Method for detecting genetically modified organisms

Abstract

To provide a method for detecting genetically modified organisms.SOLUTION: Provided is a method for detecting genetically modified organisms, the method comprising: amplifying at least a portion of the nucleic acid sequence contained in a sample that may contain at least one genetically modified organism by PCR using a primer that specifically amplifies a recombinant gene derived from the genetically modified organism and a primer that specifically amplifies the endogenous genes common to the species corresponding to the genetically modified organism and is configured such that the amplification length of the amplification product of the endogenous gene is 123% or more of the amplification length of the amplification product of the recombinant gene; and determining whether the abundance ratio of genetically modified organisms in the sample is lower than a reference value based on the PCR results.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to gene technology and relates to a method for detecting a recombinant.

Background Art

[0002] For the quantification of recombinants contained in samples such as foods, polymerase chain reaction (PCR) is widely used (see, for example, Patent Documents 1 to 7 and Non-Patent Documents 1 to 4). On the other hand, as the sample is processed, it is known that deoxyribonucleic acid (DNA) contained in the sample is decomposed and fragmented by heat, pH change, physical force, and the like. It has also been confirmed that when DNA is fragmented, an error occurs in the quantification value of the recombinant by PCR.

[0003] For example, when a soybean raw material containing 5% of a recombinant is processed into tofu, soy milk, and boiled beans, depending on the processing method and the amount of processing, the abundance ratio of the recombinant in the processed product quantified by PCR may be higher or lower than the actual 5% (see, for example, Non-Patent Document 5 and Patent Document 8). Therefore, even if the abundance ratio of the recombinant in the processed product quantified by PCR is higher than the reference value, the raw material of the processed product may not actually contain a recombinant of the reference value or more. Also, even if the abundance ratio of the recombinant in the processed product quantified by PCR is lower than the reference value, the raw material of the processed product may actually contain a recombinant of the reference value or more.

Prior Art Documents

Patent Documents

[0004]

Patent Document 1

Patent Document 2

Patent Document 3

Patent Document 4

[0005] [Non-Patent Document 1] Takeshi Ogasawara et al., "Fragmentation of DNAs of Processed Foods Made from Genetically Modified Soybeans," Jpn, J. Food Chem., 2003, 10, 155-160 [Non-Patent Document 2] Food Labeling Standards Q&A Appendix "Matters Concerning Genetically Modified Foods," Consumer Affairs Agency, Revised March 30, 2015 [Non-Patent Document 3] Food Labeling Standards Appendix, "Testing Methods for Genetically Modified Foods that Have Underwent Safety Review," Consumer Affairs Agency, revised March 28, 2019. [Non-Patent Document 4] Abstracts of Presentations at the 113th Annual Meeting of the Japanese Society for Food Hygiene, p. 73 [Non-Patent Document 5] Tomoaki Yoshimura et al., "Comparative Studies of the Quantification of Genetically Modified Organisms in Foods Processed from Maize and Soy Using Trial Producing," J. Agric. Chem., 2005, 53, 2060-2069 [Non-Patent Document 6] Did you know? The Genetically Modified Organism Labeling System, Consumer Affairs Agency [Non-Patent Document 7] Food Labeling Standards Appendix "Testing Methods for Genetically Modified Foods that Have Underwent Safety Review" (Consumer Affairs Agency), revised September 15, 2021. [Non-Patent Document 8] Genetically modified foods and testing status, Kanagawa Prefectural Institute of Public Health News No. 200, September 2020 issue. [Non-Patent Document 9] First Study Meeting on the Genetically Modified Organism Labeling System, April 26, 2017, Document 4, Summary of the Survey on the Actual Status of Separate Production and Distribution Management Related to the Genetically Modified Organism Labeling System, Consumer Affairs Agency [Overview of the project] [Problems that the invention aims to solve]

[0006] In Japan, there is a system for labeling genetically modified foods (see, for example, Non-Patent Documents 6 and 7). The system for labeling genetically modified foods is stipulated in the Food Labeling Standards. The Food Labeling Standards have been revised, and the revised Food Labeling Standards will come into effect on April 1, 2023. Under the Food Labeling Standards before the revision, soybeans and corn that have undergone segregated production and distribution management to keep unintentional contamination to 5% or less, as well as processed foods made from them, could be labeled as "segregated non-genetically modified" or "non-genetically modified."

[0007] Under the revised food labeling standards, soybeans and corn that have undergone segregated production and distribution management and are deemed free from genetically modified organisms, as well as processed foods made from them, can be labeled as "non-genetically modified" or "non-genetically modified." Soybeans and corn that have undergone segregated production and distribution management and have kept unintentional contamination to 5% or less, as well as processed foods made from them, can be labeled as having undergone appropriate segregated production and distribution management.

[0008] In most cases, the contamination of grains produced under segregated production and distribution management is well below the standard value (Non-Patent Documents 8, 9). Even if the relative abundance of genetically modified organisms in a sample cannot be accurately quantified, there is a need for a method to prove that the amount of genetically modified organisms in a sample is below the standard value. Therefore, one of the objectives of the present invention is to provide a method for detecting genetically modified organisms that proves that the amount of genetically modified organisms in a sample is below the standard value. [Means for solving the problem]

[0009] According to an aspect of the present invention, a method for detecting genetically modified organisms is provided, comprising: amplifying at least a portion of the nucleic acid sequence contained in a sample that may contain at least one genetically modified organism by PCR using a primer that specifically amplifies a recombinant gene derived from the genetically modified organism and a primer that specifically amplifies an endogenous gene commonly possessed by the organism species corresponding to the genetically modified organism, and configured such that the amplification length of the amplification product of the endogenous gene is 123% or more of the amplification length of the amplification product of the recombinant gene; and determining, based on the PCR results, whether the relative abundance of genetically modified organisms in the sample is lower than a reference value.

