Novel genomic tools for specific and real-time detection of biothreat and frequently encountered foodborne pathogens.

The bacterial genera Escherichia, Salmonella, Shigella, Vibrio, Yersinia, and Francisella include important food safety and biothreat agents. By extensive mining of the whole genome and protein databases of diverse, closely and distantly related bacterial species and strains, we have identified novel genome regions, which we utilized to develop a rapid detection platform for these pathogens. The specific genomic targets we have identified to design the primers in Francisella tularensis subsp. tularensis, F. tularensis subsp. novicida, Shigella dysenteriae, Salmonella enterica serovar Typhimurium, Vibrio cholerae, Yersinia pestis, and Yersinia pseudotuberculosis contained either known genes or putative proteins. Primer sets were designed from the target regions for use in real-time PCR assays to detect specific biothreat pathogens at species or strain levels. The primer sets were first tested by in silico PCR against whole-genome sequences of different species, subspecies, or strains and then by in vitro PCR against genomic DNA preparations from 23 strains representing six biothreat agents (Escherichia coli O157:H7 strain EDL 933, Shigella dysenteriae, S. enterica serovar Typhi, F. tularensis subsp. tularensis, V. cholerae, and Y. pestis) and six foodborne pathogens (Salmonella Typhimurium, Salmonella Saintpaul, Shigella sonnei, F. tularensis subsp. novicida, Vibrio parahaemolyticus, and Y. pseudotuberculosis). Each pathogen was specifically identifiable at the genus and species levels. Sensitivity assays performed with purified DNA showed the lowest detection limit of 128 fg of DNA/μl for F. tularensis subsp. tularensis. A preliminary test to detect Shigella organisms in a milk matrix also enabled the detection of 6 to 60 CFU/ml. These new tools could ultimately be used to develop platforms to simultaneously detect these pathogens.