Submitted to: American Society for Microbiology
Publication Type: Abstract Only
Publication Acceptance Date: 5/22/2003
Publication Date: 5/22/2003
Citation: HARRIGAN, K.A., HARKINS, K.R., WESLEY, I.V. EVALUATING PNA (PEPTIDE NUCLEIC ACID) RRNA SPECIFIC PROBES FOR USE IN THE DETECTION OF LISTERIA SPP. AND SALMONELLA SPP. IN POULTRY. AMERICAN SOCIETY FOR MICROBIOLOGY. 2003. ABSTRACT P. 502.
Technical Abstract: Listeria and Salmonella are foodborne pathogens that are of particular concern to the poultry industry. A typical test requires a 24-48 hour enrichment step prior to using most rapid kits on the market today. The majority of these kits employ antibodies as the basis for detecting these pathogens. An alternative method for tagging pathogens uses labeled probes in a fluorescence in situ hybridization (FISH) assay designed to bind to conserved rRNA sequences within a specific bacteria genus. Initial evaluation of PNA probes has shown that they can tag low levels (1-10 cfu/10 mL) of bacteria that have been enriched for 6-18 hours. Listeria specific FISH was shown for 6 species: L. monocytogenes (ATCC 19114, ATCC 19111) L. welshimeri, L. seeligeri, L. ivanovii, L. innocua, and L. grayi. Specificity of this rRNA specific probe has also been tested against Bacillus, E.coli, Salmonella, Staphylococcus, and Streptococcus, with no hybridization occurring. A real world poultry wash sample was tested for low level L. monocytogenes detection in spiked and non-spiked samples. For both samples, 0.5 mL (10k cfu of natural contaminants) of the poultry wash was added to 4.5 mL Fraser broth. One to 10 cfu of L. monocytogenes was added to the positive control. Samples were enriched at 30 C for 18 hours and then taken through the FISH process, followed by presence absence analysis on the RBD2100. A definitive population was observed in the spiked samples, and no population was observed in the negative control. This suggests there was no cross-reactivity to any of the other organisms found in the poultry wash. A similar assay is being developed for Salmonella. Preliminary data suggested that the growth kinetics of Salmonella should require a shorter enrichment time (6-8 hours) prior to presence absence testing. The Salmonella PNA probe will be tested for sensitivity using S. typhimirium, S. paratyphi A, S cholerasuis, S. entertidius, and several species obtained from Iowa State University. Preliminary tests also indicate that stringency of the assay can be increased to decrease the hybridization to other organisms that are genetically similar to Salmonella like E. coli O157:H7.