JP7181522B2 - Primer set and method for amplifying target nucleotide sequence using same - Google Patents

Description

The present invention relates to a primer set and a method for amplifying a target nucleotide sequence using the same. More particularly, the present invention relates to a method of amplifying very short target sequences consisting of 25 nucleotides by polymerase chain reaction, and primer sets used in said method.

Since commercial cultivation began in 1996, genetically modified (GM) crops have been used in many countries and regions. The cultivated area was 1.7 million hectares in 1996, but is reported to have reached 180 million hectares in 2016, more than 100 times that amount.

While GM crops are useful, not a few consumers are reluctant to use such crops as food. In Japan, a labeling system for GM foods has been established from the viewpoint of providing information to consumers. The contents are stipulated in the Food Labeling Act, and the items to be labeled are "soybeans", "corn", "potatoes", "rapeseed", and "cotton seeds" that have undergone safety screening procedures by the Ministry of Health, Labor and Welfare. , ``alfalfa'', ``sugar beet'', and ``papaya'', and 33 types of processed foods using these as raw materials. In order to ensure the effectiveness of such a food labeling system, the existence of a highly reliable GM inspection method is indispensable. As for the GM test method, the polymerase chain reaction (PCR) targeting DNA of the recombinant gene is used as a standard method all over the world. PCR is a technique that uses DNA polymerase to amplify a specific DNA sequence in large amounts, and is used in various studies and tests. Japan's safety-examined GM detection methods are listed in the Consumer Affairs Agency's "Testing Methods for Safety-examined Genetically Modified Foods" (hereinafter referred to as "Consumer Affairs Agency Manual"). At present, the Consumer Affairs Agency manual provides inspection methods capable of detecting 3 strains of GM soybeans, 13 strains of GM corn, and virus-resistant papaya from Hawaii.

However, as new GM crops are being developed one after another and undergoing safety reviews, the types of GM crops subject to detection continue to increase, and basically will not decrease. In Japan, as of May 2018, 343 types of GM crops can be commercialized as foods. A screening method that targets and detects sequences common to GM crops is effective for the efficient detection of ever-increasing GM crops. Previous screening tests have targeted consensus sequences, such as the cauliflower mosaic virus 35S promoter (P35S), that have been introduced into many GM crops (Non-Patent Document 1). However, GM varieties without P35S are also increasing rapidly. Currently, strains that do not have P35S are handled by developing individual specific detection methods, but simply adding test methods in line with the increase in the number of detection targets will increase the burden of testing. Since it will continue to increase, drastic measures are required.

At present, the most widely used GM plant production technology in the world is the Agrobacterium method using Agrobacterium, which is a fungus of root cancer. In the Agrobacterium method, the T-DNA region on the Ti plasmid is inserted into the plant nuclear genome. Therefore, since the sequence derived from Agrobacterium T-DNA is introduced into the genome of the host crop together with the recombinant gene of interest, it is considered that it can be a very effective target sequence for GM detection.

In this regard, as a result of the present inventors' analysis of databases and the like this time, sequences near the right border sequence (Right Border, RB) located at the end of T-DNA are found in many plants such as soybean, maize, cotton, and rapeseed. It was found to be commonly introduced in the genomic DNA of GM crops. However, the sequence (SEQ ID NO: 1, 5′-GAAGGCGGGGAAACGACAATCTGATC-3′: hereinafter also referred to as “RB flanking sequence”) that is common and completely conserved among the GM crops consists of only 25 nucleotides ( 25 bases) (see Figure 1).

Regarding PCR, there are already various textbook experimental protocols, and Molecular Cloning (Non-Patent Document 2), which is a representative example, describes the length of the primer as usually 20 to 25 nucleotides, Furthermore, it is described that non-specific binding to template DNA tends to occur when primers with a length of less than 18 nucleotides are used. That is, in PCR, a forward primer of about 20 nucleotides, a reverse primer of about 20 nucleotides, and a space of at least several nucleotides between the primers, so that the PCR target has a length of at least 40 nucleotides. It is common technical knowledge to Therefore, it has been considered extremely difficult to target a sequence consisting of 25 nucleotides as described above, to specifically amplify and detect the sequence by PCR.

"(Attachment) Inspection Method for Foods to which Recombinant DNA Technology Has Been Verified for Safety", November 16, 2012, Internet (URL: http://www.caa.go.jp/foods/pdf/syokuhin961_1. pdf) Green, M.; et al., Molecular Cloning: A Laboratory Manual, 4th ed. Part 1 essentials, Chapter 7 Polymerase Chain Reaction, pp. 456-457, published by Cold spring harbor laboratory

The present invention has been made in view of the problems of the prior art, and aims to provide a method for amplifying a very short target sequence consisting of 25 nucleotides by PCR, and a primer set used in the method. do. Another object of the present invention is to enable the detection of transgenic plants produced by the Agrobacterium method by detecting RB flanking sequences using the above method and primer set.

As a result of intensive studies to achieve the above object, the present inventors found that a forward primer consisting of the same sequence as the portion consisting of 13 nucleotides from the 5' end of the RB neighborhood sequence, and a forward primer from the 3' end of the RB neighborhood sequence When PCR is performed using a reverse primer (combination of RBF2 and RBR2 shown in FIG. 2A) consisting of a sequence complementary to a portion consisting of 12 nucleotides, an extremely short RB flanking sequence of 25 nucleotides is specifically I found that it can be amplified.

On the other hand, the 3′ side is longer than the forward primer by 1 nucleotide and the 3′ side is longer by 4 nucleotides than the reverse primer, and the forward primer and the reverse primer overlap by 5 nucleotides (RBF and RB flanking sequences could not be specifically amplified when using a combination with RBR).

In addition, a forward primer and a reverse primer designed so that no overlap occurs in the RB neighborhood sequence (a forward primer consisting of 13 nucleotides and a reverse primer consisting of 12 nucleotides (combination of RBF and RBR3 shown in FIG. 2A), 14 nucleotides to A forward primer consisting of and a reverse primer consisting of 11 nucleotides (a combination of RBF4 and RBR4 shown in FIG. 2A), a forward primer consisting of 15 nucleotides and a reverse primer consisting of 10 nucleotides (a combination of RBF5 and RBR5 shown in FIG. 2B) , a forward primer consisting of 11 nucleotides and a reverse primer consisting of 14 nucleotides (a combination of RBF6 and RBF6 shown in FIG. 2B), a forward primer consisting of 10 nucleotides and a reverse primer consisting of 15 nucleotides (RBF7 and RBR7 shown in FIG. 2B). )), the RB flanking sequence could not be amplified, unlike the case where the forward primer consisting of 12 nucleotides and the reverse primer consisting of 13 nucleotides were used.

Furthermore, when using a forward primer consisting of 12 nucleotides and a reverse primer consisting of 12 nucleotides that is 1 nucleotide shorter on the 5' side than the reverse primer consisting of 13 nucleotides (a combination of RBF2 and RBR8 shown in FIG. 2C). Moreover, despite shortening the reverse primer by only 1 nucleotide, it was not possible to amplify the 24-nucleotide flanking RB sequence.

Furthermore, even when using a forward primer consisting of 12 nucleotides and a reverse primer consisting of 12 nucleotides (combination of RBF8 and RBR3 shown in FIG. 2C), which are different from the RBF2 and RBR8, the RB vicinity of 24 nucleotides It was not possible to amplify the sequence.

Based on the above, it was found that the primer set capable of amplifying the highly conserved RB flanking sequences in GM plants is only one type, the forward primer consisting of 12 nucleotides and the reverse primer consisting of 13 nucleotides.

In addition, the target nucleotide sequence was replaced with a plant endogenous sequence different from the RB flanking sequence, and as a result of further extensive research, it was found that under the following conditions, even if the target sequence is as short as 25 nucleotides, PCR The present inventors have found that amplification can be carried out at , and have completed the present invention.
(1) The total number of guanines and cytosines in a target sequence consisting of 25 nucleotides is at least 13 nucleotides.
(2) One of the forward primer and the reverse primer has a chain length of 12 nucleotides, and the other has a chain length of 13 nucleotides. Each primer also contains at least a total of 5 nucleotides of guanine and cytosine.

