Fieldable genotyping of Bacillus anthracis and Yersinia pestis based on 25-loci Multi Locus VNTR Analysis

doi:10.1186/1471-2180-8-21

BMC Microbiology

Volume 8

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Ciammaruconi A
Grassi S
De Santis R
Faggioni G
Pittiglio V
D'Amelio R
Carattoli A
Cassone A
Vergnaud G
Lista F

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Ciammaruconi A
Grassi S
De Santis R
Faggioni G
Pittiglio V
D'Amelio R
Carattoli A
Cassone A
Vergnaud G
Lista F
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Methodology article

Fieldable genotyping of Bacillus anthracis and Yersinia pestis based on 25-loci Multi Locus VNTR Analysis

Andrea Ciammaruconi¹ , Saverio Grassi¹ , Riccardo De Santis¹ , Giovanni Faggioni¹ , Valentina Pittiglio¹ , Raffaele D'Amelio²^,3 , Alessandra Carattoli⁶ , Antonio Cassone⁶ , Gilles Vergnaud⁴^,5 and Florigio Lista¹^,3

¹Histology and Molecular Biology Section, Army Medical Research Center, Via Santo Stefano Rotondo 4, 00184 Rome, Italy

²Direzione Generale della Sanità Militare, Via Santo Stefano Rotondo 4, 00184 Rome, Italy

³Dipartimento di Scienze Mediche, II Facoltà di Medicina e Chirurgia Università "La Sapienza", Via di Grottarossa 1039, 00189 Rome, Italy

⁴Division of Analytical Microbiology, Centre d'Etudes du Bouchet, BP3, 91710 Vert le Petit (France)

⁵Institut de Génétique et Microbiologie, Univ Paris-Sud Orsay, F-91405, France; CNRS, Orsay, F-91405, France

⁶Department of Infectious, Parasitic, and Immunomediated Diseases, Istituto Superiore di Sanità, Viale Regina Elena, 299, 00161 Rome, Italy

author email corresponding author email

BMC Microbiology 2008, 8:21doi:10.1186/1471-2180-8-21


Published:	29 January 2008

Abstract

Background

Anthrax and plague are diseases caused by Bacillus anthracis and Yersinia pestis respectively. These bacteria are etiological agents for worldwide zoonotic diseases and are considered among the most feared potential bioterror agents. Strain differentiation is difficult for these microorganisms because of their high intraspecies genome homogeneity. Moreover, fast strain identification and comparison with known genotypes may be crucial for naturally occurring outbreaks versus bioterrorist events discrimination.

Results

Thirty-nine B. anthracis and ten Y. pestis strains, representative of the species genetic diversity, were genotyped by Agilent 2100 Bioanalyzer using previously described Multiple Locus VNTR Analysis assays (MLVA). Results were compared to previous data obtained by standard genotyping system (capillary electrophoresis on automatic sequencer) and, when necessary, direct amplicon sequencing. A reference comparison table containing actual fragment sizes, sequencer sizes and Agilent sizes was produced.

Conclusion

In this report an automated DNA electrophoresis apparatus which provides a cheaper alternative compared to capillary electrophoresis approaches was applied for genotyping of B. anthracis and Y. pestis. This equipment, uses pre-cast gels and provides easy transportation, low maintenance and overall general logistic requirements and costs, is easy to set up and provides rapid analysis. This platform is a candidate for on-site MLVA genotyping of biothreat agents as well as other bacterial pathogens. It is an alternative to the more expensive and demanding capillary electrophoresis methods, and to the less expensive but more time-consuming classical gel electrophoresis approach.