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Background  Brucellosis, caused by members of the genus  Brucella , remains one of the world's major zoonotic diseases. Six species have classically been recognised within the family  Brucella  largely based on a combination of classical microbiology and host specificity, although more recently additional isolations of novel  Brucella  have been reported from various marine mammals and voles. Classical identification to species level is based on a biotyping approach that is lengthy, requires extensive and hazardous culturing and can be difficult to interpret. Here we describe a simple and rapid approach to identification of  Brucella  isolates to the species level based on real-time PCR analysis of species-specific single nucleotide polymorphisms (SNPs) that were identified following a robust and extensive phylogenetic analysis of the genus.    Results  Seven pairs of short sequence Minor Groove Binding (MGB) probes were designed corresponding to SNPs shown to possess an allele specific for each of the six classical  Brucella  spp and the marine mammal  Brucella . Assays were optimised to identical reaction parameters in order to give a multiple outcome assay that can differentiate all the classical species and  Brucella  isolated from marine mammals. The scope of the assay was confirmed by testing of over 300 isolates of  Brucella , all of which typed as predicted when compared to other phenotypic and genotypic approaches. The assay is sensitive being capable of detecting and differentiating down to 15 genome equivalents. We further describe the design and testing of assays based on three additional SNPs located within the 16S rRNA gene that ensure positive discrimination of  Brucella  from close phylogenetic relatives on the same platform.    Conclusion  The multiple-outcome assay described represents a new tool for the rapid, simple and unambiguous characterisation of  Brucella  to the species level. Furthermore, being based on a robust phylogenetic framework, the assay provides a platform that can readily be extended in the future to incorporate newly identified  Brucella  groups, to further type at the subspecies level, or to include markers for additional useful characteristics.

Rapid identification of Brucella isolates to the species level by real time PCR based single nucleotide polymorphism (SNP) analysis