Ultrasensitive and Visual Detection of Pseudorabies Virus based on CRISPR-Cas12b

Aujeszky's disease (AD) is an acute infectious disease that infects pigs or other animals, resulting in significant economic losses and posing a threat to human health. Reliable and rapid detection methods are essential for the prevention and control of AD. In this study, a platform based on recombinase aided amplification (RAA) and CRISPR-Cas12b has been established, optimized, and evaluated for the rapid detection of Pseudorabies Virus (PRV). To enhance the usability and simplicity of the assay, we have developed PRV RAA-Cas12b methods for fluorescence-based detection and visualization. There was no cross-reaction with PRV Bartha-K61 strain or other common swine infectious viruses. The analytical sensitivity of the assay was determined to be 15 copies/µL by real-time fluorescence read-out with a 95% probability. The limit of detection (LOD) for the visual PRV RAA-Cas12b assay was 140 copies/µL with a 95% probability. Thirty-one clinical tissue samples were detected and compared with PRV qPCR to evaluate the diagnostic performance of the method. The diagnostic coincidence rate of the two methods was 100 %. Taking together, we established a rapid and convenient RAA-Cas12b method for the detection of wild-type PRV. This method holds a broad application prospect in the point-of-care testing (POCT).