KR100687765B1 - - Quantitative detection of living modified soybean ingredients by real-time PCR using SYBR Green I - Google Patents

Abstract

The present invention is genetically modified using a pair of 5-enol-pyruvyl-shikimate-3-phosphatesynthase (EPPS) gene-specific primers suitable for real-time PCR (SYBR Green I stain) and primer pairs for lectin gene amplification. Provided are quantitative methods, compositions and kits for analyzing the incorporation rate of beans.

LMO, Real-time PCR, SYBR Green I

Description

Quantitative detection of living modified soybean ingredients by real-time PCR using SYBR Green I}             

1 is a test of the melting curve (melting curve) analysis of the amplification of non-specific PCR product in the SYBR Green I method.

Figure 2 is a concentration experiment of the primer pairs of various combinations in the process of selecting a primer pair suitable for the quantitative amplification of the gene EPSPS and the endogenous lectin (A) concentration combination of the primers suitable for quantification, and (B) of the inappropriate primer pairs Representative diagram of amplification curve and melting curve in concentration combination. (A) shows no peak at NTC (no template control) and one melting curve peak at 92oC, while (B) shows amplification at NTC and several peaks of melting curve near 92oC.

FIG. 3 shows a standard curve of IRMM standard soybean powder by SYBR Green I method using RRS (SEQ ID NO: 1) and RRS4 (SEQ ID NO: 4), Lectin1 (SEQ ID NO: 11), and Lectin2 (SEQ ID NO: 12) primer sets. The sample is analyzed.

The present invention provides a gene using a primer pair for 5-enol-pyruvyl-shikimate-3-phosphate synthase (EPPS) gene and primer pair for lectin gene amplification suitable for real-time PCR using SYBR Green I stain. Quantitative methods, compositions and kits for analyzing the incorporation rate of modified soybeans.

Currently, cultivation of genetically modified crops (LMO or GMO) is on the rise for the purpose of increasing the production of crops resistant to diseases and pests, reducing the cost and labor of herbicide-resistant GM crops (Nap et al. , (2003) Plant J. 33: 1-18).

The most commercialized genetically modified crops to date include corn and soybeans (Bio Safety White Paper, (2004) Korea Research Institute of Bioscience and Biotechnology; Nap et al., (2003) Plant J. 33: 1-18), It is widely applied. It is also tried in many crops such as red pepper, rice and perilla.

However, unlike conventional breeding methods, genetically modified crops artificially introduce genes of distantly related microorganisms, plants, and animals, which are not naturally crossed, to raise concerns about potential risks to humans and the environment. Has been called up. This concern has led to the enactment of regulatory laws in many countries, including Korea, that allow genetically modified agricultural products and foods to be distributed separately from general agricultural products and foods. In Korea, from March 2001, mandatory labeling of soybeans, corn, and bean sprouts has been made to indicate whether or not genetically modified agricultural products are produced (Ministry of Agriculture and Forestry Notice No. 2000-31). From July 2001, processed foods also contain soybeans, corn, In the case of potatoes, the genetic recombination shall be indicated on the product container or package (KFDA No. 2000-43).

One of the most widely used quantitative assays for GM crops is the real-time PCR method, which measures the fluorescence value in each cycle of the PCR, completes the graph with the fluorescence value per cycle, and then falls within the exponential phase range. The standard curve is calculated using the Ct (threshold cycle) value of the standard sample, and then the LMO content is measured by substituting the Ct value of the unknown sample.

