KR101612044B1 - Standard Plasmid for Detection of Genetically Modified Organism, Qualitative detection method using the same and Detection Kit comprising the same - Google Patents

Abstract

The present invention relates to a method for quantitative analysis of a foreign gene introduced into a genetic modification using a standard plasmid and a standard plasmid for assaying a GM variant, and a kit for quantitative analysis of a genetic modification including a standard plasmid. The standard plasmids according to the present invention are cry1Ab It can be used for the screening of dielectrics containing genes, It is very useful as a reference material for analyzing whether or not the GM8 maize MON810 is mixed or mixed in the world.

Description

TECHNICAL FIELD [0001] The present invention relates to a standard plasmid for assaying a genetic variant, an assay method using the same, and a kit for assay, which comprises using the same and Detection Kit as a qualitative detection method,

The present invention relates to a standard plasmid for assaying a genetic modification, a quantitative analysis method of a foreign introduced gene incorporated into a genetic modification using the standard plasmid, and a kit for assaying a genetic modification including the standard plasmid.

Genetically modified foods are food products derived from GMOs (genetically modified organisms) that are the latest technology to improve the shelf life, nutrient content, color, flavor and texture of food. GMO is a collective term for creatures that increase the productivity by artificially separating and combining the useful genes of animals, plants and microorganisms, unlike conventional breeding by breeding of crops. Generally, to increase the productivity of GM crops, genes related to herbicide resistance, insect resistance, tolerance and cold tolerance are introduced into agricultural products. Today, there are many GMO foods on the market, with soybeans being the most common, with corn, papaya, pumpkin, and more. On the other hand, there is a controversy about the human health of GMOs, and consumers and farmers are increasingly concerned about GMO globally. Thus, the commercialization of GM crops requires environmental risk, human and food safety assessments and establishes a strong regulatory system including GMO obligatory markers in the countries importing genetically modified agricultural products. In Korea, there is a labeling system to label transgenic plants when transgenic plants contain more than 3% of genetically modified plants, and other countries have similar tolerance values. Therefore, besides the qualitative analysis method for measuring whether or not the GMO is mixed, an accurate analysis technique for quantitatively measuring the mixing ratio is needed.

Quantitative assays for GMOs are largely classified into protein analysis and DNA analysis. In general, the ELISA method using a protein expressed from a gene introduced into a transgenic plant has a limitation in that the detection intensity is lower than that of the PCR method, and protein denaturation may occur due to heat treatment. Therefore, a technology for quantifying a gene introduced into a transgenic plant using a real-time PCR method that quantitatively monitors the amount of amplification product together with amplification of the gene is currently mainly used. In addition to the primer set required for qualitative PCR, data can be obtained as a ratio of the intrinsic gene and the foreign gene to the reference material by real-time PCR using a fluorescent probe and CRM (certified reference material) or a standard plasmid as a standard material have.

The accuracy and precision of analysis of GMO tolerance values became very important according to the implementation of GMO markers. In order to accurately and precisely analyze tolerance values, the standard materials used for analysis are very important. Currently, reference materials are standard materials or genetically modified plants produced by mixing seeds of genetically modified plants and non-GMO seeds of each crop by weight in a conventional manner, Standard plasmids are used which are prepared by inserting an internal gene and an amplicon of an introduced gene extracted from a sample containing a certain amount of plants. In addition, in the case of corn, the amount of expression varies depending on the variety. In addition, when analyzing a sample containing various GM varieties, it is not possible to use a specific reference material. In particular, in the case of processed foods or raw materials, Or destruction occurs. Therefore, the method of protein detection also has limitations in GMO assay. In other words, standard materials for analysis of corn genetic variants should be able to preserve long-term stability and stability and homogeneity in itself, Is required.

SUMMARY OF THE INVENTION The present invention has been proposed in order to solve the problems of the conventional art as described above,

It is an object of the present invention to provide a standard plasmid for assaying a genetic modification, particularly a genetic modification containing the cry1Ab gene.

It is another object of the present invention to provide a method for quantitatively analyzing a GM variant using the standard plasmid, particularly a method for quantitatively analyzing a foreign gene introduced into a genetic modification containing a cry1Ab gene.

It is still another object of the present invention to provide a kit for assaying a genetic modification comprising the standard plasmid, particularly a kit for assaying a genetic modification containing the cry1Ab gene.

It is still another object of the present invention to provide a kit for assaying a genetic modification comprising the protein expressed from the standard plasmid, particularly a kit for assaying a genetic modification containing the cry1Ab gene.

According to an aspect of the present invention, there is provided a standard plasmid for black of a gene variant that contains the ss Ⅱb gene and cry1Ab gene.

According to the present invention, the cry1Ab gene comprises a 5 'flanking sequence and a 3' flanking sequence. The standard plasmid for assaying a genetic modification is provided.

The cry1Ab gene contained in the standard plasmid includes the nucleotide sequence of SEQ ID NO: 1, and the ssIIb gene includes the nucleotide sequence of SEQ ID NO: 2, but is not limited thereto.

The standard plasmid for the assay of the GM variant of the present invention includes the sequence of the GM maize MON810 confirmation sequence ( cry1Ab gene sequence) and the inherent gene ( ssIIb , Starch Synthase IIb) provided by the Food and Drug Administration and IRMM. ERM-AD413, a standard plasmid for analysis of commercially available conventional GM corn MON810, was used for the ssIIb I'm including a gene instead hmg (high mobility group) gene, as compared with the genetic composition is not suitable for domestic test method, standard plasmid of the present invention are set forth in the endogenous gene KFDA revolution ssIIb Gene.

