Establishment of in-house quantitative real-time RT-PCR assay for HIV-1 viral load measurement: Application to evaluate efficacy of ART in Ghanaian patients in an urban setting

Abstract

The aim of this study was to establish and apply a real-time quantitative reverse transcription polymerase chain reaction (RT-PCR) for human immunodeficiency virus (HIV) RNA quantification in patients on antiretroviral treatment (ART) in Ghana, where recombinant strains including CRF02_AG are prevalent. The primers and TaqMan probe concentrations as well as reaction temperatures were optimized to establish an efficient in-house quantitative assay system for HIV RNA, a tool for HIV viral load measurement in patients. Then an already established HIV-specific PCR amplicon (HIV-1 NL4-3) was used as an external standard to estimate the linearity, amplification efficiency, analytical sensitivity and reproducibility of the in-house real time quantitative assay. Finally, the assay was applied to quantify the viral load in clinical samples of HIV patients on ART. The real time quantitative assay was shown to have good linearity (R2=1.0), high amplification efficiency (E=1.91), high sensitivity (180 copies/ml), and high reproducibility (variation coefficient range, from 1.25% to 3.58%). Analytical specificity and sensitivity of the assay in clinical samples was 96.7% and 95.0%, respectively. The established tool is reliable and covers all relevant genotypes including rare and recombinant forms that circulate in the sub-region. It could therefore allow general monitoring of antiretroviral therapy in patients living in resource-limited settings due to its simplicity, rapidity and less-labour intensiveness. © 2014 Barnor JS, et al.