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Many foods including poultry products have a short shelf life in the supermarket. Meats need to be shipped shortly after processing. Current monitoring schemes involve enrichments and/or plating samples onto differential and selective media. Microbial testing requires sensitive and rapid methods for detection of biological hazards. There are a considerable number of possible foodborne pathogens that a food conglomerate or exporter/importer may be required to test for in their product(s). The challenges to developing single DNA-based method for detection of multiple pathogens, has been the compatibility of multiple primers in a multiplex PCR. There is however one universal bacterial gene, rrn, that contains universal, as well as signature sequences that differentiate bacteria taxonomically down to family, genus, and species level. Macroarray, the combination of PCR with DNA: DNA hybridization technology, allows investigators the ability to identify a myriad of organisms in a single test. There exist signature sequences within rrn that can distinguish foodborne pathogens (Salmonella enterica, Campylobacter jejuni, etc.) from innocuous normal flora bacteria. These signature sequences can be used as oligonucleotides to capture complementary sequences present among myriad of PCR products amplified using universal or phyla-specific rrn primers. Identity is determined based on the positioning of these signature-specific oligonucleotides and signal produced upon hybridization of the labeled PCR amplicon with its complementary sequence on the membrane. <P>
The objectives of this proposal are to: <OL> <LI>
Develop macroarray for detection of Salmonella enterica, Campylobacter sp., C. jejuni, C. coli, Staphylococcus aureus, Clostridium perfringens, and Listeria monocytogenes; <LI>Validate utility of macroarray for detecting Salmonella and Campylobacter in poultry.
NON-TECHNICAL SUMMARY: There are a numerous foodborne pathogens a company may be required to test to determine the relative safety of their product. Due to the shelf life of foods, it is important to quickly evaluate safety of food. Macroarray, the combination of PCR with DNA: DNA hybridization technology has the potential to quickly detect a myriad of organisms in a single test.
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APPROACH: spotted onto a specified location on the membrane. Positioning of control oligonucleotides will be done for purposes of orientation, for easy identification of other genus or species-signals produced following hybridization and detection steps discussed below. DNA extracted from each sample, pure culture, carcass rinse, etc., will serve as template in PCR reaction using universal 16S rRNA primers and digoxigenin-labeled nucleotides. The purified PCR product will be denatured and allowed to hybridize to the macroarray filters. In the initial development phase, the following organisms will serve as template in labeling 16S rRNA, PCR amplicons: S. typhimurium, E. coli, C. jejuni, S. aureus, S. epidermidis, Lactobacillus acidophilus, L. monocytogenes, and C. perfringens. Annealing/wash temperatures as well as the ionic strength of hybridization/wash buffers will be altered, one variable at a time, that optimizes conditions for detecting the major foodborne pathogens and maintains the specificity of the capture oligonucleotides. Digoxigenin-labeled PCR amplicon captured by oligonucleotides spotted on membrane will be detected using anti-digoxigenin, alkaline phosphatase antibody conjugate and colorimetric alkaline phosphatase substrate. Optimal conditions for macroarray are those in which PCR products from the major foodborne pathogens section only hybridize to their corresponding capture oligonucleotide spotted at a predetermined position on the membrane as well as the universal bacterial kingdom oligonucleotide. Once the specificity and sensitivity of macroarray has been optimized, we will evaluate this molecular detection method against conventional microbiological methods for detection of Salmonella and Campylobacter from retail poultry meats.
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PROGRESS: 2001/10 TO 2003/10<BR>
We have been striving towards the development of rapid screening methods for identifying samples contaminated with foodborne pathogens. USDA formula funded grants have funded this work as well as a USDA NRI grant for developing O157 specific PCR. Our last research project has resulted in the development of multiplex PCR-ELISA capable of detecting three major foodborne pathogens: Salmonella enterica, C. jejuni and E. coli O157:H7. Unfortunately, we have had many experimental setbacks in developing our 16S rDNA-based macroarray, a single assay for multiple pathogens. We had been unable to detect hybridization of dig-labeled PCR products to membrane-bound 18-25mer, oligonucleotides,. We have tried varying several experimental parameters including modifying hybridization and wash temperatures, salt and oligonucleotide concentration. No discernable signal was detected. There were no problems with either PCR amplification or incorporation of dig-labeled nucleotides into PCR amplicon. The problem that we are encountering may have to do with the length of the oligonucleotide as the capture probe. In reviewing literature on microarrays, the bound-capture probe is either PCR amplicon or a 50-mer, oligonucleotide that is spotted onto the membrane or glass slide. Since our ultimate goal is to develop a single test for detecting major foodborne pathogens, Campylobacter, Listeria, Staphylococcus aureus and Clostridium perfringens, we had proceeded as follows. PCR amplicons were generated as membrane bound capture probes using universal 16S or 23 S rDNA primers for Salmonella, E. coli, Klebsiella, and S. aureus. This time signal was detected. However, cross-hybridization was observed between the dig-labeled PCR product and several regions of the membrane spotted with PCR amplicons from closely related species. The dig-labeled product was not bound to membrane where PCR 16S rDNA amplicon from un-related bacteria was spotted.
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IMPACT: 2001/10 TO 2003/10<BR>
This macroarray does have promise for identifying multiple foodborne pathogens but will require further refinement requiring empirical optimization of hybridization and wash temperatures and other conditions necessary in achieving the greatest specificity for this test.