结核分枝杆菌异烟肼耐药基因突变的研究

Studies on mutation of isoniazid resistant genes in M. tuberculosis

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H3 7RV 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 2 5 %。结论 :多数结核分枝杆菌耐INH是由于其katG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 。

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 H 37 R V 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.