显色法芯片检测结核分枝杆菌利福平和异烟肼耐药基因  

Detection of resistance genes of rifampin and isoniazid in Mycobacterium tuberculosis by using gene chips

作　　者：郭乃洲 王万相 严爱华 马达 周步全 景奉香[3] 唐向荣[3] 

机构地区：[1]盐城市第一人民医院检验科,224006 [2]江苏省盐城市第二人民医院检验科 [3]中国科学院上海微系统与信息技术研究所

出　　处：《中华微生物学和免疫学杂志》2006年第6期567-570,共4页Chinese Journal of Microbiology and Immunology

摘　　要：目的建立显色法芯片检测结核分枝杆菌耐药基因的方法。方法设计4对地高辛标记引物,扩增结核分枝杆菌rpoB、katG、inhA和ahpC 4个基因部分片段,根据结核分枝杆菌利福平(RFP)和异烟肼(INH)4条耐药相关基因上8个位点的25种单核苷酸多态性设计探针制作芯片,扩增产物与芯片杂交,显色法判断结果;用该法检测46株结核分枝杆菌临床分离株。结果扩增产物琼脂糖电泳可见4条大小分别为165、181、245、315 bp DNA条带;结核分枝杆菌菌液浓度为1．6×103／ml时,该法仍可检测到各位点野生及突变信号;19种非结核分枝杆菌标准株和9种非分枝杆菌标准株扩增产物无DNA条带,与芯片杂交亦无信号,H37Rv结核分枝杆菌标准株芯片检测各位点均为野生型;5株结核分枝杆菌临床分离株重复检测5次,结果完全一致;6份PCR产物的测序结果与芯片检测结果完全一致;46株结核分枝杆菌临床分离株中,RFP耐药株28株,芯片检出突变株24株,突变率为85．7％,RFP敏感株18株,芯片检出突变株2株。INH耐药株31株,芯片检出突变株20株,突变率为64．5％,INH敏感株15株,芯片检出突变株4株。结论显色法芯片检测结核分枝杆菌耐药基因具有较高的敏感性和特异性,无需特殊仪器设备,有一定的推广应用价值。Objective To establish a method for detection of the resistance genes in Mycobaaerium tuberculosis by genes chips. Methods Designed four primers characterized by digoxin, four gene fragments of rpoB, katG, inhA and ahpC in Mycobacterium tuberculosis were amplified. Probes were designed and the gene chip was fabricated according to the 25 single nucleoticle polymorphisms of 8 sites on 4 genes associated with RFP and INH resistance, amplified products were hybridized with chips. Results Four kinds of amplification products were identified and contain 165, 181, 245 and 315 bp respectively. The method may detect the signals of wildness and mutation in every sites even when the concentration of Mycobacterium tuberculosis was 1.6 × 10^3/ml. The amplified products of 19 non-Mycobacterium tuberculosis standard strains and 9 non-Mycobacterium standard strains didn＇t have signals when hybridized with chips. Every site of standard strain of H37Rv Mycobaaerium tuberculosis was wildness. Five clinical isolates of Mycobacterium tuberculosis were detected five times repeatedly, the results were correspondent. In 46 clinical isolates, 28 strains were resisted to RFP, in which 24 strains were mutated, and the percent of mutation was 85.7% ; Eighteen strains were sensitive to RFP, 2 strains were mutated; Thirty-one strains were resisted to INH and 20 strains were mutated, the percent of mutation was 64.5 % ; Fifteen strains were sensitive to INH, 4 strains were mutated. Conclusion Coloration gene chip for detecting resistance gene in Mycobaacterium tuberculosis is highly sensitive and specific, and is technically feasible for clinical application.

关 键 词：显色法 芯片 结核分枝杆菌 利福平 异烟肼 耐药 

分 类 号：R450[医药卫生—治疗学]