Background

Polymerase chain reaction (PCR) is a powerful tool widely used to amplify specific target regions of DNA. Since its invention in the mid-1980s [1, 2], PCR has become a fundamental technique employed in various fields. A notable application of PCR is in the diagnosis of pathogenic diseases. It allows for the amplification of universal DNA barcodes, facilitating the identification of causative pathogens [3, 4]. Given that multiple copies of DNA are generated from the DNA template, the preparation of this template is essential for performing PCR. For filamentous fungi, the steps for DNA extraction and purification have been prerequisites for PCR amplification. Most fungi possess a rigid cell wall, predominantly constituted of polysaccharides like chitin and glucans [5, 6]. This results in inefficient fungal DNA extraction. The procedure for extracting DNA from fungal sources requires laborious and time-consuming steps involving the grinding of freeze-dried mycelia [7]. Additionally, the fungal DNA extraction process involves multiple stages using toxic chemicals like phenol and chloroform. A technique known as colony PCR involves directly adding cells into the PCR mixture, and it has found widespread application in bacterial and yeast research [8]. In the case of filamentous fungi, previous studies have suggested various PCR methods to minimize fungal DNA extraction steps. The methods proposed by Alshahni et al. and Walch et al. eliminated the need for fungal genomic DNA extraction [9, 10]. However, these methods incorporated additional steps, such as using lysis buffer or bovine serum albumin (BSA), to lyse fungal cell walls. Despite these advancements, the adoption of these methods for filamentous fungi has been limited due to their reduced PCR efficiency and the need for a cell lysis process.

Here, we introduce a direct PCR procedure tailored for filamentous fungi. This approach eliminates the need for the fungal DNA extraction process and the use of additional reagents such as BSA or proteinase. Our method involves adding a small amount of mycelium directly into the PCR mixture, followed by heat shock and vortexing. These heat shock and vortexing steps are key in breaking the fungal cell walls and membrane, releasing the genomic DNA for PCR amplification. This method will expedite the molecular identification of filamentous fungi and facilitate rapid diagnosis of fungal diseases.