovii which gave characteristic colonies on Brilliancetrade Listeria
Agar but the confirmatory tests provide a clear discrimination between
Listeria monocytogenes and Listeria ivanovi species
Rillettes
Lettuce
Raw milk
Smoked salmon
Raw egg
Process water
x (CFUg)
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Summary Report (Version 1)
Listeria Precis Enumeration
212 Practicability
1 Packaging Ready to use Brilliance Listeria agar plates are available
(x 10 plates)
2 Reagent volume
3 Storage conditionsndash
Shelf life of products
not opened
The plates are stored at 6 - 12degC in darkness conditions The
expiration date is mentioned on the package and on the plates It
is around 3 months
4 Modalities of use
after first use
5 Required
equipments No specific equipment is required
6 Reagents Ready to use plates
7 Training 05 day
8 Real time handling
Reference method
9 min for one sample
55 min for 10 samples
Alternative method
7 min for one sample
35 minutes for 10 samples
9 Time to result
Negative results
Reference method Day 2
Alternative method Day 2
Negative results after confirmation or positive results
Reference method Day 4 ndash Day 7
Alternative method Day 4 ndash Day 7
Positive results
Reference method Day 4 ndash Day 7
Alternative method Day 2 ndash Day 3 (with OBIS Mono Test)
or Day 4 ndash Day 7 (by the classical tests described in the
standardised method)
10 Technician skill Identical to that required for the reference method
11 Steps common to
the reference method Mother suspension revivification step and decimal dilutions
12 Traceability
13 Maintenance
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Listeria Precis Enumeration
213 Inter-laboratory study
2131 Organisation
12 laboratories participated to this study Pasteurised milk was inoculated by
Listeria monocytogenes 12a isolated from raw milk The targeted
inoculation levels were
- 0 CFUml
- 50 CFU ml
- 500 CFU ml
- 5 000 CFU ml
Each laboratory received eight flasks of 10 ml sample ie two flasks per
inoculation level Furthermore one non-inoculated sample has been added
to the package for total viable count microflora (NF ISO 4833 method)
Detailed instructions were transmitted to the collaborators by the expert
laboratory
Collaborative study laboratories and the expert laboratory carried out the
analyses with the alternative and reference methods
2132 Verification of experimental parameters
Strain stability during transport
In order to evaluate the Listeria monocytogenes strain stability during
transport bacterial count of all inoculated flasks were checked at different
time ie inoculation time after 24 h of conservation at 2deg - 8degC Results are
reported in table 11
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Summary Report (Version 1)
Listeria Precis Enumeration
Table 11 ndash Listeria monocytogenes count
by ISO 11290-2A1 method (log CFUml)
Day of the analyses Level contamination Reference method
Day 0
(29052006)
1 45
2 440
3 4 500
Day 1
(30052006)
1 60
2 430
3 4 900
No evolution of the strain was observed after storage for 24 h at 2degC ndash 8degC
Contamination level
The real contamination levels are provided in table 12
Table 12 ndash Real contamination level (CFUml)
Target inoculation
(CFU ml) Samples Ndeg
Real contamination levels
(CFUml)
0 1 - 8 0
50 5 - 6 45
500 3 - 7 440
5000 2 - 4 4 500
Sample temperature at receipt
Measured temperatures at receipt are listed in table 13
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Summary Report (Version 1)
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Table 13 - Sample temperature on receipt
Laboratories Receipt
day
Hour
receipt
Measured temperature
at receipt (degC)
Measured temperature by the
temperature probe (degC)
A
B
C
D
E
F
G
H
I
J
K
L
M
30052006
10h45
10h15
11h15
10h00
09h30
8h30
11h50
10h00
08h30
14h45
08h30
09h45
08h45
82
55
45
63
60
62
81
38
48
-
14
67
76
03
03
-13
03
04
-14
05
04
01
05
-13
04
Conclusion
The temperatures during the transport were correct for all the labs except for
Lab J which didnrsquot provide the information
All the labs received their packages with several broken flasks
Three labs (B D and L) received some damaged samples but proceeded to
analyses
All the labs started the analyses at Day 1 except Lab J
2133 Results
Aerobic mesophilic flora
The count of aerobic mesophilic flora varied from 94 102 CFUml to
33 103 CFUml
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Listeria Precis Enumeration
Expert lab results
The results obtained by the expert lab are the following
Table 14
Target inoculation level (CFUml)
Reference method ISO 11290-2
Alternative method Listeria Precis Enumeration
Rep 1 Rep 2 Rep 1 Rep 2
lt 10 lt 10 lt 10 lt 10 lt10
50 50 (Ne) 60 (Ne) 50 (Ne) 30 (Ne)
500 470 390 490 500
5 000 4 700 5 100 5 900 3 400
Ne estimated number
The obtained results are in agreement with those expected
Collaborator Labs results
A synthesis of the results obtained by the collaborator labs is provided
Table 15 (CFUml) and Table 16 (log CFUml)
Table 15 ndash Synthesis of results obtained by the alternative
and the reference methods (CFUml)
Labs Level 1 Level 2 Level 2 bis Level 3
Reference method
Alternative method
Reference method
Alternative method
Reference method
Alternative method
Reference method
Alternative method
A 40 40 70 40 480 410 210 340 1000 300 400 300 4900 4600 4900 5100
B 40 15 40 20 350 300 360 450 350 650 800 600 4100 4700 5100 3700
C 50 20 20 70 440 430 480 450 300 650 300 500 4600 5400 4600 5000
D 40 60 40 40 490 460 600 540 250 450 600 1000 4900 4900 4400 4600
E 50 40 50 20 490 520 530 560 750 550 400 200 5400 4500 4100 5400
F 60 20 60 50 370 250 300 130 300 350 600 300 4400 4700 4900 5900
G 70 35 120 50 440 390 570 370 450 550 700 700 5000 4500 4000 4400
H 110 40 100 90 520 440 450 540 150 650 300 300 4200 4700 5600 4800
L 35 60 20 40 390 390 170 380 650 350 800 200 5300 4600 5600 5900
M 45 45 30 40 450 490 550 410 400 550 800 180 4900 4900 6400 4800
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Listeria Precis Enumeration
Table 16 ndash Synthesis of results obtained by the alternative
and the reference methods (log CFUml)
Labs Level 1 Level 2 Level 2 bis Level 3
Reference method
Alternative method
Reference method
Alternative method
Reference method
Alternative method
Reference method
Alternative method
A 1602 1602 1845 1602 2681 2613 2322 2531 3000 2477 2602 2477 3690 3663 3690 3708
B 1602 1176 1602 1301 2544 2477 2556 2653 2544 2813 2903 2778 3613 3672 3708 3568
C 1699 1301 1301 1845 2643 2633 2681 2653 2477 2813 2477 2699 3663 3732 3663 3699
D 1602 1778 1602 1602 2690 2663 2778 2732 2398 2653 2778 3000 3690 3690 3643 3663
E 1699 1602 1699 1301 2690 2716 2724 2748 2875 2740 2602 2301 3732 3653 3613 3732
F 1778 1301 1778 1699 2568 2398 2477 2114 2477 2544 2778 2477 3643 3672 3690 3771
G 1845 1544 2079 1699 2643 2591 2756 2568 2653 2740 2845 2845 3699 3653 3602 3643
H 2041 1602 2000 1954 2716 2643 2653 2732 2176 2813 2477 2477 3623 3672 3748 3681
L 1544 1778 1301 1602 2591 2591 2230 2580 2813 2544 2903 2301 3724 3663 3748 3771
M 1653 1653 1477 1602 2653 2690 2740 2613 2602 2740 2903 2255 3690 3690 3806 3681
Level 2 results obtained taking account the 3 plates inoculated with the mother-suspension
Level 2 bis results obtained taking account the plates inoculated with 01 ml of the mother-suspension
Scrutiny of the measurement results for consistency
In order to identify the measurement results or laboratories that could be
inconsistent two graphical consistency techniques were realised Mandelrsquos
h-ank-statistics Mandel indicators h and k at 5 significance highlighted
some possible inconsistent data (See tables and graphics in Appendix 3 and
Appendix 4)
Mandelrsquos values Number of values observed over the threshold
Reference method Alternative method
h gt 1 Lab F Level 2 Lab F Level 2
h gt 5 Lab F Level 2
Lab B Levels 1 and 2
Lab A Level 2
Lab D Level 3
Lab F Level 2
Lab L Level 2
kgt 1 Lab F Level 3
k gt 5 Lab F Level 2 Lab C Level 2
Lab F Level 3
Lab L Levels 1 and 3
Lab M Level 1
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2638 November 19 2014
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Listeria Precis Enumeration
The emphasized results appear (i) coherent with the targeted contamination
levels (ii) concordant between both methods
All the results were thus kept for the statistical analysis
Comparison of the trueness and precision characteristics of
the reference method and alternative methods
The statistical values are summarised hereafter
Level p
Reference method Alternative method
Repeatability ratio
Reproducibility ratio Median
Repeatability standard deviation
Reproducibility standard deviation
Median Repeatability
standard deviation
Reproducibility standard deviation
1 10 16519 02415 02425 16021 02045 02433 0847 1007
2 10 26551 00543 00659 26719 00914 01325 1682 2011
2 bis 10 26581 02607 02607 25951 01900 01953 0729 0749
3 10 18671 00378 00378 18671 00478 00611 1264 1617
Bias of the alternative method
In order to estimate the bias of the alternative method with regard to the reference method for every
level Dij and t are calculated according to the following equations
RefAlt ijYijYDij
Diffp
Dijit
)2(
)(median
If the t value is superior to 2 the alternative method is significantly biased
with regard to the reference method
The values are given in table 17
Table 17 ndash t (d) values obtained by level
Level Bias t (d) Conclusion
1 0068 114 Non significant bias
2 0021 082 Non significant bias
2 bis - 0037 035 Non significant bias
3 - 0037 043 Non significant bias
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2738 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
The statistical test concludes to a non significant bias for all the inoculation
levels The bias varies from ndash 0037 to 0068 log CFUml
Comparison of the repeatability standard deviations
If the ratio Srj Alt Srj Ref of the repeatability standard deviations of the
alternative method and the reference method is larger than 2 the precision
under repeatability conditions of the alternative method is considered to be
lower than that of the reference method If this ratio is smaller than 05 the
precision under repeatability conditions of the alternative method is
considered to be greater than that of the reference method
The ratio values are given in table 18
Table 18
Level
Repeatability standard deviation
Reference method Alternative method Alternative method
reference method ratio
1 0242 0205 0847
2 0054 0091 1682
2 bis 0261 0190 0729
3 0038 0048 1264
Level Repeatability limit
Reference method Alternative method
1 0676 0573
2 0152 0256
2 bis 0730 0532
3 0106 0134
The ratios of the repeatability standard deviations are below 2 in all cases
The precision under repeatability conditions of the alternative method is
equivalent to that of the reference method
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2838 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Comparison of the reproducibility standard deviations
If the ratio Srj Alt Srj Ref of the reproducibility standard deviations of the alternative method and the
reference method is larger than 2 the precision under reproducibility conditions of the alternative
method is considered to be lower than that of the reference method If this ratio is smaller than 05 the
precision under reproducibility conditions of the alternative method is considered to be greater than
that of the reference method
The ratio values are given in table 19
Table 19
Level
Reproducibility standard deviation
Reference method Alternative method Alternative method
reference method ratio
1 0242 0243 1007
2 0066 0133 2011
2 bis 0261 0195 0749
3 0038 0061 1617
Level Reproducibility limit
Reference method Alternative method
1 0676 0681
2 0185 0371
2 bis 0730 0547
3 0106 0171
The ratios of the reproducibility standard deviations are below or close to 2
The precision under reproducibility conditions of the alternative method is
equivalent to that of the reference method
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2938 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
214 Conclusion
Method comparison study
The Listeria PrecisTM enumeration method shows satisfying linearity and
relative accuracy
The alternative method shows satisfying inclusivity and exclusivity results
Two Listeria monocytogenes strains gave negative results with the
alternative method These strains grew weakly on the reference method
media (OampA Palcam and Oxford)
The limits of repeatability of the alternative method are similar to those of the
reference method
The bias between both methods are low between - 0133 Log CFUg and
- 0008 Log CFUg
The Listeria PrecisTM enumeration method offers the saving for confirmatory
tests
Positive results are obtained with the alternative method in 2-3 days (with
OBIS Mono test) or in 4-7 days (by the classical tests described in the
standardized method) against 4-7 days for the reference method
Inter-laboratory study
The bias between the reference and the alternative methods shows satisfying
and correct values
- level 1 (100 - 1 000 CFUg) bias = + 007 log CFUg
- level 2 (1 000 - 10 000 CFUg) bias = 002 log CFUg
- level 2 bis (1 000 - 10 000 CFUg) bias = - 004 log CFUg
- level 3 (10 000 - 100 000 CFUg) bias = - 004 log CFUg
The repeatability and the reproducibility of the alternative method look similar
to those of the reference method
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3038 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
22 Extension study (realised by ASEPT 2007)
An extension study was performed in 2007 for a new confirmatory test OBIS
MONO
150 Listeria monocytogenes strains and 100 non-target strains were isolated
onto TSAYE and BrillianceTM Listeria Agar plates All the Listeria
monocytogenes and the non-target strains gave the expected results
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3138 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Appendix 1 ndash Flow diagram of the Listeria PrecisTM method
x g sample + 9 x ml buffered peptone water
Revivification step for 1 h plusmn 5 min at 20degC plusmn 1degC
Streaking 1 ml onto 3 Brilliance Listeria Agar plates (mother suspension)
or 01 ml onto 1 Brilliance Listeria Agar plates (mother suspension and decimal dilutions)
Incubation for 48 h plusmn 3 h at 37degC plusmn 1degC
Confirmatory tests on at least one typical colony
- by the tests described in the ISO method
- by applying the OBIS mono test on an isolated colony
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3238 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Appendix 2 ndash ISO 11290-2 (1998) amp ISO 11290-2A1 (2004) Horizontal
method for the detection and enumeration of Listeria monocytogenes -
Part 2 enumeration of Listeria monocytogenes in foods
10 g cheese + 90 ml buffered peptone water or Half Fraser Broth (without supplements) (110 dilution)
Stomach 1 min 30 seconds
Keep at 20degC plusmn 2degC for 1 h plusmn 5 min
Decimal dilutions in 9 ml peptone salt tubes
Dilution 110
Dilution 1100
Dilution 11000
1 ml on 3 Chromogenic Listeria medium (ISO) plates (x 2) (-1)
01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-2)
01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-3)
01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-4)
Incubation at 37degC plusmn 1degC
24 h - 48 h
Numerate typical colonies at 24 h
(bluegreen surrounded by an opaque halo)
Numerate at 48 h
Streak typical colonies on TSYEA
(5 coloniesplate 2 successive dilutions)
Confirmation tests
Catalase
Gram stain
For confirmation of L monocytogenes
Haemolysis
Carbohydrate (rhamnose and xylose) utilisation
CAMP Test
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3338 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Appendix 3 ndash Mandelrsquos graphics
Laboratoire h Ref
Niveau 1 h Ref
Niveau 2 h Ref
Niveau 3 h Alt
Niveau 1 h Alt
Niveau 2 h Alt
Niveau 3 h5 h1 h5 h1
A -050 -015 083 062 -212 -039 198 299 -198 -299
B -261 -270 021 -077 -058 173 198 299 -198 -299
C -151 -031 -014 -015 -004 -005 198 299 -198 -299
D 038 040 -137 000 072 207 198 299 -198 -299
E -001 090 155 -052 056 -101 198 299 -198 -299
F -112 -321 -153 070 -325 023 198 299 -198 -299
G 042 -071 040 147 -009 176 198 299 -198 -299
H 169 046 -169 192 018 -083 198 299 -198 -299
L 009 -120 021 -077 -231 005 198 299 -198 -299
M 001 031 014 -032 004 -011 198 299 -198 -299
-300
-200
-100
000
100
200
300
A B C D E F G H L M
Vale
urs
h d
e M
an
del(
rob
uste
s)
Laboratoires
Meacutethode de reacutefeacuterence analyse robuste
h Ref Niveau 1
h Ref Niveau 2
h Ref Niveau 3
h 5=198
h 1=299
-300
-200
-100
000
100
200
300
A B C D E F G H L M
Vale
urs
de h
de M
an
del(
rob
uste
s)
Laboratoires
Meacutethode alternative analyse robuste
h Alt Niveau 1
h Alt Niveau 2
h Alt Niveau 2
h 1=299
h 5=198
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3438 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Laboratoire k Ref
Niveau 1 k Ref
Niveau 2 k Ref
Niveau 3 k Alt
Niveau 1 k Alt
Niveau 2 k Alt
Niveau 3 k1 k5
A 000 089 142 084 162 046 259 181
B 125 087 073 104 075 046 259 181
C 117 013 091 188 022 083 259 181
D 052 036 069 000 035 083 259 181
E 028 034 037 138 019 112 259 181
F 140 222 018 027 281 112 259 181
G 088 068 024 131 145 000 259 181
H 129 094 173 016 061 000 259 181
L 069 000 073 104 270 224 259 181
M 000 048 038 043 099 241 259 181
000
050
100
150
200
250
300
350
400
A B C D E F G H L M
Vale
urs
k d
e M
an
del(
rob
uste
s)
Laboratoires
Meacutethode de reacutefeacuterence analyse robuste
k Ref Niveau 1k Ref Niveau 2k Ref Niveau 3
k 1=259
k 5=181
000
050
100
150
200
250
300
A B C D E F G H L M
Vale
urs
k d
e M
an
del
(ro
bu
ste
s)
Laboratoires
Meacutethode alternative analyse robuste
k Alt Niveau 3
k Alt Niveau 1
k Alt Niveau 2
k 1=259
k 5=181
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3538 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Appendix 4 ndash Statistical results
Level 1
Nombre de laboratoires (p) 10
A 1602 1602 1602 000 0000 0000 -0495 0000 A 1845 1602 1724 017 0122 -0122 0621 0840 0122
B 1602 1176 1389 030 0213 -0213 -2613 1247 B 1602 1301 1452 021 0151 -0151 -0769 1041 0062
C 1699 1301 1500 028 0199 -0199 -1510 1165 C 1301 1845 1573 038 -0272 0272 -0148 1881 0073
D 1602 1778 1690 012 -0088 0088 0380 0516 D 1602 1602 1602 000 0000 0000 0000 0000 -0088
E 1699 1602 1651 007 0048 -0048 -0013 0284 E 1699 1301 1500 028 0199 -0199 -0522 1376 -0151
F 1778 1301 1540 034 0239 -0239 -1116 1397 F 1778 1699 1739 006 0040 -0040 0698 0274 0199
G 1845 1544 1695 021 0151 -0151 0425 0881 G 2079 1699 1889 027 0190 -0190 1467 1315 0194
H 2041 1602 1822 031 0220 -0220 1689 1286 H 2000 1954 1977 003 0023 -0023 1917 0158 0155
L 1544 1778 1661 017 -0117 0117 0092 0685 L 1301 1602 1452 021 -0151 0151 -0769 1041 -0210
M 1653 1653 1653 000 0000 0000 0013 0000 M 1477 1602 1540 009 -0062 0062 -0319 0432 -0114
m 165186 m 160206 mediane D 00678
Cn(2p) 1867 Cn(2p) 1867 Cp 16101
Cn(p) 16101 Cn(p) 16101 Qn(D) 0092926
n=2p n 20 n=2p n 20 Qdiff 01496169
f 11 f 11 t 114
l 55 l 55
n=p n 10 n=p n 10
f 6 f 6
l 15 l 15
Qn(2p) 0091465 Qn(2p) 007745
Qn(p) 0062469 Qn(p) 0121519
Qintra 017078 Qintra 014461
Qinter 010058 Qinter 019565
Sr(eacutecart-type de reacutepeacutetabiliteacute) 02415 Sr 02045
RSDr(coefficient de variation de la reacutepeacutetabiliteacute)1462 RSDr 1277
r(repeatability limit)) 0676 r 0573
SL(eacutecart-type interlaboratoire) -001905 SLlt0 SL 001737
SR(eacutecart-type de reproductibiliteacute) 02415 SR 02433
RSDR(coefficient de variation de la reproductibiliteacute)0146 RSDR 0152
R(limite de reproductibiliteacute)) 06762 R(limite de reproductibiliteacute))068123
h k h MoyenneLaboratoires
Niveau 1
si LaboratoiresDeviationMoyenne Dk DeviationsiAlt
Niveau 1
Ref
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3638 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Level 2
Nombre de laboratoires (p) 10
A 2681 2613 2647 005 0034 -0034 -0151 0891 A 2322 2531 2427 015 -0105 0105 -2119 1620 -0220
B 2544 2477 2511 005 0033 -0033 -2699 0872 B 2556 2653 2605 007 -0048 0048 -0581 0750 0094
C 2643 2633 2638 001 0005 -0005 -0311 0130 C 2681 2653 2667 002 0014 -0014 -0040 0217 0029
D 2690 2663 2676 002 0014 -0014 0400 0357 D 2778 2732 2755 003 0023 -0023 0721 0354 0079
E 2690 2716 2703 002 -0013 0013 0897 0336 E 2724 2748 2736 002 -0012 0012 0556 0185 0033
F 2568 2398 2483 012 0085 -0085 -3214 2217 F 2477 2114 2296 026 0182 -0182 -3255 2811 -0188
G 2643 2591 2617 004 0026 -0026 -0707 0682 G 2756 2568 2662 013 0094 -0094 -0085 1453 0045
H 2716 2643 2680 005 0036 -0036 0460 0945 H 2653 2732 2693 006 -0040 0040 0181 0613 0013
L 2591 2591 2591 000 0000 0000 -1196 0000 L 2230 2580 2405 025 -0175 0175 -2307 2704 -0186
M 2653 2690 2672 003 -0018 0018 0311 0481 M 2740 2613 2677 009 0064 -0064 0040 0987 0005
m 2655082 m 26719001 mediane D 00209
Cn(2p) 1867 Cn(2p) 1867 Cp 16101
Cn(p) 16101 Cn(p) 16101 Qn(D) 0039911
n=2p n 20 n=2p n 20 Qdiff 00642591
f 11 f 11 t 082
l 55 l 55
n=p n 10 n=p n 10
f 6 f 6
l 15 l 15
Qn(2p) 0020570 Qn(2p) 00345985
Qn(p) 00332437 Qn(p) 00718158
Qintra 003841 Qintra 006460
Qinter 005352 Qinter 011563
Sr(eacutecart-type de reacutepeacutetabiliteacute) 00543 Sr 00914
RSDr(coefficient de variation de la reacutepeacutetabiliteacute)205 RSDr 342
r(repeatability limit)) 0152 r 0256
SL(eacutecart-type interlaboratoire) 000139 SL 000920
SR(eacutecart-type de reproductibiliteacute) 00659 SR 01325
RSDR(coefficient de variation de la reproductibiliteacute)0025 RSDR 0050
R(limite de reproductibiliteacute)) 018446 R(limite de reproductibiliteacute))03708618
Laboratoires
Niveau 2
Moyenne si Laboratoires
Niveau 2
DRef Alt Moyenne si Deviation h Deviation h k k
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3738 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Level 2 bis
Nombre de laboratoires (p) 10
A 3000 2477 2739 037 0261 -0261 0832 1418 A 2602 2477 2540 009 0062 -0062 -0391 0465 -0199
B 2544 2813 2678 019 -0134 0134 0211 0729 B 2903 2778 2841 009 0062 -0062 1732 0465 0162
C 2477 2813 2645 024 -0168 0168 -0135 0911 C 2477 2699 2588 016 -0111 0111 -0049 0826 -0057
D 2398 2653 2526 018 -0128 0128 -1370 0692 D 2778 3000 2889 016 -0111 0111 2074 0826 0363
E 2875 2740 2808 010 0067 -0067 1547 0365 E 2602 2301 2452 021 0151 -0151 -1012 1120 -0356
F 2477 2544 2511 005 -0033 0033 -1525 0182 F 2778 2477 2628 021 0151 -0151 0230 1120 0117
G 2653 2740 2697 006 -0044 0044 0400 0236 G 2845 2845 2845 000 0000 0000 1764 0000 0148
H 2176 2813 2495 045 -0318 0318 -1692 1727 H 2477 2477 2477 000 0000 0000 -0832 0000 -0017
L 2813 2544 2678 019 0134 -0134 0211 0729 L 2903 2301 2602 043 0301 -0301 0049 2241 -0076
M 2602 2740 2671 010 -0069 0069 0135 0375 M 2903 2255 2579 046 0324 -0324 -0112 2411 -0092
m 2658114 m 259505 mediane D -00372
Cn(2p) 1867 Cn(2p) 1867 Cp 16101
Cn(p) 16101 Cn(p) 16101 Qn(D) 01656915
n=2p n 20 n=2p n 20 Qdiff 02667753
f 11 f 11 t 035
l 55 l 55
n=p n 10 n=p n 10
f 6 f 6
l 15 l 15
Qn(2p) 0098745 Qn(2p) 007195
Qn(p) 006007 Qn(p) 008805
Qintra 018437 Qintra 013435
Qinter 009672 Qinter 014176
Sr(eacutecart-type de reacutepeacutetabiliteacute) 02607 Sr 01900
RSDr(coefficient de variation de la reacutepeacutetabiliteacute)981 RSDr 732
r(repeatability limit)) 0730 r 0532
SL(eacutecart-type interlaboratoire) -002464 SLlt0 SL 000205
SR(eacutecart-type de reproductibiliteacute) 02607 SR 01953
RSDR(coefficient de variation de la reproductibiliteacute)0098 RSDR 0075
R(limite de reproductibiliteacute)) 072996 R(limite de reproductibiliteacute))054686
Laboratoires
Niveau 2 bis
Moyenne si Laboratoires
Niveau 2 bis
DRef Alt Moyenne si Deviation h Deviation h k k
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3838 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Level 3
Nombre de laboratoires (p) 10
A 3690 3663 3676 002 0014 -0014 -0311 0513 A 3690 3708 3699 001 -0009 0009 0354 0257 0022
B 3613 3672 3642 004 -0030 0030 -1851 1109 B 3708 3568 3638 010 0070 -0070 -0844 2061 -0005
C 3663 3732 3698 005 -0035 0035 0645 1302 C 3663 3699 3681 003 -0018 0018 0000 0536 -0017
D 3690 3690 3690 000 0000 0000 0311 0000 D 3643 3663 3653 001 -0010 0010 -0545 0286 -0037
E 3732 3653 3693 006 0040 -0040 0429 1480 E 3613 3732 3673 008 -0060 0060 -0162 1769 -0020
F 3643 3672 3658 002 -0014 0014 -1157 0536 F 3690 3771 3731 006 -0040 0040 0975 1193 0073
G 3699 3653 3676 003 0023 -0023 -0328 0856 G 3602 3643 3623 003 -0021 0021 -1141 0612 -0053
H 3623 3672 3648 003 -0024 0024 -1615 0913 H 3748 3681 3715 005 0033 -0033 0664 0990 0067
L 3724 3663 3694 004 0031 -0031 0461 1150 L 3748 3771 3760 002 -0011 0011 1544 0335 0066
M 3690 3690 3690 000 0000 0000 0311 0000 M 3806 3681 3744 009 0062 -0062 1234 1848 0054
m 368334 m 368086 mediane D 00089
Cn(2p) 1867 Cn(2p) 1867 Cp 16101
Cn(p) 16101 Cn(p) 16101 Qn(D) 00325359
n=2p n 20 n=2p n 20 Qdiff 00523852
f 11 f 11 t 043
l 55 l 55
n=p n 10 n=p n 10
f 6 f 6
l 15 l 15
Qn(2p) 0014323 Qn(2p) 001811
Qn(p) 0013719 Qn(p) 003164
Qintra 002674 Qintra 003381
Qinter 002209 Qinter 005094
Sr(eacutecart-type de reacutepeacutetabiliteacute) 00378 Sr 00478
RSDr(coefficient de variation de la reacutepeacutetabiliteacute)103 RSDr 130
r(repeatability limit)) 0106 r 0134
SL(eacutecart-type interlaboratoire) -000023 SLlt0 SL 000145
SR(eacutecart-type de reproductibiliteacute) 00378 SR 00611
RSDR(coefficient de variation de la reproductibiliteacute)0010 RSDR 0017
R(limite de reproductibiliteacute)) 0105894 R(limite de reproductibiliteacute))017119
Ref Alt DLaboratoires
Niveau 3
Moyenne si Deviation h k Laboratoires
Niveau 3
Moyenne si Deviation h k
Thermo Fisher Scientific
ADRIA Deacuteveloppement 538 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
2 VALIDATION STUDY RESULTS
21 Initial validation (realised by ASEPT 2006)
211 Method Comparison Study
2111 Linearity
Linearity is the ability of the method when used with a given matrix to give results that are in proportion
to the amount of analyte present in the sample that is an increase in analyte corresponds to a linear or
proportional increase in results
Food matrices and protocols
Six matrixstrain pairs were analysed with five contamination levels and two
replicates At least 50 analyses were performed by the alternative and the
standard methods
The contamination levels the samples and the inoculated strains are
presented in the table below
Table 1
Samples Strains Contamination levels
(CFUg)
Rillettes Listeria monocytogenes 4e 10 ndash 50
50 ndash 100
100 ndash 500
500 ndash 1 000
1 000 ndash 10 000
Raw milk Listeria monocytogenes 12a
Lettuce Listeria monocytogenes 12a
Smoked salmon Listeria monocytogenes 12b
Raw egg product Listeria monocytogenes 12b
Process water Listeria monocytogenes 12a
The samples were individually inoculated and were analysed in duplicate by
the alternative and the reference methods
Results
The bi-dimensional graphs are shown figure 1
Thermo Fisher Scientific
ADRIA Deacuteveloppement 638 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Figure 1 ndash Bi-dimensional graphs
Rillettes Raw milk
Smoked salmon Raw egg product
Lettuce Process water
Thermo Fisher Scientific
ADRIA Deacuteveloppement 738 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Statistical interpretations
Statistical interpretation results are provided in Table 2
Table 2 ndash Statistical interpretations
Matrix R Selected
regression
RobF Critical
value
P Correlation
coefficient
Regression equation
Rillettes 180 GMFR 609 541 4 09960 log (Alt) = 09887 log (Ref) + 01141
Lettuce 108 GMFR 000 541 100 09992 Log (Alt) = 10081 log (Ref) - 00257
Raw milk 102 GMFR 000 541 100 09997 Log (Alt) = 10188 log (Ref) - 00737
Smoked salmon 1048 OLS1 1806 541 263 09984 Log (Alt) = 10406 log (Ref) - 01418
Raw egg product 130 GMFR 548 541 49 09958 Log (Alt) = 09775 log (Ref) - 00161
Process water 184 GMFR 0079 541 969 09985 Log (Alt) = 09680 log (Ref) + 01317
GMFR = orthogonal linear regression
OLS1 = ordinary least-square linear regression
x ndash axis = reference method
y ndash axis = alternative method
OLS1 = ordinary least-square linear regression
x axis = alternative method
y axis = reference method
Statistical Interpretation
P gt 5 not significant 1 lt P lt 5 significant
P lt 1 highly significant
The regressions straight lines are shown figure 2
Thermo Fisher Scientific
ADRIA Deacuteveloppement 838 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Figure 2 ndash Regression straight lines
Lineacuteariteacute - Œuf
Process water Process water (2) Process water
Smoked salmon (GMFR) Smoked salmon (OLS 1)
Raw milk Raw egg product
Rillettes Lettuce
Thermo Fisher Scientific
ADRIA Deacuteveloppement 938 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Discussion
The linearity is accepted in all cases with
P values 5 and correlation coefficients superior to 099
For the matrix ldquoRillettesldquo the linearity test is significant (P = 4) with a high
correlation coefficient value (0996)
The Listeria PrecisTM method shows satisfying linearity
2112 Relative accuracy
The accuracy is the closeness of agreement between a test result and the accepted reference value
The bias is the difference between the expectation of the test results and an accepted reference value
Number and nature of the samples
The repartition per category and types is provided in table 3
Table 3 ndash Number and nature of the samples
Categories Types Number of samples
Analysed Exploited (1)
Meat products
Raw meat products 6 4
Ready to cook products 4 2
Ready to eat delicatessen 10 4
Total 20 10
Dairy products
Raw milk cheeses 7 4
Raw milks 4 3
Other dairy products 5 5
Total 16 12
Fishery products
Raw fishes 5 4
Processed fishes 7 2
Ready-to-eat products 6 4
Total 18 10
Vegetables
Raw vegetables 5 4
Frozen vegetables 3 3
Lettuce raw vegetables 5 5
Total 13 12
Egg products
Raw egg 5 5
Pastries 3 3
Pasteurised egg products 4 3
Total 12 11
Environmental samples
Wipes swabs 8 4
Process water 3 3
Dusts 3 3
Total 14 10
TOTAL 93 65
(1) Some data cannot be exploited because enumeration was under the detection level for at least one of the
method
Thermo Fisher Scientific
ADRIA Deacuteveloppement 1038 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Artificial contamination of the samples
Artificial contaminations were realised by spiking after performing injury
protocols on pure cultures or by cross-contaminations
56 samples were artificially contaminated 53 gave exploitable results among
a total of 65 samples 8150 of the samples were artificially contaminated
The injury protocol and the strains inoculated are provided in Table 4
Table 4
Stress Ndeg Injury protocol Strain Origin
A 45 min at 50 degC
48
LM -H123
LM-H170
Smoked salmon
Lettuce
Lettuce
B 30 min at 50 degC
and 2 h at ndash20degC
18
43
LM-O1
Raw milk (environment)
Environment
Egg product
C 30 min at 55degC
18
48
LM-H123
Raw milk
Smoked salmon
Lettuce
D 30 min at 50degC
et 2 h at ndash80degC
34
LM-H171
LM-O1
Rillettes
Lettuce
Egg product
E 30 min at 60 degC LM-O1 Egg product
Confirmatory tests
For the reference and the alternative methods confirmations were realised
by the tests described in the ISO 12290-2A1 standard For the alternative
method only one colony was confirmed while 5 colonies were tested for the
reference method
Results
Samples were analysed in duplicate by the reference and the alternative
methods
The contamination range is provided in table 5 per tested category
Thermo Fisher Scientific
ADRIA Deacuteveloppement 1138 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Table 5 ndash Contamination range
Food category Contamination level (log CFUg)
Meat products 100 ndash 404
Dairy products 148 ndash 408
Fishery products 100 ndash 384
Vegetables 170 ndash 511
Egg products 130 ndash 499
Environmental samples 170 ndash 511
All categories 100 ndash 511
Bi-dimensional graphs are given figure 3
Figure 3 ndash Bi-dimensional graphs
Graphe bi-dimensionnel Produits carneacutes
Fishery products
Vegetables
Egg products
Meat products
Thermo Fisher Scientific
ADRIA Deacuteveloppement 1238 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Statistical interpretation according to EN ISO16140 method
The results of the statistical interpretation are given in Table 6
Table 6 ndash Statistical interpretation results
Category n R Regression
used a t(a) b t(b) Critical T
P
Ordinate
at 0
Slope
at 1
Meat products 10 158 GMFR -0455 3321 115 3123 2306 001 001
Dairy products 12 065 GMFR -0027 0385 1002 0108 2228 092 071
Fishery products 10 130 GMFR -0305 1024 1034 0312 2306 076 034
Vegetables 12 086 GMFR -0015 0128 0993 0191 2228 085 090
Egg products 11 158 GMFR -0038 0866 1001 0090 2262 093 041
Environmental
samples 10 176 GMFR -0139 265 1034 231 2306 005 003
All products 65 118 GMFR -0182 3042 1040 2145 2000 004 000
All products
(estimated number) 65 118 GMFR -0182 3042 1040 2145 2000 004 000
All products (non
estimated results) 20 115 GMFR -0367 151 1138 1141 2101 027 015
Repeatability limits obtained for the alternative and reference methods as
well as bias obtained are presented in Table 7
Dairy products Environmental samples
Log (Ref) Log (Ref)
All categories
Thermo Fisher Scientific
ADRIA Deacuteveloppement 1338 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Table 7
Categories Bias
Repeatability limits
Rob r
Ref Alt
Meat products 0008 0314 0343
Dairy products -0031 0136 0130
Fishery products -0133 0220 0214
Vegetables -0041 0180 0319
Egg products -0026 0219 0206
Environmental samples -0007 0198 0119
All products -0032 0196 0215
Regression straight lines (graph and equation representations) for each food
category and for all products are presented figure 4
Thermo Fisher Scientific
ADRIA Deacuteveloppement 1438 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Figure 4 ndash Regression straight lines
Meat products
Fishery products
Dairy products
Vegetables
Thermo Fisher Scientific
ADRIA Deacuteveloppement 1538 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Environmental samples
Egg products
All products (estimated number)
Thermo Fisher Scientific
ADRIA Deacuteveloppement 1638 November 19 2014
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Listeria Precis Enumeration
The hypotheses that the ordinates are close to 0 and the slopes close to 1
are approved in all cases except for
- Meat products (slope P = 1 ordinate P = 1)
- Environmental samples (ordinate P = 3)
- All products (ordinate P = 0)
The bias between the two methods vary from ndash 0133 to 0008 log CFU For
all categories the bias is ndash 0032 log CFU The repeatability limits of the
alternative and the reference method are similar For all products the values
are respectively 0215 and 0196 log CFU For all products these
interpretations are provided taking into account data with low contamination
level or not (estimated colonies number)
For all food products the regression straight line is the following
log (Alt) = 1040 log Ref ndash 0182
The Listeria PrecisTM method shows satisfying relative accuracy
All products
All products (Level 2)
Thermo Fisher Scientific
ADRIA Deacuteveloppement 1738 November 19 2014
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Listeria Precis Enumeration
2113 Detection limit (LOD) and quantification limit (LOQ)
The critical level is the defined as the smallest amount which can be detected (not null) but not
quantified as an exact value Below this value it cannot be sure that the true value is not null
The detection limit is defined as being higher than the critical level because it involves a power the
probability 1- which has to be well over 50 for example 95
The quantification limit is defined as the smallest amount of analyte (that is the lowest actual number of
organisms) which can be measured and quantified with defined precision and accuracy under the
experimental conditions by the method under validation
Protocol
The detection limit of the alternative method was realised with pure cultures
Three different inoculation levels were tested with six replicates per level ie
18 analyses were done by the alternative method
Listeria monocytogenes 18 was used for the detection limit of the alternative
method
Quantification limit was calculated for six independent blank samples
determinations
Results
These data are intrinsic to the method used and are presented in the
following tables
Table 8 ndash Inoculation 1 ml on 3 plates
Level (CFUml) Positive samples number Standard deviation Bias
0 06
05 26 1211 0
1 36 0816 05
5 66 2483 85
Thermo Fisher Scientific
ADRIA Deacuteveloppement 1838 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Table 9 ndash Inoculation 01 ml on 1 plate
Level (CFUml) Positive samples number Standard deviation Bias
0 06
5 36 0548 05
10 46 1414 2
50 66 2258 55
Table 10
Formulas Obtained values
LC 165 S0 + X0 18
LOD 33 S0 + X0 32
LOQ 10 S0 + X0 87
2114 Relative Sensitivity
The relative sensitivity is defined as the ability of the alternative method to detect two different amounts
of analyte measured by the reference method within a given matrix at a specified average value or
over the whole measurement range that is it is the minimal quantity variation (increase of the analyte
concentration x) which gives a significant variation of the measured signal (response y)
These data are intrinsic to the used method and are obtained from the results
of the linearity study
Sensitivity patterns obtained for tested (matrixstrain) pairs are presented in
figure 5
Thermo Fisher Scientific
ADRIA Deacuteveloppement 1938 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Figure 5 ndash Sensitivity patterns for the tested (matrixstrain) pairs
2115 SpecificitySelectivity
The specificity is defined as the degree to which a method is affected (or not) by the other components
present in a multi-component sample That is the ability of a method to measure exactly a given
analyte or its amount within the sample without interference from non-target components such as a
matrix effect or background noise
The selectivity is defined as a measure of the degree of non-interference in the presence of non-target
analytes A method is selective if it can be used to detect the analyte under examination and that a
guarantee can be provided that the detected signal can only be a product by that specific analyte
The specificity and selectivity study was performed for the Listeria PrecisTM
detection method no additional test was realised for the enumeration
method The results were the following
- Among the Listeria monocytogenes strains tested 2 strains (Listeria
monocytogenes CIP 7834 and Listeria monocytogenes CIP 105459) gave
negative results These 2 strains were tested twice by running 48 h
incubation time of the compared agars Positive results were then
observed by the alternative method Pale characteristic colonies were
observed on the ISO agar by streaking the Half Fraser broth only
- No cross reaction was observed with the non-target strains except with
Listeria ivanovii which gave characteristic colonies on Brilliancetrade Listeria
Agar but the confirmatory tests provide a clear discrimination between
Listeria monocytogenes and Listeria ivanovi species
Rillettes
Lettuce
Raw milk
Smoked salmon
Raw egg
Process water
x (CFUg)
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2038 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
212 Practicability
1 Packaging Ready to use Brilliance Listeria agar plates are available
(x 10 plates)
2 Reagent volume
3 Storage conditionsndash
Shelf life of products
not opened
The plates are stored at 6 - 12degC in darkness conditions The
expiration date is mentioned on the package and on the plates It
is around 3 months
4 Modalities of use
after first use
5 Required
equipments No specific equipment is required
6 Reagents Ready to use plates
7 Training 05 day
8 Real time handling
Reference method
9 min for one sample
55 min for 10 samples
Alternative method
7 min for one sample
35 minutes for 10 samples
9 Time to result
Negative results
Reference method Day 2
Alternative method Day 2
Negative results after confirmation or positive results
Reference method Day 4 ndash Day 7
Alternative method Day 4 ndash Day 7
Positive results
Reference method Day 4 ndash Day 7
Alternative method Day 2 ndash Day 3 (with OBIS Mono Test)
or Day 4 ndash Day 7 (by the classical tests described in the
standardised method)
10 Technician skill Identical to that required for the reference method
11 Steps common to
the reference method Mother suspension revivification step and decimal dilutions
12 Traceability
13 Maintenance
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2138 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
213 Inter-laboratory study
2131 Organisation
12 laboratories participated to this study Pasteurised milk was inoculated by
Listeria monocytogenes 12a isolated from raw milk The targeted
inoculation levels were
- 0 CFUml
- 50 CFU ml
- 500 CFU ml
- 5 000 CFU ml
Each laboratory received eight flasks of 10 ml sample ie two flasks per
inoculation level Furthermore one non-inoculated sample has been added
to the package for total viable count microflora (NF ISO 4833 method)
Detailed instructions were transmitted to the collaborators by the expert
laboratory
Collaborative study laboratories and the expert laboratory carried out the
analyses with the alternative and reference methods
2132 Verification of experimental parameters
Strain stability during transport
In order to evaluate the Listeria monocytogenes strain stability during
transport bacterial count of all inoculated flasks were checked at different
time ie inoculation time after 24 h of conservation at 2deg - 8degC Results are
reported in table 11
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2238 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Table 11 ndash Listeria monocytogenes count
by ISO 11290-2A1 method (log CFUml)
Day of the analyses Level contamination Reference method
Day 0
(29052006)
1 45
2 440
3 4 500
Day 1
(30052006)
1 60
2 430
3 4 900
No evolution of the strain was observed after storage for 24 h at 2degC ndash 8degC
Contamination level
The real contamination levels are provided in table 12
Table 12 ndash Real contamination level (CFUml)
Target inoculation
(CFU ml) Samples Ndeg
Real contamination levels
(CFUml)
0 1 - 8 0
50 5 - 6 45
500 3 - 7 440
5000 2 - 4 4 500
Sample temperature at receipt
Measured temperatures at receipt are listed in table 13
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2338 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Table 13 - Sample temperature on receipt
Laboratories Receipt
day
Hour
receipt
Measured temperature
at receipt (degC)
Measured temperature by the
temperature probe (degC)
A
B
C
D
E
F
G
H
I
J
K
L
M
30052006
10h45
10h15
11h15
10h00
09h30
8h30
11h50
10h00
08h30
14h45
08h30
09h45
08h45
82
55
45
63
60
62
81
38
48
-
14
67
76
03
03
-13
03
04
-14
05
04
01
05
-13
04
Conclusion
The temperatures during the transport were correct for all the labs except for
Lab J which didnrsquot provide the information
All the labs received their packages with several broken flasks
Three labs (B D and L) received some damaged samples but proceeded to
analyses
All the labs started the analyses at Day 1 except Lab J
2133 Results
Aerobic mesophilic flora
The count of aerobic mesophilic flora varied from 94 102 CFUml to
33 103 CFUml
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2438 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Expert lab results
The results obtained by the expert lab are the following
Table 14
Target inoculation level (CFUml)
Reference method ISO 11290-2
Alternative method Listeria Precis Enumeration
Rep 1 Rep 2 Rep 1 Rep 2
lt 10 lt 10 lt 10 lt 10 lt10
50 50 (Ne) 60 (Ne) 50 (Ne) 30 (Ne)
500 470 390 490 500
5 000 4 700 5 100 5 900 3 400
Ne estimated number
The obtained results are in agreement with those expected
Collaborator Labs results
A synthesis of the results obtained by the collaborator labs is provided
Table 15 (CFUml) and Table 16 (log CFUml)
Table 15 ndash Synthesis of results obtained by the alternative
and the reference methods (CFUml)
Labs Level 1 Level 2 Level 2 bis Level 3
Reference method
Alternative method
Reference method
Alternative method
Reference method
Alternative method
Reference method
Alternative method
A 40 40 70 40 480 410 210 340 1000 300 400 300 4900 4600 4900 5100
B 40 15 40 20 350 300 360 450 350 650 800 600 4100 4700 5100 3700
C 50 20 20 70 440 430 480 450 300 650 300 500 4600 5400 4600 5000
D 40 60 40 40 490 460 600 540 250 450 600 1000 4900 4900 4400 4600
E 50 40 50 20 490 520 530 560 750 550 400 200 5400 4500 4100 5400
F 60 20 60 50 370 250 300 130 300 350 600 300 4400 4700 4900 5900
G 70 35 120 50 440 390 570 370 450 550 700 700 5000 4500 4000 4400
H 110 40 100 90 520 440 450 540 150 650 300 300 4200 4700 5600 4800
L 35 60 20 40 390 390 170 380 650 350 800 200 5300 4600 5600 5900
M 45 45 30 40 450 490 550 410 400 550 800 180 4900 4900 6400 4800
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2538 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Table 16 ndash Synthesis of results obtained by the alternative
and the reference methods (log CFUml)
Labs Level 1 Level 2 Level 2 bis Level 3
Reference method
Alternative method
Reference method
Alternative method
Reference method
Alternative method
Reference method
Alternative method
A 1602 1602 1845 1602 2681 2613 2322 2531 3000 2477 2602 2477 3690 3663 3690 3708
B 1602 1176 1602 1301 2544 2477 2556 2653 2544 2813 2903 2778 3613 3672 3708 3568
C 1699 1301 1301 1845 2643 2633 2681 2653 2477 2813 2477 2699 3663 3732 3663 3699
D 1602 1778 1602 1602 2690 2663 2778 2732 2398 2653 2778 3000 3690 3690 3643 3663
E 1699 1602 1699 1301 2690 2716 2724 2748 2875 2740 2602 2301 3732 3653 3613 3732
F 1778 1301 1778 1699 2568 2398 2477 2114 2477 2544 2778 2477 3643 3672 3690 3771
G 1845 1544 2079 1699 2643 2591 2756 2568 2653 2740 2845 2845 3699 3653 3602 3643
H 2041 1602 2000 1954 2716 2643 2653 2732 2176 2813 2477 2477 3623 3672 3748 3681
L 1544 1778 1301 1602 2591 2591 2230 2580 2813 2544 2903 2301 3724 3663 3748 3771
M 1653 1653 1477 1602 2653 2690 2740 2613 2602 2740 2903 2255 3690 3690 3806 3681
Level 2 results obtained taking account the 3 plates inoculated with the mother-suspension
Level 2 bis results obtained taking account the plates inoculated with 01 ml of the mother-suspension
Scrutiny of the measurement results for consistency
In order to identify the measurement results or laboratories that could be
inconsistent two graphical consistency techniques were realised Mandelrsquos
h-ank-statistics Mandel indicators h and k at 5 significance highlighted
some possible inconsistent data (See tables and graphics in Appendix 3 and
Appendix 4)
Mandelrsquos values Number of values observed over the threshold
Reference method Alternative method
h gt 1 Lab F Level 2 Lab F Level 2
h gt 5 Lab F Level 2
Lab B Levels 1 and 2
Lab A Level 2
Lab D Level 3
Lab F Level 2
Lab L Level 2
kgt 1 Lab F Level 3
k gt 5 Lab F Level 2 Lab C Level 2
Lab F Level 3
Lab L Levels 1 and 3
Lab M Level 1
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2638 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
The emphasized results appear (i) coherent with the targeted contamination
levels (ii) concordant between both methods
All the results were thus kept for the statistical analysis
Comparison of the trueness and precision characteristics of
the reference method and alternative methods
The statistical values are summarised hereafter
Level p
Reference method Alternative method
Repeatability ratio
Reproducibility ratio Median
Repeatability standard deviation
Reproducibility standard deviation
Median Repeatability
standard deviation
Reproducibility standard deviation
1 10 16519 02415 02425 16021 02045 02433 0847 1007
2 10 26551 00543 00659 26719 00914 01325 1682 2011
2 bis 10 26581 02607 02607 25951 01900 01953 0729 0749
3 10 18671 00378 00378 18671 00478 00611 1264 1617
Bias of the alternative method
In order to estimate the bias of the alternative method with regard to the reference method for every
level Dij and t are calculated according to the following equations
RefAlt ijYijYDij
Diffp
Dijit
)2(
)(median
If the t value is superior to 2 the alternative method is significantly biased
with regard to the reference method
The values are given in table 17
Table 17 ndash t (d) values obtained by level
Level Bias t (d) Conclusion
1 0068 114 Non significant bias
2 0021 082 Non significant bias
2 bis - 0037 035 Non significant bias
3 - 0037 043 Non significant bias
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2738 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
The statistical test concludes to a non significant bias for all the inoculation
levels The bias varies from ndash 0037 to 0068 log CFUml
Comparison of the repeatability standard deviations
If the ratio Srj Alt Srj Ref of the repeatability standard deviations of the
alternative method and the reference method is larger than 2 the precision
under repeatability conditions of the alternative method is considered to be
lower than that of the reference method If this ratio is smaller than 05 the
precision under repeatability conditions of the alternative method is
considered to be greater than that of the reference method
The ratio values are given in table 18
Table 18
Level
Repeatability standard deviation
Reference method Alternative method Alternative method
reference method ratio
1 0242 0205 0847
2 0054 0091 1682
2 bis 0261 0190 0729
3 0038 0048 1264
Level Repeatability limit
Reference method Alternative method
1 0676 0573
2 0152 0256
2 bis 0730 0532
3 0106 0134
The ratios of the repeatability standard deviations are below 2 in all cases
The precision under repeatability conditions of the alternative method is
equivalent to that of the reference method
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2838 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Comparison of the reproducibility standard deviations
If the ratio Srj Alt Srj Ref of the reproducibility standard deviations of the alternative method and the
reference method is larger than 2 the precision under reproducibility conditions of the alternative
method is considered to be lower than that of the reference method If this ratio is smaller than 05 the
precision under reproducibility conditions of the alternative method is considered to be greater than
that of the reference method
The ratio values are given in table 19
Table 19
Level
Reproducibility standard deviation
Reference method Alternative method Alternative method
reference method ratio
1 0242 0243 1007
2 0066 0133 2011
2 bis 0261 0195 0749
3 0038 0061 1617
Level Reproducibility limit
Reference method Alternative method
1 0676 0681
2 0185 0371
2 bis 0730 0547
3 0106 0171
The ratios of the reproducibility standard deviations are below or close to 2
The precision under reproducibility conditions of the alternative method is
equivalent to that of the reference method
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2938 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
214 Conclusion
Method comparison study
The Listeria PrecisTM enumeration method shows satisfying linearity and
relative accuracy
The alternative method shows satisfying inclusivity and exclusivity results
Two Listeria monocytogenes strains gave negative results with the
alternative method These strains grew weakly on the reference method
media (OampA Palcam and Oxford)
The limits of repeatability of the alternative method are similar to those of the
reference method
The bias between both methods are low between - 0133 Log CFUg and
- 0008 Log CFUg
The Listeria PrecisTM enumeration method offers the saving for confirmatory
tests
Positive results are obtained with the alternative method in 2-3 days (with
OBIS Mono test) or in 4-7 days (by the classical tests described in the
standardized method) against 4-7 days for the reference method
Inter-laboratory study
The bias between the reference and the alternative methods shows satisfying
and correct values
- level 1 (100 - 1 000 CFUg) bias = + 007 log CFUg
- level 2 (1 000 - 10 000 CFUg) bias = 002 log CFUg
- level 2 bis (1 000 - 10 000 CFUg) bias = - 004 log CFUg
- level 3 (10 000 - 100 000 CFUg) bias = - 004 log CFUg
The repeatability and the reproducibility of the alternative method look similar
to those of the reference method
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3038 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
22 Extension study (realised by ASEPT 2007)
An extension study was performed in 2007 for a new confirmatory test OBIS
MONO
150 Listeria monocytogenes strains and 100 non-target strains were isolated
onto TSAYE and BrillianceTM Listeria Agar plates All the Listeria
monocytogenes and the non-target strains gave the expected results
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3138 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Appendix 1 ndash Flow diagram of the Listeria PrecisTM method
x g sample + 9 x ml buffered peptone water
Revivification step for 1 h plusmn 5 min at 20degC plusmn 1degC
Streaking 1 ml onto 3 Brilliance Listeria Agar plates (mother suspension)
or 01 ml onto 1 Brilliance Listeria Agar plates (mother suspension and decimal dilutions)
Incubation for 48 h plusmn 3 h at 37degC plusmn 1degC
Confirmatory tests on at least one typical colony
- by the tests described in the ISO method
- by applying the OBIS mono test on an isolated colony
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3238 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Appendix 2 ndash ISO 11290-2 (1998) amp ISO 11290-2A1 (2004) Horizontal
method for the detection and enumeration of Listeria monocytogenes -
Part 2 enumeration of Listeria monocytogenes in foods
10 g cheese + 90 ml buffered peptone water or Half Fraser Broth (without supplements) (110 dilution)
Stomach 1 min 30 seconds
Keep at 20degC plusmn 2degC for 1 h plusmn 5 min
Decimal dilutions in 9 ml peptone salt tubes
Dilution 110
Dilution 1100
Dilution 11000
1 ml on 3 Chromogenic Listeria medium (ISO) plates (x 2) (-1)
01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-2)
01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-3)
01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-4)
Incubation at 37degC plusmn 1degC
24 h - 48 h
Numerate typical colonies at 24 h
(bluegreen surrounded by an opaque halo)
Numerate at 48 h
Streak typical colonies on TSYEA
(5 coloniesplate 2 successive dilutions)
Confirmation tests
Catalase
Gram stain
For confirmation of L monocytogenes
Haemolysis
Carbohydrate (rhamnose and xylose) utilisation
CAMP Test
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3338 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Appendix 3 ndash Mandelrsquos graphics
Laboratoire h Ref
Niveau 1 h Ref
Niveau 2 h Ref
Niveau 3 h Alt
Niveau 1 h Alt
Niveau 2 h Alt
Niveau 3 h5 h1 h5 h1
A -050 -015 083 062 -212 -039 198 299 -198 -299
B -261 -270 021 -077 -058 173 198 299 -198 -299
C -151 -031 -014 -015 -004 -005 198 299 -198 -299
D 038 040 -137 000 072 207 198 299 -198 -299
E -001 090 155 -052 056 -101 198 299 -198 -299
F -112 -321 -153 070 -325 023 198 299 -198 -299
G 042 -071 040 147 -009 176 198 299 -198 -299
H 169 046 -169 192 018 -083 198 299 -198 -299
L 009 -120 021 -077 -231 005 198 299 -198 -299
M 001 031 014 -032 004 -011 198 299 -198 -299
-300
-200
-100
000
100
200
300
A B C D E F G H L M
Vale
urs
h d
e M
an
del(
rob
uste
s)
Laboratoires
Meacutethode de reacutefeacuterence analyse robuste
h Ref Niveau 1
h Ref Niveau 2
h Ref Niveau 3
h 5=198
h 1=299
-300
-200
-100
000
100
200
300
A B C D E F G H L M
Vale
urs
de h
de M
an
del(
rob
uste
s)
Laboratoires
Meacutethode alternative analyse robuste
h Alt Niveau 1
h Alt Niveau 2
h Alt Niveau 2
h 1=299
h 5=198
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3438 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Laboratoire k Ref
Niveau 1 k Ref
Niveau 2 k Ref
Niveau 3 k Alt
Niveau 1 k Alt
Niveau 2 k Alt
Niveau 3 k1 k5
A 000 089 142 084 162 046 259 181
B 125 087 073 104 075 046 259 181
C 117 013 091 188 022 083 259 181
D 052 036 069 000 035 083 259 181
E 028 034 037 138 019 112 259 181
F 140 222 018 027 281 112 259 181
G 088 068 024 131 145 000 259 181
H 129 094 173 016 061 000 259 181
L 069 000 073 104 270 224 259 181
M 000 048 038 043 099 241 259 181
000
050
100
150
200
250
300
350
400
A B C D E F G H L M
Vale
urs
k d
e M
an
del(
rob
uste
s)
Laboratoires
Meacutethode de reacutefeacuterence analyse robuste
k Ref Niveau 1k Ref Niveau 2k Ref Niveau 3
k 1=259
k 5=181
000
050
100
150
200
250
300
A B C D E F G H L M
Vale
urs
k d
e M
an
del
(ro
bu
ste
s)
Laboratoires
Meacutethode alternative analyse robuste
k Alt Niveau 3
k Alt Niveau 1
k Alt Niveau 2
k 1=259
k 5=181
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3538 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Appendix 4 ndash Statistical results
Level 1
Nombre de laboratoires (p) 10
A 1602 1602 1602 000 0000 0000 -0495 0000 A 1845 1602 1724 017 0122 -0122 0621 0840 0122
B 1602 1176 1389 030 0213 -0213 -2613 1247 B 1602 1301 1452 021 0151 -0151 -0769 1041 0062
C 1699 1301 1500 028 0199 -0199 -1510 1165 C 1301 1845 1573 038 -0272 0272 -0148 1881 0073
D 1602 1778 1690 012 -0088 0088 0380 0516 D 1602 1602 1602 000 0000 0000 0000 0000 -0088
E 1699 1602 1651 007 0048 -0048 -0013 0284 E 1699 1301 1500 028 0199 -0199 -0522 1376 -0151
F 1778 1301 1540 034 0239 -0239 -1116 1397 F 1778 1699 1739 006 0040 -0040 0698 0274 0199
G 1845 1544 1695 021 0151 -0151 0425 0881 G 2079 1699 1889 027 0190 -0190 1467 1315 0194
H 2041 1602 1822 031 0220 -0220 1689 1286 H 2000 1954 1977 003 0023 -0023 1917 0158 0155
L 1544 1778 1661 017 -0117 0117 0092 0685 L 1301 1602 1452 021 -0151 0151 -0769 1041 -0210
M 1653 1653 1653 000 0000 0000 0013 0000 M 1477 1602 1540 009 -0062 0062 -0319 0432 -0114
m 165186 m 160206 mediane D 00678
Cn(2p) 1867 Cn(2p) 1867 Cp 16101
Cn(p) 16101 Cn(p) 16101 Qn(D) 0092926
n=2p n 20 n=2p n 20 Qdiff 01496169
f 11 f 11 t 114
l 55 l 55
n=p n 10 n=p n 10
f 6 f 6
l 15 l 15
Qn(2p) 0091465 Qn(2p) 007745
Qn(p) 0062469 Qn(p) 0121519
Qintra 017078 Qintra 014461
Qinter 010058 Qinter 019565
Sr(eacutecart-type de reacutepeacutetabiliteacute) 02415 Sr 02045
RSDr(coefficient de variation de la reacutepeacutetabiliteacute)1462 RSDr 1277
r(repeatability limit)) 0676 r 0573
SL(eacutecart-type interlaboratoire) -001905 SLlt0 SL 001737
SR(eacutecart-type de reproductibiliteacute) 02415 SR 02433
RSDR(coefficient de variation de la reproductibiliteacute)0146 RSDR 0152
R(limite de reproductibiliteacute)) 06762 R(limite de reproductibiliteacute))068123
h k h MoyenneLaboratoires
Niveau 1
si LaboratoiresDeviationMoyenne Dk DeviationsiAlt
Niveau 1
Ref
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3638 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Level 2
Nombre de laboratoires (p) 10
A 2681 2613 2647 005 0034 -0034 -0151 0891 A 2322 2531 2427 015 -0105 0105 -2119 1620 -0220
B 2544 2477 2511 005 0033 -0033 -2699 0872 B 2556 2653 2605 007 -0048 0048 -0581 0750 0094
C 2643 2633 2638 001 0005 -0005 -0311 0130 C 2681 2653 2667 002 0014 -0014 -0040 0217 0029
D 2690 2663 2676 002 0014 -0014 0400 0357 D 2778 2732 2755 003 0023 -0023 0721 0354 0079
E 2690 2716 2703 002 -0013 0013 0897 0336 E 2724 2748 2736 002 -0012 0012 0556 0185 0033
F 2568 2398 2483 012 0085 -0085 -3214 2217 F 2477 2114 2296 026 0182 -0182 -3255 2811 -0188
G 2643 2591 2617 004 0026 -0026 -0707 0682 G 2756 2568 2662 013 0094 -0094 -0085 1453 0045
H 2716 2643 2680 005 0036 -0036 0460 0945 H 2653 2732 2693 006 -0040 0040 0181 0613 0013
L 2591 2591 2591 000 0000 0000 -1196 0000 L 2230 2580 2405 025 -0175 0175 -2307 2704 -0186
M 2653 2690 2672 003 -0018 0018 0311 0481 M 2740 2613 2677 009 0064 -0064 0040 0987 0005
m 2655082 m 26719001 mediane D 00209
Cn(2p) 1867 Cn(2p) 1867 Cp 16101
Cn(p) 16101 Cn(p) 16101 Qn(D) 0039911
n=2p n 20 n=2p n 20 Qdiff 00642591
f 11 f 11 t 082
l 55 l 55
n=p n 10 n=p n 10
f 6 f 6
l 15 l 15
Qn(2p) 0020570 Qn(2p) 00345985
Qn(p) 00332437 Qn(p) 00718158
Qintra 003841 Qintra 006460
Qinter 005352 Qinter 011563
Sr(eacutecart-type de reacutepeacutetabiliteacute) 00543 Sr 00914
RSDr(coefficient de variation de la reacutepeacutetabiliteacute)205 RSDr 342
r(repeatability limit)) 0152 r 0256
SL(eacutecart-type interlaboratoire) 000139 SL 000920
SR(eacutecart-type de reproductibiliteacute) 00659 SR 01325
RSDR(coefficient de variation de la reproductibiliteacute)0025 RSDR 0050
R(limite de reproductibiliteacute)) 018446 R(limite de reproductibiliteacute))03708618
Laboratoires
Niveau 2
Moyenne si Laboratoires
Niveau 2
DRef Alt Moyenne si Deviation h Deviation h k k
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3738 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Level 2 bis
Nombre de laboratoires (p) 10
A 3000 2477 2739 037 0261 -0261 0832 1418 A 2602 2477 2540 009 0062 -0062 -0391 0465 -0199
B 2544 2813 2678 019 -0134 0134 0211 0729 B 2903 2778 2841 009 0062 -0062 1732 0465 0162
C 2477 2813 2645 024 -0168 0168 -0135 0911 C 2477 2699 2588 016 -0111 0111 -0049 0826 -0057
D 2398 2653 2526 018 -0128 0128 -1370 0692 D 2778 3000 2889 016 -0111 0111 2074 0826 0363
E 2875 2740 2808 010 0067 -0067 1547 0365 E 2602 2301 2452 021 0151 -0151 -1012 1120 -0356
F 2477 2544 2511 005 -0033 0033 -1525 0182 F 2778 2477 2628 021 0151 -0151 0230 1120 0117
G 2653 2740 2697 006 -0044 0044 0400 0236 G 2845 2845 2845 000 0000 0000 1764 0000 0148
H 2176 2813 2495 045 -0318 0318 -1692 1727 H 2477 2477 2477 000 0000 0000 -0832 0000 -0017
L 2813 2544 2678 019 0134 -0134 0211 0729 L 2903 2301 2602 043 0301 -0301 0049 2241 -0076
M 2602 2740 2671 010 -0069 0069 0135 0375 M 2903 2255 2579 046 0324 -0324 -0112 2411 -0092
m 2658114 m 259505 mediane D -00372
Cn(2p) 1867 Cn(2p) 1867 Cp 16101
Cn(p) 16101 Cn(p) 16101 Qn(D) 01656915
n=2p n 20 n=2p n 20 Qdiff 02667753
f 11 f 11 t 035
l 55 l 55
n=p n 10 n=p n 10
f 6 f 6
l 15 l 15
Qn(2p) 0098745 Qn(2p) 007195
Qn(p) 006007 Qn(p) 008805
Qintra 018437 Qintra 013435
Qinter 009672 Qinter 014176
Sr(eacutecart-type de reacutepeacutetabiliteacute) 02607 Sr 01900
RSDr(coefficient de variation de la reacutepeacutetabiliteacute)981 RSDr 732
r(repeatability limit)) 0730 r 0532
SL(eacutecart-type interlaboratoire) -002464 SLlt0 SL 000205
SR(eacutecart-type de reproductibiliteacute) 02607 SR 01953
RSDR(coefficient de variation de la reproductibiliteacute)0098 RSDR 0075
R(limite de reproductibiliteacute)) 072996 R(limite de reproductibiliteacute))054686
Laboratoires
Niveau 2 bis
Moyenne si Laboratoires
Niveau 2 bis
DRef Alt Moyenne si Deviation h Deviation h k k
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3838 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Level 3
Nombre de laboratoires (p) 10
A 3690 3663 3676 002 0014 -0014 -0311 0513 A 3690 3708 3699 001 -0009 0009 0354 0257 0022
B 3613 3672 3642 004 -0030 0030 -1851 1109 B 3708 3568 3638 010 0070 -0070 -0844 2061 -0005
C 3663 3732 3698 005 -0035 0035 0645 1302 C 3663 3699 3681 003 -0018 0018 0000 0536 -0017
D 3690 3690 3690 000 0000 0000 0311 0000 D 3643 3663 3653 001 -0010 0010 -0545 0286 -0037
E 3732 3653 3693 006 0040 -0040 0429 1480 E 3613 3732 3673 008 -0060 0060 -0162 1769 -0020
F 3643 3672 3658 002 -0014 0014 -1157 0536 F 3690 3771 3731 006 -0040 0040 0975 1193 0073
G 3699 3653 3676 003 0023 -0023 -0328 0856 G 3602 3643 3623 003 -0021 0021 -1141 0612 -0053
H 3623 3672 3648 003 -0024 0024 -1615 0913 H 3748 3681 3715 005 0033 -0033 0664 0990 0067
L 3724 3663 3694 004 0031 -0031 0461 1150 L 3748 3771 3760 002 -0011 0011 1544 0335 0066
M 3690 3690 3690 000 0000 0000 0311 0000 M 3806 3681 3744 009 0062 -0062 1234 1848 0054
m 368334 m 368086 mediane D 00089
Cn(2p) 1867 Cn(2p) 1867 Cp 16101
Cn(p) 16101 Cn(p) 16101 Qn(D) 00325359
n=2p n 20 n=2p n 20 Qdiff 00523852
f 11 f 11 t 043
l 55 l 55
n=p n 10 n=p n 10
f 6 f 6
l 15 l 15
Qn(2p) 0014323 Qn(2p) 001811
Qn(p) 0013719 Qn(p) 003164
Qintra 002674 Qintra 003381
Qinter 002209 Qinter 005094
Sr(eacutecart-type de reacutepeacutetabiliteacute) 00378 Sr 00478
RSDr(coefficient de variation de la reacutepeacutetabiliteacute)103 RSDr 130
r(repeatability limit)) 0106 r 0134
SL(eacutecart-type interlaboratoire) -000023 SLlt0 SL 000145
SR(eacutecart-type de reproductibiliteacute) 00378 SR 00611
RSDR(coefficient de variation de la reproductibiliteacute)0010 RSDR 0017
R(limite de reproductibiliteacute)) 0105894 R(limite de reproductibiliteacute))017119
Ref Alt DLaboratoires
Niveau 3
Moyenne si Deviation h k Laboratoires
Niveau 3
Moyenne si Deviation h k
Thermo Fisher Scientific
ADRIA Deacuteveloppement 638 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Figure 1 ndash Bi-dimensional graphs
Rillettes Raw milk
Smoked salmon Raw egg product
Lettuce Process water
Thermo Fisher Scientific
ADRIA Deacuteveloppement 738 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Statistical interpretations
Statistical interpretation results are provided in Table 2
Table 2 ndash Statistical interpretations
Matrix R Selected
regression
RobF Critical
value
P Correlation
coefficient
Regression equation
Rillettes 180 GMFR 609 541 4 09960 log (Alt) = 09887 log (Ref) + 01141
Lettuce 108 GMFR 000 541 100 09992 Log (Alt) = 10081 log (Ref) - 00257
Raw milk 102 GMFR 000 541 100 09997 Log (Alt) = 10188 log (Ref) - 00737
Smoked salmon 1048 OLS1 1806 541 263 09984 Log (Alt) = 10406 log (Ref) - 01418
Raw egg product 130 GMFR 548 541 49 09958 Log (Alt) = 09775 log (Ref) - 00161
Process water 184 GMFR 0079 541 969 09985 Log (Alt) = 09680 log (Ref) + 01317
GMFR = orthogonal linear regression
OLS1 = ordinary least-square linear regression
x ndash axis = reference method
y ndash axis = alternative method
OLS1 = ordinary least-square linear regression
x axis = alternative method
y axis = reference method
Statistical Interpretation
P gt 5 not significant 1 lt P lt 5 significant
P lt 1 highly significant
The regressions straight lines are shown figure 2
Thermo Fisher Scientific
ADRIA Deacuteveloppement 838 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Figure 2 ndash Regression straight lines
Lineacuteariteacute - Œuf
Process water Process water (2) Process water
Smoked salmon (GMFR) Smoked salmon (OLS 1)
Raw milk Raw egg product
Rillettes Lettuce
Thermo Fisher Scientific
ADRIA Deacuteveloppement 938 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Discussion
The linearity is accepted in all cases with
P values 5 and correlation coefficients superior to 099
For the matrix ldquoRillettesldquo the linearity test is significant (P = 4) with a high
correlation coefficient value (0996)
The Listeria PrecisTM method shows satisfying linearity
2112 Relative accuracy
The accuracy is the closeness of agreement between a test result and the accepted reference value
The bias is the difference between the expectation of the test results and an accepted reference value
Number and nature of the samples
The repartition per category and types is provided in table 3
Table 3 ndash Number and nature of the samples
Categories Types Number of samples
Analysed Exploited (1)
Meat products
Raw meat products 6 4
Ready to cook products 4 2
Ready to eat delicatessen 10 4
Total 20 10
Dairy products
Raw milk cheeses 7 4
Raw milks 4 3
Other dairy products 5 5
Total 16 12
Fishery products
Raw fishes 5 4
Processed fishes 7 2
Ready-to-eat products 6 4
Total 18 10
Vegetables
Raw vegetables 5 4
Frozen vegetables 3 3
Lettuce raw vegetables 5 5
Total 13 12
Egg products
Raw egg 5 5
Pastries 3 3
Pasteurised egg products 4 3
Total 12 11
Environmental samples
Wipes swabs 8 4
Process water 3 3
Dusts 3 3
Total 14 10
TOTAL 93 65
(1) Some data cannot be exploited because enumeration was under the detection level for at least one of the
method
Thermo Fisher Scientific
ADRIA Deacuteveloppement 1038 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Artificial contamination of the samples
Artificial contaminations were realised by spiking after performing injury
protocols on pure cultures or by cross-contaminations
56 samples were artificially contaminated 53 gave exploitable results among
a total of 65 samples 8150 of the samples were artificially contaminated
The injury protocol and the strains inoculated are provided in Table 4
Table 4
Stress Ndeg Injury protocol Strain Origin
A 45 min at 50 degC
48
LM -H123
LM-H170
Smoked salmon
Lettuce
Lettuce
B 30 min at 50 degC
and 2 h at ndash20degC
18
43
LM-O1
Raw milk (environment)
Environment
Egg product
C 30 min at 55degC
18
48
LM-H123
Raw milk
Smoked salmon
Lettuce
D 30 min at 50degC
et 2 h at ndash80degC
34
LM-H171
LM-O1
Rillettes
Lettuce
Egg product
E 30 min at 60 degC LM-O1 Egg product
Confirmatory tests
For the reference and the alternative methods confirmations were realised
by the tests described in the ISO 12290-2A1 standard For the alternative
method only one colony was confirmed while 5 colonies were tested for the
reference method
Results
Samples were analysed in duplicate by the reference and the alternative
methods
The contamination range is provided in table 5 per tested category
Thermo Fisher Scientific
ADRIA Deacuteveloppement 1138 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Table 5 ndash Contamination range
Food category Contamination level (log CFUg)
Meat products 100 ndash 404
Dairy products 148 ndash 408
Fishery products 100 ndash 384
Vegetables 170 ndash 511
Egg products 130 ndash 499
Environmental samples 170 ndash 511
All categories 100 ndash 511
Bi-dimensional graphs are given figure 3
Figure 3 ndash Bi-dimensional graphs
Graphe bi-dimensionnel Produits carneacutes
Fishery products
Vegetables
Egg products
Meat products
Thermo Fisher Scientific
ADRIA Deacuteveloppement 1238 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Statistical interpretation according to EN ISO16140 method
The results of the statistical interpretation are given in Table 6
Table 6 ndash Statistical interpretation results
Category n R Regression
used a t(a) b t(b) Critical T
P
Ordinate
at 0
Slope
at 1
Meat products 10 158 GMFR -0455 3321 115 3123 2306 001 001
Dairy products 12 065 GMFR -0027 0385 1002 0108 2228 092 071
Fishery products 10 130 GMFR -0305 1024 1034 0312 2306 076 034
Vegetables 12 086 GMFR -0015 0128 0993 0191 2228 085 090
Egg products 11 158 GMFR -0038 0866 1001 0090 2262 093 041
Environmental
samples 10 176 GMFR -0139 265 1034 231 2306 005 003
All products 65 118 GMFR -0182 3042 1040 2145 2000 004 000
All products
(estimated number) 65 118 GMFR -0182 3042 1040 2145 2000 004 000
All products (non
estimated results) 20 115 GMFR -0367 151 1138 1141 2101 027 015
Repeatability limits obtained for the alternative and reference methods as
well as bias obtained are presented in Table 7
Dairy products Environmental samples
Log (Ref) Log (Ref)
All categories
Thermo Fisher Scientific
ADRIA Deacuteveloppement 1338 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Table 7
Categories Bias
Repeatability limits
Rob r
Ref Alt
Meat products 0008 0314 0343
Dairy products -0031 0136 0130
Fishery products -0133 0220 0214
Vegetables -0041 0180 0319
Egg products -0026 0219 0206
Environmental samples -0007 0198 0119
All products -0032 0196 0215
Regression straight lines (graph and equation representations) for each food
category and for all products are presented figure 4
Thermo Fisher Scientific
ADRIA Deacuteveloppement 1438 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Figure 4 ndash Regression straight lines
Meat products
Fishery products
Dairy products
Vegetables
Thermo Fisher Scientific
ADRIA Deacuteveloppement 1538 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Environmental samples
Egg products
All products (estimated number)
Thermo Fisher Scientific
ADRIA Deacuteveloppement 1638 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
The hypotheses that the ordinates are close to 0 and the slopes close to 1
are approved in all cases except for
- Meat products (slope P = 1 ordinate P = 1)
- Environmental samples (ordinate P = 3)
- All products (ordinate P = 0)
The bias between the two methods vary from ndash 0133 to 0008 log CFU For
all categories the bias is ndash 0032 log CFU The repeatability limits of the
alternative and the reference method are similar For all products the values
are respectively 0215 and 0196 log CFU For all products these
interpretations are provided taking into account data with low contamination
level or not (estimated colonies number)
For all food products the regression straight line is the following
log (Alt) = 1040 log Ref ndash 0182
The Listeria PrecisTM method shows satisfying relative accuracy
All products
All products (Level 2)
Thermo Fisher Scientific
ADRIA Deacuteveloppement 1738 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
2113 Detection limit (LOD) and quantification limit (LOQ)
The critical level is the defined as the smallest amount which can be detected (not null) but not
quantified as an exact value Below this value it cannot be sure that the true value is not null
The detection limit is defined as being higher than the critical level because it involves a power the
probability 1- which has to be well over 50 for example 95
The quantification limit is defined as the smallest amount of analyte (that is the lowest actual number of
organisms) which can be measured and quantified with defined precision and accuracy under the
experimental conditions by the method under validation
Protocol
The detection limit of the alternative method was realised with pure cultures
Three different inoculation levels were tested with six replicates per level ie
18 analyses were done by the alternative method
Listeria monocytogenes 18 was used for the detection limit of the alternative
method
Quantification limit was calculated for six independent blank samples
determinations
Results
These data are intrinsic to the method used and are presented in the
following tables
Table 8 ndash Inoculation 1 ml on 3 plates
Level (CFUml) Positive samples number Standard deviation Bias
0 06
05 26 1211 0
1 36 0816 05
5 66 2483 85
Thermo Fisher Scientific
ADRIA Deacuteveloppement 1838 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Table 9 ndash Inoculation 01 ml on 1 plate
Level (CFUml) Positive samples number Standard deviation Bias
0 06
5 36 0548 05
10 46 1414 2
50 66 2258 55
Table 10
Formulas Obtained values
LC 165 S0 + X0 18
LOD 33 S0 + X0 32
LOQ 10 S0 + X0 87
2114 Relative Sensitivity
The relative sensitivity is defined as the ability of the alternative method to detect two different amounts
of analyte measured by the reference method within a given matrix at a specified average value or
over the whole measurement range that is it is the minimal quantity variation (increase of the analyte
concentration x) which gives a significant variation of the measured signal (response y)
These data are intrinsic to the used method and are obtained from the results
of the linearity study
Sensitivity patterns obtained for tested (matrixstrain) pairs are presented in
figure 5
Thermo Fisher Scientific
ADRIA Deacuteveloppement 1938 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Figure 5 ndash Sensitivity patterns for the tested (matrixstrain) pairs
2115 SpecificitySelectivity
The specificity is defined as the degree to which a method is affected (or not) by the other components
present in a multi-component sample That is the ability of a method to measure exactly a given
analyte or its amount within the sample without interference from non-target components such as a
matrix effect or background noise
The selectivity is defined as a measure of the degree of non-interference in the presence of non-target
analytes A method is selective if it can be used to detect the analyte under examination and that a
guarantee can be provided that the detected signal can only be a product by that specific analyte
The specificity and selectivity study was performed for the Listeria PrecisTM
detection method no additional test was realised for the enumeration
method The results were the following
- Among the Listeria monocytogenes strains tested 2 strains (Listeria
monocytogenes CIP 7834 and Listeria monocytogenes CIP 105459) gave
negative results These 2 strains were tested twice by running 48 h
incubation time of the compared agars Positive results were then
observed by the alternative method Pale characteristic colonies were
observed on the ISO agar by streaking the Half Fraser broth only
- No cross reaction was observed with the non-target strains except with
Listeria ivanovii which gave characteristic colonies on Brilliancetrade Listeria
Agar but the confirmatory tests provide a clear discrimination between
Listeria monocytogenes and Listeria ivanovi species
Rillettes
Lettuce
Raw milk
Smoked salmon
Raw egg
Process water
x (CFUg)
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2038 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
212 Practicability
1 Packaging Ready to use Brilliance Listeria agar plates are available
(x 10 plates)
2 Reagent volume
3 Storage conditionsndash
Shelf life of products
not opened
The plates are stored at 6 - 12degC in darkness conditions The
expiration date is mentioned on the package and on the plates It
is around 3 months
4 Modalities of use
after first use
5 Required
equipments No specific equipment is required
6 Reagents Ready to use plates
7 Training 05 day
8 Real time handling
Reference method
9 min for one sample
55 min for 10 samples
Alternative method
7 min for one sample
35 minutes for 10 samples
9 Time to result
Negative results
Reference method Day 2
Alternative method Day 2
Negative results after confirmation or positive results
Reference method Day 4 ndash Day 7
Alternative method Day 4 ndash Day 7
Positive results
Reference method Day 4 ndash Day 7
Alternative method Day 2 ndash Day 3 (with OBIS Mono Test)
or Day 4 ndash Day 7 (by the classical tests described in the
standardised method)
10 Technician skill Identical to that required for the reference method
11 Steps common to
the reference method Mother suspension revivification step and decimal dilutions
12 Traceability
13 Maintenance
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Summary Report (Version 1)
Listeria Precis Enumeration
213 Inter-laboratory study
2131 Organisation
12 laboratories participated to this study Pasteurised milk was inoculated by
Listeria monocytogenes 12a isolated from raw milk The targeted
inoculation levels were
- 0 CFUml
- 50 CFU ml
- 500 CFU ml
- 5 000 CFU ml
Each laboratory received eight flasks of 10 ml sample ie two flasks per
inoculation level Furthermore one non-inoculated sample has been added
to the package for total viable count microflora (NF ISO 4833 method)
Detailed instructions were transmitted to the collaborators by the expert
laboratory
Collaborative study laboratories and the expert laboratory carried out the
analyses with the alternative and reference methods
2132 Verification of experimental parameters
Strain stability during transport
In order to evaluate the Listeria monocytogenes strain stability during
transport bacterial count of all inoculated flasks were checked at different
time ie inoculation time after 24 h of conservation at 2deg - 8degC Results are
reported in table 11
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2238 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Table 11 ndash Listeria monocytogenes count
by ISO 11290-2A1 method (log CFUml)
Day of the analyses Level contamination Reference method
Day 0
(29052006)
1 45
2 440
3 4 500
Day 1
(30052006)
1 60
2 430
3 4 900
No evolution of the strain was observed after storage for 24 h at 2degC ndash 8degC
Contamination level
The real contamination levels are provided in table 12
Table 12 ndash Real contamination level (CFUml)
Target inoculation
(CFU ml) Samples Ndeg
Real contamination levels
(CFUml)
0 1 - 8 0
50 5 - 6 45
500 3 - 7 440
5000 2 - 4 4 500
Sample temperature at receipt
Measured temperatures at receipt are listed in table 13
Thermo Fisher Scientific
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Summary Report (Version 1)
Listeria Precis Enumeration
Table 13 - Sample temperature on receipt
Laboratories Receipt
day
Hour
receipt
Measured temperature
at receipt (degC)
Measured temperature by the
temperature probe (degC)
A
B
C
D
E
F
G
H
I
J
K
L
M
30052006
10h45
10h15
11h15
10h00
09h30
8h30
11h50
10h00
08h30
14h45
08h30
09h45
08h45
82
55
45
63
60
62
81
38
48
-
14
67
76
03
03
-13
03
04
-14
05
04
01
05
-13
04
Conclusion
The temperatures during the transport were correct for all the labs except for
Lab J which didnrsquot provide the information
All the labs received their packages with several broken flasks
Three labs (B D and L) received some damaged samples but proceeded to
analyses
All the labs started the analyses at Day 1 except Lab J
2133 Results
Aerobic mesophilic flora
The count of aerobic mesophilic flora varied from 94 102 CFUml to
33 103 CFUml
Thermo Fisher Scientific
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Listeria Precis Enumeration
Expert lab results
The results obtained by the expert lab are the following
Table 14
Target inoculation level (CFUml)
Reference method ISO 11290-2
Alternative method Listeria Precis Enumeration
Rep 1 Rep 2 Rep 1 Rep 2
lt 10 lt 10 lt 10 lt 10 lt10
50 50 (Ne) 60 (Ne) 50 (Ne) 30 (Ne)
500 470 390 490 500
5 000 4 700 5 100 5 900 3 400
Ne estimated number
The obtained results are in agreement with those expected
Collaborator Labs results
A synthesis of the results obtained by the collaborator labs is provided
Table 15 (CFUml) and Table 16 (log CFUml)
Table 15 ndash Synthesis of results obtained by the alternative
and the reference methods (CFUml)
Labs Level 1 Level 2 Level 2 bis Level 3
Reference method
Alternative method
Reference method
Alternative method
Reference method
Alternative method
Reference method
Alternative method
A 40 40 70 40 480 410 210 340 1000 300 400 300 4900 4600 4900 5100
B 40 15 40 20 350 300 360 450 350 650 800 600 4100 4700 5100 3700
C 50 20 20 70 440 430 480 450 300 650 300 500 4600 5400 4600 5000
D 40 60 40 40 490 460 600 540 250 450 600 1000 4900 4900 4400 4600
E 50 40 50 20 490 520 530 560 750 550 400 200 5400 4500 4100 5400
F 60 20 60 50 370 250 300 130 300 350 600 300 4400 4700 4900 5900
G 70 35 120 50 440 390 570 370 450 550 700 700 5000 4500 4000 4400
H 110 40 100 90 520 440 450 540 150 650 300 300 4200 4700 5600 4800
L 35 60 20 40 390 390 170 380 650 350 800 200 5300 4600 5600 5900
M 45 45 30 40 450 490 550 410 400 550 800 180 4900 4900 6400 4800
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2538 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Table 16 ndash Synthesis of results obtained by the alternative
and the reference methods (log CFUml)
Labs Level 1 Level 2 Level 2 bis Level 3
Reference method
Alternative method
Reference method
Alternative method
Reference method
Alternative method
Reference method
Alternative method
A 1602 1602 1845 1602 2681 2613 2322 2531 3000 2477 2602 2477 3690 3663 3690 3708
B 1602 1176 1602 1301 2544 2477 2556 2653 2544 2813 2903 2778 3613 3672 3708 3568
C 1699 1301 1301 1845 2643 2633 2681 2653 2477 2813 2477 2699 3663 3732 3663 3699
D 1602 1778 1602 1602 2690 2663 2778 2732 2398 2653 2778 3000 3690 3690 3643 3663
E 1699 1602 1699 1301 2690 2716 2724 2748 2875 2740 2602 2301 3732 3653 3613 3732
F 1778 1301 1778 1699 2568 2398 2477 2114 2477 2544 2778 2477 3643 3672 3690 3771
G 1845 1544 2079 1699 2643 2591 2756 2568 2653 2740 2845 2845 3699 3653 3602 3643
H 2041 1602 2000 1954 2716 2643 2653 2732 2176 2813 2477 2477 3623 3672 3748 3681
L 1544 1778 1301 1602 2591 2591 2230 2580 2813 2544 2903 2301 3724 3663 3748 3771
M 1653 1653 1477 1602 2653 2690 2740 2613 2602 2740 2903 2255 3690 3690 3806 3681
Level 2 results obtained taking account the 3 plates inoculated with the mother-suspension
Level 2 bis results obtained taking account the plates inoculated with 01 ml of the mother-suspension
Scrutiny of the measurement results for consistency
In order to identify the measurement results or laboratories that could be
inconsistent two graphical consistency techniques were realised Mandelrsquos
h-ank-statistics Mandel indicators h and k at 5 significance highlighted
some possible inconsistent data (See tables and graphics in Appendix 3 and
Appendix 4)
Mandelrsquos values Number of values observed over the threshold
Reference method Alternative method
h gt 1 Lab F Level 2 Lab F Level 2
h gt 5 Lab F Level 2
Lab B Levels 1 and 2
Lab A Level 2
Lab D Level 3
Lab F Level 2
Lab L Level 2
kgt 1 Lab F Level 3
k gt 5 Lab F Level 2 Lab C Level 2
Lab F Level 3
Lab L Levels 1 and 3
Lab M Level 1
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2638 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
The emphasized results appear (i) coherent with the targeted contamination
levels (ii) concordant between both methods
All the results were thus kept for the statistical analysis
Comparison of the trueness and precision characteristics of
the reference method and alternative methods
The statistical values are summarised hereafter
Level p
Reference method Alternative method
Repeatability ratio
Reproducibility ratio Median
Repeatability standard deviation
Reproducibility standard deviation
Median Repeatability
standard deviation
Reproducibility standard deviation
1 10 16519 02415 02425 16021 02045 02433 0847 1007
2 10 26551 00543 00659 26719 00914 01325 1682 2011
2 bis 10 26581 02607 02607 25951 01900 01953 0729 0749
3 10 18671 00378 00378 18671 00478 00611 1264 1617
Bias of the alternative method
In order to estimate the bias of the alternative method with regard to the reference method for every
level Dij and t are calculated according to the following equations
RefAlt ijYijYDij
Diffp
Dijit
)2(
)(median
If the t value is superior to 2 the alternative method is significantly biased
with regard to the reference method
The values are given in table 17
Table 17 ndash t (d) values obtained by level
Level Bias t (d) Conclusion
1 0068 114 Non significant bias
2 0021 082 Non significant bias
2 bis - 0037 035 Non significant bias
3 - 0037 043 Non significant bias
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2738 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
The statistical test concludes to a non significant bias for all the inoculation
levels The bias varies from ndash 0037 to 0068 log CFUml
Comparison of the repeatability standard deviations
If the ratio Srj Alt Srj Ref of the repeatability standard deviations of the
alternative method and the reference method is larger than 2 the precision
under repeatability conditions of the alternative method is considered to be
lower than that of the reference method If this ratio is smaller than 05 the
precision under repeatability conditions of the alternative method is
considered to be greater than that of the reference method
The ratio values are given in table 18
Table 18
Level
Repeatability standard deviation
Reference method Alternative method Alternative method
reference method ratio
1 0242 0205 0847
2 0054 0091 1682
2 bis 0261 0190 0729
3 0038 0048 1264
Level Repeatability limit
Reference method Alternative method
1 0676 0573
2 0152 0256
2 bis 0730 0532
3 0106 0134
The ratios of the repeatability standard deviations are below 2 in all cases
The precision under repeatability conditions of the alternative method is
equivalent to that of the reference method
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2838 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Comparison of the reproducibility standard deviations
If the ratio Srj Alt Srj Ref of the reproducibility standard deviations of the alternative method and the
reference method is larger than 2 the precision under reproducibility conditions of the alternative
method is considered to be lower than that of the reference method If this ratio is smaller than 05 the
precision under reproducibility conditions of the alternative method is considered to be greater than
that of the reference method
The ratio values are given in table 19
Table 19
Level
Reproducibility standard deviation
Reference method Alternative method Alternative method
reference method ratio
1 0242 0243 1007
2 0066 0133 2011
2 bis 0261 0195 0749
3 0038 0061 1617
Level Reproducibility limit
Reference method Alternative method
1 0676 0681
2 0185 0371
2 bis 0730 0547
3 0106 0171
The ratios of the reproducibility standard deviations are below or close to 2
The precision under reproducibility conditions of the alternative method is
equivalent to that of the reference method
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2938 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
214 Conclusion
Method comparison study
The Listeria PrecisTM enumeration method shows satisfying linearity and
relative accuracy
The alternative method shows satisfying inclusivity and exclusivity results
Two Listeria monocytogenes strains gave negative results with the
alternative method These strains grew weakly on the reference method
media (OampA Palcam and Oxford)
The limits of repeatability of the alternative method are similar to those of the
reference method
The bias between both methods are low between - 0133 Log CFUg and
- 0008 Log CFUg
The Listeria PrecisTM enumeration method offers the saving for confirmatory
tests
Positive results are obtained with the alternative method in 2-3 days (with
OBIS Mono test) or in 4-7 days (by the classical tests described in the
standardized method) against 4-7 days for the reference method
Inter-laboratory study
The bias between the reference and the alternative methods shows satisfying
and correct values
- level 1 (100 - 1 000 CFUg) bias = + 007 log CFUg
- level 2 (1 000 - 10 000 CFUg) bias = 002 log CFUg
- level 2 bis (1 000 - 10 000 CFUg) bias = - 004 log CFUg
- level 3 (10 000 - 100 000 CFUg) bias = - 004 log CFUg
The repeatability and the reproducibility of the alternative method look similar
to those of the reference method
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3038 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
22 Extension study (realised by ASEPT 2007)
An extension study was performed in 2007 for a new confirmatory test OBIS
MONO
150 Listeria monocytogenes strains and 100 non-target strains were isolated
onto TSAYE and BrillianceTM Listeria Agar plates All the Listeria
monocytogenes and the non-target strains gave the expected results
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3138 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Appendix 1 ndash Flow diagram of the Listeria PrecisTM method
x g sample + 9 x ml buffered peptone water
Revivification step for 1 h plusmn 5 min at 20degC plusmn 1degC
Streaking 1 ml onto 3 Brilliance Listeria Agar plates (mother suspension)
or 01 ml onto 1 Brilliance Listeria Agar plates (mother suspension and decimal dilutions)
Incubation for 48 h plusmn 3 h at 37degC plusmn 1degC
Confirmatory tests on at least one typical colony
- by the tests described in the ISO method
- by applying the OBIS mono test on an isolated colony
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3238 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Appendix 2 ndash ISO 11290-2 (1998) amp ISO 11290-2A1 (2004) Horizontal
method for the detection and enumeration of Listeria monocytogenes -
Part 2 enumeration of Listeria monocytogenes in foods
10 g cheese + 90 ml buffered peptone water or Half Fraser Broth (without supplements) (110 dilution)
Stomach 1 min 30 seconds
Keep at 20degC plusmn 2degC for 1 h plusmn 5 min
Decimal dilutions in 9 ml peptone salt tubes
Dilution 110
Dilution 1100
Dilution 11000
1 ml on 3 Chromogenic Listeria medium (ISO) plates (x 2) (-1)
01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-2)
01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-3)
01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-4)
Incubation at 37degC plusmn 1degC
24 h - 48 h
Numerate typical colonies at 24 h
(bluegreen surrounded by an opaque halo)
Numerate at 48 h
Streak typical colonies on TSYEA
(5 coloniesplate 2 successive dilutions)
Confirmation tests
Catalase
Gram stain
For confirmation of L monocytogenes
Haemolysis
Carbohydrate (rhamnose and xylose) utilisation
CAMP Test
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3338 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Appendix 3 ndash Mandelrsquos graphics
Laboratoire h Ref
Niveau 1 h Ref
Niveau 2 h Ref
Niveau 3 h Alt
Niveau 1 h Alt
Niveau 2 h Alt
Niveau 3 h5 h1 h5 h1
A -050 -015 083 062 -212 -039 198 299 -198 -299
B -261 -270 021 -077 -058 173 198 299 -198 -299
C -151 -031 -014 -015 -004 -005 198 299 -198 -299
D 038 040 -137 000 072 207 198 299 -198 -299
E -001 090 155 -052 056 -101 198 299 -198 -299
F -112 -321 -153 070 -325 023 198 299 -198 -299
G 042 -071 040 147 -009 176 198 299 -198 -299
H 169 046 -169 192 018 -083 198 299 -198 -299
L 009 -120 021 -077 -231 005 198 299 -198 -299
M 001 031 014 -032 004 -011 198 299 -198 -299
-300
-200
-100
000
100
200
300
A B C D E F G H L M
Vale
urs
h d
e M
an
del(
rob
uste
s)
Laboratoires
Meacutethode de reacutefeacuterence analyse robuste
h Ref Niveau 1
h Ref Niveau 2
h Ref Niveau 3
h 5=198
h 1=299
-300
-200
-100
000
100
200
300
A B C D E F G H L M
Vale
urs
de h
de M
an
del(
rob
uste
s)
Laboratoires
Meacutethode alternative analyse robuste
h Alt Niveau 1
h Alt Niveau 2
h Alt Niveau 2
h 1=299
h 5=198
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3438 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Laboratoire k Ref
Niveau 1 k Ref
Niveau 2 k Ref
Niveau 3 k Alt
Niveau 1 k Alt
Niveau 2 k Alt
Niveau 3 k1 k5
A 000 089 142 084 162 046 259 181
B 125 087 073 104 075 046 259 181
C 117 013 091 188 022 083 259 181
D 052 036 069 000 035 083 259 181
E 028 034 037 138 019 112 259 181
F 140 222 018 027 281 112 259 181
G 088 068 024 131 145 000 259 181
H 129 094 173 016 061 000 259 181
L 069 000 073 104 270 224 259 181
M 000 048 038 043 099 241 259 181
000
050
100
150
200
250
300
350
400
A B C D E F G H L M
Vale
urs
k d
e M
an
del(
rob
uste
s)
Laboratoires
Meacutethode de reacutefeacuterence analyse robuste
k Ref Niveau 1k Ref Niveau 2k Ref Niveau 3
k 1=259
k 5=181
000
050
100
150
200
250
300
A B C D E F G H L M
Vale
urs
k d
e M
an
del
(ro
bu
ste
s)
Laboratoires
Meacutethode alternative analyse robuste
k Alt Niveau 3
k Alt Niveau 1
k Alt Niveau 2
k 1=259
k 5=181
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3538 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Appendix 4 ndash Statistical results
Level 1
Nombre de laboratoires (p) 10
A 1602 1602 1602 000 0000 0000 -0495 0000 A 1845 1602 1724 017 0122 -0122 0621 0840 0122
B 1602 1176 1389 030 0213 -0213 -2613 1247 B 1602 1301 1452 021 0151 -0151 -0769 1041 0062
C 1699 1301 1500 028 0199 -0199 -1510 1165 C 1301 1845 1573 038 -0272 0272 -0148 1881 0073
D 1602 1778 1690 012 -0088 0088 0380 0516 D 1602 1602 1602 000 0000 0000 0000 0000 -0088
E 1699 1602 1651 007 0048 -0048 -0013 0284 E 1699 1301 1500 028 0199 -0199 -0522 1376 -0151
F 1778 1301 1540 034 0239 -0239 -1116 1397 F 1778 1699 1739 006 0040 -0040 0698 0274 0199
G 1845 1544 1695 021 0151 -0151 0425 0881 G 2079 1699 1889 027 0190 -0190 1467 1315 0194
H 2041 1602 1822 031 0220 -0220 1689 1286 H 2000 1954 1977 003 0023 -0023 1917 0158 0155
L 1544 1778 1661 017 -0117 0117 0092 0685 L 1301 1602 1452 021 -0151 0151 -0769 1041 -0210
M 1653 1653 1653 000 0000 0000 0013 0000 M 1477 1602 1540 009 -0062 0062 -0319 0432 -0114
m 165186 m 160206 mediane D 00678
Cn(2p) 1867 Cn(2p) 1867 Cp 16101
Cn(p) 16101 Cn(p) 16101 Qn(D) 0092926
n=2p n 20 n=2p n 20 Qdiff 01496169
f 11 f 11 t 114
l 55 l 55
n=p n 10 n=p n 10
f 6 f 6
l 15 l 15
Qn(2p) 0091465 Qn(2p) 007745
Qn(p) 0062469 Qn(p) 0121519
Qintra 017078 Qintra 014461
Qinter 010058 Qinter 019565
Sr(eacutecart-type de reacutepeacutetabiliteacute) 02415 Sr 02045
RSDr(coefficient de variation de la reacutepeacutetabiliteacute)1462 RSDr 1277
r(repeatability limit)) 0676 r 0573
SL(eacutecart-type interlaboratoire) -001905 SLlt0 SL 001737
SR(eacutecart-type de reproductibiliteacute) 02415 SR 02433
RSDR(coefficient de variation de la reproductibiliteacute)0146 RSDR 0152
R(limite de reproductibiliteacute)) 06762 R(limite de reproductibiliteacute))068123
h k h MoyenneLaboratoires
Niveau 1
si LaboratoiresDeviationMoyenne Dk DeviationsiAlt
Niveau 1
Ref
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3638 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Level 2
Nombre de laboratoires (p) 10
A 2681 2613 2647 005 0034 -0034 -0151 0891 A 2322 2531 2427 015 -0105 0105 -2119 1620 -0220
B 2544 2477 2511 005 0033 -0033 -2699 0872 B 2556 2653 2605 007 -0048 0048 -0581 0750 0094
C 2643 2633 2638 001 0005 -0005 -0311 0130 C 2681 2653 2667 002 0014 -0014 -0040 0217 0029
D 2690 2663 2676 002 0014 -0014 0400 0357 D 2778 2732 2755 003 0023 -0023 0721 0354 0079
E 2690 2716 2703 002 -0013 0013 0897 0336 E 2724 2748 2736 002 -0012 0012 0556 0185 0033
F 2568 2398 2483 012 0085 -0085 -3214 2217 F 2477 2114 2296 026 0182 -0182 -3255 2811 -0188
G 2643 2591 2617 004 0026 -0026 -0707 0682 G 2756 2568 2662 013 0094 -0094 -0085 1453 0045
H 2716 2643 2680 005 0036 -0036 0460 0945 H 2653 2732 2693 006 -0040 0040 0181 0613 0013
L 2591 2591 2591 000 0000 0000 -1196 0000 L 2230 2580 2405 025 -0175 0175 -2307 2704 -0186
M 2653 2690 2672 003 -0018 0018 0311 0481 M 2740 2613 2677 009 0064 -0064 0040 0987 0005
m 2655082 m 26719001 mediane D 00209
Cn(2p) 1867 Cn(2p) 1867 Cp 16101
Cn(p) 16101 Cn(p) 16101 Qn(D) 0039911
n=2p n 20 n=2p n 20 Qdiff 00642591
f 11 f 11 t 082
l 55 l 55
n=p n 10 n=p n 10
f 6 f 6
l 15 l 15
Qn(2p) 0020570 Qn(2p) 00345985
Qn(p) 00332437 Qn(p) 00718158
Qintra 003841 Qintra 006460
Qinter 005352 Qinter 011563
Sr(eacutecart-type de reacutepeacutetabiliteacute) 00543 Sr 00914
RSDr(coefficient de variation de la reacutepeacutetabiliteacute)205 RSDr 342
r(repeatability limit)) 0152 r 0256
SL(eacutecart-type interlaboratoire) 000139 SL 000920
SR(eacutecart-type de reproductibiliteacute) 00659 SR 01325
RSDR(coefficient de variation de la reproductibiliteacute)0025 RSDR 0050
R(limite de reproductibiliteacute)) 018446 R(limite de reproductibiliteacute))03708618
Laboratoires
Niveau 2
Moyenne si Laboratoires
Niveau 2
DRef Alt Moyenne si Deviation h Deviation h k k
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3738 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Level 2 bis
Nombre de laboratoires (p) 10
A 3000 2477 2739 037 0261 -0261 0832 1418 A 2602 2477 2540 009 0062 -0062 -0391 0465 -0199
B 2544 2813 2678 019 -0134 0134 0211 0729 B 2903 2778 2841 009 0062 -0062 1732 0465 0162
C 2477 2813 2645 024 -0168 0168 -0135 0911 C 2477 2699 2588 016 -0111 0111 -0049 0826 -0057
