Recent sequence variation in probe binding site affected detection of respiratory syncytial virus group B by real-time RT-PCR

Everlyn Kamau, Charles N. Agoti, Clement S. Lewa, John Oketch, Betty E. Owor, Grieven P. Otieno, Anne Bett, Patricia Cane, D. James Nokes

    Research output: Contribution to journalArticlepeer-review

    26 Citations (Scopus)

    Abstract

    Background Direct immuno-fluorescence test (IFAT) and multiplex real-time RT-PCR have been central to RSV diagnosis in Kilifi, Kenya. Recently, these two methods showed discrepancies with an increasing number of PCR undetectable RSV-B viruses. Objectives Establish if mismatches in the primer and probe binding sites could have reduced real-time RT-PCR sensitivity. Study design Nucleoprotein (N) and glycoprotein (G) genes were sequenced for real-time RT-PCR positive and negative samples. Primer and probe binding regions in N gene were checked for mismatches and phylogenetic analyses done to determine molecular epidemiology of these viruses. New primers and probe were designed and tested on the previously real-time RT-PCR negative samples. Results N gene sequences revealed 3 different mismatches in the probe target site of PCR negative, IFAT positive viruses. The primers target sites had no mismatches. Phylogenetic analysis of N and G genes showed that real-time RT-PCR positive and negative samples fell into distinct clades. Newly designed primers-probe pair improved detection and recovered previous PCR undetectable viruses. Conclusions An emerging RSV-B variant is undetectable by a quite widely used real-time RT-PCR assay due to polymorphisms that influence probe hybridization affecting PCR accuracy.

    Original languageEnglish
    Pages (from-to)21-25
    Number of pages5
    JournalJournal of Clinical Virology
    Volume88
    DOIs
    Publication statusPublished - 1 Mar 2017

    Keywords

    • Mismatches
    • Primer
    • Probe
    • RSV
    • RT-PCR
    • Real-time

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