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Indeed, the TaqMan chemistry [ 27 ] uses three specific oligonucleotides, that is, two primers and a probe [ 29 — 31 ]. As long as the two dyes are in each other's vicinity, the quencher prevents the emission of fluorescence of the reporter. The fluorescence from the freely released reporter is detected and monitored. The fluorescence increases proportionally to the DNA quantity present in the reaction with each amplification cycle. This correlates with the increase in the copy number of the amplified target. The use of three oligonucleotides allows a more specific detection.

However, at the same time it reduces the flexibility of the system as it will not allow detecting mutated sequences e. In addition, TaqMan amplicons need to be longer as both primers and probe need to be designed in a conserved region. This is an additional drawback when working with processed samples in which the DNA might be degraded. Taqman real-time PCR is also a technology that allows multiplexing by using different fluorescent dyes for different targets to be simultaneously detected in one sample [ 32 — 34 ]. However, seeing the limitation of the number of fluorescent dyes that can be detected by the current real-time PCR instruments and the fact that multiplex PCR becomes more prone to false positives if more than five to 10 real-time PCR systems are to be combined [ 18 ], only a maximum of targets is possible.

One should further guarantee that the detection and quantification of the different targets in a single tube is not impaired, that is, the sensitivity and PCR efficiency should be equal as when the reactions are run in simplex. In addition, attention should be paid to the design of the different primers so that they cannot interact with each other, that is, primer dimer formation. Apart from real-time PCR, new alternative and advanced technologies have been proposed including the use of high-throughput systems or platforms for the detection of multiple targets, for example, microarrays, MIPC, PCR combined with capillary gel electrophoresis fingerprinting , and next generation sequencing [ 12 , 18 , 35 — 38 ] and references therein.

However, at the present stage they are often more expensive, difficult to standardise and validate, and require extensive, specialised work and equipment. They are still at the proof of concept stage and therefore not applicable in routine testing at the moment. Consequently, these methods will not be discussed as this paper focuses on upcoming challenges for GMO detection in food and feed by enforcement laboratories. The genetically modified plants that have been developed and commercialised so far, are mainly transformed using a transgenic insert.

This cassette consists of a regulatory promoter region, a coding sequence trait , and a terminator scheme in Figure 1. Evolution of the currently GM events authorised in the EU and their respective markers. This list is voluntarily not exhaustive but aims at giving the general trend in GMO evolution see text and JRC Compendium [ 23 ] for details. The traits were also limited to genes conferring herbicide tolerance HT and insect resistance IR and were introduced into few commodity crops such as maize, soybean, and oilseed rape.

The main HT sequences are the bacterial phosphinotricin- N -acetyltransferases from Streptomyces viridochromogenes pat and from Streptomyces hygroscopicus bar [ 41 ] and the 5-enolpyruvylshikimatephosphate synthase epsps from the Agrobacterium tumefaciens strain CP4 or from plant origin in casu petunia [ 42 , 43 ]. At the present time, this first-generation of traits and regulatory elements remains prominent in commercial crops and their derived food and feed products. In more recent years, new regulatory sequences have been introduced [ 44 , 45 ] such as the cauliflower mosaic virus 35 S terminator t35S , the figworth mosaic virus promoter pFMV; [ 46 ] , the Agrobacterium tumefaciens nopaline synthase promoter pNOS , the rice actin promoter pAct; [ 47 ] , and the maize ubiquitine promoter pUbiZM; [ 48 ].

Moreover, more species like rice, cotton, sugarbeet, and potato are currently used for transformation. In the coming years, an even larger panel of genes encoding various traits and new plant species are being genetically modified and brought onto the feed and food market. The diversity of transgenes and species used can be illustrated by the following examples.

Hawaiian researchers have developed a papaya Carica papaya resistant to the papaya ringspot virus [ 49 ] which was commercialised in and is being exported to Canada since They further collaborated with other countries such as Brazil, Jamaica, and Thailand to develop resistant transgenic papaya suitable for these countries [ 50 , 51 ]. Also eggplant Solanum melongena L. Cassava Manihot esculenta Crantz , on the other hand has been modified to reduce the amylase content in the starch [ 53 ] which is important for industrial purposes such as paper and textile [ 54 ].

