Primer composition, micro-fluidic chip, kit and method for simultaneously detecting multiple transgenic soybean lines and / or transgenic regulatory elements

Abstract

The invention provides a primer composition, a micro-fluidic chip, a kit and a method for simultaneously detecting multiple transgenic soybean lines and / or transgenic regulatory elements. The primer composition comprises the following primer groups: a primer group used for detecting a cauliflower mosaic virus 35S promoter, a primer group used for detecting a pea ribulose diphosphate carboxylase gene terminator, a primer group used for detecting a transgenic soybean line GMB151, a primer group used for detecting a transgenic soybean line DP356043, a primer group used for detecting a transgenic soybean line GMB152, a primer group used for detecting a transgenic soybean line DP356043 and a primer group used for detecting a transgenic soybean line DP356043. The primer group is used for detecting a transgenic soybean strain DAS-68416-4, the primer group is used for detecting a transgenic soybean strain DBN-09004-6, and the primer group gene is used for detecting a transgenic soybean strain IND-00410-5. On the basis of an LAMP (Loop-Mediated Isothermal Amplification) means and in combination with a microfluidic technology, five transgenic soybean lines and two transgenic regulatory elements can be synchronously detected at most by single sample adding.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant gene detection, and in particular relates to a primer composition, a microfluidic chip, a kit and a method for simultaneously detecting multiple transgenic soybean lines and / or transgenic regulatory elements. Background Art

[0002] Genetically modified organisms (GMOs) refer to organisms whose genomes have been altered through genetic engineering techniques, including genetically modified plants, animals and microorganisms. Among them, genetically modified plants (mainly crops) are cultivated by transferring exogenous target genes into plants using genetic engineering techniques. They have excellent characteristics such as insect resistance, herbicide resistance, stress resistance and nutritional improvement. With the continuous development of genetic modification technology, the degree of industrialization and commercialization of genetically modified crops has continued to deepen, and as a result, the safety of genetically modified products has also attracted more attention from the public. Before it is determined whether genetically modified crops and their products have potential adverse effects on human health and the ecological environment, their detection and labeling are very important to consumers.

[0003] Soybean (Glycine max (L.) Merr.) is an annual herbaceous plant of the genus Glycine in the family Leguminosae. It is a crop with seeds rich in plant protein, with a protein content of 35% to 40%. It is most commonly used to make various soy products, extract soybean oil, brew soy sauce and extract protein. Due to its high nutritional value, it is called "the king of beans", "meat in the field", "green milk", etc., and is the most respected food by nutritionists among hundreds of natural foods. Soybean is an excellent crop with comprehensive nutrition and economic efficiency. It is the main source of edible oil and plant protein in the world, and one of the main feed raw materials for poultry and livestock farming.

[0004] Loop-mediated isothermal amplification (LAMP) is a new in vitro isothermal amplification technology for specific nucleic acid fragments invented by Japanese researchers Notomi et al. in 2000. Its characteristics are that 4 to 6 specific primers are designed for the 6 regions of the target gene, and under the action of strand displacement DNA polymerase (Bst DNA polymerase), isothermal amplification is performed at 60 to 65°C, and 10 9 ~10 10 It can amplify nucleic acid at a speed of 200 times, and has the characteristics of simple operation, strong specificity, and easy detection of products.

[0005] Microfluidic chips provide a method for accurately controlling and processing trace samples, and have the characteristics of low cost, small size and high throughput. In addition, the chip design is flexible, the entire system is easy to operate and controllable; the sample volume is greatly reduced compared to microarray chips; the closed operating environment increases the anti-pollution ability of the detection; and the enhanced material transfer efficiency can shorten the reaction time and improve the sensitivity.

[0006] Existing genetically modified soybean nucleic acid detection methods can usually only detect a limited number of target gene sequences, and generally have the defects of complex operation, long time consumption and high cost, which is difficult to meet the actual needs of rapid screening and detection in the genetically modified soybean industry. Based on the current status of genetically modified soybean detection, providing a simple, fast, efficient and low-cost gene detection method has become an urgent problem to be solved. Summary of the invention

[0007] The object of the present invention is to overcome the deficiencies in the prior art, and to provide a new primer combination and a microfluidic chip, a detection kit and a corresponding detection method comprising the primer combination. Thus, the present invention is based on the LAMP method, and in combination with microfluidic technology, a single target gene sequence is detected independently in multiple independent reaction pools, and the circuit design process and pressure drive of the microfluidic chip can also be used to drive the entire detection reaction to complete. Compared with the ordinary tubular LAMP, it has the advantages of detecting multiple target genes with a single sample addition, and has the characteristics of simple operation, rapidity, high efficiency, and low cost.

[0008] After a large number of experiments and studies, the inventors of the present invention selected Lectin (soybean endogenous gene); two transgenic regulatory elements: pCaMV35S (transgenic regulatory element: cauliflower mosaic virus 35S promoter) and tE9 (transgenic regulatory element: pea ribulose bisphosphate carboxylase gene terminator); and five transgenic soybean lines, namely GMB151 (transgenic soybean line: insect resistance, herbicide tolerance), DP356043 (transgenic soybean line: herbicide tolerance), DAS-68416-4 (transgenic soybean line: herbicide tolerance), DBN-09004-6 (transgenic soybean line: herbicide tolerance) and IND-00410-5 (transgenic soybean line: quality improvement, herbicide tolerance) as detection targets, and combined the two technologies of LAMP and microfluidic chip, so that efficient and accurate detection of multiple transgenic soybean lines and transgenic regulatory elements can be achieved simultaneously with only a single sample addition. The advantages of the present invention are simple operation, low equipment requirements, and suitability for rapid and highly sensitive detection of soybean genetically modified components in a field environment.

[0009] The objective of the present invention is achieved through the following technical solutions:

[0010] In a first aspect, the present invention provides a primer composition for simultaneously detecting multiple transgenic soybean lines and / or transgenic regulatory elements by loop-mediated isothermal amplification, comprising the following primer sets: a primer set for detecting cauliflower mosaic virus 35S promoter (pCaMV35S), a primer set for detecting pea ribulose bisphosphate carboxylase gene terminator (tE9), a primer set for detecting transgenic soybean line GMB151, a primer set for detecting transgenic soybean line DP356043, a primer set for detecting transgenic soybean line DAS-68416-4, a primer set for detecting transgenic soybean line DBN-09004-6, and a primer set for detecting transgenic soybean line IND-00410-5.

[0011] According to some preferred embodiments of the present invention, the primer composition further comprises a primer set for detecting soybean Lectin gene.

[0012] According to some embodiments of the present invention, the primer set for detecting soybean Lectin gene comprises:

[0013] The outer primer pair Lectin-F3 and Lectin-B3 (Lectin-F3 / B3), whose nucleotide sequences are shown in SEQ ID NOs. 1 and 2, respectively;

[0014] The inner primer pair Lectin-FIP and Lectin-BIP (Lectin-FIP / BIP), the nucleotide sequences of which are shown in SEQ ID NOs. 3 and 4, respectively;

[0015] The nucleotide sequences of the loop primer pair Lectin-LF and Lectin-LB (Lectin-LF / LB) are shown in SEQ ID NOs. 5 and 6, respectively.

[0016] According to some embodiments of the present invention, the molar ratio of the outer primer pair Lectin-F3 / B3, the inner primer pair Lectin-FIP / BIP and the loop primer pair Lectin-LF / LB is 1:3~9:1~5, preferably 1:8:4.

[0017] According to some embodiments of the present invention, the primer set for detecting cauliflower mosaic virus 35S promoter (pCaMV35S) comprises:

[0018] The outer primer pair pCaMV35S-F3 and pCaMV35S-B3 (pCaMV35S-F3 / B3), whose nucleotide sequences are shown in SEQ ID NOs. 7 and 8, respectively;

[0019] The inner primer pair pCaMV35S-FIP and pCaMV35S-BIP (pCaMV35S-FIP / BIP), whose nucleotide sequences are shown in SEQ ID NOs. 9 and 10, respectively; and

[0020] The nucleotide sequences of the loop primer pair pCaMV35S-LF and pCaMV35S-LB (pCaMV35S-LF / LB) are shown in SEQ ID NOs. 11 and 12, respectively.

[0021] According to some embodiments of the present invention, the molar ratio of the outer primer pair pCaMV35S-F3 / B3, the inner primer pair pCaMV35S-FIP / BIP and the loop primer pair pCaMV35S-LF / LB is 1:3~9:1~5, preferably 1:8:4.

[0022] According to some embodiments of the present invention, the primer set for detecting the pea ribulose bisphosphate carboxylase gene terminator (tE9) comprises:

[0023] The outer primer pair tE9-F3 and tE9-B3 (tE9-F3 / B3), whose nucleotide sequences are shown in SEQ ID NOs. 13 and 14, respectively;

[0024] The inner primer pair tE9-FIP and tE9-BIP (tE9-FIP / BIP), whose nucleotide sequences are shown in SEQ ID NOs. 15 and 16, respectively; and

[0025] The nucleotide sequences of the loop primer pair tE9-LF and tE9-LB (tE9-LF / LB) are shown in SEQ ID NOs. 17 and 18, respectively.

[0026] According to some embodiments of the present invention, the molar ratio of the outer primer pair tE9-F3 / B3, the inner primer pair tE9-FIP / BIP and the loop primer pair tE9-LF / LB is 1:3~9:1~5, preferably 1:8:4.

[0027] According to some embodiments of the present invention, the primer set for detecting transgenic soybean line GMB151 comprises:

[0028] The outer primer pair GMB151-F3 and GMB151-B3 (GMB151-F3 / B3), whose nucleotide sequences are shown in SEQ ID NOs. 19 and 20, respectively;

[0029] The inner primer pair GMB151-FIP and GMB151-BIP (GMB151-FIP / BIP), whose nucleotide sequences are shown in SEQ ID NOs. 21 and 22, respectively; and

[0030] The nucleotide sequences of the loop primer pair GMB151-LF and GMB151-LB (GMB151-LF / LB) are shown in SEQ ID NOs. 23 and 24, respectively.