[0010] In the above method, if the ratio of genetically modified organisms is lower than the standard value, the sample may be determined to be free of genetically modified organisms.

[0011] In the above method, at least a portion of the nucleic acid sequence in the sample may be degraded.

[0012] In the above method, the sample may be processed.

[0013] The above method may further include determining the relative abundance of genetically modified organisms in the sample based on the results of PCR.

[0014] In the above method, at least a part of the nucleic acid sequence in the sample is decomposed, and the above method may further include determining that the abundance ratio of the recombinant in the sample before the nucleic acid sequence is decomposed is lower than the determined abundance ratio of the recombinant.

[0015] In the above method, the sample is processed, and the above method may further include determining that the abundance ratio of the recombinant in the sample before processing is lower than the determined abundance ratio of the recombinant in the processed sample.

[0016] In the above method, the abundance ratio of the recombinant in the sample may be determined based on the amount of the recombinant gene present in the sample determined based on the result of PCR and the amount of the endogenous gene present in the sample determined based on the result of PCR.

[0017] In the above method, the abundance ratio of the recombinant may be determined based on the following formula (1). C=(N G / N E )×(1 / R)×100 (1) In formula (1), C represents the abundance ratio (%) of the recombinant, N G represents the amount of the recombinant gene in the sample, N E represents the amount of the endogenous gene in the sample, and R represents the internal standard ratio.

[0018] In the above method, the internal standard ratio may be given by the following formula (2). R=N G100 / N E100 (2) In formula (2), N G100 represents the amount of the recombinant gene in 100% of the recombinant, and N E100 represents the amount of the endogenous gene in 100% of the recombinant.

[0019] In the above method, the internal standard ratio may be given by the following formula (3). R=N Gx / N Ex ×100 / x (3) In equation (3), N Gx This is the amount of recombinant gene in the certified reference material of x% of the genetically modified organism, N Ex This represents the amount of endogenous genes in a certified reference material containing x% of genetically modified organisms.

[0020] In the method described above, the ratio of genetically modified organisms in the sample may be determined to be lower than the standard value based on the number of cycles at which the amplification product of the recombinant gene reaches the threshold and the number of cycles at which the amplification product of the endogenous gene reaches the threshold.

[0021] In the method described above, the relative abundance of genetically modified organisms in the sample may be determined to be lower than the standard value based on the difference between the number of cycles at which the amplification product of the recombinant gene reaches the threshold and the number of cycles at which the amplification product of the endogenous gene reaches the threshold.

[0022] In the above method, the amplification length of the amplification product of the endogenous gene may be 125% or more of the amplification length of the amplification product of the recombinant gene.

[0023] In the above method, the amplification length of the recombinant gene amplification product may be between 40 bp and 1000 bp.

[0024] In the above method, PCR may be quantitative PCR.

[0025] In the above method, PCR may be real-time PCR.

[0026] In the above method, PCR may be multiplex PCR.

[0027] In the method described above, the species of organism may be a plant.

[0028] Furthermore, according to an aspect of the present invention, a kit for detecting genetically modified organisms is provided, comprising: a PCR primer that specifically amplifies recombinant genes derived from genetically modified organisms; and a PCR primer configured to specifically amplify endogenous genes commonly possessed by the organisms corresponding to the genetically modified organisms, such that the amplification length of the amplification product of the endogenous genes is 123% or more of the amplification length of the amplification product of the recombinant genes.

[0029] The above kit may also be a kit for detecting genetically modified organisms in a sample.

[0030] In the above kit, at least a portion of the nucleic acid sequence in the sample may be degraded.

[0031] In the above kit, the sample may be processed.

[0032] In the above kit, the amplification length of the endogenous gene amplification product may be 125% or more of the amplification length of the recombinant gene amplification product.

[0033] In the above kit, the amplification length of the recombinant gene amplification product may be between 40 bp and 1000 bp.

[0034] In the above kit, PCR may be quantitative PCR.

[0035] In the above kit, PCR may be performed as real-time PCR.

[0036] In the above kit, PCR may be multiplex PCR.

[0037] In the above kit, the species of organism may be a plant. [Effects of the Invention]

[0038] According to the present invention, it is possible to provide a method for detecting genetically modified organisms that proves that the number of genetically modified organisms in a sample is below a certain threshold. [Brief explanation of the drawing]

[0039] [Figure 1] This figure schematically shows the relationship between the number of nucleic acid sequences in the sample according to the embodiment and the copy number of the amplified product. [Figure 2] This figure schematically shows the relationship between ΔCq and the relative abundance of genetically modified organisms according to the embodiment. [Figure 3] This figure schematically shows the relationship between ΔCq and the relative abundance of genetically modified organisms according to the embodiment. [Figure 4] This diagram schematically shows the relationship between the ΔCq of the unprocessed product and the ΔCq of the processed product according to the embodiment. [Figure 5] This diagram schematically shows the relationship between the ΔCq of the unprocessed product and the ΔCq of the processed product according to the embodiment. [Figure 6] This diagram schematically shows the relationship between the ΔCq of the unprocessed product and the ΔCq of the processed product according to the embodiment. [Modes for carrying out the invention]

[0040] Embodiments of the present invention are described below. However, these embodiments should not be understood as limiting the present invention. Various alternative embodiments, examples, and operational techniques should become apparent to those skilled in the art from this disclosure. It should be understood that the present invention encompasses various embodiments and the like that are not described herein.

