Quantification of HIV-1 RNA load by one-tube-one-step RT PCR and real time PCR assay with TaqMan probe.

ABSTRACT

OBJECTIVES: To develop a less expensive assay to calculate HIV-1 viral load for use in resource-limited countries. MATERIAL AND METHOD: An In-house One-tube-one-step Viral Load Assay (IOVA) was developed by using real-time PCR-based with TaqMan probe. Primers and probe were designed from the conserved region of sequences from all HIV subtypes. A standard curve was generated from reference virus in various dilutions. IOVA was applied on 105 HIV-positive and 25 HIV-negative samples and compared with the results from ROCHE AMPLICLOR. RESULTS: IOVA measured HIV RNA in the samples ranging from 125 to 2 x 10(6) copies/mL. The coefficient of variation of intra- and inter-assay ranged from 0.68% to 7.89%. The sensitivity, specificity, positive and negative predictive values were 92%, 100%, 100% and 79.5% respectively. The parallel quantitative analysis showed high correlation (r=0.95) between IOVA and AMPLICOR. CONCLUSION: A new HIV-1 viral load assay was developed and validated. It was reliable and less expensive.