PS 11-128 - Detection of Bighead carp (Hypothalmichthys nobilis) eDNA using a rapid loop-mediated isothermal amplification (LAMP) assay during an eDNA degradation experiment

Abstract: Rapid eDNA assays have great promise for detecting invasive species at ports and inspection stations. The typical workflow may require the samples to be collected during the inspection and analyzed after the inspection is completed. Inspections may vary in length, so understanding the rate of degradation of the eDNA is important in interpreting results and minimizing false-negative rates. Most degradation studies have focused on quantitative PCR (qPCR). Loop-mediated isothermal amplification assays require 5 or 6 primers and amplify a region typically greater than 200 bp, whereas qPCR only uses 2 primers and a probe and targets only around 100 bp. The greater number of primers needed to bind for LAMP and increased length of the target sequence may confer greater sensitivity to eDNA degradation and reactions typically are faster to amplification. We previously developed a single LAMP assay that detects eDNA from Grass Carp, Black Carp, Bighead Carp, and Silver Carp (invasive carps). To determine if degradation could impact detection, we conducted an experiment using water sampled from tanks containing Bighead Carp. Samples were incubated in the dark at room temperature for 2, 24, 72, 168, and 336 hours after sampling. We were able to detect eDNA with LAMP at our final sampling when qPCR indicated that concentrations were very low. Our results will inform protocol development for point-of-use eDNA tools to maximize efficiency while maintaining accurate detection of invasive species at ports of entry.