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Determination of clonality and relatedness of Vibrio cholerae isolates by genomic fingerprinting, using long-range repetitive element sequence-based PCR
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Document Title
Determination of clonality and relatedness of Vibrio cholerae isolates by genomic fingerprinting, using long-range repetitive element sequence-based PCR
Author
Chokesajjawatee N, Zo YG, Colwell RR
Name from Authors Collection
Affiliations
University System of Maryland; University of Maryland College Park; University System of Maryland; University of Maryland Baltimore; National Science & Technology Development Agency - Thailand; National Center Genetic Engineering & Biotechnology (BIOTEC)
Type
Article
Source Title
APPLIED AND ENVIRONMENTAL MICROBIOLOGY
ISSN
0099-2240
Year
2008
Volume
74
Issue
17
Page
5392-5401
Open Access
Green Published
Publisher
AMER SOC MICROBIOLOGY
DOI
10.1128/AEM.00151-08
Format
Abstract
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 (similar to 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, 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.
Funding Sponsor
National Institutes of Health [1R01A139129-01]; National Science Foundation [01200677]
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WOS