Detection of genetically modified rice: a construct-specific real-time PCR method based on DNA sequences from transgenic Bt rice

Genetically modified rice varieties developed in China are close to approval for agricultural cultivation and production. However, so far no method has been reported for specific detection of transgenic varieties of this crop. In the present study, rice seeds assumed to consist of field-tested Bt rice (‘Anti-pest Shanyou 63’ and ‘Anti-pest Jinyou 63’) were used as reference material to determine transgenic DNA sequences. The transition between the cryIA(b) and cryIA(c) fusion gene and the nopaline synthase terminator (nos) sequence was used to develop a construct-specific real-time PCR based detection method. This Bt rice specific detection system was combined with a recently published quantitative real-time PCR method for the rice-specific (Oryza sativa L.) reference gene gos9. The complete PCR assay for detection of transgenic Bt rice was in-house validated and the limit of quantification was found to be below 0.1% Bt rice relative to the rice content. Application of the PCR assay should allow more precise detection of transgenic rice varieties in imported food products which are so far not approved in the EU.     

A construct-specific qualitative and quantitative PCR detection method of transgenic maize BVLA430101

Transgenic phytase maize (Zea mays L.) line BVLA430101 was the first transgenic maize obtained the security certification in 2009 in China. However, the construct of the phytase gene expression cassette and the specific detection method have not been reported yet. In this study, the phytase gene expression cassette was identified, which include maize legumin promoter, signal peptide, phytase gene, and maize legumin terminator. The construct-specific qualitative and quantitative PCR methods of BVLA430101 maize were established based on the transition of signal peptide and phytase gene using a maize taxon-specific gene zSSIIb as the endogenous gene. The detection limit for the conventional qualitative PCR was 200 haploid genome copies of BVLA430101. The absolute limit of quantification of the real-time PCR was about 20 haploid genome copies. In addition, two known BVLA430101 contents (5 and 1%) of mixed genomic DNA (V/V) were quantified using the developed real-time PCR detection system, which indicated that the developed quantitative method can be employed reliably for transgenic phytase maize BVLA430101 measurement.     

Screening of genetically modified organisms and specific detection of Bt176 maize in flours and starches by PCR-enzyme linked immunosorbent assay

Polymerase chain reaction (PCR)-enzyme linked immunosorbent assays (ELISAs) targeting either the 35S promoter or the Bt176 specific junction sequence were developed to screen for the presence of genetically modified organisms (GMOs) and specifically detect Bt176 maize in flours and starches. Two additional PCR-ELISA assays were developed to validate the results: one, based on the detection of the maize alcohol dehydrogenase 1 promoter specifically detected the presence of maize, and the other, based on the detection of a conserved sequence of plants ( 26S ribosomal RNA gene), validated the extracted DNA amplification. The PCR-ELISA assays developed here were highly specific and found to be as sensitive as the reference Southern hybridisation assay. The PCR-ELISA tests were at least 6 times more sensitive than gel electrophoresis and allowed 0.1% GMOs to be detected in Bt176, Bt11, Mon810 maize and Roundup Ready soybean. The PCR-ELISA tests are a method of choice for GMO screening and identifying Bt176 maize in flours and native starches. They may offer a cheaper alternative to the expensive real-time PCR assays and may be useful in laboratory GMO monitoring.     

A cotton‐specific gene,stearoyl‐ACP desaturase,used as a reference for qualitative and real‐time quantitative polymerase chain reaction detection of genetically modified organisms

Biotechnology has permitted the modification of agricultural materials in a very precise way to improve productivity and yields. Polymerase chain reaction (PCR)‐based methods have been the first choice of most analytical laboratories for routine use in the detection of genetically modified organisms (GMO) and their derived products. These methods rely on the amplification of transgenic sequences and quantification of the transgenic DNA by comparison with an amplified reference gene. This paper describes the specific primers and probe for the cotton stearoyl‐ACP desaturase (sad1) gene, and PCR cycling conditions suitable for the use of this sequence, which acts as an endogenous reference gene in both qualitative and quantitative PCR assays. The two methods were tested with 18 cotton varieties and identical amplification products were obtained with all of them. No amplification products were detected when DNA samples from other species, including soybean, rapeseed, tobacco, maize, tomato, potato, cucumber, pea, red pepper, sunflower, sesame, rice, peach, banana, apple, pumpkin, barley and carrot, were used as templates, which demonstrates that this system is specific for cotton. In real‐time quantitative PCR analysis, the detection limit was as low as 6 pg of DNA, which indicates that this method is suitable for application to processed food samples that contain very low copies of target DNA. Southern blot analysis confirmed that the sad1 gene was a single copy in the tested cotton varieties. Copyright © 2006 Society of Chemical Industry     

