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
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Hundreds of new methods developed each year
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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?
Introduction
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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
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The Three S’s
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Sensitivity
What is the sensitivity of current method
 What degree of sensitivity is needed
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Specificity
What is the false positive rate
 What is the false negative rate
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Speed
What is speed of current method (samples
processed/day)
 How quickly are results needed
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Performance Criteria
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Costs
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What
What
What
What
is
is
is
is
cost of current method
cost of instrumentation
cost of disposables/reagents
the cost per test
Reagents
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Prep time
Stability
Availability
Consistency (Quality Control)
Performance Criteria
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Versatility
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Product only
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Environmental samples only
Pathogens only
Microorganisms only
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Variety of food matrixes
Bacteria and/or Fungi
Acceptability of method by scientific community
and/or Regulators
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AOAC, AOAC-RI, USDA-FSIS, FDA, AFNOR
Performance Criteria
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Vendor company reputation
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Training
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First product on market
Vendor provided training on site
How much, how long
Technical Service
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Speed of service
Availability of service (24-7)
Service contract required
Technical Evaluation
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Objective
Justification (benefit of method to company)
Acceptance Criteria
Material and Methods
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Microorganisms
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Test Media/Conditions
Genus, species, source
Inoculum preparation
Inoculation Procedure
Statistical Analysis
Results
Next Steps
Case Study #1
Dichloran-Rose Bengal Agar Yeast
and Mold Method Evaluation
Dichloran-Rose Bengal Agar Yeast
and Mold Method Evaluation
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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 Evaluation
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Microorganisms:
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Mold Cultures
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Yeast Cultures
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A.niger, Penicillium spp., and Paecilomyces spp.
Z.ballii, S.cerevisiae, and a plant isolate
Inoculum Preparation:
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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
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Molds serially diluted
Yeast adjusted to a spec reading of 1.00, then serially diluted
Dichloran-Rose Bengal Agar Yeast
and Mold Method Evaluation
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Material and Methods:
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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
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Counted at 3 and 5 days
DRBA incubated at 30C and 35C
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Counted at 2, 3, 4, and 5 days
Dichloran-Rose Bengal Agar Yeast
and Mold Method Evaluation
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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 Evaluation
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Results:
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DRB at 2 days-30C was statistically equivalent to
aPDA at 5 days for mold recovery
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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 Evaluation
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Conclusion:
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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
Rapid Check Salmonella
Test Kit Evaluation
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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 Evaluation
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Acceptance Criteria:
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Cost
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Speed; shorter time to results vs. PCR?
Sensitivity; greater or equivalent to PCR?
Specificity; greater or equivalent to PCR
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 Evaluation
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Organisms and Inoculum Preparation:
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A cocktail of 5 Salmonella spp.
A cocktail of 7 non-Salmonella spp.
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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 Evaluation
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Methods:
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Inoculation of samples
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Pre-enrichment of samples
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With Salmonella
With non-Salmonella strains
With both
Traditional medium; Lactose for 24 hours
SDI medium for 5 hours
Secondary enrichment
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Tetrathionate for 24 hours
Rapid Check Salmonella
Test Kit Evaluation
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Methods (cont):
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BAX PCR analysis
3 hour re-growth
 Cell lysis
 4-8 hour PCR cycle
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SDI lateral flow assay
Load 150ul onto SDI cartridge
 Develop for 10 minutes
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Rapid Check Salmonella
Test Kit Evaluation
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Results:
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Sensitivity
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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
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No cross reactivity with non-Salmonella organisms with
either method
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Rapid Check Salmonella
Test
Kit
Evaluation
Results: Speed
BAX-PCR
SDI w/ 5 hour
medium
SDI
Enrichment
24 hours
5 hours
24 hours
Secondary
18 hours
18 hours
18 hours
Re-growth
3 hours
0 hours
0 hours
Cell lysis
0.5 hours
0 hours
0 hours
Analysis
time
8 hours
10 minutes
10 minutes
Total
53.5 hours
23 hours, 10 minutes
42 hours, 10 minutes
Rapid Check Salmonella
Test Kit Evaluation
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Conclusions:
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SDI shown to be as sensitive as BAX-PCR
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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 Evaluation
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Conclusions:
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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
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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
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Sensitivity
Specificity
Speed
Cost
Lab space