[0041] The genetically modified organism detection method according to the embodiment includes amplifying at least a portion of the nucleic acid sequence contained in a sample that may contain at least one genetically modified organism by polymerase chain reaction (PCR) using a primer that specifically amplifies recombinant genes derived from the genetically modified organism and a primer that specifically amplifies endogenous genes commonly possessed by the organisms corresponding to the genetically modified organism, and is configured such that the amplification length of the amplification product of the endogenous gene is 123% or more of the amplification length of the amplification product of the recombinant gene; and proving, based on the PCR results, that the relative abundance of genetically modified organisms in the sample is lower than a reference value.

[0042] The sample may include, for example, plant materials. Examples of plants include soybeans, wheat, barley, and corn. The sample may include materials consisting of genetically modified organisms and materials consisting of non-genetically modified organisms of the same species as the genetically modified organisms. Endogenous genes (endogenous DNA sequences) are genes (DNA sequences) that are universally present in both genetically modified and non-genetically modified organisms, provided they belong to the same species. Genetically modified organisms and non-genetically modified organisms of the same species both possess the same endogenous genes. For example, genetically modified soybeans and non-genetically modified soybeans both possess the same endogenous genes. Non-genetically modified organisms do not possess the recombinant genes (recombinant DNA sequences) that genetically modified organisms possess.

[0043] The sample may be processed, and at least a portion of the nucleic acid sequence, such as DNA, contained in the sample may be fragmented by degradation. Degradation includes degradation by heating, enzymatic degradation, acid degradation, alkali degradation, physical degradation, and degradation by fermentation by microorganisms such as bacteria. The nucleic acid sequence contained in the sample is extracted from the sample before PCR is performed.

[0044] The primers are PCR primers. The PCR may be quantitative PCR such as real-time PCR, or multiplex PCR. The type of PCR is not limited as long as it is possible to determine from the PCR results whether the relative abundance of recombinants in the sample is lower than a reference value. In this disclosure, amplification length refers to the sequence length of the amplification product by PCR. Examples of design sites for primers that specifically amplify recombinant genes include transgenes not present in the non-recombinant genome, promoters and terminators flanking the transgene, boundary regions of these sequences, and boundary regions between the non-recombinant genome and these sequences. The promoter is the region necessary for the expression of the transgene, and the terminator is the region necessary for the termination of the expression of the transgene.

[0045] The amplification length of the endogenous gene amplification product is 123% or greater, 125% or greater, 127% or greater, 129% or greater, 131% or greater, 133% or greater, or 134% or greater compared to the amplification length of the recombinant gene amplification product. Furthermore, the amplification length of the endogenous gene amplification product is 2500% or less, 1000% or less, or 500% or less compared to the amplification length of the recombinant gene amplification product.

[0046] The amplification length of the recombinant gene amplification product is, for example, 40 bp or more, 50 bp or more, 60 bp or more, 70 bp or more, 80 bp or more, 90 bp or more, or 100 bp or more. Alternatively, the amplification length of the recombinant gene amplification product is, for example, 1000 bp or less, 400 bp or less, 200 bp or less, 190 bp or less, 180 bp or less, 170 bp or less, 160 bp or less, or 150 bp or less.

[0047] If the PCR is a quantitative PCR such as real-time PCR, the ratio of genetically modified organisms in the sample is determined based on the amount of recombinant genes present in the sample, which is determined based on the PCR results, and the amount of endogenous genes present in the sample, which is also determined based on the PCR results.

[0048] For example, the relative abundance of genetically modified organisms in a sample is calculated based on the following equation (4). C=(N G / N E ) × (1 / R) × 100 (4) In equation (4), C is the percentage of genetically modified organisms present in the sample, and N is the percentage of genetically modified organisms present. G This is the amount of recombinant gene in the sample determined by quantitative PCR, N E is the amount of endogenous genes in the sample determined by quantitative PCR, and R represents the internal standard ratio.

[0049] The internal standard ratio is the ratio of the amount of recombinant genes in a 100% pure genetically modified organism to the amount of endogenous genes in a 100% pure genetically modified organism, and is given by equation (5) below. R=N G100 / N E100 (5) In equation (5), R is the internal standard ratio, NG100 This is the amount of recombinant genes in 100% of the genetically modified organism, N E100 This represents the amount of endogenous genes in 100% of the genetically modified organism. Furthermore, the internal standard ratio can also be determined by using certified reference materials, etc., whose relative abundance of genetically modified organisms is known. In this case, the internal standard ratio R is given by equation (6) below. R=N Gx / N Ex ×100 / x(6) In equation (6), R is the internal standard ratio, N Gx This is the amount of recombinant gene in the certified reference material of x% of the genetically modified organism, N Ex This represents the amount of endogenous genes in a certified reference material containing x% of genetically modified organisms.

[0050] The internal standard ratio is unique to each recombinant strain and is considered to exhibit a constant value. Therefore, a previously obtained or publicly available value may be used as the internal standard ratio. However, since the internal standard ratio may vary depending on the type of quantitative PCR instrument used, it is preferable to use the internal standard ratio value determined by the same quantitative PCR instrument used to analyze the sample.

[0051] Here, as shown in Figure 1(a), if the sample is unprocessed and the nucleic acid sequence in the sample is not degraded, the copy number of the template nucleic acid sequence theoretically corresponds to the number of molecules of the template nucleic acid sequence in the sample, regardless of the amplification length of the amplification product. In contrast, as shown in Figure 1(b), if the sample is processed and the nucleic acid sequence in the sample is degraded, the copy number of the template nucleic acid sequence tends to decrease. Furthermore, the longer the amplification length of the amplification product, the greater the tendency for the copy number of the template nucleic acid sequence to decrease.

[0052] In this embodiment, the amplification length of the endogenous gene amplification product is set to be 123% or longer than the amplification length of the recombinant gene amplification product. Therefore, the degree of decrease in the copy number of the endogenous gene template nucleic acid sequence due to sample processing and degradation of the nucleic acid sequence in the sample is greater than the degree of decrease in the copy number of the recombinant gene template nucleic acid sequence due to sample processing and degradation of the nucleic acid sequence in the sample.

