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Traceability of genetically modified organisms
Abstract
EU regulations stipulate the labeling of food products containing genetically modified organisms (GMOs) unless the GMO content is due to adventitious and unintended 'contamination' and not exceeding the 1% level at ingredient basis. In addition, member states have to ensure full traceability at all stages of the placing on the market of GMOs. Both requirements ensure consumers 'right to know', facilitate enforcement of regulatory requirements and are of importance for environmental monitoring and postmarket surveillance. Besides administrative procedures, such as used in quality certification systems, the significance of adequate molecular methods becomes more and more apparent. During the last decade a considerable number of molecular methods have been developed and validated that enable the detection, identification and quantification of GMO impurities. Most of them rely on the PCR technology and can only detect one specific stretch of DNA. It can, however, be anticipated that in the near future the situation will become more complex. The number of GMO varieties, including 'stacked-gene' varieties, which will enter the European Market will increase and it is likely that these varieties will harbor more variable constructs. New tools will be necessary to keep up with these developments. One of the most promising techniques is microarray analysis. This technique enables the screening for a large number of different GMOs within a single experiment.
... c o m / l o c a t e / f o o d c h e m gene, naturally present in the genome of the analyzed species (i.e., zein in Zea mays and lectin in Glycine max) (Meyer, Chardonnens, Hubner, & Luthy, 1996; Meyer & Jaccaud, 1997 ). Despite the interesting aspects of these studies, most of the reported work refers to raw material, such as flours or seeds, or to the final product of food processing (Aarts, van Rie, & Kok, 2002; Ahmed, 2002; Di Pinto et al., 2007; Engel, Moreano, Ehlert, & Busch, 2006; Germini et al., 2005; Passamano & Pighini, 2006; Peano et al., 2005). A systematic study on the traceability of GM DNA along a food processing chain has never been reported, and only laboratory model studies have been conducted on different processing procedures (Bauer, Weller, Hames, & Hertel, 2003). ...
... It is thus of primary importance to verify the possibility to amplify (amplificability) the extracted DNA. The DNA degradation can be caused, for example, by heating treatments (Ahmed, 2002) and exposure to pH changes (Aarts et al., 2002). Finally, another important factor which could affect the amplification step, leading also to false negative results should be taken into account, which is the presence of different inhibitors of the enzyme Taq polymerase such as proteins, fats, polysaccharides, polyphenolics and other compounds that may be present in DNA extracted from food matrices (Corbisier et al., 2007; Di Pinto et al., 2007; Lüthy, 1999; Peano et al., 2005). ...
The development of analytical methods for genetically modified organisms (GMO) screening is of great interest. In particular, since even highly processed GMO-derived food products are covered by new European legislations, a great effort has been devoted to the application of the analytical tests to these products.This work describes a polymerase chain reaction-based qualitative screening assay and a biosensor-based approach to detect transgenes in a Roundup Ready® soybean processing line. Roundup Ready® soybean was specifically analyzed in eight types of processed materials – seeds, crushed seeds, expander, crude flour, proteic flour, crude oil, degummed oil and lecithin – all derived from the same initial source and produced during the manufacturing process. Specific combinations of primers were used to differentiate sequences from the whole insert. The amplification of “marker” fragments with a maximum length of 500 bp was successfully achieved both in raw material (seeds) and in partially (crushed seeds, crude and proteic flours) and highly (crude and degummed oils and fluid lecithin) processed materials.Moreover, the extraction procedure was optimised and the polymerase chain reaction-electrophoresis analysis has been implemented by a biosensor-based approach.
... The rules on labelling are associated with obligatory information for all food stuffs (Borges et al., 2018). Aarts et al. (2002) have further highlighted that GM products should be labelled to ensure traceability of these products at all stages of the food supply chain. The primary goals of these standards are to safeguard consumers' health and safety, as well as to guarantee that food traders implement fair international and regional trading procedures. ...
The study was carried out to investigate the implications of the Namibian biosafety regulations on Namibian food and feed importers. After the Biosafety Act, 2006 (Act No. 7 of 2006), the biosafety regulation was gazetted in 2016, which saw the implementation of the national framework, the impact of food and feed importers was not known. The objective of the study was to assess the adoption and application of the national biosafety labelling regulations by food and feed importers. In addition, the impacts of these regulations on Namibian food and feed importers were assessed. The study used a structured online and hard copy survey questionnaire based on responses from 340 Namibian importers of food and feed products from eight identified Namibian regions: Khomas, Erongo, Kavango West, Kavango East, Omusati, Oshana, Ohangwena, Oshikoto, and Zambezi who have the knowledge required for the adoption and application of the Namibian biosafety labelling regulations. Using the Mann-Whitney test, the study confirmed that individuals who are aware of the biosafety Act, 2006 (Act No. 7 of 2006) are less likely to agree with statements such as experiencing problems in fulfilling requirements under the biosafety regulations. It was further concluded that there is a need to reduce the current administrative burdens for handling applications and improve dialogue between regulators and the food and feed importing industry while increasing the competence of regulators and creating more labelling regulation awareness for food and feed importers. The study further suggests that public awareness is required beyond food and feed importers.
... Another method used to regulate the GMOs and GM foods were the product traceability; the ability to track back the GMO batches and their history from the seed production to its supply chain and sale. The identity preservation (IP) system, a formalized policy, traces the GMOs and GM crops from the seed production to the processing chain [20]. Such a traceability system is crucial as it traces back any unintended gene spillage of accidental releases of GM products to the environment. ...
Genetically modified organisms (GMOs) and genetically modified (GM) foods were doubted for creating an adverse health problem for humans and animals besides causing severe environmental impacts. However, such allegations were mostly made based on inaccurate scientific data. Consequently, GM foods face blatant rejections from the public member. On the other hand, there is a rising concern for sustainable food supply to the increasing world population, and if no mitigation is taken, it may lead to severe malnutrition problems in future. As GM foods can be an ideal solution for world hunger and malnourishment problems, the misconceptions surrounding them must be first resolved. Every country that approved GMOs and GM foods has set up regulatory bodies besides enacting laws and ordinances to safeguard its citizens. In Malaysia, the Malaysian Biosafety Act and other governing bodies such as National Biosafety Board (NBB) and Genetic Modification Advisory Committee (GMAC) have played a pivotal role in ensuring the containment of GMOs and GM foods from accidental release to the environment. Despite the well-regulated plot, Malaysians are still reluctant to accept GMOs and GM foods. Thus, everystakeholder and media must play a crucialrole to curb GMOs' negative image from spreading to the public.
... Основною причиною такої необхідності є забезпечення споживачу права вибору споживання харчової продукції, яка містить ГМО. В зв'язку з чим обіг ГМО в більшості країн світу є контрольованим (наприклад -Регламенти ЄС 1829/2003, Регламент ЄС 1830/2003 Регламент ЄС 258/27) [2]. В Україні нормативно-правовим документом який регулює обігу ГМО є Закон України № 1103 «Про державну систему біобезпеки при створенні, випробуванні, транспортуванні та використанні генетично модифікованих організмів» від 31.05.2007 ...
... Основною причиною такої необхідності є забезпечення споживачу права вибору споживання харчової продукції, яка містить ГМО. В зв'язку з чим обіг ГМО в більшості країн світу є контрольованим (наприклад -Регламенти ЄС 1829/2003, Регламент ЄС 1830/2003 Регламент ЄС 258/27) [2]. В Україні нормативно-правовим документом який регулює обігу ГМО є Закон України № 1103 «Про державну систему біобезпеки при створенні, випробуванні, транспортуванні та використанні генетично модифікованих організмів» від 31.05.2007 ...
Keywords: quality and safety of AIC products, molecular genetics methods of research, genetically modified plants (GMOs), falsification of the species composition of meat products.
