耐药结核分枝杆菌的分子流行病学研究与耐药相关基因的分子生物学快速检测
发布时间：2018-11-09 16:02
【摘要】：近年来由于不规范抗结核治疗导致耐药菌尤其是耐多药菌(Multidrug-resistant tuberculsis,MDR-TB)的出现成为抗结核治疗中的难点。从基因水平阐明结核杆菌耐药性变异的机理,并在此基础上建立敏感、快速和特异的检测方法是当务之急。 目前研究已经发现和异烟肼(isoniazid,INH)及利福平(rifampin,RFP)耐药密切相关的是katG基因和rpoB基因。这两个基因的突变分别可以解释50%以上的异烟肼耐药和95%以上的利福平耐药。katG基因变异的特点是以S315T突变为常见,占了绝大多数,而rpoB基因的突变则集中在核心突变区。根据现有的资料,不同国家和地区的结核分枝杆菌基因突变特点有所不同,有必要对我国结核杆菌基因突变情况进行研究,并分析其突变特征。在此基础上建立的分子生物学快速检测耐药的方法才符合我国实际情况,具有实际应用价值。 本研究采用了一种较为简单的聚合酶链反应—限制性酶切片段多态性(polymerase chain reaction-restriction fragment length polymorphism,PCR-RFLP)方法对临床耐药结核杆菌菌株中的katG基因最为常见的S315T突变进行了筛选,并对未发现S315T突变的耐异烟肼结核杆菌的katG基因直接测序以研究KatG的基因变异。此外,还应用直接测序法对rpoB基因的突变情况作了分析。除对异烟肼与利福平耐药相关基因谱作分析外,我们还采用一种新的多位点可变数目串联重复序列分析法(Multiple loci Variable Number of Tandem Repeats Analysis,MLVA)方法对结核分枝杆菌的基因指纹作了分析,对该方法在我国的应用情况作了初步探讨,并研究基因型与耐药性之间的关系。在以上工作的基础上,本研究第二部分建立了基因特异性多重聚合酶链反应(multiplex allele specific polymerase chain reaction,MAS-PCR)以检测临床常见的katG基因和rpoB基因突变,用于对异烟肼和利福平耐药菌的快速检测。 第一部分 耐药结核分枝杆菌的分子流行病学研究 一、结核分枝杆菌的耐药表型与基因型的相关性研究 耐药基因谱的研究包括对katG和rpoB基因突变的分析。方法是对429株结核杆菌行核酸抽提后首先以PCR-RFLP方法检测有无S315T突变,对未发现S315T突变的异烟肼耐药株取部分(48株)行katG全基因测序以了解其他位点的变异情况。对96株结核杆菌rpoB基因的核心突变区以直接测序法分析突变情况。结果发现191株INH敏感株均未发现315位突变,katG基因扩增阳性的407株INH耐药株中,76.9%(166/216)存在315位丝氨酸的点突变。低度耐药株中的S315T突变率76.9%(113/147),显著高于高度耐药株中的S315T突变率40.6%(28/69)。有2株异烟肼高度耐药株中存在katG基因片段的缺失。48株异烟肼耐药株测序结果发
[Abstract]:In recent years, the emergence of drug-resistant bacteria, especially multi-drug resistant bacteria (Multidrug-resistant tuberculsis,MDR-TB), has become a difficult point in anti-tuberculosis treatment due to nonstandard anti-tuberculosis therapy. It is urgent to elucidate the mechanism of drug resistance variation of Mycobacterium tuberculosis at gene level and to establish a sensitive, rapid and specific detection method. So far, it has been found that isoniazid (isoniazid,INH) and rifampicin (rifampin,RFP) resistance are closely related to katG gene and rpoB gene. More than 50% of isoniazid resistance and 95% of rifampicin resistance can be explained by the mutation of these two genes respectively. The characteristic of katG gene mutation is that S315T mutation is the most common mutation, while the mutation of rpoB gene is concentrated in the core mutation region. According to the available data, the mutation characteristics of Mycobacterium tuberculosis gene in different countries and regions are different. It is necessary to study the mutation of Mycobacterium tuberculosis gene in China and analyze its mutation characteristics. On this basis, the method of rapid detection of drug resistance by molecular biology is in line with the actual situation in China and has practical application value. In this study, a simple polymerase chain reaction-restriction fragment polymorphism (polymerase chain reaction-restriction fragment length polymorphism,PCR-RFLP) method was used to screen the most common S315T mutation of katG gene in drug-resistant Mycobacterium tuberculosis strains. The katG gene of isoniazid resistant Mycobacterium tuberculosis with no S315T mutation was sequenced to study the variation of KatG gene. In addition, the mutation of rpoB gene was analyzed by direct sequencing. In addition to the analysis of the gene profiles of isoniazid and rifampicin resistance, a new multilocus variable number tandem repeat (Multiple loci Variable Number of Tandem Repeats Analysis,MLVA) method was used to analyze the gene fingerprints of Mycobacterium tuberculosis. The application of this method in China was discussed, and the relationship between genotypes and drug resistance was studied. On the basis of the above work, the second part of this study established a gene specific multiplex polymerase chain reaction (multiplex allele specific polymerase chain reaction,MAS-PCR) to detect common clinical mutations of katG gene and rpoB gene. For rapid detection of isoniazid and rifampicin resistant bacteria. Part I Molecular Epidemiology of Mycobacterium tuberculosis Correlation between phenotype and genotypes of Mycobacterium tuberculosis the analysis of katG and rpoB gene mutations is included in the study of drug resistance gene profile. Methods after nucleic acid extraction of 429 strains of Mycobacterium tuberculosis, the S315T mutation was detected by PCR-RFLP method, and the partial (48 strains) of isoniazid resistant strains with no S315T mutation were sequenced to find out the variation of other loci. The core mutations of rpoB gene of 96 strains of Mycobacterium tuberculosis were analyzed by direct sequencing. The results showed that none of the 191 INH susceptible strains was found to have a 315-position mutation, and 76.9% (166 / 216) of 407 INH resistant strains that were amplified by katG gene had a point mutation of 315 serine. The mutation rate of S315T in low resistant strains was 76.9% (113 / 147), which was significantly higher than that in high resistant strains (40.6%, 28 / 69). There were two isoniazid highly resistant strains with the deletion of katG gene fragment. 48 isoniazid resistant strains were sequenced.
