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

Rapid detection of SM/EMB/LVFX/AMK/CPM resistance by DNA microarray for clinical isolates from spinal tuberculosis

目的制备一种用于检测链霉素（streptomycin, SM）、乙胺丁醇（ethambutol, EMB）、左氧氟沙星（levofloxacin, LVFX）、阿米卡星（amikacin, AMK）、卷曲霉素（capreomycin, CMP）耐药基因的新型微阵列芯片，测试其快速检测脊柱结核临床分离株耐药的效果。方法 根据SM、EMB、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。结果检测时间18～23（20.1±1.2）h。与表型药敏结果对照，基因芯片检测SM、EMB、LVFX、AMK、CPM耐药的敏感度分别为90.36%、61.90%、94.59%、66.00%、65.52%，其对应的特异度分别为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，生物安全性高，能够满足临床耐多药结核快速诊断需要。

Objective To detect the feasibility and efficiency of newly self-designed DNA microarray for rapid detection of resistance to streptomycin （SM）, ethambutol （EMB）, levofloxacin （LVFX）, amikacin （AMK） and capreomycin （CMP） in spinal tuberculosis. Methods The new DNA microarray was designed to test the resistance-determining regions in rrs and rpsL （SM）, embB （EMB）, gyrA and gyrB （LVFX）, and rrs （AMK and CMP）. A set of 111 drug-resistant Mycobacterium tuberculosis strains, and 26 drug sensitive Mycobacterium tuberculosis strains with full clinical data and sensitivity test results were tested by the new DNA microarray and by DNA sequencing after inactivation, nucleic acid extraction, PCR amplification, chip hybridization, washing and scanning interpretation. The sensitivity and specificity were evaluated. Results The average detection time was 20.1±1.2 h （ranged from 18 to 23 h）. The sensitivity of the MTBDR test was 90.36%, 61.90%, 94.59%, 66.00% and 65.52% for SM/EMB/LVFX/AMK/CPM respectively, and their specificity was 70.37%, 58.11%, 58.00%, 91.95% and 80.56%, respectively. All 26 drug sensitive Mycobacterium tuberculosis strains were identified as wild type, and the specificity of these wild type strains was 100% for all 5 drugs. Conclusion This new DNA microarray is a rapid approach of good biosafety and high sensitivity and specificity in resistance test of first and second line MTB drug for the isolated Mycobacterium tuberculosis strains, and is satisfying in clinical practice for rapid diagnosis of drug-resistant Mycobacterium tuberculosis strains.