应用基因芯片系统快速检测多耐药脊柱结核临床-第三军医大学学报.docVIP

201311042 论著基因芯片法快速检测脊柱结核临床分离株SM、EMB、LVFX、AMK、CPM耐药 程 鹏1，张泽华1，李力韬1，张冠斌2，郭 永2，罗 飞1，周 强1，代 飞1，何清义1，许建中1（400038重庆，第三军医大学西南医院骨科1；102206北京，生物芯片北京国家工程研究中心1） 制备一种用于检测链霉素乙胺丁醇左氧氟沙星阿米卡星卷曲霉素耐药基因的新型微阵列芯片，快速检测脊柱结核临床分离株耐药的。方法根据SMEMB、LVFX、AMK、CPM 5种药物的常见耐药基因及突变位点（rpsL、rrs、embB、rrs及gyrA），制备DNA微阵列芯片，脊柱结核临床分离株耐药菌株111株及敏感菌株26株，均具备完整的临床信息及药敏结果，依次进行灭活、核酸提取、PCR扩增、芯片杂交、洗涤与扫描判读，与表型药敏结果对照，计算敏感度、特异度及95% CI。结果平均检测时间20（18～23）h。与表型药敏结果对照，61.90%，94.59%，66.00%，65.52%，70.37%，58.11%，58.00%，91.95%，80.56%；在26株全敏感菌株中，结果均显示为野生型，对5种抗结核药物耐药检测的特异度均为100%。结论基因芯片法检测结核耐药耗时短，一线、二线耐药检测的敏感度、特异度令人满意，生物安全性，。关键词基因芯片；脊柱结核；耐药Rapid Detection of SM/EMB/LVFX/AMK/CPM Resistance by DNA Microarray for Clinical Isolates from Spinal Tuberculosis Cheng Peng1，Zhang Zehua1，Li Litao1，ZhangGuanbin2，GuoYong2，Luo Fei1，ZhouQiang1，DaiFei1，HeQingyi1，Xu Jianzhong11Department of Orthopaedics，Southwest Hospital， Third Military Medical University，ChongQing，Chinahe National Engineering Research Center for Beijing Biochip Technology，Beijing，China [Abstract]Objective We designed a new DNA microarray for rapid detection of resistance to streptomycin，ethambutol，levofloxacin，amikacin and capreomycin， to assess the feasibility of DNA chip technology in drug resistance testing in spinal tuberculosis. MethodsThe new DNA microarray was designed to test the resistance-determining regions in rrs and rpsL （streptomycin）， embB （ethambutol） ，gyrA and gyrB （levofloxacin）， and rrs （amikacin and capreomycin）. A set of 111 drug-resistant M. tuberculosis strains， and 26 drug sensitive M. tuberculosis strains were tested by the new DNA microarray and by DNA sequencing. The sensitivity and specificity were evaluated. Result The average detection time was 20.1 ±1.2h(ranged from 18～23h）.The sensitivity of the MTBDR test SM/EMB/LVFX/AMK/CPM were as follows：61.90%，94.59%，66.00%，65.52%， respectivelyspecificity was 70.37%，58.11%，58.00%，91.95%，80.56%， respectivelyThe specificity of the wild type drug sensitive M. tuberculosis test was 100% for all 5 drug