Development of two multiplex real-time PCR assays for simultaneous detection and differentiation of monkeypox virus IIa, IIb, and I clades and the B.1 lineage

Shuting Huo; Yuda Chen; Roujian Lu; Zhongxian Zhang; Gaoqian Zhang; Li Zhao; Yao Deng; Changcheng Wu; Wenjie Tan

doi:10.1016/j.bsheal.2022.10.005

Development of two multiplex real-time PCR assays for simultaneous detection and differentiation of monkeypox virus IIa, IIb, and I clades and the B.1 lineage

Biosaf Health. 2022 Dec;4(6):392-398. doi: 10.1016/j.bsheal.2022.10.005. Epub 2022 Nov 4.

Authors

Shuting Huo¹, Yuda Chen^{1

2}, Roujian Lu¹, Zhongxian Zhang^{1

2}, Gaoqian Zhang^{1

3}, Li Zhao¹, Yao Deng¹, Changcheng Wu¹, Wenjie Tan^{1

2

3}

Affiliations

¹ NHC Key Laboratory of Biosafety, National Institute for Viral Disease Control and Prevention, Chinese Center for Disease Control and Prevention, Beijing 100052, China.
² School of Public Health, Baotou Medical College, Baotou 014030, China.
³ Department of Microbiology, Basic Medical College, Inner Mongolia Medical University, Hohhot 010010, China.

Abstract

An ongoing multi-country outbreak of monkeypox was reported in May 2022 with several deaths, affecting 107 countries of all six World Health Organization (WHO) regions. The WHO has declared the current monkeypox outbreak to be a Public Health Emergency of International Concern. It is, thus, necessary to rapidly and accurately detect and distinguish different monkeypox virus (MPXV) clades. We designed primers and probes based on the alignment of 138 complete genomes of poxviruses. In Panel 1, we mixed one pair of primers and three probes to detect and differentiate the MPXV Western Africa (IIa, IIb clade) and Congo Basin (I clade) and other orthopoxviruses. In Panel 2, we mixed one pair of primers and two probes to detect the 2022 MPXV (B.1 lineage and its descendant lineages). In addition, we tested the specificity and sensitivity of the assay using real-time PCR. In Panel 1, the assay reproducibly identified various concentrations of two plasmids of the monkeypox virus, whereas other orthopoxviruses did not cross-react. In Panel 2, the probe annealed well to MPXV B.1 and showed the expected linearity. These two multiple real-time assays are inclusive and highly specific for identifying different clades of MPXV.

Keywords: Diagnostic accuracy; Monkeypox virus; Monkeypox virus B.1 lineage; Real-time PCR.