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  • Title: Quantification of HIV-1 viral RNA and proviral DNA by isotopic competitive PCR.
    Author: Gratzl S, Moroni C, Hirsch HH.
    Journal: J Virol Methods; 1997 Jul; 66(2):269-82. PubMed ID: 9255738.
    Abstract:
    A quantitative isotopic competitive PCR (icPCR) assay was established using 32P-labeled primers targeting the HIV-1 gag gene followed by quantification using a phosphoimager. The detection limit varied from 3 to 10 molecules of DNA and 10 to 100 molecules of RNA per reaction. The icPCR quantification of HIV-1 DNA copies correlated well with the cell number of 8E5/LAV cells bearing a single provirus (r2 = 0.95). Provirus quantification was applied to overnight infected donor PBMCs, thereby determining infectious virus titres in culture supernatants as a rapid alternative to limiting dilution culture. Parallel quantification of the HIV-1 RNA indicated the infectious virus fraction to be 0.3%. In 39 HIV-1-infected patients with clinical stages A (n = 17), B (n = 15), and C (n = 7), the HIV-1 RNA in the plasma was determined ranging from 100 to 90600 RNA copies/ml. The results of icPCR and a commercial assay (ROCHE Amplicor HIV-1 Monitor) correlated well (r = 0.97). In 13 additional patients, the plasma viral load per ml was compared with 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 suitable for the measurement of proviral and viral load in experimental and clinical settings.
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