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PubMed2004

A comparative study of the allergenic potency of wild-type and glyphosate-tolerant gene-modified soybean cultivars.

A large proportion of soybean cultivars grown in the USA are now genetically modified varieties and concern has been raised about the safety of these products for consumers. A study of the impact on allergenic potency in soybeans, comparable except for the newly introduced gene (CP4 EPSPS), was performed using soybean-sensitized patients. The allergenicity of 18 different (10 GM and 8 WT) soybean extracts was examined blindly by the following three methods: A) Sera from patients with specific IgE against soybean were used to determine concentrations inducing 50% RAST inhibition; B) Histamine release induced by the extracts was examined using blood from sensitized patients; C) SPT was performed on sensitized patients with all 18 extracts. All three methods showed variations in the allergenic potency between the individual extracts but allergenic potential was not affected by presence of the transgene. By using standard in vitro methods and SPT for determination of allergenicity we were not able to detect any significant difference in the allergenic potency between GM and WT soybeans.

Food Hypersensitivity↗
PubMedDate not supplied

Production of certified reference materials for the detection of genetically modified organisms.

Certified reference materials (CRMs) are an essenIial tool in the quality assurance of analytical measurements. They are produced, certified, and used in accordance with relevant ISO (International Organization for Standardization) and BCR (Community Bureau of Reference) guidelines. The Institute for Reference Materials and Measurements (IRMM; Geel, Belgium) has produced the first powdery genetically modified organism (GMO) CRMs in cooperation with the Institute for Health and Consumer Protection (Ispra, Italy). Until now, different weight percentages in the range of 0-5% for 4 GMOs in Europe were produced and certified: Bt (Bacillus thuringiensis)-11 and Bt-176 maize, Roundup Ready soybean, and MON810 maize. Bt-11 and Bt-176 maize and Roundup Ready soybean were produced by IRMM on behalf of Fluka Chemie AG (Buchs, Switzerland). Characterization of used base material is the first step in production and is especially important for GMO CRMs. The production of powdery GMO CRMs and methods used for production control are described. Thorough control of homogeneity and stability are essential for certification of reference materials and ensure validity of the certificate for each bottle of a batch throughout a defined shelf-life. Because production of reference materials and their maintenance are very labor- and cost-intensive tasks, the usefulness of new types of GMO CRMs must be estimated carefully.

DNA, Plant↗
PubMed2003

[Laboratory-performance study of the notified methods to detect genetically modified maize (CBH351) and potato (NewLeaf Plus and NewLeaf Y)].

To investigate the key factors affecting the reliability of the analytical results, a laboratory-performance study was attempted for the notified methods to detect genetically modified (GM) maize (CBH351) and GM potato (NewLeaf Plus and NewLeaf Y). The test samples were designed as three pairs of blind duplicates, which included 0%, 0.1% and 1.0% GM maize (CBH351) or GM potato (NewLeaf Plus or NewLeaf Y). Fourteen laboratories participated in the study. The test samples were sent to the participating laboratories along with the protocol. The data were collected from all laboratories and statistically analyzed. For the 0% sample of the CBH351 maize, one laboratory reported a false-positive result. It was considered that contamination could have occurred via the common use of equipment or tools for the test. For the 0.1% samples of the NewLeaf Plus potato or NewLeaf Y potato, on the other hand, three laboratories reported false-negative results. It was presumed that these results were due to changes of the conditions of the electrophoresis and agarose-gel staining. The other laboratories reported appropriate results. It was considered that the method employed in this study was suitable for the assessment of laboratory performance.

DNA, Plant↗
PubMed2004

A histochemical method using a substrate of beta-glucuronidase for detection of genetically modified papaya.

