Monitoring of genetically modified food and feed in the Tunisian market using qualitative and quantitative real-time PCR

Genetically modified organisms (GMO) invade more and more the agricultural production in the world. Although there are no legislations on GM labeling and cultivation of GM crops in Tunisia, the present study aims to check the status of GMO in Tunisian market using qualitative and quantitative real time-PCR (QRT-PCR). Three-hundred-sixty five samples were collected and different DNA extraction methods were adapted and optimized. Specific primers targeting 35S promoter from Cauliflower mosaic virus (CaMV) and nopaline synthase terminator from Agrobacterium tumefaciens (At) were used for the detection of the GMO insert and Taxon specific primers for the detection of plant species. Validated Taqman® probes (EU-RL) targeting event specific regions of the maize events MON810, Bt11, and the soybean event RRS were used for the quantification studies. Seven food and feed products showed different amounts of RRS (1.9%), MON810 (2.1%), and Bt11 (1.6%). The results demonstrate for the first time the presence of GMO in Tunisian markets reinforcing the need for the development of accurate quantitative methods in routine analyses.     

Efficient retrieval of recombinant sequences of GM plants by Cauliflower Mosaic Virus 35S promoter-based bidirectional LT-RADE

Identification of genetically modified (GM) plants remains a difficult task, especially when few information on the GM content in a sample is available. The CaMV P-35S is a commonly applied genetic element in GM plants and represents as such a suitable starting point for the tracing of GM plants. Here, the application of a CaMV P-35S LT-RADE genome walking method on five different GM crops is documented: MON810 maize, LLRICE62 rice, T45 rapeseed, A2704-12 soybean and LLCOTTON25 cotton. Two sets of oligonucleotide primers are presented as a potential forward and reverse genome walking strategy with genomic DNA as template. The method’s applicability for assessing the recombinant nature of P-35S positive signals is discussed in detail. This study demonstrates the general use of LT-RADE in molecular characterization of GMOs retrieving several previously unknown transgenic insert nucleotide sequences. Current limitations and future developments are discussed.     

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.     

Development and in‐house validation of the event‐specific qualitative and quantitative PCR detection methods for genetically modified cotton MON15985

BACKGROUND: To implement genetically modified organism (GMO) labeling regulations, an event‐specific analysis method based on the junction sequence between exogenous integration and host genomic DNA has become the preferential approach for GMO identification and quantification. RESULTS: In this study, specific primers and TaqMan probes based on the revealed 5′‐end junction sequence of GM cotton MON15985 were designed, and qualitative and quantitative polymerase chain reaction (PCR) assays were established employing the designed primers and probes. In the qualitative PCR assay, the limit of detection (LOD) was 0.5 g kg^?1 in 100 ng total cotton genomic DNA, corresponding to about 17 copies of haploid cotton genomic DNA, and the LOD and limit of quantification (LOQ) for quantitative PCR assay were 10 and 17 copies of haploid cotton genomic DNA, respectively. Furthermore, the developed quantitative PCR assays were validated in‐house by five different researchers. Also, five practical samples with known GM contents were quantified using the developed PCR assay in in‐house validation, and the bias between the true and quantification values ranged from 2.06% to 12.59%. CONCLUSION: This study shows that the developed qualitative and quantitative PCR methods are applicable for the identification and quantification of GM cotton MON15985 and its derivates. Copyright © 2009 Society of Chemical Industry     

Investigation of the use of rolling circle amplification for the detection of GM food

We describe a study on the use of rolling circle amplification (RCA) for detecting GM event-specific motifs within short PCR amplicons, synthetic oligonucleotides, and extracted plant genomic DNA targets, as an alternative to the polymerase chain reaction (PCR). PCR-based detection has limitations that include the cost of reagents and equipment, and the potential for erroneous amplification of a contaminant. Our results reveal that RCA enables discrimination between the wild type (wt) and GM motifs when the sequences are within short PCR amplicons or synthetic oligonucleotides, but not within plant genomic DNA. These findings highlight the potential problem with implying the success of an assay when illustrated using model systems, rather than with the plant genomic target DNAs. The GM motifs selected for our studies were within Roundup Ready^TM Soya (RRS) and MON810 maize. Although knowledge of the target sequence is a prerequisite for the function of this assay, the potential of using RCA is explored.     

