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

Stelzer, Moritz; Ord, James; Neyrinck, Sabrina; Steiner, Jonas; Hartikainen, Hanna; Brys, Rein; Schmidt-Posthaus, Heike (2024). Optimal detection protocol of Tetracapsuloides bryosalmonae by environmental DNA : A comparison of qPCR and ddPCR approaches. Environmental DNA, 6(1) Wiley 10.1002/edn3.501

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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.

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