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Critical Factors Affecting Digital PCR Quantification Accuracy in HBV DNA Reference Material for In Vitro

Ning Ma1,2,3,4, Manyu Li1,2,3,4, Xiaotian Hao1,2,3,4

  • 1Division I of In Vitro Diagnostics for Infectious Diseases, Institute for In Vitro Diagnostics Control, National Institutes for Food and Drug Control, Beijing 100050, China.

Analytical Chemistry
|October 9, 2025
PubMed
Summary

Digital PCR offers precise nucleic acid quantification for reference materials. Nucleic acid extraction kits significantly impact accuracy, more than different digital PCR platforms, highlighting the need for standardized protocols.

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Area of Science:

  • Molecular Biology
  • Biotechnology
  • Clinical Diagnostics

Background:

  • Digital PCR (dPCR) is crucial for absolute nucleic acid quantification.
  • Its application in reference material characterization, including in vitro diagnostic standards, is widespread.
  • Systematic evaluation of dPCR platform and extraction kit impacts on quantification is lacking.

Purpose of the Study:

  • To establish a specific dPCR assay for Hepatitis B Virus (HBV).
  • To systematically evaluate the influence of dPCR platforms, primer-probe sets, and extraction kits on reference material quantification.
  • To provide insights for standardizing dPCR protocols in reference material quantification.

Main Methods:

  • Development of a highly specific and reproducible dPCR assay for HBV.
  • Comparative analysis of quantification results across different commercial dPCR platforms.
  • Assessment of primer-probe set and nucleic acid extraction kit variability.
  • Calculation of interplatform and inter-kit coefficients of variation (CV).

Main Results:

  • Consistent quantitative measurements were observed across various dPCR platforms (mean interplatform CV: 9.05%).
  • Nucleic acid extraction efficiency showed the most significant impact on quantification accuracy (mean CV across kits: 76.66%).
  • Primer-probe design also contributed substantially to measurement uncertainty.

Conclusions:

  • While dPCR platforms show consistency, nucleic acid extraction kits introduce significant variability in reference material quantification.
  • Standardization of nucleic acid extraction protocols is critical for accurate dPCR-based reference material characterization.
  • These findings are essential for improving the reliability and comparability of dPCR measurements.