Development of real-time PCR method for the detection and the quantification of a new endogenous reference gene in sugar beet “Beta vulgaris L.”: GMO application

Key message

Here, we describe a new developed quantitative real-time PCR method for the detection and quantification of a new specific endogenous reference gene used in GMO analysis.

Abstract

The key requirement of this study was the identification of a new reference gene used for the differentiation of the four genomic sections of the sugar beet (Beta vulgaris L.) (Beta, Corrollinae, Nanae and Procumbentes) suitable for quantification of genetically modified sugar beet. A specific qualitative polymerase chain reaction (PCR) assay was designed to detect the sugar beet amplifying a region of the adenylate transporter (ant) gene only from the species of the genomic section I of the genus Beta (cultivated and wild relatives) and showing negative PCR results for 7 species of the 3 other sections, 8 related species and 20 non-sugar beet plants. The sensitivity of the assay was 15 haploid genome copies (HGC). A quantitative real-time polymerase chain reaction (QRT-PCR) assay was also performed, having high linearity (R ² > 0.994) over sugar beet standard concentrations ranging from 20,000 to 10 HGC of the sugar beet DNA per PCR. The QRT-PCR assay described in this study was specific and more sensitive for sugar beet quantification compared to the validated test previously reported in the European Reference Laboratory. This assay is suitable for GMO quantification in routine analysis from a wide variety of matrices.     

Safety assessment of lepidopteran insect-protected transgenic rice with <Emphasis Type="Italic">cry2A*</Emphasis> gene

Numerous genetically modified (GM) crops expressing proteins for insect resistance have been commercialized following extensive testing demonstrating that the foods obtained from them are as safe as that obtained from their corresponding non-GM varieties. In this paper, we report the outcome of safety studies conducted on a newly developed insect-resistant GM rice expressing the cry2A* gene by a subchronic oral toxicity study on rats. GM rice and non-GM rice were incorporated into the diet at levels of 30, 50, and 70 % (w/w), No treatment-related adverse or toxic effects were observed based on an examination of the daily clinical signs, body weight, food consumption, hematology, serum biochemistry, and organ weight or based on gross and histopathological examination. These results demonstrate that the GM rice with cry2A* gene is as safe for food as conventional non-GM rice.     

实时荧光定量PCR方法快速检测转基因大豆

针对转基因大豆中普遍含有的35S启动子进行引物设计,以双链DNA染料SYBR GreenⅠ为荧光标记物,利用实时荧光定量PCR方法对大豆样品进行检测。该法检测转基因大豆的检测低限为0.005 nmol/L的35S启动子,线性范围达3个数量级,可快速区分转基因大豆和非转基因大豆,具有快速、简便、灵敏、安全、高通量、低成本等优点,可推广用于转基因植物产品的快速定量检测。     

Haemagglutinin inTriticale

Saline extracts of several varieties ofTriticale had haemagglutinin activity against rabbit, rat and fowl erythrocytes. In contrast to the wheat germ lectin theTriticale lectin was inactive against human B, 0 blood group type erythrocytes and rather high concentrations of the lectin are needed to agglutinate human A blood group type erythrocytes. TheTriticale lectin was purified about 20-fold with a 10% recovery of activity from one of the varieties (DTS 138) by (NH₄)₂SO₄ fractionation followed sequentially by chromatography on DEAE-cellulose and sulphopropyl-Sephadex. Approximately 4 μg of the purified lectin caused visible agglutination with trypsinised rabbit erythrocytes. Among a variety of sugars tested D-glucose, D-mannose and N-acetyl-D-glucosamine (2·5-7·5mM) caused inhibition of agglutination.     

Event-specific qualitative and quantitative PCR detection of the GMO carnation (Dianthus caryophyllus) variety Moonlite based upon the 5'-transgene integration sequence

To ensure the implementation of genetically modified organism (GMO)-labeling regulations, an event-specific detection method was developed based on the junction sequence of an exogenous integrant in the transgenic carnation variety Moonlite. The 5'-transgene integration sequence was isolated by thermal asymmetric interlaced PCR. Based upon the 5'-transgene integration sequence, the event-specific primers and TaqMan probe were designed to amplify the fragments, which spanned the exogenous DNA and carnation genomic DNA. Qualitative and quantitative PCR assays were developed employing the designed primers and probe. The detection limit of the qualitative PCR assay was 0.05% for Moonlite in 100 ng total carnation genomic DNA, corresponding to about 79 copies of the carnation haploid genome; the limit of detection and quantification of the quantitative PCR assay were estimated to be 38 and 190 copies of haploid carnation genomic DNA, respectively. Carnation samples with different contents of genetically modified components were quantified and the bias between the observed and true values of three samples were lower than the acceptance criterion (<25%) of the GMO detection method. These results indicated that these event-specific methods would be useful for the identification and quantification of the GMO carnation Moonlite.     