A major method developed over the last decade to measure the copy number of a particular gene or DNA fragment in the expression of a gene or in a mixture of samples has been to target the fluorescence values of fluorescence dyes to target DNA fragments. The method of using real-time PCR to measure in real time during the specific PCR has been the mainstream. Examples of DNA-binding fluorophores include SYBR Green I, EtBr, Rhodamine, etc. (Higuchi et al., (1992) Bio / Technology 10: 413-417, 1992; Higuchi, (1993) Bio / Technology 11: 1026-1030; Wittwer et al., (1997) BioTechniques 22: 130-138). The method developed so far is based on the TaqMan probe method, which reads the intrinsic fluorescence value of 5 'terminal reporter fluorescent dye separated from quencher dye as PCR proceeds using 5' nuclease activity (Applied Biosystems, Foster City, CA, USA; Holland et al. (1991) PNAS 88: 7276-7280), the loof portion is the target specific sequence and the hairpin structure of the probe is complementary to the PCR process. Thus, when binding to a target, the dyes at both ends separate and emit fluorescence (Kramaer et al., (1996) Nature Biotech. 14: 303-308), and donor dyes are bound to the 3 'end of the upstream oligoprobe. The acceptor dye is bound to the 5 'end of the downstream oligo probe with respect to the adjacent nucleotide sequence. When both probes bind to the target according to the PCR process, the two dyes are close to each other. Hybridization Probe Method (Livak et al., (1995) PCR Methods Appl. 4: 357-362), a universal hairpin primer (Uniprimer ^TM ) attached to Z-shaped sequences attached to gene-specific oligonucleotides is attached to when coupled to a PCR amplification product Ampliflour ^TM universal amplification and detection system that emits fluorescence ^{(Ampliflour TM universal amplification and detection system} ) (Intergen Co. Purchase, NY, USA; Nazarenko , etc., (1997) Nucleic Acid Research 25 : 2516 -2521), using a pair of primers as in conventional PCR to take advantage of the characteristics of the SYBR Green I dye that fluoresces by specifically intercalating the dye in the double helix of the PCR product DNA Methods of using the SYBR Green I dye (Molecular Probes, OR, USA; Morrison et al., (1998) BioTechniques 24: 954-962).

Real-time PCR using SYBR Green I has the advantage of being inexpensive because it does not require attaching fluorescent dyes to primers or probes. However, the real-time PCR method using SYBR Green I should be a condition that only one type of PCR product is amplified, and requires a sensitive primer condition that does not occur dimer formation between non-specific products or primers occurring in conventional PCR. Because of these problems, Terry et al. (2002. Journal of AOAC International 85: 938-944) suggested that the SYBR Green I method is not suitable for quantification of round-up ready beans, while Hernandez et al. (2004. Journal of Cereal Science 39). : 99-107) suggested that the method can be used to quantify GA21 transgenic corn, comparable to the TaqMan method. In conclusion, for the successful application of SYBR Green I, it is very important to design primer pairs that only amplify specific PCR products and do not form nonspecific counterparts, and find suitable PCR conditions that do not form dimers between primers.

The present invention is to quantify the incorporation rate of Roundy Up Ready soybean, the most widely grown genetically modified soybean in the world, by real-time PCR using SYBR Green I, to produce EPSPS and lectin-specific PCR primers. After reviewing the suitable reaction conditions of the PCR primers, it was confirmed that the incorporation rate of the round-up ready beans can be effectively quantitatively analyzed by performing the real-time PCR using the selected primer pair, and completed the present invention.

 One object of the present invention, EPSPS (5-enol-pyruvyl-shikimate-3- phosphate synthase) gene selected from SEQ ID NO: 1 and 3, SEQ ID NO: 1 and 4, SEQ ID NO: 7 and 8 or SEQ ID NO: 9 and 10 A method of quantitatively analyzing the incorporation rate of transgenic soybeans by performing real-time PCR using SYBR Green I with a pair of specific primers and a pair of lectin gene specific primers selected from SEQ ID NOs: 11 and 12 and SEQ ID NOs: 13 and 14 To provide.

Still another object of the present invention is a pair of EPSPS gene specific primers selected from SEQ ID NO: 1 and 3, SEQ ID NO: 1 and 4, SEQ ID NO: 7 and 8, or SEQ ID NO: 9 and 10, SEQ ID NO: 11 and 12, and sequence To provide a real-time PCR composition for quantitative analysis of the incorporation rate of genetically modified soybean including a pair of lectin gene specific primers selected from Nos. 13 and 14.

Still another object of the present invention is a pair of EPSPS gene specific primers selected from SEQ ID NO: 1 and 3, SEQ ID NO: 1 and 4, SEQ ID NO: 7 and 8, or SEQ ID NO: 9 and 10, SEQ ID NO: 11 and 12, and sequence To provide a real-time PCR kit for quantitative analysis of incorporation rate of genetically modified soybean including a pair of lectin gene specific primers selected from Nos. 13 and 14.

