HRCA技术联合DNA芯片检测结核分枝杆菌利福平耐药基因突变的初步分析

摘要

Objective To detect the gene mutations in rifampin-resistant mycobacterium tuberculosis by use of hyperbranched rolling cycle amplification combined with the DNA chip technology.Methods A total of 898 sputum specimens were collected to detect mycobacterium tuberculosis by direct smearing method from June 2013 to June 2015.Positive specimens were identified and isolated by modified Roche medium and hyperbranched rolling cycle amplification combined with the DNA chip method, and rifampin-resistant genes were analyzed.Results The positive rate of mycobacterium tuberculosis was 40.2% (361/898) in 898 sputum specimens, and the positive rate (98.0%, 354/361) detected by the hyperbranched rolling cycle amplification combined with the DNA chip was higher than that detected by the modified Roche culture (37.4%) (P<0.05).The result of hyperbranched rolling cycle amplification combined with the DNA chip showed that there were 85 strains mycobacterium tuberculosis with RFP drug-resistant, 31 strains (36.5%, 31/85) with simple rpoB gene mutation, 51 strains (60.0%, 51/85) with katG and rpoB gene mutation, and 3 strains (3.5%, 3/85) with inhA and rpoB gene mutation.76 strains (89.4%, 76/84) had two point mutation and 9 strains (10.6%, 9/85) had single point mutation respectively.Conclusion The hyperbranched rolling cycle amplification combined with the DNA chip technology can detect drug-resistant TB quickly and efficiently.The rpoB gene mutation is the molecular basis for rifampin drug-resistant of patients with pulmonary tuberculosis, and the patterns of mutations are diverse.%目的 对超分支滚环扩增技术(hyperbranched rolling cycle amplification, HRCA)技术联合DNA芯片检测结核分枝杆菌利福平耐药基因突变进行初步分析.方法 采用直接涂片检测在2013年至2015年期间搜集的898份痰液样本中结核分枝杆菌情况,同时采用改良罗氏培养法与HRCA技术联合DNA芯片法对阳性样本进行鉴定,并对利福平耐药基因进行初步分析.结果 989份检测的痰液样本中361份为阳性,涂阳率为40.2%.HRCA技术联合DNA芯片法阳性率为98.0%,显著高于改良罗氏培养法(35.2%),且差异具有统计学意义(P<0.05).通过HRCA技术联合DNA芯片法发现85株结核分枝杆菌对利福平耐药,其中单纯rpoB 基因突变比例为36.5%(31/344);katG基因与rpoB基因均突变比例为60.0%(51/344);inhA基因与rpoB基因突变比例均为3.5%(3/344);且二位点联合突变及单位点突变率分别为89.4%(76/85)与10.6%(9/85).结论 HRCA技术联合DNA芯片法能够有效快速检出结核分枝杆菌突变基因,rpoB基因突变为主要结核分枝杆菌利福平耐药的基础,且突变呈现多样性.