牛分枝杆菌特异性PCR检测方法的建立及初步应用

根据已发表的牛分枝杆菌的pncA的基因序列，设计和合成了一对可扩增294bp目的片段的引物，建立了特异性检测牛分枝杆菌的PCR方法。对牛分枝杆菌国际参考株和国内分离株成功扩增出294bp的特异性基因片段；对人结核分枝杆菌、副结核分枝杆菌、鸟胞内分枝杆菌和草分枝杆菌DNA的PCR扩增结果均为阴性。本PCR方法检测的敏感度可达到50pg。对10份牛分枝杆菌培养阳性和10份阴性样品的DNA分别进行了PCR检测，结果10份阳性样品中有9份样品为PCR扩增阳性，阳性符合率为90％（9／10）；而10份阴性样品则PCR扩增全部为阴性，阴性符合率为100％（10／10）。本方法可做为牛分枝杆菌的快速检测和流行病学调查的工具。

The development and preliminary application of specificity detection of Mycobacterium bovis by PCR based on the gene pncA

The pncAgene was amplified byPCR usingthe specific primers (TB- U, MB- L)that designed according to the published sequence of pncA gene of Mycobacterium bovis, the specific PCR method was developed. A 294bp fragment was specifically amplified from mycobacterium bovis, however, no product was amplified from Mycobacterium tuberculosis H37Rv strain, Mycobacterium paratuberculosis, Mycobacterium avium- intracellulare and Mycobacterium phlei. The sensitivity of the PCR assay is 50pg DNA. Upon detecting 20 clinical samples, 10culture positive samples was 9 positive usingPCR assay, the correspondence rate ofthe twomethods was 90% (9/10). 10culture negative samples was 10 negaitive using PCR assay, the correspondence rate of the two methods was 100% (10/10). Due to the high specificityand sensitivityofPCR assay, it can be served as an effective method for the rapid detection and epidemiology research of M.bovis.