论文部分内容阅读
目的建立针对艾滋病病毒I型(HIV-1)三个基因区片段的Illumina Hiseq深度测序(简称Hiseq测序)方法,并尝试用于分析抗病毒治疗急性期感染者的HIV-1准种群变化规律。方法对2例在急性期启动抗病毒治疗的HIV-1感染者进行随访检测,直至第96周。提取外周血单个核细胞(PBMC)中的脱氧核糖核酸(DNA),对基线血液样本进行HIV-1基因亚型分析。分别以gag、pol和env基因区为目的片段,对基线及随访样本进行巢式聚合酶链反应(PCR),扩增产物经一代测序确认无误后纯化、混合构建DNA文库,进行Hiseq测序。分析HIV-1准种群分布,计算基因离散率,并做系统进化树分析。结果接受抗病毒治疗后,2例感染者体内的HIV-1病毒载量下降,到第12周后无法检出;CD4+T淋巴细胞数在治疗第2、4周时有所下降,随后回升。2例感染的HIV-1毒株分别为CRF01_AE亚型和CRF07_BC亚型。核酸扩增成功的样本全部成功进行了Hiseq测序,每个目的基因区片段都获得了十万级的序列数。在基线点,频数最高的第一个准种群序列的频数占全部序列总频数的比例均超过50%。抗病毒治疗后,准种群的分布发生不同程度的变化,其中频数第一准种群频数所占比例先减小,从第12周起多数增大,第72周再减小。样本内平均基因离散率整体稳定,小范围波动。每个样本3个基因区各随访时间点与基线点之间的平均基因离散率在治疗第2周增大,随后保持稳定。系统进化树显示,不同时间的准种群序列散在分布且距离接近。结论针对HIV-1 gag、pol和env基因区的Hiseq测序方法可有效用于分析HIV-1准种群的变化规律,早期抗病毒治疗可降低病毒载量调定点且延长准种群分散时间。
Objective To develop an Illumina Hiseq sequencing (Hiseq sequencing) method for HIV-1 HIV-1 gene fragments and try to analyze the variation of HIV-1 quasi-population in the acute phase of antiviral therapy. Methods Two HIV-1 infected patients who started anti-virus therapy in the acute phase were followed up until the 96th week. DNA was extracted from peripheral blood mononuclear cells (PBMCs) and HIV-1 genotypes were analyzed for baseline blood samples. Nested polymerase chain reaction (PCR) was performed on the base line and the follow-up samples using the gag, pol and env gene regions respectively. The amplified products were purified by one-generation sequencing and then mixed to construct a DNA library for Hiseq sequencing. Analysis of HIV-1 quasi-population distribution, calculation of genetic dispersion, and phylogenetic tree analysis. Results After receiving antiviral therapy, the HIV-1 viral load decreased in 2 infected persons and could not be detected after 12 weeks. The number of CD4 + T lymphocytes decreased at the 2nd and 4th week of treatment and then rose . The two infected HIV-1 strains were CRF01_AE subtype and CRF07_BC subtype respectively. All of the successful samples for nucleic acid amplification were successfully sequenced by Hiseq, and 100,000 fragments were obtained for each target gene fragment. At the baseline point, the ratio of the frequency of the first quasi-population sequence with the highest frequency to the total sequence frequency of the entire sequence exceeds 50%. After antiviral treatment, the distribution of quasi-population varied in varying degrees, of which the proportion of the frequency of the first quasi-population frequency first decreased, the majority increased from the twelfth week and then decreased again at the 72nd week. The average gene dispersion rate in the sample is stable and fluctuates in a small range. The mean gene dispersal rate at each of the 3 gene regions at each follow-up time point and baseline was increased at 2 weeks of treatment and then remained steady. Phylogenetic tree shows that the quasi-population sequences are scattered and close at different times. Conclusion The Hiseq sequencing method targeting HIV-1 gag, pol and env genes can be used to analyze the variation of HIV-1 quasi-population. Early antiviral therapy can reduce the viral load set point and prolong quasi-population dispersal time.