Genetically Modified Organisms Quantification 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.

A convenient renewable surface plasmon resonance chip for relative quantification of genetically modified soybean in food and feed.

The cultivation of genetically modified organisms (GMO) continues to expand worldwide. Still, many consumers express concerns about the use of GMO in food or feed, and many countries have legislated on labelling systems to indicate the presence of GMO in commercial products. To deal with the increased number of GMO events and to address related regulations, alternative detection methods for GMO inspection are required. In this work, a genosensor based on Surface Plasmon Resonance under continuous flow was developed for the detection and quantification of a genetically modified soybean (event GTS 40-3-2). In a single chip, the simultaneous detection of the event-specific and the taxon-specific samples were achieved, whose detection limits were 20 pM and 16 pM, respectively. The reproducibility was 1.4%, which supports the use of the chip as a reliable and cost-effective alternative to other DNA-based techniques. The results indicate that the proposed method is a versatile tool for GMO quantification in food and feed samples.

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

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

Digital PCR as an effective tool for GMO quantification in complex matrices

The increased use of genetically modified organisms (GMOs) is accompanied by increased complexity of the matrices that contain GMOs. The most common DNA-based approach for GMO detection and quantification is real-time quantitative polymerase chain reaction (qPCR). However, as qPCR is sensitive to inhibitors and relies on standard curves for quantification, it has limited application in GMO quantification for complex matrices. To overcome this hurdle in DNA quantification, we present droplet digital PCR (ddPCR) assays that were designed to target ‘Roundup Ready’ soybean and the soybean reference gene. Three ddPCR assays were transferred from qPCR to QX100/QX200 ddPCR platforms and characterised. Together, the fitness-for-purpose study on four real-life samples and the use of a chamber-based PCR system, showed that dPCR has great potential to improve such measurements in GMO testing and monitoring of food authenticity.

Inter-laboratory analysis of selected genetically modified plant reference materials with digital PCR

Digital PCR (dPCR), as a new technology in the field of genetically modified (GM) organism (GMO) testing, enables determination of absolute target copy numbers. The purpose of our study was to test the transferability of methods designed for quantitative PCR (qPCR) to dPCR and to carry out an inter-laboratory comparison of the performance of two different dPCR platforms when determining the absolute GM copy numbers and GM copy number ratio in reference materials certified for GM content in mass fraction. Overall results in terms of measured GM% were within acceptable variation limits for both tested dPCR systems. However, the determined absolute copy numbers for individual genes or events showed higher variability between laboratories in one third of the cases, most possibly due to variability in the technical work, droplet size variability, and analysis of the raw data. GMO quantification with dPCR and qPCR was comparable. As methods originally designed for qPCR performed well in dPCR systems, already validated qPCR assays can most generally be used for dPCR technology with the purpose of GMO detection.Graphical abstractThe output of three different PCR-based platforms was assessed in an inter-laboratory comparison

Electrochemical magnetoassay coupled to PCR as a quantitative approach to detect the soybean transgenic event GTS40-3-2 in foods

Simple, cost-effective and reliable tools for the quantification of genetically modified organisms (GMO) in food and feed are highly demanded to enforce labelling legislation in the EU. Herein, we report a novel method for quantitative analysis of genetically modified soybean with the event GTS-40-3-2, also known as Roundup Ready (RR) soybean, using magnetoassays with electrochemical detection, coupled to DNA amplification by end-point polymerase chain reaction (PCR). For the proposed work, two DNA sequences were targeted via hybridisation onto magnetic beads, one specific for the transgenic event and the other for the taxon or species-specific lectin gene. Enzymatic labelling was performed to obtain an electrochemically active product measured by chronoamperometry. By optimising the number of PCR cycles, among other parameters, two magnetoassays coupled to PCR were successfully accomplished and linearity was obtained in the ranges of 53–4425 and 1093–88,496 DNA copies for the event-specific and lectin sequences, respectively. The proposed method provides accurate and precise RR soybean quantitative results, being effectively compared to those obtained by real-time PCR, as the reference method. These findings confirm the suitability of the method as an alternative tool for GMO quantification.