In one embodiment, the present invention is directed to (1) EPSPS (5-enol-pyruvyl-shikimate-3-phosphatesynthase) selected from SEQ ID NO: 1 and 3, SEQ ID NO: 1 and 4, SEQ ID NO: 7 and 8, or SEQ ID NO: 9 and 10. A) preparing a pair of specific gene primers and a pair of lectin gene specific primers selected from SEQ ID NOs: 11 and 12, and SEQ ID NOs: 13 and 14; (2) performing real-time PCR using SYBR Green I with each primer pair selected from the sample DNA as a template; (3) reading the fluorescence value of the assay sample at each cycle extension time; Measuring the EPSPS Ct / lectin Ct value; (4) real-time PCR using SYBR Green I, comprising calculating the genetic modification content by substituting the EPSPS Ct / lectin Ct value of the analyte into the standard curve obtained using the EPSPS Ct / lectin Ct value of the standard sample. It relates to a method for quantitative analysis of the mixing rate of genetically modified soybean using the method.

In the present invention, "genetically modified soybean" refers to all soybeans that have been manipulated to artificially separate and introduce useful genes of a plant or a microorganism, unlike varieties developed by conventional crop breeding. Genetically modified soybean of the present invention is resistant to gluphosate (glyphosate) that inhibits shikimic acid metabolism, strain Agrobacterium, glufosate, strain CP4 ( Agrobacterium sp, strain CP4) Refers to a soybean introduced into the EPSPS (5-enol-pyruvyl-shikimate-3-phosphate synthase) gene isolated from. Glufosate is a chemical that forms the core of the world's best-selling round-up herbicide.

In the present invention, the "mixing rate" means the ratio of the amount of genetically modified crops to the total amount of the analysis sample (unknown sample), the mixing rate of the present invention is the amount of the soybean introduced EPSPS gene in the total amount of the analysis sample The ratio of, and expressed as a percentage (%).

In order to confirm whether the EPSPS gene is introduced, there are a method of detecting a protein and a method of detecting DNA. Kits for quantifying proteins have been commercialized by ELISA methods using antibodies against EPSPS, but the expression levels of proteins vary depending on the promoter used by the developing company, and in the case of storage or processed foods, they are degraded during processing. There is a limit that can not accurately quantify the incorporation rate of genetically modified crops. On the contrary, the DNA detection method using a PCR method using an EPSPS-specific primer can directly confirm whether the gene is modified and can accurately quantify it.

In particular, the present invention relates to a real-time PCR (real-time PCR) method using a sensitive fluorescent material to accurately quantify by minimizing the error of the end-point measurement method, in particular using SYBR Green I. A widely used method for the detection of genetically modified crops using real-time PCR is the TaqMan method, which uses a probe specific for a sequence in which a fluorescent dye is attached to a primer. However, the TaqMan method is difficult to increase the cost and design of the probe due to the primer attachment of the dye. As an alternative, the method using SYBR Green I, which can be added to the PCR solution to qualitatively and quantitatively detect DNA amplification of real-time PCR, can be used. (Hernandez et al., Analytical Biochemistry 323: 164-170, 2003). The method using SYBR Green I is a method that measures the amount of fluorescence emitted when binding to the minor groove of the DNA double helix of the generated PCR product, and there is no need to design a fluorescently labeled oligonucleotide primer. Has the advantage. Since the reliability of real-time PCR results using SYBR Green I depends a lot on the primer conditions produced, proper primer preparation is very important.

In this regard, the present invention provides primer pairs suitable for quantifying the incorporation rate of transgenic soybeans by real-time PCR using SYBR Green I.

In the present invention, a "primer" is a nucleic acid sequence having a short free 3 'hydroxyl group and serves as a starting point for template strand copying under appropriate conditions. The primer is preferably a single chain oligodeoxyribonucleotide, and the primer of the present invention does not form a nonspecific PCR product in a sample in a condition that does not include NTC (no template control) and genetically modified crops. It is characterized by that it is a primer specific for the introduced EPSPS that does not form and provides reliable results.

Real-time PCR with various primer pairs showed that the primer pairs of SEQ ID NO: 1 and 3, SEQ ID NO: 1 and 4, SEQ ID NO: 7 and 8, or SEQ ID NO: 9 and 10 were primer pairs that provided highly reliable results. there was. The primers can be used at the same or unequal concentrations. At appropriate primer molar ratios, no dimers are formed between the primers, resulting in reliable PCR results. The present invention has looked at molar ratios of suitable primers that do not form dimers, and various variations of such molar ratios are within the scope of the present invention. In addition, the primer pair is applicable to various plants such as corn, soybeans, potatoes, bean sprouts, wheat, etc., which have been introduced EPSPS.

In addition, the present invention is characterized in that the lectin gene, which is an internal gene, as an internal control in order to derive a reliable result, specifically the lectin gene specific of soybean having a nucleic acid sequence of SEQ ID NO: 11 and 12 or SEQ ID NO: 13 and 14 Provide primer pairs.

