Establishment of triplex real-time quantitative PCR assay for African swine fever virus genotype I/II recombinants

African swine fever (ASFV) leads a highly contagious and lethal hemorrhagic disease, causing a huge economic loss to the global pig industry. Recently, lethal genotype I/II recombinants were isolated and characterized in China and Vietnam. To differentiate the three genotypes (I, II and I/II recombinants) of ASFV in China, a triplex quantitative PCR (qPCR) assay was developed by targeting B646L, A151R and MGF360-14L genes. The detection limitation for the A151R, MGF360-14L, and B646L genes were 10 copies/μl, demonstrating a high sensitivity. Meanwhile, no cross-reactivity was observed with nucleic acids from ASFV genotype I and II or other swine viruses, confirming a high specificity for this assay. The coefficient of assay variation was below 2%, indicating an excellent repeatability. Importantly, when piglets were challenged with ASFV genotype I/II recombinant virus, the method could be used to detect the virus shedding as early as 3 dpi. In summary, a highly sensitive and specific detection method was established, which holds significant potential for rapid diagnosis and early warning of ASFV genotype I/II recombinant infection.