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Development of a Rapid, DNA‐Based Field Test for Detection of Ranavirus, an Emerging Amphibian Disease

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 (short ssDNA) as molecular probes provides several advantages over protein‐based methods because they are more stable, easily selected and modifiable. The need of a reliable and stable probe is necessary for accurate detection tests in the field for disease monitoring. Currently, field methods are limited in the time and expertise required to obtain results. Our objective is to develop an aptamer probe with high affinity and specificity for Ranavirus virion particles and recombinant Ranavirus major capsid protein (MCP) through an iterative process known as SELEX. We developed two SELEX methods utilizing either spin concentrators or protein affinity chromatography to successfully enrich a starting library of 1014 non‐specific aptamers to those specific to Ranavirus virion particles and MCP, respectively. After 6 rounds of selection, we have isolated 24 potential Ranavirus probes and subsequently cloned into a TOPO_BluntII vector for sequencing. Aptamer sequences will be analyzed for common structural motifs between virion specific aptamers and MCP specific aptamers, to identify possible common binding sites for these two respective targets. The binding affinities of selected aptamer probes will be measured by Surface Plasma Resonance (SPR) using a streptavidin modified biosensor chip and biotinylated aptamers. We expect binding affinities of aptamers to Ranavirus targets to be within the nanomolar range, demonstrating the aptamers high affinity and specificity. Ultimately, the selected aptamers will be incorporated as molecular probes for Ranavirus into a field usable rapid pathogen test that will surpass the limitations of current field tests.