Date of Award

5-2014

Level of Access Assigned by Author

Campus-Only Dissertation

Degree Name

Doctor of Philosophy (PhD)

Department

Marine Biology

Advisor

Laurie Connell

Second Committee Member

Paul Millard

Third Committee Member

Mary-Jane Perry

Abstract

The research undertaken for this dissertation focuses on the development of a ribosomal RNA (rRNA)-based method for molecular detection of the toxin- producing marine dinoflagellate, Alexandrium spp. The assay developed uses peptide nucleic acid (PNA) probes and a portable surface plasmon resonance (SPR) biosensor instrument called SPIRIT. This method yields reliable detection of synthetic 3oligonucleotide sequences specific to the Alexandrium fundyense/A. tamarense/A. catenella species complex and to A. ostenfeldii, as well as detection of A. fundyense and A. ostenfeldii RNA extracted from cultured cells. There was no cross-reaction between non-complementary sequences and very good discrimination between negative control PNA probes and Alexandrium-specific PNA probes. The sensor surfaces used for detection in this assay were regenerated by removing the target nucleotide sequences from the immobilized probe layer with a high pH wash, allowing the system to be used for multiple assays, greatly reducing the cost per analysis. The method is streamlined, portable, and amenable to use in the field. The primary goal was to provide a means in which the organism itself could be detected and quantified prior to the formation of blooms and before toxicity occurs in shellfish. This assay could assist in future efforts of state monitoring programs to predict toxicity and engage in further testing based on presence, absence, and relative abundance of species of interest, helping to more effectively manage the safety of the shellfish industry.

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