Preferably, the cry1Ab gene contained in the standard plasmid may include a 5 'flanking sequence and a 3' flanking sequence. The standard plasmid according to the present invention comprises the cry1Ab The present invention has an advantage in that it can distinguish a target strain to be analyzed by including adjacent sequences of the 5 'region and the 3' region of the gene. The GM gene alone can not be distinguished from other varieties (for example, cry1Ab gene is GM gene of GM maize Bt11 as well as GM maize MON810), and it is judged whether or not it is the same kind as GM maize MON810 by qualitative PCR by including adjacent sequence .

Also, the event of the transgenic plant can be determined using the standard plasmid of the present invention. Therefore, the standard plasmid of the present invention is more suitable as a standard material for GMO detection because it can distinguish genetic variant events as well as GMO varieties.

More preferably, the standard plasmid may be pBlunt-12 kb MS having a genetic map as shown in Fig. In one embodiment of the present invention, the pBlunt-12kbMS plasmid comprises the forward primer SSIIb 1-5 (SEQ ID NO: 9) for securing the ssIIb insertion gene and the ssIIb gene amplified by the reverse primer SSIIb 2-3 (SEQ ID NO: 10) of PCR products and GM corn MON810 forward primer for obtaining an inserted gene GWMcry3F-1 (SEQ ID NO: 11) and amplified by the cry1Ab reverse primer AP2 (SEQ ID NO: 12) PCR products of the gene are respectively obtained and then treated with a restriction enzyme and inserted into a pCR-Blunt vector. The pBlunt-12kbMS plasmid can be used as a standard plasmid for a quantitative assay method for calculating the gene incorporation rate of the genetic modification according to the present invention.

The GM variant to be tested by the standard plasmid according to the present invention is preferably a GM maize MON810 variety, but is not limited thereto.

"GM maize MON810" of the present invention means a transgenic maize into which a cry1Ab gene, which produces a Cry1Ab protein, a Bt protein that is toxic to a pest derived from Bacillus thuringiensis ( Bt ), is introduced. Delta-endotoxins such as the Cry1Ab protein are located in the midgut epithelium of the insect's brush border and selectively bind to specific sites. After binding, the cation spores interfere with the flow of heavy ions, causing paralysis and death. cry1Ab acts only as an insecticide for insect insects and its activity is directly due to the presence of a binding site in the target insect. Since there is no such binding site on the surface of human small intestine, these proteins do not act on livestock or humans. Thus, the GM maize MON810 strain contains the cry1Ab gene and is suitable as a genetic modification of the quantitative assay according to the present invention. However, it is not necessarily limited thereto, and cry1Ab It is obvious that any genetic modification containing the gene can be the subject of the assay according to the present invention.

For another purpose of the present invention, there is provided a method for quantitative analysis of a foreign gene introduced into a genetic modification using the standard plasmid.

The quantitative analysis method

I) diluting the standard plasmid by concentration;

Ii) performing real-time PCR using the standard plasmid diluted by concentration and the DNA obtained from the biological sample as templates, respectively, using a PCR primer set for assay and a probe;

Iii) calculating a standard quantitative curve by measuring the amount of the PCR product performed using the diluted standard plasmid; And

Iv) introducing the amount of the PCR product performed on the DNA obtained from the biological sample into the standard quantitative curve to calculate the incorporation rate of the foreign introduced gene.

In the above quantitative analysis method, the GM variant is preferably a GM maize MON810 variety, but is not limited thereto.

When real-time PCR is used, the amount of intrinsic and transgenic genes can be calculated by measuring the amount of fluorescence produced in the PCR amplification process using bidirectional primers and detection probes that specifically bind to the DNA of the endogenous and transgene have. That is, the quantitative analysis method is a method of analyzing how many percent of transgenic plants are contained through the relative ratio of exogenous transgene to the inherent gene that a plant of the corresponding variety has.

According to one embodiment of the present invention, a standard curve representing the number of PCR cycles for the number of gene copies is calculated by measuring fluorescence by real-time PCR using a standard probe for detection, a primer for detecting an introduced gene, and a primer for detecting an inherent gene By comparing this to real-time PCR products from the analytical samples, the incorporation rate of transgenic plants in the sample can be analyzed.

Preferably, in the quantitative analysis method, the PCR primer set of step ii) may be a primer set of SEQ ID NOs: 3 and 4 and a primer set of SEQ ID NOs: 6 and 7, and the probes may be SEQ ID NOs: 5 and 8 But is not limited thereto. The probe refers to a detection probe in which a fluorescent substance is introduced into a base sequence for quantitative analysis during real-time PCR. The kind of the detection probe can be appropriately selected by those skilled in the art depending on the type of the inherent gene and the introduced gene.

In step iii) of the quantitative analysis method, standard quantitative curves are obtained by diluting a standard plasmid DNA at a predetermined ratio, performing real-time PCR on a plurality of standard samples having different gene copy numbers, and performing PCR on the number of gene copies obtained therefrom Can be calculated by applying the number of cycles. Specifically, the DNA of a standard substance of which the mixing ratio is known is appropriately diluted to make the number of copies at regular intervals, or DNAs of transgenic plants and unmodified plants are mixed at a predetermined ratio to prepare several samples When real-time PCR is performed for the PCR, the degree of fluorescence according to the number of PCR cycles can be measured. At this time, select the region where the fluorescence signal of the standard solution is exponentially amplified, set the threshold line (Th. Line), and set Th. The number of cycles at the point where the line and the amplification curve of the standard solution intersect is called the threshold cycle (Ct), which is the most reproducible correlation with the initial concentration of the sample (number of gene copies) This is the most important figure in the analysis. In the real-time PCR, a standard curve is created by converting the number of gene copies of the reference material at the Ct value into a log value and the number of PCR cycles with respect to the number of copies of the gene as the Y axis. When the fluorescence intensity of the analytical sample is applied to this standard curve, the number of gene copies of the analytical sample can be known.

