Journal
JOURNAL OF MICROBIOLOGICAL METHODS
Volume 58, Issue 2, Pages 213-222Publisher
ELSEVIER SCIENCE BV
DOI: 10.1016/j.mimet.2004.03.016
Keywords
MLVA; VNTR; multi-color; Escherichia coli O157; PCR multiplexing; capillary electrophoresis
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The Multiple-Locus Variable-Number Tandem-Repeats Analysis (MLVA) method is currently being used as the primary typing tool for Shiga-toxin-producing Escherichia coli (STEC) O157 isolates in our laboratory. The initial assay was performed using a single fluorescent dye and the different patterns were assigned using a gel image. Here, we present a significantly improved assay using multiple dye colors and enhanced PCR multiplexing to increase speed, and ease the interpretation of the results. the different MLVA patterns are now based on allele sizes entered as character values, thus removing the uncertainties introduced when analyzing band patterns from the gel image. We additionally propose an easy numbering scheme for the identification of separate isolates that will facilitate exchange of typing data. Seventy-two human and animal strains of Shiga-toxin-producing E. coli O157 were used for the development of the improved MLVA assay. The method is based on capillary separation of multiplexed PCR products of VNTR loci in the E. coli O157 genome labeled with multiple fluorescent dyes. The different alleles at each locus were then assigned to allele numbers, which were used for strain comparison. (C) 2004 Elsevier B.V. All rights reserved.
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