3种食源性致病菌TaqMan多重荧光定量PCR检测方法的建立

doi:10.11843/j.issn.0366-6964.2022.04.020

摘要/Abstract

摘要： 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，进行特异性、灵敏性和重复性试验，应用建立的方法检测生鲜食品中致病菌并与国家标准方法对比。结果显示：建立的多重qPCR方法特异性好，只扩增3种靶细菌；灵敏性最低检测值均为10⁴ copies·μL^-1；重复性良好，批内变异系数为0.09%～2.97%，批间变异系数为0.23%～7.82%；对120份生鲜肉样品进行检测对比，两种方法均检出2份大肠杆菌O157∶H7，21份产单核细胞李氏杆菌，多重qPCR方法检出14份沙门菌，比国标法多检出1份，建立的方法可检出所有受污染样品。综上表明，建立的多重qPCR检测方法能快速、准确检测食品中大肠杆菌O157∶H7、沙门菌和产单核细胞李氏杆菌3种食源性致病菌，为食品安全提供技术保障。

关键词: 食源性致病菌, 多重荧光定量PCR, 检测方法, 大肠杆菌O157∶H7, 沙门菌, 产单核细胞李氏杆菌

Abstract: 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.

Key words: foodborne pathogens, multiple fluorescent PCR, detection method, Escherichia coli O157∶H7, Salmonella, Listeria monocytogenes

中图分类号:

S855.1

王华健, 张宁, 杨威, 赵志强, 李茜, 陆安, 田勇, 何欣, 赵兴华, 李杰峰. 3种食源性致病菌TaqMan多重荧光定量PCR检测方法的建立[J]. 畜牧兽医学报, 2022, 53(4): 1201-1209.

WANG Huajian, ZHANG Ning, YANG Wei, ZHAO Zhiqiang, LI Qian, LU An, TIAN Yong, HE Xin, ZHAO Xinghua, LI Jiefeng. Establishment of a Multiplex TaqMan Fluorescence Quantitative PCR Method for Detection of Three Foodborne Pathogens[J]. Acta Veterinaria et Zootechnica Sinica, 2022, 53(4): 1201-1209.

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