本文選題：人類免疫缺陷病毒 + 傳播網(wǎng)絡(luò)��； 參考：《中國疾病預(yù)防控制中心》2017年碩士論文

【摘要】：對人類免疫缺陷病毒1型(HIV-1)傳播網(wǎng)絡(luò)的特征和傳播網(wǎng)絡(luò)的動態(tài)進(jìn)行深入理解和研究,將有助于HIV-1的監(jiān)測、干預(yù)和治療。目前,HIV-1傳播網(wǎng)絡(luò)分析主要通過分析HIV-1聚合酶(plol)基因區(qū)序列來實現(xiàn)。主要是通過PCR產(chǎn)物直接測序獲得序列,然后進(jìn)行傳播網(wǎng)絡(luò)的分析。而對于HIV-1新近感染者傳播網(wǎng)絡(luò)特征的描述或者網(wǎng)絡(luò)動態(tài)分析,HIV-1包膜蛋白(env)和衣殼蛋白(gag)基因片段由于其進(jìn)化速率比pol基因片段快可能更適合用來分析傳播網(wǎng)絡(luò)。PCR直接測序所獲得的信息量小,一般只能檢測優(yōu)勢準(zhǔn)種序列,不能在HIV-1準(zhǔn)種水平上進(jìn)一步推斷HIV-1傳播方向。而高通量測序技術(shù)能夠檢測個體內(nèi)病毒的低水平變異,可提示傳播方向。本研究將同時對一代測序和Hiseq高通量測序(以下簡稱Hiseq測序)的HIV-1 pol、gag和env三基因區(qū)序列進(jìn)行傳播子網(wǎng)絡(luò)分析,并探討應(yīng)用價值。目的探討HIV-1 gag、env基因區(qū)序列構(gòu)建傳播網(wǎng)絡(luò)的參數(shù);探討HIV-1pol、gag、env基因區(qū)序列在傳播子網(wǎng)絡(luò)動態(tài)分析方面的應(yīng)用價值;建立基于HIV-1pol、gag、env基因區(qū)序列準(zhǔn)種群分析的Hiseq高通量測序方法;利用Hiseq高通量測序技術(shù)進(jìn)行HIV-1 pol、gag、env基因區(qū)序列傳播子網(wǎng)絡(luò)動態(tài)分析。材料和方法1.研究對象采用單純隨機(jī)抽樣的方法,從北京佑安醫(yī)院相對封閉的MSM人群HIV-1新發(fā)感染隊列中抽取了 2010年至2012年的MSM急性期感染者100人,所有受檢者采樣前均未接受抗病毒治療。分離其外周血單核細(xì)胞(PBMC)和血漿(1.5mml/支),于-8℃分別保存?zhèn)溆谩?、實驗方法(1)從PBMC樣本中提取DNA;(2)對DNA進(jìn)行巢式PCR擴(kuò)增目的片段;(3)對PCR產(chǎn)物直接測序,并進(jìn)行基因亞型分析;(4)對pol、gag、env基因區(qū)序列進(jìn)行傳播子網(wǎng)絡(luò)動態(tài)分析;(5)針對Hiseq測序設(shè)計pol、gag、env基因區(qū)引物、優(yōu)化反應(yīng)條件,對DNA進(jìn)行巢式PCR擴(kuò)增目的片段;(6)PCR產(chǎn)物純化后,構(gòu)建DNA文庫,然后進(jìn)行Hiseq測序。(7)對Hiseq測序數(shù)據(jù)進(jìn)行進(jìn)行初步處理,對HIV準(zhǔn)種群序列進(jìn)行基因離散率、系統(tǒng)進(jìn)化分析,探究其在HIV傳播子網(wǎng)絡(luò)動態(tài)分析中的應(yīng)用價值。結(jié)果1、HIV-1毒株亞型和流行狀況本研究獲得的 HIV-1 基因亞型有 CRF01_AE、CRF07_BC、B、B'、CRF55_01B、CRF65_cpx 和未知亞型,所占比例依次為:42.7%(41/96)、25.0% (24/96)、15.6%(15/96)、1.0%(1/96)、1.0%(1/96)、3.1%(3/96)和 11.6%(11/96)。其中 CRF55_O1B 和 CRF65_cpx亞型在北京首次出現(xiàn)。2、一代測序所獲pol、gag、env三區(qū)序列用于HIV-1傳播網(wǎng)絡(luò)的探究對70份樣本的pol序列進(jìn)行傳播網(wǎng)絡(luò)分析,網(wǎng)絡(luò)包含5個傳播簇,由11個節(jié)點和7條邊組成,成簇率為15.71%(11/70)。Fisher檢驗顯示,HIV-1亞型、年齡、教育、婚姻狀況和CD4+T細(xì)胞計數(shù)等影響因素中的樣本關(guān)聯(lián)性差異無統(tǒng)計學(xué)意義(Psub=0.2058,Page=0.8652, Pedu=1.000, Pmar=1.0000, PCD4=0.7568)。固定自展值(Bootstrap) ≥90%探討gag、env基因區(qū)序列用于傳播網(wǎng)絡(luò)分析的簇內(nèi)基因距離(genetic distance,GD),Fisher檢驗顯示,gag基因區(qū)序列當(dāng)GD分別小于或等于0.5%、1.5%、2.5%、3.5%時,樣本關(guān)聯(lián)性與pol基因區(qū)序列差異無統(tǒng)計學(xué)意義(Pgag0.5=0.0257、Pgag1 5=0.7083、Pgag2.5=0.0876、Pgag3.5=0.0156,αg=0.01),env基因區(qū)序列當(dāng) GD 分別小于或等于 0.5%、1.5%、2.5%、3.5%、4.5%時樣本關(guān)聯(lián)性與GD≤1.5%和自展值≥90%時的pol基因區(qū)序列差異無統(tǒng)計學(xué)意義(Penv0.5=0.0257、Penv1.5=0.0811、Penv2.5=0.3824、Penv3.5=0.3749、Penv3.5=0.0160,αe=0.0083)。對pol、gag、env三區(qū)序列不同GD值下樣本關(guān)聯(lián)性進(jìn)行比較分析,Fisher檢驗顯示,GD≤1.5%配以自展值≥90%中,gag、env兩個基因區(qū)序列在序列樣本關(guān)聯(lián)性方面差異有統(tǒng)計學(xué)意義(Pge1.5=0.0125) ; GD≤3.0%配以自展值≥90%中,pol與gag、pol與env兩個基因區(qū)序列在序列樣本關(guān)聯(lián)性方面差異有統(tǒng)計學(xué)意義(Ppg3.0=0.0008,Ppe3.00.0001) ; GD4.5%配以自展值≥90%中,pol與 env、gag與env兩個基因區(qū)序列在序列樣本關(guān)聯(lián)性方面差異有統(tǒng)計學(xué)意義(Ppe4.50.0001,Pge4.5=0.0005)。對33份樣本的pol、gag、env序列進(jìn)行動態(tài)傳播子網(wǎng)絡(luò)分析,分別提示了在HIV-1傳播中起重要作用的兩個樣本(16014、16035)、三個樣本(16014、16017、16035)和四個樣本(16014、16017、16064、16035),同時提示了可能的傳播路徑。3、Hiseq測序用于HIV-1傳播網(wǎng)絡(luò)的方法學(xué)建立以env基因區(qū)序列為代表探索Hiseq測序適用于傳播網(wǎng)絡(luò)研究的準(zhǔn)種分析數(shù)量。選取每份樣品中最優(yōu)勢的前5、10、20準(zhǔn)種序列(分別標(biāo)示為A、B、C組),三組的樣品間平均基因離散率差異無統(tǒng)計學(xué)意義(P=0.5889),經(jīng)系統(tǒng)進(jìn)化樹分析,結(jié)果表明A組的結(jié)果與B、C兩組是一致的。因此,本研究綜合考慮后,決定選取前5個準(zhǔn)種群序列進(jìn)行傳播網(wǎng)絡(luò)分析。