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Develop a multiplex PCR assay that would provide specific detection of Campylobacter jejuni, Escherichia coli 0157:H7, Listeria monocytogenes and Salmonella in one reaction tube. Evaluate the above multiplex PCR assay for use with poultry products and other foods to detect C. jejuni, Escherichia coli 0157:H7, Listeria monocytogenes and Salmonella within the same PCR tube. To utilize pulsed-filed gel electrophoresis for developing a database system for typing strains of Campylobacter jejuni in poultry. To compare the PFGE patterns and pathogenicity of C. jejuni from poultry isolates with those of human isolates. To obtain plasmic profiles of C. jejuni isolated from poultry carcasses and humans. To determine importance of plasmic DNA in pathogenicity of C. jejuni.
NON-TECHNICAL SUMMARY: A diagnostic test that can detect 4 pathogenic bacteria at one time is being developed. Pathogenicity factors of C. jejuni are being studies. it is of critical importance to the protection of public health to have the ability to determine whether a bacterium with a specific molecular fingerprint isolated from patients suffering from a foodborne illness is identical to bacteria isolated from a suspected food source.
<P>APPROACH: In the first phase, multiplex PCR tests will be conducted using pure cultures in order to determine optimum conditions for multiplex PCR in which all four bacteria can be identified using one PCR tube. Sensitivity and specificity then will be determined. When this is completed, the multiplex PCR assay will be evaluated on a variety of commercial food products including chicken carcass washings, poultry deli meat, and fruits and vegetables. A databse of strains of Campylobacter jejuni is being set up. Pulse-field gel electrophoresis (PFGE) is being conducted on each according to established procedures. PFGE patterns of poultry isolates will be compared to those of human isolates that have been obtained from patients suffering from a foodborne illness. Using the C. jejuni isolates found in the database study that is being conducted, two sets of plasmids from each plasmid containing isolate will be purified utilizing the Quantum Prep Plasmic Miniprep Kit (Bio-Rad). The plasmids will then be evaluated for their role in pathogenicity.