Development of a multiplex fluorescence quantitative PCR for detection of genetically modified organisms

The commercially available genetically modified plants authorized worldwide and therefore the target sequences for molecular detection of genetically modified organisms (GMOs) are ever-increasing. The European Union has implemented a set of very strict procedures for approval to grow, import and/or utilize GMOs as food or food ingredients. As a result, GMO laboratories and food production industry currently are forced to apply different methods to test raw material and complex processed food products. Three exogenous genes (the 35 s promoter of the cauliflower mosaic virus (35 s), nos terminator from Agrobacterium tumefaciens (nos), and the neomycin phosphotransferase II (nptII) gene) are commonly used in GMO detection. In this paper, a multiplex quantitative real-time PCR (qPCR) system was developed which allows simultaneously detection of the three exogenous genes in one reaction tube. The determined limits for the multiplex qPCR assays were 4 copies/reaction in maize samples. The specificity of the assays was demonstrated to be 100% according to the detection results of 23 genetically modified (GM) crops and 97 complex processed food products. The validation data show the individual PCR efficiency was accredited with negligible impacts between three detection channels in 7500 fluorescence quantitative PCR machine. These results indicate that this high-throughput multiplex qPCR method which combined with a reference gene is feasible for screening of GMOs, even for the processed food.     

Screening genetically modified organisms using multiplex-PCR coupled with oligonucleotide microarray

In this research, we developed a multiplex polymerase chain reaction (multiplex-PCR) coupled with a DNA microarray system simultaneously aiming at many targets in a consecutive reaction to detect a genetically modified organism (GMO). There are a total of 20 probes for detecting a GMO in a DNA microarray which can be classified into three categories according to their purpose: the first for screening GMO from un-transgenic plants based on the common elements such as promoter, reporter and terminator genes; the second for specific gene confirmation based on the target gene sequences such as herbicide-resistance or insect-resistance genes; the third for species-specific genes which the sequences are unique for different plant species. To ensure the reliability of this method, different kinds of positive and negative controls were used in DNA microarray. Commercial GM soybean, maize, rapeseed and cotton were identified by means of this method and further confirmed by PCR analysis and sequencing. The results indicate that this method discriminates between the GMOs very quickly and in a cost-saving and more time efficient way. It can detect more than 95% of currently commercial GMO plants and the limits of detection are 0.5% for soybean and 1% for maize. This method is proved to be a new method for routine analysis of GMOs.     

Development of melting temperature-based SYBR Green I polymerase chain reaction methods for multiplex genetically modified organism detection

Commercialization of several genetically modified crops has been approved worldwide to date. Uniplex polymerase chain reaction (PCR)-based methods to identify these different insertion events have been developed, but their use in the analysis of all commercially available genetically modified organisms (GMOs) is becoming progressively insufficient. These methods require a large number of assays to detect all possible GMOs present in the sample and thereby the development of multiplex PCR systems using combined probes and primers targeted to sequences specific to various GMOs is needed for detection of this increasing number of GMOs. Here we report on the development of a multiplex real-time PCR suitable for multiple GMO identification, based on the intercalating dye SYBR Green I and the analysis of the melting curves of the amplified products. Using this method, different amplification products specific for Maximizer 176, Bt11, MON810, and GA21 maize and for GTS 40-3-2 soybean were obtained and identified by their specific Tm. We have combined amplification of these products in a number of multiplex reactions and show the suitability of the methods for identification of GMOs with a sensitivity of 0.1% in duplex reactions. The described methods offer an economic and simple alternative to real-time PCR systems based on sequence-specific probes (i.e., TaqMan chemistry). These methods can be used as selection tests and further optimized for uniplex GMO quantification.     

A Microarray-based Detection System for Genetically Modified (GM) Food Ingredients

A multiplex DNA microarray chip was developed for simultaneous identification of nine genetically modified organisms (GMOs), five plant species and three GMO screening elements, i.e. the 35S promoter, the nos terminator and the nptII gene. The chips also include several controls, such as that for the possible presence of CaMV. The on-chip detection was performed directly with PCR amplified products. Particular emphasis was placed on the reduction of the number of PCR reactions required and on the number of primers present per amplification tube. The targets were biotin labelled and the arrays were detected using a colorimetric methodology. Specificity was provided by specific capture probes designed for each GMO and for the common screening elements. The sensitivity of the assay was tested by experiments carried out in five different laboratories. The limit of detection was lower than 0.3% GMO for all tests and in general around 0.1% for most GMOs. The chip detection system complies with the requirements of current EU regulations and other countries where thresholds are established for the labelling of GMO. Serge Leimanis, Marta Hernández, Sophie Fernández: These authors contributed equally to this paper.     

