Development and interlaboratory validation of a linearized plasmid DNA certified reference material by single...

Area of Science:

• Biotechnology
• Metrology
• Molecular Biology

Background:

• Digital PCR (dPCR) is essential for gene mutation and copy number variation quantification.
• Establishing SI-traceable values for dPCR calibration is challenging due to limited certified reference materials (CRMs).
• Lack of CRMs hinders comparability of dPCR results.

Purpose of the Study:

• To develop and characterize a linearized plasmid DNA reference material (RM) for dPCR.
• To establish SI-traceable copy number concentration values for dPCR calibration.
• To assess the comparability of dPCR results using the developed RM.

Main Methods:

• Development of a linearized plasmid DNA reference material (RM).
• Characterization via interlaboratory comparison involving three national measurement institutes (NMIs).
• Orthogonal quantification using dPCR-independent single molecule counting and dPCR.
• Homogeneity and stability assessments.
• Gravimetric dilution to create a secondary CRM.
• Evaluation of four dPCR platforms using the secondary CRM.

Main Results:

• The developed RM demonstrated homogeneity and stability.
• Interlaboratory comparison showed strong agreement among NMIs (En < 1).
• Equivalence of results was established within expanded uncertainty.
• Secondary CRM evaluation revealed up to 10.5% discrepancies among dPCR platforms.
• Potential for significant overestimation in nucleic acid quantification was identified.

Conclusions:

• The developed reference material provides SI-traceable values for dPCR calibration.
• CRMs are essential for validating dPCR quantification and ensuring inter-platform comparability.
• Standardized CRMs are needed to address discrepancies in nucleic acid quantification across dPCR systems.