Primer probe composition and method for on-site detection of transgenic maize transformant

Abstract

The invention discloses a primer probe combination and a kit for on-site detection of a transgenic maize transformant, and relates to the technical field of transgenic detection. The invention provides a primer probe combination for on-site detection of a transgenic maize transformant. The primer probe combination comprises a primer pair and a probe for detecting a transgenic maize strain MON810, the primer pair and the probe are used for detecting the transgenic maize strain TC1507; the primer pair and the probe are used for detecting the transgenic maize strain Bt11; the invention discloses a primer pair and a probe for detecting corn internal reference zSSIIb. The method for detecting the transgenic maize by adopting the primer probe combination and the kit disclosed by the invention integrates sample pretreatment, nucleic acid extraction, purification, amplification and detection, has better sensitivity and accuracy, is simple, convenient, rapid and time-saving to operate, and plays an important role in on-site rapid detection of transgenic maize research and safety management.

Description

Technical Field

[0001] The present invention relates to the technical field of transgenic detection, and in particular to a primer-probe combination and a kit for on-site detection of transgenic corn lines MON810, TC1507 and Bt11. Background Art

[0002] GM corn is a genetically modified crop. Specific corn varieties have been genetically modified to exhibit desirable agricultural traits, including insect and herbicide resistance. Currently, several countries are growing corn varieties that combine these traits. GM corn has also sparked controversy over its potential health effects, potential impacts on other insects, and potential impacts on other plants through gene flow.

[0003] The genetically modified corn line MON810 was developed by Monsanto Far East Ltd. Monsanto used a gene gun to introduce the Cry1Ab gene from Bacillus thuringiensis into the maize genome, resulting in insect-resistant MON810. MON810 protects against lepidopteran pests such as the European corn borer (ECB, Ostrinia furnacalis), the Southwestern corn borer (SWCB, Diatraea grandiosella), and the Asian corn borer (ACB, Ostrinia furnacalis).

[0004] The transgenic corn line TC1507, jointly developed by DuPont China Group Co., Ltd. and Dow AgroSciences China Ltd., was developed by Pioneer using transgenic corn plants from Mycogen that harbor the Cry1F gene. The Cry1F gene comes from Bacillus thuringiensis, a common Gram-positive soil microorganism that produces an insecticidal protein that is highly selective against specific organisms.

[0005] The transgenic corn line Bt11 was developed by Syngenta. It is a transgenic corn variety with both insect resistance and herbicide resistance. The corn was introduced with the Cry1Ab gene and pat gene. The Cry1Ab comes from Bacillus thuringiensis and encodes the Cry1Ab insecticidal protein, which can effectively resist lepidopteran pests such as corn borer; the pat gene comes from Streptomyces chlorogenes and encodes glufosinate acetyltransferase, which gives the corn the characteristic of glufosinate herbicide resistance.

[0006] Regarding nucleic acid detection of genetically modified corn, the main shortcomings of existing technologies for detecting genetically modified corn include: 1) the operation process is cumbersome, requiring separate steps such as sample pretreatment, DNA extraction, and PCR amplification; 2) high requirements for laboratory conditions and operator skills; 3) long detection cycle (usually 2-3 hours); 4) inability to achieve rapid on-site detection.

[0007] Based on the above problems, the inventors have developed for several years and disclosed a new type of on-site rapid nucleic acid detection equipment in the prior application CN202110057507.0. The equipment is a "handheld real-time fluorescence PCR all-in-one machine P1000F". After temperature control optimization and upgrading, it can complete rapid nucleic acid detection within 35 minutes, and can achieve on-site automated detection of "sample in, result out". In order to solve the problem of sample pre-treatment, the inventors have developed a sample processing tube (ZL202320319535.X) that is easy to operate and fast. On the basis of sample processing and detection equipment, the present invention further develops an invention that can better cooperate with the equipment, namely a primer probe combination for the joint detection of three genetically modified corn lines and an integrated closed microfluidic chip kit and application. The kit performs nucleic acid extraction, purification, amplification and detection of samples on an integrated chip, which is applied to rapid nucleic acid detection on site. Summary of the Invention

[0008] The purpose of the present invention is to provide a primer probe combination and a kit for on-site detection of transgenic corn transformants. The primer probe combination and the kit of the present invention are used to detect transgenic corn with good sensitivity and accuracy. The method is simple to operate, fast and time-saving.

