Porcine enteric coronaviruses (CoVs), including swine acute diarrhea syndrome coronavirus (SADS-CoV), porcine epidemic diarrhea virus (PEDV), porcine deltacoronavirus (PDCoV), and porcine transmissible gastroenteritis virus (TGEV), are major pathogens causing porcine viral diarrhea syndrome (VDS), which brings significant economic losses to the swine industry; distinguishing between these clinically similar viruses has become a serious challenge. We developed a highly specific and interference-resistant porcine CoV multiplex digital PCR (dPCR) assay. The assay exhibited robust anti-interference capabilities, as the concentrations of the four viruses did not affect their accurate quantification. The coefficients of variation (CV%) of intra-batch and inter-batch repeatability for all target viruses were less than 11%. The limit of quantification (LoQ) of this dPCR assay reached 7.5 copies/reaction for each target, and it was one order of magnitude more sensitive than qPCR. The limits of detection (LoD) for SADS-CoV, PEDV, PDCoV, and TGEV were 2.72, 3.00, 3.56, and 3.19 copies/reaction, respectively. A total of 408 known samples were used for validation tests, and the results were highly consistent with the known conditions, showing a compliance rate of 97–100%. The diagnostic specificity (Dsp) of the method was 99–100%. In conclusion, the developed multiplex dPCR assay is highly suitable for early detection and quarantine in four porcine CoVs. The results indicate that this dPCR method is characterized by high specificity, anti-interference capabilities, repeatability, and high sensitivity. It also demonstrates a high compliance rate and diagnostic specificity in sample detection. This multiplex dPCR will contribute to the control of porcine enteric CoV-caused VDS and provide clues for subsequent research.

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.