Triplex digital PCR assays for the quantification of intact proviral HIV-1 DNA.

• We present a detailed protocol for a novel triplex dPCR assay for the quantification of the intact HIV-1 DNA reservoir. • We assessed and evaluated the performance of these assays on HIV-1 control cell line material and HIV-1 patient samples. • The triplex assays were implemented on the Naica dPCR platform (Stilla). 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. [ABSTRACT FROM AUTHOR]