A one-pot asymmetric ERA-CRISPR/Cas12a assay for visual detection of white spot syndrome virus in shrimp

Introduction White spot syndrome virus (WSSV) threatens shrimp aquaculture. PCR is sensitive but laboratory dependent. Isothermal amplification with CRISPR/Cas12a offers an alternative, but one-pot integration is limited by competition between double-stranded amplicons and crRNA for Cas12a. Methods A one-pot assay (OAERA-Cas12a) integrating asymmetric enzymatic recombinase amplification (aERA) with CRISPR/Cas12a was developed. Key parameters were optimized. Analytical specificity was tested against common non-target shrimp pathogens. Sensitivity was determined using plasmid standards. Diagnostic performance was evaluated on 57 samples from farm submissions and a proficiency-testing programme. qPCR was used for comparison. Results Optimal conditions were determined to be 1:120 primer ratio, 1 µM Cas12a, 4:1 Cas12a-to-crRNA, and 46 °C. No cross-reactivity was observed in the tested panel. The approximate plasmid-based LoD was determined to be 10 copies/µL (24/25 technical replicates, 96%, exact 95% CI 79.7–99.9%); 25/25 replicates were positive at 50 copies/µL. Because the lower CI bound was below 95%, a ≥95% detection probability was not established with high precision. The readout was completed within 15 min. Agreement with qPCR was observed for OAERA-Cas12a: 9/9 positive (100%, 95% CI 66.4-100%), 48/48 negative (100%, 95% CI 92.6-100%), and 57/57 overall (100%, 95% CI 93.7-100%). Conclusions OAERA-Cas12a enables laboratory-based WSSV detection in a simple one-pot format. These results provide a basis for further development toward field deployment and point-of-care testing. Independent validation remains necessary to establish field reliability, point-of-care performance, and suitability for routine surveillance.