Genetically Modified Organisms Content Research Articles

Simple, Precise, and Less Biased GMO Quantification by Multiplexed Genetic Element-Specific Digital PCR.

To provide the consumer with choices of genetically modified organisms (GMO) or non-GMO, official food labeling systems were established in many countries. Because the threshold GMO content values were set to distinguish between "non-GMO" and "GMO" designations, GMO content quantification methods are required for ensuring the appropriateness of labeling. As the number of GMOs is continuously increasing around the world, we set out to develop a low-cost, simple and less biased analytical strategy to cover all necessary detection targets. Digital PCR methods are advantageous compared to the conventional quantitative real-time PCR methods. We developed a digital PCR-based GMO quantification method to evaluate the GMO content in maize grains. To minimize the analytical workload, we adopted multiplex digital PCR targeting the 35S promoter and the nopaline synthase terminator, which are genetic elements commonly introduced in many GMOs. Our method is significantly simpler and more precise than the conventional real-time PCR-based methods. Additionally, we found that this method enables quantification of the copy number of GMO DNA without double counting multiple elements (35S promoter and nopaline synthase terminator) tandemly placed in a recombinant DNA construct. This is the first report on the development of a genetically modified maize quantification method using a multiplexed genetic element-specific digital PCR method. The tandem effect we report here is quite useful for reducing the bias in the analytical results. Multiplexed genetic element-specific digital PCR can simplify weight-based GMO quantification and thus should prove useful in light of the continuous increase in the number of GM events.

GmoDetector: An accurate and efficient GMO identification approach and its applications

The rapid increase of genetically modified organisms (GMOs) entering the food and feed markets, and the contamination of donor (micro)organisms of transgenic elements make it more challenging for the existing GMO detection. In this study, we developed a high-throughput and contamination-removal GMO detection approach named as GmoDetector. GmoDetector targeted 64 common transgenic elements and 76 GMO-specific events collected from 251 singular GM events, and combined with next generation sequencing (NGS) and target enrichment technology to detect various GMOs. As a result, GmoDetector was able to exclude the donor (micro)organism contamination, and detect the authorized and unauthorized GMOs (UGMOs) in any forms of food or feed, such as processed or unprocessed. The sensitivity of GmoDetector is as low as 0.1% (GMO content), which has met the GMO labeling threshold for all countries. Therefore, GmoDetector is a robust tool for accurate and efficient detection of the authorized and UGMOs.

Development and assessment of a duplex droplet digital PCR method for quantification of GM rice Kemingdao.

The implementation of genetically modified organism (GMO) labeling policies requires accurate quantitative methods to measure the GMO content in test samples. A Kemingdao/phospholipase D (KMD/PLD) duplex ddPCR method was established with rice genomic DNA (gDNA) of homozygous KMD as template by optimizing the annealing temperature and cycle number. Duplex ddPCR showed a linear response over the dynamic range from 68 to 175,000 copies, covering four orders of magnitude. The limit of detection (LOD) and limit of quantification (LOQ) for duplex ddPCR were determined to be 9 copies and 34 copies of the rice haploid genome, respectively. A very high dilution factor would result in unacceptable bias and coefficients of variation for determining copy number of the gDNA solution, and more than 1000 copies of the DNA template in one reaction is preferred to obtain accurate quantitative results by duplex PCR. Five blinded DNA samples with copy number ratio of 10%, 5%, 1%, 0.1%, and 0.05%, and three blinded real-life matrix samples with mass fraction of 5%, 1%, and 0.5% were quantified by duplex ddPCR, simplex ddPCR, and qPCR. These three methods all gave comparable GMO content and copy numbers within the required precision, but the duplex ddPCR showed the narrowest uncertainty interval and provided the highest precision in comparison to simplex ddPCR and qPCR. The ddPCR is a more appealing and reliable technology for the accurate quantification of GMO content than simplex ddPCR and qPCR considering the uncertainty and precision of quantitative results, the time consumption of generating droplets, and the cost of ddPCR reagents.

