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Multilocus repetitive DNA sequences for genotyping bacillus anthracis and related bacteria

United States Patent Application

View the Complete Application at the US Patent & Trademark Office
Los Alamos National Laboratory - Visit the Technology Transfer Division Website
Bacillus anthracis is one the most molecularly homogeneous pathogens described, which makes strain discrimination particularly difficult. The present invention includes a molecular-typing method based upon rapidly evolving variable number tandem repeat (VNTR) loci. Multiple-locus VNTR analysis (MLVA) combines the information from multiple alleles at several marker loci. PCR amplification products from eight VNTR regions are detected and sized using fluorescently labeled primers. Five of these eight loci were discovered by characterization of AFLP markers (vrrC.sub.1, vrrC.sub.2, vrrB.sub.1, vrrB.sub.2 and CG3); two were discovered from complete plasmid nucleotide sequences (pXO1-aat, pXO2-at); and, one was previously known (vrrA). 425 isolates were characterized to identify 89 distinct genotypes. VNTR markers frequently had multiple alleles (from 2 to 8) and diversity (D) values between 0.3 and 0.8. UPGMA cluster analysis identified six genetically distinct groups that appear to represent genetic clones. Some of these clones show worldwide distribution, while others are restricted to particular geographic regions. The present method is also applicable to related bacteria. An additional 28 loci having variable repeat units have been identified by examining the B. anthracis DNA sequence, but these loci have not yet been utilized in the identification of B. anthracis strains.
Keim, Paul S. (Flagstaff, AZ), Jackson, Paul J. (Los Alamos, NM)
09/ 842,552
April 26, 2001
[0002] This invention was made with government support under Contract No. W-7405-ENG-36 awarded by the U. S. Department of Energy to The Regents of The University of California. The government has certain rights in the invention.