Multiple-Locus Variable-Number Tandem-Repeats Analysis of Escherichia coli O157 using PCR multiplexing and multi-colored capillary electrophoresis

Journal of Microbiological Methods
Bjørn-Arne LindstedtGeorg Kapperud

Abstract

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.

References

Feb 24, 2001·Nature·N T PernaF R Blattner

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Citations

Jun 9, 2012·Journal of Microbiological Methods·Satoko MiyaBon Kimura
Sep 5, 2008·Foodborne Pathogens and Disease·A SöderströmY Andersson
Dec 5, 2009·Foodborne Pathogens and Disease·Brendan HealySéamus Fanning
Jun 7, 2011·Applied and Environmental Microbiology·C SekseA M Urdahl
Mar 30, 2010·Applied and Environmental Microbiology·Michael B CooleyRobert Mandrell
Jan 5, 2006·Applied and Environmental Microbiology·Emma L SprostonNorval J C Strachan
Dec 8, 2004·Journal of Clinical Microbiology·Vincent RamisseGilles Vergnaud
Mar 7, 2006·Journal of Clinical Microbiology·Thibaud KoesslerJacques Schrenzel
Nov 23, 2006·BMC Veterinary Research·Florence B GilbertBernard Poutrel
Sep 7, 2013·Euro Surveillance : Bulletin Européen Sur Les Maladies Transmissibles = European Communicable Disease Bulletin·C A NadonUNKNOWN MLVA Harmonization Working Group
Oct 5, 2013·BioMed Research International·A V BustamanteP M A Lucchesi
Oct 7, 2011·International Journal of Medical Microbiology : IJMM·Bjørn-Arne Lindstedt
May 10, 2011·International Journal of Food Microbiology·Shu ChenJoseph Odumeru
Sep 22, 2009·Journal of Microbiological Methods·Amee R MangesPatrick Boerlin
May 7, 2008·International Journal of Food Microbiology·Satoko MiyaMartin Wiedmann
Apr 1, 2008·Journal of Microbiological Methods·Alexander J NederbragtMary J Anderson-Glenna
Oct 4, 2012·APMIS : Acta Pathologica, Microbiologica, Et Immunologica Scandinavica·Anne Margrete UrdahlYngvild Wasteson
Mar 4, 2014·Diagnostic Microbiology and Infectious Disease·Juliana GonzálezAna Victoria Bustamante
Oct 19, 2010·Journal of Microbiological Methods·Monica RawalBjørn-Arne Lindstedt
Apr 4, 2015·Journal of Food Protection·Nydia E Reyes-RodríguezAna V Bustamante

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