HIV-1 RNA was extracted from plasma with QIAamp Viral RNA Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer’s protocol with on-column DNase I treatment. HIV-1 plasma viral load was quantified with RNA UltraSense One-Step Quantitative RT-PCR System (Invitrogen, Waltham, MA) using the following conditions: 50°C for 15 min; 95°C for 2 min; 40 cycles at 95°C for 15 sec and 60°C for 1 min. The primers and probe target the HIV-1 gag as previously described [17 (link)]. AcroMetrix HIV-1 panel (ThermoFisher Scientific, Fremont, CA) was used for standard curve.
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