PCR multiplex sur le LightCycler utilisant des sondes d'hydrolyse et d'hybridation : application à la quantification du provirus VIH-1

Abstract

Quantification of HIV provirus DNA by aid of PCR multiplex using LIGHT Cycler. The quantitation of HIV provirus DNA in peripheral blood mononuclear cells (PBMCs) may be useful to gain insight into the natural history of infection and the continued efficacy of antiretroviral therapy. A quantitative PCR with the Light Cycler strategy has been developed to evaluate HIV DNA in PBMCs together with the amplification of cellular gene (β-globin) used as an internal control. Fluorescent hydrolysis probe and hybridization probes were used to detect HIV DNA and the β-globin sequence, respectively, in the same run. Co-amplification with β-globin did not interfere with HIV-1 quantification and the sensitivity of HIV detection was 5 copies. This assay was compared with an HIV DNA competitive PCR and the correlation between the two methods was good (Δ log = 0.19). The possibility to use simultaneously fluorescent hydrolysis and hybridization probes enlarges the analytic ability of real time PCR LightCycler technology.