D 2398 2653 2526 018 -0128 0128 -1370 0692 D 2778 3000 2889 016 -0111 0111 2074 0826 0363
E 2875 2740 2808 010 0067 -0067 1547 0365 E 2602 2301 2452 021 0151 -0151 -1012 1120 -0356
F 2477 2544 2511 005 -0033 0033 -1525 0182 F 2778 2477 2628 021 0151 -0151 0230 1120 0117
G 2653 2740 2697 006 -0044 0044 0400 0236 G 2845 2845 2845 000 0000 0000 1764 0000 0148
H 2176 2813 2495 045 -0318 0318 -1692 1727 H 2477 2477 2477 000 0000 0000 -0832 0000 -0017
L 2813 2544 2678 019 0134 -0134 0211 0729 L 2903 2301 2602 043 0301 -0301 0049 2241 -0076
M 2602 2740 2671 010 -0069 0069 0135 0375 M 2903 2255 2579 046 0324 -0324 -0112 2411 -0092
m 2658114 m 259505 mediane D -00372
Cn(2p) 1867 Cn(2p) 1867 Cp 16101
Cn(p) 16101 Cn(p) 16101 Qn(D) 01656915
n=2p n 20 n=2p n 20 Qdiff 02667753
f 11 f 11 t 035
l 55 l 55
n=p n 10 n=p n 10
f 6 f 6
l 15 l 15
Qn(2p) 0098745 Qn(2p) 007195
Qn(p) 006007 Qn(p) 008805
Qintra 018437 Qintra 013435
Qinter 009672 Qinter 014176
Sr(eacutecart-type de reacutepeacutetabiliteacute) 02607 Sr 01900
RSDr(coefficient de variation de la reacutepeacutetabiliteacute)981 RSDr 732
r(repeatability limit)) 0730 r 0532
SL(eacutecart-type interlaboratoire) -002464 SLlt0 SL 000205
SR(eacutecart-type de reproductibiliteacute) 02607 SR 01953
RSDR(coefficient de variation de la reproductibiliteacute)0098 RSDR 0075
R(limite de reproductibiliteacute)) 072996 R(limite de reproductibiliteacute))054686
Laboratoires
Niveau 2 bis
Moyenne si Laboratoires
Niveau 2 bis
DRef Alt Moyenne si Deviation h Deviation h k k
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3838 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Level 3
Nombre de laboratoires (p) 10
A 3690 3663 3676 002 0014 -0014 -0311 0513 A 3690 3708 3699 001 -0009 0009 0354 0257 0022
B 3613 3672 3642 004 -0030 0030 -1851 1109 B 3708 3568 3638 010 0070 -0070 -0844 2061 -0005
C 3663 3732 3698 005 -0035 0035 0645 1302 C 3663 3699 3681 003 -0018 0018 0000 0536 -0017
D 3690 3690 3690 000 0000 0000 0311 0000 D 3643 3663 3653 001 -0010 0010 -0545 0286 -0037
E 3732 3653 3693 006 0040 -0040 0429 1480 E 3613 3732 3673 008 -0060 0060 -0162 1769 -0020
F 3643 3672 3658 002 -0014 0014 -1157 0536 F 3690 3771 3731 006 -0040 0040 0975 1193 0073
G 3699 3653 3676 003 0023 -0023 -0328 0856 G 3602 3643 3623 003 -0021 0021 -1141 0612 -0053
H 3623 3672 3648 003 -0024 0024 -1615 0913 H 3748 3681 3715 005 0033 -0033 0664 0990 0067
L 3724 3663 3694 004 0031 -0031 0461 1150 L 3748 3771 3760 002 -0011 0011 1544 0335 0066
M 3690 3690 3690 000 0000 0000 0311 0000 M 3806 3681 3744 009 0062 -0062 1234 1848 0054
m 368334 m 368086 mediane D 00089
Cn(2p) 1867 Cn(2p) 1867 Cp 16101
Cn(p) 16101 Cn(p) 16101 Qn(D) 00325359
n=2p n 20 n=2p n 20 Qdiff 00523852
f 11 f 11 t 043
l 55 l 55
n=p n 10 n=p n 10
f 6 f 6
l 15 l 15
Qn(2p) 0014323 Qn(2p) 001811
Qn(p) 0013719 Qn(p) 003164
Qintra 002674 Qintra 003381
Qinter 002209 Qinter 005094
Sr(eacutecart-type de reacutepeacutetabiliteacute) 00378 Sr 00478
RSDr(coefficient de variation de la reacutepeacutetabiliteacute)103 RSDr 130
r(repeatability limit)) 0106 r 0134
SL(eacutecart-type interlaboratoire) -000023 SLlt0 SL 000145
SR(eacutecart-type de reproductibiliteacute) 00378 SR 00611
RSDR(coefficient de variation de la reproductibiliteacute)0010 RSDR 0017
R(limite de reproductibiliteacute)) 0105894 R(limite de reproductibiliteacute))017119
Ref Alt DLaboratoires
Niveau 3
Moyenne si Deviation h k Laboratoires
Niveau 3
Moyenne si Deviation h k
Thermo Fisher Scientific
ADRIA Deacuteveloppement 738 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Statistical interpretations
Statistical interpretation results are provided in Table 2
Table 2 ndash Statistical interpretations
Matrix R Selected
regression
RobF Critical
value
P Correlation
coefficient
Regression equation
Rillettes 180 GMFR 609 541 4 09960 log (Alt) = 09887 log (Ref) + 01141
Lettuce 108 GMFR 000 541 100 09992 Log (Alt) = 10081 log (Ref) - 00257
Raw milk 102 GMFR 000 541 100 09997 Log (Alt) = 10188 log (Ref) - 00737
Smoked salmon 1048 OLS1 1806 541 263 09984 Log (Alt) = 10406 log (Ref) - 01418
Raw egg product 130 GMFR 548 541 49 09958 Log (Alt) = 09775 log (Ref) - 00161
Process water 184 GMFR 0079 541 969 09985 Log (Alt) = 09680 log (Ref) + 01317
GMFR = orthogonal linear regression
OLS1 = ordinary least-square linear regression
x ndash axis = reference method
y ndash axis = alternative method
OLS1 = ordinary least-square linear regression
x axis = alternative method
y axis = reference method
Statistical Interpretation
P gt 5 not significant 1 lt P lt 5 significant
P lt 1 highly significant
The regressions straight lines are shown figure 2
Thermo Fisher Scientific
ADRIA Deacuteveloppement 838 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Figure 2 ndash Regression straight lines
Lineacuteariteacute - Œuf
Process water Process water (2) Process water
Smoked salmon (GMFR) Smoked salmon (OLS 1)
Raw milk Raw egg product
Rillettes Lettuce
Thermo Fisher Scientific
ADRIA Deacuteveloppement 938 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Discussion
The linearity is accepted in all cases with
P values 5 and correlation coefficients superior to 099
For the matrix ldquoRillettesldquo the linearity test is significant (P = 4) with a high
correlation coefficient value (0996)
The Listeria PrecisTM method shows satisfying linearity
2112 Relative accuracy
The accuracy is the closeness of agreement between a test result and the accepted reference value
The bias is the difference between the expectation of the test results and an accepted reference value
Number and nature of the samples
The repartition per category and types is provided in table 3
Table 3 ndash Number and nature of the samples
Categories Types Number of samples
Analysed Exploited (1)
Meat products
Raw meat products 6 4
Ready to cook products 4 2
Ready to eat delicatessen 10 4
Total 20 10
Dairy products
Raw milk cheeses 7 4
Raw milks 4 3
Other dairy products 5 5
Total 16 12
Fishery products
Raw fishes 5 4
Processed fishes 7 2
Ready-to-eat products 6 4
Total 18 10
Vegetables
Raw vegetables 5 4
Frozen vegetables 3 3
Lettuce raw vegetables 5 5
Total 13 12
Egg products
Raw egg 5 5
Pastries 3 3
Pasteurised egg products 4 3
Total 12 11
Environmental samples
Wipes swabs 8 4
Process water 3 3
Dusts 3 3
Total 14 10
TOTAL 93 65
(1) Some data cannot be exploited because enumeration was under the detection level for at least one of the
method
Thermo Fisher Scientific
ADRIA Deacuteveloppement 1038 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Artificial contamination of the samples
Artificial contaminations were realised by spiking after performing injury
protocols on pure cultures or by cross-contaminations
56 samples were artificially contaminated 53 gave exploitable results among
a total of 65 samples 8150 of the samples were artificially contaminated
The injury protocol and the strains inoculated are provided in Table 4
Table 4
Stress Ndeg Injury protocol Strain Origin
A 45 min at 50 degC
48
LM -H123
LM-H170
Smoked salmon
Lettuce
Lettuce
B 30 min at 50 degC
and 2 h at ndash20degC
18
43
LM-O1
Raw milk (environment)
Environment
Egg product
C 30 min at 55degC
18
48
LM-H123
Raw milk
Smoked salmon
Lettuce
D 30 min at 50degC
et 2 h at ndash80degC
34
LM-H171
LM-O1
Rillettes
Lettuce
Egg product
E 30 min at 60 degC LM-O1 Egg product
Confirmatory tests
For the reference and the alternative methods confirmations were realised
by the tests described in the ISO 12290-2A1 standard For the alternative
method only one colony was confirmed while 5 colonies were tested for the
reference method
Results
Samples were analysed in duplicate by the reference and the alternative
methods
The contamination range is provided in table 5 per tested category
Thermo Fisher Scientific
ADRIA Deacuteveloppement 1138 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Table 5 ndash Contamination range
Food category Contamination level (log CFUg)
Meat products 100 ndash 404
Dairy products 148 ndash 408
Fishery products 100 ndash 384
Vegetables 170 ndash 511
Egg products 130 ndash 499
Environmental samples 170 ndash 511
All categories 100 ndash 511
Bi-dimensional graphs are given figure 3
Figure 3 ndash Bi-dimensional graphs
Graphe bi-dimensionnel Produits carneacutes
Fishery products
Vegetables
Egg products
Meat products
Thermo Fisher Scientific
ADRIA Deacuteveloppement 1238 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Statistical interpretation according to EN ISO16140 method
The results of the statistical interpretation are given in Table 6
Table 6 ndash Statistical interpretation results
Category n R Regression
used a t(a) b t(b) Critical T
P
Ordinate
at 0
Slope
at 1
Meat products 10 158 GMFR -0455 3321 115 3123 2306 001 001
Dairy products 12 065 GMFR -0027 0385 1002 0108 2228 092 071
Fishery products 10 130 GMFR -0305 1024 1034 0312 2306 076 034
Vegetables 12 086 GMFR -0015 0128 0993 0191 2228 085 090
Egg products 11 158 GMFR -0038 0866 1001 0090 2262 093 041
Environmental
samples 10 176 GMFR -0139 265 1034 231 2306 005 003
All products 65 118 GMFR -0182 3042 1040 2145 2000 004 000
All products
(estimated number) 65 118 GMFR -0182 3042 1040 2145 2000 004 000
All products (non
estimated results) 20 115 GMFR -0367 151 1138 1141 2101 027 015
Repeatability limits obtained for the alternative and reference methods as
well as bias obtained are presented in Table 7
Dairy products Environmental samples
Log (Ref) Log (Ref)
All categories
Thermo Fisher Scientific
ADRIA Deacuteveloppement 1338 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Table 7
Categories Bias
Repeatability limits
Rob r
Ref Alt
Meat products 0008 0314 0343
Dairy products -0031 0136 0130
Fishery products -0133 0220 0214
Vegetables -0041 0180 0319
Egg products -0026 0219 0206
Environmental samples -0007 0198 0119
All products -0032 0196 0215
Regression straight lines (graph and equation representations) for each food
category and for all products are presented figure 4
Thermo Fisher Scientific
ADRIA Deacuteveloppement 1438 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Figure 4 ndash Regression straight lines
Meat products
Fishery products
Dairy products
Vegetables
Thermo Fisher Scientific
ADRIA Deacuteveloppement 1538 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Environmental samples
Egg products
All products (estimated number)
Thermo Fisher Scientific
ADRIA Deacuteveloppement 1638 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
The hypotheses that the ordinates are close to 0 and the slopes close to 1
are approved in all cases except for
- Meat products (slope P = 1 ordinate P = 1)
- Environmental samples (ordinate P = 3)
- All products (ordinate P = 0)
The bias between the two methods vary from ndash 0133 to 0008 log CFU For
all categories the bias is ndash 0032 log CFU The repeatability limits of the
alternative and the reference method are similar For all products the values
are respectively 0215 and 0196 log CFU For all products these
interpretations are provided taking into account data with low contamination
level or not (estimated colonies number)
For all food products the regression straight line is the following
log (Alt) = 1040 log Ref ndash 0182
The Listeria PrecisTM method shows satisfying relative accuracy
All products
All products (Level 2)
Thermo Fisher Scientific
ADRIA Deacuteveloppement 1738 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
2113 Detection limit (LOD) and quantification limit (LOQ)
The critical level is the defined as the smallest amount which can be detected (not null) but not
quantified as an exact value Below this value it cannot be sure that the true value is not null
The detection limit is defined as being higher than the critical level because it involves a power the
probability 1- which has to be well over 50 for example 95
The quantification limit is defined as the smallest amount of analyte (that is the lowest actual number of
organisms) which can be measured and quantified with defined precision and accuracy under the
experimental conditions by the method under validation
Protocol
The detection limit of the alternative method was realised with pure cultures
Three different inoculation levels were tested with six replicates per level ie
18 analyses were done by the alternative method
Listeria monocytogenes 18 was used for the detection limit of the alternative
method
Quantification limit was calculated for six independent blank samples
determinations
Results
These data are intrinsic to the method used and are presented in the
following tables
Table 8 ndash Inoculation 1 ml on 3 plates
Level (CFUml) Positive samples number Standard deviation Bias
0 06
05 26 1211 0
1 36 0816 05
5 66 2483 85
Thermo Fisher Scientific
ADRIA Deacuteveloppement 1838 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Table 9 ndash Inoculation 01 ml on 1 plate
Level (CFUml) Positive samples number Standard deviation Bias
0 06
5 36 0548 05
10 46 1414 2
50 66 2258 55
Table 10
Formulas Obtained values
LC 165 S0 + X0 18
LOD 33 S0 + X0 32
LOQ 10 S0 + X0 87
2114 Relative Sensitivity
The relative sensitivity is defined as the ability of the alternative method to detect two different amounts
of analyte measured by the reference method within a given matrix at a specified average value or
over the whole measurement range that is it is the minimal quantity variation (increase of the analyte
concentration x) which gives a significant variation of the measured signal (response y)
These data are intrinsic to the used method and are obtained from the results
of the linearity study
Sensitivity patterns obtained for tested (matrixstrain) pairs are presented in
figure 5
Thermo Fisher Scientific
ADRIA Deacuteveloppement 1938 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Figure 5 ndash Sensitivity patterns for the tested (matrixstrain) pairs
2115 SpecificitySelectivity
The specificity is defined as the degree to which a method is affected (or not) by the other components
present in a multi-component sample That is the ability of a method to measure exactly a given
analyte or its amount within the sample without interference from non-target components such as a
matrix effect or background noise
The selectivity is defined as a measure of the degree of non-interference in the presence of non-target
analytes A method is selective if it can be used to detect the analyte under examination and that a
guarantee can be provided that the detected signal can only be a product by that specific analyte
The specificity and selectivity study was performed for the Listeria PrecisTM
detection method no additional test was realised for the enumeration
method The results were the following
- Among the Listeria monocytogenes strains tested 2 strains (Listeria
monocytogenes CIP 7834 and Listeria monocytogenes CIP 105459) gave
negative results These 2 strains were tested twice by running 48 h
incubation time of the compared agars Positive results were then
observed by the alternative method Pale characteristic colonies were
observed on the ISO agar by streaking the Half Fraser broth only
- No cross reaction was observed with the non-target strains except with
Listeria ivanovii which gave characteristic colonies on Brilliancetrade Listeria
Agar but the confirmatory tests provide a clear discrimination between
Listeria monocytogenes and Listeria ivanovi species
Rillettes
Lettuce
Raw milk
Smoked salmon
Raw egg
Process water
x (CFUg)
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2038 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
212 Practicability
1 Packaging Ready to use Brilliance Listeria agar plates are available
(x 10 plates)
2 Reagent volume
3 Storage conditionsndash
Shelf life of products
not opened
The plates are stored at 6 - 12degC in darkness conditions The
expiration date is mentioned on the package and on the plates It
is around 3 months
4 Modalities of use
after first use
5 Required
equipments No specific equipment is required
6 Reagents Ready to use plates
7 Training 05 day
8 Real time handling
Reference method
9 min for one sample
55 min for 10 samples
Alternative method
7 min for one sample
35 minutes for 10 samples
9 Time to result
Negative results
Reference method Day 2
Alternative method Day 2
Negative results after confirmation or positive results
Reference method Day 4 ndash Day 7
Alternative method Day 4 ndash Day 7
Positive results
Reference method Day 4 ndash Day 7
Alternative method Day 2 ndash Day 3 (with OBIS Mono Test)
or Day 4 ndash Day 7 (by the classical tests described in the
standardised method)
10 Technician skill Identical to that required for the reference method
11 Steps common to
the reference method Mother suspension revivification step and decimal dilutions
12 Traceability
13 Maintenance
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2138 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
213 Inter-laboratory study
2131 Organisation
12 laboratories participated to this study Pasteurised milk was inoculated by
Listeria monocytogenes 12a isolated from raw milk The targeted
inoculation levels were
- 0 CFUml
- 50 CFU ml
- 500 CFU ml
- 5 000 CFU ml
Each laboratory received eight flasks of 10 ml sample ie two flasks per
inoculation level Furthermore one non-inoculated sample has been added
to the package for total viable count microflora (NF ISO 4833 method)
Detailed instructions were transmitted to the collaborators by the expert
laboratory
Collaborative study laboratories and the expert laboratory carried out the
analyses with the alternative and reference methods
2132 Verification of experimental parameters
Strain stability during transport
In order to evaluate the Listeria monocytogenes strain stability during
transport bacterial count of all inoculated flasks were checked at different
time ie inoculation time after 24 h of conservation at 2deg - 8degC Results are
reported in table 11
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2238 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Table 11 ndash Listeria monocytogenes count
by ISO 11290-2A1 method (log CFUml)
Day of the analyses Level contamination Reference method
Day 0
(29052006)
1 45
2 440
3 4 500
Day 1
(30052006)
1 60
2 430
3 4 900
No evolution of the strain was observed after storage for 24 h at 2degC ndash 8degC
Contamination level
The real contamination levels are provided in table 12
Table 12 ndash Real contamination level (CFUml)
Target inoculation
(CFU ml) Samples Ndeg
Real contamination levels
(CFUml)
0 1 - 8 0
50 5 - 6 45
500 3 - 7 440
5000 2 - 4 4 500
Sample temperature at receipt
Measured temperatures at receipt are listed in table 13
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2338 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Table 13 - Sample temperature on receipt
Laboratories Receipt
day
Hour
receipt
Measured temperature
at receipt (degC)
Measured temperature by the
temperature probe (degC)
A
B
C
D
E
F
G
H
I
J
K
L
M
30052006
10h45
10h15
11h15
10h00
09h30
8h30
11h50
10h00
08h30
14h45
08h30
09h45
08h45
82
55
45
63
60
62
81
38
48
-
14
67
76
03
03
-13
03
04
-14
05
04
01
05
-13
04
Conclusion
The temperatures during the transport were correct for all the labs except for
Lab J which didnrsquot provide the information
All the labs received their packages with several broken flasks
Three labs (B D and L) received some damaged samples but proceeded to
analyses
All the labs started the analyses at Day 1 except Lab J
2133 Results
Aerobic mesophilic flora
The count of aerobic mesophilic flora varied from 94 102 CFUml to
33 103 CFUml
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2438 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Expert lab results
The results obtained by the expert lab are the following
Table 14
Target inoculation level (CFUml)
Reference method ISO 11290-2
Alternative method Listeria Precis Enumeration
Rep 1 Rep 2 Rep 1 Rep 2
lt 10 lt 10 lt 10 lt 10 lt10
50 50 (Ne) 60 (Ne) 50 (Ne) 30 (Ne)
500 470 390 490 500
5 000 4 700 5 100 5 900 3 400
Ne estimated number
The obtained results are in agreement with those expected
Collaborator Labs results
A synthesis of the results obtained by the collaborator labs is provided
Table 15 (CFUml) and Table 16 (log CFUml)
Table 15 ndash Synthesis of results obtained by the alternative
and the reference methods (CFUml)
Labs Level 1 Level 2 Level 2 bis Level 3
Reference method
Alternative method
Reference method
Alternative method
Reference method
Alternative method
Reference method
Alternative method
A 40 40 70 40 480 410 210 340 1000 300 400 300 4900 4600 4900 5100
B 40 15 40 20 350 300 360 450 350 650 800 600 4100 4700 5100 3700
C 50 20 20 70 440 430 480 450 300 650 300 500 4600 5400 4600 5000
D 40 60 40 40 490 460 600 540 250 450 600 1000 4900 4900 4400 4600
E 50 40 50 20 490 520 530 560 750 550 400 200 5400 4500 4100 5400
F 60 20 60 50 370 250 300 130 300 350 600 300 4400 4700 4900 5900
G 70 35 120 50 440 390 570 370 450 550 700 700 5000 4500 4000 4400
H 110 40 100 90 520 440 450 540 150 650 300 300 4200 4700 5600 4800
L 35 60 20 40 390 390 170 380 650 350 800 200 5300 4600 5600 5900
M 45 45 30 40 450 490 550 410 400 550 800 180 4900 4900 6400 4800
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2538 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Table 16 ndash Synthesis of results obtained by the alternative
and the reference methods (log CFUml)
Labs Level 1 Level 2 Level 2 bis Level 3
Reference method
Alternative method
Reference method
Alternative method
Reference method
Alternative method
Reference method
Alternative method
A 1602 1602 1845 1602 2681 2613 2322 2531 3000 2477 2602 2477 3690 3663 3690 3708
B 1602 1176 1602 1301 2544 2477 2556 2653 2544 2813 2903 2778 3613 3672 3708 3568
C 1699 1301 1301 1845 2643 2633 2681 2653 2477 2813 2477 2699 3663 3732 3663 3699
D 1602 1778 1602 1602 2690 2663 2778 2732 2398 2653 2778 3000 3690 3690 3643 3663
E 1699 1602 1699 1301 2690 2716 2724 2748 2875 2740 2602 2301 3732 3653 3613 3732
F 1778 1301 1778 1699 2568 2398 2477 2114 2477 2544 2778 2477 3643 3672 3690 3771
G 1845 1544 2079 1699 2643 2591 2756 2568 2653 2740 2845 2845 3699 3653 3602 3643
H 2041 1602 2000 1954 2716 2643 2653 2732 2176 2813 2477 2477 3623 3672 3748 3681
L 1544 1778 1301 1602 2591 2591 2230 2580 2813 2544 2903 2301 3724 3663 3748 3771
M 1653 1653 1477 1602 2653 2690 2740 2613 2602 2740 2903 2255 3690 3690 3806 3681
Level 2 results obtained taking account the 3 plates inoculated with the mother-suspension
Level 2 bis results obtained taking account the plates inoculated with 01 ml of the mother-suspension
Scrutiny of the measurement results for consistency
In order to identify the measurement results or laboratories that could be
inconsistent two graphical consistency techniques were realised Mandelrsquos
h-ank-statistics Mandel indicators h and k at 5 significance highlighted
some possible inconsistent data (See tables and graphics in Appendix 3 and
Appendix 4)
Mandelrsquos values Number of values observed over the threshold
Reference method Alternative method
h gt 1 Lab F Level 2 Lab F Level 2
h gt 5 Lab F Level 2
Lab B Levels 1 and 2
Lab A Level 2
Lab D Level 3
Lab F Level 2
Lab L Level 2
kgt 1 Lab F Level 3
k gt 5 Lab F Level 2 Lab C Level 2
Lab F Level 3
Lab L Levels 1 and 3
Lab M Level 1
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2638 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
The emphasized results appear (i) coherent with the targeted contamination
levels (ii) concordant between both methods
All the results were thus kept for the statistical analysis
Comparison of the trueness and precision characteristics of
the reference method and alternative methods
The statistical values are summarised hereafter
Level p
Reference method Alternative method
Repeatability ratio
Reproducibility ratio Median
Repeatability standard deviation
Reproducibility standard deviation
Median Repeatability
standard deviation
Reproducibility standard deviation
1 10 16519 02415 02425 16021 02045 02433 0847 1007
2 10 26551 00543 00659 26719 00914 01325 1682 2011
2 bis 10 26581 02607 02607 25951 01900 01953 0729 0749
3 10 18671 00378 00378 18671 00478 00611 1264 1617
Bias of the alternative method
In order to estimate the bias of the alternative method with regard to the reference method for every
level Dij and t are calculated according to the following equations
RefAlt ijYijYDij
Diffp
Dijit
)2(
)(median
If the t value is superior to 2 the alternative method is significantly biased
with regard to the reference method
The values are given in table 17
Table 17 ndash t (d) values obtained by level
Level Bias t (d) Conclusion
1 0068 114 Non significant bias
2 0021 082 Non significant bias
2 bis - 0037 035 Non significant bias
3 - 0037 043 Non significant bias
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2738 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
The statistical test concludes to a non significant bias for all the inoculation
levels The bias varies from ndash 0037 to 0068 log CFUml
Comparison of the repeatability standard deviations
If the ratio Srj Alt Srj Ref of the repeatability standard deviations of the
alternative method and the reference method is larger than 2 the precision
under repeatability conditions of the alternative method is considered to be
lower than that of the reference method If this ratio is smaller than 05 the
precision under repeatability conditions of the alternative method is
considered to be greater than that of the reference method
The ratio values are given in table 18
Table 18
Level
Repeatability standard deviation
Reference method Alternative method Alternative method
reference method ratio
1 0242 0205 0847
2 0054 0091 1682
2 bis 0261 0190 0729
3 0038 0048 1264
Level Repeatability limit
Reference method Alternative method
1 0676 0573
2 0152 0256
2 bis 0730 0532
3 0106 0134
The ratios of the repeatability standard deviations are below 2 in all cases
The precision under repeatability conditions of the alternative method is
equivalent to that of the reference method
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2838 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Comparison of the reproducibility standard deviations
If the ratio Srj Alt Srj Ref of the reproducibility standard deviations of the alternative method and the
reference method is larger than 2 the precision under reproducibility conditions of the alternative
method is considered to be lower than that of the reference method If this ratio is smaller than 05 the
precision under reproducibility conditions of the alternative method is considered to be greater than
that of the reference method
The ratio values are given in table 19
Table 19
Level
Reproducibility standard deviation
Reference method Alternative method Alternative method
reference method ratio
1 0242 0243 1007
2 0066 0133 2011
2 bis 0261 0195 0749
3 0038 0061 1617
Level Reproducibility limit
Reference method Alternative method
1 0676 0681
2 0185 0371
2 bis 0730 0547
3 0106 0171
The ratios of the reproducibility standard deviations are below or close to 2
The precision under reproducibility conditions of the alternative method is
equivalent to that of the reference method
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2938 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
214 Conclusion
Method comparison study
The Listeria PrecisTM enumeration method shows satisfying linearity and
relative accuracy
The alternative method shows satisfying inclusivity and exclusivity results
Two Listeria monocytogenes strains gave negative results with the
alternative method These strains grew weakly on the reference method
media (OampA Palcam and Oxford)
The limits of repeatability of the alternative method are similar to those of the
reference method
The bias between both methods are low between - 0133 Log CFUg and
- 0008 Log CFUg
The Listeria PrecisTM enumeration method offers the saving for confirmatory
tests
Positive results are obtained with the alternative method in 2-3 days (with
OBIS Mono test) or in 4-7 days (by the classical tests described in the
standardized method) against 4-7 days for the reference method
Inter-laboratory study
The bias between the reference and the alternative methods shows satisfying
and correct values
- level 1 (100 - 1 000 CFUg) bias = + 007 log CFUg
- level 2 (1 000 - 10 000 CFUg) bias = 002 log CFUg
- level 2 bis (1 000 - 10 000 CFUg) bias = - 004 log CFUg
- level 3 (10 000 - 100 000 CFUg) bias = - 004 log CFUg
The repeatability and the reproducibility of the alternative method look similar
to those of the reference method
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3038 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
22 Extension study (realised by ASEPT 2007)
An extension study was performed in 2007 for a new confirmatory test OBIS
MONO
150 Listeria monocytogenes strains and 100 non-target strains were isolated
onto TSAYE and BrillianceTM Listeria Agar plates All the Listeria
monocytogenes and the non-target strains gave the expected results
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3138 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Appendix 1 ndash Flow diagram of the Listeria PrecisTM method
x g sample + 9 x ml buffered peptone water
Revivification step for 1 h plusmn 5 min at 20degC plusmn 1degC
Streaking 1 ml onto 3 Brilliance Listeria Agar plates (mother suspension)
or 01 ml onto 1 Brilliance Listeria Agar plates (mother suspension and decimal dilutions)
Incubation for 48 h plusmn 3 h at 37degC plusmn 1degC
Confirmatory tests on at least one typical colony
- by the tests described in the ISO method
- by applying the OBIS mono test on an isolated colony
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3238 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Appendix 2 ndash ISO 11290-2 (1998) amp ISO 11290-2A1 (2004) Horizontal
method for the detection and enumeration of Listeria monocytogenes -
Part 2 enumeration of Listeria monocytogenes in foods
10 g cheese + 90 ml buffered peptone water or Half Fraser Broth (without supplements) (110 dilution)
Stomach 1 min 30 seconds
Keep at 20degC plusmn 2degC for 1 h plusmn 5 min
Decimal dilutions in 9 ml peptone salt tubes
Dilution 110
Dilution 1100
Dilution 11000
1 ml on 3 Chromogenic Listeria medium (ISO) plates (x 2) (-1)
01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-2)
01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-3)
01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-4)
Incubation at 37degC plusmn 1degC
24 h - 48 h
Numerate typical colonies at 24 h
(bluegreen surrounded by an opaque halo)
Numerate at 48 h
Streak typical colonies on TSYEA
(5 coloniesplate 2 successive dilutions)
Confirmation tests
Catalase
Gram stain
For confirmation of L monocytogenes
Haemolysis
Carbohydrate (rhamnose and xylose) utilisation
CAMP Test
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3338 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Appendix 3 ndash Mandelrsquos graphics
Laboratoire h Ref
Niveau 1 h Ref
Niveau 2 h Ref
Niveau 3 h Alt
Niveau 1 h Alt
Niveau 2 h Alt
Niveau 3 h5 h1 h5 h1
A -050 -015 083 062 -212 -039 198 299 -198 -299
B -261 -270 021 -077 -058 173 198 299 -198 -299
C -151 -031 -014 -015 -004 -005 198 299 -198 -299
D 038 040 -137 000 072 207 198 299 -198 -299
E -001 090 155 -052 056 -101 198 299 -198 -299
F -112 -321 -153 070 -325 023 198 299 -198 -299
G 042 -071 040 147 -009 176 198 299 -198 -299
H 169 046 -169 192 018 -083 198 299 -198 -299
L 009 -120 021 -077 -231 005 198 299 -198 -299
M 001 031 014 -032 004 -011 198 299 -198 -299
-300
-200
-100
000
100
200
300
A B C D E F G H L M
Vale
urs
h d
e M
an
del(
rob
uste
s)
Laboratoires
Meacutethode de reacutefeacuterence analyse robuste
h Ref Niveau 1
h Ref Niveau 2
h Ref Niveau 3
h 5=198
h 1=299
-300
-200
-100
000
100
200
300
A B C D E F G H L M
Vale
urs
de h
de M
an
del(
rob
uste
s)
Laboratoires
Meacutethode alternative analyse robuste
h Alt Niveau 1
h Alt Niveau 2
h Alt Niveau 2
h 1=299
h 5=198
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3438 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Laboratoire k Ref
Niveau 1 k Ref
Niveau 2 k Ref
Niveau 3 k Alt
Niveau 1 k Alt
Niveau 2 k Alt
Niveau 3 k1 k5
A 000 089 142 084 162 046 259 181
B 125 087 073 104 075 046 259 181
C 117 013 091 188 022 083 259 181
D 052 036 069 000 035 083 259 181
E 028 034 037 138 019 112 259 181
F 140 222 018 027 281 112 259 181
G 088 068 024 131 145 000 259 181
H 129 094 173 016 061 000 259 181
L 069 000 073 104 270 224 259 181
M 000 048 038 043 099 241 259 181
000
050
100
150
200
250
300
350
400
A B C D E F G H L M
Vale
urs
k d
e M
an
del(
rob
uste
s)
Laboratoires
Meacutethode de reacutefeacuterence analyse robuste
k Ref Niveau 1k Ref Niveau 2k Ref Niveau 3
k 1=259
k 5=181
000
050
100
150
200
250
300
A B C D E F G H L M
Vale
urs
k d
e M
an
del
(ro
bu
ste
s)
Laboratoires
Meacutethode alternative analyse robuste
k Alt Niveau 3
k Alt Niveau 1
k Alt Niveau 2
k 1=259
k 5=181
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3538 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Appendix 4 ndash Statistical results
Level 1
Nombre de laboratoires (p) 10
A 1602 1602 1602 000 0000 0000 -0495 0000 A 1845 1602 1724 017 0122 -0122 0621 0840 0122
B 1602 1176 1389 030 0213 -0213 -2613 1247 B 1602 1301 1452 021 0151 -0151 -0769 1041 0062
C 1699 1301 1500 028 0199 -0199 -1510 1165 C 1301 1845 1573 038 -0272 0272 -0148 1881 0073
D 1602 1778 1690 012 -0088 0088 0380 0516 D 1602 1602 1602 000 0000 0000 0000 0000 -0088
E 1699 1602 1651 007 0048 -0048 -0013 0284 E 1699 1301 1500 028 0199 -0199 -0522 1376 -0151
F 1778 1301 1540 034 0239 -0239 -1116 1397 F 1778 1699 1739 006 0040 -0040 0698 0274 0199
G 1845 1544 1695 021 0151 -0151 0425 0881 G 2079 1699 1889 027 0190 -0190 1467 1315 0194
H 2041 1602 1822 031 0220 -0220 1689 1286 H 2000 1954 1977 003 0023 -0023 1917 0158 0155
L 1544 1778 1661 017 -0117 0117 0092 0685 L 1301 1602 1452 021 -0151 0151 -0769 1041 -0210
M 1653 1653 1653 000 0000 0000 0013 0000 M 1477 1602 1540 009 -0062 0062 -0319 0432 -0114
m 165186 m 160206 mediane D 00678
Cn(2p) 1867 Cn(2p) 1867 Cp 16101
Cn(p) 16101 Cn(p) 16101 Qn(D) 0092926
n=2p n 20 n=2p n 20 Qdiff 01496169
f 11 f 11 t 114
l 55 l 55
n=p n 10 n=p n 10
f 6 f 6
l 15 l 15
Qn(2p) 0091465 Qn(2p) 007745
Qn(p) 0062469 Qn(p) 0121519
Qintra 017078 Qintra 014461
Qinter 010058 Qinter 019565
Sr(eacutecart-type de reacutepeacutetabiliteacute) 02415 Sr 02045
RSDr(coefficient de variation de la reacutepeacutetabiliteacute)1462 RSDr 1277
r(repeatability limit)) 0676 r 0573
SL(eacutecart-type interlaboratoire) -001905 SLlt0 SL 001737
SR(eacutecart-type de reproductibiliteacute) 02415 SR 02433
RSDR(coefficient de variation de la reproductibiliteacute)0146 RSDR 0152
R(limite de reproductibiliteacute)) 06762 R(limite de reproductibiliteacute))068123
h k h MoyenneLaboratoires
Niveau 1
si LaboratoiresDeviationMoyenne Dk DeviationsiAlt
Niveau 1
Ref
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3638 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Level 2
Nombre de laboratoires (p) 10
A 2681 2613 2647 005 0034 -0034 -0151 0891 A 2322 2531 2427 015 -0105 0105 -2119 1620 -0220
B 2544 2477 2511 005 0033 -0033 -2699 0872 B 2556 2653 2605 007 -0048 0048 -0581 0750 0094
C 2643 2633 2638 001 0005 -0005 -0311 0130 C 2681 2653 2667 002 0014 -0014 -0040 0217 0029
D 2690 2663 2676 002 0014 -0014 0400 0357 D 2778 2732 2755 003 0023 -0023 0721 0354 0079
E 2690 2716 2703 002 -0013 0013 0897 0336 E 2724 2748 2736 002 -0012 0012 0556 0185 0033
F 2568 2398 2483 012 0085 -0085 -3214 2217 F 2477 2114 2296 026 0182 -0182 -3255 2811 -0188
G 2643 2591 2617 004 0026 -0026 -0707 0682 G 2756 2568 2662 013 0094 -0094 -0085 1453 0045
H 2716 2643 2680 005 0036 -0036 0460 0945 H 2653 2732 2693 006 -0040 0040 0181 0613 0013
L 2591 2591 2591 000 0000 0000 -1196 0000 L 2230 2580 2405 025 -0175 0175 -2307 2704 -0186
M 2653 2690 2672 003 -0018 0018 0311 0481 M 2740 2613 2677 009 0064 -0064 0040 0987 0005
m 2655082 m 26719001 mediane D 00209
Cn(2p) 1867 Cn(2p) 1867 Cp 16101
Cn(p) 16101 Cn(p) 16101 Qn(D) 0039911
n=2p n 20 n=2p n 20 Qdiff 00642591
f 11 f 11 t 082
l 55 l 55
n=p n 10 n=p n 10
f 6 f 6
l 15 l 15
Qn(2p) 0020570 Qn(2p) 00345985
Qn(p) 00332437 Qn(p) 00718158
Qintra 003841 Qintra 006460
Qinter 005352 Qinter 011563
Sr(eacutecart-type de reacutepeacutetabiliteacute) 00543 Sr 00914
RSDr(coefficient de variation de la reacutepeacutetabiliteacute)205 RSDr 342
r(repeatability limit)) 0152 r 0256
SL(eacutecart-type interlaboratoire) 000139 SL 000920
SR(eacutecart-type de reproductibiliteacute) 00659 SR 01325
RSDR(coefficient de variation de la reproductibiliteacute)0025 RSDR 0050
R(limite de reproductibiliteacute)) 018446 R(limite de reproductibiliteacute))03708618
Laboratoires
Niveau 2
Moyenne si Laboratoires
Niveau 2
DRef Alt Moyenne si Deviation h Deviation h k k
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3738 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Level 2 bis
Nombre de laboratoires (p) 10
A 3000 2477 2739 037 0261 -0261 0832 1418 A 2602 2477 2540 009 0062 -0062 -0391 0465 -0199
B 2544 2813 2678 019 -0134 0134 0211 0729 B 2903 2778 2841 009 0062 -0062 1732 0465 0162
C 2477 2813 2645 024 -0168 0168 -0135 0911 C 2477 2699 2588 016 -0111 0111 -0049 0826 -0057
D 2398 2653 2526 018 -0128 0128 -1370 0692 D 2778 3000 2889 016 -0111 0111 2074 0826 0363
E 2875 2740 2808 010 0067 -0067 1547 0365 E 2602 2301 2452 021 0151 -0151 -1012 1120 -0356
F 2477 2544 2511 005 -0033 0033 -1525 0182 F 2778 2477 2628 021 0151 -0151 0230 1120 0117
G 2653 2740 2697 006 -0044 0044 0400 0236 G 2845 2845 2845 000 0000 0000 1764 0000 0148
H 2176 2813 2495 045 -0318 0318 -1692 1727 H 2477 2477 2477 000 0000 0000 -0832 0000 -0017
L 2813 2544 2678 019 0134 -0134 0211 0729 L 2903 2301 2602 043 0301 -0301 0049 2241 -0076
M 2602 2740 2671 010 -0069 0069 0135 0375 M 2903 2255 2579 046 0324 -0324 -0112 2411 -0092
m 2658114 m 259505 mediane D -00372
Cn(2p) 1867 Cn(2p) 1867 Cp 16101
Cn(p) 16101 Cn(p) 16101 Qn(D) 01656915
n=2p n 20 n=2p n 20 Qdiff 02667753
f 11 f 11 t 035
l 55 l 55
n=p n 10 n=p n 10
f 6 f 6
l 15 l 15
Qn(2p) 0098745 Qn(2p) 007195
Qn(p) 006007 Qn(p) 008805
Qintra 018437 Qintra 013435
Qinter 009672 Qinter 014176
Sr(eacutecart-type de reacutepeacutetabiliteacute) 02607 Sr 01900
RSDr(coefficient de variation de la reacutepeacutetabiliteacute)981 RSDr 732
r(repeatability limit)) 0730 r 0532
SL(eacutecart-type interlaboratoire) -002464 SLlt0 SL 000205
SR(eacutecart-type de reproductibiliteacute) 02607 SR 01953
RSDR(coefficient de variation de la reproductibiliteacute)0098 RSDR 0075
R(limite de reproductibiliteacute)) 072996 R(limite de reproductibiliteacute))054686
Laboratoires
Niveau 2 bis
Moyenne si Laboratoires
Niveau 2 bis
DRef Alt Moyenne si Deviation h Deviation h k k
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3838 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Level 3
Nombre de laboratoires (p) 10
A 3690 3663 3676 002 0014 -0014 -0311 0513 A 3690 3708 3699 001 -0009 0009 0354 0257 0022
B 3613 3672 3642 004 -0030 0030 -1851 1109 B 3708 3568 3638 010 0070 -0070 -0844 2061 -0005
C 3663 3732 3698 005 -0035 0035 0645 1302 C 3663 3699 3681 003 -0018 0018 0000 0536 -0017
D 3690 3690 3690 000 0000 0000 0311 0000 D 3643 3663 3653 001 -0010 0010 -0545 0286 -0037
E 3732 3653 3693 006 0040 -0040 0429 1480 E 3613 3732 3673 008 -0060 0060 -0162 1769 -0020
F 3643 3672 3658 002 -0014 0014 -1157 0536 F 3690 3771 3731 006 -0040 0040 0975 1193 0073
G 3699 3653 3676 003 0023 -0023 -0328 0856 G 3602 3643 3623 003 -0021 0021 -1141 0612 -0053
H 3623 3672 3648 003 -0024 0024 -1615 0913 H 3748 3681 3715 005 0033 -0033 0664 0990 0067
L 3724 3663 3694 004 0031 -0031 0461 1150 L 3748 3771 3760 002 -0011 0011 1544 0335 0066
M 3690 3690 3690 000 0000 0000 0311 0000 M 3806 3681 3744 009 0062 -0062 1234 1848 0054
m 368334 m 368086 mediane D 00089
Cn(2p) 1867 Cn(2p) 1867 Cp 16101
Cn(p) 16101 Cn(p) 16101 Qn(D) 00325359
n=2p n 20 n=2p n 20 Qdiff 00523852
f 11 f 11 t 043
l 55 l 55
n=p n 10 n=p n 10
f 6 f 6
l 15 l 15
Qn(2p) 0014323 Qn(2p) 001811
Qn(p) 0013719 Qn(p) 003164
Qintra 002674 Qintra 003381
Qinter 002209 Qinter 005094
Sr(eacutecart-type de reacutepeacutetabiliteacute) 00378 Sr 00478
RSDr(coefficient de variation de la reacutepeacutetabiliteacute)103 RSDr 130
r(repeatability limit)) 0106 r 0134
SL(eacutecart-type interlaboratoire) -000023 SLlt0 SL 000145
SR(eacutecart-type de reproductibiliteacute) 00378 SR 00611
RSDR(coefficient de variation de la reproductibiliteacute)0010 RSDR 0017
R(limite de reproductibiliteacute)) 0105894 R(limite de reproductibiliteacute))017119
Ref Alt DLaboratoires
Niveau 3
Moyenne si Deviation h k Laboratoires
Niveau 3
Moyenne si Deviation h k
Thermo Fisher Scientific
ADRIA Deacuteveloppement 838 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Figure 2 ndash Regression straight lines
Lineacuteariteacute - Œuf
Process water Process water (2) Process water
Smoked salmon (GMFR) Smoked salmon (OLS 1)
Raw milk Raw egg product
Rillettes Lettuce
Thermo Fisher Scientific
ADRIA Deacuteveloppement 938 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Discussion
The linearity is accepted in all cases with
P values 5 and correlation coefficients superior to 099
For the matrix ldquoRillettesldquo the linearity test is significant (P = 4) with a high
correlation coefficient value (0996)
The Listeria PrecisTM method shows satisfying linearity
2112 Relative accuracy
The accuracy is the closeness of agreement between a test result and the accepted reference value
The bias is the difference between the expectation of the test results and an accepted reference value
Number and nature of the samples
The repartition per category and types is provided in table 3
Table 3 ndash Number and nature of the samples
Categories Types Number of samples
Analysed Exploited (1)
Meat products
Raw meat products 6 4
Ready to cook products 4 2
Ready to eat delicatessen 10 4
Total 20 10
Dairy products
Raw milk cheeses 7 4
Raw milks 4 3
Other dairy products 5 5
Total 16 12
Fishery products
Raw fishes 5 4
Processed fishes 7 2
Ready-to-eat products 6 4
Total 18 10
Vegetables
Raw vegetables 5 4
Frozen vegetables 3 3
Lettuce raw vegetables 5 5
Total 13 12
Egg products
Raw egg 5 5
Pastries 3 3
Pasteurised egg products 4 3
Total 12 11
Environmental samples
Wipes swabs 8 4
Process water 3 3
Dusts 3 3
Total 14 10
TOTAL 93 65
(1) Some data cannot be exploited because enumeration was under the detection level for at least one of the
method
Thermo Fisher Scientific
ADRIA Deacuteveloppement 1038 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Artificial contamination of the samples
Artificial contaminations were realised by spiking after performing injury
protocols on pure cultures or by cross-contaminations
56 samples were artificially contaminated 53 gave exploitable results among
a total of 65 samples 8150 of the samples were artificially contaminated
The injury protocol and the strains inoculated are provided in Table 4
Table 4
Stress Ndeg Injury protocol Strain Origin
A 45 min at 50 degC
48
LM -H123
LM-H170
Smoked salmon
Lettuce
Lettuce
B 30 min at 50 degC
and 2 h at ndash20degC
18
43
LM-O1
Raw milk (environment)
Environment
Egg product
C 30 min at 55degC
18
48
LM-H123
Raw milk
Smoked salmon
Lettuce
D 30 min at 50degC
et 2 h at ndash80degC
34
LM-H171
LM-O1
Rillettes
Lettuce
Egg product
E 30 min at 60 degC LM-O1 Egg product
Confirmatory tests
For the reference and the alternative methods confirmations were realised
by the tests described in the ISO 12290-2A1 standard For the alternative
method only one colony was confirmed while 5 colonies were tested for the
reference method
Results
Samples were analysed in duplicate by the reference and the alternative
methods
The contamination range is provided in table 5 per tested category
Thermo Fisher Scientific
ADRIA Deacuteveloppement 1138 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Table 5 ndash Contamination range
Food category Contamination level (log CFUg)
Meat products 100 ndash 404
Dairy products 148 ndash 408
Fishery products 100 ndash 384
Vegetables 170 ndash 511
Egg products 130 ndash 499
Environmental samples 170 ndash 511
All categories 100 ndash 511
Bi-dimensional graphs are given figure 3
Figure 3 ndash Bi-dimensional graphs
Graphe bi-dimensionnel Produits carneacutes
Fishery products
Vegetables
Egg products
Meat products
Thermo Fisher Scientific
ADRIA Deacuteveloppement 1238 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Statistical interpretation according to EN ISO16140 method
The results of the statistical interpretation are given in Table 6
Table 6 ndash Statistical interpretation results
Category n R Regression
used a t(a) b t(b) Critical T
P
Ordinate
at 0
Slope
at 1
Meat products 10 158 GMFR -0455 3321 115 3123 2306 001 001
Dairy products 12 065 GMFR -0027 0385 1002 0108 2228 092 071
Fishery products 10 130 GMFR -0305 1024 1034 0312 2306 076 034
Vegetables 12 086 GMFR -0015 0128 0993 0191 2228 085 090
Egg products 11 158 GMFR -0038 0866 1001 0090 2262 093 041
Environmental
samples 10 176 GMFR -0139 265 1034 231 2306 005 003
All products 65 118 GMFR -0182 3042 1040 2145 2000 004 000
All products
(estimated number) 65 118 GMFR -0182 3042 1040 2145 2000 004 000
All products (non
estimated results) 20 115 GMFR -0367 151 1138 1141 2101 027 015
Repeatability limits obtained for the alternative and reference methods as
well as bias obtained are presented in Table 7
Dairy products Environmental samples
Log (Ref) Log (Ref)
All categories
Thermo Fisher Scientific
ADRIA Deacuteveloppement 1338 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Table 7
Categories Bias
Repeatability limits
Rob r
Ref Alt
Meat products 0008 0314 0343
Dairy products -0031 0136 0130
Fishery products -0133 0220 0214
Vegetables -0041 0180 0319
Egg products -0026 0219 0206
Environmental samples -0007 0198 0119
All products -0032 0196 0215
Regression straight lines (graph and equation representations) for each food
category and for all products are presented figure 4
Thermo Fisher Scientific
ADRIA Deacuteveloppement 1438 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Figure 4 ndash Regression straight lines
Meat products
Fishery products
Dairy products
Vegetables
Thermo Fisher Scientific
ADRIA Deacuteveloppement 1538 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Environmental samples
Egg products
All products (estimated number)
Thermo Fisher Scientific
ADRIA Deacuteveloppement 1638 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
The hypotheses that the ordinates are close to 0 and the slopes close to 1
are approved in all cases except for
- Meat products (slope P = 1 ordinate P = 1)
- Environmental samples (ordinate P = 3)
- All products (ordinate P = 0)
The bias between the two methods vary from ndash 0133 to 0008 log CFU For
all categories the bias is ndash 0032 log CFU The repeatability limits of the
alternative and the reference method are similar For all products the values
are respectively 0215 and 0196 log CFU For all products these
interpretations are provided taking into account data with low contamination
level or not (estimated colonies number)
For all food products the regression straight line is the following
log (Alt) = 1040 log Ref ndash 0182
The Listeria PrecisTM method shows satisfying relative accuracy
All products
All products (Level 2)
Thermo Fisher Scientific
ADRIA Deacuteveloppement 1738 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
2113 Detection limit (LOD) and quantification limit (LOQ)
The critical level is the defined as the smallest amount which can be detected (not null) but not
quantified as an exact value Below this value it cannot be sure that the true value is not null
The detection limit is defined as being higher than the critical level because it involves a power the
probability 1- which has to be well over 50 for example 95
The quantification limit is defined as the smallest amount of analyte (that is the lowest actual number of
organisms) which can be measured and quantified with defined precision and accuracy under the
experimental conditions by the method under validation
Protocol
The detection limit of the alternative method was realised with pure cultures
Three different inoculation levels were tested with six replicates per level ie
18 analyses were done by the alternative method
Listeria monocytogenes 18 was used for the detection limit of the alternative
method
Quantification limit was calculated for six independent blank samples
determinations
Results
These data are intrinsic to the method used and are presented in the
following tables
Table 8 ndash Inoculation 1 ml on 3 plates
Level (CFUml) Positive samples number Standard deviation Bias
0 06
05 26 1211 0
1 36 0816 05
5 66 2483 85
Thermo Fisher Scientific
ADRIA Deacuteveloppement 1838 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Table 9 ndash Inoculation 01 ml on 1 plate
Level (CFUml) Positive samples number Standard deviation Bias
0 06
5 36 0548 05
10 46 1414 2
50 66 2258 55
Table 10
Formulas Obtained values
LC 165 S0 + X0 18
LOD 33 S0 + X0 32
LOQ 10 S0 + X0 87
2114 Relative Sensitivity
The relative sensitivity is defined as the ability of the alternative method to detect two different amounts
of analyte measured by the reference method within a given matrix at a specified average value or
over the whole measurement range that is it is the minimal quantity variation (increase of the analyte
concentration x) which gives a significant variation of the measured signal (response y)
These data are intrinsic to the used method and are obtained from the results
of the linearity study
Sensitivity patterns obtained for tested (matrixstrain) pairs are presented in
figure 5
Thermo Fisher Scientific
ADRIA Deacuteveloppement 1938 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Figure 5 ndash Sensitivity patterns for the tested (matrixstrain) pairs
2115 SpecificitySelectivity
The specificity is defined as the degree to which a method is affected (or not) by the other components
present in a multi-component sample That is the ability of a method to measure exactly a given
analyte or its amount within the sample without interference from non-target components such as a
matrix effect or background noise
The selectivity is defined as a measure of the degree of non-interference in the presence of non-target
analytes A method is selective if it can be used to detect the analyte under examination and that a
guarantee can be provided that the detected signal can only be a product by that specific analyte
The specificity and selectivity study was performed for the Listeria PrecisTM
detection method no additional test was realised for the enumeration
method The results were the following
- Among the Listeria monocytogenes strains tested 2 strains (Listeria
monocytogenes CIP 7834 and Listeria monocytogenes CIP 105459) gave
negative results These 2 strains were tested twice by running 48 h
incubation time of the compared agars Positive results were then
observed by the alternative method Pale characteristic colonies were
observed on the ISO agar by streaking the Half Fraser broth only
- No cross reaction was observed with the non-target strains except with
Listeria ivanovii which gave characteristic colonies on Brilliancetrade Listeria
Agar but the confirmatory tests provide a clear discrimination between
Listeria monocytogenes and Listeria ivanovi species
Rillettes
Lettuce
Raw milk
Smoked salmon
Raw egg
Process water
x (CFUg)
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2038 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
212 Practicability
1 Packaging Ready to use Brilliance Listeria agar plates are available
(x 10 plates)
2 Reagent volume
3 Storage conditionsndash
Shelf life of products
not opened
The plates are stored at 6 - 12degC in darkness conditions The
expiration date is mentioned on the package and on the plates It
is around 3 months
4 Modalities of use
after first use
5 Required
equipments No specific equipment is required
6 Reagents Ready to use plates
7 Training 05 day
8 Real time handling
Reference method
9 min for one sample
55 min for 10 samples
Alternative method
7 min for one sample
35 minutes for 10 samples
9 Time to result
Negative results
Reference method Day 2
Alternative method Day 2
Negative results after confirmation or positive results
Reference method Day 4 ndash Day 7
Alternative method Day 4 ndash Day 7
Positive results
Reference method Day 4 ndash Day 7
Alternative method Day 2 ndash Day 3 (with OBIS Mono Test)
or Day 4 ndash Day 7 (by the classical tests described in the
standardised method)
10 Technician skill Identical to that required for the reference method
11 Steps common to
the reference method Mother suspension revivification step and decimal dilutions
12 Traceability
13 Maintenance
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2138 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
213 Inter-laboratory study
2131 Organisation
12 laboratories participated to this study Pasteurised milk was inoculated by
Listeria monocytogenes 12a isolated from raw milk The targeted
inoculation levels were
- 0 CFUml
- 50 CFU ml
- 500 CFU ml
- 5 000 CFU ml
Each laboratory received eight flasks of 10 ml sample ie two flasks per
inoculation level Furthermore one non-inoculated sample has been added
to the package for total viable count microflora (NF ISO 4833 method)
Detailed instructions were transmitted to the collaborators by the expert
laboratory
Collaborative study laboratories and the expert laboratory carried out the
analyses with the alternative and reference methods
2132 Verification of experimental parameters
Strain stability during transport
In order to evaluate the Listeria monocytogenes strain stability during
transport bacterial count of all inoculated flasks were checked at different
time ie inoculation time after 24 h of conservation at 2deg - 8degC Results are
reported in table 11
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2238 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Table 11 ndash Listeria monocytogenes count
by ISO 11290-2A1 method (log CFUml)
Day of the analyses Level contamination Reference method
Day 0
(29052006)
1 45
2 440
3 4 500
Day 1
(30052006)
1 60
2 430
3 4 900
No evolution of the strain was observed after storage for 24 h at 2degC ndash 8degC
Contamination level
The real contamination levels are provided in table 12
Table 12 ndash Real contamination level (CFUml)
Target inoculation
(CFU ml) Samples Ndeg
Real contamination levels
(CFUml)
0 1 - 8 0
50 5 - 6 45
500 3 - 7 440
5000 2 - 4 4 500
Sample temperature at receipt
Measured temperatures at receipt are listed in table 13
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2338 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Table 13 - Sample temperature on receipt
Laboratories Receipt
day
Hour
receipt
Measured temperature
at receipt (degC)
Measured temperature by the
temperature probe (degC)
A
B
C
D
E
F
G
H
I
J
K
L
M
30052006
10h45
10h15
11h15
10h00
09h30
8h30
11h50
10h00
08h30
14h45
08h30
09h45
08h45
82
55
45
63
60
62
81
38
48
-
14
67
76
03
03
-13
03
04
-14
05
04
01
05
-13
04
Conclusion
The temperatures during the transport were correct for all the labs except for
Lab J which didnrsquot provide the information
All the labs received their packages with several broken flasks
Three labs (B D and L) received some damaged samples but proceeded to
analyses
All the labs started the analyses at Day 1 except Lab J
2133 Results
Aerobic mesophilic flora
The count of aerobic mesophilic flora varied from 94 102 CFUml to
33 103 CFUml
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2438 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Expert lab results
The results obtained by the expert lab are the following
Table 14
Target inoculation level (CFUml)
Reference method ISO 11290-2
Alternative method Listeria Precis Enumeration
Rep 1 Rep 2 Rep 1 Rep 2
lt 10 lt 10 lt 10 lt 10 lt10
50 50 (Ne) 60 (Ne) 50 (Ne) 30 (Ne)
500 470 390 490 500
5 000 4 700 5 100 5 900 3 400
Ne estimated number
The obtained results are in agreement with those expected
Collaborator Labs results
A synthesis of the results obtained by the collaborator labs is provided
Table 15 (CFUml) and Table 16 (log CFUml)
Table 15 ndash Synthesis of results obtained by the alternative
and the reference methods (CFUml)
Labs Level 1 Level 2 Level 2 bis Level 3
Reference method
Alternative method
Reference method
Alternative method
Reference method
Alternative method
Reference method
Alternative method
A 40 40 70 40 480 410 210 340 1000 300 400 300 4900 4600 4900 5100
B 40 15 40 20 350 300 360 450 350 650 800 600 4100 4700 5100 3700
C 50 20 20 70 440 430 480 450 300 650 300 500 4600 5400 4600 5000
D 40 60 40 40 490 460 600 540 250 450 600 1000 4900 4900 4400 4600
E 50 40 50 20 490 520 530 560 750 550 400 200 5400 4500 4100 5400
F 60 20 60 50 370 250 300 130 300 350 600 300 4400 4700 4900 5900
G 70 35 120 50 440 390 570 370 450 550 700 700 5000 4500 4000 4400
H 110 40 100 90 520 440 450 540 150 650 300 300 4200 4700 5600 4800
L 35 60 20 40 390 390 170 380 650 350 800 200 5300 4600 5600 5900
M 45 45 30 40 450 490 550 410 400 550 800 180 4900 4900 6400 4800
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2538 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Table 16 ndash Synthesis of results obtained by the alternative
and the reference methods (log CFUml)
Labs Level 1 Level 2 Level 2 bis Level 3
Reference method
Alternative method
Reference method
Alternative method
Reference method
Alternative method
Reference method
Alternative method
A 1602 1602 1845 1602 2681 2613 2322 2531 3000 2477 2602 2477 3690 3663 3690 3708
B 1602 1176 1602 1301 2544 2477 2556 2653 2544 2813 2903 2778 3613 3672 3708 3568
C 1699 1301 1301 1845 2643 2633 2681 2653 2477 2813 2477 2699 3663 3732 3663 3699
D 1602 1778 1602 1602 2690 2663 2778 2732 2398 2653 2778 3000 3690 3690 3643 3663
E 1699 1602 1699 1301 2690 2716 2724 2748 2875 2740 2602 2301 3732 3653 3613 3732
F 1778 1301 1778 1699 2568 2398 2477 2114 2477 2544 2778 2477 3643 3672 3690 3771
G 1845 1544 2079 1699 2643 2591 2756 2568 2653 2740 2845 2845 3699 3653 3602 3643
H 2041 1602 2000 1954 2716 2643 2653 2732 2176 2813 2477 2477 3623 3672 3748 3681
L 1544 1778 1301 1602 2591 2591 2230 2580 2813 2544 2903 2301 3724 3663 3748 3771
M 1653 1653 1477 1602 2653 2690 2740 2613 2602 2740 2903 2255 3690 3690 3806 3681
Level 2 results obtained taking account the 3 plates inoculated with the mother-suspension
Level 2 bis results obtained taking account the plates inoculated with 01 ml of the mother-suspension
Scrutiny of the measurement results for consistency
In order to identify the measurement results or laboratories that could be
inconsistent two graphical consistency techniques were realised Mandelrsquos
h-ank-statistics Mandel indicators h and k at 5 significance highlighted
some possible inconsistent data (See tables and graphics in Appendix 3 and
Appendix 4)
Mandelrsquos values Number of values observed over the threshold
Reference method Alternative method
h gt 1 Lab F Level 2 Lab F Level 2
h gt 5 Lab F Level 2
Lab B Levels 1 and 2
Lab A Level 2
Lab D Level 3
Lab F Level 2
Lab L Level 2
kgt 1 Lab F Level 3
k gt 5 Lab F Level 2 Lab C Level 2
Lab F Level 3
Lab L Levels 1 and 3
Lab M Level 1
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2638 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
The emphasized results appear (i) coherent with the targeted contamination
levels (ii) concordant between both methods
All the results were thus kept for the statistical analysis
Comparison of the trueness and precision characteristics of
the reference method and alternative methods
The statistical values are summarised hereafter
Level p
Reference method Alternative method
Repeatability ratio
Reproducibility ratio Median
Repeatability standard deviation
Reproducibility standard deviation
Median Repeatability
standard deviation
Reproducibility standard deviation
1 10 16519 02415 02425 16021 02045 02433 0847 1007
2 10 26551 00543 00659 26719 00914 01325 1682 2011
2 bis 10 26581 02607 02607 25951 01900 01953 0729 0749
3 10 18671 00378 00378 18671 00478 00611 1264 1617
Bias of the alternative method
In order to estimate the bias of the alternative method with regard to the reference method for every
level Dij and t are calculated according to the following equations
RefAlt ijYijYDij
Diffp
Dijit
)2(
)(median
If the t value is superior to 2 the alternative method is significantly biased
with regard to the reference method
The values are given in table 17
Table 17 ndash t (d) values obtained by level
Level Bias t (d) Conclusion
1 0068 114 Non significant bias
2 0021 082 Non significant bias
2 bis - 0037 035 Non significant bias
3 - 0037 043 Non significant bias
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2738 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
The statistical test concludes to a non significant bias for all the inoculation
levels The bias varies from ndash 0037 to 0068 log CFUml
Comparison of the repeatability standard deviations
If the ratio Srj Alt Srj Ref of the repeatability standard deviations of the
alternative method and the reference method is larger than 2 the precision
under repeatability conditions of the alternative method is considered to be
lower than that of the reference method If this ratio is smaller than 05 the
precision under repeatability conditions of the alternative method is
considered to be greater than that of the reference method
The ratio values are given in table 18
Table 18
Level
Repeatability standard deviation
Reference method Alternative method Alternative method
reference method ratio
1 0242 0205 0847
2 0054 0091 1682
2 bis 0261 0190 0729
3 0038 0048 1264
Level Repeatability limit
Reference method Alternative method
1 0676 0573
2 0152 0256
2 bis 0730 0532
3 0106 0134
The ratios of the repeatability standard deviations are below 2 in all cases
The precision under repeatability conditions of the alternative method is
equivalent to that of the reference method
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2838 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Comparison of the reproducibility standard deviations
If the ratio Srj Alt Srj Ref of the reproducibility standard deviations of the alternative method and the
reference method is larger than 2 the precision under reproducibility conditions of the alternative
method is considered to be lower than that of the reference method If this ratio is smaller than 05 the
precision under reproducibility conditions of the alternative method is considered to be greater than
that of the reference method
The ratio values are given in table 19
Table 19
Level
Reproducibility standard deviation
Reference method Alternative method Alternative method
reference method ratio
1 0242 0243 1007
2 0066 0133 2011
2 bis 0261 0195 0749
3 0038 0061 1617
Level Reproducibility limit
Reference method Alternative method
1 0676 0681
2 0185 0371
2 bis 0730 0547
3 0106 0171
The ratios of the reproducibility standard deviations are below or close to 2
The precision under reproducibility conditions of the alternative method is
equivalent to that of the reference method
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2938 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
214 Conclusion
Method comparison study
The Listeria PrecisTM enumeration method shows satisfying linearity and
relative accuracy
The alternative method shows satisfying inclusivity and exclusivity results
Two Listeria monocytogenes strains gave negative results with the
alternative method These strains grew weakly on the reference method
media (OampA Palcam and Oxford)
The limits of repeatability of the alternative method are similar to those of the
reference method
The bias between both methods are low between - 0133 Log CFUg and
- 0008 Log CFUg
The Listeria PrecisTM enumeration method offers the saving for confirmatory
tests
Positive results are obtained with the alternative method in 2-3 days (with
OBIS Mono test) or in 4-7 days (by the classical tests described in the
standardized method) against 4-7 days for the reference method
Inter-laboratory study
The bias between the reference and the alternative methods shows satisfying
and correct values
- level 1 (100 - 1 000 CFUg) bias = + 007 log CFUg
- level 2 (1 000 - 10 000 CFUg) bias = 002 log CFUg
- level 2 bis (1 000 - 10 000 CFUg) bias = - 004 log CFUg
- level 3 (10 000 - 100 000 CFUg) bias = - 004 log CFUg
The repeatability and the reproducibility of the alternative method look similar
to those of the reference method
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3038 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
22 Extension study (realised by ASEPT 2007)
An extension study was performed in 2007 for a new confirmatory test OBIS
MONO
150 Listeria monocytogenes strains and 100 non-target strains were isolated
onto TSAYE and BrillianceTM Listeria Agar plates All the Listeria
monocytogenes and the non-target strains gave the expected results
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3138 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Appendix 1 ndash Flow diagram of the Listeria PrecisTM method
x g sample + 9 x ml buffered peptone water
Revivification step for 1 h plusmn 5 min at 20degC plusmn 1degC
Streaking 1 ml onto 3 Brilliance Listeria Agar plates (mother suspension)
or 01 ml onto 1 Brilliance Listeria Agar plates (mother suspension and decimal dilutions)
Incubation for 48 h plusmn 3 h at 37degC plusmn 1degC
Confirmatory tests on at least one typical colony
- by the tests described in the ISO method
- by applying the OBIS mono test on an isolated colony
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3238 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Appendix 2 ndash ISO 11290-2 (1998) amp ISO 11290-2A1 (2004) Horizontal
method for the detection and enumeration of Listeria monocytogenes -
Part 2 enumeration of Listeria monocytogenes in foods
10 g cheese + 90 ml buffered peptone water or Half Fraser Broth (without supplements) (110 dilution)
Stomach 1 min 30 seconds
Keep at 20degC plusmn 2degC for 1 h plusmn 5 min
Decimal dilutions in 9 ml peptone salt tubes
Dilution 110
Dilution 1100
Dilution 11000
1 ml on 3 Chromogenic Listeria medium (ISO) plates (x 2) (-1)
01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-2)
01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-3)
01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-4)
Incubation at 37degC plusmn 1degC
24 h - 48 h
Numerate typical colonies at 24 h
(bluegreen surrounded by an opaque halo)
Numerate at 48 h
Streak typical colonies on TSYEA
(5 coloniesplate 2 successive dilutions)
Confirmation tests
Catalase
Gram stain
For confirmation of L monocytogenes
Haemolysis
Carbohydrate (rhamnose and xylose) utilisation
CAMP Test
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3338 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Appendix 3 ndash Mandelrsquos graphics
Laboratoire h Ref
Niveau 1 h Ref
Niveau 2 h Ref
Niveau 3 h Alt
Niveau 1 h Alt
Niveau 2 h Alt
Niveau 3 h5 h1 h5 h1
A -050 -015 083 062 -212 -039 198 299 -198 -299
B -261 -270 021 -077 -058 173 198 299 -198 -299
C -151 -031 -014 -015 -004 -005 198 299 -198 -299
D 038 040 -137 000 072 207 198 299 -198 -299
E -001 090 155 -052 056 -101 198 299 -198 -299
F -112 -321 -153 070 -325 023 198 299 -198 -299
G 042 -071 040 147 -009 176 198 299 -198 -299
H 169 046 -169 192 018 -083 198 299 -198 -299
L 009 -120 021 -077 -231 005 198 299 -198 -299
M 001 031 014 -032 004 -011 198 299 -198 -299
-300
-200
-100
000
100
200
300
A B C D E F G H L M
Vale
urs
h d
e M
an
del(
rob
uste
s)
Laboratoires
Meacutethode de reacutefeacuterence analyse robuste
h Ref Niveau 1
h Ref Niveau 2
h Ref Niveau 3
h 5=198
h 1=299
-300
-200
-100
000
100
200
300
A B C D E F G H L M
Vale
urs
de h
de M
an
del(
rob
uste
s)
Laboratoires
Meacutethode alternative analyse robuste
h Alt Niveau 1
h Alt Niveau 2
h Alt Niveau 2
h 1=299
h 5=198
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3438 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Laboratoire k Ref
Niveau 1 k Ref
Niveau 2 k Ref
Niveau 3 k Alt
Niveau 1 k Alt
Niveau 2 k Alt
Niveau 3 k1 k5
A 000 089 142 084 162 046 259 181
B 125 087 073 104 075 046 259 181
C 117 013 091 188 022 083 259 181
D 052 036 069 000 035 083 259 181
E 028 034 037 138 019 112 259 181
F 140 222 018 027 281 112 259 181
G 088 068 024 131 145 000 259 181
H 129 094 173 016 061 000 259 181
L 069 000 073 104 270 224 259 181
M 000 048 038 043 099 241 259 181
000
050
100
150
200
250
300
350
400
A B C D E F G H L M
Vale
urs
k d
e M
an
del(
rob
uste
s)
Laboratoires
Meacutethode de reacutefeacuterence analyse robuste
k Ref Niveau 1k Ref Niveau 2k Ref Niveau 3
k 1=259
k 5=181
000
050
100
150
200
250
300
A B C D E F G H L M
Vale
urs
k d
e M
an
del
(ro
bu
ste
s)
Laboratoires
Meacutethode alternative analyse robuste
k Alt Niveau 3
k Alt Niveau 1
k Alt Niveau 2
k 1=259
k 5=181
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3538 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Appendix 4 ndash Statistical results
Level 1
Nombre de laboratoires (p) 10
A 1602 1602 1602 000 0000 0000 -0495 0000 A 1845 1602 1724 017 0122 -0122 0621 0840 0122
B 1602 1176 1389 030 0213 -0213 -2613 1247 B 1602 1301 1452 021 0151 -0151 -0769 1041 0062
C 1699 1301 1500 028 0199 -0199 -1510 1165 C 1301 1845 1573 038 -0272 0272 -0148 1881 0073
D 1602 1778 1690 012 -0088 0088 0380 0516 D 1602 1602 1602 000 0000 0000 0000 0000 -0088
E 1699 1602 1651 007 0048 -0048 -0013 0284 E 1699 1301 1500 028 0199 -0199 -0522 1376 -0151
F 1778 1301 1540 034 0239 -0239 -1116 1397 F 1778 1699 1739 006 0040 -0040 0698 0274 0199
G 1845 1544 1695 021 0151 -0151 0425 0881 G 2079 1699 1889 027 0190 -0190 1467 1315 0194
H 2041 1602 1822 031 0220 -0220 1689 1286 H 2000 1954 1977 003 0023 -0023 1917 0158 0155
L 1544 1778 1661 017 -0117 0117 0092 0685 L 1301 1602 1452 021 -0151 0151 -0769 1041 -0210
M 1653 1653 1653 000 0000 0000 0013 0000 M 1477 1602 1540 009 -0062 0062 -0319 0432 -0114
m 165186 m 160206 mediane D 00678
Cn(2p) 1867 Cn(2p) 1867 Cp 16101
Cn(p) 16101 Cn(p) 16101 Qn(D) 0092926
n=2p n 20 n=2p n 20 Qdiff 01496169
f 11 f 11 t 114
l 55 l 55
n=p n 10 n=p n 10
f 6 f 6
l 15 l 15
Qn(2p) 0091465 Qn(2p) 007745
Qn(p) 0062469 Qn(p) 0121519
Qintra 017078 Qintra 014461
Qinter 010058 Qinter 019565
Sr(eacutecart-type de reacutepeacutetabiliteacute) 02415 Sr 02045
RSDr(coefficient de variation de la reacutepeacutetabiliteacute)1462 RSDr 1277
r(repeatability limit)) 0676 r 0573
SL(eacutecart-type interlaboratoire) -001905 SLlt0 SL 001737
SR(eacutecart-type de reproductibiliteacute) 02415 SR 02433
RSDR(coefficient de variation de la reproductibiliteacute)0146 RSDR 0152
R(limite de reproductibiliteacute)) 06762 R(limite de reproductibiliteacute))068123
h k h MoyenneLaboratoires
Niveau 1
si LaboratoiresDeviationMoyenne Dk DeviationsiAlt
Niveau 1
Ref
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3638 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Level 2
Nombre de laboratoires (p) 10
A 2681 2613 2647 005 0034 -0034 -0151 0891 A 2322 2531 2427 015 -0105 0105 -2119 1620 -0220
B 2544 2477 2511 005 0033 -0033 -2699 0872 B 2556 2653 2605 007 -0048 0048 -0581 0750 0094
C 2643 2633 2638 001 0005 -0005 -0311 0130 C 2681 2653 2667 002 0014 -0014 -0040 0217 0029
D 2690 2663 2676 002 0014 -0014 0400 0357 D 2778 2732 2755 003 0023 -0023 0721 0354 0079
E 2690 2716 2703 002 -0013 0013 0897 0336 E 2724 2748 2736 002 -0012 0012 0556 0185 0033
F 2568 2398 2483 012 0085 -0085 -3214 2217 F 2477 2114 2296 026 0182 -0182 -3255 2811 -0188
G 2643 2591 2617 004 0026 -0026 -0707 0682 G 2756 2568 2662 013 0094 -0094 -0085 1453 0045
H 2716 2643 2680 005 0036 -0036 0460 0945 H 2653 2732 2693 006 -0040 0040 0181 0613 0013
L 2591 2591 2591 000 0000 0000 -1196 0000 L 2230 2580 2405 025 -0175 0175 -2307 2704 -0186
M 2653 2690 2672 003 -0018 0018 0311 0481 M 2740 2613 2677 009 0064 -0064 0040 0987 0005
m 2655082 m 26719001 mediane D 00209
Cn(2p) 1867 Cn(2p) 1867 Cp 16101
Cn(p) 16101 Cn(p) 16101 Qn(D) 0039911
n=2p n 20 n=2p n 20 Qdiff 00642591
f 11 f 11 t 082
l 55 l 55
n=p n 10 n=p n 10
f 6 f 6
l 15 l 15
Qn(2p) 0020570 Qn(2p) 00345985
Qn(p) 00332437 Qn(p) 00718158
Qintra 003841 Qintra 006460
Qinter 005352 Qinter 011563
Sr(eacutecart-type de reacutepeacutetabiliteacute) 00543 Sr 00914
RSDr(coefficient de variation de la reacutepeacutetabiliteacute)205 RSDr 342
r(repeatability limit)) 0152 r 0256
SL(eacutecart-type interlaboratoire) 000139 SL 000920
SR(eacutecart-type de reproductibiliteacute) 00659 SR 01325
RSDR(coefficient de variation de la reproductibiliteacute)0025 RSDR 0050
R(limite de reproductibiliteacute)) 018446 R(limite de reproductibiliteacute))03708618
Laboratoires
Niveau 2
Moyenne si Laboratoires
Niveau 2
DRef Alt Moyenne si Deviation h Deviation h k k
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3738 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Level 2 bis
Nombre de laboratoires (p) 10
A 3000 2477 2739 037 0261 -0261 0832 1418 A 2602 2477 2540 009 0062 -0062 -0391 0465 -0199
B 2544 2813 2678 019 -0134 0134 0211 0729 B 2903 2778 2841 009 0062 -0062 1732 0465 0162
C 2477 2813 2645 024 -0168 0168 -0135 0911 C 2477 2699 2588 016 -0111 0111 -0049 0826 -0057
D 2398 2653 2526 018 -0128 0128 -1370 0692 D 2778 3000 2889 016 -0111 0111 2074 0826 0363
E 2875 2740 2808 010 0067 -0067 1547 0365 E 2602 2301 2452 021 0151 -0151 -1012 1120 -0356
F 2477 2544 2511 005 -0033 0033 -1525 0182 F 2778 2477 2628 021 0151 -0151 0230 1120 0117
G 2653 2740 2697 006 -0044 0044 0400 0236 G 2845 2845 2845 000 0000 0000 1764 0000 0148
H 2176 2813 2495 045 -0318 0318 -1692 1727 H 2477 2477 2477 000 0000 0000 -0832 0000 -0017
L 2813 2544 2678 019 0134 -0134 0211 0729 L 2903 2301 2602 043 0301 -0301 0049 2241 -0076
M 2602 2740 2671 010 -0069 0069 0135 0375 M 2903 2255 2579 046 0324 -0324 -0112 2411 -0092
m 2658114 m 259505 mediane D -00372
Cn(2p) 1867 Cn(2p) 1867 Cp 16101
Cn(p) 16101 Cn(p) 16101 Qn(D) 01656915
n=2p n 20 n=2p n 20 Qdiff 02667753
f 11 f 11 t 035
l 55 l 55
n=p n 10 n=p n 10
f 6 f 6
l 15 l 15
Qn(2p) 0098745 Qn(2p) 007195
Qn(p) 006007 Qn(p) 008805
Qintra 018437 Qintra 013435
Qinter 009672 Qinter 014176
Sr(eacutecart-type de reacutepeacutetabiliteacute) 02607 Sr 01900
RSDr(coefficient de variation de la reacutepeacutetabiliteacute)981 RSDr 732
r(repeatability limit)) 0730 r 0532
SL(eacutecart-type interlaboratoire) -002464 SLlt0 SL 000205
SR(eacutecart-type de reproductibiliteacute) 02607 SR 01953
RSDR(coefficient de variation de la reproductibiliteacute)0098 RSDR 0075
R(limite de reproductibiliteacute)) 072996 R(limite de reproductibiliteacute))054686
Laboratoires
Niveau 2 bis
Moyenne si Laboratoires
Niveau 2 bis
DRef Alt Moyenne si Deviation h Deviation h k k
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3838 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Level 3
Nombre de laboratoires (p) 10
A 3690 3663 3676 002 0014 -0014 -0311 0513 A 3690 3708 3699 001 -0009 0009 0354 0257 0022
B 3613 3672 3642 004 -0030 0030 -1851 1109 B 3708 3568 3638 010 0070 -0070 -0844 2061 -0005
C 3663 3732 3698 005 -0035 0035 0645 1302 C 3663 3699 3681 003 -0018 0018 0000 0536 -0017
D 3690 3690 3690 000 0000 0000 0311 0000 D 3643 3663 3653 001 -0010 0010 -0545 0286 -0037
E 3732 3653 3693 006 0040 -0040 0429 1480 E 3613 3732 3673 008 -0060 0060 -0162 1769 -0020
F 3643 3672 3658 002 -0014 0014 -1157 0536 F 3690 3771 3731 006 -0040 0040 0975 1193 0073
G 3699 3653 3676 003 0023 -0023 -0328 0856 G 3602 3643 3623 003 -0021 0021 -1141 0612 -0053
H 3623 3672 3648 003 -0024 0024 -1615 0913 H 3748 3681 3715 005 0033 -0033 0664 0990 0067
L 3724 3663 3694 004 0031 -0031 0461 1150 L 3748 3771 3760 002 -0011 0011 1544 0335 0066
M 3690 3690 3690 000 0000 0000 0311 0000 M 3806 3681 3744 009 0062 -0062 1234 1848 0054
m 368334 m 368086 mediane D 00089
Cn(2p) 1867 Cn(2p) 1867 Cp 16101
Cn(p) 16101 Cn(p) 16101 Qn(D) 00325359
n=2p n 20 n=2p n 20 Qdiff 00523852
f 11 f 11 t 043
l 55 l 55
n=p n 10 n=p n 10
f 6 f 6
l 15 l 15
Qn(2p) 0014323 Qn(2p) 001811
Qn(p) 0013719 Qn(p) 003164
Qintra 002674 Qintra 003381
Qinter 002209 Qinter 005094
Sr(eacutecart-type de reacutepeacutetabiliteacute) 00378 Sr 00478
RSDr(coefficient de variation de la reacutepeacutetabiliteacute)103 RSDr 130
r(repeatability limit)) 0106 r 0134
SL(eacutecart-type interlaboratoire) -000023 SLlt0 SL 000145
SR(eacutecart-type de reproductibiliteacute) 00378 SR 00611
RSDR(coefficient de variation de la reproductibiliteacute)0010 RSDR 0017
R(limite de reproductibiliteacute)) 0105894 R(limite de reproductibiliteacute))017119
Ref Alt DLaboratoires
Niveau 3
Moyenne si Deviation h k Laboratoires
Niveau 3
Moyenne si Deviation h k
Thermo Fisher Scientific
ADRIA Deacuteveloppement 938 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Discussion
The linearity is accepted in all cases with
P values 5 and correlation coefficients superior to 099
For the matrix ldquoRillettesldquo the linearity test is significant (P = 4) with a high
correlation coefficient value (0996)
The Listeria PrecisTM method shows satisfying linearity
2112 Relative accuracy
The accuracy is the closeness of agreement between a test result and the accepted reference value
The bias is the difference between the expectation of the test results and an accepted reference value
Number and nature of the samples
The repartition per category and types is provided in table 3
Table 3 ndash Number and nature of the samples
Categories Types Number of samples
Analysed Exploited (1)
Meat products
Raw meat products 6 4
Ready to cook products 4 2
Ready to eat delicatessen 10 4
Total 20 10
Dairy products
Raw milk cheeses 7 4
Raw milks 4 3
Other dairy products 5 5
Total 16 12
Fishery products
Raw fishes 5 4
Processed fishes 7 2
Ready-to-eat products 6 4
Total 18 10
Vegetables
Raw vegetables 5 4
Frozen vegetables 3 3
Lettuce raw vegetables 5 5
Total 13 12
Egg products
Raw egg 5 5
Pastries 3 3
Pasteurised egg products 4 3
Total 12 11
Environmental samples
Wipes swabs 8 4
Process water 3 3
Dusts 3 3
Total 14 10
TOTAL 93 65
(1) Some data cannot be exploited because enumeration was under the detection level for at least one of the
method
Thermo Fisher Scientific
ADRIA Deacuteveloppement 1038 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Artificial contamination of the samples
Artificial contaminations were realised by spiking after performing injury
protocols on pure cultures or by cross-contaminations
56 samples were artificially contaminated 53 gave exploitable results among
a total of 65 samples 8150 of the samples were artificially contaminated
The injury protocol and the strains inoculated are provided in Table 4
Table 4
Stress Ndeg Injury protocol Strain Origin
A 45 min at 50 degC
48
LM -H123
LM-H170
Smoked salmon
Lettuce
Lettuce
B 30 min at 50 degC
and 2 h at ndash20degC
18
43
LM-O1
Raw milk (environment)
Environment
Egg product
C 30 min at 55degC
18
48
LM-H123
Raw milk
Smoked salmon
Lettuce
D 30 min at 50degC
et 2 h at ndash80degC
34
LM-H171
LM-O1
Rillettes
Lettuce
Egg product
E 30 min at 60 degC LM-O1 Egg product
Confirmatory tests
For the reference and the alternative methods confirmations were realised
by the tests described in the ISO 12290-2A1 standard For the alternative
method only one colony was confirmed while 5 colonies were tested for the
reference method
Results
Samples were analysed in duplicate by the reference and the alternative
methods
The contamination range is provided in table 5 per tested category
Thermo Fisher Scientific
ADRIA Deacuteveloppement 1138 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Table 5 ndash Contamination range
Food category Contamination level (log CFUg)
Meat products 100 ndash 404
Dairy products 148 ndash 408
Fishery products 100 ndash 384
Vegetables 170 ndash 511
Egg products 130 ndash 499
Environmental samples 170 ndash 511
All categories 100 ndash 511
Bi-dimensional graphs are given figure 3
Figure 3 ndash Bi-dimensional graphs
Graphe bi-dimensionnel Produits carneacutes
Fishery products
Vegetables
Egg products
Meat products
Thermo Fisher Scientific
ADRIA Deacuteveloppement 1238 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Statistical interpretation according to EN ISO16140 method
The results of the statistical interpretation are given in Table 6
Table 6 ndash Statistical interpretation results
Category n R Regression
used a t(a) b t(b) Critical T
P
Ordinate
at 0
Slope
at 1
Meat products 10 158 GMFR -0455 3321 115 3123 2306 001 001
Dairy products 12 065 GMFR -0027 0385 1002 0108 2228 092 071
Fishery products 10 130 GMFR -0305 1024 1034 0312 2306 076 034
Vegetables 12 086 GMFR -0015 0128 0993 0191 2228 085 090
Egg products 11 158 GMFR -0038 0866 1001 0090 2262 093 041
Environmental
samples 10 176 GMFR -0139 265 1034 231 2306 005 003
All products 65 118 GMFR -0182 3042 1040 2145 2000 004 000
All products
(estimated number) 65 118 GMFR -0182 3042 1040 2145 2000 004 000
All products (non
estimated results) 20 115 GMFR -0367 151 1138 1141 2101 027 015
Repeatability limits obtained for the alternative and reference methods as
well as bias obtained are presented in Table 7
Dairy products Environmental samples
Log (Ref) Log (Ref)
All categories
Thermo Fisher Scientific
ADRIA Deacuteveloppement 1338 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Table 7
Categories Bias
Repeatability limits
Rob r
Ref Alt
Meat products 0008 0314 0343
Dairy products -0031 0136 0130
Fishery products -0133 0220 0214
Vegetables -0041 0180 0319
Egg products -0026 0219 0206
Environmental samples -0007 0198 0119
All products -0032 0196 0215
Regression straight lines (graph and equation representations) for each food
category and for all products are presented figure 4
Thermo Fisher Scientific
ADRIA Deacuteveloppement 1438 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Figure 4 ndash Regression straight lines
Meat products
Fishery products
Dairy products
Vegetables
Thermo Fisher Scientific
ADRIA Deacuteveloppement 1538 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Environmental samples
Egg products
All products (estimated number)
Thermo Fisher Scientific
ADRIA Deacuteveloppement 1638 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
The hypotheses that the ordinates are close to 0 and the slopes close to 1
are approved in all cases except for
- Meat products (slope P = 1 ordinate P = 1)
- Environmental samples (ordinate P = 3)
- All products (ordinate P = 0)
The bias between the two methods vary from ndash 0133 to 0008 log CFU For
all categories the bias is ndash 0032 log CFU The repeatability limits of the
alternative and the reference method are similar For all products the values
are respectively 0215 and 0196 log CFU For all products these
interpretations are provided taking into account data with low contamination
level or not (estimated colonies number)
For all food products the regression straight line is the following
log (Alt) = 1040 log Ref ndash 0182
The Listeria PrecisTM method shows satisfying relative accuracy
All products
All products (Level 2)
Thermo Fisher Scientific
ADRIA Deacuteveloppement 1738 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
2113 Detection limit (LOD) and quantification limit (LOQ)
The critical level is the defined as the smallest amount which can be detected (not null) but not
quantified as an exact value Below this value it cannot be sure that the true value is not null
The detection limit is defined as being higher than the critical level because it involves a power the
probability 1- which has to be well over 50 for example 95
The quantification limit is defined as the smallest amount of analyte (that is the lowest actual number of
organisms) which can be measured and quantified with defined precision and accuracy under the
experimental conditions by the method under validation
Protocol
The detection limit of the alternative method was realised with pure cultures
Three different inoculation levels were tested with six replicates per level ie
18 analyses were done by the alternative method
Listeria monocytogenes 18 was used for the detection limit of the alternative
method
Quantification limit was calculated for six independent blank samples
determinations
Results
These data are intrinsic to the method used and are presented in the
following tables
Table 8 ndash Inoculation 1 ml on 3 plates
Level (CFUml) Positive samples number Standard deviation Bias
0 06
05 26 1211 0
1 36 0816 05
5 66 2483 85
Thermo Fisher Scientific
ADRIA Deacuteveloppement 1838 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Table 9 ndash Inoculation 01 ml on 1 plate
Level (CFUml) Positive samples number Standard deviation Bias
0 06
5 36 0548 05
10 46 1414 2
50 66 2258 55
Table 10
Formulas Obtained values
LC 165 S0 + X0 18
LOD 33 S0 + X0 32
LOQ 10 S0 + X0 87
2114 Relative Sensitivity
The relative sensitivity is defined as the ability of the alternative method to detect two different amounts
of analyte measured by the reference method within a given matrix at a specified average value or
over the whole measurement range that is it is the minimal quantity variation (increase of the analyte
concentration x) which gives a significant variation of the measured signal (response y)
These data are intrinsic to the used method and are obtained from the results
of the linearity study
Sensitivity patterns obtained for tested (matrixstrain) pairs are presented in
figure 5
Thermo Fisher Scientific
ADRIA Deacuteveloppement 1938 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Figure 5 ndash Sensitivity patterns for the tested (matrixstrain) pairs
2115 SpecificitySelectivity
The specificity is defined as the degree to which a method is affected (or not) by the other components
present in a multi-component sample That is the ability of a method to measure exactly a given
analyte or its amount within the sample without interference from non-target components such as a
matrix effect or background noise
The selectivity is defined as a measure of the degree of non-interference in the presence of non-target
analytes A method is selective if it can be used to detect the analyte under examination and that a
guarantee can be provided that the detected signal can only be a product by that specific analyte
The specificity and selectivity study was performed for the Listeria PrecisTM
detection method no additional test was realised for the enumeration
method The results were the following
- Among the Listeria monocytogenes strains tested 2 strains (Listeria
monocytogenes CIP 7834 and Listeria monocytogenes CIP 105459) gave
negative results These 2 strains were tested twice by running 48 h
incubation time of the compared agars Positive results were then
observed by the alternative method Pale characteristic colonies were
observed on the ISO agar by streaking the Half Fraser broth only
- No cross reaction was observed with the non-target strains except with
Listeria ivanovii which gave characteristic colonies on Brilliancetrade Listeria
Agar but the confirmatory tests provide a clear discrimination between
Listeria monocytogenes and Listeria ivanovi species
Rillettes
Lettuce
Raw milk
Smoked salmon
Raw egg
Process water
x (CFUg)
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2038 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
212 Practicability
1 Packaging Ready to use Brilliance Listeria agar plates are available
(x 10 plates)
2 Reagent volume
3 Storage conditionsndash
Shelf life of products
not opened
The plates are stored at 6 - 12degC in darkness conditions The
expiration date is mentioned on the package and on the plates It
is around 3 months
4 Modalities of use
after first use
5 Required
equipments No specific equipment is required
6 Reagents Ready to use plates
7 Training 05 day
8 Real time handling
Reference method
9 min for one sample
55 min for 10 samples
Alternative method
7 min for one sample
35 minutes for 10 samples
9 Time to result
Negative results
Reference method Day 2
Alternative method Day 2
Negative results after confirmation or positive results
Reference method Day 4 ndash Day 7
Alternative method Day 4 ndash Day 7
Positive results
Reference method Day 4 ndash Day 7
Alternative method Day 2 ndash Day 3 (with OBIS Mono Test)
or Day 4 ndash Day 7 (by the classical tests described in the
standardised method)
10 Technician skill Identical to that required for the reference method
11 Steps common to
the reference method Mother suspension revivification step and decimal dilutions
12 Traceability
13 Maintenance
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2138 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
213 Inter-laboratory study
2131 Organisation
12 laboratories participated to this study Pasteurised milk was inoculated by
Listeria monocytogenes 12a isolated from raw milk The targeted
inoculation levels were
- 0 CFUml
- 50 CFU ml
- 500 CFU ml
- 5 000 CFU ml
Each laboratory received eight flasks of 10 ml sample ie two flasks per
inoculation level Furthermore one non-inoculated sample has been added
to the package for total viable count microflora (NF ISO 4833 method)
Detailed instructions were transmitted to the collaborators by the expert
laboratory
Collaborative study laboratories and the expert laboratory carried out the
analyses with the alternative and reference methods
2132 Verification of experimental parameters
Strain stability during transport
In order to evaluate the Listeria monocytogenes strain stability during
transport bacterial count of all inoculated flasks were checked at different
time ie inoculation time after 24 h of conservation at 2deg - 8degC Results are
reported in table 11
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2238 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Table 11 ndash Listeria monocytogenes count
by ISO 11290-2A1 method (log CFUml)
Day of the analyses Level contamination Reference method
Day 0
(29052006)
1 45
2 440
3 4 500
Day 1
(30052006)
1 60
2 430
3 4 900
No evolution of the strain was observed after storage for 24 h at 2degC ndash 8degC
Contamination level
The real contamination levels are provided in table 12
Table 12 ndash Real contamination level (CFUml)
Target inoculation
(CFU ml) Samples Ndeg
Real contamination levels
(CFUml)
0 1 - 8 0
50 5 - 6 45
500 3 - 7 440
5000 2 - 4 4 500
Sample temperature at receipt
Measured temperatures at receipt are listed in table 13
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2338 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Table 13 - Sample temperature on receipt
Laboratories Receipt
day
Hour
receipt
Measured temperature
at receipt (degC)
Measured temperature by the
temperature probe (degC)
A
B
C
D
E
F
G
H
I
J
K
L
M
30052006
10h45
10h15
11h15
10h00
09h30
8h30
11h50
10h00
08h30
14h45
08h30
09h45
08h45
82
55
45
63
60
62
81
38
48
-
14
67
76
03
03
-13
03
04
-14
05
04
01
05
-13
04
Conclusion
The temperatures during the transport were correct for all the labs except for
Lab J which didnrsquot provide the information
All the labs received their packages with several broken flasks
Three labs (B D and L) received some damaged samples but proceeded to
analyses
All the labs started the analyses at Day 1 except Lab J
2133 Results
Aerobic mesophilic flora
The count of aerobic mesophilic flora varied from 94 102 CFUml to
33 103 CFUml
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2438 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Expert lab results
The results obtained by the expert lab are the following
Table 14
Target inoculation level (CFUml)
Reference method ISO 11290-2
Alternative method Listeria Precis Enumeration
Rep 1 Rep 2 Rep 1 Rep 2
lt 10 lt 10 lt 10 lt 10 lt10
50 50 (Ne) 60 (Ne) 50 (Ne) 30 (Ne)
500 470 390 490 500
5 000 4 700 5 100 5 900 3 400
Ne estimated number
The obtained results are in agreement with those expected
Collaborator Labs results
A synthesis of the results obtained by the collaborator labs is provided
Table 15 (CFUml) and Table 16 (log CFUml)
Table 15 ndash Synthesis of results obtained by the alternative
and the reference methods (CFUml)
Labs Level 1 Level 2 Level 2 bis Level 3
Reference method
Alternative method
Reference method
Alternative method
Reference method
Alternative method
Reference method
Alternative method
A 40 40 70 40 480 410 210 340 1000 300 400 300 4900 4600 4900 5100
B 40 15 40 20 350 300 360 450 350 650 800 600 4100 4700 5100 3700
C 50 20 20 70 440 430 480 450 300 650 300 500 4600 5400 4600 5000
D 40 60 40 40 490 460 600 540 250 450 600 1000 4900 4900 4400 4600
E 50 40 50 20 490 520 530 560 750 550 400 200 5400 4500 4100 5400
F 60 20 60 50 370 250 300 130 300 350 600 300 4400 4700 4900 5900
G 70 35 120 50 440 390 570 370 450 550 700 700 5000 4500 4000 4400
H 110 40 100 90 520 440 450 540 150 650 300 300 4200 4700 5600 4800
L 35 60 20 40 390 390 170 380 650 350 800 200 5300 4600 5600 5900
M 45 45 30 40 450 490 550 410 400 550 800 180 4900 4900 6400 4800
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2538 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Table 16 ndash Synthesis of results obtained by the alternative
and the reference methods (log CFUml)
Labs Level 1 Level 2 Level 2 bis Level 3
Reference method
Alternative method
Reference method
Alternative method
Reference method
Alternative method
Reference method
Alternative method
A 1602 1602 1845 1602 2681 2613 2322 2531 3000 2477 2602 2477 3690 3663 3690 3708
B 1602 1176 1602 1301 2544 2477 2556 2653 2544 2813 2903 2778 3613 3672 3708 3568
C 1699 1301 1301 1845 2643 2633 2681 2653 2477 2813 2477 2699 3663 3732 3663 3699
D 1602 1778 1602 1602 2690 2663 2778 2732 2398 2653 2778 3000 3690 3690 3643 3663
E 1699 1602 1699 1301 2690 2716 2724 2748 2875 2740 2602 2301 3732 3653 3613 3732
F 1778 1301 1778 1699 2568 2398 2477 2114 2477 2544 2778 2477 3643 3672 3690 3771
G 1845 1544 2079 1699 2643 2591 2756 2568 2653 2740 2845 2845 3699 3653 3602 3643
H 2041 1602 2000 1954 2716 2643 2653 2732 2176 2813 2477 2477 3623 3672 3748 3681
L 1544 1778 1301 1602 2591 2591 2230 2580 2813 2544 2903 2301 3724 3663 3748 3771
M 1653 1653 1477 1602 2653 2690 2740 2613 2602 2740 2903 2255 3690 3690 3806 3681
Level 2 results obtained taking account the 3 plates inoculated with the mother-suspension
Level 2 bis results obtained taking account the plates inoculated with 01 ml of the mother-suspension
Scrutiny of the measurement results for consistency
In order to identify the measurement results or laboratories that could be
inconsistent two graphical consistency techniques were realised Mandelrsquos
h-ank-statistics Mandel indicators h and k at 5 significance highlighted
some possible inconsistent data (See tables and graphics in Appendix 3 and
Appendix 4)
Mandelrsquos values Number of values observed over the threshold
Reference method Alternative method
h gt 1 Lab F Level 2 Lab F Level 2
h gt 5 Lab F Level 2
Lab B Levels 1 and 2
Lab A Level 2
Lab D Level 3
Lab F Level 2
Lab L Level 2
kgt 1 Lab F Level 3
k gt 5 Lab F Level 2 Lab C Level 2
Lab F Level 3
Lab L Levels 1 and 3
Lab M Level 1
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2638 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
The emphasized results appear (i) coherent with the targeted contamination
levels (ii) concordant between both methods
All the results were thus kept for the statistical analysis
Comparison of the trueness and precision characteristics of
the reference method and alternative methods
The statistical values are summarised hereafter
Level p
Reference method Alternative method
Repeatability ratio
Reproducibility ratio Median
Repeatability standard deviation
Reproducibility standard deviation
Median Repeatability
standard deviation
Reproducibility standard deviation
1 10 16519 02415 02425 16021 02045 02433 0847 1007
2 10 26551 00543 00659 26719 00914 01325 1682 2011
2 bis 10 26581 02607 02607 25951 01900 01953 0729 0749
3 10 18671 00378 00378 18671 00478 00611 1264 1617
Bias of the alternative method
In order to estimate the bias of the alternative method with regard to the reference method for every
level Dij and t are calculated according to the following equations
RefAlt ijYijYDij
Diffp
Dijit
)2(
)(median
If the t value is superior to 2 the alternative method is significantly biased
with regard to the reference method
The values are given in table 17
Table 17 ndash t (d) values obtained by level
Level Bias t (d) Conclusion
1 0068 114 Non significant bias
2 0021 082 Non significant bias
2 bis - 0037 035 Non significant bias
3 - 0037 043 Non significant bias
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2738 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
The statistical test concludes to a non significant bias for all the inoculation
levels The bias varies from ndash 0037 to 0068 log CFUml
Comparison of the repeatability standard deviations
If the ratio Srj Alt Srj Ref of the repeatability standard deviations of the
alternative method and the reference method is larger than 2 the precision
under repeatability conditions of the alternative method is considered to be
lower than that of the reference method If this ratio is smaller than 05 the
precision under repeatability conditions of the alternative method is
considered to be greater than that of the reference method
The ratio values are given in table 18
Table 18
Level
Repeatability standard deviation
Reference method Alternative method Alternative method
reference method ratio
1 0242 0205 0847
2 0054 0091 1682
2 bis 0261 0190 0729
3 0038 0048 1264
Level Repeatability limit
Reference method Alternative method
1 0676 0573
2 0152 0256
2 bis 0730 0532
3 0106 0134
The ratios of the repeatability standard deviations are below 2 in all cases
The precision under repeatability conditions of the alternative method is
equivalent to that of the reference method
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2838 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Comparison of the reproducibility standard deviations
If the ratio Srj Alt Srj Ref of the reproducibility standard deviations of the alternative method and the
reference method is larger than 2 the precision under reproducibility conditions of the alternative
method is considered to be lower than that of the reference method If this ratio is smaller than 05 the
precision under reproducibility conditions of the alternative method is considered to be greater than
that of the reference method
