A convenient renewable surface plasmon resonance chip for relative quantification of genetically modified soybean in food and feed.

The cultivation of genetically modified organisms (GMO) continues to expand worldwide. Still, many consumers express concerns about the use of GMO in food or feed, and many countries have legislated on labelling systems to indicate the presence of GMO in commercial products. To deal with the increased number of GMO events and to address related regulations, alternative detection methods for GMO inspection are required. In this work, a genosensor based on Surface Plasmon Resonance under continuous flow was developed for the detection and quantification of a genetically modified soybean (event GTS 40-3-2). In a single chip, the simultaneous detection of the event-specific and the taxon-specific samples were achieved, whose detection limits were 20 pM and 16 pM, respectively. The reproducibility was 1.4%, which supports the use of the chip as a reliable and cost-effective alternative to other DNA-based techniques. The results indicate that the proposed method is a versatile tool for GMO quantification in food and feed samples.

Potential risk evaluation for unintended entry of genetically modified plant Propagating material in Europe through import of seeds and animal feed - the experience of Latvia.

Significant attention has been drawn to the adventitious and technically unavoidable presence of genetically modified (GM) organisms in the food and feed imported into the European Union (EU), while the potential presence of GM seeds in material for cultivation is less studied. Here we report a study from an EU member state, Latvia, during years 2017-2018 regarding monitoring for the presence of GM seeds in certified seed and animal feed material. Eighty-two and 28 samples of seeds intended for cultivation were analyzed in 2017 and 2018, respectively. One soybean sample contained MON40-3-2 soybean seeds (0.09 ± 0.01%) and one maize sample contained MON810 maize seeds (0.08 ± 0.01%). In addition, 102 samples of feed imported from outside of the EU or produced locally were also analyzed for the presence of genetically modified organisms (GMOs) and viability of grains. One oilseed rape cake sample contained GT73 (1.04 ± 0.01%) and one soybean cake sample contained MON40-3-2 (<0.045%). One sample of declared MON40-3-2 GM soybean cake was confirmed to be positive, with MON40-3-2 content of 94.78 ± 10.01%. One soybean sample submitted by feed producer and originating from Argentina contained 54.9 ± 1.1% of MON40-3-2 and one rapeseed sample originating from Ukraine contained 5.30 ± 3.95% of GT73. Although only two seed samples contained low levels of GMOs authorized in the EU for food and feed uses, this study reinforced the need to maintain regular monitoring programs that assist farmers in their efforts to comply with the current EU GMO legislation.

A label survey to identify ingredients potentially containing GM organisms to estimate intake exposure in Brazil.

OBJECTIVE: To identify ingredients from products and by-products derived from GM crops in packaged food products and to analyse the presence of these ingredients in the foods most commonly consumed by the Brazilian population. DESIGN: Cross-sectional study. SETTING: A search of the scientific literature to identify the use of products and by-products derived from GM crops in foods in Brazil and a study of food labels in a supermarket belonging to one of the ten largest supermarket chains in Brazil. SUBJECTS: To identify the ingredients present in packaged food products and their nomenclatures, the labels of all packaged food products available for sale in a supermarket were analysed. Subsequently, the presence of potential GM ingredients in the foods most commonly consumed by the Brazilian population was analysed. RESULTS: A total of twenty-eight GM crops' by-products with applications in the food industry (from soyabeans, corn, cotton and a yeast) were identified. Such by-products are presented as food ingredients or additives on food labels with 101 distinct nomenclatures. Most of the variety (63·8 %) and the quantity (64·5 %) of the foods most commonly consumed by Brazilians may contain a least one GM ingredient. CONCLUSIONS: The presence of at least one potential GM ingredient was observed in more than half of the variety of foods most commonly consumed by the Brazilian population. Such ingredients were identified with distinct nomenclatures and incomplete descriptions, which may make it difficult to identify potential GM foods and confuse consumers when making food choices.

A high-throughput liquid bead array-based screening technology for Bt presence in GMO manipulation.

The number of species and planting areas of genetically modified organisms (GMOs) has been rapidly developed during the past ten years. For the purpose of GMO inspection, quarantine and manipulation, we have now devised a high-throughput Bt-based GMOs screening method based on the liquid bead array. This novel method is based on the direct competitive recognition between biotinylated antibodies and beads-coupled antigens, searching for Bt presence in samples if it contains Bt Cry1 Aa, Bt Cry1 Ab, Bt Cry1 Ac, Bt Cry1 Ah, Bt Cry1 B, Bt Cry1 C, Bt Cry1 F, Bt Cry2 A, Bt Cry3 or Bt Cry9 C. Our method has a wide GMO species coverage so that more than 90% of the whole commercialized GMO species can be identified throughout the world. Under our optimization, specificity, sensitivity, repeatability and availability validation, the method shows a high specificity and 10-50 ng/mL sensitivity of quantification. We then assessed more than 1800 samples in the field and food market to prove capacity of our method in performing a high throughput screening work for GMO manipulation. Our method offers an applicant platform for further inspection and research on GMO plants.

