JP5229864B2 - Quantitative detection method for genetically modified organisms - Google Patents

Description

  The present invention relates to a method for quantitative detection of a gene recombinant contained in soybean or processed soybean food and a nucleic acid molecule used in the method. More specifically, the present invention relates to a method for quantitatively detecting a genetically modified product contained in soybean or processed soybean food using the LAMP method or ABC-LAMP method, as well as the nucleic acid primer and nucleic acid used in the method. The present invention relates to various nucleic acid molecules such as probes and competitive nucleic acids.

Agricultural crops developed using genetic recombination technology are being put into practical use worldwide. In Japan, genetically modified organisms are recognized for 59 varieties of crops such as soybean and corn. Examples of genetically engineered crops that are actually distributed include, for example, the Bt11 line progeny (Novartis), the Event176 line progeny (Novartis), the MON810 line progeny (Monsanto), and the GA21 line. Progeny varieties (Monsanto), T25 line varieties (Aventis), and soybean, Roundup Ready Soybean line varieties (Monsanto) and the like.
Genetically modified crops are developed for the purpose of imparting industrially favorable traits such as pest resistance and herbicide resistance to natural crops. The main technique is to isolate a gene that expresses the trait from other organisms that originally have such an industrially favorable trait and introduce the gene into a target crop in a form that can be expressed. Therefore, such transgenes are included in the DNA of genetically modified crops.

  The European Community (EU) has established regulations regarding the labeling of genetically modified crops and their processed foods (Non-Patent Document 1), and Japan has also established a system for labeling of genetically modified crops and their processed foods. With this as a trigger (Non-patent Document 2), the food industry and related industries are required to grasp and recognize the presence or content of recombinant DNA in food raw crops and foods. For this reason, there is a need for a technique for confirming the presence and content of genetically modified organisms in foods, feeds, and these raw material crops, particularly the abundance ratio in the raw materials.

  As a technique for detecting a gene recombinant, for example, a method for amplifying the recombinant gene by polymerase chain reaction (hereinafter referred to as “PCR”) and detecting it (for example, Non-Patent Document 3); Quantitative detection technology for replacement soybeans and corn (for example, non-patent documents 4, 5, 6 and 7); Quantitative detection technology for recombinant soybeans and corn by quantitative PCR using fluorescent probes (real-time PCR) (for example, Non-Patent Documents 8, 9 and 10); a method for quantifying the content of a gene recombinant by measuring a protein produced from recombinant DNA using the Enzyme Linked Immuno Sorvent Assay (ELISA) method (for example, Patent document 11) can be mentioned. Recently, as a method for accurately quantifying the abundance ratio of a gene recombinant in a population including a plurality of gene recombinant strains using a quantitative PCR method, a method using an internal standard (Patent Document 1). In particular, the former method (Patent Document 1) is adopted as a standard method for testing and analyzing genetically modified foods in Japan. This method is a method of measuring the copy number of endogenous genes and recombinant genes in crops by PCR, and converting and quantifying the amount of genetically modified genes mixed from the ratio.

The conventional quantitative PCR method defined as the standard method requires more than 1 hour for amplification and detection, and there is an expensive real-time PCR device that integrates a thermal cycler and a fluorescence detection device for PCR. There was a problem that it was necessary. Therefore, development of a method for detecting a recombinant gene quickly, simply and at low cost has been desired.
[Non-Patent Document 1]
Regulation (EU) No.EC/258/97, Council Regulation (EC), No.1139 / 98
[Non-Patent Document 2]
Notification No. 1775 (June 10, 2000) Food and Marketing Bureau, Ministry of Agriculture, Forestry and Fisheries of Japan, Tokyo, Japan
[Non-Patent Document 3]
Hupfer C., Mayer J., Hotzel H., Sachse K., Engel KH, Eur. Food Res. Technol., 209, 301-304 (1999)
[Non-Patent Document 4]
Hardegger M., Brodmann P., Herrmann A., Eur. Food Res. Technol., 209, 83-87 (1999)
[Non-Patent Document 5]
[Non-Patent Document 6]
[Non-Patent Document 7]
Hupfer C., Hotzel H., Sachse K., Moreano F., Engel KH, Eur. Food Res. Technol., 212, 95-99 (2000)
[Non-Patent Document 8]
[Non-patent document 9]
Wurz A., Bluth A., Zelts P., Pfeifer C., Willmund R., Food Control, 10, 385-389 (1999)
[Non-Patent Document 10]
Berdal KG, Holst-Jensen A., Eur. Food Res. Technol., 213, 432-438 (2001)
[Non-Patent Document 11]
WO 02/34943 JP 2001-136983 A

  An object of the present invention is to provide an improved method that solves the above-described problems of the conventional method, focusing on the LAMP method or the ABC-LAMP method, and quantitatively detecting the presence of genetically modified products contained in soybeans or processed soybean foods. And a nucleic acid molecule (primer, probe, competing gene) suitable for the method, and in particular, a thermal cycler and a fluorescence detection device, such as the method for detecting a recombinant by the PCR method, are integrated. The present invention provides an accurate means for detecting a recombinant gene quickly, simply and at a low cost without requiring an expensive real-time PCR device.

  As a result of diligent research, the inventors have found a probe and a competitive gene for detecting a recombinant gene in a progeny variety of Roundup Ready Soybean strain using LAMP method or ABC-LAMP method as genetically modified soybean. The present invention has been completed by newly designing and developing a method for detecting and quantifying the above-mentioned recombinant gene using these. That is, the present invention is as follows.

(1) From FIP primer, F3 primer, LPF primer, BIP primer, B3 primer and LPB primer used for detecting and quantifying recombinant gene of transgenic soybean Roundup Ready Soybean strain by LAMP method or ABC-LAMP method The primer set described above, wherein each primer has a base sequence shown below or a base sequence complementary to the sequence.
FIP primer: GTG GTC CCA AAG ATG GAG GAG ATA TCA CAT CAA TCC ACT TGC
F3 primer: GTC ATC CCT TAC GTC AGT
LPF primer: CGT TCC AAC CAC GTC TTC AA
BIP primer: TGT CGG CAG AGG CAT CTT TTA AAA TAA ACA TAG GGA ACC CAA
B3 primer: TCA AAA TAA GAT CAT ACA TAC AGG
LPB primer: AGG AGC CAC CTT CCT TTT CC
(2) Competitive nucleic acid comprising the nucleotide sequence shown in SEQ ID NO: 9 or a complementary nucleotide sequence to be used for detecting and quantifying a recombinant gene of genetically modified soybean Roundup Ready Soybean strain by ABC-LAMP method .
(3) A nucleic acid probe used for detecting and quantifying a recombinant gene of genetically modified soybean Roundup Ready Soybean by the ABC-LAMP method, the base sequence shown in SEQ ID NO: 7 or a base sequence complementary to the sequence The above-mentioned nucleic acid probe, characterized in that the end thereof is labeled with a fluorescent dye whose fluorescence intensity changes depending on the proximity to or separation from a guanine base in the nucleic acid.
(4) A primer set consisting of FIP primer, F3 primer, LPF primer, BIP primer, B3 primer and LPB primer used for detecting and quantifying the Le1 gene sequence derived from soybean by LAMP method or ABC-LAMP method. Each primer has a base sequence shown below or a base sequence complementary to the sequence, and the above primer set.
FIP primer: AGC AAA AGA CCA AGA AAG CAC CTT CTC TTC CCG AGT GGG
F3 primer: CCA GCA ATA TCC TCT CCG A
LPF primer: GGC AGC AGA GAA CCC TAT CC
BIP primer: TTT GCC ACA CGC TAG CAA TTA CAC TTT CTT CCT TCG ATC TGT
B3 primer: TCA CTA GCG ATC GAG TAG TG
LPB primer: GTT GCA TGA GGC CAT CTA AAT GT
(5) A competitive nucleic acid comprising the nucleotide sequence shown in SEQ ID NO: 19 or a complementary nucleotide sequence to be used for detecting and quantifying the Le1 gene sequence derived from soybean by ABC-LAMP method.
(6) A nucleic acid probe used for detecting and / or quantifying the Le1 gene sequence derived from soybean by ABC-LAMP method, comprising a base sequence shown in SEQ ID NO: 18 or a base sequence complementary to the sequence, The above-mentioned nucleic acid probe, characterized in that the terminal is labeled with a fluorescent dye whose fluorescence intensity changes depending on proximity to or separation from a guanine base in the nucleic acid.
(7) A method for detecting and / or quantifying a recombinant gene sequence of a Roundup Ready Soybean strain contained in a test soybean sample using ABC-LAMP method, the nucleic acid extracted from the test soybean sample, Performing an isothermal amplification reaction in the presence of the primer set according to (1), the competitive nucleic acid according to (2) above, and the nucleic acid probe according to (3) above, and measuring the fluorescence intensity before and after the reaction. A method for detecting and / or quantifying a recombinant gene sequence of a Roundup Ready Soybean strain.
(8) A method for detecting and / or quantifying a Le1 gene sequence derived from soybean using ABC-LAMP method, the nucleic acid extracted from a test soybean sample, the primer set according to (4) above, The Le1 gene derived from soybean, wherein an isothermal amplification reaction is performed in the presence of the competitive nucleic acid according to (5) and the nucleic acid probe according to (6), and the fluorescence intensity before and after the reaction is measured. Sequence detection and / or quantification methods.
(9) Recombination of a Roundup Ready Soybean strain characterized by combining at least the primer set according to (1) above, the competitive nucleic acid according to (2) above and the nucleic acid probe according to (3) above Reagent kit for gene detection and / or quantification.
(10) A Le1 gene sequence derived from soybean, characterized by combining at least the primer set according to (4) above, the competitive nucleic acid according to (5) above and the nucleic acid probe according to (6) above. Reagent kit for detection or quantification.

  According to the present invention, a nucleic acid primer, a nucleic acid probe, and a competitive nucleic acid that are extremely suitable for detecting and / or quantifying a recombinant gene of Roundup Ready Soybean strain in soybean or soybean processed food using ABC-LAMP method are provided. By carrying out the ABC-LAMP method using these, the recombinant gene can be detected and / or quantified accurately, simply and at low cost. The nucleic acid primer is also a useful primer in the LAMP method. On the other hand, the present invention also provides a nucleic acid primer, a nucleic acid probe, and a competitive nucleic acid that are extremely suitable for simultaneously detecting and / or quantifying the Le1 gene sequence derived from soybean using the ABC-LAMP method. Of course, this nucleic acid probe is also a useful primer in the LAMP method. The quantitative value of Le1 gene obtained as a result of performing LAMP method or ABC-LAMP method using these was obtained by gradually quantifying the recombinant gene of the above Roundup Ready Soybean strain as the quantitative value of the internal standard gene. It is possible to calculate the internal standard ratio and use this internal standard ratio to determine, for example, the mixing rate of the recombinant soybean in the recombinant food.

  Soybeans from the Roundup Ready Soybean line have been introduced with a gene that confers resistance to glyphosate herbicides. Because of its resistance to this herbicide, various weeds that grow during the cultivation of the dice can be weeded with glyphosate herbicides, regardless of the type, greatly reducing the time and effort of herbicides. Can be reduced. However, some have questioned food safety. The resistance-conferring gene is a resistance gene CP4EPSPS derived from the soil bacterium Agrobacterium genus. Roundup Ready Soybean soybeans use the CaMV 35S promoter for expression of this CP4EPSPS gene. In the present invention, the boundary region sequence (35S promotor / plant junction region, AJ308514) between this promoter region and soybean genome is roundup. Ready Soybean system detection target.