The ratio values are given in table 19
Table 19
Level
Reproducibility standard deviation
Reference method Alternative method Alternative method
reference method ratio
1 0242 0243 1007
2 0066 0133 2011
2 bis 0261 0195 0749
3 0038 0061 1617
Level Reproducibility limit
Reference method Alternative method
1 0676 0681
2 0185 0371
2 bis 0730 0547
3 0106 0171
The ratios of the reproducibility standard deviations are below or close to 2
The precision under reproducibility conditions of the alternative method is
equivalent to that of the reference method
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2938 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
214 Conclusion
Method comparison study
The Listeria PrecisTM enumeration method shows satisfying linearity and
relative accuracy
The alternative method shows satisfying inclusivity and exclusivity results
Two Listeria monocytogenes strains gave negative results with the
alternative method These strains grew weakly on the reference method
media (OampA Palcam and Oxford)
The limits of repeatability of the alternative method are similar to those of the
reference method
The bias between both methods are low between - 0133 Log CFUg and
- 0008 Log CFUg
The Listeria PrecisTM enumeration method offers the saving for confirmatory
tests
Positive results are obtained with the alternative method in 2-3 days (with
OBIS Mono test) or in 4-7 days (by the classical tests described in the
standardized method) against 4-7 days for the reference method
Inter-laboratory study
The bias between the reference and the alternative methods shows satisfying
and correct values
- level 1 (100 - 1 000 CFUg) bias = + 007 log CFUg
- level 2 (1 000 - 10 000 CFUg) bias = 002 log CFUg
- level 2 bis (1 000 - 10 000 CFUg) bias = - 004 log CFUg
- level 3 (10 000 - 100 000 CFUg) bias = - 004 log CFUg
The repeatability and the reproducibility of the alternative method look similar
to those of the reference method
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3038 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
22 Extension study (realised by ASEPT 2007)
An extension study was performed in 2007 for a new confirmatory test OBIS
MONO
150 Listeria monocytogenes strains and 100 non-target strains were isolated
onto TSAYE and BrillianceTM Listeria Agar plates All the Listeria
monocytogenes and the non-target strains gave the expected results
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3138 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Appendix 1 ndash Flow diagram of the Listeria PrecisTM method
x g sample + 9 x ml buffered peptone water
Revivification step for 1 h plusmn 5 min at 20degC plusmn 1degC
Streaking 1 ml onto 3 Brilliance Listeria Agar plates (mother suspension)
or 01 ml onto 1 Brilliance Listeria Agar plates (mother suspension and decimal dilutions)
Incubation for 48 h plusmn 3 h at 37degC plusmn 1degC
Confirmatory tests on at least one typical colony
- by the tests described in the ISO method
- by applying the OBIS mono test on an isolated colony
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3238 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Appendix 2 ndash ISO 11290-2 (1998) amp ISO 11290-2A1 (2004) Horizontal
method for the detection and enumeration of Listeria monocytogenes -
Part 2 enumeration of Listeria monocytogenes in foods
10 g cheese + 90 ml buffered peptone water or Half Fraser Broth (without supplements) (110 dilution)
Stomach 1 min 30 seconds
Keep at 20degC plusmn 2degC for 1 h plusmn 5 min
Decimal dilutions in 9 ml peptone salt tubes
Dilution 110
Dilution 1100
Dilution 11000
1 ml on 3 Chromogenic Listeria medium (ISO) plates (x 2) (-1)
01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-2)
01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-3)
01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-4)
Incubation at 37degC plusmn 1degC
24 h - 48 h
Numerate typical colonies at 24 h
(bluegreen surrounded by an opaque halo)
Numerate at 48 h
Streak typical colonies on TSYEA
(5 coloniesplate 2 successive dilutions)
Confirmation tests
Catalase
Gram stain
For confirmation of L monocytogenes
Haemolysis
Carbohydrate (rhamnose and xylose) utilisation
CAMP Test
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3338 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Appendix 3 ndash Mandelrsquos graphics
Laboratoire h Ref
Niveau 1 h Ref
Niveau 2 h Ref
Niveau 3 h Alt
Niveau 1 h Alt
Niveau 2 h Alt
Niveau 3 h5 h1 h5 h1
A -050 -015 083 062 -212 -039 198 299 -198 -299
B -261 -270 021 -077 -058 173 198 299 -198 -299
C -151 -031 -014 -015 -004 -005 198 299 -198 -299
D 038 040 -137 000 072 207 198 299 -198 -299
E -001 090 155 -052 056 -101 198 299 -198 -299
F -112 -321 -153 070 -325 023 198 299 -198 -299
G 042 -071 040 147 -009 176 198 299 -198 -299
H 169 046 -169 192 018 -083 198 299 -198 -299
L 009 -120 021 -077 -231 005 198 299 -198 -299
M 001 031 014 -032 004 -011 198 299 -198 -299
-300
-200
-100
000
100
200
300
A B C D E F G H L M
Vale
urs
h d
e M
an
del(
rob
uste
s)
Laboratoires
Meacutethode de reacutefeacuterence analyse robuste
h Ref Niveau 1
h Ref Niveau 2
h Ref Niveau 3
h 5=198
h 1=299
-300
-200
-100
000
100
200
300
A B C D E F G H L M
Vale
urs
de h
de M
an
del(
rob
uste
s)
Laboratoires
Meacutethode alternative analyse robuste
h Alt Niveau 1
h Alt Niveau 2
h Alt Niveau 2
h 1=299
h 5=198
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3438 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Laboratoire k Ref
Niveau 1 k Ref
Niveau 2 k Ref
Niveau 3 k Alt
Niveau 1 k Alt
Niveau 2 k Alt
Niveau 3 k1 k5
A 000 089 142 084 162 046 259 181
B 125 087 073 104 075 046 259 181
C 117 013 091 188 022 083 259 181
D 052 036 069 000 035 083 259 181
E 028 034 037 138 019 112 259 181
F 140 222 018 027 281 112 259 181
G 088 068 024 131 145 000 259 181
H 129 094 173 016 061 000 259 181
L 069 000 073 104 270 224 259 181
M 000 048 038 043 099 241 259 181
000
050
100
150
200
250
300
350
400
A B C D E F G H L M
Vale
urs
k d
e M
an
del(
rob
uste
s)
Laboratoires
Meacutethode de reacutefeacuterence analyse robuste
k Ref Niveau 1k Ref Niveau 2k Ref Niveau 3
k 1=259
k 5=181
000
050
100
150
200
250
300
A B C D E F G H L M
Vale
urs
k d
e M
an
del
(ro
bu
ste
s)
Laboratoires
Meacutethode alternative analyse robuste
k Alt Niveau 3
k Alt Niveau 1
k Alt Niveau 2
k 1=259
k 5=181
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3538 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Appendix 4 ndash Statistical results
Level 1
Nombre de laboratoires (p) 10
A 1602 1602 1602 000 0000 0000 -0495 0000 A 1845 1602 1724 017 0122 -0122 0621 0840 0122
B 1602 1176 1389 030 0213 -0213 -2613 1247 B 1602 1301 1452 021 0151 -0151 -0769 1041 0062
C 1699 1301 1500 028 0199 -0199 -1510 1165 C 1301 1845 1573 038 -0272 0272 -0148 1881 0073
D 1602 1778 1690 012 -0088 0088 0380 0516 D 1602 1602 1602 000 0000 0000 0000 0000 -0088
E 1699 1602 1651 007 0048 -0048 -0013 0284 E 1699 1301 1500 028 0199 -0199 -0522 1376 -0151
F 1778 1301 1540 034 0239 -0239 -1116 1397 F 1778 1699 1739 006 0040 -0040 0698 0274 0199
G 1845 1544 1695 021 0151 -0151 0425 0881 G 2079 1699 1889 027 0190 -0190 1467 1315 0194
H 2041 1602 1822 031 0220 -0220 1689 1286 H 2000 1954 1977 003 0023 -0023 1917 0158 0155
L 1544 1778 1661 017 -0117 0117 0092 0685 L 1301 1602 1452 021 -0151 0151 -0769 1041 -0210
M 1653 1653 1653 000 0000 0000 0013 0000 M 1477 1602 1540 009 -0062 0062 -0319 0432 -0114
m 165186 m 160206 mediane D 00678
Cn(2p) 1867 Cn(2p) 1867 Cp 16101
Cn(p) 16101 Cn(p) 16101 Qn(D) 0092926
n=2p n 20 n=2p n 20 Qdiff 01496169
f 11 f 11 t 114
l 55 l 55
n=p n 10 n=p n 10
f 6 f 6
l 15 l 15
Qn(2p) 0091465 Qn(2p) 007745
Qn(p) 0062469 Qn(p) 0121519
Qintra 017078 Qintra 014461
Qinter 010058 Qinter 019565
Sr(eacutecart-type de reacutepeacutetabiliteacute) 02415 Sr 02045
RSDr(coefficient de variation de la reacutepeacutetabiliteacute)1462 RSDr 1277
r(repeatability limit)) 0676 r 0573
SL(eacutecart-type interlaboratoire) -001905 SLlt0 SL 001737
SR(eacutecart-type de reproductibiliteacute) 02415 SR 02433
RSDR(coefficient de variation de la reproductibiliteacute)0146 RSDR 0152
R(limite de reproductibiliteacute)) 06762 R(limite de reproductibiliteacute))068123
h k h MoyenneLaboratoires
Niveau 1
si LaboratoiresDeviationMoyenne Dk DeviationsiAlt
Niveau 1
Ref
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3638 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Level 2
Nombre de laboratoires (p) 10
A 2681 2613 2647 005 0034 -0034 -0151 0891 A 2322 2531 2427 015 -0105 0105 -2119 1620 -0220
B 2544 2477 2511 005 0033 -0033 -2699 0872 B 2556 2653 2605 007 -0048 0048 -0581 0750 0094
C 2643 2633 2638 001 0005 -0005 -0311 0130 C 2681 2653 2667 002 0014 -0014 -0040 0217 0029
D 2690 2663 2676 002 0014 -0014 0400 0357 D 2778 2732 2755 003 0023 -0023 0721 0354 0079
E 2690 2716 2703 002 -0013 0013 0897 0336 E 2724 2748 2736 002 -0012 0012 0556 0185 0033
F 2568 2398 2483 012 0085 -0085 -3214 2217 F 2477 2114 2296 026 0182 -0182 -3255 2811 -0188
G 2643 2591 2617 004 0026 -0026 -0707 0682 G 2756 2568 2662 013 0094 -0094 -0085 1453 0045
H 2716 2643 2680 005 0036 -0036 0460 0945 H 2653 2732 2693 006 -0040 0040 0181 0613 0013
L 2591 2591 2591 000 0000 0000 -1196 0000 L 2230 2580 2405 025 -0175 0175 -2307 2704 -0186
M 2653 2690 2672 003 -0018 0018 0311 0481 M 2740 2613 2677 009 0064 -0064 0040 0987 0005
m 2655082 m 26719001 mediane D 00209
Cn(2p) 1867 Cn(2p) 1867 Cp 16101
Cn(p) 16101 Cn(p) 16101 Qn(D) 0039911
n=2p n 20 n=2p n 20 Qdiff 00642591
f 11 f 11 t 082
l 55 l 55
n=p n 10 n=p n 10
f 6 f 6
l 15 l 15
Qn(2p) 0020570 Qn(2p) 00345985
Qn(p) 00332437 Qn(p) 00718158
Qintra 003841 Qintra 006460
Qinter 005352 Qinter 011563
Sr(eacutecart-type de reacutepeacutetabiliteacute) 00543 Sr 00914
RSDr(coefficient de variation de la reacutepeacutetabiliteacute)205 RSDr 342
r(repeatability limit)) 0152 r 0256
SL(eacutecart-type interlaboratoire) 000139 SL 000920
SR(eacutecart-type de reproductibiliteacute) 00659 SR 01325
RSDR(coefficient de variation de la reproductibiliteacute)0025 RSDR 0050
R(limite de reproductibiliteacute)) 018446 R(limite de reproductibiliteacute))03708618
Laboratoires
Niveau 2
Moyenne si Laboratoires
Niveau 2
DRef Alt Moyenne si Deviation h Deviation h k k
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3738 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Level 2 bis
Nombre de laboratoires (p) 10
A 3000 2477 2739 037 0261 -0261 0832 1418 A 2602 2477 2540 009 0062 -0062 -0391 0465 -0199
B 2544 2813 2678 019 -0134 0134 0211 0729 B 2903 2778 2841 009 0062 -0062 1732 0465 0162
C 2477 2813 2645 024 -0168 0168 -0135 0911 C 2477 2699 2588 016 -0111 0111 -0049 0826 -0057
D 2398 2653 2526 018 -0128 0128 -1370 0692 D 2778 3000 2889 016 -0111 0111 2074 0826 0363
E 2875 2740 2808 010 0067 -0067 1547 0365 E 2602 2301 2452 021 0151 -0151 -1012 1120 -0356
F 2477 2544 2511 005 -0033 0033 -1525 0182 F 2778 2477 2628 021 0151 -0151 0230 1120 0117
G 2653 2740 2697 006 -0044 0044 0400 0236 G 2845 2845 2845 000 0000 0000 1764 0000 0148
H 2176 2813 2495 045 -0318 0318 -1692 1727 H 2477 2477 2477 000 0000 0000 -0832 0000 -0017
L 2813 2544 2678 019 0134 -0134 0211 0729 L 2903 2301 2602 043 0301 -0301 0049 2241 -0076
M 2602 2740 2671 010 -0069 0069 0135 0375 M 2903 2255 2579 046 0324 -0324 -0112 2411 -0092
m 2658114 m 259505 mediane D -00372
Cn(2p) 1867 Cn(2p) 1867 Cp 16101
Cn(p) 16101 Cn(p) 16101 Qn(D) 01656915
n=2p n 20 n=2p n 20 Qdiff 02667753
f 11 f 11 t 035
l 55 l 55
n=p n 10 n=p n 10
f 6 f 6
l 15 l 15
Qn(2p) 0098745 Qn(2p) 007195
Qn(p) 006007 Qn(p) 008805
Qintra 018437 Qintra 013435
Qinter 009672 Qinter 014176
Sr(eacutecart-type de reacutepeacutetabiliteacute) 02607 Sr 01900
RSDr(coefficient de variation de la reacutepeacutetabiliteacute)981 RSDr 732
r(repeatability limit)) 0730 r 0532
SL(eacutecart-type interlaboratoire) -002464 SLlt0 SL 000205
SR(eacutecart-type de reproductibiliteacute) 02607 SR 01953
RSDR(coefficient de variation de la reproductibiliteacute)0098 RSDR 0075
R(limite de reproductibiliteacute)) 072996 R(limite de reproductibiliteacute))054686
Laboratoires
Niveau 2 bis
Moyenne si Laboratoires
Niveau 2 bis
DRef Alt Moyenne si Deviation h Deviation h k k
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3838 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Level 3
Nombre de laboratoires (p) 10
A 3690 3663 3676 002 0014 -0014 -0311 0513 A 3690 3708 3699 001 -0009 0009 0354 0257 0022
B 3613 3672 3642 004 -0030 0030 -1851 1109 B 3708 3568 3638 010 0070 -0070 -0844 2061 -0005
C 3663 3732 3698 005 -0035 0035 0645 1302 C 3663 3699 3681 003 -0018 0018 0000 0536 -0017
D 3690 3690 3690 000 0000 0000 0311 0000 D 3643 3663 3653 001 -0010 0010 -0545 0286 -0037
E 3732 3653 3693 006 0040 -0040 0429 1480 E 3613 3732 3673 008 -0060 0060 -0162 1769 -0020
F 3643 3672 3658 002 -0014 0014 -1157 0536 F 3690 3771 3731 006 -0040 0040 0975 1193 0073
G 3699 3653 3676 003 0023 -0023 -0328 0856 G 3602 3643 3623 003 -0021 0021 -1141 0612 -0053
H 3623 3672 3648 003 -0024 0024 -1615 0913 H 3748 3681 3715 005 0033 -0033 0664 0990 0067
L 3724 3663 3694 004 0031 -0031 0461 1150 L 3748 3771 3760 002 -0011 0011 1544 0335 0066
M 3690 3690 3690 000 0000 0000 0311 0000 M 3806 3681 3744 009 0062 -0062 1234 1848 0054
m 368334 m 368086 mediane D 00089
Cn(2p) 1867 Cn(2p) 1867 Cp 16101
Cn(p) 16101 Cn(p) 16101 Qn(D) 00325359
n=2p n 20 n=2p n 20 Qdiff 00523852
f 11 f 11 t 043
l 55 l 55
n=p n 10 n=p n 10
f 6 f 6
l 15 l 15
Qn(2p) 0014323 Qn(2p) 001811
Qn(p) 0013719 Qn(p) 003164
Qintra 002674 Qintra 003381
Qinter 002209 Qinter 005094
Sr(eacutecart-type de reacutepeacutetabiliteacute) 00378 Sr 00478
RSDr(coefficient de variation de la reacutepeacutetabiliteacute)103 RSDr 130
r(repeatability limit)) 0106 r 0134
SL(eacutecart-type interlaboratoire) -000023 SLlt0 SL 000145
SR(eacutecart-type de reproductibiliteacute) 00378 SR 00611
RSDR(coefficient de variation de la reproductibiliteacute)0010 RSDR 0017
R(limite de reproductibiliteacute)) 0105894 R(limite de reproductibiliteacute))017119
Ref Alt DLaboratoires
Niveau 3
Moyenne si Deviation h k Laboratoires
Niveau 3
Moyenne si Deviation h k
Thermo Fisher Scientific
ADRIA Deacuteveloppement 1038 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Artificial contamination of the samples
Artificial contaminations were realised by spiking after performing injury
protocols on pure cultures or by cross-contaminations
56 samples were artificially contaminated 53 gave exploitable results among
a total of 65 samples 8150 of the samples were artificially contaminated
The injury protocol and the strains inoculated are provided in Table 4
Table 4
Stress Ndeg Injury protocol Strain Origin
A 45 min at 50 degC
48
LM -H123
LM-H170
Smoked salmon
Lettuce
Lettuce
B 30 min at 50 degC
and 2 h at ndash20degC
18
43
LM-O1
Raw milk (environment)
Environment
Egg product
C 30 min at 55degC
18
48
LM-H123
Raw milk
Smoked salmon
Lettuce
D 30 min at 50degC
et 2 h at ndash80degC
34
LM-H171
LM-O1
Rillettes
Lettuce
Egg product
E 30 min at 60 degC LM-O1 Egg product
Confirmatory tests
For the reference and the alternative methods confirmations were realised
by the tests described in the ISO 12290-2A1 standard For the alternative
method only one colony was confirmed while 5 colonies were tested for the
reference method
Results
Samples were analysed in duplicate by the reference and the alternative
methods
The contamination range is provided in table 5 per tested category
Thermo Fisher Scientific
ADRIA Deacuteveloppement 1138 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Table 5 ndash Contamination range
Food category Contamination level (log CFUg)
Meat products 100 ndash 404
Dairy products 148 ndash 408
Fishery products 100 ndash 384
Vegetables 170 ndash 511
Egg products 130 ndash 499
Environmental samples 170 ndash 511
All categories 100 ndash 511
Bi-dimensional graphs are given figure 3
Figure 3 ndash Bi-dimensional graphs
Graphe bi-dimensionnel Produits carneacutes
Fishery products
Vegetables
Egg products
Meat products
Thermo Fisher Scientific
ADRIA Deacuteveloppement 1238 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Statistical interpretation according to EN ISO16140 method
The results of the statistical interpretation are given in Table 6
Table 6 ndash Statistical interpretation results
Category n R Regression
used a t(a) b t(b) Critical T
P
Ordinate
at 0
Slope
at 1
Meat products 10 158 GMFR -0455 3321 115 3123 2306 001 001
Dairy products 12 065 GMFR -0027 0385 1002 0108 2228 092 071
Fishery products 10 130 GMFR -0305 1024 1034 0312 2306 076 034
Vegetables 12 086 GMFR -0015 0128 0993 0191 2228 085 090
Egg products 11 158 GMFR -0038 0866 1001 0090 2262 093 041
Environmental
samples 10 176 GMFR -0139 265 1034 231 2306 005 003
All products 65 118 GMFR -0182 3042 1040 2145 2000 004 000
All products
(estimated number) 65 118 GMFR -0182 3042 1040 2145 2000 004 000
All products (non
estimated results) 20 115 GMFR -0367 151 1138 1141 2101 027 015
Repeatability limits obtained for the alternative and reference methods as
well as bias obtained are presented in Table 7
Dairy products Environmental samples
Log (Ref) Log (Ref)
All categories
Thermo Fisher Scientific
ADRIA Deacuteveloppement 1338 November 19 2014
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Listeria Precis Enumeration
Table 7
Categories Bias
Repeatability limits
Rob r
Ref Alt
Meat products 0008 0314 0343
Dairy products -0031 0136 0130
Fishery products -0133 0220 0214
Vegetables -0041 0180 0319
Egg products -0026 0219 0206
Environmental samples -0007 0198 0119
All products -0032 0196 0215
Regression straight lines (graph and equation representations) for each food
category and for all products are presented figure 4
Thermo Fisher Scientific
ADRIA Deacuteveloppement 1438 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Figure 4 ndash Regression straight lines
Meat products
Fishery products
Dairy products
Vegetables
Thermo Fisher Scientific
ADRIA Deacuteveloppement 1538 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Environmental samples
Egg products
All products (estimated number)
Thermo Fisher Scientific
ADRIA Deacuteveloppement 1638 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
The hypotheses that the ordinates are close to 0 and the slopes close to 1
are approved in all cases except for
- Meat products (slope P = 1 ordinate P = 1)
- Environmental samples (ordinate P = 3)
- All products (ordinate P = 0)
The bias between the two methods vary from ndash 0133 to 0008 log CFU For
all categories the bias is ndash 0032 log CFU The repeatability limits of the
alternative and the reference method are similar For all products the values
are respectively 0215 and 0196 log CFU For all products these
interpretations are provided taking into account data with low contamination
level or not (estimated colonies number)
For all food products the regression straight line is the following
log (Alt) = 1040 log Ref ndash 0182
The Listeria PrecisTM method shows satisfying relative accuracy
All products
All products (Level 2)
Thermo Fisher Scientific
ADRIA Deacuteveloppement 1738 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
2113 Detection limit (LOD) and quantification limit (LOQ)
The critical level is the defined as the smallest amount which can be detected (not null) but not
quantified as an exact value Below this value it cannot be sure that the true value is not null
The detection limit is defined as being higher than the critical level because it involves a power the
probability 1- which has to be well over 50 for example 95
The quantification limit is defined as the smallest amount of analyte (that is the lowest actual number of
organisms) which can be measured and quantified with defined precision and accuracy under the
experimental conditions by the method under validation
Protocol
The detection limit of the alternative method was realised with pure cultures
Three different inoculation levels were tested with six replicates per level ie
18 analyses were done by the alternative method
Listeria monocytogenes 18 was used for the detection limit of the alternative
method
Quantification limit was calculated for six independent blank samples
determinations
Results
These data are intrinsic to the method used and are presented in the
following tables
Table 8 ndash Inoculation 1 ml on 3 plates
Level (CFUml) Positive samples number Standard deviation Bias
0 06
05 26 1211 0
1 36 0816 05
5 66 2483 85
Thermo Fisher Scientific
ADRIA Deacuteveloppement 1838 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Table 9 ndash Inoculation 01 ml on 1 plate
Level (CFUml) Positive samples number Standard deviation Bias
0 06
5 36 0548 05
10 46 1414 2
50 66 2258 55
Table 10
Formulas Obtained values
LC 165 S0 + X0 18
LOD 33 S0 + X0 32
LOQ 10 S0 + X0 87
2114 Relative Sensitivity
The relative sensitivity is defined as the ability of the alternative method to detect two different amounts
of analyte measured by the reference method within a given matrix at a specified average value or
over the whole measurement range that is it is the minimal quantity variation (increase of the analyte
concentration x) which gives a significant variation of the measured signal (response y)
These data are intrinsic to the used method and are obtained from the results
of the linearity study
Sensitivity patterns obtained for tested (matrixstrain) pairs are presented in
figure 5
Thermo Fisher Scientific
ADRIA Deacuteveloppement 1938 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Figure 5 ndash Sensitivity patterns for the tested (matrixstrain) pairs
2115 SpecificitySelectivity
The specificity is defined as the degree to which a method is affected (or not) by the other components
present in a multi-component sample That is the ability of a method to measure exactly a given
analyte or its amount within the sample without interference from non-target components such as a
matrix effect or background noise
The selectivity is defined as a measure of the degree of non-interference in the presence of non-target
analytes A method is selective if it can be used to detect the analyte under examination and that a
guarantee can be provided that the detected signal can only be a product by that specific analyte
The specificity and selectivity study was performed for the Listeria PrecisTM
detection method no additional test was realised for the enumeration
method The results were the following
- Among the Listeria monocytogenes strains tested 2 strains (Listeria
monocytogenes CIP 7834 and Listeria monocytogenes CIP 105459) gave
negative results These 2 strains were tested twice by running 48 h
incubation time of the compared agars Positive results were then
observed by the alternative method Pale characteristic colonies were
observed on the ISO agar by streaking the Half Fraser broth only
- No cross reaction was observed with the non-target strains except with
Listeria ivanovii which gave characteristic colonies on Brilliancetrade Listeria
Agar but the confirmatory tests provide a clear discrimination between
Listeria monocytogenes and Listeria ivanovi species
Rillettes
Lettuce
Raw milk
Smoked salmon
Raw egg
Process water
x (CFUg)
Thermo Fisher Scientific
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Listeria Precis Enumeration
212 Practicability
1 Packaging Ready to use Brilliance Listeria agar plates are available
(x 10 plates)
2 Reagent volume
3 Storage conditionsndash
Shelf life of products
not opened
The plates are stored at 6 - 12degC in darkness conditions The
expiration date is mentioned on the package and on the plates It
is around 3 months
4 Modalities of use
after first use
5 Required
equipments No specific equipment is required
6 Reagents Ready to use plates
7 Training 05 day
8 Real time handling
Reference method
9 min for one sample
55 min for 10 samples
Alternative method
7 min for one sample
35 minutes for 10 samples
9 Time to result
Negative results
Reference method Day 2
Alternative method Day 2
Negative results after confirmation or positive results
Reference method Day 4 ndash Day 7
Alternative method Day 4 ndash Day 7
Positive results
Reference method Day 4 ndash Day 7
Alternative method Day 2 ndash Day 3 (with OBIS Mono Test)
or Day 4 ndash Day 7 (by the classical tests described in the
standardised method)
10 Technician skill Identical to that required for the reference method
11 Steps common to
the reference method Mother suspension revivification step and decimal dilutions
12 Traceability
13 Maintenance
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Listeria Precis Enumeration
213 Inter-laboratory study
2131 Organisation
12 laboratories participated to this study Pasteurised milk was inoculated by
Listeria monocytogenes 12a isolated from raw milk The targeted
inoculation levels were
- 0 CFUml
- 50 CFU ml
- 500 CFU ml
- 5 000 CFU ml
Each laboratory received eight flasks of 10 ml sample ie two flasks per
inoculation level Furthermore one non-inoculated sample has been added
to the package for total viable count microflora (NF ISO 4833 method)
Detailed instructions were transmitted to the collaborators by the expert
laboratory
Collaborative study laboratories and the expert laboratory carried out the
analyses with the alternative and reference methods
2132 Verification of experimental parameters
Strain stability during transport
In order to evaluate the Listeria monocytogenes strain stability during
transport bacterial count of all inoculated flasks were checked at different
time ie inoculation time after 24 h of conservation at 2deg - 8degC Results are
reported in table 11
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Summary Report (Version 1)
Listeria Precis Enumeration
Table 11 ndash Listeria monocytogenes count
by ISO 11290-2A1 method (log CFUml)
Day of the analyses Level contamination Reference method
Day 0
(29052006)
1 45
2 440
3 4 500
Day 1
(30052006)
1 60
2 430
3 4 900
No evolution of the strain was observed after storage for 24 h at 2degC ndash 8degC
Contamination level
The real contamination levels are provided in table 12
Table 12 ndash Real contamination level (CFUml)
Target inoculation
(CFU ml) Samples Ndeg
Real contamination levels
(CFUml)
0 1 - 8 0
50 5 - 6 45
500 3 - 7 440
5000 2 - 4 4 500
Sample temperature at receipt
Measured temperatures at receipt are listed in table 13
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Listeria Precis Enumeration
Table 13 - Sample temperature on receipt
Laboratories Receipt
day
Hour
receipt
Measured temperature
at receipt (degC)
Measured temperature by the
temperature probe (degC)
A
B
C
D
E
F
G
H
I
J
K
L
M
30052006
10h45
10h15
11h15
10h00
09h30
8h30
11h50
10h00
08h30
14h45
08h30
09h45
08h45
82
55
45
63
60
62
81
38
48
-
14
67
76
03
03
-13
03
04
-14
05
04
01
05
-13
04
Conclusion
The temperatures during the transport were correct for all the labs except for
Lab J which didnrsquot provide the information
All the labs received their packages with several broken flasks
Three labs (B D and L) received some damaged samples but proceeded to
analyses
All the labs started the analyses at Day 1 except Lab J
2133 Results
Aerobic mesophilic flora
The count of aerobic mesophilic flora varied from 94 102 CFUml to
33 103 CFUml
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Expert lab results
The results obtained by the expert lab are the following
Table 14
Target inoculation level (CFUml)
Reference method ISO 11290-2
Alternative method Listeria Precis Enumeration
Rep 1 Rep 2 Rep 1 Rep 2
lt 10 lt 10 lt 10 lt 10 lt10
50 50 (Ne) 60 (Ne) 50 (Ne) 30 (Ne)
500 470 390 490 500
5 000 4 700 5 100 5 900 3 400
Ne estimated number
The obtained results are in agreement with those expected
Collaborator Labs results
A synthesis of the results obtained by the collaborator labs is provided
Table 15 (CFUml) and Table 16 (log CFUml)
Table 15 ndash Synthesis of results obtained by the alternative
and the reference methods (CFUml)
Labs Level 1 Level 2 Level 2 bis Level 3
Reference method
Alternative method
Reference method
Alternative method
Reference method
Alternative method
Reference method
Alternative method
A 40 40 70 40 480 410 210 340 1000 300 400 300 4900 4600 4900 5100
B 40 15 40 20 350 300 360 450 350 650 800 600 4100 4700 5100 3700
C 50 20 20 70 440 430 480 450 300 650 300 500 4600 5400 4600 5000
D 40 60 40 40 490 460 600 540 250 450 600 1000 4900 4900 4400 4600
E 50 40 50 20 490 520 530 560 750 550 400 200 5400 4500 4100 5400
F 60 20 60 50 370 250 300 130 300 350 600 300 4400 4700 4900 5900
G 70 35 120 50 440 390 570 370 450 550 700 700 5000 4500 4000 4400
H 110 40 100 90 520 440 450 540 150 650 300 300 4200 4700 5600 4800
L 35 60 20 40 390 390 170 380 650 350 800 200 5300 4600 5600 5900
M 45 45 30 40 450 490 550 410 400 550 800 180 4900 4900 6400 4800
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Summary Report (Version 1)
Listeria Precis Enumeration
Table 16 ndash Synthesis of results obtained by the alternative
and the reference methods (log CFUml)
Labs Level 1 Level 2 Level 2 bis Level 3
Reference method
Alternative method
Reference method
Alternative method
Reference method
Alternative method
Reference method
Alternative method
A 1602 1602 1845 1602 2681 2613 2322 2531 3000 2477 2602 2477 3690 3663 3690 3708
B 1602 1176 1602 1301 2544 2477 2556 2653 2544 2813 2903 2778 3613 3672 3708 3568
C 1699 1301 1301 1845 2643 2633 2681 2653 2477 2813 2477 2699 3663 3732 3663 3699
D 1602 1778 1602 1602 2690 2663 2778 2732 2398 2653 2778 3000 3690 3690 3643 3663
E 1699 1602 1699 1301 2690 2716 2724 2748 2875 2740 2602 2301 3732 3653 3613 3732
F 1778 1301 1778 1699 2568 2398 2477 2114 2477 2544 2778 2477 3643 3672 3690 3771
G 1845 1544 2079 1699 2643 2591 2756 2568 2653 2740 2845 2845 3699 3653 3602 3643
H 2041 1602 2000 1954 2716 2643 2653 2732 2176 2813 2477 2477 3623 3672 3748 3681
L 1544 1778 1301 1602 2591 2591 2230 2580 2813 2544 2903 2301 3724 3663 3748 3771
M 1653 1653 1477 1602 2653 2690 2740 2613 2602 2740 2903 2255 3690 3690 3806 3681
Level 2 results obtained taking account the 3 plates inoculated with the mother-suspension
Level 2 bis results obtained taking account the plates inoculated with 01 ml of the mother-suspension
Scrutiny of the measurement results for consistency
In order to identify the measurement results or laboratories that could be
inconsistent two graphical consistency techniques were realised Mandelrsquos
h-ank-statistics Mandel indicators h and k at 5 significance highlighted
some possible inconsistent data (See tables and graphics in Appendix 3 and
Appendix 4)
Mandelrsquos values Number of values observed over the threshold
Reference method Alternative method
h gt 1 Lab F Level 2 Lab F Level 2
h gt 5 Lab F Level 2
Lab B Levels 1 and 2
Lab A Level 2
Lab D Level 3
Lab F Level 2
Lab L Level 2
kgt 1 Lab F Level 3
k gt 5 Lab F Level 2 Lab C Level 2
Lab F Level 3
Lab L Levels 1 and 3
Lab M Level 1
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Listeria Precis Enumeration
The emphasized results appear (i) coherent with the targeted contamination
levels (ii) concordant between both methods
All the results were thus kept for the statistical analysis
Comparison of the trueness and precision characteristics of
the reference method and alternative methods
The statistical values are summarised hereafter
Level p
Reference method Alternative method
Repeatability ratio
Reproducibility ratio Median
Repeatability standard deviation
Reproducibility standard deviation
Median Repeatability
standard deviation
Reproducibility standard deviation
1 10 16519 02415 02425 16021 02045 02433 0847 1007
2 10 26551 00543 00659 26719 00914 01325 1682 2011
2 bis 10 26581 02607 02607 25951 01900 01953 0729 0749
3 10 18671 00378 00378 18671 00478 00611 1264 1617
Bias of the alternative method
In order to estimate the bias of the alternative method with regard to the reference method for every
level Dij and t are calculated according to the following equations
RefAlt ijYijYDij
Diffp
Dijit
)2(
)(median
If the t value is superior to 2 the alternative method is significantly biased
with regard to the reference method
The values are given in table 17
Table 17 ndash t (d) values obtained by level
Level Bias t (d) Conclusion
1 0068 114 Non significant bias
2 0021 082 Non significant bias
2 bis - 0037 035 Non significant bias
3 - 0037 043 Non significant bias
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Listeria Precis Enumeration
The statistical test concludes to a non significant bias for all the inoculation
levels The bias varies from ndash 0037 to 0068 log CFUml
Comparison of the repeatability standard deviations
If the ratio Srj Alt Srj Ref of the repeatability standard deviations of the
alternative method and the reference method is larger than 2 the precision
under repeatability conditions of the alternative method is considered to be
lower than that of the reference method If this ratio is smaller than 05 the
precision under repeatability conditions of the alternative method is
considered to be greater than that of the reference method
The ratio values are given in table 18
Table 18
Level
Repeatability standard deviation
Reference method Alternative method Alternative method
reference method ratio
1 0242 0205 0847
2 0054 0091 1682
2 bis 0261 0190 0729
3 0038 0048 1264
Level Repeatability limit
Reference method Alternative method
1 0676 0573
2 0152 0256
2 bis 0730 0532
3 0106 0134
The ratios of the repeatability standard deviations are below 2 in all cases
The precision under repeatability conditions of the alternative method is
equivalent to that of the reference method
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Listeria Precis Enumeration
Comparison of the reproducibility standard deviations
If the ratio Srj Alt Srj Ref of the reproducibility standard deviations of the alternative method and the
reference method is larger than 2 the precision under reproducibility conditions of the alternative
method is considered to be lower than that of the reference method If this ratio is smaller than 05 the
precision under reproducibility conditions of the alternative method is considered to be greater than
that of the reference method
The ratio values are given in table 19
Table 19
Level
Reproducibility standard deviation
Reference method Alternative method Alternative method
reference method ratio
1 0242 0243 1007
2 0066 0133 2011
2 bis 0261 0195 0749
3 0038 0061 1617
Level Reproducibility limit
Reference method Alternative method
1 0676 0681
2 0185 0371
2 bis 0730 0547
3 0106 0171
The ratios of the reproducibility standard deviations are below or close to 2
The precision under reproducibility conditions of the alternative method is
equivalent to that of the reference method
Thermo Fisher Scientific
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Summary Report (Version 1)
Listeria Precis Enumeration
214 Conclusion
Method comparison study
The Listeria PrecisTM enumeration method shows satisfying linearity and
relative accuracy
The alternative method shows satisfying inclusivity and exclusivity results
Two Listeria monocytogenes strains gave negative results with the
alternative method These strains grew weakly on the reference method
media (OampA Palcam and Oxford)
The limits of repeatability of the alternative method are similar to those of the
reference method
The bias between both methods are low between - 0133 Log CFUg and
- 0008 Log CFUg
The Listeria PrecisTM enumeration method offers the saving for confirmatory
tests
Positive results are obtained with the alternative method in 2-3 days (with
OBIS Mono test) or in 4-7 days (by the classical tests described in the
standardized method) against 4-7 days for the reference method
Inter-laboratory study
The bias between the reference and the alternative methods shows satisfying
and correct values
- level 1 (100 - 1 000 CFUg) bias = + 007 log CFUg
- level 2 (1 000 - 10 000 CFUg) bias = 002 log CFUg
- level 2 bis (1 000 - 10 000 CFUg) bias = - 004 log CFUg
- level 3 (10 000 - 100 000 CFUg) bias = - 004 log CFUg
The repeatability and the reproducibility of the alternative method look similar
to those of the reference method
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Summary Report (Version 1)
Listeria Precis Enumeration
22 Extension study (realised by ASEPT 2007)
An extension study was performed in 2007 for a new confirmatory test OBIS
MONO
150 Listeria monocytogenes strains and 100 non-target strains were isolated
onto TSAYE and BrillianceTM Listeria Agar plates All the Listeria
monocytogenes and the non-target strains gave the expected results
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ADRIA Deacuteveloppement 3138 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Appendix 1 ndash Flow diagram of the Listeria PrecisTM method
x g sample + 9 x ml buffered peptone water
Revivification step for 1 h plusmn 5 min at 20degC plusmn 1degC
Streaking 1 ml onto 3 Brilliance Listeria Agar plates (mother suspension)
or 01 ml onto 1 Brilliance Listeria Agar plates (mother suspension and decimal dilutions)
Incubation for 48 h plusmn 3 h at 37degC plusmn 1degC
Confirmatory tests on at least one typical colony
- by the tests described in the ISO method
- by applying the OBIS mono test on an isolated colony
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3238 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Appendix 2 ndash ISO 11290-2 (1998) amp ISO 11290-2A1 (2004) Horizontal
method for the detection and enumeration of Listeria monocytogenes -
Part 2 enumeration of Listeria monocytogenes in foods
10 g cheese + 90 ml buffered peptone water or Half Fraser Broth (without supplements) (110 dilution)
Stomach 1 min 30 seconds
Keep at 20degC plusmn 2degC for 1 h plusmn 5 min
Decimal dilutions in 9 ml peptone salt tubes
Dilution 110
Dilution 1100
Dilution 11000
1 ml on 3 Chromogenic Listeria medium (ISO) plates (x 2) (-1)
01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-2)
01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-3)
01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-4)
Incubation at 37degC plusmn 1degC
24 h - 48 h
Numerate typical colonies at 24 h
(bluegreen surrounded by an opaque halo)
Numerate at 48 h
Streak typical colonies on TSYEA
(5 coloniesplate 2 successive dilutions)
Confirmation tests
Catalase
Gram stain
For confirmation of L monocytogenes
Haemolysis
Carbohydrate (rhamnose and xylose) utilisation
CAMP Test
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3338 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Appendix 3 ndash Mandelrsquos graphics
Laboratoire h Ref
Niveau 1 h Ref
Niveau 2 h Ref
Niveau 3 h Alt
Niveau 1 h Alt
Niveau 2 h Alt
Niveau 3 h5 h1 h5 h1
A -050 -015 083 062 -212 -039 198 299 -198 -299
B -261 -270 021 -077 -058 173 198 299 -198 -299
C -151 -031 -014 -015 -004 -005 198 299 -198 -299
D 038 040 -137 000 072 207 198 299 -198 -299
E -001 090 155 -052 056 -101 198 299 -198 -299
F -112 -321 -153 070 -325 023 198 299 -198 -299
G 042 -071 040 147 -009 176 198 299 -198 -299
H 169 046 -169 192 018 -083 198 299 -198 -299
L 009 -120 021 -077 -231 005 198 299 -198 -299
M 001 031 014 -032 004 -011 198 299 -198 -299
-300
-200
-100
000
100
200
300
A B C D E F G H L M
Vale
urs
h d
e M
an
del(
rob
uste
s)
Laboratoires
Meacutethode de reacutefeacuterence analyse robuste
h Ref Niveau 1
h Ref Niveau 2
h Ref Niveau 3
h 5=198
h 1=299
-300
-200
-100
000
100
200
300
A B C D E F G H L M
Vale
urs
de h
de M
an
del(
rob
uste
s)
Laboratoires
Meacutethode alternative analyse robuste
h Alt Niveau 1
h Alt Niveau 2
h Alt Niveau 2
h 1=299
h 5=198
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3438 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Laboratoire k Ref
Niveau 1 k Ref
Niveau 2 k Ref
Niveau 3 k Alt
Niveau 1 k Alt
Niveau 2 k Alt
Niveau 3 k1 k5
A 000 089 142 084 162 046 259 181
B 125 087 073 104 075 046 259 181
C 117 013 091 188 022 083 259 181
D 052 036 069 000 035 083 259 181
E 028 034 037 138 019 112 259 181
F 140 222 018 027 281 112 259 181
G 088 068 024 131 145 000 259 181
H 129 094 173 016 061 000 259 181
L 069 000 073 104 270 224 259 181
M 000 048 038 043 099 241 259 181
000
050
100
150
200
250
300
350
400
A B C D E F G H L M
Vale
urs
k d
e M
an
del(
rob
uste
s)
Laboratoires
Meacutethode de reacutefeacuterence analyse robuste
k Ref Niveau 1k Ref Niveau 2k Ref Niveau 3
k 1=259
k 5=181
000
050
100
150
200
250
300
A B C D E F G H L M
Vale
urs
k d
e M
an
del
(ro
bu
ste
s)
Laboratoires
Meacutethode alternative analyse robuste
k Alt Niveau 3
k Alt Niveau 1
k Alt Niveau 2
k 1=259
k 5=181
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3538 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Appendix 4 ndash Statistical results
Level 1
Nombre de laboratoires (p) 10
A 1602 1602 1602 000 0000 0000 -0495 0000 A 1845 1602 1724 017 0122 -0122 0621 0840 0122
B 1602 1176 1389 030 0213 -0213 -2613 1247 B 1602 1301 1452 021 0151 -0151 -0769 1041 0062
C 1699 1301 1500 028 0199 -0199 -1510 1165 C 1301 1845 1573 038 -0272 0272 -0148 1881 0073
D 1602 1778 1690 012 -0088 0088 0380 0516 D 1602 1602 1602 000 0000 0000 0000 0000 -0088
E 1699 1602 1651 007 0048 -0048 -0013 0284 E 1699 1301 1500 028 0199 -0199 -0522 1376 -0151
F 1778 1301 1540 034 0239 -0239 -1116 1397 F 1778 1699 1739 006 0040 -0040 0698 0274 0199
G 1845 1544 1695 021 0151 -0151 0425 0881 G 2079 1699 1889 027 0190 -0190 1467 1315 0194
H 2041 1602 1822 031 0220 -0220 1689 1286 H 2000 1954 1977 003 0023 -0023 1917 0158 0155
L 1544 1778 1661 017 -0117 0117 0092 0685 L 1301 1602 1452 021 -0151 0151 -0769 1041 -0210
M 1653 1653 1653 000 0000 0000 0013 0000 M 1477 1602 1540 009 -0062 0062 -0319 0432 -0114
m 165186 m 160206 mediane D 00678
Cn(2p) 1867 Cn(2p) 1867 Cp 16101
Cn(p) 16101 Cn(p) 16101 Qn(D) 0092926
n=2p n 20 n=2p n 20 Qdiff 01496169
f 11 f 11 t 114
l 55 l 55
n=p n 10 n=p n 10
f 6 f 6
l 15 l 15
Qn(2p) 0091465 Qn(2p) 007745
Qn(p) 0062469 Qn(p) 0121519
Qintra 017078 Qintra 014461
Qinter 010058 Qinter 019565
Sr(eacutecart-type de reacutepeacutetabiliteacute) 02415 Sr 02045
RSDr(coefficient de variation de la reacutepeacutetabiliteacute)1462 RSDr 1277
r(repeatability limit)) 0676 r 0573
SL(eacutecart-type interlaboratoire) -001905 SLlt0 SL 001737
SR(eacutecart-type de reproductibiliteacute) 02415 SR 02433
RSDR(coefficient de variation de la reproductibiliteacute)0146 RSDR 0152
R(limite de reproductibiliteacute)) 06762 R(limite de reproductibiliteacute))068123
h k h MoyenneLaboratoires
Niveau 1
si LaboratoiresDeviationMoyenne Dk DeviationsiAlt
Niveau 1
Ref
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3638 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Level 2
Nombre de laboratoires (p) 10
A 2681 2613 2647 005 0034 -0034 -0151 0891 A 2322 2531 2427 015 -0105 0105 -2119 1620 -0220
B 2544 2477 2511 005 0033 -0033 -2699 0872 B 2556 2653 2605 007 -0048 0048 -0581 0750 0094
C 2643 2633 2638 001 0005 -0005 -0311 0130 C 2681 2653 2667 002 0014 -0014 -0040 0217 0029
D 2690 2663 2676 002 0014 -0014 0400 0357 D 2778 2732 2755 003 0023 -0023 0721 0354 0079
E 2690 2716 2703 002 -0013 0013 0897 0336 E 2724 2748 2736 002 -0012 0012 0556 0185 0033
F 2568 2398 2483 012 0085 -0085 -3214 2217 F 2477 2114 2296 026 0182 -0182 -3255 2811 -0188
G 2643 2591 2617 004 0026 -0026 -0707 0682 G 2756 2568 2662 013 0094 -0094 -0085 1453 0045
H 2716 2643 2680 005 0036 -0036 0460 0945 H 2653 2732 2693 006 -0040 0040 0181 0613 0013
L 2591 2591 2591 000 0000 0000 -1196 0000 L 2230 2580 2405 025 -0175 0175 -2307 2704 -0186
M 2653 2690 2672 003 -0018 0018 0311 0481 M 2740 2613 2677 009 0064 -0064 0040 0987 0005
m 2655082 m 26719001 mediane D 00209
Cn(2p) 1867 Cn(2p) 1867 Cp 16101
Cn(p) 16101 Cn(p) 16101 Qn(D) 0039911
n=2p n 20 n=2p n 20 Qdiff 00642591
f 11 f 11 t 082
l 55 l 55
n=p n 10 n=p n 10
f 6 f 6
l 15 l 15
Qn(2p) 0020570 Qn(2p) 00345985
Qn(p) 00332437 Qn(p) 00718158
Qintra 003841 Qintra 006460
Qinter 005352 Qinter 011563
Sr(eacutecart-type de reacutepeacutetabiliteacute) 00543 Sr 00914
RSDr(coefficient de variation de la reacutepeacutetabiliteacute)205 RSDr 342
r(repeatability limit)) 0152 r 0256
SL(eacutecart-type interlaboratoire) 000139 SL 000920
SR(eacutecart-type de reproductibiliteacute) 00659 SR 01325
RSDR(coefficient de variation de la reproductibiliteacute)0025 RSDR 0050
R(limite de reproductibiliteacute)) 018446 R(limite de reproductibiliteacute))03708618
Laboratoires
Niveau 2
Moyenne si Laboratoires
Niveau 2
DRef Alt Moyenne si Deviation h Deviation h k k
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3738 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Level 2 bis
Nombre de laboratoires (p) 10
A 3000 2477 2739 037 0261 -0261 0832 1418 A 2602 2477 2540 009 0062 -0062 -0391 0465 -0199
B 2544 2813 2678 019 -0134 0134 0211 0729 B 2903 2778 2841 009 0062 -0062 1732 0465 0162
C 2477 2813 2645 024 -0168 0168 -0135 0911 C 2477 2699 2588 016 -0111 0111 -0049 0826 -0057
D 2398 2653 2526 018 -0128 0128 -1370 0692 D 2778 3000 2889 016 -0111 0111 2074 0826 0363
E 2875 2740 2808 010 0067 -0067 1547 0365 E 2602 2301 2452 021 0151 -0151 -1012 1120 -0356
F 2477 2544 2511 005 -0033 0033 -1525 0182 F 2778 2477 2628 021 0151 -0151 0230 1120 0117
G 2653 2740 2697 006 -0044 0044 0400 0236 G 2845 2845 2845 000 0000 0000 1764 0000 0148
H 2176 2813 2495 045 -0318 0318 -1692 1727 H 2477 2477 2477 000 0000 0000 -0832 0000 -0017
L 2813 2544 2678 019 0134 -0134 0211 0729 L 2903 2301 2602 043 0301 -0301 0049 2241 -0076
M 2602 2740 2671 010 -0069 0069 0135 0375 M 2903 2255 2579 046 0324 -0324 -0112 2411 -0092
m 2658114 m 259505 mediane D -00372
Cn(2p) 1867 Cn(2p) 1867 Cp 16101
Cn(p) 16101 Cn(p) 16101 Qn(D) 01656915
n=2p n 20 n=2p n 20 Qdiff 02667753
f 11 f 11 t 035
l 55 l 55
n=p n 10 n=p n 10
f 6 f 6
l 15 l 15
Qn(2p) 0098745 Qn(2p) 007195
Qn(p) 006007 Qn(p) 008805
Qintra 018437 Qintra 013435
Qinter 009672 Qinter 014176
Sr(eacutecart-type de reacutepeacutetabiliteacute) 02607 Sr 01900
RSDr(coefficient de variation de la reacutepeacutetabiliteacute)981 RSDr 732
r(repeatability limit)) 0730 r 0532
SL(eacutecart-type interlaboratoire) -002464 SLlt0 SL 000205
SR(eacutecart-type de reproductibiliteacute) 02607 SR 01953
RSDR(coefficient de variation de la reproductibiliteacute)0098 RSDR 0075
R(limite de reproductibiliteacute)) 072996 R(limite de reproductibiliteacute))054686
Laboratoires
Niveau 2 bis
Moyenne si Laboratoires
Niveau 2 bis
DRef Alt Moyenne si Deviation h Deviation h k k
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3838 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Level 3
Nombre de laboratoires (p) 10
A 3690 3663 3676 002 0014 -0014 -0311 0513 A 3690 3708 3699 001 -0009 0009 0354 0257 0022
B 3613 3672 3642 004 -0030 0030 -1851 1109 B 3708 3568 3638 010 0070 -0070 -0844 2061 -0005
C 3663 3732 3698 005 -0035 0035 0645 1302 C 3663 3699 3681 003 -0018 0018 0000 0536 -0017
D 3690 3690 3690 000 0000 0000 0311 0000 D 3643 3663 3653 001 -0010 0010 -0545 0286 -0037
E 3732 3653 3693 006 0040 -0040 0429 1480 E 3613 3732 3673 008 -0060 0060 -0162 1769 -0020
F 3643 3672 3658 002 -0014 0014 -1157 0536 F 3690 3771 3731 006 -0040 0040 0975 1193 0073
G 3699 3653 3676 003 0023 -0023 -0328 0856 G 3602 3643 3623 003 -0021 0021 -1141 0612 -0053
H 3623 3672 3648 003 -0024 0024 -1615 0913 H 3748 3681 3715 005 0033 -0033 0664 0990 0067
L 3724 3663 3694 004 0031 -0031 0461 1150 L 3748 3771 3760 002 -0011 0011 1544 0335 0066
M 3690 3690 3690 000 0000 0000 0311 0000 M 3806 3681 3744 009 0062 -0062 1234 1848 0054
m 368334 m 368086 mediane D 00089
Cn(2p) 1867 Cn(2p) 1867 Cp 16101
Cn(p) 16101 Cn(p) 16101 Qn(D) 00325359
n=2p n 20 n=2p n 20 Qdiff 00523852
f 11 f 11 t 043
l 55 l 55
n=p n 10 n=p n 10
f 6 f 6
l 15 l 15
Qn(2p) 0014323 Qn(2p) 001811
Qn(p) 0013719 Qn(p) 003164
Qintra 002674 Qintra 003381
Qinter 002209 Qinter 005094
Sr(eacutecart-type de reacutepeacutetabiliteacute) 00378 Sr 00478
RSDr(coefficient de variation de la reacutepeacutetabiliteacute)103 RSDr 130
r(repeatability limit)) 0106 r 0134
SL(eacutecart-type interlaboratoire) -000023 SLlt0 SL 000145
SR(eacutecart-type de reproductibiliteacute) 00378 SR 00611
RSDR(coefficient de variation de la reproductibiliteacute)0010 RSDR 0017
R(limite de reproductibiliteacute)) 0105894 R(limite de reproductibiliteacute))017119
Ref Alt DLaboratoires
Niveau 3
Moyenne si Deviation h k Laboratoires
Niveau 3
Moyenne si Deviation h k
Thermo Fisher Scientific
ADRIA Deacuteveloppement 1138 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Table 5 ndash Contamination range
Food category Contamination level (log CFUg)
Meat products 100 ndash 404
Dairy products 148 ndash 408
Fishery products 100 ndash 384
Vegetables 170 ndash 511
Egg products 130 ndash 499
Environmental samples 170 ndash 511
All categories 100 ndash 511
Bi-dimensional graphs are given figure 3
Figure 3 ndash Bi-dimensional graphs
Graphe bi-dimensionnel Produits carneacutes
Fishery products
Vegetables
Egg products
Meat products
Thermo Fisher Scientific
ADRIA Deacuteveloppement 1238 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Statistical interpretation according to EN ISO16140 method
The results of the statistical interpretation are given in Table 6
Table 6 ndash Statistical interpretation results
Category n R Regression
used a t(a) b t(b) Critical T
P
Ordinate
at 0
Slope
at 1
Meat products 10 158 GMFR -0455 3321 115 3123 2306 001 001
Dairy products 12 065 GMFR -0027 0385 1002 0108 2228 092 071
Fishery products 10 130 GMFR -0305 1024 1034 0312 2306 076 034
Vegetables 12 086 GMFR -0015 0128 0993 0191 2228 085 090
Egg products 11 158 GMFR -0038 0866 1001 0090 2262 093 041
Environmental
samples 10 176 GMFR -0139 265 1034 231 2306 005 003
All products 65 118 GMFR -0182 3042 1040 2145 2000 004 000
All products
(estimated number) 65 118 GMFR -0182 3042 1040 2145 2000 004 000
All products (non
estimated results) 20 115 GMFR -0367 151 1138 1141 2101 027 015
Repeatability limits obtained for the alternative and reference methods as
well as bias obtained are presented in Table 7
Dairy products Environmental samples
Log (Ref) Log (Ref)
All categories
Thermo Fisher Scientific
ADRIA Deacuteveloppement 1338 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Table 7
Categories Bias
Repeatability limits
Rob r
Ref Alt
Meat products 0008 0314 0343
Dairy products -0031 0136 0130
Fishery products -0133 0220 0214
Vegetables -0041 0180 0319
Egg products -0026 0219 0206
Environmental samples -0007 0198 0119
All products -0032 0196 0215
Regression straight lines (graph and equation representations) for each food
category and for all products are presented figure 4
Thermo Fisher Scientific
ADRIA Deacuteveloppement 1438 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Figure 4 ndash Regression straight lines
Meat products
Fishery products
Dairy products
Vegetables
Thermo Fisher Scientific
ADRIA Deacuteveloppement 1538 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Environmental samples
Egg products
All products (estimated number)
Thermo Fisher Scientific
ADRIA Deacuteveloppement 1638 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
The hypotheses that the ordinates are close to 0 and the slopes close to 1
are approved in all cases except for
- Meat products (slope P = 1 ordinate P = 1)
- Environmental samples (ordinate P = 3)
- All products (ordinate P = 0)
The bias between the two methods vary from ndash 0133 to 0008 log CFU For
all categories the bias is ndash 0032 log CFU The repeatability limits of the
alternative and the reference method are similar For all products the values
are respectively 0215 and 0196 log CFU For all products these
interpretations are provided taking into account data with low contamination
level or not (estimated colonies number)
For all food products the regression straight line is the following
log (Alt) = 1040 log Ref ndash 0182
The Listeria PrecisTM method shows satisfying relative accuracy
All products
All products (Level 2)
Thermo Fisher Scientific
ADRIA Deacuteveloppement 1738 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
2113 Detection limit (LOD) and quantification limit (LOQ)
The critical level is the defined as the smallest amount which can be detected (not null) but not
quantified as an exact value Below this value it cannot be sure that the true value is not null
The detection limit is defined as being higher than the critical level because it involves a power the
probability 1- which has to be well over 50 for example 95
The quantification limit is defined as the smallest amount of analyte (that is the lowest actual number of
organisms) which can be measured and quantified with defined precision and accuracy under the
experimental conditions by the method under validation
Protocol
The detection limit of the alternative method was realised with pure cultures
Three different inoculation levels were tested with six replicates per level ie
18 analyses were done by the alternative method
Listeria monocytogenes 18 was used for the detection limit of the alternative
method
Quantification limit was calculated for six independent blank samples
determinations
Results
These data are intrinsic to the method used and are presented in the
following tables
Table 8 ndash Inoculation 1 ml on 3 plates
Level (CFUml) Positive samples number Standard deviation Bias
0 06
05 26 1211 0
1 36 0816 05
5 66 2483 85
Thermo Fisher Scientific
ADRIA Deacuteveloppement 1838 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Table 9 ndash Inoculation 01 ml on 1 plate
Level (CFUml) Positive samples number Standard deviation Bias
0 06
5 36 0548 05
10 46 1414 2
50 66 2258 55
Table 10
Formulas Obtained values
LC 165 S0 + X0 18
LOD 33 S0 + X0 32
LOQ 10 S0 + X0 87
2114 Relative Sensitivity
The relative sensitivity is defined as the ability of the alternative method to detect two different amounts
of analyte measured by the reference method within a given matrix at a specified average value or
over the whole measurement range that is it is the minimal quantity variation (increase of the analyte
concentration x) which gives a significant variation of the measured signal (response y)
These data are intrinsic to the used method and are obtained from the results
of the linearity study
Sensitivity patterns obtained for tested (matrixstrain) pairs are presented in
figure 5
Thermo Fisher Scientific
ADRIA Deacuteveloppement 1938 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Figure 5 ndash Sensitivity patterns for the tested (matrixstrain) pairs
2115 SpecificitySelectivity
The specificity is defined as the degree to which a method is affected (or not) by the other components
present in a multi-component sample That is the ability of a method to measure exactly a given
analyte or its amount within the sample without interference from non-target components such as a
matrix effect or background noise
The selectivity is defined as a measure of the degree of non-interference in the presence of non-target
analytes A method is selective if it can be used to detect the analyte under examination and that a
guarantee can be provided that the detected signal can only be a product by that specific analyte
The specificity and selectivity study was performed for the Listeria PrecisTM
detection method no additional test was realised for the enumeration
method The results were the following
- Among the Listeria monocytogenes strains tested 2 strains (Listeria
monocytogenes CIP 7834 and Listeria monocytogenes CIP 105459) gave
negative results These 2 strains were tested twice by running 48 h
incubation time of the compared agars Positive results were then
observed by the alternative method Pale characteristic colonies were
observed on the ISO agar by streaking the Half Fraser broth only
- No cross reaction was observed with the non-target strains except with
Listeria ivanovii which gave characteristic colonies on Brilliancetrade Listeria
Agar but the confirmatory tests provide a clear discrimination between
Listeria monocytogenes and Listeria ivanovi species
Rillettes
Lettuce
Raw milk
Smoked salmon
Raw egg
Process water
x (CFUg)
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2038 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
212 Practicability
1 Packaging Ready to use Brilliance Listeria agar plates are available
(x 10 plates)
2 Reagent volume
3 Storage conditionsndash
Shelf life of products
not opened
The plates are stored at 6 - 12degC in darkness conditions The
expiration date is mentioned on the package and on the plates It
is around 3 months
4 Modalities of use
after first use
5 Required
equipments No specific equipment is required
6 Reagents Ready to use plates
7 Training 05 day
8 Real time handling
Reference method
9 min for one sample
55 min for 10 samples
Alternative method
7 min for one sample
35 minutes for 10 samples
9 Time to result
Negative results
Reference method Day 2
Alternative method Day 2
Negative results after confirmation or positive results
Reference method Day 4 ndash Day 7
Alternative method Day 4 ndash Day 7
Positive results
Reference method Day 4 ndash Day 7
Alternative method Day 2 ndash Day 3 (with OBIS Mono Test)
or Day 4 ndash Day 7 (by the classical tests described in the
standardised method)
10 Technician skill Identical to that required for the reference method
11 Steps common to
the reference method Mother suspension revivification step and decimal dilutions
12 Traceability
13 Maintenance
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2138 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
213 Inter-laboratory study
2131 Organisation
12 laboratories participated to this study Pasteurised milk was inoculated by
Listeria monocytogenes 12a isolated from raw milk The targeted
inoculation levels were
- 0 CFUml
- 50 CFU ml
- 500 CFU ml
- 5 000 CFU ml
Each laboratory received eight flasks of 10 ml sample ie two flasks per
inoculation level Furthermore one non-inoculated sample has been added
to the package for total viable count microflora (NF ISO 4833 method)
Detailed instructions were transmitted to the collaborators by the expert
laboratory
Collaborative study laboratories and the expert laboratory carried out the
analyses with the alternative and reference methods
2132 Verification of experimental parameters
Strain stability during transport
In order to evaluate the Listeria monocytogenes strain stability during
transport bacterial count of all inoculated flasks were checked at different
time ie inoculation time after 24 h of conservation at 2deg - 8degC Results are
reported in table 11
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2238 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Table 11 ndash Listeria monocytogenes count
by ISO 11290-2A1 method (log CFUml)
Day of the analyses Level contamination Reference method
Day 0
(29052006)
1 45
2 440
3 4 500
Day 1
(30052006)
1 60
2 430
3 4 900
No evolution of the strain was observed after storage for 24 h at 2degC ndash 8degC
Contamination level
The real contamination levels are provided in table 12
Table 12 ndash Real contamination level (CFUml)
Target inoculation
(CFU ml) Samples Ndeg
Real contamination levels
(CFUml)
0 1 - 8 0
50 5 - 6 45
500 3 - 7 440
5000 2 - 4 4 500
Sample temperature at receipt
Measured temperatures at receipt are listed in table 13
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2338 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Table 13 - Sample temperature on receipt
Laboratories Receipt
day
Hour
receipt
Measured temperature
at receipt (degC)
Measured temperature by the
temperature probe (degC)
A
B
C
D
E
F
G
H
I
J
K
L
M
30052006
10h45
10h15
11h15
10h00
09h30
8h30
11h50
10h00
08h30
14h45
08h30
09h45
08h45
82
55
45
63
60
62
81
38
48
-
14
67
76
03
03
-13
03
04
-14
05
04
01
05
-13
04
Conclusion
The temperatures during the transport were correct for all the labs except for
Lab J which didnrsquot provide the information
All the labs received their packages with several broken flasks
Three labs (B D and L) received some damaged samples but proceeded to
analyses
All the labs started the analyses at Day 1 except Lab J
2133 Results
Aerobic mesophilic flora
The count of aerobic mesophilic flora varied from 94 102 CFUml to
33 103 CFUml
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2438 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Expert lab results
The results obtained by the expert lab are the following
Table 14
Target inoculation level (CFUml)
Reference method ISO 11290-2
Alternative method Listeria Precis Enumeration
Rep 1 Rep 2 Rep 1 Rep 2
lt 10 lt 10 lt 10 lt 10 lt10
50 50 (Ne) 60 (Ne) 50 (Ne) 30 (Ne)
500 470 390 490 500
5 000 4 700 5 100 5 900 3 400
Ne estimated number
The obtained results are in agreement with those expected
Collaborator Labs results
A synthesis of the results obtained by the collaborator labs is provided
Table 15 (CFUml) and Table 16 (log CFUml)
Table 15 ndash Synthesis of results obtained by the alternative
and the reference methods (CFUml)
Labs Level 1 Level 2 Level 2 bis Level 3
Reference method
Alternative method
Reference method
Alternative method
Reference method
Alternative method
Reference method
Alternative method
A 40 40 70 40 480 410 210 340 1000 300 400 300 4900 4600 4900 5100
B 40 15 40 20 350 300 360 450 350 650 800 600 4100 4700 5100 3700
C 50 20 20 70 440 430 480 450 300 650 300 500 4600 5400 4600 5000
D 40 60 40 40 490 460 600 540 250 450 600 1000 4900 4900 4400 4600
E 50 40 50 20 490 520 530 560 750 550 400 200 5400 4500 4100 5400
F 60 20 60 50 370 250 300 130 300 350 600 300 4400 4700 4900 5900
G 70 35 120 50 440 390 570 370 450 550 700 700 5000 4500 4000 4400
H 110 40 100 90 520 440 450 540 150 650 300 300 4200 4700 5600 4800
L 35 60 20 40 390 390 170 380 650 350 800 200 5300 4600 5600 5900
M 45 45 30 40 450 490 550 410 400 550 800 180 4900 4900 6400 4800
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2538 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Table 16 ndash Synthesis of results obtained by the alternative
and the reference methods (log CFUml)
Labs Level 1 Level 2 Level 2 bis Level 3
Reference method
Alternative method
Reference method
Alternative method
Reference method
Alternative method
Reference method
Alternative method
A 1602 1602 1845 1602 2681 2613 2322 2531 3000 2477 2602 2477 3690 3663 3690 3708
B 1602 1176 1602 1301 2544 2477 2556 2653 2544 2813 2903 2778 3613 3672 3708 3568
C 1699 1301 1301 1845 2643 2633 2681 2653 2477 2813 2477 2699 3663 3732 3663 3699
D 1602 1778 1602 1602 2690 2663 2778 2732 2398 2653 2778 3000 3690 3690 3643 3663
E 1699 1602 1699 1301 2690 2716 2724 2748 2875 2740 2602 2301 3732 3653 3613 3732
F 1778 1301 1778 1699 2568 2398 2477 2114 2477 2544 2778 2477 3643 3672 3690 3771
G 1845 1544 2079 1699 2643 2591 2756 2568 2653 2740 2845 2845 3699 3653 3602 3643
H 2041 1602 2000 1954 2716 2643 2653 2732 2176 2813 2477 2477 3623 3672 3748 3681
L 1544 1778 1301 1602 2591 2591 2230 2580 2813 2544 2903 2301 3724 3663 3748 3771
M 1653 1653 1477 1602 2653 2690 2740 2613 2602 2740 2903 2255 3690 3690 3806 3681
Level 2 results obtained taking account the 3 plates inoculated with the mother-suspension
Level 2 bis results obtained taking account the plates inoculated with 01 ml of the mother-suspension
Scrutiny of the measurement results for consistency
In order to identify the measurement results or laboratories that could be
inconsistent two graphical consistency techniques were realised Mandelrsquos
h-ank-statistics Mandel indicators h and k at 5 significance highlighted
some possible inconsistent data (See tables and graphics in Appendix 3 and
Appendix 4)
Mandelrsquos values Number of values observed over the threshold
Reference method Alternative method
h gt 1 Lab F Level 2 Lab F Level 2
h gt 5 Lab F Level 2
Lab B Levels 1 and 2
Lab A Level 2
Lab D Level 3
Lab F Level 2
Lab L Level 2
kgt 1 Lab F Level 3
k gt 5 Lab F Level 2 Lab C Level 2
Lab F Level 3
Lab L Levels 1 and 3
Lab M Level 1
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2638 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
The emphasized results appear (i) coherent with the targeted contamination
levels (ii) concordant between both methods
All the results were thus kept for the statistical analysis
Comparison of the trueness and precision characteristics of
the reference method and alternative methods
The statistical values are summarised hereafter
Level p
Reference method Alternative method
Repeatability ratio
Reproducibility ratio Median
Repeatability standard deviation
Reproducibility standard deviation
Median Repeatability
standard deviation
Reproducibility standard deviation
1 10 16519 02415 02425 16021 02045 02433 0847 1007
2 10 26551 00543 00659 26719 00914 01325 1682 2011
2 bis 10 26581 02607 02607 25951 01900 01953 0729 0749
3 10 18671 00378 00378 18671 00478 00611 1264 1617
Bias of the alternative method
In order to estimate the bias of the alternative method with regard to the reference method for every
level Dij and t are calculated according to the following equations
RefAlt ijYijYDij
Diffp
Dijit
)2(
)(median
If the t value is superior to 2 the alternative method is significantly biased
with regard to the reference method
The values are given in table 17
Table 17 ndash t (d) values obtained by level
Level Bias t (d) Conclusion
1 0068 114 Non significant bias
2 0021 082 Non significant bias
2 bis - 0037 035 Non significant bias
3 - 0037 043 Non significant bias
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2738 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
The statistical test concludes to a non significant bias for all the inoculation
levels The bias varies from ndash 0037 to 0068 log CFUml
Comparison of the repeatability standard deviations
If the ratio Srj Alt Srj Ref of the repeatability standard deviations of the
alternative method and the reference method is larger than 2 the precision
under repeatability conditions of the alternative method is considered to be
lower than that of the reference method If this ratio is smaller than 05 the
precision under repeatability conditions of the alternative method is
considered to be greater than that of the reference method
The ratio values are given in table 18
Table 18
Level
Repeatability standard deviation
Reference method Alternative method Alternative method
reference method ratio
1 0242 0205 0847
2 0054 0091 1682
2 bis 0261 0190 0729
3 0038 0048 1264
Level Repeatability limit
Reference method Alternative method
1 0676 0573
2 0152 0256
2 bis 0730 0532
3 0106 0134
The ratios of the repeatability standard deviations are below 2 in all cases
The precision under repeatability conditions of the alternative method is
equivalent to that of the reference method
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2838 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Comparison of the reproducibility standard deviations
If the ratio Srj Alt Srj Ref of the reproducibility standard deviations of the alternative method and the
reference method is larger than 2 the precision under reproducibility conditions of the alternative
method is considered to be lower than that of the reference method If this ratio is smaller than 05 the
precision under reproducibility conditions of the alternative method is considered to be greater than
that of the reference method
The ratio values are given in table 19
Table 19
Level
Reproducibility standard deviation
Reference method Alternative method Alternative method
reference method ratio
1 0242 0243 1007
2 0066 0133 2011
2 bis 0261 0195 0749
3 0038 0061 1617
Level Reproducibility limit
Reference method Alternative method
1 0676 0681
2 0185 0371
2 bis 0730 0547
3 0106 0171
The ratios of the reproducibility standard deviations are below or close to 2
The precision under reproducibility conditions of the alternative method is
equivalent to that of the reference method
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2938 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
214 Conclusion
Method comparison study
The Listeria PrecisTM enumeration method shows satisfying linearity and
relative accuracy
The alternative method shows satisfying inclusivity and exclusivity results
Two Listeria monocytogenes strains gave negative results with the
alternative method These strains grew weakly on the reference method
media (OampA Palcam and Oxford)
The limits of repeatability of the alternative method are similar to those of the
reference method
The bias between both methods are low between - 0133 Log CFUg and
- 0008 Log CFUg
The Listeria PrecisTM enumeration method offers the saving for confirmatory
tests
Positive results are obtained with the alternative method in 2-3 days (with
OBIS Mono test) or in 4-7 days (by the classical tests described in the
standardized method) against 4-7 days for the reference method
Inter-laboratory study
The bias between the reference and the alternative methods shows satisfying
and correct values
- level 1 (100 - 1 000 CFUg) bias = + 007 log CFUg
- level 2 (1 000 - 10 000 CFUg) bias = 002 log CFUg
- level 2 bis (1 000 - 10 000 CFUg) bias = - 004 log CFUg
- level 3 (10 000 - 100 000 CFUg) bias = - 004 log CFUg
The repeatability and the reproducibility of the alternative method look similar
to those of the reference method
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3038 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
22 Extension study (realised by ASEPT 2007)
An extension study was performed in 2007 for a new confirmatory test OBIS
MONO
150 Listeria monocytogenes strains and 100 non-target strains were isolated
onto TSAYE and BrillianceTM Listeria Agar plates All the Listeria
monocytogenes and the non-target strains gave the expected results
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3138 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Appendix 1 ndash Flow diagram of the Listeria PrecisTM method
x g sample + 9 x ml buffered peptone water
Revivification step for 1 h plusmn 5 min at 20degC plusmn 1degC
Streaking 1 ml onto 3 Brilliance Listeria Agar plates (mother suspension)
or 01 ml onto 1 Brilliance Listeria Agar plates (mother suspension and decimal dilutions)
Incubation for 48 h plusmn 3 h at 37degC plusmn 1degC
Confirmatory tests on at least one typical colony
- by the tests described in the ISO method
- by applying the OBIS mono test on an isolated colony
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3238 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Appendix 2 ndash ISO 11290-2 (1998) amp ISO 11290-2A1 (2004) Horizontal
method for the detection and enumeration of Listeria monocytogenes -
Part 2 enumeration of Listeria monocytogenes in foods
10 g cheese + 90 ml buffered peptone water or Half Fraser Broth (without supplements) (110 dilution)
Stomach 1 min 30 seconds
Keep at 20degC plusmn 2degC for 1 h plusmn 5 min
Decimal dilutions in 9 ml peptone salt tubes
Dilution 110
Dilution 1100
Dilution 11000
1 ml on 3 Chromogenic Listeria medium (ISO) plates (x 2) (-1)
01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-2)
01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-3)
01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-4)
Incubation at 37degC plusmn 1degC
24 h - 48 h
Numerate typical colonies at 24 h
(bluegreen surrounded by an opaque halo)
Numerate at 48 h
Streak typical colonies on TSYEA
(5 coloniesplate 2 successive dilutions)
Confirmation tests
Catalase
Gram stain
For confirmation of L monocytogenes
Haemolysis
Carbohydrate (rhamnose and xylose) utilisation
CAMP Test
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3338 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Appendix 3 ndash Mandelrsquos graphics
Laboratoire h Ref
Niveau 1 h Ref
Niveau 2 h Ref
Niveau 3 h Alt
Niveau 1 h Alt
Niveau 2 h Alt
Niveau 3 h5 h1 h5 h1
A -050 -015 083 062 -212 -039 198 299 -198 -299
B -261 -270 021 -077 -058 173 198 299 -198 -299
C -151 -031 -014 -015 -004 -005 198 299 -198 -299
D 038 040 -137 000 072 207 198 299 -198 -299
E -001 090 155 -052 056 -101 198 299 -198 -299
F -112 -321 -153 070 -325 023 198 299 -198 -299
G 042 -071 040 147 -009 176 198 299 -198 -299
H 169 046 -169 192 018 -083 198 299 -198 -299
L 009 -120 021 -077 -231 005 198 299 -198 -299
M 001 031 014 -032 004 -011 198 299 -198 -299
-300
-200
-100
000
100
200
300
A B C D E F G H L M
Vale
urs
h d
e M
an
del(
rob
uste
s)
Laboratoires
Meacutethode de reacutefeacuterence analyse robuste
h Ref Niveau 1
h Ref Niveau 2
h Ref Niveau 3
h 5=198
h 1=299
-300
-200
-100
000
100
200
300
A B C D E F G H L M
Vale
urs
de h
de M
an
del(
rob
uste
s)
Laboratoires
Meacutethode alternative analyse robuste
h Alt Niveau 1
h Alt Niveau 2
h Alt Niveau 2
h 1=299
h 5=198
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3438 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Laboratoire k Ref
Niveau 1 k Ref
Niveau 2 k Ref
Niveau 3 k Alt
Niveau 1 k Alt
Niveau 2 k Alt
Niveau 3 k1 k5
A 000 089 142 084 162 046 259 181
B 125 087 073 104 075 046 259 181
C 117 013 091 188 022 083 259 181
D 052 036 069 000 035 083 259 181
E 028 034 037 138 019 112 259 181
F 140 222 018 027 281 112 259 181
G 088 068 024 131 145 000 259 181
H 129 094 173 016 061 000 259 181
L 069 000 073 104 270 224 259 181
M 000 048 038 043 099 241 259 181
000
050
100
150
200
250
300
350
400
A B C D E F G H L M
Vale
urs
k d
e M
an
del(
rob
uste
s)
Laboratoires
Meacutethode de reacutefeacuterence analyse robuste
k Ref Niveau 1k Ref Niveau 2k Ref Niveau 3
k 1=259
k 5=181
000
050
100
150
200
250
300
A B C D E F G H L M
Vale
urs
k d
e M
an
del
(ro
bu
ste
s)
Laboratoires
Meacutethode alternative analyse robuste
k Alt Niveau 3
k Alt Niveau 1
k Alt Niveau 2
k 1=259
k 5=181
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3538 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Appendix 4 ndash Statistical results
Level 1
Nombre de laboratoires (p) 10
A 1602 1602 1602 000 0000 0000 -0495 0000 A 1845 1602 1724 017 0122 -0122 0621 0840 0122
B 1602 1176 1389 030 0213 -0213 -2613 1247 B 1602 1301 1452 021 0151 -0151 -0769 1041 0062
C 1699 1301 1500 028 0199 -0199 -1510 1165 C 1301 1845 1573 038 -0272 0272 -0148 1881 0073
D 1602 1778 1690 012 -0088 0088 0380 0516 D 1602 1602 1602 000 0000 0000 0000 0000 -0088
E 1699 1602 1651 007 0048 -0048 -0013 0284 E 1699 1301 1500 028 0199 -0199 -0522 1376 -0151
F 1778 1301 1540 034 0239 -0239 -1116 1397 F 1778 1699 1739 006 0040 -0040 0698 0274 0199
G 1845 1544 1695 021 0151 -0151 0425 0881 G 2079 1699 1889 027 0190 -0190 1467 1315 0194
H 2041 1602 1822 031 0220 -0220 1689 1286 H 2000 1954 1977 003 0023 -0023 1917 0158 0155
L 1544 1778 1661 017 -0117 0117 0092 0685 L 1301 1602 1452 021 -0151 0151 -0769 1041 -0210
M 1653 1653 1653 000 0000 0000 0013 0000 M 1477 1602 1540 009 -0062 0062 -0319 0432 -0114
m 165186 m 160206 mediane D 00678
Cn(2p) 1867 Cn(2p) 1867 Cp 16101
Cn(p) 16101 Cn(p) 16101 Qn(D) 0092926
n=2p n 20 n=2p n 20 Qdiff 01496169
f 11 f 11 t 114
l 55 l 55
n=p n 10 n=p n 10
f 6 f 6
l 15 l 15
Qn(2p) 0091465 Qn(2p) 007745
Qn(p) 0062469 Qn(p) 0121519
Qintra 017078 Qintra 014461
Qinter 010058 Qinter 019565
Sr(eacutecart-type de reacutepeacutetabiliteacute) 02415 Sr 02045
RSDr(coefficient de variation de la reacutepeacutetabiliteacute)1462 RSDr 1277
r(repeatability limit)) 0676 r 0573
SL(eacutecart-type interlaboratoire) -001905 SLlt0 SL 001737
SR(eacutecart-type de reproductibiliteacute) 02415 SR 02433
RSDR(coefficient de variation de la reproductibiliteacute)0146 RSDR 0152
R(limite de reproductibiliteacute)) 06762 R(limite de reproductibiliteacute))068123
h k h MoyenneLaboratoires
Niveau 1
si LaboratoiresDeviationMoyenne Dk DeviationsiAlt
Niveau 1
Ref
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3638 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Level 2
Nombre de laboratoires (p) 10
A 2681 2613 2647 005 0034 -0034 -0151 0891 A 2322 2531 2427 015 -0105 0105 -2119 1620 -0220
B 2544 2477 2511 005 0033 -0033 -2699 0872 B 2556 2653 2605 007 -0048 0048 -0581 0750 0094
C 2643 2633 2638 001 0005 -0005 -0311 0130 C 2681 2653 2667 002 0014 -0014 -0040 0217 0029
D 2690 2663 2676 002 0014 -0014 0400 0357 D 2778 2732 2755 003 0023 -0023 0721 0354 0079
E 2690 2716 2703 002 -0013 0013 0897 0336 E 2724 2748 2736 002 -0012 0012 0556 0185 0033
F 2568 2398 2483 012 0085 -0085 -3214 2217 F 2477 2114 2296 026 0182 -0182 -3255 2811 -0188
G 2643 2591 2617 004 0026 -0026 -0707 0682 G 2756 2568 2662 013 0094 -0094 -0085 1453 0045
H 2716 2643 2680 005 0036 -0036 0460 0945 H 2653 2732 2693 006 -0040 0040 0181 0613 0013
L 2591 2591 2591 000 0000 0000 -1196 0000 L 2230 2580 2405 025 -0175 0175 -2307 2704 -0186
M 2653 2690 2672 003 -0018 0018 0311 0481 M 2740 2613 2677 009 0064 -0064 0040 0987 0005
m 2655082 m 26719001 mediane D 00209
Cn(2p) 1867 Cn(2p) 1867 Cp 16101
Cn(p) 16101 Cn(p) 16101 Qn(D) 0039911
n=2p n 20 n=2p n 20 Qdiff 00642591
f 11 f 11 t 082
l 55 l 55
n=p n 10 n=p n 10
f 6 f 6
l 15 l 15
Qn(2p) 0020570 Qn(2p) 00345985
Qn(p) 00332437 Qn(p) 00718158
Qintra 003841 Qintra 006460
Qinter 005352 Qinter 011563
Sr(eacutecart-type de reacutepeacutetabiliteacute) 00543 Sr 00914
RSDr(coefficient de variation de la reacutepeacutetabiliteacute)205 RSDr 342
r(repeatability limit)) 0152 r 0256
SL(eacutecart-type interlaboratoire) 000139 SL 000920
SR(eacutecart-type de reproductibiliteacute) 00659 SR 01325
RSDR(coefficient de variation de la reproductibiliteacute)0025 RSDR 0050
R(limite de reproductibiliteacute)) 018446 R(limite de reproductibiliteacute))03708618
Laboratoires
Niveau 2
Moyenne si Laboratoires
Niveau 2
DRef Alt Moyenne si Deviation h Deviation h k k
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3738 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Level 2 bis
Nombre de laboratoires (p) 10
A 3000 2477 2739 037 0261 -0261 0832 1418 A 2602 2477 2540 009 0062 -0062 -0391 0465 -0199
B 2544 2813 2678 019 -0134 0134 0211 0729 B 2903 2778 2841 009 0062 -0062 1732 0465 0162
C 2477 2813 2645 024 -0168 0168 -0135 0911 C 2477 2699 2588 016 -0111 0111 -0049 0826 -0057
D 2398 2653 2526 018 -0128 0128 -1370 0692 D 2778 3000 2889 016 -0111 0111 2074 0826 0363
E 2875 2740 2808 010 0067 -0067 1547 0365 E 2602 2301 2452 021 0151 -0151 -1012 1120 -0356
F 2477 2544 2511 005 -0033 0033 -1525 0182 F 2778 2477 2628 021 0151 -0151 0230 1120 0117
G 2653 2740 2697 006 -0044 0044 0400 0236 G 2845 2845 2845 000 0000 0000 1764 0000 0148
H 2176 2813 2495 045 -0318 0318 -1692 1727 H 2477 2477 2477 000 0000 0000 -0832 0000 -0017
L 2813 2544 2678 019 0134 -0134 0211 0729 L 2903 2301 2602 043 0301 -0301 0049 2241 -0076
M 2602 2740 2671 010 -0069 0069 0135 0375 M 2903 2255 2579 046 0324 -0324 -0112 2411 -0092
m 2658114 m 259505 mediane D -00372
Cn(2p) 1867 Cn(2p) 1867 Cp 16101
Cn(p) 16101 Cn(p) 16101 Qn(D) 01656915
n=2p n 20 n=2p n 20 Qdiff 02667753
f 11 f 11 t 035
l 55 l 55
n=p n 10 n=p n 10
f 6 f 6
l 15 l 15
Qn(2p) 0098745 Qn(2p) 007195
Qn(p) 006007 Qn(p) 008805
Qintra 018437 Qintra 013435
Qinter 009672 Qinter 014176
Sr(eacutecart-type de reacutepeacutetabiliteacute) 02607 Sr 01900
RSDr(coefficient de variation de la reacutepeacutetabiliteacute)981 RSDr 732
r(repeatability limit)) 0730 r 0532
SL(eacutecart-type interlaboratoire) -002464 SLlt0 SL 000205
SR(eacutecart-type de reproductibiliteacute) 02607 SR 01953
RSDR(coefficient de variation de la reproductibiliteacute)0098 RSDR 0075
R(limite de reproductibiliteacute)) 072996 R(limite de reproductibiliteacute))054686
Laboratoires
Niveau 2 bis
Moyenne si Laboratoires
Niveau 2 bis
DRef Alt Moyenne si Deviation h Deviation h k k
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3838 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Level 3
Nombre de laboratoires (p) 10
A 3690 3663 3676 002 0014 -0014 -0311 0513 A 3690 3708 3699 001 -0009 0009 0354 0257 0022
B 3613 3672 3642 004 -0030 0030 -1851 1109 B 3708 3568 3638 010 0070 -0070 -0844 2061 -0005
C 3663 3732 3698 005 -0035 0035 0645 1302 C 3663 3699 3681 003 -0018 0018 0000 0536 -0017
D 3690 3690 3690 000 0000 0000 0311 0000 D 3643 3663 3653 001 -0010 0010 -0545 0286 -0037
E 3732 3653 3693 006 0040 -0040 0429 1480 E 3613 3732 3673 008 -0060 0060 -0162 1769 -0020
F 3643 3672 3658 002 -0014 0014 -1157 0536 F 3690 3771 3731 006 -0040 0040 0975 1193 0073
G 3699 3653 3676 003 0023 -0023 -0328 0856 G 3602 3643 3623 003 -0021 0021 -1141 0612 -0053
H 3623 3672 3648 003 -0024 0024 -1615 0913 H 3748 3681 3715 005 0033 -0033 0664 0990 0067
L 3724 3663 3694 004 0031 -0031 0461 1150 L 3748 3771 3760 002 -0011 0011 1544 0335 0066
M 3690 3690 3690 000 0000 0000 0311 0000 M 3806 3681 3744 009 0062 -0062 1234 1848 0054
m 368334 m 368086 mediane D 00089
Cn(2p) 1867 Cn(2p) 1867 Cp 16101
Cn(p) 16101 Cn(p) 16101 Qn(D) 00325359
n=2p n 20 n=2p n 20 Qdiff 00523852
f 11 f 11 t 043
l 55 l 55
n=p n 10 n=p n 10
f 6 f 6
l 15 l 15
Qn(2p) 0014323 Qn(2p) 001811
Qn(p) 0013719 Qn(p) 003164
Qintra 002674 Qintra 003381
Qinter 002209 Qinter 005094
Sr(eacutecart-type de reacutepeacutetabiliteacute) 00378 Sr 00478
RSDr(coefficient de variation de la reacutepeacutetabiliteacute)103 RSDr 130
r(repeatability limit)) 0106 r 0134
SL(eacutecart-type interlaboratoire) -000023 SLlt0 SL 000145
SR(eacutecart-type de reproductibiliteacute) 00378 SR 00611
RSDR(coefficient de variation de la reproductibiliteacute)0010 RSDR 0017
R(limite de reproductibiliteacute)) 0105894 R(limite de reproductibiliteacute))017119
Ref Alt DLaboratoires
Niveau 3
Moyenne si Deviation h k Laboratoires
Niveau 3
Moyenne si Deviation h k
Thermo Fisher Scientific
ADRIA Deacuteveloppement 1238 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Statistical interpretation according to EN ISO16140 method
The results of the statistical interpretation are given in Table 6
Table 6 ndash Statistical interpretation results
Category n R Regression
used a t(a) b t(b) Critical T
P
Ordinate
at 0
Slope
at 1
Meat products 10 158 GMFR -0455 3321 115 3123 2306 001 001
Dairy products 12 065 GMFR -0027 0385 1002 0108 2228 092 071
Fishery products 10 130 GMFR -0305 1024 1034 0312 2306 076 034
Vegetables 12 086 GMFR -0015 0128 0993 0191 2228 085 090
Egg products 11 158 GMFR -0038 0866 1001 0090 2262 093 041
Environmental
samples 10 176 GMFR -0139 265 1034 231 2306 005 003
All products 65 118 GMFR -0182 3042 1040 2145 2000 004 000
All products
(estimated number) 65 118 GMFR -0182 3042 1040 2145 2000 004 000
All products (non
estimated results) 20 115 GMFR -0367 151 1138 1141 2101 027 015
Repeatability limits obtained for the alternative and reference methods as
well as bias obtained are presented in Table 7
Dairy products Environmental samples
Log (Ref) Log (Ref)
All categories
Thermo Fisher Scientific
ADRIA Deacuteveloppement 1338 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Table 7
Categories Bias
Repeatability limits
Rob r
Ref Alt
Meat products 0008 0314 0343
Dairy products -0031 0136 0130
Fishery products -0133 0220 0214
Vegetables -0041 0180 0319
Egg products -0026 0219 0206
Environmental samples -0007 0198 0119
All products -0032 0196 0215
Regression straight lines (graph and equation representations) for each food
category and for all products are presented figure 4
Thermo Fisher Scientific
ADRIA Deacuteveloppement 1438 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Figure 4 ndash Regression straight lines
Meat products
Fishery products
Dairy products
Vegetables
Thermo Fisher Scientific
ADRIA Deacuteveloppement 1538 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Environmental samples
Egg products
All products (estimated number)
Thermo Fisher Scientific
ADRIA Deacuteveloppement 1638 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
The hypotheses that the ordinates are close to 0 and the slopes close to 1
are approved in all cases except for
- Meat products (slope P = 1 ordinate P = 1)
- Environmental samples (ordinate P = 3)
- All products (ordinate P = 0)
The bias between the two methods vary from ndash 0133 to 0008 log CFU For
all categories the bias is ndash 0032 log CFU The repeatability limits of the
alternative and the reference method are similar For all products the values
are respectively 0215 and 0196 log CFU For all products these
interpretations are provided taking into account data with low contamination
level or not (estimated colonies number)
For all food products the regression straight line is the following
log (Alt) = 1040 log Ref ndash 0182
The Listeria PrecisTM method shows satisfying relative accuracy
All products
All products (Level 2)
Thermo Fisher Scientific
ADRIA Deacuteveloppement 1738 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
2113 Detection limit (LOD) and quantification limit (LOQ)
The critical level is the defined as the smallest amount which can be detected (not null) but not
quantified as an exact value Below this value it cannot be sure that the true value is not null
The detection limit is defined as being higher than the critical level because it involves a power the
probability 1- which has to be well over 50 for example 95
The quantification limit is defined as the smallest amount of analyte (that is the lowest actual number of
organisms) which can be measured and quantified with defined precision and accuracy under the
experimental conditions by the method under validation
Protocol
The detection limit of the alternative method was realised with pure cultures
Three different inoculation levels were tested with six replicates per level ie
18 analyses were done by the alternative method
Listeria monocytogenes 18 was used for the detection limit of the alternative
method
Quantification limit was calculated for six independent blank samples
determinations
Results
These data are intrinsic to the method used and are presented in the
following tables
Table 8 ndash Inoculation 1 ml on 3 plates
Level (CFUml) Positive samples number Standard deviation Bias
0 06
05 26 1211 0
1 36 0816 05
5 66 2483 85
Thermo Fisher Scientific
ADRIA Deacuteveloppement 1838 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Table 9 ndash Inoculation 01 ml on 1 plate
Level (CFUml) Positive samples number Standard deviation Bias
0 06
5 36 0548 05
10 46 1414 2
50 66 2258 55
Table 10
Formulas Obtained values
LC 165 S0 + X0 18
LOD 33 S0 + X0 32
LOQ 10 S0 + X0 87
2114 Relative Sensitivity
The relative sensitivity is defined as the ability of the alternative method to detect two different amounts
of analyte measured by the reference method within a given matrix at a specified average value or
over the whole measurement range that is it is the minimal quantity variation (increase of the analyte
concentration x) which gives a significant variation of the measured signal (response y)
These data are intrinsic to the used method and are obtained from the results
of the linearity study
Sensitivity patterns obtained for tested (matrixstrain) pairs are presented in
figure 5
Thermo Fisher Scientific
ADRIA Deacuteveloppement 1938 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Figure 5 ndash Sensitivity patterns for the tested (matrixstrain) pairs
2115 SpecificitySelectivity
The specificity is defined as the degree to which a method is affected (or not) by the other components
present in a multi-component sample That is the ability of a method to measure exactly a given
analyte or its amount within the sample without interference from non-target components such as a
matrix effect or background noise
The selectivity is defined as a measure of the degree of non-interference in the presence of non-target
analytes A method is selective if it can be used to detect the analyte under examination and that a
guarantee can be provided that the detected signal can only be a product by that specific analyte
The specificity and selectivity study was performed for the Listeria PrecisTM
detection method no additional test was realised for the enumeration
method The results were the following
- Among the Listeria monocytogenes strains tested 2 strains (Listeria
monocytogenes CIP 7834 and Listeria monocytogenes CIP 105459) gave
negative results These 2 strains were tested twice by running 48 h
incubation time of the compared agars Positive results were then
observed by the alternative method Pale characteristic colonies were
observed on the ISO agar by streaking the Half Fraser broth only
- No cross reaction was observed with the non-target strains except with
Listeria ivanovii which gave characteristic colonies on Brilliancetrade Listeria
Agar but the confirmatory tests provide a clear discrimination between
Listeria monocytogenes and Listeria ivanovi species
Rillettes
Lettuce
Raw milk
Smoked salmon
Raw egg
Process water
x (CFUg)
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2038 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
212 Practicability
1 Packaging Ready to use Brilliance Listeria agar plates are available
(x 10 plates)
2 Reagent volume
3 Storage conditionsndash
Shelf life of products
not opened
The plates are stored at 6 - 12degC in darkness conditions The
expiration date is mentioned on the package and on the plates It
is around 3 months
4 Modalities of use
after first use
5 Required
equipments No specific equipment is required
6 Reagents Ready to use plates
7 Training 05 day
8 Real time handling
Reference method
9 min for one sample
55 min for 10 samples
Alternative method
7 min for one sample
35 minutes for 10 samples
9 Time to result
Negative results
Reference method Day 2
Alternative method Day 2
Negative results after confirmation or positive results
Reference method Day 4 ndash Day 7
Alternative method Day 4 ndash Day 7
Positive results
Reference method Day 4 ndash Day 7
Alternative method Day 2 ndash Day 3 (with OBIS Mono Test)
or Day 4 ndash Day 7 (by the classical tests described in the
standardised method)
10 Technician skill Identical to that required for the reference method
11 Steps common to
the reference method Mother suspension revivification step and decimal dilutions
12 Traceability
13 Maintenance
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2138 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
213 Inter-laboratory study
2131 Organisation
12 laboratories participated to this study Pasteurised milk was inoculated by
Listeria monocytogenes 12a isolated from raw milk The targeted
inoculation levels were
- 0 CFUml
- 50 CFU ml
- 500 CFU ml
- 5 000 CFU ml
Each laboratory received eight flasks of 10 ml sample ie two flasks per
inoculation level Furthermore one non-inoculated sample has been added
to the package for total viable count microflora (NF ISO 4833 method)
Detailed instructions were transmitted to the collaborators by the expert
laboratory
Collaborative study laboratories and the expert laboratory carried out the
analyses with the alternative and reference methods
2132 Verification of experimental parameters
Strain stability during transport
In order to evaluate the Listeria monocytogenes strain stability during
transport bacterial count of all inoculated flasks were checked at different
time ie inoculation time after 24 h of conservation at 2deg - 8degC Results are
reported in table 11
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2238 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Table 11 ndash Listeria monocytogenes count
by ISO 11290-2A1 method (log CFUml)
Day of the analyses Level contamination Reference method
Day 0
(29052006)
1 45
2 440
3 4 500
Day 1
(30052006)
1 60
2 430
3 4 900
No evolution of the strain was observed after storage for 24 h at 2degC ndash 8degC
Contamination level
The real contamination levels are provided in table 12
Table 12 ndash Real contamination level (CFUml)
Target inoculation
(CFU ml) Samples Ndeg
Real contamination levels
(CFUml)
0 1 - 8 0
50 5 - 6 45
500 3 - 7 440
5000 2 - 4 4 500
Sample temperature at receipt
Measured temperatures at receipt are listed in table 13
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2338 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Table 13 - Sample temperature on receipt
Laboratories Receipt
day
Hour
receipt
Measured temperature
at receipt (degC)
Measured temperature by the
temperature probe (degC)
A
B
C
D
E
F
G
H
I
J
K
L
M
30052006
10h45
10h15
11h15
10h00
09h30
8h30
11h50
10h00
08h30
14h45
08h30
09h45
08h45
82
55
45
63
60
62
81
38
48
-
14
67
76
03
03
-13
03
04
-14
05
04
01
05
-13
04
Conclusion
The temperatures during the transport were correct for all the labs except for
Lab J which didnrsquot provide the information
All the labs received their packages with several broken flasks
Three labs (B D and L) received some damaged samples but proceeded to
analyses
All the labs started the analyses at Day 1 except Lab J
2133 Results
Aerobic mesophilic flora
The count of aerobic mesophilic flora varied from 94 102 CFUml to
33 103 CFUml
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2438 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Expert lab results
The results obtained by the expert lab are the following
Table 14
Target inoculation level (CFUml)
Reference method ISO 11290-2
Alternative method Listeria Precis Enumeration
Rep 1 Rep 2 Rep 1 Rep 2
lt 10 lt 10 lt 10 lt 10 lt10
50 50 (Ne) 60 (Ne) 50 (Ne) 30 (Ne)
500 470 390 490 500
5 000 4 700 5 100 5 900 3 400
Ne estimated number
The obtained results are in agreement with those expected
Collaborator Labs results
A synthesis of the results obtained by the collaborator labs is provided
Table 15 (CFUml) and Table 16 (log CFUml)
Table 15 ndash Synthesis of results obtained by the alternative
and the reference methods (CFUml)
Labs Level 1 Level 2 Level 2 bis Level 3
Reference method
Alternative method
Reference method
Alternative method
Reference method
Alternative method
Reference method
Alternative method
A 40 40 70 40 480 410 210 340 1000 300 400 300 4900 4600 4900 5100
B 40 15 40 20 350 300 360 450 350 650 800 600 4100 4700 5100 3700
C 50 20 20 70 440 430 480 450 300 650 300 500 4600 5400 4600 5000
D 40 60 40 40 490 460 600 540 250 450 600 1000 4900 4900 4400 4600
E 50 40 50 20 490 520 530 560 750 550 400 200 5400 4500 4100 5400
F 60 20 60 50 370 250 300 130 300 350 600 300 4400 4700 4900 5900
G 70 35 120 50 440 390 570 370 450 550 700 700 5000 4500 4000 4400
H 110 40 100 90 520 440 450 540 150 650 300 300 4200 4700 5600 4800
L 35 60 20 40 390 390 170 380 650 350 800 200 5300 4600 5600 5900
M 45 45 30 40 450 490 550 410 400 550 800 180 4900 4900 6400 4800
Thermo Fisher Scientific
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Summary Report (Version 1)
Listeria Precis Enumeration
Table 16 ndash Synthesis of results obtained by the alternative
and the reference methods (log CFUml)
Labs Level 1 Level 2 Level 2 bis Level 3
Reference method
Alternative method
Reference method
Alternative method
Reference method
Alternative method
Reference method
Alternative method
A 1602 1602 1845 1602 2681 2613 2322 2531 3000 2477 2602 2477 3690 3663 3690 3708
B 1602 1176 1602 1301 2544 2477 2556 2653 2544 2813 2903 2778 3613 3672 3708 3568
C 1699 1301 1301 1845 2643 2633 2681 2653 2477 2813 2477 2699 3663 3732 3663 3699
D 1602 1778 1602 1602 2690 2663 2778 2732 2398 2653 2778 3000 3690 3690 3643 3663
E 1699 1602 1699 1301 2690 2716 2724 2748 2875 2740 2602 2301 3732 3653 3613 3732
F 1778 1301 1778 1699 2568 2398 2477 2114 2477 2544 2778 2477 3643 3672 3690 3771
G 1845 1544 2079 1699 2643 2591 2756 2568 2653 2740 2845 2845 3699 3653 3602 3643
H 2041 1602 2000 1954 2716 2643 2653 2732 2176 2813 2477 2477 3623 3672 3748 3681
L 1544 1778 1301 1602 2591 2591 2230 2580 2813 2544 2903 2301 3724 3663 3748 3771
M 1653 1653 1477 1602 2653 2690 2740 2613 2602 2740 2903 2255 3690 3690 3806 3681
Level 2 results obtained taking account the 3 plates inoculated with the mother-suspension
Level 2 bis results obtained taking account the plates inoculated with 01 ml of the mother-suspension
Scrutiny of the measurement results for consistency
In order to identify the measurement results or laboratories that could be
inconsistent two graphical consistency techniques were realised Mandelrsquos
h-ank-statistics Mandel indicators h and k at 5 significance highlighted
some possible inconsistent data (See tables and graphics in Appendix 3 and
Appendix 4)
Mandelrsquos values Number of values observed over the threshold
Reference method Alternative method
h gt 1 Lab F Level 2 Lab F Level 2
h gt 5 Lab F Level 2
Lab B Levels 1 and 2
Lab A Level 2
Lab D Level 3
Lab F Level 2
Lab L Level 2
kgt 1 Lab F Level 3
k gt 5 Lab F Level 2 Lab C Level 2
Lab F Level 3
Lab L Levels 1 and 3
Lab M Level 1
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2638 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
The emphasized results appear (i) coherent with the targeted contamination
levels (ii) concordant between both methods
All the results were thus kept for the statistical analysis
Comparison of the trueness and precision characteristics of
the reference method and alternative methods
The statistical values are summarised hereafter
Level p
Reference method Alternative method
Repeatability ratio
Reproducibility ratio Median
Repeatability standard deviation
Reproducibility standard deviation
Median Repeatability
standard deviation
Reproducibility standard deviation
1 10 16519 02415 02425 16021 02045 02433 0847 1007
2 10 26551 00543 00659 26719 00914 01325 1682 2011
2 bis 10 26581 02607 02607 25951 01900 01953 0729 0749
3 10 18671 00378 00378 18671 00478 00611 1264 1617
Bias of the alternative method
In order to estimate the bias of the alternative method with regard to the reference method for every
level Dij and t are calculated according to the following equations
RefAlt ijYijYDij
Diffp
Dijit
)2(
)(median
If the t value is superior to 2 the alternative method is significantly biased
with regard to the reference method
The values are given in table 17
Table 17 ndash t (d) values obtained by level
Level Bias t (d) Conclusion
1 0068 114 Non significant bias
2 0021 082 Non significant bias
2 bis - 0037 035 Non significant bias
3 - 0037 043 Non significant bias
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2738 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
The statistical test concludes to a non significant bias for all the inoculation
levels The bias varies from ndash 0037 to 0068 log CFUml
Comparison of the repeatability standard deviations
If the ratio Srj Alt Srj Ref of the repeatability standard deviations of the
alternative method and the reference method is larger than 2 the precision
under repeatability conditions of the alternative method is considered to be
lower than that of the reference method If this ratio is smaller than 05 the
precision under repeatability conditions of the alternative method is
considered to be greater than that of the reference method
The ratio values are given in table 18