That is, the present invention more specifically provides the following.
<1> A primer set comprising a forward primer and a reverse primer for amplifying a target nucleotide sequence,
said target nucleotide sequence consists of 25 nucleotides and contains at least a total of 13 nucleotides of guanine and cytosine;
1 primer set selected from the group consisting of the following (a) and (b): (a) the forward primer consists of a sequence identical to the portion consisting of 12 nucleotides from the 5' end of the target nucleotide sequence, and comprises guanine and containing at least a total of 5 nucleotides of cytosine,
a primer set in which the reverse primer consists of a sequence complementary to a portion consisting of 13 nucleotides from the 3' end of the target nucleotide sequence and contains at least a total of 5 nucleotides of guanine and cytosine;
(b) the forward primer consists of a sequence identical to a portion consisting of 13 nucleotides from the 5' end of the target nucleotide sequence and contains at least a total of 5 nucleotides of guanine and cytosine;
A primer set in which the reverse primer comprises a sequence complementary to a portion consisting of 12 nucleotides from the 3' end of the target nucleotide sequence and contains at least a total of 5 nucleotides of guanine and cytosine.
<2> Primer set according to <1>, selected from the following groups (1) to (11): (1) derived from T-DNA of Agrobacterium, consisting of the nucleotide sequence set forth in SEQ ID NO: 1 The target nucleotide sequence is the RB flanking sequence that
A primer set comprising a forward primer consisting of the nucleotide sequence set forth in SEQ ID NO: 15 and a reverse primer consisting of the nucleotide sequence set forth in SEQ ID NO: 16,
(2) the target nucleotide sequence is a sequence derived from the soybean lectin gene consisting of the nucleotide sequence set forth in SEQ ID NO: 34;
A primer set comprising a forward primer consisting of the nucleotide sequence set forth in SEQ ID NO: 35 and a reverse primer consisting of the nucleotide sequence set forth in SEQ ID NO: 36,
(3) the target nucleotide sequence is a sequence derived from the soybean lectin gene consisting of the nucleotide sequence set forth in SEQ ID NO: 42;
A primer set comprising a forward primer consisting of the nucleotide sequence set forth in SEQ ID NO: 43 and a reverse primer consisting of the nucleotide sequence set forth in SEQ ID NO: 44,
(4) the target nucleotide sequence is a sequence derived from the soybean lectin gene consisting of the nucleotide sequence set forth in SEQ ID NO: 45;
A primer set comprising a forward primer consisting of the nucleotide sequence set forth in SEQ ID NO: 46 and a reverse primer consisting of the nucleotide sequence set forth in SEQ ID NO: 47,
(5) the target nucleotide sequence is a sequence derived from the soybean lectin gene consisting of the nucleotide sequence set forth in SEQ ID NO: 45;
A primer set comprising a forward primer consisting of the nucleotide sequence set forth in SEQ ID NO: 48 and a reverse primer consisting of the nucleotide sequence set forth in SEQ ID NO: 49,
(6) The target nucleotide sequence is a sequence derived from the soybean lectin gene, which consists of the nucleotide sequence set forth in SEQ ID NO: 53.
a primer set comprising a forward primer consisting of the nucleotide sequence set forth in SEQ ID NO: 54 and a reverse primer consisting of the nucleotide sequence set forth in SEQ ID NO: 55;
(7) The target nucleotide sequence is a sequence derived from the maize starch synthase IIb gene, which consists of the nucleotide sequence set forth in SEQ ID NO: 62.
A primer set comprising a forward primer consisting of the nucleotide sequence set forth in SEQ ID NO: 63 and a reverse primer consisting of the nucleotide sequence set forth in SEQ ID NO: 64,
(8) The target nucleotide sequence is a sequence derived from the maize starch synthase IIb gene, which consists of the nucleotide sequence set forth in SEQ ID NO: 71.
A primer set comprising a forward primer consisting of the nucleotide sequence set forth in SEQ ID NO: 72 and a reverse primer consisting of the nucleotide sequence set forth in SEQ ID NO: 73,
(9) The target nucleotide sequence is a sequence derived from the maize starch synthase IIb gene, which consists of the nucleotide sequence set forth in SEQ ID NO: 71.
A primer set comprising a forward primer consisting of the nucleotide sequence set forth in SEQ ID NO: 74 and a reverse primer consisting of the nucleotide sequence set forth in SEQ ID NO: 75;
(10) the target nucleotide sequence is a maize HMG gene-derived sequence consisting of the nucleotide sequence set forth in SEQ ID NO: 79;
A primer set comprising a forward primer consisting of the nucleotide sequence set forth in SEQ ID NO: 80 and a reverse primer consisting of the nucleotide sequence set forth in SEQ ID NO: 81,
(11) the target nucleotide sequence is a maize HMG gene-derived sequence consisting of the nucleotide sequence set forth in SEQ ID NO: 85;
A primer set comprising a forward primer consisting of the nucleotide sequence set forth in SEQ ID NO:86 and a reverse primer consisting of the nucleotide sequence set forth in SEQ ID NO:87.
<3> A method for amplifying a target nucleotide sequence in a sample, comprising:
A method comprising the step of amplifying a target nucleotide sequence in a sample as a template by polymerase chain reaction using the primer set according to <1> or <2>.
<4> A method for detecting a target nucleotide sequence in a sample, comprising:
A step of amplifying the target nucleotide sequence in the sample by the method described in <3>;
and detecting the target nucleotide sequence amplified in said step.
<5> A method for detecting a genetically modified plant produced by the Agrobacterium method,
Using the genomic DNA of a test plant as a template, a forward primer consisting of the nucleotide sequence set forth in SEQ ID NO: 15 and a reverse primer set consisting of the nucleotide sequence set forth in SEQ ID NO: 16 are used to perform a polymerase chain reaction;
A step of detecting an RB flanking sequence derived from Agrobacterium T-DNA, consisting of the nucleotide sequence set forth in SEQ ID NO: 1, amplified by the polymerase chain reaction;
A step of determining that the test plant is a transgenic plant produced by the Agrobacterium method when amplification of the RB flanking sequence is detected in the step;
How to include.

According to the present invention, it is possible to amplify very short target sequences consisting of 25 nucleotides by PCR. Moreover, according to the present invention, it is also possible to detect genetically modified (GM) plants produced by the Agrobacterium method by detecting the aforementioned RB flanking sequences.