35S promotor / plant junction region (AJ308514) nucleotide sequence (SEQ ID NO: 8)
GATAGTGGGA TTGTGCGTCA TCCCTTACGT CAGTGGAGAT ATCACATCAA
TCCACTTGCT TTGAAGACGT GGTTGGAACG TCTTCTTTTT CCACGTGCTC
CTCGTGGGTG GGGGTCCATC TTTGGGACCA CTGTCGGCAG AGGCATCTTC
AACGATGGCC TTTCCTTTAT CGCAATGATG GCATTTGTAG GAGCCACCTT
CCTTTTCCAT TTGGGTTCCC TATGTTTATT TTAACCTGTA TGTATGATCT
TATTTTGAAT GAAATGCAAT AAGTTATTTC TAGTAAAAAA AAATAAACAT
TTGATAGAAA CAAATTAAAG CATGCAAAAA TAACTCATTA GCATCGGTTA
AATTGAAGGG TTTGAATAAT TTGCACAAGG TTCTGAATTC

On the other hand, the LAMP (Loop-Mediated Isothermal AMPlification) method is known as a simple and rapid gene amplification method, and is isothermal (60 to 65 ° C) using primers designed based on the base sequence of the target gene. This is a method of performing an amplification reaction by incubation, and is used for detection of various genes. The detection and quantification of the gene is performed by visual observation of the turbidity of pyrophosphate, which is a side reaction of the amplification reaction, or by measuring the turbidity in real time.
As primers, four types of primers consisting of FIP primer, F3 primer, BIP primer, and B3 primer are required. In addition, in order to increase the starting point of DNA synthesis, a total of six primers are added by adding LPF primer and LPB primer. In some cases, the amplification efficiency is greatly increased according to this method. In the present invention, these six kinds of primers are used to increase the amplification efficiency.

However, in this LAMP method, in order to perform gene quantification, a dedicated device for measuring the increase in turbidity due to accumulation of pyrophosphate in real time is required. There are drawbacks such as lack of accuracy of quantification, and the applicants have improved this and developed ABC (Alternately Binding Quenching Probe Conpetitive) -LAMP method as a rapid, simple and accurate gene detection and quantification method doing.
This method is common to the LAMP method in that isothermal amplification is performed using the above-mentioned primers, but the LAMP method is further different in that a nucleic acid probe fluorescently labeled at the end and a competitive nucleic acid for the target gene coexist in the reaction system. In contrast, the target gene is quantified simply, rapidly and accurately by measuring the fluorescence intensity before and after the isothermal amplification.

  Each primer, nucleic acid probe and competitive nucleic acid in the primer set of the present invention gives the best results when detecting and quantifying recombinant genes in soybean of the Roundup Ready Soybean line using this ABC-LAMP method. Is designed to be

[Primer set]
LAMP primer was designed using Primer Explorer, a LAMP primer design support software in NetlLaboratoryl (http://venus.netlaboratory.com/) provided by Fujitsu Limited. However, it has been found that the design using this design support software has a problem in that the binding site of the probe used in the ABC-LAMP method is not taken into account, especially in the design of the LAMP primer used in the ABC-LAMP method.
The nucleic acid primer of the present invention is obtained by adding the following improvements.
In the LAMP reaction product, the site to which the probe of the present invention (AB-QProbe) can bind is considered to be a single-stranded loop characteristic of the LAMP reaction product. Therefore, in consideration of annealing of AB-QPobe at this site in ABC-LAMP, the primer RRS-FIP was designed to have an interval of about 60 bp between F2 and F1.
The nucleotide sequences of newly designed nucleic acid primers with improvements are as follows, and these are used as primer sets for the LAMP method and ABC-LAMP method.

It is as shown under the base sequence of the nucleic acid primer (5 ′ → 3 ′) for detecting and / or quantifying the recombinant gene of the transgenic soybean Roundup Ready Soybean line.
RRS-FIP primer: GTG GTC CCA AAG ATG GAG GAG ATA TCA CAT CAA TCC ACT TGC (SEQ ID NO: 1)
RRS-F3 primer: GTC ATC CCT TAC GTC AGT (SEQ ID NO: 2)
RRS-LPF primer: CGT TCC AAC CAC GTC TTC AA (SEQ ID NO: 3)
RRS-BIP primer: TGT CGG CAG AGG CAT CTT TTA AAA TAA ACA TAG GGA ACC CAA (SEQ ID NO: 4)
RRS-B3 primer: TCA AAA TAA GAT CAT ACA TAC AGG (SEQ ID NO: 5)
RRS-LPB primer: AGG AGC CAC CTT CCT TTT CC (SEQ ID NO: 6)

[Nucleic acid probe]
The base sequence of the nucleic acid probe designed in the present invention is shown below. This base sequence is designed to correspond to the base sequence starting from the 85th guanine of the 35S promotor / plant junction region (SEQ ID NO: 8), which is the target gene, and the cytosine base on the 5 ′ end side of the probe Is fluorescently labeled with a fluorescent dye such as BODIPY FL, FITC, or TMRITC that quenches in close proximity to the guanine base in the target gene, and the three ends are phosphorylated.

The base sequence of the nucleic acid probe (5 ′ → 3 ′) for detecting and / or quantifying the recombinant gene of the transgenic soybean Roundup Ready Soybean line is as follows.
ABQP-RRS: CTT TTT CCA CGT GCT CCT CGT GC (SEQ ID NO: 7)

  The probe used for the ABC-LAMP method is modified with a fluorescent dye such as BODIPY FL, and is designed to be quenched when annealed to the target gene. Therefore, in the ABC-LAMP method, when the probe binds to the target gene, the target in the target gene is such that the base in the target gene at the position corresponding to the 5 ′ end where the fluorescent dye is modified becomes G. A sequence is selected and a probe is designed based on this target sequence. In addition, the terminal base not modified with the fluorescent dye of AB-QProbe is C, which is a mismatch. In the ABC-LAMP method, gene amplification occurs due to an isothermal reaction, so the probe must bind to DNA at the LAMP reaction temperature. Therefore, the probe is designed so that its Tm value is close to the reaction temperature. The probe of the present invention also follows the probe design principle of the ABC-LAMP method, but the probe of the present invention (AB-QProbe) is further improved as follows.