Current and new approaches in GMO detection: challenges and solutions.

In many countries, genetically modified organisms (GMO) legislations have been established in order to guarantee the traceability of food/feed products on the market and to protect the consumer freedom of choice. Therefore, several GMO detection strategies, mainly based on DNA, have been developed to implement these legislations. Due to its numerous advantages, the quantitative PCR (qPCR) is the method of choice for the enforcement laboratories in GMO routine analysis. However, given the increasing number and diversity of GMO developed and put on the market around the world, some technical hurdles could be encountered with the qPCR technology, mainly owing to its inherent properties. To address these challenges, alternative GMO detection methods have been developed, allowing faster detections of single GM target (e.g., loop-mediated isothermal amplification), simultaneous detections of multiple GM targets (e.g., PCR capillary gel electrophoresis, microarray, and Luminex), more accurate quantification of GM targets (e.g., digital PCR), or characterization of partially known (e.g., DNA walking and Next Generation Sequencing (NGS)) or unknown (e.g., NGS) GMO. The benefits and drawbacks of these methods are discussed in this review.

A novel reference plasmid for the qualitative detection of genetically modified rice in food and feed.

Rice is one of the most important food crops in the world. Genetically modified (GM) technology has been used in rice to confer herbicide tolerance and pathogen or insect resistance. China invests heavily in research on GM rice. By the end of 2014, at least 250 transgenic rice lines had been developed in China. To monitor the presence of GM rice in food and feed, we collected information on foreign elements from 250 transgenic rice lines and found 5 elements, including the Agrobacterium tumefaciens nopaline synthase terminator (T-NOS), the cauliflower mosaic virus 35S promoter (CaMV35S), the ubiquitin gene (Ubi), the bar gene, and the hygromycin phosphotransferase gene (Hpt), that are commonly present in GM rice. Therefore, we constructed a novel plasmid (pBJGMM001) that contains fragments of these elements and two endogenous reference genes (the sucrose phosphate synthase gene, SPS, and the phosphoenolpyruvate carboxylase gene, PEPC). pBJGMM001 can serve as a standard for detecting 96% of GM rice lines in China. The primers, amplicons, reaction mixture, and PCR program were developed based on Chinese National Standards. The protocol was validated and determined to be suitable for practical use in monitoring and identifying GM rice.

Analytical method evaluation and discovery of variation within maize varieties in the context of food safety: transcript profiling and metabolomics.

Profiling techniques such as microarrays, proteomics, and metabolomics are used widely to assess the overall effects of genetic background, environmental stimuli, growth stage, or transgene expression in plants. To assess the potential regulatory use of these techniques in agricultural biotechnology, we carried out microarray and metabolomic studies of 3 different tissues from 11 conventional maize varieties. We measured technical variations for both microarrays and metabolomics, compared results from individual plants and corresponding pooled samples, and documented variations detected among different varieties with individual plants or pooled samples. Both microarray and metabolomic technologies are reproducible and can be used to detect plant-to-plant and variety-to-variety differences. A pooling strategy lowered sample variations for both microarray and metabolomics while capturing variety-to-variety variation. However, unknown genomic sequences differing between maize varieties might hinder the application of microarrays. High-throughput metabolomics could be useful as a tool for the characterization of transgenic crops. However, researchers will have to take into consideration the impact on the detection and quantitation of a wide range of metabolites on experimental design as well as validation and interpretation of results.

Bioinformatics and the allergy assessment of agricultural biotechnology products: industry practices and recommendations.

Bioinformatic tools are being increasingly utilized to evaluate the degree of similarity between a novel protein and known allergens within the context of a larger allergy safety assessment process. Importantly, bioinformatics is not a predictive analysis that can determine if a novel protein will ''become" an allergen, but rather a tool to assess whether the protein is a known allergen or is potentially cross-reactive with an existing allergen. Bioinformatic tools are key components of the 2009 CodexAlimentarius Commission's weight-of-evidence approach, which encompasses a variety of experimental approaches for an overall assessment of the allergenic potential of a novel protein. Bioinformatic search comparisons between novel protein sequences, as well as potential novel fusion sequences derived from the genome and transgene, and known allergens are required by all regulatory agencies that assess the safety of genetically modified (GM) products. The objective of this paper is to identify opportunities for consensus in the methods of applying bioinformatics and to outline differences that impact a consistent and reliable allergy safety assessment. The bioinformatic comparison process has some critical features, which are outlined in this paper. One of them is a curated, publicly available and well-managed database with known allergenic sequences. In this paper, the best practices, scientific value, and food safety implications of bioinformatic analyses, as they are applied to GM food crops are discussed. Recommendations for conducting bioinformatic analysis on novel food proteins for potential cross-reactivity to known allergens are also put forth.

A risk-based classification scheme for genetically modified foods. II: Graded testing.

