Rapid and sensitive one-tube detection of mpox virus using RPA-coupled CRISPR-Cas12 assay

Summary: Mpox is caused by a zoonotic virus belonging to the Orthopoxvirus genus and the Poxviridae family. In this study, we develop a recombinase polymerase amplification (RPA)-coupled CRISPR-Cas12a detection assay for the mpox virus. We design and test a series of CRISPR-derived RNAs(crRNAs) targeting the conserved D6R and E9L genes for orthopoxvirus and the unique N3R and N4R genes for mpox viruses. D6R crRNA-1 exhibits the most robust activity in detecting orthopoxviruses, and N4R crRNA-2 is able to distinguish the mpox virus from other orthopoxviruses. The Cas12a/crRNA assay alone presents a detection limit of 108 copies of viral DNA, whereas coupling RPA increases the detection limit to 1–10 copies. The one-tube RPA-Cas12a assay can, therefore, detect viral DNA as low as 1 copy within 30 min and holds the promise of providing point-of-care detection for mpox viral infection. Motivation: The rise of mpox cases in many previously non-endemic countries in 2022 prompted the WHO to declare mpox as a public health emergency of international concern. Since it is difficult to detect mpox at the early stage of infection, a rapid and accurate diagnostic assay for the mpox virus is essential to control its spread and enable timely treatment. Here, we present an RPA-Cas12a ribonucleoprotein-based method for rapid and sensitive detection of mpox.