Detection system of stacked genetically modified maize using multiplex PCR

A multiplex polymerase chain reaction (PCR) method was developed to identify and distinguish 3 kinds of stacked genetically modified (GM) maize (MON810× MON863, NK603×MON863, and NK603×MON810× MON863). Four primer pairs, SSIIb JHF/JHR, C3b 5′/TAP1–3′, HS01/cry-CR01, and HS01/CTP164-3′ yielded 101, 129, 194, and 314 bp amplicons, respectively, Using the genomic DNA of the 3 stacked GM maize as templates, 3 or 4 corresponding PCR amplicons were amplified with similar band intensities by the multiplex PCR. The limit of detection (LOD) was approximately 0.5% for 3 kinds of stacked GM maize, using the multiplex PCR. The detection system using multiplex PCR developed in this study may be applicable to monitoring, identifying, and distinguishing not only the stacked GM maizes but also other stacked genetically modified organisms (GMOs).     

Generation of Reference Material by the Use of Multiple Displacement Amplification (MDA) for the Detection of Genetically Modified Organisms (GMOs)

The identification of genetically modified (GM) organisms (GMOs) in unknown samples by polymerase chain reaction (PCR) requires the use of a positive control sample, containing the target sequence derived from the respective GMO. For this purpose, either DNA extracted from suitable reference material or plasmids bearing the sequence are used. In the case of isolated genomic DNA, the preparation is cost-intensive and time-consuming, and material availability may be limited. Once the sequence is cloned into a plasmid, it may be simpler and less cost-intensive to purify the DNA, but contamination risk is substantially higher. The potential of multiple displacement amplification (MDA) as a new tool to generate reference material for GMO detection was studied taking the GM maize MON810 as a case. MDA yield of maize-specific DNA and amplification efficiency in dependence of the amount of starting material were estimated using real-time PCR. Applicability of the amplified DNA for the use as reference material was tested with regard to real-time PCR performance and MDA bias. Depending on the amount of genomic DNA (gDNA) input into MDA, amplification rates in the range of 30-fold (100 ng input of gDNA) to 23,000-fold (0.1 ng input of gDNA) were achieved. Real-time PCR performance and gene representation of amplified DNA (mdaDNA) are comparable to those of gDNA. Our results demonstrate that the DNA amplified by MDA is suitable for the use as reference material for qualitative GMO analysis.     

A simple DNA extraction method suitable for PCR detection of genetically modified maize

BACKGROUND: The polymerase chain reaction (PCR) is a powerful tool that is being increasingly used for detection of transgenic DNA. PCR requires only a minute quantity of template, but sensitive and accurate testing requires DNA of sufficient purity and free from inhibitors such as plant polysaccharides. Several standard protocols are available for this purpose, but they usually involve several steps, imply destruction of the maize kernel, or are time‐consuming. Our aim was to develop a fast and simple extraction method to isolate a raw DNA‐containing solution from maize tissues suitable for use as a template in a PCR‐based detection assay with specific oligonucleotides directed to the identification of event MON810. RESULTS: The NaOH‐based DNA extraction method we report here is time‐saving (5 min) and can be used to isolate DNA‐containing solutions from a small maize leaf portion (down to 1 mg) or from a single overnight‐germinated kernel. PCR performed with selected primers yielded reproducible detection of transgenic DNA. CONCLUSION: The main advantages of the procedure are the quick extraction step, the possibility of non‐destructive testing of maize kernels, and the robustness of the PCR‐based detection, a consequence of the selection of MON810‐matching oligonucleotides yielding intense and highly specific amplicons. Copyright © 2007 Society of Chemical Industry     

5'-Nuclease PCR for quantitative event-specific detection of the genetically modified Mon810 MaisGard maize