Detection of nonauthorized genetically modified organisms using differential quantitative polymerase chain reaction: application to 35S in maize

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.     

转基因生物相关平台的构建

在转基因生物全球化形势下,我国十分重视转基因生物安全管理及转基因生物安全性风险交流。通过建设转基因生物相关平台,可以加强我国转基因生物安全评价、转基因生物检测、风险交流及信息的整理和共享,拓宽我国转基因生物安全管理服务和信息交流渠道。平台主要由五部分组成,即:转基因生物安全性评价数据库、转基因生物安全检测方法数据库、转基因生物安全管理政策法规数据库、转基因生物检测服务子平台、公众风险交流子平台。平台的访问界面友好、美观、实用,能方便快捷地为用户提供信息查询和浏览等服务。平台可于此地址浏览:http://www.shgmo.org/。     

The development and standardization of testing methods for genetically modified organisms and their derived products

As the worldwide commercialization of genetically modified organisms (GMOs) increases and consumers concern the safety of GMOs, many countries and regions are issuing labeling regulations on GMOs and their products. Analytical methods and their standardization for GM ingredients in foods and feed are essential for the implementation of labeling regulations. To date, the GMO testing methods are mainly based on the inserted DNA sequences and newly produced proteins in GMOs. This paper presents an overview of GMO testing methods as well as their standardization.     

Detecting un-authorized genetically modified organisms (GMOs) and derived materials

Genetically modified plants, in the following referred to as genetically modified organisms or GMOs, have been commercially grown for almost two decades. In 2010 approximately 10% of the total global crop acreage was planted with GMOs (James, 2011). More than 30 countries have been growing commercial GMOs, and many more have performed field trials. Although the majority of commercial GMOs both in terms of acreage and specific events belong to the four species: soybean, maize, cotton and rapeseed, there are another 20 + species where GMOs are commercialized or in the pipeline for commercialization. The number of GMOs cultivated in field trials or for commercial production has constantly increased during this time period. So have the number of species, the number of countries involved, the diversity of novel (added) genetic elements and the global trade. All of these factors contribute to the increasing complexity of detecting and correctly identifying GMO derived material. Many jurisdictions, including the European Union (EU), legally distinguish between authorized (and therefore legal) and un-authorized (and therefore illegal) GMOs. Information about the developments, field trials, authorizations, cultivation, trade and observations made in the official GMO control laboratories in different countries around the world is often limited, despite several attempts such as the OECD BioTrack for voluntary dissemination of data. This lack of information inevitably makes it challenging to detect and identify GMOs, especially the un-authorized GMOs. The present paper reviews the state of the art technologies and approaches in light of coverage, practicability, sensitivity and limitations. Emphasis is put on exemplifying practical detection of un-authorized GMOs. Although this paper has a European (EU) bias when examples are given, the contents have global relevance.     

Composting: a potentially safe process for disposal of genetically modified organisms

The widespread use of genetically modified organisms (GMOs) may result in the release of GMOs into the environment. The potential risks regarding their use and implementation of disposal methods, especially the possibility of novel genes from GMOs being transferred to natural organisms, need to be evaluated and better understood. There is an increasingly accepted public view that GMO products introduced into the environment should be degradable and should disappear after a limited period of time. Due to the risk of possible horizontal gene transfer, disposal methods for GMOs need to address destruction of both the organism and the genetic material. During the last two decades, we have developed a greater understanding of the biochemical, microbiological and molecular concepts of the composting process, such that maximum decomposition may be achieved in the shortest time with minimal negative impacts to the environment. The conditions created in a properly managed composting process environment may help in destroying GMOs and their genes, thereby reducing the risk of the spread of genetic material. When considering composting as a potential method for the disposal of GMOs, the establishment of controlled conditions providing an essentially homogenous environment appears to be an important requirement. An evaluation of composting as a safe option for disposal of GMOs is provided in this review.     

Detection limits of the strip test and PCR for genetically modified corn in Brazil

Brazilian legislation establishes a labeling limit for products that contain more than 1% material from genetically modified organisms (GMOs). We assessed the sensitivity of the lateral flow strip test in detection of the GMO corn varieties Bt11 and MON810 and the specificity and sensitivity of PCR techniques for their detection. For the strip test, the GMO seeds were mixed with conventional seeds at levels of 0.2, 0.4 and 0.8% for Bt11, and 0.4, 0.8 and 1.6% for MON810. Three different methodologies were assessed and whole seeds, their endosperm and embryonic axis were used. For the PCR technique, the GMO seeds of each of the two varieties were mixed with conventional seeds at levels of 20, 10, 5, 2, 1, and 0.5%. The seeds were ground and the DNA extracted. For detection of the GMO material, specific primers were used for MON810 and Bt11 and maize zein as an endogenous control. The sensitivity of the strip test varied for both maize varieties and methodologies. The test was positive for Bt11 only at 0.8%, in contrast with the detection limit of 0.4% indicated by the manufacturer. In the multiplex PCR, the primers proved to be specific for the different varieties. These varieties were detected in samples with one GMO seed in 100. Thus, this technique proved to be efficient in detecting contaminations equal to or greater than 1%.     