FIG. 2 is a schematic diagram showing the right border sequence (RB) located at the end of T-DNA inserted into the genomic DNA of a genetically modified (GM) crop, the sequence in the vicinity thereof, and the like. In the figure, "pBI101" indicates the RB of the T-DNA region (GenBank accession number: U12639) of pBI101 of the binary vector GUS gene vector used for gene recombination of plants and its flanking sequence (SEQ ID NO: 3). . Regarding RB and its neighboring sequences in GM crops, those derived from MON89788 (soybean) are shown in SEQ ID NO: 4, those derived from MON87701 (soybean) are shown in SEQ ID NO: 5, and those derived from MON87769 (soybean) are shown in SEQ ID NOS. :6, those from MON88017 (maize) are shown in SEQ ID NO: 7, those from MIR604 (maize) are shown in SEQ ID NO: 8, those from MIR162 (maize) are shown in SEQ ID NO: 9, The one derived from 3272 (maize) is shown in SEQ ID NO: 10, the one derived from RT73 (rapeseed) is shown in SEQ ID NO: 11, and the one derived from 531 (cotton) is shown in SEQ ID NO: 12. In addition, the boxed sequence indicates the sequence (SEQ ID NO: 1, RB flanking sequence) that we found to be commonly introduced in the genomic DNA of the GM crops. The underlined array indicates the right border array (RB). FIG. 2 is a schematic diagram showing the positional relationship between the RB flanking sequence and its complementary strand, and the forward and reverse primers designed to amplify the sequence by PCR. The RB flanking sequence is shown in SEQ ID NO:1 and the sequence of its complementary strand is shown in SEQ ID NO:2. The sequence of RBF2 is shown in SEQ ID NO: 15, the sequence of RBR2 is shown in SEQ ID NO: 16, the sequence of RBF is shown in SEQ ID NO: 13, the sequence of RBR is shown in SEQ ID NO: 14, and the sequence of RBR3 is shown in SEQ ID NO: :17, the sequence of RBF4 is shown in SEQ ID NO:18, and the sequence of RBR4 is shown in SEQ ID NO:19. FIG. 2 is a schematic diagram showing the positional relationship between the RB flanking sequence and its complementary strand, and the forward and reverse primers designed to amplify the sequence by PCR. The RB flanking sequence is shown in SEQ ID NO:1 and the sequence of its complementary strand is shown in SEQ ID NO:2. The sequence of RBF5 is shown in SEQ ID NO: 20, the sequence of RBR5 is shown in SEQ ID NO: 21, the sequence of RBF6 is shown in SEQ ID NO: 22, the sequence of RBR6 is shown in SEQ ID NO: 24, and the sequence of RBF7 is shown in SEQ ID NO: :25 and the sequence of RBR7 is shown in SEQ ID NO:25. FIG. 2 is a schematic diagram showing the positional relationship between the RB flanking sequence and its complementary strand, and the forward and reverse primers designed to amplify the sequence by PCR. The RB flanking sequence is shown in SEQ ID NO:1 and the sequence of its complementary strand is shown in SEQ ID NO:2. The sequence of RBF2 is shown in SEQ ID NO: 15, the sequence of RBR8 is shown in SEQ ID NO: 27, the sequence of RBF8 is shown in SEQ ID NO: 26, and the sequence of RBR3 is shown in SEQ ID NO: 17. 1 is a photograph showing the results of PCR (annealing temperature: 28° C.) using RBF2 primer and RBR2 primer and analysis by acrylamide electrophoresis. In the figure, lanes 1 and 7 are molecular weight markers, and lanes 2, 3, 4 and 5 are genomes extracted from non-genetically modified (non-GM) soybeans, GM soybeans MON89788, non-GM maize and GM maize MIR162, respectively. The results of PCR using DNA as a template are shown. Lane 6 shows the results of the negative control (no template DNA). The arrow indicates the position of the band of interest (RB neighborhood sequence). 1 is a photograph showing the results of PCR (annealing temperature: 38° C.) using RBF2 primer and RBR2 primer and analysis by acrylamide electrophoresis. The symbols in the figure are the same as in FIG. 3A. 1 is a photograph showing the results of PCR (annealing temperature: 52° C.) using RBF2 primer and RBR2 primer and analysis by acrylamide electrophoresis. The symbols in the figure are the same as in FIG. 3A. 1 is a photograph showing the results of PCR performed using RBF2 primer and RBR2 primer and analyzed by acrylamide electrophoresis. In the figure, lanes 1, 2, 3 and 4 show the results of PCR using genomic DNAs extracted from non-GM soybeans, GM soybeans MON89788, non-GM maize and GM maize MIR162 as templates, respectively. Lane 5 shows the results of the negative control (no template DNA). Lane 6 shows the results (positive control) of PCR using RBF and RBR primers to show the band of interest (RB flanking sequence). 1 is a photograph showing the results of PCR performed using RBF primers and RBR3 primers and analyzed by acrylamide electrophoresis. The symbols in the figure are the same as those in FIG. 4A. 1 is a photograph showing the results of PCR performed using RBF4 primer and RBR4 primer and analyzed by acrylamide electrophoresis. The symbols in the figure are the same as those in FIG. 4A. 1 is a photograph showing the results of PCR performed using RBF5 primer and RBR5 primer and analyzed by acrylamide electrophoresis. The symbols in the figure are the same as those in FIG. 4A. 1 is a photograph showing the results of PCR performed using RBF6 primer and RBR6 primer and analyzed by acrylamide electrophoresis. The symbols in the figure are the same as those in FIG. 4A. 1 is a photograph showing the results of PCR performed using RBF7 primer and RBR7 primer and analyzed by acrylamide electrophoresis. The symbols in the figure are the same as those in FIG. 4A. 1 is a photograph showing the results of PCR performed using RBF8 primer and RBR8 primer and analyzed by acrylamide electrophoresis. The symbols in the figure are the same as those in FIG. 4A. 1 is a photograph showing the results of PCR performed using RBF8 primer and RBR2 primer and analyzed by acrylamide electrophoresis. The symbols in the figure are the same as those in FIG. 4A. 1 is a photograph showing the results of PCR performed using RBF2 primer and RBR2 primer and analyzed by acrylamide electrophoresis. In the figure, lane 1 shows the results of PCR using genomic DNA extracted from non-GM soybean as a template. Lanes 2-9 show the results of PCR using genomic DNAs extracted from GM soybeans RRS, A2704-12, MON89788, DP305423, MON87701, MON87708, MON87705 and MON87769 as templates, respectively. Lanes 10, 11 and 12 show the results of PCR using genomic DNAs of rice, wheat and barley as templates, respectively. Lane 13 shows the result of negative control (no template DNA), lane 14 shows the result of PCR using RBF primer and RBR primer (positive control) to show the band of interest (RB flanking sequence). show. The arrow indicates the position of the band of interest (RB neighborhood sequence). 1 is a photograph showing the results of PCR performed using RBF2 primer and RBR2 primer and analyzed by acrylamide electrophoresis. In the figure, lane 15 shows the results of PCR using genomic DNA extracted from non-GM maize as a template. Lanes 16 to 30 are PCR results using genomic DNAs extracted from GM maize NK603, Event176, T25, GA21, MON810, TC1507, Bt11, MIR604, MON88017, DAS59122, MON863, MIR162, 3272, MON89034 and MON87460 as templates. Show the results. Lane 31 shows the result of negative control (no template DNA), and lane 32 shows the result of PCR using RBF primer and RBR primer (positive control) to show the target band (RB flanking sequence). show. The arrow indicates the position of the band of interest (RB neighborhood sequence). 1 is a photograph showing the results of PCR performed using RBF2 primer and RBR2 primer and analyzed by acrylamide electrophoresis. In the figure, lane 1 shows the results of PCR using only genomic DNA extracted from non-GM soybean as a template. Lane 2 shows the results of PCR using only GM soybean RRS genomic DNA as a template. Lanes 3-6 show genomic DNA extracted from GM soybean MON89788 mixed with genomic DNA extracted from non-GM soybean at ratios of 0.5%, 1.0%, 5.0% and 10.0%. The results of PCR using the obtained product as a template are shown. Lane 7 shows the results of PCR using only genomic DNA extracted from GM soybean MON89788 as a template. The arrow indicates the position of the band of interest (RB neighborhood sequence). 1 is a photograph showing the results of PCR performed using RBF2 primer and RBR2 primer and analyzed by acrylamide electrophoresis. In the figure, lane 8 shows the results of PCR using only genomic DNA extracted from non-GM maize as a template. Lane 9 shows the results of PCR using only the genomic DNA of GM maize MON810 as a template. In lanes 10-13, genomic DNA extracted from GM maize MIR162 was mixed with genomic DNA extracted from non-GM maize at ratios of 0.5%, 1.0%, 5.0% and 10.0%. The results of PCR using the obtained product as a template are shown. Lane 14 shows the results of PCR using only genomic DNA extracted from GM maize MIR162 as a template. Lane 15 shows the results of the negative control (no template DNA). The arrow indicates the position of the band of interest (RB neighborhood sequence). 1 is a photograph showing the results of PCR performed using RBF2 primer and RBR2 primer and analyzed by nucleic acid chromatography. In the figure, lane 1 shows the results of PCR using genomic DNA extracted from non-GM soybean as a template. Lanes 2-9 show the results of PCR using genomic DNAs extracted from GM soybeans RRS, A2704-12, MON89788, DP305423, MON87701, MON87705, MON87708 and MON87769 as templates, respectively. Lanes 10, 11 and 12 show the results of PCR using genomic DNAs of rice, wheat and barley as templates, respectively. Lane 13 shows the result of negative control (no template DNA), and lane 14 shows the result of PCR using RBF primer and RBR primer (positive control) to show the target band (RB flanking sequence). show. 1 is a photograph showing the results of PCR performed using RBF2 primer and RBR2 primer and analyzed by nucleic acid chromatography. In the figure, lane 15 shows the results of PCR using genomic DNA extracted from non-GM maize as a template. Lanes 16 to 30 are PCR results using genomic DNAs extracted from GM maize NK603, Event176, T25, GA21, MON810, TC1507, Bt11, MIR604, MON88017, DAS59122, MON863, MIR162, 3272, MON89034 and MON87460 as templates. Show the results. Lane 31 shows the result of negative control (no template DNA), and lane 32 shows the result of PCR using RBF primer and RBR primer (positive control) to show the target band (RB flanking sequence). show. 1 is a photograph showing the results of PCR performed using RBF2 primer and RBR2 primer and analyzed by nucleic acid chromatography. In the figure, lane 1 shows the results of PCR using only genomic DNA extracted from non-GM soybean as a template. Lane 2 shows the results of PCR using only GM soybean RRS genomic DNA as a template. Lanes 3-6 show genomic DNA extracted from GM soybean MON89788 mixed with genomic DNA extracted from non-GM soybean at ratios of 0.5%, 1.0%, 5.0% and 10.0%. The results of PCR using the obtained product as a template are shown. Lane 7 shows the results of PCR using only genomic DNA extracted from GM soybean MON89788 as a template. Lane 8 shows the results of the negative control (no template DNA). Lane 9 shows the results of PCR performed using RBF and RBR primers (positive control) to show the band of interest (RB flanking sequence). 1 is a photograph showing the results of PCR performed using RBF2 primer and RBR2 primer and analyzed by nucleic acid chromatography. In the figure, lane 10 shows the results of PCR using only genomic DNA extracted from non-GM maize as a template. Lanes 11-15 show genomic DNA extracted from GM maize MIR162 at ratios of 0.3%, 0.5%, 1.0%, 5.0% and 10.0% from non-GM maize. This figure shows the results of PCR using a mixture of genomic DNA obtained as a template. Lane 16 shows the results of PCR using only genomic DNA extracted from GM maize MIR162 as a template. Lane 17 shows the results of the negative control (no template DNA). Lane 18 shows the results of PCR performed using RBF and RBR primers (positive control) to show the band of interest (RB flanking sequence).

(primer set)
As shown in the examples below, the present inventors found that an extremely short target sequence (target nucleotide sequence) of 25 nucleotides can be amplified by the polymerase chain reaction (PCR) if the primer set shown below is used. I found.

That is, the present invention
A primer set comprising a forward primer and a reverse primer for amplifying a target nucleotide sequence,
said target nucleotide sequence consists of 25 nucleotides and contains at least a total of 13 nucleotides of guanine and cytosine;
1 primer set selected from the group consisting of the following (a) and (b): (a) the forward primer consists of a sequence identical to the portion consisting of 12 nucleotides from the 5' end of the target nucleotide sequence, and comprises guanine and containing at least a total of 5 nucleotides of cytosine,
a primer set in which the reverse primer consists of a sequence complementary to a portion consisting of 13 nucleotides from the 3' end of the target nucleotide sequence and contains at least a total of 5 nucleotides of guanine and cytosine;
(b) the forward primer consists of a sequence identical to a portion consisting of 13 nucleotides from the 5' end of the target nucleotide sequence and contains at least a total of 5 nucleotides of guanine and cytosine;
The reverse primer provides a primer set comprising a sequence complementary to a portion consisting of 12 nucleotides from the 3' end of the target nucleotide sequence and containing at least a total of 5 nucleotides of guanine and cytosine.

In the present invention, "target nucleotide sequence" means a nucleotide sequence to be amplified by PCR and consists of 25 nucleotides. The total number of guanines and cytosines (GC number) in the target nucleotide sequence may be at least 13 nucleotides in total, preferably 13 to 18 nucleotides, more preferably 14 to 16 nucleotides, and particularly preferably 15 nucleotides. be.

In the present invention, the "nucleotide" that constitutes the primer is usually DNA (adenine, cytosine, guanine, uracil), but other natural nucleotides (RNA) may be used as long as they can form base pairs. Non-natural nucleotides (artificial nucleotides, nucleotide analogues) may be used. Non-natural nucleotides include, for example, hexitol nucleic acid (HNA), cyclohexene nucleic acid (CeNA), peptide nucleic acid (PNA), glycol nucleic acid (GNA), threose nucleic acid (TNA), morpholino nucleic acid, tricyclo-DNA (tcDNA), 2' -O-methylated nucleic acid, 2'-MOE (2'-O-methoxyethyl) nucleic acid, 2'-AP (2'-O-aminopropyl) nucleic acid, 2'-fluorinated nucleic acid, 2'F- Bridged Nucleic Acids such as arabinonucleic acid (2'-F-ANA) and BNA (LNA) can be mentioned.

