Quantitation of HIV-1 proviral DNA relative to cellular DNA by the polymerase chain reaction

Abstract

We developed a quantitative assay for human immunodeficiency virus type 1 (HIV-1) proviral DNA sequences using the polymerase chain reaction (PCR). The relative copy numbers of HIV-1 proviral DNA molecules were determined by coamplification of an HIV-1 gag sequence and a portion of the DQ α locus of the histocompatibility (HLA) region. Because of the disparity in the copy number of cellular and HIV-1 templates, an attenuation in the efficiency of the HLA amplification was required to achieve simultaneous amplification and quantitation of both target sequences. The HIV-1 and HLA amplified products were detected by hybridization with radioactively labeled probes and the amount of probe bound to each product was determined with a radioanalytic system. Standard curves were generated by plotting the HIV-1 and HLA signals made against known copies of each target present prior to amplification. The copies of HIV-1 target relative to the number of cells in a given sample were determined by interpolation from standard curves. The procedure described here is generally applicable to the quantitation of other retroviruses.