Establishment and field validation of a rapid on-site recombinase polymerase amplification-lateral flow assay for...

Bovine respiratory disease (BRD) is the costliest bovine syndrome worldwide, inflicting annual losses of over one billion USD in North America alone. Transport stress, overcrowding and viral-bacterial synergy can drive mortality to 70%, yet laboratory-based diagnostics delay decisive treatment. We therefore developed pen-side real-time enzymatic recombinase amplification lateral-flow dipsticks (RT-ERA-LFD) assays targeting the two principal viral agents, bovine respiratory syncytial virus (BRSV) and bovine viral diarrhoea virus (BVDV), which enables their separate detection in a single tube. The BRSV nucleoprotein gene and BVDV 5'-UTR were cloned and in-vitro transcribed into quantified RNA standards to calibrate an enzymatic recombinase amplification (ERA) coupled with lateral-flow dipsticks (LFD). After primer/probe optimisation (BRSV-ERA-F1/R4/P2; BVDV-ERA-F1/R4/P1), the 40 °C, 20-min reactions detected as few as 10 template copies, showed 100% specificity against related bovine pathogens and matched real-time PCR results in 46 archived respiratory samples. In a field survey of nasal swabs from cattle farms in Jilin Province, China, BRSV was detected in 10.87% and BVDV in 8.70% of specimens, with results identical to qPCR obtained within 30 min without instrumentation. By delivering actionable infection status at the chute, the platform enables mass screening and timely intervention, effectively mitigating BRD's global economic impact.