Method for identifying homozygous and heterozygous transgenic maize nCX-1 based on multiple fluorescent PCR (Polymerase Chain Reaction) and application

Abstract

The invention provides a method for identifying homozygous and heterozygous transgenic maize nCX-1 based on multiple fluorescent PCR and application, and relates to the technical field of transgenic detection.The method comprises the steps that multiple fluorescent PCR amplification is conducted with DNA of a sample to be detected as a template, and whether the sample to be detected is the homozygous or heterozygous transgenic maize nCX-1 is judged according to a target amplification curve; the target amplification curve comprises a reference gene amplification curve, a transformant specificity amplification curve and a homozygosity identification amplification curve. Compared with a digital PCR technology, the multiplex fluorescent PCR technology has the same reliability, and is quicker, more efficient and low in consumption; the method solves the problems that the real-time fluorescent quantitative PCR identification of homozygous or heterozygous transgenic organisms is inaccurate, a standard curve needs to be established firstly and a calibrator needs to be needed; a foundation is laid for cross breeding and breeding and identification work of standard substances or raw materials, and the authenticity and reliability of material backgrounds are guaranteed.

Description

Technical Field

[0001] The present invention relates to the technical field of transgenic detection, and particularly relates to a method and application for identifying homozygous and heterozygous transgenic maize nCX-1 based on multiplex fluorescence PCR. Background Art

[0002] With the wide application of transgenic technology in the agricultural field, the research and industrialization of transgenic crops have developed rapidly. According to the statistical data released by the International Service for the Acquisition of Agri-biotech Applications, the global planting area of transgenic crops reached 206.3 million hectares in 2023. Although transgenic crops perform well in terms of variety resistance, nutritional improvement, etc., their safety issues have attracted wide public attention. And transgenic qualitative and quantitative detection technology is an important guarantee for the smooth implementation of systems such as transgenic industrialization management and label supervision.

[0003] An important material basis for the detection of transgenic organisms and their product components is the reference material for transgenic product detection, and the identification of the homozygosity of raw materials is a key link in the preparation of reference materials. At present, the main methods for identifying the homozygosity of transgenic plants are real-time fluorescence quantitative PCR method and digital PCR method. The real-time fluorescence quantitative PCR method has many advantages, such as fast and sensitive, but it has been questioned in terms of the accuracy of homozygosity. In addition, when using the real-time fluorescence quantitative PCR method for homozygosity identification, a standard curve must be established first and the amplification efficiencies of the endogenous gene and the exogenous gene are the same or very close. At the same time, this method also requires a calibrator with known homozygosity as the calibration material. Therefore, the application of the real-time fluorescence quantitative PCR method is greatly limited. The digital PCR method has the advantages of the real-time fluorescence quantitative PCR method in addition to being resistant to inhibitors and having accurate identification. However, the digital PCR method also has disadvantages such as low identification throughput and high cost.

[0004] In view of this, the present invention is specifically proposed. Summary of the Invention

[0005] One of the purposes of the present invention is to provide a method for identifying homozygous and heterozygous transgenic maize nCX-1 based on multiplex fluorescence PCR, so as to solve the technical problems in the prior art that the digital PCR has low throughput and high cost, or the real-time fluorescence quantitative PCR must first establish a standard curve and the amplification efficiencies of the endogenous gene and the exogenous gene are the same or very close, and the accuracy is questioned.

[0006] The second purpose of the present invention is to provide the application of the above method in the cross-breeding of transgenic maize nCX-1 and the identification of reference materials or raw materials.

[0007] In order to achieve the above purposes of the present invention, the following technical solutions are specifically adopted:

[0008] In a first aspect, the present invention provides a method for identifying homozygous and heterozygous transgenic maize nCX-1 based on multiplex fluorescence PCR, which includes performing multiplex fluorescence PCR amplification using the DNA of the sample to be tested as a template, and determining whether the sample to be tested is homozygous or heterozygous transgenic maize nCX-1 according to the target amplification curves.