[0053] The amount of genes in a sample is determined based on the copy number of the template nucleic acid sequence by PCR. Therefore, the degree of decrease in the amount of endogenous genes in the processed and degraded sample, as determined by quantitative PCR, relative to the actual amount of endogenous genes in the sample before processing, is greater than the degree of decrease in the amount of recombinant genes in the processed and degraded sample, as determined by quantitative PCR, relative to the actual amount of recombinant genes in the sample before processing.

[0054] Therefore, N in equation (4) above G Based on the degree of decrease, N E Since the degree of reduction is greater, the relative abundance of genetically modified organisms in the sample determined by the method according to this embodiment will be higher than the actual relative abundance of genetically modified organisms in the sample before processing. Therefore, if the relative abundance of genetically modified organisms in the sample determined by the method according to this embodiment is lower than the reference value, it is possible to conclude that the relative abundance of genetically modified organisms in the sample before processing is also always lower than the reference value, regardless of the degree of degradation of the nucleic acid sequence contained in the sample. Thus, if the relative abundance of genetically modified organisms in the sample determined by the method according to this embodiment is lower than the reference value, it is possible to conclude that genetically modified organisms were present in the sample before processing at a ratio always lower than the reference value.

[0055] Furthermore, the standard values ​​may be set arbitrarily by government agencies, local government agencies, food manufacturers, distributors, and consumer groups. In Japan, the permissible limit for unintentional contamination of genetically modified foods with soybeans and corn, even when segregated production and distribution management is implemented, is 5%, but the standard values ​​are not limited to this.

[0056] As mentioned above, PCR may also be multiplex PCR. In multiplex PCR, by adding multiple sets of primer pairs and probes to the PCR solution, it is possible to simultaneously detect endogenous genes and recombinant genes. In other words, using multiplex PCR, the number of cycles (Cq) at which the amplification product of recombinant genes in the sample reaches the threshold can be determined. GA ) and the number of cycles (Cq) at which the amplification product of the endogenous gene in the sample reaches the threshold. EA Based on this, it is possible to determine whether the ratio of genetically modified organisms in the sample is lower than the standard value. Cq GA and Cq EA This is detected, for example, as the number of cycles at which the fluorescence intensity of a probe, which reflects the amount of amplification product, reaches a threshold.

[0057] Cq is given by equation (7) below. GA and Cq EA The difference ΔCq A It is known that this has a negative correlation with the logarithm of the relative abundance of genetically modified organisms in the sample. ΔCq A =Cq GA -Cq EA (7) Therefore, as illustrated in Figure 2, the higher the proportion of genetically modified organisms in the sample, the greater the ΔCq. A The value decreases, and the lower the proportion of genetically modified organisms in the sample, the smaller ΔCq becomes. A It will get bigger.

[0058] Here, as shown in Figure 3 and equation (8) below, the ΔCq of the sample to be inspected A However, the ΔCq of a standard sample containing genetically modified organisms at the same abundance ratio as the reference value S If the value is greater than this, it is possible to determine that the proportion of genetically modified organisms in the sample being tested is below the standard value. ΔΔCq = ΔCq A -ΔCq S ≥0 (8) Furthermore, as shown in equation (9) below, the ΔCq of the sample to be tested AHowever, the ΔCq of a standard sample containing genetically modified organisms at the same abundance ratio as the reference value S If the value is smaller than the standard value, it can be determined that the proportion of genetically modified organisms in the sample being tested is higher than the standard value. ΔΔCq = ΔCq A -ΔCq S <0 (9)

[0059] According to our findings, when the nucleic acid sequence contained in the sample is degraded by processing the sample, as illustrated in Figure 4, Cq GA and Cq EA It tends to be larger. Also, Cq is better when the amplification length is longer. GA and Cq EA The degree to which it increases is large, and the amplification length is short, which is better for Cq GA and Cq EA The degree to which it increases becomes smaller. In this embodiment, the amplification length of the amplification product of the endogenous gene is set to be 123% or more longer than the amplification length of the amplification product of the recombinant gene. Compared to the case where the amplification length of the amplification product of the endogenous gene is shorter than the amplification length of the amplification product of the recombinant gene, as illustrated in Figure 5, when the amplification length of the amplification product of the endogenous gene is longer than the amplification length of the amplification product of the recombinant gene, as illustrated in Figure 6, the Cq of the recombinant gene with a short amplification length GA The degree to which it increases is small, and the amplification length is long for endogenous genes Cq EA The degree to which it becomes larger increases. Therefore, the ΔCq of the processed product A This is the ΔCq of the unprocessed product. A It will become smaller.

[0060] For the unprocessed product, the Cq of the decomposed sample GA The degree to which it increases, and the Cq of the decomposed sample EA If the degree to which is increased is equal to the degree to which is equal, then ΔCq A It does not change. In contrast, in the method according to this embodiment, the Cq of the decomposed sample GA Rather than the degree to which it becomes larger, the Cq of the decomposed sample EA Because the degree to which it increases is large, the ΔCq of the processed sample with degraded nucleic acid sequence is greater than that of the unprocessed product. AIt becomes smaller. Therefore, the ΔCq of the unprocessed product A Despite being smaller, the ΔCq of processed and degraded nucleic acid sequences in the sample A The ΔCq of the standard sample S More broadly, as shown in equation (8), if the relative abundance of genetically modified organisms in the sample being tested is determined to be below the standard value, it is possible to conclude that the relative abundance of genetically modified organisms in the sample before processing is also always lower than the standard value, regardless of the degree of degradation of the nucleic acid sequences contained in the sample.

[0061] (Examples) Examples of the present invention are described below. However, it goes without saying that the present invention is not limited to the following examples.

