KR100381930B1 - Method and Primers for Detecting Genetically Modified Organism - Google Patents

Abstract

The present invention relates to a method for detecting genetically modified organisms (Genetically Modified Organism) and primers for detection, and more particularly to 5-enolpyrovylshikimate-3-phosphate synthase (EPSPS)) or a gene characterized in that the polymerase chain reaction is carried out using a primer capable of specifically detecting a glyphosate oxidoreductase gene and a promoter to detect the engineered gene or promoter. It relates to a modified agricultural product detection method and a detection primer. By using the methods and primers of the invention, genetically modified agricultural products can be tested efficiently and quickly.

Description

Genetically modified agricultural product detection method and detection primer {Method and Primers for Detecting Genetically Modified Organism}

The present invention relates to a method for detecting genetically modified agricultural products and a primer for detection.

Genetically Modified Organism (GMO) refers to agricultural products that are developed to have traits or genes that cannot be represented by conventional breeding methods by using genetic engineering techniques for increasing production or distribution processing convenience.

GM foods, which are the source of GM food, are produced in the United States, Canada, and Argentina. Monsanto, the largest producer of genetically modified crops (GMOs), developed a product called Round-Up-Ready. Monsanto was originally an American chemical company that makes pesticides. Among the products is the herbicide 'round-up'. Monsanto has developed 'round-up-ready' genetically engineered soybeans that are more resistant to herbicides, no matter how much they sprinkle 'round-up' herbicides. Chemical companies like DuPont, Dow Chemical and Novartis are also developing GM products for the same purpose.

Since Monsanto released a 'Terminator' that farmers must buy seeds every year, and the Nature magazine reported that the monarch butterfly larvae died eating pollen from GM corn, the movement against GM food in the EU and Japan all started simultaneously. As it spreads, some media have criticized GM foods as 'Frankenstein Food' or 'Farmageddon'.

The lack of understanding of GMOs and the overreaction of some consumer groups has led many food companies not to use them. However, while large companies have apparently declared that they are not using GMFood, their investment in GMO development is not stopping but increasing.

The American Corn Growers Association (ACGA) urged US farmers to grow non-genetically modified grains to be tested as they are found to be mixing due to genetically modified seeds. Genetic ID, a pioneer in the field of genetically modified organisms (GMO), also tested 20 species of native corn in a joint statement with the ACGA, revealing that 45 percent of the genetically modified seeds were found in corn cropland. He pointed to the pollution.

Meanwhile, almost 91% of domestic demand is imported from the US, and GM soybean mixing is estimated to exceed 30%. Not only soybeans, but also Korea, which imports a lot of grain from the United States, is a big problem. The Ministry of Agriculture and Forestry announced that 82% of tofu sold by the Korea Consumer Protection Agency is mixed with GM soybeans. Recently, as consumers' anxiety amplified, the Ministry of Agriculture and Forestry recently decided to implement the GM agricultural product labeling system from March 2001.

In the future, GMO development and distribution is expected to be faster. The convenience and economical efficiency of genetically modified agricultural products will greatly increase the area of cultivation, and the demand for genetically modified agricultural products will increase rapidly. It is expected to be. Experts expect the global genetically modified agricultural market to expand to $ 20 billion in 2010.

Controversy continues over whether genetically modified agricultural products are harmful. This is because many scientists point out a number of problems that can arise from prolonged ingestion of GM crops. However, apart from the harmful status of the present situation in Korea, the establishment of a test method that can mask the whether or not genetically modified agricultural products are insufficient.

In line with the proliferation of genetically modified agricultural products and the increasing interest in GMO testing, the development of more efficient GMO testing systems is required. Currently available test methods include PCR for DNA analysis and ELISA for protein analysis. In general, the ELISA method for protein is inferior to the PCR method in the detection sensitivity, and has a limitation in the detection in foods that may cause protein denaturation due to heat treatment. Thus, there is a need for a method for testing genetically modified agricultural products with accurate reliability.

The present inventors designed a modified / engineered gene or promoter specific primer that can specifically detect the modified gene from the genetically modified agricultural products, and modified through the polymerase chain reaction using the designed primer The present invention has been completed by knowing that amplification of an engineered or engineered gene or promoter can be used to confirm the presence of genetic manipulation.

Accordingly, an object of the present invention is to provide a method for detecting genetically modified agricultural products and a primer for detection.

