Validation of real-time PCR analyses for line-specific quantitation of genetically modified maize and soybean using new reference molecules

Novel analytical methods based on real-time quantitative polymerase chain reactions by use of new reference molecules were validated in interlaboratory studies for the quantitation of genetically modified (GM) maize and soy. More than 13 laboratories from Japan, Korea, and the United States participated in the studies. The interlaboratory studies included 2 separate stages: (1) measurement tests of coefficient values, the ratio of recombinant DNA (r-DNA) sequence, and endogenous DNA sequence in the seeds of GM maize and GM soy; and (2) blind tests with 6 pairs of maize and soy samples, including different levels of GM maize or GM soy. Test results showed that the methods are applicable to the specific quantitation of the 5 lines of GM maize and one line of GM soy. After statistical treatment to remove outliers, the repeatability and reproducibility of these methods at a level of 5.0% were <13.7 and 15.9%, respectively. The quantitation limits of the methods were 0.50% for Bt11, T25, and MON810, and 0.10% for GA21, Event176, and Roundup Ready soy. The results of blind tests showed that the numerical information obtained from these methods will contribute to practical analyses for labeling systems of GM crops.     

Validation of an immunoassay for detection and quantitation of a genetically modified soybean in food and food fractions using reference materials: interlaboratory study

An immunoassay for detection of a specific genetically modified soybean (Roundup-Ready) was validated on dried soybean powder in an interlaboratory study. Different percentages of genetically modified soybeans in nonmodified soybean matrix were evaluated in a blind study. Thirty-eight laboratories from 13 countries participated. The immunoassay was evaluated for 2 endpoints: (1) To give a semiquantitative result, i.e., determination of a given sample above or below a given threshold, or (2) to compute a quantitative result, i.e., percentage of genetically modified soybeans in the sample. Semiquantitative results showed that a given sample which contained <2% genetically modified soybeans was identified as below 2% with a 99% confidence level. Quantitative use of the assay resulted in a repeatability (r) and reproducibility (R) that were computed to be RSDr = 7% and RSDR = 10%, respectively, for a sample containing 2% genetically modified soybeans. Application of this method depends on availability of appropriate reference materials for a specific food matrix. Only matrix-matched reference materials can be used for analysis of food or food fractions.     

Novel reference molecules for quantitation of genetically modified maize and soybean

New quantitation methods based on a real-time polymerase chain reaction (PCR) technique were developed for 5 lines of genetically modified (GM) maize, including MON810, Event176, Bt11, T25, and GA21, and a GM soy, Roundup Ready. Oligonucleotide DNA, including specific primers and fluorescent dye-labeled probes, were designed for PCRs. Two plasmids were constructed as reference molecules (RMs) for the detection of GM maize and GM soy. The molecules contain the DNA sequences of a specific region found in each GM line, universal sequences used in various GM lines, such as cauliflower mosaic virus 35S promoter and nopaline synthase terminator, and the endogenous DNA sequences of maize or soy. By using these plasmids, no GM maize and GM soy were required as reference materials for the qualitative and quantitative PCR technique. Test samples containing 0, 0.10, 0.50, 1.0, 5.0, and 10% GM maize or GM soy were quantitated. At the 5.0% level, the bias (mean-true value) ranged from 2.8 to 19.4% and the relative standard deviation was <5.2%. These results show that our method involving the use of these plasmids as RMs is reliable and practical for quantitation of GM maize and GM soy.     