Perceptions and Attitudes of University Students of Five Mexican Public Institutions on the Labeling of Products Made with Genetically Modified Organisms

Introduction: To know the perceptions and attitudes about the labeling of products made with genetically modified organisms (GMO), of students from five universities in central Mexico, in the following areas of knowledge: natural sciences, health sciences, chemical sciences, economic and administrative sciences and biological and agricultural sciences. Methodological design and approach: In the study, 2923 students answered a questionnaire with Likert scale of five questions. It is important to mention that the original questionnaire has 64 variables and 11 factors to measure the perceptions, attitudes, benefits and risks on the production and consumption of GMO. Results: Of the total sample 16.60% always read the labels, 85.70% agree that the label should show the characteristics of the product. 82.90% said that when making promotions should be reported on the label the characteristics of the product and finally 82.90% answered that the Mexican government should legislate a legal framework to regulate the mandatory labeling of products made with GMOs. Discussion or Conclusion. The sample only cover 5 Mexican universities. The relevant contribution of this research provides an empirical knowledge about the perceptions and attitudes regarding the labeling of products with GMOs content from a sample of the population of five public universities in central Mexico. This study is an important contribution for Mexican society, because there is no legal framework to regulate the labeling of products with GMO content and that it is essential to strengthen the training of university students with scientific information in this field. The labeling of products made with GMOs is generating relevant controversies worldwide, so it is important to know and spread the perceptions and attitudes of the sample considered in this study.

A novel quantitative technique in detecting stacked genetically modified plants by fluorescent-immunohistochemistry

Stacked genetically modified organisms (GMOs) are gaining popularity for their ability to enhance production efficiency and improve functional properties. Due to different labeling requirements across many countries, the detection and quantification of stacked GMO content is an important concern. Although existing methods can detect pure stacked GMOs, they are considerably less efficient at identifying material that contains the stacked GMO and its single-trait “parent” GM lines. Immunohistochemistry possesses superior specificity and sensitivity in detecting the target, especially proteins. As a result, a novel quantitative technique to detect stacked GMO was developed. Two fluorescent antibodies hybridized with two specific proteins, which were expressed by the single-trait “parents” separately, were employed. By observing whether both of the two fluorescent signals existed in the same cell, the stacked GMO could be easily detected. By calculating the fluorescent signals, quantification could be determined. This method reduces errors and the non-specificity caused by PCR-based methods, and is widely applicable in the quantitative and qualitative detection of stacked GMOs. In sum, it represents a new method for high throughput detection.

Zea maize reference materials for genetically modified organism detection in Mexico.

Zea maize reference materials for genetically modified organism detection in Mexico.

Rapid detection of P–35S and T-nos in genetically modified organisms by recombinase polymerase amplification combined with a lateral flow strip

Recombinase polymerase amplification (RPA) was first reported in 2006 and has recently been shown to improve the detection of genetically modified organisms (GMOs). This study developed a more efficient RPA-based and lateral flow test strip (LFD)-based platform for the detection of P–35S and T-nos for GMOs, circumventing the need for expensive instruments and technicians. Other methods were compared to our assay. The RPA forward and reverse primers were labeled with the fluorophore biotin, digoxin, and FITC at the 5′ end and quickly determined whether the sample contains P–35S and T-nos of GMOs using the RPA-LFD method at room temperature. Nine events of GMOs were collected to determine the specificity of the RPA-LFD method, including KMD1, MON531, Rf1, Kefeng6, RRS, Bt11, MON863, Bt176, and Ms1. The results showed that the limit of detection of RPA-LFD was 50 copies and 100 copies, which was consistent with the limit of detection of RPA-AGE. The specificity and stability among the nine events were consistent, respectively. Finally, the perfect fitness for the detection of the nine double-blind samples indicated that the detection method is practical. In conclusion, we developed a more sensitive, specific, and stable field screening method for determining the GMO content with large-scale field applications.

