The determination of infectious titers of samples containing virus particles is critical for clinical gene therapy applications. Currently available methods that determine the infectivity of viruses/viral vectors are time-consuming. In this study, we developed cell culture-based infection assays that rapidly detect wide ranges of DNA/RNA viruses/viral vectors. Viruses analyzed in this study included Adenovirus type 5, Adenovirus type 41, Vaccinia virus, Measles virus, Coxsackie virus B5, Respiratory Syncytial Virus, and Adenovirus-, Adeno-associated virus-, Retrovirus-, and Lentivirus-based vectors. Susceptible cells were infected for 3 h with samples containing infectious viruses/viral vectors. In developed assays, differences in Cq values (∆Cq) were measured between cells incubated with infectious virus for 3 h and the two controls, including 0 h and heat-inactivated controls. Controls were included to detect background signals. An infection assay with a ∆Cq greater than 3.3 was considered positive. The reproducible and repeatable infection assays were functioning based on virus entry into the cells. For all tested viruses/viral vectors, ∆Cq above 3.3 was detected between cells incubated with infectious virus for 3 h and at least one control, indicating the positive functioning of the assay. The 0 h and heat-inactivated controls that were crucial for the estimation of the background, were successfully applied to all viruses/viral vectors tested in this study. We have developed a rapid cell-culture-based assay for detection of infectious viruses. The assay could be applied to all tested viruses/viral vectors, and has the potential to become a valuable tool in clinical virology and infectious virus diagnostics.

Since the commercialization of the first genetically modified (GM) crops, their relevance in agricultural applications has increased. Soybean (Glycine max) ranks as the most widely cultivated GM crop globally. With the advancements in site-specific genome-editing tools, the development of GM organisms (GMO) using new genomic techniques has accelerated, particularly in agriculture. In the US, a genome-edited soybean variety, Calyno, was developed with altered fatty acid composition by inactivating two members of the fatty acid desaturase 2 (FAD2) gene family, FAD2-1A and FAD2-1B, using TALEN technology. Although deregulated in the US, Calyno soybeans are not authorized for cultivation or import in the European Union and therefore cannot be placed on the European market. To address the need for reliable detection methods in routine analysis for GMO by public authorities, two event-specific qPCR methods were developed to detect the FAD2-1A-Δ63bp and FAD2-1B-Δ23bp gene variants in the Calyno soybeans. In the absence of reference material, methods were validated in-house using synthetic plasmids carrying the target regions’ sequences, following European Network of GMO Laboratories (ENGL) guidelines. Both assays meet the minimum performance requirements for GMO testing, demonstrating high sensitivity, qPCR efficiency, specificity and robustness. An interlaboratory comparison study further confirmed the reliability of these methods. These qPCR assays provide an effective tool for detecting the FAD2-1A-Δ63bp and FAD2-1B-Δ23bp gene variants in Calyno soybeans, supporting GMO testing requirements in the European Union and ensuring accurate monitoring of GM crops.

Spelt products are popular with consumers achieving higher market prizes, making them susceptible to food adulteration with less valuable cereals. To facilitate product control, a recently developed duplex droplet digital PCR method enables the detection and quantification of common wheat (Triticum aestivum) contaminations in food products made from spelt (Triticum spelta). The duplex droplet digital PCR assay targets the γ-gliadin gene and the Q-locus of both subspecies. In this study, the method was validated in an interlaboratory ring trial involving 11 participating laboratories. Test materials containing defined proportions of spelt and common wheat were prepared and tested. The ring trial procedure included DNA extraction of the test samples and determination of subspecies proportions using droplet digital PCR. Results from the ring trial confirmed the method's capability for specific detection and quantification of common wheat in spelt, with acceptable relative measurement uncertainties, and without requiring reference material for calibration. To our knowledge, this is the first interlaboratory validation of a digital PCR method for species differentiation in food.

