Triplex digital PCR assays for the quantification of intact proviral HIV-1 DNA
| Autor: | Linos Vandekerckhove, Willem van Snippenberg, Ward De Spiegelaere, David Gleerup, Wim Trypsteen, Sofie Rutsaert |
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| Rok vydání: | 2021 |
| Předmět: |
0303 health sciences
030302 biochemistry & molecular biology HIV Infections Biology Real-Time Polymerase Chain Reaction Virology Jurkat cells General Biochemistry Genetics and Molecular Biology 03 medical and health sciences Viral replication Proviruses DNA Viral HIV-1 Humans Digital polymerase chain reaction Hiv 1 dna Molecular Biology 030304 developmental biology Subgenomic mRNA |
| Zdroj: | Methods (San Diego, Calif.). 201 |
| ISSN: | 1095-9130 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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