Sampling for Genetically Modified Organisms Content Analysisin Agricultural Products: From Analytical Sample to Test Portion

Objective: At present, Sampling standards and regulations for genetically modified organisms (GMO) are commonly based on theoretical calculations or computer simulations, and there is a lack of field data to validate these simulations. In view of this situation, we sampled agricultural products for GMO content analysis, and investigated the influence of various factors on the accuracy of the results. We have prepared a three-part series and in this part focused on the process from analytical sample to test portions. Method: Using non-transgenic maize as matrix, 12 lines of transgenic maize were used to produce standard analytical samples. After systematic sampling, the GMO contents of these samples were randomly tested, and their single relative standard deviations (RSD) were calculated as a measure of total RSD (single analysis) per sample. Results: By comparing the RSDs of various sampling methods, it was found that the results of 12 strains were basically consistent, and the data of MON810 were listed as a representative. The parameters affecting the standard deviation included the content (aAS), particle size (dAS), test portion mass (MTP) and the number of increments (nIT). Total analytical RSD could be reduced by decreasing particle size, and increasing test portion mass or the number of increments. Based on current laboratory testing conditions and current used kits, for high content analytical sample(>0.01%), more than 2 duplicate test portions with at least -100mesh particle size and 200mg mass were recommended. Conclusion: Based on the results, the recommended values of particle size, test portion mass and the number of increments for the process from analytical sample to test portions were given. These factors were independent on species or strains of the product, so the results were suitable to all species and strains, provided that the solid particles could be crushed to required particle size.

Panorama general de los organismos genéticamente modificados en Colombia y en el mundo: Capacidad nacional de detección

Los organismos genéticamente modificados (OGM) y en particular los cultivos genéticamente modificados (GM), son el resultado de la modificación de la información genética de una especie a partir del uso de la biotecnología moderna para proporcionar nuevas características que su contraparte no modificada no posee, tales como resistencia a insectos, tolerancia a herbicidas, contenido de nutrientes entre otros. La mayor parte de estos cultivos se concentran en cuatro productos: soya (Glycine max), maíz (Zea Mays), canola (Brassica napus) y algodón (Gossypium hirsutum); y los principales productores son Estados Unidos, Brasil, Argentina, India y Canadá. Por su parte, Colombia ocupa el puesto 18 con cultivos de maíz, algodón y claveles azules. La introducción de estas especies en cualquier mercado está limitada por la legislación propia del país destino, así como por los estudios que permiten establecer su efecto sobre el medio ambiente, la salud humana y animal; en este sentido, la precisión y confianza de las técnicas analíticas empleadas en la evaluación del contenido de OGM son un elemento importante para la toma de decisiones basadas en evidencias objetivas, especialmente frente al debate en torno a su uso. Este documento presenta una revisión de las tecnologías de análisis más importantes disponibles a nivel mundial, frente a las capacidades nacionales para su detección.

Ultrasensitive Single Fluorescence-Labeled Probe-Mediated Single Universal Primer-Multiplex-Droplet Digital Polymerase Chain Reaction for High-Throughput Genetically Modified Organism Screening.

As genetically modified (GM) technology develops and genetically modified organisms (GMOs) become more available, GMOs face increasing regulations and pressure to adhere to strict labeling guidelines. A singleplex detection method cannot perform the high-throughput analysis necessary for optimal GMO detection. Combining the advantages of multiplex detection and droplet digital polymerase chain reaction (ddPCR), a single universal primer-multiplex-ddPCR (SUP-M-ddPCR) strategy was proposed for accurate broad-spectrum screening and quantification. The SUP increases efficiency of the primers in PCR and plays an important role in establishing a high-throughput, multiplex detection method. Emerging ddPCR technology has been used for accurate quantification of nucleic acid molecules without a standard curve. Using maize as a reference point, four heterologous sequences ( 35S, NOS, NPTII, and PAT) were selected to evaluate the feasibility and applicability of this strategy. Surprisingly, these four genes cover more than 93% of the transgenic maize lines and serve as preliminary screening sequences. All screening probes were labeled with FAM fluorescence, which allows the signals from the samples with GMO content and those without to be easily differentiated. This fiveplex screening method is a new development in GMO screening. Utilizing an optimal amplification assay, the specificity, limit of detection (LOD), and limit of quantitation (LOQ) were validated. The LOD and LOQ of this GMO screening method were 0.1% and 0.01%, respectively, with a relative standard deviation (RSD) < 25%. This method could serve as an important tool for the detection of GM maize from different processed, commercially available products. Further, this screening method could be applied to other fields that require reliable and sensitive detection of DNA targets.

