荧光PCR探针熔解曲线技术检测痰标本结核分枝杆菌利福平耐药性的研究  

Study on the detection of rifampicin resistance of Mycobacterium tuberculosis in sputum samples using fluorescence PCR probe melting curve technique

作　　者：王嫩寒 田丽丽[1] 陈双双 赵琰枫 樊瑞芳 陈昊 任怡宣 张洁 易俊莉 杨新宇 于兰 丁北川 李传友 代小伟 Wang Nenhan;Tian Lili;Chen Shuangshuang;Zhao Yanfeng;Fan Ruifang;Chen Hao;Ren Yixuan;Zhang Jie;Yi Junli;Yang Xinyu;Yu Lan;Ding Beichuan;Li Chuanyou;Dai Xiaowei(Department of Tuberculosis Laboratory,Beijing Center for Disease Prevention and Control,Beijing 100035,China)

机构地区：[1]北京市疾病预防控制中心结核病实验室,北京100035

出　　处：《中国防痨杂志》2023年第6期583-588,共6页Chinese Journal of Antituberculosis

基　　金：北京市疾病预防控制中心自管课题(JKS2021-06)。

摘　　要：目的:评价荧光PCR探针熔解曲线(fluorescent polymerase chain reaction probe melting curve,MeltPro)技术检测肺结核患者痰标本中结核分枝杆菌耐药性的临床应用价值。方法:收集2021年5月至2022年5月北京市疾病预防控制中心结核病门诊收治的GeneXpert MTB/RIF检测阳性的初治肺结核患者的痰标本,对每份标本同时进行涂片镜检、分枝杆菌分离培养、MeltPro技术利福平耐药性检测。分析痰标本荷菌量对MeltPro技术耐药性检测结果的影响;培养阳性菌株行利福平比例法药物敏感性试验(简称“药敏试验”),比较比例法药敏试验、GeneXpert MTB/RIF和MeltPro技术检测利福平耐药率的差异;并以培养阳性菌株利福平比例法药敏试验结果为参考标准,评价MeltPro技术耐药性检测的效能。结果:研究共收集结核分枝杆菌阳性合格痰标本158份(例),经分枝杆菌分离培养获得阳性菌株130株。MeltPro技术检测痰标本结核分枝杆菌利福平耐药的成功率为79.1%(125/158),且随着痰涂片结果等级的升高,检测成功率由“阴性”的59.3%(32/54)上升至“+++”标本的100.0%(18/18);同时,随着GeneXpert MTB/RIF检测结核分枝杆菌等级的升高,检测成功率也由“极低”的64.5%(20/31)上升至“高”的92.9%(26/28),各等级间差异均有统计学意义(H=22.271,P=0.001;H=15.637,P=0.001)。MeltPro技术的利福平耐药检出率为17.6%(22/125),GeneXpert MTB/RIF方法的利福平耐药检出率为12.7%(20/158),比例法药敏试验的利福平耐药检出率为13.1%(17/130),三者比较差异无统计学意义(χ^(2)=1.537,P=0.464)。以培养阳性菌株比例法药敏试验结果为参考标准,MeltPro技术和GeneXpert MTB/RIF方法检测利福平耐药的敏感度、特异度、阳性预测值、阴性预测值、Kappa值分别为100.0%(17/17)、94.6%(88/93)、77.3%(17/22)、100.0%(88/88)、0.845和100.0%(17/17)、97.3%(110/113)、85.0%(17/20)、100.0%(110/110)、0.906。结论:MeltPro技术检测肺结�Objective:To evaluate the application value of the fluorescence PCR probe melting curve(MeltPro)technique in the detection of rifampicin-resistance of Mycobacterium tuberculosis(MTB)in sputum samples.Methods:Sputum samples of primary treated pulmonary tuberculosis(PTB)with positive GeneXpert MTB/RIF from Beijing Center for Disease Control and Prevention between May 2021 and May 2022 were collected.These sputum samples were tested using smear,mycobacterium isolation,and rifampicin-resistance by MeltPro technique.Drug sensitivity test(DST)of rifampicin was used for culturing positive strains,the differences in the detection rate of rifampicin resistance using DST,GeneXpert MTB/RIF,and MeltPro technique were compared.The proportional DST results of rifampicin were used as a standard to evaluate the drug-resistance detection efficiency of MeltPro technique.Results:A total of 158 qualified positive MTB sputum samples were collected,after mycobacterium culture,130 strains were got.The success rate of the detection by MeltPro technique for rifampicin in sputum samples was 79.1%(125/158).As the grade of sputum smear results increased,the success rate of MeltPro technique increased from 59.3%(32/54,negative)to 100.0%(18/18,positive,grade+++);the success rate also increased with the increase of MTB grade detected by GeneXpert MTB/RIF,from 64.5%(20/31,extremely low)to 92.9%(26/28,high),the differences between each grade were statistically significant(H=22.271,P=0.001;H=15.637,P=0.001).The detection rates of rifampicin resistance using MeltPro technique,GeneXpert MTB/RIF method and proportional DST were 17.6%(22/125),12.7%(20/158)and 13.1%(17/130),respectively.There was no statistically significant difference between the three methods(χ^(2)=1.537,P=0.464).Based on the results of proportional DST,the sensitivity,specificity,positive predictive value,negative predictive value,Kappa of MeltPro technique in the detection of rifampicin were 100.0%(17/17),94.6%(88/93),77.3%(17/22),100.0%(88/88),0.845,respectively,and were 100.0%(

关 键 词：分枝杆菌 结核 聚合酶链反应 抗药性 敏感性与特异性 

分 类 号：R521[医药卫生—内科学] R446.5[医药卫生—临床医学]