Multiplex quantification of 12 European Union authorized genetically modified maize lines with droplet digital polymerase chain reaction.

Presence of genetically modified organisms (GMO) in food and feed products is regulated in many countries. The European Union (EU) has implemented a threshold for labeling of products containing more than 0.9% of authorized GMOs per ingredient. As the number of GMOs has increased over time, standard-curve based simplex quantitative polymerase chain reaction (qPCR) analyses are no longer sufficiently cost-effective, despite widespread use of initial PCR based screenings. Newly developed GMO detection methods, also multiplex methods, are mostly focused on screening and detection but not quantification. On the basis of droplet digital PCR (ddPCR) technology, multiplex assays for quantification of all 12 EU authorized GM maize lines (per April first 2015) were developed. Because of high sequence similarity of some of the 12 GM targets, two separate multiplex assays were needed. In both assays (4-plex and 10-plex), the transgenes were labeled with one fluorescence reporter and the endogene with another (GMO concentration = transgene/endogene ratio). It was shown that both multiplex assays produce specific results and that performance parameters such as limit of quantification, repeatability, and trueness comply with international recommendations for GMO quantification methods. Moreover, for samples containing GMOs, the throughput and cost-effectiveness is significantly improved compared to qPCR. Thus, it was concluded that the multiplex ddPCR assays could be applied for routine quantification of 12 EU authorized GM maize lines. In case of new authorizations, the events can easily be added to the existing multiplex assays. The presented principle of quantitative multiplexing can be applied to any other domain.

Multiplex electrochemical DNA platform for femtomolar-level quantification of genetically modified soybean

Current EU regulations on the mandatory labeling of genetically modified organisms (GMOs) with a minimum content of 0.9% would benefit from the availability of reliable and rapid methods to detect and quantify DNA sequences specific for GMOs. Different genosensors have been developed to this aim, mainly intended for GMO screening. A remaining challenge, however, is the development of genosensing platforms for GMO quantification, which should be expressed as the number of event-specific DNA sequences per taxon-specific sequences. Here we report a simple and sensitive multiplexed electrochemical approach for the quantification of Roundup-Ready Soybean (RRS). Two DNA sequences, taxon (lectin) and event-specific (RR), are targeted via hybridization onto magnetic beads. Both sequences are simultaneously detected by performing the immobilization, hybridization and labeling steps in a single tube and parallel electrochemical readout. Hybridization is performed in a sandwich format using signaling probes labeled with fluorescein isothiocyanate (FITC) or digoxigenin (Dig), followed by dual enzymatic labeling using Fab fragments of anti-Dig and anti-FITC conjugated to peroxidase or alkaline phosphatase, respectively. Electrochemical measurement of the enzyme activity is finally performed on screen-printed carbon electrodes. The assay gave a linear range of 2–250pM for both targets, with LOD values of 650fM (160amol) and 190fM (50amol) for the event-specific and the taxon-specific targets, respectively. Results indicate that the method could be applied for GMO quantification below the European labeling threshold level (0.9%), offering a general approach for the rapid quantification of specific GMO events in foods.

A statistical approach to quantification of genetically modified organisms (GMO) using frequency distributions.

