Examples are repetitive intergenic palindromic sequence-PCR (REP-PCR) [10], random amplified polymorphic DNA-PCR (RAPD-PCR) [11], arbitrary primed-PCR (AP-PCR) [12], amplified fragment length polymorphism (AFLP) [13], single nucleotide polymorphism (SNP) [14, 15], and polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) [16]. Recently, a highly discriminatory INCB28060 ic50 typing system called MLVA, based on multilocus characterization of variable number of tandem repeats (VNTRs), has been proposed for selleck chemicals several pathogens such
as Haemophilus influenzae [17], Mycobacterium tuberculosis [18], Bacillus anthracis and Yersinia pestis [19–21]. The multilocus VNTR analysis methods (MLVA-15 and -16) were also described for Brucella genotyping [22, 23, 6]. The MLVA-15 is based on 15 polymorphic markers subdivided in two panels: the panel 1, consisting of 8 minisatellite markers with a good species identification capability, and the panel 2, consisting of 7 microsatellite markers with higher discriminatory power. MLVA-15 assay generates multiple band profiles or fingerprint patterns and represents a useful molecular approach for Brucella typing in spite
of the high DNA homology CB-839 among Brucella species (90%) [24]. The obtained MLVA band profiles may be resolved by different techniques ranging from low cost manual agarose gels to the more expensive capillary electrophoresis sequencing systems. Recently, 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]. In this paper we evaluate the possibility to use the Agilent 2100 Bioanalyzer equipment for MLVA typing of Brucella strains using the selected subset of 15 loci proposed by La Flèche [23]. On that account, 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 HSP90 ring trial, were de novo genotyped. 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]. The 15 VNTR markers amplified for bacterial genotyping were arranged into three duplex and nine singleplex PCR reactions. The arrangement of different loci in the same multiplex was such to avoid overlapping of VNTR markers size ranges; in this way it was possible to use a single 12-wells chip (DNA 1000 LabChip Kit) for strain analysis.
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