Applied Technology Center at the Pacific Northwest Laboratory (PNL), Office of Regulatory Science, Office of Regulatory Affairs (ORA), the U.S. Food and Drug Administration, Bothell, Washington, USA.
Foodborne Pathog Dis. 2021 Feb;18(2):114-122. doi: 10.1089/fpd.2020.2832. Epub 2020 Sep 15.
Consumption of -contaminated food is one of the most common causes of bacterial diarrhea. A previously developed quantitative polymerase chain reaction (qPCR) utilizing the SmartCycler instrument platform for identification of , , had to be modified to address the recent discontinuation of the SmartCycler system. In this study, a multiplex qPCR assay was optimized on the Applied Biosystems 7500 Fast (AB7500F) platform to continue using qPCR for the identification of three target spp. AB7500F qPCR efficiencies obtained by testing reference genomic DNA (gDNA) were 90.9%, 86.4%, and 94.6% for , , and , respectively, with all correlation coefficient values >0.99. The qPCR results exhibited 100% specificity by testing gDNA samples from 37 non-target reference strains and 86 target strains (50 , 27 , and 9 strains) in this study. The lowest detection level using gDNA was 4, 7, and 2 genome copies per reaction for , , and , respectively. With a 2-day enrichment procedure, the qPCR method correctly detected target species in a spiked food matrix (frog leg, an aquaculture product). The sensitivity in 25 g food matrix was 4 colony-forming units (CFUs) for , 3 CFUs for , and 2 CFUs for . The results suggest that this AB7500F-based qPCR has potential applications for the identification of , , and in contaminated food.
食用受污染的食物是导致细菌性腹泻的最常见原因之一。先前开发的一种利用 SmartCycler 仪器平台鉴定 、 、 的定量聚合酶链反应（qPCR），由于最近 SmartCycler 系统停产，必须进行修改。在这项研究中，优化了一种多重 qPCR 检测方法，在 Applied Biosystems 7500 Fast（AB7500F）平台上继续使用 qPCR 来鉴定三种目标 spp. 通过测试参考基因组 DNA（gDNA）获得的 AB7500F qPCR 效率分别为 、 、 ，其相关系数均大于 0.99。通过测试本研究中 37 种非目标参考菌株和 86 种目标菌株（50 株 、27 株 和 9 株 ）的 gDNA 样本，qPCR 结果显示出 100%的特异性。使用 gDNA 检测的最低检测水平分别为 、 和 的 4、7 和 2 基因组拷贝/反应。采用 2 天的富集程序，qPCR 方法可正确检测到添加食物基质（蛙腿，一种水产养殖产品）中的目标物种。在 25g 食物基质中的灵敏度分别为 、 和 的 4、3 和 2 菌落形成单位（CFU）。结果表明，这种基于 AB7500F 的 qPCR 方法有可能用于鉴定受污染食物中的 、 、 。