4、Hiseq測序所獲pol、gag、env三區(qū)序列用于HIV-1傳播網(wǎng)絡(luò)的探究對CRF01_AE亞型pol、gaag、env三區(qū)測序成功的40、42、42份樣本進(jìn)行HIV-1傳播網(wǎng)絡(luò)分析,pol基因區(qū)序列的傳播子網(wǎng)絡(luò)動態(tài)分析分別提示共獲得10個子網(wǎng)絡(luò),發(fā)現(xiàn)7個在 HIV-1 傳播中起重要作用的樣本(16001、16003、16014、16029、16082、16088、16097)和3個主要路徑。gag基因區(qū)序列共獲得16個子網(wǎng)絡(luò),發(fā)現(xiàn)10個在HIV-1傳播中起重要作用的樣本(16004、16011、16014、16032、16052、16056、16061、16082、16089、16097)和3個主要路徑。env基因區(qū)序列共獲得34個子網(wǎng)絡(luò),發(fā)現(xiàn)13個在HIV-1傳播中起重要作用的樣本(16007、16011、16018、16032、16056、16060、16061、16064、16070、16082、16085、16088、16092)和 3 個主要路徑。分析 CRF07_BC 亞型pol、gag、env三區(qū)測序成功的25、29、29份樣本,pol、gag、env三區(qū)序列分別發(fā)現(xiàn)在HIV-1傳播中起重要作用的 2 個樣本(16016、16023)、6 個樣本(16024、16038、16054、16065、16075、16090)、10 個樣本(16019、16028、16031、16038、16050、16065、16067、16069、16090、16098)和一個路徑,同時提示了可能的傳播路徑。結(jié)論1、固定自展值≥90%,獲得HIV-1gag、env基因區(qū)序列用于傳播網(wǎng)絡(luò)分析的GD值,分別為 GD≤3.5%、GD≤4.5%。2、傳播子網(wǎng)絡(luò)動態(tài)分析結(jié)果顯示,一代測序三區(qū)序列獲得的樣本關(guān)聯(lián)信息由高到低依次為env、gag、pol基因區(qū)序列。3、成功建立了基于Hiseq高通量測序技術(shù)的HIV-1 pol、gag、env基因區(qū)序列準(zhǔn)種群分析方法。4、Hiseq高通量測序的結(jié)果使傳播子網(wǎng)絡(luò)分析結(jié)果更精準(zhǔn)更明確,有助于了解HIV-1傳播子網(wǎng)絡(luò)的動態(tài)過程。5、Hiseq測序技術(shù)操作較簡便,在HIV-1傳播網(wǎng)絡(luò)分析中具有一定的應(yīng)用價值。
[Abstract]:The in-depth understanding and research of the characteristics of the human immunodeficiency virus 1 (HIV-1) transmission network and the dynamics of the transmission network will contribute to the monitoring, intervention and treatment of HIV-1. At present, the analysis of HIV-1 transmission network is mainly realized by analyzing the sequence of the HIV-1 polymerase (plol) gene region. The sequence is mainly obtained by direct sequencing of the PCR products. After the analysis of the propagation network, and for the description of the transmission network characteristics of the newly infected HIV-1 or the dynamic analysis of the network, the HIV-1 envelope protein (Env) and the capsid protein (GAG) gene fragment may be more suitable for the analysis of the information obtained by the direct sequencing of the transmission network.PCR because of its faster evolution rate than the pol gene fragment. The sequence of predominant quasispecies can not be used to further infer the direction of HIV-1 propagation at the HIV-1 quasi species level. High flux sequencing technology can detect the low level variation of the virus in the individual, and can indicate the direction of transmission. This study will simultaneously treat the HIV-1 pol, gag and env three gene regions of the first generation sequencing and Hiseq high throughput sequencing (following simple Hiseq sequencing). The sequence carries on the analysis of the propagation subnetwork and discusses the application value. Objective to explore the parameters of the HIV-1 gag and env gene region sequence to construct the transmission network; explore the application value of the HIV-1pol, gag, env gene region sequence in the dynamic analysis of the propagation subnetwork; establish the Hiseq high throughput sequencing party based on the quasi population analysis of the HIV-1pol, gag, env gene region sequence. Method: using Hiseq high throughput sequencing technology to carry out dynamic analysis of HIV-1 pol, gag, env gene region sequence transmission subnetwork. Materials and methods 1. subjects used a simple random sampling method to extract 100 people from 2010 to 2012 of MSM acute infection from the relatively closed MSM population of Beijing you an hospital. Antiviral treatment