1) In the present invention, when designing a probe, make sure that the 2 bases outside the 3 'end of the probe binding site in the target sequence (the 2 bases outside the fluorescent dye as viewed from the probe) are not guanine bases. The target sequence is selected.
Previous ABC-LAMP methods did not take this point into consideration, and the outer two bases contained guanine bases. If there is a guanine base at this position, the fluorescence may be quenched by the influence of the guanine base even when the probe binds to a competitive nucleic acid. Therefore, in the conventional ABC-LAMP method, this guanine base was replaced with a cytosine base when producing a competitive nucleic acid. This substitution of the guanine base with a cytosine base was one of the causes that caused a slight difference in the amplification efficiency of the target sequence and the competitive nucleic acid. However, the probe design for detection of this Roundup Ready Soybean soybean is considered not to be a guanine base on the outside, so there is no need to replace the guanine base at this position with a cytosine base, minimizing the number of base substitutions in competitive nucleic acid production. I was able to suppress it. Therefore, the amplification efficiency of the target sequence and the competitive nucleic acid could be as close as possible.

2) In designing the probe, the Tm (dissociation temperature) of the probe designed for detection of Roundup Ready Soybean soybean was considered so as to be higher than the Tm of the probe designed by the ABC-LAMP method. As a result, the binding force of the probe to the target sequence and the competitive nucleic acid became stronger, so that the double-stranded binding could be stabilized. Therefore, stable quantitative results can be obtained.

[Competitive nucleic acid]
The base sequence of a competitive nucleic acid (5 ′ → 3 ′) for detecting and / or quantifying the recombinant gene of the transgenic soybean Roundup Ready Soybean line designed in the present invention (only one of the double-stranded DNAs) And the binding sites of the above primers and probes are shown below. In addition, F1 and F2, and B1c and B2c represent the binding site in the competitive nucleic acid of a FIP primer and a BIP primer, respectively.

(SEQ ID NO: 9)

The above base sequence is a boundary region (35S promotor / plant junction region) between the promoter region (CaMV 35S) sequence and the soybean genome of the glyphosate resistance gene CP4EPSPS introduced into soybean of the Roundup Ready Soybean line (hereinafter referred to as target sequence). ) In which the 85th guanine base is replaced with cytosine and the 107th cytosine is changed to guanine.
The partial sequence of the boundary region (35S promotor / plant junction region) between the CaMV 35S promoror and soybean genome (only one of the double-stranded DNAs) is shown below, and the binding sites of each primer and probe in the sequence are shown below. . F1 and F2, and B1c and B2c represent binding sites in the sequences of the FIP primer and BIP primer, respectively.

(SEQ ID NO: 8)

  The reason why the 107th cytosine is changed to guanine in the competitive nucleic acid of the present invention is to allow the nucleic acid probe to bind to the target sequence and the competitive nucleic acid with the same binding force. That is, since the 85th base of the competitive nucleic acid is changed to cytosine, it does not bind to cytosine at the 5 ′ end of the nucleic acid probe, and the binding force between the nucleic acid probe and the competitive nucleic acid is equivalent to that of the nucleic acid probe. It becomes weaker than the binding between the nucleic acid probe that binds CG and the target sequence on the 5 ′ end side. On the other hand, the 3 ′ end of the nucleic acid probe is cytosine, and in a competitive nucleic acid, the base at the corresponding position is changed to guanine and can bind to CG. On the other hand, since the base at the corresponding position of the target sequence is cytosine, it cannot bind to the 3 ′ terminal base of the nucleic acid probe. As a result, the nucleic acid probe has the same overall binding force to the target gene and the competitive nucleic acid. It becomes possible to combine. Thereby, the fluorescence intensity measured by the ABC-LAMP method corresponds to the ratio between the target sequence and the competing nucleic acid, and the amount of the competitive nucleic acid used and the fluorescence intensity before and after the amplification reaction (relative fluorescence intensity) are measured. The initial amount contained in the sample of the target sequence (CaMV 35S promotor, the promoter region of the glyphosate resistance gene CP4EPSPS, and the soybean genome (35S promotor / plant junction region)), ie, Roundup Ready Soybean strain The amount of recombinant gene in soybean can be known.

  Furthermore, as described above, in the competitive nucleic acid design of the ABC-LAMP method so far, the base 2 outside the 3 ′ end of the probe binding site in the target sequence (the two bases outside the fluorescent dye side as seen from the probe) Since the sequence contains a guanine base, the competitive nucleic acid was designed and synthesized by replacing this part of the guanine base with a cytosine base. However, according to the present invention, a competitive nucleic acid for RRS was designed. Since a sequence that does not contain guanine bases in the 2 bases outside the 3 'end of the probe binding site was selected as the target sequence, there is no need to replace guanine bases with cytosine bases in the production of competitive nucleic acids. The number of substitutions can be minimized, so that the amplification efficiency of the target sequence and the competitive nucleic acid can be as close as possible.

  Since the amplification target by the LAMP method or ABC-LAMP method is double-stranded DNA, the nucleic acid primer, nucleic acid probe, and competitive nucleic acid can bind to any single-stranded DN of double-stranded DNA. These nucleic acid primers, nucleic acid probes, and competitive nucleic acids may be not only the sequences specifically shown above but also complementary sequences thereto.

Hereinafter, the LAMP method using the nucleic acid primer set of the present invention and the target sequence detection / quantification method by the ABC-LAMP method using the nucleic acid primer, nucleic acid probe and competitive nucleic acid will be described in detail.

