title: Recent sequence variation in probe binding site affected detection of respiratory syncytial virus group B by real-time RT-PCR.
abstract: 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.
  
  
  
  Establish if mismatches in the primer and probe binding sites could have reduced real-time RT-PCR sensitivity.
  
  
  
  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.
  
  
  
  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.
  
  
  
  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.
authors.affiliation: Epidemiology and Demography Department, Kenya Medical Research Institute (KEMRI) - Wellcome Trust Research Programme, Kilifi, Kenya. Electronic address: ekamau@kemri-wellcome.org.
authors.forename: Everlyn
authors.initials: E
authors.lastname: Kamau
authors.affiliation: Epidemiology and Demography Department, Kenya Medical Research Institute (KEMRI) - Wellcome Trust Research Programme, Kilifi, Kenya; School of Health and Human Sciences, Pwani University, Kilifi, Kenya.
authors.forename: Charles N
authors.initials: CN
authors.lastname: Agoti
authors.affiliation: Epidemiology and Demography Department, Kenya Medical Research Institute (KEMRI) - Wellcome Trust Research Programme, Kilifi, Kenya.
authors.forename: Clement S
authors.initials: CS
authors.lastname: Lewa
authors.affiliation: Epidemiology and Demography Department, Kenya Medical Research Institute (KEMRI) - Wellcome Trust Research Programme, Kilifi, Kenya.
authors.forename: John
authors.initials: J
authors.lastname: Oketch
authors.affiliation: Epidemiology and Demography Department, Kenya Medical Research Institute (KEMRI) - Wellcome Trust Research Programme, Kilifi, Kenya.
authors.forename: Betty E
authors.initials: BE
authors.lastname: Owor
authors.affiliation: Epidemiology and Demography Department, Kenya Medical Research Institute (KEMRI) - Wellcome Trust Research Programme, Kilifi, Kenya.
authors.forename: Grieven P
authors.initials: GP
authors.lastname: Otieno
authors.affiliation: Epidemiology and Demography Department, Kenya Medical Research Institute (KEMRI) - Wellcome Trust Research Programme, Kilifi, Kenya.
authors.forename: Anne
authors.initials: A
authors.lastname: Bett
authors.affiliation: Public Health England, Salisbury, United Kingdom.
authors.forename: Patricia A
authors.initials: PA
authors.lastname: Cane
authors.affiliation: Epidemiology and Demography Department, Kenya Medical Research Institute (KEMRI) - Wellcome Trust Research Programme, Kilifi, Kenya; School of Life Sciences and WIDER, University of Warwick, Coventry, United Kingdom.
authors.forename: D James
authors.initials: DJ
authors.lastname: Nokes
mesh_terms: D001665:Binding Sites; D017931:DNA Primers / Q000235:genetics; D005188:False Negative Reactions; D014644:Genetic Variation; D006801:Humans; D007630:Kenya; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D009698:Nucleoproteins / Q000235:genetics; D015345:Oligonucleotide Probes / Q000235:genetics*; D012367:RNA, Viral / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D018357:Respiratory Syncytial Virus Infections / Q000175:diagnosis* / Q000821:virology; D012136:Respiratory Syncytial Viruses / Q000235:genetics / Q000302:isolation & purification*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D014760:Viral Fusion Proteins / Q000235:genetics
chemical_list: D017931:DNA Primers; C502275:G glycoprotein, Respiratory syncytial virus; D009698:Nucleoproteins; D015345:Oligonucleotide Probes; D012367:RNA, Viral; D014760:Viral Fusion Proteins
keywords: Mismatches; Primer; Probe; RSV; RT-PCR; Real-time