LT-RADE: An Efficient User-Friendly Genome Walking Method Applied to the Molecular Characterization of the Insertion Site of Genetically Modified Maize MON810 and Rice LLRICE62

Information on the insertion site and characterization of the transgene(s) in genetically modified organisms (GMO) is very important for safety assessment and identification of a GMO. The generation of such information in general and in particular in emergencies or rapid alert situations involving GMO greatly benefit from the availability of simple, efficient, and rapid approaches. Here, we report on the improvement of a restriction independent method named “Rapid Amplification of genomic DNA Ends” (RADE). The method was developed using maize event MON810 genomic DNA as a model system, testing a standard Taq polymerase or a blend of polymerases (standard Taq and proofreading Tgo polymerases (LT-RADE)). Both methods produce an initial single strand DNA, followed by nested PCR steps and yield easy-to-isolate DNA fragments for further manipulation. We showed that the application of the Taq/Tgo polymerase blend significantly increased the size of the obtained PCR products. Using LT-RADE, we could successfully isolate the flanking regions of the transgenic insert of the GM maize event MON810 and confirmed the existing data on the adjacent regions of the insert. In addition, application of our approach allowed to efficiently isolate and identify, for the first time, the DNA sequences surrounding the insert of GM rice event LLRICE62.     

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.     

Development of a modular system for detection of genetically modified organisms in food based on ligation-dependent probe amplification

The application of a method based on ligation-dependent probe amplification to the simultaneous detection of different genetically modified organisms in food is described. The ligation reaction of target-specific probes with characteristic lengths and universal primer binding sites is followed by PCR amplification using fluorescein-labeled primers. The separation and the detection of DNA fragments are achieved by capillary electrophoresis via laser-induced fluorescence. The approach allows the simultaneous detection of several targets corresponding to different levels of specificity in a one-tube assay. Synthetic oligonucleotides were designed to detect (1) reference genes in the genome from maize, soya and rapeseed, (2) the CaMV 35S-promotor as screening element, (3) the construct-specific 35S-pat junction, and (4) the event-specific regions of the transgenic maize line MON 810 and of Roundup Ready soya. Specificity and sensitivity (GMO content 0.1%) of the approach were shown for all targets. This novel analytical strategy represents a flexible, modular system for the surveillance of GMO-derived products that can be readily complemented by further target sequences of interest.     

进口薯格中转基因成分的检测

目的建立了一种快速检测进出口食品中转基因成分的方法。方法本实验采用DNA提取试剂盒对薯格中的DNA进行快速提取,接着用实时荧光PCR方法对其进行转基因成分和品系的鉴定。结果通过对食品标签进行初筛,发现其标识成分中含有未标明的转基因成分。进一步对所含转基因成分的品系进行鉴定,确定薯格的外包裹玉米粉中含有多种转基因成分,包括转基因玉米TC1507、NK603、MON810、59122、MON89034等5个品系。结论本文方法可以用于加工食品中转基因玉米成分及品系的定性检测,也可以作为常规PCR定性方法的确证试验方法。     

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).     

Determination of eight genetically modified maize events by quantitative, multiplex PCR and fluorescence capillary gel electrophoresis

Specific legislation in the EU requires that foods containing more than 0.9% of genetically modified organisms (GMOs) should be labelled. This has necessitated the development of methods for detection and quantification of such materials. Here we present a robust, quantitative, 9-plex PCR method for event-specific detection of maize TC1507, MON863, MON810, T25, NK603, GA21, construct specific detection of BT11, BT176 and detection of the endogenous hmga maize reference gene. The method is suitable for quantification in the 0–2% range with a detection limit of approximately 0.1%. PCR is carried out in two stages. In the first stage, bipartite primers containing a universal 5′-sequence and a GMO specific 3′-sequence are used. In the second PCR stage only a universal primer is used. Trypsin digestion between the first and second PCR stages enhances signal strength and reproducibility. Probes hybridising to the PCR amplicons are then labelled by primer extension and detected by fluorescence capillary electrophoresis. Good agreement was observed in 76 of 80 determinations when 10 food and feed samples were analysed using the multiplex PCR assay 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.     