According to another aspect of the present invention, there is provided a kit for assaying genetic modification comprising the standard plasmid.

The GM variant that is the subject of the assay kit is preferably GM corn of the GM maize MON810 variety, but is not limited thereto.

Preferably, the assay kit may include a primer set consisting of SEQ ID NOS: 3 and 4, 6 and 7, and probes having SEQ ID NOS: 5 and 8, but is not limited thereto. The present invention provides a kit for assaying a genetic modification comprising the standard plasmid, enabling quantitative analysis by real-time PCR for genetic modification tests containing a cry1Ab gene.

The kit may additionally include reagents for transcription, amplification, and product detection and instructions therefor. For example, the kit may contain a transcriptional enzyme, a deoxynucleotide, a thermostable polymerase suitable for DNA amplification reactions, and a reagent for labeling and detecting nucleic acids.

In another aspect of the present invention, there is provided a kit for assaying a genetic modification containing a protein expressed from the standard plasmid. The standard plasmid of the present invention includes a T7 promoter, and the foreign gene c ry1Ab introduced into a standard plasmid The gene and the ss Ⅱb gene, an inherent gene, are operably linked to the T7 promoter. Thus, the Cry1Ab protein and the SSIIb protein can be expressed from the standard plasmid of the present invention in an appropriate protein expression environment. These expressed proteins can be used as a standard protein for quantitative analysis or qualitative analysis through an ELISA (Enzyme Linked Immunosorbent Assay) method or the like.

The standard plasmid according to the present invention is a < RTI ID = 0.0 > The present invention can be applied to the screening of genomes containing the gene, and thus the genotype of the transgenic plant can be discriminated by using the standard plasmid of the present invention. Especially It is very useful as a reference material for analyzing whether or not the GM8 maize MON810 is mixed or mixed in the world. Therefore, it is possible to provide an accurate and reliable quantitative analysis method and a kit for detection of the genetically modified maize MON810 using the standard plasmid according to the present invention.

1 is a block diagram of a standard plasmid according to the present invention.
2 is a graph showing a result of measurement of the concentration of standard plasmid DNA according to the present invention.
Figure 3 is an electrophoresis image to confirm the gene and orientation inserted into the vector to make a standard plasmid according to the present invention (M: molecular weight marker, MS: 12 bkMS, UC: Plasmid DNA, C: Plasmid DNA cut with enzyme, set1: ligation confirmation primer set, set2: orientation confirmation primer set)
4 is a graph showing fluorescence measurement values of standard plasmid DNA according to the present invention.
FIG. 5 shows the result of confirming homogeneity by performing real-time quantitative PCR using a standard plasmid DNA according to the present invention.
FIG. 6 shows the result of confirming stability by performing real-time quantitative PCR using a standard plasmid DNA according to the present invention.
7 (a) shows the result of quantitative analysis of an inherent gene of an unknown sample using a standard plasmid according to the present invention, (b) shows the result of quantitative analysis of an inherent gene of a standard plasmid and an unknown sample according to the present invention Real-time PCR results are shown.
8 (a) shows the result of quantitative analysis of an introduced gene of an unknown sample using a standard plasmid according to the present invention, and (b) shows the result of quantitative analysis of an introduced gene of a standard plasmid and an unknown sample according to the present invention Real-time PCR results are shown.

Hereinafter, the present invention will be described in more detail with reference to examples. It will be apparent to those skilled in the art that these embodiments are provided to understand the contents of the present invention and that the scope of the present invention is not limited to these embodiments.

< Example >

1. Standard plasmid production

Standard plasmids were prepared by linking the PCR products for the maize inherent gene (ss IIb ) and the GM gene ( cry1Ab ) and inserting them into the pCR-Blunt vector (Invitrogen, USA) (pBlunt-12kbMS, see Figure 1) PCR product of the cry1Ab gene amplified by the forward primer GWMcry3F-1 (SEQ ID NO: 11) and the reverse primer AP2 (SEQ ID NO: 12) was obtained in order to secure the insertion gene of the full gene And then inserted into the pCR-Blunt vector. Thereafter, a PCR product of the ssIIb gene amplified by the forward primer SSIIb 1-5 (SEQ ID NO: 9) and the reverse primer SSIIb 2-3 (SEQ ID NO: 10) was obtained in order to obtain the ssIIb insertion gene, And Stu I , and then the cry1Ab The plasmid pBlunt-12kbMS was prepared by inserting the gene-inserted pCR-Blunt vector using the Zero PCR cloning kit (Invitrogen, USA). The size of the ssIIb gene inserted in the pCR-Blunt vector was 1622 bp and cry1Ab Gene The size is 6922 bp, and the size of the final inserted insertion into the vector is 8544 bp. The insert inserted into the pBlunt-12 kb MS and its direction were confirmed as shown in Fig. The recombinant plasmid pBlunt-12kbMS was introduced into E. coli TOP10 cells, transformed, and cell culture was carried out in LB medium to obtain a large amount.

