AOAC INTERNATIONAL Methods Committee …

1 aoac PRE-PUBLICATION DRAFT aoac Standards development 2012 aoac INTERNATIONAL ALL RIGHTS RESERVED aoac INTERNATIONAL Methods Committee guidelines for Validation of Microbiological Methods for Food and Environmental Surfaces This document provides guidelines for the aoac validation of microbiological Methods for food and environmental surfaces. It includes terms and their definitions associated with the Official Methods of AnalysisSM and Performance Tested MethodsSM programs and validation requirements for qualitative, quantitative, and confirmatory identification Methods . The guideline working group consisted of Sharon Brunelle, Robert LaBudde, Maria Nelson, and Paul Wehling. Consensus was reached on the guidelines by the aoac Methods Committee for Microbiology and approved by the aoac Official Methods Board. EFFECTIVE DATE FOR USE: FEBRUARY 24, 2012 aoac guidelines for Validation of Microbiological Methods for Food and Environmental Surfaces Consensus by aoac Methods Committee on Microbiology on Sept.

2 18, 2011. Approved by aoac Official Methods Board on Sept. 22, 2011. Presented to the aoac INTERNATIONAL Board of Directors on Dec. 12, 2011 18-Jan-12 ( aoac Validation guidelines for Food Microbiology 2011 Pre-publication Draft) aoac Standards development 2012 aoac INTERNATIONAL 2 Table of Contents 1. SCOPE .. 5 2. APPLICABILITY .. 5 3. TERMS AND DEFINITIONS .. 6 ANALYTE .. 6 CANDIDATE METHOD .. 6 CANDIDATE METHOD 6 COLLABORATIVE STUDY (CS) .. 6 COMPOSITE TEST PORTION .. 6 CONFIRMATORY IDENTIFICATION METHOD .. 6 CONFIRMATORY PHASE .. 6 CONFIRMED RESULT .. 6 ENRICHMENT POOL .. 6 EXCLUSIVITY .. 6 FRACTIONAL RECOVERY .. 6 INCLUSIVITY .. 6 LIMIT OF DETECTION50 (LOD50) .. 6 MATCHED ANALYSES .. 6 MATRIX .. 6 METHOD DEVELOPER VALIDATION STUDY OR SINGLE LABORATORY VALIDATION STUDY .. 6 PRECISION .. 6 PRESUMPTIVE PHASE .. 6 PRESUMPTIVE RESULT .. 7 PROBABILITY OF DETECTION (POD) .. 7 QUALITATIVE METHOD .. 7 QUANTITATIVE METHOD .. 7 REFERENCE 7 7 REPEATABILITY CONDITIONS.

3 7 REPRODUCIBILITY .. 7 REPRODUCIBILITY CONDITIONS .. 7 ROBUSTNESS STUDY .. 7 SAMPLE .. 7 TEST PORTION .. 7 UNMATCHED ANALYSES .. 7 4. QUALITATIVE Methods TECHNICAL PROTOCOL FOR VALIDATION .. 8 METHOD DEVELOPER VALIDATION STUDY OR SINGLE LABORATORY VALIDATION (SLV OR PRECOLLABORATIVE) STUDY 8 Scope .. 8 Inclusivity/Exclusivity Study .. 8 Matrix Study .. 9 Robustness Study [Performance Tested Methods (PTM) submissions only] .. 12 INDEPENDENT VALIDATION STUDY .. 13 13 Reference Method .. 13 Matrices .. 13 Study Design .. 13 COLLABORATIVE STUDY (CS) .. 13 aoac guidelines for Validation of Microbiological Methods for Food and Environmental Surfaces Consensus by aoac Methods Committee on Microbiology on Sept. 18, 2011. Approved by aoac Official Methods Board on Sept. 22, 2011. Presented to the aoac INTERNATIONAL Board of Directors on Dec. 12, 2011 18-Jan-12 ( aoac Validation guidelines for Food Microbiology 2011 Pre-publication Draft) aoac Standards development 2012 aoac INTERNATIONAL 3 13 Number of Laboratories.

4 13 Reference Method .. 14 Matrix Selection .. 14 Analyte Level 14 Number of Test Portions .. 14 Test Portion Size, Compositing and Pooling .. 14 Source of Contamination .. 14 Preparation of Artificially Contaminated Samples .. 15 Preparation of Naturally Contaminated Samples .. 15 Confirmation of Test Portions .. 15 Data Analysis and Reporting .. 15 5. QUANTITATIVE Methods TECHNICAL PROTOCOL FOR VALIDATION .. 17 METHOD DEVELOPER VALIDATION STUDY OR SLV (PRECOLLABORATIVE) STUDY .. 17 17 Inclusivity/ Exclusivity .. 17 Matrix Study .. 18 Robustness Study (PTM submissions only) .. 20 INDEPENDENT VALIDATION STUDY .. 20 20 Reference Method .. 20 Matrices .. 21 Study Design .. 21 COLLABORATIVE STUDY (CS) .. 21 21 Number of Laboratories .. 21 Reference Method .. 21 Matrix Selection .. 21 Levels of Contamination .. 21 Number of Test Portions .. 22 Enumeration of Specific Microorganisms .. 22 Source of Contamination.

