Date of Award

5-2022

Document Type

Thesis

Degree Name

Master of Science (MS)

College/School

College of Science and Mathematics

Department/Program

Biology

Thesis Sponsor/Dissertation Chair/Project Chair

Kirsten Monsen-Collar

Committee Member

Nina Goodey

Committee Member

Ulrich Gubler

Committee Member

Lisa Hazard

Abstract

The current molecular diagnostic methods for detection of deadly pathogens within a population can be time consuming and expensive. When surveying a population for the presence of a disease, time is a valuable resource. Therefore, a rapid pathogen test to use in the field will allow for faster detection of disease that can lead to determining a course of action for conservation of a species. Ranavirus is a double-stranded DNA virus that can cause mass mortality events in amphibian, reptile and fish populations. The disease can have the potential to wipe out entire populations or species if not controlled, having a 90-100% mortality rate. Recent methods for the development of rapid pathogen tests have involved target detection probes using aptamers (short single stranded DNA) as molecular probes. Studies have shown that aptamers are more stable and cost-efficient than antibodies, and are also easier to produce. The objective of this study was to identify sequence motifs among aptamers that may make those aptamers more likely to bind tightly and specifically to Ranavirus. These aptamers were previously identified using a SELEX method, to select for aptamers that bind to both whole virus particles and the major capsid protein of Ranavirus. The results of the present research demonstrated successful expression and purification of FV-3 MCP, as well as the successful development of an EMSA screening protocol to determine binding of DNA aptamers to FV-3 MCP. Furthermore, bioinformatic analysis of DNA aptamers revealed reliable predicted aptamer secondary structures, indicating that these aptamers may be good candidates for EMSA screening. Our research may thus provide a better understanding of the specific sequence characteristics of aptamers with high affinity and specificity for FV-3 major capsid protein; the selected aptamers might therefore be used for the development of a rapid pathogen test that can be used in real-time in the field.

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