Quantifying HIV-1 DNA in Purified Lymphoid Cells

Lysed extracts from purified LPLs (CD45⁺ cells), bulk LPs and PBMCs from the same participants were simultaneously obtained and used to measure cell-associated HIV-1 DNA by ddPCR. A total of 2–3 μl of cell lysates were mixed with ddPCR supermix and primers/probes (FAM/HEX-ZEN-Iowa BlackFQ double-quenched probes, Integrated DNA Technologies) in a total volume of 20 μl to generate droplets, according to manufacturer’s recommendations. We managed to circumvent potential primer mismatch in individuals’ viral sequence by using two different primers/probe sets annealing to the 5’LTR and GAG conserved regions of HIV-1 genome [21 (link)–23 (link)] (Table 1). Here, ddPCR Supermix for Residual DNA quantification was used (186–4037, Bio-Rad). Annealing/extension step was set at 57°C to quantify vDNA using the C1000 Touch^™ Thermal Cycler (Bio-Rad) and subsequently analyzed using a QX100^™ droplet reader (Bio-Rad) and the QuantaSoft v.1.6 software (Bio-Rad).
PBMCs, bulk LPs and LPLs samples were quantified in duplicate or triplicate, respectively. PBMCs from HIV-negative donors were used as negative controls and assayed in each plate to set the positive/negative threshold for ddPCR analysis, and the number of those negative control wells was the same than replicas for each sample. The RPP30 cellular gene was quantified in parallel to normalize sample input (Table 1) [24 (link)].