摘要
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 triplexPCR. 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 βglobin were amplified by triplexPCR respectively. Result The sensitivity of the triplexPCRelectrophoresis 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 triplexPCR.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 triplexPCR 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.
出处
《中华结核和呼吸杂志》
CSCD
北大核心
1998年第9期547-551,共5页
Chinese Journal of Tuberculosis and Respiratory Diseases
关键词
结构分支杆菌
非结核分支杆菌
DNA
聚合酶链反应
Polymerase chain reaction
Mycobacterium tuberculosis
Mycobacterium nontuberculosis