validation of microbiological methods for use in the food industry brazilian association for food...
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Validation of Microbiological Validation of Microbiological Methods for Use in the Food Methods for Use in the Food
IndustryIndustry
Brazilian Association for Food Protection Brazilian Association for Food Protection 66thth International Symposium International Symposium
Sao Paulo, BrazilSao Paulo, BrazilJune 15June 15thth, 2007, 2007
IntroductionIntroduction
Hundreds of new methods developed each Hundreds of new methods developed each yearyear Pathogenic organismsPathogenic organisms Non-Pathogenic organismsNon-Pathogenic organisms DetectionDetection IdentificationIdentification
How do you know if you need a new method?How do you know if you need a new method? How do you decide if it is the right method How do you decide if it is the right method
for your purpose?for your purpose?
IntroductionIntroduction
Goal of methods evaluation is to find Goal of methods evaluation is to find an innovative technology that will an innovative technology that will allow for quick and efficient detection allow for quick and efficient detection and/or quanitation of pathogens and and/or quanitation of pathogens and spoilage organismsspoilage organisms
Performance CriteriaPerformance Criteria The Three S’sThe Three S’s
SensitivitySensitivity What is the sensitivity of current methodWhat is the sensitivity of current method What degree of sensitivity is neededWhat degree of sensitivity is needed
SpecificitySpecificity What is the false positive rateWhat is the false positive rate What is the false negative rateWhat is the false negative rate
SpeedSpeed What is speed of current method (samples What is speed of current method (samples
processed/day)processed/day) How quickly are results neededHow quickly are results needed
Performance CriteriaPerformance Criteria
CostsCosts What is cost of current methodWhat is cost of current method What is cost of instrumentationWhat is cost of instrumentation What is cost of disposables/reagentsWhat is cost of disposables/reagents What is the cost per testWhat is the cost per test
ReagentsReagents Prep timePrep time StabilityStability AvailabilityAvailability Consistency (Quality Control)Consistency (Quality Control)
Performance CriteriaPerformance Criteria
VersatilityVersatility Product onlyProduct only
Variety of food matrixesVariety of food matrixes Environmental samples onlyEnvironmental samples only Pathogens onlyPathogens only Microorganisms onlyMicroorganisms only
Bacteria and/or FungiBacteria and/or Fungi
Acceptability of method by scientific Acceptability of method by scientific community and/or Regulatorscommunity and/or Regulators AOAC, AOAC-RI, USDA-FSIS, FDA, AFNORAOAC, AOAC-RI, USDA-FSIS, FDA, AFNOR
Performance CriteriaPerformance Criteria
Vendor company reputationVendor company reputation First product on marketFirst product on market
TrainingTraining Vendor provided training on siteVendor provided training on site How much, how longHow much, how long
Technical ServiceTechnical Service Speed of serviceSpeed of service Availability of service (24-7)Availability of service (24-7) Service contract requiredService contract required
Technical EvaluationTechnical Evaluation
ObjectiveObjective Justification (benefit of method to company)Justification (benefit of method to company) Acceptance CriteriaAcceptance Criteria Material and MethodsMaterial and Methods
Test Media/ConditionsTest Media/Conditions MicroorganismsMicroorganisms
Genus, species, sourceGenus, species, source Inoculum preparationInoculum preparation
Inoculation ProcedureInoculation Procedure Statistical AnalysisStatistical Analysis ResultsResults Next StepsNext Steps
Case Study #1Case Study #1
Dichloran-Rose Bengal Agar Dichloran-Rose Bengal Agar Yeast and Mold Method Yeast and Mold Method
EvaluationEvaluation
Dichloran-Rose Bengal Agar Dichloran-Rose Bengal Agar Yeast and Mold Method Yeast and Mold Method
EvaluationEvaluation Objective:Objective: Determine validity of a 2 Determine validity of a 2
day yeast and mold method using day yeast and mold method using DRB agar incubated at 30C or 35CDRB agar incubated at 30C or 35C
Justification: Justification: Reduced product Reduced product holding time, resulting in significant holding time, resulting in significant cost savings to the plantcost savings to the plant
Acceptance Criteria:Acceptance Criteria: Recovery Recovery efficiencies must be equivalent to the efficiencies must be equivalent to the current 5 day PDA methodcurrent 5 day PDA method
Dichloran-Rose Bengal Agar Dichloran-Rose Bengal Agar Yeast and Mold Method Yeast and Mold Method
EvaluationEvaluation Microorganisms:Microorganisms:
Mold CulturesMold Cultures A.niger, Penicillium spp., A.niger, Penicillium spp., andand Paecilomyces spp. Paecilomyces spp.
