1. The digital MIQE guidelines update : minimum information for publication of quantitative digital PCR experiments for 2020Jim F. Huggett, Alexandra S. Whale, Ward De Spiegelaere, Afif M. Abdel Nour, Young-Kyung Bae, Vladimír Beneš, Dan Burke, Megan Cleveland, Philippe Corbisier, Alison S. Devonshire, Lianhua Dong, Daniela Drandi, Carole A. Foy, Jeremy A. Garson, Hua-Jun He, Jan Hellemans, Mikael Kubista, Antoon Lievens, Mike G. Makrigiorgos, Mojca Milavec, Reinhold D. Mueller, Tania Nolan, Denise M. O'Sullivan, Michael W. Pfaffl, Stefan Rödiger, Erica L. Romsos, Gregory L. Shipley, Valérie Taly, Andreas Untergasser, Carl T. Wittwer, Stephen A. Bustin, Jo Vandesompele, 2020, pregledni znanstveni članek Povzetek: 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.
Objavljeno v DiRROS: 06.08.2024; Ogledov: 27; Prenosov: 77
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2. Survey results on nucleic acid tests of infectious diseases : present status and need for rapid and near-patient diagnosticsJörg Neukammer, Martin Hussels, Andreas Kummrow, Alison S. Devonshire, Carole A. Foy, Jim F. Huggett, Helen C. Parkes, Jana Žel, Mojca Milavec, Heinz Schimmel, Wolfgang Unger, Müslüm Akgöz, Timothy D. McHugh, Viktorija Tomič, Hans-Peter Grunert, Heinz Zeichhardt, 2015, izvirni znanstveni članek Povzetek: This survey discusses current and emerging isothermal and rapid polymerase chain reaction (PCR) based nucleic acid amplification methods for near-patient diagnostics.
To assess the clinical need of rapid diagnostics for infectious diseases based on nucleic acid tests (NATs) we performed and analysed a questionnaire among laboratories participating in corresponding INSTAND ring trials for external quality assurance. The questions concerning new amplification technologies like isothermal nucleic acid amplification, potentially suited to significantly decrease turnaround times, were complemented by questions to evaluate the present status of NATs. Besides end-users, companies were also addressed by sending out a manufacturer specific questionnaire.
Analysis of the answers from 48 laboratories in 14 European countries revealed that a much shorter turnaround time is requested for selected pathogens compared to about 2 h or longer when applying temperature cycling amplification, i.e. PCR. In this context, most frequently mentioned were methicillin-resistant Staphylococcus aureus (MRSA), norovirus, influenza A and B viruses, cytomegalovirus (CMV) as well as hepatitis B virus (HBV) and hepatitis C virus (HCV). At present, 8% of the laboratories having participated in this survey apply isothermal amplification of nucleic acids to identify infectious pathogens.
Ključne besede: nucleic acid tests, infectious diseases, virus detection, bacteria detection, isothermal nucleic acid amplification, status report, questionnaire, NAT, PCR
Objavljeno v DiRROS: 26.07.2024; Ogledov: 100; Prenosov: 261
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3. Inter-laboratory assessment of different digital PCR platforms for quantification of human cytomegalovirus DNAJernej Pavšič, Alison S. Devonshire, Andrej Blejec, Carole A. Foy, Fran Van Heuverswyn, Gerwyn M. Jones, Heinz Schimmel, Jana Žel, Jim F. Huggett, Nicholas Redshaw, Maria Karczmarczyk, Erkan Mozioglu, Sema Akyürek, Müslüm Akgöz, Mojca Milavec, 2017, izvirni znanstveni članek Povzetek: 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.
Ključne besede: digital PCR, DNA quantification, inter-laboratory assessment, human cytomegalovirus, virus reference materials
Objavljeno v DiRROS: 24.07.2024; Ogledov: 89; Prenosov: 76
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4. An assessment of the reproducibility of reverse transcription digital PCR quantification of HIV-1Samreen Falak, Rainer Macdonald, Eloise J. Busby, Denise M. O'Sullivan, Mojca Milavec, Annabell Plauth, Martin Kammel, Heinz Zeichhardt, Hans-Peter Grunert, Andreas Kummrow, Jim F. Huggett, 2022, izvirni znanstveni članek Povzetek: Viral load monitoring in human immunodeficiency virus type 1 (HIV-1) infection is often performed using reverse transcription quantitative PCR (RT-qPCR) to observe response to treatment and identify the development of resistance. Traceability is achieved using a calibration hierarchy traceable to the International Unit (IU). IU values are determined using consensus agreement derived from estimations by different laboratories. Such a consensus approach is necessary due to the fact that there are currently no reference measurement procedures available that can independently assign a reference value to viral reference materials for molecular in vitro diagnostic tests. Digital PCR (dPCR) is a technique that has the potential to be used for this purpose. In this paper, we investigate the ability of reverse transcriptase dPCR (RT-dPCR) to quantify HIV-1 genomic RNA without calibration. Criteria investigated included the performance of HIV-1 RNA extraction steps, choice of reverse transcription approach and selection of target gene with assays performed in both single and duplex format. We developed a protocol which was subsequently applied by two independent laboratories as part of an external quality assurance (EQA) scheme for HIV-1 genome detection. Our findings suggest that RT-dPCR could be used as reference measurement procedure to aid the value assignment of HIV-1 reference materials to support routine calibration of HIV-1 viral load testing by RT-qPCR.
Objavljeno v DiRROS: 16.07.2024; Ogledov: 105; Prenosov: 51
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5. The performance of human cytomegalovirus digital PCR reference measurement procedure in seven external quality assessment schemes over four yearsMojca Milavec, Jernej Pavšič, Alexandra Bogožalec Košir, Gerwyn M. Jones, Denise M. O'Sullivan, Alison S. Devonshire, Fran Van Heuverswyn, Maria Karczmarczyk, Jannika Neeb, Annabell Plauth, Philippe Corbisier, Heinz Schimmel, Andreas Kummrow, Jörg Neukammer, Carole A. Foy, Martin Kammel, Hans-Peter Grunert, Heinz Zeichhardt, Jim F. Huggett, 2022, izvirni znanstveni članek Objavljeno v DiRROS: 15.07.2024; Ogledov: 199; Prenosov: 50
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6. Digital PCR for the characterization of reference materialsMegan H. Cleveland, Hua-Jun He, Mojca Milavec, Young-Kyung Bae, Peter M. Vallone, Jim F. Huggett, 2024, pregledni znanstveni članek Povzetek: Well-characterized reference materials support harmonization and accuracy when conducting nucleic acid-based tests (such as qPCR); digital PCR (dPCR) can measure the absolute concentration of a specific nucleic acid sequence in a background of non-target sequences, making it ideal for the characterization of nucleic acid-based reference materials. National Metrology Institutes are increasingly using dPCR to characterize and certify their reference materials, as it offers several advantages over indirect methods, such as UV-spectroscopy. While dPCR is gaining widespread adoption, it requires optimization and has certain limitations and considerations that users should be aware of when characterizing reference materials. This review highlights the technical considerations of dPCR, as well as its role when developing and characterizing nucleic acid-based reference materials.
Ključne besede: digital PCR, dPCR, reference materials, UV-spectroscopy
Objavljeno v DiRROS: 03.06.2024; Ogledov: 271; Prenosov: 103
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