BackgroundAccording to Regulation (EU) No 619/2011, trace amounts of non-authorised genetically modified organisms (GMO) in feed are tolerated within the EU if certain prerequisites are met. Tolerable traces must not exceed the so-called ‘minimum required performance limit’ (MRPL), which was defined according to the mentioned regulation to correspond to 0.1% mass fraction per ingredient. Therefore, not yet authorised GMO (and some GMO whose approvals have expired) have to be quantified at very low level following the qualitative detection in genomic DNA extracted from feed samples. As the results of quantitative analysis can imply severe legal and financial consequences for producers or distributors of feed, the quantification results need to be utterly reliable.ResultsWe developed a statistical approach to investigate the experimental measurement variability within one 96-well PCR plate. This approach visualises the frequency distribution as zygosity-corrected relative content of genetically modified material resulting from different combinations of transgene and reference gene Cq values. One application of it is the simulation of the consequences of varying parameters on measurement results. Parameters could be for example replicate numbers or baseline and threshold settings, measurement results could be for example median (class) and relative standard deviation (RSD). All calculations can be done using the built-in functions of Excel without any need for programming. The developed Excel spreadsheets are available (see section ‘Availability of supporting data’ for details). In most cases, the combination of four PCR replicates for each of the two DNA isolations already resulted in a relative standard deviation of 15% or less.ConclusionsThe aims of the study are scientifically based suggestions for minimisation of uncertainty of measurement especially in —but not limited to— the field of GMO quantification at low concentration levels. Four PCR replicates for each of the two DNA isolations seem to be a reasonable minimum number to narrow down the possible spread of results.Electronic supplementary materialThe online version of this article (doi:10.1186/s12859-014-0407-x) contains supplementary material, which is available to authorized users.

GMO quantification: valuable experience and insights for the future.

Cultivation and marketing of genetically modified organisms (GMOs) have been unevenly adopted worldwide. To facilitate international trade and to provide information to consumers, labelling requirements have been set up in many countries. Quantitative real-time polymerase chain reaction (qPCR) is currently the method of choice for detection, identification and quantification of GMOs. This has been critically assessed and the requirements for the method performance have been set. Nevertheless, there are challenges that should still be highlighted, such as measuring the quantity and quality of DNA, and determining the qPCR efficiency, possible sequence mismatches, characteristics of taxon-specific genes and appropriate units of measurement, as these remain potential sources of measurement uncertainty. To overcome these problems and to cope with the continuous increase in the number and variety of GMOs, new approaches are needed. Statistical strategies of quantification have already been proposed and expanded with the development of digital PCR. The first attempts have been made to use new generation sequencing also for quantitative purposes, although accurate quantification of the contents of GMOs using this technology is still a challenge for the future, and especially for mixed samples. New approaches are needed also for the quantification of stacks, and for potential quantification of organisms produced by new plant breeding techniques.

A specific endogenous reference for genetically modified common bean (Phaseolus vulgaris L.) DNA quantification by real-time PCR targeting lectin gene.

The Embrapa 5.1 genetically modified (GM) common bean was approved for commercialization in Brazil. Methods for the quantification of this new genetically modified organism (GMO) are necessary. The development of a suitable endogenous reference is essential for GMO quantification by real-time PCR. Based on this, a new taxon-specific endogenous reference quantification assay was developed for Phaseolus vulgaris L. Three genes encoding common bean proteins (phaseolin, arcelin, and lectin) were selected as candidates for endogenous reference. Primers targeting these candidate genes were designed and the detection was evaluated using the SYBR Green chemistry. The assay targeting lectin gene showed higher specificity than the remaining assays, and a hydrolysis probe was then designed. This assay showed high specificity for 50 common bean samples from two gene pools, Andean and Mesoamerican. For GM common bean varieties, the results were similar to those obtained for non-GM isogenic varieties with PCR efficiency values ranging from 92 to 101 %. Moreover, this assay presented a limit of detection of ten haploid genome copies. The primers and probe developed in this work are suitable to detect and quantify either GM or non-GM common bean.

A novel quadruplex real-time PCR method for simultaneous detection of Cry2Ae and two genetically modified cotton events (GHB119 and T304-40).