[0062] (Example 1) Using real-time PCR, a type of quantitative PCR, we measured the relative abundance of genetically modified organisms (GM content) in the model processed product.

[0063] (sample) A genetically modified maize strain reference material (10% MON810 certified reference material, catalog number: ERMBF413GK-1G, Sigma-Aldrich) was prepared. This genetically modified maize strain reference material was dry-heat sterilized at 130°C for 0 minutes, 30 minutes, 60 minutes, and 120 minutes to prepare model samples. Using the DNeasy Plant Maxi Kit (Qiagen), DNA to be used as a PCR template was extracted from the prepared model samples.

[0064] (Real-time PCR) The real-time PCR reaction mixture was prepared to have a final primer concentration of 0.5 μmol / L and a final probe concentration of 0.2 μmol / L. The template DNA was prepared to be 25 ng per reaction mixture. 12.5 μL of TaqMan Universal PCR Master Mix (Thermo Fisher Scientific) was used, and the volume of the reaction system was adjusted with distilled water to 25 μL.

[0065] The SSIIb gene sequence was selected as the target for amplification as an endogenous maize gene. As primer pairs for amplifying the SSIIb gene sequence, the following were prepared, as shown in Table 1: "SSIIb-3" (114 bp) described in SEQ ID NOs. 1 and 2, "SSIIb-3long1" (120 bp) described in SEQ ID NOs. 3 and 4, "SSIIb-3long2" (130 bp) described in SEQ ID NOs. 5 and 6, "SSIIb-3long3" (139 bp) described in SEQ ID NOs. 7 and 8, and "SSIIb-3long4" (151 bp) described in SEQ ID NOs. 9 and 10. In addition, a probe having the following sequence was prepared to recognize the space between the primer pairs that amplify the SSIIb gene sequence. The following sequence corresponds to SEQ ID NO. 11. 5'-FAM-AGCAAAGTCAGAGCGCTGCAATGCA-TAMRA-3'

[0066] The P35S gene sequence and the MON810 gene sequence were selected as recombinant genes for amplification. As shown in Table 2, the primer pair "P35S-1" (101 bp) described in SEQ ID NOs. 12 and 13 was prepared as the primer pair for amplifying the P35S gene sequence, and the primer pair "MON810-2" (113 bp) described in SEQ ID NOs. 14 and 15 was prepared as the primer pair for amplifying the MON810 gene sequence.

[0067] Furthermore, a probe was prepared that recognizes the gap between primer pairs that amplify the P35S gene sequence, and which has the following sequence. The following sequence corresponds to sequence number 16. 5'-FAM-CCCACTATCCTTCGCAAGACCCTTCCT-TAMRA-3'

[0068] Furthermore, a probe was prepared that recognizes the gap between primer pairs that amplify the MON810 gene sequence, and which has the following sequence. The following sequence corresponds to sequence number 17. 5'-FAM-AGATACCAAGCGGCCATGGACAACAA-TAMRA-3'

[0069] [Table 1] [Table 2]

[0070] Primer pair sets were prepared to measure the GM content of the model processed products. Specifically, as shown in Table 3, the following sets were prepared: "P35S-1 / SSIIb-3" set, "P35S-1 / SSIIb-3long1" set, "P35S-1 / SSIIb-3long2" set, "P35S-1 / SSIIb-3long3" set, and "P35S-1 / SSIIb-3long4" set. In addition, as shown in Table 4, the following sets were prepared: "MON810-2 / SSIIb-3" set, "MON810-2 / SSIIb-3long1" set, "MON810-2 / SSIIb-3long2" set, "MON810-2 / SSIIb-3long3" set, and "MON810-2 / SSIIblong4" set.

[0071] [Table 3]

[0072] [Table 4]

[0073] The subsequent analyses were conducted in accordance with the "Testing Methods for Genetically Modified Foods that Have Underwent Safety Review" (Revised September 15, 2021, Consumer Affairs Agency), an appendix to the Food Labeling Standards, to calculate the copy number of the endogenous gene SSIIb, the recombinant gene P35S, and the recombinant gene MON810 in each model processed product.

[0074] (result) The number of copies of the model parts were as shown in Tables 5 and 6.

[0075] [Table 5]

[0076] [Table 6]

[0077] The GM content (%) of the sample was calculated using the following formula (10). C=(N G / N E ) × (1 / R) × 100 (10) In equation (10), C is the GM content (%), N G N is the copy number of recombinant genes in the DNA extracted from the model processed product. E represents the copy number of endogenous genes in the DNA extracted from the model, and R represents the internal ratio.

[0078] For the P35S standard ratio, we used 0.41, which is the value obtained by dividing the number of copies amplified by "P35S-1" in a 10% MON810 certified reference material by the number of copies amplified by "SSIIb-3" and multiplying the result by 10. For the MON810 standard ratio, we used 0.45, which is the value obtained by dividing the number of copies amplified by "MON810-2" by the number of copies amplified by "SSIIb-3" and multiplying the result by 10.

[0079] The GM content (%) of each model processed product, calculated using the above formula (10), is shown in Tables 7 and 8.

[0080] [Table 7]

[0081] [Table 8] When using the P35S / SSIIb primer set, the GM content of the model processed products in Comparative Example 1 (SSIIb / P35S amplification ratio 1.13) and Comparative Example 2 (SSIIb / P35S amplification ratio 1.19) was sometimes lower after dry heat treatment than in the untreated (0 min) case. On the other hand, the GM content of the model processed products in Verification Example 1 (SSIIb / P35S amplification ratio 1.29), Verification Example 2 (SSIIb / P35S amplification ratio 1.38), and Verification Example 3 (SSIIb / P35S amplification ratio 1.50) was always higher after dry heat treatment compared to the untreated (0 min) case, and increased in stages depending on the dry heat treatment time.

