Developing a duplex ARMS-qPCR method to differentiate genotype I and II African swine fever viruses based on their B646L genes

African swine fever (ASF), caused by the African swine fever virus (ASFV), is an acute, hemorrhagic, and contagious disease of domestic pigs and wild boars. The disease is notifiable and listed by the World Organization for Animal Health (WOAH) (Wang N et? al. 2019). The outcomes of ASF infection can be peracute, acute, subacute, and chronic, depending on the virulence of ASFVs. According to the report of WOAH (. woah.org/app/uploads/2022/12/asf-report24.pdf), from January 2020 to December 2022, ASF led to more than 2 million pig losses. Currently, ASFV persists continuously in more than 23 countries and poses a serious threat to the global swine industry. ASF invaded China on 3 August, 2018, caused by genotype Ⅱ virulent Georgia-07-like ASFVs (Wen et?al. 2019; Zhao et?al. 2019; Wang et?al. 2020; Wang L et?al. 2022). An experimental study showed that Georgia-07-like ASFV HLJ/18 isolated in China is highly lethal and efficiently transmissible in domestic pigs (Zhao et?al. 2019; Jiang et?al. 2021). During the past four years, genotype Ⅱ Georgia-07-like ASFVs dominantly spread in China. However, the low virulent genotype Ⅱ and I ASFVs have been successively reported in China in 2020 and 2021, respectively (Sun et? al. 2021a, b; Shi et? al. 2022). Compared with the high virulent genotype Ⅱ HLJ/18 strain, the low virulent genotype I and Ⅱ ASFVs had lower virulence and high transmissibility in pigs and induced persistent and chronic infection showing irregular virus shedding at low levels (Sun et? al. 2021a, b; Tsegay et? al. 2022; Wang P et? al. 2022). Notably, when different genotype I and genotype Ⅱ viruses infect the same pig in the field, a novel virus may be generated through viral genome recombination, which brings new problems and challenges for the prevention and control of ASF in China. Thus, a diagnostic method that differentiates genotype I and Ⅱ ASFVs with high sensitivity and stability is urgently needed and will be helpful for the prevention and control of ASF in China.