was not accepted before the sample was sampled. The peripheral blood mononuclear cells (PBMC) and plasma (1.5mml/ branch) were isolated and the standby.2 was preserved at -8 C, and the experimental method (1) extracted DNA from the PBMC samples; (2) the DNA was amplified by nested PCR amplification, and (3) the PCR products were directly sequenced and the gene subtype analysis was carried out; (4) pol, gag, env genes (4) The region sequence carries on the dynamic analysis of the propagation subnetwork; (5) to design pol, gag, env gene region primers for Hiseq sequencing, optimize the reaction conditions, carry out nested PCR amplification for DNA, and (6) after the purification of the PCR product, the DNA library is constructed, and then Hiseq sequencing is carried out. (7) the Hiseq sequencing data are preliminarily processed to carry out genes for HIV quasi population sequence. Discrete rate, phylogenetic analysis, explore its application value in the dynamic analysis of HIV propagation subnetwork. Results 1, the subtypes of HIV-1 strains and epidemic status of HIV-1 gene subtypes are CRF01_AE, CRF07_BC, B, B', CRF55_01B, CRF65_cpx and unknown subtypes, which are followed by: 42.7% (41/96), 25% (24/96), 15.6% (15/96), 1% 96), 1% (1/96), 3.1% (3/96) and 11.6% (11/96). In which CRF55_O1B and CRF65_cpx subtypes appear for the first time in Beijing. The sequence of pol, gag, env three is used for the HIV-1 propagation network to explore the pol sequence of 70 samples. The network contains 5 propagation clusters consisting of 11 nodes and 7 edges, and the clustering rate is 15.7 1% (11/70).Fisher test showed that there was no statistical difference in the correlation between HIV-1 subtypes, age, education, marital status and CD4+T cell count (Psub=0.2058, Page=0.8652, Pedu=1.000, Pmar=1.0000, PCD4=0.7568). The fixed self spreading value (Bootstrap) > 90% explored gag, and the env gene region sequence was used for the transmission of network analysis. Genetic distance, GD, and Fisher test showed that when GD was less than or equal to or equal to 0.5%, 1.5%, 2.5%, 3.5%, there was no statistical difference between the correlation of the gag gene and the sequence of the pol gene region (Pgag0.5=0.0257, Pgag1 5=0.7083, Pgag2.5=0.0876, Pgag3.5=0.0156, and alpha). 0.5%, 1.5%, 1.5%, 2.5%, 3.5%, 4.5%, there was no statistical difference between the correlation of the sample and the pol gene region sequence of GD < 1.5% and the self spreading value > 90% (Penv0.5=0.0257, Penv1.5=0.0811, Penv2.5=0.3824, Penv3.5=0.3749, Penv3.5=0.0160, alpha e=0.0083). The results showed that GD < 1.5% was equal to the self spreading value more than 90%, and the sequence of two gene regions of gag and env was statistically significant (Pge1.5=0.0125), and GD < 3% was matched with the self spreading value more than 90%. The difference between pol and gag, pol and env two gene sequences was statistically significant (Ppg3.0=0.0008, Ppe3.00.0001). There were significant differences in the correlation between pol and env, gag and env sequences in the sequence samples (Ppe4.50.0001, Pge4.5=0.0005) with the self spreading value of more than 90% (Ppe4.50.0001, Pge4.5=0.0005). The dynamic propagation subnetwork analysis of pol, gag and env sequences of 33 samples showed that two samples played an important role in the HIV-1 propagation (16014160). 