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.     

Results of an interlaboratory assessment of a screening method of genetically modified organisms in soy beans and maize

The preliminary results on an interlaboratory trial on the detection of genetically modified organisms (GMO) are presented. The method applied is based on the detection of modified DNA using the polymerase chain reaction (PCR) for amplification. The amplified fragments analysed are derived from the 35S promotor and the NOS terminator used for modification and are present in 26 from the 28 GMOs currently already approved or under approval by the competent authorities. This method fits as a screening method indicating the presence of GMO in food. However, it does not allow an identification of the kind of GMO present in the samples. Samples of soybean and maize flour containing 0%, 0.1%, 0.5% and 2% GMO had been prepared for this study and are also already commercially available. In this paper the combined results from 27 laboratories are presented, indicating that on average the probability of false positive or false negative results is only about 1% for soybeans and below 5% for maize.     

Development and application of DNA analytical methods for the detection of GMOs in food

The principle of direct detection of recombinant DNA in food by the polymerase chain reaction (PCR) is discussed following the three main steps: DNA-extraction, amplification by PCR and verification of PCR products.

Suitable methods for genomic DNA isolation from homogenous, heterogeneous, low DNA containing matrices (e.g. lecithin), gelatinising material (e.g. starch), derivatives and finished products based on classical protocols and/or a combination with commercially available extraction kits are discussed. Various factors contribute to the degradation of DNA such as hydrolysis due to prolonged heat-treatment, nuclease activity and increased depurination and hydrolysis at low pH. The term “DNA quality” is defined as the degree of degradation of DNA (fragment size less than 400 bp in highly processed food) and by the presence or absence of potent inhibitors of the PCR and is, therefore, a key criterion. In general, no DNA is detectable in highly heat-treated food products, hydrolysed plant proteins (e.g. soya sauce), purified lecithin, starch derivatives (e.g. maltodextrins, glucose syrup) and defined chemical substances such as refined soya oil.

If the nucleotide sequence of a target gene or stretch of transgenic DNA is already known specific primers can be synthesised and the segment of rDNA amplified. Detection limits are in the range 20 pg–10 ng target DNA and 0.0001–1% mass fraction of GMO. Amplification products are then separated by agarose gel electrophoresis and the expected fragment size estimated by comparison with a DNA molecular weight marker.

Several methods are used to verify PCR results and they vary in reliability, precision and cost. They include specific cleavage of the amplification products by restriction endonucleases or the more time-consuming, but also more specific, transfer of separated PCR-products onto membranes (Southern Blot) followed by hybridisation with a DNA probe specific for the target sequence. Alternatively, PCR products may be verified by direct sequencing. Nested-PCR assays combines high specificity and sensitivity.

Methods for the screening of 35S-promoter, NOS-terminator and other marker genes used in a wide range of GMOs, the specific detection of approved products such as FlavrSavr™ tomatoes, Roundup Ready™ Soya, Bt-maize 176 and official validated methods for potatoes and genetically modified micro-organisms, that have a model character, are available. Methods to analyse new GMO products are being validated by interlaboratory tests and new techniques are in development (e.g. EC project: DMIF-GEN). However, these efforts may be hampered by the lack of availability of GMO reference material as well as specific sequence information which so far can only be obtained from the suppliers.     

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.     

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.     

Detection of GMO in food products in Brazil: the INCQS experience

Regulations for the use and labeling of genetically modified organism products and derived ingredients are being implemented worldwide, what demands reliable and accurate methods to detect genetically modified organisms (GMO) in raw materials and food products. This study aimed at monitoring products derived from GMO in the Brazilian market using detection methods for the presence of Roundup Ready soybean, Bt176 and MON 810 maize. The results demonstrate for the first time the presence of GM-soy in Brazilian food products, reinforcing the need for the development of accurate quantitative methods in routine analyses.     

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.     

