结核分枝杆菌异烟肝耐药基因突变的研究

目的：了解我国结核分枝杆菌耐异烟肼（INH）分离株katG基因突变情况，探讨快速检测结核分枝杆菌耐药基因型的分子药敏检测方法。方法：用PCR单链构象多态性（PCR－SSCP）和PCR直接测序法（PCR－DS）检测30株结核分枝杆菌临床分离株的katG基因。结果：以结核分枝杆菌H37Rv标准株对照观察所有INH敏感株的PCR－SSCP和PCR－DS图谱，均未发现异常；而在12株INH耐药株中，有4株无PCR－SSCP和PCR－DS图谱异常，占INH耐药株的33．4％；有5株在94位发生核苷酸错义突变，占INH耐药株41．7％，有3株在96位发生核苷酸同义突变，占INH耐药株的25％。结论：多数结核分枝杆菌对INH是由于其katC基因突变所致，可先用PCR－SSCP筛选突变株，再用PCR－DS方法测定其突变位点，达到快速检测结核分枝杆菌INH耐药基因型的目的。

Studies on mutation of isoniazid resistant genes in M. tuberculosis]

OBJECTIVE: To understand mutation of isoniazid-resistant genes in M. tuberculosis clinical isolates, and to develop the method for rapid detection of drug resistance. METHOD: Analyzing the katG genes in 30 M. tuberculosis clinical isolates with PCR-SSCP(single stranded conformation polymorphism) and PCR-DS (direct sequencing) techinques. RESULTS: With the reference of M. tuberculosis strain H37 Rv was used as control, all isoniazid-sensitive isolates and 4 isoniazid-resistant isolates displayed ab normal PCR-SSCP and PCR-DS patterns. But of 12 isoniazid-resistant isolates, 4(33.4%) isolates displayed abnormal katG PCR-SSCP pattern, 8 (46.7%) isolates displayed abnormal katG PCR-SSCP pattern, 5(41.7%) isolates were missense mutation at codon 94, 3(25%) isolates were cosense mutation at codon 96. CONCLUSIONS: Resistance to isoniazid in most M. tuberculosis isolates is due to the mutations on katG genes. PCR-SSCP techniques could be used as a method for rapid detection of isoniazid-resistance in M. tuberculosis.