Biosensor technology and surface plasmon resonance for real-time detection of genetically modified Roundup Ready soybean gene sequences

Biospecific interaction analysis (BIA) was performed using surface plasmon resonance (SPR) and biosensor technologies to detect genetically modified Roundup Ready soybean gene sequences. We first immobilized, on SA sensor chips, single-stranded biotinylated oligonucleotides containing soybean lectin and Roundup Ready gene sequences, and the efficiency of hybridization to oligonucleotide probes differing in length was determined. Second, we immobilized biotinylated PCR products from nontransgenic soybeans (genomes carrying only the lectin gene), as well as from genetically modified Roundup Ready soybean, and we injected the oligonucleotide probes. Furthermore, we used the sensor chips carrying either lectin and Roundup Ready soybean PCR products or 21-mer oligonucleotide as probes, and we injected both nonpurified and purified asymmetric PCR products. The results obtained show that 13 and 15 mer oligonucleotides are suitable probes to detect genetically modified Roundup Ready soybean gene sequences (either target oligonucleotides or PCR products) under standard BIA experimental conditions. By contrast, when 11 mer DNA probes were employed, no efficient hybridization was obtained. All the SPR-based formats were found to be useful for detection of Roundup Ready gene sequences, suggesting that these procedures are useful for the real-time monitoring of hybridization between target single-stranded PCR products, obtained by using as substrates DNA isolated from normal or transgenic soybeans, and oligonucleotide or PCR-generated probes, therefore enabling a one-step, nonradioactive protocol to perform detection.     

Detection and quantification of transgenes in grains by multiplex and real-time PCR

Multiplex PCR reactions were developed for detecting simultaneously the CryIA(b) and pat genes from events 176, MON810, BT11, and T25 of transgenic maize, using only two pairs of primers, one for the CryIA(b) gene and the other for the pat gene. The Roundup Ready soybean can be precisely detected by a multiplex PCR reaction using known primers, amplifying fragments of the NOS and the epsps sequences simultaneously. Transgenic events such as Roundup Ready soybean and GA21 maize, among others, can be quantified by real-time PCR using a pair of primers and a probe specifically designed for annealing to the NOS ending region. As an alternative to amplifying an endogenous gene, the addition of a foreign gene in a percentage equal to the required level of detection, in a parallel reaction, is proposed. The use of hexane to homogenize large flour samples is suggested.     

Qualitative and quantitative evaluation of the genomic DNA extracted from GMO and non-GMO foodstuffs with four different extraction methods

The presence of DNA in foodstuffs derived from or containing genetically modified organisms (GMO) is the basic requirement for labeling of GMO foods in Council Directive 2001/18/CE (Off. J. Eur. Communities 2001, L1 06/2). In this work, four different methods for DNA extraction were evaluated and compared. To rank the different methods, the quality and quantity of DNA extracted from standards, containing known percentages of GMO material and from different food products, were considered. The food products analyzed derived from both soybean and maize and were chosen on the basis of the mechanical, technological, and chemical treatment they had been subjected to during processing. Degree of DNA degradation at various stages of food production was evaluated through the amplification of different DNA fragments belonging to the endogenous genes of both maize and soybean. Genomic DNA was extracted from Roundup Ready soybean and maize MON810 standard flours, according to four different methods, and quantified by real-time Polymerase Chain Reaction (PCR), with the aim of determining the influence of the extraction methods on the DNA quantification through real-time PCR.     

A microarray platform for parallel detection of five transgenic events in foods: a combined polymerase chain reaction-ligation detection reaction-universal array method

We recently developed a multiplex polymerase chain reaction (PCR) system for the simultaneous detection of four transgenic maize (MON810, Bt176, Bt11, and GA21), one transgenic soybean (Roundup Ready), and two control genes (lectin and zein). Because PCR can lead to ambiguous interpretations due to low specificity, we have developed the ligation detection reaction (LDR) combined with a universal array as a molecular tool to confirm results of PCR analysis. Here, we describe the PCR-LDR-universal array procedure and demonstrate its specificity in revealing the presence of transgenic DNA in experimental samples, raw materials, and commercial foodstuffs.     