LAMP method A DNA sample is extracted from soybean or soybean processed food, the DNA sample, a primer set comprising the above six nucleic acid primers of the present invention, a strand displacement DNA polymerase such as Bst DNA Polymerase, and four kinds of substrates. A reaction solution composed of nucleotides (dNTPs) and a reaction buffer is incubated at a constant temperature of, for example, 60 to 65 ° C. At this time, if CaMV 35S, which is the promoter of the recombinant gene CP4EPSPS in soybean of the Roundup Ready Soybean line corresponding to the primer set, is present in the DNA sample, an isothermal amplification reaction occurs using the target sequence in the gene as a template, The target sequence is amplified.
In this amplification reaction, pyrophosphate is by-produced as the amplification reaction proceeds, and the reaction solution becomes cloudy. By visually observing the presence or absence of this cloudiness, the presence or absence of the above-described recombinant gene CaMV 35S can be detected. It can be confirmed that the soybean raw material is mixed in soybeans of the Roundup Ready Soybean line or processed foods using the soybeans.
In addition, the degree of turbidity (turbidity) increases with the progress of the reaction, and the time until the turbidity reaches a certain value is almost proportional to the amount of target gene in the initial stage. By measuring in real time and measuring the turbidity after a certain period of time, the initial target gene amount can be determined, whereby the recombinant gene CaMV 35S in soybean or soybean processed food can be quantified.

ABC-LAMP method The DNA sample used in the LAMP method, a primer set consisting of the six nucleic acid primers of the present invention, a strand displacement DNA polymerase such as Bst DNA Polymerase, and four nucleotides (dNTPs as substrates) ) And a reaction buffer, and a constant amount of each of the nucleic acid probe of the present invention and a competitive nucleic acid is added to form an isothermal amplification reaction system. The reaction temperature is the same as in the LAMP method. In this reaction system, if the recombinant gene CaMV 35S is present in the DNA sample, the target sequence in the gene is isothermally amplified together with the competitive nucleic acid, but the amplification efficiency is the same for the target sequence and the competitive nucleic acid. Is set to
When the nucleic acid probe binds to the target sequence, the cytosine base at the 5 ′ end of the nucleic acid probe forms a base pair with the guanine base at the corresponding position in the target sequence, and binds to the 5 ′ end of the nucleic acid probe. Quenches in close proximity to guanine bases. On the other hand, when a nucleic acid probe hybridizes to a competitive nucleic acid, the cytosine base at the 5 ′ end of the nucleic acid probe is not a guanine base at the corresponding position in the target sequence but a cytosine base, so it does not form a base pair. The fluorescent dye is separated from the guanine base in the competitive nucleic acid sequence, emits fluorescence, and does not quench. Therefore, in the amplification reaction system, the fluorescence intensity decreases as the target nucleic acid is amplified.

  On the other hand, if the amount of the nucleic acid probe is equal to or less than the total amount of the target sequence to be amplified and the competitive nucleic acid, each molecule of the nucleic acid probe is hybridized to either the target sequence or the competitive nucleic acid. As described above, the amplification efficiency is the same for the target sequence and the competitive nucleic acid, and the nucleic acid probe of the present invention binds to the target sequence and the competitive nucleic acid with the same binding force. The rate at which the nucleic acid probe hybridizes to the target sequence is proportional to the ratio between the target nucleic acid and the competitive nucleic acid after amplification, and the amount of the amplified competitive nucleic acid is equal to the initial content added in the reaction system. Therefore, if the fluorescence intensity of the reaction system is measured before and after the reaction, the amount of the target sequence before amplification, that is, the soybean of the Roundup Ready Soybean line or the soybean The amount of recombinant gene CaMV 35S of processed food that use can be measured.

In the present invention, for example, a nucleic acid primer for quantifying the soybean endogenous gene Le1 necessary for calculating the contamination ratio of Roundup Ready Soybean strain in processed soybean foods using the LAMP method or ABC-LAMP method , Nucleic acid probes and competitive nucleic acids, and methods for quantifying Le1 using the same.
In order to calculate the mixing ratio of soybean in the Roundup Ready Soybean line, it is necessary to determine the internal standard ratio, but the internal standard ratio can be calculated by the following formula.

This internal standard is specific to soybeans of the Roundup Ready Soybean line tested. Therefore, the soybean mixing ratio can be calculated by the following equation.

Nucleic acid primers, nucleic acid probes, and competitive nucleic acids used in the Le1 gene detection / quantification method used for calculating the contamination rate are as follows, and these are used for detection of the above-mentioned Roundup Ready Soybean line recombinant genes. It is designed based on the same design concept as the nucleic acid primer, nucleic acid probe and competitive nucleic acid, and has the same improvements as described above.

[Primer set]
The nucleotide sequences of the nucleic acid primers used for quantitative detection of the Le1 gene are as follows, and these are used as primer sets for the LAMP method and ABC-LAMP method.
Nucleotide primer (5 '→ 3') base sequence for detecting and / or quantifying the Le1 gene sequence derived from soybean
Le1-FIP: AGC AAA AGA CCA AGA AAG CAC CTT CTC TTC CCG AGT GGG (SEQ ID NO: 12)
Le1-F3: CCA GCA ATA TCC TCT CCG A (SEQ ID NO: 13)
Le1-LPF: GGC AGC AGA GAA CCC TAT CC (SEQ ID NO: 14)
Le1-BIP: TTT GCC ACA CGC TAG CAA TTA CAC TTT CTT CCT TCG ATC TGT (SEQ ID NO: 15)
Le1-B3: TCA CTA GCG ATC GAG TAG TG (SEQ ID NO: 16)
Le1-LPB: GTT GCA TGA GGC CAT CTA AAT GT (SEQ ID NO: 17)

[Nucleic acid probe]
The base sequence of the nucleic acid probe designed in the present invention is shown below. This base sequence is designed to correspond to the base sequence starting from the 168th guanine of the Le1 gene partial sequence that is the target gene, and the cytosine base on the 5 ′ end side of the probe contains the base sequence in the target gene. It is fluorescently labeled with a fluorescent dye such as BODIPY FL, FITC, or TMRITC that quenches in the vicinity of the guanine base, and the 3 ′ end is phosphorylated.
Nucleotide probe (5 '→ 3') nucleotide sequence for detecting and / or quantifying soybean-derived Le1 gene sequence
ABQP-Le1: CAT GCG ATT CCC CAG GTA TGT CC (SEQ ID NO: 18)

[Competitive nucleic acid]
The base sequences of competitive nucleic acids designed in the present invention (only one of the double-stranded DNAs is shown) and the binding sites of the above primers and probes are shown below. In addition, F1 and F2, and B1c and B2c represent the binding site in the competitive nucleic acid of a FIP primer and a BIP primer, respectively.

