Quantification of Cell-associated HIV-1 DNA

Total HIV-1 DNA was measured in purified peripheral blood mononuclear cells (PBMCs) by real-time quantitative polymerase chain reaction (qPCR). Total nucleic acids were extracted from PBMCs on the QIAsymphony platform using the DSP Virus/Pathogen Mini Kit (Qiagen) according to the manufacturer’s protocol. Primers were designed for HIV-1 long terminal repeat and the genomic reference gene pyruvate dehydrogenase as previously described [11 (link)]. Quantification of cell-associated HIV-1 DNA by qPCR was determined using 20 μL of extract and a standard curve with known copy numbers in 10-fold dilutions. The limit of quantitation is 10 copies/10⁶ PBMCs and the limit of detection is 1 copy/10⁶ PBMCs. Assays were performed in triplicate.

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Foster C., Domínguez-Rodríguez S., Tagarro A., Gkouleli T., Heaney J., Watters S., Bamford A., Fidler K., Navarro M., De Rossi A., Palma P., Nastouli E., Rossi P., Giaquinto C, & Rojo P. (2020). The CARMA Study: Early Infant Antiretroviral Therapy—Timing Impacts on Total HIV-1 DNA Quantitation 12 Years Later. Journal of the Pediatric Infectious Diseases Society, 10(3), 295-301.

Publication 2020

QIAsymphony platform DSP Virus/Pathogen Mini Kit

Assays Cell Dehydrogenase Dilutions Gene Genomic Hiv 1 Hiv long terminal repeat Nucleic acids Pathogen Peripheral blood mononuclear cells Polymerase chain reaction Primers Pyruvate Real time polymerase chain reaction Virus

Corresponding Organization : Imperial College Healthcare NHS Trust

Other organizations : Great Ormond Street Hospital, University College London, University College London Hospitals NHS Foundation Trust, Brighton and Sussex Medical School, Hospital General Universitario Gregorio Marañón, University of Padua, Bambino Gesù Children's Hospital

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Variable analysis

independent variables

None explicitly mentioned

dependent variables

Total HIV-1 DNA measured in purified peripheral blood mononuclear cells (PBMCs) by real-time quantitative polymerase chain reaction (qPCR)

control variables

Primer design for HIV-1 long terminal repeat and the genomic reference gene pyruvate dehydrogenase as previously described [11]
Standard curve with known copy numbers in 10-fold dilutions used for quantification of cell-associated HIV-1 DNA by qPCR
Limit of quantitation of 10 copies/10^6 PBMCs and limit of detection of 1 copy/10^6 PBMCs
Assays performed in triplicate

controls

Positive control: Not specified
Negative control: Not specified

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