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.     

Real-time polymerase chain reaction detection of cauliflower mosaic virus to complement the 35S screening assay for genetically modified organisms

Labeling of genetically modified organisms (GMOs) is now in place in many countries, including the European Union, in order to guarantee the consumer's choice between GM and non-GM products. Screening of samples is performed by polymerase chain reaction (PCR) amplification of regulatory sequences frequently introduced into genetically modified plants. Primers for the 35S promoter from Cauliflower mosaic virus (CaMV) are those most frequently used. In virus-infected plants or in samples contaminated with plant material carrying the virus, false-positive results can consequently occur. A system for real-time PCR using a TaqMan minor groove binder probe was designed that allows recognition of virus coat protein in the sample, thus allowing differentiation between transgenic and virus-infected samples. We measured the efficiency of PCR amplification, limits of detection and quantification, range of linearity, and repeatability of the assay in order to assess the applicability of the assay for routine analysis. The specificity of the detection system was tested on various virus isolates and plant species. All 8 CaMV isolates were successfully amplified using the designed system. No cross-reactivity was detected with DNA from 3 isolates of the closely related Carnation etched ring virus. Primers do not amplify plant DNA from available genetically modified maize and soybean lines or from different species of Brassicaceae or Solanaceae that are natural hosts for CaMV. We evaluated the assay for different food matrixes by spiking CaMV DNA into DNA from food samples and have successfully amplified CaMV from all samples. The assay was tested on rapeseed samples from routine GMO testing that were positive in the 35S screening assay, and the presence of the virus was confirmed.     

Quantification of the 35S promoter in DNA extracts from genetically modified organisms using real-time polymerase chain reaction and specificity assessment on various genetically modified organisms, part I: operating procedure

A highly sensitive quantitative real-time assay targeted on the 35S promoter of a commercial genetically modified organism (GMO) was characterized (sF/sR primers) and developed for an ABI Prism 7700 Sequence Detection System and TaqMan chemistry. The specificity assessment and performance criteria of sF/sR assay were compared to other P35S-targeted published assays. sF/sR primers amplified a 79 base pair DNA sequence located in a part of P35S that is highly conserved among many caulimovirus strains, i.e., this consensus part of CaMV P35S is likely to be present in many GM events. According to the experimental conditions, the absolute limit of detection for Bt176 corn was estimated between 0.2 and 2 copies of equivalent genome (CEG). The limit of quantification was reached below 0.1% Bt176 content. A Cauliflower Mosaic Virus control (CaMV) qualitative assay targeted on the ORF III of the viral genome was also used as a control (primers 3F/3R) to assess the presence of CaMV in plant-derived products. The specificity of this test was assessed on various CaMV strains, including the Figwort Mosaic Virus (FMV) and solanaceous CaMV strains. Considering the performance of sF/sR quantification test, the highly conserved sequence, and the small size of the amplicon, this assay was tested in a collaborative study in order to be proposed as an international standard.     

Real-time and conventional PCR detection of Liberty Link® rice varieties and transgenic soy in rice sampled in the Mexican and American retail markets

Samples of rice from Mexican and USA retail stores were analyzed for the presence of transgenic (GM) events using real-time PCR. In screening for the CaMV35S promoter sequence (35SP), positive results were found in 49 and 35% of the Mexican and American samples, respectively. In further investigations in Mexican samples, 43% were positive for P35S::bar, with two above the quantifiable limit; these were 0.07% and 0.05% GMO. Fourteen out of the sixteen positive samples were labeled as imported from the USA. In testing samples bought in American retail shops, 24% showed positive results, all below the quantifiable range. It could be deduced that P35S::bar positive samples were Liberty Link(R) (LL) rice. In distinguishing between LL601 and LL62, end-point PCR was used, corroborating the P35S::bar amplicon length difference of these events. LL62 was found in one rice sample purchased in Mexico and two in the USA samples. Its presence was verified with the 35S terminator sequence. All other LL positive samples contained LL601. None of the samples analyzed showed the presence of Bt63 rice. The LL rice varieties found have been identified as not being commercially cultivated, and so their presence requires further investigation. 35SP was also present in samples which did not have any LL rice. Maize sequences could not be detected in any of the samples; however, soybean DNA was found in Mexican and USA rice samples. The Roundup Ready(R) trait was detected in trace amounts in 16 and 6% of the rice samples bought in Mexico and the USA, respectively. Real-time PCR was shown to be the method of choice for the sensitive and rapid screening of commodities and retail samples for the detection of GM and other contamination.     

