Molecular analysis and detection of Vibrio vulnificus in its quiescent and active state
Panicker, Gitika
Ph.D., The University of Alabama at Birmingham, 2004, 234 pages
2004
บทคัดย่อ
Molecular analysis and detection of Vibrio vulnificus in its quiescent and active state
Vibrio vulnificus and several other Vibrio spp. are autochthonous microorganisms in Gulf of Mexico water (gulf water). The current study describes the development, optimization, and application of state-of-the-art DNA and protein-based detection methodologies that would allow for rapid, specific, and sensitive identification of V. vulnificus and selected clinically important Vibrio spp. in shellfish. Also, the overexpression of the DEAD-box protein identified at 4°C provided an insight into the cold-adaptive response in V. vulnificus , which questioned the existence of a quiescent state in V. vulnificus . The SYBR Green I ® and TaqMan-based real-time PCR assays exhibited specific detection of 84 V. vulnificus isolates using oligonucleotide primers for vvh . The minimum level of detection by the real-time PCR methods were 1 pg of purified genomic DNA or 10 2 -10 3 V. vulnificus in 1 g of unenriched oyster tissue homogenate or 10 ml of gulf water. This detection level was improved to one V. vulnificus following 5 h of enrichment. The entire detection method, including sample processing, enrichment, and real-time PCR amplification, was completed within 8 h, making it a rapid single-day assay. The multiplex PCR assay, using vvh and viuB as targets, successfully distinguished the clinical and the environmental strains of V. vulnificus . Its application on enriched natural oysters detected the presence of pathogenic V. vulnificus in 15% of the vvh positive samples. Next, the gene-specific DNA microarray coupled with multiplexed PCR offered a specific and sensitive (1 CFU/gm) method for comprehensive detection of total and pathogenic V. vulnificus, V. cholerae , and V. parahaemolyticus in shellfish and gulf water. This method proved to be an improvement over the Covalink(TM) NH hybridization assay. Using phage-displayed random peptide library and FACS analysis, a novel peptide was identified that could be used in the development of an "intact-cell"-based detection assay for clinically important Vibrio spp. The rapid and sensitive detection of V. vulnificus and other pathogenic Vibrio spp., in compliance with the Interstate Shellfish Sanitation Conference (ISSC) guidelines, would help ensure a steady supply of "safe-to-eat" postharvest treated oysters to consumers and thereby reduce Vibrio -associated illnesses or outbreaks.