高通量测序技术用于HIV感染溯源调查的研究
发布时间：2019-05-30 07:01
【摘要】：背景 HIV职业暴露感染及医源性感染的风险受到医务人员、公安干警和公众的高度关注。传统的HIV暴露后感染溯源调查主要依赖于流行病学调查和文件记录,其中HIV职业暴露管理相对规范,要求对暴露人定期随访检测HIV抗体,以监测是否在一定期限内发生HIV抗体阳转。然而,在特殊情况下,HIV抗体阳转的证据不足以认定本次感染来自职业暴露,比如暴露人在暴露前已感染HIV但处于检测窗口期而未被发现,或暴露后随访期内又发生了可能感染HIV的其他高危行为,这就需要进一步做分子生物学检测,包括病毒基因亚型和准种分析。由于缺乏类似职业暴露管理的报告和文件记录体系,HIV医源性感染的溯源调查更加困难,对分子生物学证据的需求也更为迫切。 已报道的HIV分子溯源技术主要有PCR产物直接测序法、PCR产物克隆法和终点有限稀释PCR法,用于分析暴露源和暴露人体内HIV毒株间的关系。前者操作较简便,但只能检出个体内HIV优势准种的核酸序列：后两者可以获得多条优势及弱势准种序列,本实验室曾将它们成功地应用于HIV感染溯源调查,但这两种方法的不足之处是操作比较繁琐。Illumina公司的Miseq高通量测序平台(以下简称Miseq测序)能够将PCR产物简单处理后直接测序,一次反应获得大量的核酸序列信息,已应用于丙型肝炎病毒的耐药突变检测等研究中。目前国内外尚未见有将该方法应用于HIV准种分析或溯源调查的报道。 目的 1、建立基于Miseq测序技术的HIV准种分析方法,并探索其用于HIV感染溯源调查的条件； 2、将该方法用于一个疑似HIV传播链的溯源调查。 材料和方法 1、研究对象： (1)本实验室保存的一个疑似HIV传播链上的3例HIV感染者(编号T1～T3)血样及部分对照血样。相关流行病学背景如下：T3通过男男同性性行为将HIV传播给T2,T2通过献血将HIV传播给T1。选用了11份对照样品(编号C3～C13),其中C3-C6与T1生活在同一城市,C7-C13与T2、T3生活在另一城市。 (2)在上述对照样品中,C6和C5疑似有传播关系(C5通过异性性接触将HIV传播给C6),本研究首先利用这2份样品及其对照(C3、C4)进行方法学研究,然后再扩大范围,研究其他样品间的传播关系。 2、实验方法： (1)从血浆样品中提取RNA,并逆转录为cDNA.或者从全血样品中提取总核酸,并进行逆转录反应。 (2)PCR产物直接测序：分别针对env.gag和pol基因区进行PCR扩增,产物纯化后直接测序。 (3)筛选Miseq测序的目的片段,设计、优化通用引物序列。 (4)Miseq测序所需的第二轮下游引物为融合引物,每份样品各不相同,分别优化PCR反应条件,再进行PCR扩增。 (5)PCR产物纯化、质量评估后构建基因文库。 (6)利用Miseq测序,进行初步数据处理。 (7)对获得的准种序列进行系统进化分析。 结果 1、方法学研究(使用样品C3-C6) (1)PCR产物直接测序：4份样品3个基因区均扩增、测序成功,env、pol和gag基因区的样品间基因离散率分析均显示C6与C5间亲缘关系较近,但无法获得关于传播方向的信息。使用env基因区所获得的样品间基因离散率明显大于pol和gag基因区,能够提供更多的进化信息。 (2)Miseq测序结果：4份样品均测序成功,获得的平均有效序列数为29045(23788-37397)条,平均代表1314(1229～1412)个独特准种。4份样品均存在准种序列的频率由高到低迅速递减的特点,频率最高(最优势)的前20个准种序列占HIV准种群总序列数的比例在41.5%～66.2%之间。 (3)样品间基因离散率分析：针对每份样品,分别选取最优势的前5、20、100、500个及全部准种序列进行分析。当取最优势的前5个准种序列时,C6与C5间的平均基因离散率为4.2%,低于C6与C3间(11.6%)、C6与C4间(18.2%),差异都有统计学意义(P0.01)；而除C6以外的其他样本间平均基因离散率都在10%以上。当取最优势的前20、100、500个或者全部准种序列时,所得结果相似,都提示C6与C5间亲缘关系较近。 (4)系统进化树分析：当取最优势的前5个准种序列分析时,C5和C6的准种序列聚为一簇,C3和C4的准种序列各自聚为一簇；C5与C6的准种间呈并列关系,不能提示传播方向。当取最优势的前20个或更多准种序列时,分析结果相似,不同之处是C5的部分准种包裹着C6的全部准种,即C5对C6存在并系关系(paraphyletic relationship),提示HIV传播的方向是从C5到C6；纳入分析的准种数越多,这种并系关系越加明显。 2、方法学应用：Miseq测序用于一个疑似HIV传播链的溯源调查(使用样品T1～T3,C7～C13) (1)Miseq测序结果：1份样品(C10)的测序结果质量较差,弃去；其余9份样品(T1-T3, C7-C9, C11-C13)测序成功,获得的平均有效序列数为22685(8637～37865)条,代表平均999(553～1660)个独特准种。9份样品均存在准种序列的频率由高到低迅速递减的特点,频率最高(最优势)的前20个准种序列占HIV准种群总序列数的比例在41.5%～68.9%之间。 (2)样品间基因离散率分析：每份样品均取最优势的前100个准种序列。T1与T2间的平均基因离散率(2.8%)、T2与T3间的平均基因离散率(2.9%)分别小于T1、T2与其他对照间的平均基因离散率(3.6%～14.0%、3.1%～13.6%,P0.01)。C12与T1、T2和T3间的基因离散率也较小,亲缘关系较近。 (3)系统进化树分析：每份样品均取最优势的前100个准种序列。T1、T2、T3和C12的准种序列聚为一簇,其余样品的准种序列各自聚为一簇。T3对T1、T2、C12存在并系关系,T2对T1存在并系关系,支持HIV由T3传播给T2、再由T2传播给T1的流行病学调查结果。然而,由于缺乏完整的流行病学信息,C12与T3间的传播关系无法确定,但他们同属于MSM人群,且居住地较近,T3直接或间接地将HIV传播给C12的可能性较大。 (4)Miseq测序所得有关HIV亲缘关系及传播方向的结论与本实验室前期使用PCR产物克隆法和EPLD-PCR法检测获得的结论相符。结论 1、建立了基于Miseq测序技术的HIV准种分析方法。 2、Miseq测序结果可用于推断HIV感染者之间的亲缘关系；对于有传播关系的感染者样品,当所用优势准种的数量达到一定程度时,可有效判定HIV的传播方向,所用的优势准种数越多,结果越明显。 3、对1个经性传播链和1个经性-输血传播链的分子溯源调查表明,Miseq测序结果都能有效支持相关的流行病学调查结果。但当流行病学资料不完整时,实验室数据应谨慎解读。 4、Miseq测序技术操作较简便、检测成本较低,在HIV溯源调查中具有较高的实用价值。
[Abstract]:background The risk of HIV occupational exposure and iatrogenic infection is highly closed by medical personnel, public security police and the public. Note: The traditional HIV post-exposure source of infection is mainly dependent on the epidemiological investigation and documentation, in which the relative standard of HIV occupational exposure management requires regular follow-up of the exposed person to detect the HIV antibody to monitor the occurrence of HIV antibody positive within a certain period of time. In exceptional cases, however, the evidence of the positive rotation of the HIV antibody is not sufficient to identify