PCR-Free Detection of Genetically Modified Organisms Using Magnetic Capture Technology and Fluorescence Cross-Correlation Spectroscopy

The safety of genetically modified organisms (GMOs) has attracted much attention recently. Polymerase chain reaction (PCR) amplification is a common method used in the identification of GMOs. However, a major disadvantage of PCR is the potential amplification of non-target DNA, causing false-positive identification. Thus, there remains a need for a simple, reliable and ultrasensitive method to identify and quantify GMO in crops. This report is to introduce a magnetic bead-based PCR-free method for rapid detection of GMOs using dual-color fluorescence cross-correlation spectroscopy (FCCS). The cauliflower mosaic virus 35S (CaMV35S) promoter commonly used in transgenic products was targeted. CaMV35S target was captured by a biotin-labeled nucleic acid probe and then purified using streptavidin-coated magnetic beads through biotin-streptavidin linkage. The purified target DNA fragment was hybridized with two nucleic acid probes labeled respectively by Rhodamine Green and Cy5 dyes. Finally, FCCS was used to detect and quantify the target DNA fragment through simultaneously detecting the fluorescence emissions from the two dyes. In our study, GMOs in genetically engineered soybeans and tomatoes were detected, using the magnetic bead-based PCR-free FCCS method. A detection limit of 50 pM GMOs target was achieved and PCR-free detection of GMOs from 5 µg genomic DNA with magnetic capture technology was accomplished. Also, the accuracy of GMO determination by the FCCS method is verified by spectrophotometry at 260 nm using PCR amplified target DNA fragment from GM tomato. The new method is rapid and effective as demonstrated in our experiments and can be easily extended to high-throughput and automatic screening format. We believe that the new magnetic bead-assisted FCCS detection technique will be a useful tool for PCR-free GMOs identification and other specific nucleic acids.     

GMDD: a database of GMO detection methods

Background  

Since more than one hundred events of genetically modified organisms (GMOs) have been developed and approved for commercialization in global area, the GMO analysis methods are essential for the enforcement of GMO labelling regulations. Protein and nucleic acid-based detection techniques have been developed and utilized for GMOs identification and quantification. However, the information for harmonization and standardization of GMO analysis methods at global level is needed.     

Mating preference in the invasion of growth enhanced fish

Fish with a transgene for growth hormone grow faster than the wild type and may have an advantage in sexual selection due to their larger size and earlier maturation. The cost in these genetically modified organisms (GMOs) is a lower viability of their offspring. The Trojan gene effect is a hypothesis that predicts that the release of such fish in nature can lead to an invasion by GMOs but ultimately decrease population size to extinction. We modelled GMO invasion with Mendelian inheritance of two alleles in one locus and the resulting mating and population dynamics of wild, GMO and hybrid genotypes. Invasion was attempted over a range of initial densities, representing scenarios from accidental escape to large-scale deliberate introduction of the transgenic genotype. Our results show that invasion strongly depends on hybrid fitness, requiring only a low initial density when GMOs and hybrids are preferred in mating. Preference against hybrids results in an invasion threshold, above which mating between GMOs are sufficiently frequent for invasion to take place. GMO invasion may decrease population size, but contrary to earlier studies on the Trojan gene effect, extinctions do not occur. This is due to the lower viability of GMOs being balanced by the decreased number of competitors reducing the effects of density dependence. The results emphasize the importance of initial density, hybrid fitness and density dependence when considering invasion through hybridization.     

Technocratic precautionary principle: Korean risk governance of genetically modified organisms

Regulations for genetically modified organisms (GMOs) in Korea fluctuate between technocracy and the precautionary principle (PP). Technocratic PP denotes the coexistence, or coproduction, of technocracy with PP – a complex ensemble of technocratic, precautionary policies, and hybrids of the two. This paper analyzes four types of PP-based policies linked to Korean GMO regulations: foresight and monitoring of risk; reverse burden of proof; public participation; and the public's right to know. Korean GMO regulations are consistent with the Cartagena Protocol on Biosafety, a type of PP, but lack long-term risk assessment as well as public participation. Technocracy is embedded both in advance informed agreements as a reverse burden of proof and in proof-based GMO labeling as a right-to-know policy. Technocratic PP results in inconsistencies between PP and technocratic epistemology and the gap between PP-based institutions and technocratic practices. Technocratic PP is therefore a typical phenomenon that occurs in the “glocalization” of risk regulation.     