[0009] The target amplification curves include the internal reference gene amplification curve, the transformant-specific amplification curve, and the homozygosity identification amplification curve.

[0010] Further, the determination of whether the sample to be tested is homozygous or heterozygous transgenic maize nCX-1 according to the target amplification curves includes:

[0011] If both the internal reference gene amplification curve and the transformant-specific amplification curve are typical amplification curves, and the homozygosity identification amplification curve is an atypical amplification curve, then the sample to be tested is homozygous;

[0012] If the internal reference gene amplification curve, the transformant-specific amplification curve, and the homozygosity identification amplification curve are all typical amplification curves, then the sample to be tested is heterozygous.

[0013] Further, the primer pair for amplifying homozygosity identification includes nCX-1-5F and nCX-1-3R, and their nucleotide sequences are shown in SEQ ID NO.1 and SEQ ID NO.2;

[0014] The probe for amplifying the homozygosity identification amplification curve includes nCX-1-3P, and its nucleotide sequence is shown in SEQ ID NO.5.

[0015] Further, the PCR primer pair for amplifying the internal reference gene includes zSSIIb-F and zSSIIb-R, and their nucleotide sequences are shown in SEQ ID NO.7 and SEQ ID NO.8;

[0016] The probe for amplifying the internal reference gene includes zSSIIb-P, and its nucleotide sequence is shown in SEQ ID NO.9.

[0017] Further, the PCR primer pair for amplifying transformant specificity includes nCX-1-5F and nCX-1-5R, and their nucleotide sequences are shown in SEQ ID NO.1 and SEQ ID NO.10;

[0018] The probe for amplifying transformant specificity includes nCX-1-5P, and its nucleotide sequence is shown in SEQ ID NO.4.

[0019] Further, in the reaction system of the multiplex fluorescent PCR, the molar ratio of nCX-1-5F, nCX-1-3R, zSSIIb-F, zSSIIb-R and nCX-1-5R is 1:1:1:1:1.

[0020] Further, the molar ratio of nCX-1-3P, zSSIIb-P and nCX-1-5P is 1:1:1.

[0021] Further, the final concentrations of nCX-1-5F, nCX-1-3R, zSSIIb-F, zSSIIb-R and nCX-1-5R are all 0.4 μM;

[0022] The final concentrations of nCX-1-3P, zSSIIb-P and nCX-1-5P are all 0.2 μM.

[0023] Further, the amplification conditions of the multiplex fluorescent PCR include pre-denaturation at 95°C for 5 min; denaturation at 95°C for 15 sec; annealing and extension at 60°C for 60 sec, for a total of 40 cycles; 10 min at 98°C, and storage at 4°C, with the heating and cooling rate maintained at 2°C / sec.

[0024] In a second aspect, the present invention provides the application of the above method in the cross-breeding of transgenic maize nCX-1 and the identification of reference materials or raw materials.

[0025] The method for identifying homozygous and heterozygous transgenic maize nCX-1 based on multiplex fluorescent PCR provided by the present invention discloses for the first time the use of multiplex fluorescent PCR technology to obtain target amplification curves, and establishes a method for identifying homozygous and heterozygous transgenic maize nCX-1; compared with digital PCR technology, multiplex fluorescent PCR technology has the same reliability and is faster, more efficient and less costly; compared with real-time fluorescence quantitative PCR method, it solves the problems of inaccurate identification, the need to establish a standard curve in advance and the need for calibration products in the identification of homozygous or heterozygous transgenic organisms; the method provided by the present invention lays a foundation for cross-breeding and the identification of the reproduction of reference materials or raw materials, and ensures the true and reliable background of the materials. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the specific embodiments of the present invention or the technical solutions in the prior art, the following will briefly introduce the drawings required for the description of the specific embodiments or the prior art. Obviously, the drawings in the following description are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0027] Figure 1Position map of primers / probes for identifying the homozygosity of transgenic maize nCX-1 provided in Example 3 of the present invention;

