One-step multiplex real time RT-PCR for the detection of bovine respiratory syncytial virus, bovine herpesvirus 1 and bovine parainfluenza virus 3

Leenadevi Thonur*, Madeleine Maley, Janice Gilray, Tara Crook, Ellie Laming, Dylan Turnbull, Mintu Nath, Kim Willoughby

*Corresponding author for this work

Research output: Contribution to journalArticle

42 Citations (Scopus)

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 10 2 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.

Original languageEnglish
Article number37
Number of pages9
JournalBMC Veterinary Research
Volume8
Early online date28 Mar 2012
DOIs
Publication statusPublished - 28 Mar 2012

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Bovine parainfluenza virus 3
Bovine respiratory syncytial virus
Bovine Herpesvirus 1
Bovine herpesvirus 1
Multiplex Polymerase Chain Reaction
Real-Time Polymerase Chain Reaction
reverse transcriptase polymerase chain reaction
Viruses
viruses
Reverse Transcriptase Polymerase Chain Reaction
quantitative polymerase chain reaction
Cattle Diseases
bovine respiratory disease
cattle
assays
nucleic acids
testing
antibodies
Antibodies
Human parainfluenza virus 3

ASJC Scopus subject areas

  • veterinary(all)

Cite this

One-step multiplex real time RT-PCR for the detection of bovine respiratory syncytial virus, bovine herpesvirus 1 and bovine parainfluenza virus 3. / Thonur, Leenadevi; Maley, Madeleine; Gilray, Janice; Crook, Tara; Laming, Ellie; Turnbull, Dylan; Nath, Mintu; Willoughby, Kim.

In: BMC Veterinary Research, Vol. 8, 37, 28.03.2012.

Research output: Contribution to journalArticle

Thonur, Leenadevi ; Maley, Madeleine ; Gilray, Janice ; Crook, Tara ; Laming, Ellie ; Turnbull, Dylan ; Nath, Mintu ; Willoughby, Kim. / One-step multiplex real time RT-PCR for the detection of bovine respiratory syncytial virus, bovine herpesvirus 1 and bovine parainfluenza virus 3. In: BMC Veterinary Research. 2012 ; Vol. 8.
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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 10 2 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.",
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note = "Acknowledgements This study was supported by funding from Scottish Government and by Quality Meat Scotland. We are grateful to George Caldow and other staff of the Scottish Agricultural Disease surveillance Centres for the generous supply of clinical material and sharing the IFAT results, to Peter F Nettleton for sharing clinical isolates and Colin J McInnes for support and guidance.",
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AU - Thonur, Leenadevi

AU - Maley, Madeleine

AU - Gilray, Janice

AU - Crook, Tara

AU - Laming, Ellie

AU - Turnbull, Dylan

AU - Nath, Mintu

AU - Willoughby, Kim

N1 - Acknowledgements This study was supported by funding from Scottish Government and by Quality Meat Scotland. We are grateful to George Caldow and other staff of the Scottish Agricultural Disease surveillance Centres for the generous supply of clinical material and sharing the IFAT results, to Peter F Nettleton for sharing clinical isolates and Colin J McInnes for support and guidance.

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Y1 - 2012/3/28

N2 - 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 10 2 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.

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