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PubMed2004

Rapid analysis of genetically modified organisms by in-house developed capillary electrophoresis chip and laser-induced fluorescence system.

A microfabricated, inexpensive, reusable glass capillary electrophoresis chip and a laser-induced fluorescence system were developed in-house for the rapid DNA-based analysis of genetically modified organisms (GMOs). The 35S promoter sequence of cauliflower mosaic virus and the terminator of the nopaline synthase (NOS) gene from Agrobacterium tumefaciens were both detected since they are present in most genetically modified organisms. The detection of genetically modified soybean in the presence of unaltered soybean was chosen as a model. Lectin, a plant-specific gene, was also detected for confirmation of the integrity of extracted DNA. The chip was composed of two glass plates, each 25 x 76 mm, thermally bonded together to form a closed structure. Photomasks with cross-topology were prepared rapidly by using polymeric material instead of chrome plates. The widths of the injection and separation channels were 30 and 70 microm, respectively, the effective separation length 4.5 cm. The glass slide was etched to a depth of 30 microm for both the injection and separation channel. The cost of the chip was less than 1 $ and required 2 days for photomask preparation and microfabrication. The separation and detection of polymerase chain reaction-amplified NOS, 35S, and lectin sequences (180, 195, and 181 bp, respectively) was completed in less than 60 s. As low as 0.1% GMO content was detectable by the proposed system after 35 and 40 amplification cycles for 35S and NOS, respectively, using 25 ng of extracted DNA as starting material. This corresponds to only 20 genome copies of genetically modified soybean.

Amino Acid Oxidoreductases↗
PubMed2005

Regulations governing veterinary medicinal products containing genetically modified organisms in the European community.

This paper describes particular aspects of the marketing of veterinary medicinal products (VMPs) that contain or consist of genetically modified micro-organisms (GMMs) or genetically modified organisms (GMOs). The regulatory requirements and the procedures applied in the European Union for each phase (pre-marketing, authorisation process, and post-authorisation labelling and monitoring) are explained. In most cases VMPs are subject to both pharmaceutical and GMO regulations. In the early stages of the process, before applications for marketing authorisation are submitted, the assessment of clinical trials and experiments in contained areas is principally the responsibility of national authorities. However, the marketing of all VMPs containing or consisting of GMOs must be authorised at European level, although the national authorities are informed and involved in the assessment process.

Animals↗
PubMed2004

Detection of genetically modified organisms by electrochemiluminescence PCR method.

With the development of biotechnology, more and more genetically modified organisms (GMOs) have entered commercial market. Because of the safety concerns, detection and characterization of GMOs have attracted much attention recently. In this study, electrochemiluminescence polymerase chain reaction (ECL-PCR) combined with hybridization technique was applied to detect the GMOs in genetically modified (GM) soybeans and papayas for the first time. Whether the soybeans and the papayas contain GM components was discriminated by detecting the Cauliflower mosaic virus 35S (CaMV35S) promoter. The experiment results show that the detection limit for CaMV35S promoter is 100 fmol, and the GM components can be clearly identified in GM soybeans and papayas. The technique may provide a new means in GMOs detection due to its simplicity and high efficiency.

Carica↗
PubMed2002

Risk assessment for release of genetically modified organisms: a virus to reduce the fertility of introduced wild mice, Mus domesticus.

Risk assessment is a key task in developing genetically modified organisms (GMOs) intended for release into the environment. A risk assessment protocol is described, focusing on genetically modified biological control agents intended to reduce fertility in mammalian pests. The protocol is being applied to development of an immunocontraceptive murine cytomegalovirus vaccine intended to reduce the frequency and extent of costly troublesome plagues of introduced house mice, Mus domesticus, in southern Australia. Success of the agent, including regulatory approval for release to target populations, will depend on demonstrated biosafety, on the biophysical consequences of releasing the agent, and on public perceptions of the consequences and ongoing risks. The proposed risk assessment protocol addresses biosafety and the biophysical and social risks. It elicits perceptions of interaction and risk from the project scientists and from representatives of interested or affected sectors of society. The perceptions are documented for examination interactively in subsequent socially inclusive formal risk assessments. Representatives of the relevant social sectors participate with the scientists, iteratively if needed, in a workshop to assess the risks of releasing the particular GMO into the environment, using a formal inductive procedure, GENHAZ, designed specifically for assessment and management of the risks of GMOs. Use of this protocol is intended to precede and complement risk assessment and risk management procedures specified by gene technology legislation and regulations.

