聚合酶链式反应检测结核杆菌的研究

以人型结核杆菌基因组DNA为模板，合成二段引物各20个碱基进行聚合酶链式反应(PCR)。经琼脂糖凝胶电泳证实，获得一条245bp扩增带。PCR检测的敏感性染色体基因组DNA为1pg，菌悬液为13个活菌／ml。在特异性试验中，人型结核杆趋，牛型结核杆菌、BCG可见此扩增带。被试的其它14种扰酸菌以及变铅青链霉菌、大肠杆菌质粒Puc19、星状诺卡氏菌、红球菌均未见该扩增带。54例肺结核痰标本3种方法检查的阳性率分别为：萋尼氏抗酸染色16．7％，培养法14．8％，PCR 37．0％。前2种检查方法分别与PCR比较，经统计学处理均有显著性差异(P<0．01)。12例非结核性肺部疾患痰标本抗酸染色和PCR均为阴性。结果表明，PCR技术是快速、敏感、特异诊断结核病的方法。

Detection of Mycobacterium tuberculosis in sputum specimens of pulmonary tuberculosis by DNA amplification]

The PCR was used to detect M. tuberculosis DNA sequences in uncultured clinical specimens. Two oligonucleotide primers with 20 bp each amplified target template DNA of M. tuberculosis. Amplified DNA product was 245 bp which was identified by agarose gel electrophoresis. The sensitivity of detection of M. tuberculosis genomic DNA and bacteria suspension by PCR was lpg and 13 viable bacteria cell/ml, respectively. In specificity experiments, only M. tuberculosis, M. bovis and BCG were positive by PCR, but all other 14 Mycobacterium tested, including streptomyces lividans and E. coli plasmid PUC19 were negative. The sensitivity of detection of M. tuberculosis by PCR was determined by comparing the fast-acid staining and culture on total 54 sputum specimens of pulmonary tuberculosis and 12 nontuberculosis lung disease. The positive rate of PCR in pulmonary tuberculosis were 37.0%, culture method showed only 14.8%, fast-acid staining were 16.7%. Nontuberculosis lung disease were negative. The results show that DNA amplification is a superior method with high degree of sensitivity and specificity for rapid diagnosis of pulmonary tuberculosis.