Yeast CulturesYeast Cultures Z.ballii, S.cerevisiae, Z.ballii, S.cerevisiae, and a plant isolateand a plant isolate
Inoculum Preparation:Inoculum Preparation: Organisms were harvested from aPDA plates by Organisms were harvested from aPDA plates by
washing with sterile water washing with sterile water 1ml from each individual mold or yeast 1ml from each individual mold or yeast
suspension was added to 20 mls DI watersuspension was added to 20 mls DI water Molds serially diluted Molds serially diluted Yeast adjusted to a spec reading of 1.00, then serially Yeast adjusted to a spec reading of 1.00, then serially
diluteddiluted
Dichloran-Rose Bengal Agar Dichloran-Rose Bengal Agar Yeast and Mold Method Yeast and Mold Method
EvaluationEvaluation Material and Methods:Material and Methods:
product was inoculated with 100 cfu/g of product was inoculated with 100 cfu/g of target organismstarget organisms
0.1ml of inoculated product surface 0.1ml of inoculated product surface plated onto each media (aPDA, DRBA)plated onto each media (aPDA, DRBA)
aPDA incubated at 25CaPDA incubated at 25C Counted at 3 and 5 daysCounted at 3 and 5 days
DRBA incubated at 30C and 35CDRBA incubated at 30C and 35C Counted at 2, 3, 4, and 5 daysCounted at 2, 3, 4, and 5 days
Dichloran-Rose Bengal Agar Dichloran-Rose Bengal Agar Yeast and Mold Method Yeast and Mold Method
EvaluationEvaluation Statistical Analysis:Statistical Analysis:
An analysis of variance (AOV) was An analysis of variance (AOV) was done to test if the total counts for done to test if the total counts for DRB at 2 and 5 days was significantly DRB at 2 and 5 days was significantly different from aPDA at 5 daysdifferent from aPDA at 5 days
Dichloran-Rose Bengal Agar Dichloran-Rose Bengal Agar Yeast and Mold Method Yeast and Mold Method
EvaluationEvaluation Results:Results: DRB at 2 days-30C was statistically equivalent DRB at 2 days-30C was statistically equivalent
to aPDA at 5 days for mold recovery to aPDA at 5 days for mold recovery Molds were pale in color; Molds were pale in color; Penicillium spp.Penicillium spp. was white was white
on DRB (green on aPDA). The other 2 test molds on DRB (green on aPDA). The other 2 test molds were pale yellowwere pale yellow
Yeast counts on DRB at 30C were significantly Yeast counts on DRB at 30C were significantly lower than counts on aPDA at 2 and 5 dayslower than counts on aPDA at 2 and 5 days
Mold and Yeast counts were significantly lower Mold and Yeast counts were significantly lower on DRB at 35C vs. aPDA on DRB at 35C vs. aPDA
Dichloran-Rose Bengal Agar Dichloran-Rose Bengal Agar Yeast and Mold Method Yeast and Mold Method
EvaluationEvaluation Conclusion:Conclusion:
Due to overall decreased recovery of Due to overall decreased recovery of yeast and mold, and the mold visual yeast and mold, and the mold visual observations; the Dichloran-Rose Bengal observations; the Dichloran-Rose Bengal Agar Yeast and Mold recovery medium Agar Yeast and Mold recovery medium is is notnot recommended. recommended.
Case Study #2Case Study #2
Rapid Check Rapid Check SalmonellaSalmonella Test Kit EvaluationTest Kit Evaluation
Rapid Check Rapid Check SalmonellaSalmonella Test Kit EvaluationTest Kit Evaluation
Objective:Objective: Determine validity of the Determine validity of the Strategic Diagnostics Inc. Rapid Check Strategic Diagnostics Inc. Rapid Check antibody lateral flow method for the antibody lateral flow method for the detection of detection of SalmonellaSalmonella in comparison in comparison to the BAX PCR test methodto the BAX PCR test method
Justification:Justification: Reduce testing cost, Reduce testing cost, false positives rate and technician timefalse positives rate and technician time
Rapid Check Rapid Check SalmonellaSalmonella Test Kit EvaluationTest Kit Evaluation
Acceptance Criteria:Acceptance Criteria: Speed; shorter time to results vs. PCR?Speed; shorter time to results vs. PCR? Sensitivity; greater or equivalent to PCR?Sensitivity; greater or equivalent to PCR? Specificity; greater or equivalent to PCRSpecificity; greater or equivalent to PCR
CostCost Less than or equal to BAX PCR systemLess than or equal to BAX PCR system Cost per testCost per test
Versatility; food products only, Versatility; food products only, environmental samples only, or both?environmental samples only, or both?
Rapid Check Rapid Check SalmonellaSalmonella Test Kit EvaluationTest Kit Evaluation
Organisms and Inoculum Preparation:Organisms and Inoculum Preparation: A cocktail of 5 A cocktail of 5 Salmonella spp.Salmonella spp. A cocktail of 7 non-A cocktail of 7 non-Salmonella spp.Salmonella spp.
