Document Type

Dissertation

Degree

Ph.D.

Degree Granting Department

Marine Science

Major Professor

John H. Paul

Keywords

Enterococcus, grouper, Karenia mikimotoi, NASBA

Abstract

The accurate identification of taxa from mixed assemblages using genetic analysis remains an important field of molecular biology research. The common principle behind the development of numerous documented genetic detection technologies is to exploit specific nucleotide sequences inherent to each taxon. This body of work focuses on practical applications of real-time nucleic acid sequence-based amplification (RT-NASBA) in marine science, and is presented in four case studies. Each study represents novel work in the genetic identification of respective taxa of interest using RT-NASBA. Two case studies documented the development of an assay targeting mitochondrial 16S rRNA to discern legally salable grouper species in the U.S. from fraudulently mislabeled surrogate fish. This technology was first validated using lab-based, benchtop instrumentation, and was then adapted into a complete field detection system. The third study documented an internally controlled RT-NASBA (IC-NASBA) assay for the detection and quantification of the harmful algal bloom-causing dinoflagellate, Karenia mikimotoi, by targeting the ribulose-1, 5-bisphosphate carboxylase-oxygenase (RuBisCO) large-subunit gene (rbcL). The final section of this dissertation details the preliminary development of an IC-NASBA assay targeting large subunit rRNA for the quantification of Enterococcus, which is a genus of bacteria commonly used as an indicator of fecal pollution in recreational marine water. My results show that RT-NASBA provides a suitable format for the accurate identification of target species from these taxa which include prokaryotes, as well as both unicellular and multicellular eukaryotes.

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