Using the EPSPS gene specific primer pair and the lectin gene specific primer pair, real-time PCR was performed using each primer pair and SYBR Green I selected from the sample DNA as a template, and at the time of extension of each cycle. Read the fluorescence values of the assay and measure the EPSPS Ct / Lectin Ct values. The values obtained above can be quantitatively analyzed by incorporating the EPSPS Ct / lectin Ct value of the analytical sample into the standard curve obtained using the EPSPS Ct / lectin Ct value of the standard sample.

In the present invention, the “standard curve” is an EPSPS Ct / lectin Ct value obtained by measuring the EPSPS Ct / lectin Ct value using the same primer from a standard sample having a known incorporation rate, and regression analysis of this value. It is a functional expression representing the linear relation of, and can be obtained through general program. The standard curve can be estimated by the method of least squares over n pairs of data, and the unknown regression equation fits to the fitted regression equation. By substituting the EPSPS Ct / Lectin Ct value of the analyte (unknown), the incorporation rate of the analyte can be calculated.

In another embodiment, a pair of EPSPS specific gene primers selected from SEQ ID NO: 1 and 3, SEQ ID NO: 1 and 4, SEQ ID NO: 7 and 8, or SEQ ID NO: 9 and 10, SEQ ID NO: 11 and 12, and SEQ ID NO: 13 It relates to a real-time PCR composition for quantitative analysis of incorporation rate of genetically modified soybeans comprising a pair of lectin gene specific primers selected from 14.

The composition may include reaction buffers (pH and magnesium concentrations vary), deoxynucleotides (dNTPs), Taq-polymerase, sterile water, SYBR Green I, and the like necessary to perform PCR reactions in addition to the primers.

In another embodiment, a pair of EPSPS specific gene primers selected from SEQ ID NO: 1 and 3, SEQ ID NO: 1 and 4, SEQ ID NO: 7 and 8, or SEQ ID NO: 9 and 10, SEQ ID NO: 11 and 12, and SEQ ID NO: 13 It relates to a real-time PCR kit for quantitative analysis of incorporation rate of genetically modified soybean including a pair of lectin gene specific primers selected from 14.

The kit for detection of the present invention consists of one or more other component compositions, solutions or devices suitable for analytical methods. Specifically, real-time PCR kits may include test tubes or other suitable containers, reaction buffers (pH and magnesium concentrations vary), deoxynucleotides (dNTPs), Taq-polymerases, sterile water, and the like.

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are only for illustrating the present invention in more detail, it will be apparent to those skilled in the art that the scope of the present invention is not limited by these examples in accordance with the gist of the present invention. .

Example 1 Primer Preparation and Real-time PCR Conditions

Primer was commissioned by a primer synthesis company (Bionia, Daejeon) by preparing a primer for amplification of the transgene in consideration of the length, GC content, Tm, etc. of the promoter, structural gene, terminator sequence introduced into the LM crop. In addition, after identifying genomic DNA sequences of endogenous genes specific to crops, primers were prepared for amplification of endogenous genes. The primers are primers that do not result in the formation of non-specific products in no template control (NTC) and 0% samples, and also without primer dimer formation.

Real time PCR was performed as follows.

First, a real-time PCR machine [iCycler (Bio-Rad) before preparing the sample. ABI7900 (Applied Biosystems)] is turned on. In addition, a 4-well master mix was prepared for the following composition for repeated experiments in 3-well samples. At this time, the PCR primary master mix of the introduced gene and the endogenous gene was removed except the amount of the sample DNA, and then divided into 1.5 ml tubes corresponding to 4-wells, and 200 ng of the sample DNA was mixed and mixed well. 25 ul was dispensed. At this time, the primer is put together the forward primer and the reverse primer, the combination of the forward primer 50nM, 300nM, 900nM and the reverse primer 50nM, 300nM, 900nM is possible. For example, the optimal concentrations of the forward primer of SEQ ID NO: 1 and the reverse primer of SEQ ID NO: 3 are 300 nM and 50 nM. In the well for the standard curve, a standard sample was added instead of the sample DNA. In addition, the well for NTC (no template control) was added distilled water.

