基于三个基因区片段的Hiseq高通量测序用于HIV-1传播方向调查  

Investigation of HIV-1 transmission direction by using Hiseq high-throughput sequencing based on three gene region fragments

作　　者：陈宇 杨锦 冯凯迪 龚渝蓉 潘品良[1] 王继宝 姚均[1] 段星 段松 邱茂锋[1] CHEN Yu;YANG Jin;FENG Kaidi;GONG Yurong;PAN Pinliang;WANG Jibao;YAO Jun;DUAN Xing;DUAN Song;QIU Maofeng(National Center for AIDS/STD Control and Prevention,Chinese Center for Disease Control and Prevention,Beijing 102206,China;Dehong Prefecture Center for Disease Prevention and Control,Yunnan Mangshi 678400;Weifang Medical University,Weifang 261053,Shandong)

机构地区：[1]中国疾病预防控制中心性病艾滋病预防控制中心,北京102206 [2]德宏傣族景颇族自治州疾病预防控制中心,云南芒市678400 [3]潍坊医学院,山东潍坊261053

出　　处：《中国艾滋病性病》2023年第4期372-377,共6页Chinese Journal of Aids & STD

基　　金：国家“十三五”科技重大专项(2017ZX10201101-002-003,2018ZX10721-102-003-002)。

摘　　要：目的 研究将基于HIV-1三个基因区(gag、pol、env)片段的Hiseq高通量测序和系统进化树分析用于HIV-1传播方向调查。方法 采集有明确HIV-1传播关系的3对夫妻的血液样本,提取全血中的DNA,进行HIV-1基因亚型分析。针对HIV-1 gag、pol、env基因区片段(长度依次为385 bp、388 bp、352 bp)分别进行巢式PCR扩增,将每份血样的三个基因区片段PCR产物纯化后混合构建DNA文库、Hiseq测序,根据测序结果分析HIV-1准种群分布,计算样本内、样本间的平均基因离散率,使用最大似然法构建所用优势准种群序列数由少到多(20、50、100、全部)时所得的系统进化树。结果 每对夫妻两人感染的HIV-1基因亚型分别相同。采用Hiseq测序,各基因区片段均获得了十万级的有效核酸序列数,代表71~793个独特准种群序列。样本内平均基因离散率分别为gag区0.5%~0.7%、pol区0.5%~1.0%、env区0.7%~4.4%,样本间平均基因离散率分别为gag区0.6%~17.3%、pol区0.6%~13.0%、env区1.6%~19.8%。利用不同数量的优势准种群序列构建系统进化树、结合不同靶基因片段的结果进行综合分析,每对夫妻两人间的传播方向均与背景信息相符。所用gag基因区片段在推断HIV-1传播方向方面的价值低于env和pol基因区片段的价值。结论 Hiseq测序可以有效用于HIV-1传播方向调查,不同基因区片段、不同优势准种群序列数分析结果的结合使用可以提高分析结果的可靠性。Objective To develop an approach for investigation of HIV-1 transmission direction by using Hiseq highthroughput sequencing based on HIV-1 gag, pol, and env gene region fragments and phylogenetic analysis. Methods Three couples with marital HIV-1 transmission were selected as the research subjects. The DNAs were extracted from their whole blood specimens. After HIV-1 subtyping, nested PCRs were performed for HIV-1 gag, pol, and env gene region fragments with lengths of 385 bp、388 bp、352 bp, respectively. The PCR products of three gene region fragments from each specimen were purified and mixed to construct a DNA library, followed by Hiseq sequencing. After data cleaning, the frequency of quasispecies from each specimen was counted and ranked. The intrapersonal and interpersonal genetic distance was calculated by using all quasispecies, and maximum-likelihood phylogenetic trees were constructed by using the top 20, 50, 100 and all quasispecies, respectively. Results The subtype of HIV-1 from each individual was the same as his or her spouse. Hiseq sequencing was successfully conducted on all 6 specimens and one hundred thousand levels of cleaned sequences representing 71 to 793 unique HIV-1 quasispecies were obtained. The intrapersonal average genetic distances on gag, pol, and env gene regions fragments were 0.5%-0.7%, 0.5%-1.0%, and 0.7%-4.4%, respectively. While the interpersonal average genetic distance on these fragments was 0.6%-17.3%, 0.6%-13.0%, and 1.6%-19.8%, respectively. The comprehensive results of phylogenetic analysis based on the various number of top quasispecies sequences and various target gene region fragments indicated that the HIV-1 transmission direction within the 3 couples was concordant with the corresponding background information. The value of the HIV-1 gag gene region fragment in identifying transmission direction was lower than those of the env and pol gene region fragments. Conclusions Hiseq high-throughput sequencing could effectively apply to investigate HIV-1 transmission dire

关 键 词：1型艾滋病病毒 分子流行病学 高通量测序 准种 系统发育分析 

分 类 号：R512.91[医药卫生—内科学] R373.9[医药卫生—临床医学]