SEQ ID NO: Primer / probe type Primer / probe base sequence 3 SSIIb 5 ' 5'-CTC CCA ATC CTT TGA CAT CTG C-3 ' 4 SSIIb 3 ' 5'-TCG ATT TCT CTC TTG GTG ACA GG-3 ' 5 SSIIb-taq 5'-FAM-AGC AAA GTC AGA GCG CTG CAA TGCA-TAMRA-3 ' 6 MON810 5 ' 5'-GAT GCC TTC TCC CTA GTG TTGA-3 ' 7 MON810 3 ' 5'-GGA TGC ACT CGT TGA TGT TTG-3 ' 8 MON810-taq 5'-FAM-AGA TAC CAA GCG GCC ATG GAC AAC AA-TAMRA-3 ' 9 SSIIb 1-5 5'-CTC CCA ATC CTT TGA CAT CTG C-3 ' 10 SSIIb 2-3 5'-GAC TTG CCT GAA CAC TAC ATC GAC-3 ' 11 GWMcry3F-1 5'-GAC GTT CTC AGA GAC AGT ATT CAA CTT TG-3 ' 12 AP2 5'-ACT ATA GGG CAC GCG TGG T-3 '

2. Plasmid DNA  extraction

Plasmid DNA (pDNA) was extracted using the plasmid maxi kit (Qiagen) from the cell obtained in Example 1 as it was or with slight modification to the method proposed by the manufacturer. Specifically, the cultured cells were harvested by centrifugation at 6000 g for 15 minutes at 4 ° C, and 10 ml buffer P1 was added to the harvested cell pellet and resuspended. After addition of 10 ml Buffer P2, invert and mix 4-6 times. After incubation for 5 minutes at room temperature (15-25 ° C), add 10 ml Buffer P3. (Inverting) and allowed to mix in ice for 20 minutes. The supernatant was then centrifuged at 20000 g or more for 30 minutes at 4 ° C (if the supernatant is not clean, further centrifugation is performed). The Qiagen-tip was equilibrated by passing 10 ml Buffer QBT through gravity, then the supernatant from step 5 was passed through the Qiagen-tip, and the Qiagen-tip was washed twice with 30 ml Buffer QC. Add 15 ml Buffer QF to the 50 ml tube to separate the DNA. 10.5 ml of isopropanol was added and the separated DNA was precipitated at ≥15000 g for 30 minutes at 4 ° C, and then the supernatant was discarded. Wash the DNA pellet with 5 ml of 70% ethanol at room temperature, centrifuge at ≥15000 g for 10 minutes, and discard the supernatant carefully. The pellet was left to dry naturally for 5-10 minutes and finally dissolved in a suitable volume of buffer (eg TE buffer, pH 8.0, 10 mM Tris, pH 8.5) to prepare the final plasmid DNA (pDNA).

3. Plasmid DNA Concentration and contamination level

In order to qualitatively evaluate and quantify the extracted plasmid DNA, absorbance measurement and fluorescence measurement using picogreen were performed. The DNA solution showed a UV absorbance spectrum of a typical DNA having a peak near 260 nm as a result of measurement of ultraviolet absorbance (see FIG. 2 and Table 2).

In addition, the extracted plasmid DNA was judged to be a good quality sample with less impurities such as proteins. The degree of protein contamination in the DNA solution can be determined as follows. When the absorbance at 260 nm is divided by the absorbance at 280 nm and a value between 1.7 and 2.0 is obtained, it is judged to be high-quality DNA with little protein contamination. All the DNA samples used in this experiment have values between these values, . In addition, the absorbance at 260 nm can be divided by the absorbance at 230 nm to determine the degree of contamination of sugars in the DNA solution (eg, polysaccharides that make up the plant tissue). If a value of 0.8 or more is obtained, it is judged to be high-quality DNA. All the DNA samples used in the present experiment had a value of 0.8 or more, and it was judged to be a high-quality sample which can be subjected to the next step with less sugar contamination (see Table 2).

tube 12kbms Stock solution concentration (ng / ul) 175.55 1/5 dilution concentration (ng / ul) 35.11 230 0.333 260 0.702 280 0.371 Protein incorporation (260/280) 1.89 Carbohydrate incorporation (260/230) 2.11

In order to confirm the concentration of the plasmid DNA, a method of measuring the concentration of ds-DNA using a fluorescent dye Pico Green ( ^TM) (Invitrogen, Carlsbad, Calif.) That specifically binds to ds-DNA To determine the amount of DNA in the solution. Specifically, a calibration curve for the fluorescence value was obtained using a lambda DNA calibrator already contained in the kit, and the concentration of the DNA was determined by substituting the fluorescence value of the sample into the calibration curve. As a result, the extracted plasmid DNA was qualitatively purity suitable and quantitatively sufficient to be used in the next step.

4. Standard plasmids stock  Produce

After calculating the concentration of the plasmid DNA by fluorescence quantification, the number of copies of the known plasmid DNA size (base pair, bp) was substituted into the following equation, and the final plasmid stock concentration was 4.00 X 10 &lt; ⁸ &gt; cp / [mu] l.

Number of copies (cp / ul) = amount x 6.022 x 10 ²³ / plasmid DNA size (bp) x 1 x 10 ⁹ x 650

After filtration in a clean bench to remove impurities, 50 μl of each was dispensed into approximately 800 cryo tubes (Nunc).

A preliminary test was performed using real-time PCR to determine if the number of copies match the expected number of copies. In this experiment, all experiments were carried out using ABI 7900HT (Applied Biosystems, USA). The basic design of the experiment and the experimental components such as primers and probes were taken from the specifications of the Food and Drug Administration. First, using commercially available plasmid DNA (Nippon Gene, Japan), which already knows the copy number, as a standard material for drawing a standard curve, and using the plasmid DNA extracted according to Example 1 and Example 2 as an assay sample, Respectively. A DNA standard material was added between 250000, 20000, 1500, 125, and 20 copies, and the results were repeated with constant intervals between concentrations. . This confirms the same trend in the results of the GM gene.

