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

(2022) METHODS. 201. p.41-48
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Abstract
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.
Keywords
General Biochemistry, Genetics and Molecular Biology, Molecular Biology, dPCR, HIV-1, Triplex, IPDA, HIV-1 DNA, Stilla, T-CELLS

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MLA
van Snippenberg, Willem, et al. “Triplex Digital PCR Assays for the Quantification of Intact Proviral HIV-1 DNA.” METHODS, vol. 201, 2022, pp. 41–48, doi:10.1016/j.ymeth.2021.05.006.
APA
van Snippenberg, W., Gleerup, D., Rutsaert, S., Vandekerckhove, L., De Spiegelaere, W., & Trypsteen, W. (2022). Triplex digital PCR assays for the quantification of intact proviral HIV-1 DNA. METHODS, 201, 41–48. https://doi.org/10.1016/j.ymeth.2021.05.006
Chicago author-date
Snippenberg, Willem van, David Gleerup, Sofie Rutsaert, Linos Vandekerckhove, Ward De Spiegelaere, and Wim Trypsteen. 2022. “Triplex Digital PCR Assays for the Quantification of Intact Proviral HIV-1 DNA.” METHODS 201: 41–48. https://doi.org/10.1016/j.ymeth.2021.05.006.
Chicago author-date (all authors)
van Snippenberg, Willem, David Gleerup, Sofie Rutsaert, Linos Vandekerckhove, Ward De Spiegelaere, and Wim Trypsteen. 2022. “Triplex Digital PCR Assays for the Quantification of Intact Proviral HIV-1 DNA.” METHODS 201: 41–48. doi:10.1016/j.ymeth.2021.05.006.
Vancouver
1.
van Snippenberg W, Gleerup D, Rutsaert S, Vandekerckhove L, De Spiegelaere W, Trypsteen W. Triplex digital PCR assays for the quantification of intact proviral HIV-1 DNA. METHODS. 2022;201:41–8.
IEEE
[1]
W. van Snippenberg, D. Gleerup, S. Rutsaert, L. Vandekerckhove, W. De Spiegelaere, and W. Trypsteen, “Triplex digital PCR assays for the quantification of intact proviral HIV-1 DNA,” METHODS, vol. 201, pp. 41–48, 2022.
@article{8713004,
  abstract     = {{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.}},
  author       = {{van Snippenberg, Willem and Gleerup, David and Rutsaert, Sofie and Vandekerckhove, Linos and De Spiegelaere, Ward and Trypsteen, Wim}},
  isbn         = {{1095-9130}},
  issn         = {{1046-2023}},
  journal      = {{METHODS}},
  keywords     = {{General Biochemistry,Genetics and Molecular Biology,Molecular Biology,dPCR,HIV-1,Triplex,IPDA,HIV-1 DNA,Stilla,T-CELLS}},
  language     = {{eng}},
  pages        = {{41--48}},
  title        = {{Triplex digital PCR assays for the quantification of intact proviral HIV-1 DNA}},
  url          = {{http://doi.org/10.1016/j.ymeth.2021.05.006}},
  volume       = {{201}},
  year         = {{2022}},
}

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