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

Abstract

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 realtime PCR analysis of species-specific single nucleotide polymorphisms (SNPs) that were identified following a robust and extensive phylogenetic analysis of the genus.

Brucellosis at the animal/ecosystem/human interface at the beginning of the 21st century

“…Further, these analyses and the availability of more robust phylogenetic histories, allowed the identification of canonical single nucleotide polymorphisms (SNPs) that could be exploited as the basis of rapid diagnostic tests. A number of SNP-based assays have recently been described that can rapidly identify Brucella isolates to the species level (Foster et al, 2008;Gopaul et al, 2008Gopaul et al, , 2010, identify vaccine strains or even identify to biovar level where biovars reflect true genetic groups (Fretin et al, 2008). A further major recent genome-driven advance has been the identification and exploitation of tandem DNA repeats as typing tools.…”

Serological and Nucleic Acid Based Detection of Brucellosis in Livestock Species and Molecular Characterization of Brucella melitensis Strains Isolated from Pakistan

“…In brief, genomic DNA was extracted from field isolates which grew on blood culture through procedures as explained earlier by Whatmore et al (2005). Based on single nucleotide polymorphism (SNP) identified by Whatmore et al (2007), species identification of field isolates and determination of possible vaccine markers were undertaken using previously described multiple outcome real-time PCR assays (Goupal et al, 2008(Goupal et al, , 2010. These competition assays were set up in final reaction volume of 12.5 µL containing 6.25 µL TaqMan genotyping mix (Applied Biosystems, Warrington, United Kingdom) using Agilent MX3005p platform (Agilent, La Jolla, CA) with working concentrations of primers, probes and cycling conditions as mentioned in above citation.…”

16S rRNA gene sequence analysis of a Brucella melitensis infection misidentified as Bergeyella zoohelcum

“…Current versions of validated sequence databases such as Microseq and RIDOM have been successfully used to identify dangerous bacterial pathogens including Brucella to the species level [15]. However various molecular approaches such as whole genome sequence comparisons, single nucleotide polymorphism (SNP) analysis of select genes [16], multilocus variable-number tandemrepeat (VNTR) analysis (MLVA), high-resolution melt (HRM) analysis, and differential real-time PCR assays have provided alternative approaches to detect and differentiate members of the genus Brucella to the species level. Overall, it has been demonstrated that the 16S rRNA in most Brucella species is highly conserved and, as a distinct marker of the species, enables rapid and accurate diagnosis of Brucella spp.…”