Rapid detection of African swine fever virus using Cas12a-based portable paper diagnostics

African swine fever virus (ASFV) is a dsDNA virus responsible for a severe, highly contagious, and lethal disease affecting both domestic and wild pigs. ASFV has brought enormous economic loss to a number of countries, and effective vaccine and therapy are still lacking. Therefore, a rapid, sensitive, and field-deployable detection of ASFV is important for disease surveillance and control. Herein, we developed a Cas12a-mediated portable paper assay to rapidly and precisely detect ASFV. We identified a robust set of crRNAs that recognized the highly conserved region of essential ASFV genes. The Cas12a-mediated detection assay showed low tolerance for mismatch mutations, and no cross-reactivity against other common swine pathogens. We further developed a paper-based assay to allow instrument-free detection of ASFV. Specifically, we applied gold nanoparticle–antibody conjugate to engineer homemade strips and combined it with Cas12a-mediated ASFV detection. This portable paper, instrument-free diagnostics, faithfully detected ASFV in swine samples, showing comparable sensitivity to the traditionally instrument-dependent qPCR method. Taking together, we developed a highly sensitive, instant, and economic Cas12a-mediated paper diagnostics of ASFV, with a great application potential for monitoring ASFV in the field.