title: Development and evaluation of a four-tube real time multiplex PCR assay covering fourteen respiratory viruses, and comparison to its corresponding single target counterparts.
abstract: BACKGROUND
  Multiplex real time PCR is increasingly used to diagnose respiratory viruses and has shown to be superior to traditional methods, like culture and antigen detection. However, comprehensive data on sensitivity, specificity and performance of the multiplex PCR compared to the single target PCR's is limited for most published respiratory multiplex real time PCR assays.
  
  
  OBJECTIVE
  Development and extensive analysis of an internally controlled multiplex real time rt-PCR for detection of respiratory viruses.
  
  
  METHODS
  The assay was validated in comparison to single-target PCR's using plasmid targets and prospectively collected nasopharyngeal aspirates.
  
  
  RESULTS
  Using plasmid targets the multiplex format was found to be as least as sensitive and specific as the single-target PCR and no competition was observed when different targets were present at different amounts in one tube. Clinical validation showed high concordance for all viruses tested except for samples with low levels of enterovirus.
  
  
  CONCLUSIONS
  This multiplex showed excellent specificities for all 14 respiratory viruses and sensitivity was high except for clinical samples with low levels of enterovirus.
authors.affiliation: Laboratory of Clinical Virology, Department of Medical Microbiology, Academic Medical Centre, University of Amsterdam, PO-Box 22600, 1100 DD, Amsterdam, The Netherlands. R.R.Jansen@amc.uva.nl
authors.forename: Rogier R
authors.initials: RR
authors.lastname: Jansen
authors.forename: Janke
authors.initials: J
authors.lastname: Schinkel
authors.forename: Sylvie
authors.initials: S
authors.lastname: Koekkoek
authors.forename: Dasja
authors.initials: D
authors.lastname: Pajkrt
authors.forename: Marcel
authors.initials: M
authors.lastname: Beld
authors.forename: Menno D
authors.initials: MD
authors.lastname: de Jong
authors.forename: Richard
authors.initials: R
authors.lastname: Molenkamp
mesh_terms: D002675:Child, Preschool; D005260:Female; D006801:Humans; D007223:Infant; D008297:Male; D025202:Molecular Diagnostic Techniques / Q000379:methods* / Q000592:standards; D009305:Nasopharynx / Q000821:virology; D010957:Plasmids; D016133:Polymerase Chain Reaction / Q000379:methods* / Q000592:standards; D012015:Reference Standards; D012141:Respiratory Tract Infections / Q000175:diagnosis* / Q000821:virology; D012680:Sensitivity and Specificity; D014773:Virology / Q000379:methods* / Q000592:standards; D014777:Virus Diseases / Q000175:diagnosis* / Q000821:virology; D014780:Viruses / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*