14.5 μl primer

SYBR Green I (Sigma, USA) 1.0 μl

Taq polymerase (Invitrogen, USA) 1.5 unit

2.5 μl buffer

0.5 μl of dNTP (10 mM)

Sample DNA 50 ng (10 ng / ul)  5.0 μl

                                             25.0 μl

All samples were melted and kept on ice. After dispensing, the plate was covered with an optical cover sheet and completely closed with a dedicated roller. The plate was spun down using a centrifuge to prevent bubbles from forming at the bottom of the well. After inputting each well information into the analysis program of a computer, the reaction was performed for 10 minutes at 95 ° C, followed by PCR in 40 cycles of 30 seconds at 95 ° C, 30 seconds at 58 ° C, and 30 seconds at 72 ° C. I read it. And the melting curve (melting curve) was drawn in 0.5 ℃ step by step from 55 ℃ to 95 ℃. After the PCR reaction was analyzed by an analysis program and the LM content was calculated.

Example 2: Selection of primer pairs suitable for SYBR Green I method by analysis of dissolution curves

In order to perform quantitative analysis using SYBR Green I, it is important to develop a primer that specifically amplifies the gene introduced into LM-soybean and a primer that specifically amplifies the endogenous gene present in soybean. Nonspecific products, such as primer dimers, were also detected by the SYBR Green I method with increasing PCR cycles. Studies were performed to change the base composition of PCR primers or to design primers at other DNA sites (Table 1). In the present invention, by designing a primer capable of amplifying the transgene of LM-soybean, experiments were performed through analysis of lysis curve plots under PCR conditions that minimize nonspecific products (FIG. 1).

RRS1 + RRS3 (SEQ ID NOs 1 and 3), RRS1 + RRS4 (SEQ ID NOs 1 and 4), RRS2 + RRS3 (SEQ ID NOs 2 and 3), gcgRRS1 + RRS3 (SEQ ID NOs 5 and 3), gcgRRS1 + RRS4 (SEQ ID NOs) 5 and 4), gcgRRS2 + RRS3 (SEQ ID NOs 6 and 3), gcgRRS2 + RRS4 (SEQ ID NOs 6 and 4), RRS27 + RRS91 (SEQ ID NOs 7 and 8), and sltmf3a + sltm2a (SEQ ID NOs 9 and 10) Among the 9 pairs of primers, dimers are formed in the case of NTC (Negative Control) without template DNA with non-specific amplification, so the primer pair, for example gcgRRS1 + RRS4 (SEQ ID NO: 5 and SEQ ID NO: 4), is a SYBR It was found not to be suitable for quantitative analysis for Green I. RRS1 + RRS3 (SEQ ID NOS: 1 and 3), RRS1 + RRS4 (SEQ ID NOs: 1 and 4), RRS27 + RRS91 (SEQ ID NOs: 7 and 8), and sltmf3a + sltm2a (SEQ ID NOs: 9 and 10), gcgRRS1 + RRS3 (SEQ ID NOS: 5 and 3) was selected as a candidate for the transgene. Lectin1 (SEQ ID NO: 11), Lectin2 (SEQ ID NO: 12), and sltm1 (SEQ ID NO: 13) sltm2 (SEQ ID NO: 14) for the soybean endogenous gene were considered suitable because no dimer formation occurred.

Example 3: Selection of primer pairs suitable for SYBR Green I method by difference in concentration of primers

In order to find a primer pair suitable for amplification of the transgene and the endogenous gene, experiments were performed with primer concentrations of all combinations of 50nM, 300nM and 900nM forward primers and 50nM, 300nM and 900nM reverse primers. In this analysis, even if the same primer pairs have different concentrations, primer dimer formation did not occur and there was no background effect, and Ct value was found to be different according to the LM incorporation rate. For example, RRS1 (SEQ ID NO: 1) and RRS3 (SEQ ID NO: 3) primer pairs were suitable for quantification at 50 nM and 300 nM concentrations, respectively, but when 300 nM and 300 nM were used, amplification curves and melting curves were not suitable for quantification. Showed (Figure 2). The result is 300 nM and 50 nM for primer pairs RRS1 (SEQ ID NO: 1) and RRS3 (SEQ ID NO: 3) for detection of EPSPS transgene, and 300 for RRS1 (SEQ ID NO: 1) and RRS4 (SEQ ID NO: 4), respectively. nM and 50 nM, 300 nM and 50 nM for RRS27 (SEQ ID NO: 7) and RRS91 (SEQ ID NO: 8), and 900 nM and 50 nM for sltmf3a (SEQ ID NO: 9) and sltm2a (SEQ ID NO: 10), respectively Lectin1 (SEQ ID NO: 11) and Lectin2 (SEQ ID NO: 12) for detection of endogenous lectin 50 nM and 50 nM, respectively, and 300 nM and 300 nM for sltm1 (SEQ ID NO: 13) and sltm2 (SEQ ID NO: 14), respectively Selected primer pairs, such as, worked suitably for quantitation in one or two combinations of screened concentrations. On the other hand, the concentration of 50 nM may work better at 60 nM. IRMM 1%, 2%, 5% LM soybeans were quantified by amplifying transgenes and endogenous genes with appropriate primer concentrations of RRS1 and RRS3 obtained through screening. 1% obtained 0.93% experimental value, 2% obtained 1.89% experimental value, and 5% obtained 4.08% experimental value. It has errors of 7%, 5.5% and 18.4%, respectively. Although the SYBR Green I method can be used for qualitative analysis using the dissolution curve method under PCR conditions that minimize nonspecific products, primer pairs that do not produce nonspecific products are expected to be applicable to quantitative analysis.