[0031] According to some embodiments of the present invention, the molar ratio of the outer primer pair GMB151-F3 / B3, the inner primer pair GMB151-FIP / BIP and the loop primer pair GMB151-LF / LB is 1:3~9:1~5, preferably 1:8:4.

[0032] According to some embodiments of the present invention, the primer set for detecting transgenic soybean line DP356043 comprises:

[0033] The outer primer pair DP356043-F3 and DP356043-B3 (DP356043-F3 / B3), whose nucleotide sequences are shown in SEQ ID NOs. 25 and 26, respectively;

[0034] The inner primer pair DP356043-FIP and DP356043-BIP (DP356043-FIP / BIP), whose nucleotide sequences are shown in SEQ ID NOs. 27 and 28, respectively; and

[0035] The nucleotide sequences of the loop primer pair DP356043-LF and DP356043-LB (DP356043-LF / LB) are shown in SEQ ID NOs. 29 and 30, respectively.

[0036] According to some embodiments of the present invention, the molar ratio of the outer primer pair DP356043-F3 / B3, the inner primer pair DP356043-FIP / BIP and the loop primer pair DP356043-LF / LB is 1:3~9:1~5, preferably 1:8:4.

[0037] According to some embodiments of the present invention, the primer set for detecting transgenic soybean line DAS-68416-4 comprises:

[0038] The outer primer pair DAS-68416-4-F3 and DAS-68416-4-B3 (DAS-68416-4-F3 / B3), whose nucleotide sequences are shown in SEQ ID NOs. 31 and 32, respectively;

[0039] The inner primer pair DAS-68416-4-FIP and DAS-68416-4-BIP (DAS-68416-4-FIP / BIP), whose nucleotide sequences are shown in SEQ ID NOs. 33 and 34, respectively; and

[0040] The nucleotide sequences of the loop primer pair DAS-68416-4-LF and DAS-68416-4-LB (DAS-68416-4-LF / LB) are shown in SEQ ID NOs. 35 and 36, respectively.

[0041] According to some embodiments of the present invention, the molar ratio of the outer primer pair DAS-68416-4-F3 / B3, the inner primer pair DAS-68416-4-FIP / BIP and the loop primer pair DAS-68416-4-LF / LB is 1:3~9:1~5, preferably 1:8:4.

[0042] According to some embodiments of the present invention, the primer set for detecting transgenic soybean line DBN-09004-6 comprises:

[0043] The outer primer pair DBN-09004-6-F3 and DBN-09004-6-B3 (DBN-09004-6-F3 / B3), whose nucleotide sequences are shown in SEQ ID NOs. 37 and 38, respectively;

[0044] The inner primer pair DBN-09004-6-FIP and DBN-09004-6-BIP (DBN-09004-6-FIP / BIP), whose nucleotide sequences are shown in SEQ ID NOs. 39 and 40, respectively; and

[0045] The nucleotide sequences of the loop primer pair DBN-09004-6-LF and DBN-09004-6-LB (DBN-09004-6-LF / LB) are shown in SEQ ID NOs. 41 and 42, respectively.

[0046] According to some embodiments of the present invention, the molar ratio of the outer primer pair DBN-09004-6-F3 / B3, the inner primer pair DBN-09004-6-FIP / BIP and the loop primer pair DBN-09004-6-LF / LB is 1:3~9:1~5, preferably 1:8:4.

[0047] According to some embodiments of the present invention, the primer set for detecting transgenic soybean line IND-00410-5 comprises:

[0048] The outer primer pair IND-00410-5-F3 and IND-00410-5-6-B3 (IND-00410-5-F3 / B3), whose nucleotide sequences are shown in SEQ ID NOs. 43 and 44, respectively;

[0049] The inner primer pair IND-00410-5-FIP and IND-00410-5-BIP (IND-00410-5-FIP / BIP), whose nucleotide sequences are shown in SEQ ID NOs. 45 and 46, respectively; and

[0050] The nucleotide sequences of the loop primer pair IND-00410-5-LF and IND-00410-5-LB (IND-00410-5-LF / LB) are shown in SEQ ID NOs. 47 and 48, respectively.

[0051] According to some embodiments of the present invention, the molar ratio of the outer primer pair IND-00410-5-F3 / B3, the inner primer pair IND-00410-5-FIP / BIP and the loop primer pair IND-00410-5-LF / LB is 1:3~9:1~5, preferably 1:8:4.

[0052] In the present invention, each target primer set includes a pair of outer primers, a pair of inner primers and a pair of loop primers, and the molar ratio between the primers in each pair of primers is 1: 1. For example, the molar ratio of pCaMV35S-F3 and pCaMV35S-B3 in the outer primer pair pCaMV35S-F3 / B3 of the pCaMV35S target is 1: 1, the molar ratio of pCaMV35S-FIP and pCaMV35S-BIP in the inner primer pair pCaMV35S-FIP / BIP is 1: 1, and the molar ratio of pCaMV35S-LF and pCaMV35S-LB in the loop primer pair pCaMV35S-LF / LB is 1: 1.

[0053] In a second aspect, the present invention provides a microfluidic chip for simultaneously detecting multiple transgenic soybean lines and / or transgenic regulatory elements, wherein each primer set in the primer composition according to the first aspect of the present invention is respectively contained in different reaction pools of the microfluidic chip.

[0054] According to some embodiments of the present invention, the microfluidic chip further comprises a reaction pool for containing a negative control and / or a positive control.

[0055] According to some preferred embodiments of the present invention, the microfluidic chip includes at least two groups, for example, four groups of reaction pools, in each group of reaction pools, each primer group in the primer composition according to the first aspect of the present invention is respectively contained in a different reaction pool of the microfluidic chip. The microfluidic chip is provided with multiple groups of reaction pools to achieve simultaneous detection of multiple samples, which is fast, simple, synchronous and efficient.

[0056] According to some embodiments of the present invention, each of the reaction pools comprises a lyophilized mixture of each primer set, a DNA loop-mediated isothermal amplification premix and a fluorescent dye, wherein the DNA loop-mediated isothermal amplification premix comprises a DNA polymerase, deoxyribonucleoside triphosphates (dNTPs) and a reaction buffer.

[0057] According to some embodiments of the present invention, each of the reaction pools further comprises a Tte UvrD helicase and an ultra-thermostable single-stranded binding protein (ET SSB); preferably, the Tte UvrD helicase is a Tte UvrD helicase that tolerates high temperatures (70°C); preferably, the ultra-thermostable single-stranded binding protein is an ultra-thermostable single-stranded binding protein that tolerates extremely high temperatures (95°C). The Tte UvrD helicase used in the present invention (e.g., a Tte UvrD helicase that tolerates high temperatures (70°C)) can efficiently open the DNA double-stranded structure during the amplification reaction and improve the reaction specificity; the ultra-thermostable single-stranded binding protein (ET SSB) used in the present invention (e.g., an ultra-thermostable single-stranded binding protein that tolerates extremely high temperatures (95°C)) can stabilize the single-stranded structure, facilitate rapid primer binding, and improve the reaction sensitivity.

[0058] The present invention introduces Tte UvrD helicase and ET SSB which are not used in conventional LAMP technology, and can achieve higher specificity and sensitivity compared with conventional LAMP technology. Among them, the basic principle of LAMP technology integrating Tte UvrD helicase and ET SSB is shown in Figure 2.

[0059] According to some embodiments of the present invention, the microfluidic chip further comprises a vent hole, a sample addition pool, a quantitative pool and a waste liquid pool.

[0060] In the microfluidic chip of the present invention, the air holes are used for air inlet and outlet, and at the same time ensure the subsequent smooth sample loading; the sample loading pool is used for liquid sampling, and the sample loading liquid is a mixture of reaction liquid (such as LAMP reaction liquid) and sample; the quantitative pool is used for liquid quantification to ensure that the reaction volume is fixed; the reaction pool is used for reaction, and a freeze-dried mixture (including a mixture freeze-dried powder of each primer set, DNA loop-mediated isothermal amplification premix and fluorescent dye) is pre-buried in the pool; the waste liquid pool is used to store excess waste liquid.

[0061] According to some preferred embodiments of the present invention, the microfluidic chip further comprises a bottom film bonded to the bottom thereof and a top film bonded to the top thereof, and the bottom film and the top film seal the microfluidic chip.

[0062] According to some preferred embodiments of the present invention, the top film further comprises a sealing film.

[0063] In a third aspect, the present invention provides a method for preparing a microfluidic chip according to the second aspect of the present invention, comprising:

[0064] (1) dissolving each primer set in the primer composition according to the first aspect of the present invention, the DNA loop-mediated isothermal amplification premix and the fluorescent dye in sterile water to obtain a mixed solution;

[0065] (2) spotting the mixed solution into the reaction wells of the microfluidic chip respectively;

[0066] (3) Covering the bottom of the chip with a base film, heating at 45-65° C., preferably 60° C. and 0.1-1 MPa, preferably 0.5 MPa for 0.5-3 minutes, preferably 2 minutes, so that the base film is bonded to the bottom of the chip;

[0067] (4) After freeze-drying, cover the top of the chip with the top film, and heat at 45-65°C, preferably 60°C and 0.1-1 MPa, preferably 0.5 MPa, for 0.5-3 minutes, preferably 2 minutes, to bond the top film to the top of the chip.

