Combination of quadruplex qPCR and next-generation sequencing for qualitative and quantitative analysis of the HIV-1 latent reservoir
Combination of quadruplex qPCR and next-generation sequencing for qualitative and quantitative analysis of the HIV-1 latent reservoir
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DOI：
10.1084/jem.20190896
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发表时间：
2019-10-01
影响因子：
15.3
通讯作者：
Nussenzweig, Michel C.
中科院分区：
文献类型：
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作者：
Gaebler, Christian;Lorenzi, Julio C. C.;Nussenzweig, Michel C.
HIV-1感染需要终生使用抗逆转录病毒药物治疗，因为存在转录不活跃的整合前病毒的潜在储存库。HIV-1治愈研究的目标是消除或在功能上沉默这个储蓄者。为此，有许多正在进行的研究，以评估免疫学方法，包括单抗疗法。评估这些研究的结果需要对储集层采取敏感和具体的措施。在这里，我们描述了一种相对高通量的结合定量聚合酶链式反应(QPCR)和下一代测序的方法。覆盖包装信号(PS)、组特异性抗原(GAG)、聚合酶(POL)和包膜(Env)的四种不同的qPCR探针被组合在一个单一的多重反应中，以检测有限稀释样本中的HIV-1基因组，然后对四种探针中的任何两种组合呈阳性的个别反应进行序列验证(Q4PCR)。这一敏感和具体的方法使得能够公正地描述艾滋病毒-1潜伏宿主的特征。
HIV-1 infection requires lifelong therapy with antiretroviral drugs due to the existence of a latent reservoir of transcriptionally inactive integrated proviruses. The goal of HIV-1 cure research is to eliminate or functionally silence this reservoir. To this end, there are numerous ongoing studies to evaluate immunological approaches, including monoclonal antibody therapies. Evaluating the results of these studies requires sensitive and specific measures of the reservoir. Here, we describe a relatively high-throughput combined quantitative PCR (qPCR) and next-generation sequencing method. Four different qPCR probes covering the packaging signal (PS), group-specific antigen (gag), polymerase (pol), and envelope (env) are combined in a single multiplex reaction to detect the HIV-1 genome in limiting dilution samples followed by sequence verification of individual reactions that are positive for combinations of any two of the four probes (Q4PCR). This sensitive and specific approach allows for an unbiased characterization of the HIV-1 latent reservoir.