Lutz Grohmann

Detecting un-authorized genetically modified organisms (GMOs) and derived materials

Genetically modified plants, in the following referred to as genetically modified organisms or GMOs, have been commercially grown for almost two decades. In 2010 approximately 10% of the total global crop acreage was planted with GMOs (James, 2011). More than 30 countries have been growing commercial GMOs, and many more have performed field trials. Although the majority of commercial GMOs both in terms of acreage and specific events belong to the four species: soybean, maize, cotton and rapeseed, there are another 20+ species where GMOs are commercialized or in the pipeline for commercialization. The number of GMOs cultivated in field trials or for commercial production has constantly increased during this time period. So have the number of species, the number of countries involved, the diversity of novel (added) genetic elements and the global trade. All of these factors contribute to the increasing complexity of detecting and correctly identifying GMO derived material. Many jurisdictions, including the European Union (EU), legally distinguish between authorized (and therefore legal) and un-authorized (and therefore illegal) GMOs. Information about the developments, field trials, authorizations, cultivation, trade and observations made in the official GMO control laboratories in different countries around the world is often limited, despite several attempts such as the OECD BioTrack for voluntary dissemination of data. This lack of information inevitably makes it challenging to detect and identify GMOs, especially the un-authorized GMOs. The present paper reviews the state of the art technologies and approaches in light of coverage, practicability, sensitivity and limitations. Emphasis is put on exemplifying practical detection of un-authorized GMOs. Although this paper has a European (EU) bias when examples are given, the contents have global relevance.

A practical approach to screen for authorised and unauthorised genetically modified plants

In routine analysis, screening methods based on real-time PCR are most commonly used for the detection of genetically modified (GM) plant material in food and feed. In this paper, it is shown that the combination of five DNA target sequences can be used as a universal screening approach for at least 81 GM plant events authorised or unauthorised for placing on the market and described in publicly available databases. Except for maize event LY038, soybean events DP-305423 and BPS-CV127-9 and cotton event 281-24-236 × 3006-210-23, at least one of the five genetic elements has been inserted in these GM plants and is targeted by this screening approach. For the detection of these sequences, fully validated real-time PCR methods have been selected. A screening table is presented that describes the presence or absence of the target sequences for most of the listed GM plants. These data have been verified either theoretically according to available databases or experimentally using available reference materials. The screening table will be updated regularly by a network of German enforcement laboratories.

Collaborative Trial Validation Studies of Real-Time PCR-Based GMO Screening Methods for Detection of the bar Gene and the ctp2-cp4epsps Construct

Polymerase Chain Reaction (PCR)-based screening methods targeting genetic elements commonly used in genetically modified (GM) plants are important tools for the detection of GM materials in food, feed, and seed samples. To expand and harmonize the screening capability of enforcement laboratories, the German Federal Office of Consumer Protection and Food Safety conducted collaborative trials for interlaboratory validation of real-time PCR methods for detection of the phosphinothricin acetyltransferase (bar) gene from Streptomyces hygroscopicus and a construct containing the 5-enolpyruvylshikimate-3-phosphate synthase gene from Agrobacterium tumefaciens sp. strain CP4 (ctp2-cp4epsps), respectively. To assess the limit of detection, precision, and accuracy of the methods, laboratories had to analyze two sets of 18 coded genomic DNA samples of events LLRice62 and MS8 with the bar method and NK603 and GT73 with the ctp2-cp4epsps method at analyte levels of 0, 0.02, and 0.1% GM content, respectively. In addition, standard DNAs were provided to the laboratories to generate calibration curves for copy number quantification of the bar and ctp2-cp4epsps target sequences present in the test samples. The study design and the results obtained are discussed with respect to the difficult issue of developing general guidelines and concepts for the collaborative trial validation of qualitative PCR screening methods.

Development and Validation of Duplex, Triplex, and Pentaplex Real-Time PCR Screening Assays for the Detection of Genetically Modified Organisms in Food and Feed

Worldwide, qualitative methods based on PCR are most commonly used as screening tools for genetically modified material in food and feed. However, the increasing number and diversity of genetically modified organisms (GMO) require effective methods for simultaneously detecting several genetic elements marking the presence of transgenic events. Herein we describe the development and validation of a pentaplex, as well as complementary triplex and duplex real-time PCR assays, for the detection of the most common screening elements found in commercialized GMOs: P-35S, T-nos, ctp2-cp4-epsps, bar, and pat. The use of these screening assays allows the coverage of many GMO events globally approved for commercialization. Each multiplex real-time PCR assay shows high specificity and sensitivity with an absolute limit of detection below 20 copies for the targeted sequences. We demonstrate by intra- and interlaboratory tests that the assays are robust as well as cost- and time-effective for GMO screening if applied in routine GMO analysis.

