100A-28 |
Developing a rapid detection system for Listeria monocytogenes using real-time PCR |
H. LUO1, A. E. Yousef, and H. H. Wang. (1) Department of Food Science and Technology, The Ohio State University, 110 Parker Food Science and Technology Building, 2015 Fyffe Road, Columbus, OH 43210 Listeria monocytogenes is a common foodborne pathogen found in various foods. The bacterium causes listeriosis particularly in high-risk population and the pathogenecity of which is related to its virulence factors. To minimize the risk of listeriosis, proper monitoring system is essential for early detection of potential problems. Conventional culturing methods are time consuming and give no indication for the strain’s capability of causing disease. Traditional PCR method is rapid but cannot distinguish strain virulence and cannot quantify the level of contamination. The objective of this study is to develop a rapid monitoring system for L. monocytogenes that can be used to quantitatively reflect its virulence expression. Multiple genes, including those on the virulence island, will be targeted for probe development. A real-time PCR technique capable of detecting up to 4 gene products will be used in developing the detection system. Based on published DNA sequences from L. monocytogenes and other Gram-positive bacteria, degenerate DNA oligonucleotides were derived based on homology and used as primers to amplify fragments from the target genes including hly, prf, and sig. Fragments from the same gene region but different strains of L. monocytogenes were further sequenced and compared for probe development. A specific DNA probe (5’CCA CGG AGA TGC AGT GAC3’) was developed based on genetic information from the sequences, fluorescence-labeled, and used in the Taqman assay. When applied in real-time PCR, this probe was able to detect the presence of representative L. monocytogenes strains without cross-reacting with strains from Listeria innocua, Lactococcus lactis, Pseudomonas putida and Escherichia coli. Multiple probing and procedures including reverse transcription to measure the expression of virulence genes using this system is currently underway. Successful development of this detection system will enable us to measure the real risk of listeriosis on a quantitative base.
Session 100A, Food Microbiology: General II
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