实时PCR在结核分枝杆菌异烟肼耐药突变分子诊断中的应用研究
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摘要
结核病是全球范围内危害最为严重的慢性传染病之一,在众多结核治疗药物中,异烟肼作为一线药物广泛使用,导致耐异烟肼结核发生率高,危害面广。本论文着重建立异烟肼耐药突变检测体系,并评价其应用,为最终建立起适合临床应用的异烟肼耐药突变检测、指导其正确使用提供合适手段。论文内容包括：耐药结核病的流行和诊治现状评述(第一章)、实时PCR探针熔解曲线法检测异烟肼耐药突变体系的建立和分析性能评价(第二章)、上述体系的多中心临床评价(第三章)、应用上述技术进行的异烟肼耐药突变流行病学调查(第四章)以及针对最常见katG S315T突变所建立的一种稀有突变定量体系(第五章),分述如下：
第一章绪论
首先概述了结核病的流行现状、临床诊断和治疗方案,描述了结核菌的生物学背景,针对性地阐述了异烟肼的作用原理和结核异烟肼耐药机制,总结了结核耐药检测尤其是耐异烟肼检测的现状及最新进展,并介绍了本实验室新近建立的探针熔解曲线分析(PMA)技术检测突变的原理,最后提出了本论文的目的、内容及意义。
第二章异烟肼耐药突变PMA检测体系的建立和性能评价
已有研究证实的异烟肼耐药突变相关区域包括katG315密码子、inhA启动子区、ahpC启动子区和inhA94密码子等四个区域,据此,我们建立了双管双色PMA检测体系,采用自行建立的核酸提取方法,按照优化的反应条件,该体系的检测限为3拷贝/反应。所有突变位点的熔点值(Tm)与对应的野生型相差在2℃以上,均可实现有效检出,标准盘的检测特异性达100%。检测5种常见突变的Tm值标准偏差SD<1℃(n=6),最低不均一耐药检测水平为30%(熔解速率设为1℃/步)。对中国食品药品检定研究院的非结核分枝杆菌(NTM)的检测结果表明,46株NTM在四个检测通道均未出现非特异信号。该体系可在三种不同机型的仪器上使用,结果无显著差异。
第三章PMA体系的多中心验证
利用北京、河南和深圳三家医院提供的1096株结核临床分离株,对PMA异烟肼耐药突变检测体系进行了多中心双盲验证。对照方法为比例法药敏试验,验证方法采用测序法。结果表明,与比例法药敏试验体系相比,PMA的临床灵敏度为90.8%(397/437),临床特异性为96.4%(635/659)。测序结果表明,PMA检出的所有突变型均发生了突变(涉及397株耐药株和24株敏感株)；在40株PMA检测无突变的异烟肼耐药株中,测序结果表明,4株为不均一耐药突变,5株发生了PMA检测区外突变,而另外31株均未检测到突变。测序结果表明,在异烟肼耐药株中,PMA共检测出23种突变类型。最常见的四种突变是katG S315T AGC→ACC、inhA启动子-15C→T、katG S315N AGC→AAC和ahpC启动子-10C→T突变,占异烟肼耐药株的87.4%(382/437),未检出inhA94位点突变。PMA检出76.5%(13/17)的不均一耐药株。因此,PMA与培养法药敏试验一致性好,结果准确,适合临床应用。
第四章厦门和漳州两地异烟肼耐药的流行病学分析
2006年6月至2009年12月,从厦门和漳州地区收集785份TB临床分离株,2008年10月至2009年12月收集146份涂阳痰标本,利用PMA对上述标本进行异烟肼耐药突变检测,突变阳性的标本测序验证以确定具体突变类型。对最终纳入分析的833例病人的910份标本的检测结果表明,厦门和漳州两地的异烟肼耐药突变率合计为19.4%(162/833),与我国18.96%的异烟肼耐药率接近,而高于全球13.9%的异烟肼耐药率；在所检出的14种异烟肼耐药突变中,katG S315T (AGC→ACC)突变占48%(77/162,含72份单独突变和5份合并突变),inhA启动子-15C→T突变占30%(48/162,含41份单独突变和7份合并突变),katG S315N (AGC→AAC)突变占8%(13/162,含11份单独突变和2份合并突变),三种类型突变合计占85%(138/162),是典型的优势突变,与国内外报道一致。其中排在第一位的katG S315T突变与高水平耐药、耐多药和广泛耐药密切相关,提示厦漳两地有9.2%(77/833)患者可能需要采用二线药物治疗,而处于第二位的inhA启动子-15C→T突变与低水平耐异烟肼相关,且同时耐受乙硫异烟胺和丙硫异烟胺,这部分患者(5.8%,48/833)可采用高剂量异烟肼治疗,但不适用含二线药乙硫异烟胺和丙硫异烟胺的治疗方案。因此,上述流行病学研究结果不仅有助于了解本地结核耐药情况,以便采取相应的防控措施,而且还为临床医生的合理用药提供了参考依据。
第五章实时PAP定量检测结核异烟肼耐药突变
实时追踪耐药突变的发生与变化对于指导临床治疗无疑具有重要意义,耐药突变的发生起初含量极低,而其不断累积的过程又必须通过定量检测才能实现,为此,需要发展一种能够选择性定量检测突变的技术。焦磷酸水解激活的聚合反应(Pyrophosphorolysis-activated polymerization, PAP)是一种特异检测稀有突变的方法。我们借鉴实时PCR模式,将其发展为实时PAP,用之于定量检测耐异烟肼TB中最常见的katG S315T (AGC→ACC)突变。利用染料法,分别建立了野生型和突变型两个实时PAP检测体系。采用质粒模板,两个实时PAP体系的检测限均为10拷贝／反应,特异性分别为5×105拷贝/反应和5×104拷贝／反应,选择性均为105：1。对71份临床涂阳痰标本进行实时PAP检测,并以测序和实时PCR检测为对照。结果表明,其中的64份标本,3种方法检测结果完全一致。在7份不一致的标本中,6份的实时PAP检出结果是存在不同含量的突变,即发生不同程度的不均一耐药(0.07%-94.2%),其中5份被实时PCR检出,2份突变含量高的标本被测序检出。另一份实时PAP和实时PCR均未检出突变的标本,测序结果表明突变类型为katG S315N (AGC→AAC),非上述两种方法的检测对象。上述结果证实,实时PAP具有检测低水平突变的能力,且结果准确,并可定量检测突变的多少,因此,有望用于追踪耐药突变的发生、发展过程,对监测耐药的发生、及时调整治疗方案有重要意义。
