应用多重聚合酶链反应法检测并鉴别结核分支杆菌与非结核分支杆菌DNA

Detection and identification of the DNA between Mycobacterium tuberculosis and Mycobacterium nontuberculosis by triplex polymerase chain reaction technique

目的提高聚合酶链反应（ＰＣＲ）检测分支杆菌ＤＮＡ的特异性和敏感性，以检测和鉴别结核分支杆菌与非结核分支杆菌ＤＮＡ。方法应用三对具有特异性的寡核苷酸引物，进行多重ＰＣＲ扩增。这三对引物分别对应于分支杆菌６５０００表面抗原、结核分支杆菌插入序列ＩＳ６１１０及人类β珠蛋白基因的部分序列，其扩增产物分别为３８３ｂｐ、１２３ｂｐ和２６８ｂｐ。结果此多重ＰＣＲ电泳检测的灵敏度为０．６ｐｇ。经多重ＰＣＲ扩增后进行凝胶电泳，结核分支杆菌（人型、牛型结核分支杆菌、ＢＣＧ）均可见３８３ｂｐ、１２３ｂｐ片段，而非结核分支杆菌（鸟、龟、瘰疬、蟾、堪萨斯、胞内、耻垢分支杆菌）仅见３８３ｂｐ片段（猿分支杆菌与结核分支杆菌相同）。与上相比较，模拟分支杆菌感染的临床标本，分别增加了一条２６８ｂｐ片段。实验中用于对照的所有细菌及肺炎支原体ＤＮＡ，经多重ＰＣＲ扩增后电泳，均无上述特异性扩增片段出现。对１８２例临床标本进行培养、涂片和多重ＰＣＲ检测。７２例非结核性标本，三者均阴性；１１０例结核性标本中，三者阳性率分别为２．７％、１３．６％和３２．７％。结论多重ＰＣＲ方法可检测并鉴别结核分支杆菌及非结核分支杆菌ＤＮＡ（猿分支杆菌除外），具有高度?

Objective To improve the specificity and sensitivity of polymerase chain reaction (PCR) technique for detecting and identification of DNA of M.tuberculosis and M.nontuberculosis. Method Three pairs of oligonucleotide primer were used in triplexPCR. A 383bp DNA fragment encoding part of the 65 000 mycobacterial surface antigen, a 123bp fragment corresponding to a specific M.tuberculosis complex sequence which was the insertion sequence 6110 (IS 6110) and a 268bp fragment for human βglobin were amplified by triplexPCR respectively. Result The sensitivity of the triplexPCRelectrophoresis for the DNA of mycobacterium was 0.6 pg. The specific bands of 383bp and 123bp among the amplified DNA from M.hominis, M.bovis, BCG and M.simiae were present in the agarose gel. By contrast, only a band of 383bp was found among the M.nontuberculosis which contained M.avium, M.chelonae, M.scrofulaceum, M.xenopi, M.kansasii, M.intracellulare and M.smegmatis. Compared with the standard strains,there was an additional 268bp band in simulated clinical samples infected by Mycobacterium.The above 3 specific bands were found neither in other 15 bacterial species tested nor in Mycoplasma pneumoniae.182 clinical samples were examined by culture,smear and triplexPCR.72 nontuberculous clinical samples were all negative. In 110 tuberculosis clinical samples, the positive rates were 2.7%,13.6% and 32.7%, respectively. Conclusion The triplexPCR possesses a high specificity and sensitivity.This method could detect and identify the DNA of M.tuberculosis and M.nontuberculosis except M.simiae. It is a valuable tool for early diagnosis and differentiation for infection of M.tuberculosis and M.nontuberculosis.