RT-qPCR Detection of Low-Copy HIV RNA with Yin-Yang Probes

Dmitry E. Kireev, Valentina M. Farzan, German A. Shipulin, Vladimir A. Korshun, Timofei S. Zatsepin

    Research output: Chapter in Book/Report/Conference proceedingChapterpeer-review

    Abstract

    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.

    Original languageEnglish
    Title of host publicationMethods in Molecular Biology
    PublisherHumana Press Inc.
    Pages27-35
    Number of pages9
    DOIs
    Publication statusPublished - 2020

    Publication series

    NameMethods in Molecular Biology
    Volume2063
    ISSN (Print)1064-3745
    ISSN (Electronic)1940-6029

    Keywords

    • Diagnostics
    • HIV-1
    • Molecular beacon
    • RT-qPCR
    • Yin-Yang probes

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