[0009] In order to achieve the above-mentioned object of the invention, the technical solution of the present invention is as follows:

[0010] In one aspect, the present invention provides a primer-probe combination for on-site detection of transgenic maize transformants, the primer-probe combination comprising: primer pairs and probes for detecting the sequences of the transgenic maize line MON810, as shown in SEQ ID NOs: 1-3, respectively; primer pairs and probes for detecting the sequences of the transgenic maize line TC1507, as shown in SEQ ID NOs: 4-6, respectively; primer pairs and probes for detecting the sequences of the transgenic maize line Bt11, as shown in SEQ ID NOs: 7-9, respectively; and primer pairs and probes for detecting the sequences of the maize internal reference zSSIIb, as shown in SEQ ID NOs: 10-12, respectively.

[0011] Specifically, the 5' end of the probe is labeled with a fluorescent reporter group, and the 3' end of the probe is labeled with a quencher group.

[0012] More specifically, the fluorescent reporter group is selected from one or more of FAM, ROX, HEX, CY5, VIC, TET, JOE, CY3, CY7, RED610, Texas Red, RED670, NED, AMCA, Pacific Blue, ATTO 425, BODIPY FL, Alexa Fluor488, Yakima Yellow, Quasar 570, Aqua Phluor593, ATTO 590, and CY5.5.

[0013] More specifically, the fluorescent reporter group is selected from one or more of ATTO425, FAM, HEX, CY3, CY5, CY5.5, VIC, JOE, and ROX.

[0014] More specifically, the quencher group is a fluorescence quencher group.

[0015] More specifically, the fluorescence quenching group is selected from one or more of 6-TAMRA, BHQ1, BHQ2, BHQ3, Dabcyl, Eclipse, MGB, QYS-7, and SQ1.

[0016] More specifically, the fluorescence quenching group is selected from one or more of BHQ2, BHQ3, and SQ1.

[0017] In another aspect, the present invention provides the use of the primer-probe combination described above in preparing products of transgenic corn line MON810, transgenic corn line TC1507 and transgenic corn line Bt11.

[0018] Specifically, the products include independent detection reagents, chips and test kits.

[0019] In another aspect, the present invention provides a kit for on-site detection of transgenic corn transformants, the kit comprising the above-mentioned primer-probe combination.

[0020] Specifically, the kit is an integrated closed microfluidic chip kit.

[0021] More specifically, the integrated closed microfluidic chip kit includes purification reagents and air-dried qPCR reagents.

[0022] Specifically, the purified reagents and air-dried qPCR reagents can be transported and stored at room temperature.

[0023] More specifically, the purification reagent includes a lysis solution, a washing solution, an eluent and magnetic beads, the lysis solution and the washing solution include guanidine hydrochloride, sodium acetate, Triton X-100 and 1,3-butanediol, the eluent includes Tris-HCl, and the magnetic beads are dried magnetic beads.

[0024] More specifically, the lysis solution and washing solution include 4.2M guanidine hydrochloride, 0.28M sodium acetate (pH 4.7), 1.4% Triton X-100 and 30% 1,3-butanediol, and the eluent includes 10mM Tris-HCl (pH 8.5).

[0025] Specifically, the kit completes the nucleic acid extraction, purification, amplification and detection of the sample on an integrated chip.

[0026] Specifically, the microfluidic chip kit can be used for the P1000F rapid nucleic acid detection device.

