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

Background: 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. Methods: 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. Results: 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). Conclusions: 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.