Development and field validation of RPA‐CRISPR‐Cas environmental DNA assays for the detection of brown trout (Salmo trutta) and Arctic char (Salvelinus alpinus)

Abstract

AbstractMolecular methods are rapidly evolving to enable nucleic acid diagnostics outside a laboratory setting. Such techniques are primarily utilizing isothermal amplification such as Recombinase Polymerase Amplification (RPA) and Loop‐Mediated Isothermal Amplification (LAMP) but are yet to be fully explored for monitoring using environmental DNA (eDNA). We previously presented an RPA‐CRISPR‐Cas approach for detection of Atlantic salmon in Ireland and Canada and in this manuscript we present a further application of this technique for monitoring of brown trout and Arctic char in the Burrishoole Catchment, Co. Mayo, Ireland. In developing these assays, we offer an alternative approach to the PCR‐based assays previously published and have evolved a streamlined approach to single‐species monitoring using RPA‐CRISPR‐Cas, reducing the fluorescence acquisition time from 2 h to 30 min. This demonstrates the applicability of using RPA‐CRISPR‐Cas assays for eDNA‐based detection beyond Atlantic salmon with the added benefit of a faster assay time without compromising detection sensitivity.