1. 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, final research report Abstract: 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.
Keywords: new genomic techniques, detection in food and feed, targeted mutagenesis, cisgenesis
Published in DiRROS: 02.09.2024; Views: 133; Downloads: 408
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2. 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, final research report Abstract: 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.
Keywords: DNA extraction, GMO, detection in food and feed
Published in DiRROS: 02.09.2024; Views: 165; Downloads: 1180
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3. Extraction of DNA from different sample types - a practical approach for GMO testingJana Žel, Tina Demšar, Dejan Štebih, Mojca Milavec, Kristina Gruden, 2015, original scientific article Abstract: Current methods based on DNA targets for the detection, identification and quantification of genetically modified organisms (GMOs) involve extraction of the DNA. Different extraction procedures have been developed for the great variety of samples from food, feed, seeds and particular plant parts. This makes the operation of routine analytical laboratories complex and workloads heavy. Here we present a decision-making system, developed over many years of GMO testing on different samples, that result in the application of only a few extraction methods for the majority of samples. Developed decision-making system enables quicker and more cost effective testing of GMOs. In addition, the performance of DNA extraction resulting from the use of the selected extraction methods is presented for use in subsequent testing of GMOs by real time PCR methods. This approach can be used as a model for similar systems based on nucleic acid analysis in food, feed, seeds and plants.
Keywords: extraction methods, genetically modified organisms, GMO, decision- making system (biology), GMO testing, cetyltrimethylammonium bromide, CTAB
Published in DiRROS: 29.07.2024; Views: 192; Downloads: 130
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4. 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, original scientific article Abstract: 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.
Keywords: digital PCR, droplet digital PCR, absolute quantification, reference materials, GMO quantification
Published in DiRROS: 24.07.2024; Views: 332; Downloads: 112
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5. Digital PCR as an effective tool for GMO quantification in complex matricesAlexandra Bogožalec Košir, Tina Demšar, Dejan Štebih, Jana Žel, Mojca Milavec, 2019, original scientific article Abstract: The increased use of genetically modified organisms (GMOs) is accompanied by increased complexity of the matrices that contain GMOs. The most common DNA-based approach for GMO detection and quantification is real-time quantitative polymerase chain reaction (qPCR). However, as qPCR is sensitive to inhibitors and relies on standard curves for quantification, it has limited application in GMO quantification for complex matrices. To overcome this hurdle in DNA quantification, we present droplet digital PCR (ddPCR) assays that were designed to target ‘Roundup Ready’ soybean and the soybean reference gene. Three ddPCR assays were transferred from qPCR to QX100/QX200 ddPCR platforms and characterised. Together, the fitness-for-purpose study on four real-life samples and the use of a chamber-based PCR system, showed that dPCR has great potential to improve such measurements in GMO testing and monitoring of food authenticity.
Keywords: genetically modified organisms, digital PCR, GMO quantification, complex matrices
Published in DiRROS: 23.07.2024; Views: 206; Downloads: 124
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6. High burden of clonal mast cell disorders and hereditary ▫$α-tryptasemia$▫ in patients who need Hymenoptera venom immunotherapyPeter Korošec, Gunter Sturm, Jonathan J. Lyons, Tinkara Pirc Marolt, Manca Svetina, Mitja Košnik, Mihaela Zidarn, Mark Kačar, Nina Frelih, Nika Lalek, Ajda Demšar Luzar, Samo Zver, Matevž Škerget, Ewa Czarnobilska, Wojciech Dyga, Sanja Popović-Grle, Miroslav Samaržija, Lisa Arzt-Gradwohl, Urban Čerpes, Grzegorz Porebski, Branko Pevec, Eva Schadelbauer, Peter Kopač, Julij Šelb, Matija Rijavec, 2024, original scientific article Abstract: Background
In patients who require venom immunotherapy (VIT), there is a need to identify underlying mast cell (MC) disorders since these may affect the risk and severity of future sting reactions and the long-term effectiveness of VIT.
Methods
1319 individuals with Hymenoptera venom allergy (HVA) who needed VIT from referral centers in Slovenia, Austria, Croatia, and Poland underwent examination for KIT p.D816V in peripheral blood leukocytes (PBL) using a highly sensitive PCR test and tryptase genotyping by digital droplet PCR. We also included 183 control individuals with large local reactions (LLRs) to Hymenoptera stings and with asymptomatic sensitization to Hymenoptera venoms.
Results
285 of 1319 individuals recommended for VIT (21.6%) were positive for KIT p.D816V in PBL, preferably those who present with severe reaction (33.9% [n = 207 of 610] with Ring-Messmer grade 3–4 vs. 11% [n = 78 of 709] with Grade 1–2; p < .0001), whereas only 1.3% (n = 2 of 152) of controls with LLR and none with asymptomatic sensitization (n = 31) had KIT p.D816V. KIT p.D816V allelic burden was higher in those with severe reaction (median 0.018% [n = 207] in Grade 3–4 vs. 0.001% [n = 78] in Grade 1–2; p < .0001), and the majority had normal baseline serum tryptase levels (69% [n = 196 of 285]). All KIT p.D816V-positive individuals (n = 41) who underwent bone marrow (BM) biopsy were found to have underlying clonal diseases, principally BM mastocytosis. HαT was also associated with severe HVA and symptoms (p < .01), and remarkably, 31.0% (n = 31 of 100) were found to have concomitant KIT p.D816V. Concomitant HαT and KIT p.D816V showed an additive effect, and having both was associated with the highest risk for severe HVA, even higher than having either HαT or KIT p.D816V alone (OR = 3.8; p < .01).