Base sequence of competitive nucleic acid (5 '→ 3') for detecting and / or quantifying the Le1 gene sequence derived from soybean
(SEQ ID NO: 19)

In the above sequence, the 168th guanine base in the partial sequence of the Le1 gene derived from soybean is replaced with cytosine, and the 146th cytosine is changed to guanine.
Regarding the detection and quantification of the Le1 gene by the LAMP method using a nucleic acid primer set and the ABC-LAMP method using the nucleic acid primer, nucleic acid probe and competitive nucleic acid, the above-mentioned Roundup Ready Soybean line in soybean This is the same as the method for detecting / quantifying the recombinant gene.
Examples of the present invention are shown below, but the present invention is not limited to these examples.

Example 1
(1) Detection of recombinant gene by LAMP method Using the following primers, perform a LAMP reaction at 62 ° C for 1 hour to detect the recombinant gene of genetically modified soybean Roundup Ready Soybean and the Le1 gene sequence derived from soybean・ Quantified.
The base sequences of the primers for detection / quantification of the recombinant gene and Le1 gene of the Roundup Ready Soybean strain used are as follows.

Primer for recombinant gene of Roundup Ready Soybean strain
RRS-FIP primer: GTG GTC CCA AAG ATG GAG GAG ATA TCA CAT CAA TCC ACT TGC (SEQ ID NO: 1)
RRS-F3 primer: GTC ATC CCT TAC GTC AGT (SEQ ID NO: 2)
RRS-LPF primer: CGT TCC AAC CAC GTC TTC AA (SEQ ID NO: 3)
RRS-BIP primer: TGT CGG CAG AGG CAT CTT TTA AAA TAA ACA TAG GGA ACC CAA (SEQ ID NO: 4)
RRS-B3 primer: TCA AAA TAA GAT CAT ACA TAC AGG (SEQ ID NO: 5)
RRS-LPB primer: AGG AGC CAC CTT CCT TTT CC (SEQ ID NO: 6)

Primer for Le1
Le1-FIP: AGC AAA AGA CCA AGA AAG CAC CTT CTC TTC CCG AGT GGG (SEQ ID NO: 12)
Le1-F3: CCA GCA ATA TCC TCT CCG A (SEQ ID NO: 13)
Le1-LPF: GGC AGC AGA GAA CCC TAT CC (SEQ ID NO: 14)
Le1-BIP: TTT GCC ACA CGC TAG CAA TTA CAC TTT CTT CCT TCG ATC TGT (SEQ ID NO: 15)
Le1-B3: TCA CTA GCG ATC GAG TAG TG (SEQ ID NO: 16)
Le1-LPB: GTT GCA TGA GGC CAT CTA AAT GT (SEQ ID NO: 17)

The composition of the LAMP reaction solution was as follows: H ₂ O 11.5 μl, 10 × LAMP Buffer 2.5 μl, dNTP mix (2.5 mM) 4.0 μl, RRS-FIP ( 40 μM) 1.0 μl, RRS-BIP (40 μM) 1.0 μl, RRS-F3 (5 μM) 1.0 μl, RRS-B3 (5 μM) 1.0 μl, RRS-LPF (20 μM) 1.0 μl, RRS-LPB (20 μM) 1.0 μl, Template DNA was 1.0 μl. For quantification of the Le1 gene, H ₂ O 11.5 μl, 10 × LAMP Buffer 2.5 μl, dNTP mix (2.5 mM) 4.0 μl, Le1-FIP (40 μM) 1.0 μl, Le1-BIP (40 μM) 1.0 μl, Le1 -F3 (5 μM) 1.0 μl, Le1-B3 (5 μM) 1.0 μl, Le1-LPF (20 μM) 1.0 μl, Le1-LPB (20 μM) 1.0 μl, Template DNA 1.0 μl. As template DNA, 1 × 10 ⁶ copies of PCR product containing the target gene and 50 ng of genomic DNA extracted from soybean powder were used. After completion of the LAMP reaction, restriction enzyme treatment was performed at 37 ° C. for 4 hours. The restriction enzymes used are HindIII and HaeIII. Agarose gel electrophoresis was performed using 5 μl of the LAMP product and the restriction enzyme-treated solution. The result of amplifying the recombinant gene of Roundup Ready Soybean strain is shown in FIG. 1, and the result of amplifying Le1 gene is shown in FIG. Specific amplification with the designed primers was confirmed for both the recombinant gene of the Roundup Ready Soybean strain and the LAMP product of the Le 1 gene.

