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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
Languages: English
Types: Article
Subjects: 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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