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目的 应用膜反向斑点杂交技术快速检测结核分枝杆菌对链霉素 (SM)的耐药性。方法 设计与合成用于检测结核分枝杆菌耐SM基因rpsL和rrs的寡核苷酸探针 ,点于硝酸纤维素膜上 ,与结核分枝杆菌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。结果 5 3株结核分枝杆菌临床分离株中 ,三种检测方法符合率为 10 0 %。 9株敏感株rpsL和rrs基因的SSCP图谱、膜杂交结果与标准株完全相同 ;44株耐SM菌株中 ,3 3株存在rpsL基因 43位密码子AAG→AGG突变 ,6株有rrs基因 5 13位A→C突变 ,1株有rrs基因 5 13位A→T突变 ,突变检出率为 90 .9%,40株耐SM菌株和 9株敏感株可用膜杂交方法检测出来 ,与传统药敏试验方法检测符合率为 49/5 3。结论 应用膜反向斑点杂交技术检测结核分枝杆菌耐SM基因型灵敏度高、特异性好、简便、快速 ,可用于临床耐药性检测
Objective To rapidly detect the resistance of Mycobacterium tuberculosis to streptomycin (SM) by membrane reverse dot blot hybridization. Methods Oligonucleotide probes were designed and synthesized for the detection of rpsL and rrs genes resistant to Mycobacterium tuberculosis, spotted on nitrocellulose membrane, and incubated with biotinylated polymerase chain reaction (PCR) of Mycobacterium tuberculosis isolates ) Products were reverse dot blot hybridized and compared with PCR-single strand conformation polymorphism (PCR-SSCP) and PCR-direct sequencing (PCR-DS). Results Among the 53 clinical isolates of Mycobacterium tuberculosis, the coincidence rates of the three methods were 100%. The SSCP patterns of rpsL and rrs genes of 9 strains of susceptible strains were identical to those of the standard strains. Among the 44 strains of SM-resistant strains, 43 strains of rpsL gene had AAG → AGG codon 43 mutations and 6 strains had rrs gene 5 13 A mutation of A → C, a mutant of rrs gene at 533 A → T mutation, mutation detection rate of 90.9%, 40 SM-resistant strains and 9 sensitive strains can be detected by membrane hybridization method, and traditional drug-susceptibility Test method to detect the coincidence rate of 49/5 3. Conclusion The detection of Mycobacterium tuberculosis resistance to SM genotype by membrane reverse dot blot hybridization has high sensitivity, specificity, simple and rapid, and can be used for clinical drug resistance testing