School of Food and Bioengineering, Zhengzhou University of Light Industry, Zhengzhou, 450000, Henan Province, China; Henan International Joint Laboratory of Food Safety, Zhengzhou, 450000, Henan Province, China; Collaborative Innovation Center of Food Production and Safety, Zhengzhou, 450000, Henan Province, China; Henan Key Laboratory of Cold Chain Food Quality and Safety Control, Zhengzhou, 450000, Henan Province, China.
School of Food and Bioengineering, Zhengzhou University of Light Industry, Zhengzhou, 450000, Henan Province, China.
Mol Cell Probes. 2020 Apr;50:101501. doi: 10.1016/j.mcp.2019.101501. Epub 2019 Dec 27.
Rapid analytical methods are urgently needed to evaluate Escherichia coli (E. coli) O157:H7 in food. In this work, a novel recombinase polymerase amplification (RPA)-based lateral flow dipstick (LFD) method was developed to detect E. coli. Briefly, suitable primers and probes were designed and screened. Then, RPA reaction parameters, including volume, time, and temperature, were optimized. The specificity and sensitivity of RPA-LFD were analyzed, and a contaminated milk sample was used to test the detection performance of the proposed method. The optimal RPA reaction conditions included a minimum volume of 10 μL, incubation time of 10 min, temperature range of 39-42 °C, the primer pair EOF4/EOR3, and the probe EOProb. RPA-LFD was highly sensitive, it could detect as little as 1 fg of the genomic DNA of E. coli O157:H7, and 19 nontarget DNA of foodborne bacteria did not yield amplification products. Finally, the limit of detection of RPA-LFD for E. coli O157:H7 in artificially contaminated raw milk was 4.4 CFU/mL. In summary, the RPA-LFD assay developed in this study is an effective tool for the rapid investigation of E. coli O157:H7 contamination in raw milk samples.
快速分析方法迫切需要评估食品中的大肠杆菌（E. coli）O157：H7。在这项工作中，开发了一种基于重组酶聚合酶扩增（RPA）的侧向流动试纸条（LFD）方法来检测大肠杆菌。简要地，设计和筛选了合适的引物和探针。然后，优化了 RPA 反应参数，包括体积、时间和温度。分析了 RPA-LFD 的特异性和灵敏度，并使用污染的牛奶样品测试了所提出方法的检测性能。最佳的 RPA 反应条件包括最小体积为 10μL、孵育时间为 10min、温度范围为 39-42°C、引物对 EOF4/EOR3 和探针 EOProb。RPA-LFD 具有很高的灵敏度，它可以检测到少至 1fg 的大肠杆菌 O157：H7 基因组 DNA，而 19 种食源性细菌的非靶 DNA 没有产生扩增产物。最后，RPA-LFD 对人工污染生奶中大肠杆菌 O157：H7 的检测限为 4.4 CFU/mL。总之，本研究中开发的 RPA-LFD 检测方法是快速调查生奶样品中大肠杆菌 O157：H7 污染的有效工具。
