CRISPR-Cas12-based field-deployable system for rapid detection of synthetic DNA sequence of the monkeypox virus genome

Qun Chen; Ijaz Gul; Changyue Liu; Zhengyang Lei; Xingyu Li; Muhammad A Raheem; Qian He; Zhang Haihui; Edwin Leeansyah; Can Y Zhang; Vijay Pandey; Ke Du; Peiwu Qin

doi:10.1002/jmv.28385

CRISPR-Cas12-based field-deployable system for rapid detection of synthetic DNA sequence of the monkeypox virus genome

J Med Virol. 2023 Jan;95(1):e28385. doi: 10.1002/jmv.28385.

Authors

Qun Chen^{1

2}, Ijaz Gul^{1

2}, Changyue Liu^{1

2}, Zhengyang Lei^{1

2}, Xingyu Li³, Muhammad A Raheem^{1

2}, Qian He^{1

2}, Zhang Haihui^{1

2}, Edwin Leeansyah^{1

2}, Can Y Zhang^{1

2}, Vijay Pandey^{1

2}, Ke Du⁴, Peiwu Qin^{1

2}

Affiliations

¹ Institute of Biopharmaceutical and Health Engineering, Tsinghua Shenzhen International Graduate School, Tsinghua University, Shenzhen, P. R. China.
² Tsinghua Shenzhen International Graduate School, Tsinghua-Berkeley Shenzhen Institute, Tsinghua University, Shenzhen, P. R. China.
³ Department of Hepatobiliary and Pancreatic Surgery II, The Third Xiangya Hospital, Central South University, Changsha, Hunan, P. R. China.
⁴ Department of Chemical and Environmental Engineering, University of California, Riverside, California, USA.

PMID: 36478250
DOI: 10.1002/jmv.28385

Abstract

The global outbreak of the monkeypox virus (MPXV) highlights the need for rapid and cost-effective MPXV detection tools to effectively monitor and control the monkeypox disease. Herein, we demonstrated a portable CRISPR-Cas-based system for naked-eye detection of MPXV. The system harnesses the high selectivity of CRISPR-Cas12 and the isothermal nucleic acid amplification potential of recombinase polymerase amplification. It can detect both the current circulating MPXV clade and the original clades. We reached a limit of detection (LoD) of 22.4 aM (13.5 copies/µl) using a microtiter plate reader, while the visual LoD of the system is 75 aM (45 copies/µl) in a two-step assay, which is further reduced to 25 aM (15 copies/µl) in a one-pot system. We compared our results with quantitative polymerase chain reaction and obtained satisfactory consistency. For clinical application, we demonstrated a sensitive and precise visual detection method with attomolar sensitivity and a sample-to-answer time of 35 min.

Keywords: CRISPR-Cas12a; detection; monkeypox endemic; monkeypox virus; recombinase polymerase amplification.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Base Sequence
CRISPR-Cas Systems
Humans
Monkeypox virus* / genetics
Mpox (monkeypox)* / diagnosis
Nucleic Acid Amplification Techniques / methods