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Improving eDNA yield and inhibitor reduction through increased water volumes and multi-filter isolation techniques

March 27, 2019

To inform management and conservation decisions, environmental DNA (eDNA) methods are used to detect genetic material shed into the water by imperiled and invasive species. Methodological enhancements are needed to reduce filter clogging, PCR inhibition, and false-negative detections when eDNA is at low concentrations. In the first of three simple experiments, we sought to ameliorate filter clogging from particulates and organic material through a scaled-up, multi-filter protocol. We combined four filters in a 5 mL Phenol-Chloroform-Isoamyl (PCI) procedure to allow for larger volumes of water (~1 L) to be filtered rapidly. Increasing the filtered water volume by four times resulted in 4.4X the yield of target DNA. Next, inhibition from organic material can reduce or block eDNA detections in PCR-based assays. To remove inhibitory compounds retained during eDNA isolation, we tested three methods to chemically strip inhibitors from eDNA molecules. The use of CTAB as a short-term (5–8 day) storage buffer, followed by a PCI isolation, resulted in the highest eDNA yields. Finally, as opposed to a linear relationship among increasing concentrations of filtered genomic eDNA, we observed a sharp change between the lower (70–280 ng) and higher (420–560 ng) amounts. This may be important for effectively precipitating eDNA during protocol testing.

Citation Information

Publication Year 2019
Title Improving eDNA yield and inhibitor reduction through increased water volumes and multi-filter isolation techniques
DOI 10.1038/s41598-019-40977-w
Authors Margaret Hunter, Jason Ferrante, Gaia Meigs-Friend, Amelia Ulmer
Publication Type Article
Publication Subtype Journal Article
Series Title Scientific Reports
Series Number
Index ID 70202977
Record Source USGS Publications Warehouse
USGS Organization Wetland and Aquatic Research Center