BackgroundTo date, over 150 genetically modified (GM) crops are widely cultivated. To comply with regulations developed for genetically modified organisms (GMOs), including labeling policies, many detection methods for GMO identification and quantification have been developed.ResultsTo detect the entrance and exit of unauthorized GM crop events in China, we developed a novel quadruplex real-time PCR method for simultaneous detection and quantification of GM cotton events GHB119 and T304-40 in cotton-derived products (based on the 5′-flanking sequence) and the insect-resistance gene Cry2Ae. The limit of detection was 10 copies for GHB119 and Cry2Ae and 25 copies for T304-40. The limit of quantification was 25 copies for GHB119 and Cry2Ae and 50 copies for T304-40. Moreover, low bias and acceptable standard deviation and relative standard deviation values were obtained in quantification analysis of six blind samples containing different GHB119 and T304-40 ingredients.ConclusionsThe developed quadruplex quantitative method could be used for quantitative detection of two GM cotton events (GHB119 and T304-40) and Cry2Ae gene ingredient in cotton derived products.

Development of Plasmid DNA Reference Material for the Quantification of Genetically Modified Common Bean Embrapa 5.1

The genetically modified (GM) common bean Embrapa 5.1 was recently approved for commercialization. The reliable detection and quantification of GM organisms is strongly dependent on validated methods as well as calibration systems. This work presents the development of a calibrant plasmid for Embrapa 5.1 common bean detection. The reaction parameters were determined and compared for both the plasmid DNA (pDNA) and the genomic DNA (gDNA). PCR efficiencies for pDNA were 81% for the construction-specific assays and 76% for the taxon-specific assay, whereas for gDNA efficiencies were 94 and 93%, respectively. The limits of detection (LOD) in both qPCR assays were 10(2) and 10(3) copies of gDNA and pDNA per PCR reaction, respectively. This is sufficient to detect 0.067 and 0.67% of GM common bean in 100 ng of DNA, respectively, which is in agreement with detecting the 1% GM content required by the Brazilian legislation.

Quantitative Analysis of Food and Feed Samples with Droplet Digital PCR

In this study, the applicability of droplet digital PCR (ddPCR) for routine analysis in food and feed samples was demonstrated with the quantification of genetically modified organisms (GMOs). Real-time quantitative polymerase chain reaction (qPCR) is currently used for quantitative molecular analysis of the presence of GMOs in products. However, its use is limited for detecting and quantifying very small numbers of DNA targets, as in some complex food and feed matrices. Using ddPCR duplex assay, we have measured the absolute numbers of MON810 transgene and hmg maize reference gene copies in DNA samples. Key performance parameters of the assay were determined. The ddPCR system is shown to offer precise absolute and relative quantification of targets, without the need for calibration curves. The sensitivity (five target DNA copies) of the ddPCR assay compares well with those of individual qPCR assays and of the chamber digital PCR (cdPCR) approach. It offers a dynamic range over four orders of magnitude, greater than that of cdPCR. Moreover, when compared to qPCR, the ddPCR assay showed better repeatability at low target concentrations and a greater tolerance to inhibitors. Finally, ddPCR throughput and cost are advantageous relative to those of qPCR for routine GMO quantification. It is thus concluded that ddPCR technology can be applied for routine quantification of GMOs, or any other domain where quantitative analysis of food and feed samples is needed.

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.

Tracing transgenic maize as affected by breadmaking process and raw material for the production of a traditional maize bread, broa

Broa is a maize bread highly consumed and appreciated, especially in the north and central zones of Portugal. In the manufacturing of broa, maize flour and maize semolina might be used, besides other cereals such as wheat and rye. Considering the needs for genetically modified organism (GMO) traceability in highly processed foods, the aim of this work was to assess DNA degradation, DNA amplification and GMO quantification along breadmaking process of broa. DNA degradation was noticed by its decrease of integrity after dough baking and in all parts of bread sampling. The PCR amplification results of extracted DNA from the three distinct maize breads (broa 1, 2 and 3) showed that sequences for maize invertase gene and for events MON810 and TC1507 were easily detected with strong products. Real-time PCR revealed that quantification of GMO was feasible in the three different breads and that sampling location of baked bread might have a limited influence since the average quantitative results of both events after baking were very close to the actual values in the case of broa 1 (prepared with maize semolina). In the other two maize breads subjected to the same baking treatment, the contents of MON810 maize were considerably underestimated, leading to the conclusion that heat-processing was not the responsible parameter for that distortion, but the size of particle and mechanical processing of raw maize play also a major role in GMO quantification.

