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.
Highlights
Nucleic acid amplification-based tests offer an important method for rapid and reliable diagnosis of infectious diseases
Clinical laboratories often use different Quantitative polymerase chain reaction (qPCR) platforms, different commercial or in-house calibration materials, and different PCR components, which can in turn result in significant variability of the reported quantitative and qualitative data between laboratories [2, 4, 5]
The mean DNA copy number concentration of the gDNA test material (±expanded standard error; k = 2.78) was estimated at 979 (±59) cp/μL (ESM, Fig. S2), and the gDNA units H1–H5 were homogenous in terms of the DNA copy number concentration (p > 0.41; analysis of variance (ANOVA) 95% confidence level)
Summary
Nucleic acid amplification-based tests offer an important method for rapid and reliable diagnosis of infectious diseases. Suitable reference materials with assigned values that are traceable to the International System of Units (SI units) or to other internationally accepted standards (e.g. international units; IU) would facilitate accurate and reproducible characterisation of reference materials at a lower level in the calibration hierarchy and/or of calibrators produced by different manufacturers [6, 7]. This should, in turn, increase agreement between end-user quantitative measurements and improve assessment of the analytical performance characteristics of quantitative and qualitative nucleic acid amplification-based methods [4].
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