Gotaq probe rt qpcr system

Viral and proviral HIV copy numbers were determined using RT-qPCR and qPCR, respectively. Viral RNA samples were tested using the GoTaq Probe RT-qPCR System (Promega Corporation, WI, USA), and proviral samples tested using the iTaq Universal Probes Supermix (Bio-Rad Laboratories, CA, USA), as per the manufacturer’s instructions. See S6 Table for qPCR cycling conditions and S7 Table for the primer sequences. HIV sequences were custom synthesised into gBlocks (Integrated DNA Technologies, IA, USA) for use as a standard at an initial concentration of 1×10⁸ copies followed by seven 10-fold dilutions. Standard curves had an R² ≥ 0.997 and an efficiency of 99.7%– 103.3%. There was no significant difference between the copies of viral RNA used for the initial RT-PCR in mother and child samples (p = 0.35; paired t-test). Viral load was not associated with the number of copies in the initial RT-PCR (p = 0.18; linear regression).