Species-specific assay development
In order to design primers specific to the S. nitida the DNA sequences for the cytochrome oxidase 1 (COI) gene for S. nitidaand the nine other co-occurring similar ram’s-horn shaped species commonly found at the Stodmarsh NNR were downloaded from Genbank and their sequences aligned using BioEdit version 7.2.5. Primers and probes were designed using PrimerBLAST with default settings except for targeting a 70-300 bp fragment and including only base pairs between 70 and 600 in the S. nitida consensus sequence as this corresponded to the most variable region on the multi-species alignment. Ten potential primer/probe combinations were generated and reduced to four using PrimerBLAST and looking for cross-species amplification (Supplemental file 2).
The four potential primer/probe combinations were tested firstly on DNA extracted from S. nitida followed by the other nine co-occurring snail species to test for cross-species reactivity. PCRs were set up in a total volume of 25 µL consisting of: 3 µL of extracted template DNA, 1 µL of each primer/probe (0.2 µmol/L forward primer; 0.4 µmol/L reverse primer; 0.1 µmol/L probe), 12.5 µL of TaqMan® Environmental Master Mix 2.0 (containing AmpliTaq GOLD DNA polymerase), and 6.5 µL ddH2O. Each sample was run as 12 replicates on a Bio-Rad CFX Connect real-time PCR machine as follows: an initial incubation for 5 minutes at 56.3⁰C then 10 minutes at 95°C; followed by 55 cycles with a melting temperature of 95°C for 30 seconds and an annealing temperature of 59.6⁰C for 1 minute.
Once specificity of primer/probe combinations was confirmed, the primer concentrations of two primer/probe combinations (primer/probe combinations 2 and 9) were optimised by independently varying final primer concentrations (the probe was held at a final concentration of 0.1 µmol/L) (Wilcox et al , 2015). The sensitivity of the assay was tested by creating a six-level standard curve dilution series (3x10-1 to 3x10-7 µg/µl). The standard curve was created by quantifying the DNA extracted fromS. nitida sample 7a on a Qubit Fluorometer (Thermo Fisher Scientific) and diluting the DNA to the desired concentrations using the elution buffer provided in the DNeasy Blood and Tissue kit (Qiagen). 12 replicates of each dilution were run using the optimised primer/probe concentrations to determine the standard curve slope, the limit of detection (LOD) and limit of quantification (LOQ).
The optimised assay using primer/probe combination 9 (Snit9F: 5’- CCACTTTTAATTGGGGCTCCG-3’; Snit9R: 5’- CCATGTGCAATAGGACCGCT-3’; Snit9P: 5’- TGAAGGAGGTGTTGGTACTGGGTG-3’, FAM/BHQ-1) was used to determine the presence/absence of S. nitida within the 22 ditch samples from Stodmarsh NNR and the 10 ditch samples from outside of the known range of S. nitida .