新型双重TaqMan qPCR法用于山羊Orf病毒和布鲁氏菌快速鉴别诊断和共感染筛查

Abstract

Orf and brucellosis are significant infectious diseases that pose serious threats to the goat farming industry. Despite their co-occurrence in goat herds, no TaqMan-based duplex qPCR assay has been reported for their simultaneous detection. In this study, we developed a duplex TaqMan real-time qPCR assay for the simultaneous detection of Orf virus (ORFV) and Brucella spp., facilitating rapid differential diagnosis and co-infection screening. Specific primers and probes targeting conserved regions of the ORFV B2L gene and Brucella spp. Bcsp31 gene were designed, and reaction conditions were optimized for multiplexing. The assay achieved limits of detection (LOD) of 32.0 copies/µL for ORFV and 32.8 copies/µL for Brucella spp. , with correlation coefficients (R²) exceeding 0.99. No cross-reactivity was observed with other common goat pathogens. Inter- and intra-assay coefficients of variation (CV) remained below 2%, confirming excellent reproducibility. Paired clinical samples from 214 goats suspected of being infected with ORFV or Brucella spp. across goat farms in Guizhou Province were collected during 2023–2024. The overall positive rates were 16.82% (36/214) for ORFV and 9.35% (20/214) for Brucella spp. The test also identified 8 cases (3.74%) of ORFV and Brucella spp. co-infection. When validated against conventional PCR, the duplex qPCR assay exhibited 100% diagnostic sensitivity (DSe), 97.1% specificity (DSp), and a kappa value of 0.93, confirming its high accuracy and reliability for clinical application. This study reported a novel TaqMan-based duplex qPCR assay for concurrent detection of two key goat pathogens, providing an efficient diagnostic tool for disease management in goat production.