Introduction.
In the Department of Molecular Biological Research, the Ukrainian Laboratory of Quality and Safety of Agricultural Products method of polymerase chain reaction is used to detection of genetically modified organisms in agricultural products and to DNA-identification of the species composition meat raw in products of the meat industry.
The goal of the work was to analyze and summarize the results of research detection of genetically modified organisms, identification of GM events, and DNA-identification of the species composition meat raw in sausages of the obtained in 2018.
Materials and methods.
To detection of genetically modified organisms and identification of GM events were used samples of the commercial direction. To study DNA-identification of the species composition meat raw in sausages we used 12 different kinds of sausages which were purchased in the trading network.
Results of research and discussion.
During 2018 were analyzed 356 samples of agricultural products
for the detection of genetically modified organisms by the real-time PCR. We discovered 70 positive samples that representing 19,7 % of the total. It has been established that over the past three years, the number of positive samples in percentage terms increases yearly. The main GM events of plants that are present in Ukraine are the GM-soybean GTS 40-3-2 and the GM-rape GT 73. GM-event GTS 40-3-2 is also identified in feeds. It says that biotechnological soybean is used to produce feed.
There were examined 12 samples different kind of sausages regarding the species composition of meat raw by real time PCR and found 5 samples(42,0 %), which did not correspond to the species composition of meat raw as declared by the producer.
Conclusions and prospects for further research.
We have shown that in agricultural market in Ukraine are present genetically modified crops such as soybean, corn and rape (GM event soybeen GTS 40-3-2 and event MON 89788, rape GT 73). We have found that 5 samples from 12 (42,0 %) the species composition of meat raw did not correspond to the species composition of meat raw as declared by the producer. The obtained data testify to the violation of the technology of production of this kind of meat products.
... Finally, I claim that the high level of environmental and health safety protection that the EU is trying to achieve through the special regulation of nanomaterials goes hand in hand with a very strong institutional commitment to the massive industrial exploitation of nanotechnology, destined to play a key role, together with other technological innovations, in stimulating a Bworld-class competitive industry^( [55], p. 2). 1 This arguably implies that the safe or controlled development of nanotechnology is practically taken for granted. I argue that this assumption is potentially problematic and contestable. ...
The European Union (EU) is strategically committed to the development of nanotechnology and its industrial exploitation. However, nanotechnology also has the potential to disrupt human health and the environment. The EU claims to be committed to the safe and responsible development of nanotechnology. In this sense, the EU has become the first governing body in the world to develop nanospecific regulations, largely due to legislative action taken by the European Parliament, which has compensated for the European Commission’s reluctance to develop special regulations for nanomaterials. Nevertheless, divergences aside, political bodies in the EU assume that nanotechnology development is controllable and take for granted that both the massive industrial use of nanomaterials and a high level of environmental and health protection are compatible. However, experiences such as the European controversy over agri-food biotechnology, which somewhat delegitimized the regulatory authority of the EU over technological safety and acceptability, arguably show that controllability assumptions are contestable on the grounds of alternative socio-economic and cultural preferences and values. Recently developed inclusive governance models on safety and innovation, such as “Responsible Research and Innovation” (RRI), widely claim that a diversity of considerations and issues are integrated into R&D processes. Even so, the possibility of more radically alternative constitutions of socio-technical safety seems to be seriously limited by the current ideology of innovation and economic imperatives of the global, knowledge-based, capitalist economy.
... It is thus of primary importance to verify the possibility to amplify (amplificability) the extracted DNA. The DNA degradation can be caused, for example, by heating treatments [59] and exposure to pH changes [60]. Furthermore different inhibitors of the Taq polymerase enzyme such as proteins, fats, polysaccharides, polyphenols, and other compounds that may be present in DNA may inhibit the DNA template amplification [61]- [64]. ...
In this paper real-time, label free sensing principle based on Surface Plasmon Resonance (SPR) and gravimetric, i.e. Quartz Crystal Microbalance (QCM) are described and some applications to food analysis problems are reported. Affinity receptors are introduced and examples using antibodies, nucleic acid probes and biomimetic receptors i.e. molecular imprinted polymers (MIP) reported. In particualr pesticide, endotoxins, pathogens, Genetically Modified Organism (GMOs) detection and food origin analysis are reviewed.
... More specific and accurate biological identification methods are being developed to support tracing food products on the basis of their food composition. Several biological identification methods have already been developed to identify the authenticity of food products on species, production type, and geographical origin (Dalvit et al., 2007;Franke et al., 2005;Pillonel et al., 2003;Schrö der, 2008;Civera, 2003;Peres et al., 2007;Prache, 2007;Voerkelius et al., 2010;Engel and Ratel, 2008;Van Ruth et al., 2010a,b;Maçatelli et al., 2009;Scholtens et al., 2010a;Aarts et al., 2002). The specificity and accuracy of these analysis methods influence the traceability of the origin of products and processing. ...
Traceability of food implies the ability to trace and follow a food, feed, or a food-producing animal or substance intended to be, or expected to be, incorporated into a food or feed, through all stages of production, processing, and distribution. The importance of traceability has grown due to the consumers' increasing attention to food safety and food quality, and due to the increasing complexity of food supply chains. There are several types of food traceability depending on how traceability is obtained and on what information it concerns, i.e., conventional traceability, genetic traceability, and geographic traceability. Optical, electronic, and biological identification methods are used to identify and transfer the information. The aim of this chapter is to explain the basic characteristics of traceability systems in the supply chain and to list the developments in this area. An extensive overview of analytical systems is described that may verify documentary information on the basis of analyses, i.e., DNA-based methods, chemical verification methods, visual markers for the determination of food and feed, and sensory analysis. It is concluded that in future new traceability systems will be developed that combine logistical data with analytical data that are derived from more informative multiplex approaches, increasingly comprising also data from different types of analytical approaches. In this way, the consumer may be even better informed.
... More specific and accurate biological identification methods are being developed to support tracing food products on the basis of their food composition. Several biological identification methods have already been developed to identify the authenticity of food products on species, production type, and geographical origin (Dalvit et al., 2007;Franke et al., 2005;Pillonel et al., 2003;Schrö der, 2008;Civera, 2003;Peres et al., 2007;Prache, 2007;Voerkelius et al., 2010;Engel and Ratel, 2008;Van Ruth et al., 2010a,b;Maçatelli et al., 2009;Scholtens et al., 2010a;Aarts et al., 2002). The specificity and accuracy of these analysis methods influence the traceability of the origin of products and processing. ...
Traceability of food implies the ability to trace and follow a food, feed, or a food-producing animal or substance intended to be, or expected to be, incorporated into a food or feed, through all stages of production, processing, and distribution. The importance of traceability has grown due to the consumers' increasing attention to food safety and food quality, and due to the increasing complexity of food supply chains. There are several types of food traceability depending on how traceability is obtained and on what information it concerns, i.e., conventional traceability, genetic traceability, and geographic traceability. Optical, electronic, and biological identification methods are used to identify and transfer the information. The aim of this chapter is to explain the basic characteristics of traceability systems in the supply chain and to list the developments in this area. An extensive overview of analytical systems is described that may verify documentary information on the basis of analyses, i.e., DNA-based methods, chemical verification methods, visual markers for the determination of food and feed, and sensory analysis. It is concluded that in future new traceability systems will be developed that combine logistical data with analytical data that are derived from more informative multiplex approaches, increasingly comprising also data from different types of analytical approaches. In this way, the consumer may be even better informed.
... To perform a transgenic analysis, a primary and basic task is to identify the existence of certain genotype. The currently used methods for transgenic product identification include protein-based methods [9], DNA-based methods [10], microscopy, spectroscopy, and chromatography [1,11]. The rationale behind NIR transgenic analysis is the spectral absorbance of molecular bonds such as C-H, C-N, and C-O that is related to the phenotypic changes (expression level) caused by genotypic changes. ...
