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

Area of Science:

• Virology
• Molecular Diagnostics
• Biotechnology

Background:

• The 2022 monkeypox virus (MPXV) outbreak highlighted a lack of reference materials and procedures.
• Accurate quantification of MPXV is crucial for diagnostics and outbreak monitoring.

Purpose of the Study:

• To develop and validate a digital PCR (dPCR) method for absolute quantification of the MPXV genome.
• To evaluate dPCR as a reference measurement procedure (RMP) for MPXV.
• To assess dPCR performance in external quality assessment (EQA) schemes and clinical samples.

Main Methods:

• Development of a dPCR assay for absolute quantification of MPXV DNA.
• Evaluation of DNA extraction kits and duplex assay performance for MPXV and Orthopoxvirus (OPXV) differentiation.
• Validation of the dPCR assay on multiple platforms and application to EQA materials and clinical samples.

Main Results:

• The dPCR assay provided reliable quantification of MPXV across a wide range (103–107 copies/mL) in EQA materials.
• Good correlation and agreement were observed between dPCR and quantitative real-time PCR (qPCR) results for clinical samples.
• The dPCR method enabled accurate value assignment for MPXV reference materials.

Conclusions:

• Digital PCR serves as a robust reference method for MPXV quantification.
• The developed dPCR assay supports calibration-free MPXV viral load testing, crucial for diagnostic laboratories.
• This method enhances the reliability of MPXV diagnostics and monitoring during outbreaks.