Identification of PCR-amplified genetically modified organisms (GMOs) DNA by peptide nucleic acid (PNA) probes in anion-exchange chromatographic analysis

PCR products obtained by selective amplification of transgenic DNA derived from food samples containing Roundup Ready soybean or Bt-176 maize have been analyzed by anion-exchange HPLC. Peptide nucleic acids (PNAs), oligonucleotide analogues known to bind to complementary single-stranded DNA with high affinity and specificity, have been used as specific probes in order to assess the identity of the peaks observed. Two different protocols were adopted in order to obtain single-stranded DNA: amplification with an excess of one primer or digestion of one DNA strand. The single-stranded DNA was mixed with the PNA probe, and the presence of a specific sequence was revealed through detection of the corresponding PNA:DNA peak with significantly different retention time. Advantages and limits of this approach are discussed. The method was tested with reference materials and subsequently applied to commercial samples.     

Detection and quantification of roundup ready soy in foods by conventional and real-time polymerase chain reaction

Transgenic soybean line GTS-40-3-2, marketed under the trade name Roundup Ready (RR) soy, was developed by Monsanto (USA) to allow for the use of glyphosate, the active ingredient of the herbicide Roundup, as a weed control agent. RR soy was first approved in Canada for environmental release and for feed products in 1995 and later for food products in 1996 and is widely grown in Canada. Consumer concern issues have resulted in proposed labeling regulations in Canada for foods derived from genetically engineered crops. One requirement for labeling is the ability to detect and accurately quantify the amount of transgenic material present in foods. Two assays were evaluated. A conventional qualitative Polymerase Chain Reaction (PCR) assay to detect the presence of soy and RR soy and a real-time PCR to quantify the amount of RR soy present in samples that tested positive in the first assay. PCR controls consisted of certified RR soy reference material, single transgenic soybeans, and a processed food sample containing a known amount of RR soy. To test real-world applicability, a number of common grocery store food items that contain soy-based products were tested. For some samples, significant differences in amplification efficiencies during the quantitative PCR assays were observed compared to the controls, resulting in potentially large errors in quantification. A correction factor was used to try to compensate for these differences.     

Development of a seven-target multiplex PCR for the simultaneous detection of transgenic soybean and maize in feeds and foods

The detection of genetically modified organisms (GMOs) in food and feed is an important issue for all the subjects involved in raw material control, food industry, and distribution. Because the number of GMOs authorized in the EU increased during the past few years, there is a need for methods that allow a rapid screening of products. In this paper, we propose a method for the simultaneous detection of four transgenic maize (MON810, Bt11, Bt 176, and GA21) and one transgenic soybean (Roundup Ready), which allows routine control analyses to be sped up. DNA was extracted either from maize and soybean seeds and leaves or reference materials, and the recombinant DNA target sequences were detected with 7 primer pairs, accurately designed to be highly specific for each investigated transgene. Cross and negative controls were performed to ensure the specificity of each primer pair. The method was validated on an interlaboratory ring test and good analytical parameters were obtained (LOD = 0.25%, Repeatability, (r) = 1; Reproducibility, (R) = 0.9). The method was then applied to a model biscuit made of transgenic materials baked for the purpose and to real samples such as feed and foodstuffs. On account of the high recognition specificity and the good detection limits, this multiplex PCR represents a fast and reliable screening method directly applicable in all the laboratories involved in raw material and food control.     

Development of a peptide nucleic acid array platform for the detection of genetically modified organisms in food

Two previously developed platforms, a multiplex polymerase chain reaction (PCR) and a peptide nucleic acid (PNA) array, the former allowing for the simultaneous detection of five transgenes and two endogenous controls in food and feed matrices and the latter for the assessment of the identity of amplified PCR products, were combined in order to develop a PNA array device for the screening of genetically modified organisms (GMOs) in food. PNA probes were opportunely designed, synthesized, and deposited on commercial slides. The length of the probes as well as the distance of the probes from the surface were evaluated and found to be critical points. The most suitable probes were found to be 15-mer PNAs linked to the slide surface by means of two 2-(2-aminoethoxy)ethoxyacetic acids as spacers. The device was tested on a model system constituted by flour samples containing a mixture of standards at known concentrations of transgenic material, in particular Roundup Ready soybean and Bt11, Bt176, Mon810, and GA21 maize: The DNA was amplified using the specific multiplex PCR method and tested on the PNA array. The method proposed was found to be able to correctly identify every GMO present in the tested samples.     

