A CRISPR-Based Rapid Detection Assay for Crayfish Plague (Aphanomyces astaci) From Environmental Samples

Crayfish plague, caused by Aphanomyces astaci (Aa), is an infectious disease invasive in Europe, where its rapid spread has resulted in sharp declines of native crayfish species. Monitoring currently relies on a highly sensitive, but costly and time-consuming qPCR approach. Here, we designed a simplified, rapid and cost-efficient molecular assay for on-site detection of Aa. The novel rapid assay employs a combination of isothermal recombinase polymerase amplification and CRISPR-Cas12a-based detection that can be coupled with fluorescence or lateral flow visualisation. We demonstrate that the novel assay can detect A. astaci from tissue and environmental DNA with higher sensitivity than the available qPCR assay and readily distinguishes Aa from its non-pathogenic sister taxon A. fennicus. We tested two genomic marker sites for Aa that discriminate closely related oomycetes and incorporate field-deployable lateral flow and fluorescence readouts. Our work will make crayfish plague monitoring broadly accessible to practitioners and non-academic stakeholders as a tool to curb further Aa-driven loss of Europe's imperilled freshwater crustaceans and strengthen preparedness against future incursions of the pathogen in other regions.