Comparison of digital PCR platforms and semi-nested qPCR as a tool to determine the size of the HIV reservoir.
Comparison of digital PCR platforms and semi-nested qPCR as a tool to determine the size of the HIV reservoir.
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DOI：
10.1038/srep13811
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发表时间：
2015-09-09
影响因子：
4.6
通讯作者：
De Spiegelaere W
中科院分区：
文献类型：
--
作者：
Bosman KJ;Nijhuis M;van Ham PM;Wensing AM;Vervisch K;Vandekerckhove L;De Spiegelaere W
HIV在接受抗逆转录病毒治疗（ART）的患者的潜伏感染细胞中持续存在。这种持久的前病毒DNA储库是治疗失败或中断时病毒反弹的重要预测因子，并形成治愈的主要障碍。准确定量低水平的持续HIV DNA可能有助于患者监测和治疗研究。数字PCR是一种很有前途的工具，它能够以高灵敏度进行直接绝对定量。随着最近的技术进步，有几个平台可用于在临床环境中实施数字PCR。在这里，我们比较了两种数字PCR平台，Quantstudio 3D（Life Technologies）和QX 100（Bio-Rad），以及半巢式qPCR，用于检测HIV DNA系列稀释液和从ART抑制患者PBMC中分离的DNA。所有三种方法都能够检测到2.5个HIV DNA拷贝的最低水平。QX 100的优势在于具有最小的偏倚和最高的精密度、效率和定量线性。通过Bland-Altman分析，QX 100和半巢式qPCR的患者样本定量结果高度一致（0.01 ± 0.32 log 10）。然而，由于使用当前的数字PCR平台观察到假阳性信号，半巢式qPCR在低量检测的设置中可能仍然是优选的，以区分HIV DNA的存在或不存在。
HIV persists in latently infected cells of patients on antiretroviral therapy (ART). This persistent proviral DNA reservoir is an important predictor of viral rebound upon therapy failure or interruption and forms a major obstacle towards cure. Accurate quantification of the low levels of persisting HIV DNA may aid patient monitoring and cure research. Digital PCR is a promising tool that enables direct absolute quantification with high sensitivity. With recent technological advances, several platforms are available to implement digital PCR in a clinical setting. Here, we compared two digital PCR platforms, the Quantstudio 3D (Life Technologies) and the QX100 (Bio-Rad) with a semi-nested qPCR on serial HIV DNA dilutions and DNA isolated from PBMCs of ART-suppressed patients. All three methods were able to detect target to the lowest levels of 2.5 HIV DNA copies. The QX100 excelled in having the least bias and highest precision, efficiency and quantitative linearity. Patient sample quantifications by the QX100 and semi-nested qPCR were highly agreeable by Bland-Altman analysis (0.01 ± 0.32 log10). Due to the observation of false-positive signals with current digital PCR platforms however, semi-nested qPCR may still be preferred in a setup of low quantity detection to discriminate between presence or absence of HIV DNA.