SIMUL-qPCR Top 7 STEC 检测试剂盒验证：AOAC 性能验证方法标准 022001。

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

机构信息

Applied Food Diagnostics, Inc, 18 Industrial Drive, Bloomsburg, PA 17815, USA.

出版信息

J AOAC Int. 2021 Aug 20;104(4):1098-1108. doi: 10.1093/jaoacint/qsab006.

DOI:10.1093/jaoacint/qsab006

PMID:33484257

Abstract

BACKGROUND

The Simultaneous Multiplex Real Time PCR (SIMUL-qPCR) Top 7 STEC Assay Collection is a quick, reliable method for detecting top seven Shiga toxin-producing Escherichia 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.

OBJECTIVE

This report details the method validation study to validate raw beef trim, raw ground beef, and beef carcass sampling sheets.

METHOD

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.

RESULTS

Fifty top seven STEC isolates were analyzed with the SIMUL-qPCR assay. Thirty-two 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 seven 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) did not adversely affect the assay's performance, and stability testing indicated consistent results for at least one year.

CONCLUSIONS

The data presented demonstrate that the SIMUL-qPCR Top 7 STEC Assay is a reliable method for detecting the top seven STEC.

HIGHLIGHTS

The Applied Food Diagnostics, Inc. Simultaneous Multiplex Real Time PCR (SIMUL-qPCR) Top 7 STEC Assay is capable of detecting the top seven Shiga toxin-producing Escherichia coli in beef trim, ground beef, and beef carcass sampling sheets in as little as 10 hours of enrichment.

摘要

背景

Simultaneous Multiplex Real Time PCR（SIMUL-qPCR）Top 7 STEC 检测试剂盒是一种快速可靠的方法，可用于检测生牛肉切块、生绞碎牛肉和牛肉屠体采样纸上的七种志贺毒素产生大肠杆菌（STEC）。每个检测试剂盒在一次运行中多重检测多个目标，以鉴定大肠杆菌 O157：H7、O26、O45、O103、O111、O121、O145、志贺毒素和内膜蛋白基因。该试剂盒使用专门优化的专利培养基进行一步法回收和富集肠出血性大肠杆菌。

目的

本报告详细介绍了对生牛肉切块、生绞碎牛肉和牛肉屠体采样纸进行验证的方法学研究。

方法

对生牛肉切块、生绞碎牛肉和牛肉屠体采样纸进行基质研究、包容性/排他性、产品一致性/稳定性和稳健性测试，以评估该方法的性能。

结果

对 50 株七种 STEC 分离株进行了 SIMUL-qPCR 检测分析，还对 32 株包括密切相关的非大肠杆菌种和非 STEC 大肠杆菌株进行了检测。包容性表明该试剂盒检测到了志贺毒素（stx）基因、内膜蛋白（eae）基因和七种血清群。没有检测到 32 株排他性菌株。在基质研究中，候选方法和参考方法的结果没有统计学上的显著差异。关键测试参数（富集时间、提取试剂体积和提取样品体积）的微小变化不会对检测产生不利影响，稳定性测试表明至少在一年的时间内结果一致。