Highly Sensitive GMO Detection Using Real-Time PCR with a Large Amount of DNA Template: Single-Laboratory Validation.

Current genetically modified organism (GMO) detection methods allow for sensitive detection. However, a further increase in sensitivity will enable more efficient testing for large grain samples and reliable testing for processed foods. In this study, we investigated real-time PCR-based GMO detection methods using a large amount of DNA template. We selected target sequences that are commonly introduced into many kinds of GM crops, i.e., 35S promoter and nopaline synthase (NOS) terminator. This makes the newly developed method applicable to a wide range of GMOs, including some unauthorized ones. The estimated LOD of the new method was 0.005% of GM maize events; to the best of our knowledge, this method is the most sensitive among the GM maize detection methods for which the LOD was evaluated in terms of GMO content. A 10-fold increase in the DNA amount as compared with the amount used under common testing conditions gave an approximately 10-fold reduction in the LOD without PCR inhibition. Our method is applicable to various analytical samples, including processed foods. The use of other primers and fluorescence probes would permit highly sensitive detection of various recombinant DNA sequences besides the 35S promoter and NOS terminator.

SOCIAL RESPONSIBILITY RESEARCH FOOD MANUFACTURER AS IMPORTANT FACTOR INCREASING QUALITY OF PRODUCTION

The object of research is the social responsibility of producers for the quality of food. One of the most problematic places is that social responsibility leads away from the fundamental economic role of business. Some argue that this is nothing but an adornment of reality. Others say that this is an attempt to replace the role of government as a controller of powerful multinational corporations. The general scientific methods of cognition (analysis, synthesis, abstraction), generalization methods, system analysis, grouping and comparison, and the method of expert evaluations are used in the work. The research shows that consumers do not have the opportunity to test food for the presence of genetically modified components in them because there are not enough laboratories for determining genetically modified organisms (GMOs) in the country. Based on which the author of the study concludes that in Ukraine it is critical to create modern laboratories for the study of GMO content. It is recommended to use PCR (polymerase chain reaction) – an experimental method of molecular biology – to determine the concentration of GMOs in food products. This allows a significant increase in the small concentrations of certain fragments of nucleic acid (DNA) in a biological material (sample) – with real-time detection. This method allows to specifically determine the type of GMO and determines its concentration with a certainty of at least 99.95 %. Also recommendations are given on the measures that need to be taken by the state authorities to ensure the quality of food products within the framework of monitoring the level of GMOs: The State Committee for Technical Regulation and Consumer Policy of Ukraine must determine the list of food products for compulsory testing for GMOs. The Society for the Protection of Consumer Rights to initiate the establishment of personal responsibility of enterprise managers for the violation of the Law on the mandatory informing of citizens about the availability of GMOs in food.

Development and Interlaboratory Validation of a Simple Screening Method for Genetically Modified Maize Using a ΔΔC(q)-Based Multiplex Real-Time PCR Assay.

A number of genetically modified (GM) maize events have been developed and approved worldwide for commercial cultivation. A screening method is needed to monitor GM maize approved for commercialization in countries that mandate the labeling of foods containing a specified threshold level of GM crops. In Japan, a screening method has been implemented to monitor approved GM maize since 2001. However, the screening method currently used in Japan is time-consuming and requires generation of a calibration curve and experimental conversion factor (C(f)) value. We developed a simple screening method that avoids the need for a calibration curve and C(f) value. In this method, ΔC(q) values between the target sequences and the endogenous gene are calculated using multiplex real-time PCR, and the ΔΔC(q) value between the analytical and control samples is used as the criterion for determining analytical samples in which the GM organism content is below the threshold level for labeling of GM crops. An interlaboratory study indicated that the method is applicable independently with at least two models of PCR instruments used in this study.

A highly sensitive and specific method for the screening detection of genetically modified organisms based on digital PCR without pretreatment.

