Inter-laboratory assessment of different digital PCR platforms for quantification of human cytomegalovirus DNA

• DOI: 10.1007/s00216-017-0206-0
• Publication Date: 2017-01-26T09:41:10Z
• Authors: Jernej Pavšič; Alison Devonshire; Andrej Blejec; Carole A. Foy; Fran Van Heuverswyn; Gerwyn M. Jones; Heinz Schimmel; Jana Žel; Jim F. Huggett; Nicholas Redshaw; Maria Karczmarczyk; Erkan Mozioğlu; Sema Akyürek; Müslüm Akgöz; Mojca Milavec
• Keywords: Laboratory Proficiency Testing; 0303 health sciences; digital PCR; virus reference materials; DNA quantification; Cytomegalovirus; Reproducibility of Results; Real-Time Polymerase Chain Reaction; Biochemistry; Analytical Chemistry; 3. Good health; 03 medical and health sciences; info:eu-repo/classification/udc/578; human cytomegalovirus; Cytomegalovirus Infections; DNA, Viral; Humans; inter-laboratory assessment; digital PCR, DNA quantification, inter-laboratory assessment, human cytomegalovirus, virus reference materials; Research Paper

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.