RESUMO
Accurate monitoring of low levels of viral load (the number of viral particles per milliliter of plasma) in HIV-infected patients is important in terms of evaluation of the progress of antiretroviral therapy. The general approach for detection of low copy HIV RNA is reverse transcription combined with quantitative real-time PCR based on fluorescence detection. The selection of primers and the structure of fluorogenic oligonucleotide probes are crucial for sensitivity and accuracy of the assay. In this chapter, we report the RT-qPCR protocol for detection of low copy HIV RNA using double stranded Yin-Yang DNA probes containing identical fluorescent dyes on each strand of the probe. Dye residues attached to the 3'-end of an oligonucleotide and 5'-end of the complementary oligonucleotide form a self-quenched aggregate in a Yin-Yang duplex probe, and display fluorescence light up upon probe strand displacement with the target sequence amplified in the course of PCR. Among several fluorescent dyes tested (R6G, ROX, Cy5) the ROX labeled Yin-Yang probes showed better fluorescence increase and lower Ct values. All the homo Yin-Yang probes were superior to corresponding dye-quencher probes and allowed reliable detection of 10-10,000 copies of HIV RNA per mL.
Assuntos
Infecções por HIV/diagnóstico , HIV-1/genética , RNA Viral/análise , Reação em Cadeia da Polimerase em Tempo Real/métodos , Carga Viral/métodos , Sequência de Bases , Primers do DNA/genética , Corantes Fluorescentes , HIV-1/isolamento & purificação , Humanos , Sondas de Oligonucleotídeos/genética , RNA Viral/genéticaRESUMO
Nucleic acids labeled with a fluorophore/quencher pair are widely used as probes in biomedical research and molecular diagnostics. Here we synthesized novel DNA molecular beacons double labeled with the identical dyes (R6G, ROX and Cy5) at 5'- and 3'-end and studied their photo physical properties. We demonstrated that fluorescence quenching by formation of the homo dimer exciton in such molecular beacons allows using them in homogeneous assays. Further, we developed and evaluated homo Yin-Yang DNA probes labeled with identical dyes and used them for detection of low copy HIV RNA by RT-qPCR. They demonstrated improved sensitivity (LLQ: 10 vs 30 copies mL-1) in comparison to commercially available Abbott RealTime HIV-1 kit based on VIC-BHQ dyes both for model mixtures (naive human plasma with added deactivated HIV-1 virus) and for preliminarily confirmed 36 clinical samples (4 vs 1 positive ones for low-copy samples).