Collaborative Study of a T-nos Real-Time PCR Method for Screening of Genetically Modified Organisms in Food Products

Abstract:In the EU the presence of genetically modified organisms (GMO) in food is controlled by the Member States official laboratories within their national inspection and monitoring programs. The most frequently used approach to detect the presence of GMO material in different food matrices is the PCR-based screening for the CaMV 35S promoter and the nos terminator (T-nos) DNA sequence from Agrobacterium tumefaciens. A real-time PCR method for detection of T-nos and its validation in a collaborative trial study is described in this paper. The PCR system amplifies a 84 bp fragment and showed to be sensitive to detect at least 5 copies of the T-nos DNA sequence. The assay was adapted for use in real-time PCR instruments using plastic reaction vials and glass capillaries, respectively. A total of 24 laboratories participated in the study and each laboratory received 18 DNA blind samples prepared from GMO certified reference materials (0.1 % and 0.5 % NK601 maize) and from a non-GM maize flour. All samples containing NK603 maize DNA were correctly classified as T-nos positive by the participating laboratories. For the blank samples (0 % GMO) only three false-positive results were reported. A detailed evaluation of the results of the collaborative trial study is described.Zusammenfassung:In der Europäischen Gemeinschaft werden im Rahmen von nationalen Kontrollprogrammen von den zuständigen Überwachungslaboratorien der Mitgliedstaaten umfangreiche Untersuchungen von Lebensmitteln in Hinsicht auf die Anwesenheit gentechnisch veränderter Organismen (GVO) durchgeführt. Zur Untersuchung verschiedenster Lebensmittelmatrices kommen in den meisten Fällen die PCR basierten Screening- Verfahren zum Nachweis der DNA-Sequenz des CaMV 35S Promotors und des nos Terminators (T-nos) aus Agrobacterium tumefaciens zum Einsatz. In der vorliegenden Veröffentlichung wird eine real-time PCR Methode zum T-nos Nachweis und deren Validierung in einem Ringversuch beschrieben.Mit der T-nos PCR Methode wird ein nur 84 Basenpaar langes DNA-Fragment amplifiziert. In Bezug auf die Sensitivität der Methode konnte gezeigt werden, dass mindestens 5 Kopien der T-nos Sequenz nachweisbar sind. Das T-nos real-time PCR-Verfahren kann mit Geräten, die Reaktionsgefäße aus Kunststoff oder aber Glaskapillaren verwenden, durchgeführt werden. An dem Ringversuch nahmen insgesamt 24 Labore teil. Jedes Labor erhielt 18 kodierte DNA-Proben zur Untersuchung. Die Proben wurden aus zertifizierten GVO-Referenzmaterialien (0,1 % und 0,5% NK603) bzw. aus einem Maismehl, das keine GVO-Anteile enthielt, hergestellt. Alle Proben, die DNA aus NK603 Mais enthielten und daher als T-nos positiv anzugeben waren, wurden von den teilnehmenden Laboren richtig erkannt. In Hinsicht auf die negativen Proben (0 % GVO-Anteile) wurden nur drei falschpositive Ergebnisse berichtet. Die Auswertung der Ringversuchsergebnisse wird ausführlich beschrieben.

The GMOseek matrix: a decision support tool for optimizing the detection of genetically modified plants

BackgroundSince their first commercialization, the diversity of taxa and the genetic composition of transgene sequences in genetically modified plants (GMOs) are constantly increasing. To date, the detection of GMOs and derived products is commonly performed by PCR-based methods targeting specific DNA sequences introduced into the host genome. Information available regarding the GMOs’ molecular characterization is dispersed and not appropriately organized. For this reason, GMO testing is very challenging and requires more complex screening strategies and decision making schemes, demanding in return the use of efficient bioinformatics tools relying on reliable information.DescriptionThe GMOseek matrix was built as a comprehensive, online open-access tabulated database which provides a reliable, comprehensive and user-friendly overview of 328 GMO events and 247 different genetic elements (status: 18/07/2013). The GMOseek matrix is aiming to facilitate GMO detection from plant origin at different phases of the analysis. It assists in selecting the targets for a screening analysis, interpreting the screening results, checking the occurrence of a screening element in a group of selected GMOs, identifying gaps in the available pool of GMO detection methods, and designing a decision tree. The GMOseek matrix is an independent database with effective functionalities in a format facilitating transferability to other platforms. Data were collected from all available sources and experimentally tested where detection methods and certified reference materials (CRMs) were available.ConclusionsThe GMOseek matrix is currently a unique and very valuable tool with reliable information on GMOs from plant origin and their present genetic elements that enables further development of appropriate strategies for GMO detection. It is flexible enough to be further updated with new information and integrated in different applications and platforms.

