中国部分地区结核分支杆菌耐利福平、异烟肼和链酶素耐药基因的检测

Defection rifampin-resistant and Isoniazid-resistant and streptomycin-resistant Mycobacterium tuberculosis in some areas in Chain

目的研究我国部分地区耐利福平(RFP)、异烟肼(INH)和链酶素(SM)结核分支杆菌临床分离株rpoB基因、KatGr、psL基因突变情况,评价其耐药分子机制。方法采用PCR和聚合酶链反应-单链构象多态性(PCR-SSCP)对373株结核分支杆菌临床分离株(其中敏感株148株,耐RFPI、NH、SM株共225株)rpoB基因、KatG和rpsL基因进行检测。并对其中19株SSCP阴性的耐RFP株用双脱氧末端终止法进行测序分析。结果安徽省、北京市、上海市、河北省、河南省、辽宁省、吉林省结核分支杆菌耐RFP临床分离株rpoB基因突变率分别为90%、91%、90%、91%、90%、93%、91%;KatG基因突变率分别为69%、63%、71%、68%、67%、68%、65%,rpsL基因突变率71%、69%、74%、68%、70%、61%、76%,分别为经统计学处理不同地区间无显著性差异(χ2=0.46,P>0.05)。同时rpoB基因敏感株均无突变,特异性为100%;KatG基因有3株发生突变,特异性为98%。19株SSCP阴性耐药株中经测序6株在531位密码子TCG→TTG,1株526位密码子CAC→TAC,7株发生在516位密码子GAC→GTC,5株未改变。结论我国不同地区耐利福平、异烟肼和链霉素的rpoB、KatGr、psL耐药基因突变率大致相同,rpoB基因、KatG和rpsL基因突变分别是耐RFP、INH和SM结核分支杆菌耐药性产生的主要分子机制,PCR-SSCP可快速检测结核分支杆菌RFPI、NH和SM耐药性。

Objective To evaluate the characteristics of rpoB.KatG, rpsL gene mutations detection applied resistance in multidrug resistant clinical isolates of mycobaeterium tuberculosis in some areas of Chain.Methods rpoB.KatG and rpsL gene mutation of mycobacterium tuberculosis was detected with Polymerase Chain reaction-single strand conformation （PCR-SSCP） from 373 clinical isolates,including 148 drug susceptible strains and 225 INH and RFP resistance strains. PCR products of isolates that rpoB gene mutations were not found with PCR-SSCP were sequenced using double deoxidization chain terminating. Results Respectively, the rpoB mutation rate was 90% in Anhui,91% in Beijing,90% in Shanghai,91% in Hebei,90% in Henan,93% in Liaoning and 91% in Jilin. The difference had no sigmficance between the results from these areas （ X^2 = 0.31, P 〉 0.05 ）. the KatG mutation rate was 69% in Anhui,63% in Beijing,71% in Shanghai,68% in Hehei,67% in Henan,68% in Liaoning and 65% in Jilin. the rpsL mutation rate was 71% in Anhui,69% in Beijing,74% in Shanghai,68% in Hebei,70% in Henan,61% in Liaoning and 76% in Jilin.The difference had no sigmficance between the results from these areas（ X^2 = 0.46, P 〉 0.05）. Sequence analysis showed 14 isolates had rpoB gene mutations and the other 5 isolates were not found rpoB gene mutations.The most frequent rpoB gene mutations sites are Leu-531 （6 isolates,CAC→TAC）,Tyr-526（1 isolate,GAC→GTC ）and Val-516（7 isolate, CAC→TAC）. Conclusion The major mechanism of RFP, INH, SM resistant drug was rpoB, KatG and rpsL gene mutation. About 90% rifampin-resistant clinical strains of Mycobactefium tuberculosis from Chain had rpoB mutations, PCR-SSCP is able to apply to detect M. tuberculosis on drug susceptibility.