Master of Science (MS)
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.
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Bhattacharyya, Nabanita, "Detection and Characterization of Vibrio vulnificus and Vibrio parahaemolyticus Isolates: Pentaplex PCR Assay and its Application." (2012). LSU Master's Theses. 419.