Real-time polymerase chain reaction quantification of the transgenes for roundup ready corn and roundup ready soybean in soil samples

A method for quantification of recombinant DNA for Roundup Ready (RR) corn and RR soybean in soil samples is described. Soil DNA from experimental field samples was extracted using a soil DNA extraction kit with a modified protocol. For the detection and quantification of recombinant DNA of RR corn and RR soybean, a molecular beacon and two pairs of specific primers were designed to differentially target recombinant DNA in these two genetically modified crops. Soil DNA extracts were spiked with RR corn or RR soybean DNA, and recombinant DNA was quantified using real-time PCR with a molecular beacon. As few as one copy of RR corn genome or one copy of RR soybean genome was detected in the soil DNA extract.     

Event-specific real-time detection and quantification of genetically modified Roundup Ready soybean

The event-specific real-time detection and quantification of Roundup Ready soybean (RRS) using an ABI PRISM 7700 sequence detection system with light upon extension (LUX) primer was developed in this study. The event-specific primers were designed, targeting the junction of the RRS 5' integration site and the endogenous gene lectin1. Then, a standard reference plasmid was constructed that carried both of the targeted sequences for quantitative analysis. The detection limit of the LUX real-time PCR system was 0.05 ng of 100% RRS genomic DNA, which was equal to 20.5 copies. The range of quantification was from 0.1 to 100%. The sensitivity and range of quantification successfully met the requirement of the labeling rules in the European Union and Taiwan.     

Development of a screening method for genetically modified soybean by plasmid-based quantitative competitive polymerase chain reaction

A novel type of quantitative competitive polymerase chain reaction (QC-PCR) system for the detection and quantification of the Roundup Ready soybean (RRS) was developed. This system was designed based on the advantage of a fully validated real-time PCR method used for the quantification of RRS in Japan. A plasmid was constructed as a competitor plasmid for the detection and quantification of genetically modified soy, RRS. The plasmid contained the construct-specific sequence of RRS and the taxon-specific sequence of lectin1 (Le1), and both had 21 bp oligonucleotide insertion in the sequences. The plasmid DNA was used as a reference molecule instead of ground seeds, which enabled us to precisely and stably adjust the copy number of targets. The present study demonstrated that the novel plasmid-based QC-PCR method could be a simple and feasible alternative to the real-time PCR method used for the quantification of genetically modified organism contents.     

Detection of genetically modified soybean using peptide nucleic acids (PNAs) and microarray technology

Peptide nucleic acid (PNA) microarrays for the detection of Roundup Ready soybeans in food have been prepared. PNA probes are known to be more efficient and selective in binding DNA sequences than the analogous oligonucleotides and are very suitable to be used for diagnostics in food. PNAs of different lengths were carefully designed and synthesized by solid-phase synthesis on an automatic synthesizer adopting the BOC strategy. PNAs were purified by HPLC and characterized by HPLC/MS. The probes were spotted on a functionalized surface to produce a microarray to be hybridized with PCR products. DNA extracted from reference material was amplified using Cy3- and Cy5-labeled primers, and the fluorescent PCR products obtained were hybridized on the microarray. Two protocols were adopted: the hybridization with dsDNA or with ssDNA obtained by digestion with the enzyme lambda exonuclease. The best results were obtained using a 15-mer PNA probe in combination with the ssPCR product derived from enzymatic digestion. The method was applied to the analysis of a sample of certified transgenic soybean flour.     

Detection of Genetically Modified Soy in Doughs and Cookies

In many countries, including the European Union member states, Switzerland, Australia, New Zealand, and Japan, legislation has been set up for labeling of genetically modified organisms (GMOs) in food and feed products. To comply with these regulations, reliable detection methods are necessary. If the detection is based on DNA, a GMO analysis may contain several steps where qualitative and quantitative species-specific, GMO screening, GMO construct, and GMO line-specific polymerase chain reactions (PCRs) are used. A limit of detection (LOD) thereby defines to what extent a target molecule may be detected in a sample. In this study, cookies were made with variable levels of a soy sample containing 2 wt% Roundup Ready soy. For all PCRs described, detection limits based on dilution series and practical LODs were determined. The practical LODs are used to determine to what extent a GMO ingredient may be detected in a real food product. Results reveal that, due to the baking process, the overall DNA fragment length is reduced, rendering GMO analyses more difficult. Furthermore, Roundup Ready soy line-specific and real-time quantitative PCR are less sensitive than GMO screening PCRs, whereas just these PCRs are crucial in the decision-making process regarding the presence of GMOs in a food product. Moreover, high standard deviations and errors render the precise quantification of GMOs difficult.     

