基于高通量测序技术的乙肝耐药基因突变检测方法研究

发布时间：2018-02-13 22:14

本文关键词： 高通量测序乙型肝炎 Ion Torrent PGM Miseq 扩增子测序耐药突变　出处：《中国人民解放军军事医学科学院》2017年硕士论文　论文类型：学位论文

【摘要】：一、背景高通量测序(也称下一代测序技术)技术具有强大的平行测序能力,被广泛应用于生命科学研究领域。近年来随着成本的降低和应用成熟度的增加,其在临床基因检测中备受青睐。高通量测序在用药指导方面应用广泛,如肿瘤个体化治疗基因检测,相对于传统检测,更能全面覆盖基因变异情况,为患者个体化用药提供更多参考。乙型肝炎病毒(Hepatitis B virus:HBV)耐药问题严重,目前临床用于治疗慢性乙肝的药物是核苷类药物,长期治疗都会引起不同程度的耐药现象,密切监测耐药的产生,及时调整治疗方案,已经成为医患关注的重点问题。已知与耐药相关的绝大部分突变均位于HBV聚合酶基因的逆转录酶区。现有的检测HBV突变方法有聚合酶链反应(Polymerase chain reaction:PCR)直接测序法,由于灵敏度的限制,该方法只在群体中特定突变占20%以上时才能检测到;PCR产物克隆测序法,可大致确定不同序列毒株之间的相对比例有助于发现混合株,但操作繁琐且灵敏度有限。此外,实时定量PCR,限制性片段长度多态性技术,线性探针反向杂交法在临床使用中,虽然灵敏度各不相同,但都只能针对特定已知耐药位点,针对每一个位点分别设计探针/引物,效率低下。目前,下一代测序技术(Next generation sequencing:NGS)在HBV耐药检测中的应用较少,尤其是具有实用价值的NGS检测HBV耐药突变方法未见报道。本文拟建立可用于临床用药指导的HBV耐药基因位点高通量测序检测方法,该方法能够对包括HBV聚合酶基因内的全部耐药相关区段进行分析,发现患者HBV耐药位点,为HBV感染患者的个体化治疗提供依据。二、方法本文的第一部分是基于高通量测序的乙肝耐药位点检测方法的建立,首先针对耐药位点所在区域设计特异引物,并对其进行优化;然后采用融合引物(Fusion method primer)策略设计适用于Thermo Life Ion Torrent PGM平台及Illumina Miseq高通量测序平台的引物,即在设计好的目标特异引物的基础上,添加适用于高通量测序的接头和标签序列,以此引物扩增乙型肝炎病毒脱氧核糖核酸(Hepatitis B virus Deoxyribonucleic acid,HBV DNA)获得上机文库。然后进行测序操作获得目标区域的基因序列信息,并对其进行分析。本文第二部分对基于高通量测序技术的乙肝耐药突变位点检测方法进行验证和临床样本检测。还利用以上建库引物,对浓度梯度的HBV DNA标准物质进行了文库构建,以评价本方法的灵敏度;利用耐药位点区域存在差异的两种乙肝病毒基因组质粒混合模拟样对高通量测序方法的突变率检测能力进行评价,选B型和C型HBV基因组质粒按1:99,5:95,10:90三种比例混合建库,并在Ion Torrent PGM和Miseq上进行上机测序,统计B型、C型两种HBV DNA差异碱基的检测效率;采用突变引物构建HBV突变质粒标准品,突变型和野生型HBV基因组质粒按1:99,5:95,10:90三种比例混合建库,并在Ion Torrent PGM上进行测序,统计突变碱基的检测效率,对本研究设计的高通量测序方法的突变检出能力进行评价。进一步,收集了15例慢性乙肝患者血液样本,用Pure Link?Viral RNA/DNA Mini试剂盒提取HBV DNA。患者血清HBV DNA用建立的Ion Torrent PGM方法检测,样本同时进行Sanger法测序。三、结果1.对使用特异引物扩增的PCR产物进行毛细管电泳检测分析产物长度和测序验证序列,结果表明特异引物扩增特异。PCR产物分为三个片段,大小分别是165bp、324bp和135bp,覆盖的耐药位点包括L80V/I,I169T,V173L,L180M,A181T/V,T184A,S202G/I,M204I/V/S,V214A,Q215S,N236T,M250V,G1896A。2.设计了Ion Torrent PGM的扩增子引物和基于Miseq平台的引物,并对引物扩增产物进行了毛细管电泳检测,结果表明产物长度分别为244bp、467bp、320bp和296bp、559bp、218bp,一代测序证明其序列正确,表明测序引物扩增正确;对构建的质粒标准品各步产物都采用毛细管电泳检测,构建的质粒采用毛细管电泳检测和Sanger法一代测序鉴定,结果显示HBV突变质粒构建成功。3.基于Ion Torrent?PGM平台的融合引物建库方法检测灵敏度可达50IU/mL,建立了适用于Ion Torrent?PGM平台和Miseq平台的扩增子文库构建方法,该方法可对HBV50 IU/mL的血清进行建库。对模拟样中突变率在5%及其以上的位点可很好的检出(信噪比10);对模拟样中突变率在1%丰度的位点可检出9/10,(信噪比10)。15例临床样品的检出8个耐药位点,这些位点不能被临床常用的Sanger法检测到。四、结论初步建立了高通量测序检测HBV耐药突变的检测方法,为临床动态监测乙肝病毒耐药位点突变、指导合理临床用药奠定了基础。
[Abstract]:A background, high-throughput sequencing (also known as the next generation sequencing technology) technology has the strong ability of parallel sequencing, is widely used in the field of life science research. In recent years, with lower costs and application maturity increased, which favored in clinical genetic testing. High-throughput sequencing is widely used in medicine guidance, such as the individualized treatment of tumor gene detection, compared with the traditional detection, more comprehensive coverage of genetic variation, provide more reference for individualized medication. Patients with hepatitis B virus (Hepatitis B virus:HBV) resistance to the serious problem of current clinical medicine for treating chronic hepatitis B are nucleoside drugs, long-term treatment will cause varying degrees of resistance, close the resistance monitoring, timely adjust the treatment plan, has become a key problem of medical attention. Most of the known and resistance related mutations were located in HBV Reverse transcriptase region synthase gene HBV mutation detection. The existing methods of polymerase chain reaction (Polymerase chain reaction:PCR) direct sequencing method, because of the limit of the sensitivity of the method, only in the group specific mutations accounted for more than 20% can be detected; PCR cloning method, can roughly determine the relative ratio between different strains of sequence contribute to the discovery of mixed strains, but the operation is complicated and the sensitivity is limited. In addition, real-time quantitative PCR, restriction fragment length polymorphism technique, linear probes reverse hybridization in clinical use, although the sensitivity of each are not identical, but only to specific known mutation sites, for each site were designed probe / primer. The efficiency is low. At present, the next generation sequencing technology (Next generation sequencing:NGS) is seldom used in HBV resistance detection, especially the mutation detection of NGS HBV with practical value Methods have not been reported. This paper