Establishment of RPA-CRISPR/Cas12a Detection Methods for Rapid Largemouth Bass Ranavirus Surveillance

The high mortality associated with largemouth bass ranavirus (LMBRaV) disease requires rapid and sensitive diagnosis methods to prevent virus spread and subsequent outbreak. The recombinase polymerase amplification combined with the CRISPR/Cas12a system (RPA-CRISPR/Cas12a) assay and RPA-CRISPR/Cas12a, coupled with lateral flow dipstick (RPA-CRISPR/Cas12a-LFD) assay for LMBRaV, were established in this study for low viral load surveillance. The major capsid protein (mcp) gene is highly conserved and is widely used as the target sequence in LMBRaV detection. First, based on the mcp gene sequence of LMBRaV, three candidate crRNAs were designed. Among them, the crRNA-2, which exhibited the highest cleavage activity, was selected through dual evaluation of fluorescence signal intensity and LFD color development. Then, the key reaction conditions for both assays were optimized as follows: LbCas12a protein concentration of 150 nM, crRNA-2 concentration of 200 nM, and ssDNA reporters' concentration of 200 nM. Moreover, specificity evaluation showed that both combined assays specifically recognized LMBRaV, with no cross-reactivity detected against other aquatic pathogens, such as CyHV-2, GSIV, ISKNV, WSSV, GCRV II or CrERV. The detection limit was 1 copy/μL of DNA sample for both RPA-CRISPR/Cas12a and RPA-CRISPR/Cas12a-LFD assays. Finally, validation using 24 clinical samples (16 positive, eight negative) showed that both LMBRaV RPA-CRISPR/Cas12a assays achieved 100% detection rate, whereas conventional PCR detected 13 positive samples (detection rate 81.26%). ddPCR served as the reference method for clinical validation. Therefore, the LMBRaV RPA-CRISPR/Cas12a assay and the RPA-CRISPR/Cas12a-LFD assay provide sensitive, specific, and easy-to-operate methods for the rapid detection of LMBRaV.