[0027] In another aspect, the present invention provides a method for on-site detection of transgenic corn transformants, the method comprising preparation of an integrated closed microfluidic chip kit, sample pre-treatment, and rapid device detection;

[0028] The method specifically described comprises the following steps:

[0029] (1) preparing the above primer-probe combination into an integrated closed microfluidic kit;

[0030] (2) adding samples of the transgenic corn line MON810, the transgenic corn line TC1507, and the transgenic corn line Bt11 into a sample processing tube for processing to obtain a liquid to be tested;

[0031] (3) dropping the liquid to be tested into the integrated closed microfluidic reagent kit;

[0032] (4) Place the test kit containing the liquid to be tested on the testing equipment for testing and analysis.

[0033] Specifically, the step (2) is as follows: adding a sample pre-treatment solution and abrasive particles into a sample processing tube; the sample pre-treatment solution includes Tris-HCl (pH 8.0), NaCl, EDTA and SDS;

[0034] More specifically, the concentration range of Tris-HCl is 10mM-100mM, the concentration range of NaCl is 30mM-1.4M, the concentration range of EDTA is 1.5mM-20mM, and the concentration range of SDS is 0.05%-2%; the diameter of the grinding particles is 0.1 micron-1 mm.

[0035] More specifically, the concentration of the sample pretreatment solution Tris-HCl (pH 8.0) ranges from 30 mM to 55 mM, the concentration of NaCl ranges from 350 mM to 700 mM, the concentration of EDTA ranges from 5 mM to 10 mM, and the concentration of SDS ranges from 0.5% to 1%.

[0036] Specifically, the sample in step (2) is the seeds, leaves or products thereof of the transgenic corn line MON810, the transgenic corn line TC1507 and the transgenic corn line Bt11.

[0037] Specifically, the step (4) includes:

[0038] 1) Turn on the rapid detection device;

[0039] 2) Scan the QR code;

[0040] 3) Insert the integrated microfluidic chip kit;

[0041] 4) Run the test;

[0042] 5) End and check the test results and amplification curve.

[0043] According to some embodiments of the present invention, the method comprises the following steps:

[0044] (1) preparing an integrated closed microfluidic kit by combining the above primers and probes;

[0045] (2) Rapid sample pre-processing using a sample processing tube;

[0046] (3) Using purification reagents on the chip to lyse the sample and extract and purify nucleic acids;

[0047] (4) using the primer-probe combination to rapidly amplify the sample nucleic acid extracted and purified in step (3);

[0048] (5) Analyze the amplification results.

[0049] In another aspect, the present invention provides use of the above-mentioned kit or method in detecting components of transgenic corn line MON810, transgenic corn line TC1507, transgenic corn line Bt11 and their derivatives, and products thereof.

[0050] The beneficial effects of the present invention are:

[0051] The present invention provides a primer-probe combination, an integrated microfluidic chip kit, and a method for detecting genetically modified corn. This method, using the primer-probe combination and kit described herein, integrates sample pretreatment, nucleic acid extraction, purification, amplification, and detection, offering high sensitivity and accuracy. The method is simple, rapid, and time-saving. The method can simultaneously detect three transformants: MON810, TC1507, and Bt11, and plays an important role in rapid on-site detection for genetically modified corn research and safety management. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 This is the sensitivity test result of genetically modified corn MON810.

[0053] Figure 2 This is the sensitivity test result of genetically modified corn TC1507.

[0054] Figure 3 This is the sensitivity test result of genetically modified corn Bt11.

[0055] Figure 4 This is a physical picture of the integrated microfluidic chip and sample processing tube.

[0056] Figure 5 This is the detection result of the real sample.

[0057] Figure 6 This is the detection result of the real sample.

