Validation of a portable eDNA detection kit for invasive carps

Abstract

Loop-mediated isothermal amplification (LAMP) is a rapid molecular detection technique that has been used as a diagnostic tool for detecting human and animal pathogens for over 20 years and is promising for detecting environmental DNA shed by invasive species. We designed a LAMP assay to detect the invasive carps, silver carp ( Hypophthalmichthys molitrix ), bighead carp ( Hypophthalmichthys nobilis ), black carp ( Mylopharyngodon piceus ), and grass carp ( Ctenopharyngodon idella ). To determine the sensitivity of the LAMP assay, we determined limit of detection (LOD) for each invasive carp species and compared with the performance of a grass carp quantitative PCR (qPCR) assay in LOD and in a mesocosm study. We used two grass carp densities, 3 juvenile grass carp in one mesocosm and 33 juvenile grass carp in the other. Prior to adding grass carp to the mesocosms, we added 68 kg of fathead minnows ( Pimephales promelas ) to each mesocosm to simulate farm ponds used for raising bait fish. We filtered 500 mL of water per sample to compare LAMP and qPCR analysis, and we collected 50 mL grab samples that were only analyzed using qPCR to gain additional data using a higher-throughput method to monitor environmental DNA (eDNA) levels throughout the study period. No eDNA for any of the four invasive carp species was detected in water collected from the mesocosms during the three days prior to adding grass carp. Forty-eight hours after grass carp addition to mesocosms, we detected grass carp eDNA in the mesocosm containing 33 grass carp using the LAMP assay. However, we failed to detect any grass carp DNA in the mesocosm containing 3 grass carp with the LAMP assay throughout the study. We analyzed the data using an occupancy model and found that the 500 mL filter samples yielded a higher eDNA capture probability than 50 mL grab samples in the mesocosm containing three grass carp but had similar eDNA capture probability in the mesocosm containing 33 grass carp. Both LAMP and qPCR reliably detected grass carp eDNA 2 days after grass carp addition, but detections were more consistent with qPCR. The LAMP assay may have utility for certain niche uses because it can be used to rapidly analyze eDNA samples and is robust to inhibition, despite having some limitations.