The high prevalence rate of drug-resistant Mycobacterium tuberculosis (MTB) is the major challenge in control and prevention of tuberculosis. As one of the mostly often used first-line drug for TB treatment, isoniazide (INH)-resistant TB has high morbidity and mortality. The aim of this thesis is to establish new systems for detection of the INH-resistant mutations and explore their applications in clinical settings. The thesis is composed of five parts:a review on drug-resistant TB, its diagnosis and treatment (Chapter1), establishment of a real-time PCR-based melting curve analysis method for detection of INH-resistant mutations (Chapter2), a multicenter validation study on the newly established method (Chapter3), an epidemiology study on INH-resistant TB in two local cities (Chapter4) and finally, development of a quantitative method for the katG S315T mutation (Chapter5).
Chapter1. Introduction
Firstly, the epidemiology of TB and drug-resistant TB were described, the biological background of tubercle bacillus and TB diagnostic methods were reviewed. Secondly, the mechanisms of INH action and resistance were discussed. Existing methods and their future trend in the detection of drug-resistant mutations were described. The working principle of probe melting analysis (PMA) and its use in mutation detection was illustrated. Finally, the aim, contents and meaningfulness of this thesis were given.
Chapter2. Establishment and evaluation of PMA for INH-resistant mutation detection.
INH resistance associated genes are katG315, inhA promoter, ahpC promoter and inhA94. A2-tube,2-color PMA was established targeting these four gene loci. By using our own extraction method and under the optimal experimental conditions, the limit of detection of the assay was3genome copies per reaction. The melting temperature (Tm) between wild type and the mutant types were larger than2℃and the SD were smaller than1℃(n=6). Consequently, all mutations could be efficiently detected regardless of the loci. The specificity was100%in the detection of reference panel and the detection ability for the heteroresistance was30%. The results with a panel of non-tuberculosis mycobacteria (NTM) from NIFDC showed that none NTM could display signal in all the four detection windows. The established assay could be used on three different modes of machines and the results showed no significant difference.
