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

Fei Zhao; Yamei Hu; Zhangling Fan; Baoying Huang; Liang Wei; Yu Xie; Yu Huang; Shan Mei; Liming Wang; Lingwa Wang; Bin Ai; Jugao Fang; Chen Liang; Fengwen Xu; Wenjie Tan; Fei Guo

doi:10.1016/j.crmeth.2023.100620

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

Cell Rep Methods. 2023 Oct 23;3(10):100620. doi: 10.1016/j.crmeth.2023.100620. Epub 2023 Oct 16.

Authors

Fei Zhao¹, Yamei Hu¹, Zhangling Fan¹, Baoying Huang², Liang Wei¹, Yu Xie¹, Yu Huang¹, Shan Mei¹, Liming Wang³, Lingwa Wang⁴, Bin Ai³, Jugao Fang⁴, Chen Liang⁵, Fengwen Xu⁶, Wenjie Tan⁷, Fei Guo⁸

Affiliations

¹ NHC Key Laboratory of Systems Biology of Pathogens, Institute of Pathogen Biology and Center for AIDS Research, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100730, P.R. China.
² NHC Key Laboratory of Biosafety, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, P.R. China.
³ Department of Medical Oncology, Beijing Hospital, National Center of Gerontology, Institute of Geriatric Medicine, Chinese Academy of Medical Sciences, Beijing 100730, P.R. China.
⁴ Department of Otolaryngology Head and Neck Surgery, Beijing Tongren Hospital, Capital Medical University, Beijing 100730, P.R. China.
⁵ Lady Davis Institute, Jewish General Hospital, McGill University, Montreal, QC H3T 1E2, Canada.
⁶ NHC Key Laboratory of Systems Biology of Pathogens, Institute of Pathogen Biology and Center for AIDS Research, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100730, P.R. China. Electronic address: xanadufw@aliyun.com.
⁷ NHC Key Laboratory of Biosafety, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 102206, P.R. China. Electronic address: tanwj@ivdc.chinacdc.cn.
⁸ NHC Key Laboratory of Systems Biology of Pathogens, Institute of Pathogen Biology and Center for AIDS Research, Chinese Academy of Medical Sciences & Peking Union Medical College, Beijing 100730, P.R. China. Electronic address: guofei@ipb.pumc.edu.cn.

Abstract

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.

Keywords: AsCas12a; CP: Biotechnology; CP: Microbiology; CRISPR; RPA; mpox detection; one-tube.

MeSH terms

CRISPR-Cas Systems / genetics
DNA, Viral / genetics
Humans
Monkeypox virus
Mpox (monkeypox)*
Nucleotidyltransferases
Orthopoxvirus*
RNA, Guide, CRISPR-Cas Systems
Recombinases / genetics

Substances

Recombinases
DNA, Viral
Nucleotidyltransferases
RNA, Guide, CRISPR-Cas Systems