Real-time multiplex PCR: An accurate method for the detection and quantification of 35S-CaMV promoter in genetically modified maize-containing food

A very sensitive and new real-time multiplex PCR method for the quantification of genetically modified (GM) maize crops in food materials was developed and validated for an ABI Prism 7700 Sequence Detection System. In the assay described, fluorescence-labelled TaqMan probes were chosen to detect the amplified DNA fragments during PCR. In this multiplex approach, maize-specific DNA (zein) and 35S-CaMV promoter-specific DNA fragments are amplified in the same tube. The method was tested for the detection and quantification of the four maize events that are approved in Europe and contain the 35S-CaMV promoter: Bt11, Bt176, Mon810 and T25 maize. Quantification was based on a standard curve prepared from certified maize flour reference material prepared by the Institute for Reference Materials and Measurements. Quantification within the range of the standard curve (0.05-1% GM maize) and up to 100% was possible. Repeatability of the method for each GM maize event was determined; coefficients of variations ranged from 28-40%. In addition, three internal Nestlé laboratories successfully applied this method and comparable results were obtained.     

Detection of feed-derived maize DNA in goat milk and evaluation of the potential of horizontal transfer to bacteria

The presence and the transforming capacity of feed-derived DNA in milk obtained from eight lactating goats fed on maize E176 silage were evaluated. The presence of single- and multi-copy maize genes was monitored by real-time PCR and conventional PCR. Chromosomal and plastid DNA extracted directly from maize flour and silage were readily amplifiable by conventional PCR, however, only chloroplast-specific gene fragments of 199 and 532 bp were detected in about 60 and 20%, respectively, of the milk samples analysed. Quantification by real time PCR yielded 9.5 (±6.7) × 10² plant gene copies/mL of milk sediment. In contrast, all milk samples were negative for the chromosomally located maize zein gene or the E176 specific cry1Ab transgene. The minimum concentration of plant DNA required for detection was 0.01 ng/mL raw milk for the chloroplast-specific fragment and 1 ng/mL for the cry1Ab transgene. The detection limit was determined by spiking milk samples with plant DNA prior to DNA extraction. The transformation capability of DNA in milk was evaluated after constructing a marker rescue system in Acinetobacter baylyi strain BD413 based on recombinational repair of the bla _TEM gene. Two systems were developed that allowed the plant marker gene to recombine with the bacterial chromosome [A. baylyi BD413 (pUC-bla)] or plasmids [A. baylyi BD413 (pBBR1MCS-2Φ)]. The two systems showed the same efficiency of transformation, yielding 10⁻⁵ transformants per recipient cell (t/r) using plasmid pUC18 or a 1,873 bp fragment as donor DNA, and 3.5 × 10⁻¹¹ t/r using DNA isolated from flour (E176). No transformants were detected when exposing the recipient bacterium to DNA extracted from maize (E176) silage or from milk obtained from goats feed maize (E176).     

Detection of BT transgenic maize in foodstuffs

Different foodstuffs for humans and monogastric animals were analysed to detect CryIA(b)gene and quantify the CryIA(b) protein present in the transgenic maize used as an ingredient. Eight out of 32 foods obtained from the market showed to have been elaborated with transgenic Bt maize. Specific primers used to identify the transgenic event revealed that Mon810 was predominantly present in the foodstuffs. A commercial ELISA test allowed the quantification of the CryIA(b) protein in low processed foods, and found that 0.1 ppm was the highest value per gram of food. A Western blot carried out with immuno-purified polyclonal antibodies was capable of detecting both the intact or degraded CryIA(b) protein depending on the food assayed.     

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     

Estimation of the percentage of transgenic Bt maize in maize flour mixtures using perfusion and monolithic reversed-phase high-performance liquid chromatography and chemometric tools

The estimation of the percentage of transgenic Bt maize in maize flour mixtures has been achieved in this work by high-performance liquid chromatography using perfusion and monolithic columns and chemometric analysis. Principal component analysis allowed a preliminary study of the data structure. Then, linear discriminant analysis was used to develop decision rules to classify samples in the established categories (percentages of transgenic Bt maize). Finally, linear regression (LR) and multivariate regression models (namely, principal component analysis regression (PCR), partial least squares regression (PLS-1), and multiple linear regression (MLR)) were assayed for the prediction of the percentages of transgenic Bt maize present in a maize flour mixture. Using the relative areas of the protein peaks, MLR provided the best models and was able to predict the percentage of transgenic Bt maize in flour mixtures with an error of ±5.3%, ±2.3%, and ±3.8% in the predictions of Aristis Bt, DKC6575, and PR33P67, respectively.     

