荧光PCR探针熔解曲线技术检测痰标本中结核分枝杆菌异烟肼耐药性的研究  

Clinical application of fluorescence polymerase chain reaction probe melting curve technique in detecting isoniazid drug-resistance of Mycobacterium tuberculosis in sputum samples

作　　者：王嫩寒 赵琰枫 田丽丽[1] 陈双双 陈昊 任怡宣 李传友 代小伟 WANG Nenhan;ZHAO Yanfeng;TIAN Lili;CHEN Shuangshuang;CHEN Hao;REN Yixuan;LI Chuanyou;DAI Xiaowei(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：《实用医学杂志》2023年第20期2664-2670,共7页The Journal of Practical Medicine

基　　金：北京市疾病预防控制中心自管课题(编号:JKS2021-06)。

摘　　要：目的评价荧光PCR探针熔解曲线(fluorescence polymerase chain reaction probe melting curve,Melt Pro)技术检测痰标本结核分枝杆菌(Mycobacterium tuberculosis,MTB)异烟肼耐药性的应用价值。方法收集2021年5月至2022年5月在北京市疾病预防控制中心结核病门诊Gene Xpert MTB/RIF检测为MTB的(简称MTB阳性)患者痰标本,对每份标本同时进行涂片镜检、分枝杆菌分离培养、Melt Pro技术和线性探针技术(Geno Type MTBDRplus,简称Hain试验)异烟肼耐药检测。分析Melt Pro技术检测痰标本MTB异烟肼的耐药性,痰标本荷菌量对检测结果的影响;以培养阳性菌株异烟肼比例法药敏结果为参考标准,评价Melt Pro技术耐药检测效能。结果收集MTB阳性的合格痰标本157例,经分枝杆菌分离培养获得阳性菌株130株。Melt Pro技术痰标本MTB异烟肼耐药性检测成功率为84.1%(132/157)。不同痰涂片结果等级间,Melt Pro技术检测异烟肼耐药成功的比例差异存在有统计学意义(H=24.169,P=0.000),随痰涂片结果等级的升高,检测成功率由“阴性”标本的64.8%(35/54)升高到“3+”“4+”标本的100%(17/17,6/6);该比例也随Gene Xpert MTB/RIF检测MTB等级的升高而升高,由“极低”标本的54.8%(17/31),到“高”标本的96.4%(27/28),各等级间差异有统计学意义(H=27.763,P=0.000)。Melt Pro技术、Hain试验和药敏试验的异烟肼耐药检出率分别为22.0%(29/132),15.3%(19/124),和14.6%(19/130),三者差异无统计学意义(χ^(2)=2.997,P=0.223)。以培养阳性菌株比例法药敏试验结果为参考标准,Melt Pro技术检测异烟肼耐药的灵敏度、特异度、阳性预测值、阴性预测值分别为:84.6%(11/13)、91.2%(93/102)、55%(11/20)、97.9%(93/95),Kappa值为0.614;Hain试验检测异烟肼耐药的灵敏度、特异度、阳性预测值、阴性预测值分别为:55.6%(10/18)、92.3%(84/91)、58.8%(10/17)、91.3%(84/92),Kappa值为0.490。结论Melt Pro技术可直接检测痰标本MTB耐药性,快速�Objective To evaluate the application value of the fluorescence PCR probe melting curve(MeltPro)technique in the detection of isoniazid drug-resistance of Mycobacterium tuberculosis(MTB)in sputum samples.Methods In this study,we collected sputum samples of PTB that were identified as MTB by GeneXpert MTB/RIF method(MTB positive)from 2021 to 2022.These sputum samples were detected including smear,Mycobacterium culture,GenoType MTBDRplus,and isoniazid drug-resistance test by MeltPro technique.We did the proportional drug sensitivity test(DST)of isoniazid for culturing positive strains,analyzed the influence of the amount of bacteria in sputum samples on the results of MeltPro technique,compared the difference in the detection rate of isoniazid resistance by proportional DST,GenoType MTBDRplus,and MeltPro technique;The proportional DST results of isoniazid were used as a standard to evaluate the isoniazid drug-resistance detection efficiency of MeltPro technique and GenoType MTBDRplus.Results 157 qualified MTB positive sputum samples were collected,after Mycobacterium culture,we got 130 strains.The rate of success for isoniazid in sputum samples detected by MeltPro technique was84.1%(132/157).Among different grades of sputum smear results,there is a difference in the successful rate of MeltPro technique(H=24.169,P=0.000),increased with the grade of sputum smear results,the successful rate was increased from64.8%(32/54,the negative sputum)to 100.0%(17/17、6/6,the positive sputum grade 3+、4+);the successful rate also increased with the increase of MTB grade detected by GeneXpert MTB/RIF,from54.8%(17/31,the very low sputum)to 96.4%(27/28,the high sputum)and the difference between each grade was statistically significant(H=27.763,P=0.000).The detection rate of isoniazid resistance using MeltPro technique,GenoType MTBDRplus and proportional DST were 22.0%(29/132),15.3%(19/124),和14.6%(19/130),respectively.There was no statistically significant difference between the three methods(χ^(2)=2.997,P=0.223).Based on the re

关 键 词：分枝杆菌 结核 荧光PCR探针熔解曲线 耐药性检测 痰 

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