The Application of Digital PCR as a Reference Measurement Procedure to Support the Accuracy of Quality Assurance for Infectious Disease Molecular Diagnostic Testing

Published

December 26, 2024

Author(s)

Samreen Falak, Denise O'Sullivan, Megan Cleveland, Simon Cowen, Eloise Busby, Alison Devonshire, Esmeralda Valiente, Gerwyn Jones, Martin Kammel, Mojca Milavec, Laura Vierbaum, Ingo Schellenberg, Heinz Zeichhardt, Andreas Kummrow, Peter Vallone, Rainer Macdonald, Jim Huggett

Abstract

Background Nucleic Acid Amplification Tests (NAATs) are used to diagnose many infectious diseases. They are typically sensitive and specific and can be quickly developed and adapted. It is more challenging to develop standards to ensure NAATs perform within specification; reference materials take time to develop and reference measurement procedures (RMP) have, until now, not typically existed. Here, we investigated digital PCR RMP delivery of traceability for NAAT test quality assurance evaluation during 2020-2021. Methods Three National Metrology Laboratories (NMIs) applied RMPs to estimate the RNA copy number/mL in 36 independent SARS-CoV-2 External Quality Assurance (EQA) materials. The values were combined to value assign the respective materials. 22 materials were used in six rounds of EQA over 17 months for laboratories performing COVID-19 testing. Quantitative results from three NMIs were compared with EQA reference values. Results The agreement between the three NMIs showed less than twofold difference between laboratories. 22 of these materials were used for EQA with RMP RNA reference value. Participating laboratory RT-qPCR values estimation of viral RNA quantity showed good median agreement with RT-dPCR reference value; however, differences were generally between 10-50 fold between laboratories. Conclusion This work builds on earlier studies by illustrating how RT-dPCR can provide reference values for whole pathogen materials for NAAT quality assurance. RT-dPCR values guided EQA selection and provided EQA participants with results traceability to RNA copies from the RMP. This approach could be used to standardize quality assurance for NAATs where established reference materials are not available, such as in epidemics.

Citation

Clinical Chemistry

Pub Type

Journals

Download Paper

https://doi.org/10.1093/clinchem/hvae187

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Keywords

digital PCR, dPCR, SARS-Cov-2, covid-19, EQA, virus

Citation

Falak, S. , O'Sullivan, D. , Cleveland, M. , Cowen, S. , Busby, E. , Devonshire, A. , Valiente, E. , Jones, G. , Kammel, M. , Milavec, M. , Vierbaum, L. , Schellenberg, I. , Zeichhardt, H. , Kummrow, A. , Vallone, P. , Macdonald, R. and Huggett, J. (2024), The Application of Digital PCR as a Reference Measurement Procedure to Support the Accuracy of Quality Assurance for Infectious Disease Molecular Diagnostic Testing, Clinical Chemistry, [online], https://doi.org/10.1093/clinchem/hvae187, https://tsapps.nist.gov/publication/get_pdf.cfm?pub_id=957517 (Accessed October 3, 2025)