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Validation of Microbiological Methods for Use in the Food Industry

Validation of Microbiological Methods for Use in the Food Industry. Brazilian Association for Food Protection 6 th International Symposium Sao Paulo, Brazil June 15 th , 2007. Introduction. Hundreds of new methods developed each year Pathogenic organisms Non-Pathogenic organisms

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Validation of Microbiological Methods for Use in the Food Industry

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  1. Validation of Microbiological Methods for Use in the Food Industry Brazilian Association for Food Protection 6th International Symposium Sao Paulo, Brazil June 15th, 2007

  2. Introduction • Hundreds of new methods developed each year • Pathogenic organisms • Non-Pathogenic organisms • Detection • Identification • How do you know if you need a new method? • How do you decide if it is the right method for your purpose?

  3. Introduction • Goal of methods evaluation is to find an innovative technology that will allow for quick and efficient detection and/or quanitation of pathogens and spoilage organisms

  4. Performance Criteria • The Three S’s • Sensitivity • What is the sensitivity of current method • What degree of sensitivity is needed • Specificity • What is the false positive rate • What is the false negative rate • Speed • What is speed of current method (samples processed/day) • How quickly are results needed

  5. Performance Criteria • Costs • What is cost of current method • What is cost of instrumentation • What is cost of disposables/reagents • What is the cost per test • Reagents • Prep time • Stability • Availability • Consistency (Quality Control)

  6. Performance Criteria • Versatility • Product only • Variety of food matrixes • Environmental samples only • Pathogens only • Microorganisms only • Bacteria and/or Fungi • Acceptability of method by scientific community and/or Regulators • AOAC, AOAC-RI, USDA-FSIS, FDA, AFNOR

  7. Performance Criteria • Vendor company reputation • First product on market • Training • Vendor provided training on site • How much, how long • Technical Service • Speed of service • Availability of service (24-7) • Service contract required

  8. Technical Evaluation • Objective • Justification (benefit of method to company) • Acceptance Criteria • Material and Methods • Test Media/Conditions • Microorganisms • Genus, species, source • Inoculum preparation • Inoculation Procedure • Statistical Analysis • Results • Next Steps

  9. Case Study #1 Dichloran-Rose Bengal Agar Yeast and Mold Method Evaluation

  10. Dichloran-Rose Bengal Agar Yeast and Mold Method Evaluation • Objective: Determine validity of a 2 day yeast and mold method using DRB agar incubated at 30C or 35C • Justification: Reduced product holding time, resulting in significant cost savings to the plant • Acceptance Criteria: Recovery efficiencies must be equivalent to the current 5 day PDA method

  11. Dichloran-Rose Bengal Agar Yeast and Mold Method Evaluation • Microorganisms: • Mold Cultures • A.niger, Penicillium spp., and Paecilomyces spp. • Yeast Cultures • Z.ballii, S.cerevisiae, and a plant isolate • Inoculum Preparation: • Organisms were harvested from aPDA plates by washing with sterile water • 1ml from each individual mold or yeast suspension was added to 20 mls DI water • Molds serially diluted • Yeast adjusted to a spec reading of 1.00, then serially diluted

  12. Dichloran-Rose Bengal Agar Yeast and Mold Method Evaluation • Material and Methods: • product was inoculated with 100 cfu/g of target organisms • 0.1ml of inoculated product surface plated onto each media (aPDA, DRBA) • aPDA incubated at 25C • Counted at 3 and 5 days • DRBA incubated at 30C and 35C • Counted at 2, 3, 4, and 5 days

  13. Dichloran-Rose Bengal Agar Yeast and Mold Method Evaluation • Statistical Analysis:An analysis of variance (AOV) was done to test if the total counts for DRB at 2 and 5 days was significantly different from aPDA at 5 days