[0028] Figure 2 Graph showing the results of real-time fluorescence PCR screening of 6 pairs of primers / probes for transgenic maize nCX-1 provided in Example 3 of the present invention;

[0029] Figure 3 Graph of multiplex fluorescence PCR amplification of homozygous transgenic maize nCX-1 provided in Example 4 of the present invention;

[0030] Figure 4 Graph of multiplex fluorescence PCR amplification of heterozygous transgenic maize nCX-1 provided in Example 4 of the present invention;

[0031] Figure 5 Heat map of digital PCR analysis of the reference gene zSSIIb of homozygous transgenic maize nCX-1 provided in Example 5 of the present invention;

[0032] Figure 6 Heat map of digital PCR analysis of the transformant specificity of homozygous transgenic maize nCX-1 provided in Example 5 of the present invention;

[0033] Figure 7 Heat map of digital PCR analysis of the reference gene zSSIIb of heterozygous transgenic maize nCX-1 provided in Example 5 of the present invention;

[0034] Figure 8 Heat map of digital PCR analysis of the transformant specificity of heterozygous transgenic maize nCX-1 provided in Example 5 of the present invention. Detailed implementation manners

[0035] Unless otherwise defined herein, scientific and technical terms used in conjunction with the present invention shall have the meanings commonly understood by those of ordinary skill in the art. The meanings and scopes of the terms should be clear. However, in any case of potential ambiguity, the definitions provided herein shall prevail over any dictionary or extrinsic definition. In this application, unless otherwise specified, the use of "or" means "and / or". In addition, the use of the term "including" and other forms is non-restrictive.

[0036] In general, the nomenclature and the techniques used in connection with the cell and tissue culture, molecular biology, immunology, microbiology, genetics, and protein and nucleic acid chemistry and hybridization described herein are those well known and commonly used in the art. Unless otherwise indicated, the methods and techniques of the present invention are generally carried out according to conventional methods well known in the art and as described in various general and more specific references, which are cited and discussed throughout this specification. Enzymatic reactions and purification techniques are carried out according to the manufacturer's instructions, as commonly practiced in the art or as described herein. The nomenclature, as well as the laboratory procedures and techniques used in connection with the analytical chemistry, synthetic organic chemistry, and medical and pharmaceutical chemistry described herein, are those well known and commonly used in the art.

[0037] The technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are some, but not all, of the embodiments of the present invention. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without making creative efforts shall fall within the protection scope of the present invention.

[0038] On the one hand, the present invention provides a method for identifying homozygous and heterozygous transgenic maize nCX-1 based on multiplex fluorescence PCR, including performing multiplex fluorescence PCR amplification using the DNA of the sample to be tested as a template, and judging whether the sample to be tested is homozygous or heterozygous transgenic maize nCX-1 according to the target amplification curve.

[0039] The target amplification curve includes an internal reference gene amplification curve, a transformant-specific amplification curve, and a homozygosity identification amplification curve.

[0040] For the first time, the target amplification curve is obtained by using the multiplex fluorescence PCR technology, and a method for identifying homozygous and heterozygous transgenic maize nCX-1 is established; compared with the digital PCR technology, the multiplex fluorescence PCR technology has the same reliability and is faster, more efficient, and less costly; compared with the real-time fluorescence quantitative PCR method, the problems of inaccurate identification, the need to establish a standard curve in advance, and the need for calibration products in identifying homozygous or heterozygous transgenic organisms are solved; the method provided by the present invention lays a foundation for cross-breeding and the reproduction and identification of reference materials or raw materials, and ensures the authenticity and reliability of the material background.

[0041] In some specific embodiments, the judging whether the sample to be tested is homozygous or heterozygous transgenic maize nCX-1 according to the target amplification curve includes: if both the internal reference gene amplification curve and the transformant-specific amplification curve are typical amplification curves, and the homozygosity identification amplification curve is an atypical amplification curve, then the sample to be tested is homozygous; if the internal reference gene amplification curve, the transformant-specific amplification curve, and the homozygosity identification amplification curve are all typical amplification curves, then the sample to be tested is heterozygous.

