CRISPR/Cas12a-mediated ultrasensitive and on-site monkeypox viral testing

AbstractThe unexpected transmission of monkeypox virus (MPXV) from Central and West Africa to previously non-endemic locations is triggering a global panic. The ultrasensitive, rapid, and specific detection of MPXV is crucial for controlling its spreading, while such technology has rarely been reported. Herein, we proposed an MPXV assay combining recombinase-aided amplification (RAA) and CRISPR/Cas12a for the first time. This assay targeted MPXVF3Lgene and yielded a low detection limit (LOD) of 101copies/μL. Deriving from the high specificity nature of RAA and CRISPR/Cas12a, through rational optimizations of probes and conditions, this assay showed high selectivity that could distinguish MPXV from other orthopox viruses and current high-profile viruses. To facilitate on-site screening of potential MPXV carriers, a kit integrating lateral flow strips was developed, enabling naked-eye MPXV detection with a LOD of 104copies/μL. Our RAA-Cas12a-MPXV assay was able to detect MPXV without the need for sophisticated operation and expensive equipment. We envision that this RAA-Cas12a-MPXV assay can be deployed in emerging viral outbreaks for on-site surveillance of MPXV.For TOC only