3.2. PHASE 2 – IN SILICO SELECTION AND IN VITROASSESSMENT OF MOLECULAR ASSAYS
In total 23 different primer pairs were sourced from the literature (14
publications) and 62 additional primer pairs were designed and selected
for further in silico testing (total n = 85 primer pairs).
Following initial assessment of each primer pair, 42 assays were
selected for further in vitro testing, including 34 assays
targeting mitochondrial DNA 16s (n = 3) and COI (n = 31) genes. To
gather more information on gene variation and increased taxonomic
resolution for Mytilus spp and Magallana gigas , three
cytoplasmic organelle 18s gene assays (n = 3) as well as nuclear genes
(n = 5) were added to the selected assays (overview of all assays insupplementary Table S4 ).
Results from the initial panel of 42 assays showed that average amplicon
length was 104 bp and average primer length was 22.5 bp. The average
theoretical annealing temperature was 56.2°C, ranging from 52.7 to
61.7°C, with 17/83 primers outside of the 55-60°C range. Delta-G values
of the whole panel averaged -3.9, however, 49/83 primers had one or more
reported Delta-G value higher than -6.0 with 26/83 primers having a
higher value than -9.0. When blasting the primers, 62/83 were found to
match only with the target species. When taking both primers into
account, 5 out of the 42 primer pairs matched some non-European
off-target species (supplementary Table S4 ). Two primer pairs
matched with European species: ‘’M. gigas COI 2” matched withC. angulata (not occurring in Irish coastal waters) and
‘’O. edulis 16S” matched with different Ostrea species
and was therefore considered as an Ostrea specific marker. Using
the machine learning approach for predicting cross-amplification
(eDNAassay), 32 out of 42 assays were found to have off-target AMP
values below 0.5 including the highly specific nuclear Mytilus
spp markers, hence indicating high specificity potential for 71% of
selected assays (supplementary Table S5) . The three nuclear
assays targeting the Mytilus complex were Mytilus -specific
but had AMP values ranging from 0.5 to 0.675. The results from the COI
showed eight assays to have high AMP (>0.5) with off-target
species, including five Ensis -specific assays (possible
cross-amplification with other Ensis/Pharidae species; 0.5-0.9
AMP), the C. glaucum assay (possible cross amplification withParvicardium exiquum (0.8 AMP)), the Cancer pagurus assay
(possible cross amplification with Liocarcinus depurator (0.6
AMP)) and the Homarus americanus assay (possible cross
amplification with Eurynome spinosa (0.6
AMP)). Despite the potential
for cross-amplification, all 42 assays were tested in vitro , as
described below.