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| 基于RPA-CRISPR/Cas12a的美国白蛾可视化快速检测新方法 |
| New and rapid visual detection assay for Hyphantria cunea Drury based on recombinase polymerase amplification and CRISPR/Cas12a |
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| DOI： |
| 中文关键词: 美国白蛾 RPA CRISPR Cas12a 可视化 快速检测 |
| 英文关键词:Hyphantria cunea Drury RPA CRISPR Cas12a visualization rapid detection |
| 基金项目:中国检验检疫科学研究院基本科研业务费项目（2024JK023） |
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| 中文摘要: |
| 美国白蛾Hyphantria cunea Drury是我国重要林业害虫，寄主植物众多且分布范围广泛。本试验基于重组酶聚合酶扩增（RPA）和CRISPR检测技术，构建美国白蛾RPA-CRISPR/Cas12a分子检测体系。根据NCBI数据库中的美国白蛾及近缘种、近似种线粒体CO Ⅰ基因片段信息以及昆虫样本CO I基因测序比对结果为模板，设计美国白蛾特异性RPA引物和CrRNA，通过RPA扩增目标序列，结合CRISPR检测技术并对反应条件进行优化，最终该体系灵敏度可检测的最低限度1.0×10^-1 ng/μL，较RPA结合普通凝胶电泳提高了100倍，检测体系在30 min内即可实现对害虫的现场快速、可视化、精准鉴定。为美国白蛾现场检测提供理论和实践依据，有助于保护森林资源和生态环境的安全。 |
| 英文摘要: |
| Hyphantria cunea Drury is one of the most important forestry pests in China, with numerous host plants and wide distribution ranges. Based on recombinant polymerase amplification (RPA) and CRISPR detection techniques, we constructed the RPA-CRISPR/Cas12a Molecular Detection System of H. cunea. Specific RPA primers and crRNA were designed based on the CO I gene fragment data from H. cunea and its closely related and similar species in the NCBI database, as well as the sequencing results of insect samples. An RPA-CRISPR/Cas12a molecular detection system was constructed, through RPA of target sequences and the reaction conditions were optimized with CRISPR detection technology. The system demonstrates a sensitivity of up to 1.0×10-1 ng/μL, a hundredfold improvement over traditional agarose gel electrophoresis methods. The test can be completed in 30 minutes. Our study provides theoretical and practical support for visual detection of H. cunea, contributing to the protection of forest resources and ecological environments. |
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