Animals↗
PubMed2005

Suggestions for the assessment of the allergenic potential of genetically modified organisms.

The prevalence of allergic diseases has been increasing continuously and, accordingly, there is a great desire to evaluate the allergenic potential of components in our daily environment (e.g., food). Although there is almost no scientific evidence that genetically modified organisms (GMOs) exhibit increased allergenicity compared with the corresponding wild type significant concerns have been raised regarding this matter. In principle, it is possible that the allergenic potential of GMOs may be increased due to the introduction of potential foreign allergens, to potentially upregulated expression of allergenic components caused by the modification of the wild type organism or to different means of exposure. According to the current practice, the proteins to be introduced into a GMO are evaluated for their physiochemical properties, sequence homology with known allergens and occasionally regarding their allergenic activity. We discuss why these current rules and procedures cannot predict or exclude the allergenicity of a given GMO with certainty. As an alternative we suggest to improve the current evaluation by an experimental comparison of the wild-type organism with the whole GMO regarding their potential to elicit reactions in allergic individuals and to induce de novo sensitizations. We also recommend that the suggested assessment procedures be equally applied to GMOs as well as to natural cultivars in order to establish effective measures for allergy prevention.

Allergens↗
PubMed2006

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.

Animals↗
PubMed2006

Conservation biology, genetically modified organisms, and the biosafety protocol.

Concerns have been raised regarding the potential adverse effects on biological diversity of the use of living modified organisms (LMOs, which are commonly known by similar terms such as genetically modified organisms). At the international level these concerns are addressed in part by an agreement known as the Cartagena Protocol on Biosafety and include potential toxic effects of insect-resistant crops on nontarget organisms and potential ecological effects of gene flow from modified crops, fish, microorganisms, or insects to wild species or counterparts. We reviewed the protocol's main provisions, including those dealing with risk assessment and risk management, decision making on imports, documentation accompanying shipments, and liability resulting from damages caused by LMOs. A medium-term program of work has been adopted by the parties, which includes the potential contribution of conservation biologists to delivering capacity building, developing risk assessment guidance, evaluating mechanisms of potential ecological damages from LMOs, and other issues. Conservation biologists and other experts have opportunities to influence the negotiations and implementation of the protocol by providing inputs at meetings, offering expertise to governments and organizations, and participating in or developing relevant projects and initiatives. Involvement of conservation biologists in the implementation and further development of the protocol would contribute to its effectiveness.

Animals↗
PubMed2005

Microchip capillary gel electrophoresis using programmed field strength gradients for the ultra-fast analysis of genetically modified organisms in soybeans.

We have developed a novel method for the ultra-fast analysis of genetically modified organisms (GMOs) in soybeans by microchip capillary gel electrophoresis (MCGE) using programmed field strength gradients (PFSG) in a conventional glass double-T microchip. Under the programmed electric field strength and 0.3% poly(ethylene oxide) sieving matrix, the GMO in soybeans was analyzed within only 11 s of the microchip. The MCGE-PFSG method was a program that changes the electric field strength during GMO analysis, and was also applied to the ultra-fast analysis of PCR products. Compared to MCGE using a conventional and constantly applied electric field, the MCGE-PFSG analysis generated faster results without the loss of resolving power and reproducibility for specific DNA fragments (100- and 250-bp DNA) of GM-soybeans. The MCGE-PFSG technique may prove to be a new tool in the GMO analysis due to its speed, simplicity, and high efficiency.

DNA, Plant↗
PubMed2005

Role of the "National Reference Centre for Genetically Modified Organisms (GMO) detection" in the official control of food and feed.