[0042] In some specific embodiments, the primer pair for amplifying homozygosity identification includes nCX-1-5F and nCX-1-3R, and their nucleotide sequences are shown in SEQ ID NO.1 and SEQ ID NO.2; the probe for amplifying the amplification curve of homozygosity identification includes nCX-1-3P, and its nucleotide sequence is shown in SEQ ID NO.5.

[0043] In some specific embodiments, the PCR primer pair for amplifying the reference gene includes zSSIIb-F and zSSIIb-R, and their nucleotide sequences are shown in SEQ ID NO.7 and SEQ ID NO.8; the probe for amplifying the reference gene includes zSSIIb-P, and its nucleotide sequence is shown in SEQ ID NO.9.

[0044] In some specific embodiments, the PCR primer pair for amplifying transformant specificity includes nCX-1-5F and nCX-1-5R, and their nucleotide sequences are shown in SEQ ID NO.1 and SEQ ID NO.10; the probe for amplifying transformant specificity includes nCX-1-5P, and its nucleotide sequence is shown in SEQ ID NO.4.

[0045] In some specific embodiments, the molar ratio of nCX-1-5F, nCX-1-3R, zSSIIb-F, zSSIIb-R, and nCX-1-5R in the reaction system of the multiplex fluorescent PCR is 1:1:1:1:1. In some specific embodiments, the molar ratio of nCX-1-3P, zSSIIb-P, and nCX-1-5P is 1:1:1. In some specific embodiments, the final concentrations of nCX-1-5F, nCX-1-3R, zSSIIb-F, zSSIIb-R, and nCX-1-5R are all 0.4 μM; the final concentrations of nCX-1-3P, zSSIIb-P, and nCX-1-5P are all 0.2 μM.

[0046] In some specific embodiments, the amplification conditions of the multiplex fluorescent PCR include pre-denaturation at 95°C for 5 min; denaturation at 95°C for 15 sec; annealing and extension at 60°C for 60 sec, for a total of 40 cycles; 10 min at 98°C, storage at 4°C, and the heating and cooling rate is maintained at 2°C / sec.

[0047] According to another aspect of the present invention, there is also provided the application of the above method in the cross-breeding of transgenic maize nCX-1 and the identification of reference substances or raw materials.

[0048] The present invention will be further described below by way of examples. Unless otherwise specified, the materials in the examples are prepared according to existing methods or directly purchased from the market.

[0049] Example 1 Obtaining Heterozygous Transgenic Maize nCX-1 Seeds

[0050] Seeds of transgenic maize nCX-1 known to be homozygous and its non-transgenic control were provided by the Zhejiang Academy of Agricultural Sciences. The seeds of homozygous transgenic maize nCX-1 and its non-transgenic control were planted in the test base. Heterozygous transgenic maize nCX-1 seeds were obtained by crossing the homozygous transgenic maize nCX-1 with its non-transgenic control. The harvested seeds were used for subsequent experiments.

[0051] Example 2 DNA Extraction

[0052] Seeds of homozygous transgenic maize nCX-1 and heterozygous transgenic maize nCX-1 prepared in Example 1 were separately selected and ground into powder samples one by one. DNA was extracted according to the instructions of the new plant genome extraction kit. The concentration and quality of DNA were measured using an ND8000 spectrophotometer, and its integrity was determined by agarose gel electrophoresis.

[0053] Example 3 Obtaining Homozygosity Identification Primers and Probes

[0054] Based on the plant genome sequence information at the 5' end and 3' end of the exogenous insertion fragment of transgenic maize nCX-1, the primer design software Primer Premier 5.0 was used to design multiple primers and probes for homozygosity identification. The information of the primers and probes is shown in Table 1, and the specific positions of the primers are as Figure 1 shown.

