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


CONCLUSIONS
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.
Ciammaruconi
Andrea
A
Histology and Molecular Biology Section, Army Medical Research Center, Via Santo Stefano Rotondo 4, 00184 Rome, Italy. andrea.ciammaruconi@gmail.com
Grassi
Saverio
S
De Santis
Riccardo
R
Faggioni
Giovanni
G
Pittiglio
Valentina
V
D'Amelio
Raffaele
R
Carattoli
Alessandra
A
Cassone
Antonio
A
Vergnaud
Gilles
G
Lista
Florigio
F
D001408:Bacillus anthracis / Q000145:classification / Q000235:genetics*; D015373:Bacterial Typing Techniques / Q000295:instrumentation / Q000379:methods*; D004269:DNA, Bacterial / Q000235:genetics; D019075:Electrophoresis, Capillary / Q000379:methods; D014644:Genetic Variation; D016680:Genome, Bacterial; D005838:Genotype; D018598:Minisatellite Repeats / Q000235:genetics*; D015010:Yersinia pestis / Q000145:classification / Q000235:genetics*
D004269:DNA, Bacterial