转基因玉米的多重PCR-毛细管电泳-激光诱导荧光检测方法研究

采用三重PCR反应, 同时扩增CaMV 35S启动子、 hsp70 intron1和CryIA(b)基因之间序列以及Invertase基因, 扩增产物用无胶筛分毛细管电泳-激光诱导荧光检测, 从而建立了多重PCR-毛细管电泳-激光诱导荧光快速检测转基因玉米的新方法. 对影响多重PCR扩增和毛细管电泳的因素进行了优化. 在优化的条件下, 本方法可以同时检测转基因玉米样品中3种外源基因. 经序列测试证实, 三重PCR 扩增产物的序列与原基因完全一致, 表明扩增结果可靠. 该方法能检出0.05% MON810转基因玉米成分, 远低于欧盟对转基因食品规定标识的质量分数阈值(1％). 该方法对玉米及其制品的检测结果与实时荧光PCR方法的检测结果一致, 与传统的琼脂糖凝胶电泳法相比, 具有特异性高\, 快速及灵敏等优点, 适用于玉米中转基因成分以及转基因玉米MON810品系的快速筛选、 鉴定和检测, 能满足我国实施转基因食品标签法规的要求.     

Establishment of a system based on universal multiplex-PCR for screening genetically modified crops

The rapid development of many genetically modified (GM) crops in the past two decades makes it necessary to introduce an alternative strategy for routine screening and identification. In this study, we established a universal multiplex PCR detection system which will effectively reduce the number of reactions needed for sample identification. The PCR targets of this system include the six most frequently used transgenic elements: cauliflower mosaic virus (CaMV) 35S promoter, Agrobacterium tumefaciens nopaline synthase (nos) promoter, Agrobacterium tumefaciens nopaline synthase (nos) terminator, the neomycin phosphotransferase II (nptII) gene, the 5-enolpyruvylshikimate-3-phosphate synthase (CP4 epsps) gene of Agrobacterium tumefaciens strain CP4, and the phosphinothricin N-acetyltransferase (pat) gene. According to the AGBIOS database, the coverage of this detection system is 93% of commercial GM crops. This detection system could detect all certified reference materials (CRMs) at the 1.0% level. The correct combination of all the CRM amplicon patterns proved the specificity of this multiplex PCR system. Furthermore, the amplicon patterns of this multiplex PCR detection system could be used as an index of classification which will narrow the range of possible GM products. The simulation result of this multiplex PCR detection system on all commercialized 139 GM products in the AGBIOS database showed that the maximum number of PCR reactions needed to identify an unknown sample can be reduced to 13. In this study, we established a high-throughput multiplex PCR detection system with feasible sensitivity, specificity, and cost. By incorporating this detection system, the routine GM crop-detection process will meet the challenges resulting from a rapid increase in the number of GM crops in the future.     

DNA methods: critical review of innovative approaches

The presence of ingredients derived from genetically modified organisms (GMOs) in food products in the market place is subject to a number of European regulations that stipulate which product consisting of or containing GMO-derived ingredients should be labeled as such. In order to maintain these labeling requirements, a variety of different GMO detection methods have been developed to screen for either the presence of DNA or protein derived from (approved) GM varieties. Recent incidents where unapproved GM varieties entered the European market show that more powerful GMO detection and identification methods will be needed to maintain European labeling requirements in an adequate, efficient, and cost-effective way. This report discusses the current state-of-the-art as well as future developments in GMO detection.     

The detection of T-Nos,a genetic element present in GMOs,by cross-priming isothermal amplification with real-time fluorescence

An isothermal cross-priming amplification (CPA) assay for Agrobacterium tumefaciens nopaline synthase terminator (T-Nos) was established and investigated in this work. A set of six specific primers, recognizing eight distinct regions on the T-Nos sequence, was designed. The CPA assay was performed at a constant temperature, 63 °C, and detected by real-time fluorescence. The results indicated that real-time fluorescent CPA had high specificity, and the limit of detection was 1.06?×?10³ copies of rice genomic DNA, which could be detected in 40 min. Comparison of real-time fluorescent CPA and conventional polymerase chain reaction (PCR) was also performed. Results revealed that real-time fluorescent CPA had a comparable sensitivity to conventional real-time PCR and had taken a shorter time. In addition, different contents of genetically modified (GM)-contaminated rice seed powder samples were detected for practical application. The result showed real-time fluorescent CPA could detect 0.5 % GM-contaminated samples at least, and the whole reaction could be finished in 35 min. Real-time fluorescent CPA is sensitive enough to monitor labeling systems and provides an attractive method for the detection of GMO.

