VALIDATION OF ANALYTICAL METHODS FOR THE DETECTION OF MICROBIAL PATHOGENS IN FOOD Jack Rowe Certified Laboratories, Inc. Bridging Science with Service Since 1926
Dec 11, 2015
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