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.JRC.F.6-Reference Material

Assessment of Digital PCR as a Primary Reference Measurement Procedure to Support Advances in Precision Medicine

Genetic testing for point mutations guides patient management in many cancers when analysing the DNA from tumour and, increasingly, liquid biopsies. Such measurements are going to be increasingly important in supporting the implementation of precision medicine that many predict will revolutionise clinical care. However, there are currently no reference measurement procedures available to aid harmonisation of existing testing and support implementation of newer, often more complex, tests into routine use. This study assessed the accuracy of digital PCR for copy number quantification of point mutations by evaluating potential sources of uncertainty influencing measurements of a frequently occurring mutation in colorectal cancer (KRAS G12D). Concentration values for samples containing single or mixed KRAS G12D and WT plasmid templates varied by <1.2-fold and <1.3-fold when measured using five assays differing in chemistry or with five commercial dPCR platforms. Orthogonal comparison with a chemical method (ICP-MS) via P quantification also demonstrated 1.2-fold agreement between techniques. dPCR showed robust quantification for templates of differing fragment sizes (186 – 4343 bp) and high intra- and inter-laboratory precision (%CV: 2-8% and 5-10% respectively). This work supports dPCR being more widely accepted as a SI traceable reference measurement procedure for value assignment of DNA reference materials of varying sizes in an aqueous (calibration) solution for copy-number concentration and allelic frequency measurements. Such high accuracy measurements using dPCR will be able to support the implementation and harmonisation of the molecular diagnostic procedures such as those needed to support the predicted advances in precision and personalised medicine.JRC.F.6-Reference Material

Additional file 2: of The use of digital PCR to improve the application of quantitative molecular diagnostic methods for tuberculosis

Protocol for gDNA extraction and dPCR quantification of materials. (PDF 394 kb