Table 18
Level
Repeatability standard deviation
Reference method Alternative method Alternative method
reference method ratio
1 0242 0205 0847
2 0054 0091 1682
2 bis 0261 0190 0729
3 0038 0048 1264
Level Repeatability limit
Reference method Alternative method
1 0676 0573
2 0152 0256
2 bis 0730 0532
3 0106 0134
The ratios of the repeatability standard deviations are below 2 in all cases
The precision under repeatability conditions of the alternative method is
equivalent to that of the reference method
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2838 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Comparison of the reproducibility standard deviations
If the ratio Srj Alt Srj Ref of the reproducibility standard deviations of the alternative method and the
reference method is larger than 2 the precision under reproducibility conditions of the alternative
method is considered to be lower than that of the reference method If this ratio is smaller than 05 the
precision under reproducibility conditions of the alternative method is considered to be greater than
that of the reference method
The ratio values are given in table 19
Table 19
Level
Reproducibility standard deviation
Reference method Alternative method Alternative method
reference method ratio
1 0242 0243 1007
2 0066 0133 2011
2 bis 0261 0195 0749
3 0038 0061 1617
Level Reproducibility limit
Reference method Alternative method
1 0676 0681
2 0185 0371
2 bis 0730 0547
3 0106 0171
The ratios of the reproducibility standard deviations are below or close to 2
The precision under reproducibility conditions of the alternative method is
equivalent to that of the reference method
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2938 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
214 Conclusion
Method comparison study
The Listeria PrecisTM enumeration method shows satisfying linearity and
relative accuracy
The alternative method shows satisfying inclusivity and exclusivity results
Two Listeria monocytogenes strains gave negative results with the
alternative method These strains grew weakly on the reference method
media (OampA Palcam and Oxford)
The limits of repeatability of the alternative method are similar to those of the
reference method
The bias between both methods are low between - 0133 Log CFUg and
- 0008 Log CFUg
The Listeria PrecisTM enumeration method offers the saving for confirmatory
tests
Positive results are obtained with the alternative method in 2-3 days (with
OBIS Mono test) or in 4-7 days (by the classical tests described in the
standardized method) against 4-7 days for the reference method
Inter-laboratory study
The bias between the reference and the alternative methods shows satisfying
and correct values
- level 1 (100 - 1 000 CFUg) bias = + 007 log CFUg
- level 2 (1 000 - 10 000 CFUg) bias = 002 log CFUg
- level 2 bis (1 000 - 10 000 CFUg) bias = - 004 log CFUg
- level 3 (10 000 - 100 000 CFUg) bias = - 004 log CFUg
The repeatability and the reproducibility of the alternative method look similar
to those of the reference method
Thermo Fisher Scientific
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Summary Report (Version 1)
Listeria Precis Enumeration
22 Extension study (realised by ASEPT 2007)
An extension study was performed in 2007 for a new confirmatory test OBIS
MONO
150 Listeria monocytogenes strains and 100 non-target strains were isolated
onto TSAYE and BrillianceTM Listeria Agar plates All the Listeria
monocytogenes and the non-target strains gave the expected results
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3138 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Appendix 1 ndash Flow diagram of the Listeria PrecisTM method
x g sample + 9 x ml buffered peptone water
Revivification step for 1 h plusmn 5 min at 20degC plusmn 1degC
Streaking 1 ml onto 3 Brilliance Listeria Agar plates (mother suspension)
or 01 ml onto 1 Brilliance Listeria Agar plates (mother suspension and decimal dilutions)
Incubation for 48 h plusmn 3 h at 37degC plusmn 1degC
Confirmatory tests on at least one typical colony
- by the tests described in the ISO method
- by applying the OBIS mono test on an isolated colony
Thermo Fisher Scientific
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Summary Report (Version 1)
Listeria Precis Enumeration
Appendix 2 ndash ISO 11290-2 (1998) amp ISO 11290-2A1 (2004) Horizontal
method for the detection and enumeration of Listeria monocytogenes -
Part 2 enumeration of Listeria monocytogenes in foods
10 g cheese + 90 ml buffered peptone water or Half Fraser Broth (without supplements) (110 dilution)
Stomach 1 min 30 seconds
Keep at 20degC plusmn 2degC for 1 h plusmn 5 min
Decimal dilutions in 9 ml peptone salt tubes
Dilution 110
Dilution 1100
Dilution 11000
1 ml on 3 Chromogenic Listeria medium (ISO) plates (x 2) (-1)
01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-2)
01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-3)
01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-4)
Incubation at 37degC plusmn 1degC
24 h - 48 h
Numerate typical colonies at 24 h
(bluegreen surrounded by an opaque halo)
Numerate at 48 h
Streak typical colonies on TSYEA
(5 coloniesplate 2 successive dilutions)
Confirmation tests
Catalase
Gram stain
For confirmation of L monocytogenes
Haemolysis
Carbohydrate (rhamnose and xylose) utilisation
CAMP Test
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3338 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Appendix 3 ndash Mandelrsquos graphics
Laboratoire h Ref
Niveau 1 h Ref
Niveau 2 h Ref
Niveau 3 h Alt
Niveau 1 h Alt
Niveau 2 h Alt
Niveau 3 h5 h1 h5 h1
A -050 -015 083 062 -212 -039 198 299 -198 -299
B -261 -270 021 -077 -058 173 198 299 -198 -299
C -151 -031 -014 -015 -004 -005 198 299 -198 -299
D 038 040 -137 000 072 207 198 299 -198 -299
E -001 090 155 -052 056 -101 198 299 -198 -299
F -112 -321 -153 070 -325 023 198 299 -198 -299
G 042 -071 040 147 -009 176 198 299 -198 -299
H 169 046 -169 192 018 -083 198 299 -198 -299
L 009 -120 021 -077 -231 005 198 299 -198 -299
M 001 031 014 -032 004 -011 198 299 -198 -299
-300
-200
-100
000
100
200
300
A B C D E F G H L M
Vale
urs
h d
e M
an
del(
rob
uste
s)
Laboratoires
Meacutethode de reacutefeacuterence analyse robuste
h Ref Niveau 1
h Ref Niveau 2
h Ref Niveau 3
h 5=198
h 1=299
-300
-200
-100
000
100
200
300
A B C D E F G H L M
Vale
urs
de h
de M
an
del(
rob
uste
s)
Laboratoires
Meacutethode alternative analyse robuste
h Alt Niveau 1
h Alt Niveau 2
h Alt Niveau 2
h 1=299
h 5=198
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3438 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Laboratoire k Ref
Niveau 1 k Ref
Niveau 2 k Ref
Niveau 3 k Alt
Niveau 1 k Alt
Niveau 2 k Alt
Niveau 3 k1 k5
A 000 089 142 084 162 046 259 181
B 125 087 073 104 075 046 259 181
C 117 013 091 188 022 083 259 181
D 052 036 069 000 035 083 259 181
E 028 034 037 138 019 112 259 181
F 140 222 018 027 281 112 259 181
G 088 068 024 131 145 000 259 181
H 129 094 173 016 061 000 259 181
L 069 000 073 104 270 224 259 181
M 000 048 038 043 099 241 259 181
000
050
100
150
200
250
300
350
400
A B C D E F G H L M
Vale
urs
k d
e M
an
del(
rob
uste
s)
Laboratoires
Meacutethode de reacutefeacuterence analyse robuste
k Ref Niveau 1k Ref Niveau 2k Ref Niveau 3
k 1=259
k 5=181
000
050
100
150
200
250
300
A B C D E F G H L M
Vale
urs
k d
e M
an
del
(ro
bu
ste
s)
Laboratoires
Meacutethode alternative analyse robuste
k Alt Niveau 3
k Alt Niveau 1
k Alt Niveau 2
k 1=259
k 5=181
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3538 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Appendix 4 ndash Statistical results
Level 1
Nombre de laboratoires (p) 10
A 1602 1602 1602 000 0000 0000 -0495 0000 A 1845 1602 1724 017 0122 -0122 0621 0840 0122
B 1602 1176 1389 030 0213 -0213 -2613 1247 B 1602 1301 1452 021 0151 -0151 -0769 1041 0062
C 1699 1301 1500 028 0199 -0199 -1510 1165 C 1301 1845 1573 038 -0272 0272 -0148 1881 0073
D 1602 1778 1690 012 -0088 0088 0380 0516 D 1602 1602 1602 000 0000 0000 0000 0000 -0088
E 1699 1602 1651 007 0048 -0048 -0013 0284 E 1699 1301 1500 028 0199 -0199 -0522 1376 -0151
F 1778 1301 1540 034 0239 -0239 -1116 1397 F 1778 1699 1739 006 0040 -0040 0698 0274 0199
G 1845 1544 1695 021 0151 -0151 0425 0881 G 2079 1699 1889 027 0190 -0190 1467 1315 0194
H 2041 1602 1822 031 0220 -0220 1689 1286 H 2000 1954 1977 003 0023 -0023 1917 0158 0155
L 1544 1778 1661 017 -0117 0117 0092 0685 L 1301 1602 1452 021 -0151 0151 -0769 1041 -0210
M 1653 1653 1653 000 0000 0000 0013 0000 M 1477 1602 1540 009 -0062 0062 -0319 0432 -0114
m 165186 m 160206 mediane D 00678
Cn(2p) 1867 Cn(2p) 1867 Cp 16101
Cn(p) 16101 Cn(p) 16101 Qn(D) 0092926
n=2p n 20 n=2p n 20 Qdiff 01496169
f 11 f 11 t 114
l 55 l 55
n=p n 10 n=p n 10
f 6 f 6
l 15 l 15
Qn(2p) 0091465 Qn(2p) 007745
Qn(p) 0062469 Qn(p) 0121519
Qintra 017078 Qintra 014461
Qinter 010058 Qinter 019565
Sr(eacutecart-type de reacutepeacutetabiliteacute) 02415 Sr 02045
RSDr(coefficient de variation de la reacutepeacutetabiliteacute)1462 RSDr 1277
r(repeatability limit)) 0676 r 0573
SL(eacutecart-type interlaboratoire) -001905 SLlt0 SL 001737
SR(eacutecart-type de reproductibiliteacute) 02415 SR 02433
RSDR(coefficient de variation de la reproductibiliteacute)0146 RSDR 0152
R(limite de reproductibiliteacute)) 06762 R(limite de reproductibiliteacute))068123
h k h MoyenneLaboratoires
Niveau 1
si LaboratoiresDeviationMoyenne Dk DeviationsiAlt
Niveau 1
Ref
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3638 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Level 2
Nombre de laboratoires (p) 10
A 2681 2613 2647 005 0034 -0034 -0151 0891 A 2322 2531 2427 015 -0105 0105 -2119 1620 -0220
B 2544 2477 2511 005 0033 -0033 -2699 0872 B 2556 2653 2605 007 -0048 0048 -0581 0750 0094
C 2643 2633 2638 001 0005 -0005 -0311 0130 C 2681 2653 2667 002 0014 -0014 -0040 0217 0029
D 2690 2663 2676 002 0014 -0014 0400 0357 D 2778 2732 2755 003 0023 -0023 0721 0354 0079
E 2690 2716 2703 002 -0013 0013 0897 0336 E 2724 2748 2736 002 -0012 0012 0556 0185 0033
F 2568 2398 2483 012 0085 -0085 -3214 2217 F 2477 2114 2296 026 0182 -0182 -3255 2811 -0188
G 2643 2591 2617 004 0026 -0026 -0707 0682 G 2756 2568 2662 013 0094 -0094 -0085 1453 0045
H 2716 2643 2680 005 0036 -0036 0460 0945 H 2653 2732 2693 006 -0040 0040 0181 0613 0013
L 2591 2591 2591 000 0000 0000 -1196 0000 L 2230 2580 2405 025 -0175 0175 -2307 2704 -0186
M 2653 2690 2672 003 -0018 0018 0311 0481 M 2740 2613 2677 009 0064 -0064 0040 0987 0005
m 2655082 m 26719001 mediane D 00209
Cn(2p) 1867 Cn(2p) 1867 Cp 16101
Cn(p) 16101 Cn(p) 16101 Qn(D) 0039911
n=2p n 20 n=2p n 20 Qdiff 00642591
f 11 f 11 t 082
l 55 l 55
n=p n 10 n=p n 10
f 6 f 6
l 15 l 15
Qn(2p) 0020570 Qn(2p) 00345985
Qn(p) 00332437 Qn(p) 00718158
Qintra 003841 Qintra 006460
Qinter 005352 Qinter 011563
Sr(eacutecart-type de reacutepeacutetabiliteacute) 00543 Sr 00914
RSDr(coefficient de variation de la reacutepeacutetabiliteacute)205 RSDr 342
r(repeatability limit)) 0152 r 0256
SL(eacutecart-type interlaboratoire) 000139 SL 000920
SR(eacutecart-type de reproductibiliteacute) 00659 SR 01325
RSDR(coefficient de variation de la reproductibiliteacute)0025 RSDR 0050
R(limite de reproductibiliteacute)) 018446 R(limite de reproductibiliteacute))03708618
Laboratoires
Niveau 2
Moyenne si Laboratoires
Niveau 2
DRef Alt Moyenne si Deviation h Deviation h k k
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3738 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Level 2 bis
Nombre de laboratoires (p) 10
A 3000 2477 2739 037 0261 -0261 0832 1418 A 2602 2477 2540 009 0062 -0062 -0391 0465 -0199
B 2544 2813 2678 019 -0134 0134 0211 0729 B 2903 2778 2841 009 0062 -0062 1732 0465 0162
C 2477 2813 2645 024 -0168 0168 -0135 0911 C 2477 2699 2588 016 -0111 0111 -0049 0826 -0057
D 2398 2653 2526 018 -0128 0128 -1370 0692 D 2778 3000 2889 016 -0111 0111 2074 0826 0363
E 2875 2740 2808 010 0067 -0067 1547 0365 E 2602 2301 2452 021 0151 -0151 -1012 1120 -0356
F 2477 2544 2511 005 -0033 0033 -1525 0182 F 2778 2477 2628 021 0151 -0151 0230 1120 0117
G 2653 2740 2697 006 -0044 0044 0400 0236 G 2845 2845 2845 000 0000 0000 1764 0000 0148
H 2176 2813 2495 045 -0318 0318 -1692 1727 H 2477 2477 2477 000 0000 0000 -0832 0000 -0017
L 2813 2544 2678 019 0134 -0134 0211 0729 L 2903 2301 2602 043 0301 -0301 0049 2241 -0076
M 2602 2740 2671 010 -0069 0069 0135 0375 M 2903 2255 2579 046 0324 -0324 -0112 2411 -0092
m 2658114 m 259505 mediane D -00372
Cn(2p) 1867 Cn(2p) 1867 Cp 16101
Cn(p) 16101 Cn(p) 16101 Qn(D) 01656915
n=2p n 20 n=2p n 20 Qdiff 02667753
f 11 f 11 t 035
l 55 l 55
n=p n 10 n=p n 10
f 6 f 6
l 15 l 15
Qn(2p) 0098745 Qn(2p) 007195
Qn(p) 006007 Qn(p) 008805
Qintra 018437 Qintra 013435
Qinter 009672 Qinter 014176
Sr(eacutecart-type de reacutepeacutetabiliteacute) 02607 Sr 01900
RSDr(coefficient de variation de la reacutepeacutetabiliteacute)981 RSDr 732
r(repeatability limit)) 0730 r 0532
SL(eacutecart-type interlaboratoire) -002464 SLlt0 SL 000205
SR(eacutecart-type de reproductibiliteacute) 02607 SR 01953
RSDR(coefficient de variation de la reproductibiliteacute)0098 RSDR 0075
R(limite de reproductibiliteacute)) 072996 R(limite de reproductibiliteacute))054686
Laboratoires
Niveau 2 bis
Moyenne si Laboratoires
Niveau 2 bis
DRef Alt Moyenne si Deviation h Deviation h k k
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3838 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Level 3
Nombre de laboratoires (p) 10
A 3690 3663 3676 002 0014 -0014 -0311 0513 A 3690 3708 3699 001 -0009 0009 0354 0257 0022
B 3613 3672 3642 004 -0030 0030 -1851 1109 B 3708 3568 3638 010 0070 -0070 -0844 2061 -0005
C 3663 3732 3698 005 -0035 0035 0645 1302 C 3663 3699 3681 003 -0018 0018 0000 0536 -0017
D 3690 3690 3690 000 0000 0000 0311 0000 D 3643 3663 3653 001 -0010 0010 -0545 0286 -0037
E 3732 3653 3693 006 0040 -0040 0429 1480 E 3613 3732 3673 008 -0060 0060 -0162 1769 -0020
F 3643 3672 3658 002 -0014 0014 -1157 0536 F 3690 3771 3731 006 -0040 0040 0975 1193 0073
G 3699 3653 3676 003 0023 -0023 -0328 0856 G 3602 3643 3623 003 -0021 0021 -1141 0612 -0053
H 3623 3672 3648 003 -0024 0024 -1615 0913 H 3748 3681 3715 005 0033 -0033 0664 0990 0067
L 3724 3663 3694 004 0031 -0031 0461 1150 L 3748 3771 3760 002 -0011 0011 1544 0335 0066
M 3690 3690 3690 000 0000 0000 0311 0000 M 3806 3681 3744 009 0062 -0062 1234 1848 0054
m 368334 m 368086 mediane D 00089
Cn(2p) 1867 Cn(2p) 1867 Cp 16101
Cn(p) 16101 Cn(p) 16101 Qn(D) 00325359
n=2p n 20 n=2p n 20 Qdiff 00523852
f 11 f 11 t 043
l 55 l 55
n=p n 10 n=p n 10
f 6 f 6
l 15 l 15
Qn(2p) 0014323 Qn(2p) 001811
Qn(p) 0013719 Qn(p) 003164
Qintra 002674 Qintra 003381
Qinter 002209 Qinter 005094
Sr(eacutecart-type de reacutepeacutetabiliteacute) 00378 Sr 00478
RSDr(coefficient de variation de la reacutepeacutetabiliteacute)103 RSDr 130
r(repeatability limit)) 0106 r 0134
SL(eacutecart-type interlaboratoire) -000023 SLlt0 SL 000145
SR(eacutecart-type de reproductibiliteacute) 00378 SR 00611
RSDR(coefficient de variation de la reproductibiliteacute)0010 RSDR 0017
R(limite de reproductibiliteacute)) 0105894 R(limite de reproductibiliteacute))017119
Ref Alt DLaboratoires
Niveau 3
Moyenne si Deviation h k Laboratoires
Niveau 3
Moyenne si Deviation h k
Thermo Fisher Scientific
ADRIA Deacuteveloppement 1338 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Table 7
Categories Bias
Repeatability limits
Rob r
Ref Alt
Meat products 0008 0314 0343
Dairy products -0031 0136 0130
Fishery products -0133 0220 0214
Vegetables -0041 0180 0319
Egg products -0026 0219 0206
Environmental samples -0007 0198 0119
All products -0032 0196 0215
Regression straight lines (graph and equation representations) for each food
category and for all products are presented figure 4
Thermo Fisher Scientific
ADRIA Deacuteveloppement 1438 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Figure 4 ndash Regression straight lines
Meat products
Fishery products
Dairy products
Vegetables
Thermo Fisher Scientific
ADRIA Deacuteveloppement 1538 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Environmental samples
Egg products
All products (estimated number)
Thermo Fisher Scientific
ADRIA Deacuteveloppement 1638 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
The hypotheses that the ordinates are close to 0 and the slopes close to 1
are approved in all cases except for
- Meat products (slope P = 1 ordinate P = 1)
- Environmental samples (ordinate P = 3)
- All products (ordinate P = 0)
The bias between the two methods vary from ndash 0133 to 0008 log CFU For
all categories the bias is ndash 0032 log CFU The repeatability limits of the
alternative and the reference method are similar For all products the values
are respectively 0215 and 0196 log CFU For all products these
interpretations are provided taking into account data with low contamination
level or not (estimated colonies number)
For all food products the regression straight line is the following
log (Alt) = 1040 log Ref ndash 0182
The Listeria PrecisTM method shows satisfying relative accuracy
All products
All products (Level 2)
Thermo Fisher Scientific
ADRIA Deacuteveloppement 1738 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
2113 Detection limit (LOD) and quantification limit (LOQ)
The critical level is the defined as the smallest amount which can be detected (not null) but not
quantified as an exact value Below this value it cannot be sure that the true value is not null
The detection limit is defined as being higher than the critical level because it involves a power the
probability 1- which has to be well over 50 for example 95
The quantification limit is defined as the smallest amount of analyte (that is the lowest actual number of
organisms) which can be measured and quantified with defined precision and accuracy under the
experimental conditions by the method under validation
Protocol
The detection limit of the alternative method was realised with pure cultures
Three different inoculation levels were tested with six replicates per level ie
18 analyses were done by the alternative method
Listeria monocytogenes 18 was used for the detection limit of the alternative
method
Quantification limit was calculated for six independent blank samples
determinations
Results
These data are intrinsic to the method used and are presented in the
following tables
Table 8 ndash Inoculation 1 ml on 3 plates
Level (CFUml) Positive samples number Standard deviation Bias
0 06
05 26 1211 0
1 36 0816 05
5 66 2483 85
Thermo Fisher Scientific
ADRIA Deacuteveloppement 1838 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Table 9 ndash Inoculation 01 ml on 1 plate
Level (CFUml) Positive samples number Standard deviation Bias
0 06
5 36 0548 05
10 46 1414 2
50 66 2258 55
Table 10
Formulas Obtained values
LC 165 S0 + X0 18
LOD 33 S0 + X0 32
LOQ 10 S0 + X0 87
2114 Relative Sensitivity
The relative sensitivity is defined as the ability of the alternative method to detect two different amounts
of analyte measured by the reference method within a given matrix at a specified average value or
over the whole measurement range that is it is the minimal quantity variation (increase of the analyte
concentration x) which gives a significant variation of the measured signal (response y)
These data are intrinsic to the used method and are obtained from the results
of the linearity study
Sensitivity patterns obtained for tested (matrixstrain) pairs are presented in
figure 5
Thermo Fisher Scientific
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Summary Report (Version 1)
Listeria Precis Enumeration
Figure 5 ndash Sensitivity patterns for the tested (matrixstrain) pairs
2115 SpecificitySelectivity
The specificity is defined as the degree to which a method is affected (or not) by the other components
present in a multi-component sample That is the ability of a method to measure exactly a given
analyte or its amount within the sample without interference from non-target components such as a
matrix effect or background noise
The selectivity is defined as a measure of the degree of non-interference in the presence of non-target
analytes A method is selective if it can be used to detect the analyte under examination and that a
guarantee can be provided that the detected signal can only be a product by that specific analyte
The specificity and selectivity study was performed for the Listeria PrecisTM
detection method no additional test was realised for the enumeration
method The results were the following
- Among the Listeria monocytogenes strains tested 2 strains (Listeria
monocytogenes CIP 7834 and Listeria monocytogenes CIP 105459) gave
negative results These 2 strains were tested twice by running 48 h
incubation time of the compared agars Positive results were then
observed by the alternative method Pale characteristic colonies were
observed on the ISO agar by streaking the Half Fraser broth only
- No cross reaction was observed with the non-target strains except with
Listeria ivanovii which gave characteristic colonies on Brilliancetrade Listeria
Agar but the confirmatory tests provide a clear discrimination between
Listeria monocytogenes and Listeria ivanovi species
Rillettes
Lettuce
Raw milk
Smoked salmon
Raw egg
Process water
x (CFUg)
Thermo Fisher Scientific
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Summary Report (Version 1)
Listeria Precis Enumeration
212 Practicability
1 Packaging Ready to use Brilliance Listeria agar plates are available
(x 10 plates)
2 Reagent volume
3 Storage conditionsndash
Shelf life of products
not opened
The plates are stored at 6 - 12degC in darkness conditions The
expiration date is mentioned on the package and on the plates It
is around 3 months
4 Modalities of use
after first use
5 Required
equipments No specific equipment is required
6 Reagents Ready to use plates
7 Training 05 day
8 Real time handling
Reference method
9 min for one sample
55 min for 10 samples
Alternative method
7 min for one sample
35 minutes for 10 samples
9 Time to result
Negative results
Reference method Day 2
Alternative method Day 2
Negative results after confirmation or positive results
Reference method Day 4 ndash Day 7
Alternative method Day 4 ndash Day 7
Positive results
Reference method Day 4 ndash Day 7
Alternative method Day 2 ndash Day 3 (with OBIS Mono Test)
or Day 4 ndash Day 7 (by the classical tests described in the
standardised method)
10 Technician skill Identical to that required for the reference method
11 Steps common to
the reference method Mother suspension revivification step and decimal dilutions
12 Traceability
13 Maintenance
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Summary Report (Version 1)
Listeria Precis Enumeration
213 Inter-laboratory study
2131 Organisation
12 laboratories participated to this study Pasteurised milk was inoculated by
Listeria monocytogenes 12a isolated from raw milk The targeted
inoculation levels were
- 0 CFUml
- 50 CFU ml
- 500 CFU ml
- 5 000 CFU ml
Each laboratory received eight flasks of 10 ml sample ie two flasks per
inoculation level Furthermore one non-inoculated sample has been added
to the package for total viable count microflora (NF ISO 4833 method)
Detailed instructions were transmitted to the collaborators by the expert
laboratory
Collaborative study laboratories and the expert laboratory carried out the
analyses with the alternative and reference methods
2132 Verification of experimental parameters
Strain stability during transport
In order to evaluate the Listeria monocytogenes strain stability during
transport bacterial count of all inoculated flasks were checked at different
time ie inoculation time after 24 h of conservation at 2deg - 8degC Results are
reported in table 11
Thermo Fisher Scientific
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Summary Report (Version 1)
Listeria Precis Enumeration
Table 11 ndash Listeria monocytogenes count
by ISO 11290-2A1 method (log CFUml)
Day of the analyses Level contamination Reference method
Day 0
(29052006)
1 45
2 440
3 4 500
Day 1
(30052006)
1 60
2 430
3 4 900
No evolution of the strain was observed after storage for 24 h at 2degC ndash 8degC
Contamination level
The real contamination levels are provided in table 12
Table 12 ndash Real contamination level (CFUml)
Target inoculation
(CFU ml) Samples Ndeg
Real contamination levels
(CFUml)
0 1 - 8 0
50 5 - 6 45
500 3 - 7 440
5000 2 - 4 4 500
Sample temperature at receipt
Measured temperatures at receipt are listed in table 13
Thermo Fisher Scientific
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Summary Report (Version 1)
Listeria Precis Enumeration
Table 13 - Sample temperature on receipt
Laboratories Receipt
day
Hour
receipt
Measured temperature
at receipt (degC)
Measured temperature by the
temperature probe (degC)
A
B
C
D
E
F
G
H
I
J
K
L
M
30052006
10h45
10h15
11h15
10h00
09h30
8h30
11h50
10h00
08h30
14h45
08h30
09h45
08h45
82
55
45
63
60
62
81
38
48
-
14
67
76
03
03
-13
03
04
-14
05
04
01
05
-13
04
Conclusion
The temperatures during the transport were correct for all the labs except for
Lab J which didnrsquot provide the information
All the labs received their packages with several broken flasks
Three labs (B D and L) received some damaged samples but proceeded to
analyses
All the labs started the analyses at Day 1 except Lab J
2133 Results
Aerobic mesophilic flora
The count of aerobic mesophilic flora varied from 94 102 CFUml to
33 103 CFUml
Thermo Fisher Scientific
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Listeria Precis Enumeration
Expert lab results
The results obtained by the expert lab are the following
Table 14
Target inoculation level (CFUml)
Reference method ISO 11290-2
Alternative method Listeria Precis Enumeration
Rep 1 Rep 2 Rep 1 Rep 2
lt 10 lt 10 lt 10 lt 10 lt10
50 50 (Ne) 60 (Ne) 50 (Ne) 30 (Ne)
500 470 390 490 500
5 000 4 700 5 100 5 900 3 400
Ne estimated number
The obtained results are in agreement with those expected
Collaborator Labs results
A synthesis of the results obtained by the collaborator labs is provided
Table 15 (CFUml) and Table 16 (log CFUml)
Table 15 ndash Synthesis of results obtained by the alternative
and the reference methods (CFUml)
Labs Level 1 Level 2 Level 2 bis Level 3
Reference method
Alternative method
Reference method
Alternative method
Reference method
Alternative method
Reference method
Alternative method
A 40 40 70 40 480 410 210 340 1000 300 400 300 4900 4600 4900 5100
B 40 15 40 20 350 300 360 450 350 650 800 600 4100 4700 5100 3700
C 50 20 20 70 440 430 480 450 300 650 300 500 4600 5400 4600 5000
D 40 60 40 40 490 460 600 540 250 450 600 1000 4900 4900 4400 4600
E 50 40 50 20 490 520 530 560 750 550 400 200 5400 4500 4100 5400
F 60 20 60 50 370 250 300 130 300 350 600 300 4400 4700 4900 5900
G 70 35 120 50 440 390 570 370 450 550 700 700 5000 4500 4000 4400
H 110 40 100 90 520 440 450 540 150 650 300 300 4200 4700 5600 4800
L 35 60 20 40 390 390 170 380 650 350 800 200 5300 4600 5600 5900
M 45 45 30 40 450 490 550 410 400 550 800 180 4900 4900 6400 4800
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Summary Report (Version 1)
Listeria Precis Enumeration
Table 16 ndash Synthesis of results obtained by the alternative
and the reference methods (log CFUml)
Labs Level 1 Level 2 Level 2 bis Level 3
Reference method
Alternative method
Reference method
Alternative method
Reference method
Alternative method
Reference method
Alternative method
A 1602 1602 1845 1602 2681 2613 2322 2531 3000 2477 2602 2477 3690 3663 3690 3708
B 1602 1176 1602 1301 2544 2477 2556 2653 2544 2813 2903 2778 3613 3672 3708 3568
C 1699 1301 1301 1845 2643 2633 2681 2653 2477 2813 2477 2699 3663 3732 3663 3699
D 1602 1778 1602 1602 2690 2663 2778 2732 2398 2653 2778 3000 3690 3690 3643 3663
E 1699 1602 1699 1301 2690 2716 2724 2748 2875 2740 2602 2301 3732 3653 3613 3732
F 1778 1301 1778 1699 2568 2398 2477 2114 2477 2544 2778 2477 3643 3672 3690 3771
G 1845 1544 2079 1699 2643 2591 2756 2568 2653 2740 2845 2845 3699 3653 3602 3643
H 2041 1602 2000 1954 2716 2643 2653 2732 2176 2813 2477 2477 3623 3672 3748 3681
L 1544 1778 1301 1602 2591 2591 2230 2580 2813 2544 2903 2301 3724 3663 3748 3771
M 1653 1653 1477 1602 2653 2690 2740 2613 2602 2740 2903 2255 3690 3690 3806 3681
Level 2 results obtained taking account the 3 plates inoculated with the mother-suspension
Level 2 bis results obtained taking account the plates inoculated with 01 ml of the mother-suspension
Scrutiny of the measurement results for consistency
In order to identify the measurement results or laboratories that could be
inconsistent two graphical consistency techniques were realised Mandelrsquos
h-ank-statistics Mandel indicators h and k at 5 significance highlighted
some possible inconsistent data (See tables and graphics in Appendix 3 and
Appendix 4)
Mandelrsquos values Number of values observed over the threshold
Reference method Alternative method
h gt 1 Lab F Level 2 Lab F Level 2
h gt 5 Lab F Level 2
Lab B Levels 1 and 2
Lab A Level 2
Lab D Level 3
Lab F Level 2
Lab L Level 2
kgt 1 Lab F Level 3
k gt 5 Lab F Level 2 Lab C Level 2
Lab F Level 3
Lab L Levels 1 and 3
Lab M Level 1
Thermo Fisher Scientific
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Summary Report (Version 1)
Listeria Precis Enumeration
The emphasized results appear (i) coherent with the targeted contamination
levels (ii) concordant between both methods
All the results were thus kept for the statistical analysis
Comparison of the trueness and precision characteristics of
the reference method and alternative methods
The statistical values are summarised hereafter
Level p
Reference method Alternative method
Repeatability ratio
Reproducibility ratio Median
Repeatability standard deviation
Reproducibility standard deviation
Median Repeatability
standard deviation
Reproducibility standard deviation
1 10 16519 02415 02425 16021 02045 02433 0847 1007
2 10 26551 00543 00659 26719 00914 01325 1682 2011
2 bis 10 26581 02607 02607 25951 01900 01953 0729 0749
3 10 18671 00378 00378 18671 00478 00611 1264 1617
Bias of the alternative method
In order to estimate the bias of the alternative method with regard to the reference method for every
level Dij and t are calculated according to the following equations
RefAlt ijYijYDij
Diffp
Dijit
)2(
)(median
If the t value is superior to 2 the alternative method is significantly biased
with regard to the reference method
The values are given in table 17
Table 17 ndash t (d) values obtained by level
Level Bias t (d) Conclusion
1 0068 114 Non significant bias
2 0021 082 Non significant bias
2 bis - 0037 035 Non significant bias
3 - 0037 043 Non significant bias
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2738 November 19 2014
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Listeria Precis Enumeration
The statistical test concludes to a non significant bias for all the inoculation
levels The bias varies from ndash 0037 to 0068 log CFUml
Comparison of the repeatability standard deviations
If the ratio Srj Alt Srj Ref of the repeatability standard deviations of the
alternative method and the reference method is larger than 2 the precision
under repeatability conditions of the alternative method is considered to be
lower than that of the reference method If this ratio is smaller than 05 the
precision under repeatability conditions of the alternative method is
considered to be greater than that of the reference method
The ratio values are given in table 18
Table 18
Level
Repeatability standard deviation
Reference method Alternative method Alternative method
reference method ratio
1 0242 0205 0847
2 0054 0091 1682
2 bis 0261 0190 0729
3 0038 0048 1264
Level Repeatability limit
Reference method Alternative method
1 0676 0573
2 0152 0256
2 bis 0730 0532
3 0106 0134
The ratios of the repeatability standard deviations are below 2 in all cases
The precision under repeatability conditions of the alternative method is
equivalent to that of the reference method
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2838 November 19 2014
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Listeria Precis Enumeration
Comparison of the reproducibility standard deviations
If the ratio Srj Alt Srj Ref of the reproducibility standard deviations of the alternative method and the
reference method is larger than 2 the precision under reproducibility conditions of the alternative
method is considered to be lower than that of the reference method If this ratio is smaller than 05 the
precision under reproducibility conditions of the alternative method is considered to be greater than
that of the reference method
The ratio values are given in table 19
Table 19
Level
Reproducibility standard deviation
Reference method Alternative method Alternative method
reference method ratio
1 0242 0243 1007
2 0066 0133 2011
2 bis 0261 0195 0749
3 0038 0061 1617
Level Reproducibility limit
Reference method Alternative method
1 0676 0681
2 0185 0371
2 bis 0730 0547
3 0106 0171
The ratios of the reproducibility standard deviations are below or close to 2
The precision under reproducibility conditions of the alternative method is
equivalent to that of the reference method
Thermo Fisher Scientific
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Summary Report (Version 1)
Listeria Precis Enumeration
214 Conclusion
Method comparison study
The Listeria PrecisTM enumeration method shows satisfying linearity and
relative accuracy
The alternative method shows satisfying inclusivity and exclusivity results
Two Listeria monocytogenes strains gave negative results with the
alternative method These strains grew weakly on the reference method
media (OampA Palcam and Oxford)
The limits of repeatability of the alternative method are similar to those of the
reference method
The bias between both methods are low between - 0133 Log CFUg and
- 0008 Log CFUg
The Listeria PrecisTM enumeration method offers the saving for confirmatory
tests
Positive results are obtained with the alternative method in 2-3 days (with
OBIS Mono test) or in 4-7 days (by the classical tests described in the
standardized method) against 4-7 days for the reference method
Inter-laboratory study
The bias between the reference and the alternative methods shows satisfying
and correct values
- level 1 (100 - 1 000 CFUg) bias = + 007 log CFUg
- level 2 (1 000 - 10 000 CFUg) bias = 002 log CFUg
- level 2 bis (1 000 - 10 000 CFUg) bias = - 004 log CFUg
- level 3 (10 000 - 100 000 CFUg) bias = - 004 log CFUg
The repeatability and the reproducibility of the alternative method look similar
to those of the reference method
Thermo Fisher Scientific
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Summary Report (Version 1)
Listeria Precis Enumeration
22 Extension study (realised by ASEPT 2007)
An extension study was performed in 2007 for a new confirmatory test OBIS
MONO
150 Listeria monocytogenes strains and 100 non-target strains were isolated
onto TSAYE and BrillianceTM Listeria Agar plates All the Listeria
monocytogenes and the non-target strains gave the expected results
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3138 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Appendix 1 ndash Flow diagram of the Listeria PrecisTM method
x g sample + 9 x ml buffered peptone water
Revivification step for 1 h plusmn 5 min at 20degC plusmn 1degC
Streaking 1 ml onto 3 Brilliance Listeria Agar plates (mother suspension)
or 01 ml onto 1 Brilliance Listeria Agar plates (mother suspension and decimal dilutions)
Incubation for 48 h plusmn 3 h at 37degC plusmn 1degC
Confirmatory tests on at least one typical colony
- by the tests described in the ISO method
- by applying the OBIS mono test on an isolated colony
Thermo Fisher Scientific
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Summary Report (Version 1)
Listeria Precis Enumeration
Appendix 2 ndash ISO 11290-2 (1998) amp ISO 11290-2A1 (2004) Horizontal
method for the detection and enumeration of Listeria monocytogenes -
Part 2 enumeration of Listeria monocytogenes in foods
10 g cheese + 90 ml buffered peptone water or Half Fraser Broth (without supplements) (110 dilution)
Stomach 1 min 30 seconds
Keep at 20degC plusmn 2degC for 1 h plusmn 5 min
Decimal dilutions in 9 ml peptone salt tubes
Dilution 110
Dilution 1100
Dilution 11000
1 ml on 3 Chromogenic Listeria medium (ISO) plates (x 2) (-1)
01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-2)
01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-3)
01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-4)
Incubation at 37degC plusmn 1degC
24 h - 48 h
Numerate typical colonies at 24 h
(bluegreen surrounded by an opaque halo)
Numerate at 48 h
Streak typical colonies on TSYEA
(5 coloniesplate 2 successive dilutions)
Confirmation tests
Catalase
Gram stain
For confirmation of L monocytogenes
Haemolysis
Carbohydrate (rhamnose and xylose) utilisation
CAMP Test
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3338 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Appendix 3 ndash Mandelrsquos graphics
Laboratoire h Ref
Niveau 1 h Ref
Niveau 2 h Ref
Niveau 3 h Alt
Niveau 1 h Alt
Niveau 2 h Alt
Niveau 3 h5 h1 h5 h1
A -050 -015 083 062 -212 -039 198 299 -198 -299
B -261 -270 021 -077 -058 173 198 299 -198 -299
C -151 -031 -014 -015 -004 -005 198 299 -198 -299
D 038 040 -137 000 072 207 198 299 -198 -299
E -001 090 155 -052 056 -101 198 299 -198 -299
F -112 -321 -153 070 -325 023 198 299 -198 -299
G 042 -071 040 147 -009 176 198 299 -198 -299
H 169 046 -169 192 018 -083 198 299 -198 -299
L 009 -120 021 -077 -231 005 198 299 -198 -299
M 001 031 014 -032 004 -011 198 299 -198 -299
-300
-200
-100
000
100
200
300
A B C D E F G H L M
Vale
urs
h d
e M
an
del(
rob
uste
s)
Laboratoires
Meacutethode de reacutefeacuterence analyse robuste
h Ref Niveau 1
h Ref Niveau 2
h Ref Niveau 3
h 5=198
h 1=299
-300
-200
-100
000
100
200
300
A B C D E F G H L M
Vale
urs
de h
de M
an
del(
rob
uste
s)
Laboratoires
Meacutethode alternative analyse robuste
h Alt Niveau 1
h Alt Niveau 2
h Alt Niveau 2
h 1=299
h 5=198
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3438 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Laboratoire k Ref
Niveau 1 k Ref
Niveau 2 k Ref
Niveau 3 k Alt
Niveau 1 k Alt
Niveau 2 k Alt
Niveau 3 k1 k5
A 000 089 142 084 162 046 259 181
B 125 087 073 104 075 046 259 181
C 117 013 091 188 022 083 259 181
D 052 036 069 000 035 083 259 181
E 028 034 037 138 019 112 259 181
F 140 222 018 027 281 112 259 181
G 088 068 024 131 145 000 259 181
H 129 094 173 016 061 000 259 181
L 069 000 073 104 270 224 259 181
M 000 048 038 043 099 241 259 181
000
050
100
150
200
250
300
350
400
A B C D E F G H L M
Vale
urs
k d
e M
an
del(
rob
uste
s)
Laboratoires
Meacutethode de reacutefeacuterence analyse robuste
k Ref Niveau 1k Ref Niveau 2k Ref Niveau 3
k 1=259
k 5=181
000
050
100
150
200
250
300
A B C D E F G H L M
Vale
urs
k d
e M
an
del
(ro
bu
ste
s)
Laboratoires
Meacutethode alternative analyse robuste
k Alt Niveau 3
k Alt Niveau 1
k Alt Niveau 2
k 1=259
k 5=181
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3538 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Appendix 4 ndash Statistical results
Level 1
Nombre de laboratoires (p) 10
A 1602 1602 1602 000 0000 0000 -0495 0000 A 1845 1602 1724 017 0122 -0122 0621 0840 0122
B 1602 1176 1389 030 0213 -0213 -2613 1247 B 1602 1301 1452 021 0151 -0151 -0769 1041 0062
C 1699 1301 1500 028 0199 -0199 -1510 1165 C 1301 1845 1573 038 -0272 0272 -0148 1881 0073
D 1602 1778 1690 012 -0088 0088 0380 0516 D 1602 1602 1602 000 0000 0000 0000 0000 -0088
E 1699 1602 1651 007 0048 -0048 -0013 0284 E 1699 1301 1500 028 0199 -0199 -0522 1376 -0151
F 1778 1301 1540 034 0239 -0239 -1116 1397 F 1778 1699 1739 006 0040 -0040 0698 0274 0199
G 1845 1544 1695 021 0151 -0151 0425 0881 G 2079 1699 1889 027 0190 -0190 1467 1315 0194
H 2041 1602 1822 031 0220 -0220 1689 1286 H 2000 1954 1977 003 0023 -0023 1917 0158 0155
L 1544 1778 1661 017 -0117 0117 0092 0685 L 1301 1602 1452 021 -0151 0151 -0769 1041 -0210
M 1653 1653 1653 000 0000 0000 0013 0000 M 1477 1602 1540 009 -0062 0062 -0319 0432 -0114
m 165186 m 160206 mediane D 00678
Cn(2p) 1867 Cn(2p) 1867 Cp 16101
Cn(p) 16101 Cn(p) 16101 Qn(D) 0092926
n=2p n 20 n=2p n 20 Qdiff 01496169
f 11 f 11 t 114
l 55 l 55
n=p n 10 n=p n 10
f 6 f 6
l 15 l 15
Qn(2p) 0091465 Qn(2p) 007745
Qn(p) 0062469 Qn(p) 0121519
Qintra 017078 Qintra 014461
Qinter 010058 Qinter 019565
Sr(eacutecart-type de reacutepeacutetabiliteacute) 02415 Sr 02045
RSDr(coefficient de variation de la reacutepeacutetabiliteacute)1462 RSDr 1277
r(repeatability limit)) 0676 r 0573
SL(eacutecart-type interlaboratoire) -001905 SLlt0 SL 001737
SR(eacutecart-type de reproductibiliteacute) 02415 SR 02433
RSDR(coefficient de variation de la reproductibiliteacute)0146 RSDR 0152
R(limite de reproductibiliteacute)) 06762 R(limite de reproductibiliteacute))068123
h k h MoyenneLaboratoires
Niveau 1
si LaboratoiresDeviationMoyenne Dk DeviationsiAlt
Niveau 1
Ref
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3638 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Level 2
Nombre de laboratoires (p) 10
A 2681 2613 2647 005 0034 -0034 -0151 0891 A 2322 2531 2427 015 -0105 0105 -2119 1620 -0220
B 2544 2477 2511 005 0033 -0033 -2699 0872 B 2556 2653 2605 007 -0048 0048 -0581 0750 0094
C 2643 2633 2638 001 0005 -0005 -0311 0130 C 2681 2653 2667 002 0014 -0014 -0040 0217 0029
D 2690 2663 2676 002 0014 -0014 0400 0357 D 2778 2732 2755 003 0023 -0023 0721 0354 0079
E 2690 2716 2703 002 -0013 0013 0897 0336 E 2724 2748 2736 002 -0012 0012 0556 0185 0033
F 2568 2398 2483 012 0085 -0085 -3214 2217 F 2477 2114 2296 026 0182 -0182 -3255 2811 -0188
G 2643 2591 2617 004 0026 -0026 -0707 0682 G 2756 2568 2662 013 0094 -0094 -0085 1453 0045
H 2716 2643 2680 005 0036 -0036 0460 0945 H 2653 2732 2693 006 -0040 0040 0181 0613 0013
L 2591 2591 2591 000 0000 0000 -1196 0000 L 2230 2580 2405 025 -0175 0175 -2307 2704 -0186
M 2653 2690 2672 003 -0018 0018 0311 0481 M 2740 2613 2677 009 0064 -0064 0040 0987 0005
m 2655082 m 26719001 mediane D 00209
Cn(2p) 1867 Cn(2p) 1867 Cp 16101
Cn(p) 16101 Cn(p) 16101 Qn(D) 0039911
n=2p n 20 n=2p n 20 Qdiff 00642591
f 11 f 11 t 082
l 55 l 55
n=p n 10 n=p n 10
f 6 f 6
l 15 l 15
Qn(2p) 0020570 Qn(2p) 00345985
Qn(p) 00332437 Qn(p) 00718158
Qintra 003841 Qintra 006460
Qinter 005352 Qinter 011563
Sr(eacutecart-type de reacutepeacutetabiliteacute) 00543 Sr 00914
RSDr(coefficient de variation de la reacutepeacutetabiliteacute)205 RSDr 342
r(repeatability limit)) 0152 r 0256
SL(eacutecart-type interlaboratoire) 000139 SL 000920
SR(eacutecart-type de reproductibiliteacute) 00659 SR 01325
RSDR(coefficient de variation de la reproductibiliteacute)0025 RSDR 0050
R(limite de reproductibiliteacute)) 018446 R(limite de reproductibiliteacute))03708618
Laboratoires
Niveau 2
Moyenne si Laboratoires
Niveau 2
DRef Alt Moyenne si Deviation h Deviation h k k
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3738 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Level 2 bis
Nombre de laboratoires (p) 10
A 3000 2477 2739 037 0261 -0261 0832 1418 A 2602 2477 2540 009 0062 -0062 -0391 0465 -0199
B 2544 2813 2678 019 -0134 0134 0211 0729 B 2903 2778 2841 009 0062 -0062 1732 0465 0162
C 2477 2813 2645 024 -0168 0168 -0135 0911 C 2477 2699 2588 016 -0111 0111 -0049 0826 -0057
D 2398 2653 2526 018 -0128 0128 -1370 0692 D 2778 3000 2889 016 -0111 0111 2074 0826 0363
E 2875 2740 2808 010 0067 -0067 1547 0365 E 2602 2301 2452 021 0151 -0151 -1012 1120 -0356
F 2477 2544 2511 005 -0033 0033 -1525 0182 F 2778 2477 2628 021 0151 -0151 0230 1120 0117
G 2653 2740 2697 006 -0044 0044 0400 0236 G 2845 2845 2845 000 0000 0000 1764 0000 0148
H 2176 2813 2495 045 -0318 0318 -1692 1727 H 2477 2477 2477 000 0000 0000 -0832 0000 -0017
L 2813 2544 2678 019 0134 -0134 0211 0729 L 2903 2301 2602 043 0301 -0301 0049 2241 -0076
M 2602 2740 2671 010 -0069 0069 0135 0375 M 2903 2255 2579 046 0324 -0324 -0112 2411 -0092
m 2658114 m 259505 mediane D -00372
Cn(2p) 1867 Cn(2p) 1867 Cp 16101
Cn(p) 16101 Cn(p) 16101 Qn(D) 01656915
n=2p n 20 n=2p n 20 Qdiff 02667753
f 11 f 11 t 035
l 55 l 55
n=p n 10 n=p n 10
f 6 f 6
l 15 l 15
Qn(2p) 0098745 Qn(2p) 007195
Qn(p) 006007 Qn(p) 008805
Qintra 018437 Qintra 013435
Qinter 009672 Qinter 014176
Sr(eacutecart-type de reacutepeacutetabiliteacute) 02607 Sr 01900
RSDr(coefficient de variation de la reacutepeacutetabiliteacute)981 RSDr 732
r(repeatability limit)) 0730 r 0532
SL(eacutecart-type interlaboratoire) -002464 SLlt0 SL 000205
SR(eacutecart-type de reproductibiliteacute) 02607 SR 01953
RSDR(coefficient de variation de la reproductibiliteacute)0098 RSDR 0075
R(limite de reproductibiliteacute)) 072996 R(limite de reproductibiliteacute))054686
Laboratoires
Niveau 2 bis
Moyenne si Laboratoires
Niveau 2 bis
DRef Alt Moyenne si Deviation h Deviation h k k
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3838 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Level 3
Nombre de laboratoires (p) 10
A 3690 3663 3676 002 0014 -0014 -0311 0513 A 3690 3708 3699 001 -0009 0009 0354 0257 0022
B 3613 3672 3642 004 -0030 0030 -1851 1109 B 3708 3568 3638 010 0070 -0070 -0844 2061 -0005
C 3663 3732 3698 005 -0035 0035 0645 1302 C 3663 3699 3681 003 -0018 0018 0000 0536 -0017
D 3690 3690 3690 000 0000 0000 0311 0000 D 3643 3663 3653 001 -0010 0010 -0545 0286 -0037
E 3732 3653 3693 006 0040 -0040 0429 1480 E 3613 3732 3673 008 -0060 0060 -0162 1769 -0020
F 3643 3672 3658 002 -0014 0014 -1157 0536 F 3690 3771 3731 006 -0040 0040 0975 1193 0073
G 3699 3653 3676 003 0023 -0023 -0328 0856 G 3602 3643 3623 003 -0021 0021 -1141 0612 -0053
H 3623 3672 3648 003 -0024 0024 -1615 0913 H 3748 3681 3715 005 0033 -0033 0664 0990 0067
L 3724 3663 3694 004 0031 -0031 0461 1150 L 3748 3771 3760 002 -0011 0011 1544 0335 0066
M 3690 3690 3690 000 0000 0000 0311 0000 M 3806 3681 3744 009 0062 -0062 1234 1848 0054
m 368334 m 368086 mediane D 00089
Cn(2p) 1867 Cn(2p) 1867 Cp 16101
Cn(p) 16101 Cn(p) 16101 Qn(D) 00325359
n=2p n 20 n=2p n 20 Qdiff 00523852
f 11 f 11 t 043
l 55 l 55
n=p n 10 n=p n 10
f 6 f 6
l 15 l 15
Qn(2p) 0014323 Qn(2p) 001811
Qn(p) 0013719 Qn(p) 003164
Qintra 002674 Qintra 003381
Qinter 002209 Qinter 005094
Sr(eacutecart-type de reacutepeacutetabiliteacute) 00378 Sr 00478
RSDr(coefficient de variation de la reacutepeacutetabiliteacute)103 RSDr 130
r(repeatability limit)) 0106 r 0134
SL(eacutecart-type interlaboratoire) -000023 SLlt0 SL 000145
SR(eacutecart-type de reproductibiliteacute) 00378 SR 00611
RSDR(coefficient de variation de la reproductibiliteacute)0010 RSDR 0017
R(limite de reproductibiliteacute)) 0105894 R(limite de reproductibiliteacute))017119
Ref Alt DLaboratoires
Niveau 3
Moyenne si Deviation h k Laboratoires
Niveau 3
Moyenne si Deviation h k
Thermo Fisher Scientific
ADRIA Deacuteveloppement 1438 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Figure 4 ndash Regression straight lines
Meat products
Fishery products
Dairy products
Vegetables
Thermo Fisher Scientific
ADRIA Deacuteveloppement 1538 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Environmental samples
Egg products
All products (estimated number)
Thermo Fisher Scientific
ADRIA Deacuteveloppement 1638 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
The hypotheses that the ordinates are close to 0 and the slopes close to 1
are approved in all cases except for
- Meat products (slope P = 1 ordinate P = 1)
- Environmental samples (ordinate P = 3)
- All products (ordinate P = 0)
The bias between the two methods vary from ndash 0133 to 0008 log CFU For
all categories the bias is ndash 0032 log CFU The repeatability limits of the
alternative and the reference method are similar For all products the values
are respectively 0215 and 0196 log CFU For all products these
interpretations are provided taking into account data with low contamination
level or not (estimated colonies number)
For all food products the regression straight line is the following
log (Alt) = 1040 log Ref ndash 0182
The Listeria PrecisTM method shows satisfying relative accuracy
All products
All products (Level 2)
Thermo Fisher Scientific
ADRIA Deacuteveloppement 1738 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
2113 Detection limit (LOD) and quantification limit (LOQ)
The critical level is the defined as the smallest amount which can be detected (not null) but not
quantified as an exact value Below this value it cannot be sure that the true value is not null
The detection limit is defined as being higher than the critical level because it involves a power the
probability 1- which has to be well over 50 for example 95
The quantification limit is defined as the smallest amount of analyte (that is the lowest actual number of
organisms) which can be measured and quantified with defined precision and accuracy under the
experimental conditions by the method under validation
Protocol
The detection limit of the alternative method was realised with pure cultures
Three different inoculation levels were tested with six replicates per level ie
18 analyses were done by the alternative method
Listeria monocytogenes 18 was used for the detection limit of the alternative
method
Quantification limit was calculated for six independent blank samples
determinations
Results
These data are intrinsic to the method used and are presented in the
following tables
Table 8 ndash Inoculation 1 ml on 3 plates
Level (CFUml) Positive samples number Standard deviation Bias
0 06
05 26 1211 0
1 36 0816 05
5 66 2483 85
Thermo Fisher Scientific
ADRIA Deacuteveloppement 1838 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Table 9 ndash Inoculation 01 ml on 1 plate
Level (CFUml) Positive samples number Standard deviation Bias
0 06
5 36 0548 05
10 46 1414 2
50 66 2258 55
Table 10
Formulas Obtained values
LC 165 S0 + X0 18
LOD 33 S0 + X0 32
LOQ 10 S0 + X0 87
2114 Relative Sensitivity
The relative sensitivity is defined as the ability of the alternative method to detect two different amounts
of analyte measured by the reference method within a given matrix at a specified average value or
over the whole measurement range that is it is the minimal quantity variation (increase of the analyte
concentration x) which gives a significant variation of the measured signal (response y)
These data are intrinsic to the used method and are obtained from the results
of the linearity study
Sensitivity patterns obtained for tested (matrixstrain) pairs are presented in
figure 5
Thermo Fisher Scientific
ADRIA Deacuteveloppement 1938 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Figure 5 ndash Sensitivity patterns for the tested (matrixstrain) pairs
2115 SpecificitySelectivity
The specificity is defined as the degree to which a method is affected (or not) by the other components
present in a multi-component sample That is the ability of a method to measure exactly a given
analyte or its amount within the sample without interference from non-target components such as a
matrix effect or background noise
The selectivity is defined as a measure of the degree of non-interference in the presence of non-target
analytes A method is selective if it can be used to detect the analyte under examination and that a
guarantee can be provided that the detected signal can only be a product by that specific analyte
The specificity and selectivity study was performed for the Listeria PrecisTM
detection method no additional test was realised for the enumeration
method The results were the following
- Among the Listeria monocytogenes strains tested 2 strains (Listeria
monocytogenes CIP 7834 and Listeria monocytogenes CIP 105459) gave
negative results These 2 strains were tested twice by running 48 h
incubation time of the compared agars Positive results were then
observed by the alternative method Pale characteristic colonies were
observed on the ISO agar by streaking the Half Fraser broth only
- No cross reaction was observed with the non-target strains except with
Listeria ivanovii which gave characteristic colonies on Brilliancetrade Listeria
Agar but the confirmatory tests provide a clear discrimination between
Listeria monocytogenes and Listeria ivanovi species
Rillettes
Lettuce
Raw milk
Smoked salmon
Raw egg
Process water
x (CFUg)
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2038 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
212 Practicability
1 Packaging Ready to use Brilliance Listeria agar plates are available
(x 10 plates)
2 Reagent volume
3 Storage conditionsndash
Shelf life of products
not opened
The plates are stored at 6 - 12degC in darkness conditions The
expiration date is mentioned on the package and on the plates It
is around 3 months
4 Modalities of use
after first use
5 Required
equipments No specific equipment is required
6 Reagents Ready to use plates
7 Training 05 day
8 Real time handling
Reference method
9 min for one sample
55 min for 10 samples
Alternative method
7 min for one sample
35 minutes for 10 samples
9 Time to result
Negative results
Reference method Day 2
Alternative method Day 2
Negative results after confirmation or positive results
Reference method Day 4 ndash Day 7
Alternative method Day 4 ndash Day 7
Positive results
Reference method Day 4 ndash Day 7
Alternative method Day 2 ndash Day 3 (with OBIS Mono Test)
or Day 4 ndash Day 7 (by the classical tests described in the
standardised method)
10 Technician skill Identical to that required for the reference method
11 Steps common to
the reference method Mother suspension revivification step and decimal dilutions
12 Traceability
13 Maintenance
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2138 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
213 Inter-laboratory study
2131 Organisation
12 laboratories participated to this study Pasteurised milk was inoculated by
Listeria monocytogenes 12a isolated from raw milk The targeted
inoculation levels were
- 0 CFUml
- 50 CFU ml
- 500 CFU ml
- 5 000 CFU ml
Each laboratory received eight flasks of 10 ml sample ie two flasks per
inoculation level Furthermore one non-inoculated sample has been added
to the package for total viable count microflora (NF ISO 4833 method)
Detailed instructions were transmitted to the collaborators by the expert
laboratory
Collaborative study laboratories and the expert laboratory carried out the
analyses with the alternative and reference methods
2132 Verification of experimental parameters
Strain stability during transport
In order to evaluate the Listeria monocytogenes strain stability during
transport bacterial count of all inoculated flasks were checked at different
time ie inoculation time after 24 h of conservation at 2deg - 8degC Results are
reported in table 11
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2238 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Table 11 ndash Listeria monocytogenes count
by ISO 11290-2A1 method (log CFUml)
Day of the analyses Level contamination Reference method
Day 0
(29052006)
1 45
2 440
3 4 500
Day 1
(30052006)
1 60
2 430
3 4 900
No evolution of the strain was observed after storage for 24 h at 2degC ndash 8degC
Contamination level
The real contamination levels are provided in table 12
Table 12 ndash Real contamination level (CFUml)
Target inoculation
(CFU ml) Samples Ndeg
Real contamination levels
(CFUml)
0 1 - 8 0
50 5 - 6 45
500 3 - 7 440
5000 2 - 4 4 500
Sample temperature at receipt
Measured temperatures at receipt are listed in table 13
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2338 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Table 13 - Sample temperature on receipt
Laboratories Receipt
day
Hour
receipt
Measured temperature
at receipt (degC)
Measured temperature by the
temperature probe (degC)
A
B
C
D
E
F
G
H
I
J
K
L
M
30052006
10h45
10h15
11h15
10h00
09h30
8h30
11h50
10h00
08h30
14h45
08h30
09h45
08h45
82
55
45
63
60
62
81
38
48
-
14
67
76
03
03
-13
03
04
-14
05
04
01
05
-13
04
Conclusion
The temperatures during the transport were correct for all the labs except for
Lab J which didnrsquot provide the information
All the labs received their packages with several broken flasks
Three labs (B D and L) received some damaged samples but proceeded to
analyses
All the labs started the analyses at Day 1 except Lab J
2133 Results
Aerobic mesophilic flora
The count of aerobic mesophilic flora varied from 94 102 CFUml to
33 103 CFUml
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2438 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Expert lab results
The results obtained by the expert lab are the following
Table 14
Target inoculation level (CFUml)
Reference method ISO 11290-2
Alternative method Listeria Precis Enumeration
Rep 1 Rep 2 Rep 1 Rep 2
lt 10 lt 10 lt 10 lt 10 lt10
50 50 (Ne) 60 (Ne) 50 (Ne) 30 (Ne)
500 470 390 490 500
5 000 4 700 5 100 5 900 3 400
Ne estimated number
The obtained results are in agreement with those expected
Collaborator Labs results
A synthesis of the results obtained by the collaborator labs is provided
Table 15 (CFUml) and Table 16 (log CFUml)
Table 15 ndash Synthesis of results obtained by the alternative
and the reference methods (CFUml)
Labs Level 1 Level 2 Level 2 bis Level 3
Reference method
Alternative method
Reference method
Alternative method
Reference method
Alternative method
Reference method
Alternative method
A 40 40 70 40 480 410 210 340 1000 300 400 300 4900 4600 4900 5100
B 40 15 40 20 350 300 360 450 350 650 800 600 4100 4700 5100 3700
C 50 20 20 70 440 430 480 450 300 650 300 500 4600 5400 4600 5000
D 40 60 40 40 490 460 600 540 250 450 600 1000 4900 4900 4400 4600
E 50 40 50 20 490 520 530 560 750 550 400 200 5400 4500 4100 5400
F 60 20 60 50 370 250 300 130 300 350 600 300 4400 4700 4900 5900
G 70 35 120 50 440 390 570 370 450 550 700 700 5000 4500 4000 4400
H 110 40 100 90 520 440 450 540 150 650 300 300 4200 4700 5600 4800
L 35 60 20 40 390 390 170 380 650 350 800 200 5300 4600 5600 5900
M 45 45 30 40 450 490 550 410 400 550 800 180 4900 4900 6400 4800
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2538 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Table 16 ndash Synthesis of results obtained by the alternative
and the reference methods (log CFUml)
Labs Level 1 Level 2 Level 2 bis Level 3
Reference method
Alternative method
Reference method
Alternative method
Reference method
Alternative method
Reference method
Alternative method
A 1602 1602 1845 1602 2681 2613 2322 2531 3000 2477 2602 2477 3690 3663 3690 3708
B 1602 1176 1602 1301 2544 2477 2556 2653 2544 2813 2903 2778 3613 3672 3708 3568
C 1699 1301 1301 1845 2643 2633 2681 2653 2477 2813 2477 2699 3663 3732 3663 3699
D 1602 1778 1602 1602 2690 2663 2778 2732 2398 2653 2778 3000 3690 3690 3643 3663
E 1699 1602 1699 1301 2690 2716 2724 2748 2875 2740 2602 2301 3732 3653 3613 3732
F 1778 1301 1778 1699 2568 2398 2477 2114 2477 2544 2778 2477 3643 3672 3690 3771
G 1845 1544 2079 1699 2643 2591 2756 2568 2653 2740 2845 2845 3699 3653 3602 3643
H 2041 1602 2000 1954 2716 2643 2653 2732 2176 2813 2477 2477 3623 3672 3748 3681
L 1544 1778 1301 1602 2591 2591 2230 2580 2813 2544 2903 2301 3724 3663 3748 3771
M 1653 1653 1477 1602 2653 2690 2740 2613 2602 2740 2903 2255 3690 3690 3806 3681
Level 2 results obtained taking account the 3 plates inoculated with the mother-suspension
Level 2 bis results obtained taking account the plates inoculated with 01 ml of the mother-suspension
Scrutiny of the measurement results for consistency
In order to identify the measurement results or laboratories that could be
inconsistent two graphical consistency techniques were realised Mandelrsquos
h-ank-statistics Mandel indicators h and k at 5 significance highlighted
some possible inconsistent data (See tables and graphics in Appendix 3 and
Appendix 4)
Mandelrsquos values Number of values observed over the threshold
Reference method Alternative method
h gt 1 Lab F Level 2 Lab F Level 2
h gt 5 Lab F Level 2
Lab B Levels 1 and 2
Lab A Level 2
Lab D Level 3
Lab F Level 2
Lab L Level 2
kgt 1 Lab F Level 3
k gt 5 Lab F Level 2 Lab C Level 2
Lab F Level 3
Lab L Levels 1 and 3
Lab M Level 1
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2638 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
The emphasized results appear (i) coherent with the targeted contamination
levels (ii) concordant between both methods
All the results were thus kept for the statistical analysis
Comparison of the trueness and precision characteristics of
the reference method and alternative methods
The statistical values are summarised hereafter
Level p
Reference method Alternative method
Repeatability ratio
Reproducibility ratio Median
Repeatability standard deviation
Reproducibility standard deviation
Median Repeatability
standard deviation
Reproducibility standard deviation
1 10 16519 02415 02425 16021 02045 02433 0847 1007
2 10 26551 00543 00659 26719 00914 01325 1682 2011
2 bis 10 26581 02607 02607 25951 01900 01953 0729 0749
3 10 18671 00378 00378 18671 00478 00611 1264 1617
Bias of the alternative method
In order to estimate the bias of the alternative method with regard to the reference method for every
level Dij and t are calculated according to the following equations
RefAlt ijYijYDij
Diffp
Dijit
)2(
)(median
If the t value is superior to 2 the alternative method is significantly biased
with regard to the reference method
The values are given in table 17
Table 17 ndash t (d) values obtained by level
Level Bias t (d) Conclusion
1 0068 114 Non significant bias
2 0021 082 Non significant bias
2 bis - 0037 035 Non significant bias
3 - 0037 043 Non significant bias
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2738 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
The statistical test concludes to a non significant bias for all the inoculation
levels The bias varies from ndash 0037 to 0068 log CFUml
Comparison of the repeatability standard deviations
If the ratio Srj Alt Srj Ref of the repeatability standard deviations of the
alternative method and the reference method is larger than 2 the precision
under repeatability conditions of the alternative method is considered to be
lower than that of the reference method If this ratio is smaller than 05 the
precision under repeatability conditions of the alternative method is
considered to be greater than that of the reference method
The ratio values are given in table 18
Table 18
Level
Repeatability standard deviation
Reference method Alternative method Alternative method
reference method ratio
1 0242 0205 0847
2 0054 0091 1682
2 bis 0261 0190 0729
3 0038 0048 1264
Level Repeatability limit
Reference method Alternative method
1 0676 0573
2 0152 0256
2 bis 0730 0532
3 0106 0134
The ratios of the repeatability standard deviations are below 2 in all cases
The precision under repeatability conditions of the alternative method is
equivalent to that of the reference method
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2838 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Comparison of the reproducibility standard deviations
If the ratio Srj Alt Srj Ref of the reproducibility standard deviations of the alternative method and the
reference method is larger than 2 the precision under reproducibility conditions of the alternative
method is considered to be lower than that of the reference method If this ratio is smaller than 05 the
precision under reproducibility conditions of the alternative method is considered to be greater than
that of the reference method
The ratio values are given in table 19
Table 19
Level
Reproducibility standard deviation
Reference method Alternative method Alternative method
reference method ratio
1 0242 0243 1007
2 0066 0133 2011
2 bis 0261 0195 0749
3 0038 0061 1617
Level Reproducibility limit
Reference method Alternative method
1 0676 0681
2 0185 0371
2 bis 0730 0547
3 0106 0171
The ratios of the reproducibility standard deviations are below or close to 2
The precision under reproducibility conditions of the alternative method is
equivalent to that of the reference method
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2938 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
214 Conclusion
Method comparison study
The Listeria PrecisTM enumeration method shows satisfying linearity and
relative accuracy
The alternative method shows satisfying inclusivity and exclusivity results
Two Listeria monocytogenes strains gave negative results with the
alternative method These strains grew weakly on the reference method
media (OampA Palcam and Oxford)
The limits of repeatability of the alternative method are similar to those of the
reference method
The bias between both methods are low between - 0133 Log CFUg and
- 0008 Log CFUg
The Listeria PrecisTM enumeration method offers the saving for confirmatory
tests
Positive results are obtained with the alternative method in 2-3 days (with
OBIS Mono test) or in 4-7 days (by the classical tests described in the
standardized method) against 4-7 days for the reference method
Inter-laboratory study
The bias between the reference and the alternative methods shows satisfying
and correct values
- level 1 (100 - 1 000 CFUg) bias = + 007 log CFUg
- level 2 (1 000 - 10 000 CFUg) bias = 002 log CFUg
- level 2 bis (1 000 - 10 000 CFUg) bias = - 004 log CFUg
- level 3 (10 000 - 100 000 CFUg) bias = - 004 log CFUg
The repeatability and the reproducibility of the alternative method look similar
to those of the reference method
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3038 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
22 Extension study (realised by ASEPT 2007)
An extension study was performed in 2007 for a new confirmatory test OBIS
MONO
150 Listeria monocytogenes strains and 100 non-target strains were isolated
onto TSAYE and BrillianceTM Listeria Agar plates All the Listeria
monocytogenes and the non-target strains gave the expected results
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3138 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Appendix 1 ndash Flow diagram of the Listeria PrecisTM method
x g sample + 9 x ml buffered peptone water
Revivification step for 1 h plusmn 5 min at 20degC plusmn 1degC
Streaking 1 ml onto 3 Brilliance Listeria Agar plates (mother suspension)
or 01 ml onto 1 Brilliance Listeria Agar plates (mother suspension and decimal dilutions)
Incubation for 48 h plusmn 3 h at 37degC plusmn 1degC
Confirmatory tests on at least one typical colony
- by the tests described in the ISO method
- by applying the OBIS mono test on an isolated colony
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3238 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Appendix 2 ndash ISO 11290-2 (1998) amp ISO 11290-2A1 (2004) Horizontal
method for the detection and enumeration of Listeria monocytogenes -
Part 2 enumeration of Listeria monocytogenes in foods
10 g cheese + 90 ml buffered peptone water or Half Fraser Broth (without supplements) (110 dilution)
Stomach 1 min 30 seconds
Keep at 20degC plusmn 2degC for 1 h plusmn 5 min
Decimal dilutions in 9 ml peptone salt tubes
Dilution 110
Dilution 1100
Dilution 11000
1 ml on 3 Chromogenic Listeria medium (ISO) plates (x 2) (-1)
01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-2)
01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-3)
01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-4)
Incubation at 37degC plusmn 1degC
24 h - 48 h
Numerate typical colonies at 24 h
(bluegreen surrounded by an opaque halo)
Numerate at 48 h
Streak typical colonies on TSYEA
(5 coloniesplate 2 successive dilutions)
Confirmation tests
Catalase
Gram stain
For confirmation of L monocytogenes
Haemolysis
Carbohydrate (rhamnose and xylose) utilisation
CAMP Test
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3338 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Appendix 3 ndash Mandelrsquos graphics
Laboratoire h Ref
Niveau 1 h Ref
Niveau 2 h Ref
Niveau 3 h Alt
Niveau 1 h Alt
Niveau 2 h Alt
Niveau 3 h5 h1 h5 h1
A -050 -015 083 062 -212 -039 198 299 -198 -299
B -261 -270 021 -077 -058 173 198 299 -198 -299
C -151 -031 -014 -015 -004 -005 198 299 -198 -299
D 038 040 -137 000 072 207 198 299 -198 -299
E -001 090 155 -052 056 -101 198 299 -198 -299
F -112 -321 -153 070 -325 023 198 299 -198 -299
G 042 -071 040 147 -009 176 198 299 -198 -299
H 169 046 -169 192 018 -083 198 299 -198 -299
L 009 -120 021 -077 -231 005 198 299 -198 -299
M 001 031 014 -032 004 -011 198 299 -198 -299
-300
-200
-100
000
100
200
300
A B C D E F G H L M
Vale
urs
h d
e M
an
del(
rob
uste
s)
Laboratoires
Meacutethode de reacutefeacuterence analyse robuste
h Ref Niveau 1
h Ref Niveau 2
h Ref Niveau 3
h 5=198
h 1=299
-300
-200
-100
000
100
200
300
A B C D E F G H L M
Vale
urs
de h
de M
an
del(
rob
uste
s)
Laboratoires
Meacutethode alternative analyse robuste
h Alt Niveau 1
h Alt Niveau 2
h Alt Niveau 2
h 1=299
h 5=198
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3438 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Laboratoire k Ref
Niveau 1 k Ref
Niveau 2 k Ref
Niveau 3 k Alt
Niveau 1 k Alt
Niveau 2 k Alt
Niveau 3 k1 k5
A 000 089 142 084 162 046 259 181
B 125 087 073 104 075 046 259 181
C 117 013 091 188 022 083 259 181
D 052 036 069 000 035 083 259 181
E 028 034 037 138 019 112 259 181
F 140 222 018 027 281 112 259 181
G 088 068 024 131 145 000 259 181
H 129 094 173 016 061 000 259 181
L 069 000 073 104 270 224 259 181
M 000 048 038 043 099 241 259 181
000
050
100
150
200
250
300
350
400
A B C D E F G H L M
Vale
urs
k d
e M
an
del(
rob
uste
s)
Laboratoires
Meacutethode de reacutefeacuterence analyse robuste
k Ref Niveau 1k Ref Niveau 2k Ref Niveau 3
k 1=259
k 5=181
000
050
100
150
200
250
300
A B C D E F G H L M
Vale
urs
k d
e M
an
del
(ro
bu
ste
s)
Laboratoires
Meacutethode alternative analyse robuste
k Alt Niveau 3
k Alt Niveau 1
k Alt Niveau 2
k 1=259
k 5=181
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3538 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Appendix 4 ndash Statistical results
Level 1
Nombre de laboratoires (p) 10
A 1602 1602 1602 000 0000 0000 -0495 0000 A 1845 1602 1724 017 0122 -0122 0621 0840 0122
B 1602 1176 1389 030 0213 -0213 -2613 1247 B 1602 1301 1452 021 0151 -0151 -0769 1041 0062
C 1699 1301 1500 028 0199 -0199 -1510 1165 C 1301 1845 1573 038 -0272 0272 -0148 1881 0073
D 1602 1778 1690 012 -0088 0088 0380 0516 D 1602 1602 1602 000 0000 0000 0000 0000 -0088
E 1699 1602 1651 007 0048 -0048 -0013 0284 E 1699 1301 1500 028 0199 -0199 -0522 1376 -0151
F 1778 1301 1540 034 0239 -0239 -1116 1397 F 1778 1699 1739 006 0040 -0040 0698 0274 0199
G 1845 1544 1695 021 0151 -0151 0425 0881 G 2079 1699 1889 027 0190 -0190 1467 1315 0194
H 2041 1602 1822 031 0220 -0220 1689 1286 H 2000 1954 1977 003 0023 -0023 1917 0158 0155
L 1544 1778 1661 017 -0117 0117 0092 0685 L 1301 1602 1452 021 -0151 0151 -0769 1041 -0210
M 1653 1653 1653 000 0000 0000 0013 0000 M 1477 1602 1540 009 -0062 0062 -0319 0432 -0114
m 165186 m 160206 mediane D 00678
Cn(2p) 1867 Cn(2p) 1867 Cp 16101
Cn(p) 16101 Cn(p) 16101 Qn(D) 0092926
n=2p n 20 n=2p n 20 Qdiff 01496169
f 11 f 11 t 114
l 55 l 55
n=p n 10 n=p n 10
f 6 f 6
l 15 l 15
Qn(2p) 0091465 Qn(2p) 007745
Qn(p) 0062469 Qn(p) 0121519
Qintra 017078 Qintra 014461
Qinter 010058 Qinter 019565
Sr(eacutecart-type de reacutepeacutetabiliteacute) 02415 Sr 02045
RSDr(coefficient de variation de la reacutepeacutetabiliteacute)1462 RSDr 1277
r(repeatability limit)) 0676 r 0573
SL(eacutecart-type interlaboratoire) -001905 SLlt0 SL 001737
SR(eacutecart-type de reproductibiliteacute) 02415 SR 02433
RSDR(coefficient de variation de la reproductibiliteacute)0146 RSDR 0152
R(limite de reproductibiliteacute)) 06762 R(limite de reproductibiliteacute))068123
h k h MoyenneLaboratoires
Niveau 1
si LaboratoiresDeviationMoyenne Dk DeviationsiAlt
Niveau 1
Ref
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3638 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Level 2
Nombre de laboratoires (p) 10
A 2681 2613 2647 005 0034 -0034 -0151 0891 A 2322 2531 2427 015 -0105 0105 -2119 1620 -0220
B 2544 2477 2511 005 0033 -0033 -2699 0872 B 2556 2653 2605 007 -0048 0048 -0581 0750 0094
C 2643 2633 2638 001 0005 -0005 -0311 0130 C 2681 2653 2667 002 0014 -0014 -0040 0217 0029
D 2690 2663 2676 002 0014 -0014 0400 0357 D 2778 2732 2755 003 0023 -0023 0721 0354 0079
E 2690 2716 2703 002 -0013 0013 0897 0336 E 2724 2748 2736 002 -0012 0012 0556 0185 0033
F 2568 2398 2483 012 0085 -0085 -3214 2217 F 2477 2114 2296 026 0182 -0182 -3255 2811 -0188
G 2643 2591 2617 004 0026 -0026 -0707 0682 G 2756 2568 2662 013 0094 -0094 -0085 1453 0045
H 2716 2643 2680 005 0036 -0036 0460 0945 H 2653 2732 2693 006 -0040 0040 0181 0613 0013
L 2591 2591 2591 000 0000 0000 -1196 0000 L 2230 2580 2405 025 -0175 0175 -2307 2704 -0186
M 2653 2690 2672 003 -0018 0018 0311 0481 M 2740 2613 2677 009 0064 -0064 0040 0987 0005
m 2655082 m 26719001 mediane D 00209
Cn(2p) 1867 Cn(2p) 1867 Cp 16101
Cn(p) 16101 Cn(p) 16101 Qn(D) 0039911
n=2p n 20 n=2p n 20 Qdiff 00642591
f 11 f 11 t 082
l 55 l 55
n=p n 10 n=p n 10
f 6 f 6
l 15 l 15
Qn(2p) 0020570 Qn(2p) 00345985
Qn(p) 00332437 Qn(p) 00718158
Qintra 003841 Qintra 006460
Qinter 005352 Qinter 011563
Sr(eacutecart-type de reacutepeacutetabiliteacute) 00543 Sr 00914
RSDr(coefficient de variation de la reacutepeacutetabiliteacute)205 RSDr 342
r(repeatability limit)) 0152 r 0256
SL(eacutecart-type interlaboratoire) 000139 SL 000920
SR(eacutecart-type de reproductibiliteacute) 00659 SR 01325
RSDR(coefficient de variation de la reproductibiliteacute)0025 RSDR 0050
R(limite de reproductibiliteacute)) 018446 R(limite de reproductibiliteacute))03708618
Laboratoires
Niveau 2
Moyenne si Laboratoires
Niveau 2
DRef Alt Moyenne si Deviation h Deviation h k k
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3738 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Level 2 bis
Nombre de laboratoires (p) 10
A 3000 2477 2739 037 0261 -0261 0832 1418 A 2602 2477 2540 009 0062 -0062 -0391 0465 -0199
B 2544 2813 2678 019 -0134 0134 0211 0729 B 2903 2778 2841 009 0062 -0062 1732 0465 0162
C 2477 2813 2645 024 -0168 0168 -0135 0911 C 2477 2699 2588 016 -0111 0111 -0049 0826 -0057
D 2398 2653 2526 018 -0128 0128 -1370 0692 D 2778 3000 2889 016 -0111 0111 2074 0826 0363
E 2875 2740 2808 010 0067 -0067 1547 0365 E 2602 2301 2452 021 0151 -0151 -1012 1120 -0356
F 2477 2544 2511 005 -0033 0033 -1525 0182 F 2778 2477 2628 021 0151 -0151 0230 1120 0117
G 2653 2740 2697 006 -0044 0044 0400 0236 G 2845 2845 2845 000 0000 0000 1764 0000 0148
H 2176 2813 2495 045 -0318 0318 -1692 1727 H 2477 2477 2477 000 0000 0000 -0832 0000 -0017
L 2813 2544 2678 019 0134 -0134 0211 0729 L 2903 2301 2602 043 0301 -0301 0049 2241 -0076
M 2602 2740 2671 010 -0069 0069 0135 0375 M 2903 2255 2579 046 0324 -0324 -0112 2411 -0092
m 2658114 m 259505 mediane D -00372
Cn(2p) 1867 Cn(2p) 1867 Cp 16101
Cn(p) 16101 Cn(p) 16101 Qn(D) 01656915
n=2p n 20 n=2p n 20 Qdiff 02667753
f 11 f 11 t 035
l 55 l 55
n=p n 10 n=p n 10
f 6 f 6
l 15 l 15
Qn(2p) 0098745 Qn(2p) 007195
Qn(p) 006007 Qn(p) 008805
Qintra 018437 Qintra 013435
Qinter 009672 Qinter 014176
Sr(eacutecart-type de reacutepeacutetabiliteacute) 02607 Sr 01900
RSDr(coefficient de variation de la reacutepeacutetabiliteacute)981 RSDr 732
r(repeatability limit)) 0730 r 0532
SL(eacutecart-type interlaboratoire) -002464 SLlt0 SL 000205
SR(eacutecart-type de reproductibiliteacute) 02607 SR 01953
RSDR(coefficient de variation de la reproductibiliteacute)0098 RSDR 0075
R(limite de reproductibiliteacute)) 072996 R(limite de reproductibiliteacute))054686
Laboratoires
Niveau 2 bis
Moyenne si Laboratoires
Niveau 2 bis
DRef Alt Moyenne si Deviation h Deviation h k k
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3838 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Level 3
Nombre de laboratoires (p) 10
A 3690 3663 3676 002 0014 -0014 -0311 0513 A 3690 3708 3699 001 -0009 0009 0354 0257 0022
B 3613 3672 3642 004 -0030 0030 -1851 1109 B 3708 3568 3638 010 0070 -0070 -0844 2061 -0005
C 3663 3732 3698 005 -0035 0035 0645 1302 C 3663 3699 3681 003 -0018 0018 0000 0536 -0017
D 3690 3690 3690 000 0000 0000 0311 0000 D 3643 3663 3653 001 -0010 0010 -0545 0286 -0037
E 3732 3653 3693 006 0040 -0040 0429 1480 E 3613 3732 3673 008 -0060 0060 -0162 1769 -0020
F 3643 3672 3658 002 -0014 0014 -1157 0536 F 3690 3771 3731 006 -0040 0040 0975 1193 0073
G 3699 3653 3676 003 0023 -0023 -0328 0856 G 3602 3643 3623 003 -0021 0021 -1141 0612 -0053
H 3623 3672 3648 003 -0024 0024 -1615 0913 H 3748 3681 3715 005 0033 -0033 0664 0990 0067
L 3724 3663 3694 004 0031 -0031 0461 1150 L 3748 3771 3760 002 -0011 0011 1544 0335 0066
M 3690 3690 3690 000 0000 0000 0311 0000 M 3806 3681 3744 009 0062 -0062 1234 1848 0054
m 368334 m 368086 mediane D 00089
Cn(2p) 1867 Cn(2p) 1867 Cp 16101
Cn(p) 16101 Cn(p) 16101 Qn(D) 00325359
n=2p n 20 n=2p n 20 Qdiff 00523852
f 11 f 11 t 043
l 55 l 55
n=p n 10 n=p n 10
f 6 f 6
l 15 l 15
Qn(2p) 0014323 Qn(2p) 001811
Qn(p) 0013719 Qn(p) 003164
Qintra 002674 Qintra 003381
Qinter 002209 Qinter 005094
Sr(eacutecart-type de reacutepeacutetabiliteacute) 00378 Sr 00478
RSDr(coefficient de variation de la reacutepeacutetabiliteacute)103 RSDr 130
r(repeatability limit)) 0106 r 0134
SL(eacutecart-type interlaboratoire) -000023 SLlt0 SL 000145
SR(eacutecart-type de reproductibiliteacute) 00378 SR 00611
RSDR(coefficient de variation de la reproductibiliteacute)0010 RSDR 0017
R(limite de reproductibiliteacute)) 0105894 R(limite de reproductibiliteacute))017119
Ref Alt DLaboratoires
Niveau 3
Moyenne si Deviation h k Laboratoires
Niveau 3
Moyenne si Deviation h k
Thermo Fisher Scientific
ADRIA Deacuteveloppement 1538 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Environmental samples
Egg products
All products (estimated number)
Thermo Fisher Scientific
ADRIA Deacuteveloppement 1638 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
The hypotheses that the ordinates are close to 0 and the slopes close to 1
are approved in all cases except for
- Meat products (slope P = 1 ordinate P = 1)
- Environmental samples (ordinate P = 3)
- All products (ordinate P = 0)
The bias between the two methods vary from ndash 0133 to 0008 log CFU For
all categories the bias is ndash 0032 log CFU The repeatability limits of the
alternative and the reference method are similar For all products the values
are respectively 0215 and 0196 log CFU For all products these
interpretations are provided taking into account data with low contamination
level or not (estimated colonies number)
For all food products the regression straight line is the following
log (Alt) = 1040 log Ref ndash 0182
The Listeria PrecisTM method shows satisfying relative accuracy
All products
All products (Level 2)
Thermo Fisher Scientific
ADRIA Deacuteveloppement 1738 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
2113 Detection limit (LOD) and quantification limit (LOQ)
The critical level is the defined as the smallest amount which can be detected (not null) but not
quantified as an exact value Below this value it cannot be sure that the true value is not null
The detection limit is defined as being higher than the critical level because it involves a power the
probability 1- which has to be well over 50 for example 95
The quantification limit is defined as the smallest amount of analyte (that is the lowest actual number of
organisms) which can be measured and quantified with defined precision and accuracy under the
experimental conditions by the method under validation
Protocol
The detection limit of the alternative method was realised with pure cultures
Three different inoculation levels were tested with six replicates per level ie
18 analyses were done by the alternative method
Listeria monocytogenes 18 was used for the detection limit of the alternative
method
Quantification limit was calculated for six independent blank samples
determinations
Results
These data are intrinsic to the method used and are presented in the
following tables
Table 8 ndash Inoculation 1 ml on 3 plates
Level (CFUml) Positive samples number Standard deviation Bias
0 06
05 26 1211 0
1 36 0816 05
5 66 2483 85
Thermo Fisher Scientific
ADRIA Deacuteveloppement 1838 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Table 9 ndash Inoculation 01 ml on 1 plate
Level (CFUml) Positive samples number Standard deviation Bias
0 06
5 36 0548 05
10 46 1414 2
50 66 2258 55
Table 10
Formulas Obtained values
LC 165 S0 + X0 18
LOD 33 S0 + X0 32
LOQ 10 S0 + X0 87
2114 Relative Sensitivity
The relative sensitivity is defined as the ability of the alternative method to detect two different amounts
of analyte measured by the reference method within a given matrix at a specified average value or
over the whole measurement range that is it is the minimal quantity variation (increase of the analyte
concentration x) which gives a significant variation of the measured signal (response y)
These data are intrinsic to the used method and are obtained from the results
of the linearity study
Sensitivity patterns obtained for tested (matrixstrain) pairs are presented in
figure 5
Thermo Fisher Scientific
ADRIA Deacuteveloppement 1938 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Figure 5 ndash Sensitivity patterns for the tested (matrixstrain) pairs
2115 SpecificitySelectivity
The specificity is defined as the degree to which a method is affected (or not) by the other components
present in a multi-component sample That is the ability of a method to measure exactly a given
analyte or its amount within the sample without interference from non-target components such as a
matrix effect or background noise
The selectivity is defined as a measure of the degree of non-interference in the presence of non-target
analytes A method is selective if it can be used to detect the analyte under examination and that a
guarantee can be provided that the detected signal can only be a product by that specific analyte
The specificity and selectivity study was performed for the Listeria PrecisTM
detection method no additional test was realised for the enumeration
method The results were the following
- Among the Listeria monocytogenes strains tested 2 strains (Listeria
monocytogenes CIP 7834 and Listeria monocytogenes CIP 105459) gave
negative results These 2 strains were tested twice by running 48 h
incubation time of the compared agars Positive results were then
observed by the alternative method Pale characteristic colonies were
observed on the ISO agar by streaking the Half Fraser broth only
- No cross reaction was observed with the non-target strains except with
Listeria ivanovii which gave characteristic colonies on Brilliancetrade Listeria
Agar but the confirmatory tests provide a clear discrimination between
Listeria monocytogenes and Listeria ivanovi species
Rillettes
Lettuce
Raw milk
Smoked salmon
Raw egg
Process water
x (CFUg)
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2038 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
212 Practicability
1 Packaging Ready to use Brilliance Listeria agar plates are available
(x 10 plates)
2 Reagent volume
3 Storage conditionsndash
Shelf life of products
not opened
The plates are stored at 6 - 12degC in darkness conditions The
expiration date is mentioned on the package and on the plates It
is around 3 months
4 Modalities of use
after first use
5 Required
equipments No specific equipment is required
6 Reagents Ready to use plates
7 Training 05 day
8 Real time handling
Reference method
9 min for one sample
55 min for 10 samples
Alternative method
7 min for one sample
35 minutes for 10 samples
9 Time to result
Negative results
Reference method Day 2
Alternative method Day 2
Negative results after confirmation or positive results
Reference method Day 4 ndash Day 7
Alternative method Day 4 ndash Day 7
Positive results
Reference method Day 4 ndash Day 7
Alternative method Day 2 ndash Day 3 (with OBIS Mono Test)
or Day 4 ndash Day 7 (by the classical tests described in the
standardised method)
10 Technician skill Identical to that required for the reference method
11 Steps common to
the reference method Mother suspension revivification step and decimal dilutions
12 Traceability
13 Maintenance
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2138 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
213 Inter-laboratory study
2131 Organisation
12 laboratories participated to this study Pasteurised milk was inoculated by
Listeria monocytogenes 12a isolated from raw milk The targeted
inoculation levels were
- 0 CFUml
- 50 CFU ml
- 500 CFU ml
- 5 000 CFU ml
Each laboratory received eight flasks of 10 ml sample ie two flasks per
inoculation level Furthermore one non-inoculated sample has been added
to the package for total viable count microflora (NF ISO 4833 method)
Detailed instructions were transmitted to the collaborators by the expert
laboratory
Collaborative study laboratories and the expert laboratory carried out the
analyses with the alternative and reference methods
2132 Verification of experimental parameters
Strain stability during transport
In order to evaluate the Listeria monocytogenes strain stability during
transport bacterial count of all inoculated flasks were checked at different
time ie inoculation time after 24 h of conservation at 2deg - 8degC Results are
reported in table 11
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2238 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Table 11 ndash Listeria monocytogenes count
by ISO 11290-2A1 method (log CFUml)
Day of the analyses Level contamination Reference method
Day 0
(29052006)
1 45
2 440
3 4 500
Day 1
(30052006)
1 60
2 430
3 4 900
No evolution of the strain was observed after storage for 24 h at 2degC ndash 8degC
Contamination level
The real contamination levels are provided in table 12
Table 12 ndash Real contamination level (CFUml)
Target inoculation
(CFU ml) Samples Ndeg
Real contamination levels
(CFUml)
0 1 - 8 0
50 5 - 6 45
500 3 - 7 440
5000 2 - 4 4 500
Sample temperature at receipt
Measured temperatures at receipt are listed in table 13
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2338 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Table 13 - Sample temperature on receipt
Laboratories Receipt
day
Hour
receipt
Measured temperature
at receipt (degC)
Measured temperature by the
temperature probe (degC)
A
B
C
D
E
F
G
H
I
J
K
L
M
30052006
10h45
10h15
11h15
10h00
09h30
8h30
11h50
10h00
08h30
14h45
08h30
09h45
08h45
82
55
45
63
60
62
81
38
48
-
14
67
76
03
03
-13
03
04
-14
05
04
01
05
-13
04
Conclusion
The temperatures during the transport were correct for all the labs except for
Lab J which didnrsquot provide the information
All the labs received their packages with several broken flasks
Three labs (B D and L) received some damaged samples but proceeded to
analyses
All the labs started the analyses at Day 1 except Lab J
2133 Results
Aerobic mesophilic flora
The count of aerobic mesophilic flora varied from 94 102 CFUml to
33 103 CFUml
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2438 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Expert lab results
The results obtained by the expert lab are the following
Table 14
Target inoculation level (CFUml)
Reference method ISO 11290-2
Alternative method Listeria Precis Enumeration
Rep 1 Rep 2 Rep 1 Rep 2
lt 10 lt 10 lt 10 lt 10 lt10
50 50 (Ne) 60 (Ne) 50 (Ne) 30 (Ne)
500 470 390 490 500
5 000 4 700 5 100 5 900 3 400
Ne estimated number
The obtained results are in agreement with those expected
Collaborator Labs results
A synthesis of the results obtained by the collaborator labs is provided
Table 15 (CFUml) and Table 16 (log CFUml)
Table 15 ndash Synthesis of results obtained by the alternative
and the reference methods (CFUml)
Labs Level 1 Level 2 Level 2 bis Level 3
Reference method
Alternative method
Reference method
Alternative method
Reference method
Alternative method
Reference method
Alternative method
A 40 40 70 40 480 410 210 340 1000 300 400 300 4900 4600 4900 5100
B 40 15 40 20 350 300 360 450 350 650 800 600 4100 4700 5100 3700
C 50 20 20 70 440 430 480 450 300 650 300 500 4600 5400 4600 5000
D 40 60 40 40 490 460 600 540 250 450 600 1000 4900 4900 4400 4600
E 50 40 50 20 490 520 530 560 750 550 400 200 5400 4500 4100 5400
F 60 20 60 50 370 250 300 130 300 350 600 300 4400 4700 4900 5900
G 70 35 120 50 440 390 570 370 450 550 700 700 5000 4500 4000 4400
H 110 40 100 90 520 440 450 540 150 650 300 300 4200 4700 5600 4800
L 35 60 20 40 390 390 170 380 650 350 800 200 5300 4600 5600 5900
M 45 45 30 40 450 490 550 410 400 550 800 180 4900 4900 6400 4800
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2538 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Table 16 ndash Synthesis of results obtained by the alternative
and the reference methods (log CFUml)
Labs Level 1 Level 2 Level 2 bis Level 3
Reference method
Alternative method
Reference method
Alternative method
Reference method
Alternative method
Reference method
Alternative method
A 1602 1602 1845 1602 2681 2613 2322 2531 3000 2477 2602 2477 3690 3663 3690 3708
B 1602 1176 1602 1301 2544 2477 2556 2653 2544 2813 2903 2778 3613 3672 3708 3568
C 1699 1301 1301 1845 2643 2633 2681 2653 2477 2813 2477 2699 3663 3732 3663 3699
D 1602 1778 1602 1602 2690 2663 2778 2732 2398 2653 2778 3000 3690 3690 3643 3663
E 1699 1602 1699 1301 2690 2716 2724 2748 2875 2740 2602 2301 3732 3653 3613 3732
F 1778 1301 1778 1699 2568 2398 2477 2114 2477 2544 2778 2477 3643 3672 3690 3771
G 1845 1544 2079 1699 2643 2591 2756 2568 2653 2740 2845 2845 3699 3653 3602 3643
H 2041 1602 2000 1954 2716 2643 2653 2732 2176 2813 2477 2477 3623 3672 3748 3681
L 1544 1778 1301 1602 2591 2591 2230 2580 2813 2544 2903 2301 3724 3663 3748 3771
M 1653 1653 1477 1602 2653 2690 2740 2613 2602 2740 2903 2255 3690 3690 3806 3681
Level 2 results obtained taking account the 3 plates inoculated with the mother-suspension
Level 2 bis results obtained taking account the plates inoculated with 01 ml of the mother-suspension
Scrutiny of the measurement results for consistency
In order to identify the measurement results or laboratories that could be
inconsistent two graphical consistency techniques were realised Mandelrsquos
h-ank-statistics Mandel indicators h and k at 5 significance highlighted
some possible inconsistent data (See tables and graphics in Appendix 3 and
Appendix 4)
Mandelrsquos values Number of values observed over the threshold
Reference method Alternative method
h gt 1 Lab F Level 2 Lab F Level 2
h gt 5 Lab F Level 2
Lab B Levels 1 and 2
Lab A Level 2
Lab D Level 3
Lab F Level 2
Lab L Level 2
kgt 1 Lab F Level 3
k gt 5 Lab F Level 2 Lab C Level 2
Lab F Level 3
Lab L Levels 1 and 3
Lab M Level 1
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2638 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
The emphasized results appear (i) coherent with the targeted contamination
levels (ii) concordant between both methods
All the results were thus kept for the statistical analysis
Comparison of the trueness and precision characteristics of
the reference method and alternative methods
The statistical values are summarised hereafter
Level p
Reference method Alternative method
Repeatability ratio
Reproducibility ratio Median
Repeatability standard deviation
Reproducibility standard deviation
Median Repeatability
standard deviation
Reproducibility standard deviation
1 10 16519 02415 02425 16021 02045 02433 0847 1007
2 10 26551 00543 00659 26719 00914 01325 1682 2011
2 bis 10 26581 02607 02607 25951 01900 01953 0729 0749
3 10 18671 00378 00378 18671 00478 00611 1264 1617
Bias of the alternative method
In order to estimate the bias of the alternative method with regard to the reference method for every
level Dij and t are calculated according to the following equations
RefAlt ijYijYDij
Diffp
Dijit
)2(
)(median
If the t value is superior to 2 the alternative method is significantly biased
with regard to the reference method
The values are given in table 17
Table 17 ndash t (d) values obtained by level
Level Bias t (d) Conclusion
1 0068 114 Non significant bias
2 0021 082 Non significant bias
2 bis - 0037 035 Non significant bias
3 - 0037 043 Non significant bias
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2738 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
The statistical test concludes to a non significant bias for all the inoculation
levels The bias varies from ndash 0037 to 0068 log CFUml
Comparison of the repeatability standard deviations
If the ratio Srj Alt Srj Ref of the repeatability standard deviations of the
alternative method and the reference method is larger than 2 the precision
under repeatability conditions of the alternative method is considered to be
lower than that of the reference method If this ratio is smaller than 05 the
precision under repeatability conditions of the alternative method is
considered to be greater than that of the reference method
The ratio values are given in table 18
Table 18
Level
Repeatability standard deviation
Reference method Alternative method Alternative method
reference method ratio
1 0242 0205 0847
2 0054 0091 1682
2 bis 0261 0190 0729
3 0038 0048 1264
Level Repeatability limit
Reference method Alternative method
1 0676 0573
2 0152 0256
2 bis 0730 0532
3 0106 0134
The ratios of the repeatability standard deviations are below 2 in all cases
The precision under repeatability conditions of the alternative method is
equivalent to that of the reference method
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2838 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Comparison of the reproducibility standard deviations
If the ratio Srj Alt Srj Ref of the reproducibility standard deviations of the alternative method and the
reference method is larger than 2 the precision under reproducibility conditions of the alternative
method is considered to be lower than that of the reference method If this ratio is smaller than 05 the
precision under reproducibility conditions of the alternative method is considered to be greater than
that of the reference method
The ratio values are given in table 19
Table 19
Level
Reproducibility standard deviation
Reference method Alternative method Alternative method
reference method ratio
1 0242 0243 1007
2 0066 0133 2011
2 bis 0261 0195 0749
3 0038 0061 1617
Level Reproducibility limit
Reference method Alternative method
1 0676 0681
2 0185 0371
2 bis 0730 0547
3 0106 0171
The ratios of the reproducibility standard deviations are below or close to 2
The precision under reproducibility conditions of the alternative method is
equivalent to that of the reference method
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2938 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
214 Conclusion
Method comparison study
The Listeria PrecisTM enumeration method shows satisfying linearity and
relative accuracy
The alternative method shows satisfying inclusivity and exclusivity results
Two Listeria monocytogenes strains gave negative results with the
alternative method These strains grew weakly on the reference method
media (OampA Palcam and Oxford)
The limits of repeatability of the alternative method are similar to those of the
reference method
The bias between both methods are low between - 0133 Log CFUg and
- 0008 Log CFUg
The Listeria PrecisTM enumeration method offers the saving for confirmatory
tests
Positive results are obtained with the alternative method in 2-3 days (with
OBIS Mono test) or in 4-7 days (by the classical tests described in the
standardized method) against 4-7 days for the reference method
Inter-laboratory study
The bias between the reference and the alternative methods shows satisfying
and correct values
- level 1 (100 - 1 000 CFUg) bias = + 007 log CFUg
- level 2 (1 000 - 10 000 CFUg) bias = 002 log CFUg
- level 2 bis (1 000 - 10 000 CFUg) bias = - 004 log CFUg
- level 3 (10 000 - 100 000 CFUg) bias = - 004 log CFUg
The repeatability and the reproducibility of the alternative method look similar
to those of the reference method
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3038 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
22 Extension study (realised by ASEPT 2007)
An extension study was performed in 2007 for a new confirmatory test OBIS
MONO
150 Listeria monocytogenes strains and 100 non-target strains were isolated
onto TSAYE and BrillianceTM Listeria Agar plates All the Listeria
monocytogenes and the non-target strains gave the expected results
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3138 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Appendix 1 ndash Flow diagram of the Listeria PrecisTM method
x g sample + 9 x ml buffered peptone water
Revivification step for 1 h plusmn 5 min at 20degC plusmn 1degC
Streaking 1 ml onto 3 Brilliance Listeria Agar plates (mother suspension)
or 01 ml onto 1 Brilliance Listeria Agar plates (mother suspension and decimal dilutions)
Incubation for 48 h plusmn 3 h at 37degC plusmn 1degC
Confirmatory tests on at least one typical colony
- by the tests described in the ISO method
- by applying the OBIS mono test on an isolated colony
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3238 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Appendix 2 ndash ISO 11290-2 (1998) amp ISO 11290-2A1 (2004) Horizontal
method for the detection and enumeration of Listeria monocytogenes -
Part 2 enumeration of Listeria monocytogenes in foods
10 g cheese + 90 ml buffered peptone water or Half Fraser Broth (without supplements) (110 dilution)
Stomach 1 min 30 seconds
Keep at 20degC plusmn 2degC for 1 h plusmn 5 min
Decimal dilutions in 9 ml peptone salt tubes
Dilution 110
Dilution 1100
Dilution 11000
1 ml on 3 Chromogenic Listeria medium (ISO) plates (x 2) (-1)
01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-2)
01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-3)
01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-4)
Incubation at 37degC plusmn 1degC
24 h - 48 h
Numerate typical colonies at 24 h
(bluegreen surrounded by an opaque halo)
Numerate at 48 h
Streak typical colonies on TSYEA
(5 coloniesplate 2 successive dilutions)
Confirmation tests
Catalase
Gram stain
For confirmation of L monocytogenes
Haemolysis
Carbohydrate (rhamnose and xylose) utilisation
CAMP Test
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3338 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Appendix 3 ndash Mandelrsquos graphics
Laboratoire h Ref
Niveau 1 h Ref
Niveau 2 h Ref
Niveau 3 h Alt
Niveau 1 h Alt
Niveau 2 h Alt
Niveau 3 h5 h1 h5 h1
