Synthesized double stranded gene fragments are not suitable for qPCR endogenous control spike-ins

Quality control standards are paramount for eDNA methods to gain widespread acceptance. In targeted eDNA studies, there are three main stages: sample collection, DNA extraction, and amplification via PCR. During this process, positive controls that ensure procedural success and validate negative results are typically included only in the final PCR amplification stage of the workflow. To address this issue, we explored the possibility of using synthetic dsDNA gene fragment spike-ins as endogenous controls to monitor the success of the sample collection and DNA extraction phases of the workflow. We hypothesised that short fragments of assay-specific dsDNA would be suitable for an endogenous control to monitor method success. To test this, we spiked dsDNA into two matrices, river water and TE buffer, where we then filtered and extracted each matrix and assessed the recovery of the spike-in. Our findings concluded that common eDNA collection and extraction methodologies do not consistently capture and isolate dsDNA fragments as we were unable to recover any of the dsDNA spike-ins. Thus, such fragments are unsuitable for a pre-extraction endogenous control. Further, this suggests that similar size dsDNA fragments in the environment may be missed by filtration-based eDNA studies.