QUANTIFICATION OF HIV-1 VIRAL-RNA AND PROVIRAL DNA BY ISOTOPIC COMPETITIVE PCR

A quantitative isotopic competitive PCR (icPCR) assay was established using P-32-labeled primers targeting the HIV-1 gag gene followed by qu antification using a phosphoimager. The detection limit varied from 3 to 10 molecules of DNA and 10 to 100 molecules of RNA per reaction. Th e icPCR quantification of HIV-1 DNA copies correlated well with the ce ll number of 8E5/LAV cells bearing a single provirus (r(2) = 0.95). Pr ovirus quantification was applied to overnight infected donor PBMCs, t hereby determining infectious virus titres in culture supernatants as a rapid alternative to limiting dilution culture. Parallel quantificat ion of the HIV-1 RNA indicated the infectious virus fraction to be 0.3 %. In 39 HIV-l-infected patients with clinical stages A (n = 17), B (n = 15), and C (n = 7), the HIV-1 RNA in the plasma was determined rang ing from 100 to 90600 RNA copies/ml. The results of icPCR and a commer cial assay (ROCHE Amplicor HIV-I Monitor) correlated well (r = 0.97). In 13 additional patients, the plasma viral load per mi was compared w ith the proviral load per 10(6) PBMC showing a viral excess of 10-1000 -fold (mean of 85, r = 0.7, P < 0.01). It is concluded that icPCR is s uitable for the measurement of proviral and viral load in experimental and clinical settings. (C) 1997 Elsevier Science B.V.