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

Quantitative PCR (qPCR) is an important tool in pathogen detection; however, the use of different qPCR components, calibration materials and DNA extraction methods reduces the comparability between clinics, which could result in false diagnosis and discrepancies in patient care. The establishment of a metrological framework for nucleic-acid tests is expected to improve the degree of standardisation of pathogen detection and quantification methods applied in a 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) allows quantification of nucleic acids and has already been used for a myriad of applications, including pathogen quantification. Although dPCR has the potential to provide robust and accurate quantification of nucleic acids, further assessments on its actual performance characteristics should be collected before it can be implemented in a metrological framework and to allow an adequate estimation of the measurement uncertainty. Here, high repeatability and reproducibility of dPCR for quantification of DNA from human cytomegalovirus were demonstrated. Using extracted DNA and whole-virus material, each of five dPCR platforms from four laboratories demonstrated high intermediate precision between three consecutive experiments. Furthermore, discrepancies in estimated mean DNA copy-number concentrations between different laboratories were less than two-fold, with DNA extraction recognised as the main source of variability. Our results demonstrate dPCR-based methods can be very repeatable and reproducible for quantification of viral DNA, and should be considered as potent reference method candidates for implementation in a metrological framework.

PAVSIC Jernej;  DEVONSHIRE A.S.;  BLEJEC Andrej;  FOY C.A.;  VAN HEUVERSWYN Fran;  JONES Gerwyn;  SCHIMMEL Heinz;  ZEL Jana;  HUGGETT Jim;  REDSHAW Nicholas;  KARCZMARCZYK Maria;  MOZIOĞLU Erkan;  AKYÜREK Sema;  AKGOZ M;  MILAVEC Mojca; 

2017-04-28

SPRINGER HEIDELBERG

JRC102933

1618-2642,   

https://publications.jrc.ec.europa.eu/repository/handle/JRC102933,   

10.1007/s00216-017-0206-0,