This paper reports the application of near infrared (NIR) spectroscopy and pattern recognition methods to rapid and automatic discrimination of the genotypes (parent, transgenic, and parent-transgenic hybrid) of cotton plants. Diffuse reflectance NIR spectra of representative cotton seeds (n = 120) and leaves (n = 123) were measured in the range of 4000-12000 cm(-1). A practical problem when developing classification models is the degradation and even breakdown of models caused by outliers. Considering the high-dimensional nature and uncertainty of potential spectral outliers, robust principal component analysis (rPCA) was applied to each separate sample group to detect and exclude outliers. The influence of different data preprocessing methods on model prediction performance was also investigated. The results demonstrate that rPCA can effectively detect outliers and maintain the efficiency of discriminant analysis. Moreover, the classification accuracy can be significantly improved by second-order derivative and standard normal variate (SNV). The best partial least squares discriminant analysis (PLSDA) models obtained total classification accuracy of 100% and 97.6% for seeds and leaves, respectively.
... Hierbij is het van belang dat niet alleen informatie ten aanzien van de aanwezigheid van GGO's beschikbaar komt voor de eindafnemer, maar dat de eindafnemer ook eenvoudig vast kan stellen welke GGO's het betreft en in welke concentraties ze zich in bepaalde partijen bevinden. Alleen dergelijke nieuwe informatieve en transparante informatie-overdrachtssystemen zullen kunnen garanderen dat alle doelstellingen van deze traceerbaarheidsverordening gehaald kunnen worden [6]. ...
De Expertisegroep 'GGO-vrije ketens' van Wageningen UR komt tot deze conclusie dat de realisatie van diervoederketens zonder genetisch gemodificeerde organismen (GGO) steeds lastiger wordt. Binnen de huidige wettelijke kaders is bepaald dat hetpercentage GGO-bestanddelen dat onbedoeld in een partij GGO-vrije diervoeders terechtkomt maximaal 0,9%mag zijn. De biologische sector wil helemaal geen vermenging en streeft naar een percentage van 0%. Het produceren van GGO-vrije diervoeders isproblematisch omdat in belangrijke bestanddelen van diervoeders, met name soja en mais, in veel gevallen al sporen van GGO-variëteiten voorkomen. Dit zal nog verder bemoeilijkt worden als er GGO-varianten van meer gewassen hun intrede doen op de Europeseen wereldmarkt. Vermenging is dus zeer moeilijk te voorkomen en geheel GGO-vrije productie zal in de nabije toekomst alleen gerealiseerd kunnen worden met kostbare ketensystemen die ggo- en ggo-vrije productie gescheiden kunnen houden. Uit het onderzoekblijkt verder dat de mogelijkheden om GGO-vrij te produceren in de praktijk bepaald zal worden door het type veevoederproductieketens. Wanneer in bepaalde veevoeders meer gewassen verwerkt worden waarvan er wereldwijd al GGO-partijen op commerciële basisin productie zijn, zal het lastiger zijn om GGO-vrijeproductiesystemen te handhaven. Het ziet er naar uit dat de kosten voor GGO-vrije productie, moeten worden opgebracht door de sector die GGO-vrij wil leveren. Aangezien aansprakelijkheidskwesties bijonbedoelde vermenging voorlopig niet internationaal of EU-breed geregeld zullen worden, ligt het in de lijn der verwachting dat conflicten op dit terrein niet zullen uitblijven.
... The microarray technology also has the theoretic potential to detect unauthorised GM varieties that have any similarity with known (parts of) genetic constructs. Microarray systems are now under development that screen for transgenic (border) elements in approved GM varieties as well as for unique control elements for the relevant species or possible sources of contamination (GMOchips, 2003;Rudi et al., 2003;Kok et al., 2002;Aarts et al., 2002). Fig. 1 depicts the work-up of DNA samples prior to microarray analysis for the presence of DNA fragments from GM soy and maize as depicted in Fig. 2. In this way, all known and/or approved GM varieties will show a specific pattern on the array and aberrant patterns may indicate that unapproved GM varieties are present in the sample. ...
Both labelling and traceability of genetically modified organisms are current issues that are considered in trade and regulation. Currently, labelling of genetically modified foods containing detectable transgenic material is required by EU legislation. A proposed package of legislation would extend this labelling to foods without any traces of transgenics. These new legislations would also impose labelling and a traceability system based on documentation throughout the food and feed manufacture system. The regulatory issues of risk analysis and labelling are currently harmonised by Codex Alimentarius. The implementation and maintenance of the regulations necessitates sampling protocols and analytical methodologies that allow for accurate determination of the content of genetically modified organisms within a food and feed sample. Current methodologies for the analysis of genetically modified organisms are focused on either one of two targets, the transgenic DNA inserted- or the novel protein(s) expressed- in a genetically modified product. For most DNA-based detection methods, the polymerase chain reaction is employed. Items that need consideration in the use of DNA-based detection methods include the specificity, sensitivity, matrix effects, internal reference DNA, availability of external reference materials, hemizygosity versus homozygosity, extrachromosomal DNA, and international harmonisation. For most protein-based methods, enzyme-linked immunosorbent assays with antibodies binding the novel protein are employed. Consideration should be given to the selection of the antigen bound by the antibody, accuracy, validation, and matrix effects. Currently, validation of detection methods for analysis of genetically modified organisms is taking place. In addition, new methodologies are developed, including the use of microarrays, mass spectrometry, and surface plasmon resonance. Challenges for GMO detection include the detection of transgenic material in materials with varying chromosome numbers. The existing and proposed regulatory EU requirements for traceability of genetically modified products fit within a broader tendency towards traceability of foods in general and, commercially, towards products that can be distinguished from each other. Traceability systems document the history of a product and may serve the purpose of both marketing and health protection. In this framework, segregation and identity preservation systems allow for the separation of genetically modified and non-modified products from "farm to fork". Implementation of these systems comes with specific technical requirements for each particular step of the food processing chain. In addition, the feasibility of traceability systems depends on a number of factors, including unique identifiers for each genetically modified product, detection methods, permissible levels of contamination, and financial costs. In conclusion, progress has been achieved in the field of sampling, detection, and traceability of genetically modified products, while some issues remain to be solved. For success, much will depend on the threshold level for adventitious contamination set by legislation.
... Nowadays, ingredient authentication routinely involves DNA analysis with species-specific PCR markers. Using such a method, various protocols have been designed in the last 10 years (Lockley and Bardsley, 2000) to characterize animal meats (Calvo et al., 2001;Bottero et al., 2002), fish species (Asen-sio et al., 2002;Masri et al., 2002;Rego et al., 2002) and plant GMOs (Meyer, 1999;Aarts et al., 2002;Farid, 2002). ...
Canned truffle products labeled Tuber melanosporum, the famous Perigord truffle, may contain other less tasty and cheaper truffle species. To protect consumers from fraud, a PCR DNA-based method was used to unequivocally identify the nature of the product. Several rapid and simple cell lysis procedures, used in conjunction with a commercially available DNA purification kit, were evaluated for their effectiveness in recovering DNA from canned truffle. In parallel, a marker for T. melanosporum was tested on the mitochondrial rDNA. These two techniques were then combined to differentiate T. melanosporum from other truffle species like T. aestivum, T. brumale or T. indicum up to the legal threshold in canned products. These findings not only allow a comparison of the effectiveness of the different DNA extraction methods but also provide a preliminary indication of the specificity and sensitivity of the detection with the mitochondrial marker that might be attainable for truffle species in a quantitative PCR-based analysis method.