Mathematical tools to optimize the design of oligonucleotide probes and primers

The identification and quantification of specific organisms in mixed microbial communities often relies on the ability to design oligonucleotide probes and primers with high specificity and sensitivity. The design of these oligonucleotides (or “oligos” for short) shares many of the same principles in spite of their widely divergent applications. Three common molecular biology technologies that require oligonucleotide design are polymerase chain reaction (PCR), fluorescence in situ hybridization (FISH), and DNA microarrays. This article reviews techniques and software available for the design and optimization of oligos with the goal of targeting a specific group of organisms within mixed microbial communities. Strategies for enhancing specificity without compromising sensitivity are described, as well as design tools well suited for this purpose.     

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.     

Potential subchronic food safety of the stacked trait transgenic maize GH5112E-117C in Sprague-Dawley rats

The food safety of stacked trait genetically modified (GM) maize GH5112E-117C containing insect-resistance gene Cry1Ah and glyphosate-resistant gene G2-aroA was evaluated in comparison to non-GM Hi-II maize fed to Sprague-Dawley rats during a 90-day subchronic feeding study. Three different dietary concentrations (12.5, 25 and 50 %, w/w) of the GM maize were used or its corresponding non-GM maize. No biologically significant differences in the animals’ clinical signs, body weights, food consumption, hematology, clinical chemistry, organ weights and histopathology were found between the stacked trait GM maize groups, and the non-GM maize groups. The results of the 90-day subchronic feeding study demonstrated that the stacked trait GM maize GH5112E-117C is as safe as the conventional non-GM maize Hi-II.     

Assessment of Real-time PCR Based Methods for Quantification of Pollen-mediated Gene Flow from GM to Conventional Maize in a Field Study

Maize is one of the main crops worldwide and an increasing number of genetically modified (GM) maize varieties are cultivated and commercialized in many countries in parallel to conventional crops. Given the labeling rules established e.g. in the European Union and the necessary coexistence between GM and non-GM crops, it is important to determine the extent of pollen dissemination from transgenic maize to other cultivars under field conditions. The most widely used methods for quantitative detection of GMO are based on real-time PCR, which implies the results are expressed in genome percentages (in contrast to seed or grain percentages). Our objective was to assess the accuracy of real-time PCR based assays to accurately quantify the contents of transgenic grains in non-GM fields in comparison with the real cross-fertilization rate as determined by phenotypical analysis. We performed this study in a region where both GM and conventional maize are normally cultivated and used the predominant transgenic maize Mon810 in combination with a conventional maize variety which displays the characteristic of white grains (therefore allowing cross-pollination quantification as percentage of yellow grains). Our results indicated an excellent correlation between real-time PCR results and number of cross-fertilized grains at Mon810 levels of 0.1–10%. In contrast, Mon810 percentage estimated by weight of grains produced less accurate results. Finally, we present and discuss the pattern of pollen-mediated gene flow from GM to conventional maize in an example case under field conditions.     

Effect of volunteers on maize gene flow

Regulatory approvals for deliberate release of GM maize events into the environment have lead to real situations of coexistence between GM and non-GM, with some fields being cultivated with GM and conventional varieties in successive seasons. Given the common presence of volunteer plants in maize fields in temperate areas, we investigated the real impact of GM volunteers on the yield of 12 non-GM agricultural fields. Volunteer density varied from residual to around 10% of plants in the field and was largely reduced using certain cultural practices. Plant vigour was low, they rarely had cobs and produced pollen that cross-fertilized neighbour plants only at low—but variable—levels. In the worst-case scenario, the estimated content of GMO was 0.16%. The influence of GM volunteers was not enough to reach the 0.9% adventitious GM threshold but it could potentially contribute to adventitious GM levels, especially at high initial densities (i.e. above 1,000 volunteers/ha).     

