Development of a one-run real-time PCR detection system for pathogens associated with bovine respiratory disease complex

Abstract

Bovine respiratory disease complex (BRDC) is frequently found in cattle worldwide. The etiology of BRDC is complicated by infections with multiple pathogens, making identification of the causal pathogen difficult. Here, we developed a detection system by applying TaqMan real-time PCR (Dembo respiratory-PCR) to screen a broad range of microbes associated with BRDC in a single run. We selected 16 bovine respiratory pathogens (bovine viral diarrhea virus, bovine coronavirus, bovine parainfluenza virus 3, bovine respiratory syncytial virus, influenza D virus, bovine rhinitis A virus, bovine rhinitis B virus, bovine herpesvirus 1, bovine adenovirus 3, bovine adenovirus 7, Mannheimia haemolytica, Pasteurella multocida, Histophilus somni, Trueperella pyogenes, Mycoplasma bovis and Ureaplasma diversum) as detection targets and designed novel specific primer-probe sets for nine of them. The assay performance was assessed using standard curves from synthesized DNA. In addition, the sensitivity of the assay was evaluated by spiking solutions extracted from nasal swabs that were negative by Dembo respiratory-PCR for nucleic acids of pathogens or synthesized DNA. All primer-probe sets showed high sensitivity. In this study, a total of 40 nasal swab samples from cattle on six farms were tested by Dembo respiratory-PCR. Dembo respiratory-PCR can be applied as a screening system with wide detection targets.

Gap analysis ofMycoplasma bovisdisease, diagnosis and control: An aid to identify future development requirements

“…It is important to note that although multiplex detection may help in the diagnosis of mixed mollicute infections, it may introduce additional complexity into the determination of disease aetiology; multiple mycoplasma species may be found, for example, in the upper respiratory or genital tracts that are apparent commensals with questionable significance in disease (Brown et al., ). Another broad category of nucleic acid‐based methods is multipathogen panel PCR that allows simultaneous detection of M. bovis with other bacterial and viral pathogens from BRD (Kishimoto et al., ), milk ( bactotype Mastitis HP3 PCR Kit, Qiagen; PathoProof Mastitis Major‐3 Kit; Thermo Fisher Scientific) or aborted bovine clinical samples (Tramuta et al., ). In addition, several loop‐mediated isothermal amplification (LAMP) assays, which have potential utility as pen‐side tests for M. bovis detection, have been reported recently (Ashraf et al., ; Bai et al., ; Higa et al., ), but should undergo extensive validation to ensure reliability of results.…”

A novel multiplex PCR-electronic microarray assay for rapid and simultaneous detection of bovine respiratory and enteric pathogens

“…Recently several multiplex real-time PCR assays have been developed to detect pathogens associated with BRDC and BED (Horwood and Mahony, 2011;Marley et al, 2008;Thonur et al, 2012;Cho et al, 2010). Alternatively, a real-time PCR based system was described that includes multiple reactions in one-run with similar thermal cycling conditions for detection of BRDC and BED pathogens (Kishimoto et al, 2017;Tsuchiaka et al, 2016). Although these real-time PCR assays are rapid and highly sensitive, their multiplexing capability at present is restricted by the availability of limited number of compatible fluorescent dyes.…”

Evaluation of novel multiplex qPCR assays for diagnosis of pathogens associated with the bovine respiratory disease complex

“…However, the efficiency and sensitivity of a multiplex PCR can be compromised due to primer and probe sets interfering, or the competition for the components in the reaction (Kalle et al, 2014;Parker et al, 2015). In this study, the commercially-available multiplex qPCR Pneumo4B and Pneumo4V assays demonstrated that their efficiencies for seven of these target pathogens (93-106% and 91-104%, respectively) were comparable to those of published singleplex qPCR assays (84.2-101.8%) (Kishimoto et al, 2017). Pneumo4B and Pneumo4V were able to detect between 10 copies of genomic DNA and 10-50 copies of target RNA per reaction, respectively, which are comparable to previously established methods which reported of 100-250 copies per reaction (Rahpaya et al, 2018).…”

“…While serological assays such as ELISA are frequently used to indicate prior infection with M. bovis, false negative results in early infection occur as seroconversion normally takes two weeks or more (Howard et al, 1986;Petersen et al, 2018). A wide range of polymerase chain reaction (PCR) assays have been developed to test individually for each of the pathogens involved in BRD (Amer et al, 2013;Bell et al, 2014;Klem et al, 2019;Kishimoto et al, 2017;Rahpaya et al, 2018). However, testing for multiple BRD pathogens in separate assays is prohibitively expensive for livestock producers.…”