Chapter3. A multicenter validation of the PMA method
Clinical isolates (1096) collected from hospitals of Beijing, Henan and Shenzhen were used to validate the above described PMA in a double blind format. The comparison method was proportional method for drug susceptibility testing (culture method) and confirmation method was sequencing. The results showed that the clinical sensitivity of PMA was90.8%(397/437) and the specificity was96.4%(635/659) according to the culture method. The sequencing results confirmed the rightness of all the mutations detected by PMA including397INH-resistant isolates and24susceptible isolates. Forty INH-resistant isolates were detected as wild type by PMA. Sequencing analysis displayed that4of them were heteroresistance,5had mutations uncovered by PMA and31had no mutation in the four loci. Of the23types of mutation detected by PMA and identified by sequencing in INH-resistant isolates, the most common mutation types were katG S315T AGC→ACC, inhA promoter-15C→T, katG S315N AGC→AAC, and ahpC promoter-10C→T which accounted for87.4%(382/437) in INH-resistant isolates. No mutations were detected in inhA94loci. PMA successfully detected76.5%(13/17) heteroresistant samples. In conclusion, PMA could accurately detect the mutations, had good concordance with the culture method, and therefore would be suitable for clinical use.
Chapter4. Epidemiological study on INH-resistant TB in Xiamen and Zhangzhou
In this study,785clinical isolates were collected in Xiamen and Zhangzhou from June2006to December2009and146smear positive sputum samples were collected in Xiamen from October2008and December2009. All the samples were subjected to PMA for detection of INH-resistant mutations. The mutant samples were confirmed by sequencing analysis. Totally,910valid samples from833patients were successfully analyzed. The results showed that the overall mutation rate in the population was19.4%(162/833), which was close to the mutation rate of18.9%in China and higher than that of13.9%worldwide. Of the14mutation types detected, katG S315T (AGC→ACC) accounted for48%(77/162,72patients had single mutation while5patients had compound mutations), inhA promoter-15C→T accounted for30%(48/162,41patients had single mutation while7patients had compound mutations), and katG S315N (AGC→AAC) accounted for8%(13/162,11patients had single mutation and2patients had compound mutations), and altogether they contributed to85%(138/162) and were typically predominant mutations, which was in concordance with both domestic and international report. The katG S315T (AGC→ACC) was reported to be associated with high-level drug resistance, multidrug resistance and extensively drug resistance, indicating that9.2%(77/833) of the patients might need treatment with second line drugs. inhA promoter-15C→T was associated with low-level drug resistance, this portion of patients (5.8%,48/833) could be treated with high dose INH but not with either ethionamide or protionamide. Consequently, this epidemiological study was helpful not only for understanding the situation of drug-resistant TB in these regions but also for choosing suitable treatment strategy for the patients and, in the end, benefiting prevention of drug-resistant TB.
Chapter5. Quantification of mutations in INH-resistant M.tuberculosis by real-time PAP
Real-time tracking of the occurrence and accumulation of drug-resistant mutation in TB was valuable in guiding treatment. In the early beginning the mutant occurs at extremely low abundance, and the process of continuous accumulation can be only followed by a quantitative measurement. For this purpose, we developed a method that could selectively quantify rare mutations. Pyrophosphorolysis-activated polymerization (PAP) is an extremely specific method for rare mutation detection. By borrowing the concept of real-time PCR detection, we developed a real-time PAP method to quantify katG S315T (AGC→ACC) mutation, which is the most common mutation in INH-resistant MTB. Two real-time PAP systems were established for both the wild type and the mutant by using the fluorogenic dye. With plasmid DNA, the limit of detection of the two systems was10copies per reaction, specificity was5×105and5×104copies, respectively, and the selectivity was1:105. The real-time PAP systems were used to analyze71smear-positive sputum samples, and the results were compared with both sequencing and a probe-based real-time PCR method. The results showed that fully concordant results were obtained in64samples with three methods. Of the7discrepant samples,6samples were found to have mixed wild-type and mutant MTB of different level (0.07%-94.2%), and5of them were detected as mutant by real-time PCR and2were detected as mutant by sequencing. One sample missed by both real-time PAP and real-time PCR was found to harbor a different mutation, i.e., katG S315N (AGC→AAC). The above results demonstrated that real-time PAP could detect and quantify rare mutation, and thus has great potential to track the process of the mutation accumulation and help to choose an optimal treatment strategy.
