Differentiation of Salmonella strains from the SARA, SARB and SARC reference collections by using three genes PCR-RFLP and the 2100 Agilent Bioanalyzer

Abstract

Rapid molecular typing methods are important tools in surveillance and outbreak investigations of human Salmonella infections. Here we described the development of a three-genes PCR-RFLP typing method for the differentiation of Salmonella species, subspecies and serovars using the Agilent 2100 Bioanalyzer. The fliC, gnd, and mutS genes were PCR-amplified in 160 Salmonella strains representing the two Salmonella species, six subspecies, and 41 different serovars of S. enterica subspecies enterica. PCR products were individually cut with two different restriction enzymes and the resulting 930 restriction patterns were collected using the Agilent 2100 Bioanalyzer followed by cluster analysis. Both species of Salmonella were differentiated by conventional PCR. All of S. bongori tested were gnd PCR negative due to a mismatch at the 3′-end in one the PCR primers. Salmonella subspecies were differentiated into third-teen homogeneous groups representing each of the six subspecies by cluster analysis of restriction patterns generated from the mutS gene cut with AciI. S. enterica subspecies enterica serovars were further differentiated by the combination of the three target genes and five out the six sets of restriction patterns with a discriminatory power of 0.9725 by cluster analysis. The combined RFLP results of five sets of restriction patterns allowed us to assign each of the 160 strains to one of 128 restriction types. During inoculation studies we were able to identify S. Saintpaul and Typhimurium from 24 h pre-enrichment samples using the described method. The use of fliC, gnd, and mutS PCR-RFLP with the Agilent 2100 Bioanalyzer can provide an accessible and automated alternative method for differentiation of Salmonella pathogens.

HIV-1 increases TLR responses in human primary astrocytes

“…Each RT-PCR primer pair is provided with a positive control. For more precise quantification, targeted PCR reactions were carried out, and the amplified products were analyzed by automated chip-based microcapillary electrophoresis on an Agilent 2100 Bioanalyzer instrument (Agilent Technologies, Santa Clara, CA) as previously described 52 53 .…”

Genomic-Based Restriction Enzyme Selection for Specific Detection of Piscirickettsia salmonis by 16S rDNA PCR-RFLP

“…The experimental approach using Pma CI confirmed that the in silico pattern produced in P. salmonis was distinguishable to the pattern generated in co-habitant bacteria, validating our prediction (Figure 2 ). Additionally, since the technique involves an enzymatic amplification of the 16S rDNA gene, its detection limit is comparable with all other PCR-based methods used to detect P. salmonis in agarose gels, whereas its visualization is widely improved (0.25 ng, 1000-fold improved) using the ScreenTapes of TapeStation 2200 technology, as it has been reported recently (Soler-García et al, 2014 ). Moreover, the development of new ScreenTapes (high sensitivity D5000 of Agilent Technologies) promises to further improve this detection limit to 0.01 ng, which will give remarkable sensibility to all PCR based methods, including our PCR-RFLP assay.…”

Species identification and molecular typing of human Brucella isolates from Kuwait

“…The resolution of bands was further improved by the use of Agilent Bioanalyzer. In the present investigation band profiles with high quality sizing resolution (1000–7500 bp: 15%), sizing accuracy (± 10% CV) and sizing reproducibility (5% CV) were obtained, which are essential requirements in fingerprinting based differentiation [ 51 , 52 ]. In our study, the Agilent Bioanalyzer’s band profiles along with the most advanced version of BioNumerics 7.5 formed a model experiment-analysis combination.…”