[0058] Figure 7 This is the test results of genetically modified corn MON810, genetically modified corn TC1507 and genetically modified corn Bt11. DETAILED DESCRIPTION

[0059] In order to make the technical means, creative features, purpose and efficacy of the present invention easy to understand, the present invention is further illustrated below in conjunction with specific examples, but the following examples are only preferred embodiments of the present invention, not all. Based on the examples in the implementation manner, other embodiments obtained by those skilled in the art without making creative work are all within the scope of protection of the present invention. In the following examples, unless otherwise specified, the operating methods used are all conventional operating methods, the equipment used are all conventional equipment, and the equipment and materials used in each embodiment are all the same.

[0060] The microfluidic chip device for nucleic acid detection of the present invention is detailed in patent 202110055537.8, the magnetic bead drying method is detailed in patent 202110222434.6, the sample processing tube is detailed in patent 202320319535.X, and the overall device, i.e., the PCR reaction device, is detailed in patent 202110057507.0. The actual picture of the integrated microfluidic chip and sample processing tube is as follows Figure 4 shown.

[0061] The transgenic corn MON810 in the following examples was developed by Monsanto Far East Ltd.

[0062] The genetically modified corn TC1507 was jointly developed by DuPont China Group Co., Ltd. and Dow AgroSciences China Co., Ltd.

[0063] The genetically modified corn Bt11 was developed by Syngenta.

[0064] Example 1 Primer probe combination for rapid on-site detection of transgenic corn MON810, TC1507, and Bt11 transformants

[0065] Primer and probe sequences were designed based on the specific sequences of three transgenic maize transformants: MON810, TC1507, and Bt11.

[0066] Among them, the primer and probe sequences of the transgenic corn MON810 transformant are:

[0067] MON810-F:5'-TTCCCTCTGGCCGCT-3' (SEQ ID NO:1);

[0068] MON810-R:5'-CTGACTGCTCGCAAGCA-3' (SEQ ID NO:2);

[0069] MON810-P: 5'-CAGCCCACATCGATGTCCAAGGA-3' (SEQ ID NO: 3).

[0070] The primer and probe sequences for the transgenic maize TC1507 transformant are:

[0071] TC1507-F:5'-GCGCCTCTAGTTGAAGACAC-3' (SEQ ID NO:4);

[0072] TC1507-R:5'-GAAAATTAACACATACTAAATAAATGCGTCA-3' (SEQ ID NO:5);

[0073] TC1507-P: 5'-CCTCACTCCGCTTGATCTTGGCCAA-3' (SEQ ID NO: 6).

[0074] The primer and probe sequences for transgenic maize Bt11 transformants are:

[0075] Bt11-F:5'-TAATGGTTTCTTAGACGTCAGGTG-3' (SEQ ID NO:7);

[0076] Bt11-R:5'-TTTCAATAATACTAGAGGCTAACACCTACAG-3' (SEQ ID NO:8);

[0077] Bt11-P: 5'-AAACAAATAGGGGTTCCGCGCACA-3' (SEQ ID NO: 9).

[0078] The primer and probe sequences for the maize endogenous gene zSSIIb are:

[0079] zSSIIb-F:5'-CGGTGGATGCTAAGGCTGATG-3' (SEQ ID NO: 10);

[0080] zSSIIb-R:5'-AAAGGGCCAGGTTCATTATCCTC-3' (SEQ ID NO: 11);

[0081] zSSIIb-P: 5'-TAAGGAGCACTCGCCGCCGCATCTG-3' (SEQ ID NO: 12).

[0082] The primer-probe combination for transgenic corn of the present invention is used for jointly or individually detecting three transformants of transgenic corn MON810, TC1507 and Bt11.

[0083] In the embodiment, the zSSIIb probe is labeled with ATTO 425 and the quencher group is SQ1, the MON810 probe is labeled with FAM and the quencher group is SQ1, the TC1507 probe is labeled with HEX and the quencher group is SQ1, and the Bt11 probe is labeled with CY5 and the quencher group is BHQ2.

