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1.
Qualitative and quantitative analytical methods were developed for the new event of genetically modified (GM) maize, MON863. One specific primer pair was designed for the qualitative polymerase chain reaction (PCR) method. The specificity and sensitivity of the designed primers were confirmed. PCR was performed on genomic DNAs extracted from MON863, other GM events, and cereal crops. Single PCR product was obtained from MON863 by the designed primer pair. Eight test samples including GM maize MON863 were prepared at 0.01 approximately 10% levels and analyzed by PCR. Limit of detection of the method was 0.01% for GM maize MON863. On the other hand, another specific primer pair and probe were also designed for quantitative method using a real-time polymerase chain reaction. As a reference molecule, a plasmid was constructed from a taxon-specific DNA sequence for maize, a universal sequence for a cauliflower mosaic virus (CaMV) 35S promoter used in most genetically modified organisms, and a construct-specific DNA sequence for the MON863 event. Six test samples of 0.1, 0.5, 1.0, 3.0, 5.0 and 10.0% of GM maize MON863 were quantitated for the validation of this method. At the 3.0% level, the bias (mean vs true value) for MON863 was 3.0%, and its relative standard deviation was 5.5%. Limit of quantitation of the method was 0.5%. These results show that the developed PCR methods can be used to qualitatively and quantitatively detect GM maize MON863.  相似文献   

2.
The real-time PCR methods recommended in the European Union for the quantitation of genetically modified (GM) maize events NK603, GA21, and MON 863 measure the number of copies of the GM event in relation to those of the maize-specific adh1 reference gene. The study reported here revealed that the targeted 70 base pair adh1 region exhibits a single nucleotide polymorphism (SNP839) that hampers the binding of the reverse primer used in the adh1 detection method. Partial fragments of the adh1-A and adh1-F allele were cloned. By allele-specific real-time PCR, it was shown that SNP839 corresponds to a common allelic polymorphism in maize. As a result, the quantitation of the GM maize events mentioned is positively or negatively biased, depending on the adh1 genotype of sample and calibrant. Therefore, it is proposed to revise the quantitative detection methods for NK603, GA21, and MON 863 maize.  相似文献   

3.
The number of cultured hectares and commercialized genetically modified organisms (GMOs) has increased exponentially in the past 9 years. Governments in many countries have established a policy of labeling all food and feed containing or produced by GMOs. Consequently, versatile, laboratory-transferable GMO detection methods are in increasing demand. Here, we describe a qualitative PCR-based multiplex method for simultaneous detection and identification of four genetically modified maize lines: Bt11, MON810, T25, and GA21. The described system is based on the use of five primers directed to specific sequences in these insertion events. Primers were used in a single optimized multiplex PCR reaction, and sequences of the amplified fragments are reported. The assay allows amplification of the MON810 event from the 35S promoter to the hsp intron yielding a 468 bp amplicon. Amplification of the Bt11 and T25 events from the 35S promoter to the PAT gene yielded two different amplicons of 280 and 177 bp, respectively, whereas amplification of the 5' flanking region of the GA21 gave rise to an amplicon of 72 bp. These fragments are clearly distinguishable in agarose gels and have been reproduced successfully in a different laboratory. Hence, the proposed method comprises a rapid, simple, reliable, and sensitive (down to 0.05%) PCR-based assay, suitable for detection of these four GM maize lines in a single reaction.  相似文献   

4.
Polymerase Chain Reaction (PCR) techniques are increasingly used for the detection of genetically modified (GM) crops in foods. In this paper, recombinant DNAs introduced into the seven lines of GM maize, such as Event 176, Bt11, T25, MON810, GA21, DLL25, and MON802, are sequenced. On the basis of the obtained sequence, 14 primer pairs for the detection of the segments, such as promoter, terminator regions, and construct genes, were designed. To confirm the specificities of the designed primer pairs, PCR was performed on genomic DNAs extracted from GM and non-GM maize, GM and non-GM soy, and other cereal crops. Because the presence of the corresponding DNA segments was specifically detected in GM crops by the designed primer pairs, it was concluded that this method is useful for fast and easy screening of GM crops including unauthorized ones.  相似文献   

