Significant Improvement of Environmental DNA Assay by Targeting Retrotransposon Sequences Characteristic to Anguilla Eels

Environmental DNA (eDNA) analysis faces challenges regarding sensitivity and quantification accuracy, particularly in areas with low target species densities or during seasons when eDNA release decreases. Multi-copy markers such as mitochondrial genomic DNA and ribosomal DNA (rDNA) have been widely used in eDNA analysis to address this issue. However, the copy number of the DNA markers per cell remains a potential bottleneck for eDNA sensitivity. In this study, we aimed to increase the sensitivity of eDNA assays by using retrotransposons, which are abundant in the genome, as novel target markers. We developed an assay targeting UnaSINE1, a short interspersed nucleotide element (SINE) characteristic of Anguilla eels, and compared its sensitivity and accuracy with that of an established mitochondrial 16S rRNA marker. Our results demonstrated that UnaSINE1 was detected at over 100 times the copy number of the mitochondrial marker in both genomic and eDNA samples. In the river surveys, the 16S marker was positive in 32 of the 81 samples, whereas the UnaSINE1 marker was positive in 62 samples, indicating that the use of the SINE marker remarkably reduced false negatives. Furthermore, both biological and technical replicates exhibited improved positive consistency and reduced variability in quantification, leading to more robust presence/absence determination and quantitative results. Utilizing retrotransposon sequences as markers requires additional effort for sequence acquisition and organization and may limit taxonomic resolution to the genus level. However, this approach significantly improves sensitivity without increasing the labor or cost of sampling and PCR analysis, making it highly practical for eDNA studies.