结核分枝杆菌和牛分枝杆菌TaqMan~荧光PCR检测的建立

Development of TaqMan~ real-time PCR assays for the detection of Mycobacterium tuberculosis and Mycobacterium bovis

目的对结核分枝杆菌和牛分枝杆菌分别建立荧光PCR快速检测方法。结核分枝杆菌荧光PCR对H37Rv标准菌株、结核分枝杆菌临床分离株的检测结果呈典型阳性反应，牛分枝杆菌荧光PCR对牛分枝杆菌标准菌株、BCG菌株的检测结果呈典型阳性反应，对其它分枝杆菌菌株以及常见微生物样品为阴性反应。两种荧光PCR对对应阳性重组质粒模板的检测灵敏度可达单个基因拷贝，对结核分枝杆菌或BCG标准菌株的检测灵敏度可达单个菌细胞。对临床样品的检测结果显示，荧光PCR对模拟感染奶样、血样的检测灵敏度可达单个菌细胞。所建立的方法，全过程包括血样、奶样、痰液等临床样品的前处理、核酸提取和荧光PCR检测，可在5～6h内完成。采用上述荧光PCR方法从临床采集的疑似病人痰液中检出多份结核分枝杆菌阳性样品。

Two real-time PCR assays for specific detection of Mycobacterium tuberculosis and Mycobacterium boris were developed. This assay could identify M. tuberculosis or M. boris strains and clinical isolates with various mycobacterium and microorganisms. Tests on serial 10 fold dilution of recombinant plasmid containing target template showed that both of the two assays could detect single copy of target gene. The assay could detect M. tuberculosis or M. boris in culture samples and mimic infected milk as well as blood samples. The whole procedure, including clinical sample process, nucleic acid extraction and real- time PCR process, could completed within 5-6h. Milk and blood samples from cattle and sputum samples from suspected patients were collected to examine tuberculosis by the two assays, and 15 M. tuberculosis positive samples were detected.