The National Reference Centre for Genetically Modified Organisms (GMO) detection was established in 2002 within the Istituto Zooprofilattico Sperimentale Lazio e Toscana, with the aim of providing scientific and technical support to the National Health System and to the Ministry of Health within the scope of the regulation of GMO use in food and feed.The recently adopted EU legislation on GMOs (Regulation CE no. 1829/2003 and no. 1830/2003) introduced more rigorous procedures for the authorisation, labelling and analytical control of food and feed consisting, containing or derived from GMOs. The National Reference Centre, besides its institutional tasks as one of the laboratories of the Italian National Health System, collects and analyses data and results of the national official control of GMOs; carries out scientific research aimed at developing, improving, validating and harmonising detection and quantification methods, in cooperation with other scientific institutions, the Community Reference Laboratory and within the European Network of GMOs laboratories (ENGL); collaborates with the Ministry of Health in the definition of control programmes and promotes educational and training initiatives. Objectives defined for 2004-2006, activities in progress and goals already achieved are presented.

European Union↗
PubMed1999

Genetically modified organisms: an analysis of the regulatory framework currently employed within the European Union.

BACKGROUND: Genetic engineering technology is starting to bring many commercial products to the market. These genetically modified organisms (GMOs) and their derived products are subject to topical debate as to their benefits and risks. The strengths and weaknesses of the regulatory framework that controls their development and application is central to the question of whether this technology poses significant risk to the public health during this critical phase of its evolution. METHODS: A critical review was carried out of the legal framework regulating the contained use, deliberate release and some aspects of consumer protection relevant to the control of GMOs in Europe and the United Kingdom. RESULTS: The current legal framework is failing to provide a speed of adaptation commensurate with the development of the science of genetic engineering; failing to properly respond to democratic control; failing to resolve significant conflict between the protection of free markets and protection of public health and the environment; and failing to implement obligations on biodiversity. CONCLUSION: The present legal framework must be replaced. Current European Union proposals for new standards of regulation are welcome, but provide only for further incremental change, and do not address some significant fundamental flaws in our current laws.

Consumer Product Safety↗
PubMed1999

Genetically modified organisms in food-screening and specific detection by polymerase chain reaction.

PCR methods for the detection of genetically modified organisms (GMOs) were developed that can be used for screening purposes and for specific detection of glyphosate-tolerant soybean and insect-resistant maize in food. Primers were designed to amplify parts of the 35S promoter derived from Cauliflower Mosaic Virus, the NOS terminator derived from Agrobacterium tumefaciens and the antibiotic marker gene NPTII (neomycin-phosphotransferase II), to allow for general screening of foods. PCR/hybridization protocols were established for the detection of glyphosate-tolerant RoundUp Ready soybean and insect-resistant Bt-maize. Besides hybridization, confirmation of the results using restriction analysis was also possible. The described methods enabled a highly sensitive and specific detection of GMOs and thus provide a useful tool for routine analysis of raw and processed food products.

Blotting, Southern↗
PubMed2005

Multiplex polymerase chain reaction and ligation detection reaction/universal array technology for the traceability of genetically modified organisms in foods.

A multiplex polymerase chain reaction (PCR) system was developed for the simultaneous detection of target sequences in genetically modified soybean (Roundup Ready) and maize (MON810, Bt176, Bt11, and GA21). Primer pairs were designed to amplify the junction regions of the transgenic constructs analyzed and the endogenous genes of soybean (lectin) and maize (zein) were included as internal control targets to assess the efficiency of all reactions. This multiplex PCR has constituted the basis for an efficient platform for genetically modified organism traceability based on microarray technology. In particular, the ligation detection reaction combined to a universal array approach, using the multiplex PCR as target, was applied. High specificity and sensitivity were obtained.

DNA, Plant↗
PubMed2004

Metabolism of zearalenone by genetically modified organisms expressing the detoxification gene from Clonostachys rosea.

