Post on 14-Dec-2015
Detection and Enumeration of Food Pathogens with the BAX® PCR System
Thomas P. Oscar, Ph.D.Thomas P. Oscar, Ph.D.
Research Food TechnologistResearch Food Technologist
Welcome… …thank you for coming!
Detection and Enumeration of Food Pathogens with the BAX® PCR System
Thomas P. Oscar, Ph.D.Thomas P. Oscar, Ph.D.
Research Food TechnologistResearch Food Technologist
Welcome… …thank you for coming!
University of Delaware (1978-1982)
Undergraduate Research AssistantUndergraduate Research Assistant B.S. in Animal ScienceB.S. in Animal Science
Pre-Veterinary MedicinePre-Veterinary Medicine
“Interaction of Tiamulin and Monensin in Chickens”
Pennsylvania State University (1982-1984)
Graduate Research AssistantGraduate Research Assistant M.S. in Animal NutritionM.S. in Animal Nutrition
Minor in BiochemistryMinor in Biochemistry
“Characterization of the Bovine Mammary Insulin Receptor”
North Carolina State University (1984-1987)
Graduate Research & Teaching AssistantGraduate Research & Teaching Assistant Ph.D. in Animal SciencePh.D. in Animal Science
Ruminant NutritionRuminant Nutrition
“Role of Nickel in Methane Production”
University of Tennessee, Memphis (1987-1988)
NIH Post-Doctoral Research AssociateNIH Post-Doctoral Research Associate Type II Diabetes Type II Diabetes
Rat Fat Cell ModelRat Fat Cell Model
West Virginia University (1988-1992)
Assistant Professor of Animal ScienceAssistant Professor of Animal Science Growth & DevelopmentGrowth & Development Meat TechnologyMeat Technology
“Hormonal Regulation of Lipolysis in Chicken Fat Cells”
ARS, Poultry Research LaboratoryGeorgetown, DE (1992-1994)
Research Physiologist (Poultry)Research Physiologist (Poultry) Growth & DevelopmentGrowth & Development
DelmarvaPoultryIndustry
“Improve the Lean-to-Fat Ratio of Broiler Chickens”
UMES
ARS, Nutrient Conservation & Metabolism LabBeltsville, MD (1994-1995)
Research Dairy ScientistResearch Dairy Scientist Ruminant NutritionRuminant Nutrition
Beltsville Agricultural Research Center
ARS, Microbial Food Safety Research Unit UMES, Princess Anne, MD (1995-present)
Research Food TechnologistResearch Food Technologist Predictive MicrobiologyPredictive Microbiology OutreachOutreach
Feature Presentation
Current Food Safety ApproachJack-in-the-Box
HACCPHACCP No testingNo testing
Performance StandardsPerformance Standards DetectionDetection EnumerationEnumeration
C. jejuniC. jejuni
To test or not to test,that is the question
Traditional Culture MethodDetection and Enumeration
Pre-enrichmentPre-enrichment
Selective enrichmentSelective enrichment
Selective platingSelective plating
ConfirmationConfirmation
5 to 7Days
Rapid Detection Method
BAX® PCR system
24 to 30 h
102 103 104 105 106 107101100
Bailey, J.S. 1998. J. Food Prot. 61:792-795.
Sample IncubationImportant Factors
Target pathogen (< 1/ml)
•Food Factors
•Inhibitors
•Competition
•Pathogen Factors
•Injury
•Strain
•PCR Sensitivity•104 cells/mlPCR
DetectionTime
Sample SizeChicken carcass rinse
SalmonellaSalmonella Incidence Incidence 4.9% for 10 ml4.9% for 10 ml 20.5% for 270 ml20.5% for 270 ml
Surkiewicz et al., 1969. Food Tech.23:80-85.
Monte Carlo SimulationExtrapolation to other sample sizes
Pathogen Incidence = 10/100 or
10%
100, 10 g Samples
Monte Carlo SimulationExtrapolation to other sample sizes
Pathogen Incidence = 6/10 or 60%
10, 100 g Samples
Objectives
To develop a standard curve for enumerating food To develop a standard curve for enumerating food pathogens as a function of PCR detection time.pathogens as a function of PCR detection time.
To determine the effects of strain variation, meat type and To determine the effects of strain variation, meat type and microbial competition on the shape of the standard curve.microbial competition on the shape of the standard curve.
To develop a Monte Carlo simulation model for To develop a Monte Carlo simulation model for enumeration of food pathogens as a function of sample enumeration of food pathogens as a function of sample size.size.
