Abstract: Objective　A rapid visual detection method was established for Rift Valley Fever Virus (RVFV), providing technical support for the early diagnosis of RVFV, health monitoring of experimental animals, and prevention and control of RVFV epidemics.Methods　The conserved sequences of RVFV S gene were analyzed to design and screen specific reverse transcription-recombinase-aided isothermal amplification (RT-RAA) primers and gRNAs for the CRISPR/Cas12a system, thereby establishing an RT-RAA-CRISPR/Cas12a nucleic acid visual detection method. The CRISPR/Cas12a reaction system was optimized, with three parallel replicates set for each concentration; subsequently, the RT-RAA reaction conditions were optimized to improve the detection performance of the method. Thereafter, the sensitivity and specificity of this method were evaluated. All experimental results were verified through three repeated experiments.Results　An RT-RAA-CRISPR/Cas12a nucleic acid visual detection method was established targeting the RVFV S gene. Condition optimization experiments confirmed that the method achieved optimal detection performance when the working concentrations of Cas12a protein and gRNA were 0.053 μmol/L and 0.075 μmol/L, respectively, with isothermal amplification conducted at 39 ℃ for 30 minutes followed by a detection reaction at 37 ℃ for 20 minutes. Sensitivity test result showed that this method could stably detect the recombinant plasmid containing the RVFV S gene at a concentration as low as 0.5 copies/μL, demonstrating high sensitivity. Specificity experiments indicated that the method exhibited no cross-reactivity with Nipah virus RNA, recombinant plasmid containing the Lassa virus N gene, Ebola virus RNA, or novel bunyavirus RNA, showing strong specificity.Conclusion　In this study, a RT-RAA-CRISPR/Cas12a nucleic acid visual detection method for RVFV was successfully established. This method has advantages such as high sensitivity and simple operation, and is expected to be applied in primary-level laboratories. It provides technical support for the early detection of RVFV and contributes to vaccine development and epidemic prevention and control.

Key words: RVFV, RT-RAA, CRISPR/Cas12a, nucleic acid visual detection method

摘要： 目的　针对裂谷热病毒(RVFV),建立快速可视化检测方法,为RVFV早期诊断、实验动物健康监测及疫情防 控提供技术支持。方法　分析RVFV S基因保守序列,设计并筛选特异性反转录重组酶介导等温扩增(RT-RAA) 引物与CRISPR/Cas12a系统的gRNA,建立RT-RAA-CRISPR/Cas12a核酸可视化检测方法;优化CRISPR/Cas12a反 应体系,每个浓度设置3个平行,随后优化RT-RAA反应条件,以提高方法的检测性能;随后评价该方法的敏感性 及特异性。所有实验结果均经3次重复实验验证。结果　针对RVFV S基因,建立了RT-RAA-CRISPR/Cas12a核 酸可视化检测方法;通过条件优化实验确定,当Cas12a蛋白工作浓度为0.053 μmol/L、gRNA工作浓度为 0.075 μmol/L,在39℃条件下完成30 min等温扩增、随后于37 ℃进行20 min检测反应时,该方法的检测性能最 佳;敏感性实验结果显示,该方法可稳定检出浓度低至0.5 copies/μL的RVFV S基因重组质粒,具有高敏感性;特异 性实验表明,该方法与尼帕病毒RNA、拉沙病毒重组质粒(N基因)、埃博拉病毒RNA、新布尼亚病毒RNA无交叉反 应,具有强特异性。结论　本研究成功建立了RVFV RT-RAA-CRISPR/Cas12a核酸可视化检测方法,该方法具有敏感 性高、操作简单等优势,有望应用于基层实验室,为RVFV早期检测提供技术支持,助力疫苗研发及疫情防控。

关键词: 裂谷热病毒, 反转录重组酶介导等温扩增, CRISPR/Cas12a, 核酸可视化检测