GMO Screening Research Articles

Comparative assessment of three commercial kits and in house optimized PCR assays for GMO screening in food and feed

Screening strategies for GMO detection in food and feed are a crucial aspect in GMO testing laboratories for streamlining the analytical workflow and reducing turnaround time and costs. These strategies can be more or less complex or even be targeted according to the ingredients in the product, but whatever the choice, a good basic approach is generally based on the search for 35S promoter (P35S), nos-terminator (T-nos) and FMV promoter (P-FMV). In this study, we compare the singleplex real time PCR method for P35S, T-nos and P-FMV detection currently adopted by the Italian National Reference Laboratory for GM food and feed (NRL) with three commercial kits available on the market for giving a greater choice to consider the best approach suitable to the official control laboratories that are different from each other.•The NRL optimized singleplex PCR methods and the three commercial kits fully respect all the validation parameters criteria according to the minimum performance requirements (MPR) of ENGL [1]•Screening strategies for GMO detection in food and feed are a crucial aspect in GMO testing laboratories and being the commercial kits different from each other, the laboratory can choose the methods best suit their needs reducing turnaround time and costs.

Detection of genetically modified organisms by genetic markers in the local market of Al-Muthanna Province-Iraq

Genetically altered plants also called genetically modified organisms or GMOs, are contentious subjects concerning food regulation. There are several challenges concerning the potential dangers of GMOs' toxic, allergenic and dietary interactions. In this context, sixty-seven food specimens were evaluated soybeans (Glycine max L. Merr), barley (Hordeum vulgare L.), rice cereals (Oryza sativa L.), tomato (Lycopersicum esculentum L.), Corn chips, maize (Zea mays L.), and potato (Solanum tuberosum L.) for GMO content; these samples were obtained from Iraq, and DNA extraction was performed using a test kit. DNA amplification was conducted using polymerase chain reaction (PCR); subsequently, agarose gel and four primers were used to perform electrophoresis to establish the presence of augmented GMO sequences. Experimental outcomes of 67 specimens indicated that 15 specimens tested positive; they comprised three samples each of corn chips, rice, and maize, four soybean samples, and one sample for tomato and corn seed using four primers. These results showed that GMO screening is a suitable technique for detecting transgenic plants, testing and monitoring food specimens and controlling GM levels in foods.

Set-up and in-house validation of two multiplex event-specific PCR methods to simplify screening for GMOs without common screening elements

Since commercialization, the ever growing number of Genetically Modified (GM) crops make routine screening by European enforcement laboratories more expensive, labour-intensive and challenging every year. GM events, with elements for which no detection methods are available, further extend this screening because they require for event-specific methods to be included in the screening. Here, we describe the set-up and in-house validation of two tetraplex qPCR methods for simultaneous detection of six GM events that are not covered by common element-specific screening methods. The two qualitative multiplex PCR methods comprise previously developed and interlaboratory validated singleplex detection assays for GHB614 cotton, DP305423 soybean, CV127 soybean, DAS40278 maize, VCO1981 maize and 98140 maize. A plant-specific actin PCR was included in both multiplex PCR methods as a positive control. The validation data show that the assessed performance parameters are in accordance with the minimum performance requirements suggested by European Network of GMO Laboratories. The two multiplex PCR methods are reliable, ready to use and will lead to a faster and more efficient diagnostic routine GMO screening.

RNase A Enzyme Modification of Optimized SDS Protocol for DNA Extraction Suitable for Real-Time PCR Screening of GMOs

Abstract As the number of genetically modified crops increases rapidly, their accurate detection is significant for labelling and safety assessment. Currently, real-time PCR is the “golden standard” method for GMO detection. Hence, extraction of high quality DNA represents a crucial step for accurate and efficient DNA amplification. For GMO presence evaluation in the extracted DNA from raw corn kernels and roasted soybean, we used real-time PCR method, in consistent with the ISO17025 accreditation standards. As for DNA extraction, modified basic SDS protocol by adding RNase A enzyme in different steps of the protocol, with different time and temperature of incubation was used. The results showed as most suitable, the protocol where 10 µl of RNase A enzyme was added together with the lysis buffer at 65 °C for 30 minutes. Data for DNA yield and purity for roasted soybean was 469.6±3.3 µg/ml with A260/280 absorbance ratio 1.78±0.01. Suitability of DNA extracts for GMO analysis was assessed by screening for the presence of 35S promotor and Tnos terminator. Diluted extracts in concentrations 10, 1, 0.1, 0.01 and 0.0027 ng/µl, were tested in six replicates. Positive signal of amplification (LOD) was detected in all concentrations for both genetic elements in both matrices. The LOQ for 35S and Tnos for both matrices was 0.1 ng, while for Tnos in raw corn kernels was 0.01 ng. This in-house developed DNA extraction method is simple and obtains high-quality DNA suitable for GMO screening of 35S promotor and Tnos terminator in both raw and processed matrices.