[0068] Preferably, the freeze-drying comprises the following steps:

[0069] (4-1) pre-freezing the chip at 1 to 100 kPa and -60 to -40°C, preferably 100 kPa and -50°C for 30 to 200 minutes, preferably 120 minutes, and then drying at 1 to 100 Pa and -60 to -40°C, preferably 100 Pa and -50°C for 30 to 200 minutes, preferably 120 minutes;

[0070] (4-2) Continue drying under a vacuum degree of 1 to 10 Pa, preferably 1 Pa: heat up to -50 to -30 ° C, preferably -40 ° C, keep for 30 to 200 minutes, preferably 120 minutes, then heat up to -40 to -20 ° C, preferably -30 ° C, keep for 30 to 200 minutes, preferably 120 minutes, then heat up to -30 to 0 ° C, preferably -20 ° C, keep for 30 to 200 minutes, preferably 120 minutes, then heat up to 20 to 30 ° C, preferably 25 ° C.

[0071] The microfluidic chip provided by the present invention seals the chip with the bottom film and the top film as a whole through a hot pressing bonding process, wherein the bottom film is used to seal the bottom layer of the chip, and the top film (including the sealing film) is used to seal the top layer of the chip, and the sealing film is used to seal after adding the sample. The hot pressing bonding process includes the film sticking and laminating procedures of the bottom film and the top film, the material used for the bottom film can be plastic, and the material used for the top film (including the sealing film) can be plastic, both of which have good biocompatibility and strong light transmittance, and are suitable for real operations.

[0072] Specifically, the hot pressing bonding process includes attaching a complete circular film 9596R to the bottom of the chip after the spotting is completed, the film pressing temperature is 60°C, the film pressing pressure is 0.5Mpa, and the film pressing time is 2min; after the chip is freeze-dried, a circular film 4970c containing a fan-shaped sealing film is attached to the top of the chip, the film pressing temperature is 60°C, the film pressing pressure is 0.5Mpa, and the film pressing time is 2min. The freeze-drying process includes placing the chip in a freeze dryer, staying at -50°C for 120min, turning on the vacuum pump (100kPa), staying at -50°C for 120min (vacuum degree 100Pa); turning on the heating, maintaining the vacuum degree at 1Pa, heating to -40°C, maintaining for 120min, continuing to heat to -30°C, maintaining for 120min, then heating to -20°C, maintaining for 120min, heating to 25°C, and closing the freeze dryer. Among them, the bottom film material is plastic, and the top film (including the sealing film) material is plastic. After the chip is freeze-dried, vacuum the aluminum bag to package the chip, and then place it in a 25°C constant temperature box overnight.

[0073] After a large number of experiments and studies, the inventors of the present invention have found that the use of other bonding processes has disadvantages to varying degrees: (1) Laser welding has requirements on the properties of the fused material to absorb laser light, and generally requires that the two bonding materials have similar light absorption properties to a certain extent; (2) Solvent bonding has the problem that the glue easily penetrates into the channel and causes channel blockage, and this process has high requirements on the accuracy of the heating temperature and time. Some gas products may also be generated during the curing process, causing pollution to the chip cavity environment; (3) Double-sided adhesive bonding has the disadvantage that the glue penetrates into the channel and causes channel blockage, and double-sided adhesive bonding also has obvious disadvantages such as non-specific adsorption of samples or markers; (4) Single-sided adhesive film bonding has very high requirements on the product quality of the adhesive film, and the bonding pressure is difficult to control. The commonly used film bonding process is manual film scraping. This process has a great deal of uncontrollability. For places that cannot be bonded, there is a risk of leakage during the PCR amplification process; (5) Ultrasonic welding has high requirements on the welding line. The uneven welding line will lead to cold welding points, which are prone to leakage, and the cold welding points cannot be detected.

[0074] The present invention overcomes the above-mentioned shortcomings by adopting the hot pressing bonding method, and also has the following great advantages: first, the microfluidic chip and the PC membrane are bonded at the molecular level mainly by the coordination of bonding temperature, bonding pressure and bonding time, without the need to use any auxiliary adhesives, and there is no bonding blind spot, and no pollutants penetrate and adhere to the microchannel; second, the mechanical properties and thermal properties of the chip treated by hot pressing bonding remain unchanged; third, the cost of the membrane used for hot pressing is relatively low, and the machine design and manufacturing are relatively simple, which is convenient for actual operation.

[0075] In addition, the present invention adopts a pre-freezing and step-by-step temperature freeze-drying process, wherein pre-freezing solidifies free water, giving the product the same shape before and after drying; annealing reshapes the shape and size of ice crystals, prompting subsequent rapid sublimation of water; primary drying (sublimation drying) allows the solidified free water to sublime and escape, and if secondary drying is entered too early, it is easy to cause the product to collapse or melt back; secondary drying (desorption drying) removes part of the bound water, reducing the moisture content to a lower level.

[0076] In a fourth aspect, the present invention provides a kit for simultaneously detecting multiple transgenic soybean lines and / or transgenic regulatory elements, which comprises the microfluidic chip according to the second aspect of the present invention.

[0077] According to some embodiments of the present invention, the kit further comprises other reagents for loop-mediated isothermal amplification; preferably, the other reagents for loop-mediated isothermal amplification comprise Mg 2+ and enzyme-free water; More preferably, the Mg in the other reagents for loop-mediated isothermal amplification 2+ The concentration is 6~10mM.

[0078] According to some embodiments of the present invention, the kit further comprises a positive control, wherein the positive control comprises the target amplified gene fragments of the following transgenic soybean targets: soybean Lectin gene, transgenic regulatory element pCaMV35S, transgenic regulatory element tE9, transgenic soybean line GMB151, transgenic soybean line DP356043, transgenic soybean line DAS-68416-4, transgenic soybean line DBN-09004-6 and transgenic soybean line IND-00410-5; preferably, the nucleotide sequences of the target amplified gene fragments of the soybean Lectin gene, transgenic regulatory element pCaMV35S, transgenic regulatory element tE9, transgenic soybean line GMB151, transgenic soybean line DP356043, transgenic soybean line DAS-68416-4, transgenic soybean line DBN-09004-6 and transgenic soybean line IND-00410-5 are shown as SEQ ID NOs. 131 to 138, respectively. The freeze-dried plasmid powder obtained by constructing the target amplified gene fragment using the soybean Lectin gene, transgenic regulatory element pCaMV35S, transgenic regulatory element tE9, transgenic soybean line GMB151, transgenic soybean line DP356043, transgenic soybean line DAS-68416-4, transgenic soybean line DBN-09004-6 and transgenic soybean line IND-00410-5 targets is placed in the kit.

[0079] The nucleotide sequences of the target amplified gene fragments of the soybean Lectin gene, transgenic regulatory element pCaMV35S, transgenic regulatory element tE9, transgenic soybean line GMB151, transgenic soybean line DP356043, transgenic soybean line DAS-68416-4, transgenic soybean line DBN-09004-6 and transgenic soybean line IND-00410-5 of the present invention are shown in Table 1:

[0080] Table 1

[0081]

[0082] According to some embodiments of the invention, the kit further comprises a scraper.

[0083] The detection kit of the present invention, which is based on LAMP and combined with microfluidic technology, can achieve efficient and accurate detection of multiple transgenic soybean lines and / or transgenic regulatory elements at the same time with only a single sample addition. It can also be superimposed with the addition of a Tte UvrD helicase that tolerates high temperatures (70°C) and an ultra-thermostable single-stranded binding protein (ETSSB) that tolerates extremely high temperatures (95°C), thereby having higher specificity and sensitivity than using conventional LAMP technology.

[0084] In a fifth aspect, the present invention provides a method for simultaneously detecting multiple transgenic soybean lines and / or transgenic regulatory elements, which comprises using the microfluidic chip according to the second aspect of the present invention or the kit according to the fourth aspect of the present invention for detection.

[0085] According to some embodiments of the present invention, the method includes performing a loop-mediated isothermal amplification reaction on the sample to be tested using the microfluidic chip according to the second aspect of the present invention or the kit according to the fourth aspect of the present invention, and determining the presence of genetically modified soybean components in the sample to be tested based on the generated fluorescence curve.

[0086] According to some embodiments of the present invention, the temperature of the loop-mediated isothermal amplification reaction is 60°C to 65°C, preferably 62°C.

[0087] According to some embodiments of the present invention, the loop-mediated isothermal amplification reaction time is 30 to 60 minutes, preferably 45 minutes.

[0088] According to some preferred embodiments of the present invention, the mass fraction of the sample containing genetically modified soybean components is not less than 1%, more preferably not less than 0.5%, based on the mass of the sample to be tested. In other words, the minimum detection limit of the genetically modified soybean sample using the detection method of the microfluidic chip or kit of the present invention is 1% by mass, more preferably 0.5%.

[0089] In the present invention, when the soybean endogenous gene Lectin in the detected fluorescence curve shows an S-shaped curve, and the transgenic regulatory elements pCaMV35S and tE9 and any one or more of the transgenic soybean lines GMB151, DP356043, DAS-68416-4, DBN-09004-6 and IND-00410-5 detection targets show an S-shaped curve, it indicates that the sample to be tested is a soybean sample and one or more transgenic soybean lines and / or transgenic regulatory element components are present; when the transgenic regulatory elements pCaMV35S, tE9 and the transgenic soybean lines GMB151, DP356043, DAS-68416-4, DBN-09004-6 and IND-00410-5 detection targets do not show an S-shaped curve in the detected fluorescence curve, it indicates that these two transgenic regulatory elements are not present in the sample to be tested, and the sample to be tested does not contain these five transgenic soybean lines.

[0090] Compared with the prior art, the technical solution provided by the present invention has the following beneficial effects:

[0091] 1. The present invention is based on LAMP and combined with microfluidics technology, and a single sample addition can achieve simultaneous detection of up to two soybean transgenic regulatory elements and five transgenic soybean lines;

[0092] 2. The microfluidic chip of the present invention has the advantages of simple operation, uniform liquid separation, good biocompatibility, and environmental friendliness. After being further designed into multiple regions, it can detect multiple samples at the same time, and can also have the function of simultaneously detecting multiple targets by adding samples to a single region;

[0093] 3. The detection kit of the present invention can achieve amplification quickly and efficiently, so that the entire isothermal amplification reaction can be completed within 45 minutes; the detection result can be directly judged by detecting the fluorescence signal and the peak time of the fluorescence signal, and the result identification is simple; the recognition of the 6 specific regions of the target sequence of the corresponding detection target by the 4 inner and outer primers corresponding to each detection target and the improvement of the amplification efficiency by the 2 loop primers ensure the high specificity of the chip isothermal amplification, and the specificity is strong; the minimum detection limit is 0.5wt%, and the sensitivity is high;

[0094] 4. The detection kit of the present invention further uses Tte UvrD helicase that can withstand high temperatures (70°C) and ultra-thermostable single-stranded binding protein (ET SSB) that can withstand extremely high temperatures (95°C), which has higher specificity and sensitivity than conventional LAMP technology.