第一章绪论
首先概述了结核病的流行现状、临床诊断和治疗方案,描述了结核菌的生物学背景,针对性地阐述了异烟肼的作用原理和结核异烟肼耐药机制,总结了结核耐药检测尤其是耐异烟肼检测的现状及最新进展,并介绍了本实验室新近建立的探针熔解曲线分析(PMA)技术检测突变的原理,最后提出了本论文的目的、内容及意义。
第二章异烟肼耐药突变PMA检测体系的建立和性能评价
已有研究证实的异烟肼耐药突变相关区域包括katG315密码子、inhA启动子区、ahpC启动子区和inhA94密码子等四个区域,据此,我们建立了双管双色PMA检测体系,采用自行建立的核酸提取方法,按照优化的反应条件,该体系的检测限为3拷贝/反应。所有突变位点的熔点值(Tm)与对应的野生型相差在2℃以上,均可实现有效检出,标准盘的检测特异性达100%。检测5种常见突变的Tm值标准偏差SD<1℃(n=6),最低不均一耐药检测水平为30%(熔解速率设为1℃/步)。对中国食品药品检定研究院的非结核分枝杆菌(NTM)的检测结果表明,46株NTM在四个检测通道均未出现非特异信号。该体系可在三种不同机型的仪器上使用,结果无显著差异。
第三章PMA体系的多中心验证
利用北京、河南和深圳三家医院提供的1096株结核临床分离株,对PMA异烟肼耐药突变检测体系进行了多中心双盲验证。对照方法为比例法药敏试验,验证方法采用测序法。结果表明,与比例法药敏试验体系相比,PMA的临床灵敏度为90.8%(397/437),临床特异性为96.4%(635/659)。测序结果表明,PMA检出的所有突变型均发生了突变(涉及397株耐药株和24株敏感株)；在40株PMA检测无突变的异烟肼耐药株中,测序结果表明,4株为不均一耐药突变,5株发生了PMA检测区外突变,而另外31株均未检测到突变。测序结果表明,在异烟肼耐药株中,PMA共检测出23种突变类型。最常见的四种突变是katG S315T AGC→ACC、inhA启动子-15C→T、katG S315N AGC→AAC和ahpC启动子-10C→T突变,占异烟肼耐药株的87.4%(382/437),未检出inhA94位点突变。PMA检出76.5%(13/17)的不均一耐药株。因此,PMA与培养法药敏试验一致性好,结果准确,适合临床应用。
第四章厦门和漳州两地异烟肼耐药的流行病学分析
2006年6月至2009年12月,从厦门和漳州地区收集785份TB临床分离株,2008年10月至2009年12月收集146份涂阳痰标本,利用PMA对上述标本进行异烟肼耐药突变检测,突变阳性的标本测序验证以确定具体突变类型。对最终纳入分析的833例病人的910份标本的检测结果表明,厦门和漳州两地的异烟肼耐药突变率合计为19.4%(162/833),与我国18.96%的异烟肼耐药率接近,而高于全球13.9%的异烟肼耐药率；在所检出的14种异烟肼耐药突变中,katG S315T (AGC→ACC)突变占48%(77/162,含72份单独突变和5份合并突变),inhA启动子-15C→T突变占30%(48/162,含41份单独突变和7份合并突变),katG S315N (AGC→AAC)突变占8%(13/162,含11份单独突变和2份合并突变),三种类型突变合计占85%(138/162),是典型的优势突变,与国内外报道一致。其中排在第一位的katG S315T突变与高水平耐药、耐多药和广泛耐药密切相关,提示厦漳两地有9.2%(77/833)患者可能需要采用二线药物治疗,而处于第二位的inhA启动子-15C→T突变与低水平耐异烟肼相关,且同时耐受乙硫异烟胺和丙硫异烟胺,这部分患者(5.8%,48/833)可采用高剂量异烟肼治疗,但不适用含二线药乙硫异烟胺和丙硫异烟胺的治疗方案。因此,上述流行病学研究结果不仅有助于了解本地结核耐药情况,以便采取相应的防控措施,而且还为临床医生的合理用药提供了参考依据。
第五章实时PAP定量检测结核异烟肼耐药突变
实时追踪耐药突变的发生与变化对于指导临床治疗无疑具有重要意义,耐药突变的发生起初含量极低,而其不断累积的过程又必须通过定量检测才能实现,为此,需要发展一种能够选择性定量检测突变的技术。焦磷酸水解激活的聚合反应(Pyrophosphorolysis-activated polymerization, PAP)是一种特异检测稀有突变的方法。我们借鉴实时PCR模式,将其发展为实时PAP,用之于定量检测耐异烟肼TB中最常见的katG S315T (AGC→ACC)突变。利用染料法,分别建立了野生型和突变型两个实时PAP检测体系。采用质粒模板,两个实时PAP体系的检测限均为10拷贝／反应,特异性分别为5×105拷贝/反应和5×104拷贝／反应,选择性均为105：1。对71份临床涂阳痰标本进行实时PAP检测,并以测序和实时PCR检测为对照。结果表明,其中的64份标本,3种方法检测结果完全一致。在7份不一致的标本中,6份的实时PAP检出结果是存在不同含量的突变,即发生不同程度的不均一耐药(0.07%-94.2%),其中5份被实时PCR检出,2份突变含量高的标本被测序检出。另一份实时PAP和实时PCR均未检出突变的标本,测序结果表明突变类型为katG S315N (AGC→AAC),非上述两种方法的检测对象。上述结果证实,实时PAP具有检测低水平突变的能力,且结果准确,并可定量检测突变的多少,因此,有望用于追踪耐药突变的发生、发展过程,对监测耐药的发生、及时调整治疗方案有重要意义。
