100A-23

Foodborne pathogenic bacteria can be differentiated each other based on the distribution patterns of ERIC (enterobacterial repetitive intergenic consensus) elements in their genomes.  The distribution patterns of the elements for a strain can be determined by running ERIC-PCR reactions.  Thus we tried to find the optimized concentrations of major components of the reactions for six genera of foodborne pathogenic bacteria.

Our objective was to optimize the concentrations of ERIC-PCR components to determine if strains of foodborne pathogenic bacteria can be differentiated and identified by their DNA polymorphysm.

Template DNA molecules were prepared from each strain.  The oligonucleotide primers based on ERIC elements were used.  ERIC-PCR reactions were carried out with the reaction cocktails containing different concentrations of template DNA, oligonucleotide primers, dNTPs, and MgCl₂.  PCR-generated DNA fragments were separated on a 1.5% agarose gel.  Based on the resulting PCR fingerprinting patterns, the concentrations of the four major components of the reactions were optimized.

The ERIC-PCR genomic fingerprintings were obtained for one strain per genus and they showed six distinct patterns for six genera.  PCR reaction cocktails containing 1 ul of template DNA showed the optimized fingerprinting patterns.  The cocktails containing more than 2 uM oligonucleotide primers generated consistent fingerprinting patterns.  The cocktails containing 200 uM dNTPs yielded the optimized band numbers and intensities.  The cocktails containing 2 mM MgCl₂ started generating consistent fingerprinting patterns.

The PCR reaction conditions optimized for the ERIC primers may be useful to differentiate one strain from the other especially between genera of the foodborne pathogens.  Therefore, the optimized ERIC-PCR genotyping method can be effective, when used other supplementary genotryping or conventional methods, to identify isolates from an outbreak and to estimate relative degrees of genetic similarities among isolates from outbreaks to determine their clonal relationships among outbreaks.