Detection of pseudorabies virus with a real-time recombinase-aided amplification assay

Pseudorabies (PR) is an acute infectious disease of pigs caused by pseudorabies virus (PRV), which has caused great economic losses to the pig industry worldwide. Reliable and timely diagnose is crucial for the surveillance, control and eradication of PR. Here, a real-time fluorescent recombinase-aided amplification (real-time RAA) assay was established to detect PRV. Primers and probes were designed based on the conserved regions of the PRV gE gene. The assay was specific for the detection of wild-type PRV, showing no cross-reactivity with other important porcine viruses (including PRV gE-deleted vaccine strains). Analytical sensitivity of the assay was three 50% tissue culture infectious doses (TCID₅₀ ) of PRV DNA per reaction with 95% reliability, which is comparable to that of a PRV-specific real-time PCR (qPCR) assay. In diagnosis of 206 clinical tissue samples, the diagnose accordance rate between the real-time RAA assay and qPCR assay was 97.57% (201/206). Interestingly, the amplified products of real-time RAA could be visualized under a portable blue light instrument, making it possible for the rapid detection of PRV in resource-limited settings and on-site screening. Therefore, our developed real-time RAA assay is a diagnostic method for the rapid detection of PRV in the field.