The "primer" of the present invention is an oligonucleotide that anneals to a target nucleotide sequence in PCR and serves as the origin of DNA replication, even if it is an oligonucleotide consisting of only one type of nucleotide (e.g., DNA only). It may be a chimeric oligonucleotide composed of multiple nucleotides (for example, DNA and RNA), but preferably an oligonucleotide composed only of DNA.

A "forward primer" of the present invention is a primer consisting of a sequence identical to a portion consisting of 12 or 13 nucleotides from the 5' end of a target nucleotide sequence. The total number of guanines and cytosines (GC number) in the forward primer may be at least 5 nucleotides in total, preferably 5 to 10 nucleotides, more preferably 6 to 9 nucleotides, and still more preferably 7 to 8 nucleotides. is.

A "reverse primer" of the present invention is a primer consisting of a sequence complementary to a portion consisting of 13 or 12 nucleotides from the 3' end of a target nucleotide sequence. The number of GCs in the reverse primer may be at least 5 nucleotides in total, preferably 5 to 10 nucleotides, more preferably 6 to 9 nucleotides, still more preferably 7 to 8 nucleotides.

When one of the forward primer and reverse primer of the present invention consists of 12 nucleotides, the other consists of 13 nucleotides. That is, when the sequence of the forward primer of the present invention and the sequence complementary to the reverse primer of the present invention are joined together, the target nucleotide sequence consisting of 25 nucleotides described above is obtained. Therefore, the conditions for the sequences are also the conditions for these primers. In addition, the difference in Tm value between these primers is not particularly limited, and is usually 0 to 20°C. to 5°C, more preferably 0 to 3°C, and particularly preferably 0 to 1°C.

The primer set of the present invention is not particularly limited as long as it meets the above conditions. A target nucleotide sequence includes a primer set comprising a forward primer consisting of the nucleotide sequence set forth in SEQ ID NO:15 and a reverse primer consisting of the nucleotide sequence set forth in SEQ ID NO:16.

When the soybean lectin gene is to be amplified and the nucleotide sequence of SEQ ID NO: 34 in the gene is the target nucleotide sequence, the forward primer consisting of the nucleotide sequence of SEQ ID NO: 35 and the forward primer of SEQ ID NO: 36 and a primer set (1) comprising a reverse primer consisting of the nucleotide sequence of
When the nucleotide sequence of SEQ ID NO: 42 in the soybean lectin gene is the target nucleotide sequence, a forward primer consisting of the nucleotide sequence of SEQ ID NO: 43 and a reverse primer consisting of the nucleotide sequence of SEQ ID NO: 44 are used. Primer set (2), comprising
When the nucleotide sequence of SEQ ID NO: 45 in the soybean lectin gene is the target nucleotide sequence, a forward primer consisting of the nucleotide sequence of SEQ ID NO: 46 and a reverse primer consisting of the nucleotide sequence of SEQ ID NO: 47 are used. Primer set (3), comprising
When the nucleotide sequence of SEQ ID NO: 45 in the soybean lectin gene is the target nucleotide sequence, a forward primer consisting of the nucleotide sequence of SEQ ID NO: 48 and a reverse primer consisting of the nucleotide sequence of SEQ ID NO: 49 are used. Primer set (4), comprising
When the nucleotide sequence of SEQ ID NO: 53 in the soybean lectin gene is the target nucleotide sequence, a forward primer consisting of the nucleotide sequence of SEQ ID NO: 54 and a reverse primer consisting of the nucleotide sequence of SEQ ID NO: 55 are used. Primer set (5), comprising
When the nucleotide sequence of SEQ ID NO: 28 in the soybean lectin gene is the target nucleotide sequence, a forward primer consisting of the nucleotide sequence of SEQ ID NO: 29 and a reverse primer consisting of the nucleotide sequence of SEQ ID NO: 30 are used. Primer set (6) comprising
When the nucleotide sequence of SEQ ID NO: 39 in the soybean lectin gene is the target nucleotide sequence, a forward primer consisting of the nucleotide sequence of SEQ ID NO: 40 and a reverse primer consisting of the nucleotide sequence of SEQ ID NO: 41 are used. Primer set (7), comprising
When the nucleotide sequence of SEQ ID NO: 50 in the soybean lectin gene is the target nucleotide sequence, a forward primer consisting of the nucleotide sequence of SEQ ID NO: 51 and a reverse primer consisting of the nucleotide sequence of SEQ ID NO: 52 are used. and a primer set (8) comprising:

Among these, the primer sets (1) to (5) are preferred from the viewpoint of being able to amplify the target nucleotide sequence in the soybean lectin gene with higher specificity.

When the maize starch synthase IIb gene is to be amplified and the nucleotide sequence set forth in SEQ ID NO: 62 in the gene is the target nucleotide sequence, a forward primer consisting of the nucleotide sequence set forth in SEQ ID NO: 63 and SEQ ID NO: 64 and a primer set (9) comprising a reverse primer consisting of the nucleotide sequence described in
When the nucleotide sequence set forth in SEQ ID NO: 71 in the maize starch synthase IIb gene is the target nucleotide sequence, a forward primer consisting of the nucleotide sequence set forth in SEQ ID NO: 72 and a forward primer consisting of the nucleotide sequence set forth in SEQ ID NO: 73 a primer set (10) comprising a reverse primer,
When the nucleotide sequence set forth in SEQ ID NO: 71 in the maize starch synthase IIb gene is the target nucleotide sequence, a forward primer consisting of the nucleotide sequence set forth in SEQ ID NO: 74 and a forward primer consisting of the nucleotide sequence set forth in SEQ ID NO: 75 Primer set (11), comprising a reverse primer,
When the nucleotide sequence set forth in SEQ ID NO: 65 in the maize starch synthase IIb gene is the target nucleotide sequence, a forward primer consisting of the nucleotide sequence set forth in SEQ ID NO: 66 and a forward primer consisting of the nucleotide sequence set forth in SEQ ID NO: 67 Primer set (12), including a reverse primer,
When the nucleotide sequence set forth in SEQ ID NO: 68 in the maize starch synthase IIb gene is the target nucleotide sequence, a forward primer consisting of the nucleotide sequence set forth in SEQ ID NO: 69 and a forward primer consisting of the nucleotide sequence set forth in SEQ ID NO: 70 Primer set (13), which includes a reverse primer.

Among these, the primer sets (9) to (11) are preferred from the viewpoint of being able to amplify the target nucleotide sequence in the maize starch synthase IIb gene with higher specificity.

When the maize HMG gene is to be amplified and the nucleotide sequence of SEQ ID NO: 79 in the gene is the target nucleotide sequence, the forward primer consisting of the nucleotide sequence of SEQ ID NO: 80 and the nucleotide sequence of SEQ ID NO: 81 are used. and a primer set (14) comprising a reverse primer consisting of the nucleotide sequence of
When the nucleotide sequence of SEQ ID NO: 85 in the maize HMG gene is the target nucleotide sequence, a forward primer consisting of the nucleotide sequence of SEQ ID NO: 86 and a reverse primer consisting of the nucleotide sequence of SEQ ID NO: 87 are used. and a primer set (15) comprising
When the nucleotide sequence of SEQ ID NO: 76 in the maize HMG gene is the target nucleotide sequence, a forward primer consisting of the nucleotide sequence of SEQ ID NO: 77 and a reverse primer consisting of the nucleotide sequence of SEQ ID NO: 78 are used. and primer sets (16) comprising
When the nucleotide sequence of SEQ ID NO: 82 in the maize HMG gene is the target nucleotide sequence, a forward primer consisting of the nucleotide sequence of SEQ ID NO: 83 and a reverse primer consisting of the nucleotide sequence of SEQ ID NO: 84 are used. and a primer set (17) comprising:

Among these, the primer sets (14) to (15) are preferred from the viewpoint of being able to amplify the target nucleotide sequence in the maize HMG gene with higher specificity.

The primer set of the present invention can be prepared by those skilled in the art by appropriately selecting known methods. For example, based on the information of the target nucleotide sequence, the nucleotide sequences of the forward primer and the reverse primer are designed, synthesized using a commercially available automatic nucleic acid synthesizer (manufactured by Applied Biosystems, Beckman, etc.), and then A forward primer and a reverse primer can be prepared by purifying the obtained oligonucleotide using a reverse phase column or the like.

In addition, a labeling substance may be bound to the primers of the present invention in order to facilitate detection of amplification products by PCR. The "labeling substance" is not particularly limited as long as it can bind to nucleotides and can be detected by chemical or optical methods. Examples include green fluorescent protein (GFP) and allophycocyanin (APC). , fluorescent proteins such as phycoerythrin (R-PE), alkaline phosphatase (ALP), horseradish peroxidase (HRP), enzymes such as β-galactosidase (β-gal), radioactive isotopes such as ¹²⁵ I, fluorescein isothiocyanate ( FITC), rhodamine isothiocyanate (RITC) and other fluorescent dyes, colloidal metals, color labeling substances such as colored latex, avidin, biotin, DIG, and anti-DIG antibodies. When an enzyme is used as a labeling substance, a chromogenic substrate, a fluorescent substrate, a chemiluminescent substrate, or the like can be added as a substrate to enable various detections depending on the substrate. In addition, the labeling substance may be directly bound to the nucleotides constituting the primer, or may be indirectly bound via another substance.

(Target Nucleotide Sequence Amplification and Detection Method)
The present invention provides a method (method for amplifying a target nucleotide sequence in a sample) comprising a step of amplifying the sequence by polymerase chain reaction (PCR) using the target nucleotide sequence in the sample as a template and using the primer set described above. I will provide a.