this infection from occupational exposure, such as the exposure of the exposed to HIV, but in the detection window, or other high-risk lines that may be infected with HIV during the post-exposure follow-up period For this, there is a need for further molecular biology testing, including viral gene and quasi-species It is more difficult to trace the source of HIV iatrogenic infection because of the lack of a report and documentation system that is similar to the management of occupational exposure, and the need for evidence of molecular biology is even more difficult. The methods of direct sequencing of PCR products, the cloning of PCR products and the limited dilution of end-point PCR are used to analyze the relationship between the exposure source and the HIV strain in the exposed human body. The former is simple to operate, but can only detect the nucleic acid sequences of the HIV-dominant species in the body: the latter can obtain multiple advantages and weak quasi-seed sequences, which have been successfully applied to the source tracing of HIV infection, but the deficiency of these two methods is the operation ratio The Misq high-throughput sequencing platform of Illumina (hereinafter referred to as Misq sequencing) can directly sequence the PCR product, obtain a large number of nucleic acid sequence information at a time, and has been applied to the detection of drug-resistance mutation of hepatitis C virus. At present, the method is not applied to the analysis or tracing of HIV. a newspaper The objective 1 of the invention is to establish an HIV-based analysis method based on the Misq sequencing technique and to explore its use for HIV infection the condition of the traceability survey;2. The method is used for a suspected HI V-propagating Tracing to the source of the source. Materials and Methods 1, Study object: (1)3 HIV-infected persons (No.: Blood samples from T1 to T3 and part of the control blood samples. The relevant epidemiological background is as follows: T3 is transmitted to T2 by male and male sex sexual behavior, HIV was spread to T1 by blood donation.11 control samples (No. C3-C13) were used, where C3-C6 and T1 live in the same city, C7-C 13 and T2, T3 live in another city. (2) In the above-mentioned control sample, C6 and C5 are suspected to have a propagation relationship (C5 is transmitted to C6 by heterosexual contact), and this study first uses the two samples and its control (C3, C4) to carry out the methodological study, and then the scope of expansion, research the propagation relationship between other samples. Experimental method: (1) RNA is extracted from the plasma sample and is reverse transcribed into cDNA. The total nucleic acid is extracted from the whole blood sample and the reverse transcription reaction is carried out. (2) The PCR product is directly sequenced: for env.gag and PCR amplification is carried out in the pol gene region, and the product is directly sequenced after the product is purified. (3) the screen The target fragment selected for Misq sequencing was designed to optimize the universal primer sequence. (4) The second downstream primer required for the Mises sequencing was a fusion primer, each the samples are different, the PCR reaction conditions are optimized, and then P CR amplification. (5) Purification and quality evaluation of PCR products The gene library was then constructed. (6) Mises eq. sequencing and preliminary data processing is carried out. (7) For obtaining The system evolution analysis was carried out on the quasi-seed sequence. Results 