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Although rapid nucleic acid amplification strategies (PCR and RT-PCR) to detect key foodborne pathogens have been reported, these methods largely remain in developmental stages with significant methodological hurdles. Most significant of these include the following: <ol> <li>the need to utilize large realistic sample volumes (>25 mL or g) compared to requisite PCR/RT-PCR amplification volumes of 10-50 µl; <li>residual food components that inhibit PCR/RT-PCR enzymatic reactions; <li>low levels of contaminating pathogens; <li>the need to detect viable bacterial cells; and <li>the need to rapidly confirm amplification products. The investigators seek to address these issues.</ol>
Development of strategies to ensure the safety of food is critical for sustaining U.S. agriculture. Although rapid nucleic acid amplification strategies (PCR and RT-PCR) to detect key foodborne pathogens have been reported, these methods largely remain in developmental stages with significant methodological hurdles. Most significant of these include the following: 1) the need to utilize large realistic sample volumes (>25 mL or g) compared to requisite PCR/RT-PCR amplification volumes of 10-50 µl; 2) residual food components that inhibit PCR/RT-PCR enzymatic reactions; 3) low levels of contaminating pathogens; 4) the need to detect viable bacterial cells; and 5) the need to rapidly confirm amplification products. The investigators seek to address these issues. Listeria monocytogenes and enterohemorrhagic E. coli (EHEC) will be used as the target pathogens. A bacteria immobilization technique using inexpensive metal hydroxides will be applied to non-specifically concentrate bacteria from representative meat, poultry, ready-to-eat, and produce items. Recent methodological advances including fluorescent resonance energy transfer (FRET) will be applied to improve speedand sensitivity of RT-PCR to detect and confirm viable concentrated bacteria from foods. The resulting assay should yield confirmed detection of viable L. monocytogenes and EHEC from many food matrices in less than 24 hours with detection limits significantly better than those reported with other RT-PCR protocols. The developed assays will provide more rapid options for evaluating food safety.