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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

Absence of gnd gene in S. bongori strains. The gnd gene was PCR-amplified as described in the Materials and Methods from eight different strains of S. bongori. PCR products were resolved using the Agilent 7500 DNA kit and the Agilent 2100 Bioanalyzer. Negative Control 1 corresponds to PCR reagents mix + water as a template; and Negative Control 2 corresponds to DNA from a known gnd negative bacterial strain using our PCR conditions.
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Figure 2: Absence of gnd gene in S. bongori strains. The gnd gene was PCR-amplified as described in the Materials and Methods from eight different strains of S. bongori. PCR products were resolved using the Agilent 7500 DNA kit and the Agilent 2100 Bioanalyzer. Negative Control 1 corresponds to PCR reagents mix + water as a template; and Negative Control 2 corresponds to DNA from a known gnd negative bacterial strain using our PCR conditions.

Mentions: Virtual PCR was done using the designed fliC, gnd, and mutS specific gene primers against the Salmonella database (Table S1). The virtual analysis showed all (100%) of the Salmonella strains were PCR positive for the fliC gene (Table 1). However, the virtual PCR simulation predicted negative results for three out of 27 (11%) database strains for the gnd and mutS genes: S. bongori str. NCTC 12419, S. enterica subsp. arizonae 62:z4,z23:- and S. enterica subsp. enterica serovar Newport str. SL254 (Table 1). Experimental PCR confirmed fliC amplification in all the tested strains. Contrary to the PCR database predictions, S. bongori (2) and S. enterica subsp. arizonae (2) were PCR positive for the mutS gene. As previously reported, one out of the three S. enterica subsp. enterica serovar Newport (SAR B37) was PCR negative for mutS (Brown et al., 2003). In agreement with the In silico database, 100% of the S. bongori (2) were PCR negative for the gnd gene experimentally (Table 1 and Figure 2).


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)

Absence of gnd gene in S. bongori strains. The gnd gene was PCR-amplified as described in the Materials and Methods from eight different strains of S. bongori. PCR products were resolved using the Agilent 7500 DNA kit and the Agilent 2100 Bioanalyzer. Negative Control 1 corresponds to PCR reagents mix + water as a template; and Negative Control 2 corresponds to DNA from a known gnd negative bacterial strain using our PCR conditions.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

Figure 2: Absence of gnd gene in S. bongori strains. The gnd gene was PCR-amplified as described in the Materials and Methods from eight different strains of S. bongori. PCR products were resolved using the Agilent 7500 DNA kit and the Agilent 2100 Bioanalyzer. Negative Control 1 corresponds to PCR reagents mix + water as a template; and Negative Control 2 corresponds to DNA from a known gnd negative bacterial strain using our PCR conditions.
Mentions: Virtual PCR was done using the designed fliC, gnd, and mutS specific gene primers against the Salmonella database (Table S1). The virtual analysis showed all (100%) of the Salmonella strains were PCR positive for the fliC gene (Table 1). However, the virtual PCR simulation predicted negative results for three out of 27 (11%) database strains for the gnd and mutS genes: S. bongori str. NCTC 12419, S. enterica subsp. arizonae 62:z4,z23:- and S. enterica subsp. enterica serovar Newport str. SL254 (Table 1). Experimental PCR confirmed fliC amplification in all the tested strains. Contrary to the PCR database predictions, S. bongori (2) and S. enterica subsp. arizonae (2) were PCR positive for the mutS gene. As previously reported, one out of the three S. enterica subsp. enterica serovar Newport (SAR B37) was PCR negative for mutS (Brown et al., 2003). In agreement with the In silico database, 100% of the S. bongori (2) were PCR negative for the gnd gene experimentally (Table 1 and Figure 2).

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