New genomic techniques (NGT) are increasingly applied in plant research and development in many countries due to their simplicity in introducing genetic modifications that alter specific traits. First NGT products are on the market in several countries where no specific regulatory approval is required. In the European Union (EU), however, all NGT derived products are subject to the existing genetically modified organism (GMO) authori-zation regulations. This nourishes the discussion on the technical and analytical feasibility of detecting, iden-tifying and differentiating plants produced by targeted mutagenesis from conventional breeding products and spontaneous mutations in routine food/feed and seed/grain testing by enforcement laboratories. The anticipated difficulties are currently based mainly on theoretical considerations, particularly with regard to single nucleotide or very limited sequence changes. Against that background, this brief review examines concrete and practical approaches for the detection and identification of market-relevant NGT plants. Four early commercialized NGT plants have been examined as real examples to verify the feasibility of detection method design and development using routinely applied analytical approaches. In addition, we searched for potential new screening approaches and show, how a common and well conserved genetic element used in CRISPR/Cas9 applications may serve as a potential target, if this foreign DNA element is still integrated in the host genome. The conclusions to be drawn from the considered theoretical approaches and the practical examples are described in the context of key differences to the current GMO testing practice, in particular regarding the difficulty to develop methods specifically identifying NGT applications. Finally, we discuss whether the consideration of such factors could facilitate a resolution of the current dilemma between detection and the ability to identify plant NGT events.

Interpretation of whole-genome sequencing (WGS) data for foodborne outbreak investigations is complex, as the genetic diversity within processing plants and transmission events need to be considered. In this study, we analyzed 92 food-associated Listeria monocytogenes isolates by WGS-based methods. We aimed to examine the genetic diversity within meat and fish production chains and to assess the applicability of suggested thresholds for clustering of potentially related isolates. Therefore, meat-associated isolates originating from the same samples or processing plants as well as fish-associated isolates were analyzed as distinct sets. In silico serogrouping, multilocus sequence typing (MLST), core genome MLST (cgMLST), and pangenome analysis were combined with screenings for prophages and genetic traits. Isolates of the same subtypes (cgMLST types (CTs) or MLST sequence types (STs)) were additionally compared by SNP calling. This revealed the occurrence of more than one CT within all three investigated plants and within two samples. Analysis of the fish set resulted in predominant assignment of isolates from pangasius catfish and salmon to ST2 and ST121, respectively, potentially indicating persistence within the respective production chains. The approach not only allowed the detection of distinct subtypes but also the determination of differences between closely related isolates, which need to be considered when interpreting WGS data for surveillance.

A duplex droplet digital PCR (ddPCR) is described for the detection and quantification of contaminations by common wheat (Triticum aestivum) in food products made from spelt (Triticum spelta). A single nucleotide polymorphism (SNP) in the locus Q, as well as a short sequence of the gamma-gliadin gene of the wheat genome are exploited for the development of our duplex ddPCR method. Minimal variations of DNA sequences causing different probe hydrolysis efficiencies during PCR amplification can be detected and evaluated in ddPCR. By utilizing the SNP in the locus Q, all 84 tested spelt cultivars could unambiguously discriminated from common wheat cultivars. Discrimination using the gamma-gliadin gene sequence could be achieved for 10 of 84 spelt cultivars. Additionally, we could show that impurities caused by common wheat can also be detected in einkorn wheat (T. monococcum) and in emmer (T. dicoccon). For the quantification of contaminations by common wheat in food products from spelt, we prepared flours, vegetarian burger patties and bread based on defined proportions of spelt and common wheat. These materials were tested in three different laboratories using in-house DNA extraction methods and a predefined ddPCR protocol. The results indicated precise quantification of common wheat in spelt and a low relative measurement uncertainty of the ddPCR method without the further use of reference material for calibration.

Marzipan is a mixture of almonds, sugar, and water. Almonds can be replaced by apricot kernels, which results in a similar product called persipan. Depending on the prices of almonds, profit can be maximized using apricot kernels instead of almonds. If not specified on the product, this kind of substitution is illegal and is prosecuted by official food control authorities. Likewise, also commercial buyers would like to know which product they bought. Real-time PCR systems for the determination of apricot DNA are already available, however, real-time PCR requires the use of external standards, which have a direct impact on the accuracy of the measurement. Currently, such standards are not available. In contrast to real-time PCR, digital PCR does not require external standards and exhibits a smaller measurement uncertainty as shown in recent publications. Therefore, we developed a duplex droplet digital PCR system to measure the proportion of apricot in marzipan without the use of external standards. We present validation data and results of an international proficiency test, underlining the applicability of this system.