Detection methods for genetically modified crops

Due to the market introduction of genetically modified crops (GMOs) as the Roundup Ready (RR) soya and Bt corn, the European food industry came face to face with the question of the use and labeling requirements on GMO crops and its derivatives. Although even today, no defined European legislation is available, a definitive need for detection methods exists. Both DNA and protein based methods have been developed and applied for the detection of RR soya beans and its derivatives. For the CP4 synthase, synthetic peptides corresponding with the antigenic and non-homologous parts of the CP4 synthase were synthesized and mono-specific anti-CP4 synthase monoclonal antibodies were prepared by hybridoma technology. The monoclonal antibodies were able to detect the CP4 synthase in the RR soya using Western blotting analysis. Detection limits were found between 0.5% and 1%. The method is currently validated for half-and final products. The applied DNA methodology was making use of polymerase chain reactions (PCR) using sets of primers along the gene encoding the Agrobacterium CP4 synthase. DNA extraction and purification conditions were examined on a case-by-case approach for a scala of soya products (lecithin, oil, soybean meal, soy protein isolates etc.), half-products and final consumer products. Detection limits were found between 0.01% and 0.1%. In this paper a comparison will be made between the two types of methods in relation to sample preparation, sensitivity, validation and the use for half-products and final consumer products.     

Transboundary movement of genetically modified organisms in India: Current scenario and a decision support system

Genetically modified (GM) crops have benefited global agriculture by introduction of superior traits for better agronomic performance, ensuring nutritional security and mitigating climate change. In India, to meet the demand of burgeoning population and to withstand the changing climate, GM crops would play an important role. Since 1997, GM crops are being imported through Indian Council of Agricultural Research (ICAR)-National Bureau of Plant Genetic Resources (NBPGR), New Delhi, the designated nodal organization for quarantine processing and import of GMOs (referred to GM planting material in present context) for research purposes. In the present study, an attempt has been made to analyze the trend of import of GMOs. Till the end of 2015, 205 consignments of fifteen GM crops have been imported from 19 countries by public and private sector. Detailed analysis of diversity in traits of imported GM events and imported stacked traits in cotton and maize has been made. In the recent past, four consignments of GMOs have been exported for research purposes. Involvement of public/private sector in transboundary movement of GMOs was evaluated. Along with quarantine processing of imported/exported GMOs, molecular testing for specific transgenic elements as claimed by the importer/exporter is also carried out employing polymerase chain reaction (PCR) and real-time PCR based markers. Efficient detection strategies based on GMO matrix as a decision support system, loop-mediated isothermal amplification and multi-target real-time PCR-based systems have been developed. The data presented herein would provide a decision support system to check for authorized/unauthorized GMOs in food and supply chain.     

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.     

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.     

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.     

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.     

Event-specific qualitative and quantitative PCR detection of LY038 maize in mixed samples

With the development of genetically modified organisms (GMOs), labeling regulations have been introduced, which require appropriate detection methods. Event-specific qualitative and quantitative PCR detection methods have become the internationally agreed state-of-art. This paper describes the characterization and event-specific quantitative detection of LY038 maize insert with the application of reference molecule. The flanking regions were characterized by Inverse-PCR (I-PCR). Furthermore, the event-specific PCR primers and TaqMan probe were designed based on the discovered right flanking sequence. In the qualitative PCR assay, two PCR systems were established with the event-specific and specie-specific primers respectively, and the limit of detection (LOD) was 0.1% (approximates to 37 haploid genome copies). In the quantitative TaqMan real-time PCR assay, a reference molecule was constructed by recombinant PCR and standard curves were set up. By using the reference molecule, we obtained standard curves with good linearity and relatively high efficiency of PCR reaction. The results indicated the usability of the plasmid as standard material. From above results, we believed that the established event-specific qualitative and quantitative PCR systems for LY038 maize in this study were acceptable and suitable for LY038 maize detection in mixed samples.     

