Murinda Shelton E, Ibekwe A Mark, Zulkaffly Syaizul, Cruz Andrew, Park Stanley, Razak Nur, Paudzai Farah Md, Ab Samad Liana, Baquir Khairul, Muthaiyah Kokilah, Santiago Brenna, Rusli Amirul, Balkcom Sean
1 Animal and Veterinary Sciences Department, Center for Antimicrobial Research and Food Safety, California State Polytechnic University , Pomona, California.
Foodborne Pathog Dis. 2014 Jul;11(7):529-36. doi: 10.1089/fpd.2013.1663. Epub 2014 Apr 21.
Shiga toxin-producing Escherichia coli (STEC) are a major family of foodborne pathogens of public health, zoonotic, and economic significance in the United States and worldwide. To date, there are no published reports on use of recombinase polymerase amplification (RPA) for STEC detection. The primary goal of this study was to assess the potential application of RPA in detection of STEC. This study focused on designing and evaluating RPA primers and fluorescent probes for isothermal (39°C) detection of STEC. Compatible sets of candidate primers and probes were designed for detection of Shiga toxin 1 and 2 (Stx1 and 2), respectively. The sets were evaluated for specificity and sensitivity against STEC (n=12) of various stx genotypes (stx1/stx2, stx1, or stx2, respectively), including non-Stx-producing E. coli (n=28) and other genera (n=7). The primers and probes that were designed targeted amplification of the subunit A moiety of stx1 and stx2. The assay detected STEC in real time (within 5-10 min at 39°C) with high sensitivity (93.5% vs. 90%; stx1 vs. stx2), specificity (99.1% vs. 100%; stx1 vs. stx2), and predictive value (97.9% for both stx1 vs. stx2). Limits of detection of ∼ 5-50 colony-forming units/mL were achieved in serially diluted cultures grown in brain heart infusion broth. This study successfully demonstrated for the first time that RPA can be used for isothermal real-time detection of STEC.
产志贺毒素大肠杆菌（STEC）是一类在美国乃至全球都具有重大公共卫生、人畜共患病和经济意义的食源性病原体。迄今为止，尚无关于使用重组酶聚合酶扩增（RPA）检测STEC的公开报道。本研究的主要目标是评估RPA在检测STEC方面的潜在应用。本研究重点设计和评估用于等温（39°C）检测STEC的RPA引物和荧光探针。分别设计了用于检测志贺毒素1和2（Stx1和Stx2）的候选引物和探针的兼容组。针对各种stx基因型（分别为stx1/stx2、stx1或stx2）的STEC（n = 12），包括不产Stx的大肠杆菌（n = 28）和其他属（n = 7），对这些组进行了特异性和敏感性评估。所设计的引物和探针靶向扩增stx1和stx2的A亚基部分。该检测方法在39°C下实时（5 - 10分钟内）检测STEC，具有高灵敏度（stx1为93.5%，stx2为90%）、特异性（stx1为99.1%，stx2为100%）和预测值（stx1和stx2均为97.9%）。在脑心浸液肉汤中培养的系列稀释培养物中，检测限达到约5 - 50菌落形成单位/毫升。本研究首次成功证明RPA可用于STEC的等温实时检测。
