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.     

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.     

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.     

Detection of new or modified proteins in novel foods derived from GMO – future needs

The capability of immunoassays to quantitatively detect proteins, be implemented commercially on a wide scale and generate validated data for regulatory compliance in a cost-effective manner is evidenced by the existing immunoassay industry. The use of immunoassays in the food industry is well established. To enable immunoassay testing of foods for the presence of genetically modified organisms (GMO), proprietary proteins must be available for test development. Standard reference materials are required for method development, to translate test results in terms of % GMO, and to ensure uniform test performance throughout the EU. Data are presented demonstrating quantitative detection of Roundup Ready^® Soy Bean Certified Reference Material using a commercial ELISA. Strategies for successful implementation of testing are discussed.     

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.     

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.     

Comparison of simplex and duplex real-time PCR for the quantification of GMO in maize and soybean

This paper focuses on the determination of the GMO content of maize and soybean samples using real-time PCR, comparing simplex and duplex PCR. The total DNA content of samples was determined by amplifying part of a maize gene encoding a lipid transfer protein, or part of a soybean lectin gene. The transgenic DNA was quantified by amplifying part of the CaMV 35S promoter. The importance of preparation and conservation of standards as well as the relevance of DNA extraction protocol on the variability of results are discussed. For the determination of low GMO content, limitation in the number of copies of the target gene to be amplified is considered. For samples with a theoretical GMO content of 1%, corresponding to the legal threshold for labelling, the value determined by duplex real-time PCR ranged from 0.85% to 1.20%. Both real-time simplex and duplex PCRs allowed identification of GMO free samples without ambiguity.     

Crop-specific GMO matrix-multiplex PCR: A cost-efficient screening strategy for genetically modified maize and cotton events approved globally

Crop-specific GMO matrices of 199 genetically modified (GM) events, comprising 143 GM maize events with 75 genetic elements and 56 cotton events with 45 genetic elements, were developed to screen globally approved GM maize and cotton events. As per the compiled information in the matrix, frequently present genetic elements were identified using GMOSeek algorithm: eight genetic elements ([P-35S] [r-act] [T-35S] [T-nos] [pinII] [pat] [aad-1] [cp4 epsps]) in maize and four ([P-35S] [T-nos] [pat] [nptII]) in cotton. Based on the cost-efficiency, feasibility of plexing and coverage of GM events, maize-specific tetraplex PCR, targeting Cauliflower Mosaic Virus 35S promoter (P-35S), Agrobacterium tumefaciens nos terminator (T-nos), Cauliflower Mosaic Virus 35S terminator (T-35S) and endogenous alcohol dehydrogenase (Adh1) gene, with limit of detection (LOD) up to 0.5% was developed. For screening of GM cotton events, pentaplex PCR, targeting P-35S, T-nos, neomycin phosphotransferase II (nptII), phosphinothricin acetyltransferase (pat) and endogenous stearoyl-ACP desaturase (Sad1) gene, with LOD up to 0.25% was developed. Practical applicability of multiplex PCR assays was confirmed with six maize samples of proficiency testing and eight spiked cotton samples. The reported tetraplex and pentaplex PCR assays could efficiently screen 94% of maize and 93% of cotton events approved globally. The developed GMO matrices in combination with multiplex PCR could facilitate checking the GM status of seed samples or food and feed products, and monitoring for presence of authorized GMOs in food and supply chain. This approach can be easily employed by low resource GM testing laboratories in the developing countries, as the multiplex PCR assays are easy to operate with less time and cost inputs. The GMO matrices being presented herein are based on the current information of approved GM events of maize and cotton, which can be further upgraded by including new approved GM events. As most of the newly approved GM events are stacked versions of already commercialized GM events, the developed multiplex PCR assays could also be employed to screen for their presence.     

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.     

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.     

Quantitative analysis of genetically modified organisms (GMO) in processed food by PCR-based methods

Two different PCR-based approaches for the quantitative analysis of genetically modified organism (GMO) – components in foods are presented using Soybean derived samples as an example. The first method – a double competitive PCR – is well suited to determine threshold levels of GMO content in food. The other – PCR on-line measurement – is suited to determine ratios of transgenic versus non-transgenic component. Both methods provide a means to alleviate the problems of standardisation encountered with simple qualitative PCR approaches and will allow to cope with threshold levels for GMO, once issued by legislative bodies.     

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.     

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.     

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.     