[0095] 5. When the microfluidic chip or detection kit of the present invention is used for soybean sample detection, five strains of genetically modified soybeans and two genetically modified regulatory elements can be detected quickly, simply, synchronously, efficiently, highly specifically and highly sensitively under isothermal conditions, providing a new technical platform for the detection of genetically modified components of soybeans, and providing a rapid and highly accurate detection method for the circulation and port quarantine of genetically modified soybeans and their agricultural products; it can be used for grassroots agricultural product testing units and various import and export quarantine inspection and prevention and control centers to screen and detect genetically modified soybean components, increase monitoring efforts to avoid illegal planting and sales of genetically modified soybeans, and provide a practical and reliable scientific basis and technical support for inspection and supervision work and decision-making of relevant departments; it is of great significance to improve the detection ability of genetically modified products, standardize the production behavior of soybean enterprises, ensure the safety of my country's soybean industry, ensure the safety of soybean imports and exports, and avoid the return or destruction of products due to the presence of non-compliant genetically modified components. BRIEF DESCRIPTION OF THE DRAWINGS

[0096] The embodiments of the present invention are described in detail below with reference to the accompanying drawings, wherein:

[0097] FIG1 is a schematic diagram of the structure of a microfluidic chip provided in Example 1 of the present invention;

[0098] FIG2 shows a basic schematic diagram of the LAMP technology of the present invention integrating Tte UvrD helicase and ET SSB;

[0099] FIG. 3 shows the negative control (NC) and 10 4 , 10 5 and 10 6 The isothermal amplification test results of soybean Lectin gene primer set were screened using Lectin plasmid solution with a copy / mL concentration as a template;

[0100] FIG. 4 is a graph showing the negative control (NC) and 10 4 , 10 5 and 10 6 The isothermal amplification test results of the pCaMV35S primer set using the pCaMV35S plasmid solution with a copy / mL concentration as a template to screen the transgenic regulatory element;

[0101] FIG. 5 is a graph showing the negative control (NC) and 10 4 , 10 5 and 10 6The isothermal amplification test results of the tE9 primer set using the tE9 plasmid solution with a copy / mL concentration as a template to screen the transgenic regulatory element;

[0102] FIG. 6 shows the negative control (NC) and 10 4 , 10 5 and 10 6 The isothermal amplification test results of the primer set for screening the transgenic soybean line GMB151 using the GMB151 plasmid solution with a copy / mL concentration as a template;

[0103] FIG. 7 is a graph showing the negative control (NC) and 10 4 , 10 5 and 10 6 The isothermal amplification test results of the primer set of DP356043, which was used as a template to screen the transgenic soybean line DP356043, were obtained by using the DP356043 plasmid solution with a copy / mL concentration;

[0104] FIG. 8 is a graph showing the negative control (NC) and 10 4 , 10 5 and 10 6 The isothermal amplification test results of the primer set for screening the transgenic soybean line DAS-68416-4 using the DAS-68416-4 plasmid solution with a copy / mL concentration as a template;

[0105] FIG. 9 is a graph showing the negative control (NC) and 10 4 , 10 5 and 10 6 The isothermal amplification test results of the primer set for screening the transgenic soybean line DBN-09004-6 using the DBN-09004-6 plasmid solution with a copy / mL concentration as a template;

[0106] FIG. 10 is a diagram of Example 3 of the present invention with negative control (NC) and 10 4 , 10 5 and 10 6 The isothermal amplification test results of the primer set for screening the transgenic soybean line IND-00410-5 using the IND-00410-5 plasmid solution with a copy / mL concentration as a template;

[0107] 11 is the target-specific isothermal amplification detection results of soybean Lectin gene, transgenic regulatory element pCaMV35S, tE9 and transgenic soybean lines GMB151, DP356043, DAS68416-4, DBN-09004-6 and IND-00410-5 using RNase-free water as a template in Example 4 of the present invention;

[0108] FIG12 is the target-specific isothermal amplification detection result of detecting the soybean Lectin gene using the whole genome DNA of soybean, tomato, pepper, corn, rice, barley, wheat seeds and potato tubers as templates in Example 4 of the present invention;

[0109] 13 is the isothermal amplification detection result of the target specificity of the transgenic regulatory element pCaMV35S in Example 4 of the present invention using the whole genomic DNA of the transgenic regulatory elements pCaMV35S, pFMV35S, pNOS, pUbi, tE9, t35S, tNOS and tOCS as a template to detect the transgenic regulatory element pCaMV35S;

[0110] 14 is the isothermal amplification detection result of the target specificity of the transgenic regulatory element tE9 using the whole genomic DNA of the transgenic regulatory elements pCaMV35S, pFMV35S, pNOS, pUbi, tE9, t35S, tNOS and tOCS as templates in Example 4 of the present invention;

[0111] 15 is the isothermal amplification detection result of the target specificity of detecting the transgenic soybean line GMB151 using the whole genome DNA mixed solution of commercial soybean powder of five transgenic soybean lines as a template in Example 4 of the present invention;

[0112] FIG16 is an isothermal amplification detection result of the target specificity of transgenic soybean line DP356043 using a mixed solution of whole genome DNA of commercial soybean powder of five transgenic soybean lines as a template in Example 4 of the present invention;

[0113] FIG17 is the isothermal amplification detection result of the target specificity of transgenic soybean line DAS68416-4 using a mixed solution of whole genome DNA of commercial soybean powder of five transgenic soybean lines as a template in Example 4 of the present invention;

[0114] FIG18 is the isothermal amplification detection result of the target specificity of transgenic soybean line DBN-09004-6 using a mixed solution of whole genome DNA of commercial soybean powder of five transgenic soybean lines as a template in Example 4 of the present invention;

[0115] FIG19 is an isothermal amplification detection result of the target specificity of transgenic soybean line IND-00410-5 using a mixed solution of whole genome DNA of commercial soybean powder of five transgenic soybean lines as a template in Example 4 of the present invention;

[0116] FIG20 is an isothermal amplification detection result of the target sensitivity of soybean Lectin gene, transgenic regulatory element pCaMV35S, tE9 and transgenic soybean lines GMB151, DP356043, DAS68416-4, DBN-09004-6 and IND-00410-5 using detection kit 1 ( FIG20A ) and detection kit 2 ( FIG20B ) under the condition that the mass fraction of the target sample is 1% in Example 5 of the present invention;

[0117] FIG21 is an isothermal amplification detection result of the target sensitivity of soybean Lectin gene, transgenic regulatory element pCaMV35S, tE9 and transgenic soybean lines GMB151, DP356043, DAS68416-4, DBN-09004-6 and IND-00410-5 using detection kit 1 ( FIG21A ) and detection kit 2 ( FIG21B ) under the condition of 0.5% target sample mass fraction in Example 5 of the present invention;

[0118] Figure 22 shows the isothermal amplification detection results of the target sensitivity of soybean Lectin gene, transgenic regulatory element pCaMV35S, tE9 and transgenic soybean lines GMB151, DP356043, DAS68416-4, DBN-09004-6 and IND-00410-5 under the condition of target sample mass fraction of 0.1% in Example 5 of the present invention using detection kit 1 (Figure 22A) and detection kit 2 (Figure 22B). DETAILED DESCRIPTION

[0119] The present invention is described in further detail below in conjunction with specific embodiments, and the present invention can be better understood according to the following examples. However, it should be readily understood by those skilled in the art that the following examples are merely illustrative and are not intended to limit the present invention to these specific embodiments. It should be appreciated by those skilled in the art that the present invention will encompass all improvements, alternatives and equivalents included within the scope of the claims.

[0120] Approximate terms in the specification and claims of this application are used to modify quantities, which means that the present invention is not limited to the specific quantity, but also includes the modified parts that are close to the quantity and acceptable but do not cause the relevant basic functions to change. In the specification and claims of this application, range limits can be combined and / or interchanged. If not otherwise specified, these ranges also include all values ​​and sub-ranges contained therein.

[0121] In the following examples, the plasmid powder used was synthesized by GenScript Biotechnology Co., Ltd.; the whole genomic DNA of soybean, tomato, pepper, corn, rice, barley, wheat seeds and potato tubers used was obtained by extracting nucleic acid from commercial grains using a plant genomic DNA extraction kit; the whole genomic DNA of the transgenic regulatory elements pCaMV35S, pFMV35S (Scrophulariaceae mosaic virus 35S promoter), pNOS (Agrobacterium nopaline synthase gene promoter), pUbi (maize ubiquitin gene promoter), tE9, t35S (cauliflower mosaic virus terminator), tNOS (Agrobacterium nopaline synthase gene terminator), and tOCS (octopine synthase terminator) used was obtained by extracting nucleic acid from a commercial positive control using a plant genomic DNA extraction kit. The soybean meal used for extracting transgenic regulatory elements pCaMV35S and tE9 was provided free of charge by Changsha Customs of the People's Republic of China (hereinafter referred to as Changsha Customs); the non-transgenic soybean standard containing only the endogenous gene Lectin, and the transgenic soybean lines GMB151, DP356043, DAS68416-4, DBN-09004-6 and IND-00410-5 standards used were all purchased from Shenzhen Deborui Biotechnology Co., Ltd. (hereinafter referred to as Deborui); the plant genomic DNA extraction kit used was a commercial kit (DP305) purchased from Tiangen Biotechnology Co., Ltd.; the microfluidic chip supporting instrument used was the microfluidic nucleic acid analyzer BHP9609 produced by Hunan Rongjian Biotechnology Co., Ltd.

