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Journal of Clinical Microbiology, January 2007, p. 47-53, Vol. 45, No. 1
0095-1137/07/$08.00+0     doi:10.1128/JCM.01233-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Strain-Specific Single-Nucleotide Polymorphism Assays for the Bacillus anthracis Ames Strain{dagger} ,{triangledown}

Matthew N. Van Ert,1 W. Ryan Easterday,1 Tatum S. Simonson,1 Jana M. U'Ren,1 Talima Pearson,1 Leo J. Kenefic,1 Joseph D. Busch,1 Lynn Y. Huynh,1 Megan Dukerich,1 Carla B. Trim,1 Jodi Beaudry,1 Amy Welty-Bernard,1 Timothy Read,3 Claire M. Fraser,3 Jacques Ravel,3 and Paul Keim1,2*

Northern Arizona University, Department of Biological Sciences, Box 5640, Flagstaff, Arizona 86011,1 Translational Genomics Research Institute, Pathogen Genomics Division, 445 N. Fifth Street, Phoenix, Arizona 85004,2 The Institute for Genomic Research, 9712 Medical Center Drive, Rockville, Maryland 207233

Received 15 June 2006/ Returned for modification 22 July 2006/ Accepted 7 October 2006

Highly precise diagnostics and forensic assays can be developed through a combination of evolutionary analysis and the exhaustive examination of genomic sequences. In Bacillus anthracis, whole-genome sequencing efforts revealed ca. 3,500 single-nucleotide polymorphisms (SNPs) among eight different strains and evolutionary analysis provides the identification of canonical SNPs. We have previously shown that SNPs are highly evolutionarily stable, and the clonal nature of B. anthracis makes them ideal signatures for subtyping this pathogen. Here we identified SNPs that define the lineage of B. anthracis that contains the Ames strain, the strain used in the 2001 bioterrorist attacks in the United States. Sequencing and real-time PCR were used to validate these SNPs across B. anthracis strains, including (i) 88 globally and genetically diverse isolates; (ii) isolates that were shown to be genetic relatives of the Ames strain by multiple-locus variable number tandem repeat analysis (MLVA); and (iii) several different lab stocks of the Ames strain, including a clinical isolate from the 2001 letter attack. Six SNPs were found to be highly specific for the Ames strain; four on the chromosome, one on the pX01 plasmid, and one on the pX02 plasmid. All six SNPs differentiated the B. anthracis Ames strain from the 88 unique B. anthracis strains, while five of the six separated Ames from its close genetic relatives. The use of these SNPs coupled with real-time PCR allows specific and sensitive (<100 fg of template DNA) identification of the Ames strain. This evolutionary and genomics-based approach provides an effective means for the discovery of strain-specific SNPs in B. anthracis.

* Corresponding author. Mailing address: Department of Biological Sciences, Northern Arizona University, Flagstaff, AZ 86011-5640. Phone: (928) 523-1078. Fax: (928) 523-0639. E-mail: Paul.Keim{at}nau.edu.

{dagger} Supplemental material for this article may be found at http://jcm.asm.org/.

{triangledown} Published ahead of print on 8 November 2006.

Journal of Clinical Microbiology, January 2007, p. 47-53, Vol. 45, No. 1
0095-1137/07/$08.00+0     doi:10.1128/JCM.01233-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.



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