应用膜反向斑点杂交技术快速检测结核分支杆菌耐乙胺丁醇基因型的研究

Rapid Detection of Mycobacterium tuberculosis Ethambutol-resistant Genes by Membrane-reverse Dot Blot Hybridization

应用膜反向斑点杂交技术快速检测结核分支杆菌对乙胺丁醇 (EMB)耐药性。设计与合成用于检测结核分支杆菌耐EMB基因embB的寡核苷酸探针 ,点于硝酸纤维素膜上 ,与结核分支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 ,并与PCR 单链构象多态性 (PCR SSCP)和PCR 直接测序 (PCR DS)结果比较。对 81株结核分支杆菌临床分离株进行分析 ,31株EMB敏感株中 ,2 6株embB基因的SSCP图谱、膜反向斑点杂交结果与标准株 (H37Rv)完全相同 ;其余 5株SSCP图谱出现泳动变位 ,其中 3株E1b杂交阳性 ,PCR DS分析为embB基因 30 6位密码子ATG→GTG突变 ;2株E1d杂交阳性 ,PCR DS分析为embB基因 30 6位密码子ATG→ATA突变。 5 0株耐EMB菌株中 ,2 4株PCR SSCP图谱与标准菌株相同 ,E1杂交阳性 ;2 6株PCR SSCP图谱出现泳动变位 ,其中 18株E1b杂交阳性 ,2株E1c杂交阳性 ,5株E1d杂交阳性 ,1株E1e杂交阳性 ,未发现E1f杂交阳性 ,与PCR SSCP、PCR DS分析结果一致。突变检出率为 5 2 %。膜反向斑点杂交技术可能成为检测部分结核分支杆菌乙胺丁醇耐药基因型简便、快速的方法。

To study the rapid detection of resistance to ethambutol in Mycobacterium tuberculosis by reverse dot blot hybridized technique. We prepared the oligonucleotide probes of ethambutol-resistant genes (embB) and drop it on pyroxylin membrane. The target DNA fragments of M. tuberculosis clinical isolates were labeled with biotin by PCR amplification, and then hybridized with oligonucleotide probes on membrane. Polymerase chain reaction-Single stranded conformation polymorphism (PCR-SSCP) and PCR-direct sequencing (PCR-DS) techniques were used as the control. The embB gene in 81 Mycobacterium tuberculosis clinical isolates were analyzed. Of 31 ethambutol-sensitive strains, the results of SSCP spectrum and membrane hybridization in 26 sensitive strains were similar to H37Rv and positive hybridization with E1, the SSCP spectrum in another five strains was different from that of H37Rv , 3 strains were positive hybridization with E1b, PCR-DS showed the ATG→GTG mutation at codon 306 of embB, 2 strains were positive in E1d, PCR-DS showed the ATG→ATA mutation. Of 50 ethambutol-resistant strains, 24 strains were positive hybridization with probe E1 as H37Rv; 18 strains were positive hybridization with probe E1b, 2 strains were hybridized with probe E1c, 5 strains were hybridized with E1d, 1 strains were hybridized with E1e, all strains were negative hybridization with E1f. Mutation rate was 52%. The results coincided with PCR-SSCP and PCR-DS. The membrane-reverse dot blot hybridized technique was simple and rapid and could be used to detect ethambutol-resistance of partial Mycobacterium tuberculosis clinical strains.