Rapid Visual Detection of Cyprinid Herpesvirus 2 Based on One-Tube RPA-CRISPR/Cas12a System

Cyprinid herpesvirus 2 (CyHV-2) is a highly lethal pathogen responsible for substantial economic losses in farmed cyprinids worldwide. However, rapid and field-deployable diagnostic tools remain limited. Here, we developed a one-tube recombinase polymerase amplification (RPA)-CRISPR/Cas12a-based fluorescence assay for the visual detection of CyHV-2. The system integrates RPA pre-amplification targeting the highly conserved helicase gene hphS, Cas12a-mediated specific recognition and collateral cleavage of a fluorophore-quencher-labeled ssDNA reporter, and visual readout under a portable blue light transilluminator. After optimizing the concentrations of primers, crRNA, Cas12a and the reaction time, the detection limit of this method reached 1 copy/μL, and it showed no cross-reaction with other common aquatic pathogens. Clinical evaluation using 100 tissue samples demonstrated 100% concordance with quantitative real-time PCR (qPCR). This method does not require expensive equipment and can be completed within approximately one hour. This RPA-CRISPR/Cas12a platform offers a rapid and specific solution for on-site CyHV-2 surveillance, especially suitable for aquaculture environments with limited resources, and is expected to have broader applications in the detection of aquatic pathogens.