高通量測序技術用于HIV感染溯源調查的研究
發(fā)布時間：2019-05-30 07:01
【摘要】：背景 HIV職業(yè)暴露感染及醫(yī)源性感染的風險受到醫(yī)務人員、公安干警和公眾的高度關注。傳統(tǒng)的HIV暴露后感染溯源調查主要依賴于流行病學調查和文件記錄,其中HIV職業(yè)暴露管理相對規(guī)范,要求對暴露人定期隨訪檢測HIV抗體,以監(jiān)測是否在一定期限內發(fā)生HIV抗體陽轉。然而,在特殊情況下,HIV抗體陽轉的證據不足以認定本次感染來自職業(yè)暴露,比如暴露人在暴露前已感染HIV但處于檢測窗口期而未被發(fā)現,或暴露后隨訪期內又發(fā)生了可能感染HIV的其他高危行為,這就需要進一步做分子生物學檢測,包括病毒基因亞型和準種分析。由于缺乏類似職業(yè)暴露管理的報告和文件記錄體系,HIV醫(yī)源性感染的溯源調查更加困難,對分子生物學證據的需求也更為迫切。 已報道的HIV分子溯源技術主要有PCR產物直接測序法、PCR產物克隆法和終點有限稀釋PCR法,用于分析暴露源和暴露人體內HIV毒株間的關系。前者操作較簡便,但只能檢出個體內HIV優(yōu)勢準種的核酸序列：后兩者可以獲得多條優(yōu)勢及弱勢準種序列,本實驗室曾將它們成功地應用于HIV感染溯源調查,但這兩種方法的不足之處是操作比較繁瑣。Illumina公司的Miseq高通量測序平臺(以下簡稱Miseq測序)能夠將PCR產物簡單處理后直接測序,一次反應獲得大量的核酸序列信息,已應用于丙型肝炎病毒的耐藥突變檢測等研究中。目前國內外尚未見有將該方法應用于HIV準種分析或溯源調查的報道。 目的 1、建立基于Miseq測序技術的HIV準種分析方法,并探索其用于HIV感染溯源調查的條件； 2、將該方法用于一個疑似HIV傳播鏈的溯源調查。 材料和方法 1、研究對象： (1)本實驗室保存的一個疑似HIV傳播鏈上的3例HIV感染者(編號T1～T3)血樣及部分對照血樣。相關流行病學背景如下：T3通過男男同性性行為將HIV傳播給T2,T2通過獻血將HIV傳播給T1。選用了11份對照樣品(編號C3～C13),其中C3-C6與T1生活在同一城市,C7-C13與T2、T3生活在另一城市。 (2)在上述對照樣品中,C6和C5疑似有傳播關系(C5通過異性性接觸將HIV傳播給C6),本研究首先利用這2份樣品及其對照(C3、C4)進行方法學研究,然后再擴大范圍,研究其他樣品間的傳播關系。 2、實驗方法： (1)從血漿樣品中提取RNA,并逆轉錄為cDNA.或者從全血樣品中提取總核酸,并進行逆轉錄反應。 (2)PCR產物直接測序：分別針對env.gag和pol基因區(qū)進行PCR擴增,產物純化后直接測序。 (3)篩選Miseq測序的目的片段,設計、優(yōu)化通用引物序列。 (4)Miseq測序所需的第二輪下游引物為融合引物,每份樣品各不相同,分別優(yōu)化PCR反應條件,再進行PCR擴增。 (5)PCR產物純化、質量評估后構建基因文庫。 (6)利用Miseq測序,進行初步數據處理。 (7)對獲得的準種序列進行系統(tǒng)進化分析。 結果 1、方法學研究(使用樣品C3-C6) (1)PCR產物直接測序：4份樣品3個基因區(qū)均擴增、測序成功,env、pol和gag基因區(qū)的樣品間基因離散率分析均顯示C6與C5間親緣關系較近,但無法獲得關于傳播方向的信息。使用env基因區(qū)所獲得的樣品間基因離散率明顯大于pol和gag基因區(qū),能夠提供更多的進化信息。 (2)Miseq測序結果：4份樣品均測序成功,獲得的平均有效序列數為29045(23788-37397)條,平均代表1314(1229～1412)個獨特準種。4份樣品均存在準種序列的頻率由高到低迅速遞減的特點,頻率最高(最優(yōu)勢)的前20個準種序列占HIV準種群總序列數的比例在41.5%～66.2%之間。 (3)樣品間基因離散率分析：針對每份樣品,分別選取最優(yōu)勢的前5、20、100、500個及全部準種序列進行分析。