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PubMedDate not supplied

IUPAC collaborative trial study of a method to detect genetically modified soy beans and maize in dried powder.

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.

Agrobacterium tumefaciens↗

PubMed1998

The EC novel foods Regulation--a UK perspective.

On 15 May 1997 the EC novel foods Regulation came into effect introducing a statutory pre-market approval system for novel foods across the whole of the European Union. A novel food is defined as a food which has not been consumed to a significant degree and includes foods containing or obtained from genetically modified organisms. The Regulation envisages an initial safety assessment at Member State level, although centralized procedures are available to resolve any objections between Member States. The most controversial aspect of the Regulation relates to the provisions for labelling genetically modified foods. Within the framework of the Regulation there is scope for labelling to be considered on a case by case basis, although the UK is pressing for all foods which may contain genetically modified material to be clearly labelled. To ensure that all Member States follow a consistent approach to the safety assessment of novel foods, the Commission has published a series of guidelines to accompany the regulation. The UK already has a well-developed system for assessing the safety of novel foods dating back to the approval of the first novel food in the UK in 1983.

Consumer Product Safety↗

PubMed2004

Sensitive and simultaneous analysis of five transgenic maizes using multiplex polymerase chain reaction, capillary gel electrophoresis, and laser-induced fluorescence.

The benefits of using multiplex polymerase chain reaction (PCR) followed by capillary gel electrophoresis with laser-induced fluorescence (CGE-LIF) for the simultaneous detection of five transgenic maizes (Bt11, T25, MON810, GA21, and Bt176) are demonstrated. The method uses a hexaplex PCR protocol to amplify the five mentioned transgenic amplicons plus the zein gene used as reference, followed by a CGE-LIF method to analyze the six DNA fragments. CGE-LIF was demonstrated very useful and informative for optimizing multiplex PCR parameters such as time extension, PCR buffer concentration and primers concentration. The method developed is highly sensitive and allows the simultaneous detection in a single run of percentages of transgenic maize as low as 0.054% of Bt11, 0.057% of T25, 0.036% of MON810, 0.064% of GA21, and 0.018% of Bt176 in flour obtaining signals still far from the detection limit (namely, the signal/noise ratios for the corresponding DNA peaks were 41, 124, 98, 250, 252, and 473, respectively). These percentages are well below the minimum threshold marked by the European Regulation for transgenic food labeling (i.e., 0.5-0.9%). A study on the reproducibility of the multiplex PCR-CGE-LIF procedure was also performed. Thus, values of RSD lower than 0.67 and 6.80% were obtained for migration times and corrected peak areas, respectively, for the same sample and three different days (n = 12). On the other hand, the reproducibility of the whole procedure, including four different multiplex PCR amplifications, was determined to be better than 0.66 and 23.3% for migration times and corrected peak areas, respectively. Agarose gel electrophoresis (AGE) and CGE-LIF were compared in terms of resolution and sensitivity for detecting PCR products, demonstrating that CGE-LIF can solve false positives induced by artifacts from the multiplex PCR reaction that could not be addressed by AGE. Moreover, CGE-LIF provides better resolution and sensitivity. To our knowledge, these results demonstrate for the first time that multiplex PCR-CGE-LIF is a solid alternative to determine multiple genetically modified organisms in maize flours in a single run.

DNA Primers↗

PubMedDate not supplied

[Genetically modified foods. Advantages and human health risks].

One of the most important issue with which the mankind is confronting now is related to the quantitatively as well as qualitatively assurance of the food supply necessary for human species existence. In this context, by means of genetic engineering, modified genetic organisms were obtained. In the first stage, plant crops with high productivity and resistant against diseases and pests were obtained. After that, food products having modified organoleptic properties and high nutrition values were produced. The main problem concerning the long-term consumption of these products is their toxicity, which until now was not confirmed or denied. For this reason, tests are necessary to be made in order to stipulate and prevent these effects.

Consumer Product Safety↗

PubMed2004

The relevance of gene transfer to the safety of food and feed derived from genetically modified (GM) plants.

In 2000, the thematic network ENTRANSFOOD was launched to assess four different topics that are all related to the testing or assessment of food containing or produced from genetically modified organisms (GMOs). Each of the topics was linked to a European Commission (EC)-funded large shared cost action (see http://www.entransfood.com). Since the exchange of genetic information through horizontal (lateral) gene transfer (HGT) might play a more important role, in quantity and quality, than hitherto imagined, a working group dealing with HGT in the context of food and feed safety was established. This working group was linked to the GMOBILITY project (GMOBILITY, 2003) and the results of the deliberations are laid down in this review paper. HGT is reviewed in relation to the potential risks of consuming food or feed derived from transgenic crops. First, the mechanisms for obtaining transgenic crops are described. Next, HGT mechanisms and its possible evolutionary role are described. The use of marker genes is presented in detail as a special case for genes that may pose a risk. Furthermore, the exposure to GMOs and in particular to genetically modified (GM) deoxyribonucleic acid (DNA) is discussed as part of the total risk assessment. The review finishes off with a number of conclusions related to GM food and feed safety. The aim of this paper is to provide a comprehensive overview to assist risk assessors as well as regulators and the general public in understanding the safety issues related to these mechanisms.

