耐藥結(jié)核分枝桿菌的分子流行病學(xué)研究與耐藥相關(guān)基因的分子生物學(xué)快速檢測
發(fā)布時間：2018-11-09 16:02
【摘要】：近年來由于不規(guī)范抗結(jié)核治療導(dǎo)致耐藥菌尤其是耐多藥菌(Multidrug-resistant tuberculsis,MDR-TB)的出現(xiàn)成為抗結(jié)核治療中的難點。從基因水平闡明結(jié)核桿菌耐藥性變異的機理,并在此基礎(chǔ)上建立敏感、快速和特異的檢測方法是當(dāng)務(wù)之急。 目前研究已經(jīng)發(fā)現(xiàn)和異煙肼(isoniazid,INH)及利福平(rifampin,RFP)耐藥密切相關(guān)的是katG基因和rpoB基因。這兩個基因的突變分別可以解釋50%以上的異煙肼耐藥和95%以上的利福平耐藥。katG基因變異的特點是以S315T突變?yōu)槌Ｒ?占了絕大多數(shù),而rpoB基因的突變則集中在核心突變區(qū)。根據(jù)現(xiàn)有的資料,不同國家和地區(qū)的結(jié)核分枝桿菌基因突變特點有所不同,有必要對我國結(jié)核桿菌基因突變情況進行研究,并分析其突變特征。在此基礎(chǔ)上建立的分子生物學(xué)快速檢測耐藥的方法才符合我國實際情況,具有實際應(yīng)用價值。 本研究采用了一種較為簡單的聚合酶鏈反應(yīng)—限制性酶切片段多態(tài)性(polymerase chain reaction-restriction fragment length polymorphism,PCR-RFLP)方法對臨床耐藥結(jié)核桿菌菌株中的katG基因最為常見的S315T突變進行了篩選,并對未發(fā)現(xiàn)S315T突變的耐異煙肼結(jié)核桿菌的katG基因直接測序以研究KatG的基因變異。此外,還應(yīng)用直接測序法對rpoB基因的突變情況作了分析。除對異煙肼與利福平耐藥相關(guān)基因譜作分析外,我們還采用一種新的多位點可變數(shù)目串聯(lián)重復(fù)序列分析法(Multiple loci Variable Number of Tandem Repeats Analysis,MLVA)方法對結(jié)核分枝桿菌的基因指紋作了分析,對該方法在我國的應(yīng)用情況作了初步探討,并研究基因型與耐藥性之間的關(guān)系。在以上工作的基礎(chǔ)上,本研究第二部分建立了基因特異性多重聚合酶鏈反應(yīng)(multiplex allele specific polymerase chain reaction,MAS-PCR)以檢測臨床常見的katG基因和rpoB基因突變,用于對異煙肼和利福平耐藥菌的快速檢測。 第一部分 耐藥結(jié)核分枝桿菌的分子流行病學(xué)研究 一、結(jié)核分枝桿菌的耐藥表型與基因型的相關(guān)性研究 耐藥基因譜的研究包括對katG和rpoB基因突變的分析。方法是對429株結(jié)核桿菌行核酸抽提后首先以PCR-RFLP方法檢測有無S315T突變,對未發(fā)現(xiàn)S315T突變的異煙肼耐藥株取部分(48株)行katG全基因測序以了解其他位點的變異情況。對96株結(jié)核桿菌rpoB基因的核心突變區(qū)以直接測序法分析突變情況。結(jié)果發(fā)現(xiàn)191株INH敏感株均未發(fā)現(xiàn)315位突變,katG基因擴增陽性的407株INH耐藥株中,76.9%(166/216)存在315位絲氨酸的點突變。低度耐藥株中的S315T突變率76.9%(113/147),顯著高于高度耐藥株中的S315T突變率40.6%(28/69)。有2株異煙肼高度耐藥株中存在katG基因片段的缺失。48株異煙肼耐藥株測序結(jié)果發(fā)
[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.
【學(xué)位授予單位】：復(fù)旦大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2005
【分類號】：R181.3;R446.9
本文編號：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.
【學(xué)位授予單位】：復(fù)旦大學(xué)
【學(xué)位級別】：博士
【學(xué)位授予年份】：2005
【分類號】：R181.3;R446.9
【參考文獻】
相關(guān)期刊論文 前3條
1 朱中元,陳貽平,陳允鳳,王海波,張貴琛,邵寒霜;耐多藥結(jié)核桿菌分離林KatG及inhA基因變異的研究[J];海南醫(yī)學(xué);1999年04期
2 全國結(jié)核病流行病學(xué)抽樣調(diào)查技術(shù)指導(dǎo)組;2000年全國結(jié)核病流行病學(xué)抽樣調(diào)查報告[J];中國防癆雜志;2002年02期
3 程曉東,于文彬,別良峰,蘇明權(quán),張榮,郝曉柯;多重聚合酶鏈反應(yīng)-單鏈構(gòu)象多態(tài)性分析檢測耐異煙肼結(jié)核分枝桿菌[J];中華結(jié)核和呼吸雜志;2004年01期
，本文編號：2320888
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