反向斑点杂交技术在结核分枝杆菌耐药性检测中的应用研究

Application of Reverse Dot Blot Hybridization Technique on Detecting Drug Resistance of Mycobacterium Tuberculosis

目的利用反向斑点杂交技术,建立结核分枝杆菌对利福平(RFP)、异烟肼(INH)、链霉素(SM)和乙胺丁醇(EMB)耐药相关基因突变的快速检测方法。方法根据结核分枝杆菌标准株H37Rv序列,自行设计针对rpoB、katG、inhA、rpsL和embB基因中常见耐药突变区的系列寡核苷酸探针,制成膜芯片,并对64例结核分枝杆菌临床株的基因突变情况进行检测。结果在36株RFP耐药菌中,有32株被检出在rpoB基因上发生突变,突变检出率为88.89%(32/36);在39株INH耐药菌中,有30株被检出在katG或inhA基因上发生基因突变或缺失,检出率为76.92%(30/39),;在34株SM耐药菌中,有25株被检出在rpsL基因分析部位发生突变,检出率为73.5%(25/34);在32株EMB耐药菌中,有19株在embB基因分析部位发生突变,检出率为59.4%(19/32)。膜芯片检出的突变与基因测序结果一致。结论用膜芯片检测结核分枝杆菌对利福平(RFP)、异烟肼(INH)、链霉素(SM)和乙胺丁醇(EMB)四种主要一线抗结核药的耐药性,具有较高的特异性和敏感性,可作为常规药敏方法的补充,用于指导开展早期、有效的临床化疗。

Objective To develop a new reverse dot blot method for rapid detection of rpoB. katG, inhA, rpsL and embB gene mutation in clinical rifampicin resistance, isoniazide- resistance, streptomycin- resistance and ethambutol resistance Mycobacterium tuberculosis.isolates. Methods According to the preserved sequence of Mycobacterium tuberculosis H37 Rv strains, 23 oligonucleotide probes were designed specifically to detect the most common mutant sequences n rpoB, katG, inhA, rpsl. and embB genes, and analyze the drug resistance of 64 clinical Mycobacterium tuberculosis isolates. Results Of the 36 rifampicin - resistant M. tuberculosis isolates. 32 were found with rpoB gene mutation, the detection rate was 88.89% （32/36）. Of the 39 isoniazide- resistant isolates. 30 were found with katG and/or inhA gene mutation or deletion, the detection rate was 76.92% （30/39）. Of the 34 streptomycin- resistant isolates. 25 were found with rpsL gene mutation, the detection rate was 73.5 % （25/34 ）. And 19 isolates had embB gene mutation in 32 ethambutol resistant isolates. with the detection rate of 59.4 % （ 19/32 ）. The results by reverse dot blot approach were consistent with DNA sequencing. Conclusions The reverse dot blot approach is a highly sensitive and specific method for rapid detection of mutations of rifampicin, isoniazide, streptomycin and ethambutol resistant M. tuberculosiS, and might be applicable as a supplemental detection measure of drug resistance.