变性高效液相色谱法检测结核分枝杆菌gyrA基因突变

Analysis of gyrA mutation in Mycobacteria tuberculosis by denaturing HPLC

目的 分析我国结核分枝杆菌氟喹诺酮类耐药基因gyrA突变的特点并探讨变性高效液相色谱法(DHPLC)的应用价值.方法 以结核分枝杆菌临床分离株109株(常规药敏试验显示87株氧氟沙星耐药,22株敏感)为对象,测定氧氟沙星最小抑菌浓度(MIC),同时进行gyrA基因氟喹诺酮类耐药决定区域(QRDR)DNA测序及DHPLC分析.结果 发现我国耐氧氟沙星结核分枝杆菌近来出现两个新的特点:一是gyrA QRDR双位点突变率高(87株耐药株中56.3%携有双位点突变);二是出现了以往认为的仅存在于其他细菌而在结核分枝杆菌中从未报道过的Ala74Ser突变(49株双位点突变耐药株中有20.4%携有该突变).gyrA基因突变类型与MIC之间并无密切关系.DHPLC测定中除常规的M.tb H37Rv以外还引入了M.tb Erdman做为参照菌株,成功地摆脱了95位AGC:ACC自然多态性的影响.与测序法相比较,DHPLC法的敏感度与特异度均达100%.结论 我国结核分枝杆菌氟喹诺酮类耐药已很严重,gyrA基因QRDR双位点突变率高为其特点.本研究建立的结核分枝杆菌gyrA基因突变的DHPLC分析法简便快速,稳定灵敏,在大面积大样本耐药监测以及临床快速药敏检测中有应用价值.

Objective To analyze the characteristic of fluoroquinolone resistance gene gyrA mutation in Mycobacterium tuberculosis and to evaluate the value of denaturing HPLC(DHPLC) method.Methods One hundred and nine Mycobacterium tuberculosis clinical isolates among which 87 were found ofloxacin-resistant while 22 susceptible according to routine method,received minimal inhibitory concentration (MIC) test,gyrA quinolone resistance determining region (QRDR) DNA sequencing and DHPLC analysis concurrently. Results Two new characteristics of ofloxacin-resistant Mycobacterium tuberculosis were found: one was the high rate of double mutations in gyrA QRDR (56.3% of 87 ofloxacin-resistant isolates were found carrying double mutations),the other was that among these double-mutated isolates,20.4% (10/49) harbored the Ala74Ser mutation,which previously was thought to exist only in other bacteria,and had never been found in Mycobacterium tuberculosis. No close relationship was observed between gyrA mutation type and ofloxacin MIC. By introducing M.tb H37Rv and M.tb Erdman two reference strains,interference from codon 95 AGC:ACC polymorphism was successfully avoided. Compared to DNA sequencing,the sensitivity and specificity of DHPLC method were all 100%.Conclusions Fluoroquinolone-resistance of Mycobacterium tuberculosis in our country is quite severe. The characteristic is the high rate of gyrA QRDR double point mutations. DHPLC method set up in this work is simple,rapid,stable,sensitive and may be valuable in large samples of drug-resistance survillence in large area.