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

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Kamau, Everlyn, Agoti, Charles N., Lewa, Clement S., Oketch, John, Owor, Betty E., Otieno, Grieven P., Bett, Anne, Cane, Patricia A. and Nokes, D. James (2017) Recent sequence variation in probe binding site affected detection of respiratory syncytial virus group B by real-time RT-PCR. Journal of clinical virology . doi:10.1016/j.jcv.2016.12.011 ISSN 1386-6532 .

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Official URL: http://dx.doi.org/10.1016/j.jcv.2016.12.011

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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.

Item Type: Journal Article
Subjects: ?? Mismatches ??
?? Primer ??
?? Probe ??
Q Science > QR Microbiology > QR355 Virology
?? RSV ??
?? RT-PCR ??
?? Real-time ??
Divisions: Faculty of Science, Engineering and Medicine > Science > Life Sciences (2010- )
SWORD Depositor: Library Publications Router
Library of Congress Subject Headings (LCSH): Respiratory syncytial virus, Polymerase chain reaction
Journal or Publication Title: Journal of clinical virology
Publisher: Elsevier BV
ISSN: 1386-6532
Official Date: March 2017
Dates:
DateEvent
March 2017Published
5 January 2017Available
31 December 2016Accepted
DOI: 10.1016/j.jcv.2016.12.011
Status: Peer Reviewed
Publication Status: Published
Access rights to Published version: Restricted or Subscription Access
Date of first compliant deposit: 6 February 2017
Date of first compliant Open Access: 6 February 2017
Funder: Wellcome Trust (London, England)
Grant number: 102975
RIOXX Funder/Project Grant:
Project/Grant IDRIOXX Funder NameFunder ID
102975Wellcome Trusthttp://dx.doi.org/10.13039/100010269
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