1. The methodology study (using sample C3-C6) (1) PCR product was directly sequenced:4 samples of 3 gene regions were amplified, and the sequencing was successful. samples of the nv, pol and gag gene regions The analysis of the discrete rate of the inter-product gene showed that the relationship between C6 and C5 was close to that of C5, but it was not possible to obtain information about the direction of propagation. The env gene was used. The gene discrete rate of the samples obtained in the region is significantly larger than that of the pol and gag gene regions, and more evolution information can be provided. (2) Misq sequencing results:4 samples have been successfully sequenced, and the obtained average effective sequence number is 29045 (23788-37397), and the average representative 1314 ( the frequency of the quasi-seed sequence in the four samples is from high to low, and the frequency is the highest ( The ratio of the first 20 quasi-species to the total number of the HIV quasi-population was 41.5%-66.2%. The average gene discrete rate between C6 and C5 was 4.2%, lower than that of C6 and C3 (11.6%), and between C6 and C4 (18.2%). The difference was statistically significant (P0.01), while the average gene dispersion in other samples other than C6 was above 10%. Prior to taking the most advantage, The results are similar to the results obtained in the 20,100,500, or all the quasi-seed sequences. (4) The phylogenetic tree analysis: the quasi-seed sequences of C5 and C6 when the first five of the most dominant sequences are analyzed As a cluster, the quasi-seed sequences of C3 and C4 are each clustered into a cluster; the quasi-species of C5 and C6 are in a parallel relationship, and the propagation direction can not be suggested. When the first 20 or more of the most dominant sequences are taken, the analysis results are similar, except that the part of C5 is the quasi-seed of C6, that is, the C5 pair C6 is in parallel relation (parainly tic relation Ship), indicating that the direction of HIV transmission is from C5 to C6; the more the number of quasi-species included in the analysis, the more obvious the parallel relationship is. Methodology application: Misq sequencing is used for the tracing of a suspected HIV transmission chain (using samples T1-T3, C7-C13) (1) Misq sequencing results:1 sample (10) The sequencing results of (C10) were poor and discarded; the remaining 9 samples (T1-T3, C7-C9, C11-C13) were successfully sequenced, and the obtained average effective sequence number was 222685 (8637-37865), and the average effective number of the samples was 22685 (8637-37865), and the average number of the nine samples was from high to low. The first 20 quasi-species with the highest frequency (the most advantageous) account for the ratio of the total number of HIV quasi-population sequences. Between 41.5% and 68.9%, (2) the analysis of the discrete rate of the gene between T1 and T2: the average gene discrete rate between T1 and T2 (2.8%), the average gene discrete rate between T2 and T3 (2.9%) Mean gene dispersion (3.6% ~ 14) between T1, T2 and other controls .0%, 3.1% ~ 13.6%, P0.01). C12 and T1, T2 and 3) phylogenetic tree analysis: each sample the quasi-seed sequences of T1, T2, T3 and C12 are clustered into a cluster, and the quasi-seed sequences of the remaining samples are each gathered into a cluster; and the T3 pairs T1, T2, and C In the presence of a parallel relationship, T2 has a parallel relationship to T1, which supports the transmission of HIV from T3 to T2 and is then transmitted from T2 