can be used to establish the HBV resistance gene loci high-throughput sequencing detection method to guide clinical medication, the method is able to include all resistant HBV polymerase gene related to the section analysis, found that patients with HBV mutations, and provide the basis for the individualized treatment of patients with HBV infection. In two, the first part of this method is the establishment of HBV resistance loci detection method based on high-throughput sequencing, aiming at the resistance locus specific primers and its optimization; then using fusion primers (Fusion method primer) primers for Thermo Life Ion design Torrent PGM platform and Illumina Miseq sequencing platform, which is based on specific goals primers on the joint and add tag sequences suitable for high-throughput sequencing, the amplified hepatitis B virus DNA (Hepatitis B virus Deoxyribonucleic RNA, acid, HBV DNA) to get on the library. Then the sequencing operation obtained gene sequence information of the target area, and carries on the analysis. The second part of the hepatitis B drug resistance high-throughput sequencing mutation detection method is verified and the detection of clinical samples. Based on the above database using primers HBV DNA standard material on the concentration gradient of library construction, according to the sensitivity of the method were evaluated by the mutation; there are two regions of hepatitis B virus genome plasmid of mixed model mutation on high-throughput sequencing method to evaluate the rate of detection ability, B type and C type HBV genome plasmid by 1:99,5:95,10:90 three mixed database, and computer in Ion Torrent and Miseq PGM sequencing, B statistics, detection efficiency of C type two HBV DNA different bases; the mutation lead Construction of HBV mutant plasmid, the mutant and wild type HBV plasmid genome by 1:99,5:95,10:90 three mixed library, and sequenced in Ion Torrent PGM, the detection efficiency statistics of mutation, mutation of high-throughput sequencing method designed in this paper, the detection ability evaluation. Further, a collection of 15 cases of chronic hepatitis B patients blood samples, using Pure Link? Viral RNA/DNA Mini HBV extraction kit of serum HBV in patients with Ion DNA. DNA Torrent PGM method to establish the sample detection, we carried out Sanger sequencing. Results 1. of three amplified using specific primers of PCR products were the length and sequencing analysis of capillary electrophoresis, results show that specific primer.PCR amplification product is divided into three segments, the size is 165bp, 324bp and 135bp, covering the resistance loci including L80V/I, I169T, V173L, L180M, A181T/V, T184A, S202G/I, M204I/V/S, V214A, Q215S, N236T, M250V, G1896A.2. Ion Torrent PGM designed amplification primers and primers based on the Miseq platform, and the amplified products were analyzed by capillary electrophoresis. The results showed that the product length were 244bp, 467bp, 320bp and 296bp, 559bp, 218bp, generation sequencing proved that the sequence of that is correct, correct amplification of plasmid sequencing primers; standard construction materials of the products by capillary electrophoresis, plasmid using capillary electrophoresis and Sanger generation sequencing, the results showed that HBV mutant plasmids were successfully constructed based on.3. Ion Torrent? PGM platform construction method of primer fusion detection sensitivity of 50IU/mL is established suitable for Ion Torrent amplification method for constructing PGM library? Sub platform and Miseq platform, the method of serum on HBV50 of IU/mL database. The simulation in the mutation rate in 5% and These sites can be detected very well (SNR 10); the mutation rate of 9/10 detection in 1% sites of abundance in synthetic samples, (SNR 10) detected 8 cases of.15 mutation of clinical samples, these sites cannot be detected in the commonly used Sanger method to clinical. Four, conclusion the mutation detection method of high-throughput sequencing to detect HBV resistant mutations, for the clinical dynamic monitoring of hepatitis B virus resistance loci, laid the foundation for guiding reasonable clinical medication.

【学位授予单位】：中国人民解放军军事医学科学院
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：R512.62

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