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 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. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1007/s00216-017-0206-0) contains supplementary material, which is available to authorized users

The Digital MIQE Guidelines Update: Minimum Information for Publication of Quantitative Digital PCR Experiments for 2020

Digital PCR (dPCR) has developed considerably since the publication of the Minimum Information for Publication of Digital PCR Experiments (dMIQE) guidelines in 2013, with advances in instrumentation, software, applications, and our understanding of its technological potential. Yet these developments also have associated challenges; data analysis steps, including threshold setting, can be difficult and preanalytical steps required to purify, concentrate, and modify nucleic acids can lead to measurement error. To assist independent corroboration of conclusions, comprehensive disclosure of all relevant experimental details is required. To support the community and reflect the growing use of dPCR, we present an update to dMIQE, dMIQE2020, including a simplified dMIQE table format to assist researchers in providing key experimental information and understanding of the associated experimental process. Adoption of dMIQE2020 by the scientific community will assist in standardizing experimental protocols, maximize efficient utilization of resources, and further enhance the impact of this powerful technology

CCQM-K86/P113.1: Relative quantification of genomic DNA fragments extracted from a biological tissue

Key comparison CCQM-K86 was performed to demonstrate and document the capacity of interested National Metrology Institutes (NMIs) and Designated Institutes (DIs) in the determination of the relative quantity of two specific genomic DNA fragments present in a biological tissue. The study provides the support for the following measurement claim: "Quantification of the ratio of the number of copies of specified intact sequence fragments of a length in the range of 70 to 100 nucleotides in a single genomic DNA extract from ground maize seed materials "

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

Background 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

Ekstrakcija DNA iz različnih vrst vzorcev - praktični pristop za določanje GSO

Current methods based on DNA targets for the detection, identification and quantification of genetically modified organisms (GMOs) involve extraction of the DNA. Different extraction procedures have been developed for the great variety of samples from food, feed, seeds and particular plant parts. This makes the operation of routine analytical laboratories complex and workloads heavy. Here we present a decision-making system, developed over many years of GMO testing on different samples, that result in the application of only a few extraction methods for the majority of samples. Developed decision-making system enables quicker and more cost effective testing of GMOs. In addition, the performance of DNA extraction resulting from the use of the selected extraction methods is presented for use in subsequent testing of GMOs by real time PCR methods. This approach can be used as a model for similar systems based on nucleic acid analysis in food, feed, seeds and plants.Metode za določanje, identifikacijo in kvantifikacijo gensko spremenjenih organizmov (GSO) temeljijo na zaznavanju značilnih zaporedij DNA, zato je ključni del metode ekstrakcija DNA. Za ekstrakcijo DNA iz različnih vrst vzorcev, kot so živila, krma, semena in deli rastlin, so razviti različni postopki. Delo rutinskih laboratorijev je zato zelo kompleksno in obsežno. Tu predstavljamo sistem odločanja, ki smo ga razvili v mnogih letih testiranja GSO v različnih vzorcih. Z uporabo nekaj izbranih metod ekstrakcije lahko analiziramo večino vzorcev na hitrejši in finančno učinkovitejši način. Dodatno podajamo informacijo o uporabi izbranih ekstrakcijskih metod v povezavi z nadaljnjo analizo GSO s PCR v realnem času. Ta pristop se lahko uporabi kot model za podobne sisteme, ki temeljijo na analizi nukleinskih kislin v živilih, krmi, semenih in rastlinah

Digital PCR as an effective tool for GMO quantification in complex matrices

The increased use of genetically modified organisms (GMOs) is accompanied by increased complexity of the matrices that contain GMOs. The most common DNA-based approach for GMO detection and quantification is real-time quantitative polymerase chain reaction (qPCR). However, as qPCR is sensitive to inhibitors and relies on standard curves for quantification, it has limited application in GMO quantification for complex matrices. To overcome this hurdle in DNA quantification, we present droplet digital PCR (ddPCR) assays that were designed to target ‘Roundup Ready’ soybean and the soybean reference gene. Three ddPCR assays were transferred from qPCR to QX100/QX200 ddPCR platforms and characterised. Together, the fitness-for-purpose study on four real-life samples and the use of a chamber-based PCR system, showed that dPCR has great potential to improve such measurements in GMO testing and monitoring of food authenticity

Ekstrakcija DNA iz različnih vrst vzorcev -praktični pristop za določanje GSO

Current methods based on DNA targets for the detection, identification and quantification of genetically modified organisms (GMOs) involve extraction of the DNA. Different extraction procedures have been developed for the great variety of samples from food, feed, seeds and particular plant parts. This makes the operation of routine analytical laboratories complex and workloads heavy. Here we present a decision-making system, developed over many years of GMO testing on different samples, that result in the application of only a few extraction methods for the majority of samples. Developed decision-making system enables quicker and more cost effective testing of GMOs. In addition, the performance of DNA extraction resulting from the use of the selected extraction methods is presented for use in subsequent testing of GMOs by real time PCR methods. This approach can be used as a model for similar systems based on nucleic acid analysis in food, feed, seeds and plants.Metode za določanje, identifikacijo in kvantifikacijo gensko spremenjenih organizmov (GSO) temeljijo na zaznavanju značilnih zaporedij DNA, zato je ključni del metode ekstrakcija DNA. Za ekstrakcijo DNA iz različnih vrst vzorcev, kot so živila, krma, semena in deli rastlin, so razviti različni postopki. Delo rutinskih laboratorijev je zato zelo kompleksno in obsežno. Tu predstavljamo sistem odločanja, ki smo ga razvili v mnogih letih testiranja GSO v različnih vzorcih. Z uporabo nekaj izbranih metod ekstrakcije lahko analiziramo večino vzorcev na hitrejši in finančno učinkovitejši način. Dodatno podajamo informacijo o uporabi izbranih ekstrakcijskih metod v povezavi z nadaljnjo analizo GSO s PCR v realnem času. Ta pristop se lahko uporabi kot model za podobne sisteme, ki temeljijo na analizi nukleinskih kislin v živilih, krmi, semenih in rastlinah

Digital PCR method for detection and quantification of specific antimicrobial drug-resistance mutations in human cytomegalovirus

Antimicrobial drug resistance is one of the biggest threats to human health worldwide. Timely detection and quantification of infectious agents and their susceptibility to antimicrobial drugs are crucial for efficient management of resistance to antiviral drugs. In clinical settings, viral drug resistance is most often associated with prolonged treatment of chronic infections, and assessed by genotyping methodse.g., sequencing and PCR. These approaches have limitations: sequencing can be expensive and does not provide quantificationand qPCR quantification is hampered by a lack of reference materials for standard curves. In recent years, digital PCR has been introduced, which provides absolute quantification without the need for reference materials for standard curves. Using digital PCR, we have developed a rapid, sensitive and accurate method for genotyping and quantification of the most prevalent mutations that cause human cytomegalovirus resistance to ganciclovir