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

• Published in: Cell reports methods Vol. 3; no. 10; p. 100620
• Main Authors: Zhao, Fei, Hu, Yamei, Fan, Zhangling, Huang, Baoying, Wei, Liang, Xie, Yu, Huang, Yu, Mei, Shan, Wang, Liming, Wang, Lingwa, Ai, Bin, Fang, Jugao, Liang, Chen, Xu, Fengwen, Tan, Wenjie, Guo, Fei
• Format: Journal Article
• Language: English
• Published: United States Elsevier Inc 23.10.2023; Elsevier
• Subjects: AsCas12a; CP: Biotechnology; CP: Microbiology; CRISPR; CRISPR-Cas Systems - genetics; DNA, Viral - genetics; Humans; Monkeypox virus; Mpox (monkeypox); mpox detection; Nucleotidyltransferases; one-tube; Orthopoxvirus; Recombinases - genetics; RNA, Guide, CRISPR-Cas Systems; RPA; CRISPR; one-tube; RPA; mpox detection; AsCas12a; CP: Microbiology; CP: Biotechnology
• ISSN: 2667-2375; 2667-2375
• DOI: 10.1016/j.crmeth.2023.100620

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. [Display omitted] •An RPA-Cas12a system is designed for detection of orthopox and mpox viruses•The RPA-Cas12a detection system is highly sensitive, with an LOD of 1 copy DNA•The one-tube RPA-Cas12a system is able to detect mpox virus within 30 min 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. Zhao et al. present an RPA-coupled CRISPR-Cas12a detection assay for the efficient and simultaneous detection of mpox virus and orthopoxvirus infections. The work also generates a rapid and sensitive assay for the detection of mpox in a tube and holds the promise of providing point-of-care diagnosis for the viral infection.