Detection methods and performance criteria for genetically modified organisms

Detection methods for genetically modified organisms (GMOs) are necessary for many applications, from seed purity assessment to compliance of food labeling in several countries. Numerous analytical methods are currently used or under development to support these needs. The currently used methods are bioassays and protein- and DNA-based detection protocols. To avoid discrepancy of results between such largely different methods and, for instance, the potential resulting legal actions, compatibility of the methods is urgently needed. Performance criteria of methods allow evaluation against a common standard. The more-common performance criteria for detection methods are precision, accuracy, sensitivity, and specificity, which together specifically address other terms used to describe the performance of a method, such as applicability, selectivity, calibration, trueness, precision, recovery, operating range, limit of quantitation, limit of detection, and ruggedness. Performance criteria should provide objective tools to accept or reject specific methods, to validate them, to ensure compatibility between validated methods, and be used on a routine basis to reject data outside an acceptable range of variability. When selecting a method of detection, it is also important to consider its applicability, its field of applications, and its limitations, by including factors such as its ability to detect the target analyte in a given matrix, the duration of the analyses, its cost effectiveness, and the necessary sample sizes for testing. Thus, the current GMO detection methods should be evaluated against a common set of performance criteria.     

Real-time quantitative polymerase chain reaction methods for four genetically modified maize varieties and maize DNA content in food

Quantitative detection methods are needed for enforcement of the recently introduced labeling threshold for genetically modified organisms (GMOs) in food ingredients. This labeling threshold, which is set to 1% in the European Union and Switzerland, must be applied to all approved GMOs. Four different varieties of maize are approved in the European Union: the insect-resistant Bt176 maize (Maximizer), Btl 1 maize, Mon810 (YieldGard) maize, and the herbicide-tolerant T25 (Liberty Link) maize. Because the labeling must be considered individually for each ingredient, a quantitation system for the endogenous maize content is needed in addition to the GMO-specific detection systems. Quantitative real-time polymerase chain reaction detection methods were developed for the 4 approved genetically modified maize varieties and for an endogenous maize (invertase) gene system.     

Identity preservation of genetically modified organisms in the food chain: requirements,methods, and costs

The use of the technology of genetic modification (GM) in European agriculture and the food supply chain is currently controversial. Because of strong anti-GM technology sentiments, the use of ingredients derived from plants containing GM have largely been eliminated from foods manufactured for direct human consumption by the food supply chain in much of the European Union (EU). During the past year, the attention of those opposed to the technology has turned to the use of GM ingredients in livestock production systems by incorporation of GM soy and maize in animal feed. A discussion is presented of the key issues relating to this subject, focusing on how supplies of GM or non-GM products are segregated or how their identities are preserved. The discussion is centered on GM maize and soybeans into which agronomic traits, such as herbicide tolerance and/or insect resistance, have been incorporated. These are currently the only crops into which some varieties containing GM have been approved for use in the EU.     

Surface plasmon resonance for detection of genetically modified organisms in the food supply

A review is presented demonstrating that biospecific interaction analysis, using surface plasmon resonance (SPR) and biosensor technologies is a simple, rapid, and automatable approach to detect genetically modified organisms (GMOs). Using SPR, we were able to monitor in real-time the hybridization between oligonucleotide or polymerase chain reaction (PCR)-generated probes and target single-stranded PCR products obtained by using as substrates DNA isolated from normal or transgenic soybean and maize. This procedure allows a one-step, nonradioactive detection of GMOs. PCR-generated probes are far more efficient in detecting GMOs than are oligodeoxyribonucleotide probes. This is expected to be a very important parameter, because information on low percentage of GMOs is of great value. Determination of the ability of SPR-based analysis to quantify GMOs should be considered a major research field for future studies, especially for the analyses of food supplies.     

Detection of genetically modified organisms in food: critical points for quality assurance