(2) Quantification of GM soybean by real-time turbidity measurement method The recombinant gene of Roundup Ready Soybean strain and the Le1 gene derived from soybean were quantified by real-time turbidity measurement method using the designed primer set. . The composition of the LAMP reaction solution was as follows: H ₂ O 11.5 μl, 10 × LAMP Buffer 2.5 μl, dNTP mix (2.5 mM) 4.0 μl, RRS-FIP ( 40 μM) 1.0 μl, RRS-BIP (40 μM) 1.0 μl, RRS-F3 (5 μM) 1.0 μl, RRS-B3 (5 μM) 1.0 μl, RRS-LPF (20 μM) 1.0 μl, RRS-LPB (20 μM) 1.0 μl, Template DNA was 1.0 μl. For quantification of the Le1 gene, H ₂ O 11.5 μl, 10 × LAMP Buffer 2.5 μl, dNTP mix (2.5 mM) 4.0 μl, Le1-FIP (40 μM) 1.0 μl, Le1-BIP (40 μM) 1.0 μl, Le1 -F3 (5 μM) 1.0 μl, Le1-B3 (5 μM) 1.0 μl, Le1-LPF (20 μM) 1.0 μl, Le1-LPB (20 μM) 1.0 μl, Template DNA 1.0 μl. PCR products of each target gene were prepared at 10 ³ to 10 ⁷ copies / μl and used as Template DNA. The time course of turbidity in the quantification of recombinant genes in the transgenic soybean Roundup Ready Soybean line is shown in FIG. FIG. 4 shows the relationship between the time required for the turbidity to reach a certain value (Threshold time: Tt) and the number of target genes in the initial reaction. The time course of turbidity in the Le1 gene quantification is shown in FIG. FIG. 6 shows the relationship between the time required for the turbidity to reach a certain value (Threshold time: Tt) and the number of target genes in the initial reaction. The quantification of Le1 gene was possible at a concentration of 1 × 10 ³ copies or more.

The template DNA in the above quantification of Le1 was 1.0 μl, H ₂ O 12.2 μl, 10 × PCR Buffer 2.0 μl, dNTP mix (2.5 mM) 2.0 using soybean extracted genome (0.4 ng / ul) as a template. A reaction product obtained by PCR with a reaction solution composition of μl, Le1-F (10 μM) 0.8 μl, Le1-R (10 μM) 0.8 μl, and Taq DNA polymerase 0.2 μl was used. PCR reaction conditions were 94 ° C. for 2 minutes, 94 ° C. for 30 seconds, 56 ° C. for 30 seconds, and 72 ° C. for 30 seconds for 40 cycles at 72 ° C. for 2 minutes. Template DNA for RRS quantification is the same as Le1, except that RRS-F (10 μM) or RRS-R (10 μM) is used as a primer. It was.

Example 2
Quantification of GM soybean by Alternately Binding Probe Competitive (ABC) -LAMP method (1) Fluorescence quenching test In order to evaluate the fluorescence quenching accuracy of the nucleic acid probe designed by the present invention and competitive nucleic acid, in target nucleic acid and competitive nucleic acid Two short oligonucleotides each having a nucleic acid probe binding site were synthesized, and the Roundup Ready Soy system and Le1 gene detection / quantification system evaluation in the present invention were performed using the nucleic acid probe of the present invention and each of the two oligonucleotides. As a preliminary experiment, a fluorescence quenching test was performed.

1) Probes and oligonucleotides used in Roundup Ready Soy fluorescence quenching test a) Probe for detection and quantification of recombinant genes in Roundup Ready Soybean strain
ABQP-RRS: CTT TTT CCA CGT GCT CCT CGT GC (SEQ ID NO: 7)
(5 'terminal BODIPY FL labeling, 3' terminal phosphorylation)
b) Oligonucleotide having the probe binding site of a) above
RRS-TL (target oligonucleotide):
CCC ACC CAC GAG GAG CAC GTG GAA AAA GAA GAC (SEQ ID NO: 10),
RRS-CL (competitive oligonucleotide tide):
CCC ACG CAC GAG GAG CAC GTG GAA AAA CAA GAC (SEQ ID NO: 11)

2) Probes and oligonucleotides used in the fluorescence quenching test for the Le1 gene system a) Probes for detecting and quantifying the Le1 gene
ABQP-Le1: CAT GCG ATT CCC CAG GTA TGT CC (SEQ ID NO: 18)
(5 'terminal BODIPY FL labeling, 3' terminal phosphorylation)
b) Oligonucleotide having probe binding site of c) above
Le1-TL (target oligonucleotide):
GGA CTC GAC ATA CCT GGG GAA TCG CAT GAC GTG (SEQ ID NO: 20)
Le1-CL (competitive oligonucleotide):
GGA CTG GAC ATA CCT GGG GAA TCG CAT CAC GTG (SEQ ID NO: 21)

The reaction solution composition of the fluorescence quenching test of Roundup Ready Soybean system is H ₂ O 1.5 μl, 10 × LAMP Buffer 2.5 μl, dNTP mix (2.5 mM) 3.0 μl, 1% TritonX-100 2.5 μl, Betaine (5 M) 5.0 μl ABQP-RRS (7.5 μM) 0.5 μl and synthetic oligonucleotide (mixture of RRS-TL and -CL) 10 μl. The reaction composition of the fluorescence quenching test for the Le1 gene system is 1.5 μl of H ₂ O, 2.5 μl of 10 × LAMP Buffer, 3.0 μl of dNTP mix (2.5 mM), 2.5 μl of 1% TritonX-100, Betaine (5 M) 5.0 μl, ABQP-Le1 (7.5 μM) 0.5 μl, and synthetic oligonucleotide (a mixture of Le1-TL and Le1-CL) 10 μl. The result of the fluorescence quenching test of the Roundup Ready Soybean system is shown in FIG. 7, and the result of the fluorescence quenching test of the Le1 gene system is shown in FIG. It was found that both fluorescence quenching tests showed relative fluorescence intensities depending on the ratio of target nucleic acid and competing nucleic acid.

