Genotyping of Bacillus anthracis strains based on automated capillary 25-loci multiple locus variable-number tandem repeats analysis

被引:127
作者
Lista, Florigio
Faggioni, Giovanni
Valjevac, Samina
Ciammaruconi, Andrea
Vaissaire, Josee
le Doujet, Claudine
Gorge, Olivier
De Santis, Riccardo
Carattoli, Alessandra
Ciervo, Alessandra
Fasanella, Antonio
Orsini, Francesco
D'Amelio, Raffaele
Pourcel, Christine
Cassone, Antonio
Vergnaud, Gilles
机构
[1] Army Med Res Ctr, I-00184 Rome, Italy
[2] Univ Roma La Sapienza, Fac Med 2, Cattedra Allergol & Immunol Clin, Rome, Italy
[3] Ctr Etud Bouchet, Div Analyt Microbiol, F-91710 Vert Le Petit, France
[4] Univ Paris 11, Inst Genet & Microbiol, Orsay, France
[5] CNR, Lab Charbon, LNR, AFSSA,LERPAZ, F-94700 Maisons Alfort, France
[6] Ist Super Sanita, I-00161 Rome, Italy
[7] Anthrax Ref Inst Italy, Ist Zooprofilatt Sperimentale Puglia & Basilicata, I-71100 Foggia, Italy
[8] Direz Gen Sanita Militare, I-00184 Rome, Italy
关键词
D O I
10.1186/1471-2180-6-33
中图分类号
Q93 [微生物学];
学科分类号
071005 ; 100705 ;
摘要
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. Conclusion: 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.
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页数:14
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