Characterization and event specific-detection by quantitative real-time PCR of T25 maize insert

T25 is one of the 4 maize transformation events from which commercial lines have so far been authorized in Europe. It was created by polyethylene glycol-mediated transformation using a construct bearing one copy of the synthetic pat gene associated with both promoter and terminator of the 35S ribosomal gene from cauliflower mosaic virus. In this article, we report the sequencing of the whole T25 insert and the characterization of its integration site by using a genome walking strategy. Our results confirmed that one intact copy of the initial construct had been integrated in the plant genome. They also revealed, at the 5' junction of the insert, the presence of a second truncated 35S promoter, probably resulting from rearrangements which may have occurred before or during integration of the plasmid DNA. The analysis of the junction fragments showed that the integration site of the insert presented high homologies with the Huck retrotransposon family. By using one primer annealing in the maize genome and the other in the 5' end of the integrated DNA, we developed a reliable event-specific detection system for T25 maize. To provide means to comply with the European regulation, a real-time PCR test was designed for specific quantitation of T25 event by using Taqman chemistry.     

An optimal design method for preventing air bubbles in high-temperature microfluidic devices

DNA analysis with the polymerase chain reaction (PCR) has become a routine part of medical diagnostics, environmental inspections, food evaluations, and biological studies. Furthermore, the development of a microscale PCR chip is an essential component of studies aimed at integrating PCR into a micro total analysis system (μ-TAS). However, the occurrence of air bubbles in microchannels complicates this process. In this study, we investigated a new technique based on the fluid dynamics of laminar flow that utilizes a small amount of mineral oil at the beginning of sample injection to prevent air bubbles from occurring in microchannels. We also further optimized the pressure, the length of the pressurizing channel and the volume of oil, thus making our microfluidic device more useful for high-temperature PCR. Additionally, quantitative continuous-flow PCR was performed using the optimized PCR chip in order to detect genetically modified (GM) maize. DNA was extracted from GM maize, MON 810, and non-GM maize at several concentrations from 0% (w/v) to 100% (w/v). The DNA amplification signals were then analyzed on the PCR chip using a laser-based system. The signal from our microfluidic PCR chip was found to increase in direct proportion to the initial GM maize concentration.     

Characterization of genetically modified maize in weakly contaminated seed batches and identification of the origin of the adventitious contamination

So far, relatively few genetically modified plants (GMPs) have been planted in the European Union (EU). However, in France, seed batches weakly contaminated by unidentified GM materials have recently been detected among commercial maize seeds (14 seed batches positive out of 447 analyzed). We have developed a 3-step approach to precisely identify the genetic modifications detected in such maize seed batches. First, to isolate GMPs derived from the contaminated seed batches, 10 000 maize seeds of each batch were planted and screened by polymerase chain reaction (PCR) on 100-plant batches, then on 10-plant subbatches, and finally, plant by plant. In a second step, specific identification of the individual GMPs was performed. Finally, to determine the origin of the contamination, each individual GMP was analyzed by simple sequence repeat (SSR) markers. The results showed that all batches were contaminated by few GM seeds, having a GM content < 0.1%. Finally, 12 individual GMPs have been isolated from 17 plant pools that were tested positive either for P35-S and/or T-Nos. MON810 and T25 transformation events approved for cultivation in the EU were detected in 7 individual GMPs. The other seed batches were contaminated by genetically modified organisms (GMOs) that are not approved in the EU, including GA21 or the stacking MON810/T25. Presumable identification of T14 was also achieved following sequencing of 1 individual GMP. The data also showed that most of the seed batches were contaminated by several transformation events. Finally, analysis of SSR markers indicated that the contaminations were essentially due to cross-pollination in the seed production process.     

Evaluation of the reliability of maize reference assays for GMO quantification

A reliable PCR reference assay for relative genetically modified organism (GMO) quantification must be specific for the target taxon and amplify uniformly along the commercialised varieties within the considered taxon. Different reference assays for maize (Zea mays L.) are used in official methods for GMO quantification. In this study, we evaluated the reliability of eight existing maize reference assays, four of which are used in combination with an event-specific polymerase chain reaction (PCR) assay validated and published by the Community Reference Laboratory (CRL). We analysed the nucleotide sequence variation in the target genomic regions in a broad range of transgenic and conventional varieties and lines: MON 810 varieties cultivated in Spain and conventional varieties from various geographical origins and breeding history. In addition, the reliability of the assays was evaluated based on their PCR amplification performance. A single base pair substitution, corresponding to a single nucleotide polymorphism (SNP) reported in an earlier study, was observed in the forward primer of one of the studied alcohol dehydrogenase 1 (Adh1) (70) assays in a large number of varieties. The SNP presence is consistent with a poor PCR performance observed for this assay along the tested varieties. The obtained data show that the Adh1 (70) assay used in the official CRL NK603 assay is unreliable. Based on our results from both the nucleotide stability study and the PCR performance test, we can conclude that the Adh1 (136) reference assay (T25 and Bt11 assays) as well as the tested high mobility group protein gene assay, which also form parts of CRL methods for quantification, are highly reliable. Despite the observed uniformity in the nucleotide sequence of the invertase gene assay, the PCR performance test reveals that this target sequence might occur in more than one copy. Finally, although currently not forming a part of official quantification methods, zein and SSIIb assays are found to be highly reliable in terms of nucleotide stability and PCR performance and are proposed as good alternative targets for a reference assay for maize.     

