SQUASH PCR: Rapid DNA Extraction from Filamentous Fungal Biomass for PCR Analysis

This Standard Operating Procedure outlines a protocol for the extraction of DNA from fungal biomass using the Squash PCR method, a rapid and streamlined approach suitable for downstream PCR-based applications. This technique has been previously employed for DNA preparation of filamentous fungi, but its application has been limited to species of industrial interest. In this protocol, the method has been adapted and validated for use with more than 150 filamentous fungal species belonging to Ascomycota, Basidiomycota and Mucoromycota, demonstrating its effectiveness in enabling rapid genotyping and molecular screening. Representative taxa tested belong to the genera Acremonium, Agaricus, Alternaria, Aspergillus, Cordyceps, Fusarium, Ganoderma, Mortierella, Mucor, Mycena, Penicillium, Rhizopus, Trichoderma etc. For yeast species, conventional colony PCR remains sufficient to achieve comparable outcomes. In contrast to earlier protocols relying on conidial suspensions, the present method has been performed on mycelium biomass, thereby extending its applicability to a broader range of fungal taxa (i.e. the one that do not sporulate). It is important to note that the procedure described here is intended for applications where speed and simplicity are prioritized over DNA purity, such as routine diagnostic PCR or strain verification. It is not recommended for applications requiring high-integrity or high-purity DNA, such as whole-genome sequencing.