A rapid real-time PCR assay for the detection of HIV-1 proviral DNA using double-stranded primer

Abstract

In this study, a rapid real-time PCR assay to detect HIV-1 proviral DNA in whole blood was developed using a novel double-stranded primer that does not require a target-specific fluorescent probe or intercalating dye systems. Co-amplification of a human gene RNase P served as the internal control to monitor the efficiency of the DNA extraction and PCR amplification. The HIV-1 amplification efficiency was 100% and could amplify 1 copy of HIV-1 DNA 64% of the time and all attempts to amplify 4 copies were successful in less than 51 min. All 22 HIV-1 sero-positive and 20 sero-negative whole blood specimens tested were classified correctly by this assay. In addition, 22 cultured PBMC specimens infected with various HIV-1 subtypes or CRF (A = 2, AC = 1, B = 4, C = 3, D = 3, AE = 2, F = 1, BF = 2, G = 4) were amplified equally well with a similar threshold cycle (C t) number (22.9 ± 1.2). The high amplification efficiency and short PCR cycles were in part due to the short target sequence amplified by eliminating the probe-binding sequence between the primers. This assay may be useful as an alternative confirmation test in a variety of HIV testing venues.