[0055] According to the general requirements of the guidelines for the real-time fluorescence PCR method, 6 pairs of primer-probe combinations were obtained by combining real-time fluorescence PCR primers / probes, namely nCX-1-5F / nCX-1-3R / nCX-1-3P, nCX-1-5F / nCX-1-3R1 / nCX-1-3P, nCX-1-5F / nCX-1-3R / nCX-1-5P, nCX-1-5F / nCX-1-3R1 / nCX-1-5P, nCX-1-5F / nCX-1-3R / nCX-1-3P1, nCX-1-5F / nCX-1-3R1 / nCX-1-3P1. Using the genomic DNA of heterozygous transgenic maize nCX-1 as a template, the primer / probe combination screening was carried out with reference to the real-time fluorescence PCR amplification system and reaction conditions described in Announcement No. 1861-3-2012 of the Ministry of Agriculture and Rural Affairs for the detection of maize reference genes. After testing, as Figure 2The amplification curves of the primer combination nCX-1-5F / nCX-1-3R / nCX-1-3P are S-shaped, with relatively small Ct values, strong amplification signals, and the best amplification effect.

[0056] Table 1 Screening of primers / probes for homozygosity identification

[0057]

[0058] Example 4 Identification of nCX-1 homozygosity using multiplex fluorescence PCR

[0059] The primers for real-time fluorescence PCR detection of the maize reference gene zSSIIb are the primers in Announcement No. 1861-3-2012 of the Ministry of Agriculture and Rural Affairs, with an amplified fragment length of 88 bp. The 5' end of its probe is labeled with the fluorescent group HEX, and the 3' end is labeled with BHQ1. The sequences of the primers and probe of the reference gene are as follows:

[0060] zSSIIb-F: 5'-CGGTGGATGCTAAGGCTGATG-3' (SEQ ID NO.7)

[0061] zSSIIb-R: 5'-AAAGGGCCAGGTTCATTATCCTC-3' (SEQ ID NO.8)

[0062] zSSIIb-P: 5'-HEX-TAAGGAGCACTCGCCGCCGCATCTG-3'-BHQ1 (SEQ ID NO.9)

[0063] The primers for real-time fluorescence PCR detection of the transgenic maize nCX-1 transformant specificity are the primers in Announcement No. 864-2-2024 of the Ministry of Agriculture and Rural Affairs, with an amplified fragment length of 87 bp. The 5' end of its probe is labeled with the fluorescent group FAM, and the 3' end is labeled with BHQ1. The sequences of the transformant-specific detection primers and probe are as follows:

[0064] nCX-1-5F: 5'-CGCTAGTTCATCCTCTGACTGGTA-3' (SEQ ID NO.1)

[0065] nCX-1-5R: 5'-CTAAGCGTCAATTTGTTTACACCAC-3' (SEQ ID NO.10)

[0066] nCX-1-5P: 5'-FAM-CGACCATACCAGTTGCTCAGGTGCCT-3'-BHQ1 (SEQ ID NO.4)

[0067] Select the homozygosity identification primers nCX-1-5F / nCX-1-3R / nCX-1-3P of the verified transgenic maize nCX-1 in Example 3. The target fragment amplified by this primer is 104 bp.

[0068] Multiplex fluorescence PCR system: The 20 μL reaction system includes 10 μL FastStar Essential DNA Probes Master, 2 μL genomic DNA. Add the primers and probes for each target to make the final concentrations of the primers for amplifying and detecting the internal reference gene, transformant specificity, and homozygosity identification all reach 400 nM, and the final concentrations of the probes all reach 200 nM. Pipette the 20 μL reaction system of the sample to be tested into an eight-strip tube, tighten the tube cap and centrifuge for 15 S, and place it in a real-time fluorescence PCR instrument for PCR amplification.

[0069] The PCR amplification program is: pre-denaturation at 95 °C for 5 min; denaturation at 95 °C for 15 sec; annealing and extension at 60 °C for 60 sec, for a total of 40 cycles; 10 min at 98 °C, and store at 4 °C, with the heating and cooling rate maintained at 2 °C / sec.