[0082] When using the MON810 / SSIIb primer set, the GM content of the model processed products in Comparative Example 3 (SSIIb / MON810 amplification ratio 1.01), Comparative Example 4 (SSIIb / MON810 amplification ratio 1.06), and Comparative Example 5 (SSIIb / MON810 amplification ratio 1.15) was sometimes lower after dry heat treatment compared to the untreated (0 min) case. On the other hand, the GM content of the model processed products in Verification Example 4 (SSIIb / MON810 amplification ratio 1.23) and Verification Example 5 (SSIIb / MON810 amplification ratio 1.34) was always higher after dry heat treatment compared to the untreated (0 min) case, and increased in stages depending on the dry heat treatment time.

[0083] These results indicate that when GM content is measured using a primer pair set configured such that the amplification length of the endogenous gene sequence (SSIIb in this example) is longer than a predetermined ratio to the amplification length of the recombinant DNA sequence (P35S or MON810 in this example), the GM content of the processed sample is always higher than that of the sample before processing, and increases progressively with increasing degree of processing.

[0084] Therefore, it was shown that if the GM content of a processed sample measured using a method that inevitably overestimates the GM content due to processing is lower than the standard value (e.g., 5%), then the GM content of the raw material of the sample before processing or the processed sample can be determined to be lower than the standard value (e.g., 5%).

[0085] (Example 2) The GM content of the model processed product was measured using multiplex PCR.

[0086] (sample) A model prepared using the same techniques as in Example 1 was created, and DNA to be used as a template for PCR was extracted.

[0087] (Multiplex PCR) The same primer pair set as in Example 1 was prepared to measure the GM content of the model processed products. The analysis was performed in accordance with the "Testing Method for Genetically Modified Foods that Have Passed Safety Review" (Consumer Affairs Agency, revised September 15, 2021), an appendix to the Food Labeling Standards. The fluorescence intensity threshold was set to 0.2, and the Base Line was set to 3 at the Start and 15 at the End. The Cq values ​​of the endogenous gene SSIIb and the recombinant genes P35S and MON810 were calculated for each model processed product.

[0088] (result) The Cq values ​​for each of the fabricated model parts are shown in Table 9.

[0089] [Table 9]

[0090] The ΔCq values ​​(=Cq(P35S)-Cq(SSIIb) or Cq(MON810)-Cq(SSIIb)) of the model fabricated parts were as shown in Tables 10 and 11.

[0091] [Table 10]

[0092] [Table 11]

[0093] When using the P35S / SSIIb primer set, the ΔCq values ​​of the model fabricated products in Comparative Example 6 (SSIIb / P35S amplification ratio 1.13) and Comparative Example 7 (SSIIb / P35S amplification ratio 1.19) were not consistently smaller than the untreated (0 min) ΔCq value, nor did they show a decrease in proportion to the dry heat treatment time. On the other hand, the ΔCq values ​​of the model fabricated products in Verification Example 6 (SSIIb / P35S amplification ratio 1.29), Verification Example 7 (SSIIb / P35S amplification ratio 1.38), and Verification Example 8 (SSIIb / P35S amplification ratio 1.50) were consistently smaller than the untreated (0 min) ΔCq value due to dry heat treatment, and decreased stepwise in proportion to the dry heat treatment time.

[0094] When using the MON810 / SSIIb primer set, the ΔCq values ​​of the model fabricated products in Comparative Example 8 (SSIIb / MON810 amplification ratio 1.01), Comparative Example 9 (SSIIb / MON810 amplification ratio 1.06), and Comparative Example 10 (SSIIb / MON810 amplification ratio 1.15) were not consistently smaller than the untreated (0 min) ΔCq value, nor did they show a decrease in proportion to the dry heat treatment time. On the other hand, the ΔCq values ​​of the model fabricated products in Verification Example 9 (SSIIb / MON810 amplification ratio 1.23) and Verification Example 10 (SSIIb / MON810 amplification ratio 1.34) were consistently smaller with dry heat treatment compared to the untreated (0 min) ΔCq value, and decreased stepwise in proportion to the dry heat treatment.

[0095] These results indicate that when a primer pair set is used in which the amplification length of the amplification product of the endogenous gene sequence (SSIIb in this example) is set to be longer than a predetermined ratio to the amplification length of the amplification product of the recombinant DNA sequence (P35S or MON810 in this example), the ΔCq value of the processed sample is always smaller than the ΔCq value of the sample before processing, and decreases stepwise as the degree of processing increases.

[0096] Therefore, the ΔCq of the processed sample was measured using a method in which the ΔCq value is always reduced by processing. A The value is ΔCq of a standard sample (e.g., 5% GM content). SIt was shown that if the value is greater than the standard value, the GM content of the raw materials in the sample before processing or after processing can always be determined to be lower than the standard value (e.g., 5%).

Claims

1. A method for detecting genetically modified organisms, At least a portion of the nucleic acid sequence contained in a sample potentially containing at least one genetically modified organism is amplified by PCR using a primer that specifically amplifies a recombinant gene derived from the genetically modified organism, and a primer that specifically amplifies an endogenous gene common to the species corresponding to the genetically modified organism, and is configured such that the amplification length of the amplification product of the endogenous gene is 123% or more of the amplification length of the amplification product of the recombinant gene. Based on the results of the PCR, the relative abundance of the genetically modified organism in the sample is determined. It is determined that at least a portion of the nucleic acid sequence in the sample has been degraded, and that the relative abundance of the genetically modified organism in the sample before the degradation of the nucleic acid sequence is lower than the relative abundance of the genetically modified organism determined above. Methods that include...