35), three samples (160141601716035) and four samples (16014160171606416035), indicating the possible propagation path.3. The method of Hiseq sequencing for the HIV-1 transmission network is to establish the env gene region sequence as the representative of the quasi species analysis suitable for the study of the transmission network for the exploration of the Hiseq sequencing. 5,10,20 quasi species sequence (labeled A, B, C group respectively), the difference of average gene discrepancy between the three groups was not statistically significant (P=0.5889). The results of phylogenetic tree analysis showed that the results of group A were in agreement with the group of B and C two. Therefore, after comprehensive consideration, the first 5 quasi population sequences were determined to be transmitted network analysis.4, Hiseq sequencing. The pol, gag, env three region sequence was used for the HIV-1 transmission network to investigate the HIV-1 propagation network analysis on the 40,42,42 samples of the CRF01_AE subtype pol, GAAG, env three sequence, and the propagation subnetwork dynamic analysis of the pol gene region sequence showed that 10 sub networks were obtained, and 7 samples (160) were found to play an important role in the HIV-1 propagation. 16 sub networks were obtained from the sequence of 3 main path.Gag gene regions, and 10 samples (16004160111601416032160521605616061160821608916097) which played an important role in the transmission of HIV-1 and the sequence of the 3 main path.Env gene regions were found to have 34 sub networks, and 13 were found in the transmission of HIV-1. Samples of important roles (16007160111601816032160561606016061160641607016082160851608816092) and 3 main paths were used to analyze the successful 25,29,29 samples of the CRF07_BC subtype pol, gag and env three sequences. The pol, gag, env three region sequences found 2 samples (1601616023) that played an important role in the transmission of HIV-1, respectively. 6 samples (160241603816054160651607516090), 10 samples (16019160281603116038160501606516067160691609016098) and one path were used to indicate the possible propagation path. Conclusion 1, the fixed self spreading value is more than 90%, and HIV-1gag is obtained. The GD value of the env gene region sequence is used for the transmission network network analysis, which is GD < 3.5%, GD < 4.5, respectively. %.2, the dynamic analysis of the propagation subnetwork shows that the sample association information obtained by the sequence of three sequences of the first generation sequence is from high to low to Env, gag, and pol gene region sequence.3. The HIV-1 pol based on Hiseq high throughput sequencing technology is successfully established, the gag, env gene region sequence quasi population segregation method is.4, and the result of high throughput sequencing of Hiseq is the propagation subnetwork. The analysis results are more accurate and clearer. It is helpful to understand the dynamic process of the HIV-1 propagation subnetwork (.5). The Hiseq sequencing technology is more convenient and has a certain application value in the HIV-1 transmission network analysis.
【學(xué)位授予單位】：中國疾病預(yù)防控制中心
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：R512.91

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4 倪培相;;新一代高通量測序技術(shù)在微生物基因組學(xué)研究中的應(yīng)用[A];2010年中國科學(xué)院微生物研究所博士后學(xué)術(shù)年會暨第二屆博誼論壇論文摘要集[C];2011年

5 王楷[，

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