Event-specific detection of seven genetically modified soybean and maizes using multiplex-PCR coupled with oligonucleotide microarray

With the increasing development of genetically modified organism (GMO) detection techniques, the polymerase chain reaction (PCR) technique has been the mainstay for GMO detection. An oligonucleotide microarray is a glass chip to the surface of which an array of oligonucleotides was fixed as spots, each containing numerous copies of a sequence-specific probe that is complementary to a gene of interest. So it is used to detect ten or more targets synchronously. In this research, an event-specific detection strategy based on the unique and specific integration junction sequences between the host plant genome DNA and the integrated gene is being developed for its high specificity using multiplex-PCR together with oligonucleotide microarray. A commercial GM soybean (GTS 40-3-2) and six GM maize events (MON810, MON863, Bt176, Bt11, GA21, and T25) were detected by this method. The results indicate that it is a suitable method for the identification of these GM soybean and maizes.     

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.     

Event-specific qualitative and quantitative polymerase chain reaction analysis for genetically modified canola T45

Polymerase chain reaction (PCR) methods have been the main technical support for the detection of genetically modified organisms (GMOs). To date, GMO-specific PCR detection strategies have been developed basically at four different levels, such as screening-, gene-, construct-, and event-specific detection methods. Event-specific PCR detection method is the primary trend in GMO detection because of its high specificity based on the flanking sequence of exogenous integrant. GM canola, event T45, with tolerance to glufosinate ammonium is one of the commercial genetically modified (GM) canola events approved in China. In this study, the 5'-integration junction sequence between host plant DNA and the integrated gene construct of T45 canola was cloned and revealed by means of TAIL-PCR. Specific PCR primers and TaqMan probes were designed based upon the revealed sequence, and qualitative and quantitative TaqMan real-time PCR detection assays employing these primers and probe were developed. In qualitative PCR, the limit of detection (LOD) was 0.1% for T45 canola in 100 ng of genomic DNA. The quantitative PCR assay showed limits of detection and quantification (LOD and LOQ) of 5 and 50 haploid genome copies, respectively. In addition, three mixed canola samples with known GM contents were detected employing the developed real-time PCR assay, and expected results were obtained. These results indicated that the developed event-specific PCR methods can be used for identification and quantification of T45 canola and its derivates.     

Qualitative and quantitative PCR methods for detection of three lines of genetically modified potatoes

Qualitative and quantitative polymerase chain reaction (PCR) methods have been developed for the detection of genetically modified (GM) potatoes. The combination of specific primers for amplification of the promoter region of Cry3A gene, potato leafroll virus replicase gene, and potato virus Y coat protein gene allows to identify each line of NewLeaf, NewLeaf Y, and NewLeaf Plus GM potatoes. Multiplex PCR method was also established for the simple and rapid detection of the three lines of GM potato in a mixture sample. For further quantitative detection, the realtime PCR method has been developed. This method features the use of a standard plasmid as a reference molecule. Standard plasmid contains both a specific region of the transgene Cry3A and an endogenous UDP-glucose pyrophosphorylase gene of the potato. The test samples containing 0.5, 1, 3, and 5% GM potatoes were quantified by this method. At the 3.0% level of each line of GM potato, the relative standard deviations ranged from 6.0 to 19.6%. This result shows that the above PCR methods are applicable to detect GM potatoes quantitatively as well as qualitatively.     

A quantitative immunopolymerase chain reaction method for detection of vegetative insecticidal protein in genetically modified crops

Vegetative insecticidal protein (Vip) is being employed for transgenic expression in selected crops such as cotton, brinjal, and corn. For regulatory compliance, there is a need for a sensitive and reliable detection method, which can distinguish between approved and nonapproved genetically modified (GM) events and quantify GM contents as well. A quantitative immunopolymerase chain reaction (IPCR) method has been developed for the detection and quantification of Vip protein in GM crops. The developed assay displayed a detection limit of 1 ng/mL (1 ppb) and linear quantification range between 10 and 1000 ng/mL of Vip-S protein. The sensitivity of the assay was found to be 10 times higher than an analogous enzyme-linked immunosorbent assay for Vip-S protein. The results suggest that IPCR has the potential to become a standard method to quantify GM proteins.     

