Cloke Jonathan, Matheny Sharon, Swimley Michelle, Tebbs Robert, Burrell Angelia, Flannery Jonathan, Bastin Benjamin, Bird Patrick, Benzinger M Joseph, Crowley Erin, Agin James, Goins David, Salfinger Yvonne, Brodsky Michael, Fernandez Maria Cristina
Thermo Fisher Scientific, Microbiology, Wade Rd, Basingstoke, Hampshire, RG24 8PW, United Kingdom.
Thermo Fisher Scientific, 2130 Woodward St, Austin, TX 78744.
J AOAC Int. 2016 Nov 1;99(6):1537-1554. doi: 10.5740/jaoacint.16-0235. Epub 2016 Oct 1.
The Applied Biosystems™ RapidFinder™ STEC Detection Workflow (Thermo Fisher Scientific) is a complete protocol for the rapid qualitative detection of Escherichia coli (E. coli) O157:H7 and the "Big 6" non-O157 Shiga-like toxin-producing E. coli (STEC) serotypes (defined as serogroups: O26, O45, O103, O111, O121, and O145). The RapidFinder STEC Detection Workflow makes use of either the automated preparation of PCR-ready DNA using the Applied Biosystems PrepSEQ™ Nucleic Acid Extraction Kit in conjunction with the Applied Biosystems MagMAX™ Express 96-well magnetic particle processor or the Applied Biosystems PrepSEQ Rapid Spin kit for manual preparation of PCR-ready DNA. Two separate assays comprise the RapidFinder STEC Detection Workflow, the Applied Biosystems RapidFinder STEC Screening Assay and the Applied Biosystems RapidFinder STEC Confirmation Assay. The RapidFinder STEC Screening Assay includes primers and probes to detect the presence of stx1 (Shiga toxin 1), stx2 (Shiga toxin 2), eae (intimin), and E. coli O157 gene targets. The RapidFinder STEC Confirmation Assay includes primers and probes for the "Big 6" non-O157 STEC and E. coli O157:H7. The use of these two assays in tandem allows a user to detect accurately the presence of the "Big 6" STECs and E. coli O157:H7. The performance of the RapidFinder STEC Detection Workflow was evaluated in a method comparison study, in inclusivity and exclusivity studies, and in a robustness evaluation. The assays were compared to the U.S. Department of Agriculture (USDA), Food Safety and Inspection Service (FSIS) Microbiology Laboratory Guidebook (MLG) 5.09: Detection, Isolation and Identification of Escherichia coli O157:H7 from Meat Products and Carcass and Environmental Sponges for raw ground beef (73% lean) and USDA/FSIS-MLG 5B.05: Detection, Isolation and Identification of Escherichia coli non-O157:H7 from Meat Products and Carcass and Environmental Sponges for raw beef trim. No statistically significant differences were observed between the reference method and the individual or combined kits forming the candidate assay using either of the DNA preparation kits (manual or automated extraction). For the inclusivity and exclusivity evaluation, the RapidFinder STEC Detection Workflow, comprising both RapidFinder STEC screening and confirmation kits, correctly identified all 50 target organism isolates and correctly excluded all 30 nontarget strains for both of the assays evaluated. The results of these studies demonstrate the sensitivity and selectivity of the RapidFinder STEC Detection Workflow for the detection of E. coli O157:H7 and the "Big 6" STEC serotypes in both raw ground beef and beef trim. The robustness testing demonstrated that minor variations in the method parameters did not impact the accuracy of the assay and highlighted the importance of following the correct incubation temperatures.
应用生物系统公司的RapidFinder™ STEC检测工作流程（赛默飞世尔科技公司）是一种用于快速定性检测大肠杆菌O157:H7和“六大”非O157型产志贺毒素大肠杆菌（STEC）血清型（定义为血清群：O26、O45、O103、O111、O121和O145）的完整方案。RapidFinder STEC检测工作流程可使用应用生物系统公司的PrepSEQ™核酸提取试剂盒结合应用生物系统公司的MagMAX™ Express 96孔磁珠处理器自动制备PCR-ready DNA，也可使用应用生物系统公司的PrepSEQ快速离心试剂盒手动制备PCR-ready DNA。RapidFinder STEC检测工作流程由两个单独的检测组成，即应用生物系统公司的RapidFinder STEC筛查检测和应用生物系统公司的RapidFinder STEC确认检测。RapidFinder STEC筛查检测包括用于检测stx1（志贺毒素1）、stx2（志贺毒素2）、eae（紧密素）和大肠杆菌O157基因靶点的引物和探针。RapidFinder STEC确认检测包括用于“六大”非O157 STEC和大肠杆菌O157:H7的引物和探针。串联使用这两种检测可让用户准确检测“六大”STEC和大肠杆菌O157:H7的存在。在一项方法比较研究、包容性和排他性研究以及稳健性评估中对RapidFinder STEC检测工作流程的性能进行了评估。将这些检测与美国农业部（USDA）食品安全检验局（FSIS）微生物学实验室指南（MLG）5.09：从生牛肉末（73%瘦肉）的肉制品、胴体和环境海绵中检测、分离和鉴定大肠杆菌O157:H7以及USDA/FSIS-MLG 5B.05：从生牛肉修整的肉制品、胴体和环境海绵中检测、分离和鉴定非O157:H7大肠杆菌进行了比较。使用任何一种DNA制备试剂盒（手动或自动提取），在参考方法与构成候选检测的单个或组合试剂盒之间均未观察到统计学上的显著差异。对于包容性和排他性评估，由RapidFinder STEC筛查和确认试剂盒组成的RapidFinder STEC检测工作流程在评估的两种检测中均正确鉴定了所有50株目标生物体分离株，并正确排除了所有30株非目标菌株。这些研究结果证明了RapidFinder STEC检测工作流程在检测生牛肉末和牛肉修整中的大肠杆菌O157:H7和“六大”STEC血清型方面的敏感性和选择性。稳健性测试表明方法参数的微小变化不会影响检测的准确性，并突出了遵循正确孵育温度的重要性。
