# Development of a two-tube multiplex real-time fluorescent PCR for the simultaneous differentiation of the mpox virus clades and the A.1, B.1 and C.1 lineages within clade IIb

## TL;DR

Scientists developed a new PCR test to quickly and accurately identify different types of mpox virus, including newly emerging lineages.

## Contribution

A two-tube multiplex real-time PCR assay with LNA probes for simultaneous differentiation of MPXV clades and IIb lineages.

## Key findings

- The assay showed high sensitivity (33–69 copies/reaction) and specificity with low variation (CV < 5%).
- Clinical validation showed 100% concordance for clade II differentiation and 97.6% for lineage differentiation.
- The method enables efficient screening of diverse clinical samples and addresses false negatives in clade Ib detection.

## Abstract

New clades and lineages emerged with the globally prevalent of Mpox virus (MPXV), accompanied by changing clinical symptoms, pathogenesis and transmission dynamics in associated with specific clades and lineages.

Here, we developed a two tube multiplex real-time fluorescent quantitative PCR (mrt-qPCR) assay for simultaneous differentiation of MPXV clades Ia, Ib, II, and innovative binding lock nucleic acid (LNA) probes to detect A.1, B.1 and C.1 lineages within the clade IIb.

The assay demonstrated high sensitivity (33–69 copies/reaction) and specificity with expected linearity and stability. The intra-assay and intre-assay coefficients of variations (CV) were below the acceptable threshold of 5%, and the mrt-qPCR method has good stability and reproducibility. Clinical validation using 109 qPCR positive, 1 clade IIb B.1 virus strain and 15 negative specimens revealed 100% concordance for the differentiation of the three clade II and 97.60% for the differentiation the three lineages. The two tube multi-test system streamlined workflows, enabling efficient screening of diverse clinical samples (swabs from skin lessions, oropharynx and rectum, saliva and plasma).

We have established a two-tube multiplex qPCR method for detecting different clades and lineages of the MPXV. This method addresses the issue of false-negative detection of MPXV clade Ib caused by gene fragment deletion, and has also enabled the development of a rapid detection approach for the predominantly circulating clade IIb (including lineages A.1, B.1, and C.1). This cost-effective assay provides an important tool for accurate diagnosis, typing and epidemiological surveillance of MPXV.