Vegetable proteins such as soybean protein have numerous nutritional and functional characteristics, and consequently, their utilization in meat products development has dramatically increased in recent decades. Due to high demands for soybean, transgenic Roundup Ready (RR) soybean line grains were developed and widely distributed into global markets. The current study was designed to investigate the presence of transgenic soybean in meat products sold in Riyadh food retails, Saudi Arabia. After extraction of DNA from meat product samples, qualitative duplex polymerase chain reaction (PCR) was used to detect the genetically modified (GM) soybean products in the meat samples using pairs of primers targeting the lectin gene and the 35S promoter. Real-time PCR was used to quantify the percentage of RR soy products in the positive samples. The results clarified that out of 96 tested meat product samples (minced, burger, luncheon, canned, and sausages), 75 samples were positive for the presence of lectin gene, of which 42 samples representing 43.75% of total meat product samples were positive for the presence of 35S promoter. All positive samples for 35S promoter contained RR soy below 0.1%. The results of the consumer acceptance questionnaire of GM additives in meat products proved the presence of several critical aspects of concerns to consumers of meat products in different localities of Riyadh city.
This chapter offers a comparative examination of existing regulatory policies in Europe, the United States, and Brazil. The comparison aims to provide a better localization of the Brazilian policy vis-à-vis the existing regulatory policies in the world today. Considering that the United States and Europe are currently seen as offering distinct regulatory models for GMOs, the possibility of bringing the Brazilian policy closer to one of these alternatives will allow us to capture some of its main characteristics. This part of the paper may thus offer some clues as to the alignment of Brazilian policy within the global debate on GMOs. Besides noting the proximity of Brazilian policy to one of these regulatory models, some arguments that may help in understanding this proximity are also presented.
Introduction DNA-Based Methodologies for GMO Analysis Strategies for Detection, Identification, and Quantification of GMOs Application of Current Methodologies to GMO Analysis References
To clearly discriminate the results of simultaneous screening and quantification of up to 40 different targets= DNA sequences, long probes from 100 nt to 500 nt rather than smaller or similar sized synthetic ones were adopted for multiplex ligation-dependent probe amplification (MLPA). To prepare the long probes, asymmetric PCR was employed to introduce non-complementary stuffers in between the two parts of the MLPA probe with specially designed primers, then restriction enzymes were selected to digest the double-stranded DNAs, and finally polyacrylamide gel electrophoresis was used to purify the single-stranded DNAs, i.e. the long probes. By using this approach, 12 long probes were prepared and used to identify genetically modified (GM) maize. Our experimental results show that the prepared long probes were in full accordance with the designed ones, and could be assembled in 4-plex, 7-plex and 10-plex MLPA analysis without losing result specificity and accuracy, showing they were as effective and reliable in MLPA analysis as those prepared with M13 derived vectors. This novel asymmetric PCR-based approach does not need expensive equipment, special regents or complicated operations when compared with previous methods. Therefore, our new approach could make MLPA analysis more independent, efficient and economical.
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Nucleic acid taggants can potentially be used to identify, track, and attribute microbiologicals, including genetically-modified organisms or pathogens. We propose to introduce nucleic acid taggants into bacterial media, and to then recover the taggants in sterilized bacterial waste. Surprisingly, many of the steps that might be suspected to degrade the detection of a nucleic acid taggant, such as autoclaving, bacterial growth, and chemical sterilization by bleach, have relatively little effect on taggant recovery. We can detect as little as 1000 taggants per milliliter of media. Sterilization methods affect this value by less than 100-fold. The most significant affect on taggant recovery was bacterial growth: in the presence of B. subtilis taggant recovery was reduced by 430-fold, while E. coli reduced taggant recovery by only five-fold.
During recent years, arable land used for transgenic crops has undergone an exponential increase globally, and the tendency to augment not only the cultivated area but also the number of modified genetic traits is rather evident. However, public perception and confidence regarding genetically modified organisms (GMOs) have not paralleled this increase. Moreover, a general antipathy towards GMOs is nowadays detectable especially in Europe and there is an apparent lack of information for the consumer in many cases. To counterbalance the social alarm caused by the presence of GMO-derived ingredients in the commercial food chain, competent authorities have established appropriate regulations to guarantee the consumers' right to information and choice. In this regard, several countries have established a strict labelling policy for products containing or derived from GMO. In order to achieve these labelling requirements different approaches have been taken with the aim of detection and quantification of the presence of GMO traces, using either protein- or DNA-based methods. In this paper we review the different methodologies that are being applied to GMO studies. We describe the state-of-the-art instrumentation and technologies currently used, as well as the most recent advances in analytical systems for the detection, identification and quantification of GMOs. In addition to methodological considerations, important practical aspects such as the implementation of the analytical system into the food and feed chains will be considered. Finally, the importance of standardised/validated methods and future technological trends in the field are discussed.
The protracted outbreak of mad-cow disease, repeated episodes of dioxin and mycotoxin contamination, and the issue of labelling genetically modified foods have severely shaken public confidence in the food supply industry, increasing consumer concerns about the origins of the raw materials used in both animal feed and food products. The need to develop improved techniques to characterize feed and food components has consequently become more urgent, and DNA-based technologies promise to be able to meet these needs. DNA methods can establish the origin of species in meat- and fish-based products and feed ingredients, and are the most reliable analytical approaches for authenticating processed foodstuffs. The identification of ingredients in processed feed and food is mandatory not only to ensure correct labelling and assessment of value, but also to avoid health risks related to the presence of toxic contaminants or pathogenic agents.
As a step towards developing an efficient genetically modified organism (GMO) detection system, the present investigation proposes microarray as a prototype for high-throughput detection of pure GMO samples. Common T-DNA regions of the expression cassette such as 35S promoter, 35S terminator, nopaline synthase terminator (NOSt), hygromycin, and kanamycin selection marker genes were detected in the transgenic rice, tomato, and potato developed in our laboratory. For proof-of-concept purposes, market-introduced GM potato, GM maize, and cornflakes were screened. The non-GM potato and maize control detected only the presence of endogenous genes while the GM targets detected the presence of transgenic genes such as CaMV 35S promoter, 35S terminator, NOSt and nptII, pat, cp4 epsps, cry1 Ab genes on chips. Moreover, it was observed that the sensitivity of this system for serially diluted GM potato tubers was up to 0.01-0.002% of the mass fraction. Due to its high accuracy and speed, it is believed that the microarray detection system will play an important role in routine, high-throughput detection of pure GMO samples in the future.
Bestimmten sekundären Pflanzeninhaltsstoffen schreibt man eine gesundheitsfördernde Wirkung zu. In diesem Zusammenhang haben Carotinoide und Polyphenol besonderes Interesse auf sich gezogen. Genetische Veränderungen und mikrobielle Kontaminationen lassen sich am einfachsten auf DNA-Ebene mit speziellen Hybridisierungs-Techniken und der Polymerase-Kettenreaktion (PCR) nachweisen.
