Development of a Multi-Locus Real-Time PCR with a High-Resolution Melting Assay to Differentiate Wild-Type, Asian...

Lumpy skin disease virus (LSDV) affects cattle and causes significant economic damage. The live vaccine derived from an attenuated strain is effective but is associated with mild disease and skin lesions in some vaccinated cattle. Moreover, recombinant LSDV strains, particularly one with wild-type field and vaccine strains, have recently emerged and spread throughout Asian countries. A cost-effective LSDV typing method is required. We developed a multi-locus real-time PCR with a high-resolution melting (HRM) assay to differentiate between the wild-type, vaccine, and dominant Asian recombinant strains. Based on a multiple alignment analysis, we selected three target genes for the HRM assay, ORF095, ORF126, and ORF145, in which there are insertions/deletions and nucleotide substitutions between wild-type and vaccine strains, and designed primer sets for the assay. Using the synthetic DNA encoding these genes for the two strains, it was shown that the PCR amplicons intercalated with a saturating fluorescent dye could clearly differentiate between wild-type and vaccine strains in the HRM analysis for all three target genes. Further, using clinical samples, our method was able to identify recombinant strains harboring the wild-type ORF095 and ORF145 and the vaccine strain ORF126 genes. Thus, our HRM assay may provide rapid LSDV typing.