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Detection of Vibrio vulnificus in Gulf of Mexico water and shellfish using molecular methods.

V. vulnificus is a Gram-negative, halophilic bacterium that causes septicemia and at times death in susceptible individuals through the consumption of raw oysters. In this study, two experimental approaches involved in rapid and reliable detection of this pathogen in the Gulf of Mexico water and shellfish were evaluated. First, a real-time PCR with SYBR Green I fluorescent dye, using oligonucleotide primers for the specific region of the cytolysin gene (vvh) was tested on 87 V. vulnificus isolates. Results exhibited a positive amplification of the 205 bp vvh fragment in all isolates. Following which, various concentrations of V. vulnificus were seeded in oyster tissue homogenate and sterile gulf water, and enriched with 0.2% (w/v) peptone for 5 h. Real-time PCR amplification exhibited detection of [less than or equal to]1[0.sup.1] cells in 10 ml of enriched Gulf water or 1 g of oyster tissue homogenate. Next, a Phage-display assay involving the identification of peptide ligands capable of binding to cell surface receptors on target microorganisms was developed. Four biopanning reactions were carried out by incubating V. vulnificus cells with random peptides of PH.D.-7 peptide library (NEB). A 7-mer peptide sequence was identified as being specific for the detection V. vulnificus. Further, specificity studies using fluorescent microscopy were conducted to evaluate the binding specificities of other Vibrio sp. to the Alexa-Fluor 488 labeled phage displaying the peptide of interest. The development of these rapid and early detection methods would help ensure the supply of safer oysters for consumption and reduce the V. vulnificus-associated illnesses.

Gitika Panicker and Asim K. Bej, University of Alabama at Birmingham, AL-35294.
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Title Annotation:Biological Sciences
Author:Panicker, Gitika; Bej, Asim K.
Publication:Journal of the Alabama Academy of Science
Article Type:Brief Article
Date:Apr 1, 2003
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