寡核苷酸探针膜杂交法检测耐药结核菌

Rapid detection of resistant genes of clinical isolates in Mycobatterium tuberculosis by membrane oligonucleotide probes hybridization

目的应用寡核苷酸探针膜杂交法快速检测临床分离株中结核分枝杆菌对异烟肼(INH)、链霉素(SM)、乙胺丁醇(EMB)的耐药性。方法设计与合成用于检测结核分枝杆菌3种药物常见耐药基因的寡核苷酸探针,点于硝酸纤维素膜上,与结核分枝杆菌临床分离株生物素标记的聚合酶链反应(PCR)产物进行反向斑点杂交。结果26株耐INH菌株中,16株Klb杂交阳性;7株inhla杂交阳性;23株耐SM菌株中,17株rpsl基因突变型探针Slb杂交阳性,2株突变型探针rrslb杂交阳性;31株耐EMB菌株中,13株Elb杂交阳性,1株Elc杂交阳性,5株Eld杂交阳性,1株Ele杂交阳性,11株与El探针杂交。kagG、inhA、rp-sL、rrs和embB基因膜杂交突变检出率分别为61.5%,26.9%,74%,8.7%和64.5%,与PCR-DS分析结果一致。结论寡核苷酸探针膜杂交技术可能成为检测部分结核分枝杆菌耐药基因型简便、快速的方法。

Objective To study the rapid detection of resistance to INH, SM, EMB in Mycobacterium tuberculosis by membrane oligonucleotide probes hybridization. Methods We prepared the oligonucleotide probes of five resistant genes of INH, SM,EMB and drop it on nitrocellulose membrane. The target DNA fragments of M. tuberculosis clinical isolates were labeled with biotin by PCR amplification, and then hybridized with oligonucleotied probes on membrane. Results of 26 isoniazid-resistant strains, 16 strains were positive hybridization with probe KIb. 7 strains were positive hybridization with probe inhla： Of 23 streptomycin-resistant strains. 17 strains were positive hybridization with probe Sib. 2 strains were positive hybridization with probe Eib. 1 strains were positive hybridization with probe Eic, 5 strains were positive hybridization with probe Eid. 1 strains were positive hybridization with probe Eie, 11 strains were positive hybridization with probe EI：Mutation rate of katG,inhA,rpsL,rrs and embB gene was 61.5%,26.9%,74%,8.7% and 64.5% respectively. The results coincided with PCR-DS. Conclusion The membrane oligonucleotide probes hybridization was simple and rapid and could be used to detect resistance of partial Myeobacterium tuberculosis clinical strains.