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

- Sequence variation at probe target site inhibited detection of a new RSV-B variant.
- RSV-B virus diversity was consistent with real-time RT-PCR sensitivity.
- Reduced PCR insensitivity could underestimate disease prevalence in clinical settings.
- Regular check of primer and probe target sites for rapidly evolving viruses is key.

BackgroundDirect 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.ObjectivesEstablish if mismatches in the primer and probe binding sites could have reduced real-time RT-PCR sensitivity.Study designNucleoprotein (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.ResultsN 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.ConclusionsAn 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.