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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: 101; Prenosov: 64
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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: 151; Prenosov: 59
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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: 273; Prenosov: 103
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Povzetek: The study reports the successful deposition of 15–5 precipitation-hardened (PH) martensitic steel on SAF 2507 duplex stainless steel (and vice versa) to form functionally graded materials (FGMs) using powder-based, directed-energy-deposition (DED) technology. The evolution of the microstructure and the mechanical properties of the 15–5 PH/SAF 2507 functionally graded material in the as-built state were investigated systematically. The results proved that the microstructural transition zone (MTZ) is formed above the fusion line due to the dilution effect of both steels. The phase composition of the MTZ consists of ferrite and, in comparison to the base materials, an increased amount of austenite. The interface has the lowest hardness owing to the formation of a significant proportion of the austenitic phase. However, the tensile mechanical properties were not affected by the interface as failure occurred in both SAF 2507 and at the interface regions. The research presents a promising application of FGMs in a horizontal configuration to form a high - quality, metallurgical joint between heterogeneous materials. This study introduces a novel approach by additive manufacturing (AM) of heterogeneous multi-materials, merging the favorable properties of duplex SAF 2507 and martensitic 15-5 PH stainless steels through superior metallurgical bonding. The combination of SAF 2507 and 15-5 PH in a functionally graded material has not been previously explored, as the existing studies deal with the duplex stainless steels SAF 2507 and either martensitic stainless-steel 15-5 PH deposited separately by laser powder-bed-fusion (L-PBF) or DED methods. This research pioneers the investigation of these materials in tandem, paving the way for the development of novel FGMs with optimized properties for specific applications. The resulting FGM exhibits exceptional corrosion resistance and high strength, making it highly versatile for applications in diverse industries such as offshore oil and gas production, aerospace, aviation, and chemical processing equipment.
Ključne besede: directed energy deposition, functionally graded materials, miniaturized specimen testing methods, SAF 2507, 15-5 PH
Objavljeno v DiRROS: 04.04.2024; Ogledov: 268; Prenosov: 105
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Povzetek: Alkali activated materials (AAM) are, in their broadest classification, any binder systems derived by the reaction of an alkali metal source (silicates, alkali hydroxides, carbonates , sulphates) with a solid, amorphous alumosilicate powder (found in precursors such as slag, fly ash and bottom ash). A wide variety of products can be obtained by the alkali activation process and could replace traditional construction products. Among the se, alkali activated foams (AAF) represent one of the most promising materials, owing to their economically accessible alumosilicate rich source materials, including industrial waste materials, clean processing, higher added value and most importantly, pro ducts with competitive properties. In the present study, the properties of alkali activated fly ash - based foam materials were studied at room temperature as well as at elevated temperatures (up to 1200 °C ) in order to develop a durable material in terms o f mechanical properties and suitability for high temperature applications.
Ključne besede: lightweight porous insulating material, alkali activated materials, microstructural analysis
Objavljeno v DiRROS: 05.03.2024; Ogledov: 407; Prenosov: 345
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