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

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. 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. 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. 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.