[0070] Using the homozygous transgenic maize nCX-1 with a DNA amount of 50 ng as a template, amplify by the established multiplex fluorescence PCR method. The results are as Figure 3 shown, which are the amplification curves of three replicates of the homozygous transgenic maize nCX-1. The internal reference gene and transformant specificity of the homozygous sample both obtained typical amplification curves, while the homozygosity identification did not amplify a typical amplification curve.

[0071] Using the heterozygous transgenic maize nCX-1 with a DNA amount of 50 ng as a template, amplify by the established multiplex fluorescence PCR method. The results are as Figure 4 shown, which are the amplification curves of three replicates of the heterozygous transgenic maize nCX-1. The heterozygous sample amplified typical amplification curves of the internal reference gene, transformant specificity, and homozygosity, and the repeatability of the PCR results is good.

[0072] Accuracy verification in Example 5

[0073] Adopt the digital PCR method to verify the results of the established multiplex fluorescence PCR homozygosity identification method.

[0074] The primers for quantitative detection of the maize zSSIIb gene are the primers in Announcement No. 1861-3-2012 of the Ministry of Agriculture and Rural Affairs. The amplified fragment length is 88 bp. The 5' end of the probe is labeled with the fluorescent group FAM, and the 3' end is labeled with BHQ1. The sequences of the primers and probes for the internal reference gene are as follows:

[0075] zSSIIb-F: 5'-CGGTGGATGCTAAGGCTGATG-3' (SEQ ID NO.7)

[0076] zSSIIb-R: 5'-AAAGGGCCAGGTTCATTATCCTC-3' (SEQ ID NO.8)

[0077] zSSIIb-P: 5'-FAM-TAAGGAGCACTCGCCGCCGCATCTG-3'-BHQ1

[0078] (SEQ ID NO.9)

[0079] The primers for the transformant-specific real-time fluorescence PCR detection of transgenic maize nCX-1 are the primers in "Announcement No. 864-2-2024 of the Ministry of Agriculture and Rural Affairs", with an amplified fragment length of 87 bp. The 5' end of its probe is labeled with the fluorescent group FAM, and the 3' end is labeled with BHQ1. The sequences of the transformant-specific detection primers and probe are as follows:

[0080] nCX-1-5F: 5'-CGCTAGTTCATCCTCTGACTGGTA-3' (SEQ ID NO.1)

[0081] nCX-1-5R: 5'-CTAAGCGTCAATTTGTTTACACCAC-3' (SEQ ID NO.10)

[0082] nCX-1-5P: 5'-FAM-CGACCATACCAGTTGCTCAGGTGCCT-3'-BHQ1 (SEQ ID NO:4)

[0083] Digital PCR system: The 20 μL reaction system includes 10 μL of 2x ddPCR Supermix for probes, 2 μL of genomic DNA. Primers and probes are added to make their final concentrations reach 400 nM and 200 nM. The sample (20 μL) reaction system is added to 8 wells in the middle row of the DG8 cartridge using a laboratory general pipette. 70 μL of droplet generation oil is added to each of the 8 wells in the lowest row of the DG8 cartridge, and droplet generation is automatically completed in the droplet generator. The generated droplets are transferred to a 96-well plate, sealed with a heat-sealing film, and placed in a conventional PCR instrument for PCR.

[0084] The PCR amplification program is: pre-denaturation at 95°C for 10 min; denaturation at 95°C for 30 sec; annealing and extension at 60°C for 45 sec, for a total of 40 cycles; 10 min at 98°C, and preservation at 4°C, with the heating and cooling rate maintained at 2°C / sec.

[0085] Using the heterozygous and homozygous transgenic maize nCX-1 with a DNA amount of 10 ng as templates respectively, digital PCR amplification was carried out with the maize internal standard gene zSSIIb and the nCX-1 transformant-specific primers and probes. The heat map of the digital PCR analysis is as Figures 5 to 8 shown. The results are shown in Table 2. The copy number ratio of the foreign gene to the internal reference gene in the homozygous sample is close to 1.0; the copy number ratio of the foreign gene to the internal reference gene in the heterozygous sample is close to 0.5. Using the identification results of digital PCR, the accuracy of the multiplex fluorescence PCR homozygosity identification method was verified.