Zearalenone (ZEN) is converted to a nontoxic product by a lactonohydololase encoded by zhd101. An enhanced green fluorescent protein (EGFP) gene was fused to zhd101 (i.e., egfp::zhd101) and expressed in Escherichia coli. Both recombinant ZHD101 and EGFP::ZHD101 were purified to homogeneity and characterized. Maximal activity of ZHD101 toward ZEN was measured at approximately 37 to 45 degrees C and pH 10.5 (k(cat) at 30 degrees C, 0.51 s(-1)). The enzyme was irreversibly inactivated at pH values below 4.5 or by treatment with serine protease inhibitors. ZHD101 was also active against five ZEN cognates, although the efficiencies were generally low; e.g., the k(cat) was highest with zearalanone (1.5 s(-1)) and lowest with beta-zearalenol (0.075 s(-1)). EGFP::ZHD101 had properties similar to those of the individual proteins with regard to the EGFP fluorescence and lactonohydrolase activity. Fortuitously, EGFP::ZHD101 exhibited a good correlation between the fluorescence intensity and reaction velocity under various pH conditions. We therefore used egfp::zhd101 to visually monitor the lactonohydrolase activity in genetically modified organisms and evaluated the usefulness of zhd101 for in vivo detoxification of ZEN. While recombinant E. coli and transgenic rice calluses exhibited strong EGFP fluorescence and completely degraded ZEN in liquid media, recombinant Saccharomyces cerevisiae gave poor fluorescence and did not eliminate all the toxicity of the mycotoxin in the medium; i.e., the rest of ZEN was transformed into an unfavorable substrate, beta-zearalenol, by an as-yet-unidentified reductase and remained in the medium. Even so, as much as 75% of ZEN was detoxified by the yeast transformant, which is better than the detoxification system in which food-grade Lactobacillus strains are used (H. El-Nezami, N. Polychronaki, S. Salminen, and H. Mykkuäne, Appl. Environ. Microbiol. 68:3545-3549, 2002). An appropriate combination of a candidate host microbe and the codon-optimized synthetic gene may contribute significantly to establishing a mycotoxin detoxification system for food and feed.

Escherichia coli↗
PubMed2004

Detection of genetically modified organisms in foods by DNA amplification techniques.

In this article, the different DNA amplification techniques that are being used for detecting genetically modified organisms (GMOs) in foods are examined. This study intends to provide an updated overview (including works published till June 2002) on the principal applications of such techniques together with their main advantages and drawbacks in GMO detection in foods. Some relevant facts on sampling, DNA isolation, and DNA amplification methods are discussed. Moreover; these analytical protocols are discuissed from a quantitative point of view, including the newest investigations on multiplex detection of GMOs in foods and validation of methods.

DNA, Plant↗
PubMed2006

Critical points of DNA quantification by real-time PCR--effects of DNA extraction method and sample matrix on quantification of genetically modified organisms.

BACKGROUND: Real-time PCR is the technique of choice for nucleic acid quantification. In the field of detection of genetically modified organisms (GMOs) quantification of biotech products may be required to fulfil legislative requirements. However, successful quantification depends crucially on the quality of the sample DNA analyzed. Methods for GMO detection are generally validated on certified reference materials that are in the form of powdered grain material, while detection in routine laboratories must be performed on a wide variety of sample matrixes. Due to food processing, the DNA in sample matrixes can be present in low amounts and also degraded. In addition, molecules of plant origin or from other sources that affect PCR amplification of samples will influence the reliability of the quantification. Further, the wide variety of sample matrixes presents a challenge for detection laboratories. The extraction method must ensure high yield and quality of the DNA obtained and must be carefully selected, since even components of DNA extraction solutions can influence PCR reactions. GMO quantification is based on a standard curve, therefore similarity of PCR efficiency for the sample and standard reference material is a prerequisite for exact quantification. Little information on the performance of real-time PCR on samples of different matrixes is available. RESULTS: Five commonly used DNA extraction techniques were compared and their suitability for quantitative analysis was assessed. The effect of sample matrix on nucleic acid quantification was assessed by comparing 4 maize and 4 soybean matrixes. In addition 205 maize and soybean samples from routine analysis were analyzed for PCR efficiency to assess variability of PCR performance within each sample matrix. Together with the amount of DNA needed for reliable quantification, PCR efficiency is the crucial parameter determining the reliability of quantitative results, therefore it was chosen as the primary criterion by which to evaluate the quality and performance on different matrixes and extraction techniques. The effect of PCR efficiency on the resulting GMO content is demonstrated. CONCLUSION: The crucial influence of extraction technique and sample matrix properties on the results of GMO quantification is demonstrated. Appropriate extraction techniques for each matrix need to be determined to achieve accurate DNA quantification. Nevertheless, as it is shown that in the area of food and feed testing matrix with certain specificities is impossible to define strict quality controls need to be introduced to monitor PCR. The results of our study are also applicable to other fields of quantitative testing by real-time PCR.