Quantification and persistence of recombinant DNA of Roundup Ready corn and soybean in rotation

The presence of the recombinant cp4 epsps gene from Roundup Ready (RR) corn and RR soybean was quantified using real-time PCR in soil samples from a field experiment growing RR and conventional corn and soybean in rotation. RR corn and RR soybean cp4 epsps persisted in soil for up to 1 year after seeding. The concentration of recombinant DNA in soil peaked in July and August in RR corn and RR soybean plots, respectively. A small fraction of soil samples from plots seeded with conventional crops contained recombinant DNA, suggesting transgene dispersal by means of natural process or agricultural practices. This research will aid in the understanding of the persistence of recombinant DNA in agricultural cropping systems.     

A rapeseed-specific gene, acetyl-CoA carboxylase, can be used as a reference for qualitative and real-time quantitative PCR detection of transgenes from mixed food samples

Polymerase chain reaction (PCR) methods are very useful techniques for the detection and quantification of genetically modified organisms (GMOs) in food samples. These methods rely on the amplification of transgenic sequences and quantification of the transgenic DNA by comparison to an amplified reference gene. Reported here is the development of specific primers for the rapeseed (Brassica napus) BnACCg8 gene and PCR cycling conditions suitable for the use of this sequence as an endogenous reference gene in both qualitative and quantitative PCR assays. Both methods were assayed with 20 different rapeseed varieties, and identical amplification products were obtained with all of them. No amplification products were observed when DNA samples from other Brassica species, Arabidopsis thaliana, maize, and soybean were used as templates, which demonstrates that this system is specific for rapeseed. In real-time quantitative PCR analysis, the detection limit was as low as 1.25 pg of DNA, which indicates that this method is suitable for use in processed food samples which contain very low copies of target DNA.     

Chemical composition of glyphosate-tolerant soybean 40-3-2 grown in Europe remains equivalent with that of conventional soybean (Glycine max L.)

The composition of glyphosate-tolerant (Roundup Ready) soybean 40-3-2 was compared with that of conventional soybean grown in Romania in 2005 as part of a comparative safety assessment program. Samples were collected from replicated field trials, and compositional analyses were performed to measure proximates (moisture, fat, ash, protein, and carbohydrates by calculation), fiber, amino acids, fatty acids, isoflavones, raffinose, stachyose, phytic acid, trypsin inhibitor, and lectin in grain as well as proximates and fiber in forage. The mean values for all biochemical components assessed for Roundup Ready soybean 40-30-2 were similar to those of the conventional control and were within the published range observed for commercial soybean. The compositional profile of Roundup Ready soybean 40-3-2 was also compared to that of conventional soybean varieties grown in Romania by calculating a 99% tolerance interval to describe compositional variability in the population of traditional soybean varieties already on the marketplace. These comparisons, together with the history of the safe use of soybean as a common component of animal feed and human food, lead to the conclusion that Roundup Ready soybean 40-3-2 is compositionally equivalent to and as safe and nutritious as conventional soybean varieties grown commercially.     

Real-time polymerase chain reaction monitoring of recombinant DNA entry into soil from decomposing roundup ready leaf biomass

Glyphosate-tolerant, Roundup Ready (RR) soybeans account for about 57% of all genetically modified (GM) crops grown worldwide. The entry of recombinant DNA into soil from GM crops has been identified as an environmental concern due to the possibility of their horizontal transfer to soil microorganisms. RR soybeans contain recombinant gene sequences that can be differentiated from wild-type plant and microbial genes in soil by using a sequence-specific molecular beacon and real-time polymerase chain reaction (PCR). A molecular beacon-based real-time PCR system to quantify a wild-type soybean lectin ( le1) gene was designed to compare amounts of endogenous soybean genes to recombinant DNA in soil. Microcosm studies were carried out to develop methodologies for the detection of recombinant DNA from RR soybeans in soil. RR soybean leaf litterbags were imbedded in the soil under controlled environmental conditions (60% water holding capacity, 10/15 degrees C, and 8/16 h day/night) for 30 days. The soybean biomass decomposition was described using a single-phase exponential equation, and the DNA concentration in planta and in soil was quantified using real-time PCR using sequence-specific molecular beacons for the recombinant cp4 epsps and endogenous soybean lectin ( le1) genes. The biomass of RR soybean leaves was 8.6% less than nontransgenic (NT) soybean leaves after 30 days. The pooled half-disappearance time for cp4 epsps and le1 in RR and of le1 in NT soybean leaves was 1.4 days. All genes from leaves were detected in soil after 30 days. This study provides a methodology for monitoring the entry of RR and NT soybean DNA into soil from decomposing plant residues.     

Applicability of the quantification of genetically modified organisms to foods processed from maize and soy

The applicability of quantifying genetically modified (GM) maize and soy to processed foods was investigated using heat treatment processing models. The detection methods were based on real-time quantitative polymerase chain reaction (PCR) analysis. Ground seeds of insect resistant GM maize (MON810) and glyphosate tolerant Roundup Ready (RR) soy were dissolved in water and were heat treated by autoclaving for various time intervals. The calculated copy numbers of the recombinant and taxon specific deoxyribonucleic acid (DNA) sequences in the extracted DNA solution were found to decrease with time. This decrease was influenced by the PCR-amplified size. The conversion factor (Cf), which is the ratio of the recombinant DNA sequence to the taxon specific DNA sequence and is used as a constant number for calculating GM% at each event, tended to be stable when the sizes of PCR products of two DNA sequences were nearly equal. The results suggested that the size of the PCR product plays a key role in the quantification of GM organisms in processed foods. It is believed that the Cf of the endosperm (3n) is influenced by whether the GM originated from a paternal or maternal source. The embryos and endosperms were separated from the F1 generation seeds of five GM maize events, and their Cf values were measured. Both paternal and maternal GM events were identified. In these, the endosperm Cf was lower than that of the embryo, and the embryo Cf was lower than that of the endosperm. These results demonstrate the difficulties encountered in the determination of GM% in maize grains (F2 generation) and in processed foods from maize and soy.     

Quantification of genetically modified soybean by quenching probe polymerase chain reaction

Quenching probe (QProbe) polymerase chain reaction (PCR) is a simple and cost-effective real-time PCR assay in comparison with other real-time PCR assays such as the TaqMan assay. We used QProbe-PCR to quantify genetically modified (GM) soybean (Roundup Ready soybean). We designed event-specific QProbes for Le1 (soy endogenous gene) and RRS (recombinant gene), and we quantified certified reference materials containing 0.1, 0.5, 1, 2, and 5% GM soybean. The TaqMan assay was also applied to the same samples, and the results were compared. The accuracy of QProbe-PCR was similar to that of TaqMan assay. When GM soybean content was 0.5% or more, the relative standard deviations of QProbe-PCR were less than 20%. QProbe-PCR is sensitive enough to monitor labeling systems and has acceptable levels of accuracy and precision.     

Development of one novel multiple-target plasmid for duplex quantitative PCR analysis of roundup ready soybean

To enforce the labeling regulations of genetically modified organisms (GMOs), the application of reference molecules as calibrators is becoming essential for practical quantification of GMOs. However, the reported reference molecules with tandem marker multiple targets have been proved not suitable for duplex PCR analysis. In this study, we developed one unique plasmid molecule based on one pMD-18T vector with three exogenous target DNA fragments of Roundup Ready soybean GTS 40-3-2 (RRS), that is, CaMV35S, NOS, and RRS event fragments, plus one fragment of soybean endogenous Lectin gene. This Lectin gene fragment was separated from the three exogenous target DNA fragments of RRS by inserting one 2.6 kb DNA fragment with no relatedness to RRS detection targets in this resultant plasmid. Then, we proved that this design allows the quantification of RRS using the three duplex real-time PCR assays targeting CaMV35S, NOS, and RRS events employing this reference molecule as the calibrator. In these duplex PCR assays, the limits of detection (LOD) and quantification (LOQ) were 10 and 50 copies, respectively. For the quantitative analysis of practical RRS samples, the results of accuracy and precision were similar to those of simplex PCR assays, for instance, the quantitative results were at the 1% level, the mean bias of the simplex and duplex PCR were 4.0% and 4.6%, respectively, and the statistic analysis ( t-test) showed that the quantitative data from duplex and simplex PCR had no significant discrepancy for each soybean sample. Obviously, duplex PCR analysis has the advantages of saving the costs of PCR reaction and reducing the experimental errors in simplex PCR testing. The strategy reported in the present study will be helpful for the development of new reference molecules suitable for duplex PCR quantitative assays of GMOs.