High-Resolution Melting PCR as Rapid Genotyping Tool for Species.

sp. are the causative agents of brucellosis. One of the main characteristics of the genus concerns its very high genetic homogeneity. To date, classical bacteriology typing is still considered as the gold standard assay for direct diagnosis of . Molecular approaches are routinely used for the identification of at the genus level. However, genotyping is more complex, and to date, no method exists to quickly assign a strain into species and biovar levels, and new approaches are required. Next generation sequencing (NGS) opened a new era into the diagnosis of bacterial diseases. In this study, we designed a high-resolution melting (HRM) method for the rapid screening of DNA and direct assignment into one of the 12 species of the genus. This method is based on 17 relevant single nucleotide polymorphisms (SNPs), identified and selected from a whole genome SNP (wgSNP) analysis based on 988 genomes (complete and drafts). These markers were tested against the collection of the European Reference Laboratory (EU-RL) for brucellosis (1440 DNAs extracted from strains). The results confirmed the reliability of the panel of 17 SNP markers, allowing the differentiation of each species of together with biovars 1, 2, and 3 of and vaccine strain Rev1 () within 3 h, which is a considerable gain of time for brucellosis diagnosis. Therefore, this genotyping tool provides a new and quick alternative for identification based on SNPs with the HRM-PCR assay.