[0084] Example 2 A microfluidic chip kit for detecting genetically modified corn

[0085] (1) The kit includes the primer-probe combination described in Example 1, with final concentrations as shown in Table 1 below.

[0086] Table 1 Primer and probe concentrations

[0087]

[0088]

[0089] (2) The kit also includes purification reagents and air-dried reagents.

[0090] 1) The purification reagent includes 450 μL of lysis solution, 450 μL of washing solution and 850 μL of eluent, and 15 μL of magnetic beads. The lysis solution and washing solution include 4.2 M guanidine hydrochloride, 0.28 M pH 4.7 sodium acetate, 1.4% Triton X-100 and 30% 1,3-butanediol. The eluent includes 10 mM pH 8.5 Tris-HCl. The purification reagent is added to the sample loading layer in the chip. The magnetic bead drying method is described in patent 202110222434.6.

[0091] 2) The 50 μL air-dried reagent system is shown in Table 2 below.

[0092] Table 2 Air-drying reagent system

[0093] Element volume 4×Air-Dryable qPCR Mix 12.5μL Primer probe mixture 2.5 μL

[0094] Add the reagents in Table 2 above to the reaction chamber of the integrated microfluidic chip, place the chip in an air drying oven, and air-dry according to the instructions of the 4× Air-Dryable qPCR Mix.

[0095] Example 3 Preparation of sample processing tube

[0096] (1) Install the sample processing tube;

[0097] (2) Add about 1 g of diamond abrasive to a tube and add 3 mL of sample pretreatment solution (50 mM Tris-HCl (pH 8.0), 700 mM NaCl, 10 mM EDTA, 1% SDS) to the tube;

[0098] (3) Place the tube in the tooling position of a precision manual press and wrap it with aluminum foil at 160°C.

[0099] Example 4: Detection Method for Genetically Modified Corn

[0100] (1) Remove the aluminum foil protective film from the sample processing tube, place it upright, add 100 mg of the leaf or seed sample to be tested into the sample processing tube, and tighten the tube cap;

[0101] (2) Keep the tube upright and rub it 5-10 times to ensure that the sample is fully in contact with the grinding material and that the grinding is sufficient;

[0102] (3) Open the outer packaging, take out the reagent chamber and reaction layer of the chip, tear off the sealing strip on the back of the reagent chamber, hold the reagent chamber and reaction layer so that the head of the reaction layer is vertically upward, press hard to puncture the reagent chamber until the upper and lower layers of the chip are seamlessly connected, and complete the assembly;

[0103] (4) Unscrew the sample chamber cover, add 2-3 drops (50 μL) of sample grinding solution into the sample chamber, and tighten the sample chamber cover;

[0104] (5) Press and hold the device power button for more than 3 seconds to turn on the device, and click the "Test" button on the homepage;

[0105] (6) After waiting for 3 seconds, you will see the red light flashing in the scanning window and scan the chip QR code;

[0106] (7) Scan the sample information or skip according to the on-screen prompts;

[0107] (8) Follow the on-screen instructions to open the hatch, insert the chip, and close the hatch;

[0108] (9) Click the "Run" button to start the test;

[0109] (10) After the operation is completed, check the test results.

[0110] Sample processing in the sample tube takes approximately one minute; nucleic acid extraction and purification on the integrated microfluidic chip takes approximately 12 minutes; and the real-time quantitative PCR reaction protocol on the integrated microfluidic chip is: 95°C for 1 minute; (95°C for 5 seconds, 60°C for 8 seconds), 45 cycles, for amplification time of 19 minutes. The entire process of genetically modified corn testing, from sample processing, nucleic acid extraction and purification, to amplification and detection results, takes approximately 32 minutes.

[0111] Experimental Example 1 Sensitivity Detection

[0112] Samples with MON810, TC1507, and Bt11 transformant contents of 4%, 0.4%, and 0.1% (calculated based on 50 μL of sample grinding solution for 100 mg of standard) were prepared for nucleic acid amplification detection. The system components are shown in Table 2, and the test results are shown in Table 3 below. The detection curve is shown in Figure 1-Figure 3 shown.