5.
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.  相似文献   

6.
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.  相似文献   

7.
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.  相似文献   

8.
In this study, the event-specific primers for insecticide-resistant maize, MON810, and herbicide-tolerance maize, NK603, have been designed. Simplex PCR and multiplex PCR detection method have been developed. The detection limit of the multiplex PCR is 0.5% for MON810 and NK603 in 50 ng of the template for one reaction. Quantitative methods based on real-time quantitative PCR were developed for MON810 and NK603. Plasmid pMulM2 as reference molecules for the detection of MON810 and NK603 was constructed. Quantification range was from 0.5 to 100% in 100 ng of the DNA template for one reaction. The precision of real-time Q-PCR detection methods, expressed as coefficient of variation for MON810 and NK603 varied from 1.97 to 8.01% and from 3.45 to 10.94%, respectively. The range agreed with European interlaboratories test results (25%). According to the results, the methods for quantitative detection of genetically modified maize were acceptable.  相似文献   

9.
As more and more genetically modified (GM) crops are approved for commercialization and planting, the development of quick and on-spot methods for GM crops and their derivates is required. Herein, we established the polymerase chain reaction and agarose gel electrophoresis-free system for the identification of seven GM maize events (DAS-59122-7, T25, BT176, TC1507, MON810, BT11, and MON863) employing a loop-mediated isothermal amplification (LAMP) technique. The LAMP assay was performed using a set of four specific primers at 60-65 °C in less than 40 min, and the results were observed by direct visual observation. In these developed assays, the specificity targeted at each GM maize event based on the event-specific sequence was well confirmed, and the limits of detection were as low as four copies of maize haploid genomic DNA with an exception of 40 copies for MON810 assay. Furthermore, these developed assays were successfully used to test six practical samples with different GM maize events and contents (ranged from 0.0 to 2.0%). All of the results indicated that the established event-specific visual LAMP assays are more convenient, rapid, and low-cost for GM maize routine analysis.  相似文献   

10.
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.  相似文献   

11.
With the development of genetically modified organism (GMO) detection techniques, the Polymerase Chain Reaction (PCR) technique has been the mainstay for GMO detection, and real-time PCR is the most effective and important method for GMO quantification. 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. This study establishes the event-specific detection methods for TC1507 and CBH351 maizes. In addition, the event-specific TaqMan real-time PCR detection methods for another seven GM maize events (Bt11, Bt176, GA21, MON810, MON863, NK603, and T25) were systematically optimized and developed. In these PCR assays, the fluorescent quencher, TAMRA, was dyed on the T-base of the probe at the internal position to improve the intensity of the fluorescent signal. To overcome the difficulties in obtaining the certified reference materials of these GM maizes, one novel standard reference molecule containing all nine specific integration junction sequences of these GM maizes and the maize endogenous reference gene, zSSIIb, was constructed and used for quantitative analysis. The limits of detection of these methods were 20 copies for these different GM maizes, the limits of quantitation were about 20 copies, and the dynamic ranges for quantification were from 0.05 to 100% in 100 ng of DNA template. Furthermore, nine groups of the mixed maize samples of these nine GM maize events were quantitatively analyzed to evaluate the accuracy and precision. The accuracy expressed as bias varied from 0.67 to 28.00% for the nine tested groups of GM maize samples, and the precision expressed as relative standard deviations was from 0.83 to 26.20%. All of these indicated that the established event-specific real-time PCR detection systems and the reference molecule in this study are suitable for the identification and quantification of these GM maizes.  相似文献   

12.
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.  相似文献   

13.
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.  相似文献   

14.
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.  相似文献   

15.
A novel analytical procedure based on the combination of multiplex PCR, restriction analysis, and CGE-LIF to unambiguosly and simultaneously confirm the presence of multiple lines of genetically modified corn is proposed. This methodology is based on the amplification of event-specific DNA regions by multiplex PCR using 6-FAM-labeled primers. Subsequently, PCR products are digested by a mixture containing specific restriction endonucleases. Thus, restriction endonucleases selectively recognize DNA target sequences contained in the PCR products and cleave the double-stranded DNA at a given cleavage site. Next, the restriction digest is analyzed by CGE-LIF corroborating the length of the expected restriction fragments, confirming (or not) the existence of GMOs. For accurate size determination of the DNA fragments by CGE-LIF a special standard DNA mixture was produced in this laboratory for calibration. The suitability of this mixture for size determination of labeled DNA fragments is also demonstrated. The usefulness of the proposed methodology is demonstrated through the simultaneous detection and confirmatory analysis of samples containing 0.5% of GA21 and MON863 maize plus an endogenous gene of maize as control.  相似文献   

