Composition for detecting corn nucleic acid and application thereof

Abstract

The invention relates to a composition for detecting corn nucleic acid and application of the composition, belongs to the field of biological detection, and particularly relates to a real-time fluorescent quantitative detection system and a lateral flow chromatography sensor constructed by recombinase polymerase nucleic acid amplification and CRISPR / Cas12b through a simple and easy-to-operate process of single temperature change. The two-step reaction of RPA and CRISPR / Cas12b is sequentially carried out in the same reaction tube, so that the mutual interference of RPA and CRISPR / Cas12b is solved. SgRNA is designed according to an RPA product of a specific sequence of a transgenic corn DBN9936 transformant, and whether a to-be-detected sample contains a transgenic corn component or not can be rapidly detected according to an end point fluorescence value of a real-time fluorescence PCR instrument or a detection result of reading a lateral flow chromatography test strip by utilizing trans-cleavage activity of a reaction between a base complementation principle and CRISPR / Cas12b (Clustered Regularly Interspaced Short Palindromic Repeats / CRISPR / Cas12b).

Description

TECHNICAL FIELD

[0001] The present application relates to a composition for detecting corn nucleic acid and its application, and belongs to the field of biological detection, in particular to a nucleic acid detection method for detecting corn plant DBN9936. BACKGROUND

[0002] The detection of transgenic crops and food is necessary for the implementation of relevant national policies and the protection of consumers' right to know. The most commonly used method for detecting transgenic sequences in corn at home and abroad is polymerase chain reaction (PCR), which is mainly used in laboratories, so a new generation of nucleic acid detection technology with higher sensitivity and more flexible application scenarios is needed.

[0003] Recombinase polymerase amplification (RPA) is a rapid nucleic acid amplification technology that operates at a constant temperature of 37-42℃. It achieves amplification through the mediation of recombination enzymes to invade double-stranded DNA, the stabilization of templates by single-stranded binding proteins, and the chain extension of polymerase. It does not require the thermal cycling process of PCR and can be completed within 15-30 minutes. It is characterized by simple operation, speed, high sensitivity, and low equipment requirements, and is widely used in pathogen detection, food safety, environmental monitoring, and clinical point-of-care testing.

[0004] Lateral flow biosensