Abstract: With the development of intensive farming, the demand for early and rapid detection of various pathogens in large-scale pig farms has gradually increased. The aim of this study was to establish the broad-spectrum magnetic nanoparticle visualization detection (UNVs) field detection technology for porcine reproductive and respiratory syndrome virus (PRRSV), classical swine fever virus (CSFV), porcine circovirus type 2 (PCV2), porcine epidemic diarrhea virus (PEDV), porcine pseudorabies virus (PRV), transmissible gastroenteritis virus (TGEV), and porcine parvovirus (PPV). Based on the highly conserved regions of different viruses, probes and primers were designed, and sequence-specific identification probes (enrichment probes and hybridization probes) with high specificity and enrichment and hybridization efficiency for the target pathogen genes were obtained through the screening of probes and optimization of conditions, which were coupled with ferric oxide magnetic particles and nanogold, respectively, to prepare functionalized magnetic beads specific for each pathogen and functionalized nanogold. The functionalized magnetic beads were used as carriers to enrich nucleic acid signals, and functionalized nanogold was used as a bridge to amplify signals, and the dual probes were used to simultaneously detect pathogenic nucleic acids, combined with the enzymatic visualization reaction of alkaline phosphatase to detect signals, to establish a convenient diagnostic technique for on-site visualization of UNVS for seven kinds of viruses, including PCV2, PRV, PPV, PEDV, TGEV, PRRSV, and CSFV. The results showed that the nucleic acid probes designed for screening did not cross-react with other pathogens of pigs and had good specificity. The established technique for the visualization of UNVs for PCV2, PRV, PPV, PEDV, TGEV, PRRSV and CSFV pathogens has a detection limit of 10³ copies·mL^-1 serum sample or 10³ copies·g ^-1 stool sample, which basically achieves the sensitivity of PCR or RT-PCR detection, the maximum coefficient of variation within and between groups is less than 5%, the colorimetric results are stable without large differences, there is good reproducibility, and the detection of a single sample can be completed in less than 4 h. The results of simultaneous testing and retesting of 499 clinical samples (serum and fecal samples from pigs suspected of having PCV2, PRV, PPV, PEDV, TGEV, PRRSV and CSFV) from Shaanxi Province by applying the technique and PCR assay showed that the established visualization method for UNVs against seven pathogens not only had the same sensitivity as ordinary PCR, but even had a higher detection rate compared with ordinary PCR. In this study, we successfully established a visual detection technique for UNVs against PCV2, PRV, PPV, PEDV, TGEV, PRRSV and CSFV pathogens, which has the advantages of high sensitivity, wide detection range as well as good specificity and reliability, providing a rapid, accurate and sensitive diagnostic technique for a variety of pathogens in pigs.

Key words: porcine virus disease, magnetic nanoparticle, alkaline phosphatase, visualization, field test