Specificity
Each species/assay combination (26 *42 = 1092 combinations) yielded at
least four qPCR data points (i.e. assay in duplicate tested against the
target in duplicate). When assessing assay performance, consistency in
amplification across all four qPCR reactions was considered. The overall
results are shown in Figure 2 . In total, separating Bivalvia
and Malacostraca, AMP values were generated for 276 reaction
combinations. 12 of these reactions showed minor but positive
amplification when the AMP value was below 0.3. 3 out of 55 reactions
were positive with AMP values of 0.3-0.4 whilst 0 out of 5 reactions
with AMP-values of 0.5 amplified. Of the higher AMP-values,Mytilus edulis assay reacted positively on the obtained M.
trossulus specimen from Scotland, however, the obtained oligo based on
reference data remained negative resulting in it being considered an
artifact. The M. galloprovincialis assay amplified M.
edulis target (100000 copies) <10 copies (AMP=0.7). Following
removal of the below 0.2 AMP value reactions (as they were considered
artifacts), of the 42 assays tested 38 showed satisfactory levels of
specificity to the intended target species, whereby they either
amplified only the target or produced a distinct melt temperature. The 4
remaining assays amplified other targets with similar melt curves,
indicating a lack of specificity. Out of these 4 assays, 2 assaysHamericanus COI, and Oedulis 16S showed substantial
amplification in other closely related off targets (signal strength
above 10 copies/µL). The other 2 assays (M. galloprovincialis(PAPM), Mytilus 3 (18S) showed positive but weak amplification
(signal strength below 10 copies/µL). Both Mytilus andEnsis species complexes could be successfully resolved based on
inspecting melt curves and cross-referencing amplification (or lack
thereof) of multiple assays. Specifically, the Mytilus markers on
the PAPM gene showed a range of melt curves, including 81°C
(Mytilus edulis ), 79-80°C (Mytilus trossulus ), and 79°C
(Mytilus galloprovincialis ). Similarly, the Ensis species
(COI) marker generated distinct melt curves at 75°C (Ensis
siliqua ) and 77°C (Ensis ensis ), hence providing some level of
resolution within these species complex. Based on the highest AMP-values
tested, detected and the in vitro testing 29 out of 42 assays were found
to have no risk of false positives (Table 3,supplementary Table S6 ).. A total of 6/42 assays were found to
have a risk that could be eliminated by another assay present on the
panel. Furthermore, a total of 4/42 assays were considered non-specific
and therefore the detection of C. pagarus and C. glaucumcannot be confirmed if the absence of Liocarcinus depurator andParvicardium exiguum is uncertain. The remainder of the target
species could be successfully detected by the newly developed HT-qPCR
panel
(Table 3, supplementary Table S6 ).