  14. Dichloran-Rose Bengal Agar Yeast and Mold Method Evaluation • Results: • DRB at 2 days-30C was statistically equivalent to aPDA at 5 days for mold recovery • Molds were pale in color; Penicillium spp. was white on DRB (green on aPDA). The other 2 test molds were pale yellow • Yeast counts on DRB at 30C were significantly lower than counts on aPDA at 2 and 5 days • Mold and Yeast counts were significantly lower on DRB at 35C vs. aPDA

  15. Dichloran-Rose Bengal Agar Yeast and Mold Method Evaluation • Conclusion: • Due to overall decreased recovery of yeast and mold, and the mold visual observations; the Dichloran-Rose Bengal Agar Yeast and Mold recovery medium is not recommended.

  16. Case Study #2 Rapid Check SalmonellaTest Kit Evaluation

  17. Rapid Check SalmonellaTest Kit Evaluation • Objective: Determine validity of the Strategic Diagnostics Inc. Rapid Check antibody lateral flow method for the detection of Salmonella in comparison to the BAX PCR test method • Justification: Reduce testing cost, false positives rate and technician time

  18. Rapid Check SalmonellaTest Kit Evaluation • Acceptance Criteria: • Speed; shorter time to results vs. PCR? • Sensitivity; greater or equivalent to PCR? • Specificity; greater or equivalent to PCR • Cost • Less than or equal to BAX PCR system • Cost per test • Versatility; food products only, environmental samples only, or both?

  19. Rapid Check SalmonellaTest Kit Evaluation • Organisms and Inoculum Preparation: • A cocktail of 5 Salmonella spp. • A cocktail of 7 non-Salmonella spp. • E.coli (2), Citrobacter, Bacillus, Klebsiella, Enterobacter (2) • Individual cultures grown overnight in BHI at 35C • Salmonella strains pooled, diluted to 100cfu/ml • Non-Salmonella strains pooled, diluted to 1,000 cfu/ml

  20. Rapid Check SalmonellaTest Kit Evaluation • Methods: • Inoculation of samples • With Salmonella • With non-Salmonella strains • With both • Pre-enrichment of samples • Traditional medium; Lactose for 24 hours • SDI medium for 5 hours • Secondary enrichment • Tetrathionate for 24 hours

  21. Rapid Check SalmonellaTest Kit Evaluation • Methods (cont): • BAX PCR analysis • 3 hour re-growth • Cell lysis • 4-8 hour PCR cycle • SDI lateral flow assay • Load 150ul onto SDI cartridge • Develop for 10 minutes

  22. Rapid Check SalmonellaTest Kit Evaluation • Results: • Sensitivity • Results were more consistent with SDI when recovering at the threshold level (1000 cfu/ml in the TT broth) • Equivalent results with both methods above the threshold level • SDI 5 hour pre-incubation media did not consistently support growth above the threshold level (acceptance criteria) • Specificity • No cross reactivity with non-Salmonella organisms with either method

  23. Rapid Check SalmonellaTest Kit Evaluation • Results: Speed

  24. Rapid Check SalmonellaTest Kit Evaluation • Conclusions: • SDI shown to be as sensitive as BAX-PCR • 5 hour medium not recommended • No cross reactivity observed with SDI • SDI gave results sooner than PCR • PCR has more steps, more prone to technician error • Some degree of subjectivity with SDI • SDI easier to use; 1 step inoculation of 1 single cartridge

  25. Rapid Check SalmonellaTest Kit Evaluation • Conclusions: • SDI can be successfully used for food and environmental samples • No additional equipment needed (heat blocks, thermal cycler) • Cost per test of SDI less than BAX-PCR • SDI approved for use in place of PCR • Appropriate for use by labs analyzing a smaller number of samples

  26. Value of Method Validation • Need to validate method on your intended product; rule out matrix interference • Determine minimum regulatory requirements (AOAC, AFNOR, etc) • Determine what is the right method for your lab based on volume of testing and number of technicians • Base selection of methodology on need • Sensitivity • Specificity • Speed • Cost • Lab space

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