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

作　　者：孙新城 李爽 李侠颖 周杰 曹亚新 王宏伟 张晓根 SUN Xin-Cheng;LI Shuang;LI Xia-Ying;ZHOU Jie;CAO Ya-Xin;WANG Hong-Wei;ZHANG Xiao-Gen(College of Food and Bioengineering,Zhengzhou University of Light Industry,Zhengzhou 450001,China;Food Laboratory of Zhongyuan·Zhengzhou University of Light Industry,Luohe 462300,China;Henan Provincial Key Laboratory of Cold Chain Food Quality Safety and Control,Zhengzhou 450001,China;Collaborative Innovation Center of Food Production and Safety,Henan Province,Zhengzhou 450001,China;College of Food Engineering Vocational Zhengzhou,Xinzheng 450001,China)

机构地区：[1]郑州轻工业大学食品与生物工程学院,郑州450001 [2]中原食品实验室·郑州轻工业大学,漯河462300 [3]河南省冷链食品质量安全与控制重点实验室,郑州450001 [4]食品生产与安全河南省协同创新中心,郑州450001 [5]郑州食品工程职业学院,新郑450001

出　　处：《食品安全质量检测学报》2024年第9期37-43,共7页Journal of Food Safety and Quality

基　　金：河南省科技攻关项目(242102111045);河南省重点研发项目(222102520041);河南省科技研发联合基金项目(222103810020);河南省人社厅留学回国人员择优支持项目(2020039);郑州市协同创新专项(2021ZDPY0202)。

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:H73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10^(1)、10^(2)、10^(2)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1.56×10^(2)CFU/25 g的沙门氏菌,2.13×10^(2)CFU/25 g的单增李斯特菌,2.32×10^(2)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:H73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。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 10^(1),10^(2),10^(2)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(1.56×10^(2)CFU/25 g),Listeria monocytogenes(2.13×10^(2)CFU/25 g)and Escherichia coli O157:H7(2.32×10^(2)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.

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 

分 类 号：TS251.7[轻工技术与工程—农产品加工及贮藏工程] O657.3[轻工技术与工程—食品科学与工程]