Catalytic hydrogen transfer over magnesia, Part XVIII. , Chemoselective reduction of formyl and acetyl derivatives of furan and thiophene

Chemoselective reduction of formyl and acetyl derivatives of furan (F) and thiophene (T) to the corresponding carbinols by 2-propanol has been performed in vapour phase over MgO as a catalyst. Formyl derivatives of (F) and (T) have been reduced to the corresponding carbinols with selectivity 96 and 71%, respectively. Above 573 K the reduction of (T)-CHO was accompanied by the formation of thiophene whose yield increased with temperature (36% at 723 K). Acetyl derivatives of (F) and (T) have been reduced to the corresponding carbinols which at higher temperatures underwent dehydration to vinyl derivatives and were further reduced to ethyl derivatives. This revised version was published online in June 2006 with corrections to the Cover Date.     

Near-infrared spectroscopy quantitative determination of Pefloxacin mesylate concentration in pharmaceuticals by using partial least squares and principal component regression multivariate calibration

Pefloxacin mesylate, a broad-spectrum antibacterial fluoroquinolone, has been widely used in clinical practice. Therefore, it is very important to detect the concentration of Pefloxacin mesylate. In this research, the near-infrared spectroscopy (NIRS) has been applied to quantitatively analyze on 108 injection samples, which was divided into a calibration set containing 89 samples and a prediction set containing 19 samples randomly. In order to get a satisfying result, partial least square (PLS) regression and principal components regression (PCR) have been utilized to establish quantitative models. Also, the process of establishing the models, parameters of the models, and prediction results were discussed in detail. In the PLS regression, the values of the coefficient of determination (R²) and root mean square error of cross-validation (RMSECV) of PLS regression are 0.9263 and 0.00119, respectively. For comparison, though applying PCR method to get the values of R² and RMSECV we obtained are 0.9685 and 0.00108, respectively. And the values of the standard error of prediction set (SEP) of PLS and PCR models are 0.001480 and 0.001140. The result of the prediction set suggests that these two quantitative analysis models have excellent generalization ability and prediction precision. However, for this PFLX injection samples, the PCR quantitative analysis model achieved more accurate results than the PLS model. The experimental results showed that NIRS together with PCR method provide rapid and accurate quantitative analysis of PFLX injection samples. Moreover, this study supplied technical support for the further analysis of other injection samples in pharmaceuticals.     

Optimization and Verification of Droplet Digital PCR Even-Specific Methods for the Quantification of GM Maize DAS1507 and NK603

In recent years, digital polymerase chain reaction (dPCR), a new molecular biology technique, has been gaining in popularity. Among many other applications, this technique can also be used for the detection and quantification of genetically modified organisms (GMOs) in food and feed. It might replace the currently widely used real-time PCR method (qPCR), by overcoming problems related to the PCR inhibition and the requirement of certified reference materials to be used as a calibrant. In theory, validated qPCR methods can be easily transferred to the dPCR platform. However, optimization of the PCR conditions might be necessary. In this study, we report the transfer of two validated qPCR methods for quantification of maize DAS1507 and NK603 events to the droplet dPCR (ddPCR) platform. After some optimization, both methods have been verified according to the guidance of the European Network of GMO Laboratories (ENGL) on analytical method verification (ENGL working group on “Method Verification.” (2011) Verification of Analytical Methods for GMO Testing When Implementing Interlaboratory Validated Methods). Digital PCR methods performed equally or better than the qPCR methods. Optimized ddPCR methods confirm their suitability for GMO determination in food and feed.     

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.     

