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中国防痨杂志 ›› 2020, Vol. 42 ›› Issue (9): 998-1001.doi: 10.3969/j.issn.1000-6621.2020.09.022

• 短篇论著 • 上一篇    下一篇

荧光PCR探针熔解曲线法检测结核分枝杆菌耐药性的价值

李爱芳, 崔晓利, 康磊, 雷静, 党丽云, 杨翰()   

  1. 710100 西安市胸科医院
  • 收稿日期:2020-04-21 出版日期:2020-09-10 发布日期:2020-09-18
  • 通信作者: 杨翰 E-mail:xajhyanghan@163.com

Value of fluorescence PCR probe melting curve method in detecting resistance of Mycobacterium tuberculosis

LI Ai-fang, CUI Xiao-li, KANG Lei, LEI Jing, DANG Li-yun, YANG Han()   

  1. Xi'an Chest Hospital, Xi’an 710100,China
  • Received:2020-04-21 Online:2020-09-10 Published:2020-09-18
  • Contact: YANG Han E-mail:xajhyanghan@163.com

摘要:

搜集全部2017年9月至2019年8月西安市胸科医院确诊的结核病住院患者546例作为研究对象,评价荧光聚合酶链反应(polymerase chain reaction, PCR)探针熔解曲线法(简称“熔解曲线法”)检测结核分枝杆菌(MTB)耐药性的临床应用价值。收集患者痰标本,每份标本量均不少于2ml。经菌种鉴定,最终纳入531例(株)为研究对象。每例患者采用同一标本进行BACTEC MGIT 960液体培养药物敏感性试验(简称“MGIT 960药敏试验”)和熔解曲线法耐药基因检测,对MGIT 960药敏试验和熔解曲线法检测利福平、异烟肼耐药性结果不一致者采用基因芯片法耐药基因检测对熔解曲线法结果进行验证。以MGIT 960药敏试验检测结果为参考标准,熔解曲线法检测肺结核患者痰标本MTB对链霉素耐药性的敏感度、特异度、符合率和Kappa值分别为86.67% (39/45)、99.38% (483/486)、98.31% (522/531)和0.887;检测肺结核患者痰标本MTB对异烟肼耐药性的敏感度、特异度、符合率和Kappa值分别为69.23% (54/78)、98.68% (447/453)、94.35% (501/531)和0.751;检测肺结核患者痰标本MTB对利福平耐药性的敏感度、特异度、符合率和Kappa值分别为87.50% (42/48)、96.89% (468/483)、96.05% (510/531)和0.778;检测肺结核患者痰标本MTB对乙胺丁醇耐药性的敏感度、特异度、符合率和Kappa值分别为50.00% (9/18)、98.83% (507/513)、97.18% (516/531)和0.531。在熔解曲线法检测结果为耐药而MGIT 960药敏试验为敏感的30例患者中,熔解曲线法检测结果显示:对异烟肼耐药的6例均为katG 315位密码子突变;对利福平耐药的15例中,ropB 507-512、ropB 521-528、 ropB 529-533位密码子突变分别为3例、3例、9例;对链霉素耐药的3例均为rrs 513-517位点突变;对乙胺丁醇耐药的6例均为embB 306位密码子突变。基因芯片法耐药基因检测结果显示:对异烟肼耐药的6例中katG 315 (AGC→ACC)突变和katG 315 (AGC→AAC)突变各3例;对利福平耐药的15例中,ropB基因511位CTG→CCG突变、526位CAC→TAC突变和531位TCG→TGG突变各3例、3例、9例,与熔解曲线法检测结果一致。可见,荧光PCR探针熔解曲线法对链霉素、异烟肼、利福平、乙胺丁醇耐药性突变位点检测具有较好的检测效能,可用于临床上对一线抗结核药品耐药情况的快速筛查。

关键词: 结核,抗多种药物性, 聚合酶链反应, 微生物敏感性试验, DNA探针, 微阵列分析, 对比研究, 数据说明,统计

Abstract:

A total of 546 patients with pulmonary tuberculosis who were hospitalized in Xi’an Chest Hospital between September 2017 to August 2019 were selected as subjects to evaluate the clinical application value of fluorescence PCR probe melting curve method (melting curve method) in detecting drug resistance of Mycobacterium tuberculosis (MTB). The sputum samples of the subjects with the amount larger than 2 ml were collected. After strain identification, 531 cases (strains) were finally included as the research objects. The same sputum sample of each subjects was performed drug resistance gene detection using BACTEC MGIT 960 liquid culture drug susceptibility test (960 liquid drug susceptibility test) and fluorescence PCR probe melting curve method. For subjects with inconsistent results of MGIT 960 drug sensitivity test and melting curve method for rifampicin and isoniazid resistance, the melting curve method was verified by gene chip method to detect drug resistance gene. Based on the results of MGIT 960 liquid drug susceptibility test as reference standard, the sensitivity, specificity, coincidence rate and Kappa value of the melting curve method in detecting streptomycin resistance were 86.67% (39/45), 99.38% (483/486), 98.31% (522/531) and 0.887; the sensitivity, specificity, coincidence rate and Kappa value of the melting curve method for detecting isoniazid resistance were 69.23% (54/78), 98.68% (447/453), 94.35% (501/531) and 0.751; the sensitivity, specificity, coincidence rate and Kappa value of the melting curve method for the detection of rifampicin resistance were 87.50% (42/48), 96.89% (468/483), 96.05% (510/531) and 0.778; the sensitivity, specificity, coincidence rate and Kappa value of the melting curve method for detecting ethambutol resistance were 50.00% (9/18), 98.83% (507/513), 97.18% (516/531) and 0.531. Among the 30 cases that were resistant by the melting curve method and sensitive by the MGIT 960 liquid drug sensitivity method, the melting curve method showed that the 6 cases resistant to isoniazid were katG 315 codon mutation; among the 15 cases resistant to rifampicin, the mutations at codons of ropB 507-512, ropB 521-528, and ropB 529-533 were 3 cases, 3 cases, 9 cases, respectively; the 3 cases resistant to streptomycin were rrs 513-517 codon mutation; the 6 cases resistant to ethambutol were embB 306 codon mutation. The results of gene chip detection of drug resistance genes revealed that among the 6 cases of resistance to isoniazid, there were 3 cases of katG 315 (AGC→ACC) mutation and 3 cases of katG 315 (AGC→AAC) mutation; and among the 15 cases of resistance to rifampicin, there were 3 cases of ropB 511 CTG→CCG mutation, 3 cases of ropB 526 CAC→TAC mutation and 9 cases of ropB 531 TCG→TGG mutation, which were consistent with the results of melting curve method. Fluorescence PCR probe melting curve method has good efficiency for the detection of streptomycin, isoniazid, rifampicin, ethambutol resistance mutation sites, and it can be used for rapid screening of clinical resistance to first-line anti-tuberculosis drugs.

Key words: Tuberculosis,multidrug-resistant, Polymerase chain reaction, Microbial susceptibility tests, DNA probes, Microarray analysis, Comparative study, Data interpretation,statistical