Animal Feed↗

PubMed2001

Safety considerations of DNA in food.

Recombinant DNA techniques are capable of introducing genetic changes into food organisms that are more predictable than those introduced through conventional breeding techniques. This review discusses whether the consumption of DNA in approved novel foods and novel food ingredients derived from genetically modified organisms (GMOs) can be regarded as being as safe as the consumption of DNA in existing foods. It concludes that DNA from GMOs is equivalent to DNA from existing food organisms that has always been consumed with human diets. Any risks associated with the consumption of DNA will remain, irrespective of its origin, because the body handles all DNA in the same way. The breakdown of DNA during food processing and passage through the gastrointestinal tract reduces the likelihood that intact genes capable of encoding foreign proteins will be transferred to gut microflora. The review does not specifically address food safety issues arising from the consumption of viable genetically modified microorganisms but it shows that the likelihood of transfer and functional integration of DNA from ingested food by gut microflora and/or human cells is minimal. Information reviewed does not indicate any safety concerns associated with the ingestion of DNA per se from GMOs resulting from the use of currently available recombinant DNA techniques in the food chain.

Consumer Product Safety↗

PubMed2002

Gene transfer between Salmonella enterica serovar Typhimurium inside epithelial cells.

Virulence and antibiotic resistance genes transfer between bacteria by bacterial conjugation. Conjugation also mediates gene transfer from bacteria to eukaryotic organisms, including yeast and human cells. Predicting when and where genes transfer by conjugation could enhance our understanding of the risks involved in the release of genetically modified organisms, including those being developed for use as vaccines. We report here that Salmonella enterica serovar Typhimurium conjugated inside cultured human cells. The DNA transfer from donor to recipient bacteria was proportional to the probability that the two types of bacteria occupied the same cell, which was dependent on viable and invasive bacteria and on plasmid tra genes. Based on the high frequencies of gene transfer between bacteria inside human cells, we suggest that such gene transfers occur in situ. The implications of gene transfer between bacteria inside human cells, particularly in the context of antibiotic resistance, are discussed.

Biological Evolution↗

PubMed1999

Will we be taught ethics by our clones? The mutations of the living, from endocrine disruptors to genetics.

Considering the worldwide threat to health and reproduction related to endocrine disruptors (by-products of the chemical industry); considering the untrammelled development of the industrialization and engineering of the living, ethics and gynaecology/obstetrics itself is at a crossroads. Endocrine disruptors (derived from organochlorines and persistent organic pollutants such as PCBs, dioxins and furans, and pesticides such as aldrin, chlordane and DDT), are prime suspects in the deterioration of fertility and intellectual faculties and possibly a key factor in endometriosis, breast cancer and prostate cancer. The long-term and pernicious impacts of endocrine disruptors show our poor understanding of the complexities of life's mechanisms. Paradoxically, with our short-term perspectives and predilection for a technological fix, the problem posed by endocrine disruptors may accelerate the use of reproductive technologies such as ICSI and even cloning, as well as the dissemination of genetically modified organisms. The cure could be worse than the disease. Given the gravity of the challenge to humanity related to the chemical erosion of human health, the mutation of human conception introduced by reproductive technologies and by the drive to genetically modify nature and even human nature, we must urgently re-evaluate the direction in which our societies are headed and the reliance on profit-oriented technology to save us from ourselves. In these circumstances, the collective exercise of wisdom, prudence and responsibility towards the essence and integrity of humanity has become, more than ever, an ethical, and perhaps even a survival, imperative.

Cloning, Organism↗

PubMed2003

Quantitation of Bt-176 maize genomic sequences by surface plasmon resonance-based biospecific interaction analysis of multiplex polymerase chain reaction (PCR).

