CRISPR-based platform for rapid, sensitive and field-deployable detection of scale drop disease virus in Asian sea bass (Lates calcarifer)

Abstract

Scale drop disease virus (SDDV) is a major pathogen of Asian sea bass that has emerged in many countries across the Asia Pacific since 1992 and carries the potential to cause drastic economic losses to the aquaculture sector. The lack of an approved vaccine for SDDV necessitates timely prevention as the first line of defence against the disease, but current diagnostic platforms still face challenges that render them incompatible with field applications, particularly in resource-limited settings. Here, we developed a novel detection platform for SDDV based on a CRISPR-Cas12a-based nucleic acid detection technology combined with recombinase polymerase amplification (RPA-Cas12a). Using the viral adenosine triphosphatase (SDDV-ATPase) gene as a target, we achieved the detection limit of 40 copies per reaction and high specificity for SDDV. The coupling with fluorescence and lateral flow readouts enables naked-eye visualization and straightforward data interpretation requiring minimal scientific background. Compared with semi-nested PCR in field sample evaluation, our RPA-Cas12a assay is more sensitive and capable of detecting SDDV in asymptomatic fish. Importantly, the entire workflow can be carried out at a constant temperature of 37°C within an hour from start to finish, thus removing the need for an expensive thermal cycling apparatus and long turnaround times associated with PCR-based methods. Therefore, owing to its high accuracy, rapidity and user-friendliness, the developed RPA-Cas12a platform shows the potential for diagnosis of SDDV at point of need and could be a valuable tool to help protect fish farming communities from large-scale epidemics.