Establishment of an SYBR Green-based real-time PCR assay for porcine circovirus type 4 detection.

Porcine circovirus 4 (PCV4) is a novel circovirus first discovered in China in April 2019. Here, we established an SYBR Green I-based real-time PCR for quantitative detection of PCV4. A pair of specific primers was designed based on the conserved region of Cap of PCV4. The standard curve of the established real-time PCR. assay showed a good linear relationship. The sensitivity of the established real-time PCR was 100 times greater than that of conventional PCR, and the detection limit of the assay was 3 × 101 copies. There was no cross-reactivity with other swine DNA viruses, showing good specificity. The intra-group variation coefficient was 0.37-0.78 %, and the inter-group variation coefficient was 0.57-0.94%, indicating that the assay has good repeatability. Moreover, the analysis of clinical samples showed that the positive detection rate of PCV4 was 10.71% (18/168), while that of conventional PCR was 8.93% (15/168). Interestingly, co-infection with PCV2 or PCV3, or both PCV2 and PCV3, was also detected. In conclusion, the established SYBR Green I-based real-time PCR may be a cost-effective and rapid method for PCV4 clinical diagnosis.