Feeding genetically modified maize (MON810) to dairy cows: comparison of gene expression pattern of markers for apoptosis, inflammation and cell cycle

Public concern has been expressed on the use of genetically modified (gm) maize MON810 as animal feed and for human consumption. Several studies have been conducted on potential effects of feeding MON810 to livestock focusing on animal performance, animal health and fate of recombinant DNA or protein. Though, no information on effects of feeding gm maize on the gene expression level is available so far. From 2005 until 2007, a study with 36 lactating dairy cows fed gm maize (N?=?18, MON810) or the near-isogenic counterpart (N?=?18) was carried out, investigating the fate of recombinant DNA and protein. After a period of 25?months, ten cows fed transgenic maize and seven cows fed near-isogenic maize were slaughtered due to operational reasons. In a short follow-up study, tissues of the gastrointestinal tract and samples from liver were used for gene expression analysis of major genes of the inflammation, cell cycle and apoptosis pathways. Statistical analysis of the examined gene expression pattern revealed no significant difference in the gene expression profile of cows fed transgenic or near-isogenic feed ration. Therefore, it can be assumed that compared to near-isogenic feed, gm maize MON810 does not have any effect on major genes involved in apoptosis, inflammation and cell cycle in the gastrointestinal tract and in the liver of dairy cows.     

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.     

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.     

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.     

Genetically modified maize and soybean on the Egyptian food market

The results of a survey study on food samples produced from genetically modified soybean and maize collected from the Egyptian market are presented. Forty samples of soybean and 40 samples of maize products have been gathered randomly from markets in Cairo and Giza. The genetic modification was detected by polymerase chain reaction (PCR) using official detection methods according to section 35 of the German Foodstuffs Act. Samples were investigated for the presence of material derived from the following genetically modified organisms (GMOs) all of which are approved for food use in Europe: Roundup Ready soybean (RRS) and maize lines Bt176, Bt11, T25 and MON810. In addition, samples were examined in qualitative and quantitative analysis for the presence of material derived from the transgenic maize line StarLink (Aventis) which was approved for animal feed use exclusively in the US. Twenty % of 40 investigated soy samples contained Roundup Ready soybean; 15% of 40 maize samples tested positive for Bt176 and 12.5% positive for Bt11 maize. Furthermore, the presence of StarLink maize could clearly be demonstrated in four samples mixed with Bt176 and Bt11. The percentage of StarLink was less than 1% in quantitative analysis. The maize lines T25 and MON810 were not detected.     

环介导等温扩增法检测转基因玉米MON89034

根据MON89034外源插入片段与植物基因组序列设计特异性引物,筛选最佳引物并对反应体系和反应条件进行优化,最终建立转基因玉米MON89034转化体特异性LAMP检测方法。对该方法进行了特异性、灵敏度、稳定性和重复性测试。结果表明:该方法能够特异性检测出MON89034玉米;检测其灵敏度达到1 pg;以转基因玉米MON89034 DNA标准品质量分数为1.00%,0.10%,0.05%的样品为模板,其稳定性好、重复性高,假阴性率为0。本试验设计的LAMP方法适用于特异性检测转基因玉米MON89034。     

转基因玉米品系及转化体成分四重实时荧光PCR快速鉴定

Bt11转基因玉米品系具有抗草铵膦除草剂,鳞翅目昆虫抗性的耐除草剂抗虫玉米,MIR162和Mon89034是鳞翅目昆虫抗性的单抗虫玉米,均是国内外出入境监管主要关注的转基因玉米品系。本研究通过靶标基因筛选,转基因阳性样品采集,核酸样本制备,多重引物和荧光探针组合筛选,反应体系优化以及方法学验证等过程开发建立了四重荧光定量PCR检测技术。结果表明该技术的使用可实现一个反应管中同时检测MIR162、Bt11、Mon89034三个玉米品系的特异性基因序列和一个编码玉米淀粉合成酶异构体zSTSII-2 (zSSIIb) 玉米内源基因。通过阳性对照,阴性对照和空白对照特异性S曲线与对应的阈值大小分析可判定样品中是否含有这三个转基因玉米品系及其转化体成分。经方法学特异性检测,结果与转基因检测金标准的单实时荧光PCR结果一致;经平行样和灵敏度测试,最低检测限为18个拷贝;经标准曲线扩增分析,四个基因的扩增效率均在90%~110%范围内,扩增效果良好。该方法从DNA提取到报告结果不足3小时,可缩短检测时限,节约试剂耗材成本,操作简单易行,满足高通量特征,可为市场流通产品品系和转基因成分的实时监管和快速鉴定提供技术支撑。     