Adventitious presence of transgenic events in the maize supply chain in Peru: A case study

Cultivation and trade of transgenic or genetically modified organisms (GMO) and commodities has become widespread worldwide. In particular, production of transgenic crops has seen an accelerated growth along with a complex regulatory process. Current Peruvian legislation prohibits import of transgenic seeds and cultivation of transgenic crops in National territory but allows import of GMO-derived products and commodities. In addition, there is legislation that mandates the labeling of food products containing transgenic ingredients but the labeling threshold is still under discussion and the enforcement of this law is on hold. In this context, we evaluated adventitious presence of transgenic events in locally traded yellow maize using PCR- and immuno-based detection methods. Our results indicated that contamination during the distribution system of lots derived from non-transgenic maize was unavoidable and generally below 1.0% (w/w). Transgenic event MON810 was found in truck-loads of nationally grown maize. In general, frequencies of GMO-derived targets in whole-grain lots were 2.2% (GMO content ≥ 1%), 16.4% (GMO content ≤ 1%) and 81.3% (GMO content below our detection levels). When samples of de-germinated maize where evaluated, frequencies were 25.6% (GMO content > 0.9%), 65.1% (GMO content ≤ 0.9%) and 9.3% (GMO content below our detection levels). We believe this information will aid policy makers in establishing a suitable threshold for trade and product labeling as well as to conduct further investigation on other crops and scenarios.     

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.     

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.     

Strategies to assure the absence of GMO in food products application process in a confectionery firm

This article describes the strategies and the instruments applied in the development and implementation of a system aimed at assuring the production of food which is neither made of nor derived from genetically modified raw material in a confectionery firm. It was chosen to develop a specific planning method partially taken from HACCP and FMEA techniques, mainly as far as the systematic and rigorous analysis of risks is concerned (in this case, the contamination of raw material and/or their genetically modified derivatives along the food chain). The result is a management system model that food organizations can use to assure the traceability of all ingredients incorporated into their food products and, in the same time, to guarantee that these ingredients do not contain or consist or derive from genetically modified organisms (GMOs).     

Fluorophore double stranded probe-multiplex quantitative PCR method for detecting transgenic component of promoter derived from Cauliflower Mosaic Virus and nos terminator derived from Agrobacterium tumefaciens simultaneously

The article is to establish multiplex PCR method for quantitative detecting transgenic component promoter derived from Cauliflower Mosaic Virus (CaMV 35S) and nos terminator derived from Agrobacterium tumefaciens (Tnos) in foods. According to the specific sequence of CaMV 35S and Tnos which have been used in genetically modified organisms (GMOs) frequently, and the sequence of soybean endogenous lectin gene, three pairs of primers and corresponding fluorophore double stranded probes (FDSP) were designed to allow for quantitative detecting of GMOs. FDSP designed with maximal specificity also showed the greatest detection sensitivity, and the ease in design, the simple single-dye labeling chemistry. FDSP-multiplex quantitative PCR (FDSP-MQPCR) methods were established for the detection of transgenic component CaMV 35S and Tnos simultaneously. Ten soybean flour samples were tested with FDSP-MQPCR method. The method gives five positive-samples with quantitative results in 5 h, and accuracy rate is above 97.0%. The described methods enabled a sensitive, specific, simple, and accurate detection of transgenic component and thus provide a useful tool for quantitative analysis of raw and processed food products. FDSP-MQPCR method has not only improved detection efficiency and result credibility, but also has guaranteed the better accuracy and repetitiveness.     

Auto-microfluidic thin-film chip for genetically modified maize detection

As a thin-film chip method, reverse dot blot hybridization (RDBH) has been employed to detect hazardous substances, but an automatic RDBH instrument with low workload, high accuracy and stability is still urgently needed. This paper presents our newly-developed auto-microfluidic thin-film chip (AMTC) method for multiplex screening of genetically modified (GM) maize. With specific DNA probes for genetically modified (GM) maize being immobilized on a square nylon thin-film, it was placed into a micro-reaction cell of the AMTC device. Then biotin-labeled PCR products with target DNA fragments for template amplification were added to the micro-reaction cell using a microfluidic system. When the PCR products passed the square nylon thin-film, the target DNA fragments were captured by the complementary action of DNA, where the signal was visualized with streptavidin link-coupled alkaline phosphatase color development kit. The sensitivity of GM maize detection reached 0.1% quality percentage and its stability and consistency could satisfy the requirements for practical applications. Performance advantages of the ATMC are manifold, being embodied in aspects such as easy and straightforward operation, low costs and less workload.