In regions where lumpy skin disease virus (LSDV) is endemic, infections lead to major financial setbacks due to poor hide condition, decreased fertility, progressive weight loss, and lower milk production. To curb LSDV outbreaks in China, a heterologous live attenuated goat pox virus (GTPV) vaccine (AV41 strain) has been employed. However, diagnostic approaches that can reliably tell LSDV apart from GTPV vaccine strains remain scarce. This study designed a real-time recombinase polymerase amplification (RPA) assay using primers and probes based on conserved fragments of the ORF132 gene to enable quick and specific detection of LSDV. The reaction required only 20 minutes at a steady 39 °C. The limit of detection was 15 copies/μL, with no detectable cross-reactivity to nucleic acids from goat pox virus, infectious bovine rhinotracheitis virus, Pasteurella multocida, or healthy bovine tissue. When 43 clinical specimens were tested alongside the World Organisation for Animal Health (WOAH) real-time PCR method, a kappa coefficient of 0.94 was achieved. These outcomes verify that this real-time RPA assay is both sensitive and highly specific, offering a practical tool for LSDV diagnosis and surveillance in China.