Example 4 Analysis of Unknown Sample Using Genomic DNA and Plasmid DNA as Standard for LMO Quantitative Analysis

Analysis of IRMM 1, 2 and 5% LM-Soybean samples using transgenes RRS1 (SEQ ID NO: 1) and RRS4 (SEQ ID NO: 4) shows that IRMM 1% is 1.1%, 2% is 1.5% and 5% is 3.8 Experimental values were found in%, with errors of 10%, 25% and 24%, respectively (Figure 3 and Table 2). In this example, the endogenous positive control group used a pair of endogenous genes Lectin1 (SEQ ID NO: 11) and Lectin2 (SEQ ID NO: 12). On the other hand, the analysis using the TaqMan probe method (Applied Biosystems, Foster City, CA, USA; Holland et al., (1991) PNAS 88: 7276-7280) yielded experimental values of 1.27, 2.43 and 4.74%, respectively, 27% and 21.5. Errors of% and 5.2% are shown (Table 2). In conclusion, the method using SYBR Green I gave a similar error range to the conventional TaqMan probe method.

When using the EPSPS (5-enol-pyruvyl-shikimate-3-phosphate synthase) gene-specific primer pair and the primer pair for lectin gene amplification, EPSPS-introduced various plants such as corn, soybean, potato, bean sprouts and wheat Can be efficiently quantified by real-time PCR.

Attach sequence list electronic file  

Claims (6)

(1) a pair of EPSPS (5-enol-pyruvyl-shikimate-3-phosphatesynthase) gene specific primers selected from the group consisting of SEQ ID NOs: 1 and 3, SEQ ID NOs: 1 and 4, and SEQ ID NOs: 7 and 8 for detecting EPSPS; Preparing a pair of lectin gene specific primers SEQ ID NOs: 11 and 12; (2) performing real-time PCR using SYBR Green I with each primer pair selected from the sample DNA as a template; (3) reading the fluorescence value of the assay sample and measuring the EPSPS Ct / lectin Ct value at each extension time of the cycle; (4) real-time PCR method using SYBR Green I, comprising calculating the amount of genetic modification by substituting the EPSPS Ct / lectin Ct value of the analytical sample into the standard curve obtained using the EPSPS Ct / lectin Ct value of the standard sample. Incorporation rate quantitative analysis method of genetically modified soybeans. The method of claim 1, wherein a primer pair is used at a concentration at which nonspecific amplification and primer dimer are not formed. A pair of EPSPS gene specific primers selected from the group consisting of SEQ ID NOs: 1 and 3, SEQ ID NOs: 1 and 4, and SEQ ID NOs: 7 and 8 for detecting EPSPS and a pair of lectin gene specific primers of SEQ ID NOs: 11 and 12 Real-time PCR composition for quantitative analysis of the mixing rate of genetically modified soybeans. 4. The composition of claim 3, wherein primer pairs are used at a concentration at which nonspecific amplification and primer dimers are not formed. A pair of EPSPS gene specific primers selected from the group consisting of SEQ ID NOs: 1 and 3, SEQ ID NOs: 1 and 4, and SEQ ID NOs: 7 and 8 for detecting EPSPS and a pair of lectin gene specific primers of SEQ ID NOs: 11 and 12 Real-time PCR kit for quantitative analysis of mixing rate of genetically modified soybeans. 6. The kit of claim 5, wherein primer pairs are used at a concentration where nonspecific amplification and primer dimers are not formed.

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