A -050 -015 083 062 -212 -039 198 299 -198 -299
B -261 -270 021 -077 -058 173 198 299 -198 -299
C -151 -031 -014 -015 -004 -005 198 299 -198 -299
D 038 040 -137 000 072 207 198 299 -198 -299
E -001 090 155 -052 056 -101 198 299 -198 -299
F -112 -321 -153 070 -325 023 198 299 -198 -299
G 042 -071 040 147 -009 176 198 299 -198 -299
H 169 046 -169 192 018 -083 198 299 -198 -299
L 009 -120 021 -077 -231 005 198 299 -198 -299
M 001 031 014 -032 004 -011 198 299 -198 -299
-300
-200
-100
000
100
200
300
A B C D E F G H L M
Vale
urs
h d
e M
an
del(
rob
uste
s)
Laboratoires
Meacutethode de reacutefeacuterence analyse robuste
h Ref Niveau 1
h Ref Niveau 2
h Ref Niveau 3
h 5=198
h 1=299
-300
-200
-100
000
100
200
300
A B C D E F G H L M
Vale
urs
de h
de M
an
del(
rob
uste
s)
Laboratoires
Meacutethode alternative analyse robuste
h Alt Niveau 1
h Alt Niveau 2
h Alt Niveau 2
h 1=299
h 5=198
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3438 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Laboratoire k Ref
Niveau 1 k Ref
Niveau 2 k Ref
Niveau 3 k Alt
Niveau 1 k Alt
Niveau 2 k Alt
Niveau 3 k1 k5
A 000 089 142 084 162 046 259 181
B 125 087 073 104 075 046 259 181
C 117 013 091 188 022 083 259 181
D 052 036 069 000 035 083 259 181
E 028 034 037 138 019 112 259 181
F 140 222 018 027 281 112 259 181
G 088 068 024 131 145 000 259 181
H 129 094 173 016 061 000 259 181
L 069 000 073 104 270 224 259 181
M 000 048 038 043 099 241 259 181
000
050
100
150
200
250
300
350
400
A B C D E F G H L M
Vale
urs
k d
e M
an
del(
rob
uste
s)
Laboratoires
Meacutethode de reacutefeacuterence analyse robuste
k Ref Niveau 1k Ref Niveau 2k Ref Niveau 3
k 1=259
k 5=181
000
050
100
150
200
250
300
A B C D E F G H L M
Vale
urs
k d
e M
an
del
(ro
bu
ste
s)
Laboratoires
Meacutethode alternative analyse robuste
k Alt Niveau 3
k Alt Niveau 1
k Alt Niveau 2
k 1=259
k 5=181
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3538 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Appendix 4 ndash Statistical results
Level 1
Nombre de laboratoires (p) 10
A 1602 1602 1602 000 0000 0000 -0495 0000 A 1845 1602 1724 017 0122 -0122 0621 0840 0122
B 1602 1176 1389 030 0213 -0213 -2613 1247 B 1602 1301 1452 021 0151 -0151 -0769 1041 0062
C 1699 1301 1500 028 0199 -0199 -1510 1165 C 1301 1845 1573 038 -0272 0272 -0148 1881 0073
D 1602 1778 1690 012 -0088 0088 0380 0516 D 1602 1602 1602 000 0000 0000 0000 0000 -0088
E 1699 1602 1651 007 0048 -0048 -0013 0284 E 1699 1301 1500 028 0199 -0199 -0522 1376 -0151
F 1778 1301 1540 034 0239 -0239 -1116 1397 F 1778 1699 1739 006 0040 -0040 0698 0274 0199
G 1845 1544 1695 021 0151 -0151 0425 0881 G 2079 1699 1889 027 0190 -0190 1467 1315 0194
H 2041 1602 1822 031 0220 -0220 1689 1286 H 2000 1954 1977 003 0023 -0023 1917 0158 0155
L 1544 1778 1661 017 -0117 0117 0092 0685 L 1301 1602 1452 021 -0151 0151 -0769 1041 -0210
M 1653 1653 1653 000 0000 0000 0013 0000 M 1477 1602 1540 009 -0062 0062 -0319 0432 -0114
m 165186 m 160206 mediane D 00678
Cn(2p) 1867 Cn(2p) 1867 Cp 16101
Cn(p) 16101 Cn(p) 16101 Qn(D) 0092926
n=2p n 20 n=2p n 20 Qdiff 01496169
f 11 f 11 t 114
l 55 l 55
n=p n 10 n=p n 10
f 6 f 6
l 15 l 15
Qn(2p) 0091465 Qn(2p) 007745
Qn(p) 0062469 Qn(p) 0121519
Qintra 017078 Qintra 014461
Qinter 010058 Qinter 019565
Sr(eacutecart-type de reacutepeacutetabiliteacute) 02415 Sr 02045
RSDr(coefficient de variation de la reacutepeacutetabiliteacute)1462 RSDr 1277
r(repeatability limit)) 0676 r 0573
SL(eacutecart-type interlaboratoire) -001905 SLlt0 SL 001737
SR(eacutecart-type de reproductibiliteacute) 02415 SR 02433
RSDR(coefficient de variation de la reproductibiliteacute)0146 RSDR 0152
R(limite de reproductibiliteacute)) 06762 R(limite de reproductibiliteacute))068123
h k h MoyenneLaboratoires
Niveau 1
si LaboratoiresDeviationMoyenne Dk DeviationsiAlt
Niveau 1
Ref
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3638 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Level 2
Nombre de laboratoires (p) 10
A 2681 2613 2647 005 0034 -0034 -0151 0891 A 2322 2531 2427 015 -0105 0105 -2119 1620 -0220
B 2544 2477 2511 005 0033 -0033 -2699 0872 B 2556 2653 2605 007 -0048 0048 -0581 0750 0094
C 2643 2633 2638 001 0005 -0005 -0311 0130 C 2681 2653 2667 002 0014 -0014 -0040 0217 0029
D 2690 2663 2676 002 0014 -0014 0400 0357 D 2778 2732 2755 003 0023 -0023 0721 0354 0079
E 2690 2716 2703 002 -0013 0013 0897 0336 E 2724 2748 2736 002 -0012 0012 0556 0185 0033
F 2568 2398 2483 012 0085 -0085 -3214 2217 F 2477 2114 2296 026 0182 -0182 -3255 2811 -0188
G 2643 2591 2617 004 0026 -0026 -0707 0682 G 2756 2568 2662 013 0094 -0094 -0085 1453 0045
H 2716 2643 2680 005 0036 -0036 0460 0945 H 2653 2732 2693 006 -0040 0040 0181 0613 0013
L 2591 2591 2591 000 0000 0000 -1196 0000 L 2230 2580 2405 025 -0175 0175 -2307 2704 -0186
M 2653 2690 2672 003 -0018 0018 0311 0481 M 2740 2613 2677 009 0064 -0064 0040 0987 0005
m 2655082 m 26719001 mediane D 00209
Cn(2p) 1867 Cn(2p) 1867 Cp 16101
Cn(p) 16101 Cn(p) 16101 Qn(D) 0039911
n=2p n 20 n=2p n 20 Qdiff 00642591
f 11 f 11 t 082
l 55 l 55
n=p n 10 n=p n 10
f 6 f 6
l 15 l 15
Qn(2p) 0020570 Qn(2p) 00345985
Qn(p) 00332437 Qn(p) 00718158
Qintra 003841 Qintra 006460
Qinter 005352 Qinter 011563
Sr(eacutecart-type de reacutepeacutetabiliteacute) 00543 Sr 00914
RSDr(coefficient de variation de la reacutepeacutetabiliteacute)205 RSDr 342
r(repeatability limit)) 0152 r 0256
SL(eacutecart-type interlaboratoire) 000139 SL 000920
SR(eacutecart-type de reproductibiliteacute) 00659 SR 01325
RSDR(coefficient de variation de la reproductibiliteacute)0025 RSDR 0050
R(limite de reproductibiliteacute)) 018446 R(limite de reproductibiliteacute))03708618
Laboratoires
Niveau 2
Moyenne si Laboratoires
Niveau 2
DRef Alt Moyenne si Deviation h Deviation h k k
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3738 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Level 2 bis
Nombre de laboratoires (p) 10
A 3000 2477 2739 037 0261 -0261 0832 1418 A 2602 2477 2540 009 0062 -0062 -0391 0465 -0199
B 2544 2813 2678 019 -0134 0134 0211 0729 B 2903 2778 2841 009 0062 -0062 1732 0465 0162
C 2477 2813 2645 024 -0168 0168 -0135 0911 C 2477 2699 2588 016 -0111 0111 -0049 0826 -0057
D 2398 2653 2526 018 -0128 0128 -1370 0692 D 2778 3000 2889 016 -0111 0111 2074 0826 0363
E 2875 2740 2808 010 0067 -0067 1547 0365 E 2602 2301 2452 021 0151 -0151 -1012 1120 -0356
F 2477 2544 2511 005 -0033 0033 -1525 0182 F 2778 2477 2628 021 0151 -0151 0230 1120 0117
G 2653 2740 2697 006 -0044 0044 0400 0236 G 2845 2845 2845 000 0000 0000 1764 0000 0148
H 2176 2813 2495 045 -0318 0318 -1692 1727 H 2477 2477 2477 000 0000 0000 -0832 0000 -0017
L 2813 2544 2678 019 0134 -0134 0211 0729 L 2903 2301 2602 043 0301 -0301 0049 2241 -0076
M 2602 2740 2671 010 -0069 0069 0135 0375 M 2903 2255 2579 046 0324 -0324 -0112 2411 -0092
m 2658114 m 259505 mediane D -00372
Cn(2p) 1867 Cn(2p) 1867 Cp 16101
Cn(p) 16101 Cn(p) 16101 Qn(D) 01656915
n=2p n 20 n=2p n 20 Qdiff 02667753
f 11 f 11 t 035
l 55 l 55
n=p n 10 n=p n 10
f 6 f 6
l 15 l 15
Qn(2p) 0098745 Qn(2p) 007195
Qn(p) 006007 Qn(p) 008805
Qintra 018437 Qintra 013435
Qinter 009672 Qinter 014176
Sr(eacutecart-type de reacutepeacutetabiliteacute) 02607 Sr 01900
RSDr(coefficient de variation de la reacutepeacutetabiliteacute)981 RSDr 732
r(repeatability limit)) 0730 r 0532
SL(eacutecart-type interlaboratoire) -002464 SLlt0 SL 000205
SR(eacutecart-type de reproductibiliteacute) 02607 SR 01953
RSDR(coefficient de variation de la reproductibiliteacute)0098 RSDR 0075
R(limite de reproductibiliteacute)) 072996 R(limite de reproductibiliteacute))054686
Laboratoires
Niveau 2 bis
Moyenne si Laboratoires
Niveau 2 bis
DRef Alt Moyenne si Deviation h Deviation h k k
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3838 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Level 3
Nombre de laboratoires (p) 10
A 3690 3663 3676 002 0014 -0014 -0311 0513 A 3690 3708 3699 001 -0009 0009 0354 0257 0022
B 3613 3672 3642 004 -0030 0030 -1851 1109 B 3708 3568 3638 010 0070 -0070 -0844 2061 -0005
C 3663 3732 3698 005 -0035 0035 0645 1302 C 3663 3699 3681 003 -0018 0018 0000 0536 -0017
D 3690 3690 3690 000 0000 0000 0311 0000 D 3643 3663 3653 001 -0010 0010 -0545 0286 -0037
E 3732 3653 3693 006 0040 -0040 0429 1480 E 3613 3732 3673 008 -0060 0060 -0162 1769 -0020
F 3643 3672 3658 002 -0014 0014 -1157 0536 F 3690 3771 3731 006 -0040 0040 0975 1193 0073
G 3699 3653 3676 003 0023 -0023 -0328 0856 G 3602 3643 3623 003 -0021 0021 -1141 0612 -0053
H 3623 3672 3648 003 -0024 0024 -1615 0913 H 3748 3681 3715 005 0033 -0033 0664 0990 0067
L 3724 3663 3694 004 0031 -0031 0461 1150 L 3748 3771 3760 002 -0011 0011 1544 0335 0066
M 3690 3690 3690 000 0000 0000 0311 0000 M 3806 3681 3744 009 0062 -0062 1234 1848 0054
m 368334 m 368086 mediane D 00089
Cn(2p) 1867 Cn(2p) 1867 Cp 16101
Cn(p) 16101 Cn(p) 16101 Qn(D) 00325359
n=2p n 20 n=2p n 20 Qdiff 00523852
f 11 f 11 t 043
l 55 l 55
n=p n 10 n=p n 10
f 6 f 6
l 15 l 15
Qn(2p) 0014323 Qn(2p) 001811
Qn(p) 0013719 Qn(p) 003164
Qintra 002674 Qintra 003381
Qinter 002209 Qinter 005094
Sr(eacutecart-type de reacutepeacutetabiliteacute) 00378 Sr 00478
RSDr(coefficient de variation de la reacutepeacutetabiliteacute)103 RSDr 130
r(repeatability limit)) 0106 r 0134
SL(eacutecart-type interlaboratoire) -000023 SLlt0 SL 000145
SR(eacutecart-type de reproductibiliteacute) 00378 SR 00611
RSDR(coefficient de variation de la reproductibiliteacute)0010 RSDR 0017
R(limite de reproductibiliteacute)) 0105894 R(limite de reproductibiliteacute))017119
Ref Alt DLaboratoires
Niveau 3
Moyenne si Deviation h k Laboratoires
Niveau 3
Moyenne si Deviation h k
Thermo Fisher Scientific
ADRIA Deacuteveloppement 1638 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
The hypotheses that the ordinates are close to 0 and the slopes close to 1
are approved in all cases except for
- Meat products (slope P = 1 ordinate P = 1)
- Environmental samples (ordinate P = 3)
- All products (ordinate P = 0)
The bias between the two methods vary from ndash 0133 to 0008 log CFU For
all categories the bias is ndash 0032 log CFU The repeatability limits of the
alternative and the reference method are similar For all products the values
are respectively 0215 and 0196 log CFU For all products these
interpretations are provided taking into account data with low contamination
level or not (estimated colonies number)
For all food products the regression straight line is the following
log (Alt) = 1040 log Ref ndash 0182
The Listeria PrecisTM method shows satisfying relative accuracy
All products
All products (Level 2)
Thermo Fisher Scientific
ADRIA Deacuteveloppement 1738 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
2113 Detection limit (LOD) and quantification limit (LOQ)
The critical level is the defined as the smallest amount which can be detected (not null) but not
quantified as an exact value Below this value it cannot be sure that the true value is not null
The detection limit is defined as being higher than the critical level because it involves a power the
probability 1- which has to be well over 50 for example 95
The quantification limit is defined as the smallest amount of analyte (that is the lowest actual number of
organisms) which can be measured and quantified with defined precision and accuracy under the
experimental conditions by the method under validation
Protocol
The detection limit of the alternative method was realised with pure cultures
Three different inoculation levels were tested with six replicates per level ie
18 analyses were done by the alternative method
Listeria monocytogenes 18 was used for the detection limit of the alternative
method
Quantification limit was calculated for six independent blank samples
determinations
Results
These data are intrinsic to the method used and are presented in the
following tables
Table 8 ndash Inoculation 1 ml on 3 plates
Level (CFUml) Positive samples number Standard deviation Bias
0 06
05 26 1211 0
1 36 0816 05
5 66 2483 85
Thermo Fisher Scientific
ADRIA Deacuteveloppement 1838 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Table 9 ndash Inoculation 01 ml on 1 plate
Level (CFUml) Positive samples number Standard deviation Bias
0 06
5 36 0548 05
10 46 1414 2
50 66 2258 55
Table 10
Formulas Obtained values
LC 165 S0 + X0 18
LOD 33 S0 + X0 32
LOQ 10 S0 + X0 87
2114 Relative Sensitivity
The relative sensitivity is defined as the ability of the alternative method to detect two different amounts
of analyte measured by the reference method within a given matrix at a specified average value or
over the whole measurement range that is it is the minimal quantity variation (increase of the analyte
concentration x) which gives a significant variation of the measured signal (response y)
These data are intrinsic to the used method and are obtained from the results
of the linearity study
Sensitivity patterns obtained for tested (matrixstrain) pairs are presented in
figure 5
Thermo Fisher Scientific
ADRIA Deacuteveloppement 1938 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Figure 5 ndash Sensitivity patterns for the tested (matrixstrain) pairs
2115 SpecificitySelectivity
The specificity is defined as the degree to which a method is affected (or not) by the other components
present in a multi-component sample That is the ability of a method to measure exactly a given
analyte or its amount within the sample without interference from non-target components such as a
matrix effect or background noise
The selectivity is defined as a measure of the degree of non-interference in the presence of non-target
analytes A method is selective if it can be used to detect the analyte under examination and that a
guarantee can be provided that the detected signal can only be a product by that specific analyte
The specificity and selectivity study was performed for the Listeria PrecisTM
detection method no additional test was realised for the enumeration
method The results were the following
- Among the Listeria monocytogenes strains tested 2 strains (Listeria
monocytogenes CIP 7834 and Listeria monocytogenes CIP 105459) gave
negative results These 2 strains were tested twice by running 48 h
incubation time of the compared agars Positive results were then
observed by the alternative method Pale characteristic colonies were
observed on the ISO agar by streaking the Half Fraser broth only
- No cross reaction was observed with the non-target strains except with
Listeria ivanovii which gave characteristic colonies on Brilliancetrade Listeria
Agar but the confirmatory tests provide a clear discrimination between
Listeria monocytogenes and Listeria ivanovi species
Rillettes
Lettuce
Raw milk
Smoked salmon
Raw egg
Process water
x (CFUg)
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2038 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
212 Practicability
1 Packaging Ready to use Brilliance Listeria agar plates are available
(x 10 plates)
2 Reagent volume
3 Storage conditionsndash
Shelf life of products
not opened
The plates are stored at 6 - 12degC in darkness conditions The
expiration date is mentioned on the package and on the plates It
is around 3 months
4 Modalities of use
after first use
5 Required
equipments No specific equipment is required
6 Reagents Ready to use plates
7 Training 05 day
8 Real time handling
Reference method
9 min for one sample
55 min for 10 samples
Alternative method
7 min for one sample
35 minutes for 10 samples
9 Time to result
Negative results
Reference method Day 2
Alternative method Day 2
Negative results after confirmation or positive results
Reference method Day 4 ndash Day 7
Alternative method Day 4 ndash Day 7
Positive results
Reference method Day 4 ndash Day 7
Alternative method Day 2 ndash Day 3 (with OBIS Mono Test)
or Day 4 ndash Day 7 (by the classical tests described in the
standardised method)
10 Technician skill Identical to that required for the reference method
11 Steps common to
the reference method Mother suspension revivification step and decimal dilutions
12 Traceability
13 Maintenance
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2138 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
213 Inter-laboratory study
2131 Organisation
12 laboratories participated to this study Pasteurised milk was inoculated by
Listeria monocytogenes 12a isolated from raw milk The targeted
inoculation levels were
- 0 CFUml
- 50 CFU ml
- 500 CFU ml
- 5 000 CFU ml
Each laboratory received eight flasks of 10 ml sample ie two flasks per
inoculation level Furthermore one non-inoculated sample has been added
to the package for total viable count microflora (NF ISO 4833 method)
Detailed instructions were transmitted to the collaborators by the expert
laboratory
Collaborative study laboratories and the expert laboratory carried out the
analyses with the alternative and reference methods
2132 Verification of experimental parameters
Strain stability during transport
In order to evaluate the Listeria monocytogenes strain stability during
transport bacterial count of all inoculated flasks were checked at different
time ie inoculation time after 24 h of conservation at 2deg - 8degC Results are
reported in table 11
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2238 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Table 11 ndash Listeria monocytogenes count
by ISO 11290-2A1 method (log CFUml)
Day of the analyses Level contamination Reference method
Day 0
(29052006)
1 45
2 440
3 4 500
Day 1
(30052006)
1 60
2 430
3 4 900
No evolution of the strain was observed after storage for 24 h at 2degC ndash 8degC
Contamination level
The real contamination levels are provided in table 12
Table 12 ndash Real contamination level (CFUml)
Target inoculation
(CFU ml) Samples Ndeg
Real contamination levels
(CFUml)
0 1 - 8 0
50 5 - 6 45
500 3 - 7 440
5000 2 - 4 4 500
Sample temperature at receipt
Measured temperatures at receipt are listed in table 13
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2338 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Table 13 - Sample temperature on receipt
Laboratories Receipt
day
Hour
receipt
Measured temperature
at receipt (degC)
Measured temperature by the
temperature probe (degC)
A
B
C
D
E
F
G
H
I
J
K
L
M
30052006
10h45
10h15
11h15
10h00
09h30
8h30
11h50
10h00
08h30
14h45
08h30
09h45
08h45
82
55
45
63
60
62
81
38
48
-
14
67
76
03
03
-13
03
04
-14
05
04
01
05
-13
04
Conclusion
The temperatures during the transport were correct for all the labs except for
Lab J which didnrsquot provide the information
All the labs received their packages with several broken flasks
Three labs (B D and L) received some damaged samples but proceeded to
analyses
All the labs started the analyses at Day 1 except Lab J
2133 Results
Aerobic mesophilic flora
The count of aerobic mesophilic flora varied from 94 102 CFUml to
33 103 CFUml
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2438 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Expert lab results
The results obtained by the expert lab are the following
Table 14
Target inoculation level (CFUml)
Reference method ISO 11290-2
Alternative method Listeria Precis Enumeration
Rep 1 Rep 2 Rep 1 Rep 2
lt 10 lt 10 lt 10 lt 10 lt10
50 50 (Ne) 60 (Ne) 50 (Ne) 30 (Ne)
500 470 390 490 500
5 000 4 700 5 100 5 900 3 400
Ne estimated number
The obtained results are in agreement with those expected
Collaborator Labs results
A synthesis of the results obtained by the collaborator labs is provided
Table 15 (CFUml) and Table 16 (log CFUml)
Table 15 ndash Synthesis of results obtained by the alternative
and the reference methods (CFUml)
Labs Level 1 Level 2 Level 2 bis Level 3
Reference method
Alternative method
Reference method
Alternative method
Reference method
Alternative method
Reference method
Alternative method
A 40 40 70 40 480 410 210 340 1000 300 400 300 4900 4600 4900 5100
B 40 15 40 20 350 300 360 450 350 650 800 600 4100 4700 5100 3700
C 50 20 20 70 440 430 480 450 300 650 300 500 4600 5400 4600 5000
D 40 60 40 40 490 460 600 540 250 450 600 1000 4900 4900 4400 4600
E 50 40 50 20 490 520 530 560 750 550 400 200 5400 4500 4100 5400
F 60 20 60 50 370 250 300 130 300 350 600 300 4400 4700 4900 5900
G 70 35 120 50 440 390 570 370 450 550 700 700 5000 4500 4000 4400
H 110 40 100 90 520 440 450 540 150 650 300 300 4200 4700 5600 4800
L 35 60 20 40 390 390 170 380 650 350 800 200 5300 4600 5600 5900
M 45 45 30 40 450 490 550 410 400 550 800 180 4900 4900 6400 4800
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2538 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Table 16 ndash Synthesis of results obtained by the alternative
and the reference methods (log CFUml)
Labs Level 1 Level 2 Level 2 bis Level 3
Reference method
Alternative method
Reference method
Alternative method
Reference method
Alternative method
Reference method
Alternative method
A 1602 1602 1845 1602 2681 2613 2322 2531 3000 2477 2602 2477 3690 3663 3690 3708
B 1602 1176 1602 1301 2544 2477 2556 2653 2544 2813 2903 2778 3613 3672 3708 3568
C 1699 1301 1301 1845 2643 2633 2681 2653 2477 2813 2477 2699 3663 3732 3663 3699
D 1602 1778 1602 1602 2690 2663 2778 2732 2398 2653 2778 3000 3690 3690 3643 3663
E 1699 1602 1699 1301 2690 2716 2724 2748 2875 2740 2602 2301 3732 3653 3613 3732
F 1778 1301 1778 1699 2568 2398 2477 2114 2477 2544 2778 2477 3643 3672 3690 3771
G 1845 1544 2079 1699 2643 2591 2756 2568 2653 2740 2845 2845 3699 3653 3602 3643
H 2041 1602 2000 1954 2716 2643 2653 2732 2176 2813 2477 2477 3623 3672 3748 3681
L 1544 1778 1301 1602 2591 2591 2230 2580 2813 2544 2903 2301 3724 3663 3748 3771
M 1653 1653 1477 1602 2653 2690 2740 2613 2602 2740 2903 2255 3690 3690 3806 3681
Level 2 results obtained taking account the 3 plates inoculated with the mother-suspension
Level 2 bis results obtained taking account the plates inoculated with 01 ml of the mother-suspension
Scrutiny of the measurement results for consistency
In order to identify the measurement results or laboratories that could be
inconsistent two graphical consistency techniques were realised Mandelrsquos
h-ank-statistics Mandel indicators h and k at 5 significance highlighted
some possible inconsistent data (See tables and graphics in Appendix 3 and
Appendix 4)
Mandelrsquos values Number of values observed over the threshold
Reference method Alternative method
h gt 1 Lab F Level 2 Lab F Level 2
h gt 5 Lab F Level 2
Lab B Levels 1 and 2
Lab A Level 2
Lab D Level 3
Lab F Level 2
Lab L Level 2
kgt 1 Lab F Level 3
k gt 5 Lab F Level 2 Lab C Level 2
Lab F Level 3
Lab L Levels 1 and 3
Lab M Level 1
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2638 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
The emphasized results appear (i) coherent with the targeted contamination
levels (ii) concordant between both methods
All the results were thus kept for the statistical analysis
Comparison of the trueness and precision characteristics of
the reference method and alternative methods
The statistical values are summarised hereafter
Level p
Reference method Alternative method
Repeatability ratio
Reproducibility ratio Median
Repeatability standard deviation
Reproducibility standard deviation
Median Repeatability
standard deviation
Reproducibility standard deviation
1 10 16519 02415 02425 16021 02045 02433 0847 1007
2 10 26551 00543 00659 26719 00914 01325 1682 2011
2 bis 10 26581 02607 02607 25951 01900 01953 0729 0749
3 10 18671 00378 00378 18671 00478 00611 1264 1617
Bias of the alternative method
In order to estimate the bias of the alternative method with regard to the reference method for every
level Dij and t are calculated according to the following equations
RefAlt ijYijYDij
Diffp
Dijit
)2(
)(median
If the t value is superior to 2 the alternative method is significantly biased
with regard to the reference method
The values are given in table 17
Table 17 ndash t (d) values obtained by level
Level Bias t (d) Conclusion
1 0068 114 Non significant bias
2 0021 082 Non significant bias
2 bis - 0037 035 Non significant bias
3 - 0037 043 Non significant bias
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2738 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
The statistical test concludes to a non significant bias for all the inoculation
levels The bias varies from ndash 0037 to 0068 log CFUml
Comparison of the repeatability standard deviations
If the ratio Srj Alt Srj Ref of the repeatability standard deviations of the
alternative method and the reference method is larger than 2 the precision
under repeatability conditions of the alternative method is considered to be
lower than that of the reference method If this ratio is smaller than 05 the
precision under repeatability conditions of the alternative method is
considered to be greater than that of the reference method
The ratio values are given in table 18
Table 18
Level
Repeatability standard deviation
Reference method Alternative method Alternative method
reference method ratio
1 0242 0205 0847
2 0054 0091 1682
2 bis 0261 0190 0729
3 0038 0048 1264
Level Repeatability limit
Reference method Alternative method
1 0676 0573
2 0152 0256
2 bis 0730 0532
3 0106 0134
The ratios of the repeatability standard deviations are below 2 in all cases
The precision under repeatability conditions of the alternative method is
equivalent to that of the reference method
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2838 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Comparison of the reproducibility standard deviations
If the ratio Srj Alt Srj Ref of the reproducibility standard deviations of the alternative method and the
reference method is larger than 2 the precision under reproducibility conditions of the alternative
method is considered to be lower than that of the reference method If this ratio is smaller than 05 the
precision under reproducibility conditions of the alternative method is considered to be greater than
that of the reference method
The ratio values are given in table 19
Table 19
Level
Reproducibility standard deviation
Reference method Alternative method Alternative method
reference method ratio
1 0242 0243 1007
2 0066 0133 2011
2 bis 0261 0195 0749
3 0038 0061 1617
Level Reproducibility limit
Reference method Alternative method
1 0676 0681
2 0185 0371
2 bis 0730 0547
3 0106 0171
The ratios of the reproducibility standard deviations are below or close to 2
The precision under reproducibility conditions of the alternative method is
equivalent to that of the reference method
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2938 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
214 Conclusion
Method comparison study
The Listeria PrecisTM enumeration method shows satisfying linearity and
relative accuracy
The alternative method shows satisfying inclusivity and exclusivity results
Two Listeria monocytogenes strains gave negative results with the
alternative method These strains grew weakly on the reference method
media (OampA Palcam and Oxford)
The limits of repeatability of the alternative method are similar to those of the
reference method
The bias between both methods are low between - 0133 Log CFUg and
- 0008 Log CFUg
The Listeria PrecisTM enumeration method offers the saving for confirmatory
tests
Positive results are obtained with the alternative method in 2-3 days (with
OBIS Mono test) or in 4-7 days (by the classical tests described in the
standardized method) against 4-7 days for the reference method
Inter-laboratory study
The bias between the reference and the alternative methods shows satisfying
and correct values
- level 1 (100 - 1 000 CFUg) bias = + 007 log CFUg
- level 2 (1 000 - 10 000 CFUg) bias = 002 log CFUg
- level 2 bis (1 000 - 10 000 CFUg) bias = - 004 log CFUg
- level 3 (10 000 - 100 000 CFUg) bias = - 004 log CFUg
The repeatability and the reproducibility of the alternative method look similar
to those of the reference method
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3038 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
22 Extension study (realised by ASEPT 2007)
An extension study was performed in 2007 for a new confirmatory test OBIS
MONO
150 Listeria monocytogenes strains and 100 non-target strains were isolated
onto TSAYE and BrillianceTM Listeria Agar plates All the Listeria
monocytogenes and the non-target strains gave the expected results
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3138 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Appendix 1 ndash Flow diagram of the Listeria PrecisTM method
x g sample + 9 x ml buffered peptone water
Revivification step for 1 h plusmn 5 min at 20degC plusmn 1degC
Streaking 1 ml onto 3 Brilliance Listeria Agar plates (mother suspension)
or 01 ml onto 1 Brilliance Listeria Agar plates (mother suspension and decimal dilutions)
Incubation for 48 h plusmn 3 h at 37degC plusmn 1degC
Confirmatory tests on at least one typical colony
- by the tests described in the ISO method
- by applying the OBIS mono test on an isolated colony
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3238 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Appendix 2 ndash ISO 11290-2 (1998) amp ISO 11290-2A1 (2004) Horizontal
method for the detection and enumeration of Listeria monocytogenes -
Part 2 enumeration of Listeria monocytogenes in foods
10 g cheese + 90 ml buffered peptone water or Half Fraser Broth (without supplements) (110 dilution)
Stomach 1 min 30 seconds
Keep at 20degC plusmn 2degC for 1 h plusmn 5 min
Decimal dilutions in 9 ml peptone salt tubes
Dilution 110
Dilution 1100
Dilution 11000
1 ml on 3 Chromogenic Listeria medium (ISO) plates (x 2) (-1)
01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-2)
01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-3)
01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-4)
Incubation at 37degC plusmn 1degC
24 h - 48 h
Numerate typical colonies at 24 h
(bluegreen surrounded by an opaque halo)
Numerate at 48 h
Streak typical colonies on TSYEA
(5 coloniesplate 2 successive dilutions)
Confirmation tests
Catalase
Gram stain
For confirmation of L monocytogenes
Haemolysis
Carbohydrate (rhamnose and xylose) utilisation
CAMP Test
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3338 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Appendix 3 ndash Mandelrsquos graphics
Laboratoire h Ref
Niveau 1 h Ref
Niveau 2 h Ref
Niveau 3 h Alt
Niveau 1 h Alt
Niveau 2 h Alt
Niveau 3 h5 h1 h5 h1
A -050 -015 083 062 -212 -039 198 299 -198 -299
B -261 -270 021 -077 -058 173 198 299 -198 -299
C -151 -031 -014 -015 -004 -005 198 299 -198 -299
D 038 040 -137 000 072 207 198 299 -198 -299
E -001 090 155 -052 056 -101 198 299 -198 -299
F -112 -321 -153 070 -325 023 198 299 -198 -299
G 042 -071 040 147 -009 176 198 299 -198 -299
H 169 046 -169 192 018 -083 198 299 -198 -299
L 009 -120 021 -077 -231 005 198 299 -198 -299
M 001 031 014 -032 004 -011 198 299 -198 -299
-300
-200
-100
000
100
200
300
A B C D E F G H L M
Vale
urs
h d
e M
an
del(
rob
uste
s)
Laboratoires
Meacutethode de reacutefeacuterence analyse robuste
h Ref Niveau 1
h Ref Niveau 2
h Ref Niveau 3
h 5=198
h 1=299
-300
-200
-100
000
100
200
300
A B C D E F G H L M
Vale
urs
de h
de M
an
del(
rob
uste
s)
Laboratoires
Meacutethode alternative analyse robuste
h Alt Niveau 1
h Alt Niveau 2
h Alt Niveau 2
h 1=299
h 5=198
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3438 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Laboratoire k Ref
Niveau 1 k Ref
Niveau 2 k Ref
Niveau 3 k Alt
Niveau 1 k Alt
Niveau 2 k Alt
Niveau 3 k1 k5
A 000 089 142 084 162 046 259 181
B 125 087 073 104 075 046 259 181
C 117 013 091 188 022 083 259 181
D 052 036 069 000 035 083 259 181
E 028 034 037 138 019 112 259 181
F 140 222 018 027 281 112 259 181
G 088 068 024 131 145 000 259 181
H 129 094 173 016 061 000 259 181
L 069 000 073 104 270 224 259 181
M 000 048 038 043 099 241 259 181
000
050
100
150
200
250
300
350
400
A B C D E F G H L M
Vale
urs
k d
e M
an
del(
rob
uste
s)
Laboratoires
Meacutethode de reacutefeacuterence analyse robuste
k Ref Niveau 1k Ref Niveau 2k Ref Niveau 3
k 1=259
k 5=181
000
050
100
150
200
250
300
A B C D E F G H L M
Vale
urs
k d
e M
an
del
(ro
bu
ste
s)
Laboratoires
Meacutethode alternative analyse robuste
k Alt Niveau 3
k Alt Niveau 1
k Alt Niveau 2
k 1=259
k 5=181
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3538 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Appendix 4 ndash Statistical results
Level 1
Nombre de laboratoires (p) 10
A 1602 1602 1602 000 0000 0000 -0495 0000 A 1845 1602 1724 017 0122 -0122 0621 0840 0122
B 1602 1176 1389 030 0213 -0213 -2613 1247 B 1602 1301 1452 021 0151 -0151 -0769 1041 0062
C 1699 1301 1500 028 0199 -0199 -1510 1165 C 1301 1845 1573 038 -0272 0272 -0148 1881 0073
D 1602 1778 1690 012 -0088 0088 0380 0516 D 1602 1602 1602 000 0000 0000 0000 0000 -0088
E 1699 1602 1651 007 0048 -0048 -0013 0284 E 1699 1301 1500 028 0199 -0199 -0522 1376 -0151
F 1778 1301 1540 034 0239 -0239 -1116 1397 F 1778 1699 1739 006 0040 -0040 0698 0274 0199
G 1845 1544 1695 021 0151 -0151 0425 0881 G 2079 1699 1889 027 0190 -0190 1467 1315 0194
H 2041 1602 1822 031 0220 -0220 1689 1286 H 2000 1954 1977 003 0023 -0023 1917 0158 0155
L 1544 1778 1661 017 -0117 0117 0092 0685 L 1301 1602 1452 021 -0151 0151 -0769 1041 -0210
M 1653 1653 1653 000 0000 0000 0013 0000 M 1477 1602 1540 009 -0062 0062 -0319 0432 -0114
m 165186 m 160206 mediane D 00678
Cn(2p) 1867 Cn(2p) 1867 Cp 16101
Cn(p) 16101 Cn(p) 16101 Qn(D) 0092926
n=2p n 20 n=2p n 20 Qdiff 01496169
f 11 f 11 t 114
l 55 l 55
n=p n 10 n=p n 10
f 6 f 6
l 15 l 15
Qn(2p) 0091465 Qn(2p) 007745
Qn(p) 0062469 Qn(p) 0121519
Qintra 017078 Qintra 014461
Qinter 010058 Qinter 019565
Sr(eacutecart-type de reacutepeacutetabiliteacute) 02415 Sr 02045
RSDr(coefficient de variation de la reacutepeacutetabiliteacute)1462 RSDr 1277
r(repeatability limit)) 0676 r 0573
SL(eacutecart-type interlaboratoire) -001905 SLlt0 SL 001737
SR(eacutecart-type de reproductibiliteacute) 02415 SR 02433
RSDR(coefficient de variation de la reproductibiliteacute)0146 RSDR 0152
R(limite de reproductibiliteacute)) 06762 R(limite de reproductibiliteacute))068123
h k h MoyenneLaboratoires
Niveau 1
si LaboratoiresDeviationMoyenne Dk DeviationsiAlt
Niveau 1
Ref
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3638 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Level 2
Nombre de laboratoires (p) 10
A 2681 2613 2647 005 0034 -0034 -0151 0891 A 2322 2531 2427 015 -0105 0105 -2119 1620 -0220
B 2544 2477 2511 005 0033 -0033 -2699 0872 B 2556 2653 2605 007 -0048 0048 -0581 0750 0094
C 2643 2633 2638 001 0005 -0005 -0311 0130 C 2681 2653 2667 002 0014 -0014 -0040 0217 0029
D 2690 2663 2676 002 0014 -0014 0400 0357 D 2778 2732 2755 003 0023 -0023 0721 0354 0079
E 2690 2716 2703 002 -0013 0013 0897 0336 E 2724 2748 2736 002 -0012 0012 0556 0185 0033
F 2568 2398 2483 012 0085 -0085 -3214 2217 F 2477 2114 2296 026 0182 -0182 -3255 2811 -0188
G 2643 2591 2617 004 0026 -0026 -0707 0682 G 2756 2568 2662 013 0094 -0094 -0085 1453 0045
H 2716 2643 2680 005 0036 -0036 0460 0945 H 2653 2732 2693 006 -0040 0040 0181 0613 0013
L 2591 2591 2591 000 0000 0000 -1196 0000 L 2230 2580 2405 025 -0175 0175 -2307 2704 -0186
M 2653 2690 2672 003 -0018 0018 0311 0481 M 2740 2613 2677 009 0064 -0064 0040 0987 0005
m 2655082 m 26719001 mediane D 00209
Cn(2p) 1867 Cn(2p) 1867 Cp 16101
Cn(p) 16101 Cn(p) 16101 Qn(D) 0039911
n=2p n 20 n=2p n 20 Qdiff 00642591
f 11 f 11 t 082
l 55 l 55
n=p n 10 n=p n 10
f 6 f 6
l 15 l 15
Qn(2p) 0020570 Qn(2p) 00345985
Qn(p) 00332437 Qn(p) 00718158
Qintra 003841 Qintra 006460
Qinter 005352 Qinter 011563
Sr(eacutecart-type de reacutepeacutetabiliteacute) 00543 Sr 00914
RSDr(coefficient de variation de la reacutepeacutetabiliteacute)205 RSDr 342
r(repeatability limit)) 0152 r 0256
SL(eacutecart-type interlaboratoire) 000139 SL 000920
SR(eacutecart-type de reproductibiliteacute) 00659 SR 01325
RSDR(coefficient de variation de la reproductibiliteacute)0025 RSDR 0050
R(limite de reproductibiliteacute)) 018446 R(limite de reproductibiliteacute))03708618
Laboratoires
Niveau 2
Moyenne si Laboratoires
Niveau 2
DRef Alt Moyenne si Deviation h Deviation h k k
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3738 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Level 2 bis
Nombre de laboratoires (p) 10
A 3000 2477 2739 037 0261 -0261 0832 1418 A 2602 2477 2540 009 0062 -0062 -0391 0465 -0199
B 2544 2813 2678 019 -0134 0134 0211 0729 B 2903 2778 2841 009 0062 -0062 1732 0465 0162
C 2477 2813 2645 024 -0168 0168 -0135 0911 C 2477 2699 2588 016 -0111 0111 -0049 0826 -0057
D 2398 2653 2526 018 -0128 0128 -1370 0692 D 2778 3000 2889 016 -0111 0111 2074 0826 0363
E 2875 2740 2808 010 0067 -0067 1547 0365 E 2602 2301 2452 021 0151 -0151 -1012 1120 -0356
F 2477 2544 2511 005 -0033 0033 -1525 0182 F 2778 2477 2628 021 0151 -0151 0230 1120 0117
G 2653 2740 2697 006 -0044 0044 0400 0236 G 2845 2845 2845 000 0000 0000 1764 0000 0148
H 2176 2813 2495 045 -0318 0318 -1692 1727 H 2477 2477 2477 000 0000 0000 -0832 0000 -0017
L 2813 2544 2678 019 0134 -0134 0211 0729 L 2903 2301 2602 043 0301 -0301 0049 2241 -0076
M 2602 2740 2671 010 -0069 0069 0135 0375 M 2903 2255 2579 046 0324 -0324 -0112 2411 -0092
m 2658114 m 259505 mediane D -00372
Cn(2p) 1867 Cn(2p) 1867 Cp 16101
Cn(p) 16101 Cn(p) 16101 Qn(D) 01656915
n=2p n 20 n=2p n 20 Qdiff 02667753
f 11 f 11 t 035
l 55 l 55
n=p n 10 n=p n 10
f 6 f 6
l 15 l 15
Qn(2p) 0098745 Qn(2p) 007195
Qn(p) 006007 Qn(p) 008805
Qintra 018437 Qintra 013435
Qinter 009672 Qinter 014176
Sr(eacutecart-type de reacutepeacutetabiliteacute) 02607 Sr 01900
RSDr(coefficient de variation de la reacutepeacutetabiliteacute)981 RSDr 732
r(repeatability limit)) 0730 r 0532
SL(eacutecart-type interlaboratoire) -002464 SLlt0 SL 000205
SR(eacutecart-type de reproductibiliteacute) 02607 SR 01953
RSDR(coefficient de variation de la reproductibiliteacute)0098 RSDR 0075
R(limite de reproductibiliteacute)) 072996 R(limite de reproductibiliteacute))054686
Laboratoires
Niveau 2 bis
Moyenne si Laboratoires
Niveau 2 bis
DRef Alt Moyenne si Deviation h Deviation h k k
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3838 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Level 3
Nombre de laboratoires (p) 10
A 3690 3663 3676 002 0014 -0014 -0311 0513 A 3690 3708 3699 001 -0009 0009 0354 0257 0022
B 3613 3672 3642 004 -0030 0030 -1851 1109 B 3708 3568 3638 010 0070 -0070 -0844 2061 -0005
C 3663 3732 3698 005 -0035 0035 0645 1302 C 3663 3699 3681 003 -0018 0018 0000 0536 -0017
D 3690 3690 3690 000 0000 0000 0311 0000 D 3643 3663 3653 001 -0010 0010 -0545 0286 -0037
E 3732 3653 3693 006 0040 -0040 0429 1480 E 3613 3732 3673 008 -0060 0060 -0162 1769 -0020
F 3643 3672 3658 002 -0014 0014 -1157 0536 F 3690 3771 3731 006 -0040 0040 0975 1193 0073
G 3699 3653 3676 003 0023 -0023 -0328 0856 G 3602 3643 3623 003 -0021 0021 -1141 0612 -0053
H 3623 3672 3648 003 -0024 0024 -1615 0913 H 3748 3681 3715 005 0033 -0033 0664 0990 0067
L 3724 3663 3694 004 0031 -0031 0461 1150 L 3748 3771 3760 002 -0011 0011 1544 0335 0066
M 3690 3690 3690 000 0000 0000 0311 0000 M 3806 3681 3744 009 0062 -0062 1234 1848 0054
m 368334 m 368086 mediane D 00089
Cn(2p) 1867 Cn(2p) 1867 Cp 16101
Cn(p) 16101 Cn(p) 16101 Qn(D) 00325359
n=2p n 20 n=2p n 20 Qdiff 00523852
f 11 f 11 t 043
l 55 l 55
n=p n 10 n=p n 10
f 6 f 6
l 15 l 15
Qn(2p) 0014323 Qn(2p) 001811
Qn(p) 0013719 Qn(p) 003164
Qintra 002674 Qintra 003381
Qinter 002209 Qinter 005094
Sr(eacutecart-type de reacutepeacutetabiliteacute) 00378 Sr 00478
RSDr(coefficient de variation de la reacutepeacutetabiliteacute)103 RSDr 130
r(repeatability limit)) 0106 r 0134
SL(eacutecart-type interlaboratoire) -000023 SLlt0 SL 000145
SR(eacutecart-type de reproductibiliteacute) 00378 SR 00611
RSDR(coefficient de variation de la reproductibiliteacute)0010 RSDR 0017
R(limite de reproductibiliteacute)) 0105894 R(limite de reproductibiliteacute))017119
Ref Alt DLaboratoires
Niveau 3
Moyenne si Deviation h k Laboratoires
Niveau 3
Moyenne si Deviation h k
Thermo Fisher Scientific
ADRIA Deacuteveloppement 1738 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
2113 Detection limit (LOD) and quantification limit (LOQ)
The critical level is the defined as the smallest amount which can be detected (not null) but not
quantified as an exact value Below this value it cannot be sure that the true value is not null
The detection limit is defined as being higher than the critical level because it involves a power the
probability 1- which has to be well over 50 for example 95
The quantification limit is defined as the smallest amount of analyte (that is the lowest actual number of
organisms) which can be measured and quantified with defined precision and accuracy under the
experimental conditions by the method under validation
Protocol
The detection limit of the alternative method was realised with pure cultures
Three different inoculation levels were tested with six replicates per level ie
18 analyses were done by the alternative method
Listeria monocytogenes 18 was used for the detection limit of the alternative
method
Quantification limit was calculated for six independent blank samples
determinations
Results
These data are intrinsic to the method used and are presented in the
following tables
Table 8 ndash Inoculation 1 ml on 3 plates
Level (CFUml) Positive samples number Standard deviation Bias
0 06
05 26 1211 0
1 36 0816 05
5 66 2483 85
Thermo Fisher Scientific
ADRIA Deacuteveloppement 1838 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Table 9 ndash Inoculation 01 ml on 1 plate
Level (CFUml) Positive samples number Standard deviation Bias
0 06
5 36 0548 05
10 46 1414 2
50 66 2258 55
Table 10
Formulas Obtained values
LC 165 S0 + X0 18
LOD 33 S0 + X0 32
LOQ 10 S0 + X0 87
2114 Relative Sensitivity
The relative sensitivity is defined as the ability of the alternative method to detect two different amounts
of analyte measured by the reference method within a given matrix at a specified average value or
over the whole measurement range that is it is the minimal quantity variation (increase of the analyte
concentration x) which gives a significant variation of the measured signal (response y)
These data are intrinsic to the used method and are obtained from the results
of the linearity study
Sensitivity patterns obtained for tested (matrixstrain) pairs are presented in
figure 5
Thermo Fisher Scientific
ADRIA Deacuteveloppement 1938 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Figure 5 ndash Sensitivity patterns for the tested (matrixstrain) pairs
2115 SpecificitySelectivity
The specificity is defined as the degree to which a method is affected (or not) by the other components
present in a multi-component sample That is the ability of a method to measure exactly a given
analyte or its amount within the sample without interference from non-target components such as a
matrix effect or background noise
The selectivity is defined as a measure of the degree of non-interference in the presence of non-target
analytes A method is selective if it can be used to detect the analyte under examination and that a
guarantee can be provided that the detected signal can only be a product by that specific analyte
The specificity and selectivity study was performed for the Listeria PrecisTM
detection method no additional test was realised for the enumeration
method The results were the following
- Among the Listeria monocytogenes strains tested 2 strains (Listeria
monocytogenes CIP 7834 and Listeria monocytogenes CIP 105459) gave
negative results These 2 strains were tested twice by running 48 h
incubation time of the compared agars Positive results were then
observed by the alternative method Pale characteristic colonies were
observed on the ISO agar by streaking the Half Fraser broth only
- No cross reaction was observed with the non-target strains except with
Listeria ivanovii which gave characteristic colonies on Brilliancetrade Listeria
Agar but the confirmatory tests provide a clear discrimination between
Listeria monocytogenes and Listeria ivanovi species
Rillettes
Lettuce
Raw milk
Smoked salmon
Raw egg
Process water
x (CFUg)
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2038 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
212 Practicability
1 Packaging Ready to use Brilliance Listeria agar plates are available
(x 10 plates)
2 Reagent volume
3 Storage conditionsndash
Shelf life of products
not opened
The plates are stored at 6 - 12degC in darkness conditions The
expiration date is mentioned on the package and on the plates It
is around 3 months
4 Modalities of use
after first use
5 Required
equipments No specific equipment is required
6 Reagents Ready to use plates
7 Training 05 day
8 Real time handling
Reference method
9 min for one sample
55 min for 10 samples
Alternative method
7 min for one sample
35 minutes for 10 samples
9 Time to result
Negative results
Reference method Day 2
Alternative method Day 2
Negative results after confirmation or positive results
Reference method Day 4 ndash Day 7
Alternative method Day 4 ndash Day 7
Positive results
Reference method Day 4 ndash Day 7
Alternative method Day 2 ndash Day 3 (with OBIS Mono Test)
or Day 4 ndash Day 7 (by the classical tests described in the
standardised method)
10 Technician skill Identical to that required for the reference method
11 Steps common to
the reference method Mother suspension revivification step and decimal dilutions
12 Traceability
13 Maintenance
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ADRIA Deacuteveloppement 2138 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
213 Inter-laboratory study
2131 Organisation
12 laboratories participated to this study Pasteurised milk was inoculated by
Listeria monocytogenes 12a isolated from raw milk The targeted
inoculation levels were
- 0 CFUml
- 50 CFU ml
- 500 CFU ml
- 5 000 CFU ml
Each laboratory received eight flasks of 10 ml sample ie two flasks per
inoculation level Furthermore one non-inoculated sample has been added
to the package for total viable count microflora (NF ISO 4833 method)
Detailed instructions were transmitted to the collaborators by the expert
laboratory
Collaborative study laboratories and the expert laboratory carried out the
analyses with the alternative and reference methods
2132 Verification of experimental parameters
Strain stability during transport
In order to evaluate the Listeria monocytogenes strain stability during
transport bacterial count of all inoculated flasks were checked at different
time ie inoculation time after 24 h of conservation at 2deg - 8degC Results are
reported in table 11
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2238 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Table 11 ndash Listeria monocytogenes count
by ISO 11290-2A1 method (log CFUml)
Day of the analyses Level contamination Reference method
Day 0
(29052006)
1 45
2 440
3 4 500
Day 1
(30052006)
1 60
2 430
3 4 900
No evolution of the strain was observed after storage for 24 h at 2degC ndash 8degC
Contamination level
The real contamination levels are provided in table 12
Table 12 ndash Real contamination level (CFUml)
Target inoculation
(CFU ml) Samples Ndeg
Real contamination levels
(CFUml)
0 1 - 8 0
50 5 - 6 45
500 3 - 7 440
5000 2 - 4 4 500
Sample temperature at receipt
Measured temperatures at receipt are listed in table 13
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2338 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Table 13 - Sample temperature on receipt
Laboratories Receipt
day
Hour
receipt
Measured temperature
at receipt (degC)
Measured temperature by the
temperature probe (degC)
A
B
C
D
E
F
G
H
I
J
K
L
M
30052006
10h45
10h15
11h15
10h00
09h30
8h30
11h50
10h00
08h30
14h45
08h30
09h45
08h45
82
55
45
63
60
62
81
38
48
-
14
67
76
03
03
-13
03
04
-14
05
04
01
05
-13
04
Conclusion
The temperatures during the transport were correct for all the labs except for
Lab J which didnrsquot provide the information
All the labs received their packages with several broken flasks
Three labs (B D and L) received some damaged samples but proceeded to
analyses
All the labs started the analyses at Day 1 except Lab J
2133 Results
Aerobic mesophilic flora
The count of aerobic mesophilic flora varied from 94 102 CFUml to
33 103 CFUml
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2438 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Expert lab results
The results obtained by the expert lab are the following
Table 14
Target inoculation level (CFUml)
Reference method ISO 11290-2
Alternative method Listeria Precis Enumeration
Rep 1 Rep 2 Rep 1 Rep 2
lt 10 lt 10 lt 10 lt 10 lt10
50 50 (Ne) 60 (Ne) 50 (Ne) 30 (Ne)
500 470 390 490 500
5 000 4 700 5 100 5 900 3 400
Ne estimated number
The obtained results are in agreement with those expected
Collaborator Labs results
A synthesis of the results obtained by the collaborator labs is provided
Table 15 (CFUml) and Table 16 (log CFUml)
Table 15 ndash Synthesis of results obtained by the alternative
and the reference methods (CFUml)
Labs Level 1 Level 2 Level 2 bis Level 3
Reference method
Alternative method
Reference method
Alternative method
Reference method
Alternative method
Reference method
Alternative method
A 40 40 70 40 480 410 210 340 1000 300 400 300 4900 4600 4900 5100
B 40 15 40 20 350 300 360 450 350 650 800 600 4100 4700 5100 3700
C 50 20 20 70 440 430 480 450 300 650 300 500 4600 5400 4600 5000
D 40 60 40 40 490 460 600 540 250 450 600 1000 4900 4900 4400 4600
E 50 40 50 20 490 520 530 560 750 550 400 200 5400 4500 4100 5400
F 60 20 60 50 370 250 300 130 300 350 600 300 4400 4700 4900 5900
G 70 35 120 50 440 390 570 370 450 550 700 700 5000 4500 4000 4400
H 110 40 100 90 520 440 450 540 150 650 300 300 4200 4700 5600 4800
L 35 60 20 40 390 390 170 380 650 350 800 200 5300 4600 5600 5900
M 45 45 30 40 450 490 550 410 400 550 800 180 4900 4900 6400 4800
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2538 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Table 16 ndash Synthesis of results obtained by the alternative
and the reference methods (log CFUml)
Labs Level 1 Level 2 Level 2 bis Level 3
Reference method
Alternative method
Reference method
Alternative method
Reference method
Alternative method
Reference method
Alternative method
A 1602 1602 1845 1602 2681 2613 2322 2531 3000 2477 2602 2477 3690 3663 3690 3708
B 1602 1176 1602 1301 2544 2477 2556 2653 2544 2813 2903 2778 3613 3672 3708 3568
C 1699 1301 1301 1845 2643 2633 2681 2653 2477 2813 2477 2699 3663 3732 3663 3699
D 1602 1778 1602 1602 2690 2663 2778 2732 2398 2653 2778 3000 3690 3690 3643 3663
E 1699 1602 1699 1301 2690 2716 2724 2748 2875 2740 2602 2301 3732 3653 3613 3732
F 1778 1301 1778 1699 2568 2398 2477 2114 2477 2544 2778 2477 3643 3672 3690 3771
G 1845 1544 2079 1699 2643 2591 2756 2568 2653 2740 2845 2845 3699 3653 3602 3643
H 2041 1602 2000 1954 2716 2643 2653 2732 2176 2813 2477 2477 3623 3672 3748 3681
L 1544 1778 1301 1602 2591 2591 2230 2580 2813 2544 2903 2301 3724 3663 3748 3771
M 1653 1653 1477 1602 2653 2690 2740 2613 2602 2740 2903 2255 3690 3690 3806 3681
Level 2 results obtained taking account the 3 plates inoculated with the mother-suspension
Level 2 bis results obtained taking account the plates inoculated with 01 ml of the mother-suspension
Scrutiny of the measurement results for consistency
In order to identify the measurement results or laboratories that could be
inconsistent two graphical consistency techniques were realised Mandelrsquos
h-ank-statistics Mandel indicators h and k at 5 significance highlighted
some possible inconsistent data (See tables and graphics in Appendix 3 and
Appendix 4)
Mandelrsquos values Number of values observed over the threshold
Reference method Alternative method
h gt 1 Lab F Level 2 Lab F Level 2
h gt 5 Lab F Level 2
Lab B Levels 1 and 2
Lab A Level 2
Lab D Level 3
Lab F Level 2
Lab L Level 2
kgt 1 Lab F Level 3
k gt 5 Lab F Level 2 Lab C Level 2
Lab F Level 3
Lab L Levels 1 and 3
Lab M Level 1
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2638 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
The emphasized results appear (i) coherent with the targeted contamination
levels (ii) concordant between both methods
All the results were thus kept for the statistical analysis
Comparison of the trueness and precision characteristics of
the reference method and alternative methods
The statistical values are summarised hereafter
Level p
Reference method Alternative method
Repeatability ratio
Reproducibility ratio Median
Repeatability standard deviation
Reproducibility standard deviation
Median Repeatability
standard deviation
Reproducibility standard deviation
1 10 16519 02415 02425 16021 02045 02433 0847 1007
2 10 26551 00543 00659 26719 00914 01325 1682 2011
2 bis 10 26581 02607 02607 25951 01900 01953 0729 0749
3 10 18671 00378 00378 18671 00478 00611 1264 1617
Bias of the alternative method
In order to estimate the bias of the alternative method with regard to the reference method for every
level Dij and t are calculated according to the following equations
RefAlt ijYijYDij
Diffp
Dijit
)2(
)(median
If the t value is superior to 2 the alternative method is significantly biased
with regard to the reference method
The values are given in table 17
Table 17 ndash t (d) values obtained by level
Level Bias t (d) Conclusion
1 0068 114 Non significant bias
2 0021 082 Non significant bias
2 bis - 0037 035 Non significant bias
3 - 0037 043 Non significant bias
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2738 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
The statistical test concludes to a non significant bias for all the inoculation
levels The bias varies from ndash 0037 to 0068 log CFUml
Comparison of the repeatability standard deviations
If the ratio Srj Alt Srj Ref of the repeatability standard deviations of the
alternative method and the reference method is larger than 2 the precision
under repeatability conditions of the alternative method is considered to be
lower than that of the reference method If this ratio is smaller than 05 the
precision under repeatability conditions of the alternative method is
considered to be greater than that of the reference method
The ratio values are given in table 18
Table 18
Level
Repeatability standard deviation
Reference method Alternative method Alternative method
reference method ratio
1 0242 0205 0847
2 0054 0091 1682
2 bis 0261 0190 0729
3 0038 0048 1264
Level Repeatability limit
Reference method Alternative method
1 0676 0573
2 0152 0256
2 bis 0730 0532
3 0106 0134
The ratios of the repeatability standard deviations are below 2 in all cases
The precision under repeatability conditions of the alternative method is
equivalent to that of the reference method
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2838 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Comparison of the reproducibility standard deviations
If the ratio Srj Alt Srj Ref of the reproducibility standard deviations of the alternative method and the
reference method is larger than 2 the precision under reproducibility conditions of the alternative
method is considered to be lower than that of the reference method If this ratio is smaller than 05 the
precision under reproducibility conditions of the alternative method is considered to be greater than
that of the reference method
The ratio values are given in table 19
Table 19
Level
Reproducibility standard deviation
Reference method Alternative method Alternative method
reference method ratio
1 0242 0243 1007
2 0066 0133 2011
2 bis 0261 0195 0749
3 0038 0061 1617
Level Reproducibility limit
Reference method Alternative method
1 0676 0681
2 0185 0371
2 bis 0730 0547
3 0106 0171
The ratios of the reproducibility standard deviations are below or close to 2
The precision under reproducibility conditions of the alternative method is
equivalent to that of the reference method
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2938 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
214 Conclusion
Method comparison study
The Listeria PrecisTM enumeration method shows satisfying linearity and
relative accuracy
The alternative method shows satisfying inclusivity and exclusivity results
Two Listeria monocytogenes strains gave negative results with the
alternative method These strains grew weakly on the reference method
media (OampA Palcam and Oxford)
The limits of repeatability of the alternative method are similar to those of the
reference method
The bias between both methods are low between - 0133 Log CFUg and
- 0008 Log CFUg
The Listeria PrecisTM enumeration method offers the saving for confirmatory
tests
Positive results are obtained with the alternative method in 2-3 days (with
OBIS Mono test) or in 4-7 days (by the classical tests described in the
standardized method) against 4-7 days for the reference method
Inter-laboratory study
The bias between the reference and the alternative methods shows satisfying
and correct values
- level 1 (100 - 1 000 CFUg) bias = + 007 log CFUg
- level 2 (1 000 - 10 000 CFUg) bias = 002 log CFUg
- level 2 bis (1 000 - 10 000 CFUg) bias = - 004 log CFUg
- level 3 (10 000 - 100 000 CFUg) bias = - 004 log CFUg
The repeatability and the reproducibility of the alternative method look similar
to those of the reference method
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3038 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
22 Extension study (realised by ASEPT 2007)
An extension study was performed in 2007 for a new confirmatory test OBIS
MONO
150 Listeria monocytogenes strains and 100 non-target strains were isolated
onto TSAYE and BrillianceTM Listeria Agar plates All the Listeria
monocytogenes and the non-target strains gave the expected results
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3138 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Appendix 1 ndash Flow diagram of the Listeria PrecisTM method
x g sample + 9 x ml buffered peptone water
Revivification step for 1 h plusmn 5 min at 20degC plusmn 1degC
Streaking 1 ml onto 3 Brilliance Listeria Agar plates (mother suspension)
or 01 ml onto 1 Brilliance Listeria Agar plates (mother suspension and decimal dilutions)
Incubation for 48 h plusmn 3 h at 37degC plusmn 1degC
Confirmatory tests on at least one typical colony
- by the tests described in the ISO method
- by applying the OBIS mono test on an isolated colony
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3238 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Appendix 2 ndash ISO 11290-2 (1998) amp ISO 11290-2A1 (2004) Horizontal
method for the detection and enumeration of Listeria monocytogenes -
Part 2 enumeration of Listeria monocytogenes in foods
10 g cheese + 90 ml buffered peptone water or Half Fraser Broth (without supplements) (110 dilution)
Stomach 1 min 30 seconds
Keep at 20degC plusmn 2degC for 1 h plusmn 5 min
Decimal dilutions in 9 ml peptone salt tubes
Dilution 110
Dilution 1100
Dilution 11000
1 ml on 3 Chromogenic Listeria medium (ISO) plates (x 2) (-1)
01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-2)
01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-3)
01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-4)
Incubation at 37degC plusmn 1degC
24 h - 48 h
Numerate typical colonies at 24 h
(bluegreen surrounded by an opaque halo)
Numerate at 48 h
Streak typical colonies on TSYEA
(5 coloniesplate 2 successive dilutions)
Confirmation tests
Catalase
Gram stain
For confirmation of L monocytogenes
Haemolysis
Carbohydrate (rhamnose and xylose) utilisation
CAMP Test
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3338 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Appendix 3 ndash Mandelrsquos graphics
Laboratoire h Ref
Niveau 1 h Ref
Niveau 2 h Ref
Niveau 3 h Alt
Niveau 1 h Alt
Niveau 2 h Alt
Niveau 3 h5 h1 h5 h1
A -050 -015 083 062 -212 -039 198 299 -198 -299
B -261 -270 021 -077 -058 173 198 299 -198 -299
C -151 -031 -014 -015 -004 -005 198 299 -198 -299
D 038 040 -137 000 072 207 198 299 -198 -299
E -001 090 155 -052 056 -101 198 299 -198 -299
F -112 -321 -153 070 -325 023 198 299 -198 -299
G 042 -071 040 147 -009 176 198 299 -198 -299
H 169 046 -169 192 018 -083 198 299 -198 -299
L 009 -120 021 -077 -231 005 198 299 -198 -299
M 001 031 014 -032 004 -011 198 299 -198 -299
-300
-200
-100
000
100
200
300
A B C D E F G H L M
Vale
urs
h d
e M
an
del(
rob
uste
s)
Laboratoires
Meacutethode de reacutefeacuterence analyse robuste
h Ref Niveau 1
h Ref Niveau 2
h Ref Niveau 3
h 5=198
h 1=299
-300
-200
-100
000
100
200
300
A B C D E F G H L M
Vale
urs
de h
de M
an
del(
rob
uste
s)
Laboratoires
Meacutethode alternative analyse robuste
h Alt Niveau 1
h Alt Niveau 2
h Alt Niveau 2
h 1=299
h 5=198
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3438 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Laboratoire k Ref
Niveau 1 k Ref
Niveau 2 k Ref
Niveau 3 k Alt
Niveau 1 k Alt
Niveau 2 k Alt
Niveau 3 k1 k5
A 000 089 142 084 162 046 259 181
B 125 087 073 104 075 046 259 181
C 117 013 091 188 022 083 259 181
D 052 036 069 000 035 083 259 181
E 028 034 037 138 019 112 259 181
F 140 222 018 027 281 112 259 181
G 088 068 024 131 145 000 259 181
H 129 094 173 016 061 000 259 181
L 069 000 073 104 270 224 259 181
M 000 048 038 043 099 241 259 181
000
050
100
150
200
250
300
350
400
A B C D E F G H L M
Vale
urs
k d
e M
an
del(
rob
uste
s)
Laboratoires
Meacutethode de reacutefeacuterence analyse robuste
k Ref Niveau 1k Ref Niveau 2k Ref Niveau 3
k 1=259
k 5=181
000
050
100
150
200
250
300
A B C D E F G H L M
Vale
urs
k d
e M
an
del
(ro
bu
ste
s)
Laboratoires
Meacutethode alternative analyse robuste
k Alt Niveau 3
k Alt Niveau 1
k Alt Niveau 2
k 1=259
k 5=181
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3538 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Appendix 4 ndash Statistical results
Level 1
Nombre de laboratoires (p) 10
A 1602 1602 1602 000 0000 0000 -0495 0000 A 1845 1602 1724 017 0122 -0122 0621 0840 0122
B 1602 1176 1389 030 0213 -0213 -2613 1247 B 1602 1301 1452 021 0151 -0151 -0769 1041 0062
C 1699 1301 1500 028 0199 -0199 -1510 1165 C 1301 1845 1573 038 -0272 0272 -0148 1881 0073
D 1602 1778 1690 012 -0088 0088 0380 0516 D 1602 1602 1602 000 0000 0000 0000 0000 -0088
E 1699 1602 1651 007 0048 -0048 -0013 0284 E 1699 1301 1500 028 0199 -0199 -0522 1376 -0151
F 1778 1301 1540 034 0239 -0239 -1116 1397 F 1778 1699 1739 006 0040 -0040 0698 0274 0199
G 1845 1544 1695 021 0151 -0151 0425 0881 G 2079 1699 1889 027 0190 -0190 1467 1315 0194
H 2041 1602 1822 031 0220 -0220 1689 1286 H 2000 1954 1977 003 0023 -0023 1917 0158 0155
L 1544 1778 1661 017 -0117 0117 0092 0685 L 1301 1602 1452 021 -0151 0151 -0769 1041 -0210
M 1653 1653 1653 000 0000 0000 0013 0000 M 1477 1602 1540 009 -0062 0062 -0319 0432 -0114
m 165186 m 160206 mediane D 00678
Cn(2p) 1867 Cn(2p) 1867 Cp 16101
Cn(p) 16101 Cn(p) 16101 Qn(D) 0092926
n=2p n 20 n=2p n 20 Qdiff 01496169
f 11 f 11 t 114
l 55 l 55
n=p n 10 n=p n 10
f 6 f 6
l 15 l 15
Qn(2p) 0091465 Qn(2p) 007745
Qn(p) 0062469 Qn(p) 0121519
Qintra 017078 Qintra 014461
Qinter 010058 Qinter 019565
Sr(eacutecart-type de reacutepeacutetabiliteacute) 02415 Sr 02045
RSDr(coefficient de variation de la reacutepeacutetabiliteacute)1462 RSDr 1277
r(repeatability limit)) 0676 r 0573
SL(eacutecart-type interlaboratoire) -001905 SLlt0 SL 001737
SR(eacutecart-type de reproductibiliteacute) 02415 SR 02433
RSDR(coefficient de variation de la reproductibiliteacute)0146 RSDR 0152
R(limite de reproductibiliteacute)) 06762 R(limite de reproductibiliteacute))068123
h k h MoyenneLaboratoires
Niveau 1
si LaboratoiresDeviationMoyenne Dk DeviationsiAlt
Niveau 1
Ref
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3638 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Level 2
Nombre de laboratoires (p) 10
A 2681 2613 2647 005 0034 -0034 -0151 0891 A 2322 2531 2427 015 -0105 0105 -2119 1620 -0220
B 2544 2477 2511 005 0033 -0033 -2699 0872 B 2556 2653 2605 007 -0048 0048 -0581 0750 0094
C 2643 2633 2638 001 0005 -0005 -0311 0130 C 2681 2653 2667 002 0014 -0014 -0040 0217 0029
D 2690 2663 2676 002 0014 -0014 0400 0357 D 2778 2732 2755 003 0023 -0023 0721 0354 0079
E 2690 2716 2703 002 -0013 0013 0897 0336 E 2724 2748 2736 002 -0012 0012 0556 0185 0033
F 2568 2398 2483 012 0085 -0085 -3214 2217 F 2477 2114 2296 026 0182 -0182 -3255 2811 -0188
G 2643 2591 2617 004 0026 -0026 -0707 0682 G 2756 2568 2662 013 0094 -0094 -0085 1453 0045
H 2716 2643 2680 005 0036 -0036 0460 0945 H 2653 2732 2693 006 -0040 0040 0181 0613 0013
L 2591 2591 2591 000 0000 0000 -1196 0000 L 2230 2580 2405 025 -0175 0175 -2307 2704 -0186
M 2653 2690 2672 003 -0018 0018 0311 0481 M 2740 2613 2677 009 0064 -0064 0040 0987 0005
m 2655082 m 26719001 mediane D 00209
Cn(2p) 1867 Cn(2p) 1867 Cp 16101
Cn(p) 16101 Cn(p) 16101 Qn(D) 0039911
n=2p n 20 n=2p n 20 Qdiff 00642591
f 11 f 11 t 082
l 55 l 55
n=p n 10 n=p n 10
f 6 f 6
l 15 l 15
Qn(2p) 0020570 Qn(2p) 00345985
Qn(p) 00332437 Qn(p) 00718158
Qintra 003841 Qintra 006460
Qinter 005352 Qinter 011563
Sr(eacutecart-type de reacutepeacutetabiliteacute) 00543 Sr 00914
RSDr(coefficient de variation de la reacutepeacutetabiliteacute)205 RSDr 342
r(repeatability limit)) 0152 r 0256
SL(eacutecart-type interlaboratoire) 000139 SL 000920
SR(eacutecart-type de reproductibiliteacute) 00659 SR 01325
RSDR(coefficient de variation de la reproductibiliteacute)0025 RSDR 0050
R(limite de reproductibiliteacute)) 018446 R(limite de reproductibiliteacute))03708618
Laboratoires
Niveau 2
Moyenne si Laboratoires
Niveau 2
DRef Alt Moyenne si Deviation h Deviation h k k
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3738 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Level 2 bis
Nombre de laboratoires (p) 10
A 3000 2477 2739 037 0261 -0261 0832 1418 A 2602 2477 2540 009 0062 -0062 -0391 0465 -0199
B 2544 2813 2678 019 -0134 0134 0211 0729 B 2903 2778 2841 009 0062 -0062 1732 0465 0162
C 2477 2813 2645 024 -0168 0168 -0135 0911 C 2477 2699 2588 016 -0111 0111 -0049 0826 -0057
D 2398 2653 2526 018 -0128 0128 -1370 0692 D 2778 3000 2889 016 -0111 0111 2074 0826 0363
E 2875 2740 2808 010 0067 -0067 1547 0365 E 2602 2301 2452 021 0151 -0151 -1012 1120 -0356
F 2477 2544 2511 005 -0033 0033 -1525 0182 F 2778 2477 2628 021 0151 -0151 0230 1120 0117
G 2653 2740 2697 006 -0044 0044 0400 0236 G 2845 2845 2845 000 0000 0000 1764 0000 0148
H 2176 2813 2495 045 -0318 0318 -1692 1727 H 2477 2477 2477 000 0000 0000 -0832 0000 -0017
L 2813 2544 2678 019 0134 -0134 0211 0729 L 2903 2301 2602 043 0301 -0301 0049 2241 -0076
M 2602 2740 2671 010 -0069 0069 0135 0375 M 2903 2255 2579 046 0324 -0324 -0112 2411 -0092
m 2658114 m 259505 mediane D -00372
Cn(2p) 1867 Cn(2p) 1867 Cp 16101
Cn(p) 16101 Cn(p) 16101 Qn(D) 01656915
n=2p n 20 n=2p n 20 Qdiff 02667753
f 11 f 11 t 035
l 55 l 55
n=p n 10 n=p n 10
f 6 f 6
l 15 l 15
Qn(2p) 0098745 Qn(2p) 007195
Qn(p) 006007 Qn(p) 008805
Qintra 018437 Qintra 013435
Qinter 009672 Qinter 014176
Sr(eacutecart-type de reacutepeacutetabiliteacute) 02607 Sr 01900
RSDr(coefficient de variation de la reacutepeacutetabiliteacute)981 RSDr 732
r(repeatability limit)) 0730 r 0532
SL(eacutecart-type interlaboratoire) -002464 SLlt0 SL 000205
SR(eacutecart-type de reproductibiliteacute) 02607 SR 01953
RSDR(coefficient de variation de la reproductibiliteacute)0098 RSDR 0075
R(limite de reproductibiliteacute)) 072996 R(limite de reproductibiliteacute))054686
Laboratoires
Niveau 2 bis
Moyenne si Laboratoires
Niveau 2 bis
DRef Alt Moyenne si Deviation h Deviation h k k
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3838 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Level 3
Nombre de laboratoires (p) 10
A 3690 3663 3676 002 0014 -0014 -0311 0513 A 3690 3708 3699 001 -0009 0009 0354 0257 0022
B 3613 3672 3642 004 -0030 0030 -1851 1109 B 3708 3568 3638 010 0070 -0070 -0844 2061 -0005
C 3663 3732 3698 005 -0035 0035 0645 1302 C 3663 3699 3681 003 -0018 0018 0000 0536 -0017
D 3690 3690 3690 000 0000 0000 0311 0000 D 3643 3663 3653 001 -0010 0010 -0545 0286 -0037
E 3732 3653 3693 006 0040 -0040 0429 1480 E 3613 3732 3673 008 -0060 0060 -0162 1769 -0020
F 3643 3672 3658 002 -0014 0014 -1157 0536 F 3690 3771 3731 006 -0040 0040 0975 1193 0073
G 3699 3653 3676 003 0023 -0023 -0328 0856 G 3602 3643 3623 003 -0021 0021 -1141 0612 -0053
H 3623 3672 3648 003 -0024 0024 -1615 0913 H 3748 3681 3715 005 0033 -0033 0664 0990 0067
L 3724 3663 3694 004 0031 -0031 0461 1150 L 3748 3771 3760 002 -0011 0011 1544 0335 0066
M 3690 3690 3690 000 0000 0000 0311 0000 M 3806 3681 3744 009 0062 -0062 1234 1848 0054
m 368334 m 368086 mediane D 00089
Cn(2p) 1867 Cn(2p) 1867 Cp 16101
Cn(p) 16101 Cn(p) 16101 Qn(D) 00325359
n=2p n 20 n=2p n 20 Qdiff 00523852
f 11 f 11 t 043
l 55 l 55
n=p n 10 n=p n 10
f 6 f 6
l 15 l 15
Qn(2p) 0014323 Qn(2p) 001811
Qn(p) 0013719 Qn(p) 003164
Qintra 002674 Qintra 003381
Qinter 002209 Qinter 005094
Sr(eacutecart-type de reacutepeacutetabiliteacute) 00378 Sr 00478
RSDr(coefficient de variation de la reacutepeacutetabiliteacute)103 RSDr 130
r(repeatability limit)) 0106 r 0134
SL(eacutecart-type interlaboratoire) -000023 SLlt0 SL 000145
SR(eacutecart-type de reproductibiliteacute) 00378 SR 00611
RSDR(coefficient de variation de la reproductibiliteacute)0010 RSDR 0017
R(limite de reproductibiliteacute)) 0105894 R(limite de reproductibiliteacute))017119
Ref Alt DLaboratoires
Niveau 3
Moyenne si Deviation h k Laboratoires
Niveau 3
Moyenne si Deviation h k
Thermo Fisher Scientific
ADRIA Deacuteveloppement 1838 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Table 9 ndash Inoculation 01 ml on 1 plate
Level (CFUml) Positive samples number Standard deviation Bias
0 06
5 36 0548 05
10 46 1414 2
50 66 2258 55
Table 10
Formulas Obtained values
LC 165 S0 + X0 18
LOD 33 S0 + X0 32
LOQ 10 S0 + X0 87
2114 Relative Sensitivity
The relative sensitivity is defined as the ability of the alternative method to detect two different amounts
of analyte measured by the reference method within a given matrix at a specified average value or
over the whole measurement range that is it is the minimal quantity variation (increase of the analyte
concentration x) which gives a significant variation of the measured signal (response y)
These data are intrinsic to the used method and are obtained from the results
of the linearity study
Sensitivity patterns obtained for tested (matrixstrain) pairs are presented in
figure 5
Thermo Fisher Scientific
ADRIA Deacuteveloppement 1938 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Figure 5 ndash Sensitivity patterns for the tested (matrixstrain) pairs
2115 SpecificitySelectivity
The specificity is defined as the degree to which a method is affected (or not) by the other components
present in a multi-component sample That is the ability of a method to measure exactly a given
analyte or its amount within the sample without interference from non-target components such as a
matrix effect or background noise
The selectivity is defined as a measure of the degree of non-interference in the presence of non-target
analytes A method is selective if it can be used to detect the analyte under examination and that a
guarantee can be provided that the detected signal can only be a product by that specific analyte
The specificity and selectivity study was performed for the Listeria PrecisTM
detection method no additional test was realised for the enumeration
method The results were the following
- Among the Listeria monocytogenes strains tested 2 strains (Listeria
monocytogenes CIP 7834 and Listeria monocytogenes CIP 105459) gave
negative results These 2 strains were tested twice by running 48 h
incubation time of the compared agars Positive results were then
observed by the alternative method Pale characteristic colonies were
observed on the ISO agar by streaking the Half Fraser broth only
- No cross reaction was observed with the non-target strains except with
Listeria ivanovii which gave characteristic colonies on Brilliancetrade Listeria
Agar but the confirmatory tests provide a clear discrimination between
Listeria monocytogenes and Listeria ivanovi species
Rillettes
Lettuce
Raw milk
Smoked salmon
Raw egg
Process water
x (CFUg)
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2038 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
212 Practicability
1 Packaging Ready to use Brilliance Listeria agar plates are available
(x 10 plates)
2 Reagent volume
3 Storage conditionsndash
Shelf life of products
not opened
The plates are stored at 6 - 12degC in darkness conditions The
expiration date is mentioned on the package and on the plates It
is around 3 months
4 Modalities of use
after first use
5 Required
equipments No specific equipment is required
6 Reagents Ready to use plates
7 Training 05 day
8 Real time handling
Reference method
9 min for one sample
55 min for 10 samples
Alternative method
7 min for one sample
35 minutes for 10 samples
9 Time to result
Negative results
Reference method Day 2
Alternative method Day 2
Negative results after confirmation or positive results
Reference method Day 4 ndash Day 7
Alternative method Day 4 ndash Day 7
Positive results
Reference method Day 4 ndash Day 7
Alternative method Day 2 ndash Day 3 (with OBIS Mono Test)
or Day 4 ndash Day 7 (by the classical tests described in the
standardised method)
10 Technician skill Identical to that required for the reference method
11 Steps common to
the reference method Mother suspension revivification step and decimal dilutions
12 Traceability
13 Maintenance
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2138 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
213 Inter-laboratory study
2131 Organisation
12 laboratories participated to this study Pasteurised milk was inoculated by
Listeria monocytogenes 12a isolated from raw milk The targeted
inoculation levels were
- 0 CFUml
- 50 CFU ml
- 500 CFU ml
- 5 000 CFU ml
Each laboratory received eight flasks of 10 ml sample ie two flasks per
inoculation level Furthermore one non-inoculated sample has been added
to the package for total viable count microflora (NF ISO 4833 method)
Detailed instructions were transmitted to the collaborators by the expert
laboratory
Collaborative study laboratories and the expert laboratory carried out the
analyses with the alternative and reference methods
2132 Verification of experimental parameters
Strain stability during transport
In order to evaluate the Listeria monocytogenes strain stability during
transport bacterial count of all inoculated flasks were checked at different
time ie inoculation time after 24 h of conservation at 2deg - 8degC Results are
reported in table 11
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2238 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Table 11 ndash Listeria monocytogenes count
by ISO 11290-2A1 method (log CFUml)
Day of the analyses Level contamination Reference method
Day 0
(29052006)
1 45
2 440
3 4 500
Day 1
(30052006)
1 60
2 430
3 4 900
No evolution of the strain was observed after storage for 24 h at 2degC ndash 8degC
Contamination level
The real contamination levels are provided in table 12
Table 12 ndash Real contamination level (CFUml)
Target inoculation
(CFU ml) Samples Ndeg
Real contamination levels
(CFUml)
0 1 - 8 0
50 5 - 6 45
500 3 - 7 440
5000 2 - 4 4 500
Sample temperature at receipt
Measured temperatures at receipt are listed in table 13
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2338 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Table 13 - Sample temperature on receipt
Laboratories Receipt
day
Hour
receipt
Measured temperature
at receipt (degC)
Measured temperature by the
temperature probe (degC)
A
B
C
D
E
F
G
H
I
J
K
L
M
30052006
10h45
10h15
11h15
10h00
09h30
8h30
11h50
10h00
08h30
14h45
08h30
09h45
08h45
82
55
45
63
60
62
81
38
48
-
14
67
76
03
03
-13
03
04
-14
05
04
01
05
-13
04
Conclusion
The temperatures during the transport were correct for all the labs except for
Lab J which didnrsquot provide the information
All the labs received their packages with several broken flasks
Three labs (B D and L) received some damaged samples but proceeded to
analyses
All the labs started the analyses at Day 1 except Lab J
2133 Results
Aerobic mesophilic flora
The count of aerobic mesophilic flora varied from 94 102 CFUml to
33 103 CFUml
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2438 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Expert lab results
The results obtained by the expert lab are the following
Table 14
Target inoculation level (CFUml)
Reference method ISO 11290-2
Alternative method Listeria Precis Enumeration
Rep 1 Rep 2 Rep 1 Rep 2
lt 10 lt 10 lt 10 lt 10 lt10
50 50 (Ne) 60 (Ne) 50 (Ne) 30 (Ne)
500 470 390 490 500
5 000 4 700 5 100 5 900 3 400
Ne estimated number
The obtained results are in agreement with those expected
Collaborator Labs results
A synthesis of the results obtained by the collaborator labs is provided
Table 15 (CFUml) and Table 16 (log CFUml)
Table 15 ndash Synthesis of results obtained by the alternative
and the reference methods (CFUml)
Labs Level 1 Level 2 Level 2 bis Level 3
Reference method
Alternative method
Reference method
Alternative method
Reference method
Alternative method
Reference method
Alternative method
A 40 40 70 40 480 410 210 340 1000 300 400 300 4900 4600 4900 5100
B 40 15 40 20 350 300 360 450 350 650 800 600 4100 4700 5100 3700
C 50 20 20 70 440 430 480 450 300 650 300 500 4600 5400 4600 5000
D 40 60 40 40 490 460 600 540 250 450 600 1000 4900 4900 4400 4600
E 50 40 50 20 490 520 530 560 750 550 400 200 5400 4500 4100 5400
F 60 20 60 50 370 250 300 130 300 350 600 300 4400 4700 4900 5900
G 70 35 120 50 440 390 570 370 450 550 700 700 5000 4500 4000 4400
H 110 40 100 90 520 440 450 540 150 650 300 300 4200 4700 5600 4800
L 35 60 20 40 390 390 170 380 650 350 800 200 5300 4600 5600 5900
M 45 45 30 40 450 490 550 410 400 550 800 180 4900 4900 6400 4800
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2538 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Table 16 ndash Synthesis of results obtained by the alternative
and the reference methods (log CFUml)
Labs Level 1 Level 2 Level 2 bis Level 3
Reference method
Alternative method
Reference method
Alternative method
Reference method
Alternative method
Reference method
Alternative method
A 1602 1602 1845 1602 2681 2613 2322 2531 3000 2477 2602 2477 3690 3663 3690 3708
B 1602 1176 1602 1301 2544 2477 2556 2653 2544 2813 2903 2778 3613 3672 3708 3568
C 1699 1301 1301 1845 2643 2633 2681 2653 2477 2813 2477 2699 3663 3732 3663 3699
D 1602 1778 1602 1602 2690 2663 2778 2732 2398 2653 2778 3000 3690 3690 3643 3663
E 1699 1602 1699 1301 2690 2716 2724 2748 2875 2740 2602 2301 3732 3653 3613 3732
F 1778 1301 1778 1699 2568 2398 2477 2114 2477 2544 2778 2477 3643 3672 3690 3771
G 1845 1544 2079 1699 2643 2591 2756 2568 2653 2740 2845 2845 3699 3653 3602 3643
H 2041 1602 2000 1954 2716 2643 2653 2732 2176 2813 2477 2477 3623 3672 3748 3681
L 1544 1778 1301 1602 2591 2591 2230 2580 2813 2544 2903 2301 3724 3663 3748 3771
M 1653 1653 1477 1602 2653 2690 2740 2613 2602 2740 2903 2255 3690 3690 3806 3681
Level 2 results obtained taking account the 3 plates inoculated with the mother-suspension
Level 2 bis results obtained taking account the plates inoculated with 01 ml of the mother-suspension
Scrutiny of the measurement results for consistency
In order to identify the measurement results or laboratories that could be
inconsistent two graphical consistency techniques were realised Mandelrsquos
h-ank-statistics Mandel indicators h and k at 5 significance highlighted
some possible inconsistent data (See tables and graphics in Appendix 3 and
Appendix 4)
Mandelrsquos values Number of values observed over the threshold
Reference method Alternative method
h gt 1 Lab F Level 2 Lab F Level 2
h gt 5 Lab F Level 2
Lab B Levels 1 and 2
Lab A Level 2
Lab D Level 3
Lab F Level 2
Lab L Level 2
kgt 1 Lab F Level 3
k gt 5 Lab F Level 2 Lab C Level 2
Lab F Level 3
Lab L Levels 1 and 3
Lab M Level 1
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2638 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
The emphasized results appear (i) coherent with the targeted contamination
levels (ii) concordant between both methods
All the results were thus kept for the statistical analysis
Comparison of the trueness and precision characteristics of
the reference method and alternative methods
The statistical values are summarised hereafter
Level p
Reference method Alternative method
Repeatability ratio
Reproducibility ratio Median
Repeatability standard deviation
Reproducibility standard deviation
Median Repeatability
standard deviation
Reproducibility standard deviation
1 10 16519 02415 02425 16021 02045 02433 0847 1007
2 10 26551 00543 00659 26719 00914 01325 1682 2011
2 bis 10 26581 02607 02607 25951 01900 01953 0729 0749
3 10 18671 00378 00378 18671 00478 00611 1264 1617
Bias of the alternative method
In order to estimate the bias of the alternative method with regard to the reference method for every
level Dij and t are calculated according to the following equations
RefAlt ijYijYDij
Diffp
Dijit
)2(
)(median
If the t value is superior to 2 the alternative method is significantly biased
with regard to the reference method
The values are given in table 17
Table 17 ndash t (d) values obtained by level
Level Bias t (d) Conclusion
1 0068 114 Non significant bias
2 0021 082 Non significant bias
2 bis - 0037 035 Non significant bias
3 - 0037 043 Non significant bias
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2738 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
The statistical test concludes to a non significant bias for all the inoculation
levels The bias varies from ndash 0037 to 0068 log CFUml
Comparison of the repeatability standard deviations
If the ratio Srj Alt Srj Ref of the repeatability standard deviations of the
alternative method and the reference method is larger than 2 the precision
under repeatability conditions of the alternative method is considered to be
lower than that of the reference method If this ratio is smaller than 05 the
precision under repeatability conditions of the alternative method is
considered to be greater than that of the reference method
The ratio values are given in table 18
Table 18
Level
Repeatability standard deviation
Reference method Alternative method Alternative method
reference method ratio
1 0242 0205 0847
2 0054 0091 1682
2 bis 0261 0190 0729
3 0038 0048 1264
Level Repeatability limit
Reference method Alternative method
1 0676 0573
2 0152 0256
2 bis 0730 0532
3 0106 0134
The ratios of the repeatability standard deviations are below 2 in all cases
The precision under repeatability conditions of the alternative method is
equivalent to that of the reference method
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2838 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Comparison of the reproducibility standard deviations
If the ratio Srj Alt Srj Ref of the reproducibility standard deviations of the alternative method and the
reference method is larger than 2 the precision under reproducibility conditions of the alternative
method is considered to be lower than that of the reference method If this ratio is smaller than 05 the
precision under reproducibility conditions of the alternative method is considered to be greater than
that of the reference method
The ratio values are given in table 19
Table 19
Level
Reproducibility standard deviation
Reference method Alternative method Alternative method
reference method ratio
1 0242 0243 1007
2 0066 0133 2011
2 bis 0261 0195 0749
3 0038 0061 1617
Level Reproducibility limit
Reference method Alternative method
1 0676 0681
2 0185 0371
2 bis 0730 0547
3 0106 0171
The ratios of the reproducibility standard deviations are below or close to 2
The precision under reproducibility conditions of the alternative method is
equivalent to that of the reference method
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2938 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
214 Conclusion
Method comparison study
The Listeria PrecisTM enumeration method shows satisfying linearity and
relative accuracy
The alternative method shows satisfying inclusivity and exclusivity results
Two Listeria monocytogenes strains gave negative results with the
alternative method These strains grew weakly on the reference method
media (OampA Palcam and Oxford)
The limits of repeatability of the alternative method are similar to those of the
reference method
The bias between both methods are low between - 0133 Log CFUg and
- 0008 Log CFUg
The Listeria PrecisTM enumeration method offers the saving for confirmatory
tests
Positive results are obtained with the alternative method in 2-3 days (with
OBIS Mono test) or in 4-7 days (by the classical tests described in the
standardized method) against 4-7 days for the reference method
Inter-laboratory study
The bias between the reference and the alternative methods shows satisfying
and correct values
- level 1 (100 - 1 000 CFUg) bias = + 007 log CFUg
- level 2 (1 000 - 10 000 CFUg) bias = 002 log CFUg
- level 2 bis (1 000 - 10 000 CFUg) bias = - 004 log CFUg
- level 3 (10 000 - 100 000 CFUg) bias = - 004 log CFUg
The repeatability and the reproducibility of the alternative method look similar
to those of the reference method
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3038 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
22 Extension study (realised by ASEPT 2007)
An extension study was performed in 2007 for a new confirmatory test OBIS
MONO
150 Listeria monocytogenes strains and 100 non-target strains were isolated
onto TSAYE and BrillianceTM Listeria Agar plates All the Listeria
monocytogenes and the non-target strains gave the expected results
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3138 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Appendix 1 ndash Flow diagram of the Listeria PrecisTM method
x g sample + 9 x ml buffered peptone water
Revivification step for 1 h plusmn 5 min at 20degC plusmn 1degC
Streaking 1 ml onto 3 Brilliance Listeria Agar plates (mother suspension)
or 01 ml onto 1 Brilliance Listeria Agar plates (mother suspension and decimal dilutions)
Incubation for 48 h plusmn 3 h at 37degC plusmn 1degC
Confirmatory tests on at least one typical colony
- by the tests described in the ISO method
- by applying the OBIS mono test on an isolated colony
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3238 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Appendix 2 ndash ISO 11290-2 (1998) amp ISO 11290-2A1 (2004) Horizontal
method for the detection and enumeration of Listeria monocytogenes -
Part 2 enumeration of Listeria monocytogenes in foods
10 g cheese + 90 ml buffered peptone water or Half Fraser Broth (without supplements) (110 dilution)
Stomach 1 min 30 seconds
Keep at 20degC plusmn 2degC for 1 h plusmn 5 min
Decimal dilutions in 9 ml peptone salt tubes
Dilution 110
Dilution 1100
Dilution 11000
1 ml on 3 Chromogenic Listeria medium (ISO) plates (x 2) (-1)
01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-2)
01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-3)
01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-4)
Incubation at 37degC plusmn 1degC
24 h - 48 h
Numerate typical colonies at 24 h
(bluegreen surrounded by an opaque halo)
Numerate at 48 h
Streak typical colonies on TSYEA
(5 coloniesplate 2 successive dilutions)
Confirmation tests
Catalase
Gram stain
For confirmation of L monocytogenes
Haemolysis
Carbohydrate (rhamnose and xylose) utilisation
CAMP Test
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3338 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Appendix 3 ndash Mandelrsquos graphics
Laboratoire h Ref
Niveau 1 h Ref
Niveau 2 h Ref
Niveau 3 h Alt
Niveau 1 h Alt
Niveau 2 h Alt
Niveau 3 h5 h1 h5 h1
A -050 -015 083 062 -212 -039 198 299 -198 -299
B -261 -270 021 -077 -058 173 198 299 -198 -299
C -151 -031 -014 -015 -004 -005 198 299 -198 -299
D 038 040 -137 000 072 207 198 299 -198 -299
E -001 090 155 -052 056 -101 198 299 -198 -299
F -112 -321 -153 070 -325 023 198 299 -198 -299
G 042 -071 040 147 -009 176 198 299 -198 -299
H 169 046 -169 192 018 -083 198 299 -198 -299
L 009 -120 021 -077 -231 005 198 299 -198 -299
M 001 031 014 -032 004 -011 198 299 -198 -299
-300
-200
-100
000
100
200
300
A B C D E F G H L M
Vale
urs
h d
e M
an
del(
rob
uste
s)
Laboratoires
Meacutethode de reacutefeacuterence analyse robuste
h Ref Niveau 1
h Ref Niveau 2
h Ref Niveau 3
h 5=198
h 1=299
-300
-200
-100
000
100
200
300
A B C D E F G H L M
Vale
urs
de h
de M
an
del(
rob
uste
s)
Laboratoires
Meacutethode alternative analyse robuste
h Alt Niveau 1
h Alt Niveau 2
h Alt Niveau 2
h 1=299
h 5=198
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3438 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Laboratoire k Ref
Niveau 1 k Ref
Niveau 2 k Ref
Niveau 3 k Alt
Niveau 1 k Alt
Niveau 2 k Alt
Niveau 3 k1 k5
A 000 089 142 084 162 046 259 181
B 125 087 073 104 075 046 259 181
C 117 013 091 188 022 083 259 181
D 052 036 069 000 035 083 259 181
E 028 034 037 138 019 112 259 181
F 140 222 018 027 281 112 259 181
G 088 068 024 131 145 000 259 181
H 129 094 173 016 061 000 259 181
L 069 000 073 104 270 224 259 181
M 000 048 038 043 099 241 259 181
000
050
100
150
200
250
300
350
400
A B C D E F G H L M
Vale
urs
k d
e M
an
del(
rob
uste
s)
Laboratoires
Meacutethode de reacutefeacuterence analyse robuste
k Ref Niveau 1k Ref Niveau 2k Ref Niveau 3
k 1=259
k 5=181
000
050
100
150
200
250
300
A B C D E F G H L M
Vale
urs
k d
e M
an
del
(ro
bu
ste
s)
Laboratoires
Meacutethode alternative analyse robuste
k Alt Niveau 3
k Alt Niveau 1
k Alt Niveau 2
k 1=259
k 5=181
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3538 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Appendix 4 ndash Statistical results
Level 1
Nombre de laboratoires (p) 10
A 1602 1602 1602 000 0000 0000 -0495 0000 A 1845 1602 1724 017 0122 -0122 0621 0840 0122
B 1602 1176 1389 030 0213 -0213 -2613 1247 B 1602 1301 1452 021 0151 -0151 -0769 1041 0062
C 1699 1301 1500 028 0199 -0199 -1510 1165 C 1301 1845 1573 038 -0272 0272 -0148 1881 0073
D 1602 1778 1690 012 -0088 0088 0380 0516 D 1602 1602 1602 000 0000 0000 0000 0000 -0088
E 1699 1602 1651 007 0048 -0048 -0013 0284 E 1699 1301 1500 028 0199 -0199 -0522 1376 -0151
F 1778 1301 1540 034 0239 -0239 -1116 1397 F 1778 1699 1739 006 0040 -0040 0698 0274 0199
G 1845 1544 1695 021 0151 -0151 0425 0881 G 2079 1699 1889 027 0190 -0190 1467 1315 0194
H 2041 1602 1822 031 0220 -0220 1689 1286 H 2000 1954 1977 003 0023 -0023 1917 0158 0155
L 1544 1778 1661 017 -0117 0117 0092 0685 L 1301 1602 1452 021 -0151 0151 -0769 1041 -0210
M 1653 1653 1653 000 0000 0000 0013 0000 M 1477 1602 1540 009 -0062 0062 -0319 0432 -0114
m 165186 m 160206 mediane D 00678
Cn(2p) 1867 Cn(2p) 1867 Cp 16101
Cn(p) 16101 Cn(p) 16101 Qn(D) 0092926
n=2p n 20 n=2p n 20 Qdiff 01496169
f 11 f 11 t 114
l 55 l 55
n=p n 10 n=p n 10
f 6 f 6
l 15 l 15
Qn(2p) 0091465 Qn(2p) 007745
Qn(p) 0062469 Qn(p) 0121519
Qintra 017078 Qintra 014461
Qinter 010058 Qinter 019565
Sr(eacutecart-type de reacutepeacutetabiliteacute) 02415 Sr 02045
RSDr(coefficient de variation de la reacutepeacutetabiliteacute)1462 RSDr 1277
r(repeatability limit)) 0676 r 0573
SL(eacutecart-type interlaboratoire) -001905 SLlt0 SL 001737
SR(eacutecart-type de reproductibiliteacute) 02415 SR 02433
RSDR(coefficient de variation de la reproductibiliteacute)0146 RSDR 0152
R(limite de reproductibiliteacute)) 06762 R(limite de reproductibiliteacute))068123
h k h MoyenneLaboratoires
Niveau 1
si LaboratoiresDeviationMoyenne Dk DeviationsiAlt
Niveau 1
Ref
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3638 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Level 2
Nombre de laboratoires (p) 10
A 2681 2613 2647 005 0034 -0034 -0151 0891 A 2322 2531 2427 015 -0105 0105 -2119 1620 -0220
B 2544 2477 2511 005 0033 -0033 -2699 0872 B 2556 2653 2605 007 -0048 0048 -0581 0750 0094
C 2643 2633 2638 001 0005 -0005 -0311 0130 C 2681 2653 2667 002 0014 -0014 -0040 0217 0029
D 2690 2663 2676 002 0014 -0014 0400 0357 D 2778 2732 2755 003 0023 -0023 0721 0354 0079
E 2690 2716 2703 002 -0013 0013 0897 0336 E 2724 2748 2736 002 -0012 0012 0556 0185 0033
F 2568 2398 2483 012 0085 -0085 -3214 2217 F 2477 2114 2296 026 0182 -0182 -3255 2811 -0188
G 2643 2591 2617 004 0026 -0026 -0707 0682 G 2756 2568 2662 013 0094 -0094 -0085 1453 0045
H 2716 2643 2680 005 0036 -0036 0460 0945 H 2653 2732 2693 006 -0040 0040 0181 0613 0013
L 2591 2591 2591 000 0000 0000 -1196 0000 L 2230 2580 2405 025 -0175 0175 -2307 2704 -0186
M 2653 2690 2672 003 -0018 0018 0311 0481 M 2740 2613 2677 009 0064 -0064 0040 0987 0005
m 2655082 m 26719001 mediane D 00209
Cn(2p) 1867 Cn(2p) 1867 Cp 16101
Cn(p) 16101 Cn(p) 16101 Qn(D) 0039911
n=2p n 20 n=2p n 20 Qdiff 00642591
f 11 f 11 t 082
l 55 l 55
n=p n 10 n=p n 10
f 6 f 6
l 15 l 15
Qn(2p) 0020570 Qn(2p) 00345985
Qn(p) 00332437 Qn(p) 00718158
Qintra 003841 Qintra 006460
Qinter 005352 Qinter 011563
Sr(eacutecart-type de reacutepeacutetabiliteacute) 00543 Sr 00914
RSDr(coefficient de variation de la reacutepeacutetabiliteacute)205 RSDr 342
r(repeatability limit)) 0152 r 0256
SL(eacutecart-type interlaboratoire) 000139 SL 000920
SR(eacutecart-type de reproductibiliteacute) 00659 SR 01325
RSDR(coefficient de variation de la reproductibiliteacute)0025 RSDR 0050
R(limite de reproductibiliteacute)) 018446 R(limite de reproductibiliteacute))03708618
Laboratoires
Niveau 2
Moyenne si Laboratoires
Niveau 2
DRef Alt Moyenne si Deviation h Deviation h k k
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3738 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Level 2 bis
Nombre de laboratoires (p) 10
A 3000 2477 2739 037 0261 -0261 0832 1418 A 2602 2477 2540 009 0062 -0062 -0391 0465 -0199
B 2544 2813 2678 019 -0134 0134 0211 0729 B 2903 2778 2841 009 0062 -0062 1732 0465 0162
C 2477 2813 2645 024 -0168 0168 -0135 0911 C 2477 2699 2588 016 -0111 0111 -0049 0826 -0057
D 2398 2653 2526 018 -0128 0128 -1370 0692 D 2778 3000 2889 016 -0111 0111 2074 0826 0363
E 2875 2740 2808 010 0067 -0067 1547 0365 E 2602 2301 2452 021 0151 -0151 -1012 1120 -0356
F 2477 2544 2511 005 -0033 0033 -1525 0182 F 2778 2477 2628 021 0151 -0151 0230 1120 0117
G 2653 2740 2697 006 -0044 0044 0400 0236 G 2845 2845 2845 000 0000 0000 1764 0000 0148
H 2176 2813 2495 045 -0318 0318 -1692 1727 H 2477 2477 2477 000 0000 0000 -0832 0000 -0017
L 2813 2544 2678 019 0134 -0134 0211 0729 L 2903 2301 2602 043 0301 -0301 0049 2241 -0076
M 2602 2740 2671 010 -0069 0069 0135 0375 M 2903 2255 2579 046 0324 -0324 -0112 2411 -0092
m 2658114 m 259505 mediane D -00372
Cn(2p) 1867 Cn(2p) 1867 Cp 16101
Cn(p) 16101 Cn(p) 16101 Qn(D) 01656915
n=2p n 20 n=2p n 20 Qdiff 02667753
f 11 f 11 t 035
l 55 l 55
n=p n 10 n=p n 10
f 6 f 6
l 15 l 15
Qn(2p) 0098745 Qn(2p) 007195
Qn(p) 006007 Qn(p) 008805
Qintra 018437 Qintra 013435
Qinter 009672 Qinter 014176
Sr(eacutecart-type de reacutepeacutetabiliteacute) 02607 Sr 01900
RSDr(coefficient de variation de la reacutepeacutetabiliteacute)981 RSDr 732
r(repeatability limit)) 0730 r 0532
SL(eacutecart-type interlaboratoire) -002464 SLlt0 SL 000205
SR(eacutecart-type de reproductibiliteacute) 02607 SR 01953
RSDR(coefficient de variation de la reproductibiliteacute)0098 RSDR 0075
R(limite de reproductibiliteacute)) 072996 R(limite de reproductibiliteacute))054686
Laboratoires
Niveau 2 bis
Moyenne si Laboratoires
Niveau 2 bis
DRef Alt Moyenne si Deviation h Deviation h k k
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3838 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Level 3
Nombre de laboratoires (p) 10
A 3690 3663 3676 002 0014 -0014 -0311 0513 A 3690 3708 3699 001 -0009 0009 0354 0257 0022
B 3613 3672 3642 004 -0030 0030 -1851 1109 B 3708 3568 3638 010 0070 -0070 -0844 2061 -0005
C 3663 3732 3698 005 -0035 0035 0645 1302 C 3663 3699 3681 003 -0018 0018 0000 0536 -0017
D 3690 3690 3690 000 0000 0000 0311 0000 D 3643 3663 3653 001 -0010 0010 -0545 0286 -0037
E 3732 3653 3693 006 0040 -0040 0429 1480 E 3613 3732 3673 008 -0060 0060 -0162 1769 -0020
F 3643 3672 3658 002 -0014 0014 -1157 0536 F 3690 3771 3731 006 -0040 0040 0975 1193 0073
G 3699 3653 3676 003 0023 -0023 -0328 0856 G 3602 3643 3623 003 -0021 0021 -1141 0612 -0053
H 3623 3672 3648 003 -0024 0024 -1615 0913 H 3748 3681 3715 005 0033 -0033 0664 0990 0067
L 3724 3663 3694 004 0031 -0031 0461 1150 L 3748 3771 3760 002 -0011 0011 1544 0335 0066
M 3690 3690 3690 000 0000 0000 0311 0000 M 3806 3681 3744 009 0062 -0062 1234 1848 0054
m 368334 m 368086 mediane D 00089
Cn(2p) 1867 Cn(2p) 1867 Cp 16101
Cn(p) 16101 Cn(p) 16101 Qn(D) 00325359
n=2p n 20 n=2p n 20 Qdiff 00523852
f 11 f 11 t 043
l 55 l 55
n=p n 10 n=p n 10
f 6 f 6
l 15 l 15
Qn(2p) 0014323 Qn(2p) 001811
Qn(p) 0013719 Qn(p) 003164
Qintra 002674 Qintra 003381
Qinter 002209 Qinter 005094
Sr(eacutecart-type de reacutepeacutetabiliteacute) 00378 Sr 00478
RSDr(coefficient de variation de la reacutepeacutetabiliteacute)103 RSDr 130
r(repeatability limit)) 0106 r 0134
SL(eacutecart-type interlaboratoire) -000023 SLlt0 SL 000145
SR(eacutecart-type de reproductibiliteacute) 00378 SR 00611
RSDR(coefficient de variation de la reproductibiliteacute)0010 RSDR 0017
R(limite de reproductibiliteacute)) 0105894 R(limite de reproductibiliteacute))017119
Ref Alt DLaboratoires
Niveau 3
Moyenne si Deviation h k Laboratoires
Niveau 3
Moyenne si Deviation h k
Thermo Fisher Scientific
ADRIA Deacuteveloppement 1938 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Figure 5 ndash Sensitivity patterns for the tested (matrixstrain) pairs
2115 SpecificitySelectivity
The specificity is defined as the degree to which a method is affected (or not) by the other components
present in a multi-component sample That is the ability of a method to measure exactly a given
analyte or its amount within the sample without interference from non-target components such as a
matrix effect or background noise
The selectivity is defined as a measure of the degree of non-interference in the presence of non-target
analytes A method is selective if it can be used to detect the analyte under examination and that a
guarantee can be provided that the detected signal can only be a product by that specific analyte
The specificity and selectivity study was performed for the Listeria PrecisTM
detection method no additional test was realised for the enumeration
