VALIDATION OF ANALYTICAL METHODS FOR THE DETECTION OF MICROBIAL PATHOGENS IN FOOD

Jack RoweCertified Laboratories, Inc.Bridging Science with Service Since 1926

AOAC Performance Tested Method (RI)

AOAC Official Methods of Analysis

Manufacturer’s Validation

‣ 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): Origins

Matrix Study

One laboratory 1-4 Foods or Surface Inclusivity, Exclusivity,

Robustness, Stability and Lot to Lot Variation

AOAC Performance Tested Method (RI)

Pre-Collaborative Study and Collaborative Study

Harmonization Collaborative Study

AOAC Official Methods of Analysis

Selection of matrices associated with pathogen

Natural vs. Artificial contamination Strain types Competing flora Fractional results

Matrix 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

Harmonization Collaborative Study

FDA BAM USDA FSIS AOAC

Method compared to Reference Method

Official Methods of Analysis2011.03

Salmonella in a Variety of Food

VIDAS Salmonella (SLM)Easy Salmonella

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

U.S. food import volume, by food group

 Food group   1999 2012

Volume of U.S. food imports 1/

Live meat animals (1000) 2/ 6,058.4 7,908.0Meats 1,439.0 1,335.8Fish and shellfish 1,677.8 2,354.2Dairy 242.5 209.8Vegetables 4,767.5 8,545.0Fruits 3/ 8,193.8 11,569.6Nuts 272.0 443.0Coffee and tea 1,709.4 2,093.8Cereals and bakery 6,687.6 10,184.4Vegetable oils 2,320.8 5,178.1Sugar and candy 322.1 3,730.6Cocoa and chocolate 923.7 1,237.4Other edible products 705.9 1,447.3Beverages (1000 KL) 4/ 2,854.9 5,820.1

        

Domestic Imports

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

Method Verification In House

Amaranth Coconut Cumin Egg Cracklet Hing Powder Tahina/Sesame Seeds Octopus

Was it Validated?

Spike level must not exceed 30 cells (25 gram sample)

Seven successful validations must occur (No false negatives or positives)

Method Verification In House

Salmonella culture Atypical or Typical

Serial dilution in Phosphate Buffer

Plated in Duplicate 1ml and 0.1ml

Selective Agar Hektoen or XLD

Spike Preparation

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 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

Spike Calculation

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

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

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

Verification

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

Final Package

Questions