应用PCR-反向斑点杂交技术快速检测结核分枝杆菌耐链霉素基因型  

Rapid Detection of Mycobacterium Tuberculosis Streptomycin-resistant Genes by PCR-reverse Dot Blot Hybridization

作　　者：梁建琴[1] 吴雪琼[1] 曹立雪[1] 张俊仙[1] 李洪敏[1] 

机构地区：[1]解放军第三零九医院结核病研究中心,北京100091

出　　处：《中国医师杂志》2004年第9期1170-1172,共3页Journal of Chinese Physician

基　　金：军队医学杰出中青年人才科研基金项目 (0 1JO2 0 )

摘　　要：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 。Objective To study the rapid detection of mycobacterium tuberculosis resistance to streptomycin by reverse dot blot hybridization technique. Methods The oligonucleotide probes of streptomycin-resistant genes (rpsL and rrs) were prepared and dropped on nitrocellulose membrane. The target DNA fragments of M. tuberculosis clinical isolates were labeled with biotin by PCR amplification, and then hybridized with the oligonucleotide probes on the membranes. PCR-SSCP and PCR-direct sequencing (PCR-DS) techniques were used to detect the target fragment of M.tuberculosis as control. Results In 53 M. tuberculosis clinical isolates, the consistent rate of three detection methods was 100%. Both the SSCP mapping of rpsL and rrs genes and the results of membrane hybridization in 9 drug-sensitive strains were identical to those in M. tuberculosis standard strain H37Rv. Of 44 streptomycin-resistant strains, 33 strains had AAG→AGG mutation at the codon 43 of rpsL gene, 6 strains had A→C mutation at the 513 site of rrs gene, 1 strain had A→T mutation at the 513 site of rrs gene, and the detection rate of the target genes mutation was 90 9%. In 53 M.tuberculosis clinical isolates, 40 resistant strains and 9 sensitive strains to streptomycin could be detected using dot blot hybridization and the consistent rate with the in vitro susceptibility test was 92 6%(49/53). Conclusion The reverse dot blot hybridization technique showed high sensitivity and specificity to detect Mycobacterium tuberculosis resistance to streptomycin. It possessed the simple and rapid characteristics, and could be used to detecte streptomycin-resistant M.tuberculosis clinical strains.

关 键 词：PCR-反向斑点杂交 快速检测 结核分枝杆菌 链霉素 基因型 耐药性 基因变异 

分 类 号：R346[医药卫生—基础医学]