Real-Time PCR Assays of Single-Nucleotide Polymorphisms Defining the Major Brucella Clades

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...

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Bibliographic Details
Published in:Journal of Clinical Microbiology 2008-01, Vol.46 (1), p.296-301
Main Authors: Foster, Jeffrey T, Okinaka, Richard T, Svensson, Rita, Shaw, Kathryn, De, Barun K, Robison, Richard A, Probert, William S, Kenefic, Leo J, Brown, William D, Keim, Paul
Format: Article
Language:English
Subjects:
Animals
Bacterial Typing Techniques - methods
Bacteriology
Biological and medical sciences
Brucella
Brucella - classification
Brucella - genetics
DNA, Bacterial - chemistry
DNA, Bacterial - genetics
Fundamental and applied biological sciences. Psychology
Genotype
Humans
Microbiology
Miscellaneous
Molecular Sequence Data
Polymerase Chain Reaction - methods
Polymorphism, Single Nucleotide
Sensitivity and Specificity
Sequence Analysis, DNA
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Summary: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.
ISSN:0095-1137
1098-660X
1098-5530
DOI:10.1128/JCM.01496-07