武卫影，马晓燕，张 伟

（河北农业大学食品科技学院，河北保定071000）

多重PCR快速检测三种食品病原菌

武卫影，马晓燕*，张 伟

（河北农业大学食品科技学院，河北保定071000）

建立一种快速、经济、实用的可以同步检测食品中金黄色葡萄球菌、沙门氏菌、小肠结肠炎耶尔森氏菌的多重聚合酶链式反应（polymerase chain reaction，PCR）的方法。根据金黄色葡萄球菌的nuc基因，沙门氏菌的invA基因，小肠结肠炎耶尔森氏菌的ail基因，分别设计了三对引物，对单个基因PCR和单管多重PCR扩增进行特异性、灵敏性实验以及优化反应体系。三对引物能特异性扩增出236、475、127bp的目的条带。建立的多重PCR同时对3种食源性致病菌进行检测具有较高的灵敏度，灵敏度金黄色葡萄球菌为102CFU/mL、沙门氏菌为102CFU/mL、小肠结肠炎耶尔森氏菌为102CFU/mL。初步建立能同步、简便、快速、灵敏地检测食品中金黄色葡萄球菌、沙门氏菌和小肠结肠炎耶尔森氏菌的三重PCR方法。

多重PCR，金黄色葡萄球菌，沙门氏菌，小肠结肠炎耶尔森氏菌

Abstract：To explore a fast，economical and practical multiplex PCR method which could detect Staphylococcus aureus，Salmonella，Yersinia enterocolitica common food-borne bacterial pathogens simultaneously.Three pairs of primers were designed respectively according to the nuc gene of Staphylococcus aureus，the invasive protein gene（invA） of Salmonella，the sequence of the ail gene of Yersinia enterocolitica.The specific and sensitive experiments were carried out to the amplification of single gene by PCR and Multiplex PCR amplification，and the reaction system were optimized.Three DNA fragments of 236，475 and 127bp were amplified by three pairs of specific primers.Three food borne bacterial pathogens were simultaneously detected by the established multiplex PCR technology.The sensitivity of this method was for Staphylococcus aureus 102CFU/mL，for salmonella 102CFU/mL，for Yersinia enterocolitica 102CFU/mL.A triple PCR assay had been established for the simultaneous sample，rapid and sensitive detection of Staphylococcus aureus，salmonella，Yersinia enterocolitica in food.

Key words：multiplex polymerase chain reaction（m-PCR）；Staphylococcus aureus；Salmonella；Yersinia enterocolitica

食物中毒事件每年都在世界范围内频繁暴发，其中大部分是食品中污染致病细菌引起的[1-2]。金黄色葡萄球菌、沙门氏菌、小肠结肠炎耶尔森氏菌是常见污染食品的致病菌，主要通过食品、饮水感染人类，导致细菌性食物中毒。目前国标中提供的检测病原菌方法为传统的分离培养，包括生化和血清学鉴定等多个复杂的过程。一般需4～7d。近年来在PCR的基础上发展了多重PCR（multiplex PCR，MPCR）技术，能在同一反应体系中同时对多个目的基因片段进行检测，进一步缩短了检测时间并提高了检测效率[3-5]。……