[0122] Example 1 Preparation of microfluidic chip

[0123] The microfluidic chip provided in this embodiment includes a chip and a bottom film and a top film (including a sealing film) bonded thereto. Figure 1 shows a schematic diagram of the structure of the microfluidic chip provided in this embodiment. The chip body is divided into four areas A / B / C / D, and each area is composed of a vent 1, a sample loading pool 2, a quantitative pool 3, a reaction pool 4, and a waste liquid pool 5. The vent 1 is used for air inlet and outlet, while ensuring smooth subsequent sample loading; the sample loading pool 2 is used for liquid loading, and the loading liquid is a mixture of isothermal amplification reaction liquid and sample; the quantitative pool 3 is used for liquid quantification to ensure that the volume of the reaction pool is fixed; the reaction pool 4 is used for reaction, and the mixture freeze-dried powder is pre-buried in the pool; the waste liquid pool 5 is used to store excess waste liquid.

[0124] In this embodiment, two different microfluidic chips are prepared. The difference between the two microfluidic chips is that the mixture freeze-dried powder pre-embedded in the reaction pool 4 is different. The pre-embedded mixture freeze-dried powder specifically includes the following two methods:

[0125] Method 1 includes: ① a primer sequence set designed according to the specific conserved gene sequence of the detection target (the detection targets are: Lectin gene, transgenic regulatory element pCaMV35S, tE9 and transgenic soybean lines GMB151, DP356043, DAS-68416-4, DBN-09004-6 and IND-00410-5 genes), ② a commercially available lyophilizable DNA LAMP Mix premix (a lyophilizable reaction solution containing Bst DNA polymerase, reaction buffer, dNTPs and other auxiliary materials) and ③ a fluorescent dye (SYBRGreen I).

[0126] Method 2 includes: ① a primer sequence set designed according to the specific conserved gene sequence of the detection target (the detection targets are: Lectin gene, transgenic regulatory element pCaMV35S, tE9, and transgenic soybean lines GMB151, DP356043, DAS-68416-4, DBN-09004-6 and IND-00410-5 genes), ② Tte UvrD helicase, ③ ET SSB, ④ commercial lyophilizable DNA LAMP Mix premix (lyophilizable reaction solution containing Bst DNA polymerase, reaction buffer, dNTPs and other auxiliary materials) and ⑤ fluorescent dye (SYBR Green I).

[0127] The Tte UvrD helicase used in this example is M1202 reagent produced by NEB, the ET SSB is M2401 reagent produced by NEB, and the commercial lyophilizable DNA LAMP Mix premix is ​​MDX119 reagent produced by Meridian, with a final concentration of 1X.

[0128] In various pre-buried primer sets, the molar ratio of the outer primer pair Lectin-F3 / B3, the inner primer pair Lectin-FIP / BIP and the loop primer pair Lectin-LF / LB is 1:8:4; the molar ratio of the outer primer pair pCaMV35S-F3 / B3, the inner primer pair pCaMV35S-FIP / BIP and the loop primer pair pCaMV35S-LF / LB is 1:8:4; the molar ratio of the outer primer pair tE9-F3 / B3, the inner primer pair tE9-FIP / BIP and the loop primer pair tE9-LF / LB is 1:8:4; the molar ratio of the outer primer pair GMB151-F3 / B3, the inner primer pair GMB151-FIP / BIP and the loop primer pair GMB151-LF / LB is 1:8:4; the molar ratio of the outer primer pair DP356043-F3 / B3, the inner primer pair The molar ratio of the primer pair DP356043-FIP / BIP and the loop primer pair DP356043-LF / LB was 1:8:4; the molar ratio of the outer primer pair DAS-68416-4-F3 / B3, the inner primer pair DAS-68416-4-FIP / BIP and the loop primer pair DAS-68416-4-LF / LB was 1:8:4; the outer primer pair DBN-09004-6 -F3 / B3, inner primer pair DBN-09004-6-FIP / BIP and loop primer pair DBN-09004-6-LF / LB have a molar ratio of 1:8:4; outer primer pair IND-00410-5-F3 / B3, inner primer pair IND-00410-5-FIP / BIP and loop primer pair IND-00410-5-LF / LB have a molar ratio of 1:8:4. Each target primer set includes a pair of outer primers, a pair of inner primers and a pair of loop primers, and the molar ratio between primers in each pair of primers is 1:1.

[0129] The primer set corresponding to the detection target, the commercial lyophilizable DNA LAMP Mix premix and the fluorescent dye (the microfluidic chip of method 2 is based on the microfluidic chip of method 1 and Tte UvrD helicase and ET SSB are added) are dissolved in sterile water to obtain a lyophilized mixture. Among them, the molar ratio between the primer pairs in each primer set is as shown above, the final concentration of Tte UvrD helicase in the microfluidic chip of method 2 is 0.1ng / μL, the final concentration of ET SSB is 5ng / μL, the final concentration of the commercial lyophilizable DNA LAMP Mix premix is ​​1X, and the final concentration of the fluorescent dye is 1X. The corresponding lyophilized mixture is spotted into the corresponding target well of the reaction pool 4 for bonding and lyophilization.

[0130] Bonding process: After the spotting is completed, a complete circular film 9596R is attached to the bottom, the lamination temperature is 60°C, the lamination pressure is 0.5MPa, and the lamination time is 2min; after the chip is freeze-dried, a circular film 4970c containing a fan-shaped sealing film is attached to the top, the lamination temperature is 60°C, the lamination pressure is 0.5MPa, and the lamination time is 2min.

[0131] Freeze-drying process: After the chip is placed in the freeze dryer, stay at -50℃ for 120min, turn on the vacuum pump, maintain the vacuum degree at 100kPa, stay at -50℃ for 120min (vacuum degree 100Pa); turn on the heating, maintain the vacuum degree at 1Pa, raise the temperature to -40℃, maintain for 120min, continue to raise the temperature to -30℃, maintain for 120min, then raise the temperature to -20℃, maintain for 120min, raise the temperature to 25℃, and turn off the freeze dryer. Among them, the bottom film material is plastic, and the top film (including the sealing film) material is plastic. After the chip is freeze-dried, take the aluminum bag and evacuate the vacuum to package the chip, and then place it in a 25℃ constant temperature box overnight to obtain two microfluidic chips.

[0132] Example 2 Preparation of detection kit

[0133] The components and compositions of the two detection kits provided in this embodiment are shown in Table 2 below:

[0134] Table 2

[0135]

[0136] Example 3 Primer screening experiment

[0137] Each set of primers is designed according to the specific conserved sequence of each target gene to be detected, wherein each set of primers includes: loop primers LF and / or LB, outer primers B3 and F3, inner primers FIP and BIP, as a primer set pre-buried in the reaction pool. In order to obtain a primer set with optimal specificity and sensitivity, the inventors of the present invention designed a primer set according to the specific conserved gene sequence of the target gene to be detected, and obtained three series of primer sets as shown in Tables 3, 4 and 5 below:

[0138] Table 3 Screening primer series 1 (the curve icons are left triangle / five-pointed star / asterisk / sphere, and the left triangle curve represents the negative control)

[0139]

[0140] Table 4 Screening primer series 2 (curve icons are right triangle / diamond / pentagon / square, right triangle curve represents negative control)

[0141]

[0142] Table 5 Screening primer series three (curve icons are cross / lower triangle / hexagon / upper triangle, the cross curve represents the negative control)

[0143]

[0144] Primer screening experiment:

[0145] Prepare the reaction system according to Table 6:

[0146] Table 6

[0147]

[0148] Two transgenic regulatory elements pCaMV35S and tE9, five transgenic soybean lines GMB151, DP356043, DAS-68416-4, DBN-09004-6 and IND-00410-5 genes, and soybean endogenous gene Lectin as detection target were prepared with enzyme-free water as solvent. 10 10 copies / mL concentration of plasmid solution. 10 The plasmid solution with a concentration of 10 copies / mL was diluted to make the concentration of the solution 10 4 copies / mL, 10 5 copies / mL and 10 6 copies / mL.

[0149] Using the mixed plasmid solution diluted in various gradients as the template and RNase-free water as the negative control (NC), the above primer combinations were placed in the detection kit 2 prepared in Example 2, mixed in the isothermal amplification reagent, and the reaction system was prepared according to Table 6 above. After adding the samples to the sample pools respectively, tear off the sealing film on the top film to fit tightly, and use a scraper to scrape in the same direction until it fits completely, and place the chip reaction well upward on the tray of the microfluidic nucleic acid analyzer BHP9609 to fix it. After centrifugation, isothermal amplification reaction was carried out at 62°C for 45 minutes. The fluorescence curve detection results are shown in Figures 3 to 10. The three series of primer sets provided in Tables 3-5 were screened for 8 detection targets, respectively. Among them, for primer sequence 1 in Table 3 above, the left triangle curve in Figure 3-10 represents the negative control, and the five-pointed star curve represents 10 of primer sequence 1. 4 Copies / mL concentration, the asterisk curve represents 10 of the primer sequence 1 5 Copies / mL concentration, the spherical curve represents 10 of the primer sequence 6 copies / mL concentration; for the primer sequence 2 in Table 4, the right triangle curve in Figure 3-10 represents the negative control, and the diamond curve represents the 10th copy / mL concentration of the primer sequence 2. 4 The pentagonal curve represents 10 copies / mL of primer sequence 2. 5 The square curve represents 10 copies / mL of primer sequence 2. 6 copies / mL concentration; for primer sequence three in Table 5, the cross curve in Figure 3-10 represents the negative control, and the lower triangle curve represents 10 copies / mL of primer sequence three 4 Copies / mL concentration, the hexagonal curve represents 10 of primer sequence three 5 copies / mL concentration, the upper triangle curve represents 10 of primer sequence three 6 The results are analyzed as follows:

[0150] FIG. 3 shows the negative control (NC) and 10 4 , 10 5 and 10 6 The isothermal amplification test results of soybean Lectin gene primer set were screened using Lectin plasmid solution with a concentration of 10 copies / mL as template. The fluorescence curve showed that the negative controls of the three series of primer sets were all negative, indicating that the primer sets themselves had no nonspecific reactions. 4 , 10 5 and 10 6 All Lectin plasmid solutions with different copy / mL concentrations can be detected, but primer series 2 and primer series 3 are detected later than primer series 1 at each concentration, indicating that primer series 1 has the highest sensitivity in detecting Lectin targets and will be used as the preferred primer set in the future.