Development of a SCAR marker for molecular detection and diagnosis of Tilletia controversa Kühn,the causal fungus of wheat dwarf bunt

Tilletia controversa Kühn (TCK) is an important quarantine pathogen that causes wheat dwarf bunt and results in devastating damage to wheat production. The fungus is difficult to be distinguished from T. caries and T. laevis, which cause wheat common bunt, based on morphological, physiological and symptomatological characteristics of the pathogens. The traditional detection of the fungus can be a long and tedious process with poor accuracy. The inter-simple sequence repeat (ISSR) technique has been used for identifying molecular markers for detection of TCK. Of 28 ISSR primers screened, ISSR-859 amplified a specific 678 bp DNA fragment from all TCK isolates but not from any isolates of the common bunt fungi or other pathogenic fungi tested. Based on the fragment sequence, a pair of sequence characterized amplified region (SCAR) primers was designed, which amplified a 372 bp DNA fragment specifically in TCK. The SCAR marker was detected using as low as 1 ng template DNA of TCK, and was also detected using broken teliospores and DNA from asymptomatic wheat samples. We developed the SYBR Green I and TaqMan Green I and TaqMan real-time polymorphism chain reaction methods to detect TCK with the detection limit of 0.1 fg with asymptomatic wheat samples. Further work is needed to develop a rapid test kit for this pathogenic fungus using the designed specific primers.     

Qualitative and quantitative analysis of genetically modified pepper

For the development of qualitative and quantitative PCR methods of genetically modified (GM) pepper developed in Korea, a capsanthin-capsorubin synthase (CCS) gene was used as the endogenous reference gene. The primer pair ccs-F/R amplifying the pepper endogenous gene gave rise to an amplicon of 102 bp. No amplified product was observed when DNA samples from 16 different plants were used as templates. The construct-specific primer pairs amplifying the junction region of the bar gene and Ti7 introduced in GM pepper gave rise to an amplicon of 182 bp. Quantitative PCR assay was performed using a TaqMan probe and a standard plasmid as a reference molecule, which contained both an endogenous and event-specific sequence. For the validation of this method, the test samples containing 0.1, 1, 3, 5, and 10% GM pepper were quantified.     

Comparison of transgenic <Emphasis Type="Italic">Gerbera hybrida</Emphasis> lines and traditional varieties shows no differences in cytotoxicity or metabolic fingerprints

Genetic modification using gene transfer (GM) is still controversial when applied to plant breeding at least in Europe. One major concern is how GM affects other genes and thus the metabolism of the plant. In this study, 225 genetically modified lines of the ornamental plant Gerbera hybrida and 42 non-GM gerbera varieties were used to investigate changes in secondary metabolism. The cytotoxicity of GM and non-GM gerbera extracts was evaluated on human cell lines derived from lung, liver, and intestinal tissues. The results indicate that the safety profile for GM gerbera lines is similar to the viability pattern for non-GM varieties-none of the extracts were toxic. In addition, metabolic fingerprints of gerbera extracts were identified using thin-layer chromatography and analysed by principal component analysis (PCA), the nearest neighbour classifier, and Fligner-Killeen test. No new compounds unique to GM lines were observed. With PCA, no separation between GM gerbera lines and varieties could be demonstrated. In the nearest neighbour classifier, 54% of the samples found the expected neighbour based on the gene constructs used for transformation. With Fligner-Killeen test, we studied if the amounts of compounds vary more in GM gerberas than in varieties. In most cases, there were no statistically significant differences between the varieties and GM lines or there was more variation among the non-GM varieties than in the GM lines. The variance of a single compound was significantly larger in transgenic gerbera lines than in varieties and of three compounds in non-GM varieties.     

Detection of gene flow from GM to non-GM watermelon in a field trial

Gene flow from genetically modified (GM) crops to conventional non-GM crops is a serious concern for protection of conventional and organic farming. Gene flow from GM watermelon developed for rootstock use, containing cucumber green mottle mosaic virus (CGMMV)-coat protein (CP) gene, to a non-GM isogenic control variety “Clhalteok” and grafted watermelon “Keumcheon” was investigated in a small scale field trial as a pilot study. Hybrids between GM and non-GM watermelons were screened from 1304 “Chalteok” seeds and 856 “Keumcheon” seeds using the duplex PCR method targeting theCGMMV- CP gene as a marker. Hybrids were found in all pollen recipient plots. The gene flow frequencies were greater for “Chaiteok” than for “KeumcheonD; with 75% outcrossing in the “Chaiteok” plot at the closest distance (0.8 m) to the GM plot. A much larger scale field trial is necessary to identify the isolation distance between GM and non-GM watermelon, as the behaviors of insect pollinators needs to be clarified in Korea.     