Molecular Characterization and Event-Specific Real-Time PCR Detection of Two Dissimilar Groups of Genetically Modified Petunia (Petunia x hybrida) Sold on the Market.

Petunia plants with unusual orange flowers were noticed on the European market and confirmed to be genetically modified (GM) by the Finnish authorities in spring 2017. Later in 2017, inspections and controls performed by several official laboratories of national competent authorities in the European Union detected several GM petunia varieties with orange flowers, but also another group of unusually colored flowers. In the latter group, a so far undetected gene coding for a flavonoid 3’5’ hydroxylase (F3’5’H) responsible for the purple color was identified by German and Dutch authorities, suggesting that the petunias found on the markets contain different genetic constructs. Here, a strategy is described for the identification of GM petunia varieties. It is based on an initial GMO screening for known elements using (real-time) PCR and subsequent identification of the insertion sites by a gene walking-like approach called ALF (amplification of linearly-enriched fragments) in combination with Sanger and MinION sequencing. The results indicate that the positively identified GM petunias can be traced back to two dissimilar GM events used for breeding of the different varieties. The test results also confirm that the transgenic petunia event RL01-17 used in the first German field trial in 1991 is not the origin of the GM petunias sold on the market. On basis of the obtained sequence data, event-specific real-time PCR confirmatory methods were developed and validated. These methods are applicable for the rapid detection and identification of GM petunias in routine analysis. In addition, a decision support system was developed for revealing the most likely origin of the GM petunia.

Ultrafast, universal and visual screening of dual genetically modified elements based on dual super PCR and a lateral flow biosensor

In this study, a cascade screening system has been developed combining Dual Super Polymerase Chain Reaction (DSPCR) with the universal Lateral Flow Biosensor (LFB) for the ultrafast, universal and visual screening of dual GM elements, taking P-35s × T-nos for example. In the design of DSPCR for universal screening, gene-specific forward primers were labelled with biotin and gene-specific reverse primers were tagged with Cy5 and digoxin, respectively. In 2.5-min, DSPCR effectively amplified the dual target fragments through our prototype facility. Then, through specific antigen-antibody binding, a universal lateral flow biosensor exported visually dual-amplified results simultaneously without cross contamination. After optimization, the detection limit allowed 0.05% GM maize, corresponding to nine copies in maize. The entire detection process could be achieved in 10 min without any large-scale instrumentation. This method may be useful for the ultrafast, universal and visual screening of dual GM elements (P-35s × T-nos) in GM crop lines and is expected to be of great promise for rapid GMO screening and point-of-care tests.

SCREENING FOR GMO IN FERMENTED SOY SAUCE

Soy sauce is worldwide popular condiment of Asian origin. With the advent of GM soybean production, soy sauce drew the interest of food safety control. Samples collected for inspection are generally of industrial grade soy sauce type, which is produced from hydrolyzed soybean and grain. Following the failure to perform RealTime PCR based GMO screening on a number of submitted samples we tested our screening system on soy sauce produced following traditional method based on fermentation. Four batches of soy sauce were produced and DNA extracted. DNA concentration ranged from 32,68 to 65,36 ng/μl. Amplification of taxon specific target was successful with rather high Ct ( > 30). Promoter P-35S sequence was not detected, but T-NOS was detected in three samples with values reaching or exceeding LOD of the method. The results show that it is possible to detect transgenic elements in traditionally produced soy sauce while DNA extraction from industrial grade soy sauce is not possible.

