Got glycogen?: Development and multispecies validation of the novel preserve, precipitate, lyse, precipitate, purify (pplpp) workflow for environmental dna extraction from Longmire's preserved water samples

Edmunds, Richard C., and Burrows, Damien (2020) Got glycogen?: Development and multispecies validation of the novel preserve, precipitate, lyse, precipitate, purify (pplpp) workflow for environmental dna extraction from Longmire's preserved water samples. Journal of Biomolecular Techniques, 31 (4). pp. 125-150.

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Abstract

Unfiltered and filtered water samples can be used to collect environmental DNA (eDNA). We developed the novel “Preserve, Precipitate, Lyse, Precipitate, Purify” (PPLPP) workflow to efficiently extract eDNA from Longmire’s preserved unfiltered and filtered water samples (44–100% recovery). The PPLPP workflow includes initial glycogen-aided isopropanol precipitation, guanidium hypochlorite and Triton X-100–based lysis, terminal glycogen-aided polyethylene glycol precipitation, and inhibitor purification. Three novel eDNA assays that exclusively target species invasive to Australia were also developed: Tilapia_v2_16S concurrently targets Oreochromis mossambicus (Mozambique tilapia) and Tilapia mariae (spotted tilapia) while R.marina_16S and C.caroliniana_matK discretely target Rhinella marina (cane toad) and Cabomba caroliniana (fanwort), respectively. All 3 assays were validated in silico before in vitro and in situ validations using PPLPP workflow extracted samples. PPLPP workflow was concurrently validated in vitro and in situ using all 3 assays. In vitro validations demonstrated that 1) glycogen inclusion increased extracellular DNA recovery by;48-fold compared with glycogen exclusion, 2) swinging-bucket centrifugation for 90 min at 3270 g is equivalent to fixed-angle centrifugation for 5–20 min at 6750 g, and 3) Zymo OneStep Inhibitor Removal Kit, Qiagen DNeasy PowerClean Pro Cleanup Kit, and silica-Zymo double purification provide effective inhibitor removal. In situ validation demonstrated 95.8 ± 2.8% (mean ± SEM) detectability across all 3 target species in Longmire’s preserved unfiltered and filtered water samples extracted using the PPLPP workflow (without phenol:chloroform:isoamyl alcohol purification) after 39 d of incubation at room temperature and 50°C. PPLPP workflow is recommended for future temperate and tropical eDNA studies that use Longmire’s to preserve unfiltered or filtered water samples.

Item ID: 67274
Item Type: Article (Research - C1)
ISSN: 1943-4731
Keywords: eDNA, Invasive species, Method, Tropical
Copyright Information: © 2020 ABRF.
Funders: National Environmental Science Program (NESP)
Projects and Grants: NESP 4.3: The Northern Australiae DNA Program–Revolutionising Aquatic Monitoring and Field Surveys in Tropical Waters
Date Deposited: 17 Mar 2021 00:10
FoR Codes: 41 ENVIRONMENTAL SCIENCES > 4103 Environmental biotechnology > 410304 Environmental biotechnology diagnostics (incl. biosensors) @ 100%
SEO Codes: 18 ENVIRONMENTAL MANAGEMENT > 1803 Fresh, ground and surface water systems and management > 180302 Control of pests, diseases and exotic species in fresh, ground and surface water @ 50%
18 ENVIRONMENTAL MANAGEMENT > 1802 Coastal and estuarine systems and management > 180204 Control of pests, diseases and exotic species in coastal and estuarine environments @ 50%
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