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

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