to the epidemiological survey of T1. However, due to the lack of complete epidemiological information, between C12 and T3 The transmission relationship cannot be determined, but they are the same as those belonging to the MSM population and where the place of residence is close, T3 is likely to spread HIV to C12 either directly or indirectly. (4) Misq sequencing income Conclusion on the relationship between HIV and the direction of transmission and the use of P in the early stage of this lab The results of the PCR method and the results of the EPLD-PCR are in accord with the results of the PCR. Conclusion 1. The results of the method for the analysis of HIV quasi-species based on the Misq sequencing technology are established. The results of Misq sequencing can be used to infer the relationship between the HIV-infected persons. and when the number of the dominant species used reaches a certain degree, the transmission direction of the HIV can be effectively judged, the more the dominant species used, the more the result is, E.3,1 Transmitted and 1 Transmitted-Transfusion The molecular tracing of the chain shows that the Misq sequencing results can effectively support relevant epidemiological findings, but when the epidemic
【学位授予单位】：中国疾病预防控制中心
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R512.91
本文编号：2488623
[Abstract]:background The risk of HIV occupational exposure and iatrogenic infection is highly closed by medical personnel, public security police and the public. Note: The traditional HIV post-exposure source of infection is mainly dependent on the epidemiological investigation and documentation, in which the relative standard of HIV occupational exposure management requires regular follow-up of the exposed person to detect the HIV antibody to monitor the occurrence of HIV antibody positive within a certain period of time. In exceptional cases, however, the evidence of the positive rotation of the HIV antibody is not sufficient to identify this infection from occupational exposure, such as the exposure of the exposed to HIV, but in the detection window, or other high-risk lines that may be infected with HIV during the post-exposure follow-up period For this, there is a need for further molecular biology testing, including viral gene and quasi-species It is more difficult to trace the source of HIV iatrogenic infection because of the lack of a report and documentation system that is similar to the management of occupational exposure, and the need for evidence of molecular biology is even more difficult. The methods of direct sequencing of PCR products, the cloning of PCR products and the limited dilution of end-point PCR are used to analyze the relationship between the exposure source and the HIV strain in the exposed human body. The former is simple to operate, but can only detect the nucleic acid sequences of the HIV-dominant species in the body: the latter can obtain multiple advantages and weak quasi-seed sequences, which have been successfully applied to the source tracing of HIV infection, but the deficiency of these two methods is the operation ratio The Misq high-throughput sequencing platform of Illumina (hereinafter referred to as Misq sequencing) can directly sequence the PCR product, obtain a large number of nucleic acid sequence information at a time, and has been applied to the detection of drug-resistance mutation of hepatitis C virus. At present, the method is not applied to the analysis or tracing of HIV. a newspaper The objective 1 of the invention is to establish an HIV-based analysis