Technical and practical considerations regarding the assay panel
development
This study showed that a HT-qPCR system substantially boosted the number
of assays that can be tested in vitro (e.g. 96 assays in a single
run) resulting in great time-effectiveness and therefore, cost
efficiency proposed a 5-level validation scale for determining the
readiness of eDNA assays, ranging from “Level 1 – Incomplete” to
“Level 5 – Operational”. When available, a HT-qPCR system could
therefore play a crucial role in transitioning many molecular assays
from Level 2 (i.e. testing assays against non-target organisms) to Level
3 and 4 (validating assays in the field), because many assays can be
tested in parallel. Level 4 differs from Level 5 as it is missing
statistical modelling for detection probability, but both these two last
levels are considered valid to provide satisfactory interpretation of
presence/absence of target species. Furthermore, the possibility to
co-screen a given sample for many species in a single qPCR run would
also result in saving precious eDNA template, hence increasing the
potential number of targets that can be tested.
Efficiency was satisfactory even though assays were combined into
communal thermal cycling conditions, confirming robustness of marker
selection steps and reagents used. However, issues with standard for
quantification delay in pools of synthetic targets were unexpected. This
study experimenting using a second quantification method, which made
quantification more accurate but needs further investigation. In future
studies comparison between synthetic and pools of genomic DNA of known
quantity from target organisms could circumvent these problems of
quantification.
Using a machine learning tool (eDNAassay, Kronenberger et al., 2022)
during the in-silico selection of markers proved to be very
useful in providing an initial screening of marker panels and in
particular, to assess potential biases due to data gaps in the reference
database used. For instance, 17 out of the 42 selected assays showed
higher affinity (i.e. higher AMP values) for species not necessarily
closely related to the target species, for example, Ensis assays
with P. pellucidus, which combined with the absence of reference
data from another closely related Pharidae species, Pharus
legumen , could lead to potential false positives. This suggests that
the specificity of some assays may be lower than expected. Nonetheless,
when selected markers were tested in vitro , the concordance
between expected and observed amplification was good, but in sporadic
instances low AMP values still reacted positive. To mitigate, in
uncertain situations, multiple markers are preferably selected for
various reasons i) to account for the possibility that intraspecific
variation prohibits amplification, ii) to increase the target
specificity in the case of markers not being fully diagnostic in
relation to other species, iii) to quantify the presence of targets more
accurately. In addition, multiple markers can also help troubleshootin vitro experiments by identifying possible contamination. This
study identified 21 different primer pairs from literature, eventually,
only five were used with four being redeveloped as adaptations on
existing primers. Most primers from the literature were either developed
for a different purpose or did not fit in the assay requirements. The
development of a multi-species panel requires strict rules to make
multiple assays work under the same circumstances. Protocol optimization
showed that extraction protocol chosen and inclusion of BSA are
recommended for genetic screening of zooplankton samples.
Assays developed using the HT-qPCR system are expected to be directly
transferable to conventional qPCR instruments ; however various aspects
especially related to quantification and sensitivity (e.g. LOD, LOQ)
should be re-estimated and confirmed when using a different instrument.
Nonetheless, as indicated above, the use of a HT-qPCR system enables the
rapid testing of multiple assays in parallel, which is a time-consuming
step in assay validation, prior to executing further downstream steps in
the laboratory.