2. A method for detecting genetically modified organisms, At least a portion of the nucleic acid sequence contained in a sample potentially containing at least one genetically modified organism is amplified by PCR using a primer that specifically amplifies a recombinant gene derived from the genetically modified organism, and a primer that specifically amplifies an endogenous gene common to the species corresponding to the genetically modified organism, and is configured such that the amplification length of the amplification product of the endogenous gene is 123% or more of the amplification length of the amplification product of the recombinant gene. Based on the results of the PCR, it is determined whether the relative abundance of the genetically modified organism in the sample is lower than the reference value. If at least a portion of the nucleic acid sequence in the sample is degraded, and the relative abundance of the genetically modified organism in the sample is lower than the reference value, then it is determined that the relative abundance of the genetically modified organism in the sample before the nucleic acid sequence was degraded was lower than the reference value. Methods that include...

3. The method according to claim 1 or 2, wherein at least a portion of the nucleic acid sequence in the sample is degraded.

4. The method according to any one of claims 1 to 3, wherein the sample is processed.

5. A method for detecting genetically modified organisms, At least a portion of the nucleic acid sequence contained in a sample potentially containing at least one genetically modified organism is amplified by PCR using a primer that specifically amplifies a recombinant gene derived from the genetically modified organism, and a primer that specifically amplifies an endogenous gene common to the species corresponding to the genetically modified organism, and is configured such that the amplification length of the amplification product of the endogenous gene is 123% or more of the amplification length of the amplification product of the recombinant gene. Based on the results of the PCR, the relative abundance of the genetically modified organism in the sample is determined. The sample has been processed, and it is determined that the relative abundance of the genetically modified organism in the sample before processing is lower than the relative abundance of the genetically modified organism determined in the processed sample. Methods that include...

6. A method for detecting genetically modified organisms, At least a portion of the nucleic acid sequence contained in a sample potentially containing at least one genetically modified organism is amplified by PCR using a primer that specifically amplifies a recombinant gene derived from the genetically modified organism, and a primer that specifically amplifies an endogenous gene common to the species corresponding to the genetically modified organism, and is configured such that the amplification length of the amplification product of the endogenous gene is 123% or more of the amplification length of the amplification product of the recombinant gene. Based on the results of the PCR, it is determined whether the relative abundance of the genetically modified organism in the sample is lower than the reference value. If the sample has been processed and the proportion of the genetically modified organism in the sample is lower than the reference value, it is determined that the proportion of the genetically modified organism in the sample before processing is lower than the reference value. Methods that include...

7. The method according to any one of claims 1 to 6, wherein the ratio of the genetically modified organism in the sample is determined based on the amount of the recombinant gene present in the sample determined based on the results of the PCR and the amount of the endogenous gene present in the sample determined based on the results of the PCR.

8. The method according to any one of claims 1 to 7, wherein the relative abundance of the genetically modified organism is determined based on the following formula (1). C=(N G / N E )×(1 / R)×100 (1) (1) In equation (1), C is the relative abundance (%) of the genetically modified organism, N G The amount of the recombinant gene in the sample, N E represents the amount of the endogenous gene in the sample, and R represents the internal standard ratio.

9. The method according to claim 8, wherein the internal standard ratio is given by the following equation (2). R=N G100 / N E100 (2) In equation (2), N G100 This is the amount of recombinant gene in 100% of the aforementioned genetically modified organism, N E100 This represents the amount of endogenous genes in 100% of the aforementioned genetically modified organism.

10. The method according to claim 8, wherein the internal standard ratio is given by the following equation (3). R=N Gx / N Ex ×100 / x (3) In equation (3), N Gx This is the amount of recombinant gene in the certified reference material of x% of the genetically modified organism, N Ex This represents the amount of endogenous genes in a certified reference material containing x% of genetically modified organisms.

11. A method for detecting genetically modified organisms, At least a portion of the nucleic acid sequence contained in a sample potentially containing at least one genetically modified organism is amplified by PCR using a primer that specifically amplifies a recombinant gene derived from the genetically modified organism, and a primer that specifically amplifies an endogenous gene common to the species corresponding to the genetically modified organism, and is configured such that the amplification length of the amplification product of the endogenous gene is 123% or more of the amplification length of the amplification product of the recombinant gene. Based on the number of cycles at which the amplification product of the recombinant gene reaches a threshold, and the number of cycles at which the amplification product of the endogenous gene reaches a threshold, it is determined whether the relative abundance of the recombinant gene in the sample is lower than a reference value. It is determined that at least a portion of the nucleic acid sequence in the sample has been degraded, and that the relative abundance of the genetically modified organism in the sample before the degradation of the nucleic acid sequence is lower than the reference value. Methods that include...

12. A method for detecting genetically modified organisms, At least a portion of the nucleic acid sequence contained in a sample potentially containing at least one genetically modified organism is amplified by PCR using a primer that specifically amplifies a recombinant gene derived from the genetically modified organism, and a primer that specifically amplifies an endogenous gene common to the species corresponding to the genetically modified organism, and is configured such that the amplification length of the amplification product of the endogenous gene is 123% or more of the amplification length of the amplification product of the recombinant gene. Based on the number of cycles at which the amplification product of the recombinant gene reaches a threshold, and the number of cycles at which the amplification product of the endogenous gene reaches a threshold, it is determined whether the relative abundance of the recombinant gene in the sample is lower than a reference value. The aforementioned sample has been processed, and it is determined that the relative abundance of the genetically modified organism in the sample before processing is lower than the reference value. Methods that include...

13. The method according to claim 11 or 12, wherein it is determined whether the relative abundance of the genetically modified organism in the sample is lower than a reference value, based on the difference between the number of cycles at which the amplification product of the recombinant gene reaches a threshold and the number of cycles at which the amplification product of the endogenous gene reaches a threshold.