IntroductionRecent DNA Methodology Sampling ProcedureExtraction and Purification of DNASpecific Detection of Genetic Material DNA Hybridization-based Detection Technique (Southern Blot)Nucleic Acid Amplification Methods using PCR Conventional PCRReal-time PCRImportant Bioinformatic ConsiderationsAlternative and Promising DNA Detection Techniques Thermal Cycling ProceduresIsothermic AmplificationDNA MicroarraysMicrofluidic and Nanoparticle TechniquesMass Spectrometry (MS) of DNASupplementary Photon-driven Monitoring MethodsNovel Biological Monitoring ApproachesConclusions and Future Prospects for GMO Detection by DNA AnalysisReferences Sampling ProcedureExtraction and Purification of DNA DNA Hybridization-based Detection Technique (Southern Blot) Conventional PCRReal-time PCRImportant Bioinformatic Considerations Thermal Cycling ProceduresIsothermic AmplificationDNA MicroarraysMicrofluidic and Nanoparticle TechniquesMass Spectrometry (MS) of DNASupplementary Photon-driven Monitoring Methods
Novel Biological Monitoring Approaches
Nucleic acids, especially DNA, are targets of qualitative and quantitative diagnostics for genetically modified organisms
(GMO) in seeds, food- and feedstuff. The amplification of the nucleic acid is an essential step for further analyses of the
target sequence. The PCR has been the method of choice for DNA amplification in most laboratories, and its real-time version
(qPCR) also enables quantitative analysis of target contents. Despite its numerous advantages, PCR technology has some limitations
such as the lack of true multiplexing properties. To alleviate the drawbacks linked to PCR technology, alternative nucleic
acid amplification methods with promising characteristics are being developed fast. These methods, their advantages, and the
inconveniences, which are not yet resolved are summarized in the paper. Special focus is given to the possibilities of using
these alternative methods for GMO detection in future, when expansion of GMOs both in diversity and frequencies will make
current GMO detection systems difficult to operate.
In the past, several multiplex PCR (mPCR) methods have been developed and validated for precise and accurate monitoring, tracing and regulation of genetically Engineered (GE) food, feed and seed. However, a complete and updated review of mPCR is still lacking. Recently, mPCR methods have been coupled with different qualitative and/or quantitative methods, such as real-time PCR, micro-array, multiplex ligase chain reaction, biosensors, and microfluidic device to raise the detection limit of accuracy, sensitivity and reproducibility. In this paper, we provide thorough information and discussion of mPCR methods including their development, application and challenges for GE crops analysis.
Commercialization of numerous genetically modified organisms (GMOs) has already been approved worldwide, and several additional GMOs are in the approval process. Many countries have adopted legislation to deal with GMO-related issues such as food safety, environmental concerns, and consumers' right of choice, making GMO traceability a necessity. The growing extent of GMO testing makes it important to study optimal GMO detection and identification strategies. This paper formally defines the problem of routine laboratory-level GMO tracking as a cost optimization problem, thus proposing a shift from "the same strategy for all samples" to "sample-centered GMO testing strategies." An algorithm (GMOtrack) for finding optimal two-phase (screening-identification) testing strategies is proposed. The advantages of cost optimization with increasing GMO presence on the market are demonstrated, showing that optimization approaches to analytic GMO traceability can result in major cost reductions. The optimal testing strategies are laboratory-dependent, as the costs depend on prior probabilities of local GMO presence, which are exemplified on food and feed samples. The proposed GMOtrack approach, publicly available under the terms of the General Public License, can be extended to other domains where complex testing is involved, such as safety and quality assurance in the food supply chain.
Current screening and event-specific polymerase chain reaction (PCR) assays for the detection and identification of genetically modified organisms (GMOs) in samples of unknown composition or for the detection of non-regulated GMOs have limitations, and alternative approaches are required. A transgenic DNA fingerprinting methodology using restriction enzyme digestion, adaptor ligation, and nested PCR was developed where individual GMOs are distinguished by the characteristic fingerprint pattern of the fragments generated. The inter-laboratory reproducibility of the amplified fragment sizes using different capillary electrophoresis platforms was compared, and reproducible patterns were obtained with an average difference in fragment size of 2.4 bp. DNA insert fingerprints for 12 different maize events, including two maize hybrids and one soy event, were generated that reflected the composition of the transgenic DNA constructs. Once produced, the fingerprint profiles were added to a database which can be readily exchanged and shared between laboratories. This approach should facilitate the process of GMO identification and characterization.
To screen for unauthorized genetically modified organisms (GMO) in the various crops, we developed a multiplex real-time polymerase chain reaction high-resolution melting-curve analysis method for the simultaneous qualitative detection of 35S promoter sequence of cauliflower mosaic virus (35SP) and the nopaline synthase terminator (NOST) in several crops. We selected suitable primer sets for the simultaneous detection of 35SP and NOST and designed the primer set for the detection of spiked ColE1 plasmid to evaluate the validity of the polymerase chain reaction (PCR) analyses. In addition, we optimized the multiplex PCR conditions using the designed primer sets and EvaGreen as an intercalating dye. The contamination of unauthorized GMO with single copy similar to NK603 maize can be detected as low as 0.1% in a maize sample. Furthermore, we showed that the present method would be applicable in identifying GMO in various crops and foods like authorized GM soybean, authorized GM potato, the biscuit which is contaminated with GM soybeans and the rice which is contaminated with unauthorized GM rice. We consider this method to be a simple and reliable assay for screening for unauthorized GMO in crops and the processing food products.
To fulfill labeling and traceability requirement of genetically modified (GM) maize for trade and regulation, it is essential to develop an event-specific detection method for monitoring the presence of transgenes. In pursuit of this purpose, we systematically optimized and established a combined event- and construct-specific multiplex polymerase chain reaction (mPCR) technique for simultaneous detection of 8 GM maize lines. Altogether 9 sets of primers were designed, including six that were event-specific for Event176, Bt11, TC1507, NK603, MON863, and Mon810; two that were construct-specific for T25 and GA21, and one for an endogenous zein gene. The transgene in each GM maize line and the endogenous zein gene could be clearly detected and distinguished according to the different sizes of PCR amplicons. The limit of detection (LOD) was approximately 0.25% (v/v), although the detection can be as sensitive as 0.1% as demonstrated by the International Seed Testing Association (ISTA) proficiency test. This study further improves the current PCR-based detection method for GM maize. The method can be used in an easy, sensitive, and cost and time effective way for the identification and quality screening of a specific GM maize line.
This paper reviews aspects relevant to detection and quantification of genetically modified (GM) material within the feed/food chain. The GM crop regulatory framework at the international level is evaluated with reference to traceability and labelling. Current analytical methods for the detection, identification, and quantification of transgenic DNA in food and feed are reviewed. These methods include quantitative real-time PCR, multiplex PCR, and multiplex real-time PCR. Particular attention is paid to methods able to identify multiple GM events in a single reaction and to the development of microdevices and microsensors, though they have not been fully validated for application.
Detection of nonauthorized genetically modified organisms (GMOs) has always presented an analytical challenge because the complete sequence data needed to detect them are generally unavailable although sequence similarity to known GMOs can be expected. A new approach, differential quantitative polymerase chain reaction (PCR), for detection of nonauthorized GMOs is presented here. This method is based on the presence of several common elements (e.g., promoter, genes of interest) in different GMOs. A statistical model was developed to study the difference between the number of molecules of such a common sequence and the number of molecules identifying the approved GMO (as determined by border-fragment-based PCR) and the donor organism of the common sequence. When this difference differs statistically from zero, the presence of a nonauthorized GMO can be inferred. The interest and scope of such an approach were tested on a case study of different proportions of genetically modified maize events, with the P35S promoter as the Cauliflower Mosaic Virus common sequence. The presence of a nonauthorized GMO was successfully detected in the mixtures analyzed and in the presence of (donor organism of P35S promoter). This method could be easily transposed to other common GMO sequences and other species and is applicable to other detection areas such as microbiology.
According to the preface of the regulation (EC No. 1139/98) labelling due to minor contamination would not be necessary, if tolerable limits for genetically modified DNA and protein can be established. For this purpose, methods that quantify genetically modified DNA in food based on real time PCR have been described. For the assessment of the results obtained by quantitative PCR methods, validation data concerning sensitivity, precision and trueness are necessary. By means of a published method for the quantification of genetically modified Roundup Ready (TM) soybeans a practical approach to achieve these validation data is presented in this paper. The data are obtained under repeatability conditions with series of DNA preparations and dilutions. By the use of this approach, the confidence interval and limits of quantification and detection can be specified for each sample.
