Accuracy of quantitative analysis of eDNA in seawater: comparison of filter pore size and real-time PCR methods

Abstract

Abstract Background A comparative analysis of environmental DNA (eDNA) quantification methods requires the specific detection of a single species and the eDNA yield from the filter must be high. Studies have collected eDNA using a relatively small (> 1.0 µm) filter, which compromises eDNA accuracy due to clogging of the filter in a large space, such as the ocean or water with high turbidity. Therefore, here we established an eDNA sampling method using a large-pore filter to minimize clogging. Methods and Results SYBR Green qPCR has been used to analyze Pseudo-nitzschia spp. However, we observed that there may be an overestimation due to a false-positive signal. Thus, a new specific TaqMan primer–probe set was developed and used in this study. The morphological detection method under conventional microscopy and the two eDNA qPCR methods (TaqMan, SYBR Green) were then compared to determine the correlation between cell abundance and Ct values. We observed that the eDNA yield was higher as the pore size increased, and the correlation between the abundance of morphologically identified Pseudo-nitzschia spp. Compared with the SYBR Green qPCR data, the TaqMan qPCR Ct values were more specifically correlated with the Pseudo-nitzschia spp. cell abundance determined by the conventional method. Conclusion These results suggest that treatment with large amounts of seawater using large hole filters can yield high DNA yields, and existing morphological identification and eDNA method relative comparisons have access to quantitative evaluation.