1 is agarose gel picture extracted genomic chromosome from soybean and corn flour,

Figure 2 is a gel electrophoresis picture showing the PCR result of amplifying the gtr (Soy-bean Glycine max glutamyl-tRNA reductase gene) gene using the extracted soybean chromosome as a template using a positive control primer of soybean,

Figure 3 is a gel electrophoresis picture showing the results of PCR experiments with the primer of the present invention using the extracted soybean chromosome as a template,

Figure 4 is a gel electrophoresis picture showing the results of multiplex PCR (Multiplex PCR) using the extracted soybean chromosome as a template using a positive control primer of the soybean and a primer for the detection of genetic modification,

Figure 5 is a gel electrophoresis picture showing the results of sequencing the PCR product amplified from genetically modified soybean.

The present invention relates to a method for detecting genetically modified agricultural products and a primer for detection.

The present invention provides a primer set forth in SEQ ID NO: 1 to SEQ ID NO: 18 useful for the detection of genetically modified agricultural products can detect whether the genetic modification.

The primer is a 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) gene or a glycosylated oxidoreductase gene or engineered or modified gene. Derived promoters.

The present invention is a gene for detecting the genetic manipulation of agricultural products by performing a polymerase chain reaction using a primer pair consisting of one species selected from SEQ ID NO: 1 to SEQ ID NO: 5 and one species selected from SEQ ID NO: 11 to SEQ ID NO: 14 It provides a method for detecting modified agricultural products.

The primer pair is specifically EPSPS-35-1 (SEQ ID NO: 3) / EPSPS-35R-1 (SEQ ID NO: 13), EPSPS-35-2 (SEQ ID NO: 4) / EPSSP-35R-1 (SEQ ID NO: 13), EPSPS Preferred is any one selected from -35-3 (SEQ ID NO: 5) / EPSPS-35R-1 (SEQ ID NO: 13).

The present invention is a gene for detecting the genetic manipulation of agricultural products by performing a polymerase chain reaction using a primer pair consisting of one species selected from SEQ ID NO: 6 to SEQ ID NO: 10 and one species selected from SEQ ID NO: 15 to SEQ ID NO: 18 It provides a method for detecting modified agricultural products.

The primer pair is GOX-P-1 (SEQ ID NO: 8) / GOX-PR-1 (SEQ ID NO: 17), GOX-P-1 (SEQ ID NO: 8) / GOX-PR-2 (SEQ ID NO: 18), GOX- P-2 (SEQ ID NO: 9) / GOX-PR-1, GOX-P-2 / GOX-PR-2, GOX-P-3 (SEQ ID NO: 10) / GOX-PR-1, GOX-P-3 / Preference is given to any one selected from GOX-PR-2.

The present invention also provides a genetically modified agricultural product detection kit comprising one primer pair selected from SEQ ID NO: 1 to SEQ ID NO: 5 and one primer pair selected from SEQ ID NO: 11 to SEQ ID NO: 14.

Finally, the present invention provides a genetically modified agricultural product detection kit comprising one primer selected from SEQ ID NO: 6 to SEQ ID NO: 10 and one primer pair selected from SEQ ID NO: 15 to SEQ ID NO: 18.

The gene modified or engineered from the genetically modified agricultural product was identified as a 5-enolpyrubilicimate-3-phosphate synthase or glyphosate oxidoreductase gene having herbicide resistance. Primers were designed based on phosphate synthase or glyphosate oxidoreductase genes and related promoters. After selecting efficient primers among the designed primers, polymerase chain reaction was performed using the selected primer pairs to confirm that the modified or engineered genes or promoters were specifically detected.

Thus, according to the methods and primers of the present invention, genetically modified agricultural products can be detected effectively.

Hereinafter, the present invention will be described in detail with reference to Examples, but the scope of the present invention is not limited only to these Examples.

Example 1 Design of Specific Primers for Testing Genetically Modified Agricultural Products

To investigate GM crops, we searched for GM crops from Monsanto, the largest producer of GM crops. As a result, all six types of genetically modified crops were searched for patents, and four of them were identified for patents. Genetically modified agricultural patents were largely related to two types of genetic manipulation. USP 5,776,760 (Glyphosate tolerant plants) and USP 5,463,175 (Glyphosate tolerant plants) were patents for glyphosate oxidoreductase genetic engineering, and USP 5,188,642 (Glyphosate- resistant plants) and USP 4,940,835 (Glyphosate-resistant plants) were patents for genetic engineering of 5-enolpyrubilicimate-3-phosphate synthase. In the Monsato patent, reference was made to each promoter and a gene sequence, and based on the above information, the sequence for each engineered gene and engineered promoter was obtained. Subsequently, specific primers that can be tested for genetic modification / modification based on the engineered gene and engineered promoter sequences were designed using a primer design program called Primer3. The results are shown in Table 1.