5 22 Preparation of Artificially Contaminated Samples .. 22 Use of Artificially and Naturally Contaminated Test Samples .. 22 Confirmation of Test Portions .. 22 Data Analysis and Reporting .. 22 6 CONFIRMATORY IDENTIFICATION Methods .. 24 METHOD DEVELOPER VALIDATION STUDY OR SLV (PRECOLLABORATIVE) STUDY .. 24 24 Inclusivity/Exclusivity Study .. 24 Robustness Study (PTM submissions only) .. 25 INDEPENDENT VALIDATION STUDY .. 25 25 Study Design .. 25 Data Analysis and Reporting .. 26 COLLABORATIVE STUDY .. 26 26 Number of Collaborators .. 26 Number of Tests .. 26 Data Analysis and Reporting .. 26 aoac guidelines for Validation of Microbiological Methods for Food and Environmental Surfaces Consensus by aoac Methods Committee on Microbiology on Sept. 18, 2011. Approved by aoac Official Methods Board on Sept. 22, 2011. Presented to the aoac INTERNATIONAL Board of Directors on Dec. 12, 2011 18-Jan-12 ( aoac Validation guidelines for Food Microbiology 2011 Pre-publication Draft) aoac Standards development 2012 aoac INTERNATIONAL 4 7 SAFETY.

6 27 8 REFERENCES .. 27 APPENDIX X-A. MPN Analysis of Contaminated Matrix .. 29 APPENDIX X-B. Raw Format Data Table Template and Example for Qualitative Method Single Laboratory and Collaborative Studies .. 31 APPENDIX X-B. Raw Format Data Table Template and Example for Qualitative Method Single Laboratory and Collaborative Studies .. 31 APPENDIX X-C. Calculation of POD and dPOD Values from Qualitative Method Single Laboratory Data32 APPENDIX X-D. Summary Data Table for Qualitative Method Single Laboratory Studies .. 35 APPENDIX X-E. Example of Graph of POD Values from Qualitative Method Single Laboratory Data .. 36 APPENDIX X-F. Calculation of LPOD and dLPOD Values from Qualitative Method Collaborative Study Data .. 37 APPENDIX X-G. Data Summary Table Template and Example for Qualitative Method Collaborative Studies .. 43 APPENDIX X-H. Logarithmic Transformation of Data from Quantitative Method Single Laboratory and Collaborative Data .. 46 aoac guidelines for Validation of Microbiological Methods for Food and Environmental Surfaces Consensus by aoac Methods Committee on Microbiology on Sept.

7 18, 2011. Approved by aoac Official Methods Board on Sept. 22, 2011. Presented to the aoac INTERNATIONAL Board of Directors on Dec. 12, 2011 18-Jan-12 ( aoac Validation guidelines for Food Microbiology 2011 Pre-publication Draft) aoac Standards development 2012 aoac INTERNATIONAL 5 Scope The purpose of this document is to provide comprehensive aoac INTERNATIONAL ( aoac ) technical guidelines for conducting microbiological validation studies of food and environmental analysis Methods submitted for aoac Official Methods of AnalysisSM (OMA) status and/or Performance Tested MethodsSM (PTM) certification. 1. Applicability These guidelines are applicable to the validation of any candidate method, whether proprietary or nonproprietary, that is submitted to aoac for OMA status or PTM certification. Circumstances, unforeseen by aoac , may necessitate divergence from the guidelines in certain cases. The PTM Program requires a Method Developer Study and an Independent Laboratory Study.

8 The OMA Program requires a Single Laboratory Validation (SLV) Study (also known as the Precollaborative Study), an Independent Validation Study and a Collaborative Study. A harmonized PTM-OMA program can be followed in which PTM certification is sought and, if successful, serves as the SLV and Independent Validation phase of the OMA program. This approach provides an interim certification while working toward OMA status. Please refer to the table below for more detail. Table 1: aoac Method Review Process aoac Program Study Requirements Relevant Guideline Sections Qualitative Quantitative Confirmatory Identification PTM Method Developer Validation Study Independent Validation Study OMA SLV (Precollaborative Validation) Study and and Independent Validation Study Collaborative Validation Study Harmonized PTM-OMA Method Developer Validation Study Independent Validation Study Collaborative Validation Study aoac guidelines for Validation of Microbiological Methods for Food and Environmental Surfaces Consensus by aoac Methods Committee on Microbiology on Sept.

9 18, 2011. Approved by aoac Official Methods Board on Sept. 22, 2011. Presented to the aoac INTERNATIONAL Board of Directors on Dec. 12, 2011 18-Jan-12 ( aoac Validation guidelines for Food Microbiology 2011 Pre-publication Draft) aoac Standards development 2012 aoac INTERNATIONAL 6 3. Terms and Definitions Analyte Microorganism or associated biochemicals ( DNA, proteins, or lipopolysaccharides) measured or detected by the method of analysis. Candidate Method The method submitted for validation. Candidate Method Result The final result of the qualitative or quantitative analysis for the candidate method. For Methods with a confirmation phase, only presumptive positive results that confirm positive are considered as positive for the candidate method. All other results are considered as negative for the candidate method. Collaborative Study (CS) A validation study performed by multiple laboratories to estimate critical candidate method performance parameters.

10 Composite Test Portion Test portions taken from multiple samples of the same matrix combined together. Confirmatory Identification Method Method of analysis whose purpose is to determine the identity of an analyte (Biological Threat Agent Method; BTAM) Confirmatory Phase A procedure specified in some qualitative assays whereby a preliminary presumptive result is confirmed by a subsequent and different method Confirmed Result The qualitative response from the confirmatory phase of a candidate method Enrichment Pool A pool comprised of aliquots from multiple test portion enrichments. Exclusivity The non-target strains, which are potentially cross-reactive, that are not detected by the method. Fractional Recovery Validation criterion that is satisfied when an unknown sample yields both positive and negative responses within a set of replicate analyses. The proportion of positive responses should fall within 25% and 75% and should ideally approximate 50% of the total number of replicates in the set.