SIGMO: A decision support System for Identification of genetically modified food or feed products

Since their introduction in 1994, more and more genetically modified (GM) crops are grown worldwide and introduced in food or feed products. In the European Union (EU), the production, trade and marketing of GM products is strictly regulated, but the situation is becoming more complex due to the increasing number and complexity of GM crops, and asynchronic approval procedures with the major GM crop producing countries. Importers and traders are obliged to assess their respective supply chains for the potential presence of authorised and unauthorised GM organisms (GMOs), where wrong decisions may lead to substantial economic losses. This article presents a decision support system SIGMO aimed at guiding producers and traders with the assessment of the likelihood that their products may comprise authorised or unauthorised GM materials. The assessment is based on traceability data about the product (nature and origin of the raw materials, transportation aspects), as well as analytical results of the presence of GMOs in the final product or its ingredients. The approach uses a combination of data-driven and model-driven decision support. SIGMO is composed of (1) a data base providing data about GMO crop species produced and approved in counties worldwide, (2) a multi-attribute model for the assessment of GMO presence in food/feed products, and (3) an on-line user interface. SIGMO helps producers and traders to better comply to valid EU GMO regulations and to better control their products and supply chains in terms of the unintended presence of (unauthorised) GMOs in a cost-effective way.     

Detection of unapproved genetically modified potatoes in Korea using multiplex polymerase chain reaction

Several genetically modified (GM) potato cultivars with improved traits such as increased amylopectin levels, decreased asparagine levels, and reduced incidence of black spot have been developed. In this study, we describe a multiplex polymerase chain reaction (PCR) method specific for four GM potato events (EH92-527-1, AM04-1020, PH05-026-0048, and E12) that are unauthorized in Korea. The UDP-glucose pyrophosphorylase (UGPase) gene was used as the endogenous reference gene. The specificity of the primer sets was evaluated using GM potatoes and other GM crops. The limit of detection in the developed multiplex PCR was confirmed to be approximately 0.04% (w/w). Thirty-three commercial products containing potato ingredients were tested by the multiplex PCR assay, and GM potato event E12 was found in one of the 33 samples. This result suggested that the developed PCR method could be used to effectively identify unauthorized GM potatoes in Korea.     

Monitoring the occurrence of genetically modified maize at a grain receiving port and along transportation routes in the Republic of Korea

The cultivation area of genetically modified (GM) crops is increasing all over the world. Though no land in the Republic of Korea is currently used for the cultivation of GM crops, GM crop imports for food and foraging purposes are continuously increasing. This may promote the unintentional escape of GM crops. This study was conducted to investigate whether imported GM maize is released into our environment during the transportation of grain in the Republic of Korea. Based on PCR analysis, most of the maize grains in the forage products were GM, and about 50% of the grains were germinated. Monitoring was conducted in two major grain receiving ports, 15 feed manufacturing plants, and 14 livestock barns in five provinces of the Republic of Korea from July to September 2007. We found many spilled maize grains around open storage areas of ports and along truck transportation routes near feed manufacturing plants. Established maize plants were not found at or around Incheon port. However, we found 18 established maize plants at the Gunsan port, 15 of which were GM. We also found eight GM maize plants around four feed manufacturing plants and in two livestock barns. Based on the event-specific PCR analysis, three maize events (NK603, Mon810, and TC1507) were identified. Though several GM maize plants were found around the port and feed manufacturing plants, most of these facilities were located inside the industrial park and were far from cultivated fields, likely rendering the impact of these GM maize on the natural environments negligible. However, most of the livestock barns were close to cultivated areas. Moreover, maize plants were cultivated for food or feed near some livestock barns. This practice may facilitate gene flow from GM maize to non-GM maize plants. Therefore, continuous monitoring is necessary to detect the occurrence of GM maize, and appropriate action should be taken to prevent genetic admixture in our environment.     

A multiplex nested PCR assay for the simultaneous detection of genetically modified soybean,maize and rice in highly processed products

The use of genetically modified organisms (GMOs) as food products becomes more and more widespread. The European Union has implemented a set of very strict procedures for the approval to grow, import and/or utilize GMOs as food or food ingredients. Thus, analytical methods for detection of the GMOs are necessary in order to verify compliance with labeling requirements. There are few effective screening methods for highly processed GM (genetically modified) products. Four genes (CP4-EPSPS, Cry1A(b), BAR, and, PAT) are common exogenous genes used in commercialized transgenic soybean, maize, and rice. In the present study, a multiplex nested polymerase chain reaction (PCR) method was developed to simultaneously detect the four exogenous genes and one endogenous gene in two runs. We tested eleven representative highly processed products samples (soya lecithin, soya protein powder, chocolate beverage, infant rice cereal, soybean refine oil, soybean salad oil, maize oil, maize protein powder, maize starch, maize jam) using the developed method, and amplicons of endogenous gene and transgenic fragments were obtained from all the processed products except for soybean refined oil, soybean salad oil and maize oil, and the sensitivity was 0.005%. These results indicate that multiplex nested PCR is appropriate for qualitative detection of transgenic soybean, maize and rice in highly processed products except for refined oil.     

