Development and Application of a Triplex qPCR Assay for the Simultaneous Detection of African Swine Fever Virus, Porcine Circovirus Type 2, and Porcine Circovirus Type 3

African swine fever virus (ASFV), porcine circovirus type 2 (PCV2), and porcine circovirus type 3 (PCV3) represent important viral agents that threaten pig health. To date, no triplex real-time quantitative PCR (qPCR) method has been described for the concurrent identification of all three viruses. In this study, a triplex qPCR system was constructed using the ASFV B646L gene, PCV2 ORF1 gene, and PCV3 ORF1 gene as amplification targets, enabling simultaneous detection of ASFV, PCV2, and PCV3. The standard curves generated for the three viral targets all yielded R2 values of 0.999. The detection thresholds for ASFV, PCV2, and PCV3 were 10 copies/μL. Specificity testing showed that the assay amplified only the three intended viral targets and did not cross-react with other prevalent swine pathogens. Intra-assay and inter-assay reproducibility analyses yielded coefficients of variation below 2%. Seventy-nine clinical samples were tested using the established triplex qPCR assay and a previously published reference method. Positive and negative results were identical between the two assays, giving a concordance rate of 100%. These findings indicate that the triplex real-time qPCR assay provides a sensitive, dependable, and specific method for detecting ASFV, PCV2, and PCV3 infections and may facilitate epidemiological surveillance, as well as pathogen prevention and control.