[0151] FIG. 4 shows the negative control (NC) and 10 4 , 10 5 and 10 6 The isothermal amplification test results of the pCaMV35S primer set using a pCaMV35S plasmid solution with a concentration of 10 copies / mL as a template for screening transgenic regulatory elements. The fluorescence curve showed that the negative controls of the three series of primer sets were all negative, indicating that the primer sets themselves had no nonspecific reactions. The primer series 2 was 10 4 The concentration of copies / mL could not be detected. Primer series 3 and primer series 1 were used to detect 10 4 , 10 5 and 10 6 All pCaMV35S plasmid solutions with different concentrations of 10 copies / mL can be detected, but the detection time of primer series three is much later than that of primer series one at all concentrations, indicating that primer series one has the highest sensitivity in detecting pCaMV35S target and will be used as the preferred primer set in the future.

[0152] FIG. 5 shows the negative control (NC) and 10 4 , 10 5 and 10 6 The isothermal amplification test results of the primer set for screening the transgenic regulatory element tE9 using the tE9 plasmid solution with a concentration of 10 copies / mL as the template. The fluorescence curve showed that the negative controls of the three series of primer sets were all negative, indicating that the primer sets themselves had no nonspecific reactions, and the primer series three was only at 10 6 Copies / mL concentration detection, primer series 2 at 10 4 The primer series 1 detected tE9 target very early at each concentration, indicating that the primer series 1 had the highest sensitivity in detecting tE9 target and was subsequently used as the preferred primer set.

[0153] FIG. 6 shows the negative control (NC) and 10 4 , 10 5 and 10 6 The isothermal amplification test results of the primer set for screening the transgenic soybean line GMB151 using the GMB151 plasmid solution with a concentration of 10 copies / mL as the template. The fluorescence curve showed that the negative controls of the three series of primer sets were all negative, indicating that the primer sets themselves had no nonspecific reactions. The primer series three and primer series two were positive at 10 4 The results showed that primer series 1 had the highest sensitivity in detecting GMB151 target and was used as the preferred primer set in the future.

[0154] FIG. 7 shows the negative control (NC) and 10 4 , 10 5 and 106 The isothermal amplification test results of the primer set of DP356043, a DP356043 plasmid solution with a concentration of 10 copies / mL, were used as templates to screen the transgenic soybean line DP356043. The fluorescence curve showed that the negative controls of the three series of primer sets were all negative, indicating that the primer sets themselves had no nonspecific reactions. The primer series 2 and primer series 3 were positive at 10 4 The DP356043 target could not be detected at any concentration, and primer series one could be detected very early at all concentrations, indicating that primer series one had the highest sensitivity in detecting the DP356043 target and would be used as the preferred primer set in the future.

[0155] FIG8 is a graph showing the negative control (NC) and 10 4 , 10 5 and 10 6 The isothermal amplification test results of the primer set of DAS-68416-4 were screened using the DAS-68416-4 plasmid solution with a concentration of 10 copies / mL as a template. The fluorescence curve showed that the negative controls of the three series of primer sets were all negative, indicating that the primer sets themselves had no nonspecific reactions. The primer series 2 and primer series 3 were positive at 10 4 The primer series 1 could detect the target DAS-68416-4 at all concentrations, indicating that the primer series 1 had the highest sensitivity in detecting the target DAS-68416-4 and would be used as the preferred primer set in the future.

[0156] FIG. 9 shows the negative control (NC) and 10 4 , 10 5 and 10 6 The isothermal amplification test results of the primer set of DBN-09004-6 were screened using the DBN-09004-6 plasmid solution with a concentration of 10 copies / mL as a template. The fluorescence curve showed that the negative controls of the three series of primer sets were all negative, indicating that the primer sets themselves had no nonspecific reactions. The primer series 2 and primer series 3 were positive at 10 4 The concentration of copies / mL was not detected, and at 10 5 and 10 6 The detection time was very late at the concentration of copies / mL. On the contrary, primer series one could detect very early at all concentrations, indicating that primer series one had the highest sensitivity in detecting the DBN-09004-6 target and would be used as the preferred primer set in the future.

[0157] FIG. 10 is a graph showing the negative control (NC) and the 4 , 10 5 and 10 6The isothermal amplification test results of the primer set of IND-00410-5, a transgenic soybean line, were screened using the IND-00410-5 plasmid solution with a concentration of 10 copies / mL as a template. The fluorescence curve showed that the negative controls of the three series of primer sets were all negative, indicating that the primer sets themselves had no nonspecific reactions. The primer series three was positive at 10 4 The primer series 2 was detected later than the primer series 1 at all concentrations, indicating that the primer series 1 had the highest sensitivity in detecting the IND-00410-5 target and was subsequently used as the preferred primer set.

[0158] Example 4 Specificity detection of the detection kit

[0159] RNase-free water was used as the nucleic acid-free sample template; whole genome DNA of soybean, tomato, pepper, corn, rice, barley, wheat seeds and potato tubers was used as the template for specific detection of the soybean endogenous gene Lectin target; whole genome DNA of transgenic regulatory elements pCaMV35S, pFMV35S, pNOS, pUbi, tE9, t35S, tNOS and tOCS was used as the template for detection of two transgenic regulatory elements (pCaMV35S, tE9); a mixed solution of whole genome DNA of commercial soybean powder of five transgenic soybean lines (GMB151, DP356043, DAS68416-4, DBN-09004-6, IND-00410-5) was used as the template for gene-specific detection of the five transgenic soybean lines (whole genome DNA of the corresponding line detection target was used as the positive control, and whole genome DNA of the detection targets of the other four lines was used as the non-specific target nucleic acid).

[0160] Prepare the reaction system according to Table 6 above, use the nucleic acid detection kit 2 prepared in Example 2 for isothermal amplification detection, respectively place the above template DNA in the isothermal amplification reagent provided in the kit and mix, add the sample to the sample pool, tear open the sealing film on the top film to fit tightly, use a scraper to scrape in the same direction until it fits completely, place the chip reaction well upward and stably on the tray of the microfluidic nucleic acid analyzer BHP9609, fix it, centrifuge it, and perform isothermal amplification reaction at 62°C for 45 minutes. The fluorescence curve detection results are shown in Figures 11 to 19, and the results are analyzed as follows:

[0161] Figure 11 shows the target-specific isothermal amplification detection results of soybean Lectin gene, transgenic regulatory element pCaMV35S, tE9 and transgenic soybean lines GMB151, DP356043, DAS68416-4, DBN-09004-6 and IND-00410-5 using RNase-free water as a template in Example 4. The fluorescence curve shows that all detection targets have no peaks, indicating that all detection targets have no isothermal amplification reaction, indicating that the isothermal amplification detection method of the present invention for detecting the target Lectin gene, transgenic regulatory element pCaMV35S, tE9 and transgenic soybean lines GMB151, DP356043, DAS68416-4, DBN-09004-6 and IND-00410-5 genes has high specificity and no reaction occurs in the absence of target gene nucleic acid.

[0162] Figure 12 shows the isothermal amplification detection results of the target specificity of the Lectin gene detected using the whole genome DNA of soybean, tomato, pepper, corn, rice, barley, wheat seeds and potato tubers as templates in Example 4. The fluorescence curve shows that only when the whole genome DNA of soybean seeds is used as a template, the Lectin gene curve is a standard S-shaped curve, and when the whole genome DNA of non-soybean seeds is used as a template, the Lectin gene curve has no peak, indicating that only the Lectin target of soybean undergoes an isothermal amplification reaction, thereby detecting the Lectin target, and no Lectin target is detected in other non-soybean seeds, indicating that the detection target Lectin gene of the present invention is an endogenous gene of soybean and has a high specificity.

[0163] Figure 13 shows the target-specific isothermal amplification detection results of the transgenic regulatory element pCaMV35S using the whole genomic DNA of the transgenic regulatory element pCaMV35S, pFMV35S, pNOS, pUbi, tE9, t35S, tNOS and tOCS as templates in Example 4. The fluorescence curve shows that only the pCaMV35S curve is a standard S-type, and the other detection target templates have no peaks, indicating that only the pCaMV35S target undergoes an isothermal amplification reaction, thereby detecting the pCaMV35S target, and the other detection targets are not detected, indicating that the isothermal amplification detection method for detecting the target pCaMV35S of the present invention has high specificity and can specifically detect the transgenic regulatory element pCaMV35S target.

[0164] Figure 14 shows the target-specific isothermal amplification detection results of transgenic regulatory element tE9 using whole genomic DNA of transgenic regulatory elements pCaMV35S, pFMV35S, pNOS, pUbi, tE9, t35S, tNOS and tOCS as templates in Example 4. The fluorescence curve shows that only the tE9 curve is a standard S-type, and no peaks appear in other detection target templates, indicating that only the tE9 target undergoes an isothermal amplification reaction, thereby detecting the tE9 target, and the other detection targets are not detected, indicating that the isothermal amplification detection method for detecting target tE9 of the present invention has high specificity and can specifically detect the regulatory element tE9 target.