Multiplex PCR system to track authorized and unauthorized genetically modified soybean events in food and feed

A diverse range of genetic elements has been used to develop genetically modified organisms (GMOs) over the last 18 years. Screening methods that target few elements, such as the Cauliflower Mosaic Virus 35S promoter (P-35S) and Agrobacterium tumefaciens nopaline terminator (T-nos), are not sufficient to screen GMOs. In the present study, a multiplex PCR system for all globally commercialized GM soybean events was developed to easily trace the events. For this purpose, screening elements of 24 GM soybean events were investigated and 9 screening targets were selected and divided into three individual triplex PCR systems: P-35S, ribulose-1,5-bisphosphate carboxylase small subunit promoter of Arabidopsis thaliana, T-nos, T-35S, pea E9 terminator, open reading frame 23 terminator of A. tumefaciens, proteinase inhibitor II terminator of potato, acetohydroxy acid synthase large subunit terminator of A. thaliana, and the revealed 3′ flanking sequences of DP-305423-1. The specificity of the assays was confirmed using thirteen GM soybean events as the respective positive/negative controls. The limit of detection of each multiplex set, as determined using certified reference materials of specific GM events, ranged from 0.03 to 0.5%, depending upon target. Furthermore, 26 food samples that contained soybean ingredients, which were purchased from the USA, China, Japan, and Korea, were analyzed, 17 of which contained one or more GM soybean events. These results suggest that the developed screening method can be used to efficiently track and identify 24 GM soybean events in food and feed.     

Loop-mediated isothermal amplification targeting insect resistant and herbicide tolerant transgenes: Monitoring for GM contamination in supply chain

Efficient detection strategies for genetically modified (GM) crops are required to effectively address some of the biosafety and post-release monitoring issues, as global adoption of GM crops has been unprecedently increased. Herbicide tolerance and insecticide resistance are the major traits in commercialized GM food crops. Visual as well as real-time detection system based on loop-mediated isothermal amplification (LAMP) targeting lepidopteron insect resistant cry1Ac, cry2Ab2 and glyphosate tolerant cp4-epsps genes has been reported. Specificity of LAMP assays were confirmed using fourteen GM events of four crops, namely, corn (MON810, NK603, Bt11, Bt176, MON89034), cotton (MON531, MON15985, GFM-cry1A, Event1, MLS9124, MON1445, MON15985 × MON88913), eggplant (EE1) and soybean (GTS40-3-2). Real-time LAMP was found sensitive enough to detect as low as 2 copies for cry1Ac and 4 copies for cry2Ab2 and cp4-epsps within 35 min using a calibration curve. The limit of detection (LOD) of visual LAMP assays was down to 0.01% (4 copies of GM content) which is lower than conventional PCR (detecting 40–400 copies depending on target). LAMP assays are faster and more user-friendly than conventional PCR and could be efficiently utilized for monitoring of GM contamination in food and feed supply chain, with high specificity and sensitivity. The developed assays, when combined with a fast DNA extraction method, will facilitate on-site detection to check the GM status of a sample or product at ports of entry and in farmers' fields.     

The SAFE FOODS Risk Analysis Framework suitable for GMOs? A case study

This paper describes the current EU regulatory framework for risk analysis of genetically modified (GM) crop cultivation and market introduction of derived food/feed. Furthermore the risk assessment strategies for GM crops and derived food/feed as designed by the European Food Safety Authority (EFSA) are described on which international agreement exists. Existing flaws in the EU regulatory framework for GMOs have been identified and proposals are put forward to improve current risk analysis procedures for GMOs by taking the SAFE FOODS Risk Analysis Framework into account. The SAFE FOODS framework describes an iterative decision-making process with four distinct stages i.e. framing, risk–benefit assessment, evaluation, and risk management which includes decision-making, and implementation, and a final review stage. Three major changes compared to current risk analyses practices are proposed, i.e. (i) the addition of a formal framing stage, during which problem formulation and the objectives of the risk analysis are established, (ii) enlargement of the scope of the risk assessment, by including the assessment of potential benefits, and an impact analysis of social and economic aspects, and (iii) addition of a formal evaluation stage, in order to weigh risks, costs and benefits and their distribution. Furthermore a broader participation of certain entities, organisations and individual citizens in specific segments of the risk analysis process, in particular in the framing and evaluation stage, is proposed. The proposed changes in current risk analyses practises may contribute to restore consumer confidence in risk analysis process of GMOs in the EU.     