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.     

Interlaboratory trial validation of an event-specific qualitative polymerase chain reaction-based detection method for genetically modified RT73 rapeseed

The qualitative event-specific polymerase chain reaction detection method of genetically modified (GM) RT73 rapeseed was developed based on the cloned 3' end flanking sequence of RT73 rapeseed integration. The specificity of the method for GM RT73 rapeseed was validated using several different GM rapeseed lines, GM maize lines, GM soybean line, non-GM rapeseed, and other non-GM crops. In this study, the developed method was validated through an interlaboratory study by 12 laboratories from 6 countries. The sensitivity of this method was evaluated using several mixed rapeseed meals with different GM RT73 rapeseed contents from 5.0 to 0.01% prepared by our laboratory. The evaluated results showed that all of the rapeseed endogenous reference high mobility group protein gene (HMG I/Y), figwort mosaic virus 35S (FMV 35S) promoter, and RT73 event-specific fragment could be detected from rapeseed samples at 0.1% (w/w) with a confidence level of more than 95%. All results from the 12 laboratories indicated that the developed method could be considered fit for the detection and identification of GM RT73 rapeseed.     

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.     

Molecular toolbox for the identification of unknown genetically modified organisms

Competent laboratories monitor genetically modified organisms (GMOs) and products derived thereof in the food and feed chain in the framework of labeling and traceability legislation. In addition, screening is performed to detect the unauthorized presence of GMOs including asynchronously authorized GMOs or GMOs that are not officially registered for commercialization (unknown GMOs). Currently, unauthorized or unknown events are detected by screening blind samples for commonly used transgenic elements, such as p35S or t-nos. If (1) positive detection of such screening elements shows the presence of transgenic material and (2) all known GMOs are tested by event-specific methods but are not detected, then the presence of an unknown GMO is inferred. However, such evidence is indirect because it is based on negative observations and inconclusive because the procedure does not identify the causative event per se. In addition, detection of unknown events is hampered in products that also contain known authorized events. Here, we outline alternative approaches for analytical detection and GMO identification and develop new methods to complement the existing routine screening procedure. We developed a fluorescent anchor-polymerase chain reaction (PCR) method for the identification of the sequences flanking the p35S and t-nos screening elements. Thus, anchor-PCR fingerprinting allows the detection of unique discriminative signals per event. In addition, we established a collection of in silico calculated fingerprints of known events to support interpretation of experimentally generated anchor-PCR GM fingerprints of blind samples. Here, we first describe the molecular characterization of a novel GMO, which expresses recombinant human intrinsic factor in Arabidopsis thaliana. Next, we purposefully treated the novel GMO as a blind sample to simulate how the new methods lead to the molecular identification of a novel unknown event without prior knowledge of its transgene sequence. The results demonstrate that the new methods complement routine screening procedures by providing direct conclusive evidence and may also be useful to resolve masking of unknown events by known events.     

Qualitative PCR method for Roundup Ready soybean: interlaboratory study

Quantitative and qualitative methods based on PCR have been developed for genetically modified organisms (GMO). Interlaboratory studies were previously conducted for GMO quantitative methods; in this study, an interlaboratory study was conducted for a qualitative method for a GM soybean, Roundup Ready soy (RR soy), with primer pairs designed for the quantitative method of RR soy studied previously. Fourteen laboratories in Japan participated. Each participant extracted DNA from 1.0 g each of the soy samples containing 0, 0.05, and 0.10% of RR soy, and performed PCR with primer pairs for an internal control gene (Le1) and RR soy followed by agarose gel electrophoresis. The PCR product amplified in this PCR system for Le1 was detected from all samples. The sensitivity, specificity, and false-negative and false-positive rates of the method were obtained from the results of RR soy detection. False-negative rates at the level of 0.05 and 0.10% of the RR soy samples were 6.0 and 2.3%, respectively, revealing that the LOD of the method was somewhat below 0.10%. The current study demonstrated that the qualitative method would be practical for monitoring the labeling system of GM soy in kernel lots.     

