Fang Taisong, Shen Jinling, Xue Junxin, Jiang Yuan, Guo Dehua, Yang Jielin, Kong Xiangxiang, Xu Xuebin, Wang Xiang
Technology Center for Animal Plant and Food Inspection and Quarantine of Shanghai Customs, Shanghai 200135, China.
Shanghai University, School of Life Sciences, Shanghai 200444, China.
J AOAC Int. 2022 Dec 22;106(1):156-164. doi: 10.1093/jaoacint/qsac101.
BACKGROUND: Escherichia coli O157:H7, being the cause of hemorrhagic colitis in humans, is recognized as one of the most dangerous and widespread foodborne pathogens. A highly specific, sensitive, and rapid E. coli O157:H7 detection method needs to be developed since the traditional detection methods are complex, costly, and time-consuming. OBJECTIVE: In this study, a recombinase aided amplification (RAA) assisted CRISPR/Cas12a (RAA-CRISPR/Cas12a) fluorescence platform for specific, sensitive, and rapid nucleic acid detection of E. coli O157:H7 was introduced. METHODS: First, the feasibility (components of CRISPR/Cas12a system) of the developed method was evaluated. Then a total of 34 bacterial strains were used for the specificity test, and gradient dilutions of extracted DNA and bacterial solutions of E. coli O157:H7 were prepared for the sensitivity test. Third, a real-time PCR assay for detection of the specific wzy gene of E. coli O157:H7 (FDA's Bacteriological Analytical Manual) was used for sensitivity comparison. Finally, analysis of RAA-CRISPR/Cas12a detection in spiked and 93 real ground beef samples was carried out. RESULTS: The developed RAA-CRISPR/Cas12a method showed high specificity, and the detection could be completed within 30 min (after 4 h enrichment in spiked ground beef samples). The limit of detection (LOD) of bacterial concentrations and genomic DNA was 5.4 × 102 CFU/mL and 7.5 × 10-4 ng/μL, respectively, which exhibited higher sensitivity than the RAA-gel electrophoresis and RT-PCR methods. Furthermore, it was shown that E. coli O157:H7 in ground beef samples could be positively detected after 4 h enrichment when the initial bacterial inoculum was 9.0 CFU/25 g. The detection results of the RAA-CRISPR/Cas12a method were 100% consistent with those of the RT-PCR and traditional culture-based methods while screening the E. coli O157:H7 from 93 local collected ground beef samples. CONCLUSIONS: The developed RAA-CRISPR/Cas12a method showed high specificity, high sensitivity, and rapid positive detection of E. coli O157:H7 from ground beef samples. HIGHLIGHTS: The RAA-CRISPR/Cas12a system proposed in this study provided an alternative molecular tool for quick, specific, sensitive, and accurate detection of E. coli O157:H7 in foods.
背景：大肠杆菌O157:H7是人类出血性结肠炎的病原体，被认为是最危险且分布广泛的食源性病原体之一。由于传统检测方法复杂、成本高且耗时，因此需要开发一种高度特异、灵敏且快速的大肠杆菌O157:H7检测方法。 目的：本研究介绍了一种用于特异性、灵敏且快速核酸检测大肠杆菌O157:H7的重组酶辅助扩增（RAA）辅助CRISPR/Cas12a（RAA-CRISPR/Cas12a）荧光平台。 方法：首先，评估所开发方法的可行性（CRISPR/Cas12a系统的组成部分）。然后，总共34株细菌用于特异性测试，并制备大肠杆菌O157:H7提取DNA和细菌溶液的梯度稀释液用于灵敏度测试。第三，使用实时荧光定量PCR检测大肠杆菌O157:H7的特异性wzy基因（美国食品药品监督管理局的《细菌学分析手册》）进行灵敏度比较。最后，对添加样品和93份实际绞碎牛肉样品进行RAA-CRISPR/Cas12a检测分析。 结果：所开发的RAA-CRISPR/Cas12a方法显示出高特异性，检测可在30分钟内完成（在添加绞碎牛肉样品中富集4小时后）。细菌浓度和基因组DNA的检测限分别为5.4×10²CFU/mL和7.5×10⁻⁴ ng/μL，其灵敏度高于RAA-凝胶电泳和RT-PCR方法。此外，当初始细菌接种量为9.0 CFU/25 g时，绞碎牛肉样品中的大肠杆菌O157:H7在富集4小时后可被阳性检测。在从93份本地采集的绞碎牛肉样品中筛选大肠杆菌O157:H7时，RAA-CRISPR/Cas12a方法的检测结果与RT-PCR和传统基于培养的方法的检测结果100%一致。 结论：所开发的RAA-CRISPR/Cas12a方法在检测绞碎牛肉样品中的大肠杆菌O157:H7时显示出高特异性、高灵敏度和快速阳性检测能力。 要点：本研究提出的RAA-CRISPR/Cas12a系统为快速、特异性、灵敏且准确地检测食品中的大肠杆菌O157:H7提供了一种替代分子工具。
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