validation of microbiological methods for use in the food industry brazilian association for food...

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)


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


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


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


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


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


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


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


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


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


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?


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


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


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


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


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


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


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

validation of microbiological methods for use in the food industry brazilian association for food...

Documents