Nokes, D. James and Kamau, Evelyn (2017) Data for Recent sequence variation in probe binding site affected detection of respiratory syncytial virus group B by real-time RT-PCR. [Dataset]

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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:	Dataset
Divisions:	Faculty of Medicine > Warwick Medical School
Type of Data:	Experimental data
Publisher:	Harvard Dataverse
Official Date:	14 May 2017
Dates:	Date Event 8 May 2017 Submitted 14 May 2017 Published
Status:	Not Peer Reviewed
Publication Status:	Published
Access rights to Published version:	Open Access
Copyright Holders:	University of Warwick
Description:	Data record consists of a single data file in PDF format. This dataset comprises of Nucleotide sequences used to match primers and probes, and for phylogenetic inferences. In our analysis, we observed inconsistencies between two routine RSV diagnostic methods applied in our labs, one method utilizes immunofluorescence microscopy and other molecular probes, and set out to determine. Sequence data cleaning and contig assembly was done using Sequencher program v.5.1; sequence alignment done using MAFFT v.1.8; phylogenetic inferences done in MEGA v1.6; custom BLAST of the primers and probes done using geneious v8.1.8 All sequence data reported is freely available in Genbank with accession numbers from KX775772 - KX775940.
RIOXX Funder/Project Grant:	Project/Grant ID RIOXX Funder Name Funder ID 102975 Wellcome Trust http://dx.doi.org/10.13039/100010269
Related URLs:	Related item in WRAP Other
Contributors:	Contribution Name Contributor ID Contact Person Nokes, D. James 35965

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