牛分枝杆菌三重PCR检测方法的建立  

Development of a multiplex PCR for detection of Mycobacterium bovis

作　　者：钱爱东[1] 王建[1] 王春芳[1] 

机构地区：[1]吉林农业大学动物科技学院,吉林长春130118

出　　处：《中国预防兽医学报》2011年第6期479-482,共4页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家科技支撑计划项目(2007BAD55B05)

摘　　要：为建立快速检测牛分枝杆菌(M.bovis)的三重PCR方法,本研究以M.bovis ValleeⅢ株染色体DNA为模板,分别以其recA、IS6110、IS1081基因特异性引物进行PCR扩增,建立检测M.bovis的recA-IS6110-IS1081三重PCR反应。通过PCR扩增获得大小约为860 bp、520 bp和340 bp的DNA片段。BLAST序列分析表明,这3个基因片段与GenBank中登录的相关基因的核苷酸序列同源性均达到99%。同时,recA、IS6110和IS1081PCR扩增的敏感性分别达到585 fg、19.5 fg和19.5 fg。特异性较强,只有M.bovis和人结核临床分离株扩增反应为阳性,为进一步研究recA、IS6110和IS1081基因以及其在牛结核病诊断中的应用奠定了基础。For developing the multiplex PCR of Mycobacterium bovis detection,the genomic DNA was extracted from M.bovis ValleeⅢ strain,the recA,IS6110 and IS1081 gene were amplified with 3 pairs of specific primers by PCR.The PCR products were approximately 860 bp,520 bp and 340 bp,respectively.The BLAST sequential analysis indicated that the nucleotide sequence homology with recA,IS6110 and IS1081 gene of M.bovis AF2122/97 reached 99% respectively.Moreover,the recA-IS6110-IS1081 gene triple PCR was constructed.The sensitivity of recA,IS6110 and IS1081 gene PCR were 585 fg,19.5 fg and 19.5 fg respectively,the specificity tests showed that there were no cross reacted with other bacteria.These results could be serve as a basis for further studies on the recA,IS6110 and IS1081 genes and their application in diagnosis of tuberculosis.

关 键 词：牛分枝杆菌 RECA基因 IS6110基因 IS1081基因 三重PCR 

分 类 号：S852.65[农业科学—基础兽医学]