Optimal detection protocol of Tetracapsuloides bryosalmonae by environmental DNA : A comparison of qPCR and ddPCR approaches

Investigation of environmental DNA (eDNA) is increasingly used to precisely and non-invasively detect and monitor pathogens. Among these, Tetracapsuloides bryosalmonae is a myxozoan endoparasite that causes proliferative kidney disease (PKD) in salmonid fish. Although the detection of T. bryosalmonae DNA in water samples has been shown to be promising and successful, method comparison and cross-validation are currently lacking. This study aims to directly compare the sensitivity of different eDNA-based methods in field and laboratory applications, and to develop an easy-to-apply and sensitive protocol to monitor T. bryosalmonae occurrence non-invasively by its eDNA in water samples. First, we tested three existing probe-based T. bryosalmonae-specific detection assays in parallel by comparing the limit of detection (LOD) and limit of quantification (LOQ) using quantitative PCR (qPCR) and digital droplet PCR (ddPCR) platforms. Second, the impact of different filter types and water volumes on the detection probability was tested by sampling water directly from riverbanks with a syringe-based protocol. The most sensitive detection protocol was the combination of the probe-based assay published by Bettge et al. run via ddPCR, resulting in a LOD of 1.65 copies/μL input (6.6 copies/reaction) and a LOQ of 3.66 copies/μL input (14.67 copies/reaction). The type of filter (Sterivex™ compared to Millex®) did not significantly influence detection probability, however, the volume of water sampled (600 mL compared to 300 mL) significantly affected the probability of capturing eDNA in a sample. Based on modeled probabilities of eDNA capture and detection, we calculated that using the Bettge et al. assay via the ddPCR platform for data collection, 95% overall detection probability could be achieved with three replicates of 600 mL filtered water with Sterivex™ filters. Based on this cross-validation of assays and detection platforms, we provide a cost-effective, straightforward, and highly sensitive laboratory analysis workflow to detect DNA of T. bryosalmonae from water samples.Peer reviewe