【学位授予单位】：复旦大学
【学位级别】：博士
【学位授予年份】：2005
【分类号】：R181.3;R446.9
本文编号：2320888
[Abstract]:In recent years, the emergence of drug-resistant bacteria, especially multi-drug resistant bacteria (Multidrug-resistant tuberculsis,MDR-TB), has become a difficult point in anti-tuberculosis treatment due to nonstandard anti-tuberculosis therapy. It is urgent to elucidate the mechanism of drug resistance variation of Mycobacterium tuberculosis at gene level and to establish a sensitive, rapid and specific detection method. So far, it has been found that isoniazid (isoniazid,INH) and rifampicin (rifampin,RFP) resistance are closely related to katG gene and rpoB gene. More than 50% of isoniazid resistance and 95% of rifampicin resistance can be explained by the mutation of these two genes respectively. The characteristic of katG gene mutation is that S315T mutation is the most common mutation, while the mutation of rpoB gene is concentrated in the core mutation region. According to the available data, the mutation characteristics of Mycobacterium tuberculosis gene in different countries and regions are different. It is necessary to study the mutation of Mycobacterium tuberculosis gene in China and analyze its mutation characteristics. On this basis, the method of rapid detection of drug resistance by molecular biology is in line with the actual situation in China and has practical application value. In this study, a simple polymerase chain reaction-restriction fragment polymorphism (polymerase chain reaction-restriction fragment length polymorphism,PCR-RFLP) method was used to screen the most common S315T mutation of katG gene in drug-resistant Mycobacterium tuberculosis strains. The katG gene of isoniazid resistant Mycobacterium tuberculosis with no S315T mutation was sequenced to study the variation of KatG gene. In addition, the mutation of rpoB gene was analyzed by direct sequencing. In addition to the analysis of the gene profiles of isoniazid and rifampicin resistance, a new multilocus variable number tandem repeat (Multiple loci Variable Number of Tandem Repeats Analysis,MLVA) method was used to analyze the gene fingerprints of Mycobacterium tuberculosis. The application of this method in China was discussed, and the relationship between genotypes and drug resistance was studied. On the basis of the above work, the second part of this study established a gene specific multiplex polymerase chain reaction (multiplex allele specific polymerase chain reaction,MAS-PCR) to detect common clinical mutations of katG gene and rpoB gene. For rapid detection of isoniazid and rifampicin resistant bacteria. Part I Molecular Epidemiology of Mycobacterium tuberculosis Correlation between phenotype and genotypes of Mycobacterium tuberculosis the analysis of katG and rpoB gene mutations is included in the study of drug resistance gene profile. Methods after nucleic acid extraction of 429 strains of Mycobacterium tuberculosis, the S315T mutation was detected by PCR-RFLP method, and the partial (48 strains) of isoniazid resistant strains with no S315T mutation were sequenced to find out the variation of other loci. The core mutations of rpoB gene of 96 strains of Mycobacterium tuberculosis were analyzed by direct sequencing. The results showed that none of the 191 INH susceptible strains was found to have a 315-position mutation, and 76.9% (166 / 216) of 407 INH resistant strains that were amplified by katG gene had a point mutation of 315 serine. The mutation rate of S315T in low resistant strains was 76.9% (113 / 147), which was significantly higher than that in high resistant strains (40.6%, 28 / 69). There were two isoniazid highly resistant strains with the deletion of katG gene fragment. 48 isoniazid resistant strains were sequenced.
【学位授予单位】：复旦大学
【学位级别】：博士
【学位授予年份】：2005
【分类号】：R181.3;R446.9
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