[0113] Table 3 Sensitivity test results

[0114]

[0115] As shown in Table 3 above, the primer combination and kit described in this application have good sensitivity and can detect MON810, TC1507, and Bt11 transformants with a content of 0.1% (calculated based on 50 μL sample grinding solution for 100 mg standard).

[0116] Experimental Example 2 Specificity Detection

[0117] The negative control (deionized water), non-transgenic corn seeds (1 grain), DP4114 corn seeds (1 grain), DBN9936 corn seeds (1 grain), DBN9858 corn standard (100 mg), GTS40-3-2 soybean standard (100 mg), DBN9004 soybean standard (100 mg), and MS1RF2 rapeseed standard (100 mg) were tested according to the above method. The specificity test results are shown in Table 4 below.

[0118] Table 4 Specificity detection results

[0119] sample Corn Reference Target detection results NTC — Negative Non-GMO corn seeds 30.79 Negative DP4114 corn seeds 30.12 Negative DBN9936 corn seeds 29.06 Negative DBN9858 corn standard 32.63 Negative GTS40-3-2 soybean standard — Negative DBN9004 soybean standard — Negative MS1RF2 rapeseed standard — Negative

[0120] As shown in Table 4, the primer-probe combination and the kit thereof of the present invention have good specificity.

[0121] Experimental Example 4: Testing of actual leaf and seed samples

[0122] MON810 corn leaves (about 100 mg), TC1507 corn leaves (about 100 mg), Bt11 corn leaves (about 100 mg), MON810 corn seeds (1 grain), TC1507 corn seeds (1 grain), and Bt11 corn seeds (1 grain) were used for the experiment.

[0123] The above-extracted samples were tested according to the system and steps of Examples 1-4. The test results are shown in Table 5 below. Figure 5-6 shown.

[0124] Table 5 Test results of leaf and seed samples

[0125] sample Corn Reference Target Ct value Test results MON810 corn leaves 28.35 29.51 Positive TC1507 corn leaves 25.63 32.13 Positive Bt11 corn leaves 29.36 30.66 Positive MON810 corn seeds 29.80 31.55 Positive TC1507 corn seeds 27.69 32.01 Positive Bt11 corn seeds 29.97 32.77 Positive

[0126] Table 5 shows that the transgenic maize primer-probe combination of the present invention can be used to detect real sample leaves and seeds, and can be used for rapid identification of maize transformants in the field.

[0127] Comparative Example 1

[0128] The difference between Comparative Example 1 and Examples 1-4 and Experimental Example 1 is only the difference in the primer and probe sequences, and all other aspects are the same. The primer and probe sequences of Comparative Example 1 are as follows:

[0129] The primer and probe sequences for the maize endogenous gene zSSIIb are:

[0130] zSSIIb-F:5'-CGGTGGATGCTAAGGCTGATG-3' (SEQ ID NO: 10);

[0131] zSSIIb-R:5'-AAAGGGCCAGGTTCATTATCCTC-3' (SEQ ID NO: 11);

[0132] zSSIIb-P: 5'-TAAGGAGCACTCGCCGCCGCATCTG-3' (SEQ ID NO: 12).

[0133] The primer and probe sequences for the transgenic maize MON810 transformants are:

[0134] MON810-F:5'-CACCACAGCCACCACTTCTC-3' (SEQ ID NO:13);

[0135] MON810-R:5'-ATTCGGAAATGAAAGAAGGCTACC-3' (SEQ ID NO:14);

[0136] MON810-P: 5'-CTCGTTCAGGTCGGTGCAGCCCA-3' (SEQ ID NO: 15).