Digital PCR has developed rapidly since it was first reported in the 1990s. It was recently reported that an improved method facilitated the detection of genetically modified organisms (GMOs). However, to use this improved method, the samples must be pretreated, which could introduce inaccuracy into the results. In our study, we explored a pretreatment-free digital PCR detection method for the screening for GMOs. We chose the CaMV35s promoter and the NOS terminator as the templates in our assay. To determine the specificity of our method, 9 events of GMOs were collected, including MON810, MON863, TC1507, MIR604, MIR162, GA21, T25, NK603 and Bt176. Moreover, the sensitivity, intra-laboratory and inter-laboratory reproducibility of our detection method were assessed. The results showed that the limit of detection of our method was 0.1%, which was lower than the labeling threshold level of the EU. The specificity and stability among the 9 events were consistent, respectively. The intra-laboratory and inter-laboratory reproducibility were both good. Finally, the perfect fitness for the detection of eight double-blind samples indicated the good practicability of our method. In conclusion, the method in our study would allow more sensitive, specific and stable screening detection of the GMO content of international trading products.

A novel trait-specific real-time PCR method enables quantification of genetically modified (GM) maize content in ground grain samples containing stacked GM maize

Stacked genetically modified (GM) maize is increasingly produced; thereby, current event-specific quantitative real-time polymerase chain reaction (qPCR) methods have led to the overestimation of GM organism (GMO) content compared with the actual weight/weight percentage of GM organism in maize samples. We developed a feasible qPCR method in which the GMO content is calculated based on the quantification of two herbicide-tolerant trait genes, 5-enolpyruvylshikimate-3-phosphate synthase from Agrobacterium sp. strain CP4 (cp4epsps) and phosphinothricin N-acetyl-transferase from Streptomyces viridochromogenes (pat) to quantify the GMO content in ground grain samples containing stacked GM maize. The GMO contents of two genes were quantified using a plasmid calibrant and summed for quantification of total GMO content. The trait-specific method revealed lower biases for examination of test samples containing stacked GM maize compared with the event-specific method. Our results clearly show that the trait-specific method is not only simple and cost-effective, but also useful in quantifying the GMO content in ground grain samples containing stacked GM maize, which are expected to be major events in the near future. The developed method would be the only feasible way to conduct the quantification of GMO content in the ground maize samples containing stacked GM maize for the verification of the labeling regulation.

Detection of the 35S promoter in transgenic maize via various isothermal amplification techniques: a practical approach.

In 2003 the European Commission introduced a 0.9% threshold for food and feed products containing genetically modified organism (GMO)-derived components. For commodities containing GMO contents higher than this threshold, labelling is mandatory. To provide a DNA-based rapid and simple detection method suitable for high-throughput screening of GMOs, several isothermal amplification approaches for the 35S promoter were tested: strand displacement amplification, nicking-enzyme amplification reaction, rolling circle amplification, loop-mediated isothermal amplification (LAMP) and helicase-dependent amplification (HDA). The assays developed were tested for specificity in order to distinguish between samples containing genetically modified (GM) maize and non-GM maize. For those assays capable of this discrimination, tests were performed to determine the lower limit of detection. A false-negative rate was determined to rule out whether GMO-positive samples were incorrectly classified as GMO-negative. A robustness test was performed to show reliable detection independent from the instrument used for amplification. The analysis of three GM maize lines showed that only LAMP and HDA were able to differentiate between the GMOs MON810, NK603, and Bt11 and non-GM maize. Furthermore, with the HDA assay it was possible to realize a detection limit as low as 0.5%. A false-negative rate of only 5% for 1% GM maize for all three maize lines shows that HDA has the potential to be used as an alternative strategy for the detection of transgenic maize. All results obtained with the LAMP and HDA assays were compared with the results obtained with a previously reported real-time PCR assay for the 35S promoter in transgenic maize. This study presents two new screening assays for detection of the 35S promoter in transgenic maize by applying the isothermal amplification approaches HDA and LAMP.

Detection of GM soybean by multiplex-touchdown PCR-microchip capillary electrophoresis with response surface methodology optimization.

