Abstract
Brucella sp. are the causative agents of brucellosis. One of the main characteristics of the Brucella genus concerns its very high genetic homogeneity. To date, classical bacteriology typing is still considered as the gold standard assay for direct diagnosis of Brucella. Molecular approaches are routinely used for the identification of Brucella 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 Brucella 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 Brucella strains). The results confirmed the reliability of the panel of 17 SNP markers, allowing the differentiation of each species of Brucella together with biovars 1, 2, and 3 of B. suis and vaccine strain Rev1 (B. melitensis) within 3 h, which is a considerable gain of time for brucellosis diagnosis. Therefore, this genotyping tool provides a new and quick alternative for Brucella identification based on SNPs with the HRM-PCR assay.
Highlights
Received: 21 December 2021Brucellosis, known as ‘undulant fever’, is a zoonotic disease caused by Gramnegative bacteria of the Brucella genus [1]
Several specific high-resolution molecular methods are currently available for species identification, such as DNA-based methods [2,20,24], restriction fragment length polymorphism PCR (RFLP) [25], multiple locus variable number of tandem repeat analysis (MLVA) [26,27], multilocus sequence typing (MLST) [28] or multilocus sequence analysis (MLSA) [29], single nucleotide polymorphisms (SNPs) typing [3], average nucleotide identity (ANI) [30], and more recently the matrix-assisted laser desorption ionization-time of flight mass spectrometry analysis (MALDI-TOF) [31]
With the panel of 17 markers designed in this study, it is possible to assign a DNA extracted from a strain into nine Brucella species in less than 3 h, which is an important gain in time in case of outbreak investigations
Summary
Brucellosis, known as ‘undulant fever’, is a zoonotic disease caused by Gramnegative bacteria of the Brucella genus [1]. Several specific high-resolution molecular methods are currently available for species identification, such as DNA-based methods (fragment analysis and sequencing) [2,20,24], restriction fragment length polymorphism PCR (RFLP) [25], multiple locus variable number of tandem repeat analysis (MLVA) [26,27], multilocus sequence typing (MLST) [28] or multilocus sequence analysis (MLSA) [29], SNP typing [3], average nucleotide identity (ANI) [30], and more recently the matrix-assisted laser desorption ionization-time of flight mass spectrometry analysis (MALDI-TOF) [31] These molecular methods allow the distinction between most species of the Brucella genus, but to date, there is no perfect molecular method allowing to quickly and correctly assign a strain into the Brucella genus at species and biovar levels. This genotyping tool will be used as routine tool in the network of EU Reference Laboratories for a quick Brucella identification
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