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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

Differentiation of Salmonella species, subspecies and serovars by PCR-RFLP and cluster analysis. The fliC, gnd, mutS genes were PCR-amplified on the 160 Salmonella strains. The PCR products were cut with specific restriction enzymes and the relationship among restriction patterns were analyzed as described in the Material and Methods. Homogeneous clusters consisting of one Salmonella species, subspecies, and serovars are identified in red.
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Figure 5: Differentiation of Salmonella species, subspecies and serovars by PCR-RFLP and cluster analysis. The fliC, gnd, mutS genes were PCR-amplified on the 160 Salmonella strains. The PCR products were cut with specific restriction enzymes and the relationship among restriction patterns were analyzed as described in the Material and Methods. Homogeneous clusters consisting of one Salmonella species, subspecies, and serovars are identified in red.

Mentions: The six restrictions patterns obtained by the digestion of the fliC, gnd, and mutS genes were analyzed in BioNumerics. We obtained best differential clustering using the combination of the fliC gene cut with HhaI and Sau3AI; gnd gene cut with AciI and AluI; the mutS gene cut with HaeII. Forty-three different clusters and eleven single serovars were identified for a total of 54 different types (Figure 5). This cluster distribution corresponds to a DP of 0.9725. The 43 clusters and the relationship among strains on each cluster are described in Table 2. Twenty-six out of 43 (60.5%) clusters consisted of different homogeneous serovar groups. Nineteen out of the 28 (68%) serovars and/or subspecies represented by more than one strain were grouped into homogeneous clusters (Table 2). In twelve out of these 19 (63.2%) homogeneous clusters representing serovars and subspecies containing more than one strain, 100% of the representing strains were grouped together. Seventeen out of 43 (39.5%) clusters were defined as Type I clusters consisting of S. enterica subsp. enterica strains. Five (29.4%), five (29.4%) and eight (47%) of the 17 Type I clusters did not share, shared one or two elements in their antigenic formula, respectively. Four out of five (80%) clusters sharing one element shared either H1 or H2 antigen. Five out of eight (62.5%) clusters sharing two elements shared the O and the H2 antigen. Thirty-seven % (3/8) shared both H1 and H2 antigens.


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)

Differentiation of Salmonella species, subspecies and serovars by PCR-RFLP and cluster analysis. The fliC, gnd, mutS genes were PCR-amplified on the 160 Salmonella strains. The PCR products were cut with specific restriction enzymes and the relationship among restriction patterns were analyzed as described in the Material and Methods. Homogeneous clusters consisting of one Salmonella species, subspecies, and serovars are identified in red.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 5: Differentiation of Salmonella species, subspecies and serovars by PCR-RFLP and cluster analysis. The fliC, gnd, mutS genes were PCR-amplified on the 160 Salmonella strains. The PCR products were cut with specific restriction enzymes and the relationship among restriction patterns were analyzed as described in the Material and Methods. Homogeneous clusters consisting of one Salmonella species, subspecies, and serovars are identified in red.
Mentions: The six restrictions patterns obtained by the digestion of the fliC, gnd, and mutS genes were analyzed in BioNumerics. We obtained best differential clustering using the combination of the fliC gene cut with HhaI and Sau3AI; gnd gene cut with AciI and AluI; the mutS gene cut with HaeII. Forty-three different clusters and eleven single serovars were identified for a total of 54 different types (Figure 5). This cluster distribution corresponds to a DP of 0.9725. The 43 clusters and the relationship among strains on each cluster are described in Table 2. Twenty-six out of 43 (60.5%) clusters consisted of different homogeneous serovar groups. Nineteen out of the 28 (68%) serovars and/or subspecies represented by more than one strain were grouped into homogeneous clusters (Table 2). In twelve out of these 19 (63.2%) homogeneous clusters representing serovars and subspecies containing more than one strain, 100% of the representing strains were grouped together. Seventeen out of 43 (39.5%) clusters were defined as Type I clusters consisting of S. enterica subsp. enterica strains. Five (29.4%), five (29.4%) and eight (47%) of the 17 Type I clusters did not share, shared one or two elements in their antigenic formula, respectively. Four out of five (80%) clusters sharing one element shared either H1 or H2 antigen. Five out of eight (62.5%) clusters sharing two elements shared the O and the H2 antigen. Thirty-seven % (3/8) shared both H1 and H2 antigens.

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