4种食源性致病菌多重PCR检测技术研究及应用.docVIP

4种食源性致病菌多重PCR检测技术研究及应用 　　摘要:为建立一种利用多重PCR技术检测和鉴定沙门氏菌(Salmonellla spp)、单核细胞增生李斯特菌(Listeria monocytogenes)、金黄色葡萄球菌(Staphylococcus aureus)和蜡样芽孢杆菌(Bacillus cereus)的方法,根据沙门氏菌侵袭力蛋白A基因(invA gene)、单核细胞增生李斯特菌蛋白转录调控基因(prfA gene)、金黄色葡萄球菌自溶素基因(alt gene)、蜡样芽孢杆菌的促旋酶B亚单位基因(gyrB gene)设计引物,进行多重PCR扩增,并对多重PCR反应体系进行优化。在此基础上建立了4种致病菌的多重PCR检测体系,同时以国标法进行对比验证。结果表明,本研究建立的多重PCR检测方法简单、快速、灵敏度高,具有很好的应用前景。 　　关键词:食源性致病菌;多重PCR;检测 　　中图分类号:TS207.4文献标识码:ADOI编码:10.3969/j.issn.1006-6500.2009.06.002 　　 　　Study and Application on Detection of Four Kinds of Food-borne Pathogenic Bacteria by Multiplex PCR 　　ZHAO Xin, WANG Yong, LAN Qing-kuo, ZHU Zhu,CHENG Yi 　　(Central Lab,Tianjin Academy of Agricultural Sciences,Tianjin 300381,China) 　　Abstract:In order to establish a rapid, sensitive and specific detection method for identification of four kinds of food-borne pathogenic bacteria including Salmonellla spp, Listeria monocytogenes, Staphylococcus aureus and Bacillus cereus by using multiplex PCR, according to invasion protein A gene(invA gene)of Salmonellla spp, transcriptional regulatory protein gene (prfA gene) of Listeria monocytogenes, autolysin gene (alt gene) ofStaphylococcus aureus and gyrase B subunit gene (gyrB gene) of Bacillus cereus, four pairs of specific primers were designed for multiplex PCR amplification. The multiplex PCR system and conditions of the amplification were optimized to make four kinds of food-borne pathogens have better amplification results. The results showed that the multiplex PCR method was rapid, specific and sensitive compared with national standard method. The multiplex PCR method developed in this study could provide an informative supplement to conventional microbiological methods for routine monitoring of food. 　　Key words:food-borne pathogenic bacteria; multiplex PCR; detection 　　 　　农产品在采集、加工、运输、销售等环节容易受到微生物的污染,因此,微生物检测是农产品及其加工品卫生检测中的一项重要内容,而食源性致病菌检验是微生物检验的重中之重,关系到消费者的生命财产安全[1]。 　　致病菌是能够引起人类及动物发生疾病、造成生命和财产巨大损失的一群微生物(主要是细菌),其中最为典型的致病菌是沙门氏菌、单增李斯特菌、金黄色葡萄球菌、大