Relative quantification of GM crops using digital PCR without certified reference materials

Abstract

The present invention relates to a method for the relative quantification of GM crops using digital PCR without using certified reference materials (CRMs). More specifically, the present invention relates to a primer and probe set composition for digital PCR for the relative quantification of transgenes in GM crops without using CRMs, which comprises a first primer and probe set capable of specifically detecting genes present in a single copy or in 2-3 copies within the genome of GM (genetically modified) crops; and a second primer and probe set capable of specifically detecting transgenes in GM crops, and uses thereof.

Description

[Technical Field]

[0001] The present invention relates to a method for the relative quantification of GM crops using digital PCR without a certified reference material (CRM). More specifically, the present invention relates to a method for the relative quantification of GM crops without the need for a CRM specific to each GM crop, by selecting genes present in a single copy or in 2-3 copies within the genome of a GM (genetically modified) crop and replacing the selected genes with a CRM. [Background technology]

[0002] Genetically modified organisms (GMOs) offer solutions to various problems facing humanity, including food shortages, environmental change, and the development of alternative energy sources. Because of their potential to create high added value, research and development into a variety of crops has been underway. Since the first genetically modified tomato was developed and commercialized, GMOs have been developed for a variety of crops, and the development of GMOs with pharmaceutical and functional value is currently expanding. With the enforcement of the Act on International Movement of Genetically Modified Organisms in 2008, import approval for GMO imports by use became mandatory for each competent authority, and post-transfer management measures are being implemented at the national level.

[0003] Meanwhile, digital PCR is a new approach to third-generation PCR (polymerase chain reaction) that enables real-time absolute quantification of target genes. Digital PCR involves distributing droplets containing sample gene templates, amplification primers, and fluorescent probes, each diluted to an average of 0.5 to 1 copy, into a single well. After emulsion PCR, wells that show a fluorescent signal are counted as "1" because they contain a sample with 1 gene copy number and show the signal after amplification. Wells without a fluorescent signal are counted as "0" because they contain a sample with 0 copy number and therefore are not amplified, allowing absolute quantification.

[0004] While the result analysis method of existing qPCR (quantitative PCR) is an analog method, the digital PCR method, which is a digital method in which the result signal has a value of "0" or "1", has the advantage of being able to analyze large volumes of samples and perform various test items at once. It also enables accurate absolute quantification by applying a single molecule counting method to DNA samples, which does not require a standard curve.

[0005] PCR techniques are used to quantify the relative amount of introduced genes in GM crops. This method involves creating a standard curve using a certified reference material (CRM) for the GM crop being analyzed, and then substituting the PCR results of the sample into the standard curve to quantify the GMO gene. However, this method has problems such as the impossibility of developing a relative quantification test method when a CRM is unavailable or does not exist, and differences in amplification efficiency due to differences in nucleic acid quality between the CRM and the sample.

[0006] Therefore, the present invention has developed a method for the relative quantification of introduced genes in GM crops, which allows the relative quantification of GMOs without CRM.

[0007] Meanwhile, Korean Patent Publication No. 2019-0136647 discloses a "method for detecting and absolutely quantifying Alexandrium species using digital PCR," and Korean Patent Publication No. 2001-0100456 discloses a "genetically modified organism detection kit and PCR primers used therein" that uses primers that can hybridize to specific sites in the 35S promoter. However, there is no mention of the "method for relative quantification of GM crops using digital PCR without certified reference materials" of the present invention, which uses a primer set that can specifically detect genes with a small number of copies (single or 2-3 copies) within the plant genome. Summary of the Invention [Problem to be solved by the invention]

[0008] The present invention was developed in response to these needs and aimed to develop a method for the relative quantification of GM crops without the need for certified reference materials (CRMs). To this end, we analyzed the genome sequence information of each crop to screen for genes present in single or two copies, designed molecular markers for digital PCR for the selected endogenous genes and transgenes, and performed digital PCR on GM crop samples. As a result, we confirmed that the endogenous genes selected through screening can replace CRMs. We analyzed the digital PCR results based on the copy number ratio of the transgene to the endogenous gene, and confirmed that the method of the present invention significantly reduced the error between the theoretically estimated and measured values ​​for the contamination rate of GMO samples compared to conventional real-time PCR methods, thereby completing the present invention. [Means for solving the problem]

[0009] To achieve this, the present invention provides a primer and probe set composition for digital PCR for relative quantification of transgenes in genetically modified (GM) crops without the need for a CRM (certified reference material), the composition comprising: a first primer and probe set capable of specifically detecting genes present in a single copy or 2-3 copies in the genome of a GM crop; and a second primer and probe set capable of specifically detecting transgenes in GM crops.

