复合PCR-SSCP检测巨噬细胞内结核分枝杆菌耐药性的探讨

Study of detecting drug resistance genes of M.tuberculosis in macrophage from patient with tuberculosis by multiplex PCR-SSCP

〔目的〕检测结核分枝杆菌耐药性。〔方法〕复合PCR -SSCP检测分离培养结核菌株及分离分纯结核病患者标本中巨噬细胞内TB -DNA的耐药基因变异情况。〔结果〕PCR检测TB -DNA阳性率为 90 .8% ;复合PCR -SSCP检测分离菌株总耐药率为 90 .0 % (18/ 2 0 ) ,其中耐单药、耐 2药和耐 3药突变率分别为 2 0 .0 % (4 / 2 0 )、4 0 .0 % (8/ 2 0 )和 30 .0 %(6 / 2 0 )。而对PCR扩增到的结核病患者标本中巨噬细胞内TB -DNA的检测总耐药率为 72 .7% (4 8/ 6 6 ) ;其中耐单药、耐2药和耐 3药突变率分别为 19.7% (13/ 6 6 )、36 .4 % (2 4 / 6 6 )和 16 .7% (11/ 6 6 )。〔结论〕复合PCR -SSCP测定分离分纯结核病患者巨噬细胞内TB -DNA及分析其耐药性变异 ,可为病原、耐药和耐多药结核病诊断的有效方法之一。

睴bjective〕 To detect the drug resistance of M.tuberculosis. 〔Methods〕 TB-DNA in macrophages isolated from specimens of the patients with tuberculosis was detected with PCR. The drug resistance was detected with multiplex single-strand conformation polymorphism analysis.〔Results〕 The positive rate of TB-DNA detected by PCR was 90.8%.The total rate of the mutation of M.tuberculosis strains was 90.0%(18/20),The rates of the mutation of single drug、two drugs and three drugs were 20.0%、(4/20)、40.0%(8/20)and 30.0%(6/20)individually. The total rate of the mutation of TB-DNA in macrophages isolated from specimens of the patients with tuberculosis was 72.7%.The rates of the mutation of single drug、two drugs and three drugs were 19.7%(13/66)、36.4%(24/66)and 16.7%(11/66)individually.〔Conclusion〕 It is a good method in clinical practice that TB-DNA and multiple-drug resistance of M.tuberculosis in separated and purified Macrophages from specimens of patients with tuberculosis are detected by multiplex PCR-SSCP.