ERP Micro December 2019

For clarity, the matrix extension and each modification will be summarized individually in the manuscript. Additional information is available in the appendix of the document.

Study Design The study was conducted according to the AOAC guidelines for validation of microbiological methods following the AOAC Official Method of Analysis SM program according to the AOAC® INTERNATIONAL Methods Committee Guidelines for Validation of Microbiological Methods for Food and Environmental Surfaces, Appendix J ( 7). The study outline consisted of an unpaired method comparison study. The Assurance® GDS for E . coli O157:H7 Tq (GDS EHEC) assay was compared to the U. S. Department of Agriculture-Food Safety and Inspection Service (USDA-FSIS) Microbiology Laboratory Guidebook (MLG) 5C.00 reference method for finely textured beef (FTB) and raw beef carcass towels (cloths) (8). The frozen finely textured beef was enriched for 2 h additional, then analyzed by the GDS EHEC assay at 10 – 18 h. All candidate and reference enrichment samples were confirmed by the USDA-FSIS MLG 5C.00 reference method. Fresh green onions were compared against the and U.S. Food and Drug Administration (FDA) Bacteriological Analytical Manual (BAM) Chapter 4A (9). The study outline consisted of an unpaired method comparison study. All raw green onion test portions for the alternative method were enriched for 8 – 16 h. All candidate and reference enrichment samples were confirmed by the FDA BAM reference method. Frozen finely textured beef was provided from a national beef producer. Carcass swab cloths were sampled by a national beef producer (see below for method) and shipped overnight. Green onions were purchased at local grocery retailer. All matrices were prescreened for natural contamination with EHEC organisms by the by the USDA-FSIS MLG 5C.00 method (or FDA BAM method for green onions). Total aerobic plate count for beef trim and FTB were determined following the USDA-FSIS MLG 3.02 Quantitative Analysis of Bacteria in Foods as Sanitary Indicators reference method (10). All samples, regardless of presumptive results, were confirmed. Final cultural confirmation was achieved by bioMérieux API® 20E (OMA 978.24) (11). The method comparison study for each matrix consisted of evaluating a total of 30 unpaired test portions. Within each sample set, there were 5 uninoculated control test portions (0 CFU/test portion), 20 low level inoculated test portions (0.2-2 CFU/test portion), and 5 high level inoculated test portions (2-10 CFU/test portion). Preparation of Inoculum and Test Portions The E. coli O157:H7 inoculum was prepared by retrieving the isolate from the frozen stock stored at -70°C into brain heart infusion (BHI) broth and incubating the culture at 35 ± 1°C for 18–24 h. Following incubation, the culture was diluted to a target level using Butterfield’s

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