[0010] The present invention also provides a digital PCR kit for the relative quantification of transgenes in GM crops without CRM, comprising the digital PCR primer and probe set composition; and reagents for carrying out an amplification reaction.

[0011] The present invention also provides a method for relatively quantifying transgenes in GM crops without CRM, which method comprises performing PCR using the digital PCR primer and probe set composition of the present invention.

[0012] The present invention also provides a marker composition for digital PCR for the relative quantification of introduced genes in GM crops containing the MEK1 (Mitogen-activated ERK kinase 1) or Ppck1 (phosphoenolpyruvate carboxylase kinase 1) gene as an active ingredient. [Effects of the Invention]

[0013] The method of the present invention enables relative quantification of GMOs without the need for certified reference materials (CRMs). By using a single or 2-3 copy number of endogenous genes present in GM crop genomes instead of CRMs, it is possible to perform relative quantification of GMO samples, for which no CRMs were previously available. This method has the advantage of not causing problems such as amplification efficiency and measurement errors due to differences in nucleic acid quality between CRMs and GMO samples, and is expected to be useful in GM crop quarantine and the inspection of foods contaminated with GM crops. [Brief explanation of the drawings]

[0014] [Figure 1] FIG. 1 is a schematic diagram showing the relative copy numbers between introduced genes and single copy endogenous genes (MEK1, Ppck1) in GM maize and GM bean. [Figure 2] Quantitative analysis of transgenes in GM maize using real-time PCR. BT11: transgene (target gene), MEK1: endogenous gene. [Figure 3] Quantitative analysis of transgenes in GM beans using real-time PCR. RRS: transgene (target gene), Ppck1: endogenous gene. [Figure 4]This shows the results of a relative quantitative analysis of the transgene (BT11) in GM corn by digital PCR. The percentages on the right side of the boxes are based on the copy number ratio and represent the contamination rate of the GM corn powder calculated based on the transgene and endogenous gene results (concentration) for each sample. [Figure 5] This shows the results of a relative quantitative analysis of the transgene (RSS) in GM beans using digital PCR. The percentages on the right side of the boxes are based on the copy number ratio and represent the contamination rate of GM bean powder calculated based on the transgene and endogenous gene results (concentration) for each sample. DETAILED DESCRIPTION OF THE INVENTION

[0015] To achieve the objectives of the present invention, the present invention provides a primer and probe set composition for digital PCR for relative quantification of transgenes in genetically modified (GM) crops without a CRM (certified reference material), the primer and probe set comprising: a first primer and probe set capable of specifically detecting genes present in a single copy or 2-3 copies in the genome of a GM crop; and a second primer and probe set capable of specifically detecting transgenes in GM crops.

[0016] In the present invention, the term "certified reference material (CRM)" refers to a homogeneous and stable substance, but which also has measurement traceability and uncertainty.

[0017] In the digital PCR primer and probe set composition according to the present invention, the GM crop is preferably, but not limited to, corn or beans.

[0018] Furthermore, in the digital PCR primer and probe set composition according to the present invention, the gene present in a single copy or in 2-3 copies within the genome is a gene used as a CRM for relative quantification of GM crops, and is a gene present in a single copy or in 2-3 copies within the plant body that is essential for the survival of the plant body.

[0019] In one embodiment of the present invention, the gene present in a single or 2-3 copies in the genome may be, but is not limited to, MEK1 (Mitogen-activated ERK kinase 1) or Ppck1 (phosphoenolpyruvate carboxylase kinase 1).

[0020] Furthermore, in the primer and probe set composition for digital PCR according to the present invention, the first primer and probe set is a molecular marker that targets an endogenous gene and is used in place of a CRM, and is preferably a primer consisting of the base sequences of SEQ ID NOs: 1 and 2 and a probe consisting of the base sequence of SEQ ID NO: 3; or a primer consisting of the base sequences of SEQ ID NOs: 7 and 8 and a probe consisting of the base sequence of SEQ ID NO: 9, but is not limited thereto.