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。     

Effect of critical processing procedures on transgenic components in quality and quantity level during soymilk processing of Roundup Ready Soybean

By taking 2.5% (w/w) transgenic Roundup Ready Soybean as raw materials, with the detecting technology of traditional PCR and real-time fluorescence quantitative PCR, this study analyzed the influence of such critical processing procedures as jordaning, siruping, blending, homogenization and sterilization in soymilk preparation on the fragment size of endogenous gene lectin and exogenous gene epsps, as well as the quantification of transgenic component in Roundup Ready Soybean, and discussed the degradation mechanism of DNA in the course of transgenic soybean processing. The results indicated that, different processing procedures differ in their impact on the degradation of endogenous gene and exogenous gene. Physical mechanical action, such as the jordaning, can make endogenous and exogenous genes degrade, and has more effect on the later. The jordaning process degraded the DNA of endogenous gene from 1883 to about 836 bp, and exogenous gene from 1512 bp to about 408 bp. In the blending procedure, exogenous gene DNA was also degraded from about 408 to 190 bp, but there was no obviously action on the endogenous gene. After the endogenous and the exogenous genes were degraded to some degree, such as 836 and 408 bp, respectively, they were not evidently affected by siruping procedure at 100 °C for 15 min. However, endogenous gene was further considerably degraded from around 836 to 162 bp in the sterilization procedure at 121 °C for 30 s. The effect of the homogenization step on endogenous and exogenous genes was similar to that of siruping procedure. In addition, jordaning and siruping greatly damaged to exogenous gene, which rapidly decreased the transgenic content from 2.5 to 1.656% and 0.435%, respectively. While the endogenous gene was not evidently affected by the blending and homogenization procedures, the exogenous gene DNA was remarkably degraded in the two procedures. Therefore, the transgenic content continued decreased to 0.377% after homogenization procedure. Hereafter, the endogenous gene was further considerably degraded more than the exogenous gene in the sterilization procedure, which caused a little bounce of the transgenic content in the final product to 0.518%.     

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     

Investigations on genetically modified maize (Bt-maize) in pig nutrition: fate of feed-ingested foreign DNA in pig bodies

The passage and fate of ingested DNA in 48 pigs fed with diets containing (n=12) parental or (n=36) transgenic (Bt) maize were examined. Pigs were fattened from an initial live weight of 24 kg to approximately 108 kg. Animals fed transgenic maize were slaughtered in groups (n=6) 4, 8, 12, 24, 48 and 72 h after feeding the last maize-containing diet. Those slaughtered at up to 12 h received no further feed, while those held for longer prior to slaughter received a diet in which maize was replaced by barley and wheat. Control animals were slaughtered at 4 and 8 h. DNA extracted from tissues and gut contents was examined by PCR for the presence of plant DNA and for any transgenic material. Recombinant DNA was detectable in the intestinal contents up to 48 h after the last feeding of a diet containing the transgenic maize. PCR amplification of plant gene spacers produced fragments of different sizes, dependent on feed source. The feed source of rectum samples depended on individual passage rate in the groups and their restriction analysis showed grain species-specific patterns. Recombinant or maize-specific DNA was not detectable in tissue samples of pigs. In contrast, plant DNA fragments were detectable in the investigated pig tissues.     

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.     

Rapid real-time PCR detection of transgenic cry1C rice using plasmid molecule as calibrator

Transgenic Bacillus thuringiensis (Bt) rice expressing cry1C gene showed a high level of resistance to leaffolders (Cnaphalocrocis medinalis) and stemborers. Till now, no detection method based on the plasmid molecule as the calibrator has been reported. In this study, one plasmid molecule containing the rice root-specific gene (gos9) endogenous sequence and the cry1C rice 5′ event-specific sequence was developed. Real-time polymerase chain reaction (PCR) method was established using the developed plasmid molecule as the calibrator. Two standard curves for gos9 and the cry1C rice 5′ event-specific sequence showed high PCR efficiency and good linear regression. Limit of quantification of the plasmid molecule in quantitative PCR assays was 40 copies. Biases for 5 and 0.25 % content samples’ quantification were ?6.01 and ?3.55 % with acceptable standard deviation and repeatability standard deviation, respectively. Comparing with genomic DNA, the plasmid molecule was suitable for cry1C rice quantification as the calibrator. Furthermore, the present study provided a reliable and stable identification and quantification system for monitoring cry1C rice.     

