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

Soler-García AA, De Jesús AJ, Taylor K, Brown EW - Front Microbiol (2014)

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Molecular Methods and Subtyping Branch, Division of Microbiology, Center for Food Safety and Applied Nutrition, US Food and Drug Administration College Park, MD, USA.

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.

No MeSH data available.


Related in: MedlinePlus

Effect of different restriction enzyme inactivation methods in the number and sizes of restriction fragments. The fliC gene of S. Typhimurium was PCR-amplified and cut with HhaI restriction enzyme as described in the Materials and Methods. Restriction enzyme activity was stop by heat inactivation, addition of 20 mM EDTA or the reaction was cleaned with a commercially available kit.
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Figure 3: Effect of different restriction enzyme inactivation methods in the number and sizes of restriction fragments. The fliC gene of S. Typhimurium was PCR-amplified and cut with HhaI restriction enzyme as described in the Materials and Methods. Restriction enzyme activity was stop by heat inactivation, addition of 20 mM EDTA or the reaction was cleaned with a commercially available kit.

Mentions: Restriction patterns were resolved using the Agilent DNA 1000 kit (Agilent Technologies). This kit reports a sizing accuracy of ±10%, depending upon the fragment size range, and a sizing resolution that varies from ±5 bp, ±5% and ±10% in the fragments ranging from 25–100, 100–500, and 500–1000 bp, respectively (Agilent Technologies). When resolving restricted DNA using the Bioanalyzer, adding EDTA and/or using heat inactivation of the restriction enzymes is recommended to avoid possible degradation of the internal DNA marker (Agilent Technologies). We tested the effect of adding 20 mM EDTA, heat inactivation, and the use of a commercially-available method for cleaning restriction digestion reactions on the resolution of restriction fragments and reproducibility of restriction patterns in the 2100 Agilent Bioanalyzer. No significant differences in the number of restriction fragments or the sizes obtained among treatments were observed (Figure 3). Although minor differences were detected among fragment sizes between 2 and 5 bp, no degradation of the 1500 and 15 bp internal markers were observed (Figure 3). Given these results, we chose the use of 20 mM EDTA for the inactivation of the restriction enzymes. Although heat inactivation is a cheaper alternative, this step adds 10–20 min to the procedure depending on the restriction enzyme in use.


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

Soler-García AA, De Jesús AJ, Taylor K, Brown EW - Front Microbiol (2014)

Effect of different restriction enzyme inactivation methods in the number and sizes of restriction fragments. The fliC gene of S. Typhimurium was PCR-amplified and cut with HhaI restriction enzyme as described in the Materials and Methods. Restriction enzyme activity was stop by heat inactivation, addition of 20 mM EDTA or the reaction was cleaned with a commercially available kit.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4127528&req=5

Figure 3: Effect of different restriction enzyme inactivation methods in the number and sizes of restriction fragments. The fliC gene of S. Typhimurium was PCR-amplified and cut with HhaI restriction enzyme as described in the Materials and Methods. Restriction enzyme activity was stop by heat inactivation, addition of 20 mM EDTA or the reaction was cleaned with a commercially available kit.
Mentions: Restriction patterns were resolved using the Agilent DNA 1000 kit (Agilent Technologies). This kit reports a sizing accuracy of ±10%, depending upon the fragment size range, and a sizing resolution that varies from ±5 bp, ±5% and ±10% in the fragments ranging from 25–100, 100–500, and 500–1000 bp, respectively (Agilent Technologies). When resolving restricted DNA using the Bioanalyzer, adding EDTA and/or using heat inactivation of the restriction enzymes is recommended to avoid possible degradation of the internal DNA marker (Agilent Technologies). We tested the effect of adding 20 mM EDTA, heat inactivation, and the use of a commercially-available method for cleaning restriction digestion reactions on the resolution of restriction fragments and reproducibility of restriction patterns in the 2100 Agilent Bioanalyzer. No significant differences in the number of restriction fragments or the sizes obtained among treatments were observed (Figure 3). Although minor differences were detected among fragment sizes between 2 and 5 bp, no degradation of the 1500 and 15 bp internal markers were observed (Figure 3). Given these results, we chose the use of 20 mM EDTA for the inactivation of the restriction enzymes. Although heat inactivation is a cheaper alternative, this step adds 10–20 min to the procedure depending on the restriction enzyme in use.

Bottom Line: 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.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.

View Article: PubMed Central - PubMed

Affiliation: Molecular Methods and Subtyping Branch, Division of Microbiology, Center for Food Safety and Applied Nutrition, US Food and Drug Administration College Park, MD, USA.

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.

No MeSH data available.


Related in: MedlinePlus