Quantification of HIV DNA Using Droplet Digital PCR Techniques.
Quantification of HIV DNA Using Droplet Digital PCR Techniques.
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DOI：
10.1002/cpmc.62
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发表时间：
2018-11
影响因子：
--
通讯作者：
Maldarelli F
中科院分区：
文献类型：
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作者：
Anderson EM;Maldarelli F
尽管在长寿细胞中进行了有效的抗逆转录病毒治疗，但艾滋病毒仍然存在，这是治愈的主要障碍。HIV复制周期中的关键步骤和逆转录病毒科家族的标志是病毒DNA整合到宿主基因组中。一旦整合，HIV的表达就受到宿主机制的调节，并且前病毒持续存在直到细胞死亡。具有复制能力的前病毒的细胞库可以存活多年，维持该库的机制正在研究中。然而，大多数整合的前病毒是有缺陷的或具有大的缺失，并且在治疗期间前病毒景观的组成仍然未知。定量HIV前病毒的方法在调查HIV持续性方面很有用，本单元介绍了一种利用下一代PCR平台（数字PCR）对各种HIV基因组靶点进行总HIV DNA定量的方法。各种HIV基因靶标的丰度反映了总体前病毒组成。在该方案中，从患者来源的细胞中分离总基因组DNA，其用作液滴数字PCR的模板，其中PCR反应被分配成约20，000个单独的液滴，PCR扩增至终点，并通过计数阳性和阴性液滴的数量进行绝对定量。使用简单的泊松校正直接计算拷贝数。此外，该方法学途径可用于获得其他DNA靶标的绝对定量。
HIV persists despite effective antiretroviral therapy in long lived cells, posing a major barrier towards a cure. A key step in the HIV replication cycle and a hallmark of the Retroviridae family is the integration of the viral DNA into the host genome. Once integrated, HIV expression is regulated by host machinery and the provirus persists until the cell dies. A reservoir of cells harboring replication competent proviruses can survive for years, and mechanisms that maintain that reservoir are under investigation. The majority of integrated proviruses, however, are defective or have large deletions, and the composition of the proviral landscape during therapy remains unknown. Methods to quantify HIV proviruses are useful in investigating HIV persistence, and presented in this unit is a method for total HIV DNA quantification of various HIV genome targets that utilizes the next generation PCR platform, digital PCR. The abundance of various HIV gene targets reflects the overall proviral composition. In this protocol, total genomic DNA is isolated from patient derived cells, which is used as a template for droplet digital PCR in which the PCR reaction is partitioned into approximately 20,000 individual droplets, PCR amplified to an end point, and subjected to absolute quantification by counting the number of positive and negative droplets. Copy number is directly calculated using straightforward Poisson correction. Additionally, this methodological approach can be used to obtain absolute quantification of other DNA targets.