Development of a Novel Loop-Mediated Isothermal Amplification Method for the Rapid Detection of Monkeypox Virus Infections

Chao Yu; Lulu Zuo; Jing Miao; Lingjing Mao; Benjamin Selekon; Ella Gonofio; Emmanuel Nakoune; Nicolas Berthet; Gary Wong

doi:10.3390/v15010084

Development of a Novel Loop-Mediated Isothermal Amplification Method for the Rapid Detection of Monkeypox Virus Infections

Viruses. 2022 Dec 28;15(1):84. doi: 10.3390/v15010084.

Authors

Chao Yu¹, Lulu Zuo^{1

2}, Jing Miao^{2

3}, Lingjing Mao^{2

3}, Benjamin Selekon⁴, Ella Gonofio⁴, Emmanuel Nakoune⁴, Nicolas Berthet^{3

5}, Gary Wong¹

Affiliations

¹ Viral Hemorrhagic Fevers Research Unit, CAS Key Laboratory of Molecular Virology and Immunology, Institut Pasteur of Shanghai, Chinese Academy of Sciences, Shanghai 200031, China.
² University of Chinese Academy of Sciences, Beijing 100049, China.
³ Centre for Microbes, Development, and Health, Institut Pasteur of Shanghai, Chinese Academy of Sciences, Unit of Discovery and Molecular Characterization of Pathogens, Shanghai 200031, China.
⁴ Laboratory of Arboviruses, Viral Hemorrhagic Fevers, Emerging viruses and Zoonoses, Institut Pasteur of Bangui, Bangui P.O. Box 923, Central African Republic.
⁵ Institut Pasteur, Unité Environnement et Risque Infectieux, Cellule d'Intervention Biologique d'Urgence, 75724 Paris, France.

Abstract

A recent outbreak of monkeypox virus (mpox) has prompted researchers to explore diagnostics as a means of impeding transmission and further spread. Rapid, sensitive, and specific methods are crucial for accurately diagnosing mpox infections. Here, we developed a loop-mediated isothermal amplification (LAMP) assay for the specific detection of mpox. The primer sets were designed to target regions in and around the N4R gene, and results showed a detection limit of 2 × 10⁰ DNA copies, which is comparable to the gold-standard qPCR method currently used to detect mpox. Particularly, the assay provides results visible to the naked eye within 30 min. This test specifically detects mpox DNA with no cross-reactivity to related DNA viruses including Varicella Zoster Virus (VZV), Hepatitis B virus (HBV), Vaccinia virus (VACV), Herpes simplex virus-1 (HSV-1), Herpes simplex virus-2 (HSV-2), Human papillomavirus-16 (HPV-16) and Human papillomavirus-18 (HPV-18). Furthermore, the LAMP assay has been evaluated using clinical samples from laboratory-confirmed mpox patients and found to be consistent with the qPCR results. Our results show that this single-tube LAMP method can contribute to diagnosis of suspected mpox infections in the field and clinic, especially in regions with limited laboratory resources.

Keywords: isothermal amplification; loop-mediated isothermal amplification; molecular diagnosis; monkeypox virus; visual detection.

Publication types

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

MeSH terms

DNA, Viral / analysis
DNA, Viral / genetics
Herpesvirus 2, Human / genetics
Herpesvirus 3, Human / genetics
Humans
Monkeypox virus* / genetics
Mpox (monkeypox)* / diagnosis
Nucleic Acid Amplification Techniques / methods
Sensitivity and Specificity

Substances

DNA, Viral

Supplementary concepts

LAMP assay

Grants and funding

This project was supported by the Ministry of Science and Technology of China (Grant No. 2021YFC0863400, 2022YFE0114700); the Alliance of International Scientific Organizations (Grant No. ANSO-CR-SP-2020-02); the Shanghai Municipal Science and Technology Major Project (Grant No. 2019SHZDZX02); G4 funding from Institut Pasteur, Fondation Merieux, and Chinese Academy of Sciences to G.W.; and the International Affairs Department of the Institut Pasteur of Paris.