A universal DNA extraction and PCR amplification method for fungal rDNA sequence-based identification

Area of Science:

• Mycology
• Molecular Biology
• Biotechnology

Background:

• Accurate identification of fungal pathogens is crucial for clinical diagnostics and research.
• Sequence-based identification methods, such as polymerase chain reaction (PCR), rely on high-quality template DNA.
• Existing DNA extraction protocols can be time-consuming, expensive, or require harsh chemicals, limiting their widespread application.

Purpose of the Study:

• To develop and standardize a rapid, inexpensive DNA extraction protocol for fungal pathogens.
• To ensure the protocol yields DNA of sufficient purity and quantity for PCR and sequencing.
• To validate the protocol's applicability across major fungal phyla, including yeasts and filamentous fungi.

Main Methods:

• A novel DNA extraction protocol combining physical and chemical lysis was developed.
• The protocol was tested on 519 clinical and culture collection strains of fungi.
• DNA suitability for PCR was assessed by targeting the ITS and D1/D2 regions for amplification and sequencing.

Main Results:

• The developed protocol successfully extracted template DNA from all tested fungal strains, representing major phyla.
• All extracted DNA templates were suitable for PCR amplification and subsequent sequencing.
• The protocol was completed in approximately 30 minutes, without using organic solvents or ethanol precipitation.
• The method minimized tube manipulations, enhancing ease of use.

Conclusions:

• This rapid and inexpensive DNA extraction protocol provides high-quality template DNA suitable for PCR and sequencing.
• The protocol is broadly applicable to diverse fungal species, including yeasts and filamentous fungi.
• Its efficiency and simplicity make it a valuable tool for fungal pathogen identification and molecular research.