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Real-time PCR assays of single-nucleotide polymorphisms defining the major Brucella clades - PubMed

Real-time PCR assays of single-nucleotide polymorphisms defining the major Brucella clades

Jeffrey T Foster et al. J Clin Microbiol. 2008 Jan.

Erratum in

  • J Clin Microbiol. 2008 Jul;46(7):2474

Abstract

Members of the genus Brucella are known worldwide as pathogens of wildlife and livestock and are the most common organisms of zoonotic infection in humans. In general, brucellae exhibit a range of host specificity in animals that has led to the identification of at least seven Brucella species. The genomes of the various Brucella species are highly conserved, which makes the differentiation of species highly challenging. However, we found single-nucleotide polymorphisms (SNPs) in housekeeping and other genes that differentiated the seven main Brucella species or clades and thus enabled us to develop real-time PCR assays based around these SNPs. Screening of a diverse panel of 338 diverse isolates with these assays correctly identified each isolate with its previously determined Brucella clade. Six of the seven clade-specific assays detected DNA concentrations of less than 10 fg, indicating a high level of sensitivity. This SNP-based approach places samples into a phylogenetic framework, allowing reliable comparisons to be made among the lineages of clonal bacteria and providing a solid basis for genotyping. These PCR assays provide a rapid and highly sensitive method of differentiating the major Brucella groups that will be valuable for clinical and forensic applications.

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Figures

FIG. 1.
FIG. 1.

Real-time PCR and allelic discrimination plots from TaqMan MGB assays for an SNP defining Brucella melitensis. Ten samples (4 B. melitensis and 6 other Brucella species samples) were run at concentrations decreasing from 1 ng/μl to 1 fg/μl. (A) Amplification curves for B. melitensis samples run in duplicate. (B) Allelic discrimination plots for all samples. Samples at the top left are B. melitensis and at the bottom right are other Brucella spp., and at the bottom left, squares near the plot origin are no-template controls (NTCs; n = 8). Numbers on the axes represent degrees of differentiation of points and are based on the threshold values of the reactions.

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