Production of glucose-fructose syrups from fermentative hydrolysates of plant wastes

A method has been developed for obtaining glucose—fructose syrups by the hydrolysis of plant wastes under the action of a complex of cellulases from a hybrid strain of a fungus, followed by isomerization of part of the glucose to fructose by immobilized streptomycete glucose isomerase. The conditions for raising the degree of isomerization of the glucose and the period of half-inactivation of the immobilized glucose isomerase have been optimized, with a simultaneous cheapening of the process. Plant raw materials that have been used for obtaining glucose–fructose syrups are maize cobs, wheat or rice straw, and reeds. Isomerization has been achieved by streptomycete cells immobilized on cotton lint. Institute of Microbiology of the Academy of Sciences, Republic of Uzbekistan, Tashkent, fax (3712) 41 71 29. Translated from Khimiya Prirodnykh Soedinenii, No. 4, pp. 449–451, July–August, 1998.     

Sensitive and simultaneous analysis of five transgenic maizes using multiplex polymerase chain reaction, capillary gel electrophoresis, and laser-induced fluorescence

The benefits of using multiplex polymerase chain reaction (PCR) followed by capillary gel electrophoresis with laser-induced fluorescence (CGE-LIF) for the simultaneous detection of five transgenic maizes (Bt11, T25, MON810, GA21, and Bt176) are demonstrated. The method uses a hexaplex PCR protocol to amplify the five mentioned transgenic amplicons plus the zein gene used as reference, followed by a CGE-LIF method to analyze the six DNA fragments. CGE-LIF was demonstrated very useful and informative for optimizing multiplex PCR parameters such as time extension, PCR buffer concentration and primers concentration. The method developed is highly sensitive and allows the simultaneous detection in a single run of percentages of transgenic maize as low as 0.054% of Bt11, 0.057% of T25, 0.036% of MON810, 0.064% of GA21, and 0.018% of Bt176 in flour obtaining signals still far from the detection limit (namely, the signal/noise ratios for the corresponding DNA peaks were 41, 124, 98, 250, 252, and 473, respectively). These percentages are well below the minimum threshold marked by the European Regulation for transgenic food labeling (i.e., 0.5-0.9%). A study on the reproducibility of the multiplex PCR-CGE-LIF procedure was also performed. Thus, values of RSD lower than 0.67 and 6.80% were obtained for migration times and corrected peak areas, respectively, for the same sample and three different days (n = 12). On the other hand, the reproducibility of the whole procedure, including four different multiplex PCR amplifications, was determined to be better than 0.66 and 23.3% for migration times and corrected peak areas, respectively. Agarose gel electrophoresis (AGE) and CGE-LIF were compared in terms of resolution and sensitivity for detecting PCR products, demonstrating that CGE-LIF can solve false positives induced by artifacts from the multiplex PCR reaction that could not be addressed by AGE. Moreover, CGE-LIF provides better resolution and sensitivity. To our knowledge, these results demonstrate for the first time that multiplex PCR-CGE-LIF is a solid alternative to determine multiple genetically modified organisms in maize flours in a single run.     

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.     

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.     

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.     

Exploring the hole mobility of oligothiophene based donors with different spatial symmetry and conjugation length of backbone: A theoretical insight

Novel oligothiophene based donors have been synthesized and modified. However, how molecular modifications influence their molecular stacking and charge carrier mobility is not clear. In this work, we have selected two high-performance oligothiophene based donors DRCN5T and DRCN6T to understand why the hole mobility of DRCN5T/PC71BM is much faster than that of DRCN6T/PC71BM in the experiment (Journal of the American Chemical Society, 2015). The computed data indicate that the maximum hole mobility of DRCN5T is about 20 times faster than that of DRCN6T. Moreover, DRCN5T has faster hole mobility than DRCN6T in almost every similar configuration. These mobility differences can be explained with the different positions and sparsity of side alkyl chains on the backbones. The work here gives a deep insight into the hole mobility in oligothiophene based OSCs and thereby may provide a guideline for future oligothiophene-based donor material design and morphology control.     

Interlaboratory study of DNA extraction from multiple ground samples, multiplex real-time PCR, and multiplex qualitative PCR for individual kernel detection system of genetically modified maize

In many countries, the labeling of grains, feed, and foodstuff is mandatory if the genetically modified (GM) organism content exceeds a certain level of approved GM varieties. We previously developed an individual kernel detection system consisting of grinding individual kernels, DNA extraction from the individually ground kernels, GM detection using multiplex real-time PCR, and GM event detection using multiplex qualitative PCR to analyze the precise commingling level and varieties of GM maize in real sample grains. We performed the interlaboratory study of the DNA extraction with multiple ground samples, multiplex real-time PCR detection, and multiplex qualitative PCR detection to evaluate its applicability, practicality, and ruggedness for the individual kernel detection system of GM maize. DNA extraction with multiple ground samples, multiplex real-time PCR, and multiplex qualitative PCR were evaluated by five laboratories in Japan, and all results from these laboratories were consistent with the expected results in terms of the commingling level and event analysis. Thus, the DNA extraction with multiple ground samples, multiplex real-time PCR, and multiplex qualitative PCR for the individual kernel detection system is applicable and practicable in a laboratory to regulate the commingling level of GM maize grain for GM samples, including stacked GM maize.     