Assessment of DNA degradation induced by thermal and UV radiation processing: Implications for quantification of genetically modified organisms

DNA quality is an important parameter for the detection and quantification of genetically modified organisms (GMO’s) using the polymerase chain reaction (PCR). Food processing leads to degradation of DNA, which may impair GMO detection and quantification. This study evaluated the effect of various processing treatments such as heating, baking, microwaving, autoclaving and ultraviolet (UV) irradiation on the relative transgenic content of MON 810 maize using pRSETMON-02, a dual target plasmid as a model system. Amongst all the processing treatments examined, autoclaving and UV irradiation resulted in the least recovery of the transgenic (CaMV 35S promoter) and taxon-specific (zein) target DNA sequences. Although a profound impact on DNA degradation was seen during the processing, DNA could still be reliably quantified by Real-time PCR. The measured mean DNA copy number ratios of the processed samples were in agreement with the expected values. Our study confirms the premise that the final analytical value assigned to a particular sample is independent of the degree of DNA degradation since the transgenic and the taxon-specific target sequences possessing approximately similar lengths degrade in parallel. The results of our study demonstrate that food processing does not alter the relative quantification of the transgenic content provided the quantitative assays target shorter amplicons and the difference in the amplicon size between the transgenic and taxon-specific genes is minimal.     

Detection of recombinant-DNA in foods from stacked genetically modified plants

A quantitative real-time multiplex polymerase chain reaction method was applied to the detection and quantification of MON863 and MON810 in stacked genetically modified maize MON 810xMON 863. The limit of detection was approximately 0,1%. The accuracy of the quantification, measured as bias from the accepted value and the relative repeatability standard deviation, which measures the intra-laboratory variability, were within 25% at each GM-level. A method verification has demonstrated that the MON 863 and the MON810 methods can be equally applied in quantification of the respective events in stacked MON810xMON 863.     

Ligation-dependent probe amplification for the simultaneous event-specific detection and relative quantification of DNA from two genetically modified organisms

The application of a ligation-dependent probe amplification (LPA) technique to the simultaneous event-specific detection and relative quantification of DNA from genetically modified organisms in foods is described. The system is based on the ligation of synthetic bipartite probes when hybridized to the corresponding target DNA sequence. The ligation products possess lengths characteristic for each target sequence. Universal primer binding sites (PBS) at the 5′ and 3′-ends enable their subsequent competitive amplification using one common pair of primers. The use of one fluorescein (FAM) labeled primer permits amplification products to be separated and detected via capillary electrophoresis. Respective probes were designed to allow the detection of reference genes in the genomes from maize and soya, as well as of event-specific junction regions in the transgenic maize line MON810 and in Roundup Ready soya. Specificity, sensitivity, and the potential of the technique for the relative quantification of recombinant DNA were assayed using mixtures of DNA extracted from certified reference maize and soybean flours. The novel strategy results in a modular system which can be complemented by further probes to broaden the range of target sequences.     

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.     

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.     

烘焙食品中转基因成分定性检测的FAPAS能力验证结果与分析

目的分析参加FAPAS烘焙食品中转基因成分定性检测能力验证的结果。方法根据FAPAS组织方提供的能力验证作业指导书、国家标准GB/T19495.3-2004转基因产品检测核酸提取纯化方法(部分步骤改进)和行业标准SN/T1204-2016《植物及其加工产品中转基因成分实时荧光PCR定性检验方法》对一份烘焙食品测试样品进行转基因成分定性检测。结果该样品检出pCaMV35S、tNOS、GTS40-3-2和MON863品系(基因),未检出Bt11、Bt176、GA21、MIR604、MON810、MON88017、MON89034、MON89788、NK603和TC1507品系。结论本实验重点对核酸提取方法进行改进并严格质控,满足了烘焙食品这类加工样品转基因定性检测要求,获得能力验证结果为满意。     

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.