Development of a real time reverse transcriptase polymerase chain reaction for the detection of bovine respiratory syncytial virus in clinical samples and its comparison with immunohistochemistry and immunofluorescence antibody testing

Willoughby, Kim; Thomson, Karen; Maley, Madeleine; Gilray, Janice; Scholes, S. F. E.; Howie, Fiona; Caldow, George; Nettleton, P. F.

doi:10.1016/j.vetmic.2007.07.005

Cited by 14 publications

(19 citation statements)

References 26 publications

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“…The mRT-qPCR also detected dual infections (BoHV-1 and BPI3 i) in three samples; in virus isolation of these samples, BoHV-1 overgrew BPI3, masking detection. These results demonstrate that the m RT-qPCR is more specific and sensitive in respiratory viral diagnosis when compared to conventional tests, as has been shown in both veterinary and human clinical pathology settings [16,22-25]. …”

Section: Discussionsupporting

confidence: 66%

“…A vaccine isolate of BRSV (Rispoval RS, Pfizer Ltd) was used for assay optimisation and eight other BRSV isolates were used for preliminary validation experiments [22,26]. A reference strain of BoHV-1 (6660) was used for the initial optimisation of assay and further five isolates were tested for validation purposes [27].…”

Section: Methodsmentioning

confidence: 99%

“…The primer and probe sequences, fluorophores and quenchers used are shown in Table 6[22,30]. As appropriate fluorophore combinations could not be designed using MGB probes to allow multiplexing with four colour detection, the reporter dyes were altered and the BRSV probe was modified to replace the minor groove binding (MGB) modification by locked nucleic acids (LNA).…”

Section: Methodsmentioning

confidence: 99%

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

et al. 2012

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BackgroundDetection 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.ResultsA 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).ConclusionsThe 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.

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Section: Discussionsupporting

confidence: 66%

Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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

et al. 2012

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“…The recent development and application of test methods based on the PCR for the direct detection of respiratory pathogens, particularly when applied on real-time platforms, represents a significant step forward in the investigation of BRD outbreaks (Willoughby and others 2008). These assays offer rapid, sensitive and cost-effective diagnostics which, alongside the updating of sampling protocols, offer improved detection rates and greater confidence in laboratory results.…”

Section: Introductionmentioning

confidence: 99%

Patterns of detection of respiratory viruses in nasal swabs from calves in Ireland: a retrospective study

O’Neill¹,

Mooney²,

Connaghan³

et al. 2014

Veterinary Record

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A retrospective analysis was conducted to investigate the prevalence and seasonality of bovine viral diarrhoea virus (BVDV), bovine coronavirus (BoCV), bovine herpesvirus-1 (BoHV-1), bovine respiratory syncytical virus (BRSV) and parainfluenza virus-3 (PI3V) in calves (aged three months and below) in Ireland. Results from real-time PCR testing, including cycle threshold values, conducted on nasal swabs (single or pooled) submitted from 1364 respiratory disease outbreaks between January 1, 2008 and December 31, 2012 were included in this study. One or more viruses were detected in 34.6 per cent of submissions, with BoCV detected most frequently (22.9 per cent), followed by BRSV (11.6 per cent), PI3 V (7.0 per cent), BoHV-1 (6.1 per cent) and BVDV (5.0 per cent). The detection rate of all viruses was higher when pooled multiple swabs were submitted from outbreaks rather than single swabs, with these differences being significant for all except BVDV. Two or more viruses were detected in 39.4 per cent of positive submissions, with BoCV and BRSV most commonly present as one of the two partners in detection. With the exception of BVDV, which was detected all year round, the others showed a clear seasonal pattern, being most commonly detected in winter and spring.

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“…BRD accompanied with irreversible lung damage by BRSV will make livestock more susceptible to other bovine diseases and will reduce economic returns for farmers [2, 4, 5]. For diagnosis of BRSV, virus isolation, immunohistochemistry/immunofluorescence antibody testing or reverse transcription (RT)-PCR procedures are necessary [1, 14], and these tests are both expensive and time consuming. The sensitivity of immunohistochemistry/immunofluorescence antibody testing is not high, and antibodies specific for viral protein are required.…”

mentioning

confidence: 99%

A New High-Speed Droplet-Real-Time Polymerase Chain Reaction Method Can Detect Bovine Respiratory Syncytial Virus in Less than 10 Min

Uehara

Matsuda

Sugano

et al. 2014

The Journal of Veterinary Medical Science

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The polymerase chain reaction (PCR) has been widely used for diagnosis of infectious diseases of domestic animals. Rapid detection of respiratory pathogens of cattle is useful for making therapeutic decisions. Therefore, we developed a new genetic-based method called droplet-real-time PCR, which can detect bovine respiratory syncytial virus (BRSV) within 10 min. Our droplet-real-time PCR markedly reduced the reaction time of reverse transcription-PCR while maintaining the same sensitivity as conventional real-time PCR, and it can be used as a rapid assay for detection of BRSV. Furthermore, our method is potentially applicable for rapid diagnosis of almost all infectious diseases, including highly pathogenic avian influenza virus.

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Development of a real time reverse transcriptase polymerase chain reaction for the detection of bovine respiratory syncytial virus in clinical samples and its comparison with immunohistochemistry and immunofluorescence antibody testing

Cited by 14 publications

References 26 publications

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

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

Patterns of detection of respiratory viruses in nasal swabs from calves in Ireland: a retrospective study

A New High-Speed Droplet-Real-Time Polymerase Chain Reaction Method Can Detect Bovine Respiratory Syncytial Virus in Less than 10 Min

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