title: Genotyping of Bacillus anthracis strains based on automated capillary 25-loci multiple locus variable-number tandem repeats analysis.
abstract: BACKGROUND
  The genome of Bacillus anthracis, the etiological agent of anthrax, is highly monomorphic which makes differentiation between strains difficult. A Multiple Locus Variable-number tandem repeats (VNTR) Analysis (MLVA) assay based on 20 markers was previously described. It has considerable discrimination power, reproducibility, and low cost, especially since the markers proposed can be typed by agarose-gel electrophoresis. However in an emergency situation, faster genotyping and access to representative databases is necessary.
  
  
  RESULTS
  Genotyping of B. anthracis reference strains and isolates from France and Italy was done using a 25 loci MLVA assay combining 21 previously described loci and 4 new ones. DNA was amplified in 4 multiplex PCR reactions and the length of the resulting 25 amplicons was estimated by automated capillary electrophoresis. The results were reproducible and the data were consistent with other gel based methods once differences in mobility patterns were taken into account. Some alleles previously unresolved by agarose gel electrophoresis could be resolved by capillary electrophoresis, thus further increasing the assay resolution. One particular locus, Bams30, is the result of a recombination between a 27 bp tandem repeat and a 9 bp tandem repeat. The analysis of the array illustrates the evolution process of tandem repeats.
  
  
  CONCLUSIONS
  In a crisis situation of suspected bioterrorism, standardization, speed and accuracy, together with the availability of reference typing data are important issues, as illustrated by the 2001 anthrax letters event. In this report we describe an upgrade of the previously published MLVA method for genotyping of B. anthracis and apply the method to the typing of French and Italian B. anthracis strain collections. The increased number of markers studied compared to reports using only 8 loci greatly improves the discrimination power of the technique. An Italian strain belonging to the B branch was described, and two new branches, D and E, are proposed. Owing to the upgrading achieved here, precise genotyping can now be produced either by automated capillary electrophoresis, or by the more accessible but slower and for some markers slightly less accurate agarose gel methodology.
authors.affiliation: Army Medical Research Center, Via Santo Stefano Rotondo 4 00184 Rome (Italy). florigio.lista@esercito.difesa.it
authors.forename: Florigio
authors.initials: F
authors.lastname: Lista
authors.forename: Giovanni
authors.initials: G
authors.lastname: Faggioni
authors.forename: Samina
authors.initials: S
authors.lastname: Valjevac
authors.forename: Andrea
authors.initials: A
authors.lastname: Ciammaruconi
authors.forename: Josée
authors.initials: J
authors.lastname: Vaissaire
authors.forename: Claudine
authors.initials: C
authors.lastname: le Doujet
authors.forename: Olivier
authors.initials: O
authors.lastname: Gorgé
authors.forename: Riccardo
authors.initials: R
authors.lastname: De Santis
authors.forename: Alessandra
authors.initials: A
authors.lastname: Carattoli
authors.forename: Alessandra
authors.initials: A
authors.lastname: Ciervo
authors.forename: Antonio
authors.initials: A
authors.lastname: Fasanella
authors.forename: Francesco
authors.initials: F
authors.lastname: Orsini
authors.forename: Raffaele
authors.initials: R
authors.lastname: D'Amelio
authors.forename: Christine
authors.initials: C
authors.lastname: Pourcel
authors.forename: Antonio
authors.initials: A
authors.lastname: Cassone
authors.forename: Gilles
authors.initials: G
authors.lastname: Vergnaud
mesh_terms: D001331:Automation; D001408:Bacillus anthracis / Q000145:classification* / Q000235:genetics*; D004269:DNA, Bacterial / Q000235:genetics; D019075:Electrophoresis, Capillary / Q000379:methods*; D005819:Genetic Markers; D005838:Genotype; D010802:Phylogeny; D020080:Tandem Repeat Sequences / Q000235:genetics*
chemical_list: D004269:DNA, Bacterial; D005819:Genetic Markers