5. Identification of homogeneity of standard plasmids

A total of 800 tubes were selected and 10 tubes were selected at uniform intervals to confirm homogeneity between the tubes.

The ratio of the transgene: endogen (GM gene: endogenous gene) of the plasmid DNA in 10 tubes analyzed to check the homogeneity of the pathogens was measured (transgene and endogen are recombined to 1: 1 each). The measured values were 0.971 to 0.985 and the average value was 0.98 ± 0.007 (see FIG. 5). In the case of recombinant ERM-AD413 produced by IRMM, 10 tubes were within the standard deviation range of each other and the authentication value and range were 1.037 ± 0.007, indicating that there was homogeneity between the strains. In the present invention, The ratio was closer to 1, so it was judged to be homogeneous.

6. Confirm stability of standard plasmid

In order to confirm whether the sample is stable during transport, it was tested at three different temperatures (-20 ° C, 4 ° C, 60 ° C) at regular intervals (2 weeks, 4 weeks) Were analyzed to see if there was a change in the ratio of the two genes.

The measurement of the ratio of transgene to endogenous sample analyzed for short-term stability was 0.974 ± 0.002 (0 time), 0.984 ± 0.005 (2 weeks, -20 ° C), 0.980 ± 0.011 (4 weeks, 4 ℃), 0.968 ± 0.004 (2 weeks, room temperature), 0.973 ± 0.012 (4 weeks, room temperature) 0.992 ± 0.008 (2 weeks, 60 ° C) for 4 weeks at -20 ° C, 4 ° C and room temperature, the GM gene and the intrinsic gene ratio were similar to those of the homogeneity test, and it was confirmed that the sample was stable at the temperature (See FIG. 6).

7. Using standard plasmids Quantitative property  Confirm

Real-time PCR was performed using the DNA obtained by diluting the pBlunt-12kbMS plasmid with the copy number of 6 steps as a template, and a standard quantitative curve was calculated using the result, and quantitative analysis was performed on the unknown sample. In addition, the quantitative curve of the commercially available Nippon Gene was calculated and compared with the pBlunt-12kbMS plasmid of the present invention as a calibrator. As a result pBlunt-12kbMS the correlation coefficient (R ^) value of the plasmid appeared to 0.9933 (ss Ⅱb) and 0.9985 (cry1Ab), the figure is not less than 0.99, Nippon Gene of the correlation coefficient (R ^) value of 0.9923 (ss Ⅱb) And 0.9923 ( cry1Ab ). Therefore, it was confirmed that the plasmid according to the present invention can be used as a standard plasmid for quantitative analysis. As shown in FIG. 7 and FIG. 8, it was confirmed that the plasmid can be used as a standard plasmid for quantitative analysis to know the% of introduced gene (GM) of unknown sample.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the invention is not limited to the disclosed exemplary embodiments. It is therefore intended that the scope of the invention be defined by the claims appended hereto and their equivalents.