A theoretical introduction to “Combinatory SYBR®Green qPCR Screening”, a matrix-based approach for the detection of materials derived from genetically modified plants

The detection of genetically modified (GM) materials in food and feed products is a complex multi-step analytical process invoking screening, identification, and often quantification of the genetically modified organisms (GMO) present in a sample. “Combinatory qPCR SYBR®Green screening” (CoSYPS) is a matrix-based approach for determining the presence of GM plant materials in products. The CoSYPS decision-support system (DSS) interprets the analytical results of SYBR®GREEN qPCR analysis based on four values: the C _t- and T _m values and the LOD and LOQ for each method. A theoretical explanation of the different concepts applied in CoSYPS analysis is given (GMO Universe, “Prime number tracing”, matrix/combinatory approach) and documented using the RoundUp Ready soy GTS40-3-2 as an example. By applying a limited set of SYBR®GREEN qPCR methods and through application of a newly developed “prime number”-based algorithm, the nature of subsets of corresponding GMO in a sample can be determined. Together, these analyses provide guidance for semi-quantitative estimation of GMO presence in a food and feed product.     

Validation of a newly developed hexaplex real-time PCR assay for screening for presence of GMOs in food, feed and seed

For years, an increasing number and diversity of genetically modified plants has been grown on a commercial scale. The need for detection and identification of these genetically modified organisms (GMOs) calls for broad and at the same time flexible high throughput testing methods. Here we describe the development and validation of a hexaplex real-time polymerase chain reaction (PCR) screening assay covering more than 100 approved GMOs containing at least one of the GMO targets of the assay. The assay comprises detection systems for Cauliflower Mosaic Virus 35S promoter, Agrobacterium tumefaciens NOS terminator, Figwort Mosaic Virus 34S promoter and two construct-specific sequences present in novel genetically modified soybean and maize that lack common screening elements. Additionally a detection system for an internal positive control (IPC) indicating the presence or absence of PCR inhibiting substances was included. The six real-time PCR systems were allocated to five detection channels showing no significant crosstalk between the detection channels. As part of an extensive validation, a limit of detection (LOD_abs) ≤ ten target copies was proven in hexaplex format. A sensitivity ≤ ten target copies of each GMO detection system was still shown in highly asymmetric target situations in the presence of 1,000 copies of all other GMO targets of each detection channel. Furthermore, the applicability to a broad sample spectrum and reliable indication of inhibition by the IPC system was demonstrated. The presented hexaplex assay offers sensitive and reliable detection of GMOs in processed and unprocessed food, feed and seed samples with high efficiency.     

A homogeneous assay for highly sensitive detection of CaMV35S promoter in transgenic soybean by förster resonance energy transfer between nitrogen-doped graphene quantum dots and Ag nanoparticles

In this work, a novel homogeneous assay for DNA quantitative analysis based on förster resonance energy transfer (FRET) was developed for cauliflwer mosaic virus 35s (CaMV35S) promoter of transgenic soybean detection. The homogenous FRET of fluorescence signal was fabricated by DNA hybridization with probe modified nitrogen-doped graphene quantum dots (NGQDs) and silver nanoparticles (AgNPs), which acted the donor-acceptor pairs for the first time. The highly efficient FRET and unique properties of the NGQDs made the proposed FRET system as a functionalized detection platform for labelling of DNA. Upon the recognition of specific target DNA (tDNA), the FRET between NGQDs and AgNPs was triggered to produce fluorescence quenching, which could be used for tDNA detection. The fabricated homogeneous FRET assay displayed a wide linear range of 0.1–500.0 nM and a low limit of detection 0.03 nM for the detection of CaMV35S (S/N = 3). This proposed biosensor revealed high specificity to detect tDNA, with acceptable intra-assay precision and excellent stability. This method was successfully applied to identify the real sample of 0.5% containing transgenic soybean, which achieved the most of national law regulations. This assay was further validated by polymerase chain reaction as the genetically modified organisms, suggesting that the proposed FRET system is a feasible tool for the further daily genetically modified organism detection.     