Collaborative trial validation of cry1Ab/Ac and Pubi-cry TaqMan-based real-time PCR assays for detection of DNA derived from genetically modified Bt plant products

Presence of genetic modifications in rice products originating from China and imported to the European Union market is detected since 2006. Neither these products from China nor any other genetically modified rice lines are approved as food or feed in the EU. The transgenic rice varieties identified contain genetic elements and constructs coding for insect-resistance genes from Bacillus thuringiensis (Bt) coding for insecticidal crystal (cry) proteins. In particular, DNA sequences coding for codon-optimised or fused cry1Ab/c genes and constructs driven by the maize ubiquitin promoter (P-ubiZM1) were identified. For improved screening and identification of genetic modifications present in Asian rice products, two TaqMan-based real-time PCR assays targeting codon-optimised cry1Ab/Ac and the Pubi-cry construct have been developed. These assays have been validated in an international collaborative trial with 17 participants from 10 countries. Based on a new mathematical–statistical model and an adjusted experimental set-up of the collaborative trial, a close examination of the limit of detection (LOD95%) and the probability of detection of the qualitative PCR assays was conducted. The evaluation of the method performance characteristics and results of the collaborative trial validation are presented.

Detection and characterization of an unknown rice event in Basmati rice products

At the end of 2011, genetic modifications in Basmati rice were discovered for the first time in products placed on the European Union market. The products originated from Pakistan or India. In the EU, no event of genetically modified rice is approved as food or feed. The samples were initially identified by positive PCR screening results. Some of the detected genetically modified DNA sequences were previously identified in insect-resistant rice varieties originating from Asia. In addition to a sequence coding for a cry1Ab/Ac gene driven by the maize ubiquitin promoter, the integration of a 35S CaMV promoter-driven cry2A gene was detected. This is the first notification of the presence of a cry2A gene in Asian rice products in the EU.

Guidelines for validation of DNA extraction methods applied in subsequent PCR analysis of food and feed products for the presence of genetically modified material

Guidelines for the validation of DNA extraction methods especially in the scope of the analysis of genetically modified sequences in food and feed products by real-time polymerase chain reaction (real-time PCR) are described. Performance testing of extracted DNA is primarily done by determination of the quantity and quality of amplifiable DNA in real-time PCR. Assessment of quality of extracted DNA includes determination of DNA concentration (e.g. fluorimetrically) and evaluation of fragmentation status by gel electrophoresis. A detailed procedure for in-house validation as well as subsequent inter-laboratory validation studies of DNA extraction methods is presented.ZusammenfassungEs werden Leitlinien für die Validierung von DNA-Extraktionsverfahren, insbesondere im Bereich der Untersuchung auf gentechnische Veränderungen mittels real-time PCR, beschrieben. Der Eignungstest extrahierter DNA erfolgt schwerpunktmäßig durch die Bestimmung amplifizierbarer DNA mittels real-time PCR. Weitere Untersuchungen sind die Bestimmung der DNA-Konzentration und des Fragmentierungsstatus mittels Gel-Elektrophorese. Eine detaillierte Beschreibung für eine Vorgehensweise zur Validierung von DNA-Extraktionsverfahren sowohl innerhalb eines Labors als auch im Ringversuch wird vorgestellt.

Detection of Genetically Modified Plants in Seeds, Food and Feed

Different techniques and analytical strategies are applied for detecting and quantifying the presence of genetically modified (GM) plants in food and feed products or in seeds. DNA-based detection is performed by qualitative PCR or by quantitative real-time PCR, whereas for protein-based detection immunoassays such as lateral flow devices and ELISA are applied. The testing strategy for GMO detection is constituted of a series of steps starting with a screening for frequently inserted genetic elements and gene constructs, followed by specific identification of the GM plant event and completing the analysis with the quantification of the relative amount of the GM plant event present in a given sample. This chapter outlines also the challenges currently emerging by stacked events or by the incidences of unauthorised GM plants. It provides Information on guidance documents and databases for validated detection methods used across routine control laboratories today.