<110> Korea Research Institute of Standards and Science <120> Standard Plasmid for Detection of Genetically Modified Organism,          Qualitative detection method using the same and Detection Kit          comprising the same <130> P12090861188 <160> 12 <170> Kopatentin 2.0 <210> 1 <211> 6922 <212> DNA <213> Artificial Sequence <220> <223> cry1A b <400> 1 cctgcatact ttcgttgtgc ttttatattg cctgagtaag tcttttgaaa tgttttaatg 60 aagcagaaaa tgttgattta tggcacacag gggaacaaaa gtctcacttg taatatgata 120 gagcaaaatc tcatttggat taagtagctg ctgtgattct tgtcgtctta ttttcttcct 180 agtgtggtgc aagtaaattg tgcttttggt caatcgcatt tggtatggta cagatcgaag 240 agtttttttt ccatgctata caattgagta tgaatatata atgccatctc agcttctgct 300 tgttgctgtc ttgcttaatt gctaaaatca tttgtggtac atacacacac gagtctggca 360 ttttttgagt atgatcgtct gcaacagctg ctgtatcttc tcacctagct gctgaaacat 420 taattcttga aacccacact gtttgactca tcagtttcat gcacctgtat gttttttgtt 480 gtacaaatgt ttttcatatg tttaccccaa atattgcaga tgtttgagat tgtgagcctt 540 gccgatgatt tgcttccgca tatccctgcg agaatcatta agttaccaac atattaccat 600 acatataaag gctcttcaac aaagaaatct gcttccatca aacaagaggg gggtggttca 660 acaggaaatg aaaggtctgg ccgtgagagg ctattgcgtg agcaacctga gcttttacaa 720 cagtttggta tggatttgtt acccacaatg acacaggtta gtgctggaat tcttttcctg 780 agtcctgaca gccatttcaa ttgctatttg tgttttgtct ttgtgtactt taatagcctg 840 tgtgcttgtt caggtgtatg gttctagtgt aaatgcacca atacgtcaca agtgtttatc 900 tatcattgga aaattaatgt actacagctc cgccgaaaca atccagtctc tccttggcac 960 aacaaacata tccaggtttt ctctctctca cctggaacac acaaaagaga tgcatgtttc 1020 attaatagaa ccgaaaaaat tctcagttca tttctagaga atgatttcag aacaatggat 1080 gttaaaagac aaaaagaaag tctctcaagt ctcaatgtac atacttcttt tccaatccct 1140 tctaagttat ttcatttcca tctgcagctt ccttgcgggc attcttgcat ggaaagatcc 1200 tcaagtgttg attcctgctc ttcagatagc agaaataatg atggaaaagc ttccagagac 1260 attttccatg ttatttgtga gggaaggtgt cgttcatgct gtggagtcac tcatatgttc 1320 agaatcttcc aacaaaatgc cttctcaggt gccacctcag gataaagata aagattctgc 1380 catgccatca cgttctagac gtcaacgccg gcgtgcgggt gctgttgcag cagaaaatag 1440 ttcattggat gaatcaaata gttccaacct tggtgttatg tgctcaacag caaccgcttc 1500 agaagctcca aacaccagcc ttcgtttcac agttagtgac catgcaaagt ctttcaaaga 1560 taaatacttc ccggcggata ctgattcaag tgatatcggg tttactgatg accttcttaa 1620 actgagggca ctctgtgcaa agttgaatac tgtctctgag aacgtcaaaa caaaagcaaa 1680 agggaaatca aaagctataa gcactaattt tttggacatc tcaattgatg ttgaagaaca 1740 attagacaaa ataatatctg aaatgctctc tgagcttagc aaagttaatg gtgtttccac 1800 atttgaattc attagaagtg gtgttgtcat agcgttgctt gactatttgt cctgtgggac 1860 atttgggaag gaaaaggtat ctgaaggaaa cctacctcag cttcgtcagc aggcccttag 1920 acgatacaaa accttcatat ctgttgccct ttctattgat catggacgag atgaaactcc 1980 catggcactg ttggttcaga aactgcaaag tgcattgtct tcgctggaac gtttccctgt 2040 tgtgctcagc cagtctagta ggattggtat tggagggtct cgtctgactt caggtcttag 2100 tgctctggct caacccttca agcttcgtct ttcccgagct caaggcgaaa aatcacttcg 2160 ggattattca tcaaatattg tgcttatcga cccatttgct agtcttgcat ctgttgaaga 2220 attcctttgg cctagagttc aacgcagtga ggttgcatcg aagcctataa ttccatctgg 2280 taataattct gaatctgggg ttcctggcac cacggctggt gcatcattaa cagctgcaat 2340 ggctcaatct ggtcggcgcc caacaacaag atcaaagtca tctgctgcag gtggtcttac 2400 atctaagaag gattctcatg atgaaagcac tagtactgcc aaaggaaaag gaaaagctat 2460 tgtaaagcca aactcagatg aatcaaaagg acctgactgc tcgcaagcaa attcggaaat 2520 gaaagaaggc taccgaaagt cctcgttcag gtcggtgcag cccacatcga tgtccaagga 2580 gaagtggtgg ctgtggtggg cacacttgcc gatcgggctg ggggcgctca gcggccagag 2640 ggaaccagta ccgggcacgt tgacggtctc gtgcttggcg ttgtagcgga tcaggtaaat 2700 ctcgaggtct tggctgtctt cgatgtagcc gcggagctgg tagcgagtgt aagccttgag 2760 cttggactca tcgatcttct ggtacaagta ggtagggtag cactcgtcga aagtgcccag 2820 gagagtcacg tagttctcct tgaacacatc gtcgccgccc tggatcgtga tgtcggtgct 2880 gccgcgccag ccgcggtcga gctgcctgtt gatgccgcgg aaattggggt cctggaggag 2940 attcctctcg tcgctgagac gcttggcatg cttcaccttc tcggacagct ccttcttctc 3000 gtcgaggcag aactcatcgg agaggcactc cacgaggttg gagacttggt cgatgtggta 3060 gtcagtgacg tcggtcttca ggccgatctg attgctggac gtgaagagct cattgacagc 3120 cttctgggct ctctccaggt cgtactcggc ttcgaaggtg acctcggctg gcacgaactc 3180 aatgcggtca atgtacacct cattgccgga attgaacacg tgggcgctca gggtgaaaac 3240 gctggagccg ttggagaagt tgaagggggt ggtgaaaccc acggtgcgga agctgccgga 3300 ttggaggttg ctgccgctgg acatggtggc ggagaagtta ccctgattga tcggcctgcc 3360 gtcgatggag gtgtggaatt gcaggttggt ggtgctagcg tagcgaatcc tgacgcggta 3420 cctctgggac aggggagcgg tgatgttgac gcggagggtg ctgatctggc ccggggaggt 3480 cctgcgcagg atgtcgccgc ccgtgaagcc tgggcccttc accacggagg tgccgctgcc 3540 caggttggtg gacttggtga gggggatttg ggtgatttgg gaggacggaa tgatattgtt 3600 gaactccgcg ctgcgatgaa tccaggagaa cataggagct ctgatgatgc tcacggacga 3660 gttgctgaag ccggagcgga acatggacac gtggctgagc ctgtgggaaa aaccctgcct 3720 ggggggcaca ttgttgttct gtggtgggat ctcgtccagg gaatccaccg tgccgctctt 3780 gcggtagaca gcggagggca ggttggagga ggtgccgtag gcgaactcag tgccatccag 3840 gcggacagc tgctggttgt tgataccgat gttgaagggc ctgcggtaca gggtggagct 3900 cagggtgcgg tagacgccct ggcccagctg agcgacgatg cgttgttgtg gagcggcgtt 3960 gcccatcgtg ccgtagagag gaaaggtaaa ctcggggccg ctgaagccga ccggggaggc 4020 catgatctgg tggccggacc agtagtactc gccgcggtgg gcatcggtgt agatagtgat 4080 gctgttgagg atgtccatca ggtgtgggct cctgatggag ccctcgatgc cctgggcgct 4140 gcccctgaag ctaccgtcga agttctccag gacggggttg gtgtagattt cgcgggtcag 4200 ttgggacacg gtgcggatcg ggtaggtgcg ggagtcgtag ttcgggaaga gggacacaat 4260 gtccaggacg gtgagggtca gctcgcgcct gaactggttg tagcgaatcc agtctctaga 4320 atcagggccc cagacgcgct ccaggccagt gttgtaccag cggacagcgt ggtcggtgta 4380 gttgccgatc agcctggtga ggtcgttgta gcggctgttg atggtggcgg cgtcgaagcc 4440 ccacctctgg ccaaacacgc tgacgtccct cagcacgctg aggtgcaggt tggcggcctg 4500 gacgtacacg gacaggagcg ggacttggta gttctggacg gcgaagagtg ggatggcggt 4560 ggtcagggcg ctgttcatgt cgttgaactg gatgcgcatc tcctcgcgga gagctgggtt 4620 agtggggtcg