Ultra-fast simultaneous analysis of genetically modified organisms in maize by microchip electrophoresis with LIF detector

This study examined the potential of microchip electrophoresis (ME) with a LIF detector using a programmed field strength gradient (PFSG) in a conventional glass double-T microchip for the ultra-fast detection and simultaneous analysis of genetically modified (GM) maize. The separation efficiency and sensitivity at various sieving gels (poly(ethylene oxide) (PEO, M(r) 8,000,000) and 2-hydroxyethylcellulose (HEC) (M(r) 250,000)) and fluorescent dye concentrations were investigated. The PCR products of both the GM and non-GM maize were analyzed within 30 s under the PFSG (470.6 V/cm for 20 s, 117.6 V/cm for 12 s, and 470.6 V/cm for 30 s) with a 2.5% HEC sieving matrix in the running buffer, 1 x Tris-borate EDTA (TBE) (pH 8.30) and 0.5 ppm ethidium bromide. The five transgenic maize varieties (Event176, MON810, Bt11, GA21, and T25) examined in this study were also clearly differentiated by ME-PFSG within 30 s in a single run without any loss of resolution. The ME-PFSG technique is a powerful tool for the ultra-fast detection and simultaneous analysis of GMOs in a variety of foods including maize.     

毛细管电泳-激光诱导荧光法分离检测DNA片段及基因扩增产物

以羟丙甲基纤维素和非交联聚丙烯酰胺浴液为筛分介质,将毛细管电泳-激光诱导荧光法用于DNA片段及基因扩增产物的分离检测。探讨了非胶筛分介质中高分子化合物的浓度、电解质的浓度、内插试剂用量等对DNA片段分离检测的影响;考察了DNA片段迁移时间和峰面积的重现性及DNA片段定量检测的关系。建立了一种快速、灵敏的DNA片段及基因扩增产物分离检测方法。     

Sample Injection for Capillary Electrophoresis on a Micro Fabricated Device/On Chip CE Injection

Abstract

Reproducible injection for capillary electrophoresis on a micro device/Lab on chip is not an easy task. Different injection designs (e.g., T‐type, double T‐type, cross) and different injection modes have been applied (pinched injection, gated injection, optically gated injection, pressure/pneumatic injection, double L injection) with various analytes. Problems and properties of the different modes are described.     

Quantitative-competitive polymerase chain reaction coupled with slab gel and capillary electrophoresis for the detection of roundup ready soybean and maize

The aim of the present study was to develop a quantitative-competitive PCR (QC-PCR) method to detect DNA from transgenic herbicide-resistant (roundup ready, RR) soybean and maize. Since no QC-PCR system for the quantification of RR maize had been published at the time of writing, a specific competitor DNA for transgenic event was developed. For the QC-PCR of RR-soybean, a commercially available competitor was employed. These internal standards were calibrated by coamplifying with mixtures containing RR-soybean and maize DNAs. The calibrated QC-PCR systems were applied to certified RR-soybean and maize flour mixtures in order to demonstrate their suitability not only for the quantification of the glyphosate resistance traits in DNA matrices, but also in practically relevant samples. In addition, a special focus of the present work was to compare the detection of QC-PCR products by slab gel and CGE with UV detection. CGE permitted the precise detection of transgenic events also below the equivalence points; while in slab gel electrophoresis, due to the low sensitivity the quantification of genetically modified DNA was allowed only at the equivalence point.     

Capillary-based fully integrated and automated system for nanoliter polymerase chain reaction analysis directly from cheek cells

A miniaturized, integrated and automated system based on capillary fluidics has been developed for nanoliter DNA analysis directly from cheek cells. All steps for DNA analysis, including injecting aqueous reagents and DNA samples, mixing the solutions together, thermal cell lysis, polymerase chain reaction (PCR), transfer and injection of PCR product, separation, sizing and detection of those products are performed in a capillary-based integrated system. A small amount of cheek cells collected by a plastic toothpick is directly dissolved in the PCR cocktail in a plastic vial or mixed on-line with a small volume of PCR cocktail (125 nl) in the capillary. After thermal cell lysis and PCR in a microthermal cycler, the DNA fragments are mixed with DNA size standards and transferred to a micro-cross for injection and separation by capillary gel electrophoresis. Programmable syringe pumps, switching valves, multiposition and freeze-thaw valves are used for microfluidic control in the entire system. This work establishes the feasibility of performing all the steps of DNA analysis from real samples in a capillary-based nanoliter integrated system.