8/19/2019 Iso 16140 Validation by Afnor on Tvc Tc and Ec Adria-2

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TEMPO® system :AFNOR validation according to the ISO 16140 standard

Danièle SOHIER 1* , Maryse RANNOU1 , Florence GORSE2

DRIA Développement , expert lab at the AFNOR validation committee, France, daniele.sohier@adria.tm.fr / 2 BioMérieux, France

TEMPO® system is a new automatedmethod associating an innovative cardwith an adapted medium to ensurerapid enumeration of several qualityindicators. It replaces serial dilutionsand tedious plate reading with a simple1/10 dilution and an automated MPN(Most Probable Number) enumerationmethod using 3x16 miniaturized tubes(Figures 1a, 1b, 1c).TEMPO® EC, TEMPO® TVC and TEMPO®

TC allow bacterial count of respectivelyE. coli, Total Viable flora and TotalColiforms. Comparison of these (three)

alternative methods with thecorrespondent ISO standard platecount methods (NF ISO 16649-2, ISO4833, ISO 4832) were done accordingto the ISO 16140 standard and AFNORtechnical rules.

Figure 1a :Tempo® Reagent

Figure 1b : Tempo® Filler

Figure 1c : Tempo® Reader

SENSITIVITY and SPECIFICITY STUDIESThe sensitivity and specificity of TEMPO® EC and TEMPO® TCwere assessed using pure cultures of 30 targeted strains and 20non-targeted strains. All TEMPO® EC results were in agreementwith those obtained with NF EN ISO 16649-2 reference method,while TEMPO® TC shows higher sensitivity and specificity thanthe ISO 4832 standard.

RELATIVE LINEARITY STUDIESFor each alternative methods, the relative linearity was investigatedby serial dilutions of pure culture mixed with 5 food types. Theinoculation levels were 100 - 500 – 1000 - 5.103 - 5.104 CFU/g. Thelinearity of TEMPO® EC, TEMPO® TVC and TEMPO® TC ischaracterised by linear correlation coefficients superior to 0.96; theequations are shown in Table 1.

RELATIVE ACCURACY STUDIESThe relative accuracy studies were performed by testing 50 naturallycontaminated food products including raw and processed meat,poultry, dairy products, seafood, vegetables, ready -to-eat meals, andpet food (Figure 3).

Figure 3 : TEMPO® EC, TEMPO® TVC and TEMPO® TC accuracystudies regressions, including all products

 According to the accuracy studies, TEMPO® EC, TEMPO® TVC andTEMPO® TC give results comparable to standard plate countmethods.

COLLABORATIVE STUDIESPasteurised milk samples were contaminated with 4 differentlevels of pure cultures and analysed by the reference andalternative methods within more than 8 laboratories for eachmethod (Figure 2a, 2b, 2c and Table 2).

Depending on the contamination levels tested, TEMPO® EC,TEMPO® TVC and TEMPO® TC methods show similar or higherlimit of repeatability and limit of reproducibility values ascompared to standard plate count methods. TEMPO® alternativemethods are characterised by a better laboratory resultsdispersion. The bias between both methods are non significant.

CONCLUSION :TEMPO®

system offers important economic savings by standardizing the analysis and minimizing the training time, thevolume of wastes and the number of operations. Workflow studies demonstrate a 2 to 3 fold reduction of the manipulation time usingTEMPO® methods as compared to correspondent ISO reference methods. According to these performance assessments, TEMPO® EC, TEMPO® TVC and TEMPO® TC methods represent valuable alternative forautomated methods for E. coli, Total Viable flora and Total Coliforms enumeration in food.

  Tempo®  methods

Product matrices Tempo® EC Tempo® TVC Tempo® TC

Dairy matrix Log Tempo® EC = 0.92 log ISO + 0.47 Log Tempo® TVC = 1.01 log ISO + 0.02 Log Tempo® TC = 1.07 log ISO - 0.13

Meat matrix Log Tempo® EC = 0.89 log ISO + 0.51 Log Tempo®  TVC = 1.01 log ISO + 0.14 Log Tempo® TC = 0.98 log ISO + 0.07

Seafood matrix Log Tempo ®  EC = 0.13 log ISO – 0.05 Log Tempo ® TVC = 1.05 log ISO – 0.14 Log Tempo® TC = 1.13 log ISO - 0.46

 Vegetable matrix Log Tempo®

  EC = 1.07 log ISO – 0.03 Log Tempo®

 TVC = 1.10 log ISO – 0.28 Log Tempo®

 TC = 1.22 log ISO - 0.72

Pet food matrix Log Tempo ®  EC = 0.16 log ISO – 0.39 Log Tempo ® TVC = 1.14 log ISO – 0.40 Log Tempo® TC = 1.05 log ISO - 0.17

Table 1 : TEMPO® EC, TEMPO® TVC, TEMPO® TC linear regressions

OLS 1 Regression

0,00

2,00

4,00

6,00

0,00 2,00 4,00 6,00

Reference method

   A   l   t  e  r  n  a   t   i  v  e  m  e   t   h  o

Log Tempo® EC = 1.00 log ISO + 0.36

OLS 1 Regression

0,00

2,00

4,00

6,00

8,00

0,00 2,00 4,00 6,00 8,00

Reference method

   A   l   t  e  r  n  a   t   i  v  e  m  e   t   h  o   d

Log Tempo® TVC = 1.03 log ISO –0.17

GMFR regression

0,000

2,000

4,000

6,000

8,000

0 ,0 0 1 ,0 0 2 , 0 0 3 , 00 4 , 0 0 5 , 00 6 ,0 0 7 , 0 0 8 , 00

Reference method

   A   l   t  e  r  n  a   t   i  v  e  m  e   t   h  o   d

Log Tempo® TC = 0.98 log ISO – 0.25

TEMPO® EC accuracy studyContamination levels :Log 1 CFU/g to Log 4.90 CFU/g

TEMPO® TVC accuracy studyContamination levels :Log 1 CFU/g to Log 6.60 CFU/g

TEMPO® TC accuracy studyContamination levels :Log 1 CFU/g to Log 6.15 CFU/g

1 000 to 10 000

100 to 1 000

10 to 100

R    A R    A R    A

Contamination level s

Figure 2 a : Repeatability limits

T E MP O® EC  T E MP O® T V C  T E MP O ® TC 

0

0,2

0,4

0, 6

0,8

1 000 to 10 000

100 to 1000

10 to 100

Contamination level s

T E MP O® EC  T E MP O

®

T V C  T E MP O ® TC 

R  A   R    A R    A 

Figure 2 b :Reproducibility limits

10 to 100

100 to 1 000

1 000 to 10 000

T E MP O® EC  T E MP O® T V C  T E MP O® TC 

R    A R  A   R    A

Contamination level s

Figure 2c: Laborator y dispersion

25.0

Table 2 :

Bias between TEMPO® EC, TEMPO® EC, TEMPO® ECand the corresponding standard method

  Tempo®   methods

Contaminationlevels Tempo®  EC Tempo®  TVC Tempo® TC

10 to 100 0.02 0 .10 -0.05

100 to 1 000 0.14 0.09 0.08

1 000 to 10 000 0.31 0.20 0.02