APPLICATION OF A COMMERCIAL KIT FOR DETECTION OF PCR PRODUCTS TO QUANTIFICATION OF HUMAN-IMMUNODEFICIENCY-VIRUS TYPE-1 RNA AND PROVIRAL DNA

Quantitative tests for human immunodeficiency virus type 1 (HIV-1) RNA in plasma and proviral DNA in peripheral blood mononuclear cells (PBM C) provide valuable information on the status of HIV-1 infection, This paper describes tests that were carried out with commercially availab le materials and an enzyme-linked immunosorbent assay reader for detec ting spectrophotometric changes, Samples consisted of 100 mu l of plas ma or 200,000 PBMC, The procedure involved sample preparation, PCR-bas ed amplification with the primer pair SK39 (biotinylated at the 5' end ) and SK38, hybridization of the cDNA PCR product to an RNA probe, cap ture of the RNA-DNA hybrid on a solid phase by means of strepavidin, b inding to an alkaline phosphatase-conjugated antibody directed against RNA-DNA hybrids, and incubation with p-nitrophenylphosphate. Spectrop hotometric changes were recorded at four intervals over a period of 20 h, The inclusion of HIV-1 RNA or proviral DNA standards in each run w as an integral part of the procedure, The dynamic ranges afforded by t hese assays-500 to 1 million RNA copies per mi and 10 to 5,000 provira l DNA copies per 10(6) PBMC-were applicable to most plasma specimens a nd to all PBMC specimens from HIV-1-infected patients, Correlations of log-transformed HIV-1 RNA and proviral DNA concentrations with those found by reference methods were, respectively, 0.88 and 0.80, The betw een-run coefficients of variation for the detection method were less t han or equal to 25% (range, 9.1 to 24.7) and less than or equal to 15% (range, 10.9 to 15.1), respectively, for HIV-1 RNA and proviral DNA, The reproducibility of the overall procedure for HIV-1 RNA in plasma ( including sample preparation, amplification, and detection) was given by a duplicate standard deviation of log(10) copies per ml of 0.11. Th us, the method was sufficiently precise to allow the detection of four fold changes in plasma HIV-1 RNA concentrations, with a power of 0.95.