Recent sequence variation in probe binding site affected detection of respiratory syncytial virus group B by real-time RT-PCR
Agoti, Charles N.
Lewa, Clement S.
Owor, Betty E.
Otieno, Grieven P.
Cane, Patricia A.
Nokes, D. James
- Publisher: Elsevier Science
Journal of Clinical Virology,
(issn: 1386-6532, eissn: 1873-5967)
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.