method The results were the following
- Among the Listeria monocytogenes strains tested 2 strains (Listeria
monocytogenes CIP 7834 and Listeria monocytogenes CIP 105459) gave
negative results These 2 strains were tested twice by running 48 h
incubation time of the compared agars Positive results were then
observed by the alternative method Pale characteristic colonies were
observed on the ISO agar by streaking the Half Fraser broth only
- No cross reaction was observed with the non-target strains except with
Listeria ivanovii which gave characteristic colonies on Brilliancetrade Listeria
Agar but the confirmatory tests provide a clear discrimination between
Listeria monocytogenes and Listeria ivanovi species
Rillettes
Lettuce
Raw milk
Smoked salmon
Raw egg
Process water
x (CFUg)
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2038 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
212 Practicability
1 Packaging Ready to use Brilliance Listeria agar plates are available
(x 10 plates)
2 Reagent volume
3 Storage conditionsndash
Shelf life of products
not opened
The plates are stored at 6 - 12degC in darkness conditions The
expiration date is mentioned on the package and on the plates It
is around 3 months
4 Modalities of use
after first use
5 Required
equipments No specific equipment is required
6 Reagents Ready to use plates
7 Training 05 day
8 Real time handling
Reference method
9 min for one sample
55 min for 10 samples
Alternative method
7 min for one sample
35 minutes for 10 samples
9 Time to result
Negative results
Reference method Day 2
Alternative method Day 2
Negative results after confirmation or positive results
Reference method Day 4 ndash Day 7
Alternative method Day 4 ndash Day 7
Positive results
Reference method Day 4 ndash Day 7
Alternative method Day 2 ndash Day 3 (with OBIS Mono Test)
or Day 4 ndash Day 7 (by the classical tests described in the
standardised method)
10 Technician skill Identical to that required for the reference method
11 Steps common to
the reference method Mother suspension revivification step and decimal dilutions
12 Traceability
13 Maintenance
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2138 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
213 Inter-laboratory study
2131 Organisation
12 laboratories participated to this study Pasteurised milk was inoculated by
Listeria monocytogenes 12a isolated from raw milk The targeted
inoculation levels were
- 0 CFUml
- 50 CFU ml
- 500 CFU ml
- 5 000 CFU ml
Each laboratory received eight flasks of 10 ml sample ie two flasks per
inoculation level Furthermore one non-inoculated sample has been added
to the package for total viable count microflora (NF ISO 4833 method)
Detailed instructions were transmitted to the collaborators by the expert
laboratory
Collaborative study laboratories and the expert laboratory carried out the
analyses with the alternative and reference methods
2132 Verification of experimental parameters
Strain stability during transport
In order to evaluate the Listeria monocytogenes strain stability during
transport bacterial count of all inoculated flasks were checked at different
time ie inoculation time after 24 h of conservation at 2deg - 8degC Results are
reported in table 11
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2238 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Table 11 ndash Listeria monocytogenes count
by ISO 11290-2A1 method (log CFUml)
Day of the analyses Level contamination Reference method
Day 0
(29052006)
1 45
2 440
3 4 500
Day 1
(30052006)
1 60
2 430
3 4 900
No evolution of the strain was observed after storage for 24 h at 2degC ndash 8degC
Contamination level
The real contamination levels are provided in table 12
Table 12 ndash Real contamination level (CFUml)
Target inoculation
(CFU ml) Samples Ndeg
Real contamination levels
(CFUml)
0 1 - 8 0
50 5 - 6 45
500 3 - 7 440
5000 2 - 4 4 500
Sample temperature at receipt
Measured temperatures at receipt are listed in table 13
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2338 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Table 13 - Sample temperature on receipt
Laboratories Receipt
day
Hour
receipt
Measured temperature
at receipt (degC)
Measured temperature by the
temperature probe (degC)
A
B
C
D
E
F
G
H
I
J
K
L
M
30052006
10h45
10h15
11h15
10h00
09h30
8h30
11h50
10h00
08h30
14h45
08h30
09h45
08h45
82
55
45
63
60
62
81
38
48
-
14
67
76
03
03
-13
03
04
-14
05
04
01
05
-13
04
Conclusion
The temperatures during the transport were correct for all the labs except for
Lab J which didnrsquot provide the information
All the labs received their packages with several broken flasks
Three labs (B D and L) received some damaged samples but proceeded to
analyses
All the labs started the analyses at Day 1 except Lab J
2133 Results
Aerobic mesophilic flora
The count of aerobic mesophilic flora varied from 94 102 CFUml to
33 103 CFUml
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2438 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Expert lab results
The results obtained by the expert lab are the following
Table 14
Target inoculation level (CFUml)
Reference method ISO 11290-2
Alternative method Listeria Precis Enumeration
Rep 1 Rep 2 Rep 1 Rep 2
lt 10 lt 10 lt 10 lt 10 lt10
50 50 (Ne) 60 (Ne) 50 (Ne) 30 (Ne)
500 470 390 490 500
5 000 4 700 5 100 5 900 3 400
Ne estimated number
The obtained results are in agreement with those expected
Collaborator Labs results
A synthesis of the results obtained by the collaborator labs is provided
Table 15 (CFUml) and Table 16 (log CFUml)
Table 15 ndash Synthesis of results obtained by the alternative
and the reference methods (CFUml)
Labs Level 1 Level 2 Level 2 bis Level 3
Reference method
Alternative method
Reference method
Alternative method
Reference method
Alternative method
Reference method
Alternative method
A 40 40 70 40 480 410 210 340 1000 300 400 300 4900 4600 4900 5100
B 40 15 40 20 350 300 360 450 350 650 800 600 4100 4700 5100 3700
C 50 20 20 70 440 430 480 450 300 650 300 500 4600 5400 4600 5000
D 40 60 40 40 490 460 600 540 250 450 600 1000 4900 4900 4400 4600
E 50 40 50 20 490 520 530 560 750 550 400 200 5400 4500 4100 5400
F 60 20 60 50 370 250 300 130 300 350 600 300 4400 4700 4900 5900
G 70 35 120 50 440 390 570 370 450 550 700 700 5000 4500 4000 4400
H 110 40 100 90 520 440 450 540 150 650 300 300 4200 4700 5600 4800
L 35 60 20 40 390 390 170 380 650 350 800 200 5300 4600 5600 5900
M 45 45 30 40 450 490 550 410 400 550 800 180 4900 4900 6400 4800
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2538 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Table 16 ndash Synthesis of results obtained by the alternative
and the reference methods (log CFUml)
Labs Level 1 Level 2 Level 2 bis Level 3
Reference method
Alternative method
Reference method
Alternative method
Reference method
Alternative method
Reference method
Alternative method
A 1602 1602 1845 1602 2681 2613 2322 2531 3000 2477 2602 2477 3690 3663 3690 3708
B 1602 1176 1602 1301 2544 2477 2556 2653 2544 2813 2903 2778 3613 3672 3708 3568
C 1699 1301 1301 1845 2643 2633 2681 2653 2477 2813 2477 2699 3663 3732 3663 3699
D 1602 1778 1602 1602 2690 2663 2778 2732 2398 2653 2778 3000 3690 3690 3643 3663
E 1699 1602 1699 1301 2690 2716 2724 2748 2875 2740 2602 2301 3732 3653 3613 3732
F 1778 1301 1778 1699 2568 2398 2477 2114 2477 2544 2778 2477 3643 3672 3690 3771
G 1845 1544 2079 1699 2643 2591 2756 2568 2653 2740 2845 2845 3699 3653 3602 3643
H 2041 1602 2000 1954 2716 2643 2653 2732 2176 2813 2477 2477 3623 3672 3748 3681
L 1544 1778 1301 1602 2591 2591 2230 2580 2813 2544 2903 2301 3724 3663 3748 3771
M 1653 1653 1477 1602 2653 2690 2740 2613 2602 2740 2903 2255 3690 3690 3806 3681
Level 2 results obtained taking account the 3 plates inoculated with the mother-suspension
Level 2 bis results obtained taking account the plates inoculated with 01 ml of the mother-suspension
Scrutiny of the measurement results for consistency
In order to identify the measurement results or laboratories that could be
inconsistent two graphical consistency techniques were realised Mandelrsquos
h-ank-statistics Mandel indicators h and k at 5 significance highlighted
some possible inconsistent data (See tables and graphics in Appendix 3 and
Appendix 4)
Mandelrsquos values Number of values observed over the threshold
Reference method Alternative method
h gt 1 Lab F Level 2 Lab F Level 2
h gt 5 Lab F Level 2
Lab B Levels 1 and 2
Lab A Level 2
Lab D Level 3
Lab F Level 2
Lab L Level 2
kgt 1 Lab F Level 3
k gt 5 Lab F Level 2 Lab C Level 2
Lab F Level 3
Lab L Levels 1 and 3
Lab M Level 1
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2638 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
The emphasized results appear (i) coherent with the targeted contamination
levels (ii) concordant between both methods
All the results were thus kept for the statistical analysis
Comparison of the trueness and precision characteristics of
the reference method and alternative methods
The statistical values are summarised hereafter
Level p
Reference method Alternative method
Repeatability ratio
Reproducibility ratio Median
Repeatability standard deviation
Reproducibility standard deviation
Median Repeatability
standard deviation
Reproducibility standard deviation
1 10 16519 02415 02425 16021 02045 02433 0847 1007
2 10 26551 00543 00659 26719 00914 01325 1682 2011
2 bis 10 26581 02607 02607 25951 01900 01953 0729 0749
3 10 18671 00378 00378 18671 00478 00611 1264 1617
Bias of the alternative method
In order to estimate the bias of the alternative method with regard to the reference method for every
level Dij and t are calculated according to the following equations
RefAlt ijYijYDij
Diffp
Dijit
)2(
)(median
If the t value is superior to 2 the alternative method is significantly biased
with regard to the reference method
The values are given in table 17
Table 17 ndash t (d) values obtained by level
Level Bias t (d) Conclusion
1 0068 114 Non significant bias
2 0021 082 Non significant bias
2 bis - 0037 035 Non significant bias
3 - 0037 043 Non significant bias
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2738 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
The statistical test concludes to a non significant bias for all the inoculation
levels The bias varies from ndash 0037 to 0068 log CFUml
Comparison of the repeatability standard deviations
If the ratio Srj Alt Srj Ref of the repeatability standard deviations of the
alternative method and the reference method is larger than 2 the precision
under repeatability conditions of the alternative method is considered to be
lower than that of the reference method If this ratio is smaller than 05 the
precision under repeatability conditions of the alternative method is
considered to be greater than that of the reference method
The ratio values are given in table 18
Table 18
Level
Repeatability standard deviation
Reference method Alternative method Alternative method
reference method ratio
1 0242 0205 0847
2 0054 0091 1682
2 bis 0261 0190 0729
3 0038 0048 1264
Level Repeatability limit
Reference method Alternative method
1 0676 0573
2 0152 0256
2 bis 0730 0532
3 0106 0134
The ratios of the repeatability standard deviations are below 2 in all cases
The precision under repeatability conditions of the alternative method is
equivalent to that of the reference method
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2838 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Comparison of the reproducibility standard deviations
If the ratio Srj Alt Srj Ref of the reproducibility standard deviations of the alternative method and the
reference method is larger than 2 the precision under reproducibility conditions of the alternative
method is considered to be lower than that of the reference method If this ratio is smaller than 05 the
precision under reproducibility conditions of the alternative method is considered to be greater than
that of the reference method
The ratio values are given in table 19
Table 19
Level
Reproducibility standard deviation
Reference method Alternative method Alternative method
reference method ratio
1 0242 0243 1007
2 0066 0133 2011
2 bis 0261 0195 0749
3 0038 0061 1617
Level Reproducibility limit
Reference method Alternative method
1 0676 0681
2 0185 0371
2 bis 0730 0547
3 0106 0171
The ratios of the reproducibility standard deviations are below or close to 2
The precision under reproducibility conditions of the alternative method is
equivalent to that of the reference method
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2938 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
214 Conclusion
Method comparison study
The Listeria PrecisTM enumeration method shows satisfying linearity and
relative accuracy
The alternative method shows satisfying inclusivity and exclusivity results
Two Listeria monocytogenes strains gave negative results with the
alternative method These strains grew weakly on the reference method
media (OampA Palcam and Oxford)
The limits of repeatability of the alternative method are similar to those of the
reference method
The bias between both methods are low between - 0133 Log CFUg and
- 0008 Log CFUg
The Listeria PrecisTM enumeration method offers the saving for confirmatory
tests
Positive results are obtained with the alternative method in 2-3 days (with
OBIS Mono test) or in 4-7 days (by the classical tests described in the
standardized method) against 4-7 days for the reference method
Inter-laboratory study
The bias between the reference and the alternative methods shows satisfying
and correct values
- level 1 (100 - 1 000 CFUg) bias = + 007 log CFUg
- level 2 (1 000 - 10 000 CFUg) bias = 002 log CFUg
- level 2 bis (1 000 - 10 000 CFUg) bias = - 004 log CFUg
- level 3 (10 000 - 100 000 CFUg) bias = - 004 log CFUg
The repeatability and the reproducibility of the alternative method look similar
to those of the reference method
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3038 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
22 Extension study (realised by ASEPT 2007)
An extension study was performed in 2007 for a new confirmatory test OBIS
MONO
150 Listeria monocytogenes strains and 100 non-target strains were isolated
onto TSAYE and BrillianceTM Listeria Agar plates All the Listeria
monocytogenes and the non-target strains gave the expected results
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3138 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Appendix 1 ndash Flow diagram of the Listeria PrecisTM method
x g sample + 9 x ml buffered peptone water
Revivification step for 1 h plusmn 5 min at 20degC plusmn 1degC
Streaking 1 ml onto 3 Brilliance Listeria Agar plates (mother suspension)
or 01 ml onto 1 Brilliance Listeria Agar plates (mother suspension and decimal dilutions)
Incubation for 48 h plusmn 3 h at 37degC plusmn 1degC
Confirmatory tests on at least one typical colony
- by the tests described in the ISO method
- by applying the OBIS mono test on an isolated colony
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3238 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Appendix 2 ndash ISO 11290-2 (1998) amp ISO 11290-2A1 (2004) Horizontal
method for the detection and enumeration of Listeria monocytogenes -
Part 2 enumeration of Listeria monocytogenes in foods
10 g cheese + 90 ml buffered peptone water or Half Fraser Broth (without supplements) (110 dilution)
Stomach 1 min 30 seconds
Keep at 20degC plusmn 2degC for 1 h plusmn 5 min
Decimal dilutions in 9 ml peptone salt tubes
Dilution 110
Dilution 1100
Dilution 11000
1 ml on 3 Chromogenic Listeria medium (ISO) plates (x 2) (-1)
01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-2)
01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-3)
01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-4)
Incubation at 37degC plusmn 1degC
24 h - 48 h
Numerate typical colonies at 24 h
(bluegreen surrounded by an opaque halo)
Numerate at 48 h
Streak typical colonies on TSYEA
(5 coloniesplate 2 successive dilutions)
Confirmation tests
Catalase
Gram stain
For confirmation of L monocytogenes
Haemolysis
Carbohydrate (rhamnose and xylose) utilisation
CAMP Test
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3338 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Appendix 3 ndash Mandelrsquos graphics
Laboratoire h Ref
Niveau 1 h Ref
Niveau 2 h Ref
Niveau 3 h Alt
Niveau 1 h Alt
Niveau 2 h Alt
Niveau 3 h5 h1 h5 h1
A -050 -015 083 062 -212 -039 198 299 -198 -299
B -261 -270 021 -077 -058 173 198 299 -198 -299
C -151 -031 -014 -015 -004 -005 198 299 -198 -299
D 038 040 -137 000 072 207 198 299 -198 -299
E -001 090 155 -052 056 -101 198 299 -198 -299
F -112 -321 -153 070 -325 023 198 299 -198 -299
G 042 -071 040 147 -009 176 198 299 -198 -299
H 169 046 -169 192 018 -083 198 299 -198 -299
L 009 -120 021 -077 -231 005 198 299 -198 -299
M 001 031 014 -032 004 -011 198 299 -198 -299
-300
-200
-100
000
100
200
300
A B C D E F G H L M
Vale
urs
h d
e M
an
del(
rob
uste
s)
Laboratoires
Meacutethode de reacutefeacuterence analyse robuste
h Ref Niveau 1
h Ref Niveau 2
h Ref Niveau 3
h 5=198
h 1=299
-300
-200
-100
000
100
200
300
A B C D E F G H L M
Vale
urs
de h
de M
an
del(
rob
uste
s)
Laboratoires
Meacutethode alternative analyse robuste
h Alt Niveau 1
h Alt Niveau 2
h Alt Niveau 2
h 1=299
h 5=198
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3438 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Laboratoire k Ref
Niveau 1 k Ref
Niveau 2 k Ref
Niveau 3 k Alt
Niveau 1 k Alt
Niveau 2 k Alt
Niveau 3 k1 k5
A 000 089 142 084 162 046 259 181
B 125 087 073 104 075 046 259 181
C 117 013 091 188 022 083 259 181
D 052 036 069 000 035 083 259 181
E 028 034 037 138 019 112 259 181
F 140 222 018 027 281 112 259 181
G 088 068 024 131 145 000 259 181
H 129 094 173 016 061 000 259 181
L 069 000 073 104 270 224 259 181
M 000 048 038 043 099 241 259 181
000
050
100
150
200
250
300
350
400
A B C D E F G H L M
Vale
urs
k d
e M
an
del(
rob
uste
s)
Laboratoires
Meacutethode de reacutefeacuterence analyse robuste
k Ref Niveau 1k Ref Niveau 2k Ref Niveau 3
k 1=259
k 5=181
000
050
100
150
200
250
300
A B C D E F G H L M
Vale
urs
k d
e M
an
del
(ro
bu
ste
s)
Laboratoires
Meacutethode alternative analyse robuste
k Alt Niveau 3
k Alt Niveau 1
k Alt Niveau 2
k 1=259
k 5=181
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3538 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Appendix 4 ndash Statistical results
Level 1
Nombre de laboratoires (p) 10
A 1602 1602 1602 000 0000 0000 -0495 0000 A 1845 1602 1724 017 0122 -0122 0621 0840 0122
B 1602 1176 1389 030 0213 -0213 -2613 1247 B 1602 1301 1452 021 0151 -0151 -0769 1041 0062
C 1699 1301 1500 028 0199 -0199 -1510 1165 C 1301 1845 1573 038 -0272 0272 -0148 1881 0073
D 1602 1778 1690 012 -0088 0088 0380 0516 D 1602 1602 1602 000 0000 0000 0000 0000 -0088
E 1699 1602 1651 007 0048 -0048 -0013 0284 E 1699 1301 1500 028 0199 -0199 -0522 1376 -0151
F 1778 1301 1540 034 0239 -0239 -1116 1397 F 1778 1699 1739 006 0040 -0040 0698 0274 0199
G 1845 1544 1695 021 0151 -0151 0425 0881 G 2079 1699 1889 027 0190 -0190 1467 1315 0194
H 2041 1602 1822 031 0220 -0220 1689 1286 H 2000 1954 1977 003 0023 -0023 1917 0158 0155
L 1544 1778 1661 017 -0117 0117 0092 0685 L 1301 1602 1452 021 -0151 0151 -0769 1041 -0210
M 1653 1653 1653 000 0000 0000 0013 0000 M 1477 1602 1540 009 -0062 0062 -0319 0432 -0114
m 165186 m 160206 mediane D 00678
Cn(2p) 1867 Cn(2p) 1867 Cp 16101
Cn(p) 16101 Cn(p) 16101 Qn(D) 0092926
n=2p n 20 n=2p n 20 Qdiff 01496169
f 11 f 11 t 114
l 55 l 55
n=p n 10 n=p n 10
f 6 f 6
l 15 l 15
Qn(2p) 0091465 Qn(2p) 007745
Qn(p) 0062469 Qn(p) 0121519
Qintra 017078 Qintra 014461
Qinter 010058 Qinter 019565
Sr(eacutecart-type de reacutepeacutetabiliteacute) 02415 Sr 02045
RSDr(coefficient de variation de la reacutepeacutetabiliteacute)1462 RSDr 1277
r(repeatability limit)) 0676 r 0573
SL(eacutecart-type interlaboratoire) -001905 SLlt0 SL 001737
SR(eacutecart-type de reproductibiliteacute) 02415 SR 02433
RSDR(coefficient de variation de la reproductibiliteacute)0146 RSDR 0152
R(limite de reproductibiliteacute)) 06762 R(limite de reproductibiliteacute))068123
h k h MoyenneLaboratoires
Niveau 1
si LaboratoiresDeviationMoyenne Dk DeviationsiAlt
Niveau 1
Ref
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3638 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Level 2
Nombre de laboratoires (p) 10
A 2681 2613 2647 005 0034 -0034 -0151 0891 A 2322 2531 2427 015 -0105 0105 -2119 1620 -0220
B 2544 2477 2511 005 0033 -0033 -2699 0872 B 2556 2653 2605 007 -0048 0048 -0581 0750 0094
C 2643 2633 2638 001 0005 -0005 -0311 0130 C 2681 2653 2667 002 0014 -0014 -0040 0217 0029
D 2690 2663 2676 002 0014 -0014 0400 0357 D 2778 2732 2755 003 0023 -0023 0721 0354 0079
E 2690 2716 2703 002 -0013 0013 0897 0336 E 2724 2748 2736 002 -0012 0012 0556 0185 0033
F 2568 2398 2483 012 0085 -0085 -3214 2217 F 2477 2114 2296 026 0182 -0182 -3255 2811 -0188
G 2643 2591 2617 004 0026 -0026 -0707 0682 G 2756 2568 2662 013 0094 -0094 -0085 1453 0045
H 2716 2643 2680 005 0036 -0036 0460 0945 H 2653 2732 2693 006 -0040 0040 0181 0613 0013
L 2591 2591 2591 000 0000 0000 -1196 0000 L 2230 2580 2405 025 -0175 0175 -2307 2704 -0186
M 2653 2690 2672 003 -0018 0018 0311 0481 M 2740 2613 2677 009 0064 -0064 0040 0987 0005
m 2655082 m 26719001 mediane D 00209
Cn(2p) 1867 Cn(2p) 1867 Cp 16101
Cn(p) 16101 Cn(p) 16101 Qn(D) 0039911
n=2p n 20 n=2p n 20 Qdiff 00642591
f 11 f 11 t 082
l 55 l 55
n=p n 10 n=p n 10
f 6 f 6
l 15 l 15
Qn(2p) 0020570 Qn(2p) 00345985
Qn(p) 00332437 Qn(p) 00718158
Qintra 003841 Qintra 006460
Qinter 005352 Qinter 011563
Sr(eacutecart-type de reacutepeacutetabiliteacute) 00543 Sr 00914
RSDr(coefficient de variation de la reacutepeacutetabiliteacute)205 RSDr 342
r(repeatability limit)) 0152 r 0256
SL(eacutecart-type interlaboratoire) 000139 SL 000920
SR(eacutecart-type de reproductibiliteacute) 00659 SR 01325
RSDR(coefficient de variation de la reproductibiliteacute)0025 RSDR 0050
R(limite de reproductibiliteacute)) 018446 R(limite de reproductibiliteacute))03708618
Laboratoires
Niveau 2
Moyenne si Laboratoires
Niveau 2
DRef Alt Moyenne si Deviation h Deviation h k k
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3738 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Level 2 bis
Nombre de laboratoires (p) 10
A 3000 2477 2739 037 0261 -0261 0832 1418 A 2602 2477 2540 009 0062 -0062 -0391 0465 -0199
B 2544 2813 2678 019 -0134 0134 0211 0729 B 2903 2778 2841 009 0062 -0062 1732 0465 0162
C 2477 2813 2645 024 -0168 0168 -0135 0911 C 2477 2699 2588 016 -0111 0111 -0049 0826 -0057
D 2398 2653 2526 018 -0128 0128 -1370 0692 D 2778 3000 2889 016 -0111 0111 2074 0826 0363
E 2875 2740 2808 010 0067 -0067 1547 0365 E 2602 2301 2452 021 0151 -0151 -1012 1120 -0356
F 2477 2544 2511 005 -0033 0033 -1525 0182 F 2778 2477 2628 021 0151 -0151 0230 1120 0117
G 2653 2740 2697 006 -0044 0044 0400 0236 G 2845 2845 2845 000 0000 0000 1764 0000 0148
H 2176 2813 2495 045 -0318 0318 -1692 1727 H 2477 2477 2477 000 0000 0000 -0832 0000 -0017
L 2813 2544 2678 019 0134 -0134 0211 0729 L 2903 2301 2602 043 0301 -0301 0049 2241 -0076
M 2602 2740 2671 010 -0069 0069 0135 0375 M 2903 2255 2579 046 0324 -0324 -0112 2411 -0092
m 2658114 m 259505 mediane D -00372
Cn(2p) 1867 Cn(2p) 1867 Cp 16101
Cn(p) 16101 Cn(p) 16101 Qn(D) 01656915
n=2p n 20 n=2p n 20 Qdiff 02667753
f 11 f 11 t 035
l 55 l 55
n=p n 10 n=p n 10
f 6 f 6
l 15 l 15
Qn(2p) 0098745 Qn(2p) 007195
Qn(p) 006007 Qn(p) 008805
Qintra 018437 Qintra 013435
Qinter 009672 Qinter 014176
Sr(eacutecart-type de reacutepeacutetabiliteacute) 02607 Sr 01900
RSDr(coefficient de variation de la reacutepeacutetabiliteacute)981 RSDr 732
r(repeatability limit)) 0730 r 0532
SL(eacutecart-type interlaboratoire) -002464 SLlt0 SL 000205
SR(eacutecart-type de reproductibiliteacute) 02607 SR 01953
RSDR(coefficient de variation de la reproductibiliteacute)0098 RSDR 0075
R(limite de reproductibiliteacute)) 072996 R(limite de reproductibiliteacute))054686
Laboratoires
Niveau 2 bis
Moyenne si Laboratoires
Niveau 2 bis
DRef Alt Moyenne si Deviation h Deviation h k k
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3838 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Level 3
Nombre de laboratoires (p) 10
A 3690 3663 3676 002 0014 -0014 -0311 0513 A 3690 3708 3699 001 -0009 0009 0354 0257 0022
B 3613 3672 3642 004 -0030 0030 -1851 1109 B 3708 3568 3638 010 0070 -0070 -0844 2061 -0005
C 3663 3732 3698 005 -0035 0035 0645 1302 C 3663 3699 3681 003 -0018 0018 0000 0536 -0017
D 3690 3690 3690 000 0000 0000 0311 0000 D 3643 3663 3653 001 -0010 0010 -0545 0286 -0037
E 3732 3653 3693 006 0040 -0040 0429 1480 E 3613 3732 3673 008 -0060 0060 -0162 1769 -0020
F 3643 3672 3658 002 -0014 0014 -1157 0536 F 3690 3771 3731 006 -0040 0040 0975 1193 0073
G 3699 3653 3676 003 0023 -0023 -0328 0856 G 3602 3643 3623 003 -0021 0021 -1141 0612 -0053
H 3623 3672 3648 003 -0024 0024 -1615 0913 H 3748 3681 3715 005 0033 -0033 0664 0990 0067
L 3724 3663 3694 004 0031 -0031 0461 1150 L 3748 3771 3760 002 -0011 0011 1544 0335 0066
M 3690 3690 3690 000 0000 0000 0311 0000 M 3806 3681 3744 009 0062 -0062 1234 1848 0054
m 368334 m 368086 mediane D 00089
Cn(2p) 1867 Cn(2p) 1867 Cp 16101
Cn(p) 16101 Cn(p) 16101 Qn(D) 00325359
n=2p n 20 n=2p n 20 Qdiff 00523852
f 11 f 11 t 043
l 55 l 55
n=p n 10 n=p n 10
f 6 f 6
l 15 l 15
Qn(2p) 0014323 Qn(2p) 001811
Qn(p) 0013719 Qn(p) 003164
Qintra 002674 Qintra 003381
Qinter 002209 Qinter 005094
Sr(eacutecart-type de reacutepeacutetabiliteacute) 00378 Sr 00478
RSDr(coefficient de variation de la reacutepeacutetabiliteacute)103 RSDr 130
r(repeatability limit)) 0106 r 0134
SL(eacutecart-type interlaboratoire) -000023 SLlt0 SL 000145
SR(eacutecart-type de reproductibiliteacute) 00378 SR 00611
RSDR(coefficient de variation de la reproductibiliteacute)0010 RSDR 0017
R(limite de reproductibiliteacute)) 0105894 R(limite de reproductibiliteacute))017119
Ref Alt DLaboratoires
Niveau 3
Moyenne si Deviation h k Laboratoires
Niveau 3
Moyenne si Deviation h k
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2038 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
212 Practicability
1 Packaging Ready to use Brilliance Listeria agar plates are available
(x 10 plates)
2 Reagent volume
3 Storage conditionsndash
Shelf life of products
not opened
The plates are stored at 6 - 12degC in darkness conditions The
expiration date is mentioned on the package and on the plates It
is around 3 months
4 Modalities of use
after first use
5 Required
equipments No specific equipment is required
6 Reagents Ready to use plates
7 Training 05 day
8 Real time handling
Reference method
9 min for one sample
55 min for 10 samples
Alternative method
7 min for one sample
35 minutes for 10 samples
9 Time to result
Negative results
Reference method Day 2
Alternative method Day 2
Negative results after confirmation or positive results
Reference method Day 4 ndash Day 7
Alternative method Day 4 ndash Day 7
Positive results
Reference method Day 4 ndash Day 7
Alternative method Day 2 ndash Day 3 (with OBIS Mono Test)
or Day 4 ndash Day 7 (by the classical tests described in the
standardised method)
10 Technician skill Identical to that required for the reference method
11 Steps common to
the reference method Mother suspension revivification step and decimal dilutions
12 Traceability
13 Maintenance
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2138 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
213 Inter-laboratory study
2131 Organisation
12 laboratories participated to this study Pasteurised milk was inoculated by
Listeria monocytogenes 12a isolated from raw milk The targeted
inoculation levels were
- 0 CFUml
- 50 CFU ml
- 500 CFU ml
- 5 000 CFU ml
Each laboratory received eight flasks of 10 ml sample ie two flasks per
inoculation level Furthermore one non-inoculated sample has been added
to the package for total viable count microflora (NF ISO 4833 method)
Detailed instructions were transmitted to the collaborators by the expert
laboratory
Collaborative study laboratories and the expert laboratory carried out the
analyses with the alternative and reference methods
2132 Verification of experimental parameters
Strain stability during transport
In order to evaluate the Listeria monocytogenes strain stability during
transport bacterial count of all inoculated flasks were checked at different
time ie inoculation time after 24 h of conservation at 2deg - 8degC Results are
reported in table 11
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2238 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Table 11 ndash Listeria monocytogenes count
by ISO 11290-2A1 method (log CFUml)
Day of the analyses Level contamination Reference method
Day 0
(29052006)
1 45
2 440
3 4 500
Day 1
(30052006)
1 60
2 430
3 4 900
No evolution of the strain was observed after storage for 24 h at 2degC ndash 8degC
Contamination level
The real contamination levels are provided in table 12
Table 12 ndash Real contamination level (CFUml)
Target inoculation
(CFU ml) Samples Ndeg
Real contamination levels
(CFUml)
0 1 - 8 0
50 5 - 6 45
500 3 - 7 440
5000 2 - 4 4 500
Sample temperature at receipt
Measured temperatures at receipt are listed in table 13
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2338 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Table 13 - Sample temperature on receipt
Laboratories Receipt
day
Hour
receipt
Measured temperature
at receipt (degC)
Measured temperature by the
temperature probe (degC)
A
B
C
D
E
F
G
H
I
J
K
L
M
30052006
10h45
10h15
11h15
10h00
09h30
8h30
11h50
10h00
08h30
14h45
08h30
09h45
08h45
82
55
45
63
60
62
81
38
48
-
14
67
76
03
03
-13
03
04
-14
05
04
01
05
-13
04
Conclusion
The temperatures during the transport were correct for all the labs except for
Lab J which didnrsquot provide the information
All the labs received their packages with several broken flasks
Three labs (B D and L) received some damaged samples but proceeded to
analyses
All the labs started the analyses at Day 1 except Lab J
2133 Results
Aerobic mesophilic flora
The count of aerobic mesophilic flora varied from 94 102 CFUml to
33 103 CFUml
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2438 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Expert lab results
The results obtained by the expert lab are the following
Table 14
Target inoculation level (CFUml)
Reference method ISO 11290-2
Alternative method Listeria Precis Enumeration
Rep 1 Rep 2 Rep 1 Rep 2
lt 10 lt 10 lt 10 lt 10 lt10
50 50 (Ne) 60 (Ne) 50 (Ne) 30 (Ne)
500 470 390 490 500
5 000 4 700 5 100 5 900 3 400
Ne estimated number
The obtained results are in agreement with those expected
Collaborator Labs results
A synthesis of the results obtained by the collaborator labs is provided
Table 15 (CFUml) and Table 16 (log CFUml)
Table 15 ndash Synthesis of results obtained by the alternative
and the reference methods (CFUml)
Labs Level 1 Level 2 Level 2 bis Level 3
Reference method
Alternative method
Reference method
Alternative method
Reference method
Alternative method
Reference method
Alternative method
A 40 40 70 40 480 410 210 340 1000 300 400 300 4900 4600 4900 5100
B 40 15 40 20 350 300 360 450 350 650 800 600 4100 4700 5100 3700
C 50 20 20 70 440 430 480 450 300 650 300 500 4600 5400 4600 5000
D 40 60 40 40 490 460 600 540 250 450 600 1000 4900 4900 4400 4600
E 50 40 50 20 490 520 530 560 750 550 400 200 5400 4500 4100 5400
F 60 20 60 50 370 250 300 130 300 350 600 300 4400 4700 4900 5900
G 70 35 120 50 440 390 570 370 450 550 700 700 5000 4500 4000 4400
H 110 40 100 90 520 440 450 540 150 650 300 300 4200 4700 5600 4800
L 35 60 20 40 390 390 170 380 650 350 800 200 5300 4600 5600 5900
M 45 45 30 40 450 490 550 410 400 550 800 180 4900 4900 6400 4800
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2538 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Table 16 ndash Synthesis of results obtained by the alternative
and the reference methods (log CFUml)
Labs Level 1 Level 2 Level 2 bis Level 3
Reference method
Alternative method
Reference method
Alternative method
Reference method
Alternative method
Reference method
Alternative method
A 1602 1602 1845 1602 2681 2613 2322 2531 3000 2477 2602 2477 3690 3663 3690 3708
B 1602 1176 1602 1301 2544 2477 2556 2653 2544 2813 2903 2778 3613 3672 3708 3568
C 1699 1301 1301 1845 2643 2633 2681 2653 2477 2813 2477 2699 3663 3732 3663 3699
D 1602 1778 1602 1602 2690 2663 2778 2732 2398 2653 2778 3000 3690 3690 3643 3663
E 1699 1602 1699 1301 2690 2716 2724 2748 2875 2740 2602 2301 3732 3653 3613 3732
F 1778 1301 1778 1699 2568 2398 2477 2114 2477 2544 2778 2477 3643 3672 3690 3771
G 1845 1544 2079 1699 2643 2591 2756 2568 2653 2740 2845 2845 3699 3653 3602 3643
H 2041 1602 2000 1954 2716 2643 2653 2732 2176 2813 2477 2477 3623 3672 3748 3681
L 1544 1778 1301 1602 2591 2591 2230 2580 2813 2544 2903 2301 3724 3663 3748 3771
M 1653 1653 1477 1602 2653 2690 2740 2613 2602 2740 2903 2255 3690 3690 3806 3681
Level 2 results obtained taking account the 3 plates inoculated with the mother-suspension
Level 2 bis results obtained taking account the plates inoculated with 01 ml of the mother-suspension
Scrutiny of the measurement results for consistency
In order to identify the measurement results or laboratories that could be
inconsistent two graphical consistency techniques were realised Mandelrsquos
h-ank-statistics Mandel indicators h and k at 5 significance highlighted
some possible inconsistent data (See tables and graphics in Appendix 3 and
Appendix 4)
Mandelrsquos values Number of values observed over the threshold
Reference method Alternative method
h gt 1 Lab F Level 2 Lab F Level 2
h gt 5 Lab F Level 2
Lab B Levels 1 and 2
Lab A Level 2
Lab D Level 3
Lab F Level 2
Lab L Level 2
kgt 1 Lab F Level 3
k gt 5 Lab F Level 2 Lab C Level 2
Lab F Level 3
Lab L Levels 1 and 3
Lab M Level 1
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2638 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
The emphasized results appear (i) coherent with the targeted contamination
levels (ii) concordant between both methods
All the results were thus kept for the statistical analysis
Comparison of the trueness and precision characteristics of
the reference method and alternative methods
The statistical values are summarised hereafter
Level p
Reference method Alternative method
Repeatability ratio
Reproducibility ratio Median
Repeatability standard deviation
Reproducibility standard deviation
Median Repeatability
standard deviation
Reproducibility standard deviation
1 10 16519 02415 02425 16021 02045 02433 0847 1007
2 10 26551 00543 00659 26719 00914 01325 1682 2011
2 bis 10 26581 02607 02607 25951 01900 01953 0729 0749
3 10 18671 00378 00378 18671 00478 00611 1264 1617
Bias of the alternative method
In order to estimate the bias of the alternative method with regard to the reference method for every
level Dij and t are calculated according to the following equations
RefAlt ijYijYDij
Diffp
Dijit
)2(
)(median
If the t value is superior to 2 the alternative method is significantly biased
with regard to the reference method
The values are given in table 17
Table 17 ndash t (d) values obtained by level
Level Bias t (d) Conclusion
1 0068 114 Non significant bias
2 0021 082 Non significant bias
2 bis - 0037 035 Non significant bias
3 - 0037 043 Non significant bias
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2738 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
The statistical test concludes to a non significant bias for all the inoculation
levels The bias varies from ndash 0037 to 0068 log CFUml
Comparison of the repeatability standard deviations
If the ratio Srj Alt Srj Ref of the repeatability standard deviations of the
alternative method and the reference method is larger than 2 the precision
under repeatability conditions of the alternative method is considered to be
lower than that of the reference method If this ratio is smaller than 05 the
precision under repeatability conditions of the alternative method is
considered to be greater than that of the reference method
The ratio values are given in table 18
Table 18
Level
Repeatability standard deviation
Reference method Alternative method Alternative method
reference method ratio
1 0242 0205 0847
2 0054 0091 1682
2 bis 0261 0190 0729
3 0038 0048 1264
Level Repeatability limit
Reference method Alternative method
1 0676 0573
2 0152 0256
2 bis 0730 0532
3 0106 0134
The ratios of the repeatability standard deviations are below 2 in all cases
The precision under repeatability conditions of the alternative method is
equivalent to that of the reference method
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2838 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Comparison of the reproducibility standard deviations
If the ratio Srj Alt Srj Ref of the reproducibility standard deviations of the alternative method and the
reference method is larger than 2 the precision under reproducibility conditions of the alternative
method is considered to be lower than that of the reference method If this ratio is smaller than 05 the
precision under reproducibility conditions of the alternative method is considered to be greater than
that of the reference method
The ratio values are given in table 19
Table 19
Level
Reproducibility standard deviation
Reference method Alternative method Alternative method
reference method ratio
1 0242 0243 1007
2 0066 0133 2011
2 bis 0261 0195 0749
3 0038 0061 1617
Level Reproducibility limit
Reference method Alternative method
1 0676 0681
2 0185 0371
2 bis 0730 0547
3 0106 0171
The ratios of the reproducibility standard deviations are below or close to 2
The precision under reproducibility conditions of the alternative method is
equivalent to that of the reference method
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2938 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
214 Conclusion
Method comparison study
The Listeria PrecisTM enumeration method shows satisfying linearity and
relative accuracy
The alternative method shows satisfying inclusivity and exclusivity results
Two Listeria monocytogenes strains gave negative results with the
alternative method These strains grew weakly on the reference method
media (OampA Palcam and Oxford)
The limits of repeatability of the alternative method are similar to those of the
reference method
The bias between both methods are low between - 0133 Log CFUg and
- 0008 Log CFUg
The Listeria PrecisTM enumeration method offers the saving for confirmatory
tests
Positive results are obtained with the alternative method in 2-3 days (with
OBIS Mono test) or in 4-7 days (by the classical tests described in the
standardized method) against 4-7 days for the reference method
Inter-laboratory study
The bias between the reference and the alternative methods shows satisfying
and correct values
- level 1 (100 - 1 000 CFUg) bias = + 007 log CFUg
- level 2 (1 000 - 10 000 CFUg) bias = 002 log CFUg
- level 2 bis (1 000 - 10 000 CFUg) bias = - 004 log CFUg
- level 3 (10 000 - 100 000 CFUg) bias = - 004 log CFUg
The repeatability and the reproducibility of the alternative method look similar
to those of the reference method
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3038 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
22 Extension study (realised by ASEPT 2007)
An extension study was performed in 2007 for a new confirmatory test OBIS
MONO
150 Listeria monocytogenes strains and 100 non-target strains were isolated
onto TSAYE and BrillianceTM Listeria Agar plates All the Listeria
monocytogenes and the non-target strains gave the expected results
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3138 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Appendix 1 ndash Flow diagram of the Listeria PrecisTM method
x g sample + 9 x ml buffered peptone water
Revivification step for 1 h plusmn 5 min at 20degC plusmn 1degC
Streaking 1 ml onto 3 Brilliance Listeria Agar plates (mother suspension)
or 01 ml onto 1 Brilliance Listeria Agar plates (mother suspension and decimal dilutions)
Incubation for 48 h plusmn 3 h at 37degC plusmn 1degC
Confirmatory tests on at least one typical colony
- by the tests described in the ISO method
- by applying the OBIS mono test on an isolated colony
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3238 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Appendix 2 ndash ISO 11290-2 (1998) amp ISO 11290-2A1 (2004) Horizontal
method for the detection and enumeration of Listeria monocytogenes -
Part 2 enumeration of Listeria monocytogenes in foods
10 g cheese + 90 ml buffered peptone water or Half Fraser Broth (without supplements) (110 dilution)
Stomach 1 min 30 seconds
Keep at 20degC plusmn 2degC for 1 h plusmn 5 min
Decimal dilutions in 9 ml peptone salt tubes
Dilution 110
Dilution 1100
Dilution 11000
1 ml on 3 Chromogenic Listeria medium (ISO) plates (x 2) (-1)
01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-2)
01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-3)
01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-4)
Incubation at 37degC plusmn 1degC
24 h - 48 h
Numerate typical colonies at 24 h
(bluegreen surrounded by an opaque halo)
Numerate at 48 h
Streak typical colonies on TSYEA
(5 coloniesplate 2 successive dilutions)
Confirmation tests
Catalase
Gram stain
For confirmation of L monocytogenes
Haemolysis
Carbohydrate (rhamnose and xylose) utilisation
CAMP Test
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3338 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Appendix 3 ndash Mandelrsquos graphics
Laboratoire h Ref
Niveau 1 h Ref
Niveau 2 h Ref
Niveau 3 h Alt
Niveau 1 h Alt
Niveau 2 h Alt
Niveau 3 h5 h1 h5 h1
A -050 -015 083 062 -212 -039 198 299 -198 -299
B -261 -270 021 -077 -058 173 198 299 -198 -299
C -151 -031 -014 -015 -004 -005 198 299 -198 -299
D 038 040 -137 000 072 207 198 299 -198 -299
E -001 090 155 -052 056 -101 198 299 -198 -299
F -112 -321 -153 070 -325 023 198 299 -198 -299
G 042 -071 040 147 -009 176 198 299 -198 -299
H 169 046 -169 192 018 -083 198 299 -198 -299
L 009 -120 021 -077 -231 005 198 299 -198 -299
M 001 031 014 -032 004 -011 198 299 -198 -299
-300
-200
-100
000
100
200
300
A B C D E F G H L M
Vale
urs
h d
e M
an
del(
rob
uste
s)
Laboratoires
Meacutethode de reacutefeacuterence analyse robuste
h Ref Niveau 1
h Ref Niveau 2
h Ref Niveau 3
h 5=198
h 1=299
-300
-200
-100
000
100
200
300
A B C D E F G H L M
Vale
urs
de h
de M
an
del(
rob
uste
s)
Laboratoires
Meacutethode alternative analyse robuste
h Alt Niveau 1
h Alt Niveau 2
h Alt Niveau 2
h 1=299
h 5=198
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3438 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Laboratoire k Ref
Niveau 1 k Ref
Niveau 2 k Ref
Niveau 3 k Alt
Niveau 1 k Alt
Niveau 2 k Alt
Niveau 3 k1 k5
A 000 089 142 084 162 046 259 181
B 125 087 073 104 075 046 259 181
C 117 013 091 188 022 083 259 181
D 052 036 069 000 035 083 259 181
E 028 034 037 138 019 112 259 181
F 140 222 018 027 281 112 259 181
G 088 068 024 131 145 000 259 181
H 129 094 173 016 061 000 259 181
L 069 000 073 104 270 224 259 181
M 000 048 038 043 099 241 259 181
000
050
100
150
200
250
300
350
400
A B C D E F G H L M
Vale
urs
k d
e M
an
del(
rob
uste
s)
Laboratoires
Meacutethode de reacutefeacuterence analyse robuste
k Ref Niveau 1k Ref Niveau 2k Ref Niveau 3
k 1=259
k 5=181
000
050
100
150
200
250
300
A B C D E F G H L M
Vale
urs
k d
e M
an
del
(ro
bu
ste
s)
Laboratoires
Meacutethode alternative analyse robuste
k Alt Niveau 3
k Alt Niveau 1
k Alt Niveau 2
k 1=259
k 5=181
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3538 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Appendix 4 ndash Statistical results
Level 1
Nombre de laboratoires (p) 10
A 1602 1602 1602 000 0000 0000 -0495 0000 A 1845 1602 1724 017 0122 -0122 0621 0840 0122
B 1602 1176 1389 030 0213 -0213 -2613 1247 B 1602 1301 1452 021 0151 -0151 -0769 1041 0062
C 1699 1301 1500 028 0199 -0199 -1510 1165 C 1301 1845 1573 038 -0272 0272 -0148 1881 0073
D 1602 1778 1690 012 -0088 0088 0380 0516 D 1602 1602 1602 000 0000 0000 0000 0000 -0088
E 1699 1602 1651 007 0048 -0048 -0013 0284 E 1699 1301 1500 028 0199 -0199 -0522 1376 -0151
F 1778 1301 1540 034 0239 -0239 -1116 1397 F 1778 1699 1739 006 0040 -0040 0698 0274 0199
G 1845 1544 1695 021 0151 -0151 0425 0881 G 2079 1699 1889 027 0190 -0190 1467 1315 0194
H 2041 1602 1822 031 0220 -0220 1689 1286 H 2000 1954 1977 003 0023 -0023 1917 0158 0155
L 1544 1778 1661 017 -0117 0117 0092 0685 L 1301 1602 1452 021 -0151 0151 -0769 1041 -0210
M 1653 1653 1653 000 0000 0000 0013 0000 M 1477 1602 1540 009 -0062 0062 -0319 0432 -0114
m 165186 m 160206 mediane D 00678
Cn(2p) 1867 Cn(2p) 1867 Cp 16101
Cn(p) 16101 Cn(p) 16101 Qn(D) 0092926
n=2p n 20 n=2p n 20 Qdiff 01496169
f 11 f 11 t 114
l 55 l 55
n=p n 10 n=p n 10
f 6 f 6
l 15 l 15
Qn(2p) 0091465 Qn(2p) 007745
Qn(p) 0062469 Qn(p) 0121519
Qintra 017078 Qintra 014461
Qinter 010058 Qinter 019565
Sr(eacutecart-type de reacutepeacutetabiliteacute) 02415 Sr 02045
RSDr(coefficient de variation de la reacutepeacutetabiliteacute)1462 RSDr 1277
r(repeatability limit)) 0676 r 0573
SL(eacutecart-type interlaboratoire) -001905 SLlt0 SL 001737
SR(eacutecart-type de reproductibiliteacute) 02415 SR 02433
RSDR(coefficient de variation de la reproductibiliteacute)0146 RSDR 0152
R(limite de reproductibiliteacute)) 06762 R(limite de reproductibiliteacute))068123
h k h MoyenneLaboratoires
Niveau 1
si LaboratoiresDeviationMoyenne Dk DeviationsiAlt
Niveau 1
Ref
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3638 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Level 2
Nombre de laboratoires (p) 10
A 2681 2613 2647 005 0034 -0034 -0151 0891 A 2322 2531 2427 015 -0105 0105 -2119 1620 -0220
B 2544 2477 2511 005 0033 -0033 -2699 0872 B 2556 2653 2605 007 -0048 0048 -0581 0750 0094
C 2643 2633 2638 001 0005 -0005 -0311 0130 C 2681 2653 2667 002 0014 -0014 -0040 0217 0029
D 2690 2663 2676 002 0014 -0014 0400 0357 D 2778 2732 2755 003 0023 -0023 0721 0354 0079
E 2690 2716 2703 002 -0013 0013 0897 0336 E 2724 2748 2736 002 -0012 0012 0556 0185 0033
F 2568 2398 2483 012 0085 -0085 -3214 2217 F 2477 2114 2296 026 0182 -0182 -3255 2811 -0188
G 2643 2591 2617 004 0026 -0026 -0707 0682 G 2756 2568 2662 013 0094 -0094 -0085 1453 0045
H 2716 2643 2680 005 0036 -0036 0460 0945 H 2653 2732 2693 006 -0040 0040 0181 0613 0013
L 2591 2591 2591 000 0000 0000 -1196 0000 L 2230 2580 2405 025 -0175 0175 -2307 2704 -0186
M 2653 2690 2672 003 -0018 0018 0311 0481 M 2740 2613 2677 009 0064 -0064 0040 0987 0005
m 2655082 m 26719001 mediane D 00209
Cn(2p) 1867 Cn(2p) 1867 Cp 16101
Cn(p) 16101 Cn(p) 16101 Qn(D) 0039911
n=2p n 20 n=2p n 20 Qdiff 00642591
f 11 f 11 t 082
l 55 l 55
n=p n 10 n=p n 10
f 6 f 6
l 15 l 15
Qn(2p) 0020570 Qn(2p) 00345985
Qn(p) 00332437 Qn(p) 00718158
Qintra 003841 Qintra 006460
Qinter 005352 Qinter 011563
Sr(eacutecart-type de reacutepeacutetabiliteacute) 00543 Sr 00914
RSDr(coefficient de variation de la reacutepeacutetabiliteacute)205 RSDr 342
r(repeatability limit)) 0152 r 0256
SL(eacutecart-type interlaboratoire) 000139 SL 000920
SR(eacutecart-type de reproductibiliteacute) 00659 SR 01325
RSDR(coefficient de variation de la reproductibiliteacute)0025 RSDR 0050
R(limite de reproductibiliteacute)) 018446 R(limite de reproductibiliteacute))03708618
Laboratoires
Niveau 2
Moyenne si Laboratoires
Niveau 2
DRef Alt Moyenne si Deviation h Deviation h k k
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3738 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Level 2 bis
Nombre de laboratoires (p) 10
A 3000 2477 2739 037 0261 -0261 0832 1418 A 2602 2477 2540 009 0062 -0062 -0391 0465 -0199
B 2544 2813 2678 019 -0134 0134 0211 0729 B 2903 2778 2841 009 0062 -0062 1732 0465 0162
C 2477 2813 2645 024 -0168 0168 -0135 0911 C 2477 2699 2588 016 -0111 0111 -0049 0826 -0057
D 2398 2653 2526 018 -0128 0128 -1370 0692 D 2778 3000 2889 016 -0111 0111 2074 0826 0363
E 2875 2740 2808 010 0067 -0067 1547 0365 E 2602 2301 2452 021 0151 -0151 -1012 1120 -0356
F 2477 2544 2511 005 -0033 0033 -1525 0182 F 2778 2477 2628 021 0151 -0151 0230 1120 0117
G 2653 2740 2697 006 -0044 0044 0400 0236 G 2845 2845 2845 000 0000 0000 1764 0000 0148
H 2176 2813 2495 045 -0318 0318 -1692 1727 H 2477 2477 2477 000 0000 0000 -0832 0000 -0017
L 2813 2544 2678 019 0134 -0134 0211 0729 L 2903 2301 2602 043 0301 -0301 0049 2241 -0076
M 2602 2740 2671 010 -0069 0069 0135 0375 M 2903 2255 2579 046 0324 -0324 -0112 2411 -0092
m 2658114 m 259505 mediane D -00372
Cn(2p) 1867 Cn(2p) 1867 Cp 16101
Cn(p) 16101 Cn(p) 16101 Qn(D) 01656915
n=2p n 20 n=2p n 20 Qdiff 02667753
f 11 f 11 t 035
l 55 l 55
n=p n 10 n=p n 10
f 6 f 6
l 15 l 15
Qn(2p) 0098745 Qn(2p) 007195
Qn(p) 006007 Qn(p) 008805
Qintra 018437 Qintra 013435
Qinter 009672 Qinter 014176
Sr(eacutecart-type de reacutepeacutetabiliteacute) 02607 Sr 01900
RSDr(coefficient de variation de la reacutepeacutetabiliteacute)981 RSDr 732
r(repeatability limit)) 0730 r 0532
SL(eacutecart-type interlaboratoire) -002464 SLlt0 SL 000205
SR(eacutecart-type de reproductibiliteacute) 02607 SR 01953
RSDR(coefficient de variation de la reproductibiliteacute)0098 RSDR 0075
R(limite de reproductibiliteacute)) 072996 R(limite de reproductibiliteacute))054686
Laboratoires
Niveau 2 bis
Moyenne si Laboratoires
Niveau 2 bis
DRef Alt Moyenne si Deviation h Deviation h k k
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3838 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Level 3
Nombre de laboratoires (p) 10
A 3690 3663 3676 002 0014 -0014 -0311 0513 A 3690 3708 3699 001 -0009 0009 0354 0257 0022
B 3613 3672 3642 004 -0030 0030 -1851 1109 B 3708 3568 3638 010 0070 -0070 -0844 2061 -0005
C 3663 3732 3698 005 -0035 0035 0645 1302 C 3663 3699 3681 003 -0018 0018 0000 0536 -0017
D 3690 3690 3690 000 0000 0000 0311 0000 D 3643 3663 3653 001 -0010 0010 -0545 0286 -0037
E 3732 3653 3693 006 0040 -0040 0429 1480 E 3613 3732 3673 008 -0060 0060 -0162 1769 -0020
F 3643 3672 3658 002 -0014 0014 -1157 0536 F 3690 3771 3731 006 -0040 0040 0975 1193 0073
G 3699 3653 3676 003 0023 -0023 -0328 0856 G 3602 3643 3623 003 -0021 0021 -1141 0612 -0053
H 3623 3672 3648 003 -0024 0024 -1615 0913 H 3748 3681 3715 005 0033 -0033 0664 0990 0067
L 3724 3663 3694 004 0031 -0031 0461 1150 L 3748 3771 3760 002 -0011 0011 1544 0335 0066
M 3690 3690 3690 000 0000 0000 0311 0000 M 3806 3681 3744 009 0062 -0062 1234 1848 0054
m 368334 m 368086 mediane D 00089
Cn(2p) 1867 Cn(2p) 1867 Cp 16101
Cn(p) 16101 Cn(p) 16101 Qn(D) 00325359
n=2p n 20 n=2p n 20 Qdiff 00523852
f 11 f 11 t 043
l 55 l 55
n=p n 10 n=p n 10
f 6 f 6
l 15 l 15
Qn(2p) 0014323 Qn(2p) 001811
Qn(p) 0013719 Qn(p) 003164
Qintra 002674 Qintra 003381
Qinter 002209 Qinter 005094
Sr(eacutecart-type de reacutepeacutetabiliteacute) 00378 Sr 00478
RSDr(coefficient de variation de la reacutepeacutetabiliteacute)103 RSDr 130
r(repeatability limit)) 0106 r 0134
SL(eacutecart-type interlaboratoire) -000023 SLlt0 SL 000145
SR(eacutecart-type de reproductibiliteacute) 00378 SR 00611
RSDR(coefficient de variation de la reproductibiliteacute)0010 RSDR 0017
R(limite de reproductibiliteacute)) 0105894 R(limite de reproductibiliteacute))017119
Ref Alt DLaboratoires
Niveau 3
Moyenne si Deviation h k Laboratoires
Niveau 3
Moyenne si Deviation h k
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2138 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
213 Inter-laboratory study
2131 Organisation
12 laboratories participated to this study Pasteurised milk was inoculated by
Listeria monocytogenes 12a isolated from raw milk The targeted
inoculation levels were
- 0 CFUml
- 50 CFU ml
- 500 CFU ml
- 5 000 CFU ml
Each laboratory received eight flasks of 10 ml sample ie two flasks per
inoculation level Furthermore one non-inoculated sample has been added
to the package for total viable count microflora (NF ISO 4833 method)
Detailed instructions were transmitted to the collaborators by the expert
laboratory
Collaborative study laboratories and the expert laboratory carried out the
analyses with the alternative and reference methods
2132 Verification of experimental parameters
Strain stability during transport
In order to evaluate the Listeria monocytogenes strain stability during
transport bacterial count of all inoculated flasks were checked at different
time ie inoculation time after 24 h of conservation at 2deg - 8degC Results are
reported in table 11
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2238 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Table 11 ndash Listeria monocytogenes count
by ISO 11290-2A1 method (log CFUml)
Day of the analyses Level contamination Reference method
Day 0
(29052006)
1 45
2 440
3 4 500
Day 1
(30052006)
1 60
2 430
3 4 900
No evolution of the strain was observed after storage for 24 h at 2degC ndash 8degC
Contamination level
The real contamination levels are provided in table 12
Table 12 ndash Real contamination level (CFUml)
Target inoculation
(CFU ml) Samples Ndeg
Real contamination levels
(CFUml)
0 1 - 8 0
50 5 - 6 45
500 3 - 7 440
5000 2 - 4 4 500
Sample temperature at receipt
Measured temperatures at receipt are listed in table 13
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2338 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Table 13 - Sample temperature on receipt
Laboratories Receipt
day
Hour
receipt
Measured temperature
at receipt (degC)
Measured temperature by the
temperature probe (degC)
A
B
C
D
E
F
G
H
I
J
K
L
M
30052006
10h45
10h15
11h15
10h00
09h30
8h30
11h50
10h00
08h30
14h45
08h30
09h45
08h45
82
55
45
63
60
62
81
38
48
-
14
67
76
03
03
-13
03
04
-14
05
04
01
05
-13
04
Conclusion
The temperatures during the transport were correct for all the labs except for
Lab J which didnrsquot provide the information
All the labs received their packages with several broken flasks
Three labs (B D and L) received some damaged samples but proceeded to
analyses
All the labs started the analyses at Day 1 except Lab J
2133 Results
Aerobic mesophilic flora
The count of aerobic mesophilic flora varied from 94 102 CFUml to
33 103 CFUml
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2438 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Expert lab results
The results obtained by the expert lab are the following
Table 14
Target inoculation level (CFUml)
Reference method ISO 11290-2
Alternative method Listeria Precis Enumeration
Rep 1 Rep 2 Rep 1 Rep 2
lt 10 lt 10 lt 10 lt 10 lt10
50 50 (Ne) 60 (Ne) 50 (Ne) 30 (Ne)
500 470 390 490 500
5 000 4 700 5 100 5 900 3 400
Ne estimated number
The obtained results are in agreement with those expected
Collaborator Labs results
A synthesis of the results obtained by the collaborator labs is provided
Table 15 (CFUml) and Table 16 (log CFUml)
Table 15 ndash Synthesis of results obtained by the alternative
and the reference methods (CFUml)
Labs Level 1 Level 2 Level 2 bis Level 3
Reference method
Alternative method
Reference method
Alternative method
Reference method
Alternative method
Reference method
Alternative method
A 40 40 70 40 480 410 210 340 1000 300 400 300 4900 4600 4900 5100
B 40 15 40 20 350 300 360 450 350 650 800 600 4100 4700 5100 3700
C 50 20 20 70 440 430 480 450 300 650 300 500 4600 5400 4600 5000
D 40 60 40 40 490 460 600 540 250 450 600 1000 4900 4900 4400 4600
E 50 40 50 20 490 520 530 560 750 550 400 200 5400 4500 4100 5400
F 60 20 60 50 370 250 300 130 300 350 600 300 4400 4700 4900 5900
G 70 35 120 50 440 390 570 370 450 550 700 700 5000 4500 4000 4400
H 110 40 100 90 520 440 450 540 150 650 300 300 4200 4700 5600 4800
L 35 60 20 40 390 390 170 380 650 350 800 200 5300 4600 5600 5900
M 45 45 30 40 450 490 550 410 400 550 800 180 4900 4900 6400 4800
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2538 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Table 16 ndash Synthesis of results obtained by the alternative
and the reference methods (log CFUml)
Labs Level 1 Level 2 Level 2 bis Level 3
Reference method
Alternative method
Reference method
Alternative method
Reference method
Alternative method
Reference method
Alternative method
A 1602 1602 1845 1602 2681 2613 2322 2531 3000 2477 2602 2477 3690 3663 3690 3708
B 1602 1176 1602 1301 2544 2477 2556 2653 2544 2813 2903 2778 3613 3672 3708 3568
C 1699 1301 1301 1845 2643 2633 2681 2653 2477 2813 2477 2699 3663 3732 3663 3699
D 1602 1778 1602 1602 2690 2663 2778 2732 2398 2653 2778 3000 3690 3690 3643 3663
E 1699 1602 1699 1301 2690 2716 2724 2748 2875 2740 2602 2301 3732 3653 3613 3732
F 1778 1301 1778 1699 2568 2398 2477 2114 2477 2544 2778 2477 3643 3672 3690 3771
G 1845 1544 2079 1699 2643 2591 2756 2568 2653 2740 2845 2845 3699 3653 3602 3643
H 2041 1602 2000 1954 2716 2643 2653 2732 2176 2813 2477 2477 3623 3672 3748 3681
L 1544 1778 1301 1602 2591 2591 2230 2580 2813 2544 2903 2301 3724 3663 3748 3771
M 1653 1653 1477 1602 2653 2690 2740 2613 2602 2740 2903 2255 3690 3690 3806 3681
Level 2 results obtained taking account the 3 plates inoculated with the mother-suspension
Level 2 bis results obtained taking account the plates inoculated with 01 ml of the mother-suspension
Scrutiny of the measurement results for consistency
In order to identify the measurement results or laboratories that could be
inconsistent two graphical consistency techniques were realised Mandelrsquos
h-ank-statistics Mandel indicators h and k at 5 significance highlighted
some possible inconsistent data (See tables and graphics in Appendix 3 and
Appendix 4)
Mandelrsquos values Number of values observed over the threshold
Reference method Alternative method
h gt 1 Lab F Level 2 Lab F Level 2
h gt 5 Lab F Level 2
Lab B Levels 1 and 2
Lab A Level 2
Lab D Level 3
Lab F Level 2
Lab L Level 2
kgt 1 Lab F Level 3
k gt 5 Lab F Level 2 Lab C Level 2
Lab F Level 3
Lab L Levels 1 and 3
Lab M Level 1
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2638 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
The emphasized results appear (i) coherent with the targeted contamination
levels (ii) concordant between both methods
All the results were thus kept for the statistical analysis
Comparison of the trueness and precision characteristics of
the reference method and alternative methods
The statistical values are summarised hereafter
Level p
Reference method Alternative method
Repeatability ratio
Reproducibility ratio Median
Repeatability standard deviation
Reproducibility standard deviation
Median Repeatability
standard deviation
Reproducibility standard deviation
1 10 16519 02415 02425 16021 02045 02433 0847 1007
2 10 26551 00543 00659 26719 00914 01325 1682 2011
2 bis 10 26581 02607 02607 25951 01900 01953 0729 0749
3 10 18671 00378 00378 18671 00478 00611 1264 1617
Bias of the alternative method
In order to estimate the bias of the alternative method with regard to the reference method for every
level Dij and t are calculated according to the following equations
RefAlt ijYijYDij
Diffp
Dijit
)2(
)(median
If the t value is superior to 2 the alternative method is significantly biased
with regard to the reference method
The values are given in table 17
Table 17 ndash t (d) values obtained by level
Level Bias t (d) Conclusion
1 0068 114 Non significant bias
2 0021 082 Non significant bias
2 bis - 0037 035 Non significant bias
3 - 0037 043 Non significant bias
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2738 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
The statistical test concludes to a non significant bias for all the inoculation
levels The bias varies from ndash 0037 to 0068 log CFUml
Comparison of the repeatability standard deviations
If the ratio Srj Alt Srj Ref of the repeatability standard deviations of the
alternative method and the reference method is larger than 2 the precision
under repeatability conditions of the alternative method is considered to be
lower than that of the reference method If this ratio is smaller than 05 the
precision under repeatability conditions of the alternative method is
considered to be greater than that of the reference method
The ratio values are given in table 18
Table 18
Level
Repeatability standard deviation
Reference method Alternative method Alternative method
reference method ratio
1 0242 0205 0847
2 0054 0091 1682
2 bis 0261 0190 0729
3 0038 0048 1264
Level Repeatability limit
Reference method Alternative method
1 0676 0573
2 0152 0256
2 bis 0730 0532
3 0106 0134
The ratios of the repeatability standard deviations are below 2 in all cases
The precision under repeatability conditions of the alternative method is
equivalent to that of the reference method
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2838 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Comparison of the reproducibility standard deviations
If the ratio Srj Alt Srj Ref of the reproducibility standard deviations of the alternative method and the
reference method is larger than 2 the precision under reproducibility conditions of the alternative
method is considered to be lower than that of the reference method If this ratio is smaller than 05 the
precision under reproducibility conditions of the alternative method is considered to be greater than
that of the reference method
The ratio values are given in table 19
Table 19
Level
Reproducibility standard deviation
Reference method Alternative method Alternative method
reference method ratio
1 0242 0243 1007
2 0066 0133 2011
2 bis 0261 0195 0749
3 0038 0061 1617
Level Reproducibility limit
Reference method Alternative method
1 0676 0681
2 0185 0371
2 bis 0730 0547
3 0106 0171
The ratios of the reproducibility standard deviations are below or close to 2
The precision under reproducibility conditions of the alternative method is
equivalent to that of the reference method
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2938 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
214 Conclusion
Method comparison study
The Listeria PrecisTM enumeration method shows satisfying linearity and
relative accuracy
The alternative method shows satisfying inclusivity and exclusivity results
Two Listeria monocytogenes strains gave negative results with the
alternative method These strains grew weakly on the reference method
media (OampA Palcam and Oxford)
The limits of repeatability of the alternative method are similar to those of the
reference method
The bias between both methods are low between - 0133 Log CFUg and
- 0008 Log CFUg
The Listeria PrecisTM enumeration method offers the saving for confirmatory
tests
Positive results are obtained with the alternative method in 2-3 days (with
OBIS Mono test) or in 4-7 days (by the classical tests described in the
standardized method) against 4-7 days for the reference method
Inter-laboratory study
The bias between the reference and the alternative methods shows satisfying
and correct values
- level 1 (100 - 1 000 CFUg) bias = + 007 log CFUg
- level 2 (1 000 - 10 000 CFUg) bias = 002 log CFUg
- level 2 bis (1 000 - 10 000 CFUg) bias = - 004 log CFUg
- level 3 (10 000 - 100 000 CFUg) bias = - 004 log CFUg
The repeatability and the reproducibility of the alternative method look similar
to those of the reference method
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3038 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
22 Extension study (realised by ASEPT 2007)
An extension study was performed in 2007 for a new confirmatory test OBIS
MONO
150 Listeria monocytogenes strains and 100 non-target strains were isolated
onto TSAYE and BrillianceTM Listeria Agar plates All the Listeria
monocytogenes and the non-target strains gave the expected results
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3138 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Appendix 1 ndash Flow diagram of the Listeria PrecisTM method
x g sample + 9 x ml buffered peptone water
Revivification step for 1 h plusmn 5 min at 20degC plusmn 1degC
Streaking 1 ml onto 3 Brilliance Listeria Agar plates (mother suspension)
or 01 ml onto 1 Brilliance Listeria Agar plates (mother suspension and decimal dilutions)
Incubation for 48 h plusmn 3 h at 37degC plusmn 1degC
Confirmatory tests on at least one typical colony
- by the tests described in the ISO method
- by applying the OBIS mono test on an isolated colony
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3238 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Appendix 2 ndash ISO 11290-2 (1998) amp ISO 11290-2A1 (2004) Horizontal
method for the detection and enumeration of Listeria monocytogenes -
Part 2 enumeration of Listeria monocytogenes in foods
10 g cheese + 90 ml buffered peptone water or Half Fraser Broth (without supplements) (110 dilution)
Stomach 1 min 30 seconds
Keep at 20degC plusmn 2degC for 1 h plusmn 5 min
Decimal dilutions in 9 ml peptone salt tubes
Dilution 110
Dilution 1100
Dilution 11000
1 ml on 3 Chromogenic Listeria medium (ISO) plates (x 2) (-1)
01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-2)
01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-3)
01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-4)
Incubation at 37degC plusmn 1degC
24 h - 48 h
Numerate typical colonies at 24 h
(bluegreen surrounded by an opaque halo)
Numerate at 48 h
Streak typical colonies on TSYEA
(5 coloniesplate 2 successive dilutions)
Confirmation tests
Catalase
Gram stain
For confirmation of L monocytogenes
Haemolysis
Carbohydrate (rhamnose and xylose) utilisation
CAMP Test
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3338 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Appendix 3 ndash Mandelrsquos graphics
Laboratoire h Ref
Niveau 1 h Ref
Niveau 2 h Ref
Niveau 3 h Alt
Niveau 1 h Alt
Niveau 2 h Alt
Niveau 3 h5 h1 h5 h1
A -050 -015 083 062 -212 -039 198 299 -198 -299
B -261 -270 021 -077 -058 173 198 299 -198 -299
C -151 -031 -014 -015 -004 -005 198 299 -198 -299
D 038 040 -137 000 072 207 198 299 -198 -299
E -001 090 155 -052 056 -101 198 299 -198 -299
F -112 -321 -153 070 -325 023 198 299 -198 -299
G 042 -071 040 147 -009 176 198 299 -198 -299
H 169 046 -169 192 018 -083 198 299 -198 -299
L 009 -120 021 -077 -231 005 198 299 -198 -299
M 001 031 014 -032 004 -011 198 299 -198 -299
-300
-200
-100
000
100
200
300
A B C D E F G H L M
Vale
urs
h d
e M
an
del(
rob
uste
s)
Laboratoires
Meacutethode de reacutefeacuterence analyse robuste
h Ref Niveau 1
h Ref Niveau 2
h Ref Niveau 3
h 5=198
h 1=299
-300
-200
-100
000
100
200
300
A B C D E F G H L M
Vale
urs
de h
de M
an
del(
rob
uste
s)
Laboratoires
Meacutethode alternative analyse robuste
h Alt Niveau 1
h Alt Niveau 2
h Alt Niveau 2
h 1=299
h 5=198
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3438 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Laboratoire k Ref
Niveau 1 k Ref
Niveau 2 k Ref
Niveau 3 k Alt
Niveau 1 k Alt
Niveau 2 k Alt
Niveau 3 k1 k5
A 000 089 142 084 162 046 259 181
B 125 087 073 104 075 046 259 181
C 117 013 091 188 022 083 259 181
D 052 036 069 000 035 083 259 181
E 028 034 037 138 019 112 259 181
F 140 222 018 027 281 112 259 181
G 088 068 024 131 145 000 259 181
H 129 094 173 016 061 000 259 181
L 069 000 073 104 270 224 259 181
M 000 048 038 043 099 241 259 181
000
050
100
150
200
250
300
350
400
A B C D E F G H L M
Vale
urs
k d
e M
an
del(
rob
uste
s)
Laboratoires
Meacutethode de reacutefeacuterence analyse robuste
k Ref Niveau 1k Ref Niveau 2k Ref Niveau 3
k 1=259
k 5=181
000
050
100
150
200
250
300
A B C D E F G H L M
Vale
urs
k d
e M
an
del
(ro
bu
ste
s)
Laboratoires
Meacutethode alternative analyse robuste
k Alt Niveau 3
k Alt Niveau 1
k Alt Niveau 2
k 1=259
k 5=181
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3538 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Appendix 4 ndash Statistical results
Level 1
Nombre de laboratoires (p) 10
A 1602 1602 1602 000 0000 0000 -0495 0000 A 1845 1602 1724 017 0122 -0122 0621 0840 0122
B 1602 1176 1389 030 0213 -0213 -2613 1247 B 1602 1301 1452 021 0151 -0151 -0769 1041 0062
C 1699 1301 1500 028 0199 -0199 -1510 1165 C 1301 1845 1573 038 -0272 0272 -0148 1881 0073
D 1602 1778 1690 012 -0088 0088 0380 0516 D 1602 1602 1602 000 0000 0000 0000 0000 -0088
E 1699 1602 1651 007 0048 -0048 -0013 0284 E 1699 1301 1500 028 0199 -0199 -0522 1376 -0151
F 1778 1301 1540 034 0239 -0239 -1116 1397 F 1778 1699 1739 006 0040 -0040 0698 0274 0199
G 1845 1544 1695 021 0151 -0151 0425 0881 G 2079 1699 1889 027 0190 -0190 1467 1315 0194
H 2041 1602 1822 031 0220 -0220 1689 1286 H 2000 1954 1977 003 0023 -0023 1917 0158 0155
L 1544 1778 1661 017 -0117 0117 0092 0685 L 1301 1602 1452 021 -0151 0151 -0769 1041 -0210
M 1653 1653 1653 000 0000 0000 0013 0000 M 1477 1602 1540 009 -0062 0062 -0319 0432 -0114
m 165186 m 160206 mediane D 00678
Cn(2p) 1867 Cn(2p) 1867 Cp 16101
Cn(p) 16101 Cn(p) 16101 Qn(D) 0092926
n=2p n 20 n=2p n 20 Qdiff 01496169
f 11 f 11 t 114
l 55 l 55
n=p n 10 n=p n 10
f 6 f 6
l 15 l 15
Qn(2p) 0091465 Qn(2p) 007745
Qn(p) 0062469 Qn(p) 0121519
Qintra 017078 Qintra 014461
Qinter 010058 Qinter 019565
Sr(eacutecart-type de reacutepeacutetabiliteacute) 02415 Sr 02045
RSDr(coefficient de variation de la reacutepeacutetabiliteacute)1462 RSDr 1277
r(repeatability limit)) 0676 r 0573
SL(eacutecart-type interlaboratoire) -001905 SLlt0 SL 001737
SR(eacutecart-type de reproductibiliteacute) 02415 SR 02433
RSDR(coefficient de variation de la reproductibiliteacute)0146 RSDR 0152
R(limite de reproductibiliteacute)) 06762 R(limite de reproductibiliteacute))068123
h k h MoyenneLaboratoires
Niveau 1
si LaboratoiresDeviationMoyenne Dk DeviationsiAlt
Niveau 1
Ref
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3638 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Level 2
Nombre de laboratoires (p) 10
A 2681 2613 2647 005 0034 -0034 -0151 0891 A 2322 2531 2427 015 -0105 0105 -2119 1620 -0220
B 2544 2477 2511 005 0033 -0033 -2699 0872 B 2556 2653 2605 007 -0048 0048 -0581 0750 0094
C 2643 2633 2638 001 0005 -0005 -0311 0130 C 2681 2653 2667 002 0014 -0014 -0040 0217 0029
D 2690 2663 2676 002 0014 -0014 0400 0357 D 2778 2732 2755 003 0023 -0023 0721 0354 0079
E 2690 2716 2703 002 -0013 0013 0897 0336 E 2724 2748 2736 002 -0012 0012 0556 0185 0033
F 2568 2398 2483 012 0085 -0085 -3214 2217 F 2477 2114 2296 026 0182 -0182 -3255 2811 -0188
G 2643 2591 2617 004 0026 -0026 -0707 0682 G 2756 2568 2662 013 0094 -0094 -0085 1453 0045
H 2716 2643 2680 005 0036 -0036 0460 0945 H 2653 2732 2693 006 -0040 0040 0181 0613 0013
L 2591 2591 2591 000 0000 0000 -1196 0000 L 2230 2580 2405 025 -0175 0175 -2307 2704 -0186
M 2653 2690 2672 003 -0018 0018 0311 0481 M 2740 2613 2677 009 0064 -0064 0040 0987 0005
m 2655082 m 26719001 mediane D 00209
Cn(2p) 1867 Cn(2p) 1867 Cp 16101
Cn(p) 16101 Cn(p) 16101 Qn(D) 0039911
n=2p n 20 n=2p n 20 Qdiff 00642591
f 11 f 11 t 082
l 55 l 55
n=p n 10 n=p n 10
f 6 f 6
l 15 l 15
Qn(2p) 0020570 Qn(2p) 00345985
Qn(p) 00332437 Qn(p) 00718158
Qintra 003841 Qintra 006460
Qinter 005352 Qinter 011563
Sr(eacutecart-type de reacutepeacutetabiliteacute) 00543 Sr 00914
RSDr(coefficient de variation de la reacutepeacutetabiliteacute)205 RSDr 342
r(repeatability limit)) 0152 r 0256
SL(eacutecart-type interlaboratoire) 000139 SL 000920
SR(eacutecart-type de reproductibiliteacute) 00659 SR 01325
RSDR(coefficient de variation de la reproductibiliteacute)0025 RSDR 0050
R(limite de reproductibiliteacute)) 018446 R(limite de reproductibiliteacute))03708618
Laboratoires
Niveau 2
Moyenne si Laboratoires
Niveau 2
DRef Alt Moyenne si Deviation h Deviation h k k
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3738 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Level 2 bis
Nombre de laboratoires (p) 10
A 3000 2477 2739 037 0261 -0261 0832 1418 A 2602 2477 2540 009 0062 -0062 -0391 0465 -0199
B 2544 2813 2678 019 -0134 0134 0211 0729 B 2903 2778 2841 009 0062 -0062 1732 0465 0162
C 2477 2813 2645 024 -0168 0168 -0135 0911 C 2477 2699 2588 016 -0111 0111 -0049 0826 -0057
D 2398 2653 2526 018 -0128 0128 -1370 0692 D 2778 3000 2889 016 -0111 0111 2074 0826 0363
E 2875 2740 2808 010 0067 -0067 1547 0365 E 2602 2301 2452 021 0151 -0151 -1012 1120 -0356
F 2477 2544 2511 005 -0033 0033 -1525 0182 F 2778 2477 2628 021 0151 -0151 0230 1120 0117
G 2653 2740 2697 006 -0044 0044 0400 0236 G 2845 2845 2845 000 0000 0000 1764 0000 0148
H 2176 2813 2495 045 -0318 0318 -1692 1727 H 2477 2477 2477 000 0000 0000 -0832 0000 -0017
L 2813 2544 2678 019 0134 -0134 0211 0729 L 2903 2301 2602 043 0301 -0301 0049 2241 -0076
M 2602 2740 2671 010 -0069 0069 0135 0375 M 2903 2255 2579 046 0324 -0324 -0112 2411 -0092
m 2658114 m 259505 mediane D -00372
Cn(2p) 1867 Cn(2p) 1867 Cp 16101
Cn(p) 16101 Cn(p) 16101 Qn(D) 01656915
n=2p n 20 n=2p n 20 Qdiff 02667753
f 11 f 11 t 035
l 55 l 55
n=p n 10 n=p n 10
f 6 f 6
l 15 l 15
Qn(2p) 0098745 Qn(2p) 007195
Qn(p) 006007 Qn(p) 008805
Qintra 018437 Qintra 013435
Qinter 009672 Qinter 014176
Sr(eacutecart-type de reacutepeacutetabiliteacute) 02607 Sr 01900
RSDr(coefficient de variation de la reacutepeacutetabiliteacute)981 RSDr 732
r(repeatability limit)) 0730 r 0532
SL(eacutecart-type interlaboratoire) -002464 SLlt0 SL 000205
SR(eacutecart-type de reproductibiliteacute) 02607 SR 01953
RSDR(coefficient de variation de la reproductibiliteacute)0098 RSDR 0075
R(limite de reproductibiliteacute)) 072996 R(limite de reproductibiliteacute))054686
Laboratoires
Niveau 2 bis
Moyenne si Laboratoires
Niveau 2 bis
DRef Alt Moyenne si Deviation h Deviation h k k
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3838 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Level 3
Nombre de laboratoires (p) 10
A 3690 3663 3676 002 0014 -0014 -0311 0513 A 3690 3708 3699 001 -0009 0009 0354 0257 0022
B 3613 3672 3642 004 -0030 0030 -1851 1109 B 3708 3568 3638 010 0070 -0070 -0844 2061 -0005
C 3663 3732 3698 005 -0035 0035 0645 1302 C 3663 3699 3681 003 -0018 0018 0000 0536 -0017
D 3690 3690 3690 000 0000 0000 0311 0000 D 3643 3663 3653 001 -0010 0010 -0545 0286 -0037
E 3732 3653 3693 006 0040 -0040 0429 1480 E 3613 3732 3673 008 -0060 0060 -0162 1769 -0020
F 3643 3672 3658 002 -0014 0014 -1157 0536 F 3690 3771 3731 006 -0040 0040 0975 1193 0073
G 3699 3653 3676 003 0023 -0023 -0328 0856 G 3602 3643 3623 003 -0021 0021 -1141 0612 -0053
H 3623 3672 3648 003 -0024 0024 -1615 0913 H 3748 3681 3715 005 0033 -0033 0664 0990 0067
L 3724 3663 3694 004 0031 -0031 0461 1150 L 3748 3771 3760 002 -0011 0011 1544 0335 0066
M 3690 3690 3690 000 0000 0000 0311 0000 M 3806 3681 3744 009 0062 -0062 1234 1848 0054
m 368334 m 368086 mediane D 00089
Cn(2p) 1867 Cn(2p) 1867 Cp 16101
Cn(p) 16101 Cn(p) 16101 Qn(D) 00325359
n=2p n 20 n=2p n 20 Qdiff 00523852
f 11 f 11 t 043
l 55 l 55
n=p n 10 n=p n 10
f 6 f 6
l 15 l 15
Qn(2p) 0014323 Qn(2p) 001811
Qn(p) 0013719 Qn(p) 003164
Qintra 002674 Qintra 003381
Qinter 002209 Qinter 005094
Sr(eacutecart-type de reacutepeacutetabiliteacute) 00378 Sr 00478
RSDr(coefficient de variation de la reacutepeacutetabiliteacute)103 RSDr 130
r(repeatability limit)) 0106 r 0134
SL(eacutecart-type interlaboratoire) -000023 SLlt0 SL 000145
SR(eacutecart-type de reproductibiliteacute) 00378 SR 00611
RSDR(coefficient de variation de la reproductibiliteacute)0010 RSDR 0017
R(limite de reproductibiliteacute)) 0105894 R(limite de reproductibiliteacute))017119
Ref Alt DLaboratoires
Niveau 3
Moyenne si Deviation h k Laboratoires
Niveau 3
Moyenne si Deviation h k
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2238 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Table 11 ndash Listeria monocytogenes count
by ISO 11290-2A1 method (log CFUml)
Day of the analyses Level contamination Reference method
Day 0
(29052006)
1 45
2 440
3 4 500
Day 1
(30052006)
1 60
2 430
3 4 900
No evolution of the strain was observed after storage for 24 h at 2degC ndash 8degC
Contamination level
The real contamination levels are provided in table 12
Table 12 ndash Real contamination level (CFUml)
Target inoculation
(CFU ml) Samples Ndeg
Real contamination levels
(CFUml)
0 1 - 8 0
50 5 - 6 45
500 3 - 7 440
5000 2 - 4 4 500
Sample temperature at receipt
Measured temperatures at receipt are listed in table 13
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2338 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Table 13 - Sample temperature on receipt
Laboratories Receipt
day
Hour
receipt
Measured temperature
at receipt (degC)
Measured temperature by the
temperature probe (degC)
A
B
C
D
E
F
G
H
I
J
K
L
M
30052006
10h45
10h15
11h15
10h00
09h30
8h30
11h50
10h00
08h30
14h45
08h30
09h45
08h45
82
55
45
63
60
62
81
38
48
-
14
67
76
03
03
-13
03
04
-14
05
04
01
05
-13
04
Conclusion
The temperatures during the transport were correct for all the labs except for
Lab J which didnrsquot provide the information
All the labs received their packages with several broken flasks
Three labs (B D and L) received some damaged samples but proceeded to
analyses
All the labs started the analyses at Day 1 except Lab J
2133 Results
Aerobic mesophilic flora
The count of aerobic mesophilic flora varied from 94 102 CFUml to
33 103 CFUml
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2438 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Expert lab results
The results obtained by the expert lab are the following
Table 14
Target inoculation level (CFUml)
Reference method ISO 11290-2
Alternative method Listeria Precis Enumeration
Rep 1 Rep 2 Rep 1 Rep 2
lt 10 lt 10 lt 10 lt 10 lt10
50 50 (Ne) 60 (Ne) 50 (Ne) 30 (Ne)
500 470 390 490 500
5 000 4 700 5 100 5 900 3 400
Ne estimated number
The obtained results are in agreement with those expected
Collaborator Labs results
A synthesis of the results obtained by the collaborator labs is provided
Table 15 (CFUml) and Table 16 (log CFUml)
Table 15 ndash Synthesis of results obtained by the alternative
and the reference methods (CFUml)
Labs Level 1 Level 2 Level 2 bis Level 3
Reference method
Alternative method
Reference method
Alternative method
Reference method
Alternative method
Reference method
Alternative method
A 40 40 70 40 480 410 210 340 1000 300 400 300 4900 4600 4900 5100
B 40 15 40 20 350 300 360 450 350 650 800 600 4100 4700 5100 3700
C 50 20 20 70 440 430 480 450 300 650 300 500 4600 5400 4600 5000
D 40 60 40 40 490 460 600 540 250 450 600 1000 4900 4900 4400 4600
E 50 40 50 20 490 520 530 560 750 550 400 200 5400 4500 4100 5400
F 60 20 60 50 370 250 300 130 300 350 600 300 4400 4700 4900 5900
G 70 35 120 50 440 390 570 370 450 550 700 700 5000 4500 4000 4400
H 110 40 100 90 520 440 450 540 150 650 300 300 4200 4700 5600 4800
L 35 60 20 40 390 390 170 380 650 350 800 200 5300 4600 5600 5900
M 45 45 30 40 450 490 550 410 400 550 800 180 4900 4900 6400 4800
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2538 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Table 16 ndash Synthesis of results obtained by the alternative
and the reference methods (log CFUml)
Labs Level 1 Level 2 Level 2 bis Level 3
Reference method
Alternative method
Reference method
Alternative method
Reference method
Alternative method
Reference method
Alternative method
A 1602 1602 1845 1602 2681 2613 2322 2531 3000 2477 2602 2477 3690 3663 3690 3708
B 1602 1176 1602 1301 2544 2477 2556 2653 2544 2813 2903 2778 3613 3672 3708 3568
C 1699 1301 1301 1845 2643 2633 2681 2653 2477 2813 2477 2699 3663 3732 3663 3699
D 1602 1778 1602 1602 2690 2663 2778 2732 2398 2653 2778 3000 3690 3690 3643 3663
E 1699 1602 1699 1301 2690 2716 2724 2748 2875 2740 2602 2301 3732 3653 3613 3732
F 1778 1301 1778 1699 2568 2398 2477 2114 2477 2544 2778 2477 3643 3672 3690 3771
G 1845 1544 2079 1699 2643 2591 2756 2568 2653 2740 2845 2845 3699 3653 3602 3643
H 2041 1602 2000 1954 2716 2643 2653 2732 2176 2813 2477 2477 3623 3672 3748 3681
L 1544 1778 1301 1602 2591 2591 2230 2580 2813 2544 2903 2301 3724 3663 3748 3771
M 1653 1653 1477 1602 2653 2690 2740 2613 2602 2740 2903 2255 3690 3690 3806 3681
Level 2 results obtained taking account the 3 plates inoculated with the mother-suspension
Level 2 bis results obtained taking account the plates inoculated with 01 ml of the mother-suspension
Scrutiny of the measurement results for consistency
In order to identify the measurement results or laboratories that could be
inconsistent two graphical consistency techniques were realised Mandelrsquos
h-ank-statistics Mandel indicators h and k at 5 significance highlighted
some possible inconsistent data (See tables and graphics in Appendix 3 and
Appendix 4)
Mandelrsquos values Number of values observed over the threshold
Reference method Alternative method
h gt 1 Lab F Level 2 Lab F Level 2
h gt 5 Lab F Level 2
Lab B Levels 1 and 2
Lab A Level 2
Lab D Level 3
Lab F Level 2
Lab L Level 2
kgt 1 Lab F Level 3
k gt 5 Lab F Level 2 Lab C Level 2
Lab F Level 3
Lab L Levels 1 and 3
Lab M Level 1
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2638 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
The emphasized results appear (i) coherent with the targeted contamination
levels (ii) concordant between both methods
All the results were thus kept for the statistical analysis
Comparison of the trueness and precision characteristics of
the reference method and alternative methods
The statistical values are summarised hereafter
Level p
Reference method Alternative method
Repeatability ratio
Reproducibility ratio Median
Repeatability standard deviation
Reproducibility standard deviation
Median Repeatability
standard deviation
Reproducibility standard deviation
1 10 16519 02415 02425 16021 02045 02433 0847 1007
2 10 26551 00543 00659 26719 00914 01325 1682 2011
2 bis 10 26581 02607 02607 25951 01900 01953 0729 0749
3 10 18671 00378 00378 18671 00478 00611 1264 1617
Bias of the alternative method
In order to estimate the bias of the alternative method with regard to the reference method for every
level Dij and t are calculated according to the following equations
RefAlt ijYijYDij
Diffp
Dijit
)2(
)(median
If the t value is superior to 2 the alternative method is significantly biased
with regard to the reference method
The values are given in table 17
Table 17 ndash t (d) values obtained by level
Level Bias t (d) Conclusion
1 0068 114 Non significant bias
2 0021 082 Non significant bias
2 bis - 0037 035 Non significant bias
3 - 0037 043 Non significant bias
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2738 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
The statistical test concludes to a non significant bias for all the inoculation
levels The bias varies from ndash 0037 to 0068 log CFUml
Comparison of the repeatability standard deviations
If the ratio Srj Alt Srj Ref of the repeatability standard deviations of the
alternative method and the reference method is larger than 2 the precision
under repeatability conditions of the alternative method is considered to be
lower than that of the reference method If this ratio is smaller than 05 the
precision under repeatability conditions of the alternative method is
considered to be greater than that of the reference method
The ratio values are given in table 18
Table 18
Level
Repeatability standard deviation
Reference method Alternative method Alternative method
reference method ratio
1 0242 0205 0847
2 0054 0091 1682
2 bis 0261 0190 0729
3 0038 0048 1264
Level Repeatability limit
Reference method Alternative method
1 0676 0573
2 0152 0256
2 bis 0730 0532
3 0106 0134
The ratios of the repeatability standard deviations are below 2 in all cases
The precision under repeatability conditions of the alternative method is
equivalent to that of the reference method
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2838 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Comparison of the reproducibility standard deviations
If the ratio Srj Alt Srj Ref of the reproducibility standard deviations of the alternative method and the
reference method is larger than 2 the precision under reproducibility conditions of the alternative
method is considered to be lower than that of the reference method If this ratio is smaller than 05 the
precision under reproducibility conditions of the alternative method is considered to be greater than
that of the reference method
The ratio values are given in table 19
Table 19
Level
Reproducibility standard deviation
Reference method Alternative method Alternative method
reference method ratio
1 0242 0243 1007
2 0066 0133 2011
2 bis 0261 0195 0749
3 0038 0061 1617
Level Reproducibility limit
Reference method Alternative method
1 0676 0681
2 0185 0371
2 bis 0730 0547
3 0106 0171
The ratios of the reproducibility standard deviations are below or close to 2
The precision under reproducibility conditions of the alternative method is
equivalent to that of the reference method
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2938 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
214 Conclusion
Method comparison study
The Listeria PrecisTM enumeration method shows satisfying linearity and
relative accuracy
The alternative method shows satisfying inclusivity and exclusivity results
Two Listeria monocytogenes strains gave negative results with the
alternative method These strains grew weakly on the reference method
media (OampA Palcam and Oxford)
The limits of repeatability of the alternative method are similar to those of the
reference method
The bias between both methods are low between - 0133 Log CFUg and
- 0008 Log CFUg
The Listeria PrecisTM enumeration method offers the saving for confirmatory
tests
Positive results are obtained with the alternative method in 2-3 days (with
OBIS Mono test) or in 4-7 days (by the classical tests described in the
standardized method) against 4-7 days for the reference method
Inter-laboratory study
The bias between the reference and the alternative methods shows satisfying
and correct values
- level 1 (100 - 1 000 CFUg) bias = + 007 log CFUg
- level 2 (1 000 - 10 000 CFUg) bias = 002 log CFUg
- level 2 bis (1 000 - 10 000 CFUg) bias = - 004 log CFUg
- level 3 (10 000 - 100 000 CFUg) bias = - 004 log CFUg
The repeatability and the reproducibility of the alternative method look similar
to those of the reference method
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3038 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
22 Extension study (realised by ASEPT 2007)
An extension study was performed in 2007 for a new confirmatory test OBIS
MONO
150 Listeria monocytogenes strains and 100 non-target strains were isolated
onto TSAYE and BrillianceTM Listeria Agar plates All the Listeria
monocytogenes and the non-target strains gave the expected results
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3138 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Appendix 1 ndash Flow diagram of the Listeria PrecisTM method
x g sample + 9 x ml buffered peptone water
Revivification step for 1 h plusmn 5 min at 20degC plusmn 1degC
Streaking 1 ml onto 3 Brilliance Listeria Agar plates (mother suspension)
or 01 ml onto 1 Brilliance Listeria Agar plates (mother suspension and decimal dilutions)
Incubation for 48 h plusmn 3 h at 37degC plusmn 1degC
Confirmatory tests on at least one typical colony
- by the tests described in the ISO method
- by applying the OBIS mono test on an isolated colony
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3238 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Appendix 2 ndash ISO 11290-2 (1998) amp ISO 11290-2A1 (2004) Horizontal
method for the detection and enumeration of Listeria monocytogenes -
Part 2 enumeration of Listeria monocytogenes in foods
10 g cheese + 90 ml buffered peptone water or Half Fraser Broth (without supplements) (110 dilution)
Stomach 1 min 30 seconds
Keep at 20degC plusmn 2degC for 1 h plusmn 5 min
Decimal dilutions in 9 ml peptone salt tubes
Dilution 110
Dilution 1100
Dilution 11000
1 ml on 3 Chromogenic Listeria medium (ISO) plates (x 2) (-1)
01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-2)
01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-3)
01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-4)
Incubation at 37degC plusmn 1degC
24 h - 48 h
Numerate typical colonies at 24 h
(bluegreen surrounded by an opaque halo)
Numerate at 48 h
Streak typical colonies on TSYEA
(5 coloniesplate 2 successive dilutions)
Confirmation tests
Catalase
Gram stain
For confirmation of L monocytogenes
Haemolysis
Carbohydrate (rhamnose and xylose) utilisation
CAMP Test
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3338 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Appendix 3 ndash Mandelrsquos graphics
Laboratoire h Ref
Niveau 1 h Ref
Niveau 2 h Ref
Niveau 3 h Alt
Niveau 1 h Alt
Niveau 2 h Alt
Niveau 3 h5 h1 h5 h1
A -050 -015 083 062 -212 -039 198 299 -198 -299
B -261 -270 021 -077 -058 173 198 299 -198 -299
C -151 -031 -014 -015 -004 -005 198 299 -198 -299
D 038 040 -137 000 072 207 198 299 -198 -299
E -001 090 155 -052 056 -101 198 299 -198 -299
F -112 -321 -153 070 -325 023 198 299 -198 -299
G 042 -071 040 147 -009 176 198 299 -198 -299
H 169 046 -169 192 018 -083 198 299 -198 -299
L 009 -120 021 -077 -231 005 198 299 -198 -299
M 001 031 014 -032 004 -011 198 299 -198 -299
-300
-200
-100
000
100
200
300
A B C D E F G H L M
Vale
urs
h d
e M
an
del(
rob
uste
s)
Laboratoires
Meacutethode de reacutefeacuterence analyse robuste
h Ref Niveau 1
h Ref Niveau 2
h Ref Niveau 3
h 5=198
h 1=299
-300
-200
-100
000
100
200
300
A B C D E F G H L M
Vale
urs
de h
de M
an
del(
rob
uste
s)
Laboratoires
Meacutethode alternative analyse robuste
h Alt Niveau 1
h Alt Niveau 2
h Alt Niveau 2
h 1=299
h 5=198
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3438 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Laboratoire k Ref
Niveau 1 k Ref
Niveau 2 k Ref
Niveau 3 k Alt
Niveau 1 k Alt
Niveau 2 k Alt
Niveau 3 k1 k5
A 000 089 142 084 162 046 259 181
B 125 087 073 104 075 046 259 181
C 117 013 091 188 022 083 259 181
D 052 036 069 000 035 083 259 181
E 028 034 037 138 019 112 259 181
F 140 222 018 027 281 112 259 181
G 088 068 024 131 145 000 259 181
H 129 094 173 016 061 000 259 181
L 069 000 073 104 270 224 259 181
M 000 048 038 043 099 241 259 181
000
050
100
150
200
250
300
350
400
A B C D E F G H L M
Vale
urs
k d
e M
an
del(
rob
uste
s)
Laboratoires
Meacutethode de reacutefeacuterence analyse robuste
k Ref Niveau 1k Ref Niveau 2k Ref Niveau 3
k 1=259
k 5=181
000
050
100
150
200
250
300
A B C D E F G H L M
Vale
urs
k d
e M
an
del
(ro
bu
ste
s)
Laboratoires
Meacutethode alternative analyse robuste
k Alt Niveau 3
k Alt Niveau 1
k Alt Niveau 2
k 1=259
k 5=181
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3538 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Appendix 4 ndash Statistical results
Level 1
Nombre de laboratoires (p) 10
A 1602 1602 1602 000 0000 0000 -0495 0000 A 1845 1602 1724 017 0122 -0122 0621 0840 0122
B 1602 1176 1389 030 0213 -0213 -2613 1247 B 1602 1301 1452 021 0151 -0151 -0769 1041 0062
C 1699 1301 1500 028 0199 -0199 -1510 1165 C 1301 1845 1573 038 -0272 0272 -0148 1881 0073
D 1602 1778 1690 012 -0088 0088 0380 0516 D 1602 1602 1602 000 0000 0000 0000 0000 -0088
E 1699 1602 1651 007 0048 -0048 -0013 0284 E 1699 1301 1500 028 0199 -0199 -0522 1376 -0151
F 1778 1301 1540 034 0239 -0239 -1116 1397 F 1778 1699 1739 006 0040 -0040 0698 0274 0199
G 1845 1544 1695 021 0151 -0151 0425 0881 G 2079 1699 1889 027 0190 -0190 1467 1315 0194
H 2041 1602 1822 031 0220 -0220 1689 1286 H 2000 1954 1977 003 0023 -0023 1917 0158 0155
L 1544 1778 1661 017 -0117 0117 0092 0685 L 1301 1602 1452 021 -0151 0151 -0769 1041 -0210
M 1653 1653 1653 000 0000 0000 0013 0000 M 1477 1602 1540 009 -0062 0062 -0319 0432 -0114
m 165186 m 160206 mediane D 00678
Cn(2p) 1867 Cn(2p) 1867 Cp 16101
Cn(p) 16101 Cn(p) 16101 Qn(D) 0092926
n=2p n 20 n=2p n 20 Qdiff 01496169
f 11 f 11 t 114
l 55 l 55
n=p n 10 n=p n 10
f 6 f 6
l 15 l 15
Qn(2p) 0091465 Qn(2p) 007745
Qn(p) 0062469 Qn(p) 0121519
Qintra 017078 Qintra 014461
Qinter 010058 Qinter 019565
Sr(eacutecart-type de reacutepeacutetabiliteacute) 02415 Sr 02045
RSDr(coefficient de variation de la reacutepeacutetabiliteacute)1462 RSDr 1277
r(repeatability limit)) 0676 r 0573
SL(eacutecart-type interlaboratoire) -001905 SLlt0 SL 001737
SR(eacutecart-type de reproductibiliteacute) 02415 SR 02433
RSDR(coefficient de variation de la reproductibiliteacute)0146 RSDR 0152
R(limite de reproductibiliteacute)) 06762 R(limite de reproductibiliteacute))068123
h k h MoyenneLaboratoires
Niveau 1
si LaboratoiresDeviationMoyenne Dk DeviationsiAlt
Niveau 1
Ref
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3638 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Level 2
Nombre de laboratoires (p) 10
A 2681 2613 2647 005 0034 -0034 -0151 0891 A 2322 2531 2427 015 -0105 0105 -2119 1620 -0220
B 2544 2477 2511 005 0033 -0033 -2699 0872 B 2556 2653 2605 007 -0048 0048 -0581 0750 0094
C 2643 2633 2638 001 0005 -0005 -0311 0130 C 2681 2653 2667 002 0014 -0014 -0040 0217 0029
D 2690 2663 2676 002 0014 -0014 0400 0357 D 2778 2732 2755 003 0023 -0023 0721 0354 0079
E 2690 2716 2703 002 -0013 0013 0897 0336 E 2724 2748 2736 002 -0012 0012 0556 0185 0033
F 2568 2398 2483 012 0085 -0085 -3214 2217 F 2477 2114 2296 026 0182 -0182 -3255 2811 -0188
G 2643 2591 2617 004 0026 -0026 -0707 0682 G 2756 2568 2662 013 0094 -0094 -0085 1453 0045
H 2716 2643 2680 005 0036 -0036 0460 0945 H 2653 2732 2693 006 -0040 0040 0181 0613 0013
L 2591 2591 2591 000 0000 0000 -1196 0000 L 2230 2580 2405 025 -0175 0175 -2307 2704 -0186
M 2653 2690 2672 003 -0018 0018 0311 0481 M 2740 2613 2677 009 0064 -0064 0040 0987 0005
m 2655082 m 26719001 mediane D 00209
Cn(2p) 1867 Cn(2p) 1867 Cp 16101
Cn(p) 16101 Cn(p) 16101 Qn(D) 0039911
n=2p n 20 n=2p n 20 Qdiff 00642591
f 11 f 11 t 082
l 55 l 55
n=p n 10 n=p n 10
f 6 f 6
l 15 l 15
Qn(2p) 0020570 Qn(2p) 00345985
Qn(p) 00332437 Qn(p) 00718158
Qintra 003841 Qintra 006460
Qinter 005352 Qinter 011563
Sr(eacutecart-type de reacutepeacutetabiliteacute) 00543 Sr 00914
RSDr(coefficient de variation de la reacutepeacutetabiliteacute)205 RSDr 342
r(repeatability limit)) 0152 r 0256
SL(eacutecart-type interlaboratoire) 000139 SL 000920
SR(eacutecart-type de reproductibiliteacute) 00659 SR 01325
RSDR(coefficient de variation de la reproductibiliteacute)0025 RSDR 0050
R(limite de reproductibiliteacute)) 018446 R(limite de reproductibiliteacute))03708618
Laboratoires
Niveau 2
Moyenne si Laboratoires
Niveau 2
DRef Alt Moyenne si Deviation h Deviation h k k
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3738 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Level 2 bis
Nombre de laboratoires (p) 10
A 3000 2477 2739 037 0261 -0261 0832 1418 A 2602 2477 2540 009 0062 -0062 -0391 0465 -0199
B 2544 2813 2678 019 -0134 0134 0211 0729 B 2903 2778 2841 009 0062 -0062 1732 0465 0162
C 2477 2813 2645 024 -0168 0168 -0135 0911 C 2477 2699 2588 016 -0111 0111 -0049 0826 -0057
D 2398 2653 2526 018 -0128 0128 -1370 0692 D 2778 3000 2889 016 -0111 0111 2074 0826 0363
E 2875 2740 2808 010 0067 -0067 1547 0365 E 2602 2301 2452 021 0151 -0151 -1012 1120 -0356
F 2477 2544 2511 005 -0033 0033 -1525 0182 F 2778 2477 2628 021 0151 -0151 0230 1120 0117
G 2653 2740 2697 006 -0044 0044 0400 0236 G 2845 2845 2845 000 0000 0000 1764 0000 0148
H 2176 2813 2495 045 -0318 0318 -1692 1727 H 2477 2477 2477 000 0000 0000 -0832 0000 -0017
L 2813 2544 2678 019 0134 -0134 0211 0729 L 2903 2301 2602 043 0301 -0301 0049 2241 -0076
M 2602 2740 2671 010 -0069 0069 0135 0375 M 2903 2255 2579 046 0324 -0324 -0112 2411 -0092
m 2658114 m 259505 mediane D -00372
Cn(2p) 1867 Cn(2p) 1867 Cp 16101
Cn(p) 16101 Cn(p) 16101 Qn(D) 01656915
n=2p n 20 n=2p n 20 Qdiff 02667753
f 11 f 11 t 035
l 55 l 55
n=p n 10 n=p n 10
f 6 f 6
l 15 l 15
Qn(2p) 0098745 Qn(2p) 007195
Qn(p) 006007 Qn(p) 008805
Qintra 018437 Qintra 013435
Qinter 009672 Qinter 014176
Sr(eacutecart-type de reacutepeacutetabiliteacute) 02607 Sr 01900
RSDr(coefficient de variation de la reacutepeacutetabiliteacute)981 RSDr 732
r(repeatability limit)) 0730 r 0532
SL(eacutecart-type interlaboratoire) -002464 SLlt0 SL 000205
SR(eacutecart-type de reproductibiliteacute) 02607 SR 01953
RSDR(coefficient de variation de la reproductibiliteacute)0098 RSDR 0075
R(limite de reproductibiliteacute)) 072996 R(limite de reproductibiliteacute))054686
Laboratoires
Niveau 2 bis
Moyenne si Laboratoires
Niveau 2 bis
DRef Alt Moyenne si Deviation h Deviation h k k
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3838 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Level 3
Nombre de laboratoires (p) 10
A 3690 3663 3676 002 0014 -0014 -0311 0513 A 3690 3708 3699 001 -0009 0009 0354 0257 0022
B 3613 3672 3642 004 -0030 0030 -1851 1109 B 3708 3568 3638 010 0070 -0070 -0844 2061 -0005
C 3663 3732 3698 005 -0035 0035 0645 1302 C 3663 3699 3681 003 -0018 0018 0000 0536 -0017
D 3690 3690 3690 000 0000 0000 0311 0000 D 3643 3663 3653 001 -0010 0010 -0545 0286 -0037
E 3732 3653 3693 006 0040 -0040 0429 1480 E 3613 3732 3673 008 -0060 0060 -0162 1769 -0020
F 3643 3672 3658 002 -0014 0014 -1157 0536 F 3690 3771 3731 006 -0040 0040 0975 1193 0073
G 3699 3653 3676 003 0023 -0023 -0328 0856 G 3602 3643 3623 003 -0021 0021 -1141 0612 -0053
H 3623 3672 3648 003 -0024 0024 -1615 0913 H 3748 3681 3715 005 0033 -0033 0664 0990 0067
L 3724 3663 3694 004 0031 -0031 0461 1150 L 3748 3771 3760 002 -0011 0011 1544 0335 0066
M 3690 3690 3690 000 0000 0000 0311 0000 M 3806 3681 3744 009 0062 -0062 1234 1848 0054
m 368334 m 368086 mediane D 00089
Cn(2p) 1867 Cn(2p) 1867 Cp 16101
Cn(p) 16101 Cn(p) 16101 Qn(D) 00325359
n=2p n 20 n=2p n 20 Qdiff 00523852
f 11 f 11 t 043
l 55 l 55
n=p n 10 n=p n 10
f 6 f 6
l 15 l 15
Qn(2p) 0014323 Qn(2p) 001811
Qn(p) 0013719 Qn(p) 003164
Qintra 002674 Qintra 003381
Qinter 002209 Qinter 005094
Sr(eacutecart-type de reacutepeacutetabiliteacute) 00378 Sr 00478
RSDr(coefficient de variation de la reacutepeacutetabiliteacute)103 RSDr 130
r(repeatability limit)) 0106 r 0134
SL(eacutecart-type interlaboratoire) -000023 SLlt0 SL 000145
SR(eacutecart-type de reproductibiliteacute) 00378 SR 00611
RSDR(coefficient de variation de la reproductibiliteacute)0010 RSDR 0017
R(limite de reproductibiliteacute)) 0105894 R(limite de reproductibiliteacute))017119
Ref Alt DLaboratoires
Niveau 3
Moyenne si Deviation h k Laboratoires
Niveau 3
Moyenne si Deviation h k
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2338 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Table 13 - Sample temperature on receipt
Laboratories Receipt
day
Hour
receipt
Measured temperature
at receipt (degC)
Measured temperature by the
temperature probe (degC)
A
B
C
D
E
F
G
H
I
J
K
L
M
30052006
