结核分枝杆菌常见耐药基因突变的检测及分析

广州医学院硕士研究生学位论文结核分枝杆菌常见耐药基因突变的检测与分析Detection and evaluation of common drug-resistance gene mutations in Mycobacterium tuberculosis专业名称: 临床检验诊断学研究生: 杨辉导师: 吴爱武教授陈心春教授二0一三年三月·广州目录中文摘要~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~1 英文摘要~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~4 英文缩略词~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 7 前言~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~8 第一部分利福平耐药结核分枝杆菌rpoB 基因的检测与分析~~~~~~~~~~~~ 111.材料和方法~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 112.结果~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~163.讨论~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~184.小结~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~20 第二部分异烟肼耐药结核分枝杆菌相关耐药基因的检测与分析~~~~~~~~~~~211.材料和方法~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 212.结果~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~273.讨论~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~304.小结~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~31 第三部分HRMA技术检测结核分枝杆菌利福平与异烟肼耐药~~~~~~~~~~~~ 321.材料和方法~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 322.结果~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~363.讨论~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~424.小结~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~44 全文总结~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 45 参考文献~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 46 综述~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~51 附录~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~59 攻读学位期间取得研究成果~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ 61 致谢~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~63 学位原创性声明~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~64 学术论文知识产权声明~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~64 关于学术论文使用授权的说明~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~64结核分枝杆菌常见耐药基因突变的检测与分析研究生：杨辉导师：吴爱武教授陈心春教授中文摘要研究背景及目的结核分枝杆菌（结核菌，Mycobacterium tuberculosis，MTB)是引起结核病的病原菌，至今仍然是单一感染因素导致死亡率最多的疾病之一。

利福平和异烟肼作为抗结核病治疗的一线药物在临床应用已50余年，耐药率逐年增多，已成为不容忽视的严重问题。

目前的研究发现结核菌发生耐药的原因是抗结核药物作用相关基因发生突变导致抗结核药物与药物作用结合位点亲和力下降所致。

结核菌培养药敏实验是目前诊断结核菌耐药的金标准，但其培养时间过长、易污染等，不能给临床医师提供相应快速的药敏结果，限制了其临床应用。

本研究拟通过对临床标本中分离的利福平（rifampicin，RIF）和异烟肼（isoniazid, INH)耐药的结核菌的耐药特征进行分析，应用分子生物学方法主要包括聚合酶链反应（polymerase chain reaction, PCR）和高分辨率融解曲线分析（high resolution melting analysis, HRMA) 技术检测其引起耐药的常见基因突变情况，分析引起结核菌耐药的常见基因的突变特征及可能的耐药机制，了解HRMA技术检测结果与结核菌培养药敏结果的差异，探讨HRMA在临床推广使用的可能性。

1. 收集已知药敏结果的临床结核菌分离株60株，其中15株对利福平敏感，45株对利福平耐药，提取DNA采用PCR方法检测其rpoB基因及其其中的利福平耐药决定区（rifampin resistant determination region, RRDR)的利福平耐药相关基因,并通过T-A克隆构建质粒后进行测序，检查其突变情况。

2. 收集已知药敏结果的临床结核菌分离株60株，其中19株对异烟肼敏感，41株对异烟肼耐药，提取DNA采用PCR方法检测其异烟肼耐药相关基因kasA 基因、katG基因和inhA基因的启动子区域,并通过T-A克隆构建质粒后进行测序，检查这些基因突变情况。

3. 应用HRMA技术检测237株临床结核菌分离株rpoB、katG基因和inhA 基因启动子的突变情况，进而分析判断其耐药机制，并与结核菌临床药敏培养结果进行比较。

实验结果1.在60株结核菌临床分离株rpoB基因检测中，15株利福平敏感结核菌均未检出突变；45株利福平耐药结核菌中有41株存在突变，突变率为91.1%（41/45）。

最易发生突变的位点为531位点，突变率为51.2%（21/41),其次为526、516位点，这3个位点总突变率共占rpoB基因RRDR突变的82.9%（34/41)。

2.在60株结核菌临床分离株katG基因、kasA基因和inhA基因启动子检测中，19株异烟肼敏感株均未检出突变。

41株异烟肼耐药菌株中，katG315位点突变的发生率为70.7%（29/41），inhA基因启动子-15位点发生C-T突变，发生这种突变的菌株占异烟肼耐药株的21.95%（9/41），kasA基因突变率较低。

3. 应用HRMA检测rpoB基因和katG基因、inhA基因的启动子突变情况并与结核菌临床药敏培养结果（金标准）进行比较。

发现对于培养结果为利福平耐药的菌株，HRMA技术检测结果符合率为95.4%，特异性为97.6%，敏感性为88.9%；对于培养结果为异烟肼耐药的菌株，HRMA技术检测结果符合率为94.9%，特异性为95.9%，敏感性为92.5%。

