561. Guidance document on multiplex real-time PCR methods : European Network of GMO LaboratoriesLutz Grohmann, Alessandra Barbante, Ronnie Eriksson, Francesco Gatto, Tzveta Georgieva, Ingrid Huber, Julie Hulin, René Köppel, Ugo Marchesi, Lucas Marmin, Marco Mazzara, Frank Narendja, Heather Owen, Elena Perri, Ingrid Scholtens, Tereza Sovová, Slawomir Sowa, Dejan Štebih, Christopher Weidner, Kamila Zdeňková, 2021, končno poročilo o rezultatih raziskav Povzetek: This document provides guidance for the development of new qualitative and quantitative multiplex real-time methods (mpPCR) and their implementation in a routine testing laboratory for food, feed and seeds. When implementing a mpPCR method, the laboratory should perform either an (in-house) validation or a verification of the method before its use.
The various method performance parameters of the specific requirements for mpPCR are described and examples are given. Several different aspects have been considered, including the selection of the fluorophores, the master mix and the real-time PCR instrument used. The technical aspects are examined and recommendations are made on the choice of the detection and quantification strategy. Finally, a separate chapter is dedicated to troubleshooting (e.g. underperforming method, no or poor signal strength, cross-talk).
In addition, a collection of existing mpPCR methods with emphasis on GMO detection is presented.
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562. Establishing thresholds : workshop on the MSFD Newly Introduced NIS (D2C1)Chiara Magliozzi, C. Bartilotti, A. Botelho, M. Bugeja, João Canning-Clode, Ana Fortič, Martina Orlando-Bonaca, 2023, končno poročilo o rezultatih raziskav Povzetek: The Workshop on ‘Establishing thresholds: workshop on the Marine Strategy Framework Directive (MSFD) Newly Introduced Non-Indigenous Species (NIS) (D2C1)’ took place online on the 24th November 2022, aiming at discussing and making progress towards establishing threshold values for the criterion D2C1. The workshop was organised by the Joint Research Centre (JRC), in collaboration with representatives of the Regional Sea Conventions (RSCs), under the MSFD Descriptor 2 Core Group, in the context of the MSFD Common Implementation Strategy Good Environmental Status Working Group. The participants to the workshop included academic and public authorities’ experts on MSFD NIS, RSCs representatives, the European Environment Agency (EEA), and Commission services. The workshop enabled sharing information on the state-of-the-art approaches on monitoring and assessment of D2C1 within Regional Seas and on the application of the time series analysis as a follow-up to Tsiamis et al. (2021) recommendations for setting threshold values. The discussion focused on ensuring comparability of approaches, how to account for uncertainties, and on areas for improvement. It was acknowledged that activities of RSCs are instrumental in coordinating the science and management of marine NIS. The various levels of monitoring across the North-East Atlantic Ocean, the Mediterranean Sea, the Baltic Sea and the EU Countries explain different methodological approaches for setting thresholds, while the limited monitoring restrict the application of these methodologies in the Black Sea. The workshop identified four main areas for improvements: i) establishment of acknowledged baselines for NIS introductions, ii) make further progress in monitoring and analysis methods, iii) agree on thresholds for new NIS introductions and, iv) ensure coherence and consistency between national and regional reports. The JRC will seek for opportunities to discuss and advance on identified areas for improvement with the MSFD national appointed NIS experts The MSFD Descriptor 2 (D2) Core Group will contribute to the discussions.
Ključne besede: Marine Strategy Framework Directive, MSFD, non-indigenous species, NIS, descriptor 2, marine biology, European Commission
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564. Detection of food and feed plant products obtained by targeted mutagenesis and cisgenesisSlawomir Sowa, Alessandra Barbante, Wim Broothaerts, Malcolm Burns, Frédéric Debode, Marzia De Giacomo, Marc De Loose, Tina Demšar, Kolja Eckermann, Marie Alice Fraiture, 2023, končno poročilo o rezultatih raziskav Povzetek: The current EU legislation on GMOs and GM food and feed requires analytical testing to support traceability of these products on the market. The European Network of GMO Laboratories has reviewed the implications of the analytical requirements when they are applied to plant products developed with the use of new genomic techniques, i.e. targeted mutagenesis and cisgenesis. This review concluded that analytical testing to support traceability is not considered feasible for all products obtained by targeted mutagenesis and cisgenesis, both due to technical restrictions and because of implementation issues.
Ključne besede: new genomic techniques, detection in food and feed, targeted mutagenesis, cisgenesis
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565. The International cookbook for wastewater practitioners : testing wastewater for public health, SARS-CoV-2Bernd Manfred Gawlik, Sara Comero, Daniel A. Deere, Gertjan Medema, Ismahane Remonnay, Jean-François Loret, Ana Maria de Roda Husman, Stephanie Rinck-Pfeiffer, Shelesh Agrawal, Ion Gutiérrez-Aguirre, 2024, končno poročilo o rezultatih raziskav Povzetek: During the 2020-2023 COVID-19 Global Public Health Emergency of International Concern, rapid progress was made with the application of Wastewater-Based Surveillance (WBS) (also known as Wastewater-Based Epidemiology (WBE)) to provide population-scale evidence of SARS-CoV-2 shedding into wastewater. The information gained was used to help monitor, communicate, and manage the COVID-19 pandemic.