GMO matrix: A cost-effective approach for screening unauthorized genetically modified events in India

Use of a pragmatic, affordable and reliable approach for screening and detection of a large number of genetically modified (GM) crops/events is the need of hour. A cost-effective matrix approach to check the GM status of food/feed products and for screening the presence of authorized and unauthorized GM events in India is being reported in the present study. A genetically modified organism (GMO) screening matrix, with the information on 106 genetic element targets for detection of 141 GM events of 21 crops, is being presented. These include commercially cultivated Bt cotton events and other GM events, under field trials during the past six years (2006–2012) in the country. The information on GM events, which were either indigenously developed or imported for research purposes, is also presented in brief. Ten most frequently present targets, viz., [P-35S] [T-nos] [Os-Msca1] [cry1Ab] [cry1Ac] [cry1C] [cry2Ab] [GA20 oxidase1] [nptII] [bar], were identified to screen these events using a GMOseek algorithm. This user-friendly screening tool is flexible for further updates with the new GM events and targets/elements. The data reported here related to the GM crops/events in India and the related GMO matrix are valuable tools to assist in the detection of accidental presence of unauthorized GM events in the food and supply chain globally, as well as in the context of the new labelling requirements for food commodities, as per the amendment to enforce GM food labelling from January 2013 in India. The reported GMO matrix approach would facilitate efficient, rapid and cost-effective preliminary screening by eliminating the need for development of specific testing methodologies for each GM event.     

A rapid loop-mediated isothermal amplification method for detection of the modified GM cry1A gene in transgenic insect-resistant cotton and rice

Among the commercial genetically modified (GM) crops, the insect-resistant GM crops are the major cultivars that cry gene is introduced into. A cry1Ab/1Ac GM fusion gene (GFM cry1A) and a GM truncated cry1Ac gene (cry1Ac-Mon) is the key foreign gene employed for construction of GM crops by China researchers and Monsanto Technology LLC respectively. Here these two genes are entitled “GM cry1A” gene and a rapid visual loop-mediated isothermal amplification (LAMP) assay method for detection of GM cry1A in transgenic insect-resistant crops was established. The LAMP assay was performed at an optimal temperature of 65 °C for 60 min in the presence of a set of four specific primers recognized six distinct sequences of the GM cry1A gene. The rough detection limit to the GM cry1A in samples is as low as 0.01% (a weight ratio of transgenic insect-resistant rice/cotton to non-transgenic rice/cotton). Comparatively, the sensitivity of this LAMP method is 10 times over that of the conventional PCR method. Fifteen cultivars/events and five Bt strains with or without cry1A gene were analyzed using the LAMP method as well as PCR method. The results demonstrate that this LAMP method shows a distinct specificity to the GM cry1A gene comparing with PCR analysis. Therefore, this LAMP method will be a potential effective tool for screening the GM cry1A gene in GM crops which are widely plant in China and other developing countries.     

A general multiplex-PCR assay for the general detection of genetically modified soya and maize

The use of genetically modified organisms (GMOs) as food and in food products is becoming more and more widespread. The European Union has implemented a set of very strict procedures for the approval to grow, import and/or utilize GMOs as food or food ingredients. Thus, analytical methods for the detection of GMOs are necessary in order to verify compliance with labelling requirements. In the past few years, different PCR-based methods for the specific detection of the most economically important GMOs have been proposed. A molecular screening method based on multiplex-PCR that involves amplification of specific soya or maize sequences from plant DNA and the amplification of 35S promoter and NOS terminator for the detection of genetically modified soya and maize was developed. The m-PCR assay discriminated the GMO very quickly, reproducibly and in a cost saving and less time-consuming way. It is a flexible assay to conduce a preliminary GMO screening for detection of genetically modified soya and maize.     

