Huaming Li, Xinyue Cao, Ruige Chen, Min Guang, Mengran Xu, Xiaomin Wu, Rongrong Yang, Liancheng Lei,  Fuxian Zhang.

Front. Microbiol., 03 November 2023; Volume 14 – 2023; https://doi.org/10.3389/fmicb.2023.1296038

Introduction: Due to the existence of grass carp reovirus (GCRV), grass carp hemorrhagic disease occurs frequently, and its high pathogenicity and infectivity are great challenges to the aquaculture industry. As a highly pathogenic pathogen, the outbreak of hemorrhagic disease often causes tremendous economic losses. Therefore, it is important to rapidly and accurately detect GCRV on site to control timely.

Methods: In this study, recombinant enzyme amplification (RPA) combined with clustered regularly interspaced short palindromic repeats (CRISPR)/Cas13a system was employed to establish a method to detect the vp7 gene of grass carp reovirus type 1. This method can be adopted for judging the results by collecting fluorescence signal, ultraviolet excitation visual fluorescence and test strip.

Results: Combined with the RPA amplification experiment, the detection limit of the RPA-CRISPR method can reach 7.2 × 10¹ copies/μL of vp7 gene per reaction, and the detection process can be completed within 1 h. In addition, this method had no cross-reaction with the other 11 common aquatic pathogens. Then, the performance of the RPA-CRISPR/Cas13a detection method was evaluated by comparing it with the real-time fluorescence quantitative PCR detection method of clinical samples. The results of RPA-CRISPR/Cas13a detection were shown to be in consistence with the results obtained from the real-time fluorescence quantitative PCR detection. The coincidence rate of this method with 26 GCRV clinical samples was 92.31%.

Discussion: In summary, this method has high sensitivity, specificity and on-site practicability for detecting GCRV type 1, and has great application potential in on-site GCRV monitoring.

See https://www.frontiersin.org/articles/10.3389/fmicb.2023.1296038/full

Figure 2. The locus diagram of each crRNA on the vp7 gene. The vp7 gene is 909 bp in length. CrRNA1 starts at 154 and ends at 181. CrRNA2 starts at 761 and ends at 788. CrRNA3 starts at 625 and ends at 652. CrRNA4 starts at 350 and ends at 324. CrRNA5 starts at 579 and ends at 606.