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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
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 | ||||||||
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| Subjects: | ?? Mismatches ?? ?? Primer ?? ?? Probe ?? Q Science > QR Microbiology > QR355 Virology ?? RSV ?? ?? RT-PCR ?? ?? Real-time ?? |
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| 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: |
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| 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: |
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