Crispr-Cas methods for rapid, sensitive, and field deployable detection of scale drop disease virus and tilapia lake virus

Abstract

Scale drop disease virus (SDDV) and tilapia lake virus (TiLV) are among the most devastating pathogens in farmed fish due to their ability to cause severe symptoms and high mortality rates. As commercial vaccination against these viruses is currently unavailable, rapid detection methods unconstrained by the shortcomings of traditional molecular diagnostic approaches will be critical in timely and effective prevention of SDDV and TiLV outbreaks. This research aims to develop CRISPR-Cas12a systems coupled with recombinase polymerase amplification (RPA) for sensitive and specific detection of SDDV and TiLV, targeting the conserved adenosine triphosphate (ATPase) and Segment 9 region, respectively. To maximize the field compatibility of these platforms, the entire workflow was optimized to enable operation at a constant temperature (37 -42 ºC) within 1 hr. Due to the nature of TiLV as an RNA virus, reverse transcription was incorporated into the workflow, yielding the assay termed RT-RPA-Cas12a. The methods developed herein achieved the detection limit of 40 and 200 copies per reaction for SDDV and TiLV, respectively. On the other hand, the RPA-Cas12a assay showed no cross-reactivity with non-target bacteria and fish pathogens. Importantly, it was verified that portable visualization methods such as lateral flow assays and smartphone-based detection were fully compatible with the CRISPR assays. IMPLICATION OF THE THESIS: As a result, both RPA-Cas12a approaches show significant potential as fish virus point-of-need diagnostic platforms. The CRISPR-Cas12a system is thus promoted as a diagnostic for field use as a result of the advantage of this accessibility. When finished, RPA in combination with Cas12a detection has the potential to be a powerful tool for the detection, monitoring, prevention, and management of virulent fish diseases.