Establishment of a Triplex qPCR Assay for Differentiating Highly Virulent Genotype I Recombinant Virus From Low-Virulence Genotype I and Genotype II African Swine Fever Viruses Circulating in China.

African swine fever virus (ASFV) poses serious threats to the global swine industry, food safety, and the economy. Since August 2018, different types of ASFVs have successively emerged in China, making ASF diagnostics more challenging. The highly virulent genotype I recombinant virus has gradually become the prevalent dominant strain and is identified by sequencing several of its genes, which is time-consuming and expensive. Here, we developed a triplex real-time quantitative PCR (qPCR) assay based on the ASFV B646L, X64R, and MGF_360-14L genes to differentiate highly virulent genotype I recombinant viruses from low-virulence genotype I and genotype II viruses in China. This method has high sensitivity and a limit of detection of 10 copies/reaction for standard plasmids, as well as good specificity without cross-reactions with the viral nucleic acids of porcine reproductive and respiratory syndrome virus (PRRSV), classical swine fever virus (CSFV), pseudorabies virus (PRV), porcine circovirus 2 (PCV 2), porcine circovirus 3 (PCV 3), porcine epidemic diarrhea virus (PEDV), transmissible gastroenteritis virus (TGEV), or porcine rotavirus (PoRV). Importantly, triplex qPCR can be used to quickly and accurately evaluate clinical samples and cell cultures infected with highly virulent genotype I virus, low-virulence genotype I virus, or genotype II virus. Thus, triplex qPCR provides an alternative tool for ASF surveillance in China.