Real-time quantitative PCR detection of genetically modified Maximizer maize and Roundup Ready soybean in some representative foods

A fast and quantitative method was developed to detect transgenic "Maximizer" maize "event 176" (Novartis) and "Roundup Ready" soybean (Monsanto) in food by real-time quantitative PCR. The use of the ABI Prism 7700 sequence detection system allowed the determination of the amplified product accumulation through a fluorogenic probe (TaqMan). Fluorescent dyes were chosen in such a way as to coamplify total and transgenic DNA in the same tube. Using real-time quantitative PCR, 2 pg of transgenic or total DNA per gram of starting sample was detected in 3 h after DNA extraction and the relative amounts of "Maximizer" maize and "Roundup Ready" soybean in some representative food products were quantified.     

International collaborative study of the endogenous reference gene LAT52 used for qualitative and quantitative analyses of genetically modified tomato

One tomato ( Lycopersicon esculentum) gene, LAT52, has been proved to be a suitable endogenous reference gene for genetically modified (GM) tomato detection in a previous study. Herein are reported the results of a collaborative ring trial for international validation of the LAT52 gene as endogenous reference gene and its analytical systems; 14 GMO detection laboratories from 8 countries were invited, and results were finally received from 13. These data confirmed the species specificity by testing 10 plant genomic DNAs, less allelic variation and stable single copy number of the LAT52 gene, among 12 different tomato cultivars. Furthermore, the limit of detection of LAT52 qualitative PCR was proved to be 0.1%, which corresponded to 11 copies of haploid tomato genomic DNA, and the limit of quantification for the quantitative PCR system was about 10 copies of haploid tomato genomic DNA with acceptable PCR efficiency and linearity. Additionally, the bias between the test and true values of 8 blind samples ranged from 1.94 to 10.64%. All of these validated results indicated that the LAT52 gene is suitable for use as an endogenous reference gene for the identification and quantification of GM tomato and its derivates.     

Simultaneous detection of eight genetically modified maize lines using a combination of event- and construct-specific multiplex-PCR technique

To fulfill labeling and traceability requirement of genetically modified (GM) maize for trade and regulation, it is essential to develop an event-specific detection method for monitoring the presence of transgenes. In pursuit of this purpose, we systematically optimized and established a combined event- and construct-specific multiplex polymerase chain reaction (mPCR) technique for simultaneous detection of 8 GM maize lines. Altogether 9 sets of primers were designed, including six that were event-specific for Event176, Bt11, TC1507, NK603, MON863, and Mon810; two that were construct-specific for T25 and GA21, and one for an endogenous zein gene. The transgene in each GM maize line and the endogenous zein gene could be clearly detected and distinguished according to the different sizes of PCR amplicons. The limit of detection (LOD) was approximately 0.25% (v/v), although the detection can be as sensitive as 0.1% as demonstrated by the International Seed Testing Association (ISTA) proficiency test. This study further improves the current PCR-based detection method for GM maize. The method can be used in an easy, sensitive, and cost and time effective way for the identification and quality screening of a specific GM maize line.     

Individual detection of genetically modified maize varieties in non-identity-preserved maize samples

In many countries, the labeling of grains and feed- and foodstuffs is mandatory if the genetically modified organism (GMO) content exceeds a certain level of approved GM varieties. The GMO content in a maize sample containing the combined-trait (stacked) GM maize as determined by the currently available methodology is likely to be overestimated. However, there has been little information in the literature on the mixing level and varieties of stacked GM maize in real sample grains. For the first time, the GMO content of non-identity-preserved (non-IP) maize samples imported from the United States has been successfully determined by using a previously developed individual kernel detection system coupled to a multiplex qualitative PCR method followed by multichannel capillary gel electrophoresis system analysis. To clarify the GMO content in the maize samples imported from the United States, determine how many stacked GM traits are contained therein, and which GM trait varieties frequently appeared in 2005, the GMO content (percent) on a kernel basis and the varieties of the GM kernels in the non-IP maize samples imported from the United States were investigated using the individual kernel analysis system. The average (+/-standard deviation) of the GMO contents on a kernel basis in five non-IP sample lots was determined to be 51.0+/-21.6%, the percentage of a single GM trait grains was 39%, and the percentage of the stacked GM trait grains was 12%. The MON810 grains and NK603 grains were the most frequent varieties in the single GM traits. The most frequent stacked GM traits were the MON810xNK603 grains. In addition, the present study would provide the answer and impact for the quantification of GM maize content in the GM maize kernels on labeling regulation.     

