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 .