The high prevalence rate of drug-resistant Mycobacterium tuberculosis (MTB) is the major challenge in control and prevention of tuberculosis. As one of the mostly often used first-line drug for TB treatment, isoniazide (INH)-resistant TB has high morbidity and mortality. The aim of this thesis is to establish new systems for detection of the INH-resistant mutations and explore their applications in clinical settings. The thesis is composed of five parts:a review on drug-resistant TB, its diagnosis and treatment (Chapter1), establishment of a real-time PCR-based melting curve analysis method for detection of INH-resistant mutations (Chapter2), a multicenter validation study on the newly established method (Chapter3), an epidemiology study on INH-resistant TB in two local cities (Chapter4) and finally, development of a quantitative method for the katG S315T mutation (Chapter5).
Chapter1. Introduction
Firstly, the epidemiology of TB and drug-resistant TB were described, the biological background of tubercle bacillus and TB diagnostic methods were reviewed. Secondly, the mechanisms of INH action and resistance were discussed. Existing methods and their future trend in the detection of drug-resistant mutations were described. The working principle of probe melting analysis (PMA) and its use in mutation detection was illustrated. Finally, the aim, contents and meaningfulness of this thesis were given.
Chapter2. Establishment and evaluation of PMA for INH-resistant mutation detection.
INH resistance associated genes are katG315, inhA promoter, ahpC promoter and inhA94. A2-tube,2-color PMA was established targeting these four gene loci. By using our own extraction method and under the optimal experimental conditions, the limit of detection of the assay was3genome copies per reaction. The melting temperature (Tm) between wild type and the mutant types were larger than2℃and the SD were smaller than1℃(n=6). Consequently, all mutations could be efficiently detected regardless of the loci. The specificity was100%in the detection of reference panel and the detection ability for the heteroresistance was30%. The results with a panel of non-tuberculosis mycobacteria (NTM) from NIFDC showed that none NTM could display signal in all the four detection windows. The established assay could be used on three different modes of machines and the results showed no significant difference.
