title: One-step multiplex real time RT-PCR for the detection of bovine respiratory syncytial virus, bovine herpesvirus 1 and bovine parainfluenza virus 3.
abstract: BACKGROUND
  Detection of respiratory viruses in veterinary species has traditionally relied on virus detection by isolation or immunofluorescence and/or detection of circulating antibody using ELISA or serum neutralising antibody tests. Multiplex real time PCR is increasingly used to diagnose respiratory viruses in humans and has proved to be superior to traditional methods. Bovine respiratory disease (BRD) is one of the most common causes of morbidity and mortality in housed cattle and virus infections can play a major role. We describe here a one step multiplex reverse transcriptase quantitative polymerase chain reaction (mRT-qPCR) to detect the viruses commonly implicated in BRD.
  
  
  RESULTS
  A mRT-qPCR assay was developed and optimised for the simultaneous detection of bovine respiratory syncytial virus (BRSV), bovine herpes virus type 1 (BoHV-1) and bovine parainfluenza virus type 3 (BPI3 i & ii) nucleic acids in clinical samples from cattle. The assay targets the highly conserved glycoprotein B gene of BoHV-1, nucleocapsid gene of BRSV and nucleoprotein gene of BPI3. This mRT-qPCR assay was assessed for sensitivity, specificity and repeatability using in vitro transcribed RNA and recent field isolates. For clinical validation, 541 samples from clinically affected animals were tested and mRT-qPCR result compared to those obtained by conventional testing using virus isolation (VI) and/or indirect fluorescent antibody test (IFAT).
  
  
  CONCLUSIONS
  The mRT-qPCR assay was rapid, highly repeatable, specific and had a sensitivity of 97% in detecting 102 copies of BRSV, BoHV-1 and BPI3 i & ii. This is the first mRT-qPCR developed to detect the three primary viral agents of BRD and the first multiplex designed using locked nucleic acid (LNA), minor groove binding (MGB) and TaqMan probes in one reaction mix. This test was more sensitive than both VI and IFAT and can replace the aforesaid methods for virus detection during outbreaks of BRD.
authors.affiliation: Moredun Research Institute, International Research Centre, Pentlands Science Park, Bush Loan, Penicuik, Midlothian EH26 0PZ, UK. Leenadevi.Thonur@moredun.ac.uk
authors.forename: Leenadevi
authors.initials: L
authors.lastname: Thonur
authors.forename: Madeleine
authors.initials: M
authors.lastname: Maley
authors.forename: Janice
authors.initials: J
authors.lastname: Gilray
authors.forename: Tara
authors.initials: T
authors.lastname: Crook
authors.forename: Ellie
authors.initials: E
authors.lastname: Laming
authors.forename: Dylan
authors.initials: D
authors.lastname: Turnbull
authors.forename: Mintu
authors.initials: M
authors.lastname: Nath
authors.forename: Kim
authors.initials: K
authors.lastname: Willoughby
mesh_terms: D000818:Animals; D002417:Cattle; D007242:Herpesvirus 1, Bovine / Q000302:isolation & purification*; D007241:Infectious Bovine Rhinotracheitis / Q000175:diagnosis / Q000821:virology; D029101:Parainfluenza Virus 3, Bovine / Q000302:isolation & purification*; D012766:Pasteurellosis, Pneumonic / Q000175:diagnosis / Q000821:virology; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods / Q000662:veterinary*; D015203:Reproducibility of Results; D018357:Respiratory Syncytial Virus Infections / Q000175:diagnosis / Q000662:veterinary / Q000821:virology; D018112:Respiratory Syncytial Virus, Bovine / Q000302:isolation & purification*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods / Q000662:veterinary*; D012680:Sensitivity and Specificity; D013997:Time Factors