Detailed Protocol to Perform Direct PCR Using Filamentous Fungal Biomass—Tips and Considerations

Abstract

The precise and rapid detection of fungi is important in various fields, including clinics, industry, and agriculture. While sequencing universal DNA barcodes remains the standard method for species identification and phylogenetic analysis, a significant bottleneck has been the labor-intensive and time-consuming sample preparation for genomic DNA extraction. To address this, we developed a direct PCR method that bypasses the DNA extraction steps, facilitating efficient target DNA amplification. Instead of extracting genomic DNA from fungal mycelium, our method involves adding a small quantity of mycelium directly to the PCR mixture, followed by a heat shock and vortexing. We found these simple adjustments to be sufficient to lyse many filamentous fungal cells, enabling target DNA amplification. This paper presents a comprehensive protocol for executing direct PCR in filamentous fungi. Beyond species identification, this direct PCR approach holds promise for diverse applications, such as diagnostic PCR for genotype screening without fungal DNA extraction. We anticipate that direct PCR will expedite research on filamentous fungi and diagnosis of fungal diseases.Key features• Eliminates the time-consuming genomic DNA extraction step for PCR, enhancing the speed of molecular identification.• Adds a small quantity of mycelium directly into the PCR mix.• Emphasizes the crucial role of heat shock and vortexing in achieving efficient target DNA amplification.• Accelerates the molecular identification of filamentous fungi and rapid diagnosis of fungal diseases.Graphical overview Direct PCR using filamentous fungal biomass