[0086] Table 2 Comparison of the identification results between digital PCR and multiplex fluorescence PCR

[0087]

[0088] Among them, "+" indicates that the amplification curve is in an "S" shape, which is a typical amplification curve, and "-" indicates that the amplification curve is not in an "S" shape, which is an atypical amplification curve.

[0089] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those of ordinary skill in the art should understand that they can still modify the technical solutions described in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A method for identifying homozygous and heterozygous transgenic corn nCX-1 based on multiplex fluorescence PCR, characterized in that: The method includes performing multiple fluorescence PCR amplification using the DNA of the sample to be tested as a template, and judging whether the sample to be tested is a homozygous or heterozygous transgenic corn nCX-1 according to the target amplification curve; The target amplification curve includes an internal reference gene amplification curve, a transformant-specific amplification curve and a homozygosity identification amplification curve.

2. The method according to claim 1, characterized in that The method of judging whether the sample to be tested is a homozygous or heterozygous transgenic corn nCX-1 according to the target amplification curve comprises: If the internal reference gene amplification curve and the transformant-specific amplification curve are both typical amplification curves, and the homozygous identification amplification curve is an atypical amplification curve, the sample to be tested is homozygous; If the internal reference gene amplification curve, the transformant-specific amplification curve, and the homozygous identification amplification curve are all typical amplification curves, the sample to be tested is heterozygous.

3. The method according to claim 2, characterized in that The primer pair used for amplification homozygosity identification includes nCX-1-5F and nCX-1-3R, whose nucleotide sequences are shown in SEQ ID NO.1 and SEQ ID NO.2; The probe used for amplifying the homozygous identification amplification curve includes nCX-1-3P, whose nucleotide sequence is shown in SEQ ID NO.

5.

4. The method according to claim 3, characterized in that The PCR primer pair used to amplify the internal reference gene includes zSSIIb-F and zSSIIb-R, whose nucleotide sequences are shown in SEQ ID NO.7 and SEQ ID NO.8; The probe used to amplify the internal reference gene includes zSSIIb-P, whose nucleotide sequence is shown in SEQ ID NO.

9.

5. The method according to claim 4, characterized in that The PCR primer pair used to amplify the transformant specificity includes nCX-1-5F and nCX-1-5R, whose nucleotide sequences are shown in SEQ ID NO.1 and SEQ ID NO.10; The probe used for amplifying the transformant specificity includes nCX-1-5P, whose nucleotide sequence is shown in SEQ ID NO.

4.

6. The method according to claim 5, characterized in that The molar ratio of nCX-1-5F, nCX-1-3R, zSSIIb-F, zSSIIb-R and nCX-1-5R in the reaction system of the multiplex fluorescence PCR is 1:1:1:1:

1.

7. The method according to claim 6, characterized in that The molar ratio of nCX-1-3P, zSSIIb-P and nCX-1-5P was 1:1:

1.

8. The method according to claim 7, characterized in that The final concentrations of nCX-1-5F, nCX-1-3R, zSSIIb-F, zSSIIb-R and nCX-1-5R were all 0.4 μM; The final concentrations of nCX-1-3P, zSSIIb-P and nCX-1-5P were all 0.2 μM.

9. The method according to claim 5, characterized in that The amplification conditions of the multiplex fluorescent PCR include pre-denaturation at 95°C for 5 minutes; denaturation at 95°C for 15 seconds; annealing and extension at 60°C for 60 seconds, a total of 40 cycles; 98°C for 10 minutes, storage at 4°C, and a heating and cooling rate of 2°C / second.

10. Use of the method according to any one of claims 1 to 9 in hybrid breeding of transgenic corn nCX-1 and identification of standard substances or raw materials.

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