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.     

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.     

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.     

Detection strategies for food authenticity and genetically modified foods

Analytical methods for authenticity testing have been described for all types of food and can give us important indications for analytical strategies to be developed for the detection and quantitation of genetically modified foods. Transgenic plants contain newly introduced traits or marker genes that are expressed and should be detectable by DNA or protein-based methods. Recent literature clearly favours PCR as the method of choice for the identification of GMO-derived food. Quantitative competitive PCR is an important extension of this method allowing to control labelling and maximum limits to be set for genetically modified crops in non-GMO-crops.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

Two detection methods of genetically modified maize and the state of its import into Japan

We detected genetically modified maize (GM-maize) with two different methods and examined the state of its import into Japan. A method using a cetyltrimethylammonium bromide buffer efficiently extracted DNA. Measurement of the extracted DNA at 260/230 nm and 260/280 nm indicated that the DNA was pure enough for polymerase chain reaction (PCR) amplification. Agarose gel electrophoresis of the PCR amplified products resolved a band at 329 bp for the zein gene and 437 and 522 bp for the introduced genes in Bt11, 619 bp in Event176, 194 bp in MON810 and 231 bp in LIBERTY maize. GM-maize was also detected by an enzyme-linked immunosorbent assay (ELISA) and there was no conflict in the results between the ELISA and the PCR method. However, the PCR method had advantages in distinguishing the lines of GM-maize. GM-maize was contained in the maize seeds imported into Japan between January and March in 2000 and MON810 counted for the largest percentage of the GM-maize.     

Regulation of genetically modified foods in Australia and New Zealand

Food standards in Australia and New Zealand build on the level of food safety that is generally accepted by the community. An explicitly cautious approach is applied in cases where there is no established history of safe human consumption, as is the case for foods produced using gene technology. Novel foods, including genetically modified (GM) foods, undergo a mandatory pre-market safety assessment and approval process. The approach used in Australia and New Zealand to assess the safety of foods produced using gene technology draws on concepts and principles that have been developed internationally.     

Qualitative and quantitative detection of genetically modified maize and soy in processed foods sold commercially in Brazil by PCR-based methods

Qualitative and quantitative polymerase-chain-reaction-based methods to detect genetically modified (gm) soy (RoundupReady^TM soy) and maize (Bt176 Maximizer maize; Bt11 maize, MON810 Yield Gard corn, T25 Liberty^R Link maize) were applied to processed foods sold commercially in Brazil. In total 100 foods containing maize and 100 foods containing soy were analysed in 2000 and again in 2001. In 2000, 13% of the soy containing products and 8% of those containing maize were shown to consist of material derived from the corresponding genetically modified organisms. In five samples of the products containing gm soy <4% and in eight samples >4% of the soy derived material was identified as coming from gm soybeans, whereas in the case of products containing gm maize five samples of the maize derived material were found to contain <4% and three samples >4% of gm maize. In 2001, gm soy was found in 21% of the soy containing foods, and gm maize was found in 9% of the maize containing products. Eight samples of the products containing gm soy were shown to contain <4% and 13 samples >4% of gm soy, the corresponding values for gm maize were five samples <4% and four samples >4%.     

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.     

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.     

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.     

Monitoring of genetically modified soybean events in sausage products in South Korea

The use of genetically modified (GM) ingredients in various types of food products has increased worldwide. In this study, qualitative real-time PCR assays targeting five GM soybean events (RRS, MON89788, A2704-12, A5547-127, and MON87701) were performed to monitor their use in sausage samples. Thirty sausage samples containing soybean ingredients on their label were collected from Korean food markets and analyzed. The endogenous lectin gene of soybean was used as an internal positive control. Real-time PCR showed that the threshold cycle (Ct) values ranged from 28.62 to 33.90 for the 30 sausage samples. Of the 30 sausage samples, 19 samples were negative for the presence of GM soybeans. In the positive samples, the results revealed three GM soybean events [Roundup Ready Soybean (RRS), A2704-12, and/or MON89788] in 11 of the 30 food samples tested. These results demonstrate that sausage products contain different GM soybean events.     

A DNA extraction and purification method using an ion-exchange resin type kit for the detection of genetically modified papaya in processed papaya products

A method for the extraction and purification of genomic DNA from processed papaya products is essential for the detection of approved genetically modified (GM) papaya, according to GM labeling regulations, and unapproved GM papaya, to restrict the import or sale of products containing it. Here, we investigated methods for the extraction of DNA from processed papaya products, including dried papaya, canned papaya and papaya jam. The extraction of DNA from dried papaya and canned papaya required a pre-digestion step, using RNase, cellulase and proteinase K. In the case of papaya jam, α-amylase was found to be indispensable to obtain DNA with high yield and purity. The DNA yield was considerably higher when an ion-exchange resin type kit (IER-100G) was used, compared with other five methods (IER-20G, QIAamp DNA Stool Mini Kit, DNeasy Plant Maxi Kit, GM Quicker 3 Kit and Wizard Cleanup Resin System). We developed a suitable method for the extraction and purification of DNA from processed papaya products, which could be used to detect GM papaya.