本文關(guān)鍵詞： 高通量測序 乙型肝炎 Ion Torrent PGM Miseq 擴增子測序 耐藥突變　出處：《中國人民解放軍軍事醫(yī)學科學院》2017年碩士論文　論文類型：學位論文

【摘要】：一、背景高通量測序(也稱下一代測序技術(shù))技術(shù)具有強大的平行測序能力,被廣泛應(yīng)用于生命科學研究領(lǐng)域。近年來隨著成本的降低和應(yīng)用成熟度的增加,其在臨床基因檢測中備受青睞。高通量測序在用藥指導方面應(yīng)用廣泛,如腫瘤個體化治療基因檢測,相對于傳統(tǒng)檢測,更能全面覆蓋基因變異情況,為患者個體化用藥提供更多參考。乙型肝炎病毒(Hepatitis B virus:HBV)耐藥問題嚴重,目前臨床用于治療慢性乙肝的藥物是核苷類藥物,長期治療都會引起不同程度的耐藥現(xiàn)象,密切監(jiān)測耐藥的產(chǎn)生,及時調(diào)整治療方案,已經(jīng)成為醫(yī)患關(guān)注的重點問題。已知與耐藥相關(guān)的絕大部分突變均位于HBV聚合酶基因的逆轉(zhuǎn)錄酶區(qū)�，F(xiàn)有的檢測HBV突變方法有聚合酶鏈反應(yīng)(Polymerase chain reaction:PCR)直接測序法,由于靈敏度的限制,該方法只在群體中特定突變占20%以上時才能檢測到;PCR產(chǎn)物克隆測序法,可大致確定不同序列毒株之間的相對比例有助于發(fā)現(xiàn)混合株,但操作繁瑣且靈敏度有限。此外,實時定量PCR,限制性片段長度多態(tài)性技術(shù),線性探針反向雜交法在臨床使用中,雖然靈敏度各不相同,但都只能針對特定已知耐藥位點,針對每一個位點分別設(shè)計探針/引物,效率低下。目前,下一代測序技術(shù)(Next generation sequencing:NGS)在HBV耐藥檢測中的應(yīng)用較少,尤其是具有實用價值的NGS檢測HBV耐藥突變方法未見報道。本文擬建立可用于臨床用藥指導的HBV耐藥基因位點高通量測序檢測方法,該方法能夠?qū)Π℉BV聚合酶基因內(nèi)的全部耐藥相關(guān)區(qū)段進行分析,發(fā)現(xiàn)患者HBV耐藥位點,為HBV感染患者的個體化治療提供依據(jù)。二、方法本文的第一部分是基于高通量測序的乙肝耐藥位點檢測方法的建立,首先針對耐藥位點所在區(qū)域設(shè)計特異引物,并對其進行優(yōu)化;然后采用融合引物(Fusion method primer)策略設(shè)計適用于Thermo Life Ion Torrent PGM平臺及Illumina Miseq高通量測序平臺的引物,即在設(shè)計好的目標特異引物的基礎(chǔ)上,添加適用于高通量測序的接頭和標簽序列,以此引物擴增乙型肝炎病毒脫氧核糖核酸(Hepatitis B virus Deoxyribonucleic acid,HBV DNA)獲得上機文庫。然后進行測序操作獲得目標區(qū)域的基因序列信息,并對其進行分析。本文第二部分對基于高通量測序技術(shù)的乙肝耐藥突變位點檢測方法進行驗證和臨床樣本檢測。還利用以上建庫引物,對濃度梯度的HBV DNA標準物質(zhì)進行了文庫構(gòu)建,以評價本方法的靈敏度;利用耐藥位點區(qū)域存在差異的兩種乙肝病毒基因組質(zhì)�；旌夏M樣對高通量測序方法的突變率檢測能力進行評價,選B型和C型HBV基因組質(zhì)粒按1:99,5:95,10:90三種比例混合建庫,并在Ion Torrent PGM和Miseq上進行上機測序,統(tǒng)計B型、C型兩種HBV DNA差異堿基的檢測效率;采用突變引物構(gòu)建HBV突變質(zhì)粒標準品,突變型和野生型HBV基因組質(zhì)粒按1:99,5:95,10:90三種比例混合建庫,并在Ion Torrent PGM上進行測序,統(tǒng)計突變堿基的檢測效率,對本研究設(shè)計的高通量測序方法的突變檢出能力進行評價。進一步,收集了15例慢性乙肝患者血液樣本,用Pure Link?Viral RNA/DNA Mini試劑盒提取HBV DNA�；颊哐錒BV DNA用建立的Ion Torrent PGM方法檢測,樣本同時進行Sanger法測序。三、結(jié)果1.對使用特異引物擴增的PCR產(chǎn)物進行毛細管電泳檢測分析產(chǎn)物長度和測序驗證序列,結(jié)果表明特異引物擴增特異。PCR產(chǎn)物分為三個片段,大小分別是165bp、324bp和135bp,覆蓋的耐藥位點包括L80V/I,I169T,V173L,L180M,A181T/V,T184A,S202G/I,M204I/V/S,V214A,Q215S,N236T,M250V,G1896A。2.設(shè)計了Ion Torrent PGM的擴增子引物和基于Miseq平臺的引物,并對引物擴增產(chǎn)物進行了毛細管電泳檢測,結(jié)果表明產(chǎn)物長度分別為244bp、467bp、320bp和296bp、559bp、218bp,一代測序證明其序列正確,表明測序引物擴增正確;對構(gòu)建的質(zhì)粒標準品各步產(chǎn)物都采用毛細管電泳檢測,構(gòu)建的質(zhì)粒采用毛細管電泳檢測和Sanger法一代測序鑒定,結(jié)果顯示HBV突變質(zhì)粒構(gòu)建成功。3.基于Ion Torrent?PGM平臺的融合引物建庫方法檢測靈敏度可達50IU/mL,建立了適用于Ion Torrent?PGM平臺和Miseq平臺的擴增子文庫構(gòu)建方法,該方法可對HBV50 IU/mL的血清進行建庫。對模擬樣中突變率在5%及其以上的位點可很好的檢出(信噪比10);對模擬樣中突變率在1%豐度的位點可檢出9/10,(信噪比10)。15例臨床樣品的檢出8個耐藥位點,這些位點不能被臨床常用的Sanger法檢測到。四、結(jié)論初步建立了高通量測序檢測HBV耐藥突變的檢測方法,為臨床動態(tài)監(jiān)測乙肝病毒耐藥位點突變、指導合理臨床用藥奠定了基礎(chǔ)。
[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.

【學位授予單位】：中國人民解放軍軍事醫(yī)學科學院
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：R512.62

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