Previous Article | Next Article 
Applied and Environmental Microbiology, September 2008, p. 5392-5401, Vol. 74, No. 17
0099-2240/08/$08.00+0 doi:10.1128/AEM.00151-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.
Determination of Clonality and Relatedness of Vibrio cholerae Isolates by Genomic Fingerprinting, Using Long-Range Repetitive Element Sequence-Based PCR
,
Nipa Chokesajjawatee,1,2,
Young-Gun Zo,1,3, and
Rita R. Colwell1,3*
Center of Marine Biotechnology, University of Maryland Biotechnology Institute, 701 E. Pratt Street, Baltimore, Maryland 21202,1 National Center for Genetic Engineering and Biotechnology, 113 Phahonyothin Road, Klong 1, Klong Luang, Pathumthani 12120, Thailand,2 Center of Bioinformatics and Computational Biology, University of Maryland Institute of Advanced Computer Studies, University of Maryland College Park, College Park, Maryland 207423
Received 16 January 2008/ Accepted 30 June 2008
A high-throughput method which is applicable for rapid screening, identification, and delineation of isolates of Vibrio cholerae, sensitive to genome variation, and capable of providing phylogenetic inferences enhances environmental monitoring of this bacterium. We have developed and optimized a method for genomic fingerprinting of V. cholerae based on long-range PCR. The method uses a primer set directed to enterobacterial repetitive intergenic consensus sequences, a high-fidelity DNA polymerase, and analysis via conventional agarose gel electrophoresis. Long (
10 kb), highly reproducible amplicons were generated from V. cholerae isolates, including those from different geographical locations and historical strains isolated during the period 1931-2000. The amplicons yielded reduced variability in their densitometric band patterns to 
10% and clonal distinction at <90% similarity. Rapid band-matching analysis was accomplished for fingerprints with 
90% similarity, discriminating O serotypes and biotypes (classical versus El Tor) as well as pathogenic and nonpathogenic strains. Compared to genome similarity measured by DNA-DNA hybridization, the results showed good correlation (r = 0.7; P < 0.001), with five times less measurement error and without bias. The method permits both phylogenetic inference and clonal differentiation of individual V. cholerae strains, enables robust, high-throughput analysis, and does not require specialized equipment to perform. With access to a curated public database furnished with appropriate analytical software applications, the method should prove useful in large-scale multilaboratory surveys, especially those designed to detect specific pathogens in the natural environment.
* Corresponding author. Mailing address: Center for Bioinformatics and Computational Biology, University of Maryland Institute of Advanced Computer Studies, University of Maryland College Park, College Park, MD 20742. Phone: (301) 405-9550. Fax: (301) 314-6654. E-mail: rcolwell{at}umiacs.umd.edu
Published ahead of print on 7 July 2008.
Supplemental material for this article may be found at http://aem.asm.org/.
N.C. and Y.-G.Z. contributed equally to the manuscript and are listed in alphabetical order.
Applied and Environmental Microbiology, September 2008, p. 5392-5401, Vol. 74, No. 17
0099-2240/08/$08.00+0 doi:10.1128/AEM.00151-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.
Copyright © 2009 by the American Society for Microbiology. For an alternate route to Journals.ASM.org, visit: http://intl-journals.asm.org | More Info»