Digital PCR as a potential reference measurement procedure to support monkeypox virus/Orthopoxvirus external quality assessment schemes.

Since May 2022, a wave of several thousand cases of monkeypox Virus (MPXV) infections has been reported across Europe. In the beginning of this outbreak, reference material (RM) and reference measurement procedure (RMP) were lacking. We developed a measurement procedure using digital PCR (dPCR) for the absolute quantification of the viral genome of MPXV. This study investigated dPCR as potential reference method for MPXV quantification and evaluated performance in the External quality assessment (EQA) and clinical samples. This included DNA extraction performance using two commercial kits, MPXV target assays performance in duplex format and assay characteristics to differentiate the MPXV to Orthopoxvirus (OPXV). The analytical performance and validation on different dPCR platforms were examined. The developed dPCR assay was applied and supported extensive EQA schemes for mokeypox/Orthopox virus genome detection program and assigned reference values to the corresponding EQA materials within the range of 103 copies/mL to 107 copies/mL. The candidate reference method was applied to clinical MPXV patients' samples. The estimated viral DNA quantity in clinical samples using quantitative real time PCR (qPCR) showed good correlation and agreement with dPCR reference value. Thus, the dPCR-based method enabled reliable value assignment of MPXV reference materials. This method offers a calibration-free alternative for labs without access to certified standards. Our findings suggest that dPCR could be used for reference measurement value assignment of MPXV reference materials to support calibration of MPXV viral load testing in molecular diagnostic testing laboratories.