Genetically modified maize is grown extensively in the world today. MaisGard (Monsanto, Yieldgard in the USA) is a genetically modified maize harbouring the Mon810 transformation event. European Community legislation requires that genetically modified organisms (GMOs) be approved before they are placed on the market. Labelling is required when more than 1% of any ingredient of a food originates from a GMO. There is consequently a need for specific, quantitative methods for detection of genetically modified foods. We have determined the DNA sequence of the 5'-flanking region of the Mon810 insert using ligation mediated PCR. A primer probe set overlapping the junction was designed and used in a quantitative, event-specific Taqman 5'-nuclease assay. Mon810 DNA was quantified relative to endogenous maize zein gene DNA. The results were expressed as the percentage of genetically modified Mon810 maize DNA relative to the total content of maize DNA.     

Validation of a Real-Time PCR On-Site Quantification Method for MON810 Maize

A rapid on-, or near-site, quantitative method for use as a pre-harvest predictive decision, or co-existence monitoring, tool for adventitious genetically modified (GM) presence has been developed. Based on a laboratory-based protocol for real-time (RT) quantification of the MON810 GM event in maize kernels, the duplex RT polymerase chain reaction method was constructed around the portable Cepheid SmartCyclerII platform, requiring only modest support infrastructure for field application. Validation through an international ring trial showed good compliance with minimum assay performance requirements as defined by the European Network of GMO Laboratories (RSD_r = 18.5%; RSD_R = 32.8; Bias = 26.7%).     

Statistical Evaluation of Real-Time PCR Protocols Applied to Quantify Genetically Modified Maize

Real-time PCR (RTi-PCR) is the technique of choice for event-specific quantification of genetically modified organisms (GMOs) by determining the amount of event with respect to a species-specific reference gene. Reference genes can be amplified from the genome extracted from Certified Reference Materials (CRMs) or from ad hoc designed plasmids. In the present study, we statistically evaluate the performance of RTi-PCR protocols for GM maize MON810 event by using both genomic DNA from conventional CRMs and a plasmid containing sequences representative of four maize species-specific reference genes. The significance of simple and interaction effects of several variables included in the experimental design on DNA quantification methods and RTi-PCR were evaluated and discussed. Statistically significant differences on Ct values may have an impact on the GMOs quantification and consequently on the compliance of GM quantification-established legal thresholds. Our results confirm the reliability of the plasmid as alternative calibrant for the calculation of GMOs copy number.     

A Qualitative Approach for the Assessment of the Genetic Stability of the MON 810 Trait in Commercial Seed Maize Varieties

Maize MON 810 is one of the European Union’s (EU) authorized genetically modified organisms (GMO) for placing on the food and feed market. The total number of MON 810 varieties registered in the European Common Catalogue of varieties of agricultural plant species has almost tripled since 2005. One of the requirements described in EU legislation, namely the genetic stability of GM seed varieties, was thus assessed by analyzing the intactness of the entire MON 810 integration and its genotypic stability in commercial varieties available on the market for at least the last 2 years. A combined strategy using qualitative analytical methods made possible to determine the presence/absence of the individual genetic elements and of the whole GM construct. The restriction fragment length polymorphism patterns obtained from amplified whole constructs by long polymerase chain reaction (PCR) were compared side by side. CryIA(b) protein expression levels were determined by enzyme-linked immunosorbent assay. Twenty-four out of the 26 analyzed varieties met the expected stability features. One variety gave negative results in all assays, and one variety contained the necessary genetic elements for expressing CryIA(b) protein although giving negative results for the long PCR product. To our knowledge, this study is the first post-marketing stability analysis performed on GM commercial seed varieties.     