method based on the Misq sequencing technique and to explore its use for HIV infection the condition of the traceability survey;2. The method is used for a suspected HI V-propagating Tracing to the source of the source. Materials and Methods 1, Study object: (1)3 HIV-infected persons (No.: Blood samples from T1 to T3 and part of the control blood samples. The relevant epidemiological background is as follows: T3 is transmitted to T2 by male and male sex sexual behavior, HIV was spread to T1 by blood donation.11 control samples (No. C3-C13) were used, where C3-C6 and T1 live in the same city, C7-C 13 and T2, T3 live in another city. (2) In the above-mentioned control sample, C6 and C5 are suspected to have a propagation relationship (C5 is transmitted to C6 by heterosexual contact), and this study first uses the two samples and its control (C3, C4) to carry out the methodological study, and then the scope of expansion, research the propagation relationship between other samples. Experimental method: (1) RNA is extracted from the plasma sample and is reverse transcribed into cDNA. The total nucleic acid is extracted from the whole blood sample and the reverse transcription reaction is carried out. (2) The PCR product is directly sequenced: for env.gag and PCR amplification is carried out in the pol gene region, and the product is directly sequenced after the product is purified. (3) the screen The target fragment selected for Misq sequencing was designed to optimize the universal primer sequence. (4) The second downstream primer required for the Mises sequencing was a fusion primer, each the samples are different, the PCR reaction conditions are optimized, and then P CR amplification. (5) Purification and quality evaluation of PCR products The gene library was then constructed. (6) Mises eq. sequencing and preliminary data processing is carried out. (7) For obtaining The system evolution analysis was carried out on the quasi-seed sequence. Results 1. The methodology study (using sample C3-C6) (1) PCR product was directly sequenced:4 samples of 3 gene regions were amplified, and the sequencing was successful. samples of the nv, pol and gag gene regions The analysis of the discrete rate of the inter-product gene showed that the relationship between C6 and C5 was close to that of C5, but it was not possible to obtain information about the direction of propagation. The env gene was used. The gene discrete rate of the samples obtained in the region is significantly larger than that of the pol and gag gene regions, and more evolution information can be provided. (2) Misq sequencing results:4 samples have been successfully sequenced, and the obtained average effective sequence number is 29045 (23788-37397), and the average representative 1314 ( the frequency of the quasi-seed sequence in the four samples is from high to low, and the frequency is the highest ( The ratio of the first 20 quasi-species to the total number of the HIV quasi-population was 41.5%-66.2%. The average gene discrete rate between C6 and C5 was 4.2%, lower than that of C6 and C3 (11.6%), and between C6 and C4 (18.2%). The difference was statistically significant (P0.01), while the average gene dispersion in other samples other than C6 was above 10%. Prior to taking the most advantage, The results are similar to the results obtained in the 20,100,500, or all the quasi-seed sequences. (4) The phylogenetic tree analysis: the quasi-seed sequences of C5 and C6 when the first five of the most dominant sequences are analyzed As a cluster, the quasi-seed