[0165] Figure 15 shows the isothermal amplification detection results of the target specificity of the transgenic soybean line GMB151 using the whole genome DNA mixed solution of commercial soybean powder of five strains of transgenic soybean as a template in Example 4. The fluorescence curve shows that only the GMB151 curve is a standard S-type, and no peaks appear in the other detection target templates, indicating that only the GMB151 target undergoes an isothermal amplification reaction, thereby detecting the GMB151 target, and the other detection targets are not detected, indicating that the isothermal amplification detection method of the detection target GMB151 of the present invention has high specificity and can specifically detect the transgenic soybean line GMB151 target.

[0166] Figure 16 shows the isothermal amplification detection results of the target specificity of transgenic soybean line DP356043 using the whole genome DNA mixed solution of commercial soybean powder of five transgenic soybean lines as templates in Example 4. The fluorescence curve shows that only the DP356043 curve is a standard S-type, and no peaks appear in other detection target templates, indicating that only the DP356043 target undergoes an isothermal amplification reaction, thereby detecting the DP356043 target, and the other detection targets are not detected, indicating that the isothermal amplification detection method for detecting the target DP356043 of the present invention has high specificity and can specifically detect the transgenic soybean line DP356043 target.

[0167] FIG17 shows the isothermal amplification detection results of the target specificity of transgenic soybean line DAS68416-4 using a mixed solution of whole genome DNA of commercial soybean powder of five transgenic soybean lines as a template in Example 4. The fluorescence curve shows that only the curve of transgenic soybean line DAS68416-4 is a standard S-type, and no peaks appear in other detection target templates, indicating that only DAS68416-4 target undergoes isothermal amplification reaction, thereby detecting DAS68416-4 target, and the other detection targets are not detected. This shows that the isothermal amplification detection method for detecting target DAS68416-4 of the present invention has high specificity and can specifically detect the target of transgenic soybean line DAS68416-4.

[0168] Figure 18 shows the target-specific isothermal amplification detection results of Example 4 using a mixed solution of whole genome DNA of commercial soybean powder of five strains of transgenic soybeans as a template to detect the transgenic soybean strain DBN-09004-6. The fluorescence curve shows that only the DBN-09004-6 curve is a standard S-type, and no peaks appear in other detection target templates, indicating that only the DBN-09004-6 target undergoes an isothermal amplification reaction, thereby detecting the DBN-09004-6 target, and the other detection targets are not detected, indicating that the isothermal amplification detection method of the present invention for detecting the target DBN-09004-6 has a high specificity and can specifically detect the transgenic soybean strain DBN-09004-6 target.

[0169] Figure 19 shows the isothermal amplification detection results of the target specificity of transgenic soybean line IND-00410-5 using the whole genome DNA mixed solution of commercial soybean powder of five transgenic soybean lines as templates in Example 4. The fluorescence curve shows that only the IND-00410-5 curve is a standard S-type, and no peaks appear in other detection target templates, indicating that only the IND-00410-5 target undergoes an isothermal amplification reaction, thereby detecting the IND-00410-5 target, and the other detection targets are not detected, indicating that the isothermal amplification detection method of the present invention for detecting the target IND-00410-5 has high specificity and can specifically detect the transgenic soybean line IND-00410-5 target.

[0170] Example 5 Sensitivity test of the detection kit and detection of genetically modified soybean samples

[0171] The soybean powder provided by Changsha Customs for extracting transgenic regulatory elements pCaMV35S and tE9, the non-transgenic soybean standard containing only the endogenous gene Lectin purchased from Deboray, and the standard powders of transgenic soybean lines GMB151, DP356043, DAS68416-4, DBN-09004-6 and IND-00410-5 were mixed at a mass fraction of 1%, 0.5% and 0.1% of each soybean powder sample, respectively. The total DNA of the sample was extracted using a commercial plant genomic DNA extraction kit (DP305), and the total DNA of the sample was mixed with the isothermal amplification reaction solution to prepare the reaction system according to Table 6 above. The nucleic acid detection kit provided in Example 2 (including the nucleic acid detection kits with and without superimposed high temperature resistant Tte UvrD helicase and extremely high temperature resistant ET SSB as comparison, all other components and preparation methods of the two are the same, see Example 2) is used for isothermal amplification detection, the above template DNA is placed in the isothermal amplification reagent provided in the kit for mixing, the sample is added to the sample pool, the sealing film on the top film is torn off to fit tightly, and the scraper is used to scrape in the same direction until it fits completely, and the chip reaction well is placed upward and fixed on the tray of the microfluidic nucleic acid analyzer BHP9609, and the isothermal amplification reaction is carried out at 62°C for 45 minutes after centrifugation. The fluorescence curve detection results are shown in Figures 20 to 22, and the results are analyzed as follows:

[0172] FIG20 is an isothermal amplification detection result showing the target sensitivity of soybean Lectin gene, transgenic regulatory element pCaMV35S, tE9 and transgenic soybean lines GMB151, DP356043, DAS68416-4, DBN-09004-6 and IND-00410-5 under the condition of 1% mass fraction of target sample using detection kit 1 (FIG20A) and detection kit 2 (FIG20B) in Example 5 of the present invention. The fluorescence curve shows that all detection target curves are standard S-shaped, indicating that for the mixed soybean powder with a target mass fraction of 1%, all detection targets have undergone isothermal amplification reaction, and all detection targets can be detected, indicating that each detection target can be stably detected at a mass fraction of 1%, but the peak time of all target curves in FIG20A is delayed compared with the corresponding target curve in FIG20B, indicating that the detection effect of the kit superimposed with Tte UvrD helicase and hyperthermostable single-stranded binding protein under this mass fraction condition is better.

[0173] Figure 21 shows the isothermal amplification detection results of the target sensitivity of detecting soybean Lectin gene, transgenic regulatory element pCaMV35S, tE9 and transgenic soybean lines GMB151, DP356043, DAS68416-4, DBN-09004-6 and IND-00410-5 under the condition of a target sample mass fraction of 0.5% in Example 5 of the present invention using detection kit 1 (Figure 21A) and detection kit 2 (Figure 21B). All detection target curves in Figure 21B showed a standard S-type, indicating that isothermal amplification reaction occurred in all detection targets of mixed soybean powder at a mass fraction of 0.5%, and all detection targets could be stably detected; the target curves of tE9, DAS68416-4 and DBN-09004-6 in Figure 21A were non-standard S-types, and the curves of all other detection targets showed a standard S-type, and the detection time of all detection targets was delayed by about 6 minutes compared with the corresponding target curves in Figure 21B, indicating that at this mass fraction, the existing detection targets tE9, DAS68416-4 and DBN-09004-6 were not stably detected, and the detection effects of each detection target were deteriorated, but after superimposing Tte UvrD helicase and ultra-thermostable single-stranded binding protein (i.e., detection kit 2), all detection targets could be stably detected at this plasmid concentration.

[0174] Figure 22 shows the isothermal amplification detection results of the target sensitivity of detecting the Lectin gene, transgenic regulatory element pCaMV35S, tE9 and transgenic soybean lines GMB151, DP356043, DAS68416-4, DBN-09004-6 and IND-00410-5 targets under the condition of a target sample mass fraction of 0.1% in Example 5 of the present invention using detection kit 1 (Figure 22A) and detection kit 2 (Figure 22B). The fluorescence curve in Figure 22B shows that DAS68416-4 is a negative curve, the target curves of tE9, DBN-09004-6, and IND-00410-5 are all non-S-type, and the targets of Lectin, pCaMV35S, GMB151, and DP356043 are amplified very late, indicating that for a 0.1% mass fraction of mixed soybean powder, the DAS68416-4 target is not amplified, the amplification of the targets of tE9, DBN-09004-6, and IND-00410-5 is not obvious, and the targets of Lectin, pCaMV35S, GMB151, and DP356043 are amplified very late. The fluorescence curves in Figure 22A show that tE9, DAS68416-4 and DBN-09004-6 are all negative curves. Except for the relatively obvious amplification of the Lectin target, the curves of other targets are slightly upturned, indicating that for the mixed soybean powder with a mass fraction of 0.1%, there is no amplification of tE9, DAS68416-4 and DBN-09004-6 targets, and the amplification of other detection targets is less obvious than that of the corresponding targets in Figure 22B, indicating that the closer to the sensitivity detection limit, the more obvious the effect of superimposing Tte UvrD helicase and ultra-thermostable single-stranded binding protein in the kit, and superimposing Tte The minimum detection sensitivity of the UvrD helicase and ultra-thermostable single-stranded binding protein detection kit II for Lectin gene, transgenic regulatory element pCaMV35S, tE9, and transgenic soybean lines GMB151, DP356043, DAS68416-4, DBN-09004-6 and IND-00410-5 targets is 0.5% (mass fraction).

[0175] In summary, the detection results show that when the detection kit one provided by the present invention detects the soybean endogenous gene Lectin, the transgenic regulatory element pCaMV35S and the transgenic soybean lines GMB151, DP356043 and IND-00410-5 gene targets, the minimum detection limit of the soybean sample is 0.5% (mass fraction); when the transgenic regulatory element tE9 and the transgenic soybean lines DAS68416-4 and DBN-09004-6 gene targets are detected, the minimum detection limit of the soybean sample is 1.0% (mass fraction); when the detection kit two provided by the present invention detects the soybean endogenous gene Lectin, two regulatory elements pCaMV35S, tE9 and five transgenic soybean lines GMB151, DP356043, DAS68416-4, DBN-09004-6 and IND-00410-5 gene targets, the minimum detection limit of the soybean sample is 0.5% (mass fraction), which has high sensitivity and is more sensitive than Tte without superposition of high temperature resistance. The nucleic acid detection kits with UvrD helicase and extremely high temperature resistant ET SSB and the nucleic acid detection kits with superimposed high temperature resistant Tte UvrD helicase and extremely high temperature resistant ET SSB were more stable and had earlier detection time at 1%, 0.5% and 0.1% mass fractions.

