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.