Disposable genosensor, a new tool for the detection of NOS-terminator, a genetic element present in GMOs

Legislation enacted worldwide to regulate the presence of genetically modified organisms (GMOs) in crops, foods and ingredients, necessitated the development of reliable and sensitive methods for GMO detection. An indicator based electrochemical disposable genosensor for the voltammetric detection of NOS-terminator, a genetic element present in GMOs is described as a possible substitute method for the common technique of gel electrophoresis and fluorescent image analysis. The biosensor relies on the immobilization of the 25-mer single stranded oligonucleotides (probe) related to NOS-terminator DNA sequence and the relative binding of this sequence with the polymerase chain reaction (PCR) amplified samples from certified reference material (CRM) of Roundup Ready soybean (Fluka) at a screen printed electrode (SPE). The extent of hybridization between the probe and target DNA is determined by using square wave voltammetry (SWV) with moving average baseline correction and methylene blue (3,7-bis(dimethylamino)phenothiazin-5-ium chloride, MB), as the hybridization indicator. The difference between the MB signals, obtained from the hybrid modified and probe modified SPEs, is used to detect GMOs from PCR amplified DNA samples. Numerous factors affecting the hybridization and indicator binding reactions are optimized to maximize the sensitivity.     

Challenges for methods to detect genetically modified DNA in foods

Qualitative detection methods for genetically modified (GM) DNA sequences in foods have evolved fast during the past years. The sensitivity of these systems is extremely high, even for processed foodstuffs. However, in future, quantitative results about the fraction of GM material in a composite food will be needed and the fast increasing number of GM foods on the market demands the development of more advanced multi-detection systems. Other challenges and problems might arise from the decreasing relevance of methods which screen for sequences commonly found in GMOs, the inability to detect GM foods for which the modified sequence is unknown, the lengthy standardisation procedures and the need to up-date continuously databases comprising commercially available GM foods and the respective detection strategies.     

Comprehensive matrices for regulatory approvals and genetic characterization of genetically modified organisms

The production of new types of genetically modified organisms (GMOs) and the use of products containing or derived from these materials are expanding globally. This poses a challenge in providing cost-effective comprehensive analyses. In this line, the state of art testing approaches rely on a matrix representing the GM events with their corresponding GM markers - DNA elements used in plants' transformation. Accordingly, this study aimed first at constructing an updated and comprehensive matrix of genetic characterization of GM events based on an extensive review of the relevant databases. Inclusive lists of 356 GM markers and 508 events in 29 plant species were compiled and organized into a matrix. The frequency of occurrence of these elements was then determined. Moreover, for the first time, a matrix representing the regulatory status of every compiled GM event was established. Remarkably, numerous inconsistencies were detected among the databases at the levels of nomenclature, events' registry, molecular characterization and regulatory approvals. Both matrices represent a useful tool for comprehensive and cost-effective analyses. The genetic matrix permits designing the most straightforward testing strategy that provides the maximum information about GMOs in a sample in the minimum number of experimental steps. Moreover, the novel regulatory matrix, allows further decreasing the number of required event-specific identification tests by giving higher probabilities to those authorized in the samples' country of origin. Finally, the genetics and regulatory matrices represent the building-block for establishing an inclusive automated database for GMOs which is instrumental for testing laboratories worldwide.     

Real-time PCR-based detection and quantification of genetically modified maize in processed feeds commercialised in Malaysia

The present study which dealt mainly with processed feeds and some maize samples sold commercially in Malaysia evaluated the implementation of a real-time PCR cycling system for singleplex screening of eight target sequences (lectin, hmg, adh1, p35S, NK603, GA21, MON810 and MON863) and quantification of four genetically modified (GM) maize events (NK603, GA21, MON810 and MON863). The effects of using proprietary glass magnetic particles to bind DNA to their surface were also investigated in terms of DNA quantity, purity, integrity, quality and its overall effect on DNA amplification. GM material was present in 26.2% feeds and 65% maize samples. All GM samples contained MON810 followed by NK603 (47.5%), GA21 (25%) and MON863 (2.5%). Single-event and multiple-events were identified in the GM samples with 50% of the GM samples containing multiple-events. The present study which represents a fast and reliable methodology would provide an overview of the presence and levels of GMOs in feeds and maize in Malaysia.     

Membrane based detection of genetically modified organisms in some representatives food

Recently, DNA-based techniques became very common for the detection of genetically modified organisms (GMOs) in food products. For rapid and easy detection of GMOs, polymerase chain reaction (PCR) screening methods, which amplify common transgenic elements, are applied in routine analysis. Incorporation of PCR and membrane method introduced in this study offer an alternative detection of GMOs. In this study, a total of 32 samples and three certified reference materials were tested for the existence of the 35S promoter of cauliflower mosaic virus (CaMV) and 5-enol-pyruvyl-shikimate-3-phosphate synthase (EPSPS) gene residues. Dot blot screening system introduced in this study can be routinely used as a semi-quantitative screening of GMOs.     

