Highly sensitive methods for quantitation of HIV-1 unspliced and multiply spliced RNA and proviral DNA based on semi-nested real-time RT-PCR

Laboratory of Experimental Virology, Department of Medical Microbiology, Center for Infection and Immunity Amsterdam (CINIMA), Academic Medical Center, University of Amsterdam, Amsterdam, The Netherlands

^* To whom correspondence should be addressed. Email: v.lukashov{at}amc.uva.nl.

	Abstract

The effectiveness of highly active antiretroviral therapy (HAART), the standard of care for the treatment of HIV-1 infection, is assessed by measuring the viral RNA load in plasma. A patient is considered to be successfully treated when the HIV-1 load in plasma stays below the detection limit of commercial assays. However, virus replication and evolution do continue in patients under HAART, which may eventually result in the development of drug-resistant HIV-1 strains and therapy failure. To monitor this low-level virus replication in peripheral blood mononuclear cells (PBMC), sensitive methods are required to measure HIV-1 molecular markers. We report the development of highly sensitive methods for quantitation of unspliced and multiply spliced HIV-1 RNA and proviral DNA in PBMC. The methods are based on innovative semi-nested real-time RT-PCR that combines the accuracy and precision of real-time PCR and the sensitivity of nested PCR. We show that the newly developed methods are superior to the conventional single-step real-time RT-PCR in their sensitivity, accuracy, dynamic range, and the power of quantitative detection of HIV-1 RNA and DNA in clinical samples. These easy-to-perform methods can be widely used in research, including clinical studies, to monitor intracellular processes of virus replication.