10h45
10h15
11h15
10h00
09h30
8h30
11h50
10h00
08h30
14h45
08h30
09h45
08h45
82
55
45
63
60
62
81
38
48
-
14
67
76
03
03
-13
03
04
-14
05
04
01
05
-13
04
Conclusion
The temperatures during the transport were correct for all the labs except for
Lab J which didnrsquot provide the information
All the labs received their packages with several broken flasks
Three labs (B D and L) received some damaged samples but proceeded to
analyses
All the labs started the analyses at Day 1 except Lab J
2133 Results
Aerobic mesophilic flora
The count of aerobic mesophilic flora varied from 94 102 CFUml to
33 103 CFUml
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2438 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Expert lab results
The results obtained by the expert lab are the following
Table 14
Target inoculation level (CFUml)
Reference method ISO 11290-2
Alternative method Listeria Precis Enumeration
Rep 1 Rep 2 Rep 1 Rep 2
lt 10 lt 10 lt 10 lt 10 lt10
50 50 (Ne) 60 (Ne) 50 (Ne) 30 (Ne)
500 470 390 490 500
5 000 4 700 5 100 5 900 3 400
Ne estimated number
The obtained results are in agreement with those expected
Collaborator Labs results
A synthesis of the results obtained by the collaborator labs is provided
Table 15 (CFUml) and Table 16 (log CFUml)
Table 15 ndash Synthesis of results obtained by the alternative
and the reference methods (CFUml)
Labs Level 1 Level 2 Level 2 bis Level 3
Reference method
Alternative method
Reference method
Alternative method
Reference method
Alternative method
Reference method
Alternative method
A 40 40 70 40 480 410 210 340 1000 300 400 300 4900 4600 4900 5100
B 40 15 40 20 350 300 360 450 350 650 800 600 4100 4700 5100 3700
C 50 20 20 70 440 430 480 450 300 650 300 500 4600 5400 4600 5000
D 40 60 40 40 490 460 600 540 250 450 600 1000 4900 4900 4400 4600
E 50 40 50 20 490 520 530 560 750 550 400 200 5400 4500 4100 5400
F 60 20 60 50 370 250 300 130 300 350 600 300 4400 4700 4900 5900
G 70 35 120 50 440 390 570 370 450 550 700 700 5000 4500 4000 4400
H 110 40 100 90 520 440 450 540 150 650 300 300 4200 4700 5600 4800
L 35 60 20 40 390 390 170 380 650 350 800 200 5300 4600 5600 5900
M 45 45 30 40 450 490 550 410 400 550 800 180 4900 4900 6400 4800
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2538 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Table 16 ndash Synthesis of results obtained by the alternative
and the reference methods (log CFUml)
Labs Level 1 Level 2 Level 2 bis Level 3
Reference method
Alternative method
Reference method
Alternative method
Reference method
Alternative method
Reference method
Alternative method
A 1602 1602 1845 1602 2681 2613 2322 2531 3000 2477 2602 2477 3690 3663 3690 3708
B 1602 1176 1602 1301 2544 2477 2556 2653 2544 2813 2903 2778 3613 3672 3708 3568
C 1699 1301 1301 1845 2643 2633 2681 2653 2477 2813 2477 2699 3663 3732 3663 3699
D 1602 1778 1602 1602 2690 2663 2778 2732 2398 2653 2778 3000 3690 3690 3643 3663
E 1699 1602 1699 1301 2690 2716 2724 2748 2875 2740 2602 2301 3732 3653 3613 3732
F 1778 1301 1778 1699 2568 2398 2477 2114 2477 2544 2778 2477 3643 3672 3690 3771
G 1845 1544 2079 1699 2643 2591 2756 2568 2653 2740 2845 2845 3699 3653 3602 3643
H 2041 1602 2000 1954 2716 2643 2653 2732 2176 2813 2477 2477 3623 3672 3748 3681
L 1544 1778 1301 1602 2591 2591 2230 2580 2813 2544 2903 2301 3724 3663 3748 3771
M 1653 1653 1477 1602 2653 2690 2740 2613 2602 2740 2903 2255 3690 3690 3806 3681
Level 2 results obtained taking account the 3 plates inoculated with the mother-suspension
Level 2 bis results obtained taking account the plates inoculated with 01 ml of the mother-suspension
Scrutiny of the measurement results for consistency
In order to identify the measurement results or laboratories that could be
inconsistent two graphical consistency techniques were realised Mandelrsquos
h-ank-statistics Mandel indicators h and k at 5 significance highlighted
some possible inconsistent data (See tables and graphics in Appendix 3 and
Appendix 4)
Mandelrsquos values Number of values observed over the threshold
Reference method Alternative method
h gt 1 Lab F Level 2 Lab F Level 2
h gt 5 Lab F Level 2
Lab B Levels 1 and 2
Lab A Level 2
Lab D Level 3
Lab F Level 2
Lab L Level 2
kgt 1 Lab F Level 3
k gt 5 Lab F Level 2 Lab C Level 2
Lab F Level 3
Lab L Levels 1 and 3
Lab M Level 1
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2638 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
The emphasized results appear (i) coherent with the targeted contamination
levels (ii) concordant between both methods
All the results were thus kept for the statistical analysis
Comparison of the trueness and precision characteristics of
the reference method and alternative methods
The statistical values are summarised hereafter
Level p
Reference method Alternative method
Repeatability ratio
Reproducibility ratio Median
Repeatability standard deviation
Reproducibility standard deviation
Median Repeatability
standard deviation
Reproducibility standard deviation
1 10 16519 02415 02425 16021 02045 02433 0847 1007
2 10 26551 00543 00659 26719 00914 01325 1682 2011
2 bis 10 26581 02607 02607 25951 01900 01953 0729 0749
3 10 18671 00378 00378 18671 00478 00611 1264 1617
Bias of the alternative method
In order to estimate the bias of the alternative method with regard to the reference method for every
level Dij and t are calculated according to the following equations
RefAlt ijYijYDij
Diffp
Dijit
)2(
)(median
If the t value is superior to 2 the alternative method is significantly biased
with regard to the reference method
The values are given in table 17
Table 17 ndash t (d) values obtained by level
Level Bias t (d) Conclusion
1 0068 114 Non significant bias
2 0021 082 Non significant bias
2 bis - 0037 035 Non significant bias
3 - 0037 043 Non significant bias
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2738 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
The statistical test concludes to a non significant bias for all the inoculation
levels The bias varies from ndash 0037 to 0068 log CFUml
Comparison of the repeatability standard deviations
If the ratio Srj Alt Srj Ref of the repeatability standard deviations of the
alternative method and the reference method is larger than 2 the precision
under repeatability conditions of the alternative method is considered to be
lower than that of the reference method If this ratio is smaller than 05 the
precision under repeatability conditions of the alternative method is
considered to be greater than that of the reference method
The ratio values are given in table 18
Table 18
Level
Repeatability standard deviation
Reference method Alternative method Alternative method
reference method ratio
1 0242 0205 0847
2 0054 0091 1682
2 bis 0261 0190 0729
3 0038 0048 1264
Level Repeatability limit
Reference method Alternative method
1 0676 0573
2 0152 0256
2 bis 0730 0532
3 0106 0134
The ratios of the repeatability standard deviations are below 2 in all cases
The precision under repeatability conditions of the alternative method is
equivalent to that of the reference method
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2838 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Comparison of the reproducibility standard deviations
If the ratio Srj Alt Srj Ref of the reproducibility standard deviations of the alternative method and the
reference method is larger than 2 the precision under reproducibility conditions of the alternative
method is considered to be lower than that of the reference method If this ratio is smaller than 05 the
precision under reproducibility conditions of the alternative method is considered to be greater than
that of the reference method
The ratio values are given in table 19
Table 19
Level
Reproducibility standard deviation
Reference method Alternative method Alternative method
reference method ratio
1 0242 0243 1007
2 0066 0133 2011
2 bis 0261 0195 0749
3 0038 0061 1617
Level Reproducibility limit
Reference method Alternative method
1 0676 0681
2 0185 0371
2 bis 0730 0547
3 0106 0171
The ratios of the reproducibility standard deviations are below or close to 2
The precision under reproducibility conditions of the alternative method is
equivalent to that of the reference method
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2938 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
214 Conclusion
Method comparison study
The Listeria PrecisTM enumeration method shows satisfying linearity and
relative accuracy
The alternative method shows satisfying inclusivity and exclusivity results
Two Listeria monocytogenes strains gave negative results with the
alternative method These strains grew weakly on the reference method
media (OampA Palcam and Oxford)
The limits of repeatability of the alternative method are similar to those of the
reference method
The bias between both methods are low between - 0133 Log CFUg and
- 0008 Log CFUg
The Listeria PrecisTM enumeration method offers the saving for confirmatory
tests
Positive results are obtained with the alternative method in 2-3 days (with
OBIS Mono test) or in 4-7 days (by the classical tests described in the
standardized method) against 4-7 days for the reference method
Inter-laboratory study
The bias between the reference and the alternative methods shows satisfying
and correct values
- level 1 (100 - 1 000 CFUg) bias = + 007 log CFUg
- level 2 (1 000 - 10 000 CFUg) bias = 002 log CFUg
- level 2 bis (1 000 - 10 000 CFUg) bias = - 004 log CFUg
- level 3 (10 000 - 100 000 CFUg) bias = - 004 log CFUg
The repeatability and the reproducibility of the alternative method look similar
to those of the reference method
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3038 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
22 Extension study (realised by ASEPT 2007)
An extension study was performed in 2007 for a new confirmatory test OBIS
MONO
150 Listeria monocytogenes strains and 100 non-target strains were isolated
onto TSAYE and BrillianceTM Listeria Agar plates All the Listeria
monocytogenes and the non-target strains gave the expected results
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3138 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Appendix 1 ndash Flow diagram of the Listeria PrecisTM method
x g sample + 9 x ml buffered peptone water
Revivification step for 1 h plusmn 5 min at 20degC plusmn 1degC
Streaking 1 ml onto 3 Brilliance Listeria Agar plates (mother suspension)
or 01 ml onto 1 Brilliance Listeria Agar plates (mother suspension and decimal dilutions)
Incubation for 48 h plusmn 3 h at 37degC plusmn 1degC
Confirmatory tests on at least one typical colony
- by the tests described in the ISO method
- by applying the OBIS mono test on an isolated colony
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3238 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Appendix 2 ndash ISO 11290-2 (1998) amp ISO 11290-2A1 (2004) Horizontal
method for the detection and enumeration of Listeria monocytogenes -
Part 2 enumeration of Listeria monocytogenes in foods
10 g cheese + 90 ml buffered peptone water or Half Fraser Broth (without supplements) (110 dilution)
Stomach 1 min 30 seconds
Keep at 20degC plusmn 2degC for 1 h plusmn 5 min
Decimal dilutions in 9 ml peptone salt tubes
Dilution 110
Dilution 1100
Dilution 11000
1 ml on 3 Chromogenic Listeria medium (ISO) plates (x 2) (-1)
01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-2)
01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-3)
01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-4)
Incubation at 37degC plusmn 1degC
24 h - 48 h
Numerate typical colonies at 24 h
(bluegreen surrounded by an opaque halo)
Numerate at 48 h
Streak typical colonies on TSYEA
(5 coloniesplate 2 successive dilutions)
Confirmation tests
Catalase
Gram stain
For confirmation of L monocytogenes
Haemolysis
Carbohydrate (rhamnose and xylose) utilisation
CAMP Test
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3338 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Appendix 3 ndash Mandelrsquos graphics
Laboratoire h Ref
Niveau 1 h Ref
Niveau 2 h Ref
Niveau 3 h Alt
Niveau 1 h Alt
Niveau 2 h Alt
Niveau 3 h5 h1 h5 h1
A -050 -015 083 062 -212 -039 198 299 -198 -299
B -261 -270 021 -077 -058 173 198 299 -198 -299
C -151 -031 -014 -015 -004 -005 198 299 -198 -299
D 038 040 -137 000 072 207 198 299 -198 -299
E -001 090 155 -052 056 -101 198 299 -198 -299
F -112 -321 -153 070 -325 023 198 299 -198 -299
G 042 -071 040 147 -009 176 198 299 -198 -299
H 169 046 -169 192 018 -083 198 299 -198 -299
L 009 -120 021 -077 -231 005 198 299 -198 -299
M 001 031 014 -032 004 -011 198 299 -198 -299
-300
-200
-100
000
100
200
300
A B C D E F G H L M
Vale
urs
h d
e M
an
del(
rob
uste
s)
Laboratoires
Meacutethode de reacutefeacuterence analyse robuste
h Ref Niveau 1
h Ref Niveau 2
h Ref Niveau 3
h 5=198
h 1=299
-300
-200
-100
000
100
200
300
A B C D E F G H L M
Vale
urs
de h
de M
an
del(
rob
uste
s)
Laboratoires
Meacutethode alternative analyse robuste
h Alt Niveau 1
h Alt Niveau 2
h Alt Niveau 2
h 1=299
h 5=198
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3438 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Laboratoire k Ref
Niveau 1 k Ref
Niveau 2 k Ref
Niveau 3 k Alt
Niveau 1 k Alt
Niveau 2 k Alt
Niveau 3 k1 k5
A 000 089 142 084 162 046 259 181
B 125 087 073 104 075 046 259 181
C 117 013 091 188 022 083 259 181
D 052 036 069 000 035 083 259 181
E 028 034 037 138 019 112 259 181
F 140 222 018 027 281 112 259 181
G 088 068 024 131 145 000 259 181
H 129 094 173 016 061 000 259 181
L 069 000 073 104 270 224 259 181
M 000 048 038 043 099 241 259 181
000
050
100
150
200
250
300
350
400
A B C D E F G H L M
Vale
urs
k d
e M
an
del(
rob
uste
s)
Laboratoires
Meacutethode de reacutefeacuterence analyse robuste
k Ref Niveau 1k Ref Niveau 2k Ref Niveau 3
k 1=259
k 5=181
000
050
100
150
200
250
300
A B C D E F G H L M
Vale
urs
k d
e M
an
del
(ro
bu
ste
s)
Laboratoires
Meacutethode alternative analyse robuste
k Alt Niveau 3
k Alt Niveau 1
k Alt Niveau 2
k 1=259
k 5=181
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3538 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Appendix 4 ndash Statistical results
Level 1
Nombre de laboratoires (p) 10
A 1602 1602 1602 000 0000 0000 -0495 0000 A 1845 1602 1724 017 0122 -0122 0621 0840 0122
B 1602 1176 1389 030 0213 -0213 -2613 1247 B 1602 1301 1452 021 0151 -0151 -0769 1041 0062
C 1699 1301 1500 028 0199 -0199 -1510 1165 C 1301 1845 1573 038 -0272 0272 -0148 1881 0073
D 1602 1778 1690 012 -0088 0088 0380 0516 D 1602 1602 1602 000 0000 0000 0000 0000 -0088
E 1699 1602 1651 007 0048 -0048 -0013 0284 E 1699 1301 1500 028 0199 -0199 -0522 1376 -0151
F 1778 1301 1540 034 0239 -0239 -1116 1397 F 1778 1699 1739 006 0040 -0040 0698 0274 0199
G 1845 1544 1695 021 0151 -0151 0425 0881 G 2079 1699 1889 027 0190 -0190 1467 1315 0194
H 2041 1602 1822 031 0220 -0220 1689 1286 H 2000 1954 1977 003 0023 -0023 1917 0158 0155
L 1544 1778 1661 017 -0117 0117 0092 0685 L 1301 1602 1452 021 -0151 0151 -0769 1041 -0210
M 1653 1653 1653 000 0000 0000 0013 0000 M 1477 1602 1540 009 -0062 0062 -0319 0432 -0114
m 165186 m 160206 mediane D 00678
Cn(2p) 1867 Cn(2p) 1867 Cp 16101
Cn(p) 16101 Cn(p) 16101 Qn(D) 0092926
n=2p n 20 n=2p n 20 Qdiff 01496169
f 11 f 11 t 114
l 55 l 55
n=p n 10 n=p n 10
f 6 f 6
l 15 l 15
Qn(2p) 0091465 Qn(2p) 007745
Qn(p) 0062469 Qn(p) 0121519
Qintra 017078 Qintra 014461
Qinter 010058 Qinter 019565
Sr(eacutecart-type de reacutepeacutetabiliteacute) 02415 Sr 02045
RSDr(coefficient de variation de la reacutepeacutetabiliteacute)1462 RSDr 1277
r(repeatability limit)) 0676 r 0573
SL(eacutecart-type interlaboratoire) -001905 SLlt0 SL 001737
SR(eacutecart-type de reproductibiliteacute) 02415 SR 02433
RSDR(coefficient de variation de la reproductibiliteacute)0146 RSDR 0152
R(limite de reproductibiliteacute)) 06762 R(limite de reproductibiliteacute))068123
h k h MoyenneLaboratoires
Niveau 1
si LaboratoiresDeviationMoyenne Dk DeviationsiAlt
Niveau 1
Ref
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3638 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Level 2
Nombre de laboratoires (p) 10
A 2681 2613 2647 005 0034 -0034 -0151 0891 A 2322 2531 2427 015 -0105 0105 -2119 1620 -0220
B 2544 2477 2511 005 0033 -0033 -2699 0872 B 2556 2653 2605 007 -0048 0048 -0581 0750 0094
C 2643 2633 2638 001 0005 -0005 -0311 0130 C 2681 2653 2667 002 0014 -0014 -0040 0217 0029
D 2690 2663 2676 002 0014 -0014 0400 0357 D 2778 2732 2755 003 0023 -0023 0721 0354 0079
E 2690 2716 2703 002 -0013 0013 0897 0336 E 2724 2748 2736 002 -0012 0012 0556 0185 0033
F 2568 2398 2483 012 0085 -0085 -3214 2217 F 2477 2114 2296 026 0182 -0182 -3255 2811 -0188
G 2643 2591 2617 004 0026 -0026 -0707 0682 G 2756 2568 2662 013 0094 -0094 -0085 1453 0045
H 2716 2643 2680 005 0036 -0036 0460 0945 H 2653 2732 2693 006 -0040 0040 0181 0613 0013
L 2591 2591 2591 000 0000 0000 -1196 0000 L 2230 2580 2405 025 -0175 0175 -2307 2704 -0186
M 2653 2690 2672 003 -0018 0018 0311 0481 M 2740 2613 2677 009 0064 -0064 0040 0987 0005
m 2655082 m 26719001 mediane D 00209
Cn(2p) 1867 Cn(2p) 1867 Cp 16101
Cn(p) 16101 Cn(p) 16101 Qn(D) 0039911
n=2p n 20 n=2p n 20 Qdiff 00642591
f 11 f 11 t 082
l 55 l 55
n=p n 10 n=p n 10
f 6 f 6
l 15 l 15
Qn(2p) 0020570 Qn(2p) 00345985
Qn(p) 00332437 Qn(p) 00718158
Qintra 003841 Qintra 006460
Qinter 005352 Qinter 011563
Sr(eacutecart-type de reacutepeacutetabiliteacute) 00543 Sr 00914
RSDr(coefficient de variation de la reacutepeacutetabiliteacute)205 RSDr 342
r(repeatability limit)) 0152 r 0256
SL(eacutecart-type interlaboratoire) 000139 SL 000920
SR(eacutecart-type de reproductibiliteacute) 00659 SR 01325
RSDR(coefficient de variation de la reproductibiliteacute)0025 RSDR 0050
R(limite de reproductibiliteacute)) 018446 R(limite de reproductibiliteacute))03708618
Laboratoires
Niveau 2
Moyenne si Laboratoires
Niveau 2
DRef Alt Moyenne si Deviation h Deviation h k k
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3738 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Level 2 bis
Nombre de laboratoires (p) 10
A 3000 2477 2739 037 0261 -0261 0832 1418 A 2602 2477 2540 009 0062 -0062 -0391 0465 -0199
B 2544 2813 2678 019 -0134 0134 0211 0729 B 2903 2778 2841 009 0062 -0062 1732 0465 0162
C 2477 2813 2645 024 -0168 0168 -0135 0911 C 2477 2699 2588 016 -0111 0111 -0049 0826 -0057
D 2398 2653 2526 018 -0128 0128 -1370 0692 D 2778 3000 2889 016 -0111 0111 2074 0826 0363
E 2875 2740 2808 010 0067 -0067 1547 0365 E 2602 2301 2452 021 0151 -0151 -1012 1120 -0356
F 2477 2544 2511 005 -0033 0033 -1525 0182 F 2778 2477 2628 021 0151 -0151 0230 1120 0117
G 2653 2740 2697 006 -0044 0044 0400 0236 G 2845 2845 2845 000 0000 0000 1764 0000 0148
H 2176 2813 2495 045 -0318 0318 -1692 1727 H 2477 2477 2477 000 0000 0000 -0832 0000 -0017
L 2813 2544 2678 019 0134 -0134 0211 0729 L 2903 2301 2602 043 0301 -0301 0049 2241 -0076
M 2602 2740 2671 010 -0069 0069 0135 0375 M 2903 2255 2579 046 0324 -0324 -0112 2411 -0092
m 2658114 m 259505 mediane D -00372
Cn(2p) 1867 Cn(2p) 1867 Cp 16101
Cn(p) 16101 Cn(p) 16101 Qn(D) 01656915
n=2p n 20 n=2p n 20 Qdiff 02667753
f 11 f 11 t 035
l 55 l 55
n=p n 10 n=p n 10
f 6 f 6
l 15 l 15
Qn(2p) 0098745 Qn(2p) 007195
Qn(p) 006007 Qn(p) 008805
Qintra 018437 Qintra 013435
Qinter 009672 Qinter 014176
Sr(eacutecart-type de reacutepeacutetabiliteacute) 02607 Sr 01900
RSDr(coefficient de variation de la reacutepeacutetabiliteacute)981 RSDr 732
r(repeatability limit)) 0730 r 0532
SL(eacutecart-type interlaboratoire) -002464 SLlt0 SL 000205
SR(eacutecart-type de reproductibiliteacute) 02607 SR 01953
RSDR(coefficient de variation de la reproductibiliteacute)0098 RSDR 0075
R(limite de reproductibiliteacute)) 072996 R(limite de reproductibiliteacute))054686
Laboratoires
Niveau 2 bis
Moyenne si Laboratoires
Niveau 2 bis
DRef Alt Moyenne si Deviation h Deviation h k k
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3838 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Level 3
Nombre de laboratoires (p) 10
A 3690 3663 3676 002 0014 -0014 -0311 0513 A 3690 3708 3699 001 -0009 0009 0354 0257 0022
B 3613 3672 3642 004 -0030 0030 -1851 1109 B 3708 3568 3638 010 0070 -0070 -0844 2061 -0005
C 3663 3732 3698 005 -0035 0035 0645 1302 C 3663 3699 3681 003 -0018 0018 0000 0536 -0017
D 3690 3690 3690 000 0000 0000 0311 0000 D 3643 3663 3653 001 -0010 0010 -0545 0286 -0037
E 3732 3653 3693 006 0040 -0040 0429 1480 E 3613 3732 3673 008 -0060 0060 -0162 1769 -0020
F 3643 3672 3658 002 -0014 0014 -1157 0536 F 3690 3771 3731 006 -0040 0040 0975 1193 0073
G 3699 3653 3676 003 0023 -0023 -0328 0856 G 3602 3643 3623 003 -0021 0021 -1141 0612 -0053
H 3623 3672 3648 003 -0024 0024 -1615 0913 H 3748 3681 3715 005 0033 -0033 0664 0990 0067
L 3724 3663 3694 004 0031 -0031 0461 1150 L 3748 3771 3760 002 -0011 0011 1544 0335 0066
M 3690 3690 3690 000 0000 0000 0311 0000 M 3806 3681 3744 009 0062 -0062 1234 1848 0054
m 368334 m 368086 mediane D 00089
Cn(2p) 1867 Cn(2p) 1867 Cp 16101
Cn(p) 16101 Cn(p) 16101 Qn(D) 00325359
n=2p n 20 n=2p n 20 Qdiff 00523852
f 11 f 11 t 043
l 55 l 55
n=p n 10 n=p n 10
f 6 f 6
l 15 l 15
Qn(2p) 0014323 Qn(2p) 001811
Qn(p) 0013719 Qn(p) 003164
Qintra 002674 Qintra 003381
Qinter 002209 Qinter 005094
Sr(eacutecart-type de reacutepeacutetabiliteacute) 00378 Sr 00478
RSDr(coefficient de variation de la reacutepeacutetabiliteacute)103 RSDr 130
r(repeatability limit)) 0106 r 0134
SL(eacutecart-type interlaboratoire) -000023 SLlt0 SL 000145
SR(eacutecart-type de reproductibiliteacute) 00378 SR 00611
RSDR(coefficient de variation de la reproductibiliteacute)0010 RSDR 0017
R(limite de reproductibiliteacute)) 0105894 R(limite de reproductibiliteacute))017119
Ref Alt DLaboratoires
Niveau 3
Moyenne si Deviation h k Laboratoires
Niveau 3
Moyenne si Deviation h k
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2438 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Expert lab results
The results obtained by the expert lab are the following
Table 14
Target inoculation level (CFUml)
Reference method ISO 11290-2
Alternative method Listeria Precis Enumeration
Rep 1 Rep 2 Rep 1 Rep 2
lt 10 lt 10 lt 10 lt 10 lt10
50 50 (Ne) 60 (Ne) 50 (Ne) 30 (Ne)
500 470 390 490 500
5 000 4 700 5 100 5 900 3 400
Ne estimated number
The obtained results are in agreement with those expected
Collaborator Labs results
A synthesis of the results obtained by the collaborator labs is provided
Table 15 (CFUml) and Table 16 (log CFUml)
Table 15 ndash Synthesis of results obtained by the alternative
and the reference methods (CFUml)
Labs Level 1 Level 2 Level 2 bis Level 3
Reference method
Alternative method
Reference method
Alternative method
Reference method
Alternative method
Reference method
Alternative method
A 40 40 70 40 480 410 210 340 1000 300 400 300 4900 4600 4900 5100
B 40 15 40 20 350 300 360 450 350 650 800 600 4100 4700 5100 3700
C 50 20 20 70 440 430 480 450 300 650 300 500 4600 5400 4600 5000
D 40 60 40 40 490 460 600 540 250 450 600 1000 4900 4900 4400 4600
E 50 40 50 20 490 520 530 560 750 550 400 200 5400 4500 4100 5400
F 60 20 60 50 370 250 300 130 300 350 600 300 4400 4700 4900 5900
G 70 35 120 50 440 390 570 370 450 550 700 700 5000 4500 4000 4400
H 110 40 100 90 520 440 450 540 150 650 300 300 4200 4700 5600 4800
L 35 60 20 40 390 390 170 380 650 350 800 200 5300 4600 5600 5900
M 45 45 30 40 450 490 550 410 400 550 800 180 4900 4900 6400 4800
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2538 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Table 16 ndash Synthesis of results obtained by the alternative
and the reference methods (log CFUml)
Labs Level 1 Level 2 Level 2 bis Level 3
Reference method
Alternative method
Reference method
Alternative method
Reference method
Alternative method
Reference method
Alternative method
A 1602 1602 1845 1602 2681 2613 2322 2531 3000 2477 2602 2477 3690 3663 3690 3708
B 1602 1176 1602 1301 2544 2477 2556 2653 2544 2813 2903 2778 3613 3672 3708 3568
C 1699 1301 1301 1845 2643 2633 2681 2653 2477 2813 2477 2699 3663 3732 3663 3699
D 1602 1778 1602 1602 2690 2663 2778 2732 2398 2653 2778 3000 3690 3690 3643 3663
E 1699 1602 1699 1301 2690 2716 2724 2748 2875 2740 2602 2301 3732 3653 3613 3732
F 1778 1301 1778 1699 2568 2398 2477 2114 2477 2544 2778 2477 3643 3672 3690 3771
G 1845 1544 2079 1699 2643 2591 2756 2568 2653 2740 2845 2845 3699 3653 3602 3643
H 2041 1602 2000 1954 2716 2643 2653 2732 2176 2813 2477 2477 3623 3672 3748 3681
L 1544 1778 1301 1602 2591 2591 2230 2580 2813 2544 2903 2301 3724 3663 3748 3771
M 1653 1653 1477 1602 2653 2690 2740 2613 2602 2740 2903 2255 3690 3690 3806 3681
Level 2 results obtained taking account the 3 plates inoculated with the mother-suspension
Level 2 bis results obtained taking account the plates inoculated with 01 ml of the mother-suspension
Scrutiny of the measurement results for consistency
In order to identify the measurement results or laboratories that could be
inconsistent two graphical consistency techniques were realised Mandelrsquos
h-ank-statistics Mandel indicators h and k at 5 significance highlighted
some possible inconsistent data (See tables and graphics in Appendix 3 and
Appendix 4)
Mandelrsquos values Number of values observed over the threshold
Reference method Alternative method
h gt 1 Lab F Level 2 Lab F Level 2
h gt 5 Lab F Level 2
Lab B Levels 1 and 2
Lab A Level 2
Lab D Level 3
Lab F Level 2
Lab L Level 2
kgt 1 Lab F Level 3
k gt 5 Lab F Level 2 Lab C Level 2
Lab F Level 3
Lab L Levels 1 and 3
Lab M Level 1
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2638 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
The emphasized results appear (i) coherent with the targeted contamination
levels (ii) concordant between both methods
All the results were thus kept for the statistical analysis
Comparison of the trueness and precision characteristics of
the reference method and alternative methods
The statistical values are summarised hereafter
Level p
Reference method Alternative method
Repeatability ratio
Reproducibility ratio Median
Repeatability standard deviation
Reproducibility standard deviation
Median Repeatability
standard deviation
Reproducibility standard deviation
1 10 16519 02415 02425 16021 02045 02433 0847 1007
2 10 26551 00543 00659 26719 00914 01325 1682 2011
2 bis 10 26581 02607 02607 25951 01900 01953 0729 0749
3 10 18671 00378 00378 18671 00478 00611 1264 1617
Bias of the alternative method
In order to estimate the bias of the alternative method with regard to the reference method for every
level Dij and t are calculated according to the following equations
RefAlt ijYijYDij
Diffp
Dijit
)2(
)(median
If the t value is superior to 2 the alternative method is significantly biased
with regard to the reference method
The values are given in table 17
Table 17 ndash t (d) values obtained by level
Level Bias t (d) Conclusion
1 0068 114 Non significant bias
2 0021 082 Non significant bias
2 bis - 0037 035 Non significant bias
3 - 0037 043 Non significant bias
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2738 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
The statistical test concludes to a non significant bias for all the inoculation
levels The bias varies from ndash 0037 to 0068 log CFUml
Comparison of the repeatability standard deviations
If the ratio Srj Alt Srj Ref of the repeatability standard deviations of the
alternative method and the reference method is larger than 2 the precision
under repeatability conditions of the alternative method is considered to be
lower than that of the reference method If this ratio is smaller than 05 the
precision under repeatability conditions of the alternative method is
considered to be greater than that of the reference method
The ratio values are given in table 18
Table 18
Level
Repeatability standard deviation
Reference method Alternative method Alternative method
reference method ratio
1 0242 0205 0847
2 0054 0091 1682
2 bis 0261 0190 0729
3 0038 0048 1264
Level Repeatability limit
Reference method Alternative method
1 0676 0573
2 0152 0256
2 bis 0730 0532
3 0106 0134
The ratios of the repeatability standard deviations are below 2 in all cases
The precision under repeatability conditions of the alternative method is
equivalent to that of the reference method
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2838 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Comparison of the reproducibility standard deviations
If the ratio Srj Alt Srj Ref of the reproducibility standard deviations of the alternative method and the
reference method is larger than 2 the precision under reproducibility conditions of the alternative
method is considered to be lower than that of the reference method If this ratio is smaller than 05 the
precision under reproducibility conditions of the alternative method is considered to be greater than
that of the reference method
The ratio values are given in table 19
Table 19
Level
Reproducibility standard deviation
Reference method Alternative method Alternative method
reference method ratio
1 0242 0243 1007
2 0066 0133 2011
2 bis 0261 0195 0749
3 0038 0061 1617
Level Reproducibility limit
Reference method Alternative method
1 0676 0681
2 0185 0371
2 bis 0730 0547
3 0106 0171
The ratios of the reproducibility standard deviations are below or close to 2
The precision under reproducibility conditions of the alternative method is
equivalent to that of the reference method
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2938 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
214 Conclusion
Method comparison study
The Listeria PrecisTM enumeration method shows satisfying linearity and
relative accuracy
The alternative method shows satisfying inclusivity and exclusivity results
Two Listeria monocytogenes strains gave negative results with the
alternative method These strains grew weakly on the reference method
media (OampA Palcam and Oxford)
The limits of repeatability of the alternative method are similar to those of the
reference method
The bias between both methods are low between - 0133 Log CFUg and
- 0008 Log CFUg
The Listeria PrecisTM enumeration method offers the saving for confirmatory
tests
Positive results are obtained with the alternative method in 2-3 days (with
OBIS Mono test) or in 4-7 days (by the classical tests described in the
standardized method) against 4-7 days for the reference method
Inter-laboratory study
The bias between the reference and the alternative methods shows satisfying
and correct values
- level 1 (100 - 1 000 CFUg) bias = + 007 log CFUg
- level 2 (1 000 - 10 000 CFUg) bias = 002 log CFUg
- level 2 bis (1 000 - 10 000 CFUg) bias = - 004 log CFUg
- level 3 (10 000 - 100 000 CFUg) bias = - 004 log CFUg
The repeatability and the reproducibility of the alternative method look similar
to those of the reference method
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3038 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
22 Extension study (realised by ASEPT 2007)
An extension study was performed in 2007 for a new confirmatory test OBIS
MONO
150 Listeria monocytogenes strains and 100 non-target strains were isolated
onto TSAYE and BrillianceTM Listeria Agar plates All the Listeria
monocytogenes and the non-target strains gave the expected results
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3138 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Appendix 1 ndash Flow diagram of the Listeria PrecisTM method
x g sample + 9 x ml buffered peptone water
Revivification step for 1 h plusmn 5 min at 20degC plusmn 1degC
Streaking 1 ml onto 3 Brilliance Listeria Agar plates (mother suspension)
or 01 ml onto 1 Brilliance Listeria Agar plates (mother suspension and decimal dilutions)
Incubation for 48 h plusmn 3 h at 37degC plusmn 1degC
Confirmatory tests on at least one typical colony
- by the tests described in the ISO method
- by applying the OBIS mono test on an isolated colony
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3238 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Appendix 2 ndash ISO 11290-2 (1998) amp ISO 11290-2A1 (2004) Horizontal
method for the detection and enumeration of Listeria monocytogenes -
Part 2 enumeration of Listeria monocytogenes in foods
10 g cheese + 90 ml buffered peptone water or Half Fraser Broth (without supplements) (110 dilution)
Stomach 1 min 30 seconds
Keep at 20degC plusmn 2degC for 1 h plusmn 5 min
Decimal dilutions in 9 ml peptone salt tubes
Dilution 110
Dilution 1100
Dilution 11000
1 ml on 3 Chromogenic Listeria medium (ISO) plates (x 2) (-1)
01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-2)
01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-3)
01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-4)
Incubation at 37degC plusmn 1degC
24 h - 48 h
Numerate typical colonies at 24 h
(bluegreen surrounded by an opaque halo)
Numerate at 48 h
Streak typical colonies on TSYEA
(5 coloniesplate 2 successive dilutions)
Confirmation tests
Catalase
Gram stain
For confirmation of L monocytogenes
Haemolysis
Carbohydrate (rhamnose and xylose) utilisation
CAMP Test
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3338 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Appendix 3 ndash Mandelrsquos graphics
Laboratoire h Ref
Niveau 1 h Ref
Niveau 2 h Ref
Niveau 3 h Alt
Niveau 1 h Alt
Niveau 2 h Alt
Niveau 3 h5 h1 h5 h1
A -050 -015 083 062 -212 -039 198 299 -198 -299
B -261 -270 021 -077 -058 173 198 299 -198 -299
C -151 -031 -014 -015 -004 -005 198 299 -198 -299
D 038 040 -137 000 072 207 198 299 -198 -299
E -001 090 155 -052 056 -101 198 299 -198 -299
F -112 -321 -153 070 -325 023 198 299 -198 -299
G 042 -071 040 147 -009 176 198 299 -198 -299
H 169 046 -169 192 018 -083 198 299 -198 -299
L 009 -120 021 -077 -231 005 198 299 -198 -299
M 001 031 014 -032 004 -011 198 299 -198 -299
-300
-200
-100
000
100
200
300
A B C D E F G H L M
Vale
urs
h d
e M
an
del(
rob
uste
s)
Laboratoires
Meacutethode de reacutefeacuterence analyse robuste
h Ref Niveau 1
h Ref Niveau 2
h Ref Niveau 3
h 5=198
h 1=299
-300
-200
-100
000
100
200
300
A B C D E F G H L M
Vale
urs
de h
de M
an
del(
rob
uste
s)
Laboratoires
Meacutethode alternative analyse robuste
h Alt Niveau 1
h Alt Niveau 2
h Alt Niveau 2
h 1=299
h 5=198
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3438 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Laboratoire k Ref
Niveau 1 k Ref
Niveau 2 k Ref
Niveau 3 k Alt
Niveau 1 k Alt
Niveau 2 k Alt
Niveau 3 k1 k5
A 000 089 142 084 162 046 259 181
B 125 087 073 104 075 046 259 181
C 117 013 091 188 022 083 259 181
D 052 036 069 000 035 083 259 181
E 028 034 037 138 019 112 259 181
F 140 222 018 027 281 112 259 181
G 088 068 024 131 145 000 259 181
H 129 094 173 016 061 000 259 181
L 069 000 073 104 270 224 259 181
M 000 048 038 043 099 241 259 181
000
050
100
150
200
250
300
350
400
A B C D E F G H L M
Vale
urs
k d
e M
an
del(
rob
uste
s)
Laboratoires
Meacutethode de reacutefeacuterence analyse robuste
k Ref Niveau 1k Ref Niveau 2k Ref Niveau 3
k 1=259
k 5=181
000
050
100
150
200
250
300
A B C D E F G H L M
Vale
urs
k d
e M
an
del
(ro
bu
ste
s)
Laboratoires
Meacutethode alternative analyse robuste
k Alt Niveau 3
k Alt Niveau 1
k Alt Niveau 2
k 1=259
k 5=181
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3538 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Appendix 4 ndash Statistical results
Level 1
Nombre de laboratoires (p) 10
A 1602 1602 1602 000 0000 0000 -0495 0000 A 1845 1602 1724 017 0122 -0122 0621 0840 0122
B 1602 1176 1389 030 0213 -0213 -2613 1247 B 1602 1301 1452 021 0151 -0151 -0769 1041 0062
C 1699 1301 1500 028 0199 -0199 -1510 1165 C 1301 1845 1573 038 -0272 0272 -0148 1881 0073
D 1602 1778 1690 012 -0088 0088 0380 0516 D 1602 1602 1602 000 0000 0000 0000 0000 -0088
E 1699 1602 1651 007 0048 -0048 -0013 0284 E 1699 1301 1500 028 0199 -0199 -0522 1376 -0151
F 1778 1301 1540 034 0239 -0239 -1116 1397 F 1778 1699 1739 006 0040 -0040 0698 0274 0199
G 1845 1544 1695 021 0151 -0151 0425 0881 G 2079 1699 1889 027 0190 -0190 1467 1315 0194
H 2041 1602 1822 031 0220 -0220 1689 1286 H 2000 1954 1977 003 0023 -0023 1917 0158 0155
L 1544 1778 1661 017 -0117 0117 0092 0685 L 1301 1602 1452 021 -0151 0151 -0769 1041 -0210
M 1653 1653 1653 000 0000 0000 0013 0000 M 1477 1602 1540 009 -0062 0062 -0319 0432 -0114
m 165186 m 160206 mediane D 00678
Cn(2p) 1867 Cn(2p) 1867 Cp 16101
Cn(p) 16101 Cn(p) 16101 Qn(D) 0092926
n=2p n 20 n=2p n 20 Qdiff 01496169
f 11 f 11 t 114
l 55 l 55
n=p n 10 n=p n 10
f 6 f 6
l 15 l 15
Qn(2p) 0091465 Qn(2p) 007745
Qn(p) 0062469 Qn(p) 0121519
Qintra 017078 Qintra 014461
Qinter 010058 Qinter 019565
Sr(eacutecart-type de reacutepeacutetabiliteacute) 02415 Sr 02045
RSDr(coefficient de variation de la reacutepeacutetabiliteacute)1462 RSDr 1277
r(repeatability limit)) 0676 r 0573
SL(eacutecart-type interlaboratoire) -001905 SLlt0 SL 001737
SR(eacutecart-type de reproductibiliteacute) 02415 SR 02433
RSDR(coefficient de variation de la reproductibiliteacute)0146 RSDR 0152
R(limite de reproductibiliteacute)) 06762 R(limite de reproductibiliteacute))068123
h k h MoyenneLaboratoires
Niveau 1
si LaboratoiresDeviationMoyenne Dk DeviationsiAlt
Niveau 1
Ref
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3638 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Level 2
Nombre de laboratoires (p) 10
A 2681 2613 2647 005 0034 -0034 -0151 0891 A 2322 2531 2427 015 -0105 0105 -2119 1620 -0220
B 2544 2477 2511 005 0033 -0033 -2699 0872 B 2556 2653 2605 007 -0048 0048 -0581 0750 0094
C 2643 2633 2638 001 0005 -0005 -0311 0130 C 2681 2653 2667 002 0014 -0014 -0040 0217 0029
D 2690 2663 2676 002 0014 -0014 0400 0357 D 2778 2732 2755 003 0023 -0023 0721 0354 0079
E 2690 2716 2703 002 -0013 0013 0897 0336 E 2724 2748 2736 002 -0012 0012 0556 0185 0033
F 2568 2398 2483 012 0085 -0085 -3214 2217 F 2477 2114 2296 026 0182 -0182 -3255 2811 -0188
G 2643 2591 2617 004 0026 -0026 -0707 0682 G 2756 2568 2662 013 0094 -0094 -0085 1453 0045
H 2716 2643 2680 005 0036 -0036 0460 0945 H 2653 2732 2693 006 -0040 0040 0181 0613 0013
L 2591 2591 2591 000 0000 0000 -1196 0000 L 2230 2580 2405 025 -0175 0175 -2307 2704 -0186
M 2653 2690 2672 003 -0018 0018 0311 0481 M 2740 2613 2677 009 0064 -0064 0040 0987 0005
m 2655082 m 26719001 mediane D 00209
Cn(2p) 1867 Cn(2p) 1867 Cp 16101
Cn(p) 16101 Cn(p) 16101 Qn(D) 0039911
n=2p n 20 n=2p n 20 Qdiff 00642591
f 11 f 11 t 082
l 55 l 55
n=p n 10 n=p n 10
f 6 f 6
l 15 l 15
Qn(2p) 0020570 Qn(2p) 00345985
Qn(p) 00332437 Qn(p) 00718158
Qintra 003841 Qintra 006460
Qinter 005352 Qinter 011563
Sr(eacutecart-type de reacutepeacutetabiliteacute) 00543 Sr 00914
RSDr(coefficient de variation de la reacutepeacutetabiliteacute)205 RSDr 342
r(repeatability limit)) 0152 r 0256
SL(eacutecart-type interlaboratoire) 000139 SL 000920
SR(eacutecart-type de reproductibiliteacute) 00659 SR 01325
RSDR(coefficient de variation de la reproductibiliteacute)0025 RSDR 0050
R(limite de reproductibiliteacute)) 018446 R(limite de reproductibiliteacute))03708618
Laboratoires
Niveau 2
Moyenne si Laboratoires
Niveau 2
DRef Alt Moyenne si Deviation h Deviation h k k
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3738 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Level 2 bis
Nombre de laboratoires (p) 10
A 3000 2477 2739 037 0261 -0261 0832 1418 A 2602 2477 2540 009 0062 -0062 -0391 0465 -0199
B 2544 2813 2678 019 -0134 0134 0211 0729 B 2903 2778 2841 009 0062 -0062 1732 0465 0162
C 2477 2813 2645 024 -0168 0168 -0135 0911 C 2477 2699 2588 016 -0111 0111 -0049 0826 -0057
D 2398 2653 2526 018 -0128 0128 -1370 0692 D 2778 3000 2889 016 -0111 0111 2074 0826 0363
E 2875 2740 2808 010 0067 -0067 1547 0365 E 2602 2301 2452 021 0151 -0151 -1012 1120 -0356
F 2477 2544 2511 005 -0033 0033 -1525 0182 F 2778 2477 2628 021 0151 -0151 0230 1120 0117
G 2653 2740 2697 006 -0044 0044 0400 0236 G 2845 2845 2845 000 0000 0000 1764 0000 0148
H 2176 2813 2495 045 -0318 0318 -1692 1727 H 2477 2477 2477 000 0000 0000 -0832 0000 -0017
L 2813 2544 2678 019 0134 -0134 0211 0729 L 2903 2301 2602 043 0301 -0301 0049 2241 -0076
M 2602 2740 2671 010 -0069 0069 0135 0375 M 2903 2255 2579 046 0324 -0324 -0112 2411 -0092
m 2658114 m 259505 mediane D -00372
Cn(2p) 1867 Cn(2p) 1867 Cp 16101
Cn(p) 16101 Cn(p) 16101 Qn(D) 01656915
n=2p n 20 n=2p n 20 Qdiff 02667753
f 11 f 11 t 035
l 55 l 55
n=p n 10 n=p n 10
f 6 f 6
l 15 l 15
Qn(2p) 0098745 Qn(2p) 007195
Qn(p) 006007 Qn(p) 008805
Qintra 018437 Qintra 013435
Qinter 009672 Qinter 014176
Sr(eacutecart-type de reacutepeacutetabiliteacute) 02607 Sr 01900
RSDr(coefficient de variation de la reacutepeacutetabiliteacute)981 RSDr 732
r(repeatability limit)) 0730 r 0532
SL(eacutecart-type interlaboratoire) -002464 SLlt0 SL 000205
SR(eacutecart-type de reproductibiliteacute) 02607 SR 01953
RSDR(coefficient de variation de la reproductibiliteacute)0098 RSDR 0075
R(limite de reproductibiliteacute)) 072996 R(limite de reproductibiliteacute))054686
Laboratoires
Niveau 2 bis
Moyenne si Laboratoires
Niveau 2 bis
DRef Alt Moyenne si Deviation h Deviation h k k
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3838 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Level 3
Nombre de laboratoires (p) 10
A 3690 3663 3676 002 0014 -0014 -0311 0513 A 3690 3708 3699 001 -0009 0009 0354 0257 0022
B 3613 3672 3642 004 -0030 0030 -1851 1109 B 3708 3568 3638 010 0070 -0070 -0844 2061 -0005
C 3663 3732 3698 005 -0035 0035 0645 1302 C 3663 3699 3681 003 -0018 0018 0000 0536 -0017
D 3690 3690 3690 000 0000 0000 0311 0000 D 3643 3663 3653 001 -0010 0010 -0545 0286 -0037
E 3732 3653 3693 006 0040 -0040 0429 1480 E 3613 3732 3673 008 -0060 0060 -0162 1769 -0020
F 3643 3672 3658 002 -0014 0014 -1157 0536 F 3690 3771 3731 006 -0040 0040 0975 1193 0073
G 3699 3653 3676 003 0023 -0023 -0328 0856 G 3602 3643 3623 003 -0021 0021 -1141 0612 -0053
H 3623 3672 3648 003 -0024 0024 -1615 0913 H 3748 3681 3715 005 0033 -0033 0664 0990 0067
L 3724 3663 3694 004 0031 -0031 0461 1150 L 3748 3771 3760 002 -0011 0011 1544 0335 0066
M 3690 3690 3690 000 0000 0000 0311 0000 M 3806 3681 3744 009 0062 -0062 1234 1848 0054
m 368334 m 368086 mediane D 00089
Cn(2p) 1867 Cn(2p) 1867 Cp 16101
Cn(p) 16101 Cn(p) 16101 Qn(D) 00325359
n=2p n 20 n=2p n 20 Qdiff 00523852
f 11 f 11 t 043
l 55 l 55
n=p n 10 n=p n 10
f 6 f 6
l 15 l 15
Qn(2p) 0014323 Qn(2p) 001811
Qn(p) 0013719 Qn(p) 003164
Qintra 002674 Qintra 003381
Qinter 002209 Qinter 005094
Sr(eacutecart-type de reacutepeacutetabiliteacute) 00378 Sr 00478
RSDr(coefficient de variation de la reacutepeacutetabiliteacute)103 RSDr 130
r(repeatability limit)) 0106 r 0134
SL(eacutecart-type interlaboratoire) -000023 SLlt0 SL 000145
SR(eacutecart-type de reproductibiliteacute) 00378 SR 00611
RSDR(coefficient de variation de la reproductibiliteacute)0010 RSDR 0017
R(limite de reproductibiliteacute)) 0105894 R(limite de reproductibiliteacute))017119
Ref Alt DLaboratoires
Niveau 3
Moyenne si Deviation h k Laboratoires
Niveau 3
Moyenne si Deviation h k
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2538 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Table 16 ndash Synthesis of results obtained by the alternative
and the reference methods (log CFUml)
Labs Level 1 Level 2 Level 2 bis Level 3
Reference method
Alternative method
Reference method
Alternative method
Reference method
Alternative method
Reference method
Alternative method
A 1602 1602 1845 1602 2681 2613 2322 2531 3000 2477 2602 2477 3690 3663 3690 3708
B 1602 1176 1602 1301 2544 2477 2556 2653 2544 2813 2903 2778 3613 3672 3708 3568
C 1699 1301 1301 1845 2643 2633 2681 2653 2477 2813 2477 2699 3663 3732 3663 3699
D 1602 1778 1602 1602 2690 2663 2778 2732 2398 2653 2778 3000 3690 3690 3643 3663
E 1699 1602 1699 1301 2690 2716 2724 2748 2875 2740 2602 2301 3732 3653 3613 3732
F 1778 1301 1778 1699 2568 2398 2477 2114 2477 2544 2778 2477 3643 3672 3690 3771
G 1845 1544 2079 1699 2643 2591 2756 2568 2653 2740 2845 2845 3699 3653 3602 3643
H 2041 1602 2000 1954 2716 2643 2653 2732 2176 2813 2477 2477 3623 3672 3748 3681
L 1544 1778 1301 1602 2591 2591 2230 2580 2813 2544 2903 2301 3724 3663 3748 3771
M 1653 1653 1477 1602 2653 2690 2740 2613 2602 2740 2903 2255 3690 3690 3806 3681
Level 2 results obtained taking account the 3 plates inoculated with the mother-suspension
Level 2 bis results obtained taking account the plates inoculated with 01 ml of the mother-suspension
Scrutiny of the measurement results for consistency
In order to identify the measurement results or laboratories that could be
inconsistent two graphical consistency techniques were realised Mandelrsquos
h-ank-statistics Mandel indicators h and k at 5 significance highlighted
some possible inconsistent data (See tables and graphics in Appendix 3 and
Appendix 4)
Mandelrsquos values Number of values observed over the threshold
Reference method Alternative method
h gt 1 Lab F Level 2 Lab F Level 2
h gt 5 Lab F Level 2
Lab B Levels 1 and 2
Lab A Level 2
Lab D Level 3
Lab F Level 2
Lab L Level 2
kgt 1 Lab F Level 3
k gt 5 Lab F Level 2 Lab C Level 2
Lab F Level 3
Lab L Levels 1 and 3
Lab M Level 1
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2638 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
The emphasized results appear (i) coherent with the targeted contamination
levels (ii) concordant between both methods
All the results were thus kept for the statistical analysis
Comparison of the trueness and precision characteristics of
the reference method and alternative methods
The statistical values are summarised hereafter
Level p
Reference method Alternative method
Repeatability ratio
Reproducibility ratio Median
Repeatability standard deviation
Reproducibility standard deviation
Median Repeatability
standard deviation
Reproducibility standard deviation
1 10 16519 02415 02425 16021 02045 02433 0847 1007
2 10 26551 00543 00659 26719 00914 01325 1682 2011
2 bis 10 26581 02607 02607 25951 01900 01953 0729 0749
3 10 18671 00378 00378 18671 00478 00611 1264 1617
Bias of the alternative method
In order to estimate the bias of the alternative method with regard to the reference method for every
level Dij and t are calculated according to the following equations
RefAlt ijYijYDij
Diffp
Dijit
)2(
)(median
If the t value is superior to 2 the alternative method is significantly biased
with regard to the reference method
The values are given in table 17
Table 17 ndash t (d) values obtained by level
Level Bias t (d) Conclusion
1 0068 114 Non significant bias
2 0021 082 Non significant bias
2 bis - 0037 035 Non significant bias
3 - 0037 043 Non significant bias
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2738 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
The statistical test concludes to a non significant bias for all the inoculation
levels The bias varies from ndash 0037 to 0068 log CFUml
Comparison of the repeatability standard deviations
If the ratio Srj Alt Srj Ref of the repeatability standard deviations of the
alternative method and the reference method is larger than 2 the precision
under repeatability conditions of the alternative method is considered to be
lower than that of the reference method If this ratio is smaller than 05 the
precision under repeatability conditions of the alternative method is
considered to be greater than that of the reference method
The ratio values are given in table 18
Table 18
Level
Repeatability standard deviation
Reference method Alternative method Alternative method
reference method ratio
1 0242 0205 0847
2 0054 0091 1682
2 bis 0261 0190 0729
3 0038 0048 1264
Level Repeatability limit
Reference method Alternative method
1 0676 0573
2 0152 0256
2 bis 0730 0532
3 0106 0134
The ratios of the repeatability standard deviations are below 2 in all cases
The precision under repeatability conditions of the alternative method is
equivalent to that of the reference method
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2838 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Comparison of the reproducibility standard deviations
If the ratio Srj Alt Srj Ref of the reproducibility standard deviations of the alternative method and the
reference method is larger than 2 the precision under reproducibility conditions of the alternative
method is considered to be lower than that of the reference method If this ratio is smaller than 05 the
precision under reproducibility conditions of the alternative method is considered to be greater than
that of the reference method
The ratio values are given in table 19
Table 19
Level
Reproducibility standard deviation
Reference method Alternative method Alternative method
reference method ratio
1 0242 0243 1007
2 0066 0133 2011
2 bis 0261 0195 0749
3 0038 0061 1617
Level Reproducibility limit
Reference method Alternative method
1 0676 0681
2 0185 0371
2 bis 0730 0547
3 0106 0171
The ratios of the reproducibility standard deviations are below or close to 2
The precision under reproducibility conditions of the alternative method is
equivalent to that of the reference method
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2938 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
214 Conclusion
Method comparison study
The Listeria PrecisTM enumeration method shows satisfying linearity and
relative accuracy
The alternative method shows satisfying inclusivity and exclusivity results
Two Listeria monocytogenes strains gave negative results with the
alternative method These strains grew weakly on the reference method
media (OampA Palcam and Oxford)
The limits of repeatability of the alternative method are similar to those of the
reference method
The bias between both methods are low between - 0133 Log CFUg and
- 0008 Log CFUg
The Listeria PrecisTM enumeration method offers the saving for confirmatory
tests
Positive results are obtained with the alternative method in 2-3 days (with
OBIS Mono test) or in 4-7 days (by the classical tests described in the
standardized method) against 4-7 days for the reference method
Inter-laboratory study
The bias between the reference and the alternative methods shows satisfying
and correct values
- level 1 (100 - 1 000 CFUg) bias = + 007 log CFUg
- level 2 (1 000 - 10 000 CFUg) bias = 002 log CFUg
- level 2 bis (1 000 - 10 000 CFUg) bias = - 004 log CFUg
- level 3 (10 000 - 100 000 CFUg) bias = - 004 log CFUg
The repeatability and the reproducibility of the alternative method look similar
to those of the reference method
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3038 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
22 Extension study (realised by ASEPT 2007)
An extension study was performed in 2007 for a new confirmatory test OBIS
MONO
150 Listeria monocytogenes strains and 100 non-target strains were isolated
onto TSAYE and BrillianceTM Listeria Agar plates All the Listeria
monocytogenes and the non-target strains gave the expected results
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3138 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Appendix 1 ndash Flow diagram of the Listeria PrecisTM method
x g sample + 9 x ml buffered peptone water
Revivification step for 1 h plusmn 5 min at 20degC plusmn 1degC
Streaking 1 ml onto 3 Brilliance Listeria Agar plates (mother suspension)
or 01 ml onto 1 Brilliance Listeria Agar plates (mother suspension and decimal dilutions)
Incubation for 48 h plusmn 3 h at 37degC plusmn 1degC
Confirmatory tests on at least one typical colony
- by the tests described in the ISO method
- by applying the OBIS mono test on an isolated colony
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3238 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Appendix 2 ndash ISO 11290-2 (1998) amp ISO 11290-2A1 (2004) Horizontal
method for the detection and enumeration of Listeria monocytogenes -
Part 2 enumeration of Listeria monocytogenes in foods
10 g cheese + 90 ml buffered peptone water or Half Fraser Broth (without supplements) (110 dilution)
Stomach 1 min 30 seconds
Keep at 20degC plusmn 2degC for 1 h plusmn 5 min
Decimal dilutions in 9 ml peptone salt tubes
Dilution 110
Dilution 1100
Dilution 11000
1 ml on 3 Chromogenic Listeria medium (ISO) plates (x 2) (-1)
01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-2)
01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-3)
01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-4)
Incubation at 37degC plusmn 1degC
24 h - 48 h
Numerate typical colonies at 24 h
(bluegreen surrounded by an opaque halo)
Numerate at 48 h
Streak typical colonies on TSYEA
(5 coloniesplate 2 successive dilutions)
Confirmation tests
Catalase
Gram stain
For confirmation of L monocytogenes
Haemolysis
Carbohydrate (rhamnose and xylose) utilisation
CAMP Test
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3338 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Appendix 3 ndash Mandelrsquos graphics
Laboratoire h Ref
Niveau 1 h Ref
Niveau 2 h Ref
Niveau 3 h Alt
Niveau 1 h Alt
Niveau 2 h Alt
Niveau 3 h5 h1 h5 h1
A -050 -015 083 062 -212 -039 198 299 -198 -299
B -261 -270 021 -077 -058 173 198 299 -198 -299
C -151 -031 -014 -015 -004 -005 198 299 -198 -299
D 038 040 -137 000 072 207 198 299 -198 -299
E -001 090 155 -052 056 -101 198 299 -198 -299
F -112 -321 -153 070 -325 023 198 299 -198 -299
G 042 -071 040 147 -009 176 198 299 -198 -299
H 169 046 -169 192 018 -083 198 299 -198 -299
L 009 -120 021 -077 -231 005 198 299 -198 -299
M 001 031 014 -032 004 -011 198 299 -198 -299
-300
-200
-100
000
100
200
300
A B C D E F G H L M
Vale
urs
h d
e M
an
del(
rob
uste
s)
Laboratoires
Meacutethode de reacutefeacuterence analyse robuste
h Ref Niveau 1
h Ref Niveau 2
h Ref Niveau 3
h 5=198
h 1=299
-300
-200
-100
000
100
200
300
A B C D E F G H L M
Vale
urs
de h
de M
an
del(
rob
uste
s)
Laboratoires
Meacutethode alternative analyse robuste
h Alt Niveau 1
h Alt Niveau 2
h Alt Niveau 2
h 1=299
h 5=198
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3438 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Laboratoire k Ref
Niveau 1 k Ref
Niveau 2 k Ref
Niveau 3 k Alt
Niveau 1 k Alt
Niveau 2 k Alt
Niveau 3 k1 k5
A 000 089 142 084 162 046 259 181
B 125 087 073 104 075 046 259 181
C 117 013 091 188 022 083 259 181
D 052 036 069 000 035 083 259 181
E 028 034 037 138 019 112 259 181
F 140 222 018 027 281 112 259 181
G 088 068 024 131 145 000 259 181
H 129 094 173 016 061 000 259 181
L 069 000 073 104 270 224 259 181
M 000 048 038 043 099 241 259 181
000
050
100
150
200
250
300
350
400
A B C D E F G H L M
Vale
urs
k d
e M
an
del(
rob
uste
s)
Laboratoires
Meacutethode de reacutefeacuterence analyse robuste
k Ref Niveau 1k Ref Niveau 2k Ref Niveau 3
k 1=259
k 5=181
000
050
100
150
200
250
300
A B C D E F G H L M
Vale
urs
k d
e M
an
del
(ro
bu
ste
s)
Laboratoires
Meacutethode alternative analyse robuste
k Alt Niveau 3
k Alt Niveau 1
k Alt Niveau 2
k 1=259
k 5=181
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3538 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Appendix 4 ndash Statistical results
Level 1
Nombre de laboratoires (p) 10
A 1602 1602 1602 000 0000 0000 -0495 0000 A 1845 1602 1724 017 0122 -0122 0621 0840 0122
B 1602 1176 1389 030 0213 -0213 -2613 1247 B 1602 1301 1452 021 0151 -0151 -0769 1041 0062
C 1699 1301 1500 028 0199 -0199 -1510 1165 C 1301 1845 1573 038 -0272 0272 -0148 1881 0073
D 1602 1778 1690 012 -0088 0088 0380 0516 D 1602 1602 1602 000 0000 0000 0000 0000 -0088
E 1699 1602 1651 007 0048 -0048 -0013 0284 E 1699 1301 1500 028 0199 -0199 -0522 1376 -0151
F 1778 1301 1540 034 0239 -0239 -1116 1397 F 1778 1699 1739 006 0040 -0040 0698 0274 0199
G 1845 1544 1695 021 0151 -0151 0425 0881 G 2079 1699 1889 027 0190 -0190 1467 1315 0194
H 2041 1602 1822 031 0220 -0220 1689 1286 H 2000 1954 1977 003 0023 -0023 1917 0158 0155
L 1544 1778 1661 017 -0117 0117 0092 0685 L 1301 1602 1452 021 -0151 0151 -0769 1041 -0210
M 1653 1653 1653 000 0000 0000 0013 0000 M 1477 1602 1540 009 -0062 0062 -0319 0432 -0114
m 165186 m 160206 mediane D 00678
Cn(2p) 1867 Cn(2p) 1867 Cp 16101
Cn(p) 16101 Cn(p) 16101 Qn(D) 0092926
n=2p n 20 n=2p n 20 Qdiff 01496169
f 11 f 11 t 114
l 55 l 55
n=p n 10 n=p n 10
f 6 f 6
l 15 l 15
Qn(2p) 0091465 Qn(2p) 007745
Qn(p) 0062469 Qn(p) 0121519
Qintra 017078 Qintra 014461
Qinter 010058 Qinter 019565
Sr(eacutecart-type de reacutepeacutetabiliteacute) 02415 Sr 02045
RSDr(coefficient de variation de la reacutepeacutetabiliteacute)1462 RSDr 1277
r(repeatability limit)) 0676 r 0573
SL(eacutecart-type interlaboratoire) -001905 SLlt0 SL 001737
SR(eacutecart-type de reproductibiliteacute) 02415 SR 02433
RSDR(coefficient de variation de la reproductibiliteacute)0146 RSDR 0152
R(limite de reproductibiliteacute)) 06762 R(limite de reproductibiliteacute))068123
h k h MoyenneLaboratoires
Niveau 1
si LaboratoiresDeviationMoyenne Dk DeviationsiAlt
Niveau 1
Ref
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3638 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Level 2
Nombre de laboratoires (p) 10
A 2681 2613 2647 005 0034 -0034 -0151 0891 A 2322 2531 2427 015 -0105 0105 -2119 1620 -0220
B 2544 2477 2511 005 0033 -0033 -2699 0872 B 2556 2653 2605 007 -0048 0048 -0581 0750 0094
C 2643 2633 2638 001 0005 -0005 -0311 0130 C 2681 2653 2667 002 0014 -0014 -0040 0217 0029
D 2690 2663 2676 002 0014 -0014 0400 0357 D 2778 2732 2755 003 0023 -0023 0721 0354 0079
E 2690 2716 2703 002 -0013 0013 0897 0336 E 2724 2748 2736 002 -0012 0012 0556 0185 0033
F 2568 2398 2483 012 0085 -0085 -3214 2217 F 2477 2114 2296 026 0182 -0182 -3255 2811 -0188
G 2643 2591 2617 004 0026 -0026 -0707 0682 G 2756 2568 2662 013 0094 -0094 -0085 1453 0045
H 2716 2643 2680 005 0036 -0036 0460 0945 H 2653 2732 2693 006 -0040 0040 0181 0613 0013
L 2591 2591 2591 000 0000 0000 -1196 0000 L 2230 2580 2405 025 -0175 0175 -2307 2704 -0186
M 2653 2690 2672 003 -0018 0018 0311 0481 M 2740 2613 2677 009 0064 -0064 0040 0987 0005
m 2655082 m 26719001 mediane D 00209
Cn(2p) 1867 Cn(2p) 1867 Cp 16101
Cn(p) 16101 Cn(p) 16101 Qn(D) 0039911
n=2p n 20 n=2p n 20 Qdiff 00642591
f 11 f 11 t 082
l 55 l 55
n=p n 10 n=p n 10
f 6 f 6
l 15 l 15
Qn(2p) 0020570 Qn(2p) 00345985
Qn(p) 00332437 Qn(p) 00718158
Qintra 003841 Qintra 006460
Qinter 005352 Qinter 011563
Sr(eacutecart-type de reacutepeacutetabiliteacute) 00543 Sr 00914
RSDr(coefficient de variation de la reacutepeacutetabiliteacute)205 RSDr 342
r(repeatability limit)) 0152 r 0256
SL(eacutecart-type interlaboratoire) 000139 SL 000920
SR(eacutecart-type de reproductibiliteacute) 00659 SR 01325
RSDR(coefficient de variation de la reproductibiliteacute)0025 RSDR 0050
R(limite de reproductibiliteacute)) 018446 R(limite de reproductibiliteacute))03708618
Laboratoires
Niveau 2
Moyenne si Laboratoires
Niveau 2
DRef Alt Moyenne si Deviation h Deviation h k k
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3738 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Level 2 bis
Nombre de laboratoires (p) 10
A 3000 2477 2739 037 0261 -0261 0832 1418 A 2602 2477 2540 009 0062 -0062 -0391 0465 -0199
B 2544 2813 2678 019 -0134 0134 0211 0729 B 2903 2778 2841 009 0062 -0062 1732 0465 0162
C 2477 2813 2645 024 -0168 0168 -0135 0911 C 2477 2699 2588 016 -0111 0111 -0049 0826 -0057
D 2398 2653 2526 018 -0128 0128 -1370 0692 D 2778 3000 2889 016 -0111 0111 2074 0826 0363
E 2875 2740 2808 010 0067 -0067 1547 0365 E 2602 2301 2452 021 0151 -0151 -1012 1120 -0356
F 2477 2544 2511 005 -0033 0033 -1525 0182 F 2778 2477 2628 021 0151 -0151 0230 1120 0117
G 2653 2740 2697 006 -0044 0044 0400 0236 G 2845 2845 2845 000 0000 0000 1764 0000 0148
H 2176 2813 2495 045 -0318 0318 -1692 1727 H 2477 2477 2477 000 0000 0000 -0832 0000 -0017
L 2813 2544 2678 019 0134 -0134 0211 0729 L 2903 2301 2602 043 0301 -0301 0049 2241 -0076
M 2602 2740 2671 010 -0069 0069 0135 0375 M 2903 2255 2579 046 0324 -0324 -0112 2411 -0092
m 2658114 m 259505 mediane D -00372
Cn(2p) 1867 Cn(2p) 1867 Cp 16101
Cn(p) 16101 Cn(p) 16101 Qn(D) 01656915
n=2p n 20 n=2p n 20 Qdiff 02667753
f 11 f 11 t 035
l 55 l 55
n=p n 10 n=p n 10
f 6 f 6
l 15 l 15
Qn(2p) 0098745 Qn(2p) 007195
Qn(p) 006007 Qn(p) 008805
Qintra 018437 Qintra 013435
Qinter 009672 Qinter 014176
Sr(eacutecart-type de reacutepeacutetabiliteacute) 02607 Sr 01900
RSDr(coefficient de variation de la reacutepeacutetabiliteacute)981 RSDr 732
r(repeatability limit)) 0730 r 0532
SL(eacutecart-type interlaboratoire) -002464 SLlt0 SL 000205
SR(eacutecart-type de reproductibiliteacute) 02607 SR 01953
RSDR(coefficient de variation de la reproductibiliteacute)0098 RSDR 0075
R(limite de reproductibiliteacute)) 072996 R(limite de reproductibiliteacute))054686
Laboratoires
Niveau 2 bis
Moyenne si Laboratoires
Niveau 2 bis
DRef Alt Moyenne si Deviation h Deviation h k k
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3838 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Level 3
Nombre de laboratoires (p) 10
A 3690 3663 3676 002 0014 -0014 -0311 0513 A 3690 3708 3699 001 -0009 0009 0354 0257 0022
B 3613 3672 3642 004 -0030 0030 -1851 1109 B 3708 3568 3638 010 0070 -0070 -0844 2061 -0005
C 3663 3732 3698 005 -0035 0035 0645 1302 C 3663 3699 3681 003 -0018 0018 0000 0536 -0017
D 3690 3690 3690 000 0000 0000 0311 0000 D 3643 3663 3653 001 -0010 0010 -0545 0286 -0037
E 3732 3653 3693 006 0040 -0040 0429 1480 E 3613 3732 3673 008 -0060 0060 -0162 1769 -0020
F 3643 3672 3658 002 -0014 0014 -1157 0536 F 3690 3771 3731 006 -0040 0040 0975 1193 0073
G 3699 3653 3676 003 0023 -0023 -0328 0856 G 3602 3643 3623 003 -0021 0021 -1141 0612 -0053
H 3623 3672 3648 003 -0024 0024 -1615 0913 H 3748 3681 3715 005 0033 -0033 0664 0990 0067
L 3724 3663 3694 004 0031 -0031 0461 1150 L 3748 3771 3760 002 -0011 0011 1544 0335 0066
M 3690 3690 3690 000 0000 0000 0311 0000 M 3806 3681 3744 009 0062 -0062 1234 1848 0054
m 368334 m 368086 mediane D 00089
Cn(2p) 1867 Cn(2p) 1867 Cp 16101
Cn(p) 16101 Cn(p) 16101 Qn(D) 00325359
n=2p n 20 n=2p n 20 Qdiff 00523852
f 11 f 11 t 043
l 55 l 55
n=p n 10 n=p n 10
f 6 f 6
l 15 l 15
Qn(2p) 0014323 Qn(2p) 001811
Qn(p) 0013719 Qn(p) 003164
Qintra 002674 Qintra 003381
Qinter 002209 Qinter 005094
Sr(eacutecart-type de reacutepeacutetabiliteacute) 00378 Sr 00478
RSDr(coefficient de variation de la reacutepeacutetabiliteacute)103 RSDr 130
r(repeatability limit)) 0106 r 0134
SL(eacutecart-type interlaboratoire) -000023 SLlt0 SL 000145
SR(eacutecart-type de reproductibiliteacute) 00378 SR 00611
RSDR(coefficient de variation de la reproductibiliteacute)0010 RSDR 0017
R(limite de reproductibiliteacute)) 0105894 R(limite de reproductibiliteacute))017119
Ref Alt DLaboratoires
Niveau 3
Moyenne si Deviation h k Laboratoires
Niveau 3
Moyenne si Deviation h k
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2638 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
The emphasized results appear (i) coherent with the targeted contamination
levels (ii) concordant between both methods
All the results were thus kept for the statistical analysis
Comparison of the trueness and precision characteristics of
the reference method and alternative methods
The statistical values are summarised hereafter
Level p
Reference method Alternative method
Repeatability ratio
Reproducibility ratio Median
Repeatability standard deviation
Reproducibility standard deviation
Median Repeatability
standard deviation
Reproducibility standard deviation
1 10 16519 02415 02425 16021 02045 02433 0847 1007
2 10 26551 00543 00659 26719 00914 01325 1682 2011
2 bis 10 26581 02607 02607 25951 01900 01953 0729 0749
3 10 18671 00378 00378 18671 00478 00611 1264 1617
Bias of the alternative method
In order to estimate the bias of the alternative method with regard to the reference method for every
level Dij and t are calculated according to the following equations
RefAlt ijYijYDij
Diffp
Dijit
)2(
)(median
If the t value is superior to 2 the alternative method is significantly biased
with regard to the reference method
The values are given in table 17
Table 17 ndash t (d) values obtained by level
Level Bias t (d) Conclusion
1 0068 114 Non significant bias
2 0021 082 Non significant bias
2 bis - 0037 035 Non significant bias
3 - 0037 043 Non significant bias
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2738 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
The statistical test concludes to a non significant bias for all the inoculation
levels The bias varies from ndash 0037 to 0068 log CFUml
Comparison of the repeatability standard deviations
If the ratio Srj Alt Srj Ref of the repeatability standard deviations of the
alternative method and the reference method is larger than 2 the precision
under repeatability conditions of the alternative method is considered to be
lower than that of the reference method If this ratio is smaller than 05 the
precision under repeatability conditions of the alternative method is
considered to be greater than that of the reference method
The ratio values are given in table 18
Table 18
Level
Repeatability standard deviation
Reference method Alternative method Alternative method
reference method ratio
1 0242 0205 0847
2 0054 0091 1682
2 bis 0261 0190 0729
3 0038 0048 1264
Level Repeatability limit
Reference method Alternative method
1 0676 0573
2 0152 0256
2 bis 0730 0532
3 0106 0134
The ratios of the repeatability standard deviations are below 2 in all cases
The precision under repeatability conditions of the alternative method is
equivalent to that of the reference method
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2838 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Comparison of the reproducibility standard deviations
If the ratio Srj Alt Srj Ref of the reproducibility standard deviations of the alternative method and the
reference method is larger than 2 the precision under reproducibility conditions of the alternative
method is considered to be lower than that of the reference method If this ratio is smaller than 05 the
precision under reproducibility conditions of the alternative method is considered to be greater than
that of the reference method
The ratio values are given in table 19
Table 19
Level
Reproducibility standard deviation
Reference method Alternative method Alternative method
reference method ratio
1 0242 0243 1007
2 0066 0133 2011
2 bis 0261 0195 0749
3 0038 0061 1617
Level Reproducibility limit
Reference method Alternative method
1 0676 0681
2 0185 0371
2 bis 0730 0547
3 0106 0171
The ratios of the reproducibility standard deviations are below or close to 2
The precision under reproducibility conditions of the alternative method is
equivalent to that of the reference method
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2938 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
214 Conclusion
Method comparison study
The Listeria PrecisTM enumeration method shows satisfying linearity and
relative accuracy
The alternative method shows satisfying inclusivity and exclusivity results
Two Listeria monocytogenes strains gave negative results with the
alternative method These strains grew weakly on the reference method
media (OampA Palcam and Oxford)
The limits of repeatability of the alternative method are similar to those of the
reference method
The bias between both methods are low between - 0133 Log CFUg and
- 0008 Log CFUg
The Listeria PrecisTM enumeration method offers the saving for confirmatory
tests
Positive results are obtained with the alternative method in 2-3 days (with
OBIS Mono test) or in 4-7 days (by the classical tests described in the
standardized method) against 4-7 days for the reference method
Inter-laboratory study
The bias between the reference and the alternative methods shows satisfying
and correct values
- level 1 (100 - 1 000 CFUg) bias = + 007 log CFUg
- level 2 (1 000 - 10 000 CFUg) bias = 002 log CFUg
- level 2 bis (1 000 - 10 000 CFUg) bias = - 004 log CFUg
- level 3 (10 000 - 100 000 CFUg) bias = - 004 log CFUg
The repeatability and the reproducibility of the alternative method look similar
to those of the reference method
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3038 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
22 Extension study (realised by ASEPT 2007)
An extension study was performed in 2007 for a new confirmatory test OBIS
MONO
150 Listeria monocytogenes strains and 100 non-target strains were isolated
onto TSAYE and BrillianceTM Listeria Agar plates All the Listeria
monocytogenes and the non-target strains gave the expected results
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3138 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Appendix 1 ndash Flow diagram of the Listeria PrecisTM method
x g sample + 9 x ml buffered peptone water
Revivification step for 1 h plusmn 5 min at 20degC plusmn 1degC
Streaking 1 ml onto 3 Brilliance Listeria Agar plates (mother suspension)
or 01 ml onto 1 Brilliance Listeria Agar plates (mother suspension and decimal dilutions)
Incubation for 48 h plusmn 3 h at 37degC plusmn 1degC
Confirmatory tests on at least one typical colony
- by the tests described in the ISO method
- by applying the OBIS mono test on an isolated colony
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3238 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Appendix 2 ndash ISO 11290-2 (1998) amp ISO 11290-2A1 (2004) Horizontal
method for the detection and enumeration of Listeria monocytogenes -
Part 2 enumeration of Listeria monocytogenes in foods
10 g cheese + 90 ml buffered peptone water or Half Fraser Broth (without supplements) (110 dilution)
Stomach 1 min 30 seconds
Keep at 20degC plusmn 2degC for 1 h plusmn 5 min
Decimal dilutions in 9 ml peptone salt tubes
Dilution 110
Dilution 1100
Dilution 11000
1 ml on 3 Chromogenic Listeria medium (ISO) plates (x 2) (-1)
01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-2)
01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-3)
01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-4)
Incubation at 37degC plusmn 1degC
24 h - 48 h
Numerate typical colonies at 24 h
(bluegreen surrounded by an opaque halo)
Numerate at 48 h
Streak typical colonies on TSYEA
(5 coloniesplate 2 successive dilutions)
Confirmation tests
Catalase
Gram stain
For confirmation of L monocytogenes
Haemolysis
Carbohydrate (rhamnose and xylose) utilisation
CAMP Test
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3338 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Appendix 3 ndash Mandelrsquos graphics
Laboratoire h Ref
Niveau 1 h Ref
Niveau 2 h Ref
Niveau 3 h Alt
Niveau 1 h Alt
Niveau 2 h Alt
Niveau 3 h5 h1 h5 h1
A -050 -015 083 062 -212 -039 198 299 -198 -299
B -261 -270 021 -077 -058 173 198 299 -198 -299
C -151 -031 -014 -015 -004 -005 198 299 -198 -299
D 038 040 -137 000 072 207 198 299 -198 -299
E -001 090 155 -052 056 -101 198 299 -198 -299
F -112 -321 -153 070 -325 023 198 299 -198 -299
G 042 -071 040 147 -009 176 198 299 -198 -299
H 169 046 -169 192 018 -083 198 299 -198 -299
L 009 -120 021 -077 -231 005 198 299 -198 -299
M 001 031 014 -032 004 -011 198 299 -198 -299
-300
-200
-100
000
100
200
300
A B C D E F G H L M
Vale
urs
h d
e M
an
del(
rob
uste
s)
Laboratoires
Meacutethode de reacutefeacuterence analyse robuste
h Ref Niveau 1
h Ref Niveau 2
h Ref Niveau 3
h 5=198
h 1=299
-300
-200
-100
000
100
200
300
A B C D E F G H L M
Vale
urs
de h
de M
an
del(
rob
uste
s)
Laboratoires
Meacutethode alternative analyse robuste
h Alt Niveau 1
h Alt Niveau 2
h Alt Niveau 2
h 1=299
h 5=198
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3438 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Laboratoire k Ref
Niveau 1 k Ref
Niveau 2 k Ref
Niveau 3 k Alt
Niveau 1 k Alt
Niveau 2 k Alt
Niveau 3 k1 k5
A 000 089 142 084 162 046 259 181
B 125 087 073 104 075 046 259 181
C 117 013 091 188 022 083 259 181
D 052 036 069 000 035 083 259 181
E 028 034 037 138 019 112 259 181
F 140 222 018 027 281 112 259 181
G 088 068 024 131 145 000 259 181
H 129 094 173 016 061 000 259 181
L 069 000 073 104 270 224 259 181
M 000 048 038 043 099 241 259 181
000
050
100
150
200
250
300
350
400
A B C D E F G H L M
Vale
urs
k d
e M
an
del(
rob
uste
s)
Laboratoires
Meacutethode de reacutefeacuterence analyse robuste
k Ref Niveau 1k Ref Niveau 2k Ref Niveau 3
k 1=259
k 5=181
000
050
100
150
200
250
300
A B C D E F G H L M
Vale
urs
k d
e M
an
del
(ro
bu
ste
s)
Laboratoires
Meacutethode alternative analyse robuste
k Alt Niveau 3
k Alt Niveau 1
k Alt Niveau 2
k 1=259
k 5=181
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3538 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Appendix 4 ndash Statistical results
Level 1
Nombre de laboratoires (p) 10
A 1602 1602 1602 000 0000 0000 -0495 0000 A 1845 1602 1724 017 0122 -0122 0621 0840 0122
B 1602 1176 1389 030 0213 -0213 -2613 1247 B 1602 1301 1452 021 0151 -0151 -0769 1041 0062
C 1699 1301 1500 028 0199 -0199 -1510 1165 C 1301 1845 1573 038 -0272 0272 -0148 1881 0073
D 1602 1778 1690 012 -0088 0088 0380 0516 D 1602 1602 1602 000 0000 0000 0000 0000 -0088
E 1699 1602 1651 007 0048 -0048 -0013 0284 E 1699 1301 1500 028 0199 -0199 -0522 1376 -0151
F 1778 1301 1540 034 0239 -0239 -1116 1397 F 1778 1699 1739 006 0040 -0040 0698 0274 0199
G 1845 1544 1695 021 0151 -0151 0425 0881 G 2079 1699 1889 027 0190 -0190 1467 1315 0194
H 2041 1602 1822 031 0220 -0220 1689 1286 H 2000 1954 1977 003 0023 -0023 1917 0158 0155
L 1544 1778 1661 017 -0117 0117 0092 0685 L 1301 1602 1452 021 -0151 0151 -0769 1041 -0210
M 1653 1653 1653 000 0000 0000 0013 0000 M 1477 1602 1540 009 -0062 0062 -0319 0432 -0114
m 165186 m 160206 mediane D 00678
Cn(2p) 1867 Cn(2p) 1867 Cp 16101
Cn(p) 16101 Cn(p) 16101 Qn(D) 0092926
n=2p n 20 n=2p n 20 Qdiff 01496169
f 11 f 11 t 114
l 55 l 55
n=p n 10 n=p n 10
f 6 f 6
l 15 l 15
Qn(2p) 0091465 Qn(2p) 007745
Qn(p) 0062469 Qn(p) 0121519
Qintra 017078 Qintra 014461
Qinter 010058 Qinter 019565
Sr(eacutecart-type de reacutepeacutetabiliteacute) 02415 Sr 02045
RSDr(coefficient de variation de la reacutepeacutetabiliteacute)1462 RSDr 1277
r(repeatability limit)) 0676 r 0573
SL(eacutecart-type interlaboratoire) -001905 SLlt0 SL 001737
SR(eacutecart-type de reproductibiliteacute) 02415 SR 02433
RSDR(coefficient de variation de la reproductibiliteacute)0146 RSDR 0152
R(limite de reproductibiliteacute)) 06762 R(limite de reproductibiliteacute))068123
h k h MoyenneLaboratoires
Niveau 1
si LaboratoiresDeviationMoyenne Dk DeviationsiAlt
Niveau 1
Ref
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3638 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Level 2
Nombre de laboratoires (p) 10
A 2681 2613 2647 005 0034 -0034 -0151 0891 A 2322 2531 2427 015 -0105 0105 -2119 1620 -0220
B 2544 2477 2511 005 0033 -0033 -2699 0872 B 2556 2653 2605 007 -0048 0048 -0581 0750 0094
C 2643 2633 2638 001 0005 -0005 -0311 0130 C 2681 2653 2667 002 0014 -0014 -0040 0217 0029
D 2690 2663 2676 002 0014 -0014 0400 0357 D 2778 2732 2755 003 0023 -0023 0721 0354 0079
E 2690 2716 2703 002 -0013 0013 0897 0336 E 2724 2748 2736 002 -0012 0012 0556 0185 0033
F 2568 2398 2483 012 0085 -0085 -3214 2217 F 2477 2114 2296 026 0182 -0182 -3255 2811 -0188
G 2643 2591 2617 004 0026 -0026 -0707 0682 G 2756 2568 2662 013 0094 -0094 -0085 1453 0045
H 2716 2643 2680 005 0036 -0036 0460 0945 H 2653 2732 2693 006 -0040 0040 0181 0613 0013
L 2591 2591 2591 000 0000 0000 -1196 0000 L 2230 2580 2405 025 -0175 0175 -2307 2704 -0186
M 2653 2690 2672 003 -0018 0018 0311 0481 M 2740 2613 2677 009 0064 -0064 0040 0987 0005
m 2655082 m 26719001 mediane D 00209
Cn(2p) 1867 Cn(2p) 1867 Cp 16101
Cn(p) 16101 Cn(p) 16101 Qn(D) 0039911
n=2p n 20 n=2p n 20 Qdiff 00642591
f 11 f 11 t 082
l 55 l 55
n=p n 10 n=p n 10
f 6 f 6
l 15 l 15
Qn(2p) 0020570 Qn(2p) 00345985
Qn(p) 00332437 Qn(p) 00718158
Qintra 003841 Qintra 006460
Qinter 005352 Qinter 011563
Sr(eacutecart-type de reacutepeacutetabiliteacute) 00543 Sr 00914
RSDr(coefficient de variation de la reacutepeacutetabiliteacute)205 RSDr 342
r(repeatability limit)) 0152 r 0256
SL(eacutecart-type interlaboratoire) 000139 SL 000920
SR(eacutecart-type de reproductibiliteacute) 00659 SR 01325
RSDR(coefficient de variation de la reproductibiliteacute)0025 RSDR 0050
R(limite de reproductibiliteacute)) 018446 R(limite de reproductibiliteacute))03708618
Laboratoires
Niveau 2
Moyenne si Laboratoires
Niveau 2
DRef Alt Moyenne si Deviation h Deviation h k k
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3738 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Level 2 bis
Nombre de laboratoires (p) 10
A 3000 2477 2739 037 0261 -0261 0832 1418 A 2602 2477 2540 009 0062 -0062 -0391 0465 -0199
B 2544 2813 2678 019 -0134 0134 0211 0729 B 2903 2778 2841 009 0062 -0062 1732 0465 0162
C 2477 2813 2645 024 -0168 0168 -0135 0911 C 2477 2699 2588 016 -0111 0111 -0049 0826 -0057
D 2398 2653 2526 018 -0128 0128 -1370 0692 D 2778 3000 2889 016 -0111 0111 2074 0826 0363
E 2875 2740 2808 010 0067 -0067 1547 0365 E 2602 2301 2452 021 0151 -0151 -1012 1120 -0356
F 2477 2544 2511 005 -0033 0033 -1525 0182 F 2778 2477 2628 021 0151 -0151 0230 1120 0117
G 2653 2740 2697 006 -0044 0044 0400 0236 G 2845 2845 2845 000 0000 0000 1764 0000 0148
H 2176 2813 2495 045 -0318 0318 -1692 1727 H 2477 2477 2477 000 0000 0000 -0832 0000 -0017
L 2813 2544 2678 019 0134 -0134 0211 0729 L 2903 2301 2602 043 0301 -0301 0049 2241 -0076
M 2602 2740 2671 010 -0069 0069 0135 0375 M 2903 2255 2579 046 0324 -0324 -0112 2411 -0092
m 2658114 m 259505 mediane D -00372
Cn(2p) 1867 Cn(2p) 1867 Cp 16101
Cn(p) 16101 Cn(p) 16101 Qn(D) 01656915
n=2p n 20 n=2p n 20 Qdiff 02667753
f 11 f 11 t 035
l 55 l 55
n=p n 10 n=p n 10
f 6 f 6
l 15 l 15
Qn(2p) 0098745 Qn(2p) 007195
Qn(p) 006007 Qn(p) 008805
Qintra 018437 Qintra 013435
Qinter 009672 Qinter 014176
Sr(eacutecart-type de reacutepeacutetabiliteacute) 02607 Sr 01900
RSDr(coefficient de variation de la reacutepeacutetabiliteacute)981 RSDr 732
r(repeatability limit)) 0730 r 0532
SL(eacutecart-type interlaboratoire) -002464 SLlt0 SL 000205
SR(eacutecart-type de reproductibiliteacute) 02607 SR 01953
RSDR(coefficient de variation de la reproductibiliteacute)0098 RSDR 0075
R(limite de reproductibiliteacute)) 072996 R(limite de reproductibiliteacute))054686
Laboratoires
Niveau 2 bis
Moyenne si Laboratoires
Niveau 2 bis
DRef Alt Moyenne si Deviation h Deviation h k k
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3838 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Level 3
Nombre de laboratoires (p) 10
A 3690 3663 3676 002 0014 -0014 -0311 0513 A 3690 3708 3699 001 -0009 0009 0354 0257 0022
B 3613 3672 3642 004 -0030 0030 -1851 1109 B 3708 3568 3638 010 0070 -0070 -0844 2061 -0005
C 3663 3732 3698 005 -0035 0035 0645 1302 C 3663 3699 3681 003 -0018 0018 0000 0536 -0017
D 3690 3690 3690 000 0000 0000 0311 0000 D 3643 3663 3653 001 -0010 0010 -0545 0286 -0037
E 3732 3653 3693 006 0040 -0040 0429 1480 E 3613 3732 3673 008 -0060 0060 -0162 1769 -0020
F 3643 3672 3658 002 -0014 0014 -1157 0536 F 3690 3771 3731 006 -0040 0040 0975 1193 0073
G 3699 3653 3676 003 0023 -0023 -0328 0856 G 3602 3643 3623 003 -0021 0021 -1141 0612 -0053
H 3623 3672 3648 003 -0024 0024 -1615 0913 H 3748 3681 3715 005 0033 -0033 0664 0990 0067
L 3724 3663 3694 004 0031 -0031 0461 1150 L 3748 3771 3760 002 -0011 0011 1544 0335 0066
M 3690 3690 3690 000 0000 0000 0311 0000 M 3806 3681 3744 009 0062 -0062 1234 1848 0054
m 368334 m 368086 mediane D 00089
Cn(2p) 1867 Cn(2p) 1867 Cp 16101
Cn(p) 16101 Cn(p) 16101 Qn(D) 00325359
n=2p n 20 n=2p n 20 Qdiff 00523852
f 11 f 11 t 043
l 55 l 55
n=p n 10 n=p n 10
f 6 f 6
l 15 l 15
Qn(2p) 0014323 Qn(2p) 001811
Qn(p) 0013719 Qn(p) 003164
Qintra 002674 Qintra 003381
Qinter 002209 Qinter 005094
Sr(eacutecart-type de reacutepeacutetabiliteacute) 00378 Sr 00478
RSDr(coefficient de variation de la reacutepeacutetabiliteacute)103 RSDr 130
r(repeatability limit)) 0106 r 0134
SL(eacutecart-type interlaboratoire) -000023 SLlt0 SL 000145
SR(eacutecart-type de reproductibiliteacute) 00378 SR 00611
RSDR(coefficient de variation de la reproductibiliteacute)0010 RSDR 0017
R(limite de reproductibiliteacute)) 0105894 R(limite de reproductibiliteacute))017119
Ref Alt DLaboratoires
Niveau 3
Moyenne si Deviation h k Laboratoires
Niveau 3
Moyenne si Deviation h k
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2738 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
The statistical test concludes to a non significant bias for all the inoculation
levels The bias varies from ndash 0037 to 0068 log CFUml
Comparison of the repeatability standard deviations
If the ratio Srj Alt Srj Ref of the repeatability standard deviations of the
alternative method and the reference method is larger than 2 the precision
under repeatability conditions of the alternative method is considered to be
lower than that of the reference method If this ratio is smaller than 05 the
precision under repeatability conditions of the alternative method is
considered to be greater than that of the reference method
The ratio values are given in table 18
Table 18
Level
Repeatability standard deviation
Reference method Alternative method Alternative method
reference method ratio
1 0242 0205 0847
2 0054 0091 1682
2 bis 0261 0190 0729
3 0038 0048 1264
Level Repeatability limit
Reference method Alternative method
1 0676 0573
2 0152 0256
2 bis 0730 0532
3 0106 0134
The ratios of the repeatability standard deviations are below 2 in all cases
The precision under repeatability conditions of the alternative method is
equivalent to that of the reference method
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2838 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Comparison of the reproducibility standard deviations
If the ratio Srj Alt Srj Ref of the reproducibility standard deviations of the alternative method and the
reference method is larger than 2 the precision under reproducibility conditions of the alternative
method is considered to be lower than that of the reference method If this ratio is smaller than 05 the
precision under reproducibility conditions of the alternative method is considered to be greater than
that of the reference method
The ratio values are given in table 19
Table 19
Level
Reproducibility standard deviation
Reference method Alternative method Alternative method
reference method ratio
1 0242 0243 1007
2 0066 0133 2011
2 bis 0261 0195 0749
3 0038 0061 1617
Level Reproducibility limit
Reference method Alternative method
1 0676 0681
2 0185 0371
2 bis 0730 0547
3 0106 0171
The ratios of the reproducibility standard deviations are below or close to 2
The precision under reproducibility conditions of the alternative method is
equivalent to that of the reference method
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2938 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
214 Conclusion
Method comparison study
The Listeria PrecisTM enumeration method shows satisfying linearity and
relative accuracy
The alternative method shows satisfying inclusivity and exclusivity results
Two Listeria monocytogenes strains gave negative results with the
alternative method These strains grew weakly on the reference method
media (OampA Palcam and Oxford)
The limits of repeatability of the alternative method are similar to those of the
reference method
The bias between both methods are low between - 0133 Log CFUg and
- 0008 Log CFUg
The Listeria PrecisTM enumeration method offers the saving for confirmatory
tests
Positive results are obtained with the alternative method in 2-3 days (with
OBIS Mono test) or in 4-7 days (by the classical tests described in the
standardized method) against 4-7 days for the reference method
Inter-laboratory study
The bias between the reference and the alternative methods shows satisfying
and correct values
- level 1 (100 - 1 000 CFUg) bias = + 007 log CFUg
- level 2 (1 000 - 10 000 CFUg) bias = 002 log CFUg
- level 2 bis (1 000 - 10 000 CFUg) bias = - 004 log CFUg
- level 3 (10 000 - 100 000 CFUg) bias = - 004 log CFUg
The repeatability and the reproducibility of the alternative method look similar
to those of the reference method
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3038 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
22 Extension study (realised by ASEPT 2007)
An extension study was performed in 2007 for a new confirmatory test OBIS
MONO
150 Listeria monocytogenes strains and 100 non-target strains were isolated
onto TSAYE and BrillianceTM Listeria Agar plates All the Listeria
monocytogenes and the non-target strains gave the expected results
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3138 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Appendix 1 ndash Flow diagram of the Listeria PrecisTM method
x g sample + 9 x ml buffered peptone water
Revivification step for 1 h plusmn 5 min at 20degC plusmn 1degC
Streaking 1 ml onto 3 Brilliance Listeria Agar plates (mother suspension)
or 01 ml onto 1 Brilliance Listeria Agar plates (mother suspension and decimal dilutions)
Incubation for 48 h plusmn 3 h at 37degC plusmn 1degC
Confirmatory tests on at least one typical colony
- by the tests described in the ISO method
- by applying the OBIS mono test on an isolated colony
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3238 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Appendix 2 ndash ISO 11290-2 (1998) amp ISO 11290-2A1 (2004) Horizontal
method for the detection and enumeration of Listeria monocytogenes -
Part 2 enumeration of Listeria monocytogenes in foods
10 g cheese + 90 ml buffered peptone water or Half Fraser Broth (without supplements) (110 dilution)
Stomach 1 min 30 seconds
Keep at 20degC plusmn 2degC for 1 h plusmn 5 min
Decimal dilutions in 9 ml peptone salt tubes
Dilution 110
Dilution 1100
Dilution 11000
1 ml on 3 Chromogenic Listeria medium (ISO) plates (x 2) (-1)
01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-2)
01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-3)
01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-4)
Incubation at 37degC plusmn 1degC
24 h - 48 h
Numerate typical colonies at 24 h
(bluegreen surrounded by an opaque halo)
Numerate at 48 h
Streak typical colonies on TSYEA
(5 coloniesplate 2 successive dilutions)
Confirmation tests
Catalase
Gram stain
For confirmation of L monocytogenes
Haemolysis
Carbohydrate (rhamnose and xylose) utilisation
CAMP Test
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3338 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Appendix 3 ndash Mandelrsquos graphics
Laboratoire h Ref
Niveau 1 h Ref
Niveau 2 h Ref
Niveau 3 h Alt
Niveau 1 h Alt
Niveau 2 h Alt
Niveau 3 h5 h1 h5 h1
A -050 -015 083 062 -212 -039 198 299 -198 -299
B -261 -270 021 -077 -058 173 198 299 -198 -299
C -151 -031 -014 -015 -004 -005 198 299 -198 -299
D 038 040 -137 000 072 207 198 299 -198 -299
E -001 090 155 -052 056 -101 198 299 -198 -299
F -112 -321 -153 070 -325 023 198 299 -198 -299
G 042 -071 040 147 -009 176 198 299 -198 -299
H 169 046 -169 192 018 -083 198 299 -198 -299
L 009 -120 021 -077 -231 005 198 299 -198 -299
M 001 031 014 -032 004 -011 198 299 -198 -299
-300
-200
-100
000
100
200
300
A B C D E F G H L M
Vale
urs
h d
e M
an
del(
rob
uste
s)
Laboratoires
Meacutethode de reacutefeacuterence analyse robuste
h Ref Niveau 1
h Ref Niveau 2
h Ref Niveau 3
h 5=198
h 1=299
-300
-200
-100
000
100
200
300
A B C D E F G H L M
Vale
urs
de h
de M
an
del(
rob
uste
s)
Laboratoires
Meacutethode alternative analyse robuste
h Alt Niveau 1
h Alt Niveau 2
h Alt Niveau 2
h 1=299
h 5=198
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3438 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Laboratoire k Ref
Niveau 1 k Ref
Niveau 2 k Ref
Niveau 3 k Alt
Niveau 1 k Alt
Niveau 2 k Alt
Niveau 3 k1 k5
A 000 089 142 084 162 046 259 181
B 125 087 073 104 075 046 259 181
C 117 013 091 188 022 083 259 181
D 052 036 069 000 035 083 259 181
E 028 034 037 138 019 112 259 181
F 140 222 018 027 281 112 259 181
G 088 068 024 131 145 000 259 181
H 129 094 173 016 061 000 259 181
L 069 000 073 104 270 224 259 181
M 000 048 038 043 099 241 259 181
000
050
100
150
200
250
300
350
400
A B C D E F G H L M
Vale
urs
k d
e M
an
del(
rob
uste
s)
Laboratoires
Meacutethode de reacutefeacuterence analyse robuste
k Ref Niveau 1k Ref Niveau 2k Ref Niveau 3
k 1=259
k 5=181
000
050
100
150
200
250
300
A B C D E F G H L M
Vale
urs
k d
e M
an
del
(ro
bu
ste
s)
Laboratoires
Meacutethode alternative analyse robuste
k Alt Niveau 3
k Alt Niveau 1
k Alt Niveau 2
k 1=259
k 5=181
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3538 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Appendix 4 ndash Statistical results
Level 1
Nombre de laboratoires (p) 10
A 1602 1602 1602 000 0000 0000 -0495 0000 A 1845 1602 1724 017 0122 -0122 0621 0840 0122
B 1602 1176 1389 030 0213 -0213 -2613 1247 B 1602 1301 1452 021 0151 -0151 -0769 1041 0062
C 1699 1301 1500 028 0199 -0199 -1510 1165 C 1301 1845 1573 038 -0272 0272 -0148 1881 0073
D 1602 1778 1690 012 -0088 0088 0380 0516 D 1602 1602 1602 000 0000 0000 0000 0000 -0088
E 1699 1602 1651 007 0048 -0048 -0013 0284 E 1699 1301 1500 028 0199 -0199 -0522 1376 -0151
F 1778 1301 1540 034 0239 -0239 -1116 1397 F 1778 1699 1739 006 0040 -0040 0698 0274 0199
G 1845 1544 1695 021 0151 -0151 0425 0881 G 2079 1699 1889 027 0190 -0190 1467 1315 0194
H 2041 1602 1822 031 0220 -0220 1689 1286 H 2000 1954 1977 003 0023 -0023 1917 0158 0155
L 1544 1778 1661 017 -0117 0117 0092 0685 L 1301 1602 1452 021 -0151 0151 -0769 1041 -0210
M 1653 1653 1653 000 0000 0000 0013 0000 M 1477 1602 1540 009 -0062 0062 -0319 0432 -0114
m 165186 m 160206 mediane D 00678
Cn(2p) 1867 Cn(2p) 1867 Cp 16101
Cn(p) 16101 Cn(p) 16101 Qn(D) 0092926
n=2p n 20 n=2p n 20 Qdiff 01496169
f 11 f 11 t 114
l 55 l 55
n=p n 10 n=p n 10
f 6 f 6
l 15 l 15
Qn(2p) 0091465 Qn(2p) 007745
Qn(p) 0062469 Qn(p) 0121519
Qintra 017078 Qintra 014461
Qinter 010058 Qinter 019565
Sr(eacutecart-type de reacutepeacutetabiliteacute) 02415 Sr 02045
RSDr(coefficient de variation de la reacutepeacutetabiliteacute)1462 RSDr 1277
r(repeatability limit)) 0676 r 0573
SL(eacutecart-type interlaboratoire) -001905 SLlt0 SL 001737
SR(eacutecart-type de reproductibiliteacute) 02415 SR 02433
RSDR(coefficient de variation de la reproductibiliteacute)0146 RSDR 0152
R(limite de reproductibiliteacute)) 06762 R(limite de reproductibiliteacute))068123
h k h MoyenneLaboratoires
Niveau 1
si LaboratoiresDeviationMoyenne Dk DeviationsiAlt
Niveau 1
Ref
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3638 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Level 2
Nombre de laboratoires (p) 10
A 2681 2613 2647 005 0034 -0034 -0151 0891 A 2322 2531 2427 015 -0105 0105 -2119 1620 -0220
B 2544 2477 2511 005 0033 -0033 -2699 0872 B 2556 2653 2605 007 -0048 0048 -0581 0750 0094
C 2643 2633 2638 001 0005 -0005 -0311 0130 C 2681 2653 2667 002 0014 -0014 -0040 0217 0029
D 2690 2663 2676 002 0014 -0014 0400 0357 D 2778 2732 2755 003 0023 -0023 0721 0354 0079
E 2690 2716 2703 002 -0013 0013 0897 0336 E 2724 2748 2736 002 -0012 0012 0556 0185 0033
F 2568 2398 2483 012 0085 -0085 -3214 2217 F 2477 2114 2296 026 0182 -0182 -3255 2811 -0188
G 2643 2591 2617 004 0026 -0026 -0707 0682 G 2756 2568 2662 013 0094 -0094 -0085 1453 0045
H 2716 2643 2680 005 0036 -0036 0460 0945 H 2653 2732 2693 006 -0040 0040 0181 0613 0013
L 2591 2591 2591 000 0000 0000 -1196 0000 L 2230 2580 2405 025 -0175 0175 -2307 2704 -0186
M 2653 2690 2672 003 -0018 0018 0311 0481 M 2740 2613 2677 009 0064 -0064 0040 0987 0005
m 2655082 m 26719001 mediane D 00209
Cn(2p) 1867 Cn(2p) 1867 Cp 16101
Cn(p) 16101 Cn(p) 16101 Qn(D) 0039911
n=2p n 20 n=2p n 20 Qdiff 00642591
f 11 f 11 t 082
l 55 l 55
n=p n 10 n=p n 10
f 6 f 6
l 15 l 15
Qn(2p) 0020570 Qn(2p) 00345985
Qn(p) 00332437 Qn(p) 00718158
Qintra 003841 Qintra 006460
Qinter 005352 Qinter 011563
Sr(eacutecart-type de reacutepeacutetabiliteacute) 00543 Sr 00914
RSDr(coefficient de variation de la reacutepeacutetabiliteacute)205 RSDr 342
r(repeatability limit)) 0152 r 0256
SL(eacutecart-type interlaboratoire) 000139 SL 000920
SR(eacutecart-type de reproductibiliteacute) 00659 SR 01325
RSDR(coefficient de variation de la reproductibiliteacute)0025 RSDR 0050
R(limite de reproductibiliteacute)) 018446 R(limite de reproductibiliteacute))03708618
Laboratoires
Niveau 2
Moyenne si Laboratoires
Niveau 2
DRef Alt Moyenne si Deviation h Deviation h k k
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3738 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Level 2 bis
Nombre de laboratoires (p) 10
A 3000 2477 2739 037 0261 -0261 0832 1418 A 2602 2477 2540 009 0062 -0062 -0391 0465 -0199
B 2544 2813 2678 019 -0134 0134 0211 0729 B 2903 2778 2841 009 0062 -0062 1732 0465 0162
C 2477 2813 2645 024 -0168 0168 -0135 0911 C 2477 2699 2588 016 -0111 0111 -0049 0826 -0057
D 2398 2653 2526 018 -0128 0128 -1370 0692 D 2778 3000 2889 016 -0111 0111 2074 0826 0363
E 2875 2740 2808 010 0067 -0067 1547 0365 E 2602 2301 2452 021 0151 -0151 -1012 1120 -0356
F 2477 2544 2511 005 -0033 0033 -1525 0182 F 2778 2477 2628 021 0151 -0151 0230 1120 0117
G 2653 2740 2697 006 -0044 0044 0400 0236 G 2845 2845 2845 000 0000 0000 1764 0000 0148
H 2176 2813 2495 045 -0318 0318 -1692 1727 H 2477 2477 2477 000 0000 0000 -0832 0000 -0017
L 2813 2544 2678 019 0134 -0134 0211 0729 L 2903 2301 2602 043 0301 -0301 0049 2241 -0076
M 2602 2740 2671 010 -0069 0069 0135 0375 M 2903 2255 2579 046 0324 -0324 -0112 2411 -0092
m 2658114 m 259505 mediane D -00372
Cn(2p) 1867 Cn(2p) 1867 Cp 16101
Cn(p) 16101 Cn(p) 16101 Qn(D) 01656915
n=2p n 20 n=2p n 20 Qdiff 02667753
f 11 f 11 t 035
l 55 l 55
n=p n 10 n=p n 10
f 6 f 6
l 15 l 15
Qn(2p) 0098745 Qn(2p) 007195
Qn(p) 006007 Qn(p) 008805
Qintra 018437 Qintra 013435
Qinter 009672 Qinter 014176
Sr(eacutecart-type de reacutepeacutetabiliteacute) 02607 Sr 01900
RSDr(coefficient de variation de la reacutepeacutetabiliteacute)981 RSDr 732
r(repeatability limit)) 0730 r 0532
SL(eacutecart-type interlaboratoire) -002464 SLlt0 SL 000205
SR(eacutecart-type de reproductibiliteacute) 02607 SR 01953
RSDR(coefficient de variation de la reproductibiliteacute)0098 RSDR 0075
R(limite de reproductibiliteacute)) 072996 R(limite de reproductibiliteacute))054686
Laboratoires
Niveau 2 bis
Moyenne si Laboratoires
Niveau 2 bis
DRef Alt Moyenne si Deviation h Deviation h k k
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3838 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Level 3
Nombre de laboratoires (p) 10
A 3690 3663 3676 002 0014 -0014 -0311 0513 A 3690 3708 3699 001 -0009 0009 0354 0257 0022
B 3613 3672 3642 004 -0030 0030 -1851 1109 B 3708 3568 3638 010 0070 -0070 -0844 2061 -0005
C 3663 3732 3698 005 -0035 0035 0645 1302 C 3663 3699 3681 003 -0018 0018 0000 0536 -0017
D 3690 3690 3690 000 0000 0000 0311 0000 D 3643 3663 3653 001 -0010 0010 -0545 0286 -0037
E 3732 3653 3693 006 0040 -0040 0429 1480 E 3613 3732 3673 008 -0060 0060 -0162 1769 -0020
F 3643 3672 3658 002 -0014 0014 -1157 0536 F 3690 3771 3731 006 -0040 0040 0975 1193 0073
G 3699 3653 3676 003 0023 -0023 -0328 0856 G 3602 3643 3623 003 -0021 0021 -1141 0612 -0053
H 3623 3672 3648 003 -0024 0024 -1615 0913 H 3748 3681 3715 005 0033 -0033 0664 0990 0067
L 3724 3663 3694 004 0031 -0031 0461 1150 L 3748 3771 3760 002 -0011 0011 1544 0335 0066
M 3690 3690 3690 000 0000 0000 0311 0000 M 3806 3681 3744 009 0062 -0062 1234 1848 0054
m 368334 m 368086 mediane D 00089
Cn(2p) 1867 Cn(2p) 1867 Cp 16101
Cn(p) 16101 Cn(p) 16101 Qn(D) 00325359
n=2p n 20 n=2p n 20 Qdiff 00523852
f 11 f 11 t 043
l 55 l 55
n=p n 10 n=p n 10
f 6 f 6
l 15 l 15
Qn(2p) 0014323 Qn(2p) 001811
Qn(p) 0013719 Qn(p) 003164
Qintra 002674 Qintra 003381
Qinter 002209 Qinter 005094
Sr(eacutecart-type de reacutepeacutetabiliteacute) 00378 Sr 00478
RSDr(coefficient de variation de la reacutepeacutetabiliteacute)103 RSDr 130
r(repeatability limit)) 0106 r 0134
SL(eacutecart-type interlaboratoire) -000023 SLlt0 SL 000145
SR(eacutecart-type de reproductibiliteacute) 00378 SR 00611
RSDR(coefficient de variation de la reproductibiliteacute)0010 RSDR 0017
R(limite de reproductibiliteacute)) 0105894 R(limite de reproductibiliteacute))017119
Ref Alt DLaboratoires
Niveau 3
Moyenne si Deviation h k Laboratoires
Niveau 3
Moyenne si Deviation h k
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2838 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Comparison of the reproducibility standard deviations
If the ratio Srj Alt Srj Ref of the reproducibility standard deviations of the alternative method and the
reference method is larger than 2 the precision under reproducibility conditions of the alternative
method is considered to be lower than that of the reference method If this ratio is smaller than 05 the
precision under reproducibility conditions of the alternative method is considered to be greater than
that of the reference method
The ratio values are given in table 19
Table 19
Level
Reproducibility standard deviation
Reference method Alternative method Alternative method
reference method ratio
1 0242 0243 1007
2 0066 0133 2011
2 bis 0261 0195 0749
3 0038 0061 1617
Level Reproducibility limit
Reference method Alternative method
1 0676 0681
2 0185 0371
2 bis 0730 0547
3 0106 0171
The ratios of the reproducibility standard deviations are below or close to 2
The precision under reproducibility conditions of the alternative method is
equivalent to that of the reference method
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2938 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
214 Conclusion
Method comparison study
The Listeria PrecisTM enumeration method shows satisfying linearity and
relative accuracy
The alternative method shows satisfying inclusivity and exclusivity results
Two Listeria monocytogenes strains gave negative results with the
alternative method These strains grew weakly on the reference method
media (OampA Palcam and Oxford)
The limits of repeatability of the alternative method are similar to those of the
reference method
The bias between both methods are low between - 0133 Log CFUg and
- 0008 Log CFUg
The Listeria PrecisTM enumeration method offers the saving for confirmatory
tests
Positive results are obtained with the alternative method in 2-3 days (with
OBIS Mono test) or in 4-7 days (by the classical tests described in the
standardized method) against 4-7 days for the reference method
Inter-laboratory study
The bias between the reference and the alternative methods shows satisfying
and correct values
- level 1 (100 - 1 000 CFUg) bias = + 007 log CFUg
- level 2 (1 000 - 10 000 CFUg) bias = 002 log CFUg
- level 2 bis (1 000 - 10 000 CFUg) bias = - 004 log CFUg
- level 3 (10 000 - 100 000 CFUg) bias = - 004 log CFUg
The repeatability and the reproducibility of the alternative method look similar
