Quantifying HIV DNA Intactness

CD4+ T cells were isolated from fresh or cryopreserved PBMCs using EasySep™ Human CD4+ T Cell Isolation Kit (#17952, StemCell technologies), after which genomic DNA was extracted using QIAamp DNA Mini Kits (#51304, Qiagen). Total and intact HIV DNA were measured simultaneously with the Rainbow proviral DNA digital PCR assay (38 (link)), using the QIAcuity digital PCR platform. This multiplex assay includes RU5 primers and probes for total HIV DNA detection (76 (link)) and the psi and env primers and probes of the IPDA assay (39 (link)). RPP30 was used as a reference gene (38 (link)). Intactness was quantified based on the detection of env and psi and corrected for shearing using a DNA Shearing Index (DSI) correction, as previously described (39 (link)). Defective HIV DNA was calculated by subtracting intact from total HIV DNA.

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De Clercq J., De Scheerder M.A., Mortier V., Verhofstede C., Vandecasteele S.J., Allard S.D., Necsoi C., De Wit S., Gerlo S, & Vandekerckhove L. (2024). Longitudinal patterns of inflammatory mediators after acute HIV infection correlate to intact and total reservoir. Frontiers in Immunology, 14, 1337316.

Publication 2023

EasySep™ Human CD4+ T Cell Isolation Kit QIAamp DNA Mini Kits

Corresponding Organization : Ghent University Hospital

Other organizations : AZ Sint-Jan, Vrije Universiteit Brussel, Universitair Ziekenhuis Brussel, Université Libre de Bruxelles, Centre Hospitalier Universitaire de Saint-Pierre

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Variable analysis

independent variables

Fresh or cryopreserved PBMCs

dependent variables

Total HIV DNA
Intact HIV DNA
Defective HIV DNA

control variables

CD4+ T cells isolated using EasySep™ Human CD4+ T Cell Isolation Kit
Genomic DNA extracted using QIAamp DNA Mini Kits
RPP30 used as a reference gene

controls

Positive control: None specified
Negative control: None specified

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