Conclusions
By employing prospective universal tryptase genotyping and examination for KIT p.D816V in PBL in large HVA populations, we have demonstrated a high burden of clonal MC disorders and HαT in patients who require VIT.
Keywords: anaphylaxis, hereditary α-tryptasemia, hypersensitivity, immunotherapy, mast cell, mastocytosis, venom
Published in DiRROS: 17.06.2024; Views: 363; Downloads: 243
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7. MRI macromolecular contrast agents as indicators of changed tumor blood flowTeodora Ivanuša, Katarina Beravs, Maja Čemažar, Vladimir Jevtič, Franci Demšar, Gregor Serša, 2001, original scientific article Abstract: Background. A rapid mapping technique derived from dynamic contrast enhanced MRI data was used to identify and characterize reduction of blood flow in fibrosarcoma SA-1 tumors treated either by application of electric pulses or vinblastine. Materials and methods. Tissue permeability surface area product (PS) and fractional blood volume (BV) were calculated on a pixel-by-pixel basis using dynamic MRI intensity data after administration of gadomer - 17 orpolylysine-Gd-DTPA; prototypic macromolecular contrast agents designed for blood pool enhancement. PS and BV values of untreated tumors were compared to those of tumors treated by local application of 8 electric pulses (amplitude/distance ratio, 1300 V/cm; duration, 100 us, frequency, 1 Hz) percutaneously to the tumor or by systemic administration of vinblastine (2.5 mg/kg). Results. Both treatments transiently, but significantly reduced tumor blood flow, application of electric pulses to the tumors being by 40% more effective in reducing tumor blood flow than systemic administration of vinblastine. PS and BV values derived with polylysine-Gd-DTPA-enhanced MRI were lower compared to those with gadomer-17, due to larger molecular size. Interestingly, Gd-DTPA-enhanced MRI did not show any significant changes of PSand BV between untreated and treated tumors. Conclusion. This study demonstrates that dynamic contrast enhanced MRI can be effectively used to qualitatively monitor tumor blood flow, and quantitatively by means of BV and PS.
Published in DiRROS: 25.01.2024; Views: 487; Downloads: 112
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9. Doktrinarni problemi diagnostičnega algoritma retroperitonealnega prostoraJurij Us, Franci-Stanko Marolt, Marjeta Stanovnik, Jože Stropnik, Erika Brenčič, Marjeta Demšar, V. Istenič, E. Baretič-Kolar, Marko Kralj, 1982, professional article Keywords: radiologija, intervencijska radiologija
Published in DiRROS: 14.09.2023; Views: 499; Downloads: 146
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10. Priporočila za obravnavo bolnikov s pljučnim rakomMartina Vrankar, Nina Boc, Izidor Kern, Aleš Rozman, Karmen Stanič, Tomaž Štupnik, Mojca Unk, Maja Ebert Moltara, Vesna Zadnik, Katja Adamič, Jernej Benedik, Marko Bitenc, Jasna But-Hadžić, Anton Crnjac, Marina Čakš, Dominik Časar, Eva Ćirić, Tanja Čufer, Ana Demšar, Rok Devjak, Goran Gačevski, Marta Globočnik Kukovica, Kristina Gornik-Kramberger, Maja Ivanetič Pantar, Marija Ivanović, Urška Janžič, Staša Jelerčič, Veronika Kloboves-Prevodnik, Mile Kovačević, Luka Ležaič, Mateja Marc-Malovrh, Katja Mohorčič, Loredana Mrak, Igor Požek, Nina Turnšek, Bogdan Vidmar, Dušanka Vidovič, Gregor Vlačić, Ana Lina Vodušek, Rok Zbačnik, Ivana Žagar, 2023, professional article Abstract: Leta 2019 so bila objavljena Priporočila za obravnavo bolnikov s pljučnim rakom, ki so v slovenski prostor vnesla prepotrebno poenotenje diagnostike in zdravljenja z namenom izboljšanja preživetja bolnikov s pljučnim rakom. Posodobitev Priporočil tri leta po izidu izvirnika prinaša največ novosti v poglavju o sistemskem zdravljenju bolnikov s pljučnim rakom. To kaže na izjemen napredek na področju razumevanja onkogeneze in biologije pljučnega raka ter s tem razvoja novih zdravil. Breme pljučnega raka ostaja veliko, saj je pljučni rak pri nas in v svetu še vedno najpogostejši vzrok smrti zaradi raka. Za vsako peto smrt zaradi raka je odgovoren pljučni rak. Skoraj tretjina bolnikov s pljučnim rakom ne prejme specifičnega onkološkega zdravljenja, bodisi zaradi slabega stanja zmogljivosti, spremljajočih bolezni ali obsega bolezni. Polovica bolnikov ima ob diagnozi razsejano bolezen, zaradi česar izboljšanje preživetja z malimi koraki sledi napredku v zdravljenju bolnikov s pljučnim rakom. Ti podatki nas opominjajo, da se bomo morali za velike premike v obravnavi bolnikov s pljučnim rakom lotiti drugačnih pristopov. Kot najbolj obetavno se ponuja zgodnje odkrivanje bolezni, ko so možnosti ozdravitve pljučnega raka najboljše. Zapisana Priporočila so usmeritev za obravnavo bolnikov s pljučnim rakom. Le s sodobnim multidisciplinarnim pristopom obravnave lahko bolniku ponudimo zdravljenje, ki mu omogoča najboljši izhod prognostično neugodne bolezni.
Keywords: pljučni rak, priporočila
Published in DiRROS: 27.07.2023; Views: 789; Downloads: 303
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