Validation of microbiological methods for use in the food industry l.jpg
This presentation is the property of its rightful owner.
Sponsored Links
1 / 26

Validation of Microbiological Methods for Use in the Food Industry PowerPoint PPT Presentation


  • 331 Views
  • Uploaded on
  • Presentation posted in: General

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

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.While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server.


- - - - - - - - - - - - - - - - - - - - - - - - - - E N D - - - - - - - - - - - - - - - - - - - - - - - - - -

Presentation Transcript


Validation of microbiological methods for use in the food industry l.jpg

Validation of Microbiological Methods for Use in the Food Industry

Brazilian Association for Food Protection

6th International Symposium

Sao Paulo, Brazil

June 15th, 2007


Introduction l.jpg

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?


Introduction3 l.jpg

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


Performance criteria l.jpg

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


Performance criteria5 l.jpg

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)


Performance criteria6 l.jpg

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


Performance criteria7 l.jpg

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


Technical evaluation l.jpg

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


Case study 1 dichloran rose bengal agar yeast and mold method evaluation l.jpg

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


Dichloran rose bengal agar yeast and mold method evaluation l.jpg

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


Dichloran rose bengal agar yeast and mold method evaluation11 l.jpg

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


Dichloran rose bengal agar yeast and mold method evaluation12 l.jpg

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


Dichloran rose bengal agar yeast and mold method evaluation13 l.jpg

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


Dichloran rose bengal agar yeast and mold method evaluation14 l.jpg

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


Dichloran rose bengal agar yeast and mold method evaluation15 l.jpg

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.


Case study 2 rapid check salmonella test kit evaluation l.jpg

Case Study #2 Rapid Check SalmonellaTest Kit Evaluation


Rapid check salmonella test kit evaluation l.jpg

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


Rapid check salmonella test kit evaluation18 l.jpg

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?


Rapid check salmonella test kit evaluation19 l.jpg

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


Rapid check salmonella test kit evaluation20 l.jpg

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


Rapid check salmonella test kit evaluation21 l.jpg

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


Rapid check salmonella test kit evaluation22 l.jpg

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


Rapid check salmonella test kit evaluation23 l.jpg

Rapid Check SalmonellaTest Kit Evaluation

  • Results: Speed


Rapid check salmonella test kit evaluation24 l.jpg

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


Rapid check salmonella test kit evaluation25 l.jpg

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


Value of method validation l.jpg

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


  • Login