 The detection of genetically modified organisms (GMOs) by the polymerase chain reaction (PCR) is a complex multiparameter problem. Therefore, a number of critical issues in respect to quality control need to be considered. For practical purposes, the PCR process itself can be divided into three subprocesses: template isolation and reaction setup (pre-PCR), PCR reaction and detection of amplification products, and data evaluation (post-PCR). Crucial factors for the pre-PCR process are the following: homogeneity of the sample to be analysed, performance of template isolation and purification in terms of yield and purity, standardized process for the estimation of concentrations of genomic DNA and all reagents used in the reaction. For the PCR itself, crucial factors to be controlled are: setup of reactions, batch to batch variations of reagents, temperature-time programs used for the PCR amplification, and the performance of different types of hardware (e.g. different brands of thermocyclers). The crucial factor for the post-PCR process is the detection of the amplification products of the PCR. The tremendous sensitivity of PCR methods requires a careful and consequent separation of the three processes in terms of hardware, laboratory space and sample handling. The avoidance of contamination is one of the most critical factors. The goal of quality assurance measures must be to ensure appropriate results at maximum sensitivity. The complexity of any PCR system used for the detection of GMOs leads to the requirement of a careful validation process for any laboratory using such methods. For qualitative analyses crucial validation parameters are: specificity, selectivity, repeatability, intermediate precision, reproducibility, limit of detection and robustness. Received: 5 October 1998 / Accepted: 22 February 1999     

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.     

Evaluation of stable isotope labelling strategies for the quantitation of CP4 EPSPS in genetically modified soya

The introduction of genetically modified (GM) crops into the market has raised a general alertness relating to the control and safety of foods. The applicability of protein separation hyphenated to mass spectrometry to identify the bacterial enolpyruvylshikimate-3-phosphate synthase (CP4 EPSPS) protein expressed in GM crops has been previously reported [M.F. Ocana, P.D. Fraser, R.K.P. Patel, J.M. Halket, P.M. Bramley, Rapid Commun. Mass Spectrom. 21 (2007) 319.]. Herein, we investigate the suitability of two strategies that employ heavy stable isotopes, i.e. AQUA and iTRAQ, to quantify different levels of CP4 EPSPS in up to four GM preparations. Both quantification strategies showed potential to determine whether the presence of GM material is above the limits established by the European Union. The AQUA quantification procedure involved protein solubilisation/fractionation and subsequent separation using SDS-PAGE. A segment of the gel in which the protein of interest was located was excised, the stable isotope labeled peptide added at a known concentration and proteolytic digestion initiated. Following recovery of the peptides, on-line separation and detection using LC-MS was carried out. A similar approach was used for the iTRAQ workflow with the exception that proteins were digested in solution and generated tryptic peptides were chemically tagged. Both procedures demonstrated the potential for quantitative detection at 0.5% (w/w) GM soya which is a level below the current European Union's threshold for food-labelling. In this context, a comparison between the two procedures is provided within the present study.     

A practical approach to screen for authorised and unauthorised genetically modified plants

In routine analysis, screening methods based on real-time PCR are most commonly used for the detection of genetically modified (GM) plant material in food and feed. In this paper, it is shown that the combination of five DNA target sequences can be used as a universal screening approach for at least 81 GM plant events authorised or unauthorised for placing on the market and described in publicly available databases. Except for maize event LY038, soybean events DP-305423 and BPS-CV127-9 and cotton event 281-24-236 × 3006-210-23, at least one of the five genetic elements has been inserted in these GM plants and is targeted by this screening approach. For the detection of these sequences, fully validated real-time PCR methods have been selected. A screening table is presented that describes the presence or absence of the target sequences for most of the listed GM plants. These data have been verified either theoretically according to available databases or experimentally using available reference materials. The screening table will be updated regularly by a network of German enforcement laboratories.     

Development and validation of real-time PCR screening methods for detection of cry1A.105 and cry2Ab2 genes in genetically modified organisms

Primers and probes were developed for the element-specific detection of cry1A.105 and cry2Ab2 genes, based on their DNA sequence as present in GM maize MON89034. Cry genes are present in many genetically modified (GM) plants and they are important targets for developing GMO element-specific detection methods. Element-specific methods can be of use to screen for the presence of GMOs in food and feed supply chains. Moreover, a combination of GMO elements may indicate the potential presence of unapproved GMOs (UGMs). Primer-probe combinations were evaluated in terms of specificity, efficiency and limit of detection. Except for specificity, the complete experiment was performed in 9 PCR runs, on 9 different days and by testing 8 DNA concentrations. The results showed a high specificity and efficiency for cry1A.105 and cry2Ab2 detection. The limit of detection was between 0.05 and 0.01 ng DNA per PCR reaction for both assays. These data confirm the applicability of these new primer-probe combinations for element detection that can contribute to the screening for GM and UGM crops in food and feed samples.     

