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

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 p...

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Autores principales: Kamau, Everlyn, Agoti, Charles N., Lewa, Clement S., Oketch, John, Owor, Betty E., Otieno, Grieven P., Bett, Anne, Cane, Patricia A., Nokes, D. James
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Elsevier Science 2017
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5331890/
https://www.ncbi.nlm.nih.gov/pubmed/28107671
http://dx.doi.org/10.1016/j.jcv.2016.12.011
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author Kamau, Everlyn
Agoti, Charles N.
Lewa, Clement S.
Oketch, John
Owor, Betty E.
Otieno, Grieven P.
Bett, Anne
Cane, Patricia A.
Nokes, D. James
author_facet Kamau, Everlyn
Agoti, Charles N.
Lewa, Clement S.
Oketch, John
Owor, Betty E.
Otieno, Grieven P.
Bett, Anne
Cane, Patricia A.
Nokes, D. James
author_sort Kamau, Everlyn
collection PubMed
description 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.
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spelling pubmed-53318902017-03-09 Recent sequence variation in probe binding site affected detection of respiratory syncytial virus group B by real-time RT-PCR Kamau, Everlyn Agoti, Charles N. Lewa, Clement S. Oketch, John Owor, Betty E. Otieno, Grieven P. Bett, Anne Cane, Patricia A. Nokes, D. James J Clin Virol Short Communication 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. Elsevier Science 2017-03 /pmc/articles/PMC5331890/ /pubmed/28107671 http://dx.doi.org/10.1016/j.jcv.2016.12.011 Text en © 2017 The Authors http://creativecommons.org/licenses/by/4.0/ This is an open access article under the CC BY license (http://creativecommons.org/licenses/by/4.0/).
spellingShingle Short Communication
Kamau, Everlyn
Agoti, Charles N.
Lewa, Clement S.
Oketch, John
Owor, Betty E.
Otieno, Grieven P.
Bett, Anne
Cane, Patricia A.
Nokes, D. James
Recent sequence variation in probe binding site affected detection of respiratory syncytial virus group B by real-time RT-PCR
title Recent sequence variation in probe binding site affected detection of respiratory syncytial virus group B by real-time RT-PCR
title_full Recent sequence variation in probe binding site affected detection of respiratory syncytial virus group B by real-time RT-PCR
title_fullStr Recent sequence variation in probe binding site affected detection of respiratory syncytial virus group B by real-time RT-PCR
title_full_unstemmed Recent sequence variation in probe binding site affected detection of respiratory syncytial virus group B by real-time RT-PCR
title_short Recent sequence variation in probe binding site affected detection of respiratory syncytial virus group B by real-time RT-PCR
title_sort recent sequence variation in probe binding site affected detection of respiratory syncytial virus group b by real-time rt-pcr
topic Short Communication
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC5331890/
https://www.ncbi.nlm.nih.gov/pubmed/28107671
http://dx.doi.org/10.1016/j.jcv.2016.12.011
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