牛奶中3种致病菌多重PCR的建立及初步应用  

Establishment and Application of a Multiplex PCR Assay for Simultaneous Detection of Three Pathogenic Bacterias in Milk

作　　者：张亚楠 焦仁刚[2] 赵贵[2] 陈锡龙[2] 黄鑫[2] 栾庆祥[2] 章厉劼[2] 彭珊 徐景峨 蒲龄 余波 ZHANG Yanan;JIAO Rengang;ZHAO Gui;CHEN Xilong;HUANG Xin;LUAN Qingxiang;ZHANG Lijie;PENG Shan;XU Jing e;PU Ling;YU Bo(Institute of Animal Husbandry and Veterinary Medicine of Guizhou Academy of Agricultural Sciences,Guiyang 550005,China;Guizhou Institute of Veterinary Drug and Feeds Control,Guiyang 550003,China)

机构地区：[1]贵州省农业科学院畜牧兽医研究所,贵阳550005 [2]贵州省兽药饲料监察所,贵阳550003

出　　处：《中国乳品工业》2020年第3期47-51,共5页China Dairy Industry

基　　金：贵州省科技计划项目(黔科合支撑[2019]2320号)。

摘　　要：为了能同时在牛奶中快速检测金黄色葡萄球菌、大肠杆菌、沙门氏菌,本研究根据GenBank中的金黄色葡萄球菌femB、大肠杆菌phoA、沙门氏菌invA基因序列,分别设计3对特异性引物(目的片段分别为233 bp、444 bp、724 bp),通过对PCR反应条件的优化建立了多重PCR方法。敏感性、特异性试验结果显示,该多重PCR对金黄色葡萄球菌、大肠杆菌、沙门氏菌的最低核酸检测量分别为13.3 pg/mL、13.3 pg/mL、133.3 pg/mL,而对牛结核杆菌、多杀性巴氏杆菌、牛支原体、单核细胞增生李斯特氏菌、志贺氏菌的扩增结果均为阴性。对25分临床样品的检测结果表明,该多重PCR检测结果与单一PCR检测结果一致。结果表明,建立的多重PCR方法具有很好的特异性和敏感性,可用于金黄色葡萄球菌、大肠杆菌和沙门氏菌的快速鉴别诊断。To establish a multiplex PCR assay(mPCR)for the rapid identification of Staphylococcus aureus,Escherichia coli and Salmonella,three pairs of specific primers were designed,respectively which refered to the femB gene sequence of Staphylococcus aureus,the phoA gene sequence of Escherichia coli and the invA gene sequence of Salmonella in GenBank.The amplified products were 233 bp,444 bp and 724 bp,respective ly.A multiplex PCR method was established by optimizing the reaction conditions of PCR.The sensitivity and specificity tests showed that mPCR was highly sensitive in Staphylococcus aureus 13.3 pg/mL、Escherichia coli 13.3 pg/mL、and Salmonella 133.3 pg/mL respectively.No band was amplified from Mycobacterium bovis,Pasteurella multocida and Mycoplasma bovis by the mPCR.25 clinical samples were detected by the mPCR.The results revealed that the established multiplex PCR method was highly specific and sensitive,and it could be used for rapid differential diagnosis of Staphylococcus aureus,Escherichia coli and Salmonella.

关 键 词：金黄色葡萄球菌 大肠杆菌 沙门氏菌 多重PCR 

分 类 号：TS252.7[轻工技术与工程—农产品加工及贮藏工程]