1. rpoB基因突变是结核菌对利福平耐药的原因之一，其主要突变位点为531、526、516。

2. katG基因和inhA基因启动子突变是结核菌对异烟肼耐药的原因之一，主要突变形式为katG315（AGC-ACC）和inhA-15（C-T）。

3.高分辨率融解曲线分析技术与结核菌培养药敏结果具有较好的一致性，具有在临床推广使用的可能。

关键词结核分枝杆菌；利福平；异烟肼；耐药；高分辨率融解曲线分析Detection and evaluation of common drug-resistance gene mutations in Mycobacterium tuberculosisMaster Degree Candidate: Hui YangTutor : Prof . Aiwu WuProf. Xinchun ChenAbstractBackground and ObjectivesTuberculosis(TB) is caused by the infection of Mycobacterium tuberculosis(MTB), and it is still one of diseases with higher mortality caused by single infectious factor.. As first-line treatment drugs, such as rifampicin and isoniazid, have been used nearly 50 years in clinical application, and drug resistance rate is increasing year by year, which become a serious problem that can not be ignored. The present study show that the reason of drug resistance is the occurrence of related gene mutations in MTB, which decreased the affinity between the drug and target gene. Mycobacterium tuberculosis drug susceptibility test is the golden standard for the diagnosis of TB drug resistance, but the shortcoming of the test is long incubation time and microorganism’s pollution, and it couldn’t provide drug resistance results accurately, so it could not be used in clinical laborator widely. This study will focus on the drugs, such as rifampicin and isoniazid(INH), resistance characteristics of Mycobacterium tuberculosis strain that were isolated from clinical specimen . molecular biological methods such as polymerase chain reaction and high resolution melting curve analysis(HRMA) technique will be used to detect and analyze thecommon mutation gene in Mycobacterium tuberculosis strains , and HRMA technology will be compared with Mycobacterium tuberculosis drug susceptibility test.Methods1. To collect 60 Mycobacterium tuberculosis strains isolated from clinical samples, among which 15 strains were sensitive to rifampin, and 45 strains were resistant to rifampicin. Genome DNA was purified, after PCR amplification, gene rpoB with rifampin resistant determination region(RRDR) will be detected,, and amplicon of gene rpoB will were cloned into T-vector and sequenced to check its gene mutation.2. To collect of 60 Mycobacterium tuberculosis strains isolated from clinical samples, including 19 strains with sensitivity to isoniazid, 41 strains with resistance to isoniazid. Genome DNA was purified, after PCR amplification, gene kasA, katG and inhA gene promoter regions that are related with isoniazid resistance of Mycobacterium tuberculosis, were detected , these gene amplicon were cloned into T-vector and sequenced to check their gene mutations.3. HRMA technology was used to detect gene mutation of gene rpoB, katG and gene inhA promoter region of 237 Mycobacterium tuberculosis strains,, and the detection results of HRMA will be compared with Mycobacterium tuberculosis drug susceptibility test(golden standard).Results1 .Among 60 Mycobacterium tuberculosis strains isolated from clinical samples, rpoB gene mutation was detected. No rpoB gene mutation was detected in 15 strains with the phenotype of rifampin sensitivity; among 45 strains with the phenotype ofrifampin resistance, the gene mutation rate was 91.1% (41/45). The most frequent mutation sites located on 531, the mutation rate was 51.2% (21/41), followed by sites 526, 516, the total mutation rate of 3 sites in gene mutation of rpoB gene with RRDR were 82.9% (34/41).2 Among 60 Mycobacterium tuberculosis strains , no gene mutation was detected in 19 strains with the phenotype of isoniazid sensitivity. Among 41strains with the phenotype of isoniazid resistance, the incidence of katG315 gene mutation was 70.7% (29/41),inhA gene promoter region -15 sites appeared C-T mutations, the C-T mutation rate were 21.95% (9/41), the frequency of kasA gene mutation was low.3 To rifampin resistance Mycobacterium tuberculosis strains, the coincident rate between the golden standard results and HRMA results was 95.4%, the specificity was 97.6%, the sensitivity was 88.9%; to INH resistance strains, the coincident rate between the golden standard results and HRMA results was 94.9%, the specificity was 95.9%, the sensitivity was 92.5%.Conclusions1 .Gene mutation of rpoB gene is one of the reasons of rifampin resistance in Mycobacterium tuberculosis strains, the main mutation sites located on 531, 526, 516.2. Gene mutation of gene katG and gene inhA promoter region is one of the causes of isoniazid resistance in Mycobacterium tuberculosis strains, the mutation form were mainly katG315 (AGC-ACC) and inhA-15 (C-T).3. The detection results of drug resistance between high resolution melting curve analysis(HRMA) technology and golden standard in Mycobacterium tuberculosis strains was coincident, HRMA could be used in clinical laboratory for detecting drugs resistance of Mycobacterium tuberculosis strains .Key wordsMycobacterium tuberculosis; Rifampicin; Isoniazid; Drugs resistance; High resolution melting analysis英文缩略词中文名称英文名称缩略词结核分枝杆菌mycobacterium tuberculosis MTB 利福平 rifampicin RIF 异烟肼 isoniazid INH 高分辨率融解曲线分析 high resolution melting analysis HRMA 耐多药结核病 multidrug resistance tuberculosis MDR-TB 单核苷酸多态性 single nucleotide polymorphism SNP耐利福平决定区 rifampicin resistance determination region RRDR 聚合酶链反应polymerase chain reaction PCR前言结核病是由结核分枝杆菌（结核菌）感染所导致的疾病。