According to the preface of the regulation (EC No. 1139/98) labelling due to minor contamination would not be necessary, if tolerable limits for genetically modified DNA and protein can be established. For this purpose, methods that quantify genetically modified DNA in food based on real time PCR have been described. For the assessment of the results obtained by quantitative PCR methods, validation data concerning sensitivity, precision and trueness are necessary. By means of a published method for the quantification of genetically modified Roundup Ready™ soybeans a practical approach to achieve these validation data is presented in this paper. The data are obtained under repeatability conditions with series of DNA preparations and dilutions. By the use of this approach, the confidence interval and limits of quantification and detection can be specified for each sample.
A method using polymerase chain reaction (PCR) was designed for the detection of genetically modified maize (GM-maize). There are four lines of GM-maize imported from the United States, and the presence of recombinant deoxyribonucleic acid (DNA) in the maize could be detected with four pairs of specific oligonucleotide primers designed from the sequences of the newly introduced genes. The maize zein gene was also detected as an internal control. This method allows specific detection of each of Bt11, Event176, MON810 and LIBERTY by using pairs of specific primers designed to amplify a segment including part: of the exogenously introduced sequence and part of the intrinsic maize sequence. The detection sensitivity was about 0.05% for Event176, MON810 and LIBERTY, and about 0.01% for Bt11. To distinguish among three insect-resistant GM-maize lines, we designed a multiplex PCR method. These three GM-maize lines were distinguishable on the basis of the expected lengths of their amplicons.
An event-specific PCR method for detection and quantification of genetically modified Roundup Ready soybean (RRS) is described in this article. The complete DNA sequence at both the right and left integration sites of this genetically modified organism has recently been determined. Based on these sequence data, transformation event-specific primer pairs were developed. These primers amplify a fragment of the unique junction region between the inserted DNA and the plant DNA and therefore act as unique identifiers. Two sensitive, qualitative PCR assays gave absolute detection limits of 5 copies of the RRS junction fragment in 100 pg of total DNA per reaction. A real-time PCR method was then developed with the LightCycler System. For determination of the RRS content, a completely new type of external calibration standard is introduced here. A fragment of the RRS specific junction region and a fragment of the endogenous soybean lectin gene were both cloned in a plasmid vector. These new diagnostic DNA fragments allow quantification of RRS in whichever type of matrix, in a range of 10-106 copies of each target.
Numerous analytical methods, both qualitative and quantitative, have been developed to determine reliably the presence and/or the amount of genetically modified organisms (GMOs) in agricultural commodities, in raw agricultural materials and in processed and refined ingredients. In addition to the "classical" methods for DNA and protein analysis, e.g. polymerase chain reaction and enzyme linked immunosorbent analysis, certain types of GMO-containing matrices can be profiled by complementary chemical analysis methods such as chromatography and near infrared spectroscopy. This review summarises the status of the most widely used GMO analysis technologies, identifies new areas of analytical investigation and discusses current needs and future challenges.
DNA-based analytical methods are often used to verify the presence of genetically modified organisms (GMOs) in food. In Switzerland,
a preliminary study, organized by a subcommission of the Swiss Food Manual, of different polymerase chain reaction (PCR) systems
for the detection of GMOs showed that the application of qualitative PCR systems can lead to interlaboratory differences of
at least a factor of 10. These differences can be diminished using internal standards (competitors). The quantitative competitive
(QC) PCR for the detection of the 35S promoter or the NOS terminator in food samples is presented. The GMO content of food
samples can be determined using QC-PCR.
In this article we describe the isolation and characterisation of the junction between insert DNA and plant DNA in the transgenic Roundup Ready soybean line event 40-3-2. Our results establish that during integration of the insert DNA several rearrangements occurred at the 3' NOS junction and that the genomic plant DNA at the pre-integration site may have been rearranged. These findings highlight the utility of characterising junction regions to fulfil the request for information regarding which DNA sequences have been incorporated in commercialised transgenic lines. Furthermore, the characterisation of junction regions is, in our opinion, the method of choice to support method development for detection and identification of plant biotechnology-derived products.
A qualitative screening method was validated for the detection of genetically modified organisms (GMOs) in various processed food matrices (cooked maize grit, infant formula, biscuits, meal of acidified soybeans). The prepared food matrices contained each 0%, 2%, 100% (10% instead of 100% in the case of biscuits) of Roundup-Ready soybeans and/or of Bt-176 maize. The method was based on the detection of the introduced DNA sequences by using the polymerase chain reaction (PCR) for amplification of DNA. Two different detection systems were applied: one based on the 35S promoter fragment which is present in GM-soybeans and in GM-maize and one based on the nos terminator sequence which is present only in GM-soybeans. Prior to the validation study, it was demonstrated that the food matrices were homogenous and that there was no cross-contamination between GMO-containing and GMO-free samples. Some laboratories had a high portion of false positive results probably due to laboratory contamination whereas all others reported nearly always correct data for all matrices investigated. Statistical analysis showed that after exclusion of the outliers, an average of 97% correct results was obtained for non-GMO containing samples (3% false positive results) and an average of 98% correct results for GMO-containing samples (2% false negative results). The validated method was shown to be suitable for screening of GMOs in processed food matrices also when extreme physical stress was applied (e.g., heating for 45 min at 100vvC or 10 min at 180vvC) or when multiple components were present, as in the case of biscuit preparations.
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.
Updated information of mechanisms for T-DNA transfer to plant cells by Agrobacterium tumefaciens is provided, focused on the role played by the different components of the virulence system. The general assessments for the establishment of efficient transformation protocols are discussed with an emphasis in the application of this methodology to monocotyledonous plants. Based on our own experience, we present the establishment of sugarcane transformation by A. tumefaciens as a model of application of this methodology to an important culture plant species, previously considered recalcitrant and inaccessible for this type of genetic manipulation.
Sequencing of DNA fragments of 130 and 200 bp using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry
for DNA ladder detection was demonstrated. With further improvement in mass resolution and detection sensitivity, mass spectrometry
shows great promise for routine DNA sequencing in the future.
Escherichia coli were separated from a mixture containing human blood cells by means of dielectrophoresis and then subjected to electronic lysis followed by proteolytic digestion on a single microfabricated bioelectronic chip. An alternating current electric field was used to direct the bacteria to 25 microlocations above individually addressable platinum microelectrodes. The platinum electrodes were 80 microns in diameter and had center-to-center spacings of 200 microns. After the isolation, the bacteria were lysed by a series of high-voltage pulses. The lysate contained a spectrum of nucleic acids including RNA, plasmid DNA, and genomic DNA. The lysate was further examined by electronically enhanced hybridization on separate bioelectronic chips. Dielectrophoretic separation of cells followed by electronic lysis and digestion on an electronically active chip may have potential as a sample preparation process for chip-based hybridization assays in an integrated DNA/RNA analysis system.
We have developed novel nucleic acid probes that recognize and report the presence of specific nucleic acids in homogeneous solutions. These probes undergo a spontaneous fluorogenic conformational change when they hybridize to their targets. Only perfectly complementary targets elicit this response, as hybridization does not occur when the target contains a mismatched nucleotide or a deletion. The probes are particularly suited for monitoring the synthesis of specific nucleic acids in real time. When used in nucleic acid amplification assays, gene detection is homogeneous and sensitive, and can be carried out in a sealed tube. When introduced into living cells, these probes should enable the origin, movement, and fate of specific mRNAs to be traced.
Molecular diagnostics is progressing from low-throughput, heterogeneous, mostly manual technologies to higher throughput, closed-tube, and automated methods. Fluorescence is the favored signaling technology for such assays, and a number of techniques rely on energy transfer between a fluorophore and a proximal quencher molecule. In these methods, dual-labeled probes hybridize to an amplicon and changes in the quenching of the fluorophore are detected. We describe a new technology that is simple to use, gives highly specific information, and avoids the major difficulties of the alternative methods. It uses a primer with an integral tail that is used to probe an extension product of the primer. The probing of a target sequence is thereby converted into a unimolecular event, which has substantial benefits in terms of kinetics, thermodynamics, assay design, and probe reliability.