A histochemical assay for detecting genetically modified (GM) papaya (derived from Line 55-1) is described. GM papaya, currently undergoing a safety assessment in Japan, was developed using a construct that included a beta-glucuronidase (GUS) reporter gene linked to a virus coat protein (CP) gene. Histochemical assay was used to visualize the blue GUS reaction product from transgenic seed embryos. Twelve embryos per fruit were extracted from the papaya seeds using a surgical knife. The embryos were incubated with the substrate 5-bromo-4-chloro-3-indolyl-beta-D-glucuronide (X-Gluc) in a 96-well microtiter plate for 10-15 hours at 37 degrees C. Seventy-five percent of GM papaya embryos should turn blue theoretically. The histochemical assay results were completely consistent with those from a qualitative polymerase chain reaction (PCR) method developed by this laboratory. Furthermore, the method was validated in a five-laboratory study. The method for detection of GM papaya is rapid and simple, and does not require use of specialized equipment.

Carica↗
PubMed2005

Germ-line enhancement of humans and non-humans.

The current difference in attitude toward germ-line enhancement in humans and nonhumans is unjustified. Society should be more cautious in modifying the genes of nonhumans and more bold in thinking about modifying our own genome. I identify four classes of arguments pertaining to germ-line enhancement: safety arguments, justice arguments, trust arguments, and naturalness arguments. The first three types are compelling, but do not distinguish between human and nonhuman cases. The final class of argument would justify a distinction between human and nonhuman germ-line enhancement; however, this type of argument fails and, therefore, the discrepancy in attitude toward human and nonhuman germ-line enhancement is unjustified.

Commodification↗
PubMed2006

Detection method for genetically modified papaya using duplex PCR.

A simple and rapid method for the identification of genetically modified (GM) papaya, derived from Line 55-1, was developed by modifying the Japanese official PCR method. Genomic DNA was directly extracted from the fresh fruit without the lyophilization step, using a commercial silica-based kit. To develop a duplex PCR method which simultaneously detects the GM papaya-specific gene and the intrinsic papain gene, the papain 2-5'/3' (amplicon size; 184 bp) primer pair for the detection of the papain gene was newly designed within the region of the products (211 bp) amplified using the papain 1-5'/-3' primer pair adopted in the Japanese official PCR method. To detect the GM papaya-specific gene, the primer pair Nos C-5'/CaM N-3' described in the Japanese official method was used. The DNA sequences of the GM papaya gene and the intrinsic papain gene were co-amplified using the PCR method in a single tube. The developed duplex PCR method allows the simultaneous detection of the products by means of agarose gel electrophoresis or microchip electrophoresis. The proposed method for GM papaya identification is simple and rapid.

Carica↗
PubMed2005

[Laboratory performance study of the quantitative detection method for genetically modified soybeans (roundup ready soybeans 40-3-2)].

To investigate important factors affecting the analytical results, a laboratory-performance study was attempted for the Japanese official methods to detect genetically modified (GM) soybeans (40-3-2). Test samples containing 0, 1 and 5% GM soya powder in non-GM soya powder was prepared. A set of 3 test samples was sent to the participating laboratories along with the protocol. The data were collected from all laboratories and statistically analyzed. In the real-time PCR detection method, the average values of the GM 1% and 5% samples were both much lower than the spiked value because the laboratories using a silica-membrane DNA extraction method underestimated the GM value. On the other hand, the laboratories using other extraction methods, such as the CTAB method obtained values close to the spiked value. These results suggest that use of the silica-membrane DNA extraction method may result in underestimation of the GM content in the real-time PCR method. In the ELISA method, the average value of 5% spiked samples appears to be slightly higher than the fortified value. But, overall, it was considered that reported values were close to the spiked level.

DNA, Plant↗
PubMed2001

[Detection of recombinant DNA from genetically modified papaya].

