Development of a Novel Duplex qPCR-HRM Assay for Rapid Detection of African Swine Fever Virus in Pigs

This study introduces an assay combining real-time qPCR and high-resolution melting (HRM) analysis for the detection of African Swine Fever Virus (ASFV). The assay utilizes two subunits of RNA polymerases (RNAP1 or NP1450 and RNAP6 or C147L) gene-targeting primers. Initially, the assay was developed on plasmids containing synthetic gene constructs and later validated on clinical samples obtained from field outbreaks. The assay demonstrated high specificity, as evidenced by the absence of amplification from closely related viruses such as porcine parvovirus type 1 (PPV1), porcine parvovirus type 2 (PPV2), and porcine circovirus (PCV). Comparative analysis between qPCR-HRM assay and TaqMan real-time PCR showed similar detection limits for both methods. The assay showed an accuracy of 100% compared to the World Organisation for Animal Health (WOAH)-recommended assay when tested on 25 representative clinical samples. In silico computational analysis revealed that the identified primer sets can detect all the ASFV genotypes circulating globally. Notably, this assay offers an advantage of differentiating strain with single base change (single nucleotide polymorphisms; SNP) using HRM peaks. The study revealed that utilizing real-time PCR with HRM is a cost-effective method of early detection of ASFV in field. This provides an alternative method for swiftly, simply, and accurately detecting ASFV with high specificity and sensitivity.