快速荧光PCR法检测包装饮用水中产气荚膜梭菌

Rapid and Sensitive Detection of Clostridium Perfringens in Packaged Drinking Water by Real-Time Fluorescent PCR

由产气荚膜梭菌引起的包装饮用水污染在世界范围内的报道越来越多。产气荚膜梭菌既是水微生物污染的一种指示菌,同时也是一种致病菌,对人类生命和健康构成潜在威胁。该研究开发了一种基于实时荧光PCR的快速检测包装饮用水中的产气荚膜梭菌新方法。该方法针对cpa基因设计引物和探针,用于检测产气荚膜梭菌。50 mL水样膜过滤后在(36±1)℃下孵育4 h,提取菌体DNA,采用实时荧光PCR检测。结果显示该方法特异性强,检出限为1 cfu/50 mL。PCR反应混合物预混冻干和核酸提取优化使整个检测过程用时≤6 h,实时荧光PCR检测结果与国家标准方法GB 8538—2016检测结果一致。因此,该实时荧光PCR检测方法具有灵敏度高、特异性强、检测时间短等特性,为日常包装饮用水中产气荚膜梭菌监管和质控提供了新的检测技术手段。

Contamination of packaged drinking water caused by Clostridium perfringens are increasingly reported worldwide.It has been potential threats to human life and health.In this study,a new rapid method based on real-time fluorescent PCR was developed to detect Clostridium perfringens in packaged drinking water.The designed primers and probes targeted cpa gene was used to detect Clostridium perfringens.After fitered with membranes,the samples were incubated for only 4 h at 36℃.The realtime PCR was applied for dectecting Clostridium perfringens in samples after DNA extration.The results showed that the established real-time fluorescent fluorescent PCR was highly specific,and the detection limit was as few as one bacterial cell in a 50 mL water sample.PCR reaction mixture premixed lyophilization and nucleic acid extraction optimization permitted the whole detection process less than 6 h.The result of real-time fluorescent PCR assay showed the same effect as the national standard methods GB 8538—2016.Therefore,the novel real-time fluorescent PCR assay allowed the sensitive and specific characterization,making it potential tool for the direct detection of Clostridium perfringens in packaged drinking water.