Development of a multiplex detection system for toxic Amanita species based on isothermal recombinase polymerase amplification and CRISPR/Cas12b technology
- 1College of Life Sciences, Hunan Normal University, Changsha, China
- 2Jiangsu Key Laboratory of Marine Biological Resources and Environment, Jiangsu Key Laboratory of Marine Pharmaceutical Compound Screening, Jiangsu Ocean University, Lianyungang, China
- 3Co-Innovation Center of Jiangsu Marine Bio-industry Technology, Jiangsu Ocean University, Lianyungang, China
Abstract
Background: Toxic Amanita species are a major cause of fatal mushroom poisoning worldwide. Their morphological similarities to the edible species of mushrooms increase the risk of accidental ingestion, highlighting the need for rapid and accurate identification.
Objective: We aimed to develop a multiplex detection system using isothermal recombinase polymerase amplification (RPA) and CRISPR/Cas12b technology for rapid and specific detection of multiple toxic Amanita species.
Methods: Species-specific RPA primers and single guide RNAs (sgRNAs) were designed and optimized for six toxic Amanita species (A. exitialis, A. pallidorosea, A. subjunquillea, A. rimosa, A. molliuscula, and A. fuligineoides). The detection system combined RPA with Cas12b-mediated fluorescence readout to evaluate the specificity and sensitivity, and the results were visualized using lateral flow strips and a microfluidic disc chip for parallel detection.
Results: The selected primer–sgRNA combinations showed high specificity values without cross-reactivity and a low detection limit of 10 pg of genomic DNA per reaction. The lateral flow strips enabled intuitive and reliable detection. Furthermore, the microfluidic disc chip enabled multiplex detection for both single-species and four-species mixed samples.
Conclusion: We successfully established a rapid, sensitive, and specific multiplex detection system for toxic Amanita species using purified genomic DNA from authenticated specimens, providing a proof-of-concept analytical strategy for informing future development of instrument–light species identification tools in mushroom poisoning cases. We also summarize the evaluations required for further optimization of the current design prior to practical application.
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BibTeX
@article{li2026development,
title = {{Development of a multiplex detection system for toxic Amanita species based on isothermal recombinase polymerase amplification and CRISPR/Cas12b technology}},
author = {Hao Li and Xinyu Ying and Zhidan Luo and Chen Lu and Ping Zhang},
journal = {Frontiers in Molecular Biosciences},
year = {2026},
month = oct,
doi = {10.3389/fmolb.2026.1832406},
url = {https://doi.org/10.3389/fmolb.2026.1832406},
note = {CC BY 4.0},
}
RIS
TY - JOUR
TI - Development of a multiplex detection system for toxic Amanita species based on isothermal recombinase polymerase amplification and CRISPR/Cas12b technology
AU - Hao Li
AU - Xinyu Ying
AU - Zhidan Luo
AU - Chen Lu
AU - Ping Zhang
PY - 2026
DA - 2026/10/12
JO - Frontiers in Molecular Biosciences
DO - 10.3389/fmolb.2026.1832406
UR - https://doi.org/10.3389/fmolb.2026.1832406
AB - Background: Toxic Amanita species are a major cause of fatal mushroom poisoning worldwide. Their morphological similarities to the edible species of mushrooms increase the risk of accidental ingestion, highlighting the need for rapid and accurate identification. Objective: We aimed to develop a multiplex detection system using isothermal recombinase polymerase amplification (RPA) and CRISPR/Cas12b technology for rapid and specific detection of multiple toxic Amanita species. Methods: Species-specific RPA primers and single guide RNAs (sgRNAs) were designed and optimized for six toxic Amanita species (A. exitialis, A. pallidorosea, A. subjunquillea, A. rimosa, A. molliuscula, and A. fuligineoides). The detection system combined RPA with Cas12b-mediated fluorescence readout to evaluate the specificity and sensitivity, and the results were visualized using lateral flow strips and a microfluidic disc chip for parallel detection. Results: The selected primer–sgRNA combinations showed high specificity values without cross-reactivity and a low detection limit of 10 pg of genomic DNA per reaction. The lateral flow strips enabled intuitive and reliable detection. Furthermore, the microfluidic disc chip enabled multiplex detection for both single-species and four-species mixed samples. Conclusion: We successfully established a rapid, sensitive, and specific multiplex detection system for toxic Amanita species using purified genomic DNA from authenticated specimens, providing a proof-of-concept analytical strategy for informing future development of instrument–light species identification tools in mushroom poisoning cases. We also summarize the evaluations required for further optimization of the current design prior to practical application.
SN - 2296-889X
ER -