AUTHOR=Soler-García Ángel A. , De Jesús Antonio J. , Taylor Kishana , Brown Eric W. 

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

JOURNAL=Frontiers in Microbiology

VOLUME=5

YEAR=2014

URL=https://www.frontiersin.org/journals/microbiology/articles/10.3389/fmicb.2014.00417

DOI=10.3389/fmicb.2014.00417

ISSN=1664-302X

ABSTRACT=<p>Rapid molecular typing methods are important tools in surveillance and outbreak investigations of human <italic>Salmonella</italic> infections. Here we described the development of a three-genes PCR-RFLP typing method for the differentiation of <italic>Salmonella</italic> species, subspecies and serovars using the Agilent 2100 Bioanalyzer. The <italic>fliC</italic>, <italic>gnd</italic>, and <italic>mutS</italic> genes were PCR-amplified in 160 <italic>Salmonella</italic> strains representing the two <italic>Salmonella</italic> species, six subspecies, and 41 different serovars of <italic>S. enterica</italic> subspecies <italic>enterica</italic>. 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 <italic>Salmonella</italic> were differentiated by conventional PCR. All of <italic>S. bongori</italic> tested were <italic>gnd</italic> PCR negative due to a mismatch at the 3′-end in one the PCR primers. <italic>Salmonella</italic> subspecies were differentiated into third-teen homogeneous groups representing each of the six subspecies by cluster analysis of restriction patterns generated from the <italic>mutS</italic> gene cut with <italic>AciI</italic>. <italic>S. enterica</italic> subspecies <italic>enterica</italic> 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 <italic>S</italic>. Saintpaul and Typhimurium from 24 h pre-enrichment samples using the described method. The use of <italic>fliC</italic>, <italic>gnd</italic>, and <italic>mutS</italic> PCR-RFLP with the Agilent 2100 Bioanalyzer can provide an accessible and automated alternative method for differentiation of <italic>Salmonella</italic> pathogens.</p>