gcctcccact cgcggaagct ctcagcgtag atttggtaga ggttgctgag 4680 gccctccagg cggctgatgg cctggttcct ggcgaactcc tcgatcctct ggttgatgag 4740 ctgctcgatt tgcaccagga aggcgtccca ctgggagggg ccaaagatgc cccagatgat 4800 gtccacgagg cccaggacga agccagcgcc tggcacgaac tcgctgagca ggaactgcgt 4860 gagggagagg gagatgtcga tgggggtgta accggtctcg atgcgctcac cgccgagcac 4920 ctcgacctca gggttgctga ggcagttgta cgggatgcac tcgttgatgt ttgggttgtt 4980 gtccatggcc gcttggtatc tgcattacaa tgaaatgagc aaagactatg tgagtaacac 5040 tggtcaacac tagggagaag gcatcgagca agatacgtat gtaaagagaa gcaatatagt 5100 gtcagttggt agatactaga taccatcagg aggtaaggag agcaacaaaa aggaaactct 5160 ctgtcagtac 5220 ttatttcttc agacaacaat atttaaaaca agtgcatctg atcttgactt atggtcacaa 5280 taaaggagca gagataaaca tcaaaatttc gtcatttata tttattcctt caggcgttaa 5340 caatttaaca gcacacaaac aaaaacagaa taggaatatc taattttggc aaataataag 5400 ctctgcagac gaacaaatta ttatagtatc gcctataata tgaatcccta tactattgac 5460 ccatgtagta tgaagcctgt gcctaaatta acagcaaact tctgaatcca agtgccctat 5520 aacaccaaca tgtgcttaaa taaataccgc taagcaccaa attacacatt tctcgtattg 5580 ctgtgtaggt tctatcttcg tttcgtacta ccatgtccct atattttgct gctacaaagg 5640 acggcaagta atcagcacag gcagaacacg atttcagagt gtaattctag atccagctaa 5700 accactctca gcaatcacca cacaagagag cattcagaga aacgtggcag taacaaaggc 5760 agagggcgga gtgagcgcgt accgaagacg gtagatctgc tagagtcagc ttgtcagcgt 5820 gtcctctcca aatgaaatga acttccttat atagaggaag ggtcttgcga aggatagtgg 5880 gattgtgcgt catcccttac gtcagtggag atatcacatc aatccacttg ctttgaagac 5940 gtggttggaa cgtcttcttt ttccacgatg ctcctcgtgg gtgggggtcc atctttggga 6000 ccactgtcgg cagaggcatc ttcaacgatg gcctttcctt tatcgcaatg atggcatttg 6060 taggagccac cttccttttc cactatcttc acaataaagt gacagatagc tgggcaatgg 6120 caaaggatgt taaacgttag agtccttcgt ccttcgaagc attatttccc ttgggatata 6180 atgattttcg ggcgaaggtt atgaaggaca taccttcatc agcacaagat ataataatga 6240 agaaggaatc atatgaagta taagagataa cataaacaat tatatattat aatcaactta 6300 tttctatttt attatcatgg aagaatagaa atgatatcaa attacagatg taccttcggc 6360 ttgaaagaag gtaaaagtat aagcgtggcg caaaagcaaa tgccaagtca gcgcgaacag 6420 tacggaaaca ctgttcatct atttataggc acgagacgca gcccatatga aattacaccc 6480 atgccctcta catttgctaa taactctata gtaatccgtc gaggtctaaa tagccttttc 6540 atctttaagt cggtttcctt ttctgctatt atctcgaagc tcttctgcgc acagcttcgg 6600 ctccgcgaca tccttcgtat tccttttgtg cttcttcaca ctgtggtttt aactcaagtc 6660 cgaagatacc tgttcatgca tcatactcca gaaactttgt taaatcatgt ttttgaggac 6720 cttcagaagc cgaaggccac caacagtagc ccctcgtaat attaatttgt tagaatgata 6780 aatttagatt gcgatatgga cgaaggccct aagccgaagg tccgaaaaaa cacctttcct 6840 ttgctagaat agcaacagtc actgacaagc gggcccttcc agttttcagc gcactaggcg 6900 tataaataag agcgcaccac ga 6922 <210> 2 <211> 1622 <212> DNA <213> Artificial Sequence <220> <223> SSllb <400> 2 tcccaatcct ttgacatctg ctccgaagca aagtcagagc gctgcaatgc aaaacggaac 60 gagtgggggc agcagcgcga gcaccgccgc gccggtgtcc ggacccaaag ctgatcatcc 120 atcagctcct gtcaccaaga gagaaatcga tgccagtgcg gtgaagccag agcccgcagg 180 tgatgatgct agaccggtgg aaagcatagg catcgctgaa ccggtggatg ctaaggctga 240 tgcagctccg gctacagatg cggcggcgag tgctccttat gacagggagg ataatgaacc 300 tggccctttg gctgggccta atgtgatgaa cgtcgtcgtg gtggcttctg aatgtgctcc 360 tttctgcaag acaggtaggt tgcatttcag ctgagtttct ccgtccaaaa taagcgactg 420 ctctgacaac cgttgccgca taggtggcct tggagatgtc gtgggtgctt tgcctaaggc 480 tctggcgagg agaggacacc gtgttatggt attcatctct ttcattttcg ttatatctat 540 gcaccctacg agagacagca gatactgttt cagctgccat gcgcctgaat gcagattctg 600 aactcttgtc ctttgcgaaa catggggaaa aaattaagat cacatagtca tacttctgta 660 accacaagtt ttgcctcagt gctctaatgt acttccacaa aaaaaaggag gaaattataa 720 ttcatatatg accagaaaag taatgcaatt ttgtgtcctg atgttaggtc gtgataccaa 780 gatatggaga gtatgccgaa gcccgggatt taggtgtaag gagacgttac aaggtagctg 840 gacaggtatg aaaaaagtgg aaacaaagtc gctgtagaac cgatcctgta tgaaagaaag 900 aaaatatgct agacctgatt tttttcctcc tgtgattttc aggattcaga agttacttat 960 tttcactctt acattgatgg agttgatttt gtattcgtag aagcccctcc cttccggcac 1020 cggcacaata atatttatgg gggagaaaga ttggtaaata ccaattttga agtgcttttg 1080 ctaacagtag aaaatcaagt tggatgcttg tcacatttat ctaaactatg tctctgaatt 1140 taatcacctg atggcaggat attttgaagc gcatgatttt gttctgcaag gccgctgttg 1200 aggtactgct agccattggt ttctaagaaa ttcacaaagt aacaaaaaaa ttatgatggc 1260 ttactggcta ataagaagat aaattcatcg ctggtaaatt ttgataagtc aacagtgacc 1320 aggccccata gaattacaca atatacaccc ttcactgttt agttaatata tttttcttct 1380 acttttccaa gctgtctatg cacctcattg aatagtttgt tatgacagac tcaattacag 1440 ctcatatggg ctatagttta ttgtggggcg aataaatctg catcatcctc tttttcaaag 1500 ttattttgaa catttgcagg ttccatggta tgctccatgt ggcggtactg tctatggtga 1560 tggcaactta gttttcattg ctaatgattg gcataccgca cttctgcctg tctatctaaa 1620 gg 1622 <210> 3 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> SSllb forward primer <400> 3 ctcccaatcc tttgacatct gc 22 <210> 4 <211> 23 <212> DNA <213> Artificial Sequence <220> <223> SSllb reverse primer <400> 4 tcgatttctc tcttggtgac agg 23 <210> 5 <211> 25 <212> DNA <213> Artificial Sequence <220> <223> SSllb taq <400> 5 agcaaagtca gagcgctgca atgca 25 <210> 6 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> MON 810 forward primer <400> 6 gatgccttct ccctagtgtt ga 22 <210> 7 <211> 21 <212> DNA <213> Artificial Sequence <220> <223> MON 810 reverse primer <400> 7 ggatgcactc gttgatgttt g 21 <210> 8 <211> 26 <212> DNA <213> Artificial Sequence <220> <223> MON 810 taq <400> 8 agataccaag cggccatgga caacaa 26 <210> 9 <211> 22 <212> DNA <213> Artificial Sequence <220> <223> SSllb 1-5 <400> 9 ctcccaatcc tttgacatct gc 22 <210> 10 <211> 24 <212> DNA <213> Artificial Sequence <220> <223> SSllb 2-3 <400> 10 gacttgcctg aacactacat cgac 24 <210> 11 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> GWMcry3F-1 <400> 11 gacgttctca gagacagtat tcaactttg 29 <210> 12 <211> 19 <212> DNA <213> Artificial Sequence <220> <223> AP2 <400> 12 actatagggc acgcgtggt 19