Background Fast, reliable and easy to handle methods are required to facilitate urgently needed point-of-care testing (POCT) in the current coronavirus pandemic. Life-threatening severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) has rapidly spread all over the world, infecting more than 33,500,000 people and killing over 1 million of them as of October 2020. Infected individuals without any symptoms might still transfer the virus to others underlining the extraordinary transmissibility of this new coronavirus. In order to identify early infections effectively, treat patients on time and control disease spreading, rapid, accurate and onsite testing methods are urgently required. Results Here we report the development of a loop-mediated isothermal amplification (LAMP) based method to detect SARS-CoV-2 genes ORF8 and N directly from pharyngeal swab samples. The established reverse transcription LAMP (RT-LAMP) assay detects SARS-CoV-2 directly from pharyngeal swab samples without previous time-consuming and laborious RNA extraction. The assay is sensitive and highly specific for SARS-CoV-2 detection, showing no cross reactivity when tested on 20 other respiratory pathogens. The assay is 12 times faster and 10 times cheaper than routine reverse transcription real-time polymerase chain reaction, depending on the assay used. Conclusion The fast and easy to handle RT-LAMP assay amplifying specifically the genomic regions ORF8 and N of SARS-CoV-2 is ideally suited for POCT at e.g. railway stations, airports or hospitals. Given the current pandemic situation, rapid, cost efficient and onsite methods like the here presented RT-LAMP assay are urgently needed to contain the viral spread.

LebensmittelchemieVolume 74, Issue S1 p. S1-023-S1-023 Poster der 71. Arbeitstagung des Regionalverbands Bayern (9.–10. März 2020, Würzburg) Quantifizierung von Tierarten in Lebensmitteln mittels digitaler PCR Satsada Vanthavongsee, Satsada Vanthavongsee Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Veterinärstraße 2, 85764 OberschleißheimSearch for more papers by this authorPatrick Guertler, Patrick Guertler Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Veterinärstraße 2, 85764 OberschleißheimSearch for more papers by this authorMarzena Maggipinto, Marzena Maggipinto Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Veterinärstraße 2, 85764 OberschleißheimSearch for more papers by this authorMelanie Pavlovic, Melanie Pavlovic Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Veterinärstraße 2, 85764 OberschleißheimSearch for more papers by this authorIngrid Huber, Ingrid Huber Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Veterinärstraße 2, 85764 OberschleißheimSearch for more papers by this author Satsada Vanthavongsee, Satsada Vanthavongsee Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Veterinärstraße 2, 85764 OberschleißheimSearch for more papers by this authorPatrick Guertler, Patrick Guertler Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Veterinärstraße 2, 85764 OberschleißheimSearch for more papers by this authorMarzena Maggipinto, Marzena Maggipinto Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Veterinärstraße 2, 85764 OberschleißheimSearch for more papers by this authorMelanie Pavlovic, Melanie Pavlovic Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Veterinärstraße 2, 85764 OberschleißheimSearch for more papers by this authorIngrid Huber, Ingrid Huber Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit, Veterinärstraße 2, 85764 OberschleißheimSearch for more papers by this author First published: 05 May 2020 https://doi.org/10.1002/lemi.202051023AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article. Volume74, IssueS1Supplement: Vorträge und Poster der 71. Arbeitstagung des Regionalverbands BayernMarch 2020Pages S1-023-S1-023 RelatedInformation

Genetically modified alfalfa is authorized for cultivation in several countries since 2005. On the other hand, cultivation in or export to the European Union is not allowed and thus neither certified reference material nor official event-specific detection methods are available. Therefore, based on patent sequence information, event-specific real-time PCR detection methods targeting the junction sequence of the alfalfa genome and the transgenic insert of the respective events J101, J163 and KK179 were developed. Newly developed plasmids were used as reference material for assay optimization and in-house validation. Plasmid standards were quantified using digital droplet PCR and LOD95%, PCR efficiency, robustness and specificity of the assays were determined using real-time PCR. A LOD95% of 10 copies per PCR reaction was observed and PCR efficiencies of 95–97 % were achieved. Different real-time PCR instruments and PCR conditions were applied to test for robustness of the assays using DNA at a concentration of 30 copies per μL for each gm alfalfa event. All replicates were positive independent of the instrument or the PCR condition. DNA from certified reference material of different genetically modified crops as well as reference materials of the three events was used to experimentally test for specificity. No unspecific amplification signal was observed for any of the assays. Validation results were in line with the “Minimum Performance Requirements for Analytical Methods of GMO Testing” of the European Network of GMO Laboratories. Furthermore, an inter-laboratory comparison study was conducted to show the transferability and applicability of the methods and to verify the assay performance parameters.

