Quantitative real-time PCR on Lightcycler® for the detection of human immunodeficiency virus type 2 (HIV-2)

Similar to HIV-1, the viral load in HIV-2 is a marker of the evolution of infection and the success of therapy. No approved or commercially distributed assay exists to determine the plasma viral load of HIV-2. We therefore developed a quantitative real-time PCR to determine the plasma RNA viral load of HIV-2, based on the reference strains ROD and EHO, which represent subtypes A and B of HIV-2, respectively. After testing several pairs of primers, a set was chosen that recognised the 5′-LTR region of a subtypes A and B consensus sequence. The quantification of the PCR reaction was done using SYBR-Green I as the fluorescent dye in a Lightcycler® system and relying on an external standard curve. The method was then optimised with reference strains, an validated further using patient samples and an inter-laboratory comparison. Good specificity was obtained for both subtypes of HIV-2, and a linear range between 10 and 106 copies of viral RNA. The limit of quantitation was 250 copies per millilitre of plasma. The coefficient of variation ranged from 0.7 to 5.6%, depending on the concentration of the target sequences.

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