Claims (12)

SEQ ID NO: 2 of the sequence GM corn MON810 variety of standard plasmid for black of a gene variant that contains the cry1Ab gene consisting of the nucleotide sequence of SEQ ID NO: 1 ssⅡb gene and consisting of. delete delete delete 2. The standard plasmid according to claim 1, wherein the standard plasmid is the restriction enzyme map disclosed in Fig. I) preparing a standard plasmid according to claim 1 by dilution by concentration;
Ii) performing real-time PCR using the standard plasmid diluted by concentration and the DNA obtained from the biological sample as templates, respectively, using a PCR primer set for assay and a probe;
Iii) calculating a standard quantitative curve by measuring the amount of the PCR product performed using the diluted standard plasmid; And
And iv) introducing the amount of the PCR product carried out on the DNA obtained from the biological sample into the standard quantitative curve to calculate the incorporation rate of the exogenous transgene, thereby quantitatively analyzing the exogenous transgene incorporated into the genetic modification . The method according to claim 6, wherein the GM variant is GM maize MON810. 7. The method according to claim 6, wherein the PCR primer set in step ii) is a primer set of SEQ ID NOs: 3 and 4 and a primer set of SEQ ID NOs: 6 and 7, Way. A kit for assaying a genetic modification comprising the standard plasmid according to claim 1. 10. The assay kit according to claim 9, wherein the genetic modification is genetically modified maize of GM maize MON810. 10. The assay kit according to claim 9, wherein the assay kit comprises a primer set consisting of SEQ ID NOS: 3 and 4, and 6 and 7, and a probe having SEQ ID NOS: 5 and 8. A kit for assaying a genetic modification comprising a protein expressed from a standard plasmid according to claim 1.

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