Example 2 Genomic DNA Extraction

In the present invention, for the genetic testing of genetically modified agricultural products, the genes are extracted from various agricultural products or foods and the extracted genes are examined by polymerase chain reaction (PCR). In the present invention for the extraction of the gene was preferably experimented using the extraction kit. The extraction kit was equipped with all the reagents required for separation purification, and using a column type purification system, it was possible to easily separate and purify high purity DNA in a short time. The extraction kit was used to isolate and purify genes from processed foods such as soybean sprouts, tofu, and milk powder as well as various agricultural products such as soybeans, corn, rice, and seeds.

In the present invention, in order to obtain accurate results, the Reference Reference Material (Round-Up-Ready Soya SB-0 (without genetically modified soybeans), SB-0.1 (with 0.1% genetically modified soybeans), SB-1 ( 1% genetically modified soybeans) and SB-5 (5% genetically modified soybeans) were purchased from Institutr for Reference Materials and Measurements, Belgium.

First, soybean flour, cornmeal, and tofu were each placed in a 1.5 ml tube of 0.1 g each, containing 1 ml of lysis buffer (8.0 M urea, 0.25% SDS, 0.25% sodium lauryl sarcinosate, 50 mM disodium EDTA, pH8.0) and Proteinase K. 10 ul (10mg / ml) was added and reacted at 60 ° C for 30 minutes.

The reaction tube was centrifuged at 3,000 to 5,000 rpm for 5 minutes, and then the supernatant was washed with the same amount of binding buffer (4 M GuHCl, 50 mM Sodium citrate, 0.3 M Sodiumacetate, 10 mM DTT, 1% tween20, pH 4.0). The mixture was transferred to a spin-column tube mounted on a collection tube, and centrifuged at 8,000 rpm for 1 minute to bind the chromosome to the filter of the column. After washing the column twice with 500 ul of Washing Buffer (20mM NaCl, 2mM Tris-HCL, pH7.7, 80% EtOH), 100 ul of Elution Buffer was added to the chromosome in a new tube. The purified chromosome was subjected to gel electrophoresis, and the results are shown in FIG. 1. In Fig. 1, M denotes a marker, 1 and 2 lanes are soybeans, 3 and 4 lanes are tofu, and 5 and 6 lanes represent genomic DNA extracted from corn flour. The purified chromosomes were then quantified and used for PCR experiments.

Example 3: Amplification of gtr Gene by Soybean Positive Control Primer

In the present invention, in order to confirm whether normal genes are extracted before genetically modified agricultural products are detected, a positive control primer was designed and PCR experiments were performed using chromosomes extracted from soybean as templates. The primer for the positive control targets the soy-bean Glycine max glutamyl-tRNA reductase gene (gtr) gene in soybean, and a pair of gtr-F (5'-gcctgtggaaatgcgtgaaaa) and gtr-R (5'-tggtggaaagggagtgaaaaagga) In the case of corn, Tub-F (5'-cgagcccagtcagccaggtc) and Tub-R (5'-gcggcgtggaagagagaagg) were used as a pair of primers for the tubulin gene.

In the case of PCR, the genomic DNA extracted in Example 2 was used as a template, and the positive control primers were amplified by inserting 20 pmoles into AccuPower PCR PreMix (Bionia Co., Ltd.), a PCR amplification kit. The amplification conditions were performed for 5 minutes at 94 ° C. for initial denaturation, 1 minute denaturation at 94 ° C. for 1 minute, annealing at 57 ° C. for 1 minute, and extension for 1 minute at 72 ° C. for 30 cycles. (last-extension) was performed for 5 minutes.

As shown in FIG. 2, 342 bp PCR product in soybean and 310 bp PCR product in corn was obtained, respectively. In addition, no PCR fragment was detected in the negative control without template DNA. In Figure 2, M is a marker, lane 1 is a negative control, lane 2 is tofu, lane 3 is SB-0, lane 4 is SB-0.1, lane 5 is SB-1, lane 6 is SB- The result of experiment with 5 is shown.

Example 4 PCR Amplification of Modified Genes from Genetically Modified Agricultural Products

