Detection of Spiked HIV1 in plasma matrix using RT-qPCR

Abstract

Sensitive recovery and detection of HIV-1 RNA from plasma are important for studies of circulating viral nucleic acids in patient samples. To assess the compatibility of human plasma with an HIV-1 PCR-based workflow, HIV-negative plasma samples were spiked with 0, 10, 50, or 100 µL of HIV-1 strain 89.6 viral stock. Samples were lysed to disrupt viral particles, and RNA was isolated by a phenol-chloroform extraction method. Reverse transcription was performed using oligo(dT) primers, followed by real-time quantitative PCR (RT-qPCR) with primer sets targeting the HIV-1 nef and env sequences in viral transcripts. Nef and env are known to remain expressed in plasma of patients on combination anti-retroviral therapies (cART). Our results showed that env and nef HIV-1 transcripts were undetected in the negative control. A dose-dependent increase in viral transcripts was observed in samples spiked with 10, 50, and 100uL of HIV-1 viral stock. Our findings demonstrate that HIV-1 RNA containing nef and env sequences can be recovered and detected from the tested plasma matrix. Conclusively, this method can be applied to detect HIV-1 viral transcripts in patient plasma samples.