Detection of processed genetically modified food using CIM monolithic columns for DNA isolation

The availability of sufficient quantities of DNA of adequate quality is crucial in polymerase chain reaction (PCR)-based methods for genetically modified food detection. In this work, the suitability of anion-exchange CIM (Convective Interaction Media; BIA Separations, Ljubljana, Slovenia) monolithic columns for isolation of DNA from food was studied. Maize and its derivates corn meal and thermally pretreated corn meal were chosen as model food. Two commercially available CIM disk columns were tested: DEAE (diethylaminoethyl) and QA (quaternary amine). Preliminary separations were performed with standard solution of salmon DNA at different pH values and different NaCl concentrations in mobile phase. DEAE groups and pH 8 were chosen for further isolations of DNA from a complex matrix-food extract. The quality and quantity of isolated DNA were tested on agarose gel electrophoresis, with UV-scanning spectrophotometry, and by amplification with real-time PCR. DNA isolated in this way was of suitable quality for further PCR analyses. The described method is also applicable for DNA isolation from processed foods with decreased DNA content. Furthermore, it is more effective and less time-consuming in comparison with the existing proposed methods for isolation of DNA from plant-derived foods.     

Rapid amplification of genetically modified organisms using a circular ferrofluid-driven PCR microchip

The use of genetically modified organisms (GMOs) as food and in food products is becoming more and more widespread. Polymerase chain reaction (PCR) technology is extensively used for the detection of GMOs in food products in order to verify compliance with labeling requirements. In this paper, we present a novel close-loop ferrofluid-driven PCR microchip for rapid amplification of GMOs. The microchip was fabricated in polymethyl methacrylate by CO₂ laser ablation and was integrated with three temperature zones. PCR solution was contained in a circular closed microchannel and was driven by magnetic force generated by an external magnet through a small oil-based ferrofluid plug. Successful amplification of genetically modified soya and maize were achieved in less than 13 min. This PCR microchip combines advantages of cycling flexibility and quick temperature transitions associated with two existing microchip PCR techniques, and it provides a cost saving and less time-consuming way to conduct preliminary screening of GMOs. Figure Schematic of the circular ferrofluid-driven PCR microchip     

Taqman-MGB实时荧光PCR定量检测转基因玉米MON863

运用实时荧光聚合酶链式反应技术(polymerase chain reaction,PCR)对转基因玉米MON863进行品种特异性检测和定量分析。通过设计玉米内源基因和外源基因边界序列特异性引物和Taqman-MGB探针,验证了内源基因的物种特异性和外源基因边界序列的品种特异性。利用已知转基因百分含量的MON863玉米作为标准品,进行荧光定量反应,建立定量标准曲线,通过标准曲线对玉米样品MON863玉米成份进行含量分析。结果表明,该方法重复性好,检测特异性强,最低检测限浓度达到0.001 ng/μL,即14个拷贝。由于使用实时荧光PCR技术,检测周期短,操作简便,可广泛运用于转基因玉米MON863的进出口检测和转基因产品的含量分析。     

Validation of an HPLC method for quantitation of MDMA in tablets

An isocratic reversed-phase high-performance liquid chromatography (HPLC) method is developed and validated for the quantitation of 3,4-methylenedioxymethamphetamine (MDMA) in tablets. The chromatographic separation is achieved with potassium phosphate buffer (pH 3.2)-acetonitrile (9:1, v/v) as mobile phase, a Chromspher B column, and UV detection at 210 nm. The calibration curve is linear from 1.4 to 111 microg/mL. The percent relative standard deviation for intra- and interday precision studies is 2.7% each. The measurement uncertainty is estimated to 9%. The method is specific and successfully used for routine quantitation of MDMA in tablets.     

