Development of a Duplex TaqMan-MGB qPCR Assay for Differential Detection of Chinese Epidemic Lumpy Skin Disease Virus Strains and Goatpox Virus

Lumpy skin disease (LSD) is a transboundary viral disease of cattle, including Asian water buffalo and yaks, and certain wild ruminants (e.g., African buffalo, giraffe, wildebeest, eland, and Arabian oryx). It is caused by lumpy skin disease virus (LSDV), a member of the genus Capripoxvirus (family Poxviridae) together with goatpox virus (GTPV) and sheeppox virus (SPPV). High nucleotide identity and serological cross-reactivity among these viruses hinder differential diagnosis. The aim of this study was to develop a Duplex TaqMan-MGB qPCR Assay for Differential Detection of Chinese Epidemic Lumpy Skin Disease Virus Strains and Goatpox Virus. We developed a duplex TaqMan-MGB real-time PCR (qPCR) assay targeting the LSDV GPCR and GTPV RPO30 loci. Virus-specific primers and MGB probes were designed, and the reaction was optimized for single-tube, two-target detection. The assay showed no cross-amplification, limits of detection of 1 × 101 copies/μL (LSDV) and 1 × 101 copies/μL (GTPV), and coefficients of variation < 1%. The assay was applied to 175 yak-derived field specimens from Qinghai–Tibet Plateau, of which 16 and 36 were positive for LSDV and GTPV, respectively. LSDV- and GTPV-positive samples showed specific amplification in the FAM and VIC channels, respectively. The duplex format enables concurrent detection and unambiguous differentiation of LSDV and GTPV and is compatible with high-throughput screening. This sensitive, specific, and reproducible assay supports surveillance and control of LSD in endemic and at-risk regions.