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A Simple Method To Detect Point Mutations in Aspergillus fumigatus cyp51A Gene Using a Surveyor Nuclease Assay
Teppei Arai1, Hidetaka Majima1,2, Akira Watanabe3
1Medical Mycology Research Center, Chiba University, Chiba, Japan.
Abstract:
One of the main mechanisms of azole resistance of Aspergillus fumigatus is thought to be a reduction in the drug's affinity for the target molecule, Cyp51A, due to its amino acid mutation(s). It is known that the azole resistance pattern is closely related to the mutation site(s) of the molecule. In this study, we tried to develop a simple and rapid detection method for cyp51A mutations using the endonuclease Surveyor nuclease. The Surveyor nuclease assay was verified using several azole-resistant strains of A. fumigatus that possess point mutations in Cyp51A. For validation of the Surveyor nuclease assay, blind tests were conducted using 48 strains of A. fumigatus (17 azole-resistant and 31 azole-susceptible strains). The Surveyor nuclease assay could rapidly detect cyp51A mutations with one primer set. Also, all the tested strains harboring different cyp51A single point mutations could be clearly distinguished from the wild type. The Surveyor nuclease assay is a simple method that can detect cyp51A mutations rapidly.
Insights
A new assay using Surveyor nuclease quickly detects mutations in the Aspergillus fumigatus cyp51A gene, which cause azole antifungal resistance. This method accurately identifies resistance-linked mutations in clinical fungal isolates.
Area of Science:
- Mycology
- Molecular Biology
- Antimicrobial Resistance
Background:
- Azole antifungals are crucial for treating Aspergillus fumigatus infections.
- Resistance to azoles in A. fumigatus is often linked to mutations in the target enzyme, Cyp51A.
- Accurate and rapid detection of these mutations is essential for guiding treatment decisions.
Purpose of the Study:
- To develop a simple and rapid method for detecting cyp51A mutations in A. fumigatus.
- To validate the efficacy of the Surveyor nuclease assay for identifying azole resistance-associated mutations.
Main Methods:
- Development and application of the Surveyor nuclease assay.
- Testing the assay on known azole-resistant and azole-susceptible A. fumigatus strains.
- Blind testing with 48 A. fumigatus isolates (17 resistant, 31 susceptible).
Main Results:
- The Surveyor nuclease assay successfully and rapidly detected cyp51A mutations using a single primer set.
- All tested strains with single point mutations in cyp51A were clearly distinguished from the wild type.
- The assay demonstrated high accuracy in identifying known resistant and susceptible strains.
Conclusions:
- The Surveyor nuclease assay provides a simple and rapid method for detecting cyp51A mutations.
- This assay can effectively identify azole resistance mechanisms in A. fumigatus.
- The validated assay has potential for clinical application in diagnosing and managing fungal infections.
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