1 / 26

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

tanner
Download Presentation

Validation of Microbiological Methods for Use in the Food Industry

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript


  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

More Related