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Journal of Clinical Microbiology, January 2008, p. 296-301, Vol. 46, No. 1
0095-1137/08/$08.00+0     doi:10.1128/JCM.01496-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

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

Jeffrey T. Foster,1 Richard T. Okinaka,2 Rita Svensson,2 Kathryn Shaw,2 Barun K. De,3 Richard A. Robison,4 William S. Probert,5 Leo J. Kenefic,1 William D. Brown,1 and Paul Keim1*

Center for Microbial Genetics and Genomics, Northern Arizona University, Flagstaff, Arizona 86011-5640,1 Bioscience Division, Los Alamos National Laboratory, Los Alamos, New Mexico 87545,2 Centers for Disease Control and Prevention, Atlanta, Georgia 30333,3 Department of Microbiology and Molecular Biology, Brigham Young University, Provo, Utah 84602,4 Microbial Diseases Laboratory, California Department of Public Health, Richmond, California 948045

Received 25 July 2007/ Returned for modification 21 October 2007/ Accepted 4 November 2007

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.

* Corresponding author. Mailing address: Center for Microbial Genetics and Genomics, Northern Arizona University, Flagstaff, AZ 86011-5640. Phone: (928) 523-1078. Fax: (928) 523-0639. E-mail: Paul.Keim{at}nau.edu

{triangledown} Published ahead of print on 21 November 2007.

Journal of Clinical Microbiology, January 2008, p. 296-301, Vol. 46, No. 1
0095-1137/08/$08.00+0     doi:10.1128/JCM.01496-07
Copyright © 2008, American Society for Microbiology. All Rights Reserved.



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