title: Inter-laboratory assessment of different digital PCR platforms for quantification of human cytomegalovirus DNA.
abstract: Quantitative PCR (qPCR) is an important tool in pathogen detection. However, the use of different qPCR components, calibration materials and DNA extraction methods reduces comparability between laboratories, which can result in false diagnosis and discrepancies in patient care. The wider establishment of a metrological framework for nucleic acid tests could improve the degree of standardisation of pathogen detection and the quantification methods applied in the clinical context. To achieve this, accurate methods need to be developed and implemented as reference measurement procedures, and to facilitate characterisation of suitable certified reference materials. Digital PCR (dPCR) has already been used for pathogen quantification by analysing nucleic acids. Although dPCR has the potential to provide robust and accurate quantification of nucleic acids, further assessment of its actual performance characteristics is needed before it can be implemented in a metrological framework, and to allow adequate estimation of measurement uncertainties. Here, four laboratories demonstrated reproducibility (expanded measurement uncertainties below 15%) of dPCR for quantification of DNA from human cytomegalovirus, with no calibration to a common reference material. Using whole-virus material and extracted DNA, an intermediate precision (coefficients of variation below 25%) between three consecutive experiments was noted. Furthermore, discrepancies in estimated mean DNA copy number concentrations between laboratories were less than twofold, with DNA extraction as the main source of variability. These data demonstrate that dPCR offers a repeatable and reproducible method for quantification of viral DNA, and due to its satisfactory performance should be considered as candidate for reference methods for implementation in a metrological framework.
authors.affiliation: Department of Biotechnology and Systems Biology, National Institute of Biology, Večna pot 111, 1000, Ljubljana, Slovenia. jernej.pavsic@nib.si.|Jožef Stefan International Postgraduate School, Jamova 39, 1000, Ljubljana, Slovenia. jernej.pavsic@nib.si.
authors.forename: Jernej
authors.initials: J
authors.lastname: Pavšič
authors.affiliation: Molecular and Cell Biology, LGC Ltd., Queens Road, Teddington, Middlesex, TW11 0LY, UK.
authors.forename: Alison
authors.initials: A
authors.lastname: Devonshire
authors.affiliation: Department of Biotechnology and Systems Biology, National Institute of Biology, Večna pot 111, 1000, Ljubljana, Slovenia.
authors.forename: Andrej
authors.initials: A
authors.lastname: Blejec
authors.affiliation: Molecular and Cell Biology, LGC Ltd., Queens Road, Teddington, Middlesex, TW11 0LY, UK.
authors.forename: Carole A
authors.initials: CA
authors.lastname: Foy
authors.affiliation: European Commission, Joint Research Centre (JRC), Directorate F. Retieseweg 111, 2440, Geel, Belgium.
authors.forename: Fran
authors.initials: F
authors.lastname: Van Heuverswyn
authors.affiliation: Molecular and Cell Biology, LGC Ltd., Queens Road, Teddington, Middlesex, TW11 0LY, UK.
authors.forename: Gerwyn M
authors.initials: GM
authors.lastname: Jones
authors.affiliation: European Commission, Joint Research Centre (JRC), Directorate F. Retieseweg 111, 2440, Geel, Belgium.
authors.forename: Heinz
authors.initials: H
authors.lastname: Schimmel
authors.affiliation: Department of Biotechnology and Systems Biology, National Institute of Biology, Večna pot 111, 1000, Ljubljana, Slovenia.
authors.forename: Jana
authors.initials: J
authors.lastname: Žel
authors.affiliation: Molecular and Cell Biology, LGC Ltd., Queens Road, Teddington, Middlesex, TW11 0LY, UK.|School of Biosciences and Medicine, Faculty of Health and Medical Science, University of Surrey, Guildford, Surrey, GU2 7XH, UK.
authors.forename: Jim F
authors.initials: JF
authors.lastname: Huggett
authors.affiliation: Molecular and Cell Biology, LGC Ltd., Queens Road, Teddington, Middlesex, TW11 0LY, UK.
authors.forename: Nicholas
authors.initials: N
authors.lastname: Redshaw
authors.affiliation: European Commission, Joint Research Centre (JRC), Directorate F. Retieseweg 111, 2440, Geel, Belgium.
authors.forename: Maria
authors.initials: M
authors.lastname: Karczmarczyk
authors.affiliation: Bioanalysis Laboratory, National Metrology Institute of Turkey, (TUBITAK UME), PO Box 54, 41470, Gebze, Kocaeli, Turkey.
authors.forename: Erkan
authors.initials: E
authors.lastname: Mozioğlu
authors.affiliation: Bioanalysis Laboratory, National Metrology Institute of Turkey, (TUBITAK UME), PO Box 54, 41470, Gebze, Kocaeli, Turkey.
authors.forename: Sema
authors.initials: S
authors.lastname: Akyürek
authors.affiliation: Bioanalysis Laboratory, National Metrology Institute of Turkey, (TUBITAK UME), PO Box 54, 41470, Gebze, Kocaeli, Turkey.
authors.forename: Müslüm
authors.initials: M
authors.lastname: Akgöz
authors.affiliation: Department of Biotechnology and Systems Biology, National Institute of Biology, Večna pot 111, 1000, Ljubljana, Slovenia.
authors.forename: Mojca
authors.initials: M
authors.lastname: Milavec
mesh_terms: D003587:Cytomegalovirus / Q000235:genetics* / Q000302:isolation & purification; D003586:Cytomegalovirus Infections / Q000175:diagnosis* / Q000235:genetics / Q000821:virology; D004279:DNA, Viral / Q000032:analysis* / Q000235:genetics; D006801:Humans; D059021:Laboratory Proficiency Testing / Q000592:standards*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results
chemical_list: D004279:DNA, Viral
keywords: DNA quantification; Digital PCR; Human cytomegalovirus; Inter-laboratory assessment; Virus reference materials