Multiple-Locus Variable-Number Tandem-Repeats Analysis of Escherichia coli O157 using PCR multiplexing and multi-colored capillary electrophoresis.
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.
Lindstedt
Bjørn-Arne
BA
Division of Infectious Diseases Control, Norwegian Institute of Public Health, Geitmyrsveien 75, P.O. Box 4404 Torshov, N-0403 Oslo, Norway. bjorn-arne.lindstedt@fhi.no
Vardund
Traute
T
Kapperud
Georg
G
D000818:Animals; D001431:Bacteriological Techniques; D001483:Base Sequence; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D019075:Electrophoresis, Capillary / Q000379:methods; D019453:Escherichia coli O157 / Q000145:classification / Q000235:genetics* / Q000302:isolation & purification; D005456:Fluorescent Dyes; D005821:Genetic Techniques; D006801:Humans; D018598:Minisatellite Repeats; D016133:Polymerase Chain Reaction / Q000379:methods
D017931:DNA Primers; D004269:DNA, Bacterial; D005456:Fluorescent Dyes