A cost-effective and simplified protocol for fungal DNA extraction using silica-based grinding, without liquid nitrogen or lyophilization
¹Laboratory of Virology, National Institute of Agricultural Research, 14 rue Ibn Temmam, PO Box 257, Kenitra, Morocco
²Laboratory of Plant, Animal, and Agro-Industry Productions, Faculty of Sciences, University Ibn Toufail, Kenitra, Morocco
*Correspondence: karima_selmaoui@yahoo.fr
⁑These authors contributed equally to this work and share first authorship
⁂These authors contributed equally to this work and share last authorship
Abstract:
Efficient DNA extraction from filamentous fungi is often hindered in developing countries because of the limited availability of liquid nitrogen and lyophilization, which are widely used for breaking down the fungal cell walls. While the production and storage of liquid nitrogen pose significant environmental concerns owing to their high carbon footprint and associated costs, the adoption of lyophilization is restricted by its substantial operational expenses. To address these challenges, a cost-effective and accessible CTAB-based DNA extraction protocol was developed, utilizing silicon dioxide as an abrasive, along with mortar and pestle grinding. This approach eliminates the dependency on liquid nitrogen and lyophilization. DNA was successfully extracted from the mycelia of Colletotrichum sp.1, Colletotrichum sp. 2, and Penicillium sp. using the developed protocol. Spectrophotometric quantification revealed high average DNA concentrations. Purity was assessed using A260/280 and A260/230 absorbance ratios, which fell within the recommended range, indicating minimal contamination and high-quality DNA. DNA integrity was further confirmed by PCR amplification using ITS1 and ITS4 primers, producing expected amplicons of 594 bp for both Colletotrichum species and 585 bp for Penicillium. This protocol provides a reliable and affordable alternative for DNA extraction from fungal mycelia, enabling broader accessibility to laboratories with limited resources.
Key words:
Colletotrichum spp., CTAB-based protocol, fungal DNA extraction, PCR amplification, Penicillium sp., silicon dioxide, spectrophotometric quantification