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RNA Detection Technology for Applications in Marine Science: Microbes to Fish Robert Michael Ulrich University of South Florida, [email protected]

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RNA Detection Technology for Applications in Marine Science: Microbes to Fish Robert Michael Ulrich University of South Florida, Bobmiu@Aol.Com University of South Florida Scholar Commons Graduate Theses and Dissertations Graduate School 6-25-2014 RNA Detection Technology for Applications in Marine Science: Microbes to Fish Robert Michael Ulrich University of South Florida, [email protected] Follow this and additional works at: https://scholarcommons.usf.edu/etd Part of the Biology Commons, and the Molecular Biology Commons Scholar Commons Citation Ulrich, Robert Michael, "RNA Detection Technology for Applications in Marine Science: Microbes to Fish" (2014). Graduate Theses and Dissertations. https://scholarcommons.usf.edu/etd/5321 This Dissertation is brought to you for free and open access by the Graduate School at Scholar Commons. It has been accepted for inclusion in Graduate Theses and Dissertations by an authorized administrator of Scholar Commons. For more information, please contact [email protected]. RNA Detection Technology for Applications in Marine Science: Microbes to Fish by Robert M. Ulrich A dissertation submitted in partial fulfillment of the requirements for the degree of Doctor of Philosophy College of Marine Science University of South Florida Major Professor: John H. Paul, Ph.D. Valerie J. Harwood, Ph.D. Mya Breitbart, Ph.D. Christopher D. Stallings, Ph.D. David E. John, Ph.D. Date of Approval June 25, 2014 Keywords: NASBA, grouper, Karenia mikimotoi, Enterococcus Copyright © 2014, Robert M. Ulrich DEDICATION This dissertation is dedicated to my fiancée, Dr. Shannon McQuaig for inspiring my return to graduate school and her continued support over the last four years. On no other porch in our little town have there been more impactful scientific discussions, nor more words of encouragement. ACKNOWLEDGMENTS I gratefully acknowledge the many people who have encouraged and advised me throughout my graduate studies. To my major advisor, John H. Paul, thank you for the opportunity to study under your esteemed tutelage. Thank you to my committee members, Valerie J. Harwood, Mya Breitbart, Christopher D. Stallings, and David E. John, for your guidance and insight. I thankfully recognize my mother, step-father, late father, sister, and brother-in-law for their endless love and support, as well as members of the McQuaig family. I appreciate members of the Paul Lab who have enriched my life in immeasurable ways. I am grateful for the generous support of the C.W. Bill Young and Sanibel-Captiva Shell Club/Mary and Al Bridell Memorial Fellowships. TABLE OF CONTENTS List of Tables ................................................................................................................................. iv List of Figures ..................................................................................................................................v Abstract .......................................................................................................................................... vi Chapter One: Introduction ...............................................................................................................1 Overview of Dissertation .....................................................................................................5 References ............................................................................................................................6 Chapter Two: Ensuring Seafood Identity: Grouper Identification by Real-Time Nucleic Acid Sequence-Based Amplification (RT-NASBA) .................................................................9 Note to Reader .....................................................................................................................9 Abstract ................................................................................................................................9 Introduction ........................................................................................................................10 Materials and Methods .......................................................................................................15 Sequencing 16S rDNA from grouper tissue ..........................................................15 Primer and molecular beacon design .....................................................................17 RNA extraction and RT-NASBA assay .................................................................18 RT-NASBA on cooked samples ............................................................................19 Phylogenetic and statistical analysis ......................................................................20 Results ................................................................................................................................21 RT-NASBA specificity ..........................................................................................21 RT-NASBA on cooked grouper samples ...............................................................24 Discussion ..........................................................................................................................27 Acknowledgments..............................................................................................................30 References ..........................................................................................................................30 Chapter Three: A Handheld Sensor Assay for the Identification of Grouper as a Safeguard against Seafood Mislabeling Fraud ..........................................................................................35 Note to Reader ...................................................................................................................35 Abstract ..............................................................................................................................35 Introduction ........................................................................................................................36 Materials and Methods .......................................................................................................41 Fish specimens .......................................................................................................41 Field RNA purification ..........................................................................................42 Multiplex RT-NASBA assay .................................................................................43 Evaluating performance of field RNA purification ...............................................44 Handheld RT-NASBA analyzer ............................................................................45 i Statistical analysis ..................................................................................................46 Results ................................................................................................................................47 Performance of field RNA purification .................................................................47 Evaluation of field QuadPyre RT-NASBA ............................................................49 Evaluating the QuadPyre assay on cooked tissue ..................................................50 Discussion ..........................................................................................................................51 Acknowledgments..............................................................................................................56 References ..........................................................................................................................57 Chapter Four: Detection and Quantification of Karenia mikimotoi using Real-Time Nucleic Acid Sequence-Based Amplification with Internal Control RNA (IC-NASBA) ....................60 Note to Reader ...................................................................................................................60 Abstract ..............................................................................................................................60 Introduction ........................................................................................................................61 Materials and Methods .......................................................................................................64 In vitro transcript....................................................................................................64 Primer and molecular beacon design .....................................................................65 Synthesis of internal control (IC) RNA .................................................................65 Sensitivity and specificity ......................................................................................66 Environmental samples ..........................................................................................68 NASBA assay ........................................................................................................69 Statistical analysis ..................................................................................................70 Results ................................................................................................................................70 Sensitivity and specificity ......................................................................................70 Environmental samples ..........................................................................................75 Discussion .........................................................................................................................76
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