三种奶牛常见病菌多重PCR诊断方法的建立  

The Establishment of Multiplex PCR Assay for Diagnosing Three Bacteria from Cows

作　　者：高瑞娜[1] 张婷[2] 

机构地区：[1]朔州职业技术学院生物工程系,山西朔州036000 [2]新乡医学院三全学院生命科学技术学院

出　　处：《畜牧兽医杂志》2015年第4期15-18,22,共5页Journal of Animal Science and Veterinary Medicine

摘　　要：根据GenBank公布的布鲁氏菌Bcsp31、牛分枝杆菌pncA、炭疽杆菌capA三种特异基因的保守序列分别设计引物,在引物前添加通用引物(Tag),建立3种奶牛常见病菌的多重实时荧光定量PCR方法;通过构建3种病菌PCR扩增目的基因的质粒用于制备标准品进行敏感性验证;利用大肠杆菌、沙门氏菌和金黄色葡萄球菌用于特异性试验,并对30份样本进行了检测。结果表明,本研究构建的多重实时荧光定量PCR检测时间仅需2h,灵敏度为100个拷贝/uL,与其它细菌无交叉反应,特异性为100%,阳性检出率为88.89%。因此,本试验成功建立了检测3种奶牛常见病菌早期诊断的方法,可以在奶牛养殖中应用。In order to establish a multiplex PCR assay which can detect three types of bacteria from cows.An universal primer（Tag）was added in primers which were designed based on the nucleotide sequences of Brucella Bcsp31,Mycobacterium bovis pncA and Bacillus anthracis capA published in GenBank.Recombinant plasmid of target genes were constructed by PCR amplification of three bacteria as standard production which was used in sensitivity test;E.coli,Salmonella and Staphylococcus aureus were used to carry out the specificity test and 30 samples which were suspected of bacteria infected were detected.The result indicated that the method only required 2hand the detection sensitivity of the multiplex assay was 100copies/μL of plasmid.There was no cross reaction with other pathogeneses and specificity was 100%.Among the samples,88.89% were detected out the pathogens.In conclusion,the method which can detect three bacteria from cows at the same time in early diagnosis was established for dairy farming applications.

关 键 词：奶牛 布鲁氏菌 牛分枝杆菌 炭疽杆菌 多重实时荧光定量PCR 

分 类 号：S854.43[农业科学—临床兽医学]