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Title:Inter-laboratory assessment of different digital PCR platforms for quantification of human cytomegalovirus DNA
Authors:ID Pavšič, Jernej (Author)
ID Devonshire, Alison S. (Author)
ID Blejec, Andrej (Author)
ID Foy, Carole A. (Author)
ID Heuverswyn, Fran Van (Author)
ID Jones, Gerwyn M. (Author)
ID Schimmel, Heinz (Author)
ID Žel, Jana (Author)
ID Huggett, Jim F. (Author)
ID Redshaw, Nicholas (Author)
ID Karczmarczyk, Maria (Author)
ID Mozioglu, Erkan (Author)
ID Akyürek, Sema (Author)
ID Akgöz, Müslüm (Author)
ID Milavec, Mojca (Author)
Files:URL URL - Source URL, visit http://dx.doi.org/10.1007/s00216-017-0206-0
 
.pdf PDF - Presentation file, download (638,49 KB)
MD5: 315D9D64042B8F34F5B431556FF38B60
 
Language:English
Typology:1.01 - Original Scientific Article
Organization:Logo NIB - National Institute of Biology
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.
Keywords:digital PCR, DNA quantification, inter-laboratory assessment, human cytomegalovirus, virus reference materials
Publication status:Published
Publication version:Version of Record
Publication date:26.01.2017
Year of publishing:2017
Number of pages:str. 2601-2614
Numbering:Vol. 409, iss. 10
PID:20.500.12556/DiRROS-19697 New window
UDC:578
ISSN on article:1618-2642
DOI:10.1007/s00216-017-0206-0 New window
COBISS.SI-ID:4223055 New window
Publication date in DiRROS:24.07.2024
Views:400
Downloads:235
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Record is a part of a journal

Title:Analytical and bioanalytical chemistry
Shortened title:Anal. bioanal. chem.
Publisher:Springer
ISSN:1618-2642
COBISS.SI-ID:6966294 New window

Document is financed by a project

Funder:ARIS - Slovenian Research and Innovation Agency
Project number:P4-0165-2015
Name:Biotehnologija in sistemska biologija rastlin

Funder:EC - European Commission
Funding programme:EURAMET and the European Union
Name:EMRP project
Acronym:INFECT MET

Funder:ARIS - Slovenian Research and Innovation Agency
Project number:1000-13-0105

Funder:Other - Other funder or multiple funders
Funding programme:Metrology Institute of the Republic of Slovenia, with financial support from the European Regional Development Fund

Licences

License:CC BY 4.0, Creative Commons Attribution 4.0 International
Link:http://creativecommons.org/licenses/by/4.0/
Description:This is the standard Creative Commons license that gives others maximum freedom to do what they want with the work as long as they credit the author.

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