(2) Using the nucleic acid primer, nucleic acid probe, and competitive nucleic acid designed in the present invention, a calibration curve for target gene quantification by the ABC-LAMP method was prepared.
The nucleic acid primer used in Example 1 was used, and the nucleic acid probe used in (1) above was used. The competitive nucleic acid used was the competitive nucleic acid represented by SEQ ID NO: 9 in the quantification of the recombinant gene of the Roundup Ready Soybean strain, and the competitive nucleic acid represented by SEQ ID NO: 19 in the quantification of the Le1 gene.
The composition of the reaction solution was as follows: H ₂ O 6.5 μl, 10 × LAMP Buffer 2.5 μl, dNTP mix (2.5 mM) 4.0 μl, RRS-FIP (40 μM) ) 1.0 μl, RRS-BIP (40 μM) 1.0 μl, RRS-F3 (5 μM) 1.0 μl, RRS-B3 (5 μM) 1.0 μl, RRS-LPF (20 μM) 1.0 μl, RRS-LPB (20 μM) 1.0 μl, 1 % TritonX-100 2.5 μl, ABQP-RRS (7.5 μM) 0.5 μl, further adjust the number of competing genes to 10 ⁴ copies / μl, target gene number to 10 ³ -10 ⁷ copies / μl, and add 1.0 μl each Added.

For quantification of Le1 gene, H ₂ O 6.5 μl, 10 × LAMP Buffer 2.5 μl, dNTP mix (2.5 mM) 4.0 μl, Le1-FIP (40 μM) 1.0 μl, Le1-BIP (40 μM) 1.0 μl, Le1 -F3 (40μM) 1.0μl, Le1-B3 (40μM) 1.0μl, Le1-LPF (40μM) 1.0μl, Le1-LPB (40μM) 1.0μl, 1% TritonX-100 2.5μl, ABQP-Le1 (7.5μM) To 0.5 μl, the number of competing genes was further adjusted to 10 ⁴ copies / μl and the target gene number was adjusted to 10 ³ −10 ⁷ copies / μl.
The reaction conditions for both genes were 1 hour at 62 ° C.
FIG. 9 shows the relationship between the relative fluorescence intensity Bs and the number of initial target genes in the quantification of the recombinant gene of the Roundup Ready Soybean strain. Similarly, FIG. 10 shows the relationship between the relative fluorescence intensity Bs and the number of initial target genes in the quantification of the Le1 gene.
It was found that the results of both genes showed relative fluorescence intensities depending on the ratio of target nucleic acid and competing nucleic acid.

It is an electrophoresis photograph showing the result of amplification of the recombinant gene by LAMP using the primer for amplification of the recombinant gene in Roundup Ready Soybean strain soybean of the present invention. It is an electrophoresis photograph showing the result of amplification of the recombinant gene by LAMP using the soybean-derived Le1 gene amplification primer of the present invention. It is a graph which shows the time-dependent change of the turbidity observed as a result of performing real-time LAMP method using the primer for recombinant gene amplification in the Roundup Ready Soybean line soybean of this invention. It is a graph which shows the relationship between Tt and the number of target genes of the reaction early stage about the recombinant gene in Roundup Ready soybean obtained by the said turbidity method. It is a graph which shows the time-dependent change of the turbidity observed as a result of performing real-time LAMP method using the primer for Le1 gene amplification of this invention. It is a graph which shows the relationship between Tt about the Le1 gene obtained by the said turbidity method, and the number of target genes at the initial stage of the reaction. It is a graph which shows the result of having performed the fluorescence-quenching test using the probe of this invention used for fixed_quantity | quantitative_assay of the recombinant gene of a Roundup Ready Soybean system | strain, and a partial arrangement | sequence of a competitive nucleic acid. It is a graph which shows the result of having performed the fluorescence-quenching test using the probe of this invention used for quantification of Le1 gene, and a partial arrangement | sequence of a competitive nucleic acid. Relative fluorescence intensity Bs and Roundup Ready obtained as a result of measuring the fluorescence intensity before and after the LAMP reaction using ABC-LAMP method using the recombinant gene of Roundup Ready Soybean strain and the primer, probe and competitive nucleic acid of the present invention. It is a graph which shows the relationship with the initial number of target genes of the recombinant gene of Soybean system | strain. Relative fluorescence intensity Bs and the number of initial target genes of Le1 gene obtained as a result of ABC-LAMP method using the Le1 gene, the primer, probe and competitive nucleic acid of the present invention and measuring the fluorescence intensity before and after the LAMP reaction It is a graph which shows the relationship.

Claims (2)

A reagent kit for detecting and / or quantifying a recombinant gene of genetically modified soybean Roundup Ready Soybean strain by ABC-LAMP method,
A primer set comprising a FIP primer, an F3 primer, an LPF primer, a BIP primer, a B3 primer, and an LPB primer having the base sequence shown below or a base sequence complementary to the sequence ;
FIP primer: GTG GTC CCA AAG ATG GAG GAG ATA TCA CAT CAA TCC ACT TGC
F3 primer: GTC ATC CCT TAC GTC AGT
LPF primer: CGT TCC AAC CAC GTC TTC AA
BIP primer: TGT CGG CAG AGG CAT CTT TTA AAA TAA ACA TAG GGA ACC CAA
B3 primer: TCA AAA TAA GAT CAT ACA TAC AGG
LPB primer: AGG AGC CAC CTT CCT TTT CC
A competitive nucleic acid comprising the base sequence shown in SEQ ID NO: 9 or a base sequence complementary to the sequence; and
It consists of the base sequence shown in SEQ ID NO: 7 or a base sequence complementary to the sequence, and is labeled with a fluorescent dye whose fluorescence intensity changes depending on proximity to or separation from a guanine base in the nucleic acid. A nucleic acid probe;
A reagent kit characterized by combining at least. A method for detecting and / or quantifying a recombinant gene sequence of a genetically modified soybean Roundup Ready Soybean strain contained in a test soybean sample using an ABC-LAMP method,
And wherein the nucleic acid extracted from a test soybean sample, the primer set according to claim 1, the presence of a competitive nucleic acid及beauty nucleic acid probes, perform isothermal amplification reaction, and measuring the fluorescence intensity before and after the reaction A method for detecting and / or quantifying a recombinant gene sequence of a Roundup Ready Soybean strain.