A multiplex real-time PCR assay for the detection and differentiation of the newly emerged porcine circovirus type 3 and continuously evolving type 2 strains in the United States

Wang, Yin; Feng, Yuan; Zheng, Wanglong; Noll, Lance; Porter, Elizabeth; Potter, Megan; Cino, Giselle; Peddireddi, Lalitha; Liu, Xuming; Anderson, Gary; Bai, Jianfa

A multiplex quantitative real-time polymerase chain reaction (mqPCR) assay was developed and validated for the detection and differentiation of porcine circovirus type 3 (PCV3) and type 2 (PCV2) strains. The assay coverage was 97.9% (184/188) for PCV3 and 99.1% (1889/1907) for PCV2 sequences that were available from the current GenBank database. The PCR amplification efficiencies were 98–99% for plasmids, and 92–96% for diagnostic samples, with correlation coefficients all greater than 0.99. The limit of detection (LOD) determined as plasmid copies per reaction was 17 for PCV3 and 14 for PCV2. The assay specifically detected the targeted viruses without cross reacting to each other or to other common porcine viruses. Among 336 swine clinical samples collected in 2018, 101 (30.1%) were PCV3 positive, 56 (16.7%) were PCV2 positive and 18 (5.4%) were co-positives. Sixty selected PCV3 positives were confirmed by Sanger sequencing, and 53 of the 56 PCV2 positive samples were tested positive by another validated PCR assay.