用多重PCR方法检测奶牛场麻雀中分枝杆菌感染情况

目的：利用可以鉴别分支杆菌属中结核分枝杆菌、牛分支杆菌、非结核分枝杆菌多重PCR方法，检测奶牛场麻雀组织中分支杆菌感染情况。方法根据结核分枝杆菌种特异基因目的片段MTP40、分枝杆菌属特异基因32kD、结核分枝杆菌复合群特异基因IS6110插入序列，设计合成三对特异性引物，扩增对32kD的506bp、MTP40的396bp和IS6110的984bp片段，以此方法检测奶牛场麻雀肺组织中分支杆菌感染情况，并对阳性结果的目的片段进行克隆测序。结果在分析的21份麻雀肺组织中，检测出2例非结核分枝杆菌阳性病料，阳性率9.52%。2例阳性样本PCR扩增得到的片段与GenBank收录的32kD基因同源性分别为95.8%和97.2%。结论麻雀肺组织中存在分支杆菌感染阳性病例提示该牛场中生物体内存在分支杆菌潜伏感染，并可能导致奶牛的潜伏感染以及干扰结核检疫。

Detection of Mycobacterium in Sparrow Tissues in a Dairy Cattle Farm with Multiplex PCR Assay

Objective To detect the Mycobacterium infection in the tissues of sparrows in a dairy cattle farm with a mul-tiplex PCR assay that can discriminate Mycobacterium tuberculosis,Mycobacterium bovis,and non-tuberculous myco-bacteria. Method Three pairs of primers were designed and synthesized based on the specific genes of Mycobacterium tuberculosis （MTP40）,the specific genes of Mycobacterium（32kD）,and the specific genes of Mycobacterium tuberculosis complex（IS6110）,and used in a multiplex PCR assay for amplification of the 506bp gene of 32kD,the 396bp gene of MTP40,and the 984bp gene of IS6110. The assay was carried out to detect the Mycobacterium infection in sparrow lung tissue in a dairy cattle farm and the positive fragments were cloned and sequenced. Result 2 of 21 spar-row lung tissue samples were positive for non-tuberculous mycobacteria with a positive rate of 9.52%. The homology of the 2 positive samples with the 32kD gene in GenBank was 95.8%and 97.2%respectively. Conclusion The presence of mycobacterial infection in sparrow lung tissues suggested that organisms in the dairy cattle farm had been latently infected with Mycobacterium, possibly causing latent infection with Mycobacterium and thus interfering with TB tests.