New trends in bioanalytical tools for the detection of genetically modified organisms: an update

Despite the controversies surrounding genetically modified organisms (GMOs), the production of GM crops is increasing, especially in developing countries. Thanks to new technologies involving genetic engineering and unprecedented access to genomic resources, the next decade will certainly see exponential growth in GMO production. Indeed, EU regulations based on the precautionary principle require any food containing more than 0.9% GM content to be labeled as such. The implementation of these regulations necessitates sampling protocols, the availability of certified reference materials and analytical methodologies that allow the accurate determination of the content of GMOs. In order to qualify for the validation process, a method should fulfil some criteria, defined as “acceptance criteria” by the European Network of GMO Laboratories (ENGL). Several methods have recently been developed for GMO detection and quantitation, mostly based on polymerase chain reaction (PCR) technology. PCR (including its different formats, e.g., double competitive PCR and real-time PCR) remains the technique of choice, thanks to its ability to detect even small amounts of transgenes in raw materials and processed foods. Other approaches relying on DNA detection are based on quartz crystal microbalance piezoelectric biosensors, dry reagent dipstick-type sensors and surface plasmon resonance sensors. The application of visible/near-infrared (vis/NIR) spectroscopy or mass spectrometry combined with chemometrics techniques has also been envisaged as a powerful GMO detection tool. Furthermore, in order to cope with the multiplicity of GMOs released onto the market, the new challenge is the development of routine detection systems for the simultaneous detection of numerous GMOs, including unknown GMOs.     

Rapid analysis of genetically modified organisms by in-house developed capillary electrophoresis chip and laser-induced fluorescence system

A microfabricated, inexpensive, reusable glass capillary electrophoresis chip and a laser-induced fluorescence system were developed in-house for the rapid DNA-based analysis of genetically modified organisms (GMOs). The 35S promoter sequence of cauliflower mosaic virus and the terminator of the nopaline synthase (NOS) gene from Agrobacterium tumefaciens were both detected since they are present in most genetically modified organisms. The detection of genetically modified soybean in the presence of unaltered soybean was chosen as a model. Lectin, a plant-specific gene, was also detected for confirmation of the integrity of extracted DNA. The chip was composed of two glass plates, each 25 x 76 mm, thermally bonded together to form a closed structure. Photomasks with cross-topology were prepared rapidly by using polymeric material instead of chrome plates. The widths of the injection and separation channels were 30 and 70 microm, respectively, the effective separation length 4.5 cm. The glass slide was etched to a depth of 30 microm for both the injection and separation channel. The cost of the chip was less than 1 $ and required 2 days for photomask preparation and microfabrication. The separation and detection of polymerase chain reaction-amplified NOS, 35S, and lectin sequences (180, 195, and 181 bp, respectively) was completed in less than 60 s. As low as 0.1% GMO content was detectable by the proposed system after 35 and 40 amplification cycles for 35S and NOS, respectively, using 25 ng of extracted DNA as starting material. This corresponds to only 20 genome copies of genetically modified soybean.     

Detection of genetic modification in cured tobacco leaf: proficiency testing using polymerase chain reaction-based methods

The Cooperation Centre for Scientific Research Relative to Tobacco (CORESTA; Paris, France) "Task Force Genetically Modified Tobacco-Detection Methods" investigated the performance of qualitative and quantitative methods based on the polymerase chain reaction (PCR) for the detection and quantitation of genetically modified (GM) tobacco. In the 4 successful rounds of proficiency testing, the cauliflower mosaic virus 35S RNA promoter (CaMV 35S) and the Agrobacterium tumefaciens nopaline synthase terminator (NOS) were selected as target sequences. Blind-coded reference materials containing from 0.1 to 5.0% and from 0.15 to 4% GM tobacco were used in 2 rounds of qualitative and quantitative PCR, respectively. Eighteen laboratories from 10 countries participated in this study. Considering all methods and 2 rounds, the different laboratories were able to detect GM tobacco at the 0.1% level in 46 out of 58 tests in qualitative assays. The results of the proficiency test indicate that both end point screening and real-time quantitative methods are suitable for the detection of genetically modified organisms in tobacco leaf samples having a GM content of 0.1% or higher. The CORESTA proficiency study represents a first step towards the interlaboratory evaluation of accuracy and precision of PCR-based GM tobacco detection, which may lead to the harmonization of analytical procedures and to the enhancement of comparability of testing results produced by different laboratories.