This paper presents a graded approach to the testing of crop-derived genetically modified (GM) foods based on concern levels in a proposed risk-based classification scheme (RBCS) and currently available testing methods. A graded approach offers the potential for more efficient use of testing resources by focusing less on lower concern GM foods, and more on higher concern foods. In this proposed approach to graded testing, products that are classified as Level I would have met baseline testing requirements that are comparable to what is widely applied to premarket assessment of GM foods at present. In most cases, Level I products would require no further testing, or very limited confirmatory analyses. For products classified as Level II or higher, additional testing would be required, depending on the type of the substance, prior dietary history, estimated exposure level, prior knowledge of toxicity of the substance, and the nature of the concern related to unintended changes in the modified food. Level III testing applies only to the assessment of toxic and antinutritional effects from intended changes and is tailored to the nature of the substance in question. Since appropriate test methods are not currently available for all effects of concern, future research to strengthen the testing of GM foods is discussed.

A risk-based classification scheme for genetically modified foods. I: Conceptual development.

The predominant paradigm for the premarket assessment of genetically modified (GM) foods reflects heightened public concern by focusing on foods modified by recombinant deoxyribonucleic acid (rDNA) techniques, while foods modified by other methods of genetic modification are generally not assessed for safety. To determine whether a GM product requires less or more regulatory oversight and testing, we developed and evaluated a risk-based classification scheme (RBCS) for crop-derived GM foods. The results of this research are presented in three papers. This paper describes the conceptual development of the proposed RBCS that focuses on two categories of adverse health effects: (1) toxic and antinutritional effects, and (2) allergenic effects. The factors that may affect the level of potential health risks of GM foods are identified. For each factor identified, criteria for differentiating health risk potential are developed. The extent to which a GM food satisfies applicable criteria for each factor is rated separately. A concern level for each category of health effects is then determined by aggregating the ratings for the factors using predetermined aggregation rules. An overview of the proposed scheme is presented, as well as the application of the scheme to a hypothetical GM food.

A risk-based classification scheme for genetically modified foods. III: Evaluation using a panel of reference foods.

This paper presents an exploratory evaluation of four functional components of a proposed risk-based classification scheme (RBCS) for crop-derived genetically modified (GM) foods in a concordance study. Two independent raters assigned concern levels to 20 reference GM foods using a rating form based on the proposed RBCS. The four components of evaluation were: (1) degree of concordance, (2) distribution across concern levels, (3) discriminating ability of the scheme, and (4) ease of use. At least one of the 20 reference foods was assigned to each of the possible concern levels, demonstrating the ability of the scheme to identify GM foods of different concern with respect to potential health risk. There was reasonably good concordance between the two raters for the three separate parts of the RBCS. The raters agreed that the criteria in the scheme were sufficiently clear in discriminating reference foods into different concern levels, and that with some experience, the scheme was reasonably easy to use. Specific issues and suggestions for improvements identified in the concordance study are discussed.

Comparison of different real-time PCR chemistries and their suitability for detection and quantification of genetically modified organisms.

BACKGROUND: The real-time polymerase chain reaction is currently the method of choice for quantifying nucleic acids in different DNA based quantification applications. It is widely used also for detecting and quantifying genetically modified components in food and feed, predominantly employing TaqMan and SYBR Green real-time PCR chemistries. In our study four alternative chemistries: Lux, Plexor, Cycling Probe Technology and LNA were extensively evaluated and compared using TaqMan chemistry as a reference system. RESULTS: Amplicons were designed on the maize invertase gene and the 5'-junction of inserted transgene and plant genomic DNA in MON 810 event. Real-time assays were subsequently compared for their efficiency in PCR amplification, limits of detection and quantification, repeatability and accuracy to test the performance of the assays. Additionally, the specificity of established assays was checked on various transgenic and non-transgenic plant species. The overall applicability of the designed assays was evaluated, adding practicability and costs issues to the performance characteristics. CONCLUSION: Although none of the chemistries significantly outperformed the others, there are certain characteristics that suggest that LNA technology is an alternative to TaqMan when designing assays for quantitative analysis. Because LNA probes are much shorter they might be especially appropriate when high specificity is required and where the design of a common TaqMan probe is difficult or even impossible due to sequence characteristics. Plexor on the other hand might be a method of choice for qualitative analysis when sensitivity, low cost and simplicity of use prevail.

Genetically Engineered Plants and Foods: A Scientist's Analysis of the Issues (Part I).

Through the use of the new tools of genetic engineering, genes can be introduced into the same plant or animal species or into plants or animals that are not sexually compatible-the latter is a distinction with classical breeding. This technology has led to the commercial production of genetically engineered (GE) crops on approximately 250 million acres worldwide. These crops generally are herbicide and pest tolerant, but other GE crops in the pipeline focus on other traits. For some farmers and consumers, planting and eating foods from these crops are acceptable; for others they raise issues related to safety of the foods and the environment. In Part I of this review some general and food issues raised regarding GE crops and foods will be addressed. Responses to these issues, where possible, cite peer-reviewed scientific literature. In Part II to appear in 2009, issues related to environmental and socioeconomic aspects of GE crops and foods will be covered.