Field application of an improved protocol for environmental DNA extraction, purification, and measurement using Sterivex filter

Abstract

Environmental DNA (eDNA) is increasingly popular as a useful non-invasive method to monitor and study biodiversity and community structure in freshwater and marine environments. To effectively extract eDNA from the filter surface is a fundamental factor determining the representativeness of the samples. We improved the eDNA extraction efficiency of an established Sterivex method by 12- to 16-fold using a larger volume of lysis buffer mix coupled with backflushing the cartridges. The DNeasy extraction column could be overloaded when the environmental sample input is high, possibly due to a higher nonspecific binding present in environmental samples, thus resulting in a relatively lower quantity measured. Therefore, we included an internal control DNA in the extraction to monitor the extraction and purification efficiencies in field samples, which is crucial for quantification of original eDNA concentration. The use of Takara Probe qPCR Mix supplemented with protein-based additives improved the robustness of the real time PCR assay on inhibitor-rich environmental samples, but prior purification by Qiagen PowerClean Pro Cleanup kit could be essential for inhibitor-rich water samples, even though the recovery rate was unexpectedly low (average 33.0%). The improved extraction and quantification complement the qualitative analyses including metabarcoding and metagenomics in field application.

Environmental DNA (eDNA) Metabarcoding in the Fish Market and Nearby Seafood Restaurants in Taiwan Reveals the Underestimation of Fish Species Diversity in Seafood

“…DNA extraction was conducted as previously described [ 24 ] with some modifications. A lysis buffer mix (PBS 220 μL, Buffer AL 200 μL, Proteinase K 20 μL; 440 μL total volume) was introduced to the cartridge through the inlet.…”

Prospects and challenges of environmental DNA (eDNA) metabarcoding in mangrove restoration in Southeast Asia

“…Therefore, field decontamination before sampling is paramount to ensure sample independence (Goldberg et al, 2016). The most widely-adopted approach to capture eDNA is by filtration, which is highly dependent on factors like pH, amount of suspended particles, filter pore size, and filtered water volume (Majaneva et al, 2018;Wong et al, 2020). For the turbid estuarine waters characteristic of mangroves in SEA, high concentrations of suspended organic and inorganic particles are associated with higher eDNA abundance but also rapid clogging of filters.…”

Development of species‐specific multiplex real‐time PCR assays for tracing the small pelagic fishes of North Pacific with environmental DNA

“…Combining the recent advance on eDNA and metabarcoding technologies, the AORI has launched a project called “Ocean DNA” to map the marine lives in the North Pacific. We have optimized an extraction method of eDNA to obtain maximum DNA quantities from standard Sterivex filters, which consolidates the basis of quantitative analysis and increases the depth of the data representation (Wong et al, 2020). Using eDNA and a specific quantitative real‐time PCR (qPCR) assay, we have previously reported the migration pattern of juvenile chum salmon in the coastal area of Japan (Minegishi et al, 2019).…”

“…Combining the recent advance on eDNA and metabarcoding technologies, the AORI has launched a project called "Ocean DNA" to map the marine lives in the North Pacific. We have optimized an extraction method of eDNA to obtain maximum DNA quantities from standard Sterivex filters, which consolidates the basis of quantitative analysis and increases the depth of the data representation (Wong et al, 2020).…”