E.coli (2), Citrobacter, Bacillus, Klebsiella, E.coli (2), Citrobacter, Bacillus, Klebsiella, Enterobacter (2)Enterobacter (2)
Individual cultures grown overnight in BHI at Individual cultures grown overnight in BHI at 35C35C
Salmonella Salmonella strains pooled, diluted to strains pooled, diluted to 100cfu/ml100cfu/ml
Non-Non-SalmonellaSalmonella strains pooled, diluted to strains pooled, diluted to 1,000 cfu/ml1,000 cfu/ml
Rapid Check Rapid Check SalmonellaSalmonella Test Kit EvaluationTest Kit Evaluation
Methods:Methods: Inoculation of samplesInoculation of samples
With With SalmonellaSalmonella With non-With non-SalmonellaSalmonella strains strains With both With both
Pre-enrichment of samplesPre-enrichment of samples Traditional medium; Lactose for 24 hoursTraditional medium; Lactose for 24 hours SDI medium for 5 hoursSDI medium for 5 hours
Secondary enrichment Secondary enrichment Tetrathionate for 24 hoursTetrathionate for 24 hours
Rapid Check Rapid Check SalmonellaSalmonella Test Kit EvaluationTest Kit Evaluation
Methods (cont):Methods (cont): BAX PCR analysisBAX PCR analysis
3 hour re-growth3 hour re-growth Cell lysis Cell lysis 4-8 hour PCR cycle4-8 hour PCR cycle
SDI lateral flow assaySDI lateral flow assay Load 150ul onto SDI cartridgeLoad 150ul onto SDI cartridge Develop for 10 minutesDevelop for 10 minutes
Rapid Check Rapid Check SalmonellaSalmonella Test Kit EvaluationTest Kit Evaluation
ResultsResults:: SensitivitySensitivity
Results were more consistent with SDI when recovering at Results were more consistent with SDI when recovering at the threshold level (1000 cfu/ml in the TT broth)the threshold level (1000 cfu/ml in the TT broth)
Equivalent results with both methods above the threshold Equivalent results with both methods above the threshold level level
SDI 5 hour pre-incubation media did not consistently SDI 5 hour pre-incubation media did not consistently support growth above the threshold level (acceptance support growth above the threshold level (acceptance criteria)criteria)
SpecificitySpecificity No cross reactivity with non-No cross reactivity with non-SalmonellaSalmonella organisms with organisms with
either methodeither method
Rapid Check Rapid Check SalmonellaSalmonella Test Kit EvaluationTest Kit Evaluation
ResultsResults: Speed: Speed
BAX-PCRBAX-PCR SDI w/ 5 hour SDI w/ 5 hour mediummedium
SDI SDI
EnrichmenEnrichmentt
24 hours24 hours 5 hours5 hours 24 hours24 hours
SecondarySecondary 18 hours18 hours 18 hours18 hours 18 hours18 hours
Re-growthRe-growth 3 hours3 hours 0 hours0 hours 0 hours0 hours
Cell lysisCell lysis 0.5 hours0.5 hours 0 hours0 hours 0 hours0 hours
Analysis Analysis timetime
8 hours8 hours 10 minutes10 minutes 10 minutes10 minutes
TotalTotal 53.5 53.5 hourshours
23 hours, 10 23 hours, 10 minutesminutes
42 hours, 10 42 hours, 10 minutesminutes
Rapid Check Rapid Check SalmonellaSalmonella Test Kit EvaluationTest Kit Evaluation
Conclusions:Conclusions: SDI shown to be as sensitive as BAX-PCRSDI shown to be as sensitive as BAX-PCR
5 hour medium not recommended5 hour medium not recommended No cross reactivity observed with SDINo cross reactivity observed with SDI SDI gave results sooner than PCRSDI gave results sooner than PCR PCR has more steps, more prone to technician PCR has more steps, more prone to technician
errorerror Some degree of subjectivity with SDISome degree of subjectivity with SDI SDI easier to use; 1 step inoculation of 1 single SDI easier to use; 1 step inoculation of 1 single
cartridgecartridge
Rapid Check Rapid Check SalmonellaSalmonella Test Kit EvaluationTest Kit Evaluation
Conclusions:Conclusions: SDI can be successfully used for food SDI can be successfully used for food
and environmental samplesand environmental samples No additional equipment needed (heat No additional equipment needed (heat
blocks, thermal cycler)blocks, thermal cycler) Cost per test of SDI less than BAX-PCRCost per test of SDI less than BAX-PCR SDI approved for use in place of PCRSDI approved for use in place of PCR Appropriate for use by labs analyzing a Appropriate for use by labs analyzing a
smaller number of samplessmaller number of samples
Value of Method ValidationValue of Method Validation
Need to validate method on your intended Need to validate method on your intended product; rule out matrix interferenceproduct; rule out matrix interference
Determine minimum regulatory requirements Determine minimum regulatory requirements (AOAC, AFNOR, etc)(AOAC, AFNOR, etc)
Determine what is the right method for your lab Determine what is the right method for your lab based on volume of testing and number of based on volume of testing and number of technicianstechnicians
Base selection of methodology on needBase selection of methodology on need SensitivitySensitivity SpecificitySpecificity SpeedSpeed CostCost Lab spaceLab space