當取最優(yōu)勢的前5個準種序列時,C6與C5間的平均基因離散率為4.2%,低于C6與C3間(11.6%)、C6與C4間(18.2%),差異都有統(tǒng)計學意義(P0.01)；而除C6以外的其他樣本間平均基因離散率都在10%以上。當取最優(yōu)勢的前20、100、500個或者全部準種序列時,所得結果相似,都提示C6與C5間親緣關系較近。 (4)系統(tǒng)進化樹分析：當取最優(yōu)勢的前5個準種序列分析時,C5和C6的準種序列聚為一簇,C3和C4的準種序列各自聚為一簇；C5與C6的準種間呈并列關系,不能提示傳播方向。當取最優(yōu)勢的前20個或更多準種序列時,分析結果相似,不同之處是C5的部分準種包裹著C6的全部準種,即C5對C6存在并系關系(paraphyletic relationship),提示HIV傳播的方向是從C5到C6；納入分析的準種數越多,這種并系關系越加明顯。 2、方法學應用：Miseq測序用于一個疑似HIV傳播鏈的溯源調查(使用樣品T1～T3,C7～C13) (1)Miseq測序結果：1份樣品(C10)的測序結果質量較差,棄去；其余9份樣品(T1-T3, C7-C9, C11-C13)測序成功,獲得的平均有效序列數為22685(8637～37865)條,代表平均999(553～1660)個獨特準種。9份樣品均存在準種序列的頻率由高到低迅速遞減的特點,頻率最高(最優(yōu)勢)的前20個準種序列占HIV準種群總序列數的比例在41.5%～68.9%之間。 (2)樣品間基因離散率分析：每份樣品均取最優(yōu)勢的前100個準種序列。T1與T2間的平均基因離散率(2.8%)、T2與T3間的平均基因離散率(2.9%)分別小于T1、T2與其他對照間的平均基因離散率(3.6%～14.0%、3.1%～13.6%,P0.01)。C12與T1、T2和T3間的基因離散率也較小,親緣關系較近。 (3)系統(tǒng)進化樹分析：每份樣品均取最優(yōu)勢的前100個準種序列。T1、T2、T3和C12的準種序列聚為一簇,其余樣品的準種序列各自聚為一簇。T3對T1、T2、C12存在并系關系,T2對T1存在并系關系,支持HIV由T3傳播給T2、再由T2傳播給T1的流行病學調查結果。然而,由于缺乏完整的流行病學信息,C12與T3間的傳播關系無法確定,但他們同屬于MSM人群,且居住地較近,T3直接或間接地將HIV傳播給C12的可能性較大。 (4)Miseq測序所得有關HIV親緣關系及傳播方向的結論與本實驗室前期使用PCR產物克隆法和EPLD-PCR法檢測獲得的結論相符。結論 1、建立了基于Miseq測序技術的HIV準種分析方法。 2、Miseq測序結果可用于推斷HIV感染者之間的親緣關系；對于有傳播關系的感染者樣品,當所用優(yōu)勢準種的數量達到一定程度時,可有效判定HIV的傳播方向,所用的優(yōu)勢準種數越多,結果越明顯。 3、對1個經性傳播鏈和1個經性-輸血傳播鏈的分子溯源調查表明,Miseq測序結果都能有效支持相關的流行病學調查結果。但當流行病學資料不完整時,實驗室數據應謹慎解讀。 4、Miseq測序技術操作較簡便、檢測成本較低,在HIV溯源調查中具有較高的實用價值。
[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
【學位授予單位】：中國疾病預防控制中心
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R512.91
本文編號：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
【學位授予單位】：中國疾病預防控制中心
【學位級別】：碩士
【學位授予年份】：2014
【分類號】：R512.91
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