Title

Discovery of DNA Aptamers for the Selection and Identification of Ranavirus

Presentation Type

Event

Start Date

27-4-2019 9:30 AM

End Date

7-5-2019 10:44 AM

Abstract

A key problem when developing molecular diagnostic techniques is procuring target detection probes. The need of a stable and selective probe is necessary for accurate target identification in disease detection. Antibodies are widely used but they are unstable at ambient temperatures and are expensive and time consuming to develop. The use of aptamers (ssDNA) as molecular probes provides several advantages over protein based methods being that they are significantly more stable, easily selected and modifiable. The development of an aptamer specific probe has been performed for Ranavirus virion particles and recombinant Ranavirus major capsid protein (MCP) through an iterative process known as SELEX. Two separate SELEX methods utilizing spin concentrators and affinity chromatography have been designed and verified for the isolation of virion specific aptamers and MCP specific aptamers, respectively. The final aptamers have been cloned and sequenced to identify common motifs between virion specific aptamers and MCP specific aptamers indicating enrichment and specificity to Ranavirus. Aptamers with Ranavirus specific motifs have been modified to contain a biotin molecule for use in binding assays. The binding affinities of aptamers will be assessed using sandwich ELISA with conjugated streptavidin Horse Radish Peroxidase as the chromogen. The ultimate goal is to incorporate selected aptamers with the highest binding affinity as molecular probes for Ranavirus into a field usable rapid pathogen test.

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Apr 27th, 9:30 AM May 7th, 10:44 AM

Discovery of DNA Aptamers for the Selection and Identification of Ranavirus

A key problem when developing molecular diagnostic techniques is procuring target detection probes. The need of a stable and selective probe is necessary for accurate target identification in disease detection. Antibodies are widely used but they are unstable at ambient temperatures and are expensive and time consuming to develop. The use of aptamers (ssDNA) as molecular probes provides several advantages over protein based methods being that they are significantly more stable, easily selected and modifiable. The development of an aptamer specific probe has been performed for Ranavirus virion particles and recombinant Ranavirus major capsid protein (MCP) through an iterative process known as SELEX. Two separate SELEX methods utilizing spin concentrators and affinity chromatography have been designed and verified for the isolation of virion specific aptamers and MCP specific aptamers, respectively. The final aptamers have been cloned and sequenced to identify common motifs between virion specific aptamers and MCP specific aptamers indicating enrichment and specificity to Ranavirus. Aptamers with Ranavirus specific motifs have been modified to contain a biotin molecule for use in binding assays. The binding affinities of aptamers will be assessed using sandwich ELISA with conjugated streptavidin Horse Radish Peroxidase as the chromogen. The ultimate goal is to incorporate selected aptamers with the highest binding affinity as molecular probes for Ranavirus into a field usable rapid pathogen test.