DNA, Plant↗
PubMed2003

PCR technology for screening and quantification of genetically modified organisms (GMOs).

Although PCR technology has obvious limitations, the potentially high degree of sensitivity and specificity explains why it has been the first choice of most analytical laboratories interested in detection of genetically modified (GM) organisms (GMOs) and derived materials. Because the products that laboratories receive for analysis are often processed and refined, the quality and quantity of target analyte (e.g. protein or DNA) frequently challenges the sensitivity of any detection method. Among the currently available methods, PCR methods are generally accepted as the most sensitive and reliable methods for detection of GM-derived material in routine applications. The choice of target sequence motif is the single most important factor controlling the specificity of the PCR method. The target sequence is normally a part of the modified gene construct, for example a promoter, a terminator, a gene, or a junction between two of these elements. However, the elements may originate from wildtype organisms, they may be present in more than one GMO, and their copy number may also vary from one GMO to another. They may even be combined in a similar way in more than one GMO. Thus, the choice of method should fit the purpose. Recent developments include event-specific methods, particularly useful for identification and quantification of GM content. Thresholds for labelling are now in place in many countries including those in the European Union. The success of the labelling schemes is dependent upon the efficiency with which GM-derived material can be detected. We will present an overview of currently available PCR methods for screening and quantification of GM-derived DNA, and discuss their applicability and limitations. In addition, we will discuss some of the major challenges related to determination of the limits of detection (LOD) and quantification (LOQ), and to validation of methods.

DNA↗
PubMed2005

High-throughput double quantitative competitive polymerase chain reaction for determination of genetically modified organisms.

Quantitative competitive polymerase chain reaction (PCR), especially the double competitive PCR methods (DC-PCR), have evolved as reliable approaches to quantification of genetically modified organisms (GMO) in food. However, DC-PCR is a low-throughput method because it requires titration of each sample with various amounts of a competitive internal standard, a protocol that involves several PCRs per sample followed by electrophoresis and densitometry. To address this drawback, we have developed a new method for GMO quantification, namely, a high-throughput double quantitative competitive PCR (HT-DCPCR). In HT-DCPCR, electrophoresis and densitometry are replaced by a rapid, microtiter well-based bioluminometric hybridization assay and there is no need for titration of each sample. The determination of GM soya was chosen as a model. We have constructed internal standards (DNA competitors) both for the 35S promoter sequence and for a plant-specific reference gene (lectin). The competitors have identical size and share the same primer binding sites with the target sequences but differ in a 24-bp internal segment. Each target sequence (35S and lectin) is coamplified with a constant amount (1000 copies) of the respective competitor. The four amplified fragments are hybridized with specific probes and captured on a universal solid phase to achieve simplicity and high throughput. The hybrids are determined by using streptavidin conjugated to the photoprotein aequorin. The ratio of the luminescence values obtained for the target and the competitor is linearly related to the starting amount of target DNA. The limit of quantification for the 35S promoter is 24 copies. The proposed method was evaluated by determining the GMO content of soybean powder certified reference materials. Also HT-DCPCR was compared to real-time PCR in a variety of real samples.

Calibration↗