Development and application of a SYBR Green I fluorescent PCR assay for the differentiation of genotypes I and II African swine fever viruses

African swine fever (ASF) is a highly fatal hemorrhagic disease affecting domestic pigs caused by African swine fever virus (ASFV). Genetic analysis of ASFV isolates to date has identified 24 geographically related genotypes with various subgroups, but only genotype I and II ASFVs have been reported outside Africa. ASFV genotype II and genotype I viruses were reported in China in 2018 and 2021, respectively. In this study, unique and highly conserved noncoding regions were found between MGF_505-9R and MGF_505-10R in the 188 genomes of ASFV genotypes I and II. A pair of primers was designed on the basis of this region. By optimizing the reaction system and conditions, a SYBR Green I fluorescence PCR assay that can distinguish between ASFV genotypes I and II was established, and the sensitivity, reproducibility and specificity were evaluated. The detection limit was 1 TCID50/0.1 mL for both genotypes, with no cross-reactivity observed with other common pig pathogens. The intra- and interbatch variation coefficients were both less than 1.2%. Clinical sample detection analysis revealed 47 positive cases out of 100, including 3 for genotype I and 44 for genotype II, aligning with results from the WOAH-recommended and national standard methods. The method developed in this study allows for the differentiation of ASFV genotypes I and II without the need for genome sequencing, offering a convenient and rapid approach for ASFV detection and genotype identification.