sequences of C3 and C4 are each clustered into a cluster; the quasi-species of C5 and C6 are in a parallel relationship, and the propagation direction can not be suggested. When the first 20 or more of the most dominant sequences are taken, the analysis results are similar, except that the part of C5 is the quasi-seed of C6, that is, the C5 pair C6 is in parallel relation (parainly tic relation Ship), indicating that the direction of HIV transmission is from C5 to C6; the more the number of quasi-species included in the analysis, the more obvious the parallel relationship is. Methodology application: Misq sequencing is used for the tracing of a suspected HIV transmission chain (using samples T1-T3, C7-C13) (1) Misq sequencing results:1 sample (10) The sequencing results of (C10) were poor and discarded; the remaining 9 samples (T1-T3, C7-C9, C11-C13) were successfully sequenced, and the obtained average effective sequence number was 222685 (8637-37865), and the average effective number of the samples was 22685 (8637-37865), and the average number of the nine samples was from high to low. The first 20 quasi-species with the highest frequency (the most advantageous) account for the ratio of the total number of HIV quasi-population sequences. Between 41.5% and 68.9%, (2) the analysis of the discrete rate of the gene between T1 and T2: the average gene discrete rate between T1 and T2 (2.8%), the average gene discrete rate between T2 and T3 (2.9%) Mean gene dispersion (3.6% ~ 14) between T1, T2 and other controls .0%, 3.1% ~ 13.6%, P0.01). C12 and T1, T2 and 3) phylogenetic tree analysis: each sample the quasi-seed sequences of T1, T2, T3 and C12 are clustered into a cluster, and the quasi-seed sequences of the remaining samples are each gathered into a cluster; and the T3 pairs T1, T2, and C In the presence of a parallel relationship, T2 has a parallel relationship to T1, which supports the transmission of HIV from T3 to T2 and is then transmitted from T2 to the epidemiological survey of T1. However, due to the lack of complete epidemiological information, between C12 and T3 The transmission relationship cannot be determined, but they are the same as those belonging to the MSM population and where the place of residence is close, T3 is likely to spread HIV to C12 either directly or indirectly. (4) Misq sequencing income Conclusion on the relationship between HIV and the direction of transmission and the use of P in the early stage of this lab The results of the PCR method and the results of the EPLD-PCR are in accord with the results of the PCR. Conclusion 1. The results of the method for the analysis of HIV quasi-species based on the Misq sequencing technology are established. The results of Misq sequencing can be used to infer the relationship between the HIV-infected persons. and when the number of the dominant species used reaches a certain degree, the transmission direction of the HIV can be effectively judged, the more the dominant species used, the more the result is, E.3,1 Transmitted and 1 Transmitted-Transfusion The molecular tracing of the chain shows that the Misq sequencing results can effectively support relevant epidemiological findings, but when the epidemic
【学位授予单位】：中国疾病预防控制中心
【学位级别】：硕士
【学位授予年份】：2014
【分类号】：R512.91
【参考文献】
中国期刊全文数据库 前3条
1 尤超;赵大球;梁乘榜;周春华;;PCR引物设计方法综述[J];现代农业科技;2011年17期
2 ;卫生部关于印发《医务人员艾滋病病毒职业暴露防护工作指导原则(试行)》的通知[J];中华人民共和国卫生部公报;2004年05期
3 梁浩;罗皓;邵一鸣;刘伟;张志勇;邢辉;卢灿健;沈菁;麦志丹;;广西HIV-1重组毒株env区快速基因分型方法的建立[J];中华实验和临床病毒学杂志;2006年03期
，本文编号：2488623
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