我国农业转基因生物安全管理现状

转基因技术可以打破物种界限,实现作物性状的定向改造,在解决粮食增产、环境污染、节水增效,以及改进农产品质量等方面显示出巨大潜力。同时,转基因技术具有潜在的风险,转基因生物的安全性受到人们的广泛关注。本文介绍了我国农业转基因生物安全管理状况,包括法规制度体系、安全评价体系和安全监管体系,旨在让人们更好地了解我国农业转基因生物安全管理知识。     

The structuring of GMO release and evaluation in EU law

Genetically modified organisms (GMOs) and their behavior in the environment are complex and can only be assessed if the different components are distinguished. This article examines, how by EU law the real causation processes from the GMO release to various endpoints are dissected, individually analysed and then again viewed in their entirety. In addition, the articles includes, how the intellectual process of assessment is divided into the steps of tiered generation, shared submission and structured evaluation of relevant knowledge. The framework proposed for such an examination allows to identify strengths and weaknesses of GMO risk assessment in the EU.     

转基因生物及其产品检测技术和标准化

随着转基因生物及其产品的大规模商业化以及消费者对其安全性的担心,很多国家和地区纷纷出台包括转基因产品标签制度在内的转基因生物安全管理的法律法规,为保障转基因产品标签制度的实施以及消费者知情权和选择权,转基因产品检测分析方法及其标准化研究受到人们广泛重视。目前,国内外常用的转基因产品检测方法主要包括针对转基因产品中的目的核酸DNA分子或者其编码的蛋白质分子,本文将对转基因生物及其产品检测方法及其标准化的进展及发展趋势做一概述。     

EFSA’s scientific activities and achievements on the risk assessment of genetically modified organisms (GMOs) during its first decade of existence: looking back and ahead

Genetically modified organisms (GMOs) and derived food and feed products are subject to a risk analysis and regulatory approval before they can enter the market in the European Union (EU). In this risk analysis process, the role of the European Food Safety Authority (EFSA), which was created in 2002 in response to multiple food crises, is to independently assess and provide scientific advice to risk managers on any possible risks that the use of GMOs may pose to human and animal health and the environment. EFSA’s scientific advice is elaborated by its GMO Panel with the scientific support of several working groups and EFSA’s GMO Unit. This review presents EFSA’s scientific activities and highlights its achievements on the risk assessment of GMOs for the first 10 years of its existence. Since 2002, EFSA has issued 69 scientific opinions on genetically modified (GM) plant market registration applications, of which 62 for import and processing for food and feed uses, six for cultivation and one for the use of pollen (as or in food), and 19 scientific opinions on applications for marketing products made with GM microorganisms. Several guidelines for the risk assessment of GM plants, GM microorganisms and GM animals, as well as on specific issues such as post-market environmental monitoring (PMEM) were elaborated. EFSA also provided scientific advice upon request of the European Commission on safeguard clause and emergency measures invoked by EU Member States, annual PMEM reports, the potential risks of new biotechnology-based plant breeding techniques, evaluations of previously assessed GMOs in the light of new scientific publications, and the use of antibiotic resistance marker genes in GM plants. Future challenges relevant to the risk assessment of GMOs are discussed. EFSA’s risk assessments of GMO applications ensure that data are analysed and presented in a way that facilitates scientifically sound decisions that protect human and animal health and the environment.     

Detection of genetically modified DNA in fresh and processed foods sold in Kuwait

Developments in genetic engineering technology have led to an increase in number of food products that contain genetically engineered crops in the global market. However, due to lack of scientific studies, the presence of genetically modified organisms (GMOs) in the Kuwaiti food market is currently ambiguous. Foods both for human and animal consumption are being imported from countries that are known to produce GM food. Therefore, an attempt has been made to screen foods sold in the Kuwaiti market to detect GMOs in the food. For this purpose, samples collected from various markets in Kuwait have been screened by SYBR green-based real time polymerase chain reaction (RT-PCR) method. Further confirmation and GMO quantification was performed by TaqMan-based RT-PCR. Results indicated that a significant number of food commodities sold in Kuwait were tested positive for the presence of GMO. Interestingly, certain processed foods were tested positive for more than one transgenic events showing complex nature of GMOs in food samples. Results of this study clearly indicate the need for well-defined legislations and regulations on the marketing of approved GM food and its labeling to protect consumer's rights.