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

Abstract

BackgroundBrucellosis, 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.ResultsThe 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.ConclusionIn 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.

Capillary electrophoresis based on nucleic acid detection for diagnosing human infectious disease

“…Such developments in MCE included: MCE integrated bacteria lysis, cleanup, PCR, and CE detection for diagnosis of bacteria [156]; Caliper LabChip 90 system performed strain genotyping in one sixth the time of other microfluidics systems (e.g. the Agilent 2100 bioanalyzer) [144]; large-size dsDNA was separated ultra rapidly and efficiently in a blended polymer matrix by MCE [157]; small DNA fragments were analyzed using MCE-AD without requiring any preconditioning or use of polyacrylamide gel in the microchannel [158]; MCE based on a short capillary and a slotted-vial array automated the sample introduction system for high speed separation of DNA fragments without increasing the separation electric field strength [159]; MCE applied to analysis of DNA with 500-5000 bp and for analysis of DNA ligation [160]; MCE employed embedded sub-micron pillar arrays to produce different retarding obstacles for the DNA molecules allowing for the separation of DNA in a free buffer solution [161]. 3.…”

“…Consequently, fluorescence detection has not yet achieved extensive application in clinical diagnosis. Although CE assays have improved greatly over those in the preliminary stages of development, their stability, reliability, and reproducibility are not as good as those provided by traditional techniques [122,143], and sometimes the accuracy of the assays requires additional corrections [32,144]. Thus, the results derived from CE analyses continue to require verification by accepted methods, such as DGGE, direct sequencing, and real-time fluorescence quantitative PCR.…”

Diagnostic Approach to Brucellosis

“…Now semi-automated blood culture techniques like such as the BACTEC™ the time to detection has been significantly reduced. Brucellae can be detected in the blood of infected patients after four days of culture or less [11]. Molecular techniques such as Polymerase chain reaction (PCR) assays can be used to amplify and detect Brucella DNA in pure cultures and in clinical specimens.…”

Applications of Microfluidics in the Agro-Food Sector: A Review

“…The microfluidic sensing device measured changes in the refractive index within specific sensing microchannels that were functionalized with anti-FMDV antibodies. Santis et al (2011) used microfluidics technology for highthroughput accurate Brucella genotyping. The microfluidic platform improved the detection of amplified DNA fragments size in terms of handling and rapidity.…”