Development and interlaboratory validation of a linearized plasmid DNA certified reference material by single molecule direct counting and digital PCR.

Digital PCR (dPCR) technology is widely utilized for various applications, including the quantification of gene mutations and copy number variations. Certified reference materials (CRMs) play a critical role in improving the comparability of dPCR results, establishing SI-traceable copy number concentration values for dPCR calibration remains a key challenge due to the limited availability of CRMs value-assigned by higher-order, independent methods. To tackle this issue, a linearized plasmid DNA reference material (RM) was developed and rigorously characterized through an interlaboratory comparison involving three national measurement institutes (NMIs) from China (NIM), South Korea (KRISS), and Japan (NMIJ). The copy number concentration of the high-concentration RM was determined using two orthogonal methods: a dPCR-independent single molecule direct counting method and dPCR. Its homogeneity and stability were confirmed over the study duration. The equivalence of results among the NMIs was assessed using the En score, all En values were <1 indicating strong agreement and consistency with the candidate reference value within the expanded uncertainty. Additionally, a secondary CRM was generated by gravimetrically diluting the high-concentration RM to a nominal concentration of 10 copies/μL. This secondary CRM was used to evaluate four distinct dPCR platforms, revealing notable discrepancies in measurement results of up to 10.5 % among the platforms, potentially leading to substantial overestimation in nucleic acid quantification. These results emphasize the necessity of utilizing CRMs with SI-traceable certified values to validate dPCR quantification results and ensure comparability across different measurement systems.