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Open Access Methodology article

High throughput MLVA-16 typing for Brucella based on the microfluidics technology

Riccardo De Santis1, Andrea Ciammaruconi1, Giovanni Faggioni1, Silvia Fillo1, Bernardina Gentile1, Elisabetta Di Giannatale2, Massimo Ancora2 and Florigio Lista1*

Author Affiliations

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

2 Istituto Zooprofilattico Sperimentale dell'Abruzzo e del Molise "G. Caporale", (Istituto G. Caporale), Teramo, Italy

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BMC Microbiology 2011, 11:60  doi:10.1186/1471-2180-11-60

Published: 24 March 2011

Abstract

Background

Brucellosis, a zoonosis caused by the genus Brucella, has been eradicated in Northern Europe, Australia, the USA and Canada, but remains endemic in most areas of the world. The strain and biovar typing of Brucella field samples isolated in outbreaks is useful for tracing back source of infection and may be crucial for discriminating naturally occurring outbreaks versus bioterrorist events, being Brucella a potential biological warfare agent. In the last years MLVA-16 has been described for Brucella spp. genotyping. The MLVA band profiles may be resolved by different techniques i.e. the manual agarose gels, the capillary electrophoresis sequencing systems or the microfluidic Lab-on-Chip electrophoresis. In this paper we described a high throughput system of MLVA-16 typing for Brucella spp. by using of the microfluidics technology.

Results

The Caliper LabChip 90 equipment was evaluated for MLVA-16 typing of sixty-three Brucella samples. Furthermore, in order to validate the system, DNA samples previously resolved by sequencing system and Agilent technology, were de novo genotyped. The comparison of the MLVA typing data obtained by the Caliper equipment and those previously obtained by the other analysis methods showed a good correlation. However the outputs were not accurate as the Caliper DNA fragment sizes showed discrepancies compared with real data and a conversion table from observed to expected data was created.

Conclusion

In this paper we described the MLVA-16 using a rapid, sophisticated microfluidics technology for detection of amplification product sizes. The comparison of the MLVA typing data produced by Caliper LabChip 90 system with the data obtained by different techniques showed a general concordance of the results. Furthermore this platform represents a significant improvement in terms of handling, data acquiring, computational efficiency and rapidity, allowing to perform the strain genotyping in a time equal to one sixth respect to other microfluidics systems as e.g. the Agilent 2100 bioanalyzer.

Finally, this platform can be considered a valid alternative to standard genotyping techniques, particularly useful dealing with a large number of samples in short time. These data confirmed that this technology represents a significative advancement in high-throughput accurate Brucella genotyping.