JP-7854642-B2 - Method for detecting genetically modified organisms

JP7854642B2JP 7854642 B2JP7854642 B2JP 7854642B2JP-7854642-B2

Inventors

真野潤一
井川豪志
野間聡
菊池洋介

Assignees

国立研究開発法人農業・食品産業技術総合研究機構
株式会社日清製粉グループ本社

Dates

Publication Date: 20260507
Application Date: 20220330

Claims (20)

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...
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...
The method according to claim 1 or 2 , wherein at least a portion of the nucleic acid sequence in the sample is degraded.
The method according to any one of claims 1 to 3 , wherein the sample is processed.
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 ...
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...
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.
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) In equation (1), C represents the relative abundance (%) of the genetically modified organism, NG represents the amount of the recombinant gene in the sample, NE represents the amount of the endogenous gene in the sample, and R represents the internal standard ratio.
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), NG100 represents the amount of recombinant genes in 100% of the genetically modified organism, and N E100 represents the amount of endogenous genes in 100% of the genetically modified organism.
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 represents the amount of recombinant genes in the certified reference material of x% of the recombinant organism, and N Ex represents the amount of endogenous genes in the certified reference material of x% of the recombinant organism.
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 ...
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...
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.
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.
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.
The method according to any one of claims 1 to 15 , wherein the PCR is quantitative PCR.
The method according to any one of claims 1 to 10 , wherein the PCR is real-time PCR.
The method according to any one of claims 11 to 13 , wherein the PCR is multiplex PCR.
The method according to any one of claims 1 to 18 , wherein the species of organism is a plant.
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 .

Description

This invention relates to genetic technology, and more particularly to a method for detecting genetically modified organisms. Polymerase chain reaction (PCR) is widely used for quantifying genetically modified organisms (GMs) in food and other samples (see, for example, Patent Documents 1 to 7 and Non-Patent Documents 1 to 4). However, it is known that as samples are processed, deoxyribonucleic acid (DNA) contained in the sample degrades and fragments due to heat, pH changes, and physical forces. Furthermore, it has been confirmed that DNA fragmentation can lead to errors in the quantification of GMs by PCR. For example, when soybean raw materials containing 5% genetically modified organisms (GMs) are processed into tofu, soy milk, and boiled beans, it has been reported that the proportion of GMs in the processed products quantified by PCR may be higher or lower than the actual 5%, depending on the processing method and amount (see, for example, Non-Patent Document 5 and Patent Document 8). Therefore, even if the proportion of GMs in the processed products quantified by PCR is higher than the standard value, the raw materials for the processed products may not actually contain GMs above the standard value. Conversely, even if the proportion of GMs in the processed products quantified by PCR is lower than the standard value, the raw materials for the processed products may actually contain GMs above the standard value. Patent No. 4291568Patent No. 4317450Patent No. 4925607Patent No. 5229864Special Publication No. 2002-536024Japanese Patent Publication No. 2013-063082Japanese Patent Publication No. 2017-143803Japanese Patent Publication No. 2021-40592 Takeshi Ogasawara et al., "Fragmentation of DNAs of Processed Foods Made from Genetically Modified Soybeans," Jpn, J. Food Chem., 2003, 10, 155-160Food Labeling Standards Q&A Appendix "Matters Concerning Genetically Modified Foods," Consumer Affairs Agency, Revised March 30, 2015Food Labeling Standards Appendix, "Testing Methods for Genetically Modified Foods that Have Underwent Safety Review," Consumer Affairs Agency, revised March 28, 2019.Abstracts of Presentations at the 113th Annual Meeting of the Japanese Society for Food Hygiene, page 73.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, 2060-2069Did you know? The Genetically Modified Organism Labeling System, Consumer Affairs AgencyFood Labeling Standards Appendix "Testing Methods for Genetically Modified Foods that Have Underwent Safety Review" (Consumer Affairs Agency), revised September 15, 2021.Genetically modified foods and testing status, Kanagawa Prefectural Institute of Public Health News No. 200, September 2020 issue.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 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.This figure schematically shows the relationship between ΔCq and the relative abundance of genetically modified organisms according to the embodiment.This figure schematically shows the relationship between ΔCq and the relative abundance of genetically modified organisms according to the embodiment.This figure schematically shows the relationship between the ΔCq of the unprocessed product and the ΔCq of the processed product according to the embodiment.This figure schematically shows the relationship between the ΔCq of the unprocessed product and the ΔCq of the processed product according to the embodiment.This figure schematically shows the relationship between the ΔCq of the unprocessed product and the ΔCq of the processed product according to the embodiment. The following describes embodiments of the present invention. However, these embodiments should not be understood as limiting the 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. The genetically modified organism detection method according to this embodiment includes amplifying at least a portion of the nucleic acid sequence contained in a sample potentially containing 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 configured to specifically amplify endogenous genes commonly possessed by the organisms corresponding to the genetically modified organism, suc