Monitoring of Bt11 and Bt176 genetically modified maize in food sold commercially in Brazil from 2005 to 2007

BACKGROUND: The first genetically modified (GM) maize lines were approved for trading in Brazil after December 2007 and they were T25, MON810, Bt11, NK603 and GA21. The polymerase chain reaction (PCR) method was employed to monitor the presence of Bt11 and nested PCR was used to detect the presence of Bt176 in 81 maize‐derived products (maize flour, corn meal, maize flour flakes and polenta) that were sold in Brazilian market from 2005 to 2007, before the release of GM maize in Brazil. RESULTS: The PCR detection limit for Bt11 was 10 g kg^?1 and for nested PCR of Bt176 it was 1 g kg^?1. All Brazilian samples analyzed showed no positive signal for these GM maize events. CONCLUSION: Bt11 and Bt176 GM maize lines were not detected by specific PCR in 81 maize‐derived food samples sold in Brazil from 2005 to 2007, before the commercial release of GM maize in Brazil. These Brazilian food industries were in compliance with the rules stipulated by the current legislation with respect to consumer requirements about GMO labeling. Copyright © 2010 Society of Chemical Industry     

Detection of eight GMO maize events by qualitative, multiplex PCR and fluorescence capillary gel electrophoresis

Specific legislation in the EU and several other countries requires that foods containing genetically modified organisms (GMOs) should be approved and labelled. This has necessitated the development of methods for detection of such materials. For screening purposes these methods should preferably enable detection of several different GMOs. Here we present a simple, robust, qualitative, nineplex PCR method for event-specific detection of maize T25, GA21, TC1507, MON863, MON810, NK603, construct specific detection of BT176, BT11 and detection of the endogenous hmga maize reference gene. PCR is carried out with primers labelled with fluorescent groups and the amplicons are detected using fluorescence capillary electrophoresis. Using mixtures of DNA from different certified reference materials, the detection limit was determined to approximately 0.1% for each GMO. Good agreement was observed in 85 of 88 determinations when eleven food and feed samples were analysed using the multiplex PCR assay and compared to results from quantitative real-time 5′-nuclease PCR. Discrepancies were only observed for one GMO at or close to the detection limit. The presented method is therefore suitable for screening purposes for food and feed containing the most common maize GMOs.     

实时荧光PCR定量检测加工产品中转基因玉米Mon810成分

采用实时荧光PCR技术，建立了定量(性)鉴定检测加工产品中转基因玉米Mon810成分的方法。实验设计的可以扩增玉米自身基因和外源基因边界序列的引物和探针具有品种和品系特异性，特异性地检测出食品、饲料等加工产品中转基因玉米Mon810成分。某些检测样品不仅检出转基因玉米Mon810成分，还同时检出其它转基因玉米品系或其它转基因品种。本研究实验建立的转基因玉米Mon810品系鉴定检测方法，即可以用于加工产品中转基因成分的定量检测(检测低限为0．1％)，也可以用于定性检测，或作为常规PCR定性检测后的确证实验方法。     

Quantitative,multiplex ligation-dependent probe amplification for the determination of eight genetically modified maize events

Specific legislation in the EU requires that foods containing more than 0.9% of genetically modified organisms (GMOs) should be labelled. To this end, we have developed a robust, quantitative, sensitive, nine-plex ligation-dependent probe amplification method, GMO-MLPA, for event-specific detection of maize TC1507, MON810, NK603, MON863, BT176, T25, GA21, construct-specific detection of BT11, and detection of the endogenous hmga maize reference gene. Ligated probes are amplified by PCR. Amplicons are detected using capillary electrophoresis. Specific GMO signals are normalised relative to the signal from the endogenous hmga gene and quantified by comparing with known standard curves. The method is suitable for quantification in the 0–2% range. Agreement was obtained in 149 of 160 determinations when 11 known mixtures of GMO and 9 food and feed samples were analysed using the GMO-MLPA method and compared to results from quantitative real-time 5′-nuclease PCR. The presented method is, therefore, suitable for quantification purposes for food and feed containing the most common maize GMOs.     

Molecular Identification of Four Genetically Modified Maize (Bt11, Bt176, Mon810 and T25) by Duplex Quantitative Real-Time PCR

In response to the increasing number of genetically modified (GM) events released on the market, control laboratories explore various strategies to simplify and reduce the number of tests needed to characterise the content in genetically modified organism (GMO) of a given sample. Lastly, multiplexing is considered as one of the possible ways to decrease the time and cost of analysis. Here, we report the development of four duplex polymerase chain reaction (PCR) tests for the identification and the quantification of four maize transformation events from which commercial lines have been authorised in Europe namely, Bt11 and Bt176 (Syngenta, DE, USA), Mon810 MaisGard? (Monsanto, MO, USA) and T25 Liberty Link? (Bayer CropScience, Monheim, Germany). The duplex PCR tests combine a maize-specific PCR test hybridising in the Adh1 locus with an event-specific detection system designed on a junction fragment for each of these four GM maize. Real-time PCR tests, suitable to comply with the European regulation, were designed by using Taqman® chemistry.     

