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

Abstract

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.

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

“…BRSV outbreaks most commonly occur in winter (Valarcher and Taylor 2007) and BRSV-related disease is highly dependent on animal age (Hagglund and others 2006). Studies by Thonur (Thonur and others 2012) reported single detections of BoHV1 (20 per cent), BRSV (10 per cent) and PI3 (3 per cent) in a subset of UK nasal calf swabs tested using multiplex RT-PCR. Forty-four percent of calves tested in a targeted Scottish study (Hotchkiss and others 2010) were positive for PI3 virus, with much lower levels (10 per cent) of BRSV detected while Pardon reported 60 and 20 per cent positive viral detections for BVDV and BRSV, respectively, in veal calves which died due to BRD (Pardon and others 2011).…”

Detection of Respiratory Viral Antigens in Cattle Lung Tissues by Direct ELISA

“…However, the ability to perform ELISA within a short time frame to detect different viral agents reduces hands-on time in the laboratory, is more efficient in differential diagnosis [27]. In this study, the samples have no examined by FAT or isolation so there is not evaluated the specificity of other tests.…”

Development and evaluation of two truncated recombinant NP antigen-based indirect ELISAs for detection of bovine parainfluenza virus type 3 antibodies in cattle

“…Several attempts have been made to develop a more effective diagnostic assay, including recent PCR approaches that can detect BPIV3 http://dx.doi.org/10.1016/j.jviromet.2015.05.015 0166-0934/© 2015 Elsevier B.V. All rights reserved. nucleic acids in clinical samples from cattle (Thonur et al, 2012). In general, serological assays, including indirect immunofluorescence assay (IF), serum neutralisation test (SNT), complement fixation (CF), hemagglutination inhibition (HI), and Enzyme-Linked Immunosorbent Assay (ELISA) have been frequently employed to detect BPIV3 infection (Adair, 1986;Key and Derbyshire, 1984;Trybala et al, 1989).…”