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

The use of digital PCR to improve the application of quantitative molecular diagnostic methods for tuberculosis

CITATION: Devonshire, A. S., et al. 2016. The use of digital PCR to improve the application of quantitative molecular diagnostic methods for tuberculosis. BMC Infectious Diseases, 16:366, doi:10.1186/s12879-016-1696-7.The original publication is available at https://bmcinfectdis.biomedcentral.comBackground: Real-time PCR (qPCR) based methods, such as the Xpert MTB/RIF, are increasingly being used to diagnose tuberculosis (TB). While qualitative methods are adequate for diagnosis, the therapeutic monitoring of TB patients requires quantitative methods currently performed using smear microscopy. The potential use of quantitative molecular measurements for therapeutic monitoring has been investigated but findings have been variable and inconclusive. The lack of an adequate reference method and reference materials is a barrier to understanding the source of such disagreement. Digital PCR (dPCR) offers the potential for an accurate method for quantification of specific DNA sequences in reference materials which can be used to evaluate quantitative molecular methods for TB treatment monitoring. Methods: To assess a novel approach for the development of quality assurance materials we used dPCR to quantify specific DNA sequences in a range of prototype reference materials and evaluated accuracy between different laboratories and instruments. The materials were then also used to evaluate the quantitative performance of qPCR and Xpert MTB/RIF in eight clinical testing laboratories. Results: dPCR was found to provide results in good agreement with the other methods tested and to be highly reproducible between laboratories without calibration even when using different instruments. When the reference materials were analysed with qPCR and Xpert MTB/RIF by clinical laboratories, all laboratories were able to correctly rank the reference materials according to concentration, however there was a marked difference in the measured magnitude. Conclusions: TB is a disease where the quantification of the pathogen could lead to better patient management and qPCR methods offer the potential to rapidly perform such analysis. However, our findings suggest that when precisely characterised materials are used to evaluate qPCR methods, the measurement result variation is too high to determine whether molecular quantification of Mycobacterium tuberculosis would provide a clinically useful readout. The methods described in this study provide a means by which the technical performance of quantitative molecular methods can be evaluated independently of clinical variability to improve accuracy of measurement results. These will assist in ultimately increasing the likelihood that such approaches could be used to improve patient management of TB.https://bmcinfectdis.biomedcentral.com/articles/10.1186/s12879-016-1696-7Publisher's versio

The use of digital PCR to improve the application of quantitative molecular diagnostic methods for tuberculosis

CITATION: Devonshire, A. S., et al. 2016. The use of digital PCR to improve the application of quantitative molecular diagnostic methods for tuberculosis. BMC Infectious Diseases, 16:366, doi:10.1186/s12879-016-1696-7.The original publication is available at https://bmcinfectdis.biomedcentral.comBackground: Real-time PCR (qPCR) based methods, such as the Xpert MTB/RIF, are increasingly being used to diagnose tuberculosis (TB). While qualitative methods are adequate for diagnosis, the therapeutic monitoring of TB patients requires quantitative methods currently performed using smear microscopy. The potential use of quantitative molecular measurements for therapeutic monitoring has been investigated but findings have been variable and inconclusive. The lack of an adequate reference method and reference materials is a barrier to understanding the source of such disagreement. Digital PCR (dPCR) offers the potential for an accurate method for quantification of specific DNA sequences in reference materials which can be used to evaluate quantitative molecular methods for TB treatment monitoring. Methods: To assess a novel approach for the development of quality assurance materials we used dPCR to quantify specific DNA sequences in a range of prototype reference materials and evaluated accuracy between different laboratories and instruments. The materials were then also used to evaluate the quantitative performance of qPCR and Xpert MTB/RIF in eight clinical testing laboratories. Results: dPCR was found to provide results in good agreement with the other methods tested and to be highly reproducible between laboratories without calibration even when using different instruments. When the reference materials were analysed with qPCR and Xpert MTB/RIF by clinical laboratories, all laboratories were able to correctly rank the reference materials according to concentration, however there was a marked difference in the measured magnitude. Conclusions: TB is a disease where the quantification of the pathogen could lead to better patient management and qPCR methods offer the potential to rapidly perform such analysis. However, our findings suggest that when precisely characterised materials are used to evaluate qPCR methods, the measurement result variation is too high to determine whether molecular quantification of Mycobacterium tuberculosis would provide a clinically useful readout. The methods described in this study provide a means by which the technical performance of quantitative molecular methods can be evaluated independently of clinical variability to improve accuracy of measurement results. These will assist in ultimately increasing the likelihood that such approaches could be used to improve patient management of TB.https://bmcinfectdis.biomedcentral.com/articles/10.1186/s12879-016-1696-7Publisher's versio