A multiplex crystal digital PCR for detection and quantitation of porcine circovirus type 2 and type 3

Abstract Porcine circovirus (PCV) has become one of the major diseases costing huge economic losses in global pig industry, and PCV type 2 (PCV2) and 3 (PCV3) infections are widespread worldwide. A multiplex crystal digital PCR (cdPCR) was developed with three pairs of specific primers and corresponding probes targeting the Rep region of PCV2, the Cap region of PCV3, and an external process control gene (EPC), respectively, after optimization of the concentration of primers and probes, and annealing temperature. The results showed that the multiplex cdPCR exhibited precise and differential detection capabilities for PCV2 and PCV3 with limit of detection of 1.39×101 and 1.27×101 copies/reaction respectively, whereas no cross-reaction with other porcine viruses. The intra-assay and inter-assay coefficients of variation (CV) were less than 8.75%, indicating good repeatability and reproducibility. Then, PCV2 and PCV3 were detected simultaneously in 40 tissue samples and 70 feed samples with cdPCR and quantitative PCR (qPCR). For tissue samples, crystal dPCR and qPCR had similar positive rates for PCV2 (52.17% vs 54.35%), PCV3 (4.35% vs 2.17%) and co-infection of both viruses (13.04% vs 10.87%). However, in feed samples, the positive detection rate of PCV2 (20%) and co-infection (12.86%) by the cdPCR was surprisingly higher than the qPCR (12.86% and 0%). Accordingly, the highly specific and sensitive multiplex crystal dPCR allowed us to accurately detect PCV2 and PCV3 simultaneously, and is particularly well suited for applications that require the detection of small amounts of input nucleic acid or samples with intensive processing and complex matrix.