Identifier

etd-07112012-025642

Degree

Master of Science (MS)

Department

Environmental Sciences

Document Type

Thesis

Abstract

The aim of the study was to develop an effective multiplex PCR protocol for simultaneous, rapid detection and characterization of two potential pathogens, Vibrio vulnificus and Vibrio parahaemolyticus and to enumerate their abundance in the environment. A Pentaplex PCR (pPCR) assay condition was developed with a combination of two species- and three pathogenic- specific PCR primer sets to simultaneously detect and characterize bacterial isolates for virulent/ non- virulent strains of V. parahaemolyticus and V. vulnificus. The pPCR assay was validated by three methods. First pPCR was tested on 300 bacterial isolates comprising of 7 reference strains, 117 V. vulnificus, 30 V. parahaemolyticus and 146 unknown bacterial species and results were compared with other reported PCR reactions. Second, 51 pPCR tested isolates were analyzed by 16S rDNA sequencing to confirm for any false negative/positive reaction. Finally, the effectiveness of the pooled five primer pairs to amplify specific genes in individual target species amongst a heterogeneous bacterial sample was validated. The pPCR assay conditions worked with 96.6 - 98.7% efficiency. The pPCR assay was tested on 782 bacterial isolates from Breton Sound and Barataria Bay water samples from selected months of 2011. Our findings showed higher occurrence of V. vulnificus (~ 49%) than that of V. parahaemolyticus (~ 12 %) and their prevalences were influenced by temperature and salinity in the Gulf waters. The pPCR tested isolates showed frequent but lower occurrence of pathogenic strains (less than 1%), in cooler months. This simple, rapid, and cost-effective assay can be applied to screen and confirm a large number of isolates from clinical/environmental samples which will help to detect, predict disease outbreaks and therefore, to develop better risk management strategies.

Date

2012

Document Availability at the Time of Submission

Secure the entire work for patent and/or proprietary purposes for a period of one year. Student has submitted appropriate documentation which states: During this period the copyright owner also agrees not to exercise her/his ownership rights, including public use in works, without prior authorization from LSU. At the end of the one year period, either we or LSU may request an automatic extension for one additional year. At the end of the one year secure period (or its extension, if such is requested), the work will be released for access worldwide.

Committee Chair

Hou, Aixin

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