[0021] In addition, in the primer and probe set composition for digital PCR according to the present invention, the second primer and probe set is a molecular marker targeting an introduced gene of a GM crop, and is preferably a primer consisting of the nucleotide sequences of SEQ ID NOs: 4 and 5 and a probe consisting of the nucleotide sequence of SEQ ID NO: 6; or a primer consisting of the nucleotide sequences of SEQ ID NOs: 10 and 11 and a probe consisting of the nucleotide sequence of SEQ ID NO: 12, but is not limited thereto.

[0022] In one embodiment of the present invention, the primers consisting of the base sequences of SEQ ID NOs: 1 and 2 and the probe consisting of the base sequence of SEQ ID NO: 3 target the MEK1 gene derived from corn, the primers consisting of the base sequences of SEQ ID NOs: 4 and 5 and the probe consisting of the base sequence of SEQ ID NO: 6 target the transgene of GM corn BT11, the primers consisting of the base sequences of SEQ ID NOs: 7 and 8 and the probe consisting of the base sequence of SEQ ID NO: 9 target the Ppck1 gene derived from soybeans, and the primers consisting of the base sequences of SEQ ID NOs: 10 and 11 and the probe consisting of the base sequence of SEQ ID NO: 12 target the transgene of GM soybean RSS.

[0023] In one embodiment of the present invention, the primers may comprise oligonucleotides consisting of a segment of 15 or more, 16 or more, or 17 or more consecutive nucleotides within SEQ ID NOs: 1, 2, 4, 5, 7, 8, 10, and 11, depending on the sequence length of each primer. For example, a primer of SEQ ID NO: 1 (18 oligonucleotides) may comprise oligonucleotides consisting of a segment of 15 or more, 16 or more, or 17 or more nucleotides within the sequence of SEQ ID NO: 1.

[0024] As used herein, a "primer" refers to a single-stranded oligonucleotide sequence that is complementary to the nucleic acid strand to be copied and can act as a starting point for the synthesis of a primer extension product. The length and sequence of the primer must allow it to initiate the synthesis of the extension product. The specific length and sequence of the primer will depend on the required complexity of the DNA or RNA target, as well as the conditions under which the primer will be used, such as temperature and ionic strength.

[0025] Herein, oligonucleotides used as primers may further contain nucleotide analogs, such as phosphorothioates, alkyl phosphorothioates, or peptide nucleic acids, or may contain intercalating agents.

[0026] As used herein, the term "probe" refers to a hybridization probe, a linear oligomer containing natural or modified monomers or linkages containing deoxyribonucleotides and ribonucleotides that can bind sequence-specifically to a complementary strand of nucleic acid. The probes of the present invention are allele-specific probes that hybridize to DNA fragments from one member of the same species where a polymorphic site exists among nucleic acid fragments from two members of the same species, but not to fragments from the other member. Preferably, the probe is single-stranded for maximum hybridization efficiency, and more preferably, is deoxyribonucleotide, but is not limited thereto.

[0027] In the present invention, the term "hybridization" means that complementary single-stranded nucleic acids form a double-stranded nucleic acid. Hybridization can occur between perfectly matched or substantially matched nucleic acid duplexes with some mismatches. Complementarity for hybridization can vary depending on hybridization conditions, particularly temperature.

[0028] In one embodiment of the present invention, the probes consisting of the base sequences of SEQ ID NOs: 3, 6, 9, and 12 are labeled with a fluorescent dye at one of the 5' and 3' ends and a quencher at the other end, preferably labeled with a fluorescent dye at the 5' end and a quencher at the 3' end, and specifically bind to products amplified by a primer set. Examples of the fluorescent dye include, but are not limited to, FAM, VIC, TET, JOE, HEX, CY3, CY5, ROX, RED610, TEXASRED, RED670, TYE563, BIOTIN, DIGOXIGENIN, and NED.

[0029] The present invention also provides a digital PCR kit for CRM-free relative quantification of transgenes in GM crops, comprising the above-mentioned digital PCR primer and probe set composition; and reagents for carrying out an amplification reaction.

[0030] In the kit according to the present invention, the reagents for carrying out the amplification reaction may include, but are not limited to, a DNA polymerase, dNTPs, and a buffer.

[0031] The kit may further include a user's guide describing optimal reaction conditions. The guide is a printed document that explains how to use the kit, for example, how to prepare the reverse transcription buffer and PCR buffer, and the suggested reaction conditions. The guide includes an information pamphlet in the form of a brochure or flyer, a label attached to the kit, and instructions on the surface of a package containing the kit. The guide also includes information that is published or provided via electronic media such as the Internet.

