van Snippenberg Willem, Gleerup David, Rutsaert Sofie, Vandekerckhove Linos, De Spiegelaere Ward, Trypsteen Wim
HIV Cure Research Centre, Department of Internal Medicine and Pediatrics, Ghent University and Ghent University Hospital, Belgium; Ghent University Digital PCR Consortium, Ghent University, Belgium.
Ghent University Digital PCR Consortium, Ghent University, Belgium; Department of Morphology, Ghent University, Belgium.
Methods. 2022 May;201:41-48. doi: 10.1016/j.ymeth.2021.05.006. Epub 2021 May 13.
The development of an HIV-1 cure is hampered by the existence of a persistent (latent) reservoir that contains a small proportion of replication-competent intact proviruses which refuels viral replication upon treatment discontinuation. Therefore, an accurate evaluation and quantification of these (intact) proviruses is essential to determine the efficacy of HIV-1 cure strategies which aim to eliminate this reservoir. Here, we present two triplex digital PCR assays which resulted from a combination of two existing methods, the IPDA (a 2-colour digital PCR based method) and Q4PCR assays (4 colour qPCR method), and tested the functionality on a three-colour digital PCR platform. In the present paper, we provide a step-by-step experimental protocol for these triplex digital PCR assays and validate their performance on a latently infected Jurkat cell-line model and HIV-1 patient samples. Our data demonstrates the potential and flexibility of increasing the number of subgenomic regions of HIV-1 within the IPDA to acquire sensitive detection of the HIV-1 reservoir while benefitting from the advantages of a dPCR setup.
HIV-1治愈方法的研发受到持续（潜伏）病毒库的阻碍，该病毒库中含有一小部分具有复制能力的完整前病毒，在停止治疗后会重新引发病毒复制。因此，准确评估和定量这些（完整的）前病毒对于确定旨在消除该病毒库的HIV-1治愈策略的疗效至关重要。在此，我们展示了两种三重数字PCR检测方法，它们是由两种现有方法（IPDA，一种基于双色数字PCR的方法和Q4PCR检测方法，四色定量PCR方法）组合而成，并在三色数字PCR平台上测试了其功能。在本文中，我们提供了这些三重数字PCR检测方法的详细实验方案，并在潜伏感染的Jurkat细胞系模型和HIV-1患者样本上验证了它们的性能。我们的数据证明了在IPDA中增加HIV-1亚基因组区域数量以实现对HIV-1病毒库的灵敏检测的潜力和灵活性，同时受益于数字PCR设置的优势。
