1. Digital PCR for direct quantification of viruses without DNA extractionJernej Pavšič, Jana Žel, Mojca Milavec, 2016, izvirni znanstveni članek Povzetek: DNA extraction before amplification is considered an essential step for quantification of viral DNA using real-time PCR (qPCR). However, this can directly affect the final measurements due to variable DNA yields and removal of inhibitors, which leads to increased inter-laboratory variability of qPCR measurements and reduced agreement on viral loads. Digital PCR (dPCR) might be an advantageous methodology for the measurement of virus concentrations, as it does not depend on any calibration material and it has higher tolerance to inhibitors. DNA quantification without an extraction step (i.e. direct quantification) was performed here using dPCR and two different human cytomegalovirus whole-virus materials. Two dPCR platforms were used for this direct quantification of the viral DNA, and these were compared with quantification of the extracted viral DNA in terms of yield and variability. Direct quantification of both whole-virus materials present in simple matrices like cell lysate or Tris-HCl buffer provided repeatable measurements of virus concentrations that were probably in closer agreement with the actual viral load than when estimated through quantification of the extracted DNA. Direct dPCR quantification of other viruses, reference materials and clinically relevant matrices is now needed to show the full versatility of this very promising and cost-efficient development in virus quantification.
Ključne besede: molecular diagnostics, direct quantification, viruses, virus reference materials, human cytomegalovirus, digital PCR
Objavljeno v DiRROS: 25.07.2024; Ogledov: 108; Prenosov: 80
 Celotno besedilo (778,33 KB)
Gradivo ima več datotek! Več... |
2. 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: 106; Prenosov: 91
Celotno besedilo (638,49 KB)
Gradivo ima več datotek! Več... |
3. Inter-laboratory analysis of selected genetically modified plant reference materials with digital PCRDavid Dobnik, Tina Demšar, Ingrid Huber, Lars Gerdes, Sylvia Broeders, Nancy Roosens, Frédéric Debode, Gilbert Berben, Jana Žel, 2018, izvirni znanstveni članek Povzetek: Digital PCR (dPCR), as a new technology in the field of genetically modified (GM) organism (GMO) testing, enables determination of absolute target copy numbers. The purpose of our study was to test the transferability of methods designed for quantitative PCR (qPCR) to dPCR and to carry out an inter-laboratory comparison of the performance of two different dPCR platforms when determining the absolute GM copy numbers and GM copy number ratio in reference materials certified for GM content in mass fraction. Overall results in terms of measured GM% were within acceptable variation limits for both tested dPCR systems. However, the determined absolute copy numbers for individual genes or events showed higher variability between laboratories in one third of the cases, most possibly due to variability in the technical work, droplet size variability, and analysis of the raw data. GMO quantification with dPCR and qPCR was comparable. As methods originally designed for qPCR performed well in dPCR systems, already validated qPCR assays can most generally be used for dPCR technology with the purpose of GMO detection.
Ključne besede: digital PCR, droplet digital PCR, absolute quantification, reference materials, GMO quantification
Objavljeno v DiRROS: 24.07.2024; Ogledov: 167; Prenosov: 67
Celotno besedilo (466,94 KB)
Gradivo ima več datotek! Več... |
4. 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: 283; Prenosov: 107
 Povezava na datoteko |
