Development of a real-time quantitative RT-PCR for the detection of HIV-2 RNA in plasma

An assay is described for the quantification of human immunodeficiency virus type 2 (HIV-2) RNA in EDTA plasma based on RT-PCR using the Taqman real-time PCR detection method. As standard, an electron microscopically counted virus stock of HIV-2 strain NIHZ was used. The lower detection limit is 5 # 102 HIV-2 RNA copies per ml of EDTA plasma. The assay is linear within the range required (5 # 102-106 HIV-2 RNA copies/ml of EDTA plasma) with an intra assay variability of 2.5% and an inter-assay variability ranging from 2% at 106 copies to 7.5% at the lower detection limit. Three primer/probe combinations were developed to circumvent false negative samples due to nucleotide variation in the target sequence. Using these primer/probe sets enabled the detection of HIV-2 DNA sequences from all HIV-2 seropositive individuals and two out of five dual human immunodeficiency virus type 1 (HIV-1) and HIV-2 seropositive individuals visiting the University Hospital Rotterdam.