LebensmittelchemieVolume 73, Issue S1 p. S123-S123 Lebensmittel(bio)technologie (ATW-L) Genome Editing – Überblick und Auswirkung des EuGH-Urteils Christin-Kirsty Baillie, Christin-Kirsty Baillie Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit (LGL), LH, Veterinärstr. 2, 85764 OberschleißheimSearch for more papers by this authorPatrick Guertler, Patrick Guertler Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit (LGL), LH, Veterinärstr. 2, 85764 OberschleißheimSearch for more papers by this authorArmin Baiker, Armin Baiker Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit (LGL), LH, Veterinärstr. 2, 85764 OberschleißheimSearch for more papers by this authorUlrich Busch, Ulrich Busch Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit (LGL), LH, Veterinärstr. 2, 85764 OberschleißheimSearch for more papers by this author Christin-Kirsty Baillie, Christin-Kirsty Baillie Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit (LGL), LH, Veterinärstr. 2, 85764 OberschleißheimSearch for more papers by this authorPatrick Guertler, Patrick Guertler Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit (LGL), LH, Veterinärstr. 2, 85764 OberschleißheimSearch for more papers by this authorArmin Baiker, Armin Baiker Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit (LGL), LH, Veterinärstr. 2, 85764 OberschleißheimSearch for more papers by this authorUlrich Busch, Ulrich Busch Bayerisches Landesamt für Gesundheit und Lebensmittelsicherheit (LGL), LH, Veterinärstr. 2, 85764 OberschleißheimSearch for more papers by this author First published: 05 May 2020 https://doi.org/10.1002/lemi.201951123AboutPDF ToolsRequest permissionExport citationAdd to favoritesTrack citation ShareShare Give accessShare full text accessShare full-text accessPlease review our Terms and Conditions of Use and check box below to share full-text version of article.I have read and accept the Wiley Online Library Terms and Conditions of UseShareable LinkUse the link below to share a full-text version of this article with your friends and colleagues. Learn more.Copy URL Share a linkShare onFacebookTwitterLinkedInRedditWechat No abstract is available for this article. Volume73, IssueS1Supplement: 48. Deutscher LebensmittelchemikertagSeptember 2019Pages S123-S123 RelatedInformation

[This corrects the article DOI: 10.1016/j.bdq.2018.12.001.].

Analysis of non-transgenic seed samples for the presence of genetically modified maize MON863 revealed unexpected amplification signals using an official qPCR method. These amplification signals only occured when using other master mix products as in the original validation process. DNA sequence data from an unspecific amplicon could be mapped to mitochondrial maize DNA reference sequence. Oligo sequence analysis revealed that forward primer and probe both can hybridize to the mitochondrial maize DNA leading to unspecific amplification signals in qPCR. Therefore, we designed and validated a new qPCR method for event MON863 with a LOD of 5 copies per reaction. The method shows high specificity as no unspecific amplification signal was detected after analysis of reference material for different genetically modified crops and conventional maize samples. Robustness tests were performed in two different laboratories and no effects on method performance could be observed when using different master mixes and qPCR devices, as well as with variation in oligonucleotide concentrations.

Bei der Zulassung von gentechnisch veränderten (gv) Pflanzen (GVP) in der Europäischen Union müssen Hersteller dieser Pflanzen neben Referenzmaterial und geeigneten Nachweismethoden auch Informationen über die gentechnische Veränderung bereitstellen. Bei nicht zugelassenen GVP fehlen Informationen über den genetischen Aufbau teilweise oder völlig. In einem Projekt am Bayerischen Landesamt für Gesundheit und Lebensmittelsicherheit (LGL) soll daher ein Workflow zur Charakterisierung von neuen und unbekannten GVP mittels Next Generation Sequencing (NGS) entwickelt werden. Für die Entwicklung wurden zwei gv Maislinien (MON88017, MON89034) verwendet, für die bereits detaillierte Sequenzinformationen zum inserierten DNA-Fragment und den flankierenden Regionen vorliegen. Eine entwickelte automatisierte DNA-Extraktionsmethode wurde für die Isolierung der DNA verwendet. Die isolierte DNA wurde genutzt, um die Präparation von NGS-Libraries zu etablieren. Die Libraries wurden mittels real-time PCR quantifiziert und anschließend am Genzentrum der Ludwig-Maximilians-Universität sequenziert. Die Auswertung der Daten erfolgt derzeit am LGL.