Development and application of a general plasmid reference material for GMO screening

The use of analytical controls is essential when performing GMO detection through screening tests. Additionally, the presence of taxon-specific sequences is analyzed mostly for quality control during GMO detection. In this study, 11 commonly used genetic elements involving three promoters (P-35S, P-FMV35S and P-NOS), four marker genes (Bar, NPTII, HPT and Pmi), and four terminators (T-NOS, T-35S, T-g7 and T-e9), together with the reference gene fragments from six major crops of maize, soybean, rapeseed, rice, cotton and wheat, were co-integrated into the same single plasmid to construct a general reference plasmid pBI121-Screening. The suitability test of pBI121-Screening plasmid as reference material indicated that the non-target sequence on the pBI121-Screening plasmid did not affect the PCR amplification efficiencies of screening methods and taxon-specific methods. The sensitivity of screening and taxon-specific assays ranged from 5 to 10 copies of pBI121-Screening plasmid, meeting the sensitivity requirement of GMO detection. The construction of pBI121-Screening solves the lack of a general positive control for screening tests, thereby reducing the workload and cost of preparing a plurality of the positive control.

Real-time and visual loop-mediated isothermal amplification: Efficient GMO screening targeting pat and pmi marker genes

Rapid and efficient screening assays based on loop-mediated isothermal amplification (LAMP) were developed, targeting two marker genes, namely, phosphinothricin-N-acetyltransferase (pat) and phosphomannose isomerase (pmi). These marker genes are being employed in more than 35% of GM events approved globally. Specificity of developed visual and real-time LAMP assays was confirmed using seven GM events of two crops, viz., maize (3272, 59122, Bt11, Bt176, MIR604, TC1507), and cotton (Widestrike™). In visual LAMP, positive amplification can be directly analyzed by the colorimetric change from orange to green, whereas real-time LAMP is based on the monitoring of fluorescence signals as amplification and annealing curves. Visual LAMP was found sensitive enough to detect up to 0.05% GM content for pat and 0.1% for pmi within 40 min. Real-time LAMP efficiently detected up to 0.01% GM content within 30 min. Practical applicability of developed assays was confirmed using proficiency test samples of maize. LAMP assays for pmi gene have been reported for the first time. Due to portability of systems, the developed LAMP assays when combined with a fast DNA extraction method could facilitate on-site GMO screening.

Development and applicability of a ready-to-use PCR system for GMO screening

With the growing number of GMOs introduced to the market, testing laboratories have seen their workload increase significantly. Ready-to-use multi-target PCR-based detection systems, such as pre-spotted plates (PSP), reduce analysis time while increasing capacity. This paper describes the development and applicability to GMO testing of a screening strategy involving a PSP and its associated web-based Decision Support System. The screening PSP was developed to detect all GMOs authorized in the EU in one single PCR experiment, through the combination of 16 validated assays. The screening strategy was successfully challenged in a wide inter-laboratory study on real-life food/feed samples. The positive outcome of this study could result in the adoption of a PSP screening strategy across the EU; a step that would increase harmonization and quality of GMO testing in the EU. Furthermore, this system could represent a model for other official control areas where high-throughput DNA-based detection systems are needed.

Detection of genetically modified organisms in soy products sold in Turkish market

PCR-based technique for GMO detection is the most reliable choice because of its high sensitivity and specificity. As a candidate of the European Union, Turkey must comply with the rules for launching into the market, traceability, and labeling of GMOs as established by EU legislation. Therefore, the objective of this study is to assess soybean products in the Turkish market to verify compliance with legislation using qualitative Polymerase Chain Reaction (PCR) assay to detect the presence of GM soybean and to quantify its amount of GM soybean in the samples tested positive using real-time PCR. DNA extracted by the modified CTAB method was properly used for PCR amplification of food materials. The amplification of a 118 bp DNA fragment of the lectin gene from soybean by PCR was successfully achieved in all samples. The GMO screening was based on the detection of 35S promoter and NOS terminator sequences. The GM positive samples were subjected to detection of Roundup ReadyTM soybean (RR) using quantitative real-time PCR. It was found that 100% of the tested food samples contained less than 0.1 per cent of EPSPS gene.

