The Validation of the SIMUL-qPCR Top 7 STEC Assay Collection: AOAC Performance Tested MethodSM 022001

Abstract

The Simultaneous Multiplex Real Time PCR (SIMUL-qPCR) Top 7 STEC Assay Collection is a quick, reliable method for detecting Top 7 Shiga toxin-producing E. coli (STEC) in raw beef trim, raw ground beef, and beef carcass sampling sheets. Each assay multiplexes several targets in one run to identify E. coli O157: H7, O26, O45, O103, O111, O121, O145, Shiga toxin and intimin genes. This collection uses specifically optimized proprietary media for single-step recovery and enrichment of Enterohemorrhagic E. coli (EHEC). This report details the method validation study to validate raw beef trim, raw ground beef, and beef carcass sampling sheets. Matrix studies for raw beef trim, raw ground beef, and beef carcass sampling sheets, inclusivity/exclusivity, product consistency/stability, and robustness testing were conducted to assess the method's performance. 50 Top 7 STEC isolates were analyzed with the SIMUL-qPCR Assay. 32 isolates, including closely related non-E. coli species and E. coli non-STEC strains, were also tested. Inclusivity showed the collection detected the Shiga toxin (stx) gene, intimin (eae) gene, and Top 7 serogroups. None of the 32 exclusivity strains were detected. The candidate and reference methods' results had no statistically significant differences during matrix studies. Small variations in critical test parameters (enrichment time, extraction reagent volume, and extracted sample volume) didn't adversely affect the assay's performance, and stability testing indicated consistent results for at least one year. The data presented demonstrate that the SIMUL-qPCR Top 7 STEC Assay is a reliable method for detecting the Top 7 STEC.