Detection
The limit of detection (LOD) and limit of quantification (LOQ) of primer/probe combination 9 were found to be 3x10-4ng/µL and 3x10-5 ng/µL respectively. The LOD and LOQ have various definitions in the eDNA literature, here LOD is defined as the lowest standard concentration at which 95% of technical replicates amplify and LOQ is the lowest standard concentration for which the coefficient of variation (CV; equal to the standard deviation quantity divided by the mean quantity of a group of replicates) value is <35% (Klymus et al . 2019). The LOD corresponded to a Ct value of 37.47 which encompassed 100% of the Ct values in this study i.e. all positive amplifications were above the limit of detection. Detection in a water sample was indicated by at least 1 of 12 positive qPCR replicates (Biggs et al. 2015).
All Stodmarsh NNR ditch water samples and ditch samples from outside the known range of S. nitida were subjected to inhibition testing, all samples except two from Stodmarsh NNR (136 and 146) did not cause inhibition of qPCR. Samples 136 and 146 caused complete inhibition of the qPCR inhibition assay.
All Stodmarsh NNR ditch water samples and ditch samples from outside the known range of S. nitida were then subjected to the optimised assay for S. nitida detection, with seven ditches from Stodmarsh NNR being positive for S. nitida DNA (Table 1). One ditch (ditch 34) with very low positivity (1/12), was re-tested to confirm positivity (3/12 on repeat). All remaining ditches were negative for S. nitida DNA, therefore other than those ditches where S. nitidawas found by manual survey - ditches 62, 70 and 108 (Table 1) - S. nitida is likely to be absent.