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.

Testing the Robustness of Validated Methods for Quantitative Detection of GMOs Across qPCR Instruments

The number of real-time polymerase chain reaction (qPCR) instruments available has greatly increased over recent years. However, little information is available on the performance of validated quantitative methods when tested in different instruments. A study has been designed to evaluate the robustness of three validated methods for genetically modified organisms (GMO) quantification across six real-time platforms from four different suppliers. The performance of three validated methods for event-specific detection of Bt11 maize, DAS 59122 maize and MON 89788 soybean was evaluated on six qPCR platforms (ABI 7900 HT, ABI Prism® 7700 and ABI 7500 from Applied Biosystems; LightCycler® 480 from Roche; Mx3005P® from Stratagene; and iQ™5 from Bio-Rad). Method performance criteria were compared against European Network of GMO Laboratories method performance requirements for analytical methods of GMO testing. The latter comparison indicates that the criteria are fulfilled for most of the platforms and levels of GMO concentrations, though with minor exceptions. The analysis of variance (one-way) indicated that the quantification of GMOs was affected by the platforms, which did not respond consistently across GM levels. A two-way analysis of variance confirmed that there were significant interactions between platforms and GM levels. The pairwise comparison of the platforms' performance indicated that some deviate significantly from others, though differences between methods were observed.

Applicability of Plasmid Calibrant pTC1507 in Quantification of TC1507 Maize: An Interlaboratory Study

To enforce the labeling regulations of genetically modified organisms (GMOs), the application of DNA plasmids as calibrants is becoming essential for the practical quantification of GMOs. This study reports the construction of plasmid pTC1507 for a quantification assay of genetically modified (GM) maize TC1507 and the collaborative ring trial in international validation of its applicability as a plasmid calibrant. pTC1507 includes one event-specific sequence of TC1507 maize and one unique sequence of maize endogenous gene zSSIIb. A total of eight GMO detection laboratories worldwide were invited to join the validation process, and test results were returned from all eight participants. Statistical analysis of the returned results showed that real-time PCR assays using pTC1507 as calibrant in both GM event-specific and endogenous gene quantifications had high PCR efficiency (ranging from 0.80 to 1.15) and good linearity (ranging from 0.9921 to 0.9998). In a quantification assay of five blind samples, the bias between the test values and true values ranged from 2.6 to 24.9%. All results indicated that the developed pTC1507 plasmid is applicable for the quantitative analysis of TC1507 maize and can be used as a suitable substitute for dried powder certified reference materials (CRMs).

Testing the interaction between analytical modules: an example with Roundup Ready®soybean line GTS 40-3-2

BackgroundThe modular approach to analysis of genetically modified organisms (GMOs) relies on the independence of the modules combined (i.e. DNA extraction and GM quantification). The validity of this assumption has to be proved on the basis of specific performance criteria.ResultsAn experiment was conducted using, as a reference, the validated quantitative real-time polymerase chain reaction (PCR) module for detection of glyphosate-tolerant Roundup Ready® GM soybean (RRS). Different DNA extraction modules (CTAB, Wizard and Dellaporta), were used to extract DNA from different food/feed matrices (feed, biscuit and certified reference material [CRM 1%]) containing the target of the real-time PCR module used for validation. Purity and structural integrity (absence of inhibition) were used as basic criteria that a DNA extraction module must satisfy in order to provide suitable template DNA for quantitative real-time (RT) PCR-based GMO analysis. When performance criteria were applied (removal of non-compliant DNA extracts), the independence of GMO quantification from the extraction method and matrix was statistically proved, except in the case of Wizard applied to biscuit. A fuzzy logic-based procedure also confirmed the relatively poor performance of the Wizard/biscuit combination.ConclusionsFor RRS, this study recognises that modularity can be generally accepted, with the limitation of avoiding combining highly processed material (i.e. biscuit) with a magnetic-beads system (i.e. Wizard).