HIV-1 Quantitation by Real-Time RT-PCR 101 MOLECULAR BIOTECHNOLOGY Volume 32, 2006 RESEARCH 101 Molecular Biotechnology © 2006 Humana Press Inc. All rights of any nature whatsoever reserved. 1073–6085/2006/32:2/101–110/$30.00 *Author to whom all correspondence and reprint requests should be addressed. 1 Department of Molecular Medicine and Hematology, National Health Laboratory Services, University of the Witwatersrand, Faculty of Health Science, Johannesburg, South Africa. E-mail: rekhn@hsc.pg.wits.ac.za or rekhnat11@yahoo.com. 2 International AIDS Vaccine Initiative, Johannesburg, South Africa; 3 Department of Molecular Medicine and Hematology, University of the Witwatersrand, Faculty of Health Science, Johannesburg, South Africa. Abstract Fluorogenic LUX Primer for Quantitation of HIV-1 by Real-Time RT-PCR Natela Rekhviashvili, 1,* Gwynneth Stevens, 2 Lesley Scott, 3 and Wendy Stevens 1,3 Measurement of HIV-1 viral load in plasma is an important marker of disease progression and efficacy of antiretroviral therapy. Real-time polymerase chain reaction (PCR) offers an opportunity to develop more affordable alternative viral load assays. This article reports on the development of a novel real-time reverse- transcriptase (RT)-PCR assay for quantitation of HIV-1 RNA copies. This assay utilizes the LightCycler ® (version2) real-time PCR platform and light upon extension (LUX) primer for specific detection of amplicons. An external standard (ES) for quantitation of viral RNA represents an in vitro transcribed RNA. The LUX assay shows a wide linear (R 2 = 0.99) dynamic range from 4 × 10 6 to 4 × 10 2 copies/mL. Analyti- cal sensitivity of the assay is 4 × 10 2 copies/mL of ES RNA. Intra- and inter-assay variability of the LUX assay was less than 0.5log 10 copies of ES RNA (i.e., no clinically significant variability was found). Virol- ogy quality assurance (VQA) HIV-1 RNA copy controls were used to validate ES and preliminarily evalu- ate the assay performance. This feasibility study demonstrated that the LUX assay is sensitive, reproducible, and compares well to the Roche Amplicor tests used for characterization of the RNA copy controls. These results suggest further evaluation of the LUX assay using a large cohort of well-characterized samples from HIV-1 positive individuals. Index Entries: HIV-1 viral load; real-time reverse-transcriptase (RT)-polymerase chain reaction (PCR); quantitation; LUX; affordable. 1. Introduction The implementation of effective antiretroviral (ARV) therapy for HIV-1 in resource-limited set- tings requires both laboratory infrastructure and the development of cost-effective techniques for diagnosis and monitoring of patients on therapy to support the clinical management of infected individuals (1,2). In particular, determination of HIV-1 RNA burden in plasma has been deemed valuable for assessing the efficiency of ARV therapy and for monitoring disease progression (3, 4). Although a number of “gold standard” HIV-1 viral load tests have been well validated and already have Food and Drug Administration (FDA) approval, they remain largely unaffordable for routine patient monitoring in developing coun- tries. In South Africa, approx 5.3 million adults and children are estimated to be HIV infected (5) and thus a staggering number of individuals are likely to require ARV therapy. Thus, much effort is being expended to develop more affordable, high- throughput HIV-1 viral load-testing alternatives. An option under evaluation is real-time poly- merase chain reaction (PCR) technology, which has become a powerful tool in the molecular diagnostics armamentarium, offering improved quantitation of nucleic acids and a number of detection formats that enhance both assay speci-