多重荧光定量聚合酶链反应法检测肉制品中的3种食源性致病菌

Determination of 3 kinds of food-borne pathogens in meat products by multiple fluorescence quantitative polymerase chain reaction

中文关键词:  沙门氏菌  单增李斯特菌  大肠杆菌O157:H7  多重荧光定量聚合酶链反应法

英文关键词:Salmonella  Listeria monocytogenes  Escherichia coli O157:H7  multiple fluorescence quantitative polymerase chain reaction

中文摘要:

      目的 建立多重荧光定量聚合酶链反应法(quantitative polymerase chain reaction, qPCR)快速检测肉制品中沙门氏菌、单增李斯特菌、大肠杆菌O157:H7 3种食源性致病菌的方法。方法 根据沙门氏菌的invA基因、单增李斯特菌的hemolysin基因、大肠杆菌O157:H7的rfbE基因序列分别设计特异性引物及探针, 通过优化反应体系, 测定其灵敏度、特异性和重复性, 建立了可同时检测上述3种食源性致病菌多重qPCR方法。结果 该方法只扩增3种靶细菌, 对其他供试菌不扩增; 沙门氏菌、单增李斯特菌、大肠杆菌O157:H7的检出限分别为101、102、102拷贝数/μL, 并且拥有良好的重复性和特异性。人工污染的猪肉样品经SLE培养基富集8 h后, 分别可以检测到初始菌液浓度为1.56×102 CFU/25 g的沙门氏菌, 2.13×102 CFU/25 g的单增李斯特菌, 2.32×102 CFU/25 g的大肠杆菌O157:H7。结论 所建立的多重qPCR灵敏度高、特异性强、重复性好, 可同时检测肉制品中沙门氏菌、单增李斯特菌和大肠杆菌O157:H7 3种食源性致病菌, 在提高食品安全和保护人类健康方面有重要意义。

英文摘要:

      Objective To establish a method for rapid determination of Salmonella, Listeria monocytogenes and Escherichia coli O157:H7 in meat products by multiple fluorescence quantitative polymerase chain reaction (qPCR). Methods Specific primers and probes were designed based on the conservative sequences of Salmonella invA gene, Listeria monocytogenes hemolysin gene and Escherichia coli O157:H7 rfbE gene. By optimizing the reaction system and determining its sensitivity, specificity and repeatability, a multiplex qPCR method that could simultaneously detect the above 3 kinds of foodborne pathogens was established. Results The method only amplified 3 kinds of target bacteria and did not amplify other tested bacteria. The limits of detection of Salmonella, Listeria monocytogenes and Escherichia coli O157:H7 were 101, 102, 102 copies/μL, respectively, and had good repeatability and specificity. After the artificially contaminated pork samples were enriched in SLE medium for 8 h, the initial concentration of Salmonella (1.56×102 CFU/25 g), Listeria monocytogenes (2.13×102 CFU/25 g) and Escherichia coli O157:H7 (2.32×102 CFU/25 g) could be detected, respectively. Conclusion The established multiple qPCR has high sensitivity, strong specificity and good repeatability, and can simultaneously detect Salmonella, Listeria monocytogenes and Escherichia coli O157:H7 in meat products, which has important significance in improving food safety and protecting human health.