In addition to these single trait GMO, a new trend of gene stacking has been observed [ 55 ]. This implies the introduction of different transgenic inserts into the same plant through conventional crossbreeding, co-, or retransformation to combine different traits [ 55 , 56 ]. In , 12 countries have been growing events with two or more traits. It is the first plant GMO with eight combined events for multiple modes of insect resistance and herbicide tolerance.

It is currently available for maize, but cotton, soybean, and speciality crop variations are to be released [ 59 ]. In the above described GMO, the introduced genetic elements are mainly coming from a noncrossable organism so-called donor organism. These elements are further combined with plant-specific markers such as the rice actin promoter and the maize ubiquitin promoter to construct the transgenic insert.

However, other types of modifications in the genome can be introduced to alter the plant's characteristics. Several of these techniques have already been adopted by commercial plant breeders especially in the USA. One of these is the introduction of a DNA sequence which is completely derived from the recipient species itself in a process that is called cisgenesis [ 60 ]. This technique together with oligonucleotide directed mutagenesis ODM and agro-infiltration are the most used new breeding techniques [ 60 ]. They are still mainly applied at research level. It should however be noted that discussions are still ongoing whether the organisms created by these plant breeding methods will fall under the current GMO regulation or not.

Due to the broad range of GMO, authorised and unauthorised, possibly present on the worldwide and local market, it rapidly becomes unrealistic to use a one-by-one based identification strategy. This step is necessary and an important task of the enforcement laboratories to unequivocally identify each GMO, that is, by targeting the DNA fragment on the junction between the plant genome and the transgene event-specific method; [ 61 ]. Already now, strategies like simplex or multiplex real-time PCR limited to targets when using the real-time PCR technology for the identification of known junction sequences i.

In the near future this will become simply impossible. Moreover, no event-specific methods are available for the UGM, especially the unexpected ones coming from field trials, and so they are undetectable with such kind of methodology. Only in the positive cases, a second step will specifically identify which individual event s is are present in the sample. The most common recombinant elements in the current GM crops are p35S and tNOS [ 44 , 45 , 62 ], both transcription regulating sequences.

Consequently, in order to assess the presence of GM material in a product, a screening PCR for those generic recombinant markers is often performed. This basic screening was very efficient when only few GMO were present on the food and feed market. However, as the number of GM events is growing exponentially, limiting the screening to those common targets with high-coverage power has the disadvantage that a too large number of GM needs to be confirmed using the event-specific detection methods.

Therefore, additional targets GM-specific elements such as HR genes pat , bar , epsps and IR genes cry gene family can be added to reduce the number of putative GM events to be identified in the sample due to a higher discriminative power. Several detection methods have been published targeting such types of elements [ 68 — 70 ]. This makes the simultaneous use in a single run in a well plate format impossible by enforcement laboratories. To reduce the number of analyses and facilitate high-throughput analysis some multiplex real-time PCR screening strategies have been developed. However, these require multichannel detection devices and often include costly detection probes for at least some of the targets [ 32 — 34 ].

Furthermore, the multiplex strategy misses the flexibility of a modular system, that is, each time a new method needs to be added, the complete system needs to be validated. In this screening approach, a limited set of simplex real-time PCR methods targeting various types of elements endogenous sequences, generic markers, and GM-specific elements is selected in such a way that multiple GMO can be detected within a single analytical run.

A careful selection of the markers was performed allowing developing an approach wherein not only most GMO are detected but also discriminated [ 28 , 68 , 72 , 73 ]. Special attention was given to the size of the amplicon, that is, the methods were developed to amplify a short amplicon around bp that is particularly important to detect transgenes in processed food where the DNA might be degraded. Many of the elements used in transgenic constructs are derived from donor organisms such as bacteria and viruses.

One such element is the 35 S promoter of the cauliflower mosaic virus CaMV which has Brassica plants as its natural host. To discriminate between the p35S present in a GM event and the one due to its possible natural presence, an additional marker CRT was developed.

Genetically engineered food: methods and detection.

This marker targets the reverse transcriptase gene of CaMV and will thus allow the detection of the virus. As this approach is modular, three additional markers were developed to answer the actual need in GMO detection. Two are targeting the promoters of the figworth mosaic virus pFMV and the A. An example of the efficient use of this decision support system is shown in Figure 2 for the detection of maize event NK The following steps are performed. As this decision support system is a modular tool, screening methods targeting new sequences present in new GM events can be added at any moment.