[0032] The present invention also provides a method for relatively quantifying a transgene in a GM crop without CRM, comprising the step of performing PCR using the digital PCR primer and probe set composition of the present invention.

[0033] The method further specifically comprises: isolating genomic DNA from the GM crop sample; dispensing a reaction solution containing DNA polymerase and the primer and probe set composition for digital PCR using the isolated genomic DNA as a template into each well of a microfluidic digital PCR plate having tens of thousands of partition cells per well; amplifying a target nucleic acid sequence in each well via PCR; and determining the amount of the target nucleic acid by counting the number of cells having the amplified target nucleic acid, but is not limited thereto.

[0034] The method of the present invention includes isolating genomic DNA from a GM crop sample. The genomic DNA can be isolated using methods known in the art, such as the CTAB method using a Wizard prep kit (Promega). Using the isolated genomic DNA as a template, a target sequence can be amplified by performing an amplification reaction using a primer and probe set composition for digital PCR according to one embodiment of the present invention.

[0035] In the present invention, the principle of digital PCR (dPCR) is roughly comprised of division, amplification, and counting. After dispensing and dividing a sample containing DNA into a nanoplate consisting of wells divided into tens of thousands of cells (approximately 26,000 cells), PCR amplification is performed, and absolute quantification is performed by counting the cells in which the desired fragment has been amplified and the cells in which it has not.

[0036] The relative quantification method of the present invention involves identifying endogenous genes present in small numbers, for example, a single copy or 2-3 copies, in the genome of a plant, developing a primer set that can specifically detect the gene, and using the primer set as an internal control and as a substitute for CRM.

[0037] The present invention also provides a marker composition for digital PCR for the relative quantification of transgenes in GM crops containing the MEK1 (Mitogen-activated ERK kinase 1) or Ppck1 (phosphoenolpyruvate carboxylase kinase 1) gene as an active ingredient.

[0038] In this invention, we have confirmed that the MEK1 or Ppck1 gene selected to replace CRM in GM maize or GM beans can function as a CRM for relative quantification, and therefore, these genes will be used as markers for digital PCR for the relative quantification of introduced genes in GM crops.

[0039] The present invention will be described in detail below with reference to examples. However, the following examples are merely illustrative of the present invention and are not intended to limit the scope of the present invention.

[0040] Materials and Methods 1. GMO samples and genomic DNA extraction In this invention, a relative quantification method was tested on GM corn and beans. Corn is a crop that produces offspring by cross-pollination, while beans are produced by self-pollination. Powder samples of GM corn and GM beans used in this invention were purchased from Sigma (Tables 1 and 3). Genomic DNA was extracted from each purchased sample using a QIAamp PowerFecal Pro DNA Kit (cat. no. ID 51804) (Tables 2 and 4).

[0041] [Table 1]

[0042] [Table 2]

[0043] [Table 3]

[0044] [Table 4]

[0045] 2. Preparation of primer and probe sets for digital PCR Primers and probes for detecting endogenous and target genes (transgenes) for qualitative and quantitative analysis of GM corn BT11 were prepared as shown in Table 5 below, with reference to NCBI Reference Sequence: NM_001371620.1. Primers and probes for detecting endogenous and target genes for qualitative and quantitative analysis of GM soybean RRS were prepared as shown in Table 6 below, with reference to GenBank accession no. DQ657244.1.

[0046] [Table 5]

[0047] [Table 6]

[0048] Example 1. Quantitative analysis of transgenes in GM crops using real-time PCR Quantitative analysis was performed using real-time PCR with the extracted genomic DNA and the prepared primer and probe sets. The experiment was performed using a CFX96™ Real-Time PCR Detection System after forming a reaction solution containing 20 ng of template DNA, 4.8 μl of each primer and probe, 10 μl of a polymerase mixture containing dNTPs, and 24.2 μl of RNase-free water.

[0049] [Table 7]

[0050] As a result, as shown in Figures 2 and 3, it was confirmed that the Ct values ​​of the target introduced genes (BT11 and RRS) increased as the rate of GM crop contamination in the sample decreased, while the Ct values ​​of the endogenous genes (MEK1 and Ppck1) remained unchanged.

[0051] Example 2. Quantitative analysis of transgenes in GM crops using digital PCR Relative quantification by digital PCR was analyzed using the same samples and primer / probe sets used in Example 1. Digital PCR was performed using a QIAcuity instrument with a Nanoplate 26K 24-well plate, and the results were analyzed using the QIAcuity Software Suite 1.2.18 program. The digital PCR reaction mixture was the same as in Table 8 below, and the amplification conditions were the same as in Table 7.

