Development of a SYBR green-based real-time quantitative PCR assay to detect PCV3 in pigs

•We established a SYBR green-based real-time qPCR assay to detect PCV3.•We compared the LOD between gel-based assay and SYBR green-based qPCR assay.•The melt curve temperature of PCV3 was 82.5°C.•The CV of intra-assay was up to 1.83% and the CV of inter-assay was up to 2.27%.•The assay in this study was of high sensitivity, specificity and reproducibility. Porcine circovirus 3 (PCV3) has been reported in cases of porcine dermatitis and nephropathy syndrome, reproductive failure, cardiac and multi-systemic inflammation. A SYBR green-based real-time quantitative PCR (qPCR) assay was established in this study to detect PCV3 in 203 clinical samples from suckling piglets affected by congenital tremors in China. The limit of detection (LOD) of PCV3 was 1.73×104 copies/μL for gel electrophoresis and 1.73×102 copies/μL for SYBR green-based real-time qPCR. The melt curve analysis showed a single melt peak at 82.5°C.The intra-assay coefficient of variation was up to 1.83% and the inter-assay coefficient of variation was up to 2.27%. The PCV3 positive detection rate of 203 clinical samples for the SYBR green-based real-time qPCR and the conventional PCR was 86.70% (176/203) and 26.60% (54/203), respectively. Each tissue detected in the SYBR green-based real-time qPCR showed a higher positive rate than that detected in the conventional PCR. These results indicated that the SYBR green-based real-time qPCR assay is a powerful method with high sensitivity, specificity and reproducibility for epidemiological investigations of PCV3.