应用多重PCR方法快速鉴定结核分枝杆菌与非结核分枝杆菌

目的 探讨应用多重PCR技术快速鉴定结核分枝杆菌与非结核分枝杆菌的可靠性.方法 根据结核分枝杆菌种的MTP40基因序列(396bp),分枝杆菌属的32kD基因序列(506bp),结核分枝杆菌复合体群的IS6110序列(984bp),采用3对特异性引物(PT1,T2;MT1,T2;IS5,S6)在同一反应体系和条件下对92株结核杆菌临床分离株和5株非结核分枝杆菌临床分离株进行扩增,并与标准菌株进行比较.结果 92株结核分枝杆菌临床分离株中,0株扩增出与结核分枝杆菌H37RV标准株相同的396bp、506bp、984bp 3条DNA片段,敏感性达97.8%,特异性为100%;5株非结核分枝杆菌临床分离株均扩增出506bp DNA片段,敏感性达100%,特异性为100%.结论 应用多重PCR方法,对结核分枝杆菌及非结核分枝杆菌进行鉴定,结果快速、准确、可靠,为临床结核病与非结核分枝杆菌病的快速诊断提供了有效的手段,具有很好的临床应用价值.

Application of multiplex PCR in rapid identification of Mycobacterium tuberculosis and non-tuberculosis Mycobacteria

Objective To investigate the feasibility of multiplex PCR for rapid identification of Mycobacterium tuberculosis and non-tuberculosis Mycobacteria. Methods According to MTP40 gene sequence of Mycobacterium tuberculosis, 32kD gene sequence of Mycobacterium and IS6110 insertion sequence gene sequence of Mycobacterium tuberculosis complex, three specific pairs of primers (PT1-PT2, MT1-MT2 and IS5-IS6) for Mycobacterium were designed, and the target DNA for MTP40, 32kD and IS6110 was 396bp, 506bp and 984bp, respectively. The genome of 92 Mycobacterium tuberculosis clinically isolated strains and 5 non-tuberculosis Mycobacteria clinical strains were amplified in the same system, and the results were compared with reference strains. Results Among 92 clinical strains of Mycobacterium tuberculosis, the DNA fragments of 396bp, 506bp and 984bp were found in 90 Mycobacterium tuberculosis clinical strains, as well as in the reference strain H37Rv; the sensitivity of multiplex PCR for Mycobacterium tuberculosis was 97.8%, and the specificity was 100.0%. The DNA fragments of 506bp were all found in 5 non-tuberculosis Mycobacteria clinical strains, the sensitivity and specificity for non-tuberculosis Mycobacterium were both 100.0%. Conclusion The multiplex PCR is a rapid, sensitive and specific method for identification of Mycobacterium tuberculosis and non-tuberculosis Mycobacteria, and it may provide an effective way for clinical diagnosis of Mycobacterium tuberculosis and non-tuberculosis Mycobacteria, therefore useful in clinical application.