Variations of tissue DNA density and nuclear DNA content in soybean lines and their impacts on the GMO quantification

The present study investigated the DNA density of the embryo, cotyledon, and seed coat of each soybean from 15 soybean cultivars, and evaluated the impacts of variations of tissue DNA density and nuclear DNA content in soybean lines on GMO quantification. The results have shown that DNA densities and DNA quantity ratios among the various tissues of soybean are significantly different from each other and have insignificant influence on the transgenic copy number and therefore on GMO quantification. The nature of the CRM and the transgenic material present in the analyzed sample has prominent impact on transgenic genome copies, thus on GMO quantification. The DNA densities of the various soybean-derived products are also distinctly different from that of the whole soybean, but which does not result in the similar differences on the GMO ratio among them. Nuclear DNA content of soybean is different from cultivar to cultivar. Results shows that variation of nuclear DNA content in soybean lines has a great impact on the accurate determination of GMO. In some extreme situations, the deviation amplitude can reach 26%, which is intolerable for accurate determination.     

The implications for mandatory GM labelling under the Consumer Protection Act in South Africa

The South African Consumer Protection Act 68 of 2008 was passed into law, which among other things, mandates the labelling of genetically modified (GM) ingredients in packed goods. The aim of this study was to evaluate the potential impact of mandatory GM food labelling in terms of the Consumer Protection Act by determining what products currently on the market would be implicated. A total of 46 food products from different companies was selected and sampled randomly with an emphasis on those containing canola, maize and soybean, since GM varieties for these crops have been approved in South Africa in terms of the Genetically Modified Organisms (GMO) Act of 1997. The products were screened for the presence of genetic modification and, if positive, quantified. Genetic modification was detected in 50% of products tested, including seven out of 14 (50%) products labelled to indicate an absence of genetic modification. The results from this study indicate that at a 5% GM labelling threshold, 19 out of the 46 products tested would need to be labelled for their GM content. Of the 14 products labelled to indicate an absence of GM, five exceeded the 1% threshold for non-GM labelling. Many companies are concerned about the cost effectiveness of mandatory labelling. However, the Consumer Protection Act makes a provision for a “may contain” clause that if used relieves the producer of the financial considerations to verify the GM content of a product. Although the Consumer Protection Act does not make any provision for monitoring, consumers or consumer groups have recourse against companies in contravention of the Consumer Protection Act through the provision of the National Consumer Commissioner (NCC).     

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.     

An event-specific qualitative and real-time PCR detection of 98140 maize in mixed samples

The purpose of this study is to establish a reliable detection method specific for event 98140, a type of multiple herbicide-resistant corn. Based on the 3′- flanking sequence of event 98140, qualitative and quantitative PCR detection assays were developed. The results revealed the LOD of 0.05% of GM maize for qualitative PCR assay and 4 transgenic haploid genome copies for quantitative PCR assay used in this study. The LOQ was 20 transgenic haploid genome copies, and based on this, as low as 0.05% of 98140 genomic DNA could be accurately and quantitatively detected by means of the quantitative method. Two mixed corn samples, with known 98140 contents (2% and 0.5%), were used to verify the developed real-time PCR system, and the expected results were observed. The results indicated that the developed event-specific PCR methods could be used for the identification and quantification of maize line 98140.     

Occurrence of aflatoxin contamination in maize throughout the supply chain in the Democratic Republic of Congo

Aflatoxin occurs ubiquitously in maize and is both an economic and a public health concern due to its carcinogenic effects. Nevertheless, in developing countries such as the Democratic Republic of Congo, there is still a dearth of information regarding its natural occurrence, exposure to both humans and animals and strategies for controlling its proliferation or contamination of food crops such as maize. The present study comparatively assessed the aflatoxin occurrence in maize samples collected in the DRC throughout the food supply chain. The analytical method used during this experiment was found to be accurate with a recovery ranging between 70.65 and 98.20%, and also precise (RSD<15%) except for AFG₁, which showed an RSD of 18.05%. Moreover, the limits of detection (LOD) and quantification (LOQ) for aflatoxin ranged from 0.31 to 0.69 μg/kg and from 1 to 2 μg/kg, respectively. The results also revealed that in the pre-harvest period, 32% of maize samples (out of a total of 50) were positive, with aflatoxins at a range of 1.5–51.23 μg/kg for AFB1 and 3.1–103.89 μg/kg for total aflatoxin. As the supply chain progresses, the contamination of maize samples also increases, with 100% of the collected maize samples found to contain aflatoxins at 300 times higher than the maximum limit of 10 μg/kg for total aflatoxin as set by the WHO. The aflatoxin content of maize samples was found to increase drastically at up to 500 times between the city store and the distribution system at market level. This was confirmed with a statistical analysis showing a significant difference (p < 0.01) between city store/market samples and pre-harvest, harvest, farm store and after-transportation samples.     

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.