License DNA sequencing for E. coli O157:H7.

Since 1982 E. 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. Investigations conducted by the U.S. Centers for Disease Control indicate that EHEC is the third most frequent cause of foodborne illness in the United States. 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.

Current techniques for detecting this pathogen are either expensive, time-consuming, cumbersome, have low specificity and sensitivity, or require extensive training to undertake. In addition, all require prior enrichment steps and increase the amount of time needed to obtain final confirmatory results. USDA-ARS scientists have discovered oligonucleotide sequences--primers that were derived from DNA sequences contained within a 60-MDa plasmid. These specifically amplify a DNA fragment of the plasmid found in all EHEC strains. The technology is available for licensing.

You can use the polymerase chain reaction (PCR) to amplify just a few copies of the target DNA, 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. This advance requires only a small sample and just a fraction of the time required by traditional techniques.

These DNA sequences are useful research tools for designing 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 make possible rapid testing of large numbers of samples. Such tests can be used to detect the organism in foods.

Investigators also developed a multiplex PCR assay for specific detection and identification of E. coli O157:H7. PCR primers for the 60-MDa plasmid were used in combination with primers for amplifying a fragment of the EAEA gene and for conserved sequences of Shiga-like toxin I and II (SLT-I, SLT-II) genes. You can simultaneously amplify the three different DNA sequences by using the multiplex PCR.

Patent. 5,652,102--Assay for enterohemorrhagic Escherichia coli O157:H7 by the polymerase chain reaction. Issued: July 29, 1997. Inventors: Pina Fratamico et al. Assigned to USDA. Primers specific for enterohemorrhagic Escherichia coli (EHEC) O157:H7 bacteria have been designed. These are useful for detecting the bacteria using the PCR. The primers were derived from DNA sequences contained within a 60-MDa plasmid that is present in most EHEC. The primers may also be used in combination with primers derived from other sequences of significance.

Further information. Pina M. Fratamico, Microbial Food Safety, USDA-ARS Eastern Regional Research Center, 600 East Mermaid Lane, Wyndmoor, PA 19038; phone: [phone]; fax: [phone]; email: [e-mail]. Licensing: C.G. Crawford; phone: [phone]; email: [e-mail].