validation of analytical methods for the detection of microbial pathogens in food
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VALIDATION OF ANALYTICAL METHODS FOR THE DETECTION OF MICROBIAL PATHOGENS IN FOOD. Jack Rowe Certified Laboratories, Inc. Bridging Science with Service Since 1926. Manufacturer’s Validation. AOAC Performance Tested Method (RI) AOAC Official Methods of Analysis.

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validation of analytical methods for the detection of microbial pathogens in food
VALIDATION OF ANALYTICAL METHODS FOR THE DETECTION OF MICROBIAL PATHOGENS IN FOOD

Jack Rowe

Certified Laboratories, Inc.

Bridging Science with Service Since 1926

manufacturer s validation
Manufacturer’s Validation
  • AOAC Performance Tested Method (RI)
  • AOAC Official Methods of Analysis
aoac performance tested method ri origins
AOAC Performance Tested Method (RI): Origins
    • Rapid increase in Proprietary Methods
      • Speed to market critical
    • AOAC OMA
    • Higher Cost, More Time
  • Performance Tested Methods
    • Kicked off in 1991
    • Opportunity to achieve some level of validation in less time
    • Often serves as a “pre-collaborative” until full OMA is completed
aoac performance tested method ri
AOAC Performance Tested Method (RI)

Matrix Study

  • One laboratory
  • 1-4 Foods or Surface
  • Inclusivity, Exclusivity, Robustness, Stability and Lot to Lot Variation
aoac official methods of analysis
AOAC Official Methods of Analysis
  • Pre-Collaborative Study and Collaborative Study
  • Harmonization Collaborative Study
matrix study
Matrix Study
  • Selection of matrices associated with pathogen
  • Natural vs. Artificial contamination
  • Strain types
  • Competing flora
  • Fractional results
harmonization collaborative study
Harmonization Collaborative Study
  • Quantitative
  • 8 laboratories minimum, 1-6 matrices
  • 4 inoculation levels/matrix
  • 2 test portions per level per matrix per method
  • Qualitative
    • 10 laboratories minimum, 1-6 matrices,
    • 3 inoculation levels per matrix,
    • 6 test portions per level per matrix per method
    • Method compared to Reference Method
vidas salmonella slm easy salmonella
VIDAS Salmonella (SLM)Easy Salmonella

Official Methods of Analysis

2011.03

Salmonella in a Variety of Food

slide11
Foods/Eggs and Egg Products,Eggs and Egg Products/Liquid Eggs,Eggs and Egg Products/Eggs,Dairy Products/Ice Cream,Vegetables/Spinach,Spinach/Fresh Spinach,Fish/Shrimp,Nuts and Nut Products/Peanut Butter,Peanuts/Peanut Butter,Meat and Meat Products/Turkey,Meat and Meat Products/Pork,Meat and Meat Products/Roast Beef,Meat and Meat Products/Pork Sausage,Meat and Meat Products/Chicken,Dairy Products/Milk,Milk/Whole Milk,Foods/Fish,Nuts and Nut Products/Pecans,Baked Goods/Cake Mix,Grains/Dry Pasta,Spices and Condiments/Black Pepper,Milk/Dried Milk,Eggs and Egg Products/Dried Egg Yolks,Cacao Bean and Its Products/Chocolate,Foods/Fruits and Fruit Products,Fruit Juices/Orange Juice,Pet Foods,Water

:

Validated Matrices

method verification in house
Method Verification In House
  • FDA will accept all matrices validated by the manufacturer for the AOAC study
  • Other matrices must be verified by laboratory

to determine if the method is suitable for the matrix

was it validated
Was it Validated?
  • Amaranth
  • Coconut
  • Cumin
  • Egg Cracklet
  • Hing Powder
  • Tahina/Sesame Seeds
  • Octopus
method verification in house1
Method Verification In House

Spike level must not exceed 30 cells

(25 gram sample)

Seven successful validations must occur

(No false negatives or positives)

spike preparation
Spike Preparation
  • Salmonella culture

Atypical or Typical

  • Serial dilution in Phosphate Buffer
  • Plated in Duplicate 1ml and 0.1ml
  • Selective Agar

Hektoen or XLD

spike calculation
Spike Calculation
  • Count plates after incubation per BAM

Example

Dilution 4 125 Dilution 5 14

Dilution 4 113 Dilution 5 12

125 + 113 / 2 = 119 cfu/ml

spike calculation1
Spike Calculation
  • Dilution 4 tube contains 119 cfu/ml
  • X ml x 119 cfu/ml = 30 cfu
  • 30 cfu/119 cfu/ml = X ml
  • X = 0.252 or 250 ul
verification set up
Verification Set Up

Prepare sample per BAM (or BioMerieux)

instructions

Weigh out 25 gram sample and spike with calculated volume

Plate the calculated spike volume in

duplicate on selective agar

verification
Verification
  • Incubate per normal protocol
  • Confirm counts from spike volume do not exceed 30 cells
  • Control sample must produce a negative result. If the sample is “presumptive positive” it contains an unspecified amount of Salmonella and demonstration of < 30 cfu/ 25 gram sample is not possible
  • Abandon Verification
verification1
Verification
  • All controls must give appropriate reactions
  • Positive Cultural Control
  • Negative Cultural Control
  • Diluent Blank
  • The Presumptive Positive spike must be confirmed following reference method
final package
Final Package
  • Biochemical Identification of Spike, Negative and Positive Control
  • Media preparation logs for all media used
  • Autoclave Sterilization Records for all media
  • Media Sterility records for all media used
  • Balance Calibration Check Records
  • Temperature Records for all Appliances
  • Water Quality Records
  • Confirmation serves as Document of Productivity
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