Development of a qualitative, multiplex real-time PCR kit for screening of genetically modified organisms (GMOs)

The number of commercially available genetically modified organisms (GMOs) and therefore the diversity of possible target sequences for molecular detection techniques are constantly increasing. As a result, GMO laboratories and the food production industry currently are forced to apply many different methods to reliably test raw material and complex processed food products. Screening methods have become more and more relevant to minimize the analytical effort and to make a preselection for further analysis (e.g., specific identification or quantification of the GMO). A multiplex real-time PCR kit was developed to detect the 35S promoter of the cauliflower mosaic virus, the terminator of the nopaline synthase gene of Agrobacterium tumefaciens, the 35S promoter from the figwort mosaic virus, and the bar gene of the soil bacterium Streptomyces hygroscopicus as the most widely used sequences in GMOs. The kit contains a second assay for the detection of plant-derived DNA to control the quality of the often processed and refined sample material. Additionally, the plant-specific assay comprises a homologous internal amplification control for inhibition control. The determined limits of detection for the five assays were 10 target copies/reaction. No amplification products were observed with DNAs of 26 bacterial species, 25 yeasts, 13 molds, and 41 not genetically modified plants. The specificity of the assays was further demonstrated to be 100% by the specific amplification of DNA derived from reference material from 22 genetically modified crops. The applicability of the kit in routine laboratory use was verified by testing of 50 spiked and unspiked food products. The herein described kit represents a simple and sensitive GMO screening method for the reliable detection of multiple GMO-specific target sequences in a multiplex real-time PCR reaction.     

Comparison of two methods for ginsenosides quantitation

The present study provides a comparison of two liquid chromatography–tandem mass spectrometry methods for ginsenosides analysis. The two methods have the same liquid chromatography separation procedure, and both use tandem mass spectrometry detection. However, one method uses multiple reaction monitoring transitions commonly recommended in the literature starting with [M + Na]⁺ as the molecular ions and with detection of specific fragment ions from the molecules M, while the other is an original method using [M + Cs]⁺ as molecular ions and Cs⁺ as fragment ion. The method using [M + Cs]⁺ as molecular ion has a very high sensitivity allowing the measurement of concentrations in the injecting solutions as low as 4 ng/ml with peaks at this concentration showing signal to noise ratio of 20 or higher. The procedures were utilized for the measurement of eight ginsenosides (Rb1, Rb2, Rc, Rd, Re, Rf (S), Rg1, and Rg2), although the method using [M + Cs]⁺ has the potential for measuring other ginsenosides. As an application, the ginsenosides were measured in several types of ginseng root, several dietary supplements containing ginseng extracts, four energy drinks, and a sample of ashwagandha.     

Using multiple PCR and CE with chemiluminescence detection for simultaneous qualitative and quantitative analysis of genetically modified organism

In this paper, an ultrasensitive CE-CL detection system coupled with a novel double-on-column coaxial flow detection interface was developed for the detection of PCR products. A reliable procedure based on this system had been demonstrated for qualitative and quantitative analysis of genetically modified organism-the detection of Roundup Ready Soy (RRS) samples was presented as an example. The promoter, terminator, function and two reference genes of RRS were amplified with multiplex PCR simultaneously. After that, the multiplex PCR products were labeled with acridinium ester at the 5'-terminal through an amino modification and then analyzed by the proposed CE-CL system. Reproducibility of analysis times and peak heights for the CE-CL analysis were determined to be better than 0.91 and 3.07% (RSD, n=15), respectively, for three consecutive days. It was shown that this method could accurately and qualitatively detect RRS standards and the simulative samples. The evaluation in terms of quantitative analysis of RRS provided by this new method was confirmed by comparing our assay results with those of the standard real-time quantitative PCR (RT-QPCR) using SYBR Green I dyes. The results showed a good coherence between the two methods. This approach demonstrated the possibility for accurate qualitative and quantitative detection of GM plants in a single run.