On-site detection and differentiation of African swine fever virus variants using an orthogonal CRISPR-Cas12b/Cas13a-based assay
Zhe Wang, Yu Wang, Ying Zhang, Guosong Qin, Wenbo Sun, Aiping Wang, Yanfang Wang, Gaiping Zhang, Zhao Jianguo, Gaiping Zhang, Zhao Jianguo
Abstract
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.