Comparison of tuber proteomes of potato varieties, landraces, and genetically modified lines

Crop improvement by genetic modification remains controversial, one of the major issues being the potential for unintended effects. Comparative safety assessment includes targeted analysis of key nutrients and antinutritional factors, but broader scale-profiling or "omics" methods could increase the chances of detecting unintended effects. Comparative assessment should consider the extent of natural variation and not simply compare genetically modified (GM) lines and parental controls. In this study, potato (Solanum tuberosum) proteome diversity has been assessed using a range of diverse non-GM germplasm. In addition, a selection of GM potato lines was compared to assess the potential for unintended differences in protein profiles. Clear qualitative and quantitative differences were found in the protein patterns of the varieties and landraces examined, with 1,077 of 1,111 protein spots analyzed showing statistically significant differences. The diploid species Solanum phureja could be clearly differentiated from tetraploid (Solanum tuberosum) genotypes. Many of the proteins apparently contributing to genotype differentiation are involved in disease and defense responses, the glycolytic pathway, and sugar metabolism or protein targeting/storage. Only nine proteins out of 730 showed significant differences between GM lines and their controls. There was much less variation between GM lines and their non-GM controls compared with that found between different varieties and landraces. A number of proteins were identified by mass spectrometry and added to a potato tuber two-dimensional protein map.     

Quantification of genetically modified soybeans using a combination of a capillary-type real-time PCR system and a plasmid reference standard

Because the labeling of grains and feed- and foodstuffs is mandatory if the genetically modified organism (GMO) content exceeds a certain level of approved genetically modified varieties in many countries, there is a need for a rapid and useful method of GMO quantification in food samples. In this study, a rapid detection system was developed for Roundup Ready Soybean (RRS) quantification using a combination of a capillary-type real-time PCR system, a LightCycler real-time PCR system, and plasmid DNA as the reference standard. In addition, we showed for the first time that the plasmid and genomic DNA should be similar in the established detection system because the PCR efficiencies of using plasmid DNA and using genomic DNA were not significantly different. The conversion factor (Cf) to calculate RRS content (%) was further determined from the average value analyzed in three laboratories. The accuracy and reproducibility of this system for RRS quantification at a level of 5.0% were within a range from 4.46 to 5.07% for RRS content and within a range from 2.0% to 7.0% for the relative standard deviation (RSD) value, respectively. This system rapidly monitored the labeling system and had allowable levels of accuracy and precision.     

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.     

Regulatory challenges for GM crops in developing economies: the African experience

Globally, transgenic or genetically modified (GM) crops are considered regulated products that are subject to regulatory oversight during trans-boundary movement, testing and environmental release. In Africa, regulations for transgenic crops are based on the outcomes of the historic Earth Summit Conference held in Rio, Brazil two decades ago, namely, the adoption of the Convention on Biological Diversity (CBD) and the subsequent adoption of the Cartagena Protocol on Biosafety. To exploit the potential benefits of transgenic crops while safeguarding the potential risks on human health and environment, most African countries have signed and ratified the CBD and the Cartagena Protocol on Biosafety. Consequently, these countries are required to take appropriate legal, administrative and other measures to ensure that the handling and utilization of living modified organisms are undertaken in a manner that reduces the risks to humans and the environment. These countries are also expected to provide regulatory oversight on transgenic crops through functional national biosafety frameworks (NBFs). While in principle this approach is ideal, NBFs in most African countries are steeped in a host of policy, legal and operational challenges that appear to be at cross-purposes with the noble efforts of seeking to access, test and deliver promising GM crops for use by resource-limited farmers in Africa. In this paper we discuss the regulatory challenges faced during the development and commercialization of GM crops based on experiences from countries in Sub-Saharan Africa.     

Potential use of an arthropod database to support the non-target risk assessment and monitoring of transgenic plants

Worldwide, plants obtained through genetic modification are subject to a risk analysis and regulatory approval before they can enter the market. An area of concern addressed in environmental risk assessments is the potential of genetically modified (GM) plants to adversely affect non-target arthropods and the valued ecosystem services they provide. Environmental risk assessments are conducted case-by-case for each GM plant taking into account the plant species, its trait(s), the receiving environments into which the GM plant is to be released and its intended uses, and the combination of these characteristics. To facilitate the non-target risk assessment of GM plants, information on arthropods found in relevant agro-ecosystems in Europe has been compiled in a publicly available database of bio-ecological information during a project commissioned by the European Food Safety Authority (EFSA). Using different hypothetical GM maize case studies, we demonstrate how the information contained in the database can assist in identifying valued species that may be at risk and in selecting suitable species for laboratory testing, higher-tier studies, as well as post-market environmental monitoring.