If this is not the case, one might suspect the presence of an UGM in the sample and further confirmation of the event would be needed. This might require first the use of alternative and advanced tools such as anchor-PCR fingerprinting [ 79 ] followed by confirmatory sequencing of the suspected amplified fragment s or DNA walking [ 80 ] to identify the junction between the transgene and the plant genome for which later on a real-time PCR event-specific method can be developed.

Alternatively, the next generation sequencing technology can be used to screen whole genomes for foreign DNA and their respective junctions. The roadmap for these ad hoc scenarios should be drawn based upon the evaluation of the perceived risk for health, environment, and economy. The number of GMO cultivated for commercial or research purposes continues to increase worldwide. Not only these will add to the complexity of detecting authorised GMO by the enforcement laboratories in food and feed samples, but also the occurrence of UGM which will be steadily increasing in the coming years.

Unintended escapes and intermingling with UGM can never be ruled out [ 81 ]. Most likely there will also be a growing number of GMO with traits of industrial or pharmaceutical relevance but not intended for food or feed use. These could also enter the agricultural supply chains and cause ethical and religious concern or even pose a significant risk to human and animal health [ 38 ]. Therefore, there is an evident need for a continuous development of appropriate detection methods and strategies. However, gene stacking poses a new challenge for GMO detection laboratories as there is no way except when the analysis is done on individual seeds and plants to discriminate between the presence of the GMO separately in a sample or combined as a stacked event [ 16 , 55 , 82 ].

In response to the increasing diversity of GMO on the market, new screening markers for the new species-specific sequences e. The challenge will be more in collecting the massive amount of data sequences especially for UGM to develop standardised real-time PCR methods and then combining the obtained results in an appropriate decision system, like CoSYPS, for an efficient manner of GMO testing. Eventually, also the event-specific methods should be developed, to unambiguously identify the GMO in a second step. Samples containing a mutation form heteroduplexes in the post-PCR fragment mix.

These are identified as differential melting temperature curves in comparison to homoduplexes e. HRM can be complemented with confirmation of the mutation by sequencing. However, the screening methodology will no longer fully be suited for the detection of new types of genetic modifications that are under development through new plant breeding techniques. In the case of cisgenesis, for example, detection of the inserted elements alone can no longer be used as evidence of the genetic modification. However, the order of the different elements and the insertion loci into the plant genome still may offer an opportunity for the detection of these modifications [ 38 ].

Provided this information is available, these event-specific sequences can be exploited to develop new real-time PCR-based methods if needed. Also for other types of genetic modifications introduced by these new plant breeding techniques e. Without prior knowledge, detection of these small modifications would be unlikely to be used in routine laboratories as more complex technologies are needed e.

For the others, the detection of the genetic modification is currently not possible e. In addition, crops resulting from most of the techniques cannot be distinguished from conventionally bred crops or from crops produced by natural genetic variation, and identification is therefore not possible [ 60 ].

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These new advances in molecular biotechnology pose challenges to regulators as to whether they fall within the scope of their regulatory authority. In , the European Commission, with the cooperation of different member states, published an overview of these techniques [ 60 ]. At the moment it is, however, not clear yet if these new breeding techniques lead to organisms that will be classified under the currently used GMO definition [ 85 ] and thus the GMO legislation. If they would be classified as GMO, they would require control and traceability.

As the current methodologies will be insufficient, new approaches, probably involving a combination of different analytical methods, need to be developed. If they are not classified as GMO, the currently used detection methods would remain appropriate for now. Nevertheless, to anticipate the increasing number of GM events, the development of additional screening markers, potentially in multiplex set-ups which might involve technologies beyond the real-time PCR platforms , should be continued to arrive in the near future to a robust and cost-efficient solution to the GMO challenge.

Europe PMC requires Javascript to function effectively. Recent Activity. The snippet could not be located in the article text. This may be because the snippet appears in a figure legend, contains special characters or spans different sections of the article. J Biomed Biotechnol. Published online Oct PMID: Sylvia R. Broeders , Sigrid C. De Keersmaecker , and Nancy H. Sigrid C. De Keersmaecker. Nancy H. Roosens: eb. Received Mar 30; Accepted Sep Broeders et al.

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Abstract Biotech crops are the fastest adopted crop technology in the history of modern agriculture. Introduction Commercialisation of biotech or genetically modified GM crops was started in Evolution of GMO The genetically modified plants that have been developed and commercialised so far, are mainly transformed using a transgenic insert. Open in a separate window. Figure 1. Evolution of the Screening Strategy in Response to the GMO Complexity Due to the broad range of GMO, authorised and unauthorised, possibly present on the worldwide and local market, it rapidly becomes unrealistic to use a one-by-one based identification strategy.