Materials and Methods
SalmonellaSalmonella Typhimurium 14028Typhimurium 14028
WorthingtonWorthington
Starter culturesStarter cultures 3737°C for 23 h at 150 opm°C for 23 h at 150 opm
Brain heart infusion brothBrain heart infusion broth
Inoculated Pack Study Pre-enrichment Samples
SampleSample 25 g of chicken + 225 ml of buffered peptone water25 g of chicken + 225 ml of buffered peptone water
InoculumInoculum 10100.70.7 to 10 to 1066 CFU CFU
IncubationIncubation 3737°C without shaking°C without shaking
SamplingSampling 0, 2, 4, 6, 8, 10, 12, 24 h0, 2, 4, 6, 8, 10, 12, 24 h
PCR Detection Time Score
PCR AnalysisPCR Analysis BAXBAX® System® System
One gel per sampleOne gel per sample
Scoring SystemScoring System 0 = no band0 = no band
1 = faint band1 = faint band
2 = < full band2 = < full band
3 = full band3 = full band
0 2 4 6 8 10 12 24 MWSubsample (h)
Score 0 0 1 2 3 3 3 3
Example
TotalScore
15
DatasetSterile breast meat and Typhimurium 14028
Strain Chicken Dilution VolumeLog
CFU/mlLog CFU PCR Score
s2 sBM -8 0.5 9.89 1.65 11s2 sBM -5 1 10.03 5.01 17s2 sBM -6 0.5 10.01 3.70 15s2 sBM -9 0.5 10.03 0.71 11s2 sBM -5 0.5 10.05 4.72 18s2 sBM -7 0.5 10.01 2.70 14s2 sBM -4 1 9.97 5.99 18s2 sBM -9 1 9.97 0.99 11s2 sBM -6 1 9.94 3.97 16s2 sBM -4 0.5 9.97 5.69 21s2 sBM -8 1 9.94 1.97 14s2 sBM -7 1 9.97 2.99 14
0 1 2 3 4 5 6 70
5
10
15
20
25
Thigh
Breast
Breast
Thigh
Salmonella Typhimurium (log number/25 g)
PC
R d
etec
tion
time
scor
e
Type of Chicken MeatSterile cooked (autoclaved) chicken meat
8 12 16 20 24 28 32 36 40 44 480
10
20
30
40
50Breast
Breast
Thigh
Thigh
Temperature ( C)
Lag
Tim
e (h
)
Previous StudySalmonella Typhimurium 14028
Oscar, 2002. Int. J. Food Microbiol. 76:177-190.
0 10 20 30 40 50 60 700
1
2
3
4
5
6
7
8
9
10
11
Time (h)
Salm
onel
la T
yphi
mur
ium
AT
CC
140
28(l
og C
FU/g
)
8 12 16 20 24 28 32 36 40 44 480.0
0.2
0.4
0.6
0.8
1.0Breast
Breast
Thigh
Thigh
Temperature ( C)
Gro
wth
Rat
e(l
og C
FU/h
)
Previous StudySalmonella Typhimurium 14028
Oscar, 2002. Int. J. Food Microbiol. 76:177-190.
Conclusion
Dilution may minimize effects of the food matrix Dilution may minimize effects of the food matrix on PCR detection time score. on PCR detection time score.
Strain Variation117 Salmonella Isolates
Chicken Operations
0 1 2 30
1
2
3
4
5
S1S2S5S7S14S17S20S22
S26S30S31S33S38S44S52S62
Log cycle increase
Tim
e, h
Strain variation at 40°Cin brain heart infusion broth
Oscar, 1998. J. Food Prot. 61:964-968.
TyphimuriumWorthington
Previous Study
0 1 2 3 4 5 6 70
4
8
12
16
20
24
Worthington
Typhimurium
Typhimurium
Worthington
Salmonella spp. (log number/25 g)
PC
R d
etec
tion
time
scor
e
ResultsNaturally contaminated breast skin
Generation Time
Variation among 45 strains of Variation among 45 strains of S.S. Enteritidis was: Enteritidis was: 22% at 922% at 9°C °C 4% at 37°C4% at 37°C
Fehlhaber and Kruger, 1998. J. Appl. Microbiol. 84: 945-949.
Conclusion
Strain variation may not greatly affect PCR Strain variation may not greatly affect PCR detection time score under optimal growth detection time score under optimal growth conditions.conditions.
Microbial Competition
0 1 2 3 4 5 6 70
4
8
12
16
20
24 Naturally contaminated breast skinSterile cooked breast or thigh meat
Salmonella spp. (log number/25 g)
PC
R d
etec
tion
time
scor
e
Microbial CompetitionSalmonella Typhimurium DT104
0 10 20 30 40 504
5
6
7
8
9
10
11
12
DT104; nsBM14028; sBM
Temperature ( C)
MP
D (
log
CF
U/g
)
S. Enteritidis
10 20 30 40 500.0
0.2
0.4
0.6
0.8
GFPParent
Temperature ( C)
SGR
(lo
g/h)
Microbial CompetitionGreen fluorescent protein
Conclusion
Microbial competition affected PCR detection Microbial competition affected PCR detection time score and thus, needs to be incorporated into time score and thus, needs to be incorporated into the standard curve.the standard curve.
Monte Carlo Simulation Modeling
0 1 2 3 4 5 6 70
5
10
15
20
25
Y = 1 + 4.89X - 0.31X2
R2 = 0.9611Most likely
Maximum
Minimum
Salmonella spp. (log number/25 g)
PC
R d
etec
tion
time
scor
e
Final Standard Curve95% Prediction Interval
Pert(1.4, 2.1, 2.9)X <= 1.6578
5.0%X <= 2.5890
95.0%
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.2 1.4 1.6 1.8 2 2.2 2.4 2.6 2.8 3
Simulation ModelExcel + @Risk
Sample Type of chicken PCR detection time score1 Thigh skin 02 Thigh skin 03 Thigh skin 14 Breast skin 05 Breast skin 06 Breast skin 07 Breast meat 08 Breast meat 09 Breast meat 010 Breast meat 411 Breast meat 012 Breast meat 0
Naturally Contaminated ChickenNot inoculated with Salmonella
Sample size, g Incidence, % Minimum Median Maximum
25 16.7 1 2 16
50 30.8 1 3 22
100 51.5 1 3 33
200 76.7 1 5 42
400 94.1 1 8 70
500 97.4 1 9 81
Distribution of contamination (number of Salmonella )
Effect of Sample SizeSimulation results
Conclusion
Linear extrapolation of detection and enumeration Linear extrapolation of detection and enumeration results is not appropriate.results is not appropriate.
Future ResearchEnumeration
Automated BAXAutomated BAX® System® System Cycle threshold rather than band width score.Cycle threshold rather than band width score.
Other Pathogens and FoodsOther Pathogens and Foods
The End
Thank you for your attention!
I will be glad to answer your questions
The End
Thank you for your attention!
I will be glad to answer your questions