Two quantitative multiplex real-time PCR systems for the efficient GMO screening of food products

According to the EU and Swiss food legislation, only deregulated traits of transgenic plants are allowed to be imported and sold to the consumer. In order to control imports of soya and maize products from retailers, efficient and reliable methods for the detection and quantification are a prerequisite. The screening for specific DNA elements characteristic of transgenic plants is crucial for further analysis and has a major impact on the efficiency of the whole analysis workflow. To allow laboratories to efficiently and reliably screen food products for transgenic plant products, two novel multiplex real-time polymerase chain reaction (PCR) systems were developed and validated. One system determines DNA contents from maize, soya, cauliflower mosaic virus (CaMV) 35S promoter (P35S), NOS terminator from Agrobacterium tumefaciens and CaMV, and the second PCR system simultaneously detects DNA sequences from figwort mosaic virus promoter (PFMV), from bar gene of Streptomyces hygroscopicus, from gene coding for phosphinothricin acetyltransferase (PAT) and from a DNA construct of enolpyruvyl shikimate phosphate synthase gene (CP4-EPSPS) and Arabidopsis thaliana (CPT2). The tests exhibit good specificity and a limit of detection of at least 0.1 % for all analytes.

A survey on genetically modified maize in foods commercialised in Portugal

Maize, the second most important genetically modified (GM) crop, has the highest number of authorised GM events for food and feed in the EU. To provide consumer's information, labelling for food products containing more than 0.9% of GM material is demanded by the actual EU legislation. Analysis of foods is then essential to detect and quantify GM maize material and verify the compliance with labelling information. The aim of the present work was to assess the presence of GM maize in a range of processed foods commercialised in Portugal between 2007 and 2010. For this purpose, screening of GM material was carried out by qualitative PCR targeting the 35S promoter and the NOS terminator, followed by the specific detection of Bt11, MON810, Bt176, GA21, MON863, NK603, TC1507 (also known as DAS1507), DAS59122 and MIR604 events. The identified maize events were confirmed and quantified by real-time PCR with hydrolysis probes. The overall results of GMO screening were 30% for 35S promoter, 10% for NOS terminator and 25% for identified events. The most frequently detected events were MON810, TC1507 and NK603, with one sample containing GA21, while the other events were not detected in any of the analysed foods. The quantitative results suggest the need for a more severe control since 4% of the analysed foods contained more than the threshold for labelling and none of them declared the presence of GMO.

Near infrared and fluorescence spectroscopic methods and electronic nose technology for monitoring foods

There is a clear need for application of proper methods for measuring food quality and safety in the globalized food-webs. Numerous instrumental methods have been established in the course of the 20th century and are developing further, together with data analysis techniques, for such purposes. Among them, near-infrared and fluorescence spectroscopic methods and chemical sensor arrays called electronic noses show particular promise for rapid, non-destructive, non-invasive and cost-effective ways for assessing changes and enhancing control during processing and storage of foods. Their key advantages as analytical tools are 1) their relatively high speed of analysis, 2) the lack of a need to carry out complex sample preparation or processing, 3) their relatively low cost, and 4) their suitability for on-line monitoring or quality control. The present survey attempts to demonstrate examples from the above areas, limiting itself mainly to monitoring some quality indices which contribute to the functionality or acceptability of foods as affected by alternative processing technologies, or loss of freshness/microbial safety, or developing spoilage during storage and marketing. These instrumental methods are correlative techniques: they must be calibrated first against (traditional) reference properties, and the instrumental data are evaluated with the help of chemometric methods. Near-infrared (NIR) spectroscopy can be used in either the reflectance or the transmittance mode. NIR spectra transformed to mathematical derivatives allows subtle spectrum changes to be resolved. Selected examples from the extensive NIRS literature relate to assessment of the quality of frozen fish, predicting cooking loss of chicken patties, detecting complex physico-chemical changes of minced meat as a function of the intensity of high hydrostatic pressure treatment, comparing changes of NIR spectrometric “fingerprints” caused by gamma radiation or high pressure pasteurization of liquid egg white. Changes of NIR spectra reflect several parameters which suit the evaluation of loss of freshness, and onset of spoilage of various foods. NIR spectroscopy shows an application potential for rapid detection of bacterial or mould contamination. It may serve as a tool for detecting initial stages of mobilization processes during germination of cereal grains, or even for GMO screening. Spectrofluorometic measurements have shown potential, e.g. to monitor lipid oxidation and development of meat rancidity, to differentiate between raw and processed milks, and to monitor fish and egg freshness. Electronic noses containing chemical sensor arrays offer a rapid method for evaluation of head-space volatiles of food samples, important for characterizing quality and safety. Such gas sensors may be able to classify storage time, and determine spoilage, either earlier or at the same time as the human senses, or “sniffing out” bacterial pathogens or (toxigenic) fungal growth on certain foods. Electronic nose sensing is also a promising method for detecting quality changes of fruit- and vegetable products non-destructively. In relation to some examples to be presented in the paper, certain software developments as qualitative classification tools made by Hungarian scientists will be pointed out.

