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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 and subspecies by PCR-RFLP and cluster analysis. The mutS gene was PCR-amplified on the 160 Salmonella strains. The PCR product was cut with AciI. Restriction patterns were analyzed as described in the Materials and Methods. The relationship among restriction patterns was analyzed by cluster analysis using BioNumerics. The mutS-AciI banding pattern is shown. Homogeneous clusters consisting of Salmonella subspecies are indicated.
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Figure 4: Differentiation of Salmonella species and subspecies by PCR-RFLP and cluster analysis. The mutS gene was PCR-amplified on the 160 Salmonella strains. The PCR product was cut with AciI. Restriction patterns were analyzed as described in the Materials and Methods. The relationship among restriction patterns was analyzed by cluster analysis using BioNumerics. The mutS-AciI banding pattern is shown. Homogeneous clusters consisting of Salmonella subspecies are indicated.

Mentions: The relationship among restriction patterns was analyzed by cluster analysis. A total of possible 63 individual and multiple combinations were analyzed. Dendrograms were drawn using BioNumerics (Applied Maths). S. enterica is comprised of six subspecies: enterica (I), salamae (II), arizonae (IIIa), diarizonae (IIIb), houtenae (IV), and indica (VI). For simplicity, S. bongori is still commonly referred to as subsp. V (Tindall et al., 2005). Some derivatives of S. enterica subsp. houtenae (IV) have been reported and identified as subgroup VII (Boyd et al., 1996). Based on biotype these are very similar to subsp. IV but can be distinguished by multilocus enzyme electrophoresis (Boyd et al., 1996). To establish whether PCR-RFLP has the potential to differentiate among Salmonella subspecies we conducted cluster analysis as described in the Material and Methods Section. Based on the distribution of subspecies and the number of members in each subspecies group (Figure 1A), we expected that the best clustering would consist of seven to eight clusters depending on whether the derivatives of subsp. IV could be separated in two distinct clusters. These theoretical subspecies clustering show a discriminatory power (DP) equal to 0.167 (Hunter and Gaston, 1988; Hunter, 1990). The current RFLP cluster analysis showed that restriction patterns obtained cutting the mutS gene with the restriction enzyme AciI was indeed sufficient to differentiate the different subspecies of Salmonella (Figure 4). S. enterica subsp. II, IIIa, IIIb, IV, V, and VI were grouped in single homogeneous clusters (Figure 4). S. enterica subsp. I was grouped into six homogeneous clusters consisting of 109, 2, 15, 3, 5, and 12 members, respectively. This clustering corresponds to a DP of 0.5219. This suggests that Salmonella subspecies can be differentiated by mutS-AciI RFLP cluster analysis.


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 and subspecies by PCR-RFLP and cluster analysis. The mutS gene was PCR-amplified on the 160 Salmonella strains. The PCR product was cut with AciI. Restriction patterns were analyzed as described in the Materials and Methods. The relationship among restriction patterns was analyzed by cluster analysis using BioNumerics. The mutS-AciI banding pattern is shown. Homogeneous clusters consisting of Salmonella subspecies are indicated.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 4: Differentiation of Salmonella species and subspecies by PCR-RFLP and cluster analysis. The mutS gene was PCR-amplified on the 160 Salmonella strains. The PCR product was cut with AciI. Restriction patterns were analyzed as described in the Materials and Methods. The relationship among restriction patterns was analyzed by cluster analysis using BioNumerics. The mutS-AciI banding pattern is shown. Homogeneous clusters consisting of Salmonella subspecies are indicated.
Mentions: The relationship among restriction patterns was analyzed by cluster analysis. A total of possible 63 individual and multiple combinations were analyzed. Dendrograms were drawn using BioNumerics (Applied Maths). S. enterica is comprised of six subspecies: enterica (I), salamae (II), arizonae (IIIa), diarizonae (IIIb), houtenae (IV), and indica (VI). For simplicity, S. bongori is still commonly referred to as subsp. V (Tindall et al., 2005). Some derivatives of S. enterica subsp. houtenae (IV) have been reported and identified as subgroup VII (Boyd et al., 1996). Based on biotype these are very similar to subsp. IV but can be distinguished by multilocus enzyme electrophoresis (Boyd et al., 1996). To establish whether PCR-RFLP has the potential to differentiate among Salmonella subspecies we conducted cluster analysis as described in the Material and Methods Section. Based on the distribution of subspecies and the number of members in each subspecies group (Figure 1A), we expected that the best clustering would consist of seven to eight clusters depending on whether the derivatives of subsp. IV could be separated in two distinct clusters. These theoretical subspecies clustering show a discriminatory power (DP) equal to 0.167 (Hunter and Gaston, 1988; Hunter, 1990). The current RFLP cluster analysis showed that restriction patterns obtained cutting the mutS gene with the restriction enzyme AciI was indeed sufficient to differentiate the different subspecies of Salmonella (Figure 4). S. enterica subsp. II, IIIa, IIIb, IV, V, and VI were grouped in single homogeneous clusters (Figure 4). S. enterica subsp. I was grouped into six homogeneous clusters consisting of 109, 2, 15, 3, 5, and 12 members, respectively. This clustering corresponds to a DP of 0.5219. This suggests that Salmonella subspecies can be differentiated by mutS-AciI RFLP cluster analysis.

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