Abstract
BackgroundBrucellosis 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.ResultsSeventeen 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.ConclusionIn 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.
Highlights
Brucellosis is an important zoonosis caused by the genus Brucella
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
In order to set up this method, we analyzed, by MLVA-15 [23] on Agilent 2100 Bioanalyzer, seventeen Brucella strains previously identified as B. melitensis biovar 3 by direct sequencing [27]
Summary
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. A rapid and inexpensive method based on the Lab on a chip technology (Agilent Technologies) has been proposed This miniaturized platform for electrophoresis applications is able to size and quantitate PCR fragments, and was previously used for studying the genetic variability of bcIA gene of Bacillus cereus [25] and for genotyping of Bacillus anthracis and Yersinia pestis [26]. We analyzed by Agilent technology the collection of seventeen human Brucella isolates whose MLVA fingerprinting profiles were previously resolved by capillary electrophoresis sequencing system [27] and results compared, while twelve DNA samples, provided in 2007 for a MLVA VNTR ring trial, were de novo genotyped
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