GMO Detection Methodology

GMO detection is an essential part of the European regulatory framework concerning the environmental release of GMOs. While Directive 2001/18/EC is not strictly prescribing the detection methodology in case of an experimental release of GMOs in field trials, GMO authorisations for cultivation and placing on the market as food and feed according to Regulation (EC) No. 1829/2003 are rigidly coupled to particular requirements. For example, applicants have to deliver reference materials, methods for sampling, for matrix-specific DNA-extraction and for detection and event-specific quantification of the notified GMO. Method requirements and detailed implementation guidelines for the scientific assessment and validation of the reference materials and the method protocols are defined by Regulation (EC) No. 641/2004. The Community Reference Laboratory (CRL) located at the IHCP/JRC in Ispra has to handle the core tasks with respect to the assessment of the GMO detection method, e. g. intraand inter-laboratory validation according to specified method performance and acceptance criteria defined by the ENGL (European Network of GMO Laboratories). Furthermore, currently applied GMO methodologies are mainly based on the technical guidance and definitions given in Recommendation 2004/787/EC, e. g. use of DNA-based analytical strategies and the unit-of-measurement for GMO quantification (DNA copy number based). Although a certain degree of harmonisation between current detection methodologies and legal requirements has been achieved in the EU, the presentation wants to address some of the challenging issues, e. g. correct identification and quantification of stacked events, efficient methodologies for GMO screening and problems arising from the low level presence of unauthorised events.

Surveying the RR soy content of commercially available food products in Hungary

The main goal of our research was to establish to what extent Hungarian consumers are exposed to consuming genetically modified foods. To achieve this goal, 251 food samples were bought from the Hungarian food market and examined by the Central Food Research Institute (91 samples) and by the National Institute for Food Safety and Nutrition (160 samples) with PCR. The GMO screening was based on the detection of 35S promoter and nos terminator sequences. The GM positive samples were subjected to event specific detection of RR soy with quantitative real-time PCR. According to the results 38% of the examined samples indicated presence of RR soy in soy-containing foods and 6% of these samples contained RR soy above the 0.9% threshold. These results demonstrate for the first time the assessment of the presence of GMO in the Hungarian food market for large sample numbers.

PCR detection of genetically modified soy bean and maize in food/feed stuffs

Public concern about possible negative health effects due to consumption of GM plants, together with concerns about possible environmental dangers, have led the European Commission to limit strictly the distribution of GM plants in the EU. The EU regulations No 49 and 50/2000 require the food industry to label, as GMO containing, those food products not certified free of transgenic plant material. Foods are considered "clean" and may be left unlabeled, if the GM content does not exceed 1%. The presence of GM in food and feed products can be evaluated by detecting the transgenic DNA (PCR technique), or by detecting proteins derived from this DNA (immunological methods). At the Institute of Molecular Genetics and Genetic Engineering both qualitative and quantitative PCR-based tests are standardized. Commonly used primers are designed from the CaMV35S promoter for the answer yes/no in general GMO screening procedure. Specific modifications, as in herbicide tolerant RoundUp Ready Soybean or insect resistant maize (Bt176 corn), are identified by primers designed from specific genes e.g. the EPSPS - gene from A.tumefaciens (for soybean) and the CrylA gene from B.thuringiensis (for maize). The experimentally determined detection limit is 1pg, which allows detection of O,01% GM DNA in test-samples and meets criteria prescribed for the EU laboratories accredited to issue a certificate.