Related Experiment Video
Updated: Mar 8, 2026
Author Spotlight: Advancements in Multiplex Detection of Respiratory Viruses
Published on: November 10, 2023
Recent sequence variation in probe binding site affected detection of respiratory syncytial virus group B by
Everlyn Kamau1, Charles N Agoti2, Clement S Lewa1
1Epidemiology and Demography Department, Kenya Medical Research Institute (KEMRI) - Wellcome Trust Research Programme, Kilifi, Kenya.
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.
