Droplet digital polymerase chain reaction (ddPCR)

The concentration of HIV-1 DNA copies in each sample was determined using the following primers and probes spanning HXB2 coordinates 684–810 (Malnati et al., 2008): LTRgagF: 5’-TCTCGACGCAGGACTCG-3’, LTRgagR: 5’- TACTGACGCTCTCGCACC- 3’, and LTRgagProbe: 5’/56-FAM/CTCTCTCCT/ZEN/TCTAGCCTC/31ABkFQ/- 3’. Cell concentration was determined using the following RPP30 primers and probes: RPP30-F: 5’- GATTTGGACCTGCGAGCG-3’, RPP30-R: 5’-GCGGCTGTCTCCACAAGT-3’, RPP30-Probe: 5’-/56-FAM/CTGACCTGA/ZEN/AGGCTCT/31ABkFQ/- 3’ (Hindson et al., 2011). LTRgag ddPCR reactions contained the following: 10 μl ddPCR Supermix for Probes (no dUTP) (Bio-Rad), 1 μl each primer (LTRgagF and LTRgagR) at a concentration of 20 μM, 0.35 μl of LTRgag probe at a concentration of 20 μM, 6.65 μl DEPC-treated water (Thermo Fisher Scientific), and 3 μl of undiluted DNA for a total volume of 22 μl. RPP30 ddPCR reactions contained the following: 10 μl ddPCR Supermix for Probes (no dUTP) (Bio-Rad), 1 μl each primer (RPP30-F and RPP30-R) at a concentration of 20 μM, 0.35 μl of RPP30 probe at a concentration of 20 μM, 8.65 μl DEPC-treated water (Thermo Fisher Scientific), and 1 μl of DNA diluted 1:100 in DEPC-treated water. 8E5 DNA was used as a positive control and no-template controls were included in each run. Samples were run in duplicate and the values averaged.

Droplets were generated using the QX200 Automated Droplet Generator (Bio-Rad), in a final volume of 40 μl. Following droplet generation, plates were sealed with a pierceable foil seal and immediately subjected to the following PCR conditions on a C1000 Touch Thermal Cycler with 96-Deep Well Reaction Module (Bio-Rad): 95°C for 10 min, then 45 cycles of 95°C for 30 s followed by 60°C for 1 min, a final extension of 98°C for 10 min and a 4°C hold. Following thermal cycling, plates were read on the QX200 Droplet Reader (Bio-Rad) with the following set-up: Experiment = Rare Event Detection, Mix = ddPCR Supermix for Probes (no dUTP), Target 1 = FAM, Target 2 = VIC.

Analysis of ddPCR data used QuantaSoft (Bio-Rad) with thresholds for the detection of each target set using the negative controls. The same threshold was used for all reactions of a particular target (either LTRgag or RPP30) in a given run. The concentration of HIV-1 copies / μl of sample was calculated as follows: Quantasoft LTRgag concentration ×(total volume of PCR reaction/volume of DNA added). The concentration of cells/μl of sample was calculated as follows: Quantasoft RPP30 concentration x (total volume of PCR reaction/volume of DNA added) x dilution factor x 0.5.