On-site detection and differentiation of African swine fever virus variants using an orthogonal CRISPR-Cas12b/Cas13a-based assay

The African swine fever virus (ASFV) and its variants have induced substantial economic losses in China, prompting a critical need for efficient detection methods. Several PCR-based methods have been developed to discriminate between wild-type ASFV and gene-deleted variants. However, the requirement for sophisticated equipment and skilled operators limits their use in field settings. Here, we developed a CRISPR-Cas12b/Cas13a-based detection assay that can identify ASFV variants with minimal equipment requirements and a short turnaround time. The assay utilizes the distinct DNA/RNA collateral cleavage preferences of Cas12b/Cas13a to detect two amplified targets from multiplex recombinase polymerase amplification (RPA) in a single tube, and the results can be visualized through fluorescent or lateral-flow readouts. When tested with clinical samples in field settings, our assay successfully detected all ASFV-positive samples in less than 60 min. This assay provides a rapid on-site surveillance tool for detecting ASFV and its emerging variants.