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Abstract: 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.
Keywords: digital PCR, DNA quantification, inter-laboratory assessment, human cytomegalovirus, virus reference materials
Published in DiRROS: 24.07.2024; Views: 119; Downloads: 98
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Abstract: DNA metabarcoding combines DNA barcoding with high-throughput sequencing to identify different taxa within environmental communities. The ITS has already been proposed and widely used as universal barcode marker for plants, but a comprehensive, updated and accurate reference dataset of plant ITS sequences has not been available so far. Here, we constructed reference datasets of Viridiplantae ITS1, ITS2 and entire ITS sequences including both Chlorophyta and Streptophyta. The sequences were retrieved from NCBI, and the ITS region was extracted. The sequences underwent identity check to remove misidentified records and were clustered at 99% identity to reduce redundancy and computational effort. For this step, we developed a script called ‘better clustering for QIIME’ (bc4q) to ensure that the representative sequences are chosen according to the composition of the cluster at a different taxonomic level. The three datasets obtained with the bc4q script are PLANiTS1 (100 224 sequences), PLANiTS2 (96 771 sequences) and PLANiTS (97 550 sequences), and all are pre-formatted for QIIME, being this the most used bioinformatic pipeline for metabarcoding analysis. Being curated and updated reference databases, PLANiTS1, PLANiTS2 and PLANiTS are proposed as a reliable, pivotal first step for a general standardization of plant DNA metabarcoding studies. The bc4q script is presented as a new tool useful in each research dealing with sequences clustering.
Keywords: DNA metabarcoding, reference datasets
Published in DiRROS: 19.07.2024; Views: 135; Downloads: 89
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Keywords: quality of life, multiple myeloma, reference data
Published in DiRROS: 05.07.2024; Views: 178; Downloads: 47
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Abstract: The Life Cycle Assessment of a building is a complex analysis that also involves the use of the predicted Reference Service Life (RSL) of the building components and materials, as well as the predicted RSL of the whole building. The RSL values of individual materials and building components can be obtained from different sources and are not exactly comparable. In the present study, the influence of selected RLS values on an LCA assessment was evaluated. Three different RSL databases were used as the sources of the data to estimate the environmental impacts of selected building components (internal wooden door and external finishing coat). Two scenarios were presented. In the first scenario a building component can be reused in another building, while in the second scenario the reuse of the building component is not possible. The study showed that dependent on the selected RSL database, the results can differ by up to a factor of five. Therefore, it is very important to describe clearly the maintenance scenarios for a building in order to have a reliable comparison of the results of LCA assessments.
Keywords: reference service life, life cycle assessment, refurbishment
Published in DiRROS: 08.03.2024; Views: 330; Downloads: 234
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Abstract: We determined the size of differences in stand and tree growth in semi-natural forests with respect to 16 reference soil groups. The forest area of Slovenia (11.8 thousand km2) was used as the study area, and reference soil units were derived from the national soil map at a 1:25,000 scale consisting of 10,781 polygons with an average size of 117.95 ha. Stand growth was defined as periodic stand basal area increment, while the growth of Norway spruce, silver fir, Scots pine, European beech and sessile oak trees was estimated by the periodic diameter increment of 238,349 dominant trees on 67,061 permanent sampling plots. A linear fixed-effects model and linear mixed-effect models were used for studying stand and tree growth in different site, stand and tree conditions. The soil unit was the dummy variable with Dystric Cambisols set as the reference category. Soil contributed 4.3 % to the explained variability of basal area increment and 4–27 % to the explained variability of the diameter increment of the five tree species. Soil was a stronger driver of stand and tree growth than climate or topography. Stand and tree species production rate on soil units was in the interval of −28 % to +5 % and −47 % to +14 % of that on the reference soil unit, respectively. Stand growth was the highest on Eutric Gleysols and the lowest on Histosols, and tree species generally exhibited the highest and the lowest growth rates on different soil units. We suggest that soil should be considered in growth models and studied interrelatedly with climatic, site and stand variables.
Keywords: reference soil groups, FAO soil unit, natural forest, stand growth, tree growth
Published in DiRROS: 29.12.2022; Views: 740; Downloads: 408
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