Sven Pecoraro

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.

GMOfinder—A GMO Screening Database

The development and cultivation of genetically modified crops is still increasing globally. Food and feed imports from outside the European Union (EU) will subsequently require more effort from the responsible authorities in monitoring the compliance with effective labelling regulations. The aim of this study was the development of the GMOfinder, a database for collection and interpretation of information related to the screening for genetically modified organisms (GMOs). Different genetic elements (e.g. promoters, terminators, structural genes) are artificially introduced into plants to establish new genetic modifications. The introduced elements may vary between GMO events, depending on the intended trait(s). Screening for such inserted elements with (real-time) polymerase chain reaction is a common first step to analyse samples for the presence of any genetical modification. From the pattern of detectable and nondetectable elements, valuable conclusions about the identity of putative present GMO event(s) can be drawn with the GMOfinder. Information about selected genetic elements from the literature, applications for authorisation and other (web) sources were systematically integrated in a tabular matrix format. Special care was taken to additionally record the sources of the information, thus facilitating evaluation of screening results, and tracing of possible errors in the matrix. The GMOfinder accesses data from the element matrix with implemented algorithms and facilitates to interpret the outcome of screenings. Such a preselection helps to systematically narrow down the candidates for subsequent identification reactions. Optional display of events with potentially masked elements completes the included features.

Optimization of digital droplet polymerase chain reaction for quantification of genetically modified organisms.

Graphical abstract

DUPLEX POLYMERASE CHAIN REACTION (PCR) FOR THE SIMULTANEOUS DETECTION OF CRYIA(B) AND THE MAIZE UBIQUITIN PROMOTER IN THE TRANSGENIC RICE LINE KMD1

ABSTRACT This study describes a combination of screening and gene-specific methods for the detection of GMO in food and feed. Using specific primers, the maize ubiquitin promoter and the cryIA(b) gene, which encodes for a delta-endotoxin transferring insect resistance, can be detected simultaneously by a duplex PCR in samples containing the transgenic rice line Kemingdao1 (KMD1). This method is also suitable for the screening for Bt11 maize.

Molecular characterization of an unauthorized genetically modified Bacillus subtilis production strain identified in a vitamin B2 feed additive

Highlights • Genetically modified Bacillus subtilis identified in a vitamin B2 product.• Whole genome sequencing runs are performed for characterization of the isolated strain.• Complex modifications of the genome are identified.• Four putative recombinant plasmids are characterized.• Real-time PCR methods are developed and available for testing vitamin B2 products.

A statistical approach to quantification of genetically modified organisms (GMO) using frequency distributions

BackgroundAccording to Regulation (EU) No 619/2011, trace amounts of non-authorised genetically modified organisms (GMO) in feed are tolerated within the EU if certain prerequisites are met. Tolerable traces must not exceed the so-called ‘minimum required performance limit’ (MRPL), which was defined according to the mentioned regulation to correspond to 0.1% mass fraction per ingredient. Therefore, not yet authorised GMO (and some GMO whose approvals have expired) have to be quantified at very low level following the qualitative detection in genomic DNA extracted from feed samples. As the results of quantitative analysis can imply severe legal and financial consequences for producers or distributors of feed, the quantification results need to be utterly reliable.ResultsWe developed a statistical approach to investigate the experimental measurement variability within one 96-well PCR plate. This approach visualises the frequency distribution as zygosity-corrected relative content of genetically modified material resulting from different combinations of transgene and reference gene Cq values. One application of it is the simulation of the consequences of varying parameters on measurement results. Parameters could be for example replicate numbers or baseline and threshold settings, measurement results could be for example median (class) and relative standard deviation (RSD). All calculations can be done using the built-in functions of Excel without any need for programming. The developed Excel spreadsheets are available (see section ‘Availability of supporting data’ for details). In most cases, the combination of four PCR replicates for each of the two DNA isolations already resulted in a relative standard deviation of 15% or less.ConclusionsThe aims of the study are scientifically based suggestions for minimisation of uncertainty of measurement especially in —but not limited to— the field of GMO quantification at low concentration levels. Four PCR replicates for each of the two DNA isolations seem to be a reasonable minimum number to narrow down the possible spread of results.

Official food control laboratories in Germany: results of GMO analyses from 2012 to 2016

The testing of food for genetic modificationsis an important area in official food control. In Europe, specific requirements for labelling and authorisation of genetically modified organisms (GMO) and products thereof in food and feed are defined in the Regulations (EC) No. 1829/2003 and 1830/2003. In Germany, GMO analysis of food samples taken within the official food control is performed by the laboratories of the Federal States. For more than 10 years, results of these analyses have been summarized and evaluated. This collection of data was originally done in the context of interpretation of individual results according to the labelling requirements defined by the Regulations 1829/ 2003 and 1830/2003. According to those regulations, specific GMO labelling in food containing material is not necessary if it contains, consists or is produced from GMO of\ 0.9% of the food ingredients—considered individually or food consisting of a single ingredient—provided that this presence is adventitious or technically unavoidable. If GM constituents are detectable in food, ‘‘operators must be in a position to supply evidence to satisfy the competent authorities that they have taken appropriate steps to avoid the presence of such material’’. Experts of official food control in Germany have described an approach for the interpretation of the undefined term ‘‘technically unavoidable’’ (Waiblinger et al. 2007). Generally, whether ingredients derived of genetically modified plants (GMP) can be avoided or not depends on the circumstance of cultivation, the plant species (e.g. soybean and maize), and separation of commodity flows, traceability, availability and demand. To evaluate whether Journal of Consumer Protection and Food Safety Journal für Verbraucherschutz und Lebensmittelsicherheit