Surface plasmon resonance (SPR) based biosensors have been described for the identification of genetically modified organisms (GMO) by biospecific interaction analysis (BIA). This paper describes the design and testing of an SPR-based BIA protocol for quantitative determinations of GMOs. Biotinylated multiplex Polymerase Chain Reaction (PCR) products from nontransgenic maize as well as maize powders containing 0.5 and 2% genetically modified Bt-176 sequences were immobilized on different flow cells of a sensor chip. After immobilization, different oligonucleotide probes recognizing maize zein and Bt-176 sequences were injected. The results obtained were compared with Southern blot analysis and with quantitative real-time PCR assays. It was demonstrated that sequential injections of Bt-176 and zein probes to sensor chip flow cells containing multiplex PCR products allow discrimination between PCR performed using maize genomic DNA containing 0.5% Bt-176 sequences and that performed using maize genomic DNA containing 2% Bt-176 sequences. The efficiency of SPR-based BIA in discriminating material containing different amounts of Bt-176 maize is comparable to real-time quantitative PCR and much more reliable than Southern blotting, which in the past has been used for semiquantitative purposes. Furthermore, the approach allows the BIA assay to be repeated several times on the same multiplex PCR product immobilized on the sensor chip, after washing and regeneration of the flow cell. Finally, it is emphasized that the presented strategy to quantify GMOs could be proposed for all of the SPR-based, commercially available biosensors. Some of these optical SPR-based biosensors use, instead of flow-based sensor chips, stirred microcuvettes, reducing the costs of the experimentation.

DNA, Plant↗

PubMed2006

Loss of agro-biodiversity, uncertainty, and perceived control: a comparative risk perception study in Austria and China.

The biogeographical centers of origin of important food crops-called Vavilov centers-are considered to be crucial sources of genetic diversity for present and future crop-breeding programs and thus for human food safety worldwide. Global environmental change and more intensified modes of crop production may cause genetic erosion (loss of traditional crop varieties and loss of crop wild relatives), especially in Vavilov centers. The present study focused on how the risk of genetic erosion (or loss of agro-biodiversity) is perceived in comparison to 16 other risk topics by experts and lay people in Austria and China. The most striking result was that genetic erosion was perceived to be an exceptionally unknown and uncertain risk topic, given that only genetically modified organisms (GMOs) were perceived as being even more uncertain. As a consequence of the high uncertainty, the idea of applying the precautionary principle to further prevent genetic erosion is discussed. An unprecedented finding-one that differs from Austrian participants-is that the Chinese have a higher perceived control over all risk topics. The increased perception of controllability in China is discussed in light of the theory of reflexive modernization. This theory strives to explain the increased critical attitude in Western countries such as Austria toward scientific innovations and toward the idea that everything can be calculated and mastered at will. By revealing different notions of risk perception, this research also provides additional scientific input to risk communication efforts for public education.

Agriculture↗

PubMed2001

Recent production of candidate reference materials at IRMM.

In the execution of its mission to promote a common European measurement system in support of EU policies, IRMM's Reference Materials Unit is currently involved in preparation of proficiency-testing samples and candidate reference materials. Recent work related to bovine spongiform encephalopathy in cows, genetically modified organisms, and a variety of environmental materials is described.

Animals↗

PubMed2005

[Animal transgenesis for necessities of xenotransplantation].

The presence in humans of xenoreactive antibodies directed against swine Gal antigen present on the surface of xenograft donor cells, leads to the complement activation and immediate xenograft rejection--as a consequence of hyperacute immunological reaction. The graft of genetically modified organ of a swine depleted of at alpha1,3-galactosylotransferase enzyme that is responsible for Gal antigen origin, would be tolerated with simultaneous administration of medicines decreasing other less severe immunological reactions. To prevent hyperacute rejection it is also possible to modify swine genome by human genes controlling enzymatic cascade of complement or modifying the set of donor's cell surface proteins. The removal or significant reduction of number of Gal antigen molecules prevents complement activation. In this situation, the main reason for the graft rejection is malfunction of the regulation of coagulation system inside the graft and cellular reactions involving macrophages, neutrophiles, NK cells and lymphocytes T. Minor genetic differences between the acceptor's organism and the donor of grafting organ can lead to so called delayed xenograft rejection. As our knowledge about reactions taking place inside grafted tissue will increase it would be possible to introduce genes controlling coagulation system into swine genome.

Animals↗

PubMed1997

Bacterial communities of the rhizosphere and endorhiza associated with field-grown cucumber plants inoculated with a plant growth-promoting rhizobacterium or its genetically modified derivative.

The future use of genetically modified microorganisms in the environment will be dependent on the ability to asses potential or theoretical risks associated with their introduction into natural ecosystems. To assess potential risks, several ecological parameters must be examined, including the impact of the introduced genetically modified organism on the microbial communities associated with the environment into which the introduction will occur. A 2-year field study was established to examine whether the indigenous bacterial communities of the rhizosphere and endorhiza (internal root tissues) were affected differently by the introduction of an unaltered wild type and its genetically modified derivative. Treatments consisted of the wild-type strain Pseudomonas fluorescens 89B-27 and a bioluminescent derivative GEM-8 (89B-27::Tn4431). Cucumber root or seed samples were taken 0, 7, 14, 21, 35, and 70 days after planting (DAP) in 1994 and 0, 7, 14, 28, 42, and 70 DAP in 1995. Samples were processed to examine the bacterial communities of both the rhizosphere ad endorhiza. Over 7200 bacterial colonies were isolated from the rhizosphere Community structure at the genus level was assessed using genera richness and Hill's diversity numbers, N1 and N2. The aerobic-heterotrophic bacterial community structure at the genus level did not significantly vary between treatments but did differ temporally. The data indicate that the introduction of the genetically modified derivative of 89B-27 did not pose a greater environmental risk than its unaltered wild type with respect to aerobic-heterotrophic bacterial community structure.