[0176] From the above experimental results, the detection method of the present invention can detect five strain targets of transgenic soybean and two transgenic regulatory element targets quickly, simply, synchronously, efficiently, highly specifically and highly sensitively under isothermal conditions.

[0177] Although various aspects and embodiments of the present invention are disclosed herein, other aspects and embodiments will be apparent to those skilled in the art. The various aspects and embodiments disclosed herein are for illustrative purposes only and not for limiting purposes. The scope and spirit of the present invention are determined solely by the appended claims.

Claims

1. A primer combination for simultaneously detecting multiple transgenic soybean lines and / or transgenic regulatory elements using loop-mediated isothermal amplification, characterized in that: The primer composition comprises the following primer sets: a primer set for detecting the 35S promoter of cauliflower mosaic virus, a primer set for detecting the terminator of pea ribulose bisphosphate carboxylase gene, a primer set for detecting the transgenic soybean line GMB151, a primer set for detecting the transgenic soybean line DP356043, a primer set for detecting the transgenic soybean line DAS-68416-4, a primer set for detecting the transgenic soybean line DBN-09004-6 and a primer set for detecting the transgenic soybean line IND-00410-5.

2. The primer composition according to claim 1, characterized in that The primer composition also includes a primer set for detecting soybean Lectin gene.

3. The primer composition according to claim 2, characterized in that The primer set for detecting soybean Lectin gene comprises: The outer primer pair Lectin-F3 and Lectin-B3, whose nucleotide sequences are shown in SEQ ID NOs. 1 and 2, respectively; The inner primer pair Lectin-FIP and Lectin-BIP, whose nucleotide sequences are shown in SEQ ID NOs. 3 and 4, respectively; The nucleotide sequences of the loop primer pair Lectin-LF and Lectin-LB are shown in SEQ ID NOs. 5 and 6, respectively.

4. The primer composition according to any one of claims 1 to 3, characterized in that The primer set for detecting the cauliflower mosaic virus 35S promoter comprises: The outer primer pair pCaMV35S-F3 and pCaMV35S-B3, whose nucleotide sequences are shown in SEQ ID NOs. 7 and 8, respectively; The inner primer pair pCaMV35S-FIP and pCaMV35S-BIP, whose nucleotide sequences are shown in SEQ ID NOs. 9 and 10, respectively; and The loop primer pair pCaMV35S-LF and pCaMV35S-LB, whose nucleotide sequences are shown in SEQ ID NOs. 11 and 12, respectively; The primer set for detecting the terminator of pea ribulose bisphosphate carboxylase gene comprises: The outer primer pair tE9-F3 and tE9-B3, whose nucleotide sequences are shown in SEQ ID NOs. 13 and 14, respectively; The inner primer pair tE9-FIP and tE9-BIP, whose nucleotide sequences are shown in SEQ ID NOs. 15 and 16, respectively; and Loop primer pair tE9-LF and tE9-LB, whose nucleotide sequences are shown in SEQ ID NOs. 17 and 18, respectively; The primer set for detecting the transgenic soybean line GMB151 comprises: The outer primer pair GMB151-F3 and GMB151-B3, whose nucleotide sequences are shown in SEQ ID NOs. 19 and 20, respectively; The inner primer pair GMB151-FIP and GMB151-BIP, whose nucleotide sequences are shown in SEQ ID NOs. 21 and 22, respectively; and The loop primer pair GMB151-LF and GMB151-LB, whose nucleotide sequences are shown in SEQ ID NOs. 23 and 24, respectively; The primer set for detecting transgenic soybean line DP356043 includes: The outer primer pair DP356043-F3 and DP356043-B3, whose nucleotide sequences are shown in SEQ ID NOs. 25 and 26, respectively; The inner primer pair DP356043-FIP and DP356043-BIP, whose nucleotide sequences are shown in SEQ ID NOs. 27 and 28, respectively; and The loop primer pair DP356043-LF and DP356043-LB, whose nucleotide sequences are shown in SEQ ID NOs. 29 and 30, respectively; The primer set for detecting the transgenic soybean line DAS-68416-4 includes: The outer primer pair DAS-68416-4-F3 and DAS-68416-4-B3, whose nucleotide sequences are shown in SEQ ID NOs. 31 and 32, respectively; The inner primer pair DAS-68416-4-FIP and DAS-68416-4-BIP, whose nucleotide sequences are shown in SEQ ID NOs. 33 and 34, respectively; and Loop primer pair DAS-68416-4-LF and DAS-68416-4-LB, whose nucleotide sequences are shown in SEQ ID NOs. 35 and 36, respectively; The primer set for detecting the transgenic soybean line DBN-09004-6 includes: The outer primer pair DBN-09004-6-F3 and DBN-09004-6-B3, whose nucleotide sequences are shown in SEQ ID NOs. 37 and 38, respectively; The inner primer pair DBN-09004-6-FIP and DBN-09004-6-BIP, whose nucleotide sequences are shown in SEQ ID NOs. 39 and 40, respectively; and Loop primer pair DBN-09004-6-LF and DBN-09004-6-LB, whose nucleotide sequences are shown in SEQ ID NOs. 41 and 42, respectively; The primer set for detecting transgenic soybean line IND-00410-5 includes: The outer primer pair IND-00410-5-F3 and IND-00410-5-6-B3, whose nucleotide sequences are shown in SEQ ID NOs. 43 and 44, respectively; The inner primer pair IND-00410-5-FIP and IND-00410-5-BIP, whose nucleotide sequences are shown in SEQ ID NOs. 45 and 46, respectively; and The nucleotide sequences of the loop primer pair IND-00410-5-LF and IND-00410-5-LB are shown in SEQ ID NOs. 47 and 48, respectively.

5. A microfluidic chip for simultaneously detecting multiple transgenic soybean strains and / or transgenic regulatory elements, characterized in that: Each primer set in the primer composition according to any one of claims 1 to 4 is respectively contained in different reaction pools of the microfluidic chip.

6. The microfluidic chip according to claim 5, characterized in that: The microfluidic chip comprises at least two groups of reaction pools, and in each group of reaction pools, each primer set in the primer composition according to any one of claims 1 to 4 is respectively contained in a different reaction pool of the microfluidic chip.

7. The microfluidic chip according to claim 5, characterized in that: Each of the reaction pools also contains a Tte UvrD helicase and an ultrathermostable single-stranded binding protein.

8. The microfluidic chip according to claim 5, characterized in that: The microfluidic chip also includes a vent hole, a sample addition pool, a quantitative pool and a waste liquid pool.

9. The microfluidic chip according to any one of claims 5 to 8, characterized in that: The microfluidic chip further comprises a bottom film bonded to the bottom thereof and a top film bonded to the top thereof, and the bottom film and the top film seal the microfluidic chip.

10. A method for preparing a microfluidic chip according to any one of claims 5 to 9, characterized in that: The preparation method comprises: (1) dissolving each primer set in the primer composition according to any one of claims 1 to 4, the DNA loop-mediated isothermal amplification premix and the fluorescent dye in sterile water to obtain a mixed solution; (2) spotting the mixed solution into the reaction wells of the microfluidic chip respectively; (3) Cover the bottom of the chip with the base film and heat it at 45~65℃ and 0.1~1Mpa for 0.5~3 minutes to bond the base film to the bottom of the chip; (4) After freeze-drying, cover the top of the chip with the top film and heat it at 45~65℃ and 0.1~1Mpa for 0.5~3 minutes to bond the top film to the top of the chip.

11. The preparation method according to claim 10, characterized in that: The freeze-drying comprises the following steps: (4-1) Pre-freeze the chip at 1-100 kPa and -60--40°C for 30-200 minutes, and then dry it at 1-100 Pa and -60--40°C for 30-200 minutes; (4-2) Continue drying under a vacuum degree of 1 to 10 Pa: heat to -50 to -30°C, maintain for 30 to 200 minutes, then heat to -40 to -20°C, maintain for 30 to 200 minutes, then heat to -30 to 0°C, maintain for 30 to 200 minutes, and then heat to 20 to 30°C.

12. A kit for simultaneously detecting multiple transgenic soybean lines and / or transgenic regulatory elements, characterized in that: The kit comprises the microfluidic chip according to any one of claims 5 to 9.

13. The kit according to claim 12, characterized in that The kit also contains a positive control, wherein the positive control contains the target amplified gene fragments of the following transgenic soybean targets: soybean Lectin gene, transgenic regulatory element pCaMV35S, transgenic regulatory element tE9, transgenic soybean line GMB151, transgenic soybean line DP356043, transgenic soybean line DAS-68416-4, transgenic soybean line DBN-09004-6 and transgenic soybean line IND-00410-5.

14. The kit according to claim 13, characterized in that The nucleotide sequences of the target amplified gene fragments of the soybean Lectin gene, transgenic regulatory element pCaMV35S, transgenic regulatory element tE9, transgenic soybean line GMB151, transgenic soybean line DP356043, transgenic soybean line DAS-68416-4, transgenic soybean line DBN-09004-6 and transgenic soybean line IND-00410-5 are shown in SEQ ID NOs. 131 to 138, respectively.

15. A method for simultaneously detecting multiple transgenic soybean lines and / or transgenic regulatory elements, characterized in that: The method comprises using the microfluidic chip according to any one of claims 5 to 9 or the kit according to any one of claims 12 to 14 for detection.

16. The method according to claim 15, characterized in that The method comprises using the microfluidic chip according to any one of claims 5 to 9 or the kit according to any one of claims 12 to 14 to perform a loop-mediated isothermal amplification reaction on the sample to be tested, and determining the presence of genetically modified soybean components in the sample to be tested based on the generated fluorescence curve.

17. The method according to claim 15 or 16, characterized in that Based on the mass of the sample to be tested, the mass fraction of the sample containing genetically modified soybean ingredients shall not be less than 1%.

18. The method according to claim 15 or 16, characterized in that Based on the mass of the sample to be tested, the mass fraction of the sample containing genetically modified soybean ingredients shall not be less than 0.5%.

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