A method using polymerase chain reaction (PCR) was developed to detect the genetically modified (GM) papaya (55-1 line), of which the mandatory safety assessment has not been finished in Japan because of insufficient data. The papaya intrinsic papain gene was used as an internal control. The results of PCR amplification of the papain gene segment indicated that a commercial silica membrane type kit (QIAGEN DNeasy plant mini) was useful for extraction of DNA from papaya fruit, but not for extraction from canned papaya fruit. On the other hand, a commercial ion-exchange type kit (QIAGEN Genomic-tip) provided enough purified DNA for PCR from canned papaya fruit. Compared with the parental line and other commercial non-GM papayas, the DNA from GM papaya fruit provided specific amplification bands in PCR with five primer pairs (Nos. 2-6) including beta-glucuronidase and neomycin phosphotransferase II gene-specific ones. On the other hand, the primer pairs recognizing these genes showed false-positive results when we used DNAs from canned papaya. Therefore, we recommend that the primer pairs (Nos. 5 and 6) recognizing the sequences derived from two different species of organism should be used in order to detect specifically the GM papaya in canned fruits.

Carica↗
PubMed2004

Assessing the survival of transgenic plant DNA in the human gastrointestinal tract.

The inclusion of genetically modified (GM) plants in the human diet has raised concerns about the possible transfer of transgenes from GM plants to intestinal microflora and enterocytes. The persistence in the human gut of DNA from dietary GM plants is unknown. Here we study the survival of the transgene epsps from GM soya in the small intestine of human ileostomists (i.e., individuals in which the terminal ileum is resected and digesta are diverted from the body via a stoma to a colostomy bag). The amount of transgene that survived passage through the small bowel varied among individuals, with a maximum of 3.7% recovered at the stoma of one individual. The transgene did not survive passage through the intact gastrointestinal tract of human subjects fed GM soya. Three of seven ileostomists showed evidence of low-frequency gene transfer from GM soya to the microflora of the small bowel before their involvement in these experiments. As this low level of epsps in the intestinal microflora did not increase after consumption of the meal containing GM soya, we conclude that gene transfer did not occur during the feeding experiment.

Base Sequence↗
PubMedDate not supplied

Assessing the allergenicity of proteins introduced into genetically modified crops using specific human IgE assays.

Global commercial production of genetically modified (GM) crops has grown to over 67 million hectares annually, primarily of herbicide-tolerant and insect protection crop varieties. GM crops are produced by the insertion of specific genes that either encode a protein, or a regulatory RNA sequence. A comprehensive safety evaluation is conducted for each new commercial GM crop, including an assessment of the potential allergenicity of any newly introduced protein. If the gene was derived from an allergenic organism, or the protein sequence is highly similar to a known allergen, immunoassays, e.g., Western blot assays and enzyme-linked immunosorbent assay tests, are performed to identify protein-specific IgE binding by sera of individuals allergic to the gene source, or the source of the sequence-matched allergen. Although such assays are commonly used to identify previously unknown allergens, criteria have not been established to demonstrate that a protein is unlikely to cause allergic reactions. This review discusses factors that affect the predictive value of these tests, including clinical selection criteria for serum donors, selection of blocking reagents to reduce nonspecific antibody binding, inhibition assays to verify specificity of binding, and scientifically justified limits of detection (sensitivity) in the absence of information regarding biological thresholds.

Allergens↗
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↗
PubMed2002

[Study on the identification of genomic modified foods].

Nucleotide-based amplification method is an important system for the identification of genomic modified foods (GMF). Roundup Ready Soybeans (Monsanto company), Bt 176 GM maize (Novartis/Ciba-Geigy company) and Cecropin D capsicum was used as material to search for the feasibility of investigating the safety of GMF by PCR method. Primers specific for inserted genes and crop endogenous genes in Roundup Ready Soybeans, Bt 176 maize and Cecropin D capsicum were applied. The discrimination system for GM soybeans, GM maize and GM capsicum from the counterpart of non-GM products and the detection system for correlating marker genes and transgenes are established. The method was easy and fast, and the corresponding results fixed the standard or declared data.

Capsicum↗
PubMed2005

Preparation of monoclonal antibody against HPT and its application to detecting marker protein in genetically modified rice.

