Detection and Enumeration of Food Pathogens with the BAX® PCR System Thomas P. Oscar, Ph.D. Research Food Technologist Welcome……thank you for coming!

Detection and Enumeration of Food Pathogens with the BAX® PCR System Thomas P. Oscar, Ph.D. Research Food Technologist Welcome……thank you for coming!

University of Delaware ( ) Undergraduate Research Assistant Undergraduate Research Assistant  B.S. in Animal Science  Pre-Veterinary Medicine “Interaction of Tiamulin and Monensin in Chickens”

Pennsylvania State University ( ) Graduate Research Assistant  M.S. in Animal Nutrition  Minor in Biochemistry “Characterization of the Bovine Mammary Insulin Receptor”

North Carolina State University ( ) Graduate Research & Teaching Assistant Graduate Research & Teaching Assistant  Ph.D. in Animal Science  Ruminant Nutrition “Role of Nickel in Methane Production”

University of Tennessee, Memphis ( ) NIH Post-Doctoral Research Associate  Type II Diabetes  Rat Fat Cell Model

West Virginia University ( ) Assistant Professor of Animal Science Assistant Professor of Animal Science  Growth & Development  Meat Technology “Hormonal Regulation of Lipolysis in Chicken Fat Cells”

ARS, Poultry Research Laboratory Georgetown, DE ( ) Research Physiologist (Poultry)  Growth & Development Delmarva Poultry Industry “Improve the Lean-to-Fat Ratio of Broiler Chickens” UMES

ARS, Nutrient Conservation & Metabolism Lab Beltsville, MD ( ) Research Dairy Scientist Research Dairy Scientist  Ruminant Nutrition Beltsville Agricultural Research Center

ARS, Microbial Food Safety Research Unit UMES, Princess Anne, MD (1995-present) Research Food Technologist  Predictive Microbiology  Outreach

Feature Presentation

Current Food Safety Approach Jack-in-the-Box HACCP HACCP  No testing Performance Standards Performance Standards  Detection  Enumeration  C. jejuni To test or not to test, that is the question

Traditional Culture Method Detection and Enumeration Pre-enrichment Pre-enrichment Selective enrichment Selective enrichment Selective plating Selective plating Confirmation Confirmation 5 to 7 Days

Rapid Detection Method BAX ® PCR system 24 to 30 h Bailey, J.S J. Food Prot. 61:

Sample Incubation Important Factors Target pathogen (< 1/ml) Food Factors Inhibitors Competition Pathogen Factors Injury Strain PCR Sensitivity 10 4 cells/ml PCR Detection Time

Sample Size Chicken carcass rinse Salmonella Incidence  4.9% for 10 ml  20.5% for 270 ml Surkiewicz et al., Food Tech.23:80-85.

Monte Carlo Simulation Extrapolation to other sample sizes Pathogen Incidence = 10/100 or 10% 100, 10 g Samples

Monte Carlo Simulation Extrapolation to other sample sizes Pathogen Incidence = 6/10 or 60% 10, 100 g Samples

Objectives To develop a standard curve for enumerating food pathogens as a function of PCR detection time. To develop a standard curve for enumerating food pathogens as a function of PCR detection time. To determine the effects of strain variation, meat type and microbial competition on the shape of the standard curve. To determine the effects of strain variation, meat type and microbial competition on the shape of the standard curve. To develop a Monte Carlo simulation model for enumeration of food pathogens as a function of sample size. To develop a Monte Carlo simulation model for enumeration of food pathogens as a function of sample size.

Materials and Methods Salmonella Salmonella  Typhimurium  Worthington Starter cultures Starter cultures  37°C for 23 h at 150 opm  Brain heart infusion broth

Inoculated Pack Study Pre-enrichment Samples Sample Sample  25 g of chicken ml of buffered peptone water Inoculum Inoculum  to 10 6 CFU Incubation Incubation  37°C without shaking Sampling Sampling  0, 2, 4, 6, 8, 10, 12, 24 h

PCR Detection Time Score PCR Analysis PCR Analysis  BAX® System  One gel per sample Scoring System  0 = no band  1 = faint band  2 = < full band  3 = full band

MWSubsample (h) Score Example Total Score 15

Dataset Sterile breast meat and Typhimurium 14028

Type of Chicken Meat Sterile cooked (autoclaved) chicken meat

Previous Study Salmonella Typhimurium Oscar, Int. J. Food Microbiol. 76:

Previous Study Salmonella Typhimurium Oscar, Int. J. Food Microbiol. 76:

Conclusion Dilution may minimize effects of the food matrix on PCR detection time score. Dilution may minimize effects of the food matrix on PCR detection time score.

Strain Variation 117 Salmonella Isolates Chicken Operations

Strain variation at 40°C in brain heart infusion broth Oscar, J. Food Prot. 61: Typhimurium Worthington Previous Study

Results Naturally contaminated breast skin

Generation Time Variation among 45 strains of S. Enteritidis was: Variation among 45 strains of S. Enteritidis was:  22% at 9°C  4% at 37°C Fehlhaber and Kruger, J. Appl. Microbiol. 84:

Conclusion Strain variation may not greatly affect PCR detection time score under optimal growth conditions. Strain variation may not greatly affect PCR detection time score under optimal growth conditions.

Microbial Competition

Microbial Competition Salmonella Typhimurium DT104

Microbial Competition Green fluorescent protein

Conclusion Microbial competition affected PCR detection time score and thus, needs to be incorporated into the standard curve. Microbial competition affected PCR detection time score and thus, needs to be incorporated into the standard curve.

Monte Carlo Simulation Modeling

Final Standard Curve 95% Prediction Interval

Simulation Model Excel

Naturally Contaminated Chicken Not inoculated with Salmonella

Effect of Sample Size Simulation results

Conclusion Linear extrapolation of detection and enumeration results is not appropriate. Linear extrapolation of detection and enumeration results is not appropriate.

Future Research Enumeration Automated BAX® System Automated BAX® System  Cycle threshold rather than band width score. Other Pathogens and Foods Other 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