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 10(6) 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.
Ruelle
Jean
J
Unité de Virologie-AIDS Reference Laboratory, Université Catholique de Louvain, Avenue Hippocrate 54 (5492), 1200 Brussels, Belgium.
Mukadi
Benoît Kabamba
BK
Schutten
Martin
M
Goubau
Patrick
P
D001483:Base Sequence; D016384:Consensus Sequence; D017931:DNA Primers; D015498:HIV-2 / Q000235:genetics / Q000302:isolation & purification*; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D012367:RNA, Viral / Q000097:blood / Q000235:genetics; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D019562:Viral Load
D017931:DNA Primers; D012367:RNA, Viral