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The African swine fever virus (ASFV), particularly the genotypes I and II strains that have spread beyond Africa, continues to threaten global swine production and food security. Currently, African swine fever (ASF) control strategies rely heavily on biosecurity measures underpinned with molecular diagnostics. To address the need for genotyping, the present study developed a tetra-primer amplification refractory mutation system PCR (ARMS-PCR) assay capable of discriminating genotypes I and II ASFVs, as well as their mixed infections. The assay utilizes a multiplex of four primers: two outer universal primers and two allele-specific inner primers targeting a conserved single nucleotide polymorphism (SNP, G1656A) of the B646L gene, specific to genotypes I and II ASFVs. The tetra-primer ARMS-PCR assay demonstrated a detection limit of 100 copies per reaction, which is comparable to that of the WOAH-recommended PCR method, and no cross-reactivity was observed with eight other porcine viruses. Furthermore, clinical validation confirmed that the assay demonstrated a 97.22% coincidence rate (35/36) with the standard WOAH-recommended PCR method. In summary, the tetra-primer ARMS-PCR assay provides a practical, specific, and cost-effective tool for ASF epidemiological surveillance, well-suited for resource-limited regions and those with cocirculation of genotypes I and II ASFVs.

Keywords: ASFV; SNP; differential detection; genotype I and II; tetra-primer ARMS-PCR.