14. The method according to any one of claims 1 to 13, wherein the amplification length of the amplification product of the endogenous gene is 125% or more of the amplification length of the amplification product of the recombinant gene.

15. The method according to any one of claims 1 to 14, wherein the amplification length of the amplification product of the recombinant gene is 40 bp or more and 1000 bp or less.

16. The method according to any one of claims 1 to 15, wherein the PCR is quantitative PCR.

17. The method according to any one of claims 1 to 10, wherein the PCR is real-time PCR.

18. The method according to any one of claims 11 to 13, wherein the PCR is multiplex PCR.

19. The method according to any one of claims 1 to 18, wherein the species of organism is a plant.

20. A kit for detecting genetically modified organisms, PCR primers that specifically amplify recombinant genes derived from genetically modified organisms, A PCR primer configured to specifically amplify an endogenous gene commonly possessed by the organism corresponding to the genetically modified organism, and such that the amplification length of the amplification product of the endogenous gene is 123% or more of the amplification length of the amplification product of the recombinant gene, A kit that includes, Based on the PCR results, the relative abundance of the genetically modified organism in the sample that may contain the genetically modified organism is determined. To determine that at least a portion of the nucleic acid sequence contained in the sample has been degraded, and that the relative abundance of the genetically modified organism in the sample before the degradation of the nucleic acid sequence is lower than the relative abundance of the genetically modified organism determined, kit.

21. A kit for detecting genetically modified organisms, PCR primers that specifically amplify recombinant genes derived from genetically modified organisms, A PCR primer configured to specifically amplify an endogenous gene commonly possessed by the organism corresponding to the genetically modified organism, and such that the amplification length of the amplification product of the endogenous gene is 123% or more of the amplification length of the amplification product of the recombinant gene, A kit that includes, Based on the PCR results, the relative abundance of the genetically modified organism in the sample that may contain the genetically modified organism is determined. The above sample has been processed, and in order to determine that the relative abundance of the genetically modified organism in the sample before processing is lower than the relative abundance of the genetically modified organism determined in the processed sample, kit.

22. A kit for detecting genetically modified organisms, PCR primers that specifically amplify recombinant genes derived from genetically modified organisms, A PCR primer configured to specifically amplify an endogenous gene commonly possessed by the organism corresponding to the genetically modified organism, and such that the amplification length of the amplification product of the endogenous gene is 123% or more of the amplification length of the amplification product of the recombinant gene, A kit that includes, Based on the PCR results, it is determined whether the relative abundance of the genetically modified organism in the sample that may contain the genetically modified organism is lower than the reference value. If at least a portion of the nucleic acid sequence contained in the sample is degraded, and the relative abundance of the genetically modified organism in the sample is lower than the reference value, then to determine that the relative abundance of the genetically modified organism in the sample before the nucleic acid sequence was degraded is lower than the reference value, kit.

23. A kit for detecting genetically modified organisms, PCR primers that specifically amplify recombinant genes derived from genetically modified organisms, A PCR primer configured to specifically amplify an endogenous gene commonly possessed by the organism corresponding to the genetically modified organism, and such that the amplification length of the amplification product of the endogenous gene is 123% or more of the amplification length of the amplification product of the recombinant gene, A kit that includes, Based on the PCR results, it is determined whether the relative abundance of the genetically modified organism in the sample that may contain the genetically modified organism is lower than the reference value. If the sample has been processed and the relative abundance of the genetically modified organism in the sample is lower than the reference value, then, in order to determine that the relative abundance of the genetically modified organism in the sample before processing is lower than the reference value, kit.

24. A kit for detecting genetically modified organisms, PCR primers that specifically amplify recombinant genes derived from genetically modified organisms, The endogenous genes commonly possessed by the species corresponding to the aforementioned genetically modified organism are specifically amplified. A PCR primer configured such that the amplification length of the amplification product of the endogenous gene is 123% or more of the amplification length of the amplification product of the recombinant gene, A kit that includes, Based on the number of cycles at which the amplification product of the recombinant gene reaches a threshold, and the number of cycles at which the amplification product of the endogenous gene reaches a threshold, it is determined whether the relative abundance of the recombinant organism in a sample that may contain the recombinant organism is lower than a reference value. In order to determine that at least a portion of the nucleic acid sequence contained in the sample has been degraded, and that the relative abundance of the genetically modified organism in the sample before the degradation of the nucleic acid sequence is lower than the reference value, kit.

25. A kit for detecting genetically modified organisms, PCR primers that specifically amplify recombinant genes derived from genetically modified organisms, The endogenous genes commonly possessed by the species corresponding to the aforementioned genetically modified organism are specifically amplified. A PCR primer configured such that the amplification length of the amplification product of the endogenous gene is 123% or more of the amplification length of the amplification product of the recombinant gene, A kit that includes, Based on the number of cycles at which the amplification product of the recombinant gene reaches a threshold, and the number of cycles at which the amplification product of the endogenous gene reaches a threshold, it is determined whether the relative abundance of the recombinant organism in a sample that may contain the recombinant organism is lower than a reference value. The above sample has been processed, and in order to determine that the relative abundance of the genetically modified organism in the sample before processing is lower than the reference value, kit.

26. The kit according to any one of claims 20 to 25, wherein the amplification length of the amplification product of the endogenous gene is 125% or more of the amplification length of the amplification product of the recombinant gene.

27. The kit according to any one of claims 20 to 26, wherein the amplification length of the amplification product of the recombinant gene is 40 bp or more and 1000 bp or less.

28. The kit according to any one of claims 20 to 23, wherein the PCR is quantitative PCR.

29. The kit according to any one of claims 20 to 23, wherein the PCR is real-time PCR.

30. The kit according to claim 24 or 25, wherein the PCR is multiplex PCR.

31. The kit according to any one of claims 20 to 30, wherein the species of organism is a plant.

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