核酸质谱检测结核分枝杆菌耐药方法的建立  

Establishment of MassARRAY system for detection of drug resistance of Mycobacterium tuberculosis

作　　者：余艳芳 赵开顺 屠春林 陈娓 梁海鹰 易清清 YU Yan-fang;ZHAO Kai-shun;TU Chun-lin;CHEN Wei;LIANG Hai-ying;YI Qing-qing(Department of Respiratory Medicine,Shanghai Jiading District Central Hospital Affiliated to Shanghai University of Medicine&Health Sciences,Shanghai 201800,China;Department of Pulmonology,Shanghai Jiading District Central Hospital Affiliated to Shanghai University of Medicine&Health Sciences,Shanghai 201800,China;Department of Central Laboratory,Shanghai Jiading District Central Hospital Affiliated to Shanghai University of Medicine&Health Sciences,Shanghai 201800,China)

机构地区：[1]上海健康医学院附属嘉定区中心医院呼吸内科,上海201800 [2]上海健康医学院附属嘉定区中心医院肺科,上海201800 [3]上海健康医学院附属嘉定区中心医院中心实验室,上海201800

出　　处：《临床肺科杂志》2021年第1期74-81,共8页Journal of Clinical Pulmonary Medicine

基　　金：上海市嘉定区卫生健康系统科研项目(No.2017ZD05);上海市嘉定区科委科研项目(No.JDKW-2019-W03)。

摘　　要：目的建立核酸飞行质谱(MassARRAY)方法检测结核耐药基因突变检测体系,并评估其检测临床耐药结核病的应用价值。方法收集2016年1月-2019年6日上海市嘉定区中心医院肺科肺结核有治疗经历患者的菌培养样本55例,其中药物敏感样本10例,45例耐多药样本中,耐异烟肼、耐氟喹诺酮类(左氧氟沙星、莫西沙星)的样本45例、耐二线类注射药物(阿米卡星、卷曲霉素)的样本23例;供试样本均提取核酸,先用一代测序验证质谱检测的准确性,再根据临床药敏试验结果,验证质谱检测复治样本的有效性,主要评价指标为检测的灵敏度、特异性和一致性。结果与一代测序相比,该体系检测临床菌株异烟肼、氟喹诺酮类药物和二线注射类药物的灵敏度分别为100%(37/37)、100%(34/34)和100%(10/10),特异性分别为100%(18/18)、100%(21/21)和100%(45/45),总一致率100%,一致性检验Kappa值为1。以最低抑菌浓度法(MIC)为金标准,该体系检测临床菌株异烟肼、氟喹诺酮类药物和二线注射类药物的灵敏度分别为82.2%(37/45)、75.6%(34/45)和45.5%(10/22),特异性分别为100%(10/10)、100%(10/10)和100%(33/33),两种方法的总一致率为89.1%,一致性检验Kappa值为0.703。结论质谱检测基因突变与最低抑菌浓度法检测结核菌耐药总体具有一定的一致性,可快速检测结核分枝杆菌的耐药性,有望成为复治患者辅助临床治疗的新手段。Objective To establish the detection system of drug-resistant gene mutation of Mycobacterium tuberculosis(MTB)by MALDI-TOF MS(MassARRAY),and to evaluate its application value in the detection of clinical drug-resistant tuberculosis.Methods Bacterial culture of 55 patients with previously treated were collected from pulmonary tuberculosis retreatment patients in Shanghai Jiading District Central Hospital from sanuary 2016 to June 2019,including 10 drug sensitive samples,and 45 multi drug resistant samples.Among them,there were 45 isoniazid resistant,fluoroquinolones resistant(levofloxacin,moxifloxacin)resistant samples,and 23 second-line injection resistant(amikacin,capreomycin)resistant samples.Nucleic acid was extracted and measured with the first generation to verify the accuracy of MALDI-TOF MS(MassARRAY)detection.According to the results of clinical drug sensitivity test,the effectiveness of mass spectrometry detection of retreatment samples was verified.The main evaluation indicators were the sensitivity,specificity and consistency of detection.Results Compared with the first-generation sequencing,the sensitivity of the system to detect clinical strains isoniazid,fluoroquinolones and second-line injection drugs was 100%(37/37),100%(34/34)and 100%(10/10),the specificity was 100%(18/18),100%(21/21)and 100%(45/45),the total consistency was 100%,and the kappa value of the consistency test was 1.Taking Minimum Inhibitory Concentration(MIC)as the gold standard,the sensitivity of the system for the detection of clinical strains isoniazid,fluoroquinolones and second-line injection drugs was 82.2%(37/45),75.6%(34/45)and 45.5%(10/22),the specificity was 100%(10/10),100%(10/10)and 100%(33/33),the total consistency of the two methods was 89.1%,and the kappa value of the consistency test was 0.703.Conclusion The detection of gene mutation by MALDI-TOF MS(MassARRAY)is consistent with the detection of drug resistance by MIC method in clinical tubercle bacillus(TB)isolates,which can quickly detect the drug resistance of Myc

关 键 词：结核分枝杆菌 核酸飞行质谱 复治患者 耐药 

分 类 号：R52[医药卫生—内科学]