The present invention also provides a method comprising the steps of amplifying a target nucleotide sequence in a sample by the above method and detecting the target nucleotide sequence amplified in the step (a method for detecting a target nucleotide sequence in a sample ) are also provided.

In the present invention, the "sample" is not particularly limited as long as it can contain the target nucleotide sequence. etc.). Nucleotides to be subjected to PCR can be isolated from these by any method, for example, dissolution treatment with a surfactant (CTAB, etc.), sonication, shaking agitation using glass beads, French press, and the like. is mentioned. Purification of nucleotides can be performed by, for example, phenol extraction, chromatography, ion exchange, gel electrophoresis, density-dependent centrifugation, and the like. More specifically, samples to be subjected to PCR include double-stranded nucleic acids such as genomic DNA and PCR fragments isolated by the above method, and single-stranded cDNAs such as cDNA prepared from total RNA or mRNA by reverse transcription reaction. Nucleic acids are included.

In the present invention, the term "template" means a nucleotide on the base of complementary strand synthesis in PCR. A complementary strand having a sequence complementary to the template has the meaning of a strand corresponding to the template, but the relationship between the two is only relative. That is, the strand synthesized as the complementary strand can function again as a template. In addition, the "target nucleotide sequence" to be amplified or detected in the present invention includes not only the sequence concerned but also the complementary strand of the sequence.

In the present invention, "polymerase chain reaction (PCR)" can amplify a target nucleotide sequence by repeating temperature changes. More specifically, a step of annealing a forward primer and a reverse primer to a target nucleotide sequence, then performing an elongation reaction with a DNA polymerase starting from the primers, PCR is composed of a cycle consisting of these three steps, the step of dissociating the main strand by heat denaturation to form a single strand.

The temperature in the annealing step is not particularly limited as long as it is a temperature at which the annealing occurs and can be maintained, and from the viewpoint of suppressing nonspecific amplification products, it is preferably 25 to 65 ° C., more preferably 35. to 60°C, more preferably 45 to 55°C, and particularly preferably 52°C. Although the retention time is not particularly limited, it is preferably 1 to 60 seconds, more preferably 10 to 50 seconds, even more preferably 20 to 40 seconds, and particularly preferably 30 seconds. Also, the annealing temperature may be the same or different in all cycles.

The temperature in the elongation step is not particularly limited as long as it is a temperature at which a complementary strand can be synthesized. The retention time is not particularly limited, but is preferably 1 to 60 seconds, more preferably 5 to 40 seconds, even more preferably 10 to 20 seconds, and particularly preferably 15 seconds. Also, the annealing temperature may be the same or different in all cycles. The annealing temperature may be the same temperature as the elongation reaction temperature, but should not be set higher than the elongation reaction temperature.

The temperature in the heat denaturation step is not particularly limited as long as it can dissociate the double strands, and is usually 80 to 100°C, preferably 90 to 98°C, and particularly preferably 95°C. Although the holding time is not particularly limited, it is preferably 1 to 30 seconds, more preferably 1 to 20 seconds, still more preferably 1 to 10 seconds, and particularly preferably 5 seconds. Also, the annealing temperature may be the same or different in all cycles. The annealing temperature may be the same temperature as the elongation reaction temperature, but should not be set higher than the elongation reaction temperature.

In PCR, the number of cycles is not particularly limited as long as the target nucleotide sequence can be amplified to the extent that it can be detected in the method described later, but it is usually 10 to 50 cycles, preferably 20 to 40 cycles, more preferably 35 cycles. is.

The composition of the reaction solution for such PCR is not particularly limited as long as it contains the composition essential for performing PCR. In other words, it is sufficient that the reaction solution contains a composition necessary for amplifying DNA. That is, the reaction solution contains, for example, the primer set described above and the DNA polymerase described later, as well as substrates such as dNTPs, divalent ions (e.g., magnesium ions, calcium ions), and monovalent ions (e.g., sodium ions, potassium ions), or salts for providing them (eg, magnesium sulfate, magnesium acetate, magnesium chloride) and buffers (eg, Tris-HCl buffer, phosphate buffer). In addition to these, the PCR reaction solution according to the present invention may contain organic solvents (e.g., ethanol, methanol, acetone), organic acids (e.g., formic acid, acetic acid, benzoic acid), surfactants (e.g., sodium lauryl sulfate). (SDS), Triton X-100), amino acids (e.g., aspartic acid, glutamic acid, lysine, tryptophan), sugars (e.g., glucose, xylose, galactose), dimethylsulfoxide, betaine, DNA-binding proteins, etc., as additives. You can

The "DNA polymerase" used in PCR may be any polymerase that has the activity of synthesizing a complementary strand of DNA to a target nucleotide sequence (DNA-dependent DNA polymerase). However, a thermostable DNA polymerase is preferred. The thermostable DNA polymerase is not particularly limited, and includes DNA polymerases having at least one of 5'→3' exonuclease activity, 3'→5' exonuclease activity (proofreading activity) and TdT activity. . Polymerases having 5′→3′ exonuclease activity and TdT activity include Pol-type DNA polymerases, more specifically Taq polymerase and Tth polymerase. Polymerases having proofreading activity include α-type DNA polymerases, and more specifically, Pfu polymerase and KOD polymerase. Moreover, the thermostable DNA polymerase of the present invention includes not only natural polymerases but also artificially modified polymerases. Modified DNA polymerases include, for example, Taq polymerase with proofreading activity added (ExTaq polymerase).

Among these, the DNA polymerase according to the present invention is preferably a thermostable DNA polymerase having 5′→3′ exonuclease activity and 3′→5′ exonuclease activity. Specifically, ExTaq polymerase is mentioned. In PCR, both of these activities can also be exhibited by using a mixture of multiple types of DNA polymerases (for example, Pol-type DNA polymerase and α-type DNA polymerase).

In the present invention, the target nucleotide sequence amplified by PCR can be detected by those skilled in the art by appropriately using known techniques. Known techniques include electrophoresis (e.g., acrylamide gel electrophoresis for developing amplification products), nucleic acid chromatography (for example, STH method of TBA), intercalation method, and quencher-mediated fluorescence detection method.

(Kits for amplifying or detecting target nucleotide sequences)
The present invention can also provide kits for amplifying or detecting target nucleotide sequences that include the primer sets described above. The kit contains the above DNA polymerase and other PCR reaction mixture compositions (substrate (dNTP), the ions or salts for providing them, buffers, additives) and instructions for use of the kit. . In addition, when a labeling substance is bound to the primer of the present invention, the kit may also contain a substrate for detecting the labeling substance. In addition, depending on the detection method, a carrier for developing the amplified product by PCR (e.g., polyacrylamide gel in electrophoresis method, chromatography test paper in nucleic acid chromatography method) and solvent, fluorescent substance (e.g., in intercalation method Intercalators), fluorescent and quencher-conjugated probes in quencher-mediated fluorescence detection methods are also optionally included in the kit. Additionally, DNA molecular weight markers and positive controls may also be included in the kit to verify the amplified target nucleotide sequence.

(Method for detecting transgenic plants)
As shown in the examples below, the present inventors found that in the genomic DNA of a transgenic plant produced by the Agrobacterium method, the RB flanking sequence derived from the T-DNA of Agrobacterium (SEQ ID NO: 1 The described nucleotide sequences) were found to be commonly inserted. Furthermore, this extremely short 25-nucleotide flanking RB sequence can be detected by PCR using a forward primer consisting of the nucleotide sequence set forth in SEQ ID NO: 15 and a reverse primer set consisting of the nucleotide sequence set forth in SEQ ID NO: 16. The inventors also found that

Accordingly, the present invention provides
A method for detecting a genetically modified plant produced by the Agrobacterium method, comprising:
Using the genomic DNA of a test plant as a template, a forward primer consisting of the nucleotide sequence set forth in SEQ ID NO: 15 and a reverse primer set consisting of the nucleotide sequence set forth in SEQ ID NO: 16 are used to perform a polymerase chain reaction;
A step of detecting an RB flanking sequence derived from Agrobacterium T-DNA, consisting of the nucleotide sequence set forth in SEQ ID NO: 1, amplified by the polymerase chain reaction;
A step of determining that the test plant is a transgenic plant produced by the Agrobacterium method when amplification of the RB flanking sequence is detected in the step;
Also provided is a method comprising:

The "test plant" is not particularly limited as long as it is a plant that may be a genetically modified plant or a plant that may be contaminated with a genetically modified plant, but soybeans, corn, Potatoes, rapeseed, cotton, alfalfa, sugar beets, and papaya. The method for preparing the genomic DNA to be used in the method of the present invention is as shown in (Method for Amplification and Detection of Target Nucleotide Sequence) above. Examples include seeds, fruits, leaves, stems and roots of the test plant.

In addition, the method for detecting the amplification product obtained by the polymerase chain reaction (PCR) and PCR is also as shown in the above (Method for amplification and detection of target nucleotide sequence). When amplification of the RB flanking sequence is detected, the test plant is determined to be a transgenic plant produced by the Agrobacterium method.

The "genetically modified plant produced by the Agrobacterium method" determined by the method of the present invention is preferably an RB derived from the Agrobacterium T-DNA consisting of the nucleotide sequence set forth in SEQ ID NO: 1. Genetically modified plants in which the flanking sequence is inserted into the genomic DNA, specifically, genetically modified soybeans such as MON89788, MON87701, MON87708 and MON87769, gene sets such as MIR162, MIR604, MON88017, DAS59122 and 3272 Replaced corn.