Development of a certified reference material for genetically modified potato with altered starch composition

The presence of genetically modified organisms (GMOs) in food and feed products is subject to regulation in the European Union (EU) and elsewhere. As part of the EU authorization procedure for GMOs intended for food and feed use, reference materials must be produced for the quality control of measurements to quantify the GMOs. Certified reference materials (CRMs) are available for a range of herbicide- and insect-resistant genetically modified crops such as corn, soybean, and cotton. Here the development of the first CRM for a GMO that differs from its non-GMO counterpart in a major compositional constituent, that is, starch, is described. It is shown that the modification of the starch composition of potato (Solanum tuberosum L.) tubers, together with other characteristics of the delivered materials, have important consequences for the certification strategy. Moreover, the processing and characterization of the EH92-527-1 potato material required both new and modified procedures, different from those used routinely for CRMs produced from genetically modified seeds.     

Event specific qualitative and quantitative polymerase chain reaction detection of genetically modified MON863 maize based on the 5'-transgene integration sequence

Because of the genetically modified organisms (GMOs) labeling policies issued in many countries and areas, polymerase chain reaction (PCR) methods were developed for the execution of GMO labeling policies, such as screening, gene specific, construct specific, and event specific PCR detection methods, which have become a mainstay of GMOs detection. The event specific PCR detection method is the primary trend in GMOs detection because of its high specificity based on the flanking sequence of the exogenous integrant. This genetically modified maize, MON863, contains a Cry3Bb1 coding sequence that produces a protein with enhanced insecticidal activity against the coleopteran pest, corn rootworm. In this study, the 5'-integration junction sequence between the host plant DNA and the integrated gene construct of the genetically modified maize MON863 was revealed by means of thermal asymmetric interlaced-PCR, and the specific PCR primers and TaqMan probe were designed based upon the revealed 5'-integration junction sequence; the conventional qualitative PCR and quantitative TaqMan real-time PCR detection methods employing these primers and probes were successfully developed. In conventional qualitative PCR assay, the limit of detection (LOD) was 0.1% for MON863 in 100 ng of maize genomic DNA for one reaction. In the quantitative TaqMan real-time PCR assay, the LOD and the limit of quantification were eight and 80 haploid genome copies, respectively. In addition, three mixed maize samples with known MON863 contents were detected using the established real-time PCR systems, and the ideal results indicated that the established event specific real-time PCR detection systems were reliable, sensitive, and accurate.     

Ultrasensitive detection of genetically modified maize DNA by capillary gel electrophoresis with laser-induced fluorescence using different fluorescent intercalating dyes

In this work, four different fluorescent intercalating dyes are compared for the ultrasensitive CGE-LIF detection of DNA from transgenic maize in flours. The fluorescent intercalating dyes compared are YOPRO-1, SYBR-Green-I, Ethidium bromide (EthBr), and EnhanCE. For all the four dyes optimum concentrations are established, and efficient separations of DNA fragments ranging in size from 80 to 1000 bp are obtained. The comparative study demonstrates that SYBR-Green-I and YOPRO-1 provide better limits of detection (LODs) than EnhanCE or EthBr (i.e., LODs are, respectively, 700, 1000, 11300, and 97400 zmol, calculated for a 200-bp DNA fragment). Separations using YOPRO-1 are faster than those using SYBR-Green-I (30 min vs 47 min for the analysis of the 80-1000 bp DNA fragments). Also, separations using YOPRO-1 are more efficient than those using SYBR-Green-I (e.g., 2.4 x 10(6) plates/m vs 1.6 x 10(6) plates/m, respectively, calculated for the 200-bp fragment). Also, buffer depletion and cost per analysis are worse with SYBR-Green-I than with YOPRO-1. Therefore, YOPRO-1 was selected as the preferred intercalating dye. Using this fluorescent compound, analysis time reproducibility for the CGE-LIF separation of the DNA fragments is determined to be better than 1.7% (% RSD, n = 10) within the same day, and better than 1.9% (% RSD, n = 30) for three different days. Moreover, the fluorescence signal obtained using this dye is shown to vary linearly with the DNA concentration in the range studied, i.e., 1-500 ng/microL. It is demonstrated that by using this method 0.01% of transgenic maize can be detected in flour by direct injection of the PCR-amplified sample.     

