Feb 11, 2025

Public workspaceEnvironmental DNA (e-DNA) extraction from soil

DOI
dx.doi.org/10.17504/protocols.io.dm6gp9295vzp/v1
  • Nuttapon Pombubpa1,2,
  • Ariya Chindamporn3,2,
  • Nattapol Kraisitudomsook4,2,
  • Neil Andrew Robert Gow5,2,
  • Syahriar Nur Maulana Malik Ibrahim1,2,
  • Fikran Aranda Fahrudin6,2
  • 1Department of Microbiology, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand;
  • 2Southeast Asia Antifungal Resistance Monitoring Initiative (SEA-ARMi), Department of Microbiology, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand;
  • 3Department of Microbiology, Faculty of Medicine, Chulalongkorn University, Bangkok 10330, Thailand;
  • 4Department of Biology, Faculty of Science and Technology, Muban Chombueng Rajabhat University, Ratchaburi 70150, Thailand;
  • 5Medical Research Council Centre for Medical Mycology, University of Exeter, Exeter, United Kingdom;
  • 6Program of Biotechnology, Faculty of Science, Chulalongkorn University, Bangkok 10330, Thailand
Syahriar Nur Maulana Malik Ibrahim
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DOI: dx.doi.org/10.17504/protocols.io.dm6gp9295vzp/v1
Protocol Citation: Nuttapon Pombubpa, Ariya Chindamporn, Nattapol Kraisitudomsook, Neil Andrew Robert Gow, Syahriar Nur Maulana Malik Ibrahim, Fikran Aranda Fahrudin 2025. Environmental DNA (e-DNA) extraction from soil. protocols.io https://dx.doi.org/10.17504/protocols.io.dm6gp9295vzp/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: January 22, 2025
Last Modified: February 11, 2025
Protocol Integer ID: 118861
Keywords: WHO FPPL, e-DNA, Environmental DNA, Filamentous fungi, Metagenomic, Soil
Funders Acknowledgements:
FAILSAFE Awarded projects - Seed Grant
Grant ID: FAILSAFE FR1-23
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Abstract
Soil harbors diverse microorganisms, including fungi that play crucial environmental roles but can also harm plants, animals, and humans through infections or spore exposure. In 2022, the WHO released several pathogenic fungi, including Fusarium group, Mucorales and Eumycetoma agents, which are prevalent in Thailand's agricultural soils and have caused significant human infections. Despite this, the genetic mechanisms behind their infections and antifungal resistance remain poorly understood, particularly in Southeast Asia. Antifungal resistance to major drug classes, such as azoles, echinocandins, and polyenes, is increasing, driven by specific resistance genes. To address these challenges, we propose establishing a Southeast Asian (SEA) network of mycologists to study fungal pathogens, focusing on collecting soil samples, extracting fungal genomic DNA, and creating a comprehensive genome database. This initiative will improve detection and management of drug-resistant fungi, enhance clinical treatments, and strengthen agricultural biosecurity. By leveraging genomic technologies and fostering international collaboration, the project aims to develop targeted strategies against fungal infections and resistance, benefiting public health, food security, and sustainable agriculture across SEA. Establishing a fungal biobank and expert network will support long-term efforts to combat antifungal resistance, providing critical resources for swift and accurate responses to fungal threats.
Attachments
Icon for Hand book DNeasy Power soil pro.pdf
Hand book DNeasy Pow...
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Materials
  1. Soil Sample: 0.5 g
  2. Micropipette and Tips: 1000-10 µL
  3. DNeasy PowerSoil Pro Kit (QIAGEN)
  4. Eppendorf Tubes
  5. Agarose and Electrophoresis Set
  6. TBE Buffer
  7. DNA Ladder
Soil Sample Preparation:
Soil Sample Preparation:
Weigh 0.5 g of soil (remove debris like stones or large particles).
DNA Extraction Using the DNeasy PowerSoil Kit:
DNA Extraction Using the DNeasy PowerSoil Kit:
Add the soil sample into the PowerBead Pro Tube.
Add Solution CD1 to the tube as per kit instructions.
Homogenize the sample using one of the following Vortex Adapter method.
Cell Lysis
Cell Lysis
Ensure proper mixing and cell lysis through homogenization.
Inhibitor Removal
Inhibitor Removal
Add Solution CD2 to remove impurities.
Add Solution CD3, then load the sample into the MB Spin Column.
Washing
Washing
Wash the column with Solution EA followed by Solution C5 to ensure purity.
Elution
Elution
Elute the DNA using Solution C6 to collect the extracted DNA.
DNA Qualification and Quantification:
DNA Qualification and Quantification:
Perform electrophoresis on an agarose gel to confirm DNA quality.
Use a Nanodrop spectrophotometer to measure DNA concentration and purity.
Submission of Environmental DNA:
Submission of Environmental DNA:
Send the extracted e-DNA to the FAILSAFE Project Team at Chulalongkorn University for further analysis.