3种食源性致病菌TaqMan多重荧光定量PCR检测方法的建立

旨在建立一种可同时快速检测大肠杆菌O157 ∶ H7、沙门菌和产单核细胞李氏杆菌3种食源性致病菌的TaqMan多重荧光定量PCR(qPCR)方法.针对大肠杆菌O157 ∶ H7 rfbE基因、沙门菌invA基因和产单核细胞李氏杆菌hlyA基因的保守序列分别设计特异性引物和TaqMan探针,建立多重qPCR反应体系,进行...

Establishment of a Multiplex TaqMan Fluorescence Quantitative PCR Method for Detection of Three Foodborne Pathogens

This experiment was conducted to establish a TaqMan multifluorescent quantitative PCR method for simultaneous detection of Escherichia coli O157∶H7, Salmonella and Listeria monocytogenes. Specific primers and TaqMan probes were designed according to the conservative sequences of E. coli O157∶H7 rfbE gene, Salmonella invA gene and L. monocytogenes hlyA gene, respectively, and a multiplex qPCR reaction system was established to detect their specificity, sensitivity and repeatability. The established method was used to detect pathogenic bacteria in fresh food and compared with the national standard method. The multiplex qPCR method established in the experiment has good specificity, only three kinds of target bacteria can be amplified, the lowest sensitivity detection value is 10⁴ copies·μL^-1, and the repeatability is good, the intra-assay coefficient of variation is 0.09%-2.97%, and the inter-assay coefficient of variation is 0.23%-7.82%. One hundred and twenty fresh meat samples were detected by the multiplex qPCR method and compared with the national standard method, 2 samples of E. coli O157∶H7 and 21 samples of L. monocytogenes were detected by the two methods, and 14 samples of Salmonella were detected by the multiple qPCR method, one more than the national standard method. All contaminated samples were detected by the established method. The results confirmed that the established multiple qPCR detection method can quickly and accurately detect the three foodborne pathogens, E. coli O157∶H7, Salmonella and L. monocytogenes, and provide technical support for food safety.