OBJECTIVE: To produce the monoclonal antibodies (mAbs) against hygromycin B phosphotransferase (HPT) and to develop immunoassay based on mAbs for biosafety assessment of HPT in genetically modified rice (GM rice). METHODS: BALB/c mice were immunized with purified recombinant 6His. HPT protein, and the conventional hybridoma technology was used to generate the monoclonal hybridoma cells. ELISA and Western blot were used to analyze the specificity of mAbs recognizing HPT and the cross reaction with other proteins. A double-Ab sandwich ELISA method was established to detect HPT expression level in the sck gene-modified rice plants. RESULTS: Four hybridomas, named F1, D4-2, D4-4, and D4-5, producing the mAbs against HPT were successfully obtained with the titer of ascetic mAbs ranging from 1x10(-4) to 1x10(-5). Identification of subclass showed that all the produced mAbs belonged to IgG1. Western blot showed specific binding reaction between the mAbs to the HPT proteins expressed in the GM rice. A double sandwich ELISA coated with anti-HPT polyclonal antibody was established with mAbs as sandwich antibody, which showed a sensitivity of 30ng/mL and did not crossreact with other proteins. The expression level of HPT in the leaves of sck-transformed lines was detected (80-150ng/mL). But HPT protein in the grain and seed of GM rice could not be detected using this ELISA assay. CONCLUSION: Anti-HPT mAbs prepared herein have a high specificity and can be used for rapid assay of HPT antigen. The expression level of HPT in the GM rice grain and seed is lower than our ELISA detection limit.

Animals↗
PubMed2001

PCR-based detection of genetically modified soybean and maize in raw and highly processed foodstuffs.

The PCR method has proved to be an invaluable tool for the specific and sensitive detection of genetically modified material (e.g., Roundup Ready Soybean and Bt-176 "Maximizer" Maize) in foodstuffs. The first step in the procedure, namely the purification of nucleic acids from the sample, is often the deciding factor in the production of meaningful results. In this study, we present two procedures that enable an efficient isolation of trace amounts of genetic material from both raw and highly processed foodstuffs. We show that for optimal, PCR-ready DNA purification from highly processed foodstuffs and PCR inhibitor-rich substances--such as cocoa-containing products--adapted protocols for the QIAGEN QIAamp DNA Stool Mini Kit can be utilized. For complete DNA isolation from raw foodstuffs, a protocol using the DNeasy Plant Mini Kit is presented.

DNA Primers↗
PubMed2004

Elicitation of expert judgments of uncertainty in the risk assessment of herbicide-tolerant oilseed crops.

One of the lay public's concerns about genetically modified (GM) organisms (GMO) and related emerging technologies is that not all the important risks are evaluated or even identified yet--and that ignorance of the unanticipated risks could lead to severe environmental or public health consequences. To some degree, even the scientists who participated in the analysis of the risks from GMOs (arguably the people most qualified to critique these analyses) share some of this concern. To formally explore the uncertainty in the risk assessment of a GM crop, we conducted detailed interviews of seven leading experts on GM oilseed crops to obtain qualitative and quantitative information on their understanding of the uncertainties associated with the risks to agriculture from GM oilseed crops (canola or rapeseed). The results of these elicitations revealed three issues of potential concern that are currently left outside the scope of risk assessments. These are (1) the potential loss of the agronomic and environmental benefits of glyphosate (a herbicide widely used in no-till agriculture) due to the combined problems of glyphosate-tolerant canola and wheat volunteer plants, (2) the growing problem of seed lot contamination, and (3) the potential market impacts. The elicitations also identified two areas where knowledge is insufficient. These are: the occurrence of hybridization between canola and wild relatives and the ability of the hybrids to perpetuate themselves in nature, and the fate of the herbicide-tolerance genes in soil and their interaction with soil microfauna and -flora. The methodological contribution of this work is a formal approach to analyzing the uncertainty surrounding complex problems.

Agriculture↗
PubMed2003

Safety assessment of recombinant green fluorescent protein orally administered to weaned rats.

