Development of a multiplex droplet digital PCR method for detection and differentiation of mpox virus clades

Background: The current outbreak of mpox has been declared a public health emergency of international concern by the World Health Organization. However, distinguishing symptoms of mpox virus (MPXV) infection from other orthopoxviruses is atypical, necessitating laboratory confirmatory tests to aid in clinical diagnosis. Therefore, rapid and accurate detection and differentiation of various clades of MPXV are imperative. Objective: A multiplex droplet digital PCR (ddPCR) method was developed to detect and differentiate various clades of MPXV with subsequent evaluation of its sensitivity and accessibility through the analysis of 17 clinical samples. Methods: Primers and probes for multiple ddPCR were designed by comparing multiple complete genomes of orthopoxviruses. Primer and probe concentrations, reaction conditions were tentatively optimized on the Biorad QX200 platform. Seventeen clinical samples of MPXV were detected and verified by Sanger sequencing. Results: The established ddPCR method could detect and differentiate MPXV, and the results were consistent with those of Sanger sequencing. Conclusion: Multiplex ddPCR could be used to detect and distinguish different clades of MPXV rapidly and accurately.