Chapter3. A multicenter validation of the PMA method
Clinical isolates (1096) collected from hospitals of Beijing, Henan and Shenzhen were used to validate the above described PMA in a double blind format. The comparison method was proportional method for drug susceptibility testing (culture method) and confirmation method was sequencing. The results showed that the clinical sensitivity of PMA was90.8%(397/437) and the specificity was96.4%(635/659) according to the culture method. The sequencing results confirmed the rightness of all the mutations detected by PMA including397INH-resistant isolates and24susceptible isolates. Forty INH-resistant isolates were detected as wild type by PMA. Sequencing analysis displayed that4of them were heteroresistance,5had mutations uncovered by PMA and31had no mutation in the four loci. Of the23types of mutation detected by PMA and identified by sequencing in INH-resistant isolates, the most common mutation types were katG S315T AGC→ACC, inhA promoter-15C→T, katG S315N AGC→AAC, and ahpC promoter-10C→T which accounted for87.4%(382/437) in INH-resistant isolates. No mutations were detected in inhA94loci. PMA successfully detected76.5%(13/17) heteroresistant samples. In conclusion, PMA could accurately detect the mutations, had good concordance with the culture method, and therefore would be suitable for clinical use.
Chapter4. Epidemiological study on INH-resistant TB in Xiamen and Zhangzhou
In this study,785clinical isolates were collected in Xiamen and Zhangzhou from June2006to December2009and146smear positive sputum samples were collected in Xiamen from October2008and December2009. All the samples were subjected to PMA for detection of INH-resistant mutations. The mutant samples were confirmed by sequencing analysis. Totally,910valid samples from833patients were successfully analyzed. The results showed that the overall mutation rate in the population was19.4%(162/833), which was close to the mutation rate of18.9%in China and higher than that of13.9%worldwide. Of the14mutation types detected, katG S315T (AGC→ACC) accounted for48%(77/162,72patients had single mutation while5patients had compound mutations), inhA promoter-15C→T accounted for30%(48/162,41patients had single mutation while7patients had compound mutations), and katG S315N (AGC→AAC) accounted for8%(13/162,11patients had single mutation and2patients had compound mutations), and altogether they contributed to85%(138/162) and were typically predominant mutations, which was in concordance with both domestic and international report. The katG S315T (AGC→ACC) was reported to be associated with high-level drug resistance, multidrug resistance and extensively drug resistance, indicating that9.2%(77/833) of the patients might need treatment with second line drugs. inhA promoter-15C→T was associated with low-level drug resistance, this portion of patients (5.8%,48/833) could be treated with high dose INH but not with either ethionamide or protionamide. Consequently, this epidemiological study was helpful not only for understanding the situation of drug-resistant TB in these regions but also for choosing suitable treatment strategy for the patients and, in the end, benefiting prevention of drug-resistant TB.
Chapter5. Quantification of mutations in INH-resistant M.tuberculosis by real-time PAP
Real-time tracking of the occurrence and accumulation of drug-resistant mutation in TB was valuable in guiding treatment. In the early beginning the mutant occurs at extremely low abundance, and the process of continuous accumulation can be only followed by a quantitative measurement. For this purpose, we developed a method that could selectively quantify rare mutations. Pyrophosphorolysis-activated polymerization (PAP) is an extremely specific method for rare mutation detection. By borrowing the concept of real-time PCR detection, we developed a real-time PAP method to quantify katG S315T (AGC→ACC) mutation, which is the most common mutation in INH-resistant MTB. Two real-time PAP systems were established for both the wild type and the mutant by using the fluorogenic dye. With plasmid DNA, the limit of detection of the two systems was10copies per reaction, specificity was5×105and5×104copies, respectively, and the selectivity was1:105. The real-time PAP systems were used to analyze71smear-positive sputum samples, and the results were compared with both sequencing and a probe-based real-time PCR method. The results showed that fully concordant results were obtained in64samples with three methods. Of the7discrepant samples,6samples were found to have mixed wild-type and mutant MTB of different level (0.07%-94.2%), and5of them were detected as mutant by real-time PCR and2were detected as mutant by sequencing. One sample missed by both real-time PAP and real-time PCR was found to harbor a different mutation, i.e., katG S315N (AGC→AAC). The above results demonstrated that real-time PAP could detect and quantify rare mutation, and thus has great potential to track the process of the mutation accumulation and help to choose an optimal treatment strategy.
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