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Rapid and Specific Visual Detection of Amanita fuliginea by Combining Recombinase Polymerase Amplification with
Hao Li1, Guochun Wang2,3, Xin Wu1
1College of Life Sciences, Hunan Normal University, Changsha, China.
Abstract:
Amanita fuliginea is a common lethal mushroom with high mortality rates due to its ease of ingestion and concealed early symptoms. Rapid and specific identification of this species is crucial for clinical diagnosis and treatment. However, the existing rapid detection methods for nucleic acids of A. fuliginea were still plagued by specificity issues. Herein, we developed a recombinase polymerase amplification (RPA) coupled with CRISPR/Cas12b and lateral flow strip (LFS) assay for the rapid and specific detection of A. fuliginea. The RPA-CRISPR/Cas12b-LFS assay provides visual results within 35 min and does not rely on expensive equipment. High specificity was demonstrated against other Amanita species and non-Amanita species, with a sensitivity of 31 pg of genomic DNA per reaction. In conclusion, the RPA-CRISPR/Cas12b-LFS assay presents a rapid, specific, sensitive, and convenient tool for identifying A. fuliginea, particularly suitable for primary healthcare institutions or remote areas.
Insights
A new assay combining recombinase polymerase amplification (RPA) with CRISPR/Cas12b and lateral flow strips (LFS) offers rapid and specific detection of the lethal mushroom Amanita fuliginea. This tool is crucial for timely clinical diagnosis and treatment.
Area of Science:
- Mycology
- Molecular Biology
- Biotechnology
Background:
- Amanita fuliginea is a deadly mushroom responsible for numerous fatalities.
- Its easy ingestion and subtle early symptoms complicate clinical diagnosis.
- Current rapid detection methods for A. fuliginea nucleic acids lack sufficient specificity.
Purpose of the Study:
- To develop a rapid, specific, and sensitive detection method for Amanita fuliginea.
- To overcome the limitations of existing nucleic acid detection techniques.
Main Methods:
- Development of a recombinase polymerase amplification (RPA) assay.
- Integration of CRISPR/Cas12b technology for enhanced specificity.
- Utilized lateral flow strips (LFS) for visual, equipment-free results.
Main Results:
- The developed RPA-CRISPR/Cas12b-LFS assay achieved visual results within 35 minutes.
- Demonstrated high specificity against related and unrelated mushroom species.
- Achieved a sensitivity of 31 pg of genomic DNA per reaction.
Conclusions:
- The RPA-CRISPR/Cas12b-LFS assay is a rapid, specific, sensitive, and convenient tool for identifying Amanita fuliginea.
- This method is particularly suitable for resource-limited settings like primary healthcare institutions or remote areas.
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