AUTHORS: Caden J. Jungbluth, Wisconsin Cooperative Fishery Research Unit, Molecular Conservation Genetics Lab; Amy L. Springer, Wisconsin Cooperative Fishery Research Unit, Molecular Conservation Genetics Lab; Stephen F. Spear, U.S. Geological Survey, Upper Midwest Environmental Sciences Center; Jared J. Homola, U.S. Geological Survey, Wisconsin Cooperative Fishery Research Unit
ABSTRACT: Prevention of new invasive species often hinges on early detection and quick removal. Aquatic invasive species can be especially difficult to detect without extensive sampling due to habitat complexity and low initial abundances. Point-of-use biomolecule detection methods provide a possible solution via near real-time identification of DNA or other biological materials. Loop-mediated isothermal amplification (LAMP) provides a user-friendly approach for environmental (e)DNA detection that can be easily deployed in the field and yield diagnostic results within 40 minutes. Water can be filtered and the eDNA can be rapidly extracted with a Chelex resin and a portable heat block. We are developing a LAMP assay for red swamp crayfish (Procambarus clarkii) to enable identification of introductory pathways (e.g. the pet trade) and monitoring of the spread of established invasion sites. We generated 36 candidate primer sets that we evaluated in silico for red swamp crayfish specificity using the program BLAST to identify DNA sequence alignments to other species in the NCBI database. Sixteen species-specific candidate primer sets were then optimized for temperature of the LAMP reaction and brand of master mix. The optimal set of conditions were then used to further evaluate species-specificity through test amplifications of extracted DNA of related species (i.e. other Procambarus crayfish). Finally, we performed limit of detection testing down to a concentration of 1 copy/μL of target DNA, with consistent detections present at 100 copies/μL. Field collection of eDNA water samples from known infested waterbodies is underway to identify potential environmental inhibitors to our ability to detect red swamp crayfish presence. This tool could be implemented to detect—and possibly prevent—invasions through rapid field-based monitoring in areas where red swamp crayfish are a concern for introduction and spread.
