复合PCR-膜芯片技术对结核分枝杆菌链霉素耐药性的快速检测

Rapid detection of streptomycin resistance in Mycobacterium tuberculosis by complex polymerase chain reaction-gene membrane chip

目的 应用复合聚合酶链反应 (复合 PCR) -膜芯片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 rps L和 rrs的寡核苷酸探针制作膜芯片 ,与结核分枝杆菌分离株生物素标记的 rps L和 rrs基因复合 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 2株结核分枝杆菌临床分离株中 ,9株敏感株rps L和 rrs基因的 SSCP图谱、膜芯片杂交结果与标准株完全相同 ;4 3株耐 SM菌株中 ,33株存在 rps L 基因4 3位密码子 AAG→ AGG突变 ,5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 ,突变率为 90 .7%。结论 膜芯片技术检测结核分枝杆菌耐 SM基因型灵敏度高、特异性强、简便、快速 。

Objective To study the rapid detection of Mycobacterium tuberculosis resistance to streptomycin by complex polymerase chain reaction (PCR) gene membrane chip. Methods We prepared the oligonucleotide probe of streptomycin resistant gene ( rpsL and rrs ) and gene chip on membrane. The target DNA fragment of M.tuberculosis clinical isolates was labeled with biotin by complex PCR amplification, and then hybridized with gene membrane chip. PCR Single stranded conformation polymorphism (PCR SSCP) and PCR DNA sequencing (PCR DS) techniques were used as the control. Results Of 52 M.tuberculosis clinical isolates, SSCP spectrum of rpsL and rrs gene, results of membrane chip in 9 sensitive strains were similar to M.tuberculosis strain H37Rv. Of 43 streptomycin resistant strains, 33 strains have AAG→AGG mutations at codon 43 of rpsL gene, 5 strains have A→C mutations in position 513 of rrs gene, 1 strains have A→T mutations in position 513 of rrs gene. The rate of the mutation was 90 7%. Conclusion The gene chip technique showed high sensitivity and specificity to detect Mycobacterium tuberculosis resistance to streptomycin. It was simple and rapid and could be used for detection of streptomycin resistant clinical strains.