基因芯片检测结核分枝杆菌利福平异烟肼耐药性的应用评价

Evaluation of DNA microarray for detection of rifampin and isoniazid resistance in Mycobacterium tuberculosis isolates

目的评价基因芯片检测结核分枝杆菌临床株对利福平和异烟肼耐药性的效果，探讨耐多药结核分枝杆菌基因检测合适的靶位点。方法回顾性收集深圳市慢性病防治中心2009年门诊患者痰标本菌株24株及2007至2009年深圳市耐药监测项目痰标本培养分离菌株127株。采用基因芯片检测其对利福平和异烟肼耐药性，与比例法药敏试验结果比较，评价其敏感度、特异度和准确性。Kappa检验评价芯片检测和表型药敏结果的一致性。对结核分枝杆菌利福平、异烟肼主要耐药相关基因rpoB、katG、inhA编码区、inhA启动子区和ahpC启动子区测序，分析耐药相关突变位点及频率。结果基因芯片检测利福平耐药的敏感度为94．4％，特异度为97．5％，准确性为96．0％；检测异烟肼耐药的敏感度为79．1％，特异度为100％，准确性为86．8％。耐利福平菌株中97．2％（70／72）在rpoB突变热点区域发生突变，位点包括531、526、516、511和533位密码子。耐异烟肼菌株中，70．3％（64／91）发生katG315密码子突变，11．0％（10／91）发生inhA-15突变，还有9株（9．9％）在ahpC启动子区发生突变，包括ahpC-9（4株）、ahpC-10（2株）、ahpC-6（2株）、ahpC-12（1株）和ahpC-32（1株）。结论基因芯片系统可以快速检测结核分枝杆菌对利福平和异烟肼耐药性，对利福平的检测具有良好的敏感度和特异度，检测异烟肼敏感度稍低。ahpC启动子区可以作为异烟肼耐药突变检测靶基因，联合筛选katG315密码子、inhA-15和ahpC启动子区突变可以显著提高异烟肼耐药性检测敏感度。

Objective To evaluate the performance of DNA microarray for rapid detection resistance to rifampin and isoniazid in Mycobacterium tuberculosis clinical isolates and identify suitable target sites for molecular genetic test. Methods Twenty-four clinical Mycobacterium tuberculosis isolates were collected retrospectively from Shenzhen Center for Chronic Disease Control in 2009 and 127 isolates from project on anti-tuberculosis drug resistance surveillance in Shenzhen during 2007 to 2009. Drug susceptibility to rifampin and isoniazid of the stains were determined by DNA microarray, and results were compared to that obtained with reference proportion method drug susceptibility testing for sensitivity, specificity and accuracy. The consistency of microarray and phenotypic susceptibility testing was evaluated by Kappa test. Genetic mutations in rpoB, katG, inhA, regulatory region of inhA, and regulatory region of ahpC were investigated by DNA sequencing to assess proper loci for rapid molecular diagnosis. Results Compared against results of proportion method, the sensitivity, specificity and accuracy of the DNA microarray assay for rifampin resistance were 94.4%, 97.5% and 96.0% respectively, and for isoniazid resistance were 79. 1%, 100% and 86. 8% respectively. Mutations in resistance-determining region of rpoB were observed in 97.2% （70/72） of the isolates resistant to rifampin, which contributed in the 531, 526,516,511 and 533 codon region. Mutations in katG315 codon, inhA-15, and ahpC regulatory region were found in 70.3% （64/ 91 ）, 11. 0% （10/91） and 9. 9% （9/90） of the isolates resistant to isoniazid, respectively. Mutations of ahpC promoter region consists of ahpC-9 （4 strains） , ahpC-10 （2 strains） , ahpC-6 （2 strains） , ahpC-12 （1 strain）, and ahpC-32 （1 strain）. Conclusions DNA nficroarray provided a rapid method for the detection of drug-resistant Myeobacteriurn tuberculosis isolates, and demonstrated good performance except less sensitive for isoniazid resistance. The mutations in ahpC regulator7 region might be good target loci for detection of isoniazid-resistant Myvobacteriurn tuberculosis, so screening the region may significantly improve the sensitivity for molecular genetic tests.