Rapid and significant innovations in the field of WBS were realised in parallel in multiple jurisdictions globally. Global collaboration resulted in step-change improvements in multiple aspects of WBS simultaneously, including: health sector leadership; integration with public health surveillance; social and ethical guidance; high-level tactical wastewater catchment sampling strategies; wastewater sampling techniques; transport and storage of samples; laboratory analysis; normalisation and standardisation; data analysis and interpretation; and communication of results.
Innovations in WBS relating to COVID-19 continue to take place, and the benefits of those improvements are now being applied to other targets, such as other pathogens and genes. There are many journal articles and other publications describing these innovations. However, valuable lessons from the COVID-19 pandemic for WBS have not been systematically documented into a practical and readily digestible format.
The purpose of this ‘Cookbook’ is to provide practical and proven ‘recipes’ for WBS for SARS-CoV-2 for two reasons: firstly to help those considering new or extended WBS programmes targeting SARS-CoV-2; and secondly to provide a valuable record of what has been learned from the 2020- 2023 period to help inform WBS programmes to respond to future pandemics and for surveillance of other targets.
The Cookbook is structured into eight parts: 1. Introduction; 2. High-level Overview; 3. Health Sector Perspectives; 4. Methods and Methodologies - From data to action; 5. Methods and Methodologies - Measurement and testing; 6. Methods and Methodologies - Sampling; 6. Methods and Methodologies - Transportation protocols; 8. Conclusions. Multiple authors have contributed to each part, primarily from the European Union, as well as others from the global community of practice.
The Cookbook has been drafted by a global team of authors, with one or more lead authors identified for each chapter and working with their co-authors. The chapters were then subjected to extensive peer review from the external review group. These authors and reviewers are acknowledged in the Cookbook.
Ključne besede: coronavirus disease, disease surveillance, innovation, public health, report, sampling, standardisation, testing, wastewater
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566. Guidance on the selection and use of DNA extraction methods : JRC technical reportTheo W. Prins, Wim Broothaerts, Malcolm Burns, Tina Demšar, Sophia Edelmann, Nina Papazova, Verena Peterseil, Isabel Taverniers, 2024, končno poročilo o rezultatih raziskav Povzetek: DNA extraction is at the forefront of further analytical measurements on DNA targets and affects the downstream results. This report from the European Network of GMO Laboratories (ENGL) provides guidance on the selection and use of fit-for-purpose DNA extraction methods. It focusses on DNA extraction in the context of official controls on the presence and content of genetically modified organisms in food and feed. It provides guidance on protocols and selection support systems, validation approaches, assessment of DNA quality parameters and examples of practical solutions derived from collective experiences. There are many variations on the theme of DNA extraction, but there is no single protocol that works adequately across all food and feed matrices. Before using a new method in the laboratory, or in case of modifications to a protocol, validation or verification is needed to show that a chosen method is fit for purpose for use in routine analysis. This guidance is aimed to help the DNA analysis laboratories in fulfilling the standardisation requirements and support their daily operations.
Ključne besede: DNA extraction, GMO, detection in food and feed
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567. Poročilo o preskusu št.: LVG 2024-105 : vzorec št. 2024/00649Barbara Piškur, Patricija Podkrajšek, Špela Hočevar, 2024, izvedensko mnenje, arbitražna odločba Ključne besede: varstvo gozdov, morfološke analize, Geosmithia morbida, bolezen tisočerih rakov, qPCR, Juglans, program preiskav
Objavljeno v DiRROS: 30.08.2024; Ogledov: 289; Prenosov: 0
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568. Metrological evaluation of DNA extraction method effects on the bacterial microbiome and resistome in sputumAleksander Benčič, Nataša Toplak, Simon Koren, Alexandra Bogožalec Košir, Mojca Milavec, Viktorija Tomič, Dane Lužnik, Tanja Dreo, 2024, izvirni znanstveni članek Povzetek: Targeted high-throughput sequencing (HTS) has revolutionized the way we look at bacterial communities. It can be used for the species-specific detection of bacteria as well as for the determination of the microbiome and resistome and can be applied to samples from almost any environment. However, the results of targeted HTS can be influenced by many factors, which poses a major challenge for its use in clinical diagnostics. In this study, we investigated the impact of the DNA extraction method on the determination of the bacterial microbiome and resistome by targeted HTS using principles from metrology and diagnostics such as repeatability and analytical sensitivity. Sputum samples spiked with Acinetobacter baumannii, Klebsiella pneumoniae, and Pseudomonas aeruginosa at three different concentrations (103–106 cells/mL) were used. DNA was extracted from each sample on 2 separate days in three replicates each using three different extraction methods based on cetrimonium bromide, magnetic beads, and silica membranes. All three spiked bacteria were detected in sputum, and the DNA extraction method had no significant effect on detection. However, the DNA extraction method had significant effects on the composition of the microbiome and the resistome. The sequencing results were repeatable in the majority of cases. The silica membrane-based DNA extraction kit provided the most repeatable results and the highest diversity of the microbiome and resistome. Targeted HTS has been shown to be a reliable tool for determining the microbiome and resistome; however, the method of DNA extraction should be carefully selected to minimize its impact on the results.
Ključne besede: targeted high-throughput sequencing, bacterial microbiome, resistome, bacteria detection, DNA extraction, metrology, diagnostics, repeatability
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569. Degradation processes of two compound layers on nitrided surfaces during the wear test by ‘‘block on hot al cylinder’’Martin Lamut, Jaka Burja, Milan Terčelj, Goran Kugler, David Bombač, 2024, izvirni znanstveni članek Ključne besede: wear, nitrided steel, hot-work tool steel, thermal fatigue, aluminium extrusion, tool surface degradation
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