Several proposed biotechnological applications of green fluorescent protein (GFP) are likely to result in its introduction into the food supply of domestic animals and humans. We fed pure GFP and diets containing transgenic canola expressing GFP to young male rats for 26 d to evaluate the potential toxicity and allergenicity of GFP. Animals (n = 8 per group) were fed either AIN-93G (control), control diet plus 1.0 mg of purified GFP daily, modified control diet with 200 g/kg canola (Brassica rapa cv Westar), or control diet with 200 g/kg transgenic canola containing one of two levels of GFP. Ingestion of GFP did not affect growth, food intake, relative weight of intestine or other organs, or activities of hepatic enzymes in serum. Comparison of the amino acid sequence of GFP to known food allergens revealed that the greatest number of consecutive amino acid matches between GFP and any food allergen was four, suggesting the absence of common allergen epitopes. Moreover, GFP was rapidly degraded during simulated gastric digestion. These data indicate that GFP is a low allergenicity risk and provide preliminary indications that GFP is not likely to represent a health risk.

Administration, Oral↗
PubMedDate not supplied

A conceptual framework for the design of environmental post-market monitoring of genetically modified plants.

Genetically modified plants (GMPs) may soon be cultivated commercially in several member countries of the European Union (EU). According to EU Directive 2001/18/EC, post-market monitoring (PMM) for commercial GMP cultivation must be implemented, in order to detect and prevent adverse effects on human health and the environment. However, no general PMM strategies for GMP cultivation have been established so far. We present a conceptual framework for the design of environmental PMM for GMP cultivation based on current EU legislation and common risk analysis procedures. We have established a comprehensive structure of the GMP approval process, consisting of pre-market risk assessment (PMRA) as well as PMM. Both programs can be distinguished conceptually due to principles inherent to risk analysis procedures. The design of PMM programs should take into account the knowledge gained during approval for commercialization of a specific GMP and the decisions made in the environmental risk assessments (ERAs). PMM is composed of case-specific monitoring (CSM) and general surveillance. CSM focuses on anticipated effects of a specific GMP. Selection of case-specific indicators for detection of ecological exposure and effects, as well as definition of effect sizes, are important for CSM. General surveillance is designed to detect unanticipated effects on general safeguard subjects, such as natural resources, which must not be adversely affected by human activities like GMP cultivation. We have identified clear conceptual differences between CSM and general surveillance, and propose to adopt separate frameworks when developing either of the two programs. Common to both programs is the need to put a value on possible ecological effects of GMP cultivation. The structure of PMM presented here will be of assistance to industry, researchers, and regulators, when assessing GMPs during commercialization.

Consumer Product Safety↗
PubMed2003

The 5-enolpyruvylshikimate-3-phosphate synthase of glyphosate-tolerant soybean expressed in Escherichia coli shows no severe allergenicity.

The recombinant gene was amplified from the chromosomal DNA of genetically-modified (GM) soybeans and identified as epsps encoding 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) which renders glyphosate resistance. The epsps structural gene was introduced in the pET28(a) plasmid for its expression in Escherichia coli BL21(DE3). It was confirmed that the maximal productivity of the EPSPS protein was achieved when cultivating the recombinant strain in a LB broth for 2 h after supplementing 1 mM isopropylbeta-D-thiogalactopyranoside (IPTG) in a 2 h-culture broth. Since the expressed EPSPS protein was found as an insoluble form in the inclusion body, it was extracted by 6 M urea after sonication, and then purified through immobilized nickel-affinity column chromatography to isolate EPSPS having a molecular mass of 57 kDa. When incubated in simulated gastric fluid containing pepsin at pH 1.5, the purified EPSPS protein was completely digested within 1 min. In addition, the passive cutaneous anaphylaxis reaction of the purified EPSPS protein was not observed in the Sprague Dawley rat system that was administered either orally or subcutaneously. Furthermore, treatment of the EPSPS protein to the culture of the sensitized peritoneal mast cells, or unsensitized but antisera-labeled mast cells, showed neither a remarkable change in the histamine release nor a cytokine production, including interleukin-4 (IL-4) and tumor necrosis factor-alpha (TNF-alpha). Thus, it can be concluded that the EPSPS protein in the GM soybean showed no significant allergenicity in the Sprague Dawley rats.

3-Phosphoshikimate 1-Carboxyvinyltransferase↗