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<p><i>Amanita fuliginea</i> 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 <i>A. fuliginea</i> 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 <i>A. fuliginea</i>. The RPA-CRISPR/Cas12b-LFS assay provides visual results within 35&#xa0;min and does not rely on expensive equipment. High specificity was demonstrated against other <i>Amanita</i> species and non-<i>Amanita</i> species, with a sensitivity of 31&#xa0;pg of genomic DNA per reaction. In conclusion, the RPA-CRISPR/Cas12b-LFS assay presents a rapid, specific, sensitive, and convenient tool for identifying <i>A. fuliginea</i>, particularly suitable for primary healthcare institutions or remote areas.</p>

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Rapid and Specific Visual Detection of Amanita fuliginea by Combining Recombinase Polymerase Amplification with CRISPR/Cas12b and Lateral Flow Strip

  • 期刊论文

  • 发表日期：

    2025年8月13日

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  • Hao Li,
  • Guochun Wang,
  • Xin Wu,
  • Xinyu Ying,
  • Zhidan Luo,
  • Chen Lu,
  • Ping Zhang

摘要

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.