Assessing Copy Number of MON 810 Integrations in Commercial Seed Maize Varieties by 5′ Event-Specific Real-Time PCR Validated Method Coupled to 2^{ - \Delta \Delta CT} Analysis

The objective of the present study was to assess the applicability of the MON 810 5′ event-specific method validated by the Community Reference Laboratory for Genetically Modified Food and Feed that is commonly used for quantitative purposes. This 5′ event-specific/hmg-taxon gene real-time polymerase chain reaction (PCR) protocol coupled to analysis was the chosen approach to determine the MON 810 insert copy number per haploid genome across 26 genetically modified commercial maize varieties. Variety DK 513 containing one copy integration per haploid genome was used as calibrator in each assay. Complementary data from end-point real-time PCRs that targeted specifically the MON 810 insert were also analyzed. Global results assessed and guaranteed the genetic intactness of the transgenic integration per haploid genome for 24 out of the 26 commercial varieties studied, which showed no significant differences between values respect to the calibrator value. Conversely, two varieties showed no intact transgenic insert in their genomes. This validated analytical method was suitable for MON 810 detection and quantification purposes.     

Simple,sensitive, accurate multiplex quantitative competitive PCR with capillary electrophoresis detection for the determination of genetically modified maize

Legislation in the EU requires that foods containing more than 0.9% of genetically modified organisms (GMOs) should be labelled. To this end, we have developed a simple and accurate capillary electrophoresis multiplex quantitative competitive PCR (ce-mqcPCR) method for event-specific quantification of the five novel GM maize events DAS59122, LY038, MON88017, MIR604 and Event 3272. The method combines the simplicity of constructing multiple competitors in silico with the high resolution and sensitivity of fluorescence capillary electrophoresis and the use of an internal template reference amplicon. The competitors are synthesised commercially and added in equal amounts as a restriction enzyme-digested plasmid insert to the multiplex PCR. Quantification is performed by analysing the relative amounts of GMO and GMO competitor fragment pairs after capillary electrophoresis and correcting for differences in maize DNA by comparing with the internal reference gene amplicon. Since the competitors employ the same primers as their corresponding targets, all existing qualitative multiplex PCRs can in principle easily be converted to quantitative assays without changing primer sets or amplification conditions. The ce-mqcPCR method correctly determined 120 GMO templates in known mixed samples. No false-positive or false-negative signals were obtained.     

Detection of genetically modified organisms in foreign-made processed foods containing corn and potato

Investigations of the validity of labeling regarding genetically modified (GM) products were conducted using polymerase chain reaction (PCR) methods for foreign-made processed foods made from corn and potato purchased in the Tokyo area and in the USA. Several kinds of GM crops were detected in 12 of 32 samples of processed corn samples. More than two GM events for which safety reviews have been completed in Japan were simultaneously detected in 10 samples. GM events MON810 and Bt11 were most frequently detected in the samples by qualitative PCR methods. MON810 was detected in 11 of the 12 samples, and Bt11 was detected in 6 of the 12 samples. In addition, Roundup Ready soy was detected in one of the 12 samples. On the other hand, CBH351, for which the safety assessment was withdrawn in Japan, was not detected in any of the 12 samples. A trial quantitative analysis was performed on six of the GM maize qualitatively positive samples. The estimated amounts of GM maize in these samples ranged from 0.2 to 2.8%, except for one sample, which contained 24.1%. For this sample, the total amount found by event-specific quantitative analysis was 23.8%. Additionally, Roundup Ready soy was detected in one sample of 21 potato-processed foods, although GM potatoes were not detected in any sample.