This paper presents results of a collaborative trial study (IUPAC project No. 650/93/97) involving 29 laboratories in 13 countries applying a method for detecting genetically modified organisms (GMOs) in food. The method is based on using the polymerase chain reaction to determine the 35S promotor and the NOS terminator for detection of GMOs. reference materials were produced that were derived from genetically modified soy beans and maize. Correct identification of samples containing 2% GMOs is achievable for both soy beans and maize. For samples containing 0.5% genetically modified soy beans, analysis of the 35S promotor resulted also in a 100% correct classification. However, 3 false-negative results (out of 105 samples analyzed) were reported for analysis of the NOS terminator, which is due to the lower sensitivity of this method. Because of the bigger genomic DNA of maize, the probability of encountering false-negative results for samples containing 0.5% GMOs is greater for maize than for soy beans. For blank samples (0% GMO), only 2 false-positive results for soy beans and one for maize were reported. These results appeared as very weak signals and were most probably due to contamination of laboratory equipment.
Three different biosensors for detection of Genetically Modified Organisms (GMOs) are presented. The sensing principle is based on the affinity interaction between nucleic acids: the probe is immobilised on the sensor surface and the target analyte is free in solution. The immobilised probes are specific for most inserted sequences in GMOs: the promoter P35S and the terminator TNOS. Electrochemical methods with screen-printed electrodes, piezoelectric and optical (SPR) transduction principles were applied.
Seven lines of genetically modified (GM) maize have been authorized in Japan as foods and feeds imported from the USA. We improved a multiplex PCR method described in the previous report in order to distinguish the five lines of GM maize. Genomic DNA was extracted from GM maize with a silica spin column kit, which could reduce experimental time and improve safety in the laboratory and potentially in the environment. We sequenced recombinant DNA (r-DNA) introduced into GM maize, and re-designed new primer pairs to increase the specificity of PCR to distinguish five lines of GM maize by multiplex PCR. A primer pair for the maize intrinsic zein gene (Ze1) was also designed to confirm the presence of amplifiable maize DNA. The lengths of PCR products using these six primer pairs were different. The Ze1 and the r-DNAs from the five lines of GM maize were qualitatively detected in one tube. The specific PCR bands were distinguishable from each other on the basis of the expected length. The r-DNA could be detected from maize samples containing 0.5% of each of the five lines of GM maize. The sensitivity would be acceptable to secure the verification of non-GMO materials and to monitor the reliability of the labeling system.
The emerging role of single-nucleotide polymorphisms (SNPs) in clinical association and pharmacogenetic studies has created a need for high-throughput genotyping technologies. We describe a novel method for multiplexed genotyping of SNPs that employs PCR amplification on microspheres. Oligonucleotide PCR primers were designed for each polymorphic locus such that one of the primers contained a recognition site for BbvI (a type IIS restriction enzyme), followed by 11 nucleotides of locus-specific sequence, which reside immediately upstream of the polymorphic site. Following amplification, this configuration allows for any SNP to be exposed by BbvI digestion and interrogated via primer extension, four-color minisequencing. Primers containing 5' acrylamide groups were attached covalently to the solid support through copolymerization into acrylamide beads. Highly multiplexed solid-phase amplification using human genomic DNA was demonstrated with 57 beads in a single reaction. Multiplexed amplification and minisequencing reactions using bead sets representing eight polymorphic loci were carried out with genomic DNA from eight individuals. Sixty-three of 64 genotypes were accurately determined by this method when compared to genotypes determined by restriction-enzyme digestion of PCR products. This method provides an accurate, robust approach toward multiplexed genotyping that may facilitate the use of SNPs in such diverse applications as pharmacogenetics and genome-wide association studies for complex genetic diseases.
Worldwide, the use of genetically modified plants is increasingly important. The European Community (EC) has already approved herbicide tolerant soybeans (Roundup Ready Soybeans, RRS, Monsanto) and insect tolerant corn (Bt-corn, Novartis), and further approvals of genetically modified plants for food production (tomatoes, radicchio, rapeseed, soybeans and corn) are pending.
A piezoelectric affinity sensor, based on DNA hybridisation has been studied for applications to Genetically Modified Organisms (GMOs) detection. The thiol/dextran modified surfaces were coupled to streptavidin for immobilising 5'-biotinyltead probes (25-mer). The probes sequences were respectively internal to the amplified product of P35S and T-NOS. These target sequences were chosen on the base of their wide presence in GMOs. The system has been optimised using synthetic complementary oligonucleotides (25-mer) and the specificity of the system tested with a non-complementary oligonucleotide (23-mer). The hybridisation study was performed also with samples of DNA isolated from CRM (Certified Reference Materials) soybean powder containing 2% of transgenic material and amplified by PCR. Non amplified genomic or plasmidic DNA was also used. The developed system was very specific, binding only the complementary DNA strand. The CV% was 20% both with synthetic oligonucleotides and PCR amplified samples. The sensor signal was independent of the sample dilution but the system is still at a semi-quantitative level.
There is an increasing need for analytical methods for the detection of genetically modified organisms (GMOs) detection in food due to the growth of use of GMOs, or their derivatives, in the food industry. This paper aims to briefly introduce the reader to GMOs, to describe the state of the art in detection methods for GMOs, and to provide the reader with the associated terminology, mainly borrowed from molecular biology. The main characteristics of GMO are illustrated and future developments using the biosensor approach for detection are presented.
To evaluate and characterize the stability of traits transferred viaAgrobacterium transformation, foreign gene expression must be examined in sexually derived progeny. The objective of this study was to analyze three transgenic peanut plants, 1-10, 12-1, and 17-1, for the inheritance and expression of their foreign genes. Segregation ratios for the introduced genes in T2 plants gave either 100% or 3:1 expression of the -glucuronidase (GUS) gene, demonstrating recovery of both homozygous and heterozygous T1 plants. Fluorometric GUS assay in T1 and T2 generations of all three plants showed that the GUS gene was stably expressed in the progeny. DNA analyses showed 100% concordance between the presence of the foreign gene and enzyme activity. Our results demonstrate that transgenes in peanut introduced byAgrobacterium can be inherited in a Mendelian manner.
Recently, the DNA sequences of both the 5' and 3' sites of integration of the transgene in Roundup Ready soya have been described. We have utilised this information to design two event-specific assays for the quantitative detection of this genetically modified (GM) organism in food matrices using real time PCR. One assay has been designed to use TaqMan technology for detection using the ABI PRISM 7700 and the second to utilise Scorpion primers for use on the Roche Diagnostics LightCycler detection system. In both cases, the Roundup Ready gene was quantified relative to the endogenous soya lectin gene and results expressed as the percentage of GM soya relative to total soya content using standards of known GM content.
Today DNA-based techniques are very common for the detection of genetically modified organisms (GMOs) in food products. For
fast and easy detection of GMOs, polymerase chain reaction (PCR) screening methods, which amplify common transgenic elements,
are applied in routine analysis. These techniques do not allow differentiation between GMOs and the natural occurrence of
transgenic elements, such as the 35S-promoter of cauliflower mosaic virus (CaMV) or the NOS-terminator of Agrobacterium tumefaciens, and thus may result in false-positive detection of GMOs. In this study we evaluated three different existing 35S screening
systems and report the development of two new CaMV-specific PCR systems. These PCR systems based on CaMV-specific genes allow
the identification of positively screened 35S food samples as naturally virus-infected products or plants. Seven food samples
tested positive in routine 35S screening analysis and negative in GMO specific systems were investigated using the new virus-specific
PCR systems. In all seven samples CaMV was detected.
