Application of a commercial kit for detection of PCR products to quantification of human immunodeficiency virus type 1 RNA and proviral DNA

Abstract

Quantitative tests for human immunodeficiency virus type 1 (HIV-1) RNA in plasma and proviral DNA in peripheral blood mononuclear cells (PBMC) provide valuable information on the status of HIV-1 infection. This paper describes tests that were carried out with commercially available materials and an enzyme-linked immunosorbent assay reader for detecting spectrophotometric changes. Samples consisted of 100 microliters of plasma or 200,000 PBMC. The procedure involved sample preparation, PCR-based amplification with the primer pair SK39 (biotinylated at the 5' end) and SK38, hybridization of the cDNA PCR product to an RNA probe, capture of the RNA-DNA hybrid on a solid phase by means of strepavidin, binding to an alkaline phosphatase-conjugated antibody directed against RNA-DNA hybrids, and incubation with p-nitrophenylphosphate. Spectrophotometric 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 was an integral part of the procedure. The dynamic ranges afforded by these assays--500 to 1 million RNA copies per ml and 10 to 5,000 proviral DNA copies per 10(6) PBMC--were applicable to most plasma specimens and 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 between-run coefficients of variation for the detection method were < or = 25% (range, 9.1 to 24.7) and < or = 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 log10 copies per ml of 0.11. Thus, the method was sufficiently precise to allow the detection of fourfold changes in plasma HIV-1 RNA concentrations, with a power of 0.95.