A multiplex real-time PCR assay for identification porcine circovirus 2, atypical porcine pestivirus, and classical swine fever virus

BACKGROUND: Porcine circovirus 2 (PCV2), atypical porcine pestivirus (APPV), and classical swine fever virus (CSFV) are significant pathogens causing congenital tremor in swine, resulting in substantial economic losses worldwide. METHODS: A TaqMan multiplex real-time PCR method was developed to detect PCV2, APPV, and CSFV, simultaneously. This method was then applied to investigate 476 clinical samples from newborn piglets with congenital tremor symptoms. RESULTS: This method exhibited high specificity with no cross-reactivity with other viruses. Furthermore, the assay demonstrated remarkable sensitivity, with a detection limit of 22.6 copies/µL for each pathogen, and excellent repeatability, as evidenced by intra- and inter-assay variability coefficients of less than 2%. The assay was applied to 476 clinical samples, revealing single infection rates of 36.97% for PCV2, 46.43% for APPV, and 14.71% for CSFV. Mixed infection rates were observed at 18.48% for PCV2 + APPV, 5.67% for PCV2 + CSFV, 5.25% for APPV + CSFV, and 3.57% for PCV2 + APPV + CSFV. CONCLUSIONS: A multiplex real-time PCR assay was created to simultaneously detect PCV2, APPV, and CSFV. Testing on clinical samples showed it to be a quick, sensitive, and specific tool for differentiating viruses linked to congenital tremor, highlighting its strong potential for diagnosing swine diseases.