[0052] [Table 8]

[0053] The analysis revealed that the concentration of the transgene BT11 gene in GM corn samples decreased depending on the GMO contamination rate, while the concentration of the endogenous gene MEK1 remained unchanged regardless of the GMO contamination rate (Figure 4). As shown in Figure 1, the copy number ratio of transgenes to endogenous genes in corn is 1:2. Therefore, the GM corn powder contamination rate was calculated based on the transgene and endogenous gene concentrations in a sample containing 1% GM corn powder, and was found to be 0.974% ((8.9 / 1827.1) x 2 x 100), a difference of only 2.6% between the theoretical and actual values.

[0054] The results for the GM bean samples also showed a similar trend to that for the GM corn, but the concentration of the introduced RRS gene decreased with the GMO contamination rate, while the concentration of the endogenous Ppck1 gene remained unchanged regardless of the GMO contamination rate (Figure 5). Furthermore, based on the 1:1 copy number ratio of introduced genes to endogenous genes in beans (Figure 1), the contamination rate of GM bean powder was calculated based on the introduced gene and endogenous gene concentrations for a sample containing 1% GM bean powder, resulting in a value of 0.94% ((19.7 / 2096.9) x 100), a difference of 6.1% between the theoretical and actual values.

[0055] Based on the above results, it was confirmed that the MEK1 or Ppck1 gene selected in the present invention to replace CRM in GM corn or GM soybean can function as CRM for relative quantification.

Claims

1. A primer and probe set composition for digital PCR for relative quantification of transgenes in GM (genetically modified) crops without a CRM (certified reference material), comprising: a first primer and probe set capable of specifically detecting genes present in a single copy or in 2-3 copies within the genome of a GM crop; and a second primer and probe set capable of specifically detecting transgenes in GM crops.

2. The digital PCR primer and probe set composition according to claim 1, wherein the GM crop is corn or beans.

3. The primer and probe set composition for digital PCR according to claim 1, wherein the gene present in a single or two copies within the genome is a MEK1 (Mitogen-activated ERK kinase 1) or Ppck1 (phosphoenolpyruvate carboxylase kinase 1) gene.

4. The primer and probe set composition for digital PCR according to claim 1, characterized in that the first primer and probe set is a primer consisting of the base sequences of SEQ ID NOs: 1 and 2 and a probe consisting of the base sequence of SEQ ID NO: 3; or a primer consisting of the base sequences of SEQ ID NOs: 7 and 8 and a probe consisting of the base sequence of SEQ ID NO:

9.

5. The primer and probe set composition for digital PCR according to claim 1, characterized in that the second primer and probe set is a primer consisting of the base sequences of SEQ ID NOs: 4 and 5 and a probe consisting of the base sequence of SEQ ID NO: 6; or a primer consisting of the base sequences of SEQ ID NOs: 10 and 11 and a probe consisting of the base sequence of SEQ ID NO:

12.

6. A digital PCR kit for relative quantification of transgenes in GM crops without a certified reference material (CRM), comprising the digital PCR primer and probe set composition of claim 1 and reagents for carrying out an amplification reaction.

7. 7. The kit of claim 6, wherein the reagents for carrying out the amplification reaction include a DNA polymerase, dNTPs, and a buffer.

8. A method for relatively quantifying introduced genes in genetically modified (GM) crops without a certified reference material (CRM), comprising the step of performing PCR using the digital PCR primer and probe set composition of claim 1.

9. The method comprises: isolating genomic DNA from a GM crop sample; Dispensing a reaction solution containing the isolated genomic DNA as a template, a DNA polymerase, and the primer and probe set composition for digital PCR according to claim 1 into each well of a microfluidic digital PCR plate having tens of thousands of partition cells per well; amplifying a target nucleic acid sequence in each well via PCR; 9. The method for relative quantification of transgenes in GM crops without CRM, as described in claim 8, characterized in that it includes a step of counting the number of cells containing the amplified target nucleic acid to determine the amount of the target nucleic acid.

10. A marker composition for digital PCR for the relative quantification of transgenes in GM crops containing the MEK1 (Mitogen-activated ERK kinase 1) or Ppck1 (phosphoenolpyruvate carboxylase kinase 1) gene as an active ingredient.

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