Two Pentaplex Real-Time Fluorescent PCR Assays for Simultaneous Detection of Ten Ovine Respiratory and Reproductive Pathogens

We developed and evaluated two pentaplex real-time fluorescent PCR assays (Panel 1/Panel 2) for simultaneous detection of ten ovine pathogens. Panel 1 targets respiratory pathogens: Bluetongue virus (BTV), Foot-and-mouth disease virus (FMDV), Schmallenberg virus (SBV), Peste des petits ruminants virus (PPRV), and Sheep pox virus-Goat pox virus (SPPV-GTPV). Panel 2 targets reproductive pathogens: Border disease virus (BDV), Brucella spp. (Bru), Toxoplasma gondii (T. gondii), Chlamydia abortus (C. abortus), and Caprine arthritis-encephalitis virus (CAEV). Each panel uses six fluorescent channels for detection. Validation using reference pathogens and plasmids confirmed that the assay can effectively detect the target pathogens. The Limit of Detection at 95% probability (LOD95) ranged from 2.9 to 6.1 copies/μL for all targets, with correlation coefficients (R2) greater than 0.99. The intra-assay and inter-assay coefficients of variation (CV) were less than 1.5% at both high (105 copies/μL) and low (103 copies/μL) template concentrations. Cross-reactivity tests demonstrated high specificity with no cross-detection among different targets. Validation using 620 clinical samples collected from multiple provinces in China showed strong concordance with reference methods. This multiplex assay provides a rapid, sensitive, specific, and reliable tool for the differential detection and surveillance of major ovine respiratory and reproductive pathogens.