In order to test the modified genes of GM crops, they were tested with SB-5 containing 5% transgenic soybeans. Dozens of primers designed in the present invention were tested to show three primer pairs (EPSPS-35-1 / EPSPS-35R-1, EPSPS 35-2 / EPSPS-35R-1 and EPSPS-) that showed the optimal test efficiency. 35-3 / EPSPS-35R-1) was selected. PCR reaction conditions were performed after initial denaturation at 94 ° C for 5 minutes, denaturation at 94 ° C for 1 minute, annealing at 57 ° C for 1 minute, and extension for 1 minute at 72 ° C for 30 cycles. Extension (last-extension) was carried out for 5 minutes. The product of the PCR reaction was electrophoresed and the results are shown in FIG. 3. In FIG. 3, M represents a marker, lane 1 represents the result of experiments with known primer pairs EU-35S-1 (5-gctcctacaaatgccatca) / EU-35S-2 (5-gatagtgggattgtgcgtca), and lane 2 is known. Test results with the primer pair EU-NOS-1 (5-gaatcctgttgccggtcttg) / EU-NOS-2 (5-ttatcctagtttgcgcgcta), lane 3 is EPSPS-35-1 / EPSPS-35R-1, and lane 4 is Lane 5 shows the results of experiments with EPSPS-35-3 / EPSPS-35R-1. As a result, it was confirmed that genetically modified soybean was effectively detected by obtaining PCR products of 195bp, 180bp, 248bp, 146bp, and 88bp, respectively.

Example 5 Multiplex Polymerase Chain Reaction (Multiplex PCR)

In the present invention, a gene amplification PCR kit comprising a primer pair of the present invention was prepared to perform a polymerase chain reaction. The kit was pre-mixed with all the components needed for the test, lyophilized in one portion, added with special stabilizing agents to increase the specificity and stability of the PCR reaction mixture, as well as to detect trace amounts of genes. As a result of increased stability, long-term storage (1 month at room temperature, 2 years or more during frozen storage) was possible. In addition, confirmation dye was added to enable electrophoresis immediately after the reaction.

Multiplex PCR (Multiplex PCR) experiments were performed under the amplification conditions of Examples 3 and 4 using the positive control primer of soybean and the primer for detecting the transgenic soybean at the same time, and the results are shown in FIG. 4. As shown in FIG. 4, 342 bp positive control amplification products appeared constant in all samples, and five pairs of modified gene amplification products were identified for each sample. Each lane is the same as shown in FIG. 3.

Example 6 Sequencing

After the PCR amplification products were purified, the PCR products amplified by PCR directed sequencing were examined to determine whether they were related to the modified gene. Direct sequencing of the purified PCR product using primers used in PCR was carried out on electrophoresis on 6% modified acrylamide gel containing 8M Urea and silver staining of the glass plate with gel was performed. 5 is shown. As a result of reading the nucleotide sequence of the gene, it was found that the amplified gene in the experiment was part of the 5-enolpyrubilicimate-3-phosphate synthase (EPSPS) gene, which is a gene used for genetically modified agricultural products.

The genetically modified agricultural product detecting method and primer for detecting the present invention can more easily and accurately examine the presence or absence of genetic manipulation from foods such as soybeans, corn, seeds, and soybean sprouts, tofu, and powdered milk.

As described above, according to the genetically modified agricultural product detection method and the detection primer of the present invention, it is possible to accurately examine the presence or absence of genetic manipulation.

Claims (6)

Genetically modified agricultural products for detecting the genetic modification of the agricultural product by performing a polymerase chain reaction using a primer pair consisting of one selected from SEQ ID NO: 1 to SEQ ID NO: 5 and one selected from SEQ ID NO: 11 to SEQ ID NO: 14 Detection method. The method of claim 2, wherein the primer pair is EPSPS-35-1 (SEQ ID NO: 3) / EPSPS-35R-1 (SEQ ID NO: 13), EPSPS-35-2 (SEQ ID NO: 4) / EPSSP-35R-1, EPSPS -35-3 (SEQ ID NO: 5) / EPSPS-35R-1. Genetically modified agricultural products for detecting the genetic modification of the agricultural product by performing a polymerase chain reaction using a primer pair consisting of one selected from SEQ ID NO: 6 to SEQ ID NO: 10 and one selected from SEQ ID NO: 15 to SEQ ID NO: 18 Detection method. The primer pair of claim 3, wherein the primer pair is GOX-P-1 (SEQ ID NO: 8) / GOX-PR-1 (SEQ ID NO: 17), GOX-P-1 (SEQ ID NO: 8) / GOX-PR-2 (SEQ ID NO: 5) 18), GOX-P-2 (SEQ ID NO: 9) / GOX-PR-1, GOX-P-2 / GOX-PR-2, GOX-P-3 (SEQ ID NO: 10) / GOX-PR-1, The method selected from GOX-P-3 / GOX-PR-2. Genetically modified agricultural product detection kit comprising one primer selected from SEQ ID NO: 1 to SEQ ID NO: 5 and one primer pair selected from SEQ ID NO: 11 to SEQ ID NO: 14. Genetically modified agricultural product detection kit comprising one primer selected from SEQ ID NO: 6 to SEQ ID NO: 10 and one primer pair selected from SEQ ID NO: 15 to SEQ ID NO: 18.