to those of the reference method
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3038 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
22 Extension study (realised by ASEPT 2007)
An extension study was performed in 2007 for a new confirmatory test OBIS
MONO
150 Listeria monocytogenes strains and 100 non-target strains were isolated
onto TSAYE and BrillianceTM Listeria Agar plates All the Listeria
monocytogenes and the non-target strains gave the expected results
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3138 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Appendix 1 ndash Flow diagram of the Listeria PrecisTM method
x g sample + 9 x ml buffered peptone water
Revivification step for 1 h plusmn 5 min at 20degC plusmn 1degC
Streaking 1 ml onto 3 Brilliance Listeria Agar plates (mother suspension)
or 01 ml onto 1 Brilliance Listeria Agar plates (mother suspension and decimal dilutions)
Incubation for 48 h plusmn 3 h at 37degC plusmn 1degC
Confirmatory tests on at least one typical colony
- by the tests described in the ISO method
- by applying the OBIS mono test on an isolated colony
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3238 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Appendix 2 ndash ISO 11290-2 (1998) amp ISO 11290-2A1 (2004) Horizontal
method for the detection and enumeration of Listeria monocytogenes -
Part 2 enumeration of Listeria monocytogenes in foods
10 g cheese + 90 ml buffered peptone water or Half Fraser Broth (without supplements) (110 dilution)
Stomach 1 min 30 seconds
Keep at 20degC plusmn 2degC for 1 h plusmn 5 min
Decimal dilutions in 9 ml peptone salt tubes
Dilution 110
Dilution 1100
Dilution 11000
1 ml on 3 Chromogenic Listeria medium (ISO) plates (x 2) (-1)
01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-2)
01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-3)
01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-4)
Incubation at 37degC plusmn 1degC
24 h - 48 h
Numerate typical colonies at 24 h
(bluegreen surrounded by an opaque halo)
Numerate at 48 h
Streak typical colonies on TSYEA
(5 coloniesplate 2 successive dilutions)
Confirmation tests
Catalase
Gram stain
For confirmation of L monocytogenes
Haemolysis
Carbohydrate (rhamnose and xylose) utilisation
CAMP Test
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3338 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Appendix 3 ndash Mandelrsquos graphics
Laboratoire h Ref
Niveau 1 h Ref
Niveau 2 h Ref
Niveau 3 h Alt
Niveau 1 h Alt
Niveau 2 h Alt
Niveau 3 h5 h1 h5 h1
A -050 -015 083 062 -212 -039 198 299 -198 -299
B -261 -270 021 -077 -058 173 198 299 -198 -299
C -151 -031 -014 -015 -004 -005 198 299 -198 -299
D 038 040 -137 000 072 207 198 299 -198 -299
E -001 090 155 -052 056 -101 198 299 -198 -299
F -112 -321 -153 070 -325 023 198 299 -198 -299
G 042 -071 040 147 -009 176 198 299 -198 -299
H 169 046 -169 192 018 -083 198 299 -198 -299
L 009 -120 021 -077 -231 005 198 299 -198 -299
M 001 031 014 -032 004 -011 198 299 -198 -299
-300
-200
-100
000
100
200
300
A B C D E F G H L M
Vale
urs
h d
e M
an
del(
rob
uste
s)
Laboratoires
Meacutethode de reacutefeacuterence analyse robuste
h Ref Niveau 1
h Ref Niveau 2
h Ref Niveau 3
h 5=198
h 1=299
-300
-200
-100
000
100
200
300
A B C D E F G H L M
Vale
urs
de h
de M
an
del(
rob
uste
s)
Laboratoires
Meacutethode alternative analyse robuste
h Alt Niveau 1
h Alt Niveau 2
h Alt Niveau 2
h 1=299
h 5=198
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3438 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Laboratoire k Ref
Niveau 1 k Ref
Niveau 2 k Ref
Niveau 3 k Alt
Niveau 1 k Alt
Niveau 2 k Alt
Niveau 3 k1 k5
A 000 089 142 084 162 046 259 181
B 125 087 073 104 075 046 259 181
C 117 013 091 188 022 083 259 181
D 052 036 069 000 035 083 259 181
E 028 034 037 138 019 112 259 181
F 140 222 018 027 281 112 259 181
G 088 068 024 131 145 000 259 181
H 129 094 173 016 061 000 259 181
L 069 000 073 104 270 224 259 181
M 000 048 038 043 099 241 259 181
000
050
100
150
200
250
300
350
400
A B C D E F G H L M
Vale
urs
k d
e M
an
del(
rob
uste
s)
Laboratoires
Meacutethode de reacutefeacuterence analyse robuste
k Ref Niveau 1k Ref Niveau 2k Ref Niveau 3
k 1=259
k 5=181
000
050
100
150
200
250
300
A B C D E F G H L M
Vale
urs
k d
e M
an
del
(ro
bu
ste
s)
Laboratoires
Meacutethode alternative analyse robuste
k Alt Niveau 3
k Alt Niveau 1
k Alt Niveau 2
k 1=259
k 5=181
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3538 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Appendix 4 ndash Statistical results
Level 1
Nombre de laboratoires (p) 10
A 1602 1602 1602 000 0000 0000 -0495 0000 A 1845 1602 1724 017 0122 -0122 0621 0840 0122
B 1602 1176 1389 030 0213 -0213 -2613 1247 B 1602 1301 1452 021 0151 -0151 -0769 1041 0062
C 1699 1301 1500 028 0199 -0199 -1510 1165 C 1301 1845 1573 038 -0272 0272 -0148 1881 0073
D 1602 1778 1690 012 -0088 0088 0380 0516 D 1602 1602 1602 000 0000 0000 0000 0000 -0088
E 1699 1602 1651 007 0048 -0048 -0013 0284 E 1699 1301 1500 028 0199 -0199 -0522 1376 -0151
F 1778 1301 1540 034 0239 -0239 -1116 1397 F 1778 1699 1739 006 0040 -0040 0698 0274 0199
G 1845 1544 1695 021 0151 -0151 0425 0881 G 2079 1699 1889 027 0190 -0190 1467 1315 0194
H 2041 1602 1822 031 0220 -0220 1689 1286 H 2000 1954 1977 003 0023 -0023 1917 0158 0155
L 1544 1778 1661 017 -0117 0117 0092 0685 L 1301 1602 1452 021 -0151 0151 -0769 1041 -0210
M 1653 1653 1653 000 0000 0000 0013 0000 M 1477 1602 1540 009 -0062 0062 -0319 0432 -0114
m 165186 m 160206 mediane D 00678
Cn(2p) 1867 Cn(2p) 1867 Cp 16101
Cn(p) 16101 Cn(p) 16101 Qn(D) 0092926
n=2p n 20 n=2p n 20 Qdiff 01496169
f 11 f 11 t 114
l 55 l 55
n=p n 10 n=p n 10
f 6 f 6
l 15 l 15
Qn(2p) 0091465 Qn(2p) 007745
Qn(p) 0062469 Qn(p) 0121519
Qintra 017078 Qintra 014461
Qinter 010058 Qinter 019565
Sr(eacutecart-type de reacutepeacutetabiliteacute) 02415 Sr 02045
RSDr(coefficient de variation de la reacutepeacutetabiliteacute)1462 RSDr 1277
r(repeatability limit)) 0676 r 0573
SL(eacutecart-type interlaboratoire) -001905 SLlt0 SL 001737
SR(eacutecart-type de reproductibiliteacute) 02415 SR 02433
RSDR(coefficient de variation de la reproductibiliteacute)0146 RSDR 0152
R(limite de reproductibiliteacute)) 06762 R(limite de reproductibiliteacute))068123
h k h MoyenneLaboratoires
Niveau 1
si LaboratoiresDeviationMoyenne Dk DeviationsiAlt
Niveau 1
Ref
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3638 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Level 2
Nombre de laboratoires (p) 10
A 2681 2613 2647 005 0034 -0034 -0151 0891 A 2322 2531 2427 015 -0105 0105 -2119 1620 -0220
B 2544 2477 2511 005 0033 -0033 -2699 0872 B 2556 2653 2605 007 -0048 0048 -0581 0750 0094
C 2643 2633 2638 001 0005 -0005 -0311 0130 C 2681 2653 2667 002 0014 -0014 -0040 0217 0029
D 2690 2663 2676 002 0014 -0014 0400 0357 D 2778 2732 2755 003 0023 -0023 0721 0354 0079
E 2690 2716 2703 002 -0013 0013 0897 0336 E 2724 2748 2736 002 -0012 0012 0556 0185 0033
F 2568 2398 2483 012 0085 -0085 -3214 2217 F 2477 2114 2296 026 0182 -0182 -3255 2811 -0188
G 2643 2591 2617 004 0026 -0026 -0707 0682 G 2756 2568 2662 013 0094 -0094 -0085 1453 0045
H 2716 2643 2680 005 0036 -0036 0460 0945 H 2653 2732 2693 006 -0040 0040 0181 0613 0013
L 2591 2591 2591 000 0000 0000 -1196 0000 L 2230 2580 2405 025 -0175 0175 -2307 2704 -0186
M 2653 2690 2672 003 -0018 0018 0311 0481 M 2740 2613 2677 009 0064 -0064 0040 0987 0005
m 2655082 m 26719001 mediane D 00209
Cn(2p) 1867 Cn(2p) 1867 Cp 16101
Cn(p) 16101 Cn(p) 16101 Qn(D) 0039911
n=2p n 20 n=2p n 20 Qdiff 00642591
f 11 f 11 t 082
l 55 l 55
n=p n 10 n=p n 10
f 6 f 6
l 15 l 15
Qn(2p) 0020570 Qn(2p) 00345985
Qn(p) 00332437 Qn(p) 00718158
Qintra 003841 Qintra 006460
Qinter 005352 Qinter 011563
Sr(eacutecart-type de reacutepeacutetabiliteacute) 00543 Sr 00914
RSDr(coefficient de variation de la reacutepeacutetabiliteacute)205 RSDr 342
r(repeatability limit)) 0152 r 0256
SL(eacutecart-type interlaboratoire) 000139 SL 000920
SR(eacutecart-type de reproductibiliteacute) 00659 SR 01325
RSDR(coefficient de variation de la reproductibiliteacute)0025 RSDR 0050
R(limite de reproductibiliteacute)) 018446 R(limite de reproductibiliteacute))03708618
Laboratoires
Niveau 2
Moyenne si Laboratoires
Niveau 2
DRef Alt Moyenne si Deviation h Deviation h k k
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3738 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Level 2 bis
Nombre de laboratoires (p) 10
A 3000 2477 2739 037 0261 -0261 0832 1418 A 2602 2477 2540 009 0062 -0062 -0391 0465 -0199
B 2544 2813 2678 019 -0134 0134 0211 0729 B 2903 2778 2841 009 0062 -0062 1732 0465 0162
C 2477 2813 2645 024 -0168 0168 -0135 0911 C 2477 2699 2588 016 -0111 0111 -0049 0826 -0057
D 2398 2653 2526 018 -0128 0128 -1370 0692 D 2778 3000 2889 016 -0111 0111 2074 0826 0363
E 2875 2740 2808 010 0067 -0067 1547 0365 E 2602 2301 2452 021 0151 -0151 -1012 1120 -0356
F 2477 2544 2511 005 -0033 0033 -1525 0182 F 2778 2477 2628 021 0151 -0151 0230 1120 0117
G 2653 2740 2697 006 -0044 0044 0400 0236 G 2845 2845 2845 000 0000 0000 1764 0000 0148
H 2176 2813 2495 045 -0318 0318 -1692 1727 H 2477 2477 2477 000 0000 0000 -0832 0000 -0017
L 2813 2544 2678 019 0134 -0134 0211 0729 L 2903 2301 2602 043 0301 -0301 0049 2241 -0076
M 2602 2740 2671 010 -0069 0069 0135 0375 M 2903 2255 2579 046 0324 -0324 -0112 2411 -0092
m 2658114 m 259505 mediane D -00372
Cn(2p) 1867 Cn(2p) 1867 Cp 16101
Cn(p) 16101 Cn(p) 16101 Qn(D) 01656915
n=2p n 20 n=2p n 20 Qdiff 02667753
f 11 f 11 t 035
l 55 l 55
n=p n 10 n=p n 10
f 6 f 6
l 15 l 15
Qn(2p) 0098745 Qn(2p) 007195
Qn(p) 006007 Qn(p) 008805
Qintra 018437 Qintra 013435
Qinter 009672 Qinter 014176
Sr(eacutecart-type de reacutepeacutetabiliteacute) 02607 Sr 01900
RSDr(coefficient de variation de la reacutepeacutetabiliteacute)981 RSDr 732
r(repeatability limit)) 0730 r 0532
SL(eacutecart-type interlaboratoire) -002464 SLlt0 SL 000205
SR(eacutecart-type de reproductibiliteacute) 02607 SR 01953
RSDR(coefficient de variation de la reproductibiliteacute)0098 RSDR 0075
R(limite de reproductibiliteacute)) 072996 R(limite de reproductibiliteacute))054686
Laboratoires
Niveau 2 bis
Moyenne si Laboratoires
Niveau 2 bis
DRef Alt Moyenne si Deviation h Deviation h k k
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3838 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Level 3
Nombre de laboratoires (p) 10
A 3690 3663 3676 002 0014 -0014 -0311 0513 A 3690 3708 3699 001 -0009 0009 0354 0257 0022
B 3613 3672 3642 004 -0030 0030 -1851 1109 B 3708 3568 3638 010 0070 -0070 -0844 2061 -0005
C 3663 3732 3698 005 -0035 0035 0645 1302 C 3663 3699 3681 003 -0018 0018 0000 0536 -0017
D 3690 3690 3690 000 0000 0000 0311 0000 D 3643 3663 3653 001 -0010 0010 -0545 0286 -0037
E 3732 3653 3693 006 0040 -0040 0429 1480 E 3613 3732 3673 008 -0060 0060 -0162 1769 -0020
F 3643 3672 3658 002 -0014 0014 -1157 0536 F 3690 3771 3731 006 -0040 0040 0975 1193 0073
G 3699 3653 3676 003 0023 -0023 -0328 0856 G 3602 3643 3623 003 -0021 0021 -1141 0612 -0053
H 3623 3672 3648 003 -0024 0024 -1615 0913 H 3748 3681 3715 005 0033 -0033 0664 0990 0067
L 3724 3663 3694 004 0031 -0031 0461 1150 L 3748 3771 3760 002 -0011 0011 1544 0335 0066
M 3690 3690 3690 000 0000 0000 0311 0000 M 3806 3681 3744 009 0062 -0062 1234 1848 0054
m 368334 m 368086 mediane D 00089
Cn(2p) 1867 Cn(2p) 1867 Cp 16101
Cn(p) 16101 Cn(p) 16101 Qn(D) 00325359
n=2p n 20 n=2p n 20 Qdiff 00523852
f 11 f 11 t 043
l 55 l 55
n=p n 10 n=p n 10
f 6 f 6
l 15 l 15
Qn(2p) 0014323 Qn(2p) 001811
Qn(p) 0013719 Qn(p) 003164
Qintra 002674 Qintra 003381
Qinter 002209 Qinter 005094
Sr(eacutecart-type de reacutepeacutetabiliteacute) 00378 Sr 00478
RSDr(coefficient de variation de la reacutepeacutetabiliteacute)103 RSDr 130
r(repeatability limit)) 0106 r 0134
SL(eacutecart-type interlaboratoire) -000023 SLlt0 SL 000145
SR(eacutecart-type de reproductibiliteacute) 00378 SR 00611
RSDR(coefficient de variation de la reproductibiliteacute)0010 RSDR 0017
R(limite de reproductibiliteacute)) 0105894 R(limite de reproductibiliteacute))017119
Ref Alt DLaboratoires
Niveau 3
Moyenne si Deviation h k Laboratoires
Niveau 3
Moyenne si Deviation h k
Thermo Fisher Scientific
ADRIA Deacuteveloppement 2938 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
214 Conclusion
Method comparison study
The Listeria PrecisTM enumeration method shows satisfying linearity and
relative accuracy
The alternative method shows satisfying inclusivity and exclusivity results
Two Listeria monocytogenes strains gave negative results with the
alternative method These strains grew weakly on the reference method
media (OampA Palcam and Oxford)
The limits of repeatability of the alternative method are similar to those of the
reference method
The bias between both methods are low between - 0133 Log CFUg and
- 0008 Log CFUg
The Listeria PrecisTM enumeration method offers the saving for confirmatory
tests
Positive results are obtained with the alternative method in 2-3 days (with
OBIS Mono test) or in 4-7 days (by the classical tests described in the
standardized method) against 4-7 days for the reference method
Inter-laboratory study
The bias between the reference and the alternative methods shows satisfying
and correct values
- level 1 (100 - 1 000 CFUg) bias = + 007 log CFUg
- level 2 (1 000 - 10 000 CFUg) bias = 002 log CFUg
- level 2 bis (1 000 - 10 000 CFUg) bias = - 004 log CFUg
- level 3 (10 000 - 100 000 CFUg) bias = - 004 log CFUg
The repeatability and the reproducibility of the alternative method look similar
to those of the reference method
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3038 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
22 Extension study (realised by ASEPT 2007)
An extension study was performed in 2007 for a new confirmatory test OBIS
MONO
150 Listeria monocytogenes strains and 100 non-target strains were isolated
onto TSAYE and BrillianceTM Listeria Agar plates All the Listeria
monocytogenes and the non-target strains gave the expected results
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3138 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Appendix 1 ndash Flow diagram of the Listeria PrecisTM method
x g sample + 9 x ml buffered peptone water
Revivification step for 1 h plusmn 5 min at 20degC plusmn 1degC
Streaking 1 ml onto 3 Brilliance Listeria Agar plates (mother suspension)
or 01 ml onto 1 Brilliance Listeria Agar plates (mother suspension and decimal dilutions)
Incubation for 48 h plusmn 3 h at 37degC plusmn 1degC
Confirmatory tests on at least one typical colony
- by the tests described in the ISO method
- by applying the OBIS mono test on an isolated colony
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3238 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Appendix 2 ndash ISO 11290-2 (1998) amp ISO 11290-2A1 (2004) Horizontal
method for the detection and enumeration of Listeria monocytogenes -
Part 2 enumeration of Listeria monocytogenes in foods
10 g cheese + 90 ml buffered peptone water or Half Fraser Broth (without supplements) (110 dilution)
Stomach 1 min 30 seconds
Keep at 20degC plusmn 2degC for 1 h plusmn 5 min
Decimal dilutions in 9 ml peptone salt tubes
Dilution 110
Dilution 1100
Dilution 11000
1 ml on 3 Chromogenic Listeria medium (ISO) plates (x 2) (-1)
01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-2)
01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-3)
01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-4)
Incubation at 37degC plusmn 1degC
24 h - 48 h
Numerate typical colonies at 24 h
(bluegreen surrounded by an opaque halo)
Numerate at 48 h
Streak typical colonies on TSYEA
(5 coloniesplate 2 successive dilutions)
Confirmation tests
Catalase
Gram stain
For confirmation of L monocytogenes
Haemolysis
Carbohydrate (rhamnose and xylose) utilisation
CAMP Test
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3338 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Appendix 3 ndash Mandelrsquos graphics
Laboratoire h Ref
Niveau 1 h Ref
Niveau 2 h Ref
Niveau 3 h Alt
Niveau 1 h Alt
Niveau 2 h Alt
Niveau 3 h5 h1 h5 h1
A -050 -015 083 062 -212 -039 198 299 -198 -299
B -261 -270 021 -077 -058 173 198 299 -198 -299
C -151 -031 -014 -015 -004 -005 198 299 -198 -299
D 038 040 -137 000 072 207 198 299 -198 -299
E -001 090 155 -052 056 -101 198 299 -198 -299
F -112 -321 -153 070 -325 023 198 299 -198 -299
G 042 -071 040 147 -009 176 198 299 -198 -299
H 169 046 -169 192 018 -083 198 299 -198 -299
L 009 -120 021 -077 -231 005 198 299 -198 -299
M 001 031 014 -032 004 -011 198 299 -198 -299
-300
-200
-100
000
100
200
300
A B C D E F G H L M
Vale
urs
h d
e M
an
del(
rob
uste
s)
Laboratoires
Meacutethode de reacutefeacuterence analyse robuste
h Ref Niveau 1
h Ref Niveau 2
h Ref Niveau 3
h 5=198
h 1=299
-300
-200
-100
000
100
200
300
A B C D E F G H L M
Vale
urs
de h
de M
an
del(
rob
uste
s)
Laboratoires
Meacutethode alternative analyse robuste
h Alt Niveau 1
h Alt Niveau 2
h Alt Niveau 2
h 1=299
h 5=198
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3438 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Laboratoire k Ref
Niveau 1 k Ref
Niveau 2 k Ref
Niveau 3 k Alt
Niveau 1 k Alt
Niveau 2 k Alt
Niveau 3 k1 k5
A 000 089 142 084 162 046 259 181
B 125 087 073 104 075 046 259 181
C 117 013 091 188 022 083 259 181
D 052 036 069 000 035 083 259 181
E 028 034 037 138 019 112 259 181
F 140 222 018 027 281 112 259 181
G 088 068 024 131 145 000 259 181
H 129 094 173 016 061 000 259 181
L 069 000 073 104 270 224 259 181
M 000 048 038 043 099 241 259 181
000
050
100
150
200
250
300
350
400
A B C D E F G H L M
Vale
urs
k d
e M
an
del(
rob
uste
s)
Laboratoires
Meacutethode de reacutefeacuterence analyse robuste
k Ref Niveau 1k Ref Niveau 2k Ref Niveau 3
k 1=259
k 5=181
000
050
100
150
200
250
300
A B C D E F G H L M
Vale
urs
k d
e M
an
del
(ro
bu
ste
s)
Laboratoires
Meacutethode alternative analyse robuste
k Alt Niveau 3
k Alt Niveau 1
k Alt Niveau 2
k 1=259
k 5=181
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3538 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Appendix 4 ndash Statistical results
Level 1
Nombre de laboratoires (p) 10
A 1602 1602 1602 000 0000 0000 -0495 0000 A 1845 1602 1724 017 0122 -0122 0621 0840 0122
B 1602 1176 1389 030 0213 -0213 -2613 1247 B 1602 1301 1452 021 0151 -0151 -0769 1041 0062
C 1699 1301 1500 028 0199 -0199 -1510 1165 C 1301 1845 1573 038 -0272 0272 -0148 1881 0073
D 1602 1778 1690 012 -0088 0088 0380 0516 D 1602 1602 1602 000 0000 0000 0000 0000 -0088
E 1699 1602 1651 007 0048 -0048 -0013 0284 E 1699 1301 1500 028 0199 -0199 -0522 1376 -0151
F 1778 1301 1540 034 0239 -0239 -1116 1397 F 1778 1699 1739 006 0040 -0040 0698 0274 0199
G 1845 1544 1695 021 0151 -0151 0425 0881 G 2079 1699 1889 027 0190 -0190 1467 1315 0194
H 2041 1602 1822 031 0220 -0220 1689 1286 H 2000 1954 1977 003 0023 -0023 1917 0158 0155
L 1544 1778 1661 017 -0117 0117 0092 0685 L 1301 1602 1452 021 -0151 0151 -0769 1041 -0210
M 1653 1653 1653 000 0000 0000 0013 0000 M 1477 1602 1540 009 -0062 0062 -0319 0432 -0114
m 165186 m 160206 mediane D 00678
Cn(2p) 1867 Cn(2p) 1867 Cp 16101
Cn(p) 16101 Cn(p) 16101 Qn(D) 0092926
n=2p n 20 n=2p n 20 Qdiff 01496169
f 11 f 11 t 114
l 55 l 55
n=p n 10 n=p n 10
f 6 f 6
l 15 l 15
Qn(2p) 0091465 Qn(2p) 007745
Qn(p) 0062469 Qn(p) 0121519
Qintra 017078 Qintra 014461
Qinter 010058 Qinter 019565
Sr(eacutecart-type de reacutepeacutetabiliteacute) 02415 Sr 02045
RSDr(coefficient de variation de la reacutepeacutetabiliteacute)1462 RSDr 1277
r(repeatability limit)) 0676 r 0573
SL(eacutecart-type interlaboratoire) -001905 SLlt0 SL 001737
SR(eacutecart-type de reproductibiliteacute) 02415 SR 02433
RSDR(coefficient de variation de la reproductibiliteacute)0146 RSDR 0152
R(limite de reproductibiliteacute)) 06762 R(limite de reproductibiliteacute))068123
h k h MoyenneLaboratoires
Niveau 1
si LaboratoiresDeviationMoyenne Dk DeviationsiAlt
Niveau 1
Ref
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3638 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Level 2
Nombre de laboratoires (p) 10
A 2681 2613 2647 005 0034 -0034 -0151 0891 A 2322 2531 2427 015 -0105 0105 -2119 1620 -0220
B 2544 2477 2511 005 0033 -0033 -2699 0872 B 2556 2653 2605 007 -0048 0048 -0581 0750 0094
C 2643 2633 2638 001 0005 -0005 -0311 0130 C 2681 2653 2667 002 0014 -0014 -0040 0217 0029
D 2690 2663 2676 002 0014 -0014 0400 0357 D 2778 2732 2755 003 0023 -0023 0721 0354 0079
E 2690 2716 2703 002 -0013 0013 0897 0336 E 2724 2748 2736 002 -0012 0012 0556 0185 0033
F 2568 2398 2483 012 0085 -0085 -3214 2217 F 2477 2114 2296 026 0182 -0182 -3255 2811 -0188
G 2643 2591 2617 004 0026 -0026 -0707 0682 G 2756 2568 2662 013 0094 -0094 -0085 1453 0045
H 2716 2643 2680 005 0036 -0036 0460 0945 H 2653 2732 2693 006 -0040 0040 0181 0613 0013
L 2591 2591 2591 000 0000 0000 -1196 0000 L 2230 2580 2405 025 -0175 0175 -2307 2704 -0186
M 2653 2690 2672 003 -0018 0018 0311 0481 M 2740 2613 2677 009 0064 -0064 0040 0987 0005
m 2655082 m 26719001 mediane D 00209
Cn(2p) 1867 Cn(2p) 1867 Cp 16101
Cn(p) 16101 Cn(p) 16101 Qn(D) 0039911
n=2p n 20 n=2p n 20 Qdiff 00642591
f 11 f 11 t 082
l 55 l 55
n=p n 10 n=p n 10
f 6 f 6
l 15 l 15
Qn(2p) 0020570 Qn(2p) 00345985
Qn(p) 00332437 Qn(p) 00718158
Qintra 003841 Qintra 006460
Qinter 005352 Qinter 011563
Sr(eacutecart-type de reacutepeacutetabiliteacute) 00543 Sr 00914
RSDr(coefficient de variation de la reacutepeacutetabiliteacute)205 RSDr 342
r(repeatability limit)) 0152 r 0256
SL(eacutecart-type interlaboratoire) 000139 SL 000920
SR(eacutecart-type de reproductibiliteacute) 00659 SR 01325
RSDR(coefficient de variation de la reproductibiliteacute)0025 RSDR 0050
R(limite de reproductibiliteacute)) 018446 R(limite de reproductibiliteacute))03708618
Laboratoires
Niveau 2
Moyenne si Laboratoires
Niveau 2
DRef Alt Moyenne si Deviation h Deviation h k k
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3738 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Level 2 bis
Nombre de laboratoires (p) 10
A 3000 2477 2739 037 0261 -0261 0832 1418 A 2602 2477 2540 009 0062 -0062 -0391 0465 -0199
B 2544 2813 2678 019 -0134 0134 0211 0729 B 2903 2778 2841 009 0062 -0062 1732 0465 0162
C 2477 2813 2645 024 -0168 0168 -0135 0911 C 2477 2699 2588 016 -0111 0111 -0049 0826 -0057
D 2398 2653 2526 018 -0128 0128 -1370 0692 D 2778 3000 2889 016 -0111 0111 2074 0826 0363
E 2875 2740 2808 010 0067 -0067 1547 0365 E 2602 2301 2452 021 0151 -0151 -1012 1120 -0356
F 2477 2544 2511 005 -0033 0033 -1525 0182 F 2778 2477 2628 021 0151 -0151 0230 1120 0117
G 2653 2740 2697 006 -0044 0044 0400 0236 G 2845 2845 2845 000 0000 0000 1764 0000 0148
H 2176 2813 2495 045 -0318 0318 -1692 1727 H 2477 2477 2477 000 0000 0000 -0832 0000 -0017
L 2813 2544 2678 019 0134 -0134 0211 0729 L 2903 2301 2602 043 0301 -0301 0049 2241 -0076
M 2602 2740 2671 010 -0069 0069 0135 0375 M 2903 2255 2579 046 0324 -0324 -0112 2411 -0092
m 2658114 m 259505 mediane D -00372
Cn(2p) 1867 Cn(2p) 1867 Cp 16101
Cn(p) 16101 Cn(p) 16101 Qn(D) 01656915
n=2p n 20 n=2p n 20 Qdiff 02667753
f 11 f 11 t 035
l 55 l 55
n=p n 10 n=p n 10
f 6 f 6
l 15 l 15
Qn(2p) 0098745 Qn(2p) 007195
Qn(p) 006007 Qn(p) 008805
Qintra 018437 Qintra 013435
Qinter 009672 Qinter 014176
Sr(eacutecart-type de reacutepeacutetabiliteacute) 02607 Sr 01900
RSDr(coefficient de variation de la reacutepeacutetabiliteacute)981 RSDr 732
r(repeatability limit)) 0730 r 0532
SL(eacutecart-type interlaboratoire) -002464 SLlt0 SL 000205
SR(eacutecart-type de reproductibiliteacute) 02607 SR 01953
RSDR(coefficient de variation de la reproductibiliteacute)0098 RSDR 0075
R(limite de reproductibiliteacute)) 072996 R(limite de reproductibiliteacute))054686
Laboratoires
Niveau 2 bis
Moyenne si Laboratoires
Niveau 2 bis
DRef Alt Moyenne si Deviation h Deviation h k k
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3838 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Level 3
Nombre de laboratoires (p) 10
A 3690 3663 3676 002 0014 -0014 -0311 0513 A 3690 3708 3699 001 -0009 0009 0354 0257 0022
B 3613 3672 3642 004 -0030 0030 -1851 1109 B 3708 3568 3638 010 0070 -0070 -0844 2061 -0005
C 3663 3732 3698 005 -0035 0035 0645 1302 C 3663 3699 3681 003 -0018 0018 0000 0536 -0017
D 3690 3690 3690 000 0000 0000 0311 0000 D 3643 3663 3653 001 -0010 0010 -0545 0286 -0037
E 3732 3653 3693 006 0040 -0040 0429 1480 E 3613 3732 3673 008 -0060 0060 -0162 1769 -0020
F 3643 3672 3658 002 -0014 0014 -1157 0536 F 3690 3771 3731 006 -0040 0040 0975 1193 0073
G 3699 3653 3676 003 0023 -0023 -0328 0856 G 3602 3643 3623 003 -0021 0021 -1141 0612 -0053
H 3623 3672 3648 003 -0024 0024 -1615 0913 H 3748 3681 3715 005 0033 -0033 0664 0990 0067
L 3724 3663 3694 004 0031 -0031 0461 1150 L 3748 3771 3760 002 -0011 0011 1544 0335 0066
M 3690 3690 3690 000 0000 0000 0311 0000 M 3806 3681 3744 009 0062 -0062 1234 1848 0054
m 368334 m 368086 mediane D 00089
Cn(2p) 1867 Cn(2p) 1867 Cp 16101
Cn(p) 16101 Cn(p) 16101 Qn(D) 00325359
n=2p n 20 n=2p n 20 Qdiff 00523852
f 11 f 11 t 043
l 55 l 55
n=p n 10 n=p n 10
f 6 f 6
l 15 l 15
Qn(2p) 0014323 Qn(2p) 001811
Qn(p) 0013719 Qn(p) 003164
Qintra 002674 Qintra 003381
Qinter 002209 Qinter 005094
Sr(eacutecart-type de reacutepeacutetabiliteacute) 00378 Sr 00478
RSDr(coefficient de variation de la reacutepeacutetabiliteacute)103 RSDr 130
r(repeatability limit)) 0106 r 0134
SL(eacutecart-type interlaboratoire) -000023 SLlt0 SL 000145
SR(eacutecart-type de reproductibiliteacute) 00378 SR 00611
RSDR(coefficient de variation de la reproductibiliteacute)0010 RSDR 0017
R(limite de reproductibiliteacute)) 0105894 R(limite de reproductibiliteacute))017119
Ref Alt DLaboratoires
Niveau 3
Moyenne si Deviation h k Laboratoires
Niveau 3
Moyenne si Deviation h k
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3038 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
22 Extension study (realised by ASEPT 2007)
An extension study was performed in 2007 for a new confirmatory test OBIS
MONO
150 Listeria monocytogenes strains and 100 non-target strains were isolated
onto TSAYE and BrillianceTM Listeria Agar plates All the Listeria
monocytogenes and the non-target strains gave the expected results
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3138 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Appendix 1 ndash Flow diagram of the Listeria PrecisTM method
x g sample + 9 x ml buffered peptone water
Revivification step for 1 h plusmn 5 min at 20degC plusmn 1degC
Streaking 1 ml onto 3 Brilliance Listeria Agar plates (mother suspension)
or 01 ml onto 1 Brilliance Listeria Agar plates (mother suspension and decimal dilutions)
Incubation for 48 h plusmn 3 h at 37degC plusmn 1degC
Confirmatory tests on at least one typical colony
- by the tests described in the ISO method
- by applying the OBIS mono test on an isolated colony
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3238 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Appendix 2 ndash ISO 11290-2 (1998) amp ISO 11290-2A1 (2004) Horizontal
method for the detection and enumeration of Listeria monocytogenes -
Part 2 enumeration of Listeria monocytogenes in foods
10 g cheese + 90 ml buffered peptone water or Half Fraser Broth (without supplements) (110 dilution)
Stomach 1 min 30 seconds
Keep at 20degC plusmn 2degC for 1 h plusmn 5 min
Decimal dilutions in 9 ml peptone salt tubes
Dilution 110
Dilution 1100
Dilution 11000
1 ml on 3 Chromogenic Listeria medium (ISO) plates (x 2) (-1)
01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-2)
01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-3)
01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-4)
Incubation at 37degC plusmn 1degC
24 h - 48 h
Numerate typical colonies at 24 h
(bluegreen surrounded by an opaque halo)
Numerate at 48 h
Streak typical colonies on TSYEA
(5 coloniesplate 2 successive dilutions)
Confirmation tests
Catalase
Gram stain
For confirmation of L monocytogenes
Haemolysis
Carbohydrate (rhamnose and xylose) utilisation
CAMP Test
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3338 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Appendix 3 ndash Mandelrsquos graphics
Laboratoire h Ref
Niveau 1 h Ref
Niveau 2 h Ref
Niveau 3 h Alt
Niveau 1 h Alt
Niveau 2 h Alt
Niveau 3 h5 h1 h5 h1
A -050 -015 083 062 -212 -039 198 299 -198 -299
B -261 -270 021 -077 -058 173 198 299 -198 -299
C -151 -031 -014 -015 -004 -005 198 299 -198 -299
D 038 040 -137 000 072 207 198 299 -198 -299
E -001 090 155 -052 056 -101 198 299 -198 -299
F -112 -321 -153 070 -325 023 198 299 -198 -299
G 042 -071 040 147 -009 176 198 299 -198 -299
H 169 046 -169 192 018 -083 198 299 -198 -299
L 009 -120 021 -077 -231 005 198 299 -198 -299
M 001 031 014 -032 004 -011 198 299 -198 -299
-300
-200
-100
000
100
200
300
A B C D E F G H L M
Vale
urs
h d
e M
an
del(
rob
uste
s)
Laboratoires
Meacutethode de reacutefeacuterence analyse robuste
h Ref Niveau 1
h Ref Niveau 2
h Ref Niveau 3
h 5=198
h 1=299
-300
-200
-100
000
100
200
300
A B C D E F G H L M
Vale
urs
de h
de M
an
del(
rob
uste
s)
Laboratoires
Meacutethode alternative analyse robuste
h Alt Niveau 1
h Alt Niveau 2
h Alt Niveau 2
h 1=299
h 5=198
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3438 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Laboratoire k Ref
Niveau 1 k Ref
Niveau 2 k Ref
Niveau 3 k Alt
Niveau 1 k Alt
Niveau 2 k Alt
Niveau 3 k1 k5
A 000 089 142 084 162 046 259 181
B 125 087 073 104 075 046 259 181
C 117 013 091 188 022 083 259 181
D 052 036 069 000 035 083 259 181
E 028 034 037 138 019 112 259 181
F 140 222 018 027 281 112 259 181
G 088 068 024 131 145 000 259 181
H 129 094 173 016 061 000 259 181
L 069 000 073 104 270 224 259 181
M 000 048 038 043 099 241 259 181
000
050
100
150
200
250
300
350
400
A B C D E F G H L M
Vale
urs
k d
e M
an
del(
rob
uste
s)
Laboratoires
Meacutethode de reacutefeacuterence analyse robuste
k Ref Niveau 1k Ref Niveau 2k Ref Niveau 3
k 1=259
k 5=181
000
050
100
150
200
250
300
A B C D E F G H L M
Vale
urs
k d
e M
an
del
(ro
bu
ste
s)
Laboratoires
Meacutethode alternative analyse robuste
k Alt Niveau 3
k Alt Niveau 1
k Alt Niveau 2
k 1=259
k 5=181
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3538 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Appendix 4 ndash Statistical results
Level 1
Nombre de laboratoires (p) 10
A 1602 1602 1602 000 0000 0000 -0495 0000 A 1845 1602 1724 017 0122 -0122 0621 0840 0122
B 1602 1176 1389 030 0213 -0213 -2613 1247 B 1602 1301 1452 021 0151 -0151 -0769 1041 0062
C 1699 1301 1500 028 0199 -0199 -1510 1165 C 1301 1845 1573 038 -0272 0272 -0148 1881 0073
D 1602 1778 1690 012 -0088 0088 0380 0516 D 1602 1602 1602 000 0000 0000 0000 0000 -0088
E 1699 1602 1651 007 0048 -0048 -0013 0284 E 1699 1301 1500 028 0199 -0199 -0522 1376 -0151
F 1778 1301 1540 034 0239 -0239 -1116 1397 F 1778 1699 1739 006 0040 -0040 0698 0274 0199
G 1845 1544 1695 021 0151 -0151 0425 0881 G 2079 1699 1889 027 0190 -0190 1467 1315 0194
H 2041 1602 1822 031 0220 -0220 1689 1286 H 2000 1954 1977 003 0023 -0023 1917 0158 0155
L 1544 1778 1661 017 -0117 0117 0092 0685 L 1301 1602 1452 021 -0151 0151 -0769 1041 -0210
M 1653 1653 1653 000 0000 0000 0013 0000 M 1477 1602 1540 009 -0062 0062 -0319 0432 -0114
m 165186 m 160206 mediane D 00678
Cn(2p) 1867 Cn(2p) 1867 Cp 16101
Cn(p) 16101 Cn(p) 16101 Qn(D) 0092926
n=2p n 20 n=2p n 20 Qdiff 01496169
f 11 f 11 t 114
l 55 l 55
n=p n 10 n=p n 10
f 6 f 6
l 15 l 15
Qn(2p) 0091465 Qn(2p) 007745
Qn(p) 0062469 Qn(p) 0121519
Qintra 017078 Qintra 014461
Qinter 010058 Qinter 019565
Sr(eacutecart-type de reacutepeacutetabiliteacute) 02415 Sr 02045
RSDr(coefficient de variation de la reacutepeacutetabiliteacute)1462 RSDr 1277
r(repeatability limit)) 0676 r 0573
SL(eacutecart-type interlaboratoire) -001905 SLlt0 SL 001737
SR(eacutecart-type de reproductibiliteacute) 02415 SR 02433
RSDR(coefficient de variation de la reproductibiliteacute)0146 RSDR 0152
R(limite de reproductibiliteacute)) 06762 R(limite de reproductibiliteacute))068123
h k h MoyenneLaboratoires
Niveau 1
si LaboratoiresDeviationMoyenne Dk DeviationsiAlt
Niveau 1
Ref
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3638 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Level 2
Nombre de laboratoires (p) 10
A 2681 2613 2647 005 0034 -0034 -0151 0891 A 2322 2531 2427 015 -0105 0105 -2119 1620 -0220
B 2544 2477 2511 005 0033 -0033 -2699 0872 B 2556 2653 2605 007 -0048 0048 -0581 0750 0094
C 2643 2633 2638 001 0005 -0005 -0311 0130 C 2681 2653 2667 002 0014 -0014 -0040 0217 0029
D 2690 2663 2676 002 0014 -0014 0400 0357 D 2778 2732 2755 003 0023 -0023 0721 0354 0079
E 2690 2716 2703 002 -0013 0013 0897 0336 E 2724 2748 2736 002 -0012 0012 0556 0185 0033
F 2568 2398 2483 012 0085 -0085 -3214 2217 F 2477 2114 2296 026 0182 -0182 -3255 2811 -0188
G 2643 2591 2617 004 0026 -0026 -0707 0682 G 2756 2568 2662 013 0094 -0094 -0085 1453 0045
H 2716 2643 2680 005 0036 -0036 0460 0945 H 2653 2732 2693 006 -0040 0040 0181 0613 0013
L 2591 2591 2591 000 0000 0000 -1196 0000 L 2230 2580 2405 025 -0175 0175 -2307 2704 -0186
M 2653 2690 2672 003 -0018 0018 0311 0481 M 2740 2613 2677 009 0064 -0064 0040 0987 0005
m 2655082 m 26719001 mediane D 00209
Cn(2p) 1867 Cn(2p) 1867 Cp 16101
Cn(p) 16101 Cn(p) 16101 Qn(D) 0039911
n=2p n 20 n=2p n 20 Qdiff 00642591
f 11 f 11 t 082
l 55 l 55
n=p n 10 n=p n 10
f 6 f 6
l 15 l 15
Qn(2p) 0020570 Qn(2p) 00345985
Qn(p) 00332437 Qn(p) 00718158
Qintra 003841 Qintra 006460
Qinter 005352 Qinter 011563
Sr(eacutecart-type de reacutepeacutetabiliteacute) 00543 Sr 00914
RSDr(coefficient de variation de la reacutepeacutetabiliteacute)205 RSDr 342
r(repeatability limit)) 0152 r 0256
SL(eacutecart-type interlaboratoire) 000139 SL 000920
SR(eacutecart-type de reproductibiliteacute) 00659 SR 01325
RSDR(coefficient de variation de la reproductibiliteacute)0025 RSDR 0050
R(limite de reproductibiliteacute)) 018446 R(limite de reproductibiliteacute))03708618
Laboratoires
Niveau 2
Moyenne si Laboratoires
Niveau 2
DRef Alt Moyenne si Deviation h Deviation h k k
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3738 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Level 2 bis
Nombre de laboratoires (p) 10
A 3000 2477 2739 037 0261 -0261 0832 1418 A 2602 2477 2540 009 0062 -0062 -0391 0465 -0199
B 2544 2813 2678 019 -0134 0134 0211 0729 B 2903 2778 2841 009 0062 -0062 1732 0465 0162
C 2477 2813 2645 024 -0168 0168 -0135 0911 C 2477 2699 2588 016 -0111 0111 -0049 0826 -0057
D 2398 2653 2526 018 -0128 0128 -1370 0692 D 2778 3000 2889 016 -0111 0111 2074 0826 0363
E 2875 2740 2808 010 0067 -0067 1547 0365 E 2602 2301 2452 021 0151 -0151 -1012 1120 -0356
F 2477 2544 2511 005 -0033 0033 -1525 0182 F 2778 2477 2628 021 0151 -0151 0230 1120 0117
G 2653 2740 2697 006 -0044 0044 0400 0236 G 2845 2845 2845 000 0000 0000 1764 0000 0148
H 2176 2813 2495 045 -0318 0318 -1692 1727 H 2477 2477 2477 000 0000 0000 -0832 0000 -0017
L 2813 2544 2678 019 0134 -0134 0211 0729 L 2903 2301 2602 043 0301 -0301 0049 2241 -0076
M 2602 2740 2671 010 -0069 0069 0135 0375 M 2903 2255 2579 046 0324 -0324 -0112 2411 -0092
m 2658114 m 259505 mediane D -00372
Cn(2p) 1867 Cn(2p) 1867 Cp 16101
Cn(p) 16101 Cn(p) 16101 Qn(D) 01656915
n=2p n 20 n=2p n 20 Qdiff 02667753
f 11 f 11 t 035
l 55 l 55
n=p n 10 n=p n 10
f 6 f 6
l 15 l 15
Qn(2p) 0098745 Qn(2p) 007195
Qn(p) 006007 Qn(p) 008805
Qintra 018437 Qintra 013435
Qinter 009672 Qinter 014176
Sr(eacutecart-type de reacutepeacutetabiliteacute) 02607 Sr 01900
RSDr(coefficient de variation de la reacutepeacutetabiliteacute)981 RSDr 732
r(repeatability limit)) 0730 r 0532
SL(eacutecart-type interlaboratoire) -002464 SLlt0 SL 000205
SR(eacutecart-type de reproductibiliteacute) 02607 SR 01953
RSDR(coefficient de variation de la reproductibiliteacute)0098 RSDR 0075
R(limite de reproductibiliteacute)) 072996 R(limite de reproductibiliteacute))054686
Laboratoires
Niveau 2 bis
Moyenne si Laboratoires
Niveau 2 bis
DRef Alt Moyenne si Deviation h Deviation h k k
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3838 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Level 3
Nombre de laboratoires (p) 10
A 3690 3663 3676 002 0014 -0014 -0311 0513 A 3690 3708 3699 001 -0009 0009 0354 0257 0022
B 3613 3672 3642 004 -0030 0030 -1851 1109 B 3708 3568 3638 010 0070 -0070 -0844 2061 -0005
C 3663 3732 3698 005 -0035 0035 0645 1302 C 3663 3699 3681 003 -0018 0018 0000 0536 -0017
D 3690 3690 3690 000 0000 0000 0311 0000 D 3643 3663 3653 001 -0010 0010 -0545 0286 -0037
E 3732 3653 3693 006 0040 -0040 0429 1480 E 3613 3732 3673 008 -0060 0060 -0162 1769 -0020
F 3643 3672 3658 002 -0014 0014 -1157 0536 F 3690 3771 3731 006 -0040 0040 0975 1193 0073
G 3699 3653 3676 003 0023 -0023 -0328 0856 G 3602 3643 3623 003 -0021 0021 -1141 0612 -0053
H 3623 3672 3648 003 -0024 0024 -1615 0913 H 3748 3681 3715 005 0033 -0033 0664 0990 0067
L 3724 3663 3694 004 0031 -0031 0461 1150 L 3748 3771 3760 002 -0011 0011 1544 0335 0066
M 3690 3690 3690 000 0000 0000 0311 0000 M 3806 3681 3744 009 0062 -0062 1234 1848 0054
m 368334 m 368086 mediane D 00089
Cn(2p) 1867 Cn(2p) 1867 Cp 16101
Cn(p) 16101 Cn(p) 16101 Qn(D) 00325359
n=2p n 20 n=2p n 20 Qdiff 00523852
f 11 f 11 t 043
l 55 l 55
n=p n 10 n=p n 10
f 6 f 6
l 15 l 15
Qn(2p) 0014323 Qn(2p) 001811
Qn(p) 0013719 Qn(p) 003164
Qintra 002674 Qintra 003381
Qinter 002209 Qinter 005094
Sr(eacutecart-type de reacutepeacutetabiliteacute) 00378 Sr 00478
RSDr(coefficient de variation de la reacutepeacutetabiliteacute)103 RSDr 130
r(repeatability limit)) 0106 r 0134
SL(eacutecart-type interlaboratoire) -000023 SLlt0 SL 000145
SR(eacutecart-type de reproductibiliteacute) 00378 SR 00611
RSDR(coefficient de variation de la reproductibiliteacute)0010 RSDR 0017
R(limite de reproductibiliteacute)) 0105894 R(limite de reproductibiliteacute))017119
Ref Alt DLaboratoires
Niveau 3
Moyenne si Deviation h k Laboratoires
Niveau 3
Moyenne si Deviation h k
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3138 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Appendix 1 ndash Flow diagram of the Listeria PrecisTM method
x g sample + 9 x ml buffered peptone water
Revivification step for 1 h plusmn 5 min at 20degC plusmn 1degC
Streaking 1 ml onto 3 Brilliance Listeria Agar plates (mother suspension)
or 01 ml onto 1 Brilliance Listeria Agar plates (mother suspension and decimal dilutions)
Incubation for 48 h plusmn 3 h at 37degC plusmn 1degC
Confirmatory tests on at least one typical colony
- by the tests described in the ISO method
- by applying the OBIS mono test on an isolated colony
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3238 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Appendix 2 ndash ISO 11290-2 (1998) amp ISO 11290-2A1 (2004) Horizontal
method for the detection and enumeration of Listeria monocytogenes -
Part 2 enumeration of Listeria monocytogenes in foods
10 g cheese + 90 ml buffered peptone water or Half Fraser Broth (without supplements) (110 dilution)
Stomach 1 min 30 seconds
Keep at 20degC plusmn 2degC for 1 h plusmn 5 min
Decimal dilutions in 9 ml peptone salt tubes
Dilution 110
Dilution 1100
Dilution 11000
1 ml on 3 Chromogenic Listeria medium (ISO) plates (x 2) (-1)
01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-2)
01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-3)
01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-4)
Incubation at 37degC plusmn 1degC
24 h - 48 h
Numerate typical colonies at 24 h
(bluegreen surrounded by an opaque halo)
Numerate at 48 h
Streak typical colonies on TSYEA
(5 coloniesplate 2 successive dilutions)
Confirmation tests
Catalase
Gram stain
For confirmation of L monocytogenes
Haemolysis
Carbohydrate (rhamnose and xylose) utilisation
CAMP Test
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3338 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Appendix 3 ndash Mandelrsquos graphics
Laboratoire h Ref
Niveau 1 h Ref
Niveau 2 h Ref
Niveau 3 h Alt
Niveau 1 h Alt
Niveau 2 h Alt
Niveau 3 h5 h1 h5 h1
A -050 -015 083 062 -212 -039 198 299 -198 -299
B -261 -270 021 -077 -058 173 198 299 -198 -299
C -151 -031 -014 -015 -004 -005 198 299 -198 -299
D 038 040 -137 000 072 207 198 299 -198 -299
E -001 090 155 -052 056 -101 198 299 -198 -299
F -112 -321 -153 070 -325 023 198 299 -198 -299
G 042 -071 040 147 -009 176 198 299 -198 -299
H 169 046 -169 192 018 -083 198 299 -198 -299
L 009 -120 021 -077 -231 005 198 299 -198 -299
M 001 031 014 -032 004 -011 198 299 -198 -299
-300
-200
-100
000
100
200
300
A B C D E F G H L M
Vale
urs
h d
e M
an
del(
rob
uste
s)
Laboratoires
Meacutethode de reacutefeacuterence analyse robuste
h Ref Niveau 1
h Ref Niveau 2
h Ref Niveau 3
h 5=198
h 1=299
-300
-200
-100
000
100
200
300
A B C D E F G H L M
Vale
urs
de h
de M
an
del(
rob
uste
s)
Laboratoires
Meacutethode alternative analyse robuste
h Alt Niveau 1
h Alt Niveau 2
h Alt Niveau 2
h 1=299
h 5=198
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3438 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Laboratoire k Ref
Niveau 1 k Ref
Niveau 2 k Ref
Niveau 3 k Alt
Niveau 1 k Alt
Niveau 2 k Alt
Niveau 3 k1 k5
A 000 089 142 084 162 046 259 181
B 125 087 073 104 075 046 259 181
C 117 013 091 188 022 083 259 181
D 052 036 069 000 035 083 259 181
E 028 034 037 138 019 112 259 181
F 140 222 018 027 281 112 259 181
G 088 068 024 131 145 000 259 181
H 129 094 173 016 061 000 259 181
L 069 000 073 104 270 224 259 181
M 000 048 038 043 099 241 259 181
000
050
100
150
200
250
300
350
400
A B C D E F G H L M
Vale
urs
k d
e M
an
del(
rob
uste
s)
Laboratoires
Meacutethode de reacutefeacuterence analyse robuste
k Ref Niveau 1k Ref Niveau 2k Ref Niveau 3
k 1=259
k 5=181
000
050
100
150
200
250
300
A B C D E F G H L M
Vale
urs
k d
e M
an
del
(ro
bu
ste
s)
Laboratoires
Meacutethode alternative analyse robuste
k Alt Niveau 3
k Alt Niveau 1
k Alt Niveau 2
k 1=259
k 5=181
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3538 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Appendix 4 ndash Statistical results
Level 1
Nombre de laboratoires (p) 10
A 1602 1602 1602 000 0000 0000 -0495 0000 A 1845 1602 1724 017 0122 -0122 0621 0840 0122
B 1602 1176 1389 030 0213 -0213 -2613 1247 B 1602 1301 1452 021 0151 -0151 -0769 1041 0062
C 1699 1301 1500 028 0199 -0199 -1510 1165 C 1301 1845 1573 038 -0272 0272 -0148 1881 0073
D 1602 1778 1690 012 -0088 0088 0380 0516 D 1602 1602 1602 000 0000 0000 0000 0000 -0088
E 1699 1602 1651 007 0048 -0048 -0013 0284 E 1699 1301 1500 028 0199 -0199 -0522 1376 -0151
F 1778 1301 1540 034 0239 -0239 -1116 1397 F 1778 1699 1739 006 0040 -0040 0698 0274 0199
G 1845 1544 1695 021 0151 -0151 0425 0881 G 2079 1699 1889 027 0190 -0190 1467 1315 0194
H 2041 1602 1822 031 0220 -0220 1689 1286 H 2000 1954 1977 003 0023 -0023 1917 0158 0155
L 1544 1778 1661 017 -0117 0117 0092 0685 L 1301 1602 1452 021 -0151 0151 -0769 1041 -0210
M 1653 1653 1653 000 0000 0000 0013 0000 M 1477 1602 1540 009 -0062 0062 -0319 0432 -0114
m 165186 m 160206 mediane D 00678
Cn(2p) 1867 Cn(2p) 1867 Cp 16101
Cn(p) 16101 Cn(p) 16101 Qn(D) 0092926
n=2p n 20 n=2p n 20 Qdiff 01496169
f 11 f 11 t 114
l 55 l 55
n=p n 10 n=p n 10
f 6 f 6
l 15 l 15
Qn(2p) 0091465 Qn(2p) 007745
Qn(p) 0062469 Qn(p) 0121519
Qintra 017078 Qintra 014461
Qinter 010058 Qinter 019565
Sr(eacutecart-type de reacutepeacutetabiliteacute) 02415 Sr 02045
RSDr(coefficient de variation de la reacutepeacutetabiliteacute)1462 RSDr 1277
r(repeatability limit)) 0676 r 0573
SL(eacutecart-type interlaboratoire) -001905 SLlt0 SL 001737
SR(eacutecart-type de reproductibiliteacute) 02415 SR 02433
RSDR(coefficient de variation de la reproductibiliteacute)0146 RSDR 0152
R(limite de reproductibiliteacute)) 06762 R(limite de reproductibiliteacute))068123
h k h MoyenneLaboratoires
Niveau 1
si LaboratoiresDeviationMoyenne Dk DeviationsiAlt
Niveau 1
Ref
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3638 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Level 2
Nombre de laboratoires (p) 10
A 2681 2613 2647 005 0034 -0034 -0151 0891 A 2322 2531 2427 015 -0105 0105 -2119 1620 -0220
B 2544 2477 2511 005 0033 -0033 -2699 0872 B 2556 2653 2605 007 -0048 0048 -0581 0750 0094
C 2643 2633 2638 001 0005 -0005 -0311 0130 C 2681 2653 2667 002 0014 -0014 -0040 0217 0029
D 2690 2663 2676 002 0014 -0014 0400 0357 D 2778 2732 2755 003 0023 -0023 0721 0354 0079
E 2690 2716 2703 002 -0013 0013 0897 0336 E 2724 2748 2736 002 -0012 0012 0556 0185 0033
F 2568 2398 2483 012 0085 -0085 -3214 2217 F 2477 2114 2296 026 0182 -0182 -3255 2811 -0188
G 2643 2591 2617 004 0026 -0026 -0707 0682 G 2756 2568 2662 013 0094 -0094 -0085 1453 0045
H 2716 2643 2680 005 0036 -0036 0460 0945 H 2653 2732 2693 006 -0040 0040 0181 0613 0013
L 2591 2591 2591 000 0000 0000 -1196 0000 L 2230 2580 2405 025 -0175 0175 -2307 2704 -0186
M 2653 2690 2672 003 -0018 0018 0311 0481 M 2740 2613 2677 009 0064 -0064 0040 0987 0005
m 2655082 m 26719001 mediane D 00209
Cn(2p) 1867 Cn(2p) 1867 Cp 16101
Cn(p) 16101 Cn(p) 16101 Qn(D) 0039911
n=2p n 20 n=2p n 20 Qdiff 00642591
f 11 f 11 t 082
l 55 l 55
n=p n 10 n=p n 10
f 6 f 6
l 15 l 15
Qn(2p) 0020570 Qn(2p) 00345985
Qn(p) 00332437 Qn(p) 00718158
Qintra 003841 Qintra 006460
Qinter 005352 Qinter 011563
Sr(eacutecart-type de reacutepeacutetabiliteacute) 00543 Sr 00914
RSDr(coefficient de variation de la reacutepeacutetabiliteacute)205 RSDr 342
r(repeatability limit)) 0152 r 0256
SL(eacutecart-type interlaboratoire) 000139 SL 000920
SR(eacutecart-type de reproductibiliteacute) 00659 SR 01325
RSDR(coefficient de variation de la reproductibiliteacute)0025 RSDR 0050
R(limite de reproductibiliteacute)) 018446 R(limite de reproductibiliteacute))03708618
Laboratoires
Niveau 2
Moyenne si Laboratoires
Niveau 2
DRef Alt Moyenne si Deviation h Deviation h k k
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3738 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Level 2 bis
Nombre de laboratoires (p) 10
A 3000 2477 2739 037 0261 -0261 0832 1418 A 2602 2477 2540 009 0062 -0062 -0391 0465 -0199
B 2544 2813 2678 019 -0134 0134 0211 0729 B 2903 2778 2841 009 0062 -0062 1732 0465 0162
C 2477 2813 2645 024 -0168 0168 -0135 0911 C 2477 2699 2588 016 -0111 0111 -0049 0826 -0057
D 2398 2653 2526 018 -0128 0128 -1370 0692 D 2778 3000 2889 016 -0111 0111 2074 0826 0363
E 2875 2740 2808 010 0067 -0067 1547 0365 E 2602 2301 2452 021 0151 -0151 -1012 1120 -0356
F 2477 2544 2511 005 -0033 0033 -1525 0182 F 2778 2477 2628 021 0151 -0151 0230 1120 0117
G 2653 2740 2697 006 -0044 0044 0400 0236 G 2845 2845 2845 000 0000 0000 1764 0000 0148
H 2176 2813 2495 045 -0318 0318 -1692 1727 H 2477 2477 2477 000 0000 0000 -0832 0000 -0017
L 2813 2544 2678 019 0134 -0134 0211 0729 L 2903 2301 2602 043 0301 -0301 0049 2241 -0076
M 2602 2740 2671 010 -0069 0069 0135 0375 M 2903 2255 2579 046 0324 -0324 -0112 2411 -0092
m 2658114 m 259505 mediane D -00372
Cn(2p) 1867 Cn(2p) 1867 Cp 16101
Cn(p) 16101 Cn(p) 16101 Qn(D) 01656915
n=2p n 20 n=2p n 20 Qdiff 02667753
f 11 f 11 t 035
l 55 l 55
n=p n 10 n=p n 10
f 6 f 6
l 15 l 15
Qn(2p) 0098745 Qn(2p) 007195
Qn(p) 006007 Qn(p) 008805
Qintra 018437 Qintra 013435
Qinter 009672 Qinter 014176
Sr(eacutecart-type de reacutepeacutetabiliteacute) 02607 Sr 01900
RSDr(coefficient de variation de la reacutepeacutetabiliteacute)981 RSDr 732
r(repeatability limit)) 0730 r 0532
SL(eacutecart-type interlaboratoire) -002464 SLlt0 SL 000205
SR(eacutecart-type de reproductibiliteacute) 02607 SR 01953
RSDR(coefficient de variation de la reproductibiliteacute)0098 RSDR 0075
R(limite de reproductibiliteacute)) 072996 R(limite de reproductibiliteacute))054686
Laboratoires
Niveau 2 bis
Moyenne si Laboratoires
Niveau 2 bis
DRef Alt Moyenne si Deviation h Deviation h k k
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3838 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Level 3
Nombre de laboratoires (p) 10
A 3690 3663 3676 002 0014 -0014 -0311 0513 A 3690 3708 3699 001 -0009 0009 0354 0257 0022
B 3613 3672 3642 004 -0030 0030 -1851 1109 B 3708 3568 3638 010 0070 -0070 -0844 2061 -0005
C 3663 3732 3698 005 -0035 0035 0645 1302 C 3663 3699 3681 003 -0018 0018 0000 0536 -0017
D 3690 3690 3690 000 0000 0000 0311 0000 D 3643 3663 3653 001 -0010 0010 -0545 0286 -0037
E 3732 3653 3693 006 0040 -0040 0429 1480 E 3613 3732 3673 008 -0060 0060 -0162 1769 -0020
F 3643 3672 3658 002 -0014 0014 -1157 0536 F 3690 3771 3731 006 -0040 0040 0975 1193 0073
G 3699 3653 3676 003 0023 -0023 -0328 0856 G 3602 3643 3623 003 -0021 0021 -1141 0612 -0053
H 3623 3672 3648 003 -0024 0024 -1615 0913 H 3748 3681 3715 005 0033 -0033 0664 0990 0067
L 3724 3663 3694 004 0031 -0031 0461 1150 L 3748 3771 3760 002 -0011 0011 1544 0335 0066
M 3690 3690 3690 000 0000 0000 0311 0000 M 3806 3681 3744 009 0062 -0062 1234 1848 0054
m 368334 m 368086 mediane D 00089
Cn(2p) 1867 Cn(2p) 1867 Cp 16101
Cn(p) 16101 Cn(p) 16101 Qn(D) 00325359
n=2p n 20 n=2p n 20 Qdiff 00523852
f 11 f 11 t 043
l 55 l 55
n=p n 10 n=p n 10
f 6 f 6
l 15 l 15
Qn(2p) 0014323 Qn(2p) 001811
Qn(p) 0013719 Qn(p) 003164
Qintra 002674 Qintra 003381
Qinter 002209 Qinter 005094
Sr(eacutecart-type de reacutepeacutetabiliteacute) 00378 Sr 00478
RSDr(coefficient de variation de la reacutepeacutetabiliteacute)103 RSDr 130
r(repeatability limit)) 0106 r 0134
SL(eacutecart-type interlaboratoire) -000023 SLlt0 SL 000145
SR(eacutecart-type de reproductibiliteacute) 00378 SR 00611
RSDR(coefficient de variation de la reproductibiliteacute)0010 RSDR 0017
R(limite de reproductibiliteacute)) 0105894 R(limite de reproductibiliteacute))017119
Ref Alt DLaboratoires
Niveau 3
Moyenne si Deviation h k Laboratoires
Niveau 3
Moyenne si Deviation h k
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3238 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Appendix 2 ndash ISO 11290-2 (1998) amp ISO 11290-2A1 (2004) Horizontal
method for the detection and enumeration of Listeria monocytogenes -
Part 2 enumeration of Listeria monocytogenes in foods
10 g cheese + 90 ml buffered peptone water or Half Fraser Broth (without supplements) (110 dilution)
Stomach 1 min 30 seconds
Keep at 20degC plusmn 2degC for 1 h plusmn 5 min
Decimal dilutions in 9 ml peptone salt tubes
Dilution 110
Dilution 1100
Dilution 11000
1 ml on 3 Chromogenic Listeria medium (ISO) plates (x 2) (-1)
01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-2)
01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-3)
01 ml on 1 Chromogenic Listeria medium (ISO) plate (x 2) (-4)
Incubation at 37degC plusmn 1degC
24 h - 48 h
Numerate typical colonies at 24 h
(bluegreen surrounded by an opaque halo)
Numerate at 48 h
Streak typical colonies on TSYEA
(5 coloniesplate 2 successive dilutions)
Confirmation tests
Catalase
Gram stain
For confirmation of L monocytogenes
Haemolysis
Carbohydrate (rhamnose and xylose) utilisation
CAMP Test
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3338 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Appendix 3 ndash Mandelrsquos graphics
Laboratoire h Ref
Niveau 1 h Ref
Niveau 2 h Ref
Niveau 3 h Alt
Niveau 1 h Alt
Niveau 2 h Alt
Niveau 3 h5 h1 h5 h1
A -050 -015 083 062 -212 -039 198 299 -198 -299
B -261 -270 021 -077 -058 173 198 299 -198 -299
C -151 -031 -014 -015 -004 -005 198 299 -198 -299
D 038 040 -137 000 072 207 198 299 -198 -299
E -001 090 155 -052 056 -101 198 299 -198 -299
F -112 -321 -153 070 -325 023 198 299 -198 -299
G 042 -071 040 147 -009 176 198 299 -198 -299
H 169 046 -169 192 018 -083 198 299 -198 -299
L 009 -120 021 -077 -231 005 198 299 -198 -299
M 001 031 014 -032 004 -011 198 299 -198 -299
-300
-200
-100
000
100
200
300
A B C D E F G H L M
Vale
urs
h d
e M
an
del(
rob
uste
s)
Laboratoires
Meacutethode de reacutefeacuterence analyse robuste
h Ref Niveau 1
h Ref Niveau 2
h Ref Niveau 3
h 5=198
h 1=299
-300
-200
-100
000
100
200
300
A B C D E F G H L M
Vale
urs
de h
de M
an
del(
rob
uste
s)
Laboratoires
Meacutethode alternative analyse robuste
h Alt Niveau 1
h Alt Niveau 2
h Alt Niveau 2
h 1=299
h 5=198
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3438 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Laboratoire k Ref
Niveau 1 k Ref
Niveau 2 k Ref
Niveau 3 k Alt
Niveau 1 k Alt
Niveau 2 k Alt
Niveau 3 k1 k5
A 000 089 142 084 162 046 259 181
B 125 087 073 104 075 046 259 181
C 117 013 091 188 022 083 259 181
D 052 036 069 000 035 083 259 181
E 028 034 037 138 019 112 259 181
F 140 222 018 027 281 112 259 181
G 088 068 024 131 145 000 259 181
H 129 094 173 016 061 000 259 181
L 069 000 073 104 270 224 259 181
M 000 048 038 043 099 241 259 181
000
050
100
150
200
250
300
350
400
A B C D E F G H L M
Vale
urs
k d
e M
an
del(
rob
uste
s)
Laboratoires
Meacutethode de reacutefeacuterence analyse robuste
k Ref Niveau 1k Ref Niveau 2k Ref Niveau 3
k 1=259
k 5=181
000
050
100
150
200
250
300
A B C D E F G H L M
Vale
urs
k d
e M
an
del
(ro
bu
ste
s)
Laboratoires
Meacutethode alternative analyse robuste
k Alt Niveau 3
k Alt Niveau 1
k Alt Niveau 2
k 1=259
k 5=181
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3538 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Appendix 4 ndash Statistical results
Level 1
Nombre de laboratoires (p) 10
A 1602 1602 1602 000 0000 0000 -0495 0000 A 1845 1602 1724 017 0122 -0122 0621 0840 0122
B 1602 1176 1389 030 0213 -0213 -2613 1247 B 1602 1301 1452 021 0151 -0151 -0769 1041 0062
C 1699 1301 1500 028 0199 -0199 -1510 1165 C 1301 1845 1573 038 -0272 0272 -0148 1881 0073
D 1602 1778 1690 012 -0088 0088 0380 0516 D 1602 1602 1602 000 0000 0000 0000 0000 -0088
E 1699 1602 1651 007 0048 -0048 -0013 0284 E 1699 1301 1500 028 0199 -0199 -0522 1376 -0151
F 1778 1301 1540 034 0239 -0239 -1116 1397 F 1778 1699 1739 006 0040 -0040 0698 0274 0199
G 1845 1544 1695 021 0151 -0151 0425 0881 G 2079 1699 1889 027 0190 -0190 1467 1315 0194
H 2041 1602 1822 031 0220 -0220 1689 1286 H 2000 1954 1977 003 0023 -0023 1917 0158 0155
L 1544 1778 1661 017 -0117 0117 0092 0685 L 1301 1602 1452 021 -0151 0151 -0769 1041 -0210
M 1653 1653 1653 000 0000 0000 0013 0000 M 1477 1602 1540 009 -0062 0062 -0319 0432 -0114
m 165186 m 160206 mediane D 00678
Cn(2p) 1867 Cn(2p) 1867 Cp 16101
Cn(p) 16101 Cn(p) 16101 Qn(D) 0092926
n=2p n 20 n=2p n 20 Qdiff 01496169
f 11 f 11 t 114
l 55 l 55
n=p n 10 n=p n 10
f 6 f 6
l 15 l 15
Qn(2p) 0091465 Qn(2p) 007745
Qn(p) 0062469 Qn(p) 0121519
Qintra 017078 Qintra 014461
Qinter 010058 Qinter 019565
Sr(eacutecart-type de reacutepeacutetabiliteacute) 02415 Sr 02045
RSDr(coefficient de variation de la reacutepeacutetabiliteacute)1462 RSDr 1277
r(repeatability limit)) 0676 r 0573
SL(eacutecart-type interlaboratoire) -001905 SLlt0 SL 001737
SR(eacutecart-type de reproductibiliteacute) 02415 SR 02433
RSDR(coefficient de variation de la reproductibiliteacute)0146 RSDR 0152
R(limite de reproductibiliteacute)) 06762 R(limite de reproductibiliteacute))068123
h k h MoyenneLaboratoires
Niveau 1
si LaboratoiresDeviationMoyenne Dk DeviationsiAlt
Niveau 1
Ref
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3638 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Level 2
Nombre de laboratoires (p) 10
A 2681 2613 2647 005 0034 -0034 -0151 0891 A 2322 2531 2427 015 -0105 0105 -2119 1620 -0220
B 2544 2477 2511 005 0033 -0033 -2699 0872 B 2556 2653 2605 007 -0048 0048 -0581 0750 0094
C 2643 2633 2638 001 0005 -0005 -0311 0130 C 2681 2653 2667 002 0014 -0014 -0040 0217 0029
D 2690 2663 2676 002 0014 -0014 0400 0357 D 2778 2732 2755 003 0023 -0023 0721 0354 0079
E 2690 2716 2703 002 -0013 0013 0897 0336 E 2724 2748 2736 002 -0012 0012 0556 0185 0033
F 2568 2398 2483 012 0085 -0085 -3214 2217 F 2477 2114 2296 026 0182 -0182 -3255 2811 -0188
G 2643 2591 2617 004 0026 -0026 -0707 0682 G 2756 2568 2662 013 0094 -0094 -0085 1453 0045
H 2716 2643 2680 005 0036 -0036 0460 0945 H 2653 2732 2693 006 -0040 0040 0181 0613 0013
L 2591 2591 2591 000 0000 0000 -1196 0000 L 2230 2580 2405 025 -0175 0175 -2307 2704 -0186
M 2653 2690 2672 003 -0018 0018 0311 0481 M 2740 2613 2677 009 0064 -0064 0040 0987 0005
m 2655082 m 26719001 mediane D 00209
Cn(2p) 1867 Cn(2p) 1867 Cp 16101
Cn(p) 16101 Cn(p) 16101 Qn(D) 0039911
n=2p n 20 n=2p n 20 Qdiff 00642591
f 11 f 11 t 082
l 55 l 55
n=p n 10 n=p n 10
f 6 f 6
l 15 l 15
Qn(2p) 0020570 Qn(2p) 00345985
Qn(p) 00332437 Qn(p) 00718158
Qintra 003841 Qintra 006460
Qinter 005352 Qinter 011563
Sr(eacutecart-type de reacutepeacutetabiliteacute) 00543 Sr 00914
RSDr(coefficient de variation de la reacutepeacutetabiliteacute)205 RSDr 342
r(repeatability limit)) 0152 r 0256
SL(eacutecart-type interlaboratoire) 000139 SL 000920
SR(eacutecart-type de reproductibiliteacute) 00659 SR 01325
RSDR(coefficient de variation de la reproductibiliteacute)0025 RSDR 0050
R(limite de reproductibiliteacute)) 018446 R(limite de reproductibiliteacute))03708618
Laboratoires
Niveau 2
Moyenne si Laboratoires
Niveau 2
DRef Alt Moyenne si Deviation h Deviation h k k
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3738 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Level 2 bis
Nombre de laboratoires (p) 10
A 3000 2477 2739 037 0261 -0261 0832 1418 A 2602 2477 2540 009 0062 -0062 -0391 0465 -0199
B 2544 2813 2678 019 -0134 0134 0211 0729 B 2903 2778 2841 009 0062 -0062 1732 0465 0162
C 2477 2813 2645 024 -0168 0168 -0135 0911 C 2477 2699 2588 016 -0111 0111 -0049 0826 -0057
D 2398 2653 2526 018 -0128 0128 -1370 0692 D 2778 3000 2889 016 -0111 0111 2074 0826 0363
E 2875 2740 2808 010 0067 -0067 1547 0365 E 2602 2301 2452 021 0151 -0151 -1012 1120 -0356
F 2477 2544 2511 005 -0033 0033 -1525 0182 F 2778 2477 2628 021 0151 -0151 0230 1120 0117
G 2653 2740 2697 006 -0044 0044 0400 0236 G 2845 2845 2845 000 0000 0000 1764 0000 0148
H 2176 2813 2495 045 -0318 0318 -1692 1727 H 2477 2477 2477 000 0000 0000 -0832 0000 -0017
L 2813 2544 2678 019 0134 -0134 0211 0729 L 2903 2301 2602 043 0301 -0301 0049 2241 -0076
M 2602 2740 2671 010 -0069 0069 0135 0375 M 2903 2255 2579 046 0324 -0324 -0112 2411 -0092
m 2658114 m 259505 mediane D -00372
Cn(2p) 1867 Cn(2p) 1867 Cp 16101
Cn(p) 16101 Cn(p) 16101 Qn(D) 01656915
n=2p n 20 n=2p n 20 Qdiff 02667753
f 11 f 11 t 035
l 55 l 55
n=p n 10 n=p n 10
f 6 f 6
l 15 l 15
Qn(2p) 0098745 Qn(2p) 007195
Qn(p) 006007 Qn(p) 008805
Qintra 018437 Qintra 013435
Qinter 009672 Qinter 014176
Sr(eacutecart-type de reacutepeacutetabiliteacute) 02607 Sr 01900
RSDr(coefficient de variation de la reacutepeacutetabiliteacute)981 RSDr 732
r(repeatability limit)) 0730 r 0532
SL(eacutecart-type interlaboratoire) -002464 SLlt0 SL 000205
SR(eacutecart-type de reproductibiliteacute) 02607 SR 01953
RSDR(coefficient de variation de la reproductibiliteacute)0098 RSDR 0075
R(limite de reproductibiliteacute)) 072996 R(limite de reproductibiliteacute))054686
Laboratoires
Niveau 2 bis
Moyenne si Laboratoires
Niveau 2 bis
DRef Alt Moyenne si Deviation h Deviation h k k
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3838 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Level 3
Nombre de laboratoires (p) 10
A 3690 3663 3676 002 0014 -0014 -0311 0513 A 3690 3708 3699 001 -0009 0009 0354 0257 0022
B 3613 3672 3642 004 -0030 0030 -1851 1109 B 3708 3568 3638 010 0070 -0070 -0844 2061 -0005
C 3663 3732 3698 005 -0035 0035 0645 1302 C 3663 3699 3681 003 -0018 0018 0000 0536 -0017
D 3690 3690 3690 000 0000 0000 0311 0000 D 3643 3663 3653 001 -0010 0010 -0545 0286 -0037
E 3732 3653 3693 006 0040 -0040 0429 1480 E 3613 3732 3673 008 -0060 0060 -0162 1769 -0020
F 3643 3672 3658 002 -0014 0014 -1157 0536 F 3690 3771 3731 006 -0040 0040 0975 1193 0073
G 3699 3653 3676 003 0023 -0023 -0328 0856 G 3602 3643 3623 003 -0021 0021 -1141 0612 -0053
H 3623 3672 3648 003 -0024 0024 -1615 0913 H 3748 3681 3715 005 0033 -0033 0664 0990 0067
L 3724 3663 3694 004 0031 -0031 0461 1150 L 3748 3771 3760 002 -0011 0011 1544 0335 0066
M 3690 3690 3690 000 0000 0000 0311 0000 M 3806 3681 3744 009 0062 -0062 1234 1848 0054
m 368334 m 368086 mediane D 00089
Cn(2p) 1867 Cn(2p) 1867 Cp 16101
Cn(p) 16101 Cn(p) 16101 Qn(D) 00325359
n=2p n 20 n=2p n 20 Qdiff 00523852
f 11 f 11 t 043
l 55 l 55
n=p n 10 n=p n 10
f 6 f 6
l 15 l 15
Qn(2p) 0014323 Qn(2p) 001811
Qn(p) 0013719 Qn(p) 003164
Qintra 002674 Qintra 003381
Qinter 002209 Qinter 005094
Sr(eacutecart-type de reacutepeacutetabiliteacute) 00378 Sr 00478
RSDr(coefficient de variation de la reacutepeacutetabiliteacute)103 RSDr 130
r(repeatability limit)) 0106 r 0134
SL(eacutecart-type interlaboratoire) -000023 SLlt0 SL 000145
SR(eacutecart-type de reproductibiliteacute) 00378 SR 00611
RSDR(coefficient de variation de la reproductibiliteacute)0010 RSDR 0017
R(limite de reproductibiliteacute)) 0105894 R(limite de reproductibiliteacute))017119
Ref Alt DLaboratoires
Niveau 3
Moyenne si Deviation h k Laboratoires
Niveau 3
Moyenne si Deviation h k
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3338 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Appendix 3 ndash Mandelrsquos graphics
Laboratoire h Ref
Niveau 1 h Ref
Niveau 2 h Ref
Niveau 3 h Alt
Niveau 1 h Alt
Niveau 2 h Alt
Niveau 3 h5 h1 h5 h1
A -050 -015 083 062 -212 -039 198 299 -198 -299
B -261 -270 021 -077 -058 173 198 299 -198 -299
C -151 -031 -014 -015 -004 -005 198 299 -198 -299
D 038 040 -137 000 072 207 198 299 -198 -299
E -001 090 155 -052 056 -101 198 299 -198 -299
F -112 -321 -153 070 -325 023 198 299 -198 -299
G 042 -071 040 147 -009 176 198 299 -198 -299
H 169 046 -169 192 018 -083 198 299 -198 -299
L 009 -120 021 -077 -231 005 198 299 -198 -299
M 001 031 014 -032 004 -011 198 299 -198 -299
-300
-200
-100
000
100
200
300
A B C D E F G H L M
Vale
urs
h d
e M
an
del(
rob
uste
s)
Laboratoires
Meacutethode de reacutefeacuterence analyse robuste
h Ref Niveau 1
h Ref Niveau 2
h Ref Niveau 3
h 5=198
h 1=299
-300
-200
-100
000
100
200
300
A B C D E F G H L M
Vale
urs
de h
de M
an
del(
rob
uste
s)
Laboratoires
Meacutethode alternative analyse robuste
h Alt Niveau 1
h Alt Niveau 2
h Alt Niveau 2
h 1=299
h 5=198
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3438 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Laboratoire k Ref
Niveau 1 k Ref
Niveau 2 k Ref
Niveau 3 k Alt
Niveau 1 k Alt
Niveau 2 k Alt
Niveau 3 k1 k5
A 000 089 142 084 162 046 259 181
B 125 087 073 104 075 046 259 181
C 117 013 091 188 022 083 259 181
D 052 036 069 000 035 083 259 181
E 028 034 037 138 019 112 259 181
F 140 222 018 027 281 112 259 181
G 088 068 024 131 145 000 259 181
H 129 094 173 016 061 000 259 181
L 069 000 073 104 270 224 259 181
M 000 048 038 043 099 241 259 181
000
050
100
150
200
250
300
350
400
A B C D E F G H L M
Vale
urs
k d
e M
an
del(
rob
uste
s)
Laboratoires
Meacutethode de reacutefeacuterence analyse robuste
k Ref Niveau 1k Ref Niveau 2k Ref Niveau 3
k 1=259
k 5=181
000
050
100
150
200
250
300
A B C D E F G H L M
Vale
urs
k d
e M
an
del
(ro
bu
ste
s)
Laboratoires
Meacutethode alternative analyse robuste
k Alt Niveau 3
k Alt Niveau 1
k Alt Niveau 2
k 1=259
k 5=181
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3538 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Appendix 4 ndash Statistical results
Level 1
Nombre de laboratoires (p) 10
A 1602 1602 1602 000 0000 0000 -0495 0000 A 1845 1602 1724 017 0122 -0122 0621 0840 0122
B 1602 1176 1389 030 0213 -0213 -2613 1247 B 1602 1301 1452 021 0151 -0151 -0769 1041 0062
C 1699 1301 1500 028 0199 -0199 -1510 1165 C 1301 1845 1573 038 -0272 0272 -0148 1881 0073
D 1602 1778 1690 012 -0088 0088 0380 0516 D 1602 1602 1602 000 0000 0000 0000 0000 -0088
E 1699 1602 1651 007 0048 -0048 -0013 0284 E 1699 1301 1500 028 0199 -0199 -0522 1376 -0151
F 1778 1301 1540 034 0239 -0239 -1116 1397 F 1778 1699 1739 006 0040 -0040 0698 0274 0199
G 1845 1544 1695 021 0151 -0151 0425 0881 G 2079 1699 1889 027 0190 -0190 1467 1315 0194
H 2041 1602 1822 031 0220 -0220 1689 1286 H 2000 1954 1977 003 0023 -0023 1917 0158 0155
L 1544 1778 1661 017 -0117 0117 0092 0685 L 1301 1602 1452 021 -0151 0151 -0769 1041 -0210
M 1653 1653 1653 000 0000 0000 0013 0000 M 1477 1602 1540 009 -0062 0062 -0319 0432 -0114
m 165186 m 160206 mediane D 00678
Cn(2p) 1867 Cn(2p) 1867 Cp 16101
Cn(p) 16101 Cn(p) 16101 Qn(D) 0092926
n=2p n 20 n=2p n 20 Qdiff 01496169
f 11 f 11 t 114
l 55 l 55
n=p n 10 n=p n 10
f 6 f 6
l 15 l 15
Qn(2p) 0091465 Qn(2p) 007745
Qn(p) 0062469 Qn(p) 0121519
Qintra 017078 Qintra 014461
Qinter 010058 Qinter 019565
Sr(eacutecart-type de reacutepeacutetabiliteacute) 02415 Sr 02045
RSDr(coefficient de variation de la reacutepeacutetabiliteacute)1462 RSDr 1277
r(repeatability limit)) 0676 r 0573
SL(eacutecart-type interlaboratoire) -001905 SLlt0 SL 001737
SR(eacutecart-type de reproductibiliteacute) 02415 SR 02433
RSDR(coefficient de variation de la reproductibiliteacute)0146 RSDR 0152
R(limite de reproductibiliteacute)) 06762 R(limite de reproductibiliteacute))068123
h k h MoyenneLaboratoires
Niveau 1
si LaboratoiresDeviationMoyenne Dk DeviationsiAlt
Niveau 1
Ref
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3638 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Level 2
Nombre de laboratoires (p) 10
A 2681 2613 2647 005 0034 -0034 -0151 0891 A 2322 2531 2427 015 -0105 0105 -2119 1620 -0220
B 2544 2477 2511 005 0033 -0033 -2699 0872 B 2556 2653 2605 007 -0048 0048 -0581 0750 0094
C 2643 2633 2638 001 0005 -0005 -0311 0130 C 2681 2653 2667 002 0014 -0014 -0040 0217 0029
D 2690 2663 2676 002 0014 -0014 0400 0357 D 2778 2732 2755 003 0023 -0023 0721 0354 0079
E 2690 2716 2703 002 -0013 0013 0897 0336 E 2724 2748 2736 002 -0012 0012 0556 0185 0033
F 2568 2398 2483 012 0085 -0085 -3214 2217 F 2477 2114 2296 026 0182 -0182 -3255 2811 -0188
G 2643 2591 2617 004 0026 -0026 -0707 0682 G 2756 2568 2662 013 0094 -0094 -0085 1453 0045
H 2716 2643 2680 005 0036 -0036 0460 0945 H 2653 2732 2693 006 -0040 0040 0181 0613 0013
L 2591 2591 2591 000 0000 0000 -1196 0000 L 2230 2580 2405 025 -0175 0175 -2307 2704 -0186
M 2653 2690 2672 003 -0018 0018 0311 0481 M 2740 2613 2677 009 0064 -0064 0040 0987 0005
m 2655082 m 26719001 mediane D 00209
Cn(2p) 1867 Cn(2p) 1867 Cp 16101
Cn(p) 16101 Cn(p) 16101 Qn(D) 0039911
n=2p n 20 n=2p n 20 Qdiff 00642591
f 11 f 11 t 082
l 55 l 55
n=p n 10 n=p n 10
f 6 f 6
l 15 l 15
Qn(2p) 0020570 Qn(2p) 00345985
Qn(p) 00332437 Qn(p) 00718158
Qintra 003841 Qintra 006460
Qinter 005352 Qinter 011563
Sr(eacutecart-type de reacutepeacutetabiliteacute) 00543 Sr 00914
RSDr(coefficient de variation de la reacutepeacutetabiliteacute)205 RSDr 342
r(repeatability limit)) 0152 r 0256
SL(eacutecart-type interlaboratoire) 000139 SL 000920
SR(eacutecart-type de reproductibiliteacute) 00659 SR 01325
RSDR(coefficient de variation de la reproductibiliteacute)0025 RSDR 0050
R(limite de reproductibiliteacute)) 018446 R(limite de reproductibiliteacute))03708618
Laboratoires
Niveau 2
Moyenne si Laboratoires
Niveau 2
DRef Alt Moyenne si Deviation h Deviation h k k
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3738 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Level 2 bis
Nombre de laboratoires (p) 10
A 3000 2477 2739 037 0261 -0261 0832 1418 A 2602 2477 2540 009 0062 -0062 -0391 0465 -0199
B 2544 2813 2678 019 -0134 0134 0211 0729 B 2903 2778 2841 009 0062 -0062 1732 0465 0162
C 2477 2813 2645 024 -0168 0168 -0135 0911 C 2477 2699 2588 016 -0111 0111 -0049 0826 -0057
D 2398 2653 2526 018 -0128 0128 -1370 0692 D 2778 3000 2889 016 -0111 0111 2074 0826 0363
E 2875 2740 2808 010 0067 -0067 1547 0365 E 2602 2301 2452 021 0151 -0151 -1012 1120 -0356
F 2477 2544 2511 005 -0033 0033 -1525 0182 F 2778 2477 2628 021 0151 -0151 0230 1120 0117
G 2653 2740 2697 006 -0044 0044 0400 0236 G 2845 2845 2845 000 0000 0000 1764 0000 0148
H 2176 2813 2495 045 -0318 0318 -1692 1727 H 2477 2477 2477 000 0000 0000 -0832 0000 -0017
L 2813 2544 2678 019 0134 -0134 0211 0729 L 2903 2301 2602 043 0301 -0301 0049 2241 -0076
M 2602 2740 2671 010 -0069 0069 0135 0375 M 2903 2255 2579 046 0324 -0324 -0112 2411 -0092
m 2658114 m 259505 mediane D -00372
Cn(2p) 1867 Cn(2p) 1867 Cp 16101
Cn(p) 16101 Cn(p) 16101 Qn(D) 01656915
n=2p n 20 n=2p n 20 Qdiff 02667753
f 11 f 11 t 035
l 55 l 55
n=p n 10 n=p n 10
f 6 f 6
l 15 l 15
Qn(2p) 0098745 Qn(2p) 007195
Qn(p) 006007 Qn(p) 008805
Qintra 018437 Qintra 013435
Qinter 009672 Qinter 014176
Sr(eacutecart-type de reacutepeacutetabiliteacute) 02607 Sr 01900
RSDr(coefficient de variation de la reacutepeacutetabiliteacute)981 RSDr 732
r(repeatability limit)) 0730 r 0532
SL(eacutecart-type interlaboratoire) -002464 SLlt0 SL 000205
SR(eacutecart-type de reproductibiliteacute) 02607 SR 01953
RSDR(coefficient de variation de la reproductibiliteacute)0098 RSDR 0075
R(limite de reproductibiliteacute)) 072996 R(limite de reproductibiliteacute))054686
Laboratoires
Niveau 2 bis
Moyenne si Laboratoires
Niveau 2 bis
DRef Alt Moyenne si Deviation h Deviation h k k
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3838 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Level 3
Nombre de laboratoires (p) 10
A 3690 3663 3676 002 0014 -0014 -0311 0513 A 3690 3708 3699 001 -0009 0009 0354 0257 0022
B 3613 3672 3642 004 -0030 0030 -1851 1109 B 3708 3568 3638 010 0070 -0070 -0844 2061 -0005
C 3663 3732 3698 005 -0035 0035 0645 1302 C 3663 3699 3681 003 -0018 0018 0000 0536 -0017
D 3690 3690 3690 000 0000 0000 0311 0000 D 3643 3663 3653 001 -0010 0010 -0545 0286 -0037
E 3732 3653 3693 006 0040 -0040 0429 1480 E 3613 3732 3673 008 -0060 0060 -0162 1769 -0020
F 3643 3672 3658 002 -0014 0014 -1157 0536 F 3690 3771 3731 006 -0040 0040 0975 1193 0073
G 3699 3653 3676 003 0023 -0023 -0328 0856 G 3602 3643 3623 003 -0021 0021 -1141 0612 -0053
H 3623 3672 3648 003 -0024 0024 -1615 0913 H 3748 3681 3715 005 0033 -0033 0664 0990 0067
L 3724 3663 3694 004 0031 -0031 0461 1150 L 3748 3771 3760 002 -0011 0011 1544 0335 0066
M 3690 3690 3690 000 0000 0000 0311 0000 M 3806 3681 3744 009 0062 -0062 1234 1848 0054
m 368334 m 368086 mediane D 00089
Cn(2p) 1867 Cn(2p) 1867 Cp 16101
Cn(p) 16101 Cn(p) 16101 Qn(D) 00325359
n=2p n 20 n=2p n 20 Qdiff 00523852
f 11 f 11 t 043
l 55 l 55
n=p n 10 n=p n 10
f 6 f 6
l 15 l 15
Qn(2p) 0014323 Qn(2p) 001811
Qn(p) 0013719 Qn(p) 003164
Qintra 002674 Qintra 003381
Qinter 002209 Qinter 005094
Sr(eacutecart-type de reacutepeacutetabiliteacute) 00378 Sr 00478
RSDr(coefficient de variation de la reacutepeacutetabiliteacute)103 RSDr 130
r(repeatability limit)) 0106 r 0134
SL(eacutecart-type interlaboratoire) -000023 SLlt0 SL 000145
SR(eacutecart-type de reproductibiliteacute) 00378 SR 00611
RSDR(coefficient de variation de la reproductibiliteacute)0010 RSDR 0017
R(limite de reproductibiliteacute)) 0105894 R(limite de reproductibiliteacute))017119
Ref Alt DLaboratoires
Niveau 3
Moyenne si Deviation h k Laboratoires
Niveau 3
Moyenne si Deviation h k
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3438 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Laboratoire k Ref
Niveau 1 k Ref
Niveau 2 k Ref
Niveau 3 k Alt
Niveau 1 k Alt
Niveau 2 k Alt
Niveau 3 k1 k5
A 000 089 142 084 162 046 259 181
B 125 087 073 104 075 046 259 181
C 117 013 091 188 022 083 259 181
D 052 036 069 000 035 083 259 181
E 028 034 037 138 019 112 259 181
F 140 222 018 027 281 112 259 181
G 088 068 024 131 145 000 259 181
H 129 094 173 016 061 000 259 181
L 069 000 073 104 270 224 259 181
M 000 048 038 043 099 241 259 181
000
050
100
150
200
250
300
350
400
A B C D E F G H L M
Vale
urs
k d
e M
an
del(
rob
uste
s)
Laboratoires
Meacutethode de reacutefeacuterence analyse robuste
k Ref Niveau 1k Ref Niveau 2k Ref Niveau 3
k 1=259
k 5=181
000
050
100
150
200
250
300
A B C D E F G H L M
Vale
urs
k d
e M
an
del
(ro
bu
ste
s)
Laboratoires
Meacutethode alternative analyse robuste
k Alt Niveau 3
k Alt Niveau 1
k Alt Niveau 2
k 1=259
k 5=181
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3538 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Appendix 4 ndash Statistical results
Level 1
Nombre de laboratoires (p) 10
A 1602 1602 1602 000 0000 0000 -0495 0000 A 1845 1602 1724 017 0122 -0122 0621 0840 0122
B 1602 1176 1389 030 0213 -0213 -2613 1247 B 1602 1301 1452 021 0151 -0151 -0769 1041 0062
C 1699 1301 1500 028 0199 -0199 -1510 1165 C 1301 1845 1573 038 -0272 0272 -0148 1881 0073
D 1602 1778 1690 012 -0088 0088 0380 0516 D 1602 1602 1602 000 0000 0000 0000 0000 -0088
E 1699 1602 1651 007 0048 -0048 -0013 0284 E 1699 1301 1500 028 0199 -0199 -0522 1376 -0151
F 1778 1301 1540 034 0239 -0239 -1116 1397 F 1778 1699 1739 006 0040 -0040 0698 0274 0199
G 1845 1544 1695 021 0151 -0151 0425 0881 G 2079 1699 1889 027 0190 -0190 1467 1315 0194
H 2041 1602 1822 031 0220 -0220 1689 1286 H 2000 1954 1977 003 0023 -0023 1917 0158 0155
L 1544 1778 1661 017 -0117 0117 0092 0685 L 1301 1602 1452 021 -0151 0151 -0769 1041 -0210
M 1653 1653 1653 000 0000 0000 0013 0000 M 1477 1602 1540 009 -0062 0062 -0319 0432 -0114
m 165186 m 160206 mediane D 00678
Cn(2p) 1867 Cn(2p) 1867 Cp 16101
Cn(p) 16101 Cn(p) 16101 Qn(D) 0092926
n=2p n 20 n=2p n 20 Qdiff 01496169
f 11 f 11 t 114
l 55 l 55
n=p n 10 n=p n 10
f 6 f 6
l 15 l 15
Qn(2p) 0091465 Qn(2p) 007745
Qn(p) 0062469 Qn(p) 0121519
Qintra 017078 Qintra 014461
Qinter 010058 Qinter 019565
Sr(eacutecart-type de reacutepeacutetabiliteacute) 02415 Sr 02045
RSDr(coefficient de variation de la reacutepeacutetabiliteacute)1462 RSDr 1277
r(repeatability limit)) 0676 r 0573
SL(eacutecart-type interlaboratoire) -001905 SLlt0 SL 001737
SR(eacutecart-type de reproductibiliteacute) 02415 SR 02433
RSDR(coefficient de variation de la reproductibiliteacute)0146 RSDR 0152
R(limite de reproductibiliteacute)) 06762 R(limite de reproductibiliteacute))068123
h k h MoyenneLaboratoires
Niveau 1
si LaboratoiresDeviationMoyenne Dk DeviationsiAlt
Niveau 1
Ref
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3638 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Level 2
Nombre de laboratoires (p) 10
A 2681 2613 2647 005 0034 -0034 -0151 0891 A 2322 2531 2427 015 -0105 0105 -2119 1620 -0220
B 2544 2477 2511 005 0033 -0033 -2699 0872 B 2556 2653 2605 007 -0048 0048 -0581 0750 0094
C 2643 2633 2638 001 0005 -0005 -0311 0130 C 2681 2653 2667 002 0014 -0014 -0040 0217 0029
D 2690 2663 2676 002 0014 -0014 0400 0357 D 2778 2732 2755 003 0023 -0023 0721 0354 0079
E 2690 2716 2703 002 -0013 0013 0897 0336 E 2724 2748 2736 002 -0012 0012 0556 0185 0033
F 2568 2398 2483 012 0085 -0085 -3214 2217 F 2477 2114 2296 026 0182 -0182 -3255 2811 -0188
G 2643 2591 2617 004 0026 -0026 -0707 0682 G 2756 2568 2662 013 0094 -0094 -0085 1453 0045
H 2716 2643 2680 005 0036 -0036 0460 0945 H 2653 2732 2693 006 -0040 0040 0181 0613 0013
L 2591 2591 2591 000 0000 0000 -1196 0000 L 2230 2580 2405 025 -0175 0175 -2307 2704 -0186
M 2653 2690 2672 003 -0018 0018 0311 0481 M 2740 2613 2677 009 0064 -0064 0040 0987 0005
m 2655082 m 26719001 mediane D 00209
Cn(2p) 1867 Cn(2p) 1867 Cp 16101
Cn(p) 16101 Cn(p) 16101 Qn(D) 0039911
n=2p n 20 n=2p n 20 Qdiff 00642591
f 11 f 11 t 082
l 55 l 55
n=p n 10 n=p n 10
f 6 f 6
l 15 l 15
Qn(2p) 0020570 Qn(2p) 00345985
Qn(p) 00332437 Qn(p) 00718158
Qintra 003841 Qintra 006460
Qinter 005352 Qinter 011563
Sr(eacutecart-type de reacutepeacutetabiliteacute) 00543 Sr 00914
RSDr(coefficient de variation de la reacutepeacutetabiliteacute)205 RSDr 342
r(repeatability limit)) 0152 r 0256
SL(eacutecart-type interlaboratoire) 000139 SL 000920
SR(eacutecart-type de reproductibiliteacute) 00659 SR 01325
RSDR(coefficient de variation de la reproductibiliteacute)0025 RSDR 0050
R(limite de reproductibiliteacute)) 018446 R(limite de reproductibiliteacute))03708618
Laboratoires
Niveau 2
Moyenne si Laboratoires
Niveau 2
DRef Alt Moyenne si Deviation h Deviation h k k
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3738 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Level 2 bis
Nombre de laboratoires (p) 10
A 3000 2477 2739 037 0261 -0261 0832 1418 A 2602 2477 2540 009 0062 -0062 -0391 0465 -0199
B 2544 2813 2678 019 -0134 0134 0211 0729 B 2903 2778 2841 009 0062 -0062 1732 0465 0162
C 2477 2813 2645 024 -0168 0168 -0135 0911 C 2477 2699 2588 016 -0111 0111 -0049 0826 -0057
D 2398 2653 2526 018 -0128 0128 -1370 0692 D 2778 3000 2889 016 -0111 0111 2074 0826 0363
E 2875 2740 2808 010 0067 -0067 1547 0365 E 2602 2301 2452 021 0151 -0151 -1012 1120 -0356
F 2477 2544 2511 005 -0033 0033 -1525 0182 F 2778 2477 2628 021 0151 -0151 0230 1120 0117
G 2653 2740 2697 006 -0044 0044 0400 0236 G 2845 2845 2845 000 0000 0000 1764 0000 0148
H 2176 2813 2495 045 -0318 0318 -1692 1727 H 2477 2477 2477 000 0000 0000 -0832 0000 -0017
L 2813 2544 2678 019 0134 -0134 0211 0729 L 2903 2301 2602 043 0301 -0301 0049 2241 -0076
M 2602 2740 2671 010 -0069 0069 0135 0375 M 2903 2255 2579 046 0324 -0324 -0112 2411 -0092
m 2658114 m 259505 mediane D -00372
Cn(2p) 1867 Cn(2p) 1867 Cp 16101
Cn(p) 16101 Cn(p) 16101 Qn(D) 01656915
n=2p n 20 n=2p n 20 Qdiff 02667753
f 11 f 11 t 035
l 55 l 55
n=p n 10 n=p n 10
f 6 f 6
l 15 l 15
Qn(2p) 0098745 Qn(2p) 007195
Qn(p) 006007 Qn(p) 008805
Qintra 018437 Qintra 013435
Qinter 009672 Qinter 014176
Sr(eacutecart-type de reacutepeacutetabiliteacute) 02607 Sr 01900
RSDr(coefficient de variation de la reacutepeacutetabiliteacute)981 RSDr 732
r(repeatability limit)) 0730 r 0532
SL(eacutecart-type interlaboratoire) -002464 SLlt0 SL 000205
SR(eacutecart-type de reproductibiliteacute) 02607 SR 01953
RSDR(coefficient de variation de la reproductibiliteacute)0098 RSDR 0075
R(limite de reproductibiliteacute)) 072996 R(limite de reproductibiliteacute))054686
Laboratoires
Niveau 2 bis
Moyenne si Laboratoires
Niveau 2 bis
DRef Alt Moyenne si Deviation h Deviation h k k
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3838 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Level 3
Nombre de laboratoires (p) 10
A 3690 3663 3676 002 0014 -0014 -0311 0513 A 3690 3708 3699 001 -0009 0009 0354 0257 0022
B 3613 3672 3642 004 -0030 0030 -1851 1109 B 3708 3568 3638 010 0070 -0070 -0844 2061 -0005
C 3663 3732 3698 005 -0035 0035 0645 1302 C 3663 3699 3681 003 -0018 0018 0000 0536 -0017
D 3690 3690 3690 000 0000 0000 0311 0000 D 3643 3663 3653 001 -0010 0010 -0545 0286 -0037
E 3732 3653 3693 006 0040 -0040 0429 1480 E 3613 3732 3673 008 -0060 0060 -0162 1769 -0020
F 3643 3672 3658 002 -0014 0014 -1157 0536 F 3690 3771 3731 006 -0040 0040 0975 1193 0073
G 3699 3653 3676 003 0023 -0023 -0328 0856 G 3602 3643 3623 003 -0021 0021 -1141 0612 -0053
H 3623 3672 3648 003 -0024 0024 -1615 0913 H 3748 3681 3715 005 0033 -0033 0664 0990 0067
L 3724 3663 3694 004 0031 -0031 0461 1150 L 3748 3771 3760 002 -0011 0011 1544 0335 0066
M 3690 3690 3690 000 0000 0000 0311 0000 M 3806 3681 3744 009 0062 -0062 1234 1848 0054
m 368334 m 368086 mediane D 00089
Cn(2p) 1867 Cn(2p) 1867 Cp 16101
Cn(p) 16101 Cn(p) 16101 Qn(D) 00325359
n=2p n 20 n=2p n 20 Qdiff 00523852
f 11 f 11 t 043
l 55 l 55
n=p n 10 n=p n 10
f 6 f 6
l 15 l 15
Qn(2p) 0014323 Qn(2p) 001811
Qn(p) 0013719 Qn(p) 003164
Qintra 002674 Qintra 003381
Qinter 002209 Qinter 005094
Sr(eacutecart-type de reacutepeacutetabiliteacute) 00378 Sr 00478
RSDr(coefficient de variation de la reacutepeacutetabiliteacute)103 RSDr 130
r(repeatability limit)) 0106 r 0134
SL(eacutecart-type interlaboratoire) -000023 SLlt0 SL 000145
SR(eacutecart-type de reproductibiliteacute) 00378 SR 00611
RSDR(coefficient de variation de la reproductibiliteacute)0010 RSDR 0017
R(limite de reproductibiliteacute)) 0105894 R(limite de reproductibiliteacute))017119
Ref Alt DLaboratoires
Niveau 3
Moyenne si Deviation h k Laboratoires
Niveau 3
Moyenne si Deviation h k
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3538 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Appendix 4 ndash Statistical results
Level 1
Nombre de laboratoires (p) 10
A 1602 1602 1602 000 0000 0000 -0495 0000 A 1845 1602 1724 017 0122 -0122 0621 0840 0122
B 1602 1176 1389 030 0213 -0213 -2613 1247 B 1602 1301 1452 021 0151 -0151 -0769 1041 0062
C 1699 1301 1500 028 0199 -0199 -1510 1165 C 1301 1845 1573 038 -0272 0272 -0148 1881 0073
D 1602 1778 1690 012 -0088 0088 0380 0516 D 1602 1602 1602 000 0000 0000 0000 0000 -0088
E 1699 1602 1651 007 0048 -0048 -0013 0284 E 1699 1301 1500 028 0199 -0199 -0522 1376 -0151
F 1778 1301 1540 034 0239 -0239 -1116 1397 F 1778 1699 1739 006 0040 -0040 0698 0274 0199
G 1845 1544 1695 021 0151 -0151 0425 0881 G 2079 1699 1889 027 0190 -0190 1467 1315 0194
H 2041 1602 1822 031 0220 -0220 1689 1286 H 2000 1954 1977 003 0023 -0023 1917 0158 0155
L 1544 1778 1661 017 -0117 0117 0092 0685 L 1301 1602 1452 021 -0151 0151 -0769 1041 -0210
M 1653 1653 1653 000 0000 0000 0013 0000 M 1477 1602 1540 009 -0062 0062 -0319 0432 -0114
m 165186 m 160206 mediane D 00678
Cn(2p) 1867 Cn(2p) 1867 Cp 16101
Cn(p) 16101 Cn(p) 16101 Qn(D) 0092926
n=2p n 20 n=2p n 20 Qdiff 01496169
f 11 f 11 t 114
l 55 l 55
n=p n 10 n=p n 10
f 6 f 6
l 15 l 15
Qn(2p) 0091465 Qn(2p) 007745
Qn(p) 0062469 Qn(p) 0121519
Qintra 017078 Qintra 014461
Qinter 010058 Qinter 019565
Sr(eacutecart-type de reacutepeacutetabiliteacute) 02415 Sr 02045
RSDr(coefficient de variation de la reacutepeacutetabiliteacute)1462 RSDr 1277
r(repeatability limit)) 0676 r 0573
SL(eacutecart-type interlaboratoire) -001905 SLlt0 SL 001737
SR(eacutecart-type de reproductibiliteacute) 02415 SR 02433
RSDR(coefficient de variation de la reproductibiliteacute)0146 RSDR 0152
R(limite de reproductibiliteacute)) 06762 R(limite de reproductibiliteacute))068123
h k h MoyenneLaboratoires
Niveau 1
si LaboratoiresDeviationMoyenne Dk DeviationsiAlt
Niveau 1
Ref
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3638 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Level 2
Nombre de laboratoires (p) 10
A 2681 2613 2647 005 0034 -0034 -0151 0891 A 2322 2531 2427 015 -0105 0105 -2119 1620 -0220
B 2544 2477 2511 005 0033 -0033 -2699 0872 B 2556 2653 2605 007 -0048 0048 -0581 0750 0094
C 2643 2633 2638 001 0005 -0005 -0311 0130 C 2681 2653 2667 002 0014 -0014 -0040 0217 0029
D 2690 2663 2676 002 0014 -0014 0400 0357 D 2778 2732 2755 003 0023 -0023 0721 0354 0079
E 2690 2716 2703 002 -0013 0013 0897 0336 E 2724 2748 2736 002 -0012 0012 0556 0185 0033
F 2568 2398 2483 012 0085 -0085 -3214 2217 F 2477 2114 2296 026 0182 -0182 -3255 2811 -0188
G 2643 2591 2617 004 0026 -0026 -0707 0682 G 2756 2568 2662 013 0094 -0094 -0085 1453 0045
H 2716 2643 2680 005 0036 -0036 0460 0945 H 2653 2732 2693 006 -0040 0040 0181 0613 0013
L 2591 2591 2591 000 0000 0000 -1196 0000 L 2230 2580 2405 025 -0175 0175 -2307 2704 -0186
M 2653 2690 2672 003 -0018 0018 0311 0481 M 2740 2613 2677 009 0064 -0064 0040 0987 0005
m 2655082 m 26719001 mediane D 00209
Cn(2p) 1867 Cn(2p) 1867 Cp 16101
Cn(p) 16101 Cn(p) 16101 Qn(D) 0039911
n=2p n 20 n=2p n 20 Qdiff 00642591
f 11 f 11 t 082
l 55 l 55
n=p n 10 n=p n 10
f 6 f 6
l 15 l 15
Qn(2p) 0020570 Qn(2p) 00345985
Qn(p) 00332437 Qn(p) 00718158
Qintra 003841 Qintra 006460
Qinter 005352 Qinter 011563
Sr(eacutecart-type de reacutepeacutetabiliteacute) 00543 Sr 00914
RSDr(coefficient de variation de la reacutepeacutetabiliteacute)205 RSDr 342
r(repeatability limit)) 0152 r 0256
SL(eacutecart-type interlaboratoire) 000139 SL 000920
SR(eacutecart-type de reproductibiliteacute) 00659 SR 01325
RSDR(coefficient de variation de la reproductibiliteacute)0025 RSDR 0050
R(limite de reproductibiliteacute)) 018446 R(limite de reproductibiliteacute))03708618
Laboratoires
Niveau 2
Moyenne si Laboratoires
Niveau 2
DRef Alt Moyenne si Deviation h Deviation h k k
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3738 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Level 2 bis
Nombre de laboratoires (p) 10
A 3000 2477 2739 037 0261 -0261 0832 1418 A 2602 2477 2540 009 0062 -0062 -0391 0465 -0199
B 2544 2813 2678 019 -0134 0134 0211 0729 B 2903 2778 2841 009 0062 -0062 1732 0465 0162
C 2477 2813 2645 024 -0168 0168 -0135 0911 C 2477 2699 2588 016 -0111 0111 -0049 0826 -0057
D 2398 2653 2526 018 -0128 0128 -1370 0692 D 2778 3000 2889 016 -0111 0111 2074 0826 0363
E 2875 2740 2808 010 0067 -0067 1547 0365 E 2602 2301 2452 021 0151 -0151 -1012 1120 -0356
F 2477 2544 2511 005 -0033 0033 -1525 0182 F 2778 2477 2628 021 0151 -0151 0230 1120 0117
G 2653 2740 2697 006 -0044 0044 0400 0236 G 2845 2845 2845 000 0000 0000 1764 0000 0148
H 2176 2813 2495 045 -0318 0318 -1692 1727 H 2477 2477 2477 000 0000 0000 -0832 0000 -0017
L 2813 2544 2678 019 0134 -0134 0211 0729 L 2903 2301 2602 043 0301 -0301 0049 2241 -0076
M 2602 2740 2671 010 -0069 0069 0135 0375 M 2903 2255 2579 046 0324 -0324 -0112 2411 -0092
m 2658114 m 259505 mediane D -00372
Cn(2p) 1867 Cn(2p) 1867 Cp 16101
Cn(p) 16101 Cn(p) 16101 Qn(D) 01656915
n=2p n 20 n=2p n 20 Qdiff 02667753
f 11 f 11 t 035
l 55 l 55
n=p n 10 n=p n 10
f 6 f 6
l 15 l 15
Qn(2p) 0098745 Qn(2p) 007195
Qn(p) 006007 Qn(p) 008805
Qintra 018437 Qintra 013435
Qinter 009672 Qinter 014176
Sr(eacutecart-type de reacutepeacutetabiliteacute) 02607 Sr 01900
RSDr(coefficient de variation de la reacutepeacutetabiliteacute)981 RSDr 732
r(repeatability limit)) 0730 r 0532
SL(eacutecart-type interlaboratoire) -002464 SLlt0 SL 000205
SR(eacutecart-type de reproductibiliteacute) 02607 SR 01953
RSDR(coefficient de variation de la reproductibiliteacute)0098 RSDR 0075
R(limite de reproductibiliteacute)) 072996 R(limite de reproductibiliteacute))054686
Laboratoires
Niveau 2 bis
Moyenne si Laboratoires
Niveau 2 bis
DRef Alt Moyenne si Deviation h Deviation h k k
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3838 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Level 3
Nombre de laboratoires (p) 10
A 3690 3663 3676 002 0014 -0014 -0311 0513 A 3690 3708 3699 001 -0009 0009 0354 0257 0022
B 3613 3672 3642 004 -0030 0030 -1851 1109 B 3708 3568 3638 010 0070 -0070 -0844 2061 -0005
C 3663 3732 3698 005 -0035 0035 0645 1302 C 3663 3699 3681 003 -0018 0018 0000 0536 -0017
D 3690 3690 3690 000 0000 0000 0311 0000 D 3643 3663 3653 001 -0010 0010 -0545 0286 -0037
E 3732 3653 3693 006 0040 -0040 0429 1480 E 3613 3732 3673 008 -0060 0060 -0162 1769 -0020
F 3643 3672 3658 002 -0014 0014 -1157 0536 F 3690 3771 3731 006 -0040 0040 0975 1193 0073
G 3699 3653 3676 003 0023 -0023 -0328 0856 G 3602 3643 3623 003 -0021 0021 -1141 0612 -0053
H 3623 3672 3648 003 -0024 0024 -1615 0913 H 3748 3681 3715 005 0033 -0033 0664 0990 0067
L 3724 3663 3694 004 0031 -0031 0461 1150 L 3748 3771 3760 002 -0011 0011 1544 0335 0066
M 3690 3690 3690 000 0000 0000 0311 0000 M 3806 3681 3744 009 0062 -0062 1234 1848 0054
m 368334 m 368086 mediane D 00089
Cn(2p) 1867 Cn(2p) 1867 Cp 16101
Cn(p) 16101 Cn(p) 16101 Qn(D) 00325359
n=2p n 20 n=2p n 20 Qdiff 00523852
f 11 f 11 t 043
l 55 l 55
n=p n 10 n=p n 10
f 6 f 6
l 15 l 15
Qn(2p) 0014323 Qn(2p) 001811
Qn(p) 0013719 Qn(p) 003164
Qintra 002674 Qintra 003381
Qinter 002209 Qinter 005094
Sr(eacutecart-type de reacutepeacutetabiliteacute) 00378 Sr 00478
RSDr(coefficient de variation de la reacutepeacutetabiliteacute)103 RSDr 130
r(repeatability limit)) 0106 r 0134
SL(eacutecart-type interlaboratoire) -000023 SLlt0 SL 000145
SR(eacutecart-type de reproductibiliteacute) 00378 SR 00611
RSDR(coefficient de variation de la reproductibiliteacute)0010 RSDR 0017
R(limite de reproductibiliteacute)) 0105894 R(limite de reproductibiliteacute))017119
Ref Alt DLaboratoires
Niveau 3
Moyenne si Deviation h k Laboratoires
Niveau 3
Moyenne si Deviation h k
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3638 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Level 2
Nombre de laboratoires (p) 10
A 2681 2613 2647 005 0034 -0034 -0151 0891 A 2322 2531 2427 015 -0105 0105 -2119 1620 -0220
B 2544 2477 2511 005 0033 -0033 -2699 0872 B 2556 2653 2605 007 -0048 0048 -0581 0750 0094
C 2643 2633 2638 001 0005 -0005 -0311 0130 C 2681 2653 2667 002 0014 -0014 -0040 0217 0029
D 2690 2663 2676 002 0014 -0014 0400 0357 D 2778 2732 2755 003 0023 -0023 0721 0354 0079
E 2690 2716 2703 002 -0013 0013 0897 0336 E 2724 2748 2736 002 -0012 0012 0556 0185 0033
F 2568 2398 2483 012 0085 -0085 -3214 2217 F 2477 2114 2296 026 0182 -0182 -3255 2811 -0188
G 2643 2591 2617 004 0026 -0026 -0707 0682 G 2756 2568 2662 013 0094 -0094 -0085 1453 0045
H 2716 2643 2680 005 0036 -0036 0460 0945 H 2653 2732 2693 006 -0040 0040 0181 0613 0013
L 2591 2591 2591 000 0000 0000 -1196 0000 L 2230 2580 2405 025 -0175 0175 -2307 2704 -0186
M 2653 2690 2672 003 -0018 0018 0311 0481 M 2740 2613 2677 009 0064 -0064 0040 0987 0005
m 2655082 m 26719001 mediane D 00209
Cn(2p) 1867 Cn(2p) 1867 Cp 16101
Cn(p) 16101 Cn(p) 16101 Qn(D) 0039911
n=2p n 20 n=2p n 20 Qdiff 00642591
f 11 f 11 t 082
l 55 l 55
n=p n 10 n=p n 10
f 6 f 6
l 15 l 15
Qn(2p) 0020570 Qn(2p) 00345985
Qn(p) 00332437 Qn(p) 00718158
Qintra 003841 Qintra 006460
Qinter 005352 Qinter 011563
Sr(eacutecart-type de reacutepeacutetabiliteacute) 00543 Sr 00914
RSDr(coefficient de variation de la reacutepeacutetabiliteacute)205 RSDr 342
r(repeatability limit)) 0152 r 0256
SL(eacutecart-type interlaboratoire) 000139 SL 000920
SR(eacutecart-type de reproductibiliteacute) 00659 SR 01325
RSDR(coefficient de variation de la reproductibiliteacute)0025 RSDR 0050
R(limite de reproductibiliteacute)) 018446 R(limite de reproductibiliteacute))03708618
Laboratoires
Niveau 2
Moyenne si Laboratoires
Niveau 2
DRef Alt Moyenne si Deviation h Deviation h k k
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3738 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Level 2 bis
Nombre de laboratoires (p) 10
A 3000 2477 2739 037 0261 -0261 0832 1418 A 2602 2477 2540 009 0062 -0062 -0391 0465 -0199
B 2544 2813 2678 019 -0134 0134 0211 0729 B 2903 2778 2841 009 0062 -0062 1732 0465 0162
C 2477 2813 2645 024 -0168 0168 -0135 0911 C 2477 2699 2588 016 -0111 0111 -0049 0826 -0057
D 2398 2653 2526 018 -0128 0128 -1370 0692 D 2778 3000 2889 016 -0111 0111 2074 0826 0363
E 2875 2740 2808 010 0067 -0067 1547 0365 E 2602 2301 2452 021 0151 -0151 -1012 1120 -0356
F 2477 2544 2511 005 -0033 0033 -1525 0182 F 2778 2477 2628 021 0151 -0151 0230 1120 0117
G 2653 2740 2697 006 -0044 0044 0400 0236 G 2845 2845 2845 000 0000 0000 1764 0000 0148
H 2176 2813 2495 045 -0318 0318 -1692 1727 H 2477 2477 2477 000 0000 0000 -0832 0000 -0017
L 2813 2544 2678 019 0134 -0134 0211 0729 L 2903 2301 2602 043 0301 -0301 0049 2241 -0076
M 2602 2740 2671 010 -0069 0069 0135 0375 M 2903 2255 2579 046 0324 -0324 -0112 2411 -0092
m 2658114 m 259505 mediane D -00372
Cn(2p) 1867 Cn(2p) 1867 Cp 16101
Cn(p) 16101 Cn(p) 16101 Qn(D) 01656915
n=2p n 20 n=2p n 20 Qdiff 02667753
f 11 f 11 t 035
l 55 l 55
n=p n 10 n=p n 10
f 6 f 6
l 15 l 15
Qn(2p) 0098745 Qn(2p) 007195
Qn(p) 006007 Qn(p) 008805
Qintra 018437 Qintra 013435
Qinter 009672 Qinter 014176
Sr(eacutecart-type de reacutepeacutetabiliteacute) 02607 Sr 01900
RSDr(coefficient de variation de la reacutepeacutetabiliteacute)981 RSDr 732
r(repeatability limit)) 0730 r 0532
SL(eacutecart-type interlaboratoire) -002464 SLlt0 SL 000205
SR(eacutecart-type de reproductibiliteacute) 02607 SR 01953
RSDR(coefficient de variation de la reproductibiliteacute)0098 RSDR 0075
R(limite de reproductibiliteacute)) 072996 R(limite de reproductibiliteacute))054686
Laboratoires
Niveau 2 bis
Moyenne si Laboratoires
Niveau 2 bis
DRef Alt Moyenne si Deviation h Deviation h k k
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3838 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Level 3
Nombre de laboratoires (p) 10
A 3690 3663 3676 002 0014 -0014 -0311 0513 A 3690 3708 3699 001 -0009 0009 0354 0257 0022
B 3613 3672 3642 004 -0030 0030 -1851 1109 B 3708 3568 3638 010 0070 -0070 -0844 2061 -0005
C 3663 3732 3698 005 -0035 0035 0645 1302 C 3663 3699 3681 003 -0018 0018 0000 0536 -0017
D 3690 3690 3690 000 0000 0000 0311 0000 D 3643 3663 3653 001 -0010 0010 -0545 0286 -0037
E 3732 3653 3693 006 0040 -0040 0429 1480 E 3613 3732 3673 008 -0060 0060 -0162 1769 -0020
F 3643 3672 3658 002 -0014 0014 -1157 0536 F 3690 3771 3731 006 -0040 0040 0975 1193 0073
G 3699 3653 3676 003 0023 -0023 -0328 0856 G 3602 3643 3623 003 -0021 0021 -1141 0612 -0053
H 3623 3672 3648 003 -0024 0024 -1615 0913 H 3748 3681 3715 005 0033 -0033 0664 0990 0067
L 3724 3663 3694 004 0031 -0031 0461 1150 L 3748 3771 3760 002 -0011 0011 1544 0335 0066
M 3690 3690 3690 000 0000 0000 0311 0000 M 3806 3681 3744 009 0062 -0062 1234 1848 0054
m 368334 m 368086 mediane D 00089
Cn(2p) 1867 Cn(2p) 1867 Cp 16101
Cn(p) 16101 Cn(p) 16101 Qn(D) 00325359
n=2p n 20 n=2p n 20 Qdiff 00523852
f 11 f 11 t 043
l 55 l 55
n=p n 10 n=p n 10
f 6 f 6
l 15 l 15
Qn(2p) 0014323 Qn(2p) 001811
Qn(p) 0013719 Qn(p) 003164
Qintra 002674 Qintra 003381
Qinter 002209 Qinter 005094
Sr(eacutecart-type de reacutepeacutetabiliteacute) 00378 Sr 00478
RSDr(coefficient de variation de la reacutepeacutetabiliteacute)103 RSDr 130
r(repeatability limit)) 0106 r 0134
SL(eacutecart-type interlaboratoire) -000023 SLlt0 SL 000145
SR(eacutecart-type de reproductibiliteacute) 00378 SR 00611
RSDR(coefficient de variation de la reproductibiliteacute)0010 RSDR 0017
R(limite de reproductibiliteacute)) 0105894 R(limite de reproductibiliteacute))017119
Ref Alt DLaboratoires
Niveau 3
Moyenne si Deviation h k Laboratoires
Niveau 3
Moyenne si Deviation h k
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3738 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Level 2 bis
Nombre de laboratoires (p) 10
A 3000 2477 2739 037 0261 -0261 0832 1418 A 2602 2477 2540 009 0062 -0062 -0391 0465 -0199
B 2544 2813 2678 019 -0134 0134 0211 0729 B 2903 2778 2841 009 0062 -0062 1732 0465 0162
C 2477 2813 2645 024 -0168 0168 -0135 0911 C 2477 2699 2588 016 -0111 0111 -0049 0826 -0057
D 2398 2653 2526 018 -0128 0128 -1370 0692 D 2778 3000 2889 016 -0111 0111 2074 0826 0363
E 2875 2740 2808 010 0067 -0067 1547 0365 E 2602 2301 2452 021 0151 -0151 -1012 1120 -0356
F 2477 2544 2511 005 -0033 0033 -1525 0182 F 2778 2477 2628 021 0151 -0151 0230 1120 0117
G 2653 2740 2697 006 -0044 0044 0400 0236 G 2845 2845 2845 000 0000 0000 1764 0000 0148
H 2176 2813 2495 045 -0318 0318 -1692 1727 H 2477 2477 2477 000 0000 0000 -0832 0000 -0017
L 2813 2544 2678 019 0134 -0134 0211 0729 L 2903 2301 2602 043 0301 -0301 0049 2241 -0076
M 2602 2740 2671 010 -0069 0069 0135 0375 M 2903 2255 2579 046 0324 -0324 -0112 2411 -0092
m 2658114 m 259505 mediane D -00372
Cn(2p) 1867 Cn(2p) 1867 Cp 16101
Cn(p) 16101 Cn(p) 16101 Qn(D) 01656915
n=2p n 20 n=2p n 20 Qdiff 02667753
f 11 f 11 t 035
l 55 l 55
n=p n 10 n=p n 10
f 6 f 6
l 15 l 15
Qn(2p) 0098745 Qn(2p) 007195
Qn(p) 006007 Qn(p) 008805
Qintra 018437 Qintra 013435
Qinter 009672 Qinter 014176
Sr(eacutecart-type de reacutepeacutetabiliteacute) 02607 Sr 01900
RSDr(coefficient de variation de la reacutepeacutetabiliteacute)981 RSDr 732
r(repeatability limit)) 0730 r 0532
SL(eacutecart-type interlaboratoire) -002464 SLlt0 SL 000205
SR(eacutecart-type de reproductibiliteacute) 02607 SR 01953
RSDR(coefficient de variation de la reproductibiliteacute)0098 RSDR 0075
R(limite de reproductibiliteacute)) 072996 R(limite de reproductibiliteacute))054686
Laboratoires
Niveau 2 bis
Moyenne si Laboratoires
Niveau 2 bis
DRef Alt Moyenne si Deviation h Deviation h k k
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3838 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Level 3
Nombre de laboratoires (p) 10
A 3690 3663 3676 002 0014 -0014 -0311 0513 A 3690 3708 3699 001 -0009 0009 0354 0257 0022
B 3613 3672 3642 004 -0030 0030 -1851 1109 B 3708 3568 3638 010 0070 -0070 -0844 2061 -0005
C 3663 3732 3698 005 -0035 0035 0645 1302 C 3663 3699 3681 003 -0018 0018 0000 0536 -0017
D 3690 3690 3690 000 0000 0000 0311 0000 D 3643 3663 3653 001 -0010 0010 -0545 0286 -0037
E 3732 3653 3693 006 0040 -0040 0429 1480 E 3613 3732 3673 008 -0060 0060 -0162 1769 -0020
F 3643 3672 3658 002 -0014 0014 -1157 0536 F 3690 3771 3731 006 -0040 0040 0975 1193 0073
G 3699 3653 3676 003 0023 -0023 -0328 0856 G 3602 3643 3623 003 -0021 0021 -1141 0612 -0053
H 3623 3672 3648 003 -0024 0024 -1615 0913 H 3748 3681 3715 005 0033 -0033 0664 0990 0067
L 3724 3663 3694 004 0031 -0031 0461 1150 L 3748 3771 3760 002 -0011 0011 1544 0335 0066
M 3690 3690 3690 000 0000 0000 0311 0000 M 3806 3681 3744 009 0062 -0062 1234 1848 0054
m 368334 m 368086 mediane D 00089
Cn(2p) 1867 Cn(2p) 1867 Cp 16101
Cn(p) 16101 Cn(p) 16101 Qn(D) 00325359
n=2p n 20 n=2p n 20 Qdiff 00523852
f 11 f 11 t 043
l 55 l 55
n=p n 10 n=p n 10
f 6 f 6
l 15 l 15
Qn(2p) 0014323 Qn(2p) 001811
Qn(p) 0013719 Qn(p) 003164
Qintra 002674 Qintra 003381
Qinter 002209 Qinter 005094
Sr(eacutecart-type de reacutepeacutetabiliteacute) 00378 Sr 00478
RSDr(coefficient de variation de la reacutepeacutetabiliteacute)103 RSDr 130
r(repeatability limit)) 0106 r 0134
SL(eacutecart-type interlaboratoire) -000023 SLlt0 SL 000145
SR(eacutecart-type de reproductibiliteacute) 00378 SR 00611
RSDR(coefficient de variation de la reproductibiliteacute)0010 RSDR 0017
R(limite de reproductibiliteacute)) 0105894 R(limite de reproductibiliteacute))017119
Ref Alt DLaboratoires
Niveau 3
Moyenne si Deviation h k Laboratoires
Niveau 3
Moyenne si Deviation h k
Thermo Fisher Scientific
ADRIA Deacuteveloppement 3838 November 19 2014
Summary Report (Version 1)
Listeria Precis Enumeration
Level 3
Nombre de laboratoires (p) 10
A 3690 3663 3676 002 0014 -0014 -0311 0513 A 3690 3708 3699 001 -0009 0009 0354 0257 0022
B 3613 3672 3642 004 -0030 0030 -1851 1109 B 3708 3568 3638 010 0070 -0070 -0844 2061 -0005
C 3663 3732 3698 005 -0035 0035 0645 1302 C 3663 3699 3681 003 -0018 0018 0000 0536 -0017
D 3690 3690 3690 000 0000 0000 0311 0000 D 3643 3663 3653 001 -0010 0010 -0545 0286 -0037
E 3732 3653 3693 006 0040 -0040 0429 1480 E 3613 3732 3673 008 -0060 0060 -0162 1769 -0020
F 3643 3672 3658 002 -0014 0014 -1157 0536 F 3690 3771 3731 006 -0040 0040 0975 1193 0073
G 3699 3653 3676 003 0023 -0023 -0328 0856 G 3602 3643 3623 003 -0021 0021 -1141 0612 -0053
H 3623 3672 3648 003 -0024 0024 -1615 0913 H 3748 3681 3715 005 0033 -0033 0664 0990 0067
L 3724 3663 3694 004 0031 -0031 0461 1150 L 3748 3771 3760 002 -0011 0011 1544 0335 0066
M 3690 3690 3690 000 0000 0000 0311 0000 M 3806 3681 3744 009 0062 -0062 1234 1848 0054
m 368334 m 368086 mediane D 00089
Cn(2p) 1867 Cn(2p) 1867 Cp 16101
Cn(p) 16101 Cn(p) 16101 Qn(D) 00325359
n=2p n 20 n=2p n 20 Qdiff 00523852
f 11 f 11 t 043
l 55 l 55
n=p n 10 n=p n 10
f 6 f 6
l 15 l 15
Qn(2p) 0014323 Qn(2p) 001811
Qn(p) 0013719 Qn(p) 003164
Qintra 002674 Qintra 003381
Qinter 002209 Qinter 005094
Sr(eacutecart-type de reacutepeacutetabiliteacute) 00378 Sr 00478
RSDr(coefficient de variation de la reacutepeacutetabiliteacute)103 RSDr 130
r(repeatability limit)) 0106 r 0134
SL(eacutecart-type interlaboratoire) -000023 SLlt0 SL 000145
SR(eacutecart-type de reproductibiliteacute) 00378 SR 00611
RSDR(coefficient de variation de la reproductibiliteacute)0010 RSDR 0017
R(limite de reproductibiliteacute)) 0105894 R(limite de reproductibiliteacute))017119
Ref Alt DLaboratoires
Niveau 3
Moyenne si Deviation h k Laboratoires
Niveau 3
Moyenne si Deviation h k