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

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Kamau, Everlyn ; Agoti, Charles N. ; Lewa, Clement S. ; Oketch, John ; Owor, Betty E. ; Otieno, Grieven P. ; Bett, Anne ; Cane, Patricia A. ; Nokes, D. James (2017)
  • Publisher: Elsevier Science
  • Journal: Journal of Clinical Virology, volume 88, pages 21-25 (issn: 1386-6532, eissn: 1873-5967)
  • Related identifiers: pmc: PMC5331890, doi: 10.1016/j.jcv.2016.12.011
  • Subject: Mismatches | RSV | Real-time | Primer | RT-PCR | Virology | Infectious Diseases | Short Communication | QR355 | Probe

Background\ud \ud 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.\ud \ud Objectives\ud \ud Establish if mismatches in the primer and probe binding sites could have reduced real-time RT-PCR sensitivity.\ud \ud Study design\ud \ud 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.\ud \ud Results\ud \ud 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.\ud \ud Conclusions\ud \ud 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.
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