结核分枝杆菌与牛分枝杆菌PCR快速鉴别检测方法的建立  

Establishment of a multiple PCR assay for rapid differentiation of Mycobacterium tuberculosis from Mycobacterium bovis

作　　者：朱汝仪[1] 潘文[1] 赵明秋[1] 琚春梅[1] 王波[1] 陈金顶[1] 

机构地区：[1]华南农业大学兽医学院,广东广州510642

出　　处：《中国兽医科学》2010年第4期378-383,共6页Chinese Veterinary Science

基　　金：国家自然科学基金-广东省自然科学基金联合项目(U0631006);教育部"长江学者和创新团队发展计划"创新团队项目(IRT0723);广东省自然科学基金创新团队项目(5200638)

摘　　要：根据结核分枝杆菌与牛分枝杆菌基因组的比对分析结果,针对结核分枝杆菌与牛分枝杆菌153bp、RD10和TbD1的3处差异缺失区域设计引物,分别建立了结核分枝杆菌与牛分枝杆菌PCR快速鉴别检测方法。应用建立的3种方法分别对84株结核分枝杆菌、3株牛分枝杆菌、51株非结核分枝杆菌及9种其他常见菌株进行了检测。结果显示,用针对153 bp缺失片段建立的PCR方法分别在结核分枝杆菌及牛分枝杆菌中扩增出645 bp及492 bp的目的条带;用针对RD10、TbD1建立的PCR方法分别在结核分枝杆菌及牛分枝杆菌中扩增出478 bp及361 bp的目的条带、358 bp及524 bp的目的条带。这3种PCR检测方法的准确率和特异性均为100%。敏感性试验结果显示,这些检测方法对结核分枝杆菌和牛分枝杆菌基因组DNA的检测极限均为10 pg。表明,此方法可用于牛源或人源结核分枝杆菌及牛分枝杆菌的快速鉴别检测。Based on the deletion sequences of 153-bp fragment,RD10 and TbD1 in Mycobacterium tuberculosis and Mycobacterium bovis,three multiplex-PCRs（m-PCR） for differentiation of M.tuberculosis and M.bovis were developed.These PCR assays were optimized and validated using 84 M.tuberculosis clinical strains,3 M.bovis strains,51 non-tuberculous mycobacterial（NTM） strains and 9 other bacterial species strains.The PCR based on the 153-bp amplified a unique 645bp and 492bp amplicon from M.tuberculosis and M.bovis respectively.The PCR based on RD10 amplified a unique 478bp and 361bp amplicon from M.tuberculosis and M.bovis respectively,while the PCR based on TbD1 amplified a unique 358bp and 524bp amplicon from M.tuberculosis and M.bovis respectively.The accuracy of these assays was 100% in terms of specificity.Sensitivity of these assays for the detection of genomic DNA was 10pg.These methods could be confidently applied to routine differentiation of M.tuberculosis from M.bovis from both veterinary and human origins.

关 键 词：结核分枝杆菌 牛分枝杆菌 聚合酶链反应 快速鉴别检测 

分 类 号：S852.618[农业科学—基础兽医学] Q503[农业科学—兽医学]