Multiple-locus variable-number tandem-repeats analysis of Salmonella enterica subsp. enterica serovar Typhimurium using PCR multiplexing and multicolor capillary electrophoresis.
The multiple-locus variable-number tandem-repeats analysis (MLVA) method is currently being used as the primary typing tool for Salmonella enterica subsp. enterica serovar Typhimurium (S. Typhimurium) isolates in our laboratory. Our published initial MLVA was performed using a single fluorescent dye and the different patterns were assigned from gel images. Here we present a new and significantly improved assay using multiple dye colors, enhanced PCR multiplexing and the introduction of two new loci for better adaptation to capillary electrophoresis with increased speed. The different MLVA patterns are now based on allele sizes entered as character values, thus removing the uncertainties introduced when analyzing band patterns from gel images. We additionally propose an easy numbering scheme for the identification of separate isolates that will facilitate exchange of typing data. A total of 106 human, bird, animal and food isolates of S. Typhimurium, including 16 with definite type (DT) 104, were used for the development of the improved MLVA. The method is based on capillary separation of multiplexed PCR products from five VNTR loci in the S. Typhimurium genome labeled with multiple fluorescent dyes. The different alleles at each locus were then assigned 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 Nydalen, Torshov, N-0403 Oslo, Norway. bjorn-arne.lindstedt@fhi.no
Vardund
Traute
T
Aas
Lena
L
Kapperud
Georg
G
D000483:Alleles; D000818:Animals; D016172:DNA Fingerprinting / Q000379:methods; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D019075:Electrophoresis, Capillary / Q000379:methods; D005456:Fluorescent Dyes / Q000737:chemistry; D006801:Humans; D018598:Minisatellite Repeats / Q000235:genetics; D010802:Phylogeny; D016133:Polymerase Chain Reaction / Q000379:methods*; D012486:Salmonella typhimurium / Q000145:classification* / Q000235:genetics*
D004269:DNA, Bacterial; D005456:Fluorescent Dyes