Identification of Genetically Modified Zebrafish ( Danio rerio ) by Protein- and DNA-Analysis

The import, sale and possession of fluorescent transgenic zebrafish, offered under the name “GloFish^?” in U.S. aquarium shops, are not permitted in the European Union. A PCR-based method has been developed to detect transgenic zebrafish harbouring the gene (dsRed) coding for the red fluorescent protein, originally isolated from the marine sponge Discosoma striata. Two types of PCR have been performed: (i) PCR to detect amplifiable genomic zebrafish DNA was checked using primers specific for the zebrafish parvalbumin gene; (ii) PCR with primers to specifically amplify the dsRed gene. In both PCR systems, genomic DNA isolated from wild type zebrafish was used as control template, in the second PCR system, the plasmid dsRed2-N1 was used as a positive control. Applying this method to several specimens of presumed GloFish^? from traders in the Netherlands and Germany revealed the presence of transgenic fish. In addition, a rapid method for screening zebrafish suspected to be genetically modified has been developed by measuring the fluorescence of water-soluble protein.     

Novel feeds—a review of experiments at our Institute

Incorporation of the Bacillus thuringiensis (Bt) toxin gene or the glufosinate (phosphinothricin) tolerance (Pat) gene into the genetic construction of maize and sugar-beets did not significantly affect chemical composition and digestibility of crude nutrients and has no consequences on energy content, as demonstrated. This is supported by results from feeding trials carried out with poultry, pigs and ruminants. Although the rations were formulated to have high incorporation rates of transgenic or alternatively isogenic feedstuffs, their production power did not differ. From the nutritional point of view the results confirm the thesis that these types of genetic modifications (first generation of GM-plants without substantial changes in composition) do not significantly affect chemical composition and energetic feeding value. Fragments of plant DNA could be detected in some animal tissues (e.g. muscle from chicken), but fragments of transgenic DNA were not found.     

Nutritional assessment of transgenic lysine‐rich maize compared with conventional quality protein maize

BACKGROUND: The gene sb401 encoding a lysine‐rich protein has been successfully integrated into the genome of maize (Zea mays), its expression showing as increased levels of lysine and total protein in maize seeds. As part of a nutritional assessment of transgenic maize, nutritional composition, especially unintended changes in key nutrients such as proximates, amino acids, minerals and vitamins as well as in antinutrient (phytate phosphorus), and protein nutritional quality were compared between transgenic maize (inbred line 642 and hybrid line Y642) and conventional quality protein maize (QPM) Nongda 108. RESULTS: The contents of total protein, lysine, some other amino acids, several minerals and vitamin B₂ in transgenic inbred line 642 and hybrid line Y642 were significantly higher than those in conventional QPM. Water‐soluble protein and G2‐glutelin were significantly promoted in transgenic maize Y642. CONCLUSION: Insertion of the lysine‐rich sb401 gene increased the total protein and lysine content of transgenic maize varieties, leading to an improved amino acid score and therefore an improvement in the nutritive value of maize. © 2013 Society of Chemical Industry     

Development of an event-specific Real-time PCR detection method for the transgenic Bt rice line KMD1

The present study describes an event-specific quantitative Real-time PCR detection method for the transgenic Bt rice line Kemingdao 1 (KMD1). This rice line which is not approved in any country so far is likely to be approved in China in the near future. The developed primers amplify a DNA sequence spanning the integration site of the genetic construct in KMD1. DNA sequence information of this unknown site necessary for primer design was achieved using SiteFinding-PCR technique. The specificity of the detection method was shown by testing a number of different transgenic and conventional plant varieties (e.g. rice LL 601, LL 62, Bt 63). As alternative to genomic DNA, we developed double target hybrid amplicons as synthetic calibration standards in Real-time PCR. These amplicons contained both one copy of the KMD1 event-specific sequence and one copy of a sequence of the rice reference gene gos9. The limit of quantification (LOQ) of the method was tested to be 0.05%. R. Babekova and T. Funk contributed equally to this paper.