In addition, the method for detecting genetically modified plants of the present invention can also be applied to a method for confirming plants in which traces of genetic modification have been deleted in genome editing. That is, in genome editing, foreign DNA encoding the editing system (guide RNA, nuclease, etc.) is integrated into the genomic DNA of the plant by the Agrobacterium method in order to modify the target gene. After the mutation is introduced into the target gene by the editing system, the foreign DNA is usually removed from the genomic DNA by crossing with a wild-type plant or using Cre-loxp or the like. In order to confirm this removal (removal of traces of genetic recombination), it is also suitably used in the method for detecting genetically modified plants of the present invention.

EXAMPLES The present invention will be described in more detail below based on examples and comparative examples, but the present invention is not limited to the following examples. Examples and comparative examples were carried out using the materials and methods described below.

(materials and methods)
1. Plant seeds In Japan, among the GM crops that have undergone safety reviews, 8 GM soybean lines: RRS, A2704-12, MON89788, DP305423, MON87701, MON87705, MON87708, MON87769, and GM maize 15 Strains: NK603, Event176, T25, GA21, MON810, TC1507, Bt11, MIR604, MON88017, DAS59122, MON863, MIR162, 3272, MON89034, MON87460 were subjected to the following experiments.

2. DNA extraction Genomic DNA of soybean and maize was extracted using DNeasy Plant Maxi kit (manufactured by Qiagen). The extraction method was in accordance with the Consumer Affairs Agency manual (see Non-Patent Document 2 “(Attachment) Inspection Method for Foods to which Recombinant DNA Technology Has Been Verified for Safety”). The concentration of the extracted DNA was quantified based on the absorbance at 260 nm using a NanoDrop ultratrace spectrophotometer (manufactured by ThermoFisher Scientific). Extracted DNA was adjusted to 50 ng/μL and 100 ng was used as template for PCR analysis.

3. Preparation of Simulated GM Crop Contamination Samples Each GM soybean and corn quasi-GM crop adulteration sample was prepared by diluting the respective extracted DNA with the same concentration of Non-GM soybean and corn extracted DNA. did. Specifically, DNA extracted from GM soybean and GM corn and DNA extracted from Non-GM soybean and Non-GM corn are adjusted to have the same concentration, and mixed by changing the volume ratio of each of the extracted DNA solutions. Simulated spiked samples of each concentration were prepared by

4. Detection by PCR and Electrophoresis Sequences for PCR primers shown in Tables 1 to 4 below were designed, and oligo DNAs for primers were synthesized by FASMAC Co., Ltd. based on the sequences.

Then, 10 μL of a real-time PCR reagent (trade name: SYBR Premix Ex TaqII, manufactured by Takara Co., Ltd.) was mixed with a target primer set solution (final concentration of each primer: 0.3 μM), and the template DNA was added to obtain a total volume of 20 μL. A PCR reaction solution was prepared.

The PCR reaction solution is set in a PCR device, and after denaturation treatment at 95 ° C. for 30 seconds, 35 Cycle amplification reactions were performed. GeneAmp PCR system 9700 from ThermoFisher Scientific was used as a PCR device.

As a negative control, the PCR was performed without adding template DNA (No template control, NTC). The PCR was performed using the RBF and RBR primer sets.

For the resulting PCR amplification product, 10 μL of the 20 μL reaction solution was subjected to electrophoresis on a 12% acrylamide gel. As a molecular weight marker, GeneRuler Ultra Low Range DNA Ladder (manufactured by ThermoFisher Scientific) was also used for electrophoresis. Next, using a nucleic acid staining solution (product name: Gel Red, manufactured by FUJIFILM Wako Pure Chemical Industries, Ltd.), nucleic acid bands in the gel were stained and detected.

5. Detection by Nucleic Acid Chromatography The PCR amplification products were also detected by nucleic acid chromatography (STH (Single-stranded Tag Hybridization)). In the STH method, a target nucleotide sequence is amplified by PCR using a biotin-labeled primer and a single-tag DNA-added primer. When the amplified solution is mixed with avidin-coated latex (blue) and spread on a membrane strip, the strong hybridization reaction between the linearly immobilized complementary tag DNA and the single tag DNA causes the PCR amplification product to be trapped. be done. The presence or absence of the target nucleotide sequence in the specimen can be visually determined by the line becoming blue with the trapped latex-labeled PCR amplification product.

In this example, oligo DNA with a single tag DNA (F1 tag) added to the 5' end of RBF2 and oligo DNA labeled with biotin at the 5' end of RBR2 were used as a combination of primers for detecting RB flanking sequences. or oligo DNA with a single tag DNA (F2 tag) added to the 5' end of the RBF, and a primer set in which the 5' end of the RBR primer was labeled with biotin. The addition of tag DNA and biotin labeling to these primers were performed by TBA Co., Ltd.

Using these primer sets, a PCR reaction was carried out under the conditions described in "4. Detection by PCR and electrophoresis". Next, 1 μL of the 20 μL of the solution after the reaction was mixed with 10 μL of the developing solution and 1 μL of the latex solution to prepare a total volume of 20 μL (salt concentration: 70 mM) and subjected to chromatographic development. Chromatography development was performed using a strip for nucleic acid chromatography (product name: C-PAS4 (Chromatography Printed-Array strip 4), manufactured by TBA Co., Ltd.) according to the manufacturer's manual.

(Example 1) Examination of primer sequences and PCR conditions Target sequences common to GM crops in order to efficiently detect existing GM crops and to detect all growing GM crops A screening method that detects as At present, the most widely used GM plant production technology in the world is the Agrobacterium method, in which the T-DNA region on the Ti plasmid is inserted into the nuclear genome of the plant. Therefore, sequences derived from Agrobacterium T-DNA are considered to be very effective target sequences for GM detection.

Therefore, in order to identify T-DNA-derived sequences that are commonly inserted into genomic DNA of GM plants, GM crop genomes created by the Agrobacterium method were collected from patent information and Internet databases for various GM crops. Sequence information derived from the T-DNA region inserted into the DNA was obtained and alignment analysis was performed (GMO Detection method Database (GMDD) http://gmdd.sjtu.edu.cn/, WO2009/064652A1, WO2007/142840A2 reference).

As a result, as shown in FIG. 1, the only completely conserved sequence in the 5′-side border sequence containing all identifiable RBs was the RB flanking sequence consisting of 25 nucleotides. It was also revealed that the sequences are conserved not only in GM soybeans and maize, but also in various GM crops such as rapeseed and cotton.

However, regarding PCR, there are already various textbook experimental protocols, and Molecular Cloning, which is a representative example, describes the length of the primer as usually 20 to 25 nucleotides, and furthermore, 18 nucleotides. It is described that when primers with a length less than 100 are used, there is a tendency for non-specific binding to occur with the template DNA. That is, in PCR, a forward primer of about 20 nucleotides and a reverse primer of about 20 nucleotides, and a space of at least a few nucleotides between the primers, are technically designed to have a length of at least 40 nucleotides or more as a PCR target. It has become common knowledge.

That is, targeting a sequence consisting of 25 nucleotides as described above and specifically detecting the sequence was thought to be extremely difficult and unprecedented. Therefore, extensive studies were conducted to determine whether or not the 25-nucleotide flanking RB sequence can be detected by PCR.

Specifically, first, in order to detect the RB flanking sequence, a forward primer (RBF shown in Table 1 and FIG. 2A) consisting of 13 nucleotides targeting the sequence consisting of 25 nucleotides, It was examined whether the target sequence could be specifically amplified by PCR using a reverse primer (RBR, shown in Table 1 and FIG. 2A) consisting of 17 nucleotides overlapping nucleotides (GAAAC). Genomic DNA of GM soybean MON89788 or GM maize MIR162, which has already been shown to have the RB flanking sequence, was used as template DNA for PCR.

As a result, although not shown in the data, as a result of PCR using this primer set, although a clear band was detected at the position of 25 bp, it was also amplified from the reaction solution (NTC) containing no template DNA. A product was detected, and it became clear that the primer set using this primer set could not specifically amplify the target sequence (although it did not amplify in a template DNA-dependent manner, it was cleared at the 25 bp position). Since a band was detected in the following experiment, the amplification product obtained using the primer set of RBF and RBR was used as a positive control).

Next, a 12-nucleotide forward primer (“RBF2” shown in Table 1 and FIG. 2A) targeting the 25-nucleotide sequence, and a 13-nucleotide reverse primer (Table 1 and “RBR2” shown in FIG. 2A) were used to examine whether the target sequence can be specifically amplified.

As a result, as shown in FIGS. 3A to 3C, unlike the case of using the above-described primer set that overlaps with 5 nucleotides, using the primer set consisting of 12 nucleotides and 13 nucleotides, genomic DNAs derived from NTC and non-GM plants were obtained. No band indicating amplification of the target sequence at 25 nucleotides was observed when PCR was performed as a template. On the other hand, when PCR is performed using genomic DNA derived from GM plants as a template, the band is observed, and when the primer set consisting of 12 nucleotides and 13 nucleotides is used, the target sequence can be specifically detected. became clear.

In general, the annealing temperature for PCR is determined with reference to the melting temperature (Tm) value of the primers used, and if the Tm values of the primers are different, it is recommended to use the lower value as the annealing temperature. (Hiroki Nakayama, Bio Experiment Illustrated 3 PCR that really increases, published by Shujunsha, June 1996). The Tm values of RBF2 and RBR2 were 28.9° C. and 28.1° C., respectively, by the GC method (calculated values obtained at a cation concentration of 60 mM).

