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Development of strategies to ensure the safety of food is critical for sustaining U.S. agriculture. Recently developed detection methods based on DNA amplification, termed polymerase chain reaction (PCR), offer a high degree of speed, sensitivity and specificity for pathogen screening in foods. Positive results, however, must still be confirmed by time-consuming conventional methods as PCR does not differentiate viable from nonviable cells. Methodologies will be developed to rapidly detect viable enterohemorrhagic Escherichia coli (EHEC).
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A new DNA-based detection method, quantitative competitive PCR (QC-PCR), and an RNA-based detection method, RT-PCR of mRNA will be developed. The slt-I and slt-II toxin genes will be used as targets. For QC-PCR, a segment of competing DNA similar to the target DNA will be created. A known constant amount of competing DNA is then added to dilutions of extracted target DNA followed by PCR amplification. The amount of target DNA is quantified by a generated standard curve. QC-PCR at two points during enrichment will be used to determine viability. For RT-PCR, mRNA, which is a known indicator of cell viability, will be used. RT-PCR amplification of slt mRNA will be used afer a brief enrichment to determine viability. </P>
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The sensitivity of both methods with respect to viable and injured cells will be evaluated.The methods will be applied to artificially contaminated ground beef samples. The development of nucleic acid based methodologies to detect viable EHEC will be applicable to other foodborne pathogens and will provide a more rapid option for monitoring food safety.</p>