Wang, X.; Li, H.; Guo, R.; Yang, Y.; Niu, C.; Zhou, S.; Gao, H.; Jin, X.; Wang, S.; Du, M.; Cheng, X.; Zhu, L.; Dong, L.

The accurate detection of monkeypox virus (MPXV) is crucial for effective viral diagnosis and epidemic prevention. Currently, the field lacks standardized reference methods for MPXV quantification, as well as reliable pseudovirus reference materials (RMs) to ensure quality control throughout the detection process. To address this, we developed a droplet digital PCR (ddPCR) reference measurement procedure (RMP) for precise quantification of MPXV genes B6R, which demonstrates excellent performance with a wide dynamic range (11-1.24x10^4 copies/L), strong linearity (R2=0.9984), and a low limit of quantitation (11 copies/L, CV[&le;]25%). Repeatability tests confirmed high precision, with inter-laboratory CVs <10% across nine labs using different dPCR platforms. recovery efficiencies for B6R and F3L were ~69%, with uncertainties incorporated into final measurements. The homogeneity assessment showed good results (CV=2.94%), and stability studies confirmed the stability of the sample during -70 and short-term storage (4 /-20 ). Mandel\'s statistical data shows that the reproducibility between laboratories is consistent. This validated ddPCR RMP provides metrological traceability, ensuring reliable and comparable results for MPXV diagnosis and supporting public health efforts.