The surveillance of food labelling concerning genetically modified organisms (GMOs) requires DNA-based analytical techniques.
Present assay systems allow the detection of GMO in food; however, they do not permit their quantitation. In this study, we
report the development of quantitative competitive polymerase chain reaction (QC-PCR) systems for the detection and quantitation
of the Roundup Ready soybean (RRS) and the Maximizer maize (MM) in food samples. Three DNA fragments that differ from the
GMO-specific sequences by an insertion were constructed and used as internal standards in the PCR. These standards were calibrated
by co-amplifying with mixtures containing RRS DNA and MM DNA, respectively. The calibrated QC-PCR systems were applied to
nine commercial food samples containing RRS DNA and to three certified RRS flour mixtures in order to elucidate whether these
food samples contain more or less than 1% RRS DNA. Finally, the GMO contents of four samples that were found to contain more
than 1% RRS were determined by QC-PCR using various amounts of standard DNA.
This bibliographic search covers the literature till December, 1995 on microprojectile mediated plant transformation, plasmid
construct used, and the type of expression obtained, since the inception of the concept by Sanford et al., in 1987.
The discovery of the molecules that bear the genetic information of any living organisms among which DNA (DeoxyriboNucleic Acid) plays a central role has been a revolution for life sciences. Taking the advantage of the universality of the genetic code, researchers have succeeded to associate DNA sequences coming from different organisms using molecular biology techniques and to integrate foreign DNA within plants. These genetically-modified organisms (the so called GMOs) have the ability to synthesise some additional proteins which confer new properties on them. Improving the protection of agricultural crops is one of the sought advantages by the gene transfer.According to the European regulation, the new foodstuffs made from genetically modified soya or maize, must be labelled. The control of the labelling of such foodstuffs is based on the detection of the foreign DNA sequences born by the genetically-modified organisms. One of the analytical methods used for enforcement of this regulation is the Polymerase Chain Reaction (PCR) method. The principle of the PCR method is to multiply specific sequences of DNA, making them detectable. This highly sensitive method offers the advantage of detecting DNA molecules which are more thermostable than proteins.
Immunoassay methodology is currently the method of choice for the quantitative and semi-quantitative detection of many types of proteins in complex mixtures. A combination of sensitivity, specificity and cost-effectiveness in terms of analytical performance are allied to a diverse array of assay formats suitable for laboratory and field use. In the present article, the problems of setting up immunoassays for novel food proteins, and the questions that need to be answered if a successful outcome is to be achieved, are discussed. Speculation on future developments in immunochemistry leads to the conclusion that antibody technology will play an important role in detection of novel proteins from genetically modified organisms.
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.
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.
Traceability in food manufacturing can range from in-house traceability in production plants to traceability in whole or part of the production chain from raw material to consumer, and descriptors of the product and its history can be few or many as decided. Well thought-out traceability systems are fundamental to achieving optimal benefits from quality control, production control and for fulfilling consumer demands etc. In order to facilitate the discussion of a traceability strategy in food industries this Viewpoint outlines the fundamental theoretical issues of traceability systems and presents a more practical discussion of its extent.
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 ReadyTM 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 fast and quantitative method was developed to detect transgenic "Maximizer" maize "event 176" (Novartis) and "Roundup Ready" soybean (Monsanto) in food by real-time quantitative PCR. The use of the ABI Prism 7700 sequence detection system allowed the determination of the amplified product accumulation through a fluorogenic probe (TaqMan). Fluorescent dyes were chosen in such a way as to coamplify total and transgenic DNA in the same tube. Using real-time quantitative PCR, 2 pg of transgenic or total DNA per gram of starting sample was detected in 3 h after DNA extraction and the relative amounts of "Maximizer" maize and "Roundup Ready" soybean in some representative food products were quantified.
A method has been developed to distinguish between traditional soy beans and transgenic Roundup Ready soy beans, i.e. the glyphosate ('Roundup') resistant soy bean variety developed by Monsanto Company. Glyphosate resistance results from the incorporation of an Agrobacterium-derived 5-enol-pyruvyl-shikimate-3-phosphatesynthase (EPSPS) gene. The detection method developed is based on a nested Polymerase Chain Reaction (PCR) procedure. Ten femtograms of soy bean DNA can be detected, while, starting from whole soy beans, Roundup Ready DNA can be detected at a level of 1 Roundup Ready soy bean in 5000 non-GM soy beans (0.02% Roundup Ready soy bean). The method has been applied to samples of soy bean, soy-meal pellets and soy bean flour, as well as a number of processed complex products such as infant formula based on soy, tofu, tempeh, soy-based desserts, bakery products and complex meat and meat-replacing products. The results obtained are discussed with respect to practical application of the detection method developed.
Nature Biotechnology journal featuring biotechnology articles and science research papers of commercial interest in pharmaceutical, medical, and environmental sciences.
A simple yet powerful tool for providing for rapid gene identification in the clinic would be the combination of isothermal gene amplification with electronic microchip analysis. This is a first report of such a union of these technologies.
The first assay demonstrates discrimination between four bacterial pathogens. For this, one portion of the bacterial 16S rRNA gene encompassing a microheterogeneous region was isothermally amplified using Strand Displacement Amplification (SDA). Type identification was then made by "sandwich" assay format either using selective electronic hybridization of amplicons to sequence-specific capture oligonucleotides and a universal, fluorescently labeled reporter oligonucleotide, or, alternatively, sequence-specific reporters and a universal capture oligonucleotide. The second assay tested for the presence or absence of the Factor V Leiden point mutation using DNA obtained from 18 patients in a blind assay. For this, allele-specific SDA was developed. Following amplification using a sense-biotinylated primer and either the corresponding antisense wild type or mutant primer, multiple patient amplicons were targeted to specified locations on the microarray and visualized using a fluorescently labeled reporter oligonucleotide. Positive signals were scored as greater than or equal to two times the background.
Bacterial type-specific signals were between 3- to 10-fold greater than nonspecific in both assay formats. Using allele-specific SDA, 100% agreement was observed between PAGE analysis, microarray results, and clinical diagnosis in Factor V mutation analysis.
We demonstrated two model clinical assays combining amplified materials and microelectronic arrays, one potentially suitable for pathogen screening and the other for a deleterious genetic mutation.
Major recent advances in molecular amplification in the past year were initial validation of two new amplification technologies (rolling circle amplification and Invader), a significant increase in the number of molecular diagnostic assays, achievement of amplification directly on microarrays (by strand displacement amplification and rolling circle amplification), and description of two new read-out probes (Scorpions and nanoparticles).
Normal, high stearic acid and high lauric acid canola oil varieties were heated in the presence of air to allow autoxidation to occur. After the reaction, the oils were analyzed using a non-aqueous reversed-phase high-performance liquid chromatographic separation followed by detection using atmospheric pressure chemical ionization mass spectrometry. Oxidized products were separated and identified. The major autoxidation products which remained intact were epoxides and hydroperoxides. Two classes of epoxy triacylglycerols (TAGs) were formed. One class with the epoxy group replacing a site of unsaturation and one class adjacent to a site of unsaturation, as was previously reported for model TAGs. Intact oxidation products resulted mostly from oxidation of oleic acid, while oxidation products of linoleic and linolenic acid chains decomposed to yield chain-shortened species. Both neutral and polar chain-shortened products were observed. Polar chain-shortened decomposition products eluted at very short retention times and required a different chromatographic gradient to separate the molecules. This class of molecules was tentatively identified as core aldehydes. The high stearic acid canola oil yielded more intact oxidation products containing stearic acid, as expected. The high lauric acid oil produced intact oxidation products which contained lauric acid.
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