Progress in Foreign Cooperation of China's Oil Industry

In 2015, China's oil companies' overseas equity oil has grown steadily to approximately 150 milliontonnes. State-owned oil majors have turned from scale-oriented to profit-oriented by adjusting investment speed and optimizing portfolio to improve operating profit of overseas business. The overseas investment of private oil companies mainly focused on two fields: the first is to invest in countries along the Belt and Road(the Silk Road Economic Belt and the 21st-century Maritime Silk Road), and the second is to enter the oil industry of the developed countries by MA. Limited progress has been made in the foreign cooperation of domestic upstream sector, while new changes emerged in the foreign cooperation of upstream sector. Some planned projects were postponed or delayed due to domestic or international market changes. Private oil companies, however, began to cooperate with foreign companies in the downstream sector after the former got the right to import crude oil.     

Quantitative competitive PCR for the detection of genetically modified organisms in food

Present polymerase chain reaction (PCR) detection methods only allow the qualitative detection of GMO in food without quantitation of the GMO content. Clearly, the availability of quantitative detection methods for GMO analysis is an important prerequisite for the introduction of threshold limits for GMOs in food. PCR is well known to be quantitative if internal DNA standards are co-amplified together with the target DNA. This quantitative competitive (QC) PCR was first described in the early nineties and is widely used nowadays.

We have developed and evaluated QC–PCR systems for the quantitative detection of Roundup Ready^TM soybean (RRS) and Maximizer maize (MM) in food samples. Three DNA fragments differing from the GMO specific sequences by DNA insertions were constructed and used as internal standards in QC–PCR. These standards were calibrated by co-amplifying with mixtures containing defined amounts of RRS DNA and MM DNA, respectively. The calibrated QC–PCR systems were applied to several commercial food samples containing RRS and to three certified RRS flour mixtures (Fluka standards). Recently, quantitative methods for the detection of RRS were successfully tested in a collaborative study involving twelve European control laboratories. Thus, QC–PCR methods will allow to survey “de minimis thresholds” of GMOs in food.     

Prevalence of genetically modified foods (GM foods) in the United Arab Emirates

The real and/or perceived risks of genetically modified organisms (GMO) prompted food safety regulators to label the GM products. Although there are no legislations on GM labeling and cultivation of GM crops in the UAE, the present study aims to monitor the status of GM foods in the UAE market using Light cycler real time PCR technology and GMO screening kit. The yield and purity of DNA extracted by CTAB method was higher when compared to Qiagen plant kit with an exception of soya products for which Qiagen kit yielded better results.Out of 128 samples tested, 16 were positive for plant, 35S promoter and Tnos fragment. In conclusion, GMO screening assay applied in this study confirms the presence of genetically modified food in the UAE market. The rapidly growing GM market with multiple events and the threat from unapproved events signifies the value of surveillance program for monitoring the status of GM foods.     

Monitoring the occurrence of genetically modified soybean and maize in cultivated fields and along the transportation routes of the Incheon Port in South Korea

In South Korea, imported genetically modified (GM) soybean and maize have been approved for both human consumption and use in animal feed, but not for use in cultivation in fields. This study was conducted to survey the spread of GM soybean and maize in South Korea using multiplex-PCR analysis methods. Cultivated soybean, wild soybean, and maize leaf samples were collected from 26 major areas of soybean cultivation throughout eight provinces. Roadside areas near a major grain port in Incheon were also surveyed to investigate the escape and spread of GM seeds and plants. Amplification results showed that no GM soybean or maize was collected from cultivated fields. However, four GM maize plants were found in samples collected from the roadside near a grain transporting company at the Incheon Port. Based on PCR analysis using GM maize event-specific primers, it was suggested that a maize plant may be Mon810, while the other plants may be stacked events: Mon863 × Mon810 or Mon88017 × Mon810.     

Multiplex qualitative PCR assay for identification of genetically modified canola events and real-time event-specific PCR assay for quantification of the GT73 canola event

Genetically modified (GM) canola is the most widely grown oilseed crop in Canada. At this time, commercially produced GM canola cultivars in Canada have the events GT73/RT73 and Ms8xRf3. Commercial seed sale of canola cultivars containing the GM events such as OXY235 and T45 has been discontinued. Adventitious presence of GM seeds and grains in non-GM grains is a concern for international grain trade, and development of effective detection methods is important. A multiplex qualitative PCR procedure was established for the detection of the GM canola events OXY235, Ms8xRf3, T45 and GT73. The presence of the GM canola events was also successfully detected in ground spiked wheat and barley grain samples prepared at 0.1%, 0.5% and 1.0% levels (w/w). The GT73 real-time PCR assay was successfully used to quantify DNA extracted from spiked ground canola samples consisting of 5%, 1%, 0.5% and 0.1% GT73 (w/w).