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L. MA, G. Zhang, and M. P. Doyle. Center for Food Safety, Univ. of Georgia, Dept. of Food Science & Technology, 1109 Experiment St., Griffin, GA 30223-1797 Listeria monocytogenes is a leading cause of death attributed to foodborne bacterial pathogens. Lack of sufficient scientific information has led regulatory agencies to consider any strain of L. monocytogenes to be potentially pathogenic for humans although several lines of evidence indicate there are differences in virulence potential among L. monocytogenes isolates. Our long term goal is to develop a DNA microarray chip that can differentiate and identify Listeria species, including L. monocytogenes, as well as subtype and assess the virulence potential of L. monocytogenes isolates. Our initial approach in achieving such a goal was to develop a DNA microarray chip for identification of Listeria species and partial serotyping and genotyping of L. monocytogenes. In addition, the use of spacer molecules to reduce steric interference from the supporting matrix to the probes was also evaluated. A redundant and hierarchically structured set of oligonucleotide probes (17-37 mer) targeting 5 genes (16S rRNA, iap, gltA, gltB, inlB) were designed, synthesized and spotted onto epoxy-derivatized glass slides. Target DNA was amplified from purified genomic DNA via asymmetric polymerase chain reaction (PCR). Results from hybridization of the chip with target DNA from 18 L. monocytogenes strains for which serotype and genotype data were available have shown the array's ability to unambiguously serotype and genotype. The addition of 12-mer spacer molecules significantly increased the intensity of hybridization signals. The developed DNA microarray chip will enable rapid and accurate discrimination among six Listeria species and partial serotyping and genotyping of L.monocytogenes isolates. Furthermore, with the addition of more probes targeting virulence-associated genetic markers, the chip should be useful for rapid and more accurate global assessment of the virulence potential of L. monocytogenes isolates. Such differentiation may offer an additional opportunity for prioritization of treatment and control Listeriae.
Session 17, Biotechnology: General
2005 IFT Annual Meeting, July 15-20 - New Orleans, Louisiana |