[0137] The primer and probe sequences for the transgenic maize TC1507 transformant are:

[0138] TC1507-F:5'-TAGCTTCGGCCAGAATGG-3' (SEQ ID NO:16);

[0139] TC1507-R:5'-CTTTGCCAAGATCAAGCG-3' (SEQ ID NO:17);

[0140] TC1507-P: 5'-TAACTCAAGGCCCTCACTCCG-3' (SEQ ID NO: 18).

[0141] The primer and probe sequences for transgenic maize Bt11 transformants are:

[0142] Bt11-qF:5'-TGTGTGGCCATTTATCATCGAC-3' (SEQ ID NO: 19);

[0143] Bt11-qR:5'-GGGATCTCAAGAAGATCCTTTGATC-3'(SEQ ID NO:20);

[0144] Bt11-qP: 5'-ACGGGGTCTGACGCTCAGTGGA-3' (SEQ ID NO: 21).

[0145] The experimental results of Comparative Example 1 are as follows Figure 7 As shown in the figure, the standards of MON180, TC1507 and Bt11 were added respectively. It can be seen from the figure that the amplification of the Bt11 primer probe is poor, the primer probe of TC1507 shows an obvious delay, and the primer probes of MON810 and Bt11 show non-specific amplification and poor specificity.

[0146] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A primer-probe combination for on-site detection of transgenic corn transformants, characterized in that: The primer-probe combination includes: primer pairs and probes for detecting the sequence of the transgenic corn line MON810 as shown in SEQ ID NOs: 1-3, respectively; primer pairs and probes for detecting the sequence of the transgenic corn line TC1507 as shown in SEQ ID NOs: 4-6, respectively; primer pairs and probes for detecting the sequence of the transgenic corn line Bt11 as shown in SEQ ID NOs: 7-9, respectively; and primer pairs and probes for detecting the sequence of the corn internal reference zSSIIb as shown in SEQ ID NOs: 10-12, respectively.

2. Use of the primer-probe combination according to claim 1 in the preparation of a product for detecting transgenic corn line MON810, transgenic corn line TC1507 and transgenic corn line Bt11.

3. The use according to claim 2, characterized in that The products include independent detection reagents, chips and test kits.

4. A kit for on-site detection of transgenic corn transformants, characterized in that: The kit comprises the primer-probe combination according to claim 1.

5. The kit according to claim 4, characterized in that The test kit is an integrated closed microfluidic chip test kit.

6. The kit according to claim 5, characterized in that The integrated closed microfluidic chip kit includes purification reagents and air-dried qPCR reagents.

7. A method for on-site detection of transgenic corn transformants, characterized in that: The method comprises the following steps: (1) preparing the primer-probe combination described in claim 1 into an integrated closed microfluidic kit; (2) adding samples of the transgenic corn line MON810, the transgenic corn line TC1507, and the transgenic corn line Bt11 into a sample processing tube for processing to obtain a liquid to be tested; (3) dropping the liquid to be tested into the integrated closed microfluidic reagent kit; (4) Place the test kit containing the liquid to be tested on the testing equipment for testing and analysis.

8. The method according to claim 7, characterized in that The step (2) specifically comprises: adding a sample pre-treatment solution and abrasive particles into the sample processing tube; The sample pretreatment solution includes Tris-HCl (pH 8.0), NaCl, EDTA and SDS; The samples described in step (2) are seeds, leaves or products thereof of the transgenic corn line MON810, the transgenic corn line TC1507 and the transgenic corn line Bt11.

9. The method according to claim 7, characterized in that The step (4) specifically includes: 1) Turn on the rapid detection device; 2) Scan the QR code; 3) Inserting the integrated microfluidic chip kit; 4) Run the test; 5) End and check the test results and amplification curve.

10. Use of the primer-probe combination of claim 1, the kit of any one of claims 4 to 6, or the method of any one of claims 7 to 9 in detecting components of transgenic maize line MON810, transgenic maize line TC1507, and transgenic maize line Bt11, their derivatives, and products thereof.

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