Figure 2. Conclusions and Future Perspectives The number of GMO cultivated for commercial or research purposes continues to increase worldwide. References 1. James C. Executive summary. Dymond M, Hurr K. European Parliament. Official Journal of the European Union.

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Analytical methods for detection and determination of genetically modified organisms in agricultural crops and plant-derived food products. European Food Research and Technology. New approaches in GMO detection. Although gene therapy is still relatively new, it has had some successes. It has been used to treat genetic disorders such as severe combined immunodeficiency , [] and Leber's congenital amaurosis. Germline gene therapy results in any change being inheritable, which has raised concerns within the scientific community. The work was widely condemned as unethical, dangerous, and premature.

Aquaculture is a growing industry, currently providing over half the consumed fish worldwide. Fish can also be used to detect aquatic pollution or function as bioreactors. Several groups have been developing zebrafish to detect pollution by attaching fluorescent proteins to genes activated by the presence of pollutants. The fish will then glow and can be used as environmental sensors.

It was originally developed by one of the groups to detect pollution, but is now part of the ornamental fish trade, becoming the first genetically modified animal to become publicly available as a pet when in it was introduced for sale in the USA. GM fish are widely used in basic research in genetics and development. Two species of fish, zebrafish and medaka , are most commonly modified because they have optically clear chorions membranes in the egg , rapidly develop, and the one-cell embryo is easy to see and microinject with transgenic DNA.

GM fish have been developed with promoters driving an over-production of growth hormone for use in the aquaculture industry to increase the speed of development and potentially reduce fishing pressure on wild stocks. This has resulted in dramatic growth enhancement in several species, including salmon , [] trout [] and tilapia. In biological research, transgenic fruit flies Drosophila melanogaster are model organisms used to study the effects of genetic changes on development.

They also have a relatively simple genome compared to many vertebrates , with typically only one copy of each gene, making phenotypic analysis easy. Due to their significance to human health, scientists are looking at ways to control mosquitoes through genetic engineering. Malaria-resistant mosquitoes have been developed in the laboratory by inserting a gene that reduces the development of the malaria parasite [] and then use homing endonucleases to rapidly spread that gene throughout the male population known as a gene drive.

Other insect pests that make attractive targets are moths. Silkworm, the larvae stage of Bombyx mori , is an economically important insect in sericulture. Scientists are developing strategies to enhance silk quality and quantity. There is also potential to use the silk producing machinery to make other valuable proteins. Systems have been developed to create transgenic organisms in a wide variety of other animals. Chickens have been genetically modified for a variety of purposes. This includes studying embryo development , [] preventing the transmission of bird flu [] and providing evolutionary insights using reverse engineering to recreate dinosaur-like phenotypes.

GM frogs can also be used as pollution sensors, especially for endocrine disrupting chemicals. The nematode Caenorhabditis elegans is one of the major model organisms for researching molecular biology. Transgenes can also be combined with RNAi techniques to rescue phenotypes, study gene function, image cell development in real time or control expression for different tissues or developmental stages.

The gene responsible for albinism in sea cucumbers has been found and used to engineer white sea cucumbers , a rare delicacy. The technology also opens the way to investigate the genes responsible for some of the cucumbers more unusual traits, including hibernating in summer, eviscerating their intestines, and dissolving their bodies upon death.

By using microinjection and radiation scientists have now created the first genetically modified flatworms. It is of interest due to its reproductive cycle being synchronized with lunar phases, regeneration capacity and slow evolution rate. Genetically modified organisms are regulated by government agencies.

This applies to research as well as the release of genetically modified organisms, including crops and food. The development of a regulatory framework concerning genetic engineering began in , at Asilomar , California. The Asilomar meeting recommended a set of guidelines regarding the cautious use of recombinant technology and any products resulting from that technology.

Universities and research institutes generally have a special committee that is responsible for approving any experiments that involve genetic engineering. Many experiments also need permission from a national regulatory group or legislation. All staff must be trained in the use of GMOs and all laboratories must gain approval from their regulatory agency to work with GMOs. They are assigned to one of four risk categories based on their virulence, the severity of disease, the mode of transmission, and the availability of preventive measures or treatments. There are four biosafety levels that a laboratory can fall into, ranging from level 1 which is suitable for working with agents not associated with disease to level 4 working with life-threatening agents.

