Journal
JOURNAL OF CLINICAL VIROLOGY
Volume 88, Issue -, Pages 21-25Publisher
ELSEVIER
DOI: 10.1016/j.jcv.2016.12.011
Keywords
RSV; Real-time; RT-PCR; Primer; Probe; Mismatches
Categories
Funding
- Wellcome Trust [102975]
Ask authors/readers for more resources
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. (C) 2017 The Authors. Published by Elsevier B.V.
Authors
I am an author on this paper
Click your name to claim this paper and add it to your profile.
Reviews
Recommended
No Data Available