Fig. 1.

Establishment of dPCR method and reference material for detecting MPXV. The selection of annealing temperature was displayed in (a), dPCR amplification was set at the following annealing temperature gradients: 54℃, 55℃, 56℃, 57℃, and 58℃. The optimization of primer and probe concentrations were showed in (b), the different primer concentrations (300 nM, 400 nM, 500 nM, 600 nM) and probe concentrations (200 nM, 300 nM, 400 nM) were contained in orthogonal test. c The linearity range of the established dPCR method. The concentration of templates range from 38 to 9590 copies/reaction. d Homogeneity test result: eleven units were used for the homogeneity test. F tests was employed to statistically analyze the difference from intra and inter-units, and no significant distinction was observed between them. e Six qPCR kits were evaluated by the reference material. The dotted line represents the value of reference material quantified by dPCR