Development and validation of a CRISPR/Cas12a-based platform for rapid and sensitive detection of the large yellow croaker iridovirus

Abstract

Large yellow croaker iridovirus (LYCIV) poses a growing threat to large yellow croaker (Larimichthys crocea) aquaculture, requiring early diagnosis for effective prevention and control. However, current detection methods are time-consuming, labor-intensive, or require specialized equipment, hindering timely diagnosis. To address this concern, we developed a novel platform combining CRISPR/Cas12a and recombinase polymerase amplification (RPA) for rapid and sensitive LYCIV detection. Our strategy targeted the conserved ATPase gene, utilizing specific crRNA and RPA primers for selective detection. Additionally, we employed a lateral flow strip technique for intuitive visual assessment. The performance evaluation demonstrated outstanding specificity and sensitivity, with a limit of detection as low as 5 × 103 copies / reaction of iridovirus. Validation on spiked and clinical fish samples confirmed the accuracy through PCR analysis. Overall, our developed platform offers a convenient, on-site, and user-friendly tool for early diagnosis, prevention, and control of LYCIV-related diseases in large yellow croaker aquaculture.