Pre-centrifugation before DNA extraction mitigates extraction efficiency reduction of environmental DNA caused by the preservative solution (benzalkonium chloride) remaining in the filters

In the biomonitoring using environmental DNA (eDNA) analysis, the suppression of eDNA degradation has been an important issue to estimate accurately the presence of target species and their eDNA concentrations. A recently proposed eDNA preservation technique requires only the addition of benzalkonium chloride (BAC) to water samples and has been used for various studies as an alternative solution to on-site filtration. However, BAC remaining in filters may adsorb to and inhibit the function of the enzyme used to extract eDNA from filters. In this study, we tested whether eDNA yield is affected by BAC addition immediately before filtration and demonstrated that the BAC in water remaining in filters decreased the eDNA yield significantly. We found that simple pre-centrifugation is effective for removing the remaining water from the filters to prevent the eDNA yield reduction due to the use of BAC. This is the most important finding in the present study. Appropriate eDNA extraction techniques are needed when BAC is used to secure eDNA yields and expand the application of eDNA analysis for biomonitoring.