A new method to detect red spotted grouper neuro necrosis virus (RGNNV) based on CRISPR/Cas13a

Red-spotted grouper nervous necrosis virus (RGNNV) is a serious pathogen that causes high mortality in many species of fish in marine aquaculture. Larvae and juveniles are more easily infected by RGNNV, and the cumulative mortality can reach 100% post-infection. To date, a simple, convenient, and low complexity method to detect this virus does not exist. Therefore, we developed a rapid detection method for RGNNV based on the CRISPR-Cas13a system. We optimized each of the four parts of the detection system: selection of efficient CRISPR-derived RNA(crRNA), reaction temperature, control of reporter probe content, and reaction concentration ratio of Cas13a to crRNA. Our detection system was sensitive enough to detect single-stranded RGNNV at a minimum of 102 fM. We evaluated the effectiveness of the detection system by testing it on different NNVs and RGNNV-infected RGNNV fish from different regions of China and found that the system had high specificity. Overall, our results showed that the CRISPR-Cas13a detection system was a robust, specific, and validated method for RGNNV detection. Thus, it provided a theoretical basis for the field detection of RGNNV.