Triplex Crystal Digital PCR for the Detection and Differentiation of the Wild-Type Strain and the MGF505-2R and I177L Gene-Deleted Strain of African Swine Fever Virus

African swine fever (ASF) is a severe and highly contagious viral disease that affects domestic pigs and wild boars, characterized by a high fever and internal bleeding. The disease is caused by African swine fever virus (ASFV), which is prevalent worldwide and has led to significant economic losses in the global pig industry. In this study, three pairs of specific primers and TaqMan probes were designed for the ASFV B646L, MGF505-2R and I177L genes. After optimizing the reaction conditions of the annealing temperature, primer concentration and probe concentration, triplex crystal digital PCR (cdPCR) and triplex real-time quantitative PCR (qPCR) were developed for the detection and differentiation of the wild-type ASFV strain and the MGF505-2R and/or I177L gene-deleted ASFV strains. The results indicate that both triplex cdPCR and triplex qPCR were highly specific, sensitive and repeatable. The assays could detect only the B646L, MGF505-2R and I177L genes, without cross-reaction with other swine viruses (i.e., PRRSV, CSFV, PCV2, PCV3, PEDV, PDCoV and PRV). The limit of detection (LOD) of triplex cdPCR was 12 copies/reaction, and the LOD of triplex qPCR was 500 copies/reaction. The intra-assay and inter-assay coefficients of variation (CVs) for repeatability and reproducibility were less than 2.7% for triplex cdPCR and less than 1.8% for triplex qPCR. A total of 1510 clinical tissue samples were tested with both methods, and the positivity rates of ASFV were 14.17% (214/1510) with triplex cdPCR and 12.98% (196/1510) with triplex qPCR, with a coincidence rate of 98.81% between the two methods. The positivity rate for the MGF505-2R gene-deleted ASFV strains was 0.33% (5/1510), and no I177L gene-deleted ASFV strain was found. The results indicate that triplex cdPCR and triplex qPCR developed in this study can provide rapid, sensitive and accurate methods for the detection and differentiation of the ASFV B646L, MGF505-2R and I177L genes.