ESTABLISHMENT OF RAA-CRISPR/CAS12A DETECTION FOR GENOTYPE Ⅱ GRASS CARP REOVIRUS

Grass carp reovirus (GCRV) is responsible for severe hemorrhagic disease in grass carp, posing a significant threat to grass carp farming in our country. The type Ⅱ strain of grass carp reovirus (GCRV-Ⅱ) is the primary pathogen causing this disease. In order to establish a specific, sensitive, and easy-to-use detection method for GCRV-Ⅱ, we employed a recombinant enzyme-mediated strand-exchange nucleic acid amplification (RAA) technique. In this study, we combined recombinase-aided amplification (RAA) with the clustered regularly interspaced short palindromic repeats (CRISPR) system to construct a one-step, rapid, and visual detection method for the GCRV-Ⅱ virus. We began by selecting the conserved region of s6 gene (GQ896337), which is consistent among GCRV-Ⅱ strains but significantly different across various GCRV genotypes. We designed five pairs of RAA candidate primers and four crRNAs, synthesizing and screening them to identify the specific primer and crRNA combinations. Subsequently, the specificity, sensitivity, and accuracy of the reaction were evaluated. Finally, a “one-step” reaction system comprised a lower amplification phase and an upper detection phase. The results demonstrated that GCRV-Ⅱ could be detected at a constant temperature of 37℃, with a detection limit as low as 10² copies/μL. When tested on twenty-four clinical samples, the positive detection rate of this method surpassed that of the PCR method currently used in the industry. The detection method for GCRV-Ⅱ established in this study is easy to use, highly specific, sensitive, and reproducible. It is also compatible with existing equipment and allows for visual assessment under blue light.