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

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).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.Bovine respiratory disease (BRD) is a major disease problem for the cattle industry, causing huge economic losses; research on BRD has been a longstanding global priority. While the aetiology is multifactorial, infectious agents are important in the development of disease. The important infectious causes of BRD include viruses, bacteria and mycoplasma [1,2]. Aside of infectious causes, stress and environmental factors such as weaning, temperature, stocking density, dust, humidity, transportation and inadequate nutrition are also important co-factors in development of disease [3]. In economic terms, BRD leads to decreased production, higher levels of mortality and morbidity, increased veterinary and labour costs and reduced carcass value [4-6].T