Background:  The planned national rollout of anti-retroviral therapy (ARV) in South Africa will increase the number of people requiring HIV-related laboratory investigations. Serial measurements of HIV-1 viral load are important for monitoring patient’s response to ARV. We report on the development of an affordable viral-load assay using the LightCycler instrument (Roche) for real-time quantitative PCR.

Methods:  Viral RNA was extracted from 0.5 mL of plasma. One-step quantitative RT-PCR (reverse transcriptase polymerase chain reaction) was performed as a multiplex reaction. Two different sets of primers and hybridization probes were used to detect a wild type/external standard and an internal positive control sequence, respectively. Both the external standard and internal positive control were designed and cloned into the pGEM T-Easy vector (Promega). RNA transcripts for both ES and IPC were synthesized in vitro.

Results:  Hybridization probes for detection of the wild type amplicon showed high specificity for HIV-1 subtype C, which is predominant in South Africa. The internal positive control served the dual purpose of controlling for false negative results, and as a calibrator in each run. The assay demonstrated high analytical sensitivity:  3 copies per reaction of the external standard RNA transcript could be detected reproducibly. The dynamic range of the test covered 5 orders of magnitude, from 10⁷ to 10³ external standard copies per mL. Replicate testing of the external standard established the lower limit of quantitation of the assay at 1500 copies/mL.

Conclusions:  Compared to current “gold standards” for HIV viral load testing, this assay on the LightCycler is rapid and robust and the reagents are less expensive. Early evaluation of this assay shows good performance characteristics and further validation on a large number of samples is warranted.

Keywords: Viral load; Affordable; Real-time PCR