Different countries use different nomenclature to describe the levels and can have different requirements for what can be done at each level. For example, a crop not intended for food use is generally not reviewed by authorities responsible for food safety. The European Union EU differentiates between approval for cultivation within the EU and approval for import and processing. USA regulations sees them as separate and does not regulate them under the same conditions, while in Europe a GMO is any organism created using genetic engineering techniques.

One of the key issues concerning regulators is whether GM products should be labeled. The European Commission says that mandatory labeling and traceability are needed to allow for informed choice, avoid potential false advertising [] and facilitate the withdrawal of products if adverse effects on health or the environment are discovered. Labeling of GMO products in the marketplace is required in 64 countries. In Canada and the US labeling of GM food is voluntary, [] while in Europe all food including processed food or feed which contains greater than 0.

There is controversy over GMOs, especially with regard to their release outside laboratory environments. The dispute involves consumers, producers, biotechnology companies, governmental regulators, nongovernmental organizations, and scientists. Many of these concerns involve GM crops and whether food produced from them is safe and what impact growing them will have on the environment. These controversies have led to litigation, international trade disputes, and protests, and to restrictive regulation of commercial products in some countries. These include whether they may provoke an allergic reaction , whether the transgenes could transfer to human cells and whether genes not approved for human consumption could outcross into the food supply.

There is a scientific consensus [] [] [] [] that currently available food derived from GM crops poses no greater risk to human health than conventional food, [] [] [] [] [] [] but that each GM food needs to be tested on a case-by-case basis before introduction. Gene flow between GM crops and compatible plants, along with increased use of broad-spectrum herbicides , [] can increase the risk of herbicide resistant weed populations.

Other environmental and agronomic concerns include a decrease in biodiversity, an increase in secondary pests non-targeted pests and evolution of resistant insect pests. Resistance was found to be slow to evolve when best practice strategies were followed. Follow up studies have since shown that the toxicity levels encountered in the field were not high enough to harm the larvae.

Accusations that scientists are " playing God " and other religious issues have been ascribed to the technology from the beginning. They propose mandatory labeling [] [] or a moratorium on such products. Environment International. In spite of this, the number of studies specifically focused on safety assessment of GM plants is still limited. However, it is important to remark that for the first time, a certain equilibrium in the number of research groups suggesting, on the basis of their studies, that a number of varieties of GM products mainly maize and soybeans are as safe and nutritious as the respective conventional non-GM plant, and those raising still serious concerns, was observed.

Moreover, it is worth mentioning that most of the studies demonstrating that GM foods are as nutritional and safe as those obtained by conventional breeding, have been performed by biotechnology companies or associates, which are also responsible of commercializing these GM plants. Anyhow, this represents a notable advance in comparison with the lack of studies published in recent years in scientific journals by those companies. Krimsky S Archived from the original PDF on 7 February Retrieved 7 July I began this article with the testimonials from respected scientists that there is literally no scientific controversy over the health effects of GMOs.

My investigation into the scientific literature tells another story. Critical Reviews in Biotechnology. Here, we show that a number of articles some of which have strongly and negatively influenced the public opinion on GM crops and even provoked political actions, such as GMO embargo, share common flaws in the statistical evaluation of the data. Having accounted for these flaws, we conclude that the data presented in these articles does not provide any substantial evidence of GMO harm. The presented articles suggesting possible harm of GMOs received high public attention. However, despite their claims, they actually weaken the evidence for the harm and lack of substantial equivalency of studied GMOs.

We emphasize that with over published articles on GMOs over the last 10 years it is expected that some of them should have reported undesired differences between GMOs and conventional crops even if no such differences exist in reality. Journal of the Science of Food and Agriculture.

It is therefore not surprising that efforts to require labeling and to ban GMOs have been a growing political issue in the USA citing Domingo and Bordonaba, Overall, a broad scientific consensus holds that currently marketed GM food poses no greater risk than conventional food Major national and international science and medical associations have stated that no adverse human health effects related to GMO food have been reported or substantiated in peer-reviewed literature to date. Despite various concerns, today, the American Association for the Advancement of Science, the World Health Organization, and many independent international science organizations agree that GMOs are just as safe as other foods.

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How to Deal with the Upcoming Challenges in GMO Detection in Food and Feed

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