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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.
SUBMITTER: Kamau E
PROVIDER: S-EPMC5331890 | biostudies-literature | 2017 Mar
REPOSITORIES: biostudies-literature
Kamau Everlyn E Agoti Charles N CN Lewa Clement S CS Oketch John J Owor Betty E BE Otieno Grieven P GP Bett Anne A Cane Patricia A PA Nokes D James DJ
Journal of clinical virology : the official publication of the Pan American Society for Clinical Virology 20170105
<h4>Background</h4>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.<h4>Objectives</h4>Establish if mismatches in the primer and probe binding sites could have reduced real-time RT-PCR sensitivity.<h4>Study design</h4>Nucleoprotein (N) and glycoprotein (G) genes were sequenced for real-time RT-PCR positive and negat ...[more]