20.500.12556/DiRROS-21952 The application of digital PCR as a reference measurement procedure to support the accuracy of quality assurance for infectious disease molecular diagnostic testing BACKGROUND: Nucleic acid amplification tests (NAATs) assist in the diagnosis of numerous infectious diseases. They are typically sensitive and specific and can be quickly developed and adapted. Far more challenging is the development of standards to ensure NAATs are performing within specification; reference materials take time to develop and suitable reference measurement procedures (RMPs) have not been available. This study investigated digital PCR (dPCR) RMP delivery of traceability for NAAT external quality assessment (EQA). METHODS: Three National Metrology Institutes (NMIs) applied reverse transcription (RT)-dPCR as a candidate RMP to estimate the RNA quantity in 32 independent severe acute respiratory syndrome coronavirus 2 materials. The results were combined to value assign the respective materials: 21 materials were used in 6 rounds of EQA over 17 months for 61 laboratories for COVID-19 testing results compared with reference values. RESULTS: The agreement between the 3 NMIs showed <2-fold difference between laboratories. EQA laboratory reverse transcription quantitative PCR (RTqPCR) values estimation of viral RNA quantity showed good median agreement with RT-dPCR reference value; however, RT-qPCR differences were generally between 10- and 50-fold between laboratories. CONCLUSION: This work demonstrates how RT-dPCR can provide reference values for whole virus materials for NAAT quality assurance. RT-dPCR values guided EQA control material selection and provided EQA participants with traceability to RNA copy number delivered through the RMP. This approach can be used to support routine reference material use as well as to standardize quality assurance for NAATs where established reference materials are not available, such as in disease outbreaks. nucleic acid amplification tests dPCR reference measurement procedure external quality assessment SARS-CoV-2 testi s pomnoževanjem nukleinskih kislin dPCR referenčni merilni postopek zunanje ocenjevanje kakovosti SARS-CoV-2 true false true Angleški jezik Slovenski jezik Neznano 2025-04-14 08:14:09 2025-04-14 08:14:09 2025-10-24 03:50:14 0000-00-00 00:00:00 2025 0 0 Soavtorji: Denise M O’Sullivan, Megan H Cleveland, Simon Cowen, Eloise J Busby, Alison S Devonshire, Esmeralda Valiente, Gerwyn M Jones, Martin Kammel, Mojca Milavec, Laura Vierbaum, Ingo Schellenberg, Heinz Zeichhardt, Andreas Kummrow, Peter M Vallone, Rainer Macdonald, Jim F Huggett; str. 378–386 no. 3 Vol. 71 2025 0000-00-00 Zaloznikova Objavljeno NiDoloceno 0000-00-00 0000-00-00 2025-03-01 577.2 0009-9147 10.1093/clinchem/hvae187 229099011 https://academic.oup.com/clinchem/article/71/3/378/7933106 1 aed9b25f-18f7-11f0-963a-001a4af901a5 https://dirros.openscience.si/Dokument.php?lang=slv&id=31653 RAZ_Falak_Samreen_2025.pdf RAZ_Falak_Samreen_2025.pdf 1 8999D6E98A742338D0D8DED849A7FAFD f30bb426ba78dc281e6d9d329c99b12bc431d8b041a35c6a3c4fb1b11351061b 36fa2970-18f8-11f0-963a-001a4af901a5 https://dirros.openscience.si/Dokument.php?lang=slv&id=31654 hvae187_supplementary_data.docx hvae187_supplementary_data.docx Dopolnilne informacije 1 375D62AC1C889607681276A7467B013F 071ae4d0369acce51c0ac7878a9839eb5accf92448d3c2822a2b728a00d1d844 e457aab3-18f8-11f0-963a-001a4af901a5 https://dirros.openscience.si/Dokument.php?lang=slv&id=31656 https://doi.org/10.1093/clinchem/hvae187 0 4efc5c94-18f8-11f0-963a-001a4af901a5 https://dirros.openscience.si/Dokument.php?lang=slv&id=31655 Nacionalni inštitut za biologijo 0 0 0