Cucumis sativus↗

PubMed1996

Bioprocessing with genetically modified and other organisms: case studies in processing constraints.

Whereas the gene stability related considerations are important in bioprocessing with recombinant cultures, bioreactor design and scale-up require attention to the often reduced shear tolerance of the genetically altered biocatalysts relative to the corresponding wild strains. In addition, the peculiarities of expression of the rDNA product impact the downstream recovery methods. As a consequence, a bioprocessing scheme and the process machinery designed for a naturally occurring organism may need significant modifications for use with a genetically modified variety of the same organism. The case studies described highlight some of the processing constraints and consideration of general relevance.

Animals↗

PubMed2006

The occurrence of antibiotic resistance genes in Taq polymerases and a decontamination method applied to the detection of genetically modified crops.

Different antibiotic resistance (AR) genes, such as Bla, Tet and NPTII, contaminate commercially available Taq polymerases. The specificity of the AR gene PCR can be increased when using a restriction enzyme-based decontamination of polymerase. The elimination of Taq polymerase contamination allows the use of PCR tests to screen seeds (corn) and processed food for the presence of genetically modified organisms (GMO) based on the detection of AR genes. Without a decontamination procedure for AR genes, PCR screening tests should be interpreted with caution.

Crops, Agricultural↗

PubMed2005

A method for determining zygosity of transgenic zebrafish by TaqMan real-time PCR.

When producing a genetically modified organism, intended genes are often integrated into a target genome by random insertions. Subsequently, it is often desirable to know the gene copy number of the transgenic organism and the zygosity of its offspring. Because of the random insertions, the estimation can be made only by quantitative measurement of the genes. Even though TaqMan real-time PCR has been used in gene expression analysis, it is routinely used to quantify differences larger than twofold or more than one PCR cycle. In this study, we employed TaqMan quantitative PCR to determine zygosity of transgenic fluorescent zebrafish in which a homozygote and a hemizygote differ by only twofold. We measured relative quantities of the transgene by taking the threshold cycle (Ct) of both the transgene and an internal control zebrafish genomic DNA. Using scatterplots and statistical inference, we demonstrated that homozygotes and hemizygotes could be differentiated unambiguously when multiple measurements were taken. We discuss the relationship between the repetitive measurements and TaqMan precision with a statistical model. The result illustrates that the method can be extended to some areas that require even higher precision such as determining the polyploidy of an organism.

Animals↗

PubMed2000

Plant biotechnology. Italian scientists blast GMO restrictions.

While plant scientists around the world celebrate the complete sequence of the genome of the mustardlike plant Arabidopsis thaliana (see p. 2054), embattled colleagues in Italy are protesting new rules that bar all field trials involving genetically modified organisms (GMOs). The researchers hope to turn the prevailing tide by bringing their plight to the attention of colleagues around the world and exerting pressure on their government through a petition drive.

Biotechnology↗

PubMed2005

Interlaboratory transfer of a PCR multiplex method for simultaneous detection of four genetically modified maize lines: Bt11, MON810, T25, and GA21.

The number of cultured hectares and commercialized genetically modified organisms (GMOs) has increased exponentially in the past 9 years. Governments in many countries have established a policy of labeling all food and feed containing or produced by GMOs. Consequently, versatile, laboratory-transferable GMO detection methods are in increasing demand. Here, we describe a qualitative PCR-based multiplex method for simultaneous detection and identification of four genetically modified maize lines: Bt11, MON810, T25, and GA21. The described system is based on the use of five primers directed to specific sequences in these insertion events. Primers were used in a single optimized multiplex PCR reaction, and sequences of the amplified fragments are reported. The assay allows amplification of the MON810 event from the 35S promoter to the hsp intron yielding a 468 bp amplicon. Amplification of the Bt11 and T25 events from the 35S promoter to the PAT gene yielded two different amplicons of 280 and 177 bp, respectively, whereas amplification of the 5' flanking region of the GA21 gave rise to an amplicon of 72 bp. These fragments are clearly distinguishable in agarose gels and have been reproduced successfully in a different laboratory. Hence, the proposed method comprises a rapid, simple, reliable, and sensitive (down to 0.05%) PCR-based assay, suitable for detection of these four GM maize lines in a single reaction.

Base Sequence↗