Development of taxon-specific sequences of common wheat for the detection of genetically modified wheat

Qualitative and quantitative Polymerase Chain Reaction (PCR) systems aimed at the specific detection and quantification of common wheat DNA are described. Many countries have issued regulations to label foods that include genetically modified organisms (GMOs). PCR technology is widely recognized as a reliable and useful technique for the qualitative and quantitative detection of GMOs. Detection methods are needed to amplify a target GM gene, and the amplified results should be compared with those of the corresponding taxon-specific reference gene to obtain reliable results. This paper describes the development of a specific DNA sequence in the waxy-D1 gene for common wheat (Triticum aestivum L.) and the design of a specific primer pair and TaqMan probe on the waxy-D1 gene for PCR analysis. The primers amplified a product (Wx012) of 102 bp. It is indicated that the Wx012 DNA sequence is specific to common wheat, showing homogeneity in qualitative PCR results and very similar quantification accuracy along 19 distantly related common wheat varieties. In Southern blot and real-time PCR analyses, this sequence showed either a single or a low number of copy genes. In addition, by qualitative and quantitative PCR using wx012 primers and a wx012-T probe, the limits of detection of the common wheat genome were found to be about 15 copies, and the reproducibility was reliable. In consequence, the PCR system using wx012 primers and wx012-T probe is considered to be suitable for use as a common wheat-specific taxon-specific reference gene in DNA analyses, including GMO tests.     

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.     

First application of a microsphere-based immunoassay to the detection of genetically modified organisms (GMOs): quantification of Cry1Ab protein in genetically modified maize

An innovative covalent microsphere immunoassay, based on the usage of fluorescent beads coupled to a specific antibody, was developed for the quantification of the endotoxin Cry1Ab present in MON810 and Bt11 genetically modified (GM) maize lines. In particular, a specific protocol was developed to assess the presence of Cry1Ab in a very broad range of GM maize concentrations, from 0.1 to 100% [weight of genetically modified organism (GMO)/weight]. Test linearity was achieved in the range of values from 0.1 to 3%, whereas fluorescence signal increased following a nonlinear model, reaching a plateau at 25%. The limits of detection and quantification were equal to 0.018 and 0.054%, respectively. The present study describes the first application of quantitative high-throughput immunoassays in GMO analysis.     

Detection of genetically modified canola using multiplex PCR coupled with oligonucleotide microarray hybridization

A rapid method was developed for concurrent screening of transgenic elements in GM canola. This method utilizes a single multiplex PCR coupled with an oligonucleotide DNA array capable of simultaneously detecting the 12 approved GM canola lines in Canada. The assay includes construct-specific elements for identification of approved lines, common elements (e.g., CaMV 35S promoter, Agrobacterium tumefaciens nos terminator, or nptII gene) for screening of approved or unapproved lines, a canola-specific endogenous gene, and endogenous genes from heterologous crops to serve as additional controls. Oligonucleotide probes were validated individually for functionality and specificity by amplification of specific transgene sequences from appropriate GM canola lines corresponding to each probe sequence, and hybridization of amplicons to the array. Each target sequence hybridized to its corresponding oligonucleotide probe and no significant cross-hybridization was observed. The limit of detection was examined for the GM lines GT73, T45, and MS8/RF3, and was determined to be 0.1%, 0.1%, and 0.5%, respectively, well within the European food and feed labeling threshold level of 0.9% for approved GM product. Practically, the method was demonstrated to be effective for the detection of GM canola in several types of animal feed, as well as in commercial canola meal.     

Detection of genetically modified coho salmon using polymerase chain reaction (PCR) amplification

A PCR-based protocol for the identification of genetically modified salmon carrying a growth hormone transgene was developed. Several primer pairs were examined, and the primers that gave consistent results were selected to conduct routine testing. Comparison among several DNA extraction procedures, as well as different buffer compositions, led to the adoption of TriZol as the method of choice. Low potassium and high magnesium chloride concentrations were very important in the overall success of the PCR reaction, whereas buffer pH, ranging from 8.3 to 9.2, had little impact on the amplification reaction. The optimal primer annealing temperature was 52 degrees C. Although fish muscle tissues were the primary source for DNA samples, detection of the transgene was also possible in bones, skin, fins, and other organs. No benefits were achieved by the addition of additives such as dimethyl sulfoxide and betaine to the PCR reaction. This optimized PCR method was used to identify all samples tested (61 samples and 17 controls) with 100% accuracy.     

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.     

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.     

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.