CRISPR/Cas13a与RPA联用技术在鲤浮肿病毒 (CEV) 现场快速检测中的应用

Integration of RPA and CRISPR-Cas13a for on-site detection of Carpedema virus (CEV)

  • 摘要: 鲤浮肿病毒 (Carp edema virus, CEV) 是严重危害鲤科鱼类的一种重要病原体,对中国的鲤鱼养殖业及观赏鱼进出口贸易造成了重大经济损失。该病毒具有高度传染性和强致病性,已成为威胁鲤科鱼类健康的主要因素之一。为实现CEV的现场即时检测,本研究开发了一种基于重组酶聚合酶扩增 (RPA) 结合CRISPR/Cas13a系统的现场检测技术,采用恒温扩增方式,无需依赖专业仪器设备,可实现病毒特异性单分子水平的核酸检测。全部检测过程可在30~60 min内完成,最低检测限可达1.256 拷贝·μL−1,灵敏度高、特异性强;结果可通过试纸条直观呈现,实现可视化判读。这项技术适用于口岸检疫、养殖场监测和野外调查等场景,能够实现快速、精准的现场检测,显著提升检测效率并缩短检测周期,为CEV的防控提供有力的技术支撑。

     

    Abstract: Carp edema virus (CEV) causes significant economic losses in China's carp aquaculture industry and the ornamental fish import-export trade. Known for its high pathogenicity and mortality rates, CEV poses a serious threat to carp health. To achieve rapid on-site detection of CEV, we develops a diagnostic technique that combines recombinase polymerase amplification (RPA) with the CRISPR/Cas13a system. This technology enables isothermal amplification without the need for specialized equipment and operates at 37 °C. It provides specific nucleic acid detection at the single-molecule level for the virus and completes testing within 30 to 60 min. The detection limit is up to 1.256 copies·μL−1, indicating high sensitivity and specificity. Results are interpretable through lateral flow test strips. This method is suitable for port quarantine, farm monitoring, and field surveys, enabling fast, accurate, and portable on-site testing. It significantly enhances detection efficiency while reducing turnaround times, providing a reliable technical solution for the prevention and control of CEV.

     

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