The combination of the molecular technique, the multivariate strategy and microchip capillary electrophoresis (MCE) was applied to rapid and sensitive analysis of genetically modified (GM) soybean in food samples. A multiplex-touchdown polymerase chain reaction (PCR) system was developed for simultaneously amplifying three target sequences in Roundup Ready soybean (RRS). Response surface methodology was introduced to determine the optimal separation condition in MCE with good resolution and short analytical time. The detection of the PCR products of RRS was completed within 4 min under the optimal conditions. The specificity of the method was evaluated by testing non-GM soybean materials and three GM maize varieties (MON810, Bt176 and Bt11). A sensitivity of 0.1% GM organisms content was obtained, which was remarkably lower than the labeling threshold for transgenic food defined as 0.9% in the European regulation. The relative standard deviation of migration time was in the range of 0.17-0.95%. The proposed method was rapid, sensitive and specific and can be used to identify and detect GM soybean in food samples.

Adventitious presence of transgenic events in the maize supply chain in Peru: A case study

Cultivation and trade of transgenic or genetically modified organisms (GMO) and commodities has become widespread worldwide. In particular, production of transgenic crops has seen an accelerated growth along with a complex regulatory process. Current Peruvian legislation prohibits import of transgenic seeds and cultivation of transgenic crops in National territory but allows import of GMO-derived products and commodities. In addition, there is legislation that mandates the labeling of food products containing transgenic ingredients but the labeling threshold is still under discussion and the enforcement of this law is on hold. In this context, we evaluated adventitious presence of transgenic events in locally traded yellow maize using PCR- and immuno-based detection methods. Our results indicated that contamination during the distribution system of lots derived from non-transgenic maize was unavoidable and generally below 1.0% (w/w). Transgenic event MON810 was found in truck-loads of nationally grown maize. In general, frequencies of GMO-derived targets in whole-grain lots were 2.2% (GMO content≥1%), 16.4% (GMO content≤1%) and 81.3% (GMO content below our detection levels). When samples of de-germinated maize where evaluated, frequencies were 25.6% (GMO content>0.9%), 65.1% (GMO content≤0.9%) and 9.3% (GMO content below our detection levels). We believe this information will aid policy makers in establishing a suitable threshold for trade and product labeling as well as to conduct further investigation on other crops and scenarios.

Effect of high temperature and pressure on quantification of MON 810 maize

Maize MON 810 (Zea mays L.) is the only transgenic cultivar grown in the European Union countries and food products with its content higher than 0.9% must be labelled. Processing such as high temperature (121&amp;deg;C), elevated pressure (0.1 MPa), and low pH 2.25 fragmented DNA. A two order difference in the species specific gene content compared to the transgenic DNA content in plant materials used has led to false negative results in the quantification of transgenic DNA. The maize containing 4.2% of the transgene after processing appeared to be as low as 3.0% (100&amp;deg;C) and 1.9% (121&amp;deg;C, 0.1 MPa). The 2.1% amount of the transgene dropped at 100&amp;deg;C to 1.0% and at 121&amp;deg;C, 0.1 MPa to 0.6%. Determination of GMO (Genetically Modified Organism) content in processed foods may lead to incorrect statement and labelling could mislead consumers in these cases. &amp;nbsp;

Relative quantification in seed GMO analysis: state of art and bottlenecks

Reliable quantitative methods are needed to comply with current EU regulations on the mandatory labeling of genetically modified organisms (GMOs) and GMO-derived food and feed products with a minimum GMO content of 0.9 %. The implementation of EU Commission Recommendation 2004/787/EC on technical guidance for sampling and detection which meant as a helpful tool for the practical implementation of EC Regulation 1830/2003, which states that "the results of quantitative analysis should be expressed as the number of target DNA sequences per target taxon specific sequences calculated in terms of haploid genomes". This has led to an intense debate on the type of calibrator best suitable for GMO quantification. The main question addressed in this review is whether reference materials and calibrators should be matrix based or whether pure DNA analytes should be used for relative quantification in GMO analysis. The state of the art, including the advantages and drawbacks, of using DNA plasmid (compared to genomic DNA reference materials) as calibrators, is widely described. In addition, the influence of the genetic structure of seeds on real-time PCR quantitative results obtained for seed lots is discussed. The specific composition of a seed kernel, the mode of inheritance, and the ploidy level ensure that there is discordance between a GMO % expressed as a haploid genome equivalent and a GMO % based on numbers of seeds. This means that a threshold fixed as a percentage of seeds cannot be used as such for RT-PCR. All critical points that affect the expression of the GMO content in seeds are discussed in this paper.