16.
Multiplex PCR procedures were developed for simultaneously detecting multiple target sequences in genetically modified (GM) soybean (Roundup Ready), maize (event 176, Bt11, Mon810, T14/25), and canola (GT73, HCN92/28, MS8/RF3, Oxy 235). Internal control targets (invertase gene in corn, lectin and beta-actin genes in soybean, and cruciferin gene in canola) were included as appropriate to assess the efficiency of all reactions, thereby eliminating any false negatives. Primer combinations that allowed the identification of specific lines were used. In one system of identification, simultaneous amplification profiling (SAP), rather than target specific detection, was used for the identification of four GM maize lines. SAP is simple and has the potential to identify both approved and nonapproved GM lines. The template concentration was identified as a critical factor affecting efficient multiplex PCRs. In canola, 75 ng of DNA template was more effective than 50 ng of DNA for the simultaneous amplification of all targets in a reaction volume of 25 microL. Reliable identification of GM canola was achieved at a DNA concentration of 3 ng/microL, and at 0.1% for GM soybean, indicating high levels of sensitivity. Nonspecific amplification was utilized in this study as a tool for specific and reliable identification of one line of GM maize. The primer cry1A 4-3' (antisense primer) recognizes two sites on the DNA template extracted from GM transgenic maize containing event 176 (European corn borer resistant), resulting in the amplification of products of 152 bp (expected) and 485 bp (unexpected). The latter fragment was sequenced and confirmed to be Cry1A specific. The systems described herein represent simple, accurate, and sensitive GMO detection methods in which only one reaction is necessary to detect multiple GM target sequences that can be reliably used for the identification of specific lines of GMOs.  相似文献   

17.
Several genetically modified (GM) cultivars are registered in Canada although they are not currently in commercial production. The GM cultivars can be distinguished from the non-GM and other GM cultivars by analyzing the DNA nucleotide sequence at the insertion site of the transgene corresponding to a single transformation event in the plant genome. Techniques based on modified polymerase chain reaction (PCR) strategies were used to generate sequence information from the plant genome flanking the insertion site of transgenic DNA for specific GM potato events. The plant genome sequence adjacent to the transgenic insertion was used to design PCR primers, which could be used in combination with a primer annealing to one of the nearby inserted genetic elements to amplify an event specific DNA fragment. The event specific PCR fragments generated were sequenced to confirm the specificity of the method.  相似文献   

18.
To devise a rapid and reliable method for the detection and identification of genetically modified (GM) events, we developed a multiplex polymerase chain reaction (PCR) coupled with a DNA microarray system simultaneously aiming at many targets in a single reaction. The system included probes for screening gene, species reference gene, specific gene, construct-specific gene, event-specific gene, and internal and negative control genes. 18S rRNA was combined with species reference genes as internal controls to assess the efficiency of all reactions and to eliminate false negatives. Two sets of the multiplex PCR system were used to amplify four and five targets, respectively. Eight different structure genes could be detected and identified simultaneously for Roundup Ready soybean in a single microarray. The microarray specificity was validated by its ability to discriminate two GM maizes Bt176 and Bt11. The advantages of this method are its high specificity and greatly reduced false-positives and -negatives. The multiplex PCR coupled with microarray technology presented here is a rapid and reliable tool for the simultaneous detection of GM organism ingredients.  相似文献   

19.
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.  相似文献   

20.
根据七种转基因玉米的重组DNA结构分别对Bt11、Bt176 、Mon810 、Mon863 、TC1507、 GA21 和NK603设计转化体特异性引物,进行多重PCR检测。在此基础上分别设计和筛选了七种转基因玉米转化体特异性oligo探针,制备转基因玉米的寡核苷酸芯片。实验表明,该探针特异性好,同常用的凝胶电泳检测方法相比,芯片杂交的灵敏度（0.01%）,优于凝胶电泳检测（0.1%）,由于采用了多重PCR技术一次可同时检测多个基因,提高了检测的准确率和效率。  相似文献   

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