异源双链分析法快速检测结核分枝杆菌氟喹诺酮类耐药性  

Rapid Detection of Fluoroquinolone-resistance of Mycobacterium Tuberculosis Isolates by Heteroduplex Analysis

作　　者：王伟[1] 石瑞如[1] 张国龙[1] 付光宇 彭义利[1] 苑星[1] 

机构地区：[1]河南省胸科医院检验科及中美结核病研究中心,郑州450008 [2]郑州市安图绿科生物有限公司,450000

出　　处：《医学研究杂志》2010年第10期62-65,共4页Journal of Medical Research

基　　金：河南省医学科技攻关重点项目(200702015)

摘　　要：目的采用变性高效液相色谱(DHPLC)和Surveyor酶法分析结核分枝杆菌氟喹诺酮类耐药基因gyrA突变,探讨这两种异源双链分析法在结核菌耐药快速检测中的应用价值。方法 102株结核分枝杆菌临床分离株(常规药敏试验显示80株氧氟沙星耐药,22株敏感),PCR扩增gyrA基因氟喹诺酮类耐药决定区域,与参照菌株M.tbErdman的扩增产物形成异源双链,然后分别采用DHPLC和Surveyor酶法检测基因突变,同时进行DNA测序作为对照。结果 DHPLC法和Surveyor酶法与DNA测序法相比,具有同样的检出率(均为100%),80株耐药株均存在gyrA基因突变,而22株敏感株均无基因突变发生。结论 DHPLC法和Surveyor酶法均可用来快速检测结核分枝杆菌氟喹诺酮类耐药。两者均简便,稳定,灵敏,前者高通量,全自动化,但需要昂贵的设备;后者成本低,不需要特殊设备,适合我国大部分临床实验室开展。Objective To detect gyrA gene mutation and fluoroquinolone - resistance of Mycobacterium tuberculosis clinical isolates by using two types of hcteroduplex analysis ： denaturing high - performance liquid chromatography （DHPLC） and Surveyor nuclcase method and to evaluate their potential value in future clinical use. Methods One hundred and two Mycobacterium tuberculosis clinical isolates （ 80 ofloxacin - resistant, 22 ofloxacin - sensitive, by routine method） were applied for PCR amplification of quinolone resistance determining region in gyrA gene. PCR products was hybridized respectively with M. tb Erdman. Heteroduplex was analyzed by DHPLC and Surveyor nuclease methods at the same time. DNA sequencing was performed as gold standard. Results High detection rate （ 100% ） was shown both in DHPLC and in Surveyor nuclease method, compared with DNA sequencing. Eighty ofloxacin - resistant isolates were found with gyrA mutation while those 22 ofloxacin - sensitive isolates with no mutation. Conclusion DHPLC and Surveyor nuclease methods can both be regarded as promising rapid tools for analysis of fluoroquinolone - resistance of Mycobacterium tuberculosis. The former has many advantages： simple, stable, sensitive, high through - put, automatic, except the need of an expensive equipment. The latter is cost - effective with no need of special equipment, can he widely used in most of clinical laboratories in our country.

关 键 词：异源双链分析 基因突变 高效液相色谱法 核酸酶 分枝杆菌 结核 

分 类 号：R378.911[医药卫生—病原生物学]