AOAC-RI ERP Book Micro Jan 19 2017

2.5 Analysis of Candidate Method Sub-Portions After the 14 d stabilization period post-inoculation, the samples were subjected to the following enrichment procedures and testing on the MDA. 3M™ provided all 3M TM consumable supplies involved with the MDA test. In brief, 3M TM Buffered Peptone Water (BPW) was pre-heated to 41.5°C + 1°C. The 25 g sample was added to 225 mL 3M TM BPW, gently agitated and incubated at 41.5°C + 1°C for 18 h. The mixing of the enrichment broth and blueberries was performed gently with efforts to limit any bursting of blueberries. The enriched samples were then subject to analysis as outlined by the instructions for use for the 3M TM Molecular Detection Assay 2 – E. coli O157 test kit. The sample results obtained after completing the MDA wer e considered the ‘screening stage’ result for the candidate method. Concurrent with the transfer and testing on the MDA, aliquots of the enriched samples were transferred and subjected to the testing outlined in Section 2.4 for cultural confirmation. The results obtained after completing this methodology were considered the ‘confirmation stage’ result for the candidate method. 2.6 Most Probable Number Analysis As mentioned above in Section 2.3, samples of each portion were inoculated with two different levels of E. coli O157 (i.e. Portion 1, and Portion 2) and used for performing a five tube-three level MPN analysis to determine the concentration of E. coli O157 inoculated into the portion. This analysis was setup after the 14 d stabilization period in order to mimic all handling and treatment of matrices. From each 300 g sample of each portion, five 50 g samples and five 10 g samples were weighed into sterile Whirl-Pak bags and hydrated with 425 mL and 90 mL of mBPWp, respectively. Each sample was then processed according to the reference method procedures described above in Section 2.4. Note: for the MPN test samples, the set of five samples (high inoculum) and twenty samples (low inoculum) for the reference testing were also used (25 g, respectively). Samples that confirm positive for this procedure were noted and the LCF MPN Calculator Version 1.6 (Least Cost Formulations, Ltd., Virginia Beach, VA) was used to calculate the MPN/g and 95% confidence intervals for each portion using the mass of each sample and the number of reference method confirmed positives at each level of the MPN setup. 2.7 Probability of Detection Statistical Analysis After results from the “screening” and “confirmation” stages were obtained for the candidate method and “confirmed” results were obtained for the reference method, the data was statistically analyzed according to the POD methods of the 2012 version of the AOAC International Methods Committee Guidelines for Validation of Microbiological Methods for Food and Environmental Surfaces document (1). The POD for the candidate method’s “screening stage” (presumptive) results (POD CP ) were calculated for each level of inoculation, and were compared with the POD for

CONFIDENTIAL INFORMATION Page 4 of 13