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.

Foodborne bacterial pathogens, including S. aureus, L. monocytogenes, C. sakazakii, and M. bovis, pose significant threats to dairy safety and public health. Current detection methods, such as culture-based techniques and real-time quantitative PCR (qPCR), are either time-consuming or limited in absolute quantification accuracy. Herein, we developed and validated a novel quadruplex droplet digital PCR (ddPCR) assay for simultaneous detection and absolute quantification of these four pathogens in dairy products. The assay targets the femA, hly, ompA, and esxA genes, respectively, with optimized primer/probe concentrations of 500 nM/400 nM and an annealing temperature of 58 °C. The established method demonstrated high specificity, with no cross-reactivity against common dairy-associated bacteria. The limits of detection (LoDs) ranged from 7.04 to 10.31 copies/reaction, with coefficients of variation (CVs) below 14% for intra-assay and 10% for inter-assay repeatability. Notably, the ddPCR assay detected a 25% co-contamination rate compared to 13% by qPCR among 120 dairy samples, suggesting higher sensitivity for low-abundance targets. This quadruplex ddPCR platform offers a rapid, sensitive, and high-throughput solution for food safety surveillance, particularly in high-risk dairy matrices such as infant formula.