|DNA sequencing for E. coli O157:H7|
Since 1982, Escherichia coli O157:H7, also known as enterohemorrhagic E. coli (EHEC), has been linked to numerous foodborne outbreaks and sporadic cases of hemorrhagic colitis and hemolytic uremic syndrome worldwide. The most visible outbreak in the United States occurred in 1993 on the west coast involving over 500 cases and the deaths of several young children. Investigations conducted by the Center for Disease Control indicate that EHEC is the third most frequent cause of foodborne illness in the US. The most common epidemiologically-associated food is ground beef. Beef and dairy cattle carry the EHEC organism in their intestinal tracts; contamination of carcasses can occur during slaughter operations.
Present detection methodologies for this pathogen are either expensive, time-consuming, cumbersome, have low specificity and sensitivity, or require extensive training to perform. In addition, all current methods require prior enrichment steps and therefore increase the amount of time needed to obtain final confirmatory results. Scientists in the Microbial Food Safety Research Unit, ERRC, have discovered oligonucleotide sequences, primers that were derived from DNA sequences contained within a 60-MDa plasmid, which specifically amplify a DNA fragment of the plasmid found in all EHEC strains. The Polymerase Chain Reaction (PCR), can be used to amplify just a few copies of the target DNA thus eliminating the need for a time-consuming enrichment step. Various techniques including agarose gel electrophoresis or DNA hybridization with calorimetric or chemiluminescent detection can be used to detect the PCR products. Thus this development has the dual advantages of requiring only a small sample and just a fraction of the time required by traditional techniques.
These DNA sequences are useful research tools which can be used to design rapid, specific and sensitive tests that governmental regulatory agencies and the food industry can incorporate into their E. coli O157:H7 detection programs. Protocols incorporating these DNA sequences are amenable to automation and therefore rapid testing of large numbers of samples is made possible. Such tests can be used to detect the organism in foods such as ground beef, in cattle fecal specimens, and can potentially be designed to detect the pathogen on beef carcasses during inspection. A multiplex PCR assay was developed for specific detection and identification of E. coli O157:H7. PCR primers for the 60-MDa plasmid were used in combination with primers for amplification of a fragment of the eaea gene and for conserved sequences of Shiga-like toxin I and II (SLT-I, SLT-II) genes. Simultaneous amplification of the three different DNA sequences is achieved using the multiplex PCR (see figure). Fragments of 1089 bp, 225/228 bp and 166 bp of the eaeA gene, SLT-II/SLT-I genes and 60 MDa plasmid, respectively, are amplified. Rapid detection of EHEC in foods will prevent the release of contaminated foods into the marketplace consequently reducing, and perhaps even eliminating, the incidence of EHEC food-associated illnesses and their associated economic burdens.
The patent is available for licensing and the Eastern Regional Research Center is interested in exploring the possibilities of collaboration with a company on the further development of this technology through a Cooperative Research and Development Agreement.
Caption: Agarose gel stained with ethidium bromide (A) and hybridization using digoxigenin-labeled probes (B) of the PCR products obtained from E. coli O157:H7 ATCC strains 43894 (lanes a, c, e and g) and 43895 (lanes b, d,f and h). Lane (M), DNA size markers (1-kb ladder). (B) lanes a and b were hybridized with probe PMI (eaeA gene), lanes c and d with probes 428-I and 428-II (SLT-I and SLT-II genes), e and f with probe PS28 (60-MDa plasmid) and lanes g and h with all four probes.
U.S. Patent 5,652,102 issued July 29, 1997.
Dr. Pina M. Fratamico
Microbial Food Safety
Eastern Regional Research Center
600 East Mermaid Lane
Wyndmoor, PA 19038
Technology Transfer Contact:
Victor (Vic) Chavez