Lab on a chip genotyping for Brucella spp. based on 15-loci multi locus VNTR analysis

• Published in: BMC microbiology Vol. 9; no. 1; p. 66
• Main Authors: De Santis, Riccardo, Ciammaruconi, Andrea, Faggioni, Giovanni, D'Amelio, Raffaele, Marianelli, Cinzia, Lista, Florigio
• Format: Journal Article
• Language: English
• Published: England BioMed Central Ltd 07.04.2009; BioMed Central; BMC
• Subjects: Bacterial Typing Techniques - instrumentation; Bacterial Typing Techniques - methods; Brucella - classification; Brucella - genetics; Brucellosis; Brucellosis - microbiology; Diagnosis; DNA microarrays; DNA, Bacterial - analysis; Genetic aspects; Genetic Markers; Methodology article; Minisatellite Repeats - genetics; Molecular Epidemiology; Physiological aspects; Reproducibility of Results; Sensitivity and Specificity; Sequence Analysis, DNA - methods; Italy
• ISSN: 1471-2180; 1471-2180
• DOI: 10.1186/1471-2180-9-66

Brucellosis is an important zoonosis caused by the genus Brucella. In addition Brucella represents potential biological warfare agents due to the high contagious rates for humans and animals. Therefore, the strain typing epidemiological tool may be crucial for tracing back source of infection in outbreaks and discriminating naturally occurring outbreaks versus bioterroristic event. A Multiple Locus Variable-number tandem repeats (VNTR) Analysis (MLVA) assay based on 15 polymorphic markers was previously described. The obtained MLVA band profiles may be resolved by techniques ranging from low cost manual agarose gels to the more expensive capillary electrophoresis sequencing. In this paper a rapid, accurate and reproducible system, based on the Lab on a chip technology was set up for Brucella spp. genotyping. Seventeen DNA samples of Brucella strains isolated in Sicily, previously genotyped, and twelve DNA samples, provided by MLVA Brucella VNTR ring trial, were analyzed by MLVA-15 on Agilent 2100. The DNA fragment sizes produced by Agilent, compared with those expected, showed discrepancies; therefore, in order to assign the correct alleles to the Agilent DNA fragment sizes, a conversion table was produced. In order to validate the system twelve unknown DNA samples were analyzed by this method obtaining a full concordance with the VNTR ring trial results. In this paper we described a rapid and specific detection method for the characterization of Brucella isolates. The comparison of the MLVA typing data produced by Agilent system with the data obtained by standard sequencing or ethidium bromide slab gel electrophoresis showed a general concordance of the results. Therefore this platform represents a fair compromise among costs, speed and specificity compared to any conventional molecular typing technique.