Therefore, as shown in FIGS. 3A to 3C, when the annealing temperature was set to the lower one of these and PCR was performed, a band was detected at the position of 25 bp, but the target band was faint. A non-specific band was conspicuous in . On the other hand, when the annealing temperature was gradually increased, it was found that at 52°C, almost no non-specific bands were visible, and the desired 25 bp band was clearly detected. When the annealing temperature was further increased from 52°C, the 25 bp band tended to become extremely thin (data not shown), so in this detection method, the optimum annealing temperature was determined to be 52°C. .

(Comparative Example 1) Study 2 on primer sequences
As described above, in the target sequence, the forward primer and the reverse primer are arranged without overlapping, and a primer set designed so that the total number of nucleotides of these primers is the same as the target sequence (25 nucleotides) is used. As a result, the target sequence could be specifically amplified despite being as short as 25 nucleotides.

Therefore, while the total number of nucleotides in the primer set was 25 nucleotides, the length of each primer was changed to verify whether the target sequence could be specifically amplified. Specifically, for a target sequence consisting of 25 nucleotides, the forward and reverse primers are respectively 13 nucleotides (RBF) and 12 nucleotides (RBR3), 14 nucleotides (RBF4) and 11 nucleotides (RBR4), 15 nucleotides ( RBF5) and 10 nucleotides (RBR5), 11 nucleotides (RBF6) and 14 nucleotides (RBR6), and 10 nucleotides (RBF7) and 15 nucleotides (RBR7) were designed and examined. See Table 1 and FIGS. 2A and 2B for the sequences of these primers.

However, as shown in FIGS. 4B to 4F, the target sequence could not be specifically detected using any of the primer sets.

Further, the target sequence is shortened by 1 nucleotide to 24 nucleotides, and the sequence is specifically amplified and detected using a Forward primer consisting of 12 nucleotides and a Reverse primer consisting of 12 nucleotides (RBF2 and RBR8, RBF8 and RBR3). See Table 1 and FIG. 2C for the sequences of these primers.

However, as shown in FIGS. 4G and H, no band was observed at the target position using any of the primer sets, and the target sequence could not be specifically detected.

These results strongly suggested that the primer set capable of detecting the RB flanking sequence was a combination of RBF2 and RBR2 only. In particular, RBR8 did not amplify the PCR product in combination with RBF2, although the guanine (G) at the 5' end of RBR2 was only shortened by one nucleotide.

(Example 2) Confirmation of specificity of RB flanking sequence detection method As described above, a forward primer (RBF2) consisting of 12 nucleotides and a reverse primer (RBR2) consisting of 13 nucleotides that do not overlap with the primer are used. As a result, it was possible to detect GM soybean MON89788 and GM maize MIR162, which have already been found to have T-DNA flanking RB sequences.

Therefore, it was examined whether other GM crops could also be detected by PCR using a primer set of RBF2 and RBR2 (also referred to as "RB flanking sequence detection method").

As a result, as shown in FIGS. 5A and B, GM soybeans MON87701, MON87708 and MON87769, and GM maize MIR604, MON88017, DAS59122 and 3272, which have been clarified to have been produced by the Agrobacterium method, were placed at the target positions. band was recognized.

On the other hand, MON87705, MON89034 and MON87460 produced by the Agrobacterium method did not amplify the target band. However, for these GM soybeans and corn, the present inventors confirmed the sequences near the 5'-side border with reference to patent information (WO2010/037016A1, WO2007/140256A1, WO2009/111263A1), etc., but the sequence near the RB sequence was not found. That is, although these three types of GM crops were produced by the Agrobacterium method, they do not have the RB flanking sequences, and therefore, it is highly likely that they were not amplified.

Furthermore, GM soybeans RRS, A2704-12 and DP305423 produced by methods other than the Agrobacterium method such as particle gun method and electroporation method, and GM corn NK603, Event176, T25, GA21, MON810, TC1507, Bt11 and Amplification of the target band was not observed in MON863.

From the above results, it was clarified that the 25-nucleotide flanking RB sequence can be specifically detected by PCR using the 12-nucleotide forward primer (RBF2) and the 13-nucleotide reverse primer (RBR2). rice field.

In addition, among the GM soybeans and GM corn that could be detected by the PCR, MON89788, MON87701, MON87708, MON87769, MIR162, MIR604 and 3272 are P35S used for screening detection of GM crops in the Consumer Affairs Agency manual. does not have an array. In addition, Agrobacterium nopaline synthase gene terminator (TNOS) used for screening purposes alongside P35S (see Non-Patent Document 2 “(Attachment) Inspection method for safety-examined recombinant DNA technology applied food”) ) in MIR162, MIR604 and 3272, but not in the other four GM soybean lines. In addition, the terminator (tE9) of the ribulose 1,5-bisphosphate carboxylase gene of Pisum sativum (see Coruzzi, G. et al., The EMBO Journal, 1984, pp. 3, 1671-1679) is used in Monsanto's GM soybeans. Among the above GM soybeans, MON89788, MON87708, and MON87769 have tE9, but the other four lines do not have tE9 (Takabatake, R. et al., Food Chemistry, 2018, 252 , pages 390-396). Thus, it is difficult to detect all seven strains by existing screening approaches. On the other hand, according to the method of the present invention, as described above, any strain can be detected, suggesting that the method is useful in detecting GM foods.

(Example 3) Verification of the lower detection limit in the RB flanking sequence detection method In GM testing, there are many cases in which a minute amount of contaminating DNA is targeted for detection. GM soybeans and GM corn that have undergone safety screening for food labeling are exempted from labeling regarding recombinants if appropriate separate production and distribution management is in place and contamination is up to 5%. Therefore, samples containing 0.5%, 1.0%, 5.0%, 10.0% and 100% of MON89788 and MIR162 were prepared, and the availability of detection by the RB flanking sequence detection method was verified.

As a result, as shown in FIGS. 6A and B, it was shown that both MON89788 and MIR162 can be detected if they are mixed at 0.5% or more, which is 1/10 of the reference value. Therefore, it was clarified that the lower detection limit of the method for detecting RB flanking sequences of the present invention is 0.5% or less for both GM soybeans and corn.

(Example 4) Detection of RB flanking sequences by nucleic acid chromatography To simplify the method for detecting RB flanking sequences of the present invention, nucleic acid chromatography (TBA's STH method (https://www.t-bioarray.com/contents /technology.html))).

In the STH method (STH C-PAS system), a set of a primer with a biotin molecule added to the 5′ side and a primer with a special nucleotide sequence called a tag sequence added, and a test paper for nucleic acid chromatography (C- PAS) is used. When PCR is performed with the primer set, both ends of the PCR product are amplified with a biotin molecule and a tag sequence added. A DNA having a sequence complementary to the tag sequence is immobilized at a predetermined position on the C-PAS membrane, and when the PCR product is chromatographically developed, the hybridization reaction between the tag DNA and the complementary tag DNA causes PCR. Amplification products are trapped. Furthermore, by using latex-labeled avidin beads, it is possible to visually determine the presence or absence of target DNA without using electrophoresis or DNA staining.

Therefore, it was confirmed that RBF2 and RBR2 were labeled with a tag sequence (F1) and a biotin molecule, respectively, and that RB flanking sequences could be detected by the STH method using these primer sets.

As a result, as shown in FIGS. 7A and B, GM crops could be specifically detected by the STH method as well as the detection method using acrylamide electrophoresis. In addition, when the detection limit in the STH method was verified, as shown in FIGS. 8A and B, it was possible to detect both GM soybeans and GM corn up to 0.5% or less, as in the case of using acrylamide gel electrophoresis. It has been shown.

(Example 5) Examination of applicability of 25-nucleotide PCR to sequences other than RB flanking sequences (generalization of 25-nucleotide PCR)
As described above, a very short RB flanking sequence of 25 nucleotides could be detected by PCR. However, in the first place, there are almost no examples of whether or not such short sequences can be detected by PCR, not limited to sequences near RB.

Therefore, is PCR targeting a 25-nucleotide sequence using genomic DNA as a template (also referred to as "25-nucleotide PCR") applicable to nucleotide sequences other than the RB flanking sequence (PCR targeting 25-nucleotide can be generalized).

It should be noted that when performing GM detection, it is common to co-detect the crop species-specific endogenous sequences rather than targeting the recombinant DNA alone. Such a positive control makes it possible to confirm that the PCR reaction system including the reagents and equipment used is functioning normally. As an endogenous sequence, the Consumer Affairs Agency manual describes the lectin gene (Le1) for soybean (Vodkin L. et al., Cell, 1983, 34, pp. 1023-1031), and the starch synthase IIb (SSIIb) for maize (
Harn, C.; et al., Plant Molecular Biology, 1998, 37, pages 639-649). HMG (see Krech, AB et al., Gene, 1999, pp. 234, 45-50) is also frequently used in overseas testing methods as an endogenous sequence of maize. Therefore, based on the genomic DNA sequences of these Le1, SSIIb and HMG genes, 25-nucleotide targets consisting of various nucleotide sequences were selected, forward primers (12 nucleotides) and reverse primers (13 nucleotides), or forward primers (13 nucleotides). ) and a reverse primer (12 nucleotides) were designed, and the availability of specific detection was examined. Table 5 shows the results obtained.

As shown in Table 5, no amplification of the sequence was observed when the total number of GCs in the target nucleotide sequence, ie the total number of GCs in the forward and reverse primers, was 12 or less. On the other hand, amplification of the sequence was observed when the total number of GCs was 14 or more.

In addition, when the total number of GCs is 13, there are cases where it is amplified or not. was large (12.4° C.). Therefore, when the number of GCs is 13, it is considered that the balance of Tm values between primers is important. Then, from this point of view, when the results of the RB neighborhood sequence were reviewed again, the total number of GCs in the RB neighborhood sequence (target nucleotide sequence) was 13, and RBF2 (Tm value: 28.9 ° C.) and When using RBR2 (Tm value: 28.2°C), the sequence can be amplified, while using RBF (Tm value: 34.5°C) and EBR3 (Tm value: 22.1°C) The reason why it was not amplified can be well explained.

In addition, among primer sets in which amplification of the target nucleotide sequence was observed, primer sets with higher specificity for crop species include Le1F3 and Le1R3, Le1F6 and Le1R6, Le1F7 and Le1R7, Le1F9 and Le1R9, SSIIbF1 and SSIIbR1, and SSIIbF5. SSIIbR5, HMGF2 and HMGR2, HMGF6 and HMGR6 could be obtained.

In the present invention, since the target nucleotide sequence is as short as 25 nucleotides, it is difficult to predict in the design stage whether a highly specific primer set will be obtained. Therefore, in order to obtain a primer set that has high specificity and suppresses the formation of primer-dimers, several types of primer sets that are expected to be capable of amplification are designed, and among them, A method of selecting a primer set that exhibits specificity may be effective.

As explained above, according to the present invention, it is possible to amplify a very short target sequence consisting of 25 nucleotides by PCR.

In the current world of genetic analysis including PCR, there are almost no techniques that can clearly detect DNA as short as 25 nucleotides. In the conventional PCR method, as described above, two types of primers consisting of around 20 nucleotides are used, so it is practically impossible to detect targets as short as 25 nucleotides.

Since the PCR method was originally developed for the purpose of easily cloning a specific gene, improvements have been made toward the amplification of targets consisting of longer sequences. On the other hand, in genetic tests such as GM detection, PCR conditions for amplifying long sequences are not necessarily optimal for those purposes. Conversely, within the range where specificity is ensured, a shorter target may improve detection sensitivity or broaden applicability. For example, in the field of food research, it has been reported that the DNA of raw material crops is significantly fragmented in processed foods and the like, making it difficult to detect by conventional PCR (Yoshimura, T. et al., Journal of Agricultural and Food Chemistry, 2005, 53, 2060-2069; Yoshimura, T. et al., Journal of Agricultural and Food Chemistry, 2005, 53, 2052-2059). According to the present invention, it is possible to detect a much shorter sequence than conventional PCR, so it may be possible to improve the performance of inspection methods for processed foods.

Moreover, according to the present invention, it is also possible to detect genetically modified (GM) plants produced by the Agrobacterium method by detecting the aforementioned RB flanking sequences. In particular, it becomes possible to detect most of the GM crops produced by the Agrobacterium method, which is currently most used around the world, with one primer set.

In the above-described examples, GM soybeans and GM corn, which have been reviewed for safety related to food labeling, are used as the subject of the present invention. It is considered that the application to GM crops is also useful. In recent years, in particular, there have been cases of GM crops, whose safety has not been confirmed as food or from the perspective of the Cartagena Act, have flowed into Japan from overseas or are feared to flow into Japan (Ministry of Health, Labor and Welfare of Japan, “Safety Inspection method for non-examined recombinant DNA technology applied food” http://www.mhlw.go.jp/file/06-Seisakujouhou-11130500-Shokuhinanzenbu/0000171839.pdf, Japan Ministry of Agriculture, Forestry and Fisheries “2016 (Heisei 28 Q&A about unapproved genetically modified wheat in Japan discovered in the United States in July 2009” http://www.maff.go.jp/j/syouan/keikaku/soukatu/attach/pdf/index- 8. pdf, Japan Ministry of Agriculture, Forestry and Fisheries press release http://www.maff.go.jp/j/press/syouan/nouan/170510.html, Japan Ministry of Agriculture, Forestry and Fisheries press release http://www.maff.go.jp/j/press/syouan/nouan/170510.html jp/j/press/syouan/nouan/141225.html). Also, in Japan, there is no end to the cases where GM plants whose safety has not been reviewed are found to be growing in inappropriate environments (for example, Nara Institute of Science and Technology Press Release http: //www.naist.jp/pressrelease/2017/09/004001.html, Nagoya University Notice from the university "Accidents related to Type 2 use of genetically modified organisms", http://www.nagoya-u.jp/ ac.jp/info/20150522.html, National Agriculture and Food Research Organization ((former) National Institute of Agrobiological Sciences) press release http://www.naro.affrc.go. jp/archive/nias/press/2016/20160325/). These include not a few GM plants produced by the Agrobacterium method. In particular, in the above-described examples, it was confirmed that GM plants can be detected even by a simple detection method using nucleic acid chromatography. It is considered that the present invention is useful because it can determine whether a plant is a GM plant produced by the Agrobacterium method.

Therefore, the present invention is useful in genetic testing in various fields such as detection of GM crops.

Claims (4)

A primer set comprising a forward primer and a reverse primer for amplifying a target nucleotide sequence in genomic DNA , the primer set being selected from the following groups (1) to (11):
(1) The target nucleotide sequence is an RB flanking sequence derived from T-DNA of Agrobacterium, which consists of the nucleotide sequence set forth in SEQ ID NO: 1.
A forward primer consisting of the nucleotide sequence set forth in SEQ ID NO: 15 and
a primer set comprising a reverse primer consisting of the nucleotide sequence set forth in SEQ ID NO: 16;
(2) the target nucleotide sequence is a sequence derived from the soybean lectin gene consisting of the nucleotide sequence set forth in SEQ ID NO: 34;
a forward primer consisting of the nucleotide sequence set forth in SEQ ID NO: 35 and
a primer set comprising a reverse primer consisting of the nucleotide sequence set forth in SEQ ID NO: 36;
(3) the target nucleotide sequence is a sequence derived from the soybean lectin gene consisting of the nucleotide sequence set forth in SEQ ID NO: 42;
a forward primer consisting of the nucleotide sequence set forth in SEQ ID NO: 43 and
a primer set comprising a reverse primer consisting of the nucleotide sequence set forth in SEQ ID NO: 44;
(4) the target nucleotide sequence is a sequence derived from the soybean lectin gene consisting of the nucleotide sequence set forth in SEQ ID NO: 45;
a forward primer consisting of the nucleotide sequence set forth in SEQ ID NO: 46 and
a primer set comprising a reverse primer consisting of the nucleotide sequence set forth in SEQ ID NO: 47;
(5) the target nucleotide sequence is a sequence derived from the soybean lectin gene consisting of the nucleotide sequence set forth in SEQ ID NO: 45;
a forward primer consisting of the nucleotide sequence set forth in SEQ ID NO: 48 and
a primer set comprising a reverse primer consisting of the nucleotide sequence set forth in SEQ ID NO: 49;
(6) The target nucleotide sequence is a sequence derived from the soybean lectin gene, which consists of the nucleotide sequence set forth in SEQ ID NO: 53.
a forward primer consisting of the nucleotide sequence set forth in SEQ ID NO: 54 and
a primer set comprising a reverse primer consisting of the nucleotide sequence set forth in SEQ ID NO: 55;
(7) The target nucleotide sequence is a sequence derived from the maize starch synthase IIb gene, which consists of the nucleotide sequence set forth in SEQ ID NO: 62.
a forward primer consisting of the nucleotide sequence set forth in SEQ ID NO: 63 and
a primer set comprising a reverse primer consisting of the nucleotide sequence set forth in SEQ ID NO: 64;
(8) The target nucleotide sequence is a sequence derived from the maize starch synthase IIb gene, which consists of the nucleotide sequence set forth in SEQ ID NO: 71.
A forward primer consisting of the nucleotide sequence set forth in SEQ ID NO: 72 and
a primer set comprising a reverse primer consisting of the nucleotide sequence set forth in SEQ ID NO: 73;
(9) The target nucleotide sequence is a sequence derived from the maize starch synthase IIb gene, which consists of the nucleotide sequence set forth in SEQ ID NO: 71.
a forward primer consisting of the nucleotide sequence set forth in SEQ ID NO: 74 and
a primer set comprising a reverse primer consisting of the nucleotide sequence set forth in SEQ ID NO: 75;
(10) the target nucleotide sequence is a maize HMG gene-derived sequence consisting of the nucleotide sequence set forth in SEQ ID NO: 79;
A forward primer consisting of the nucleotide sequence set forth in SEQ ID NO: 80 and
a primer set comprising a reverse primer consisting of the nucleotide sequence set forth in SEQ ID NO: 81;
(11) the target nucleotide sequence is a maize HMG gene-derived sequence consisting of the nucleotide sequence set forth in SEQ ID NO: 85;
A forward primer consisting of the nucleotide sequence set forth in SEQ ID NO: 86 and
A primer set comprising a reverse primer consisting of the nucleotide sequence set forth in SEQ ID NO:87. A method for amplifying a target nucleotide sequence within genomic DNA in a sample, comprising:
A method comprising amplifying the target nucleotide sequence as a template by polymerase chain reaction using the primer set of claim 1 . A method for detecting a target nucleotide sequence within genomic DNA in a sample, comprising:
amplifying the target nucleotide sequence by the method of claim 2 ;
and detecting the target nucleotide sequence amplified in said step. A method for detecting a genetically modified plant produced by the Agrobacterium method, comprising:
Using the genomic DNA of a test plant as a template, a forward primer consisting of the nucleotide sequence set forth in SEQ ID NO: 15 and a reverse primer set consisting of the nucleotide sequence set forth in SEQ ID NO: 16 are used to perform a polymerase chain reaction;
A step of detecting an RB flanking sequence derived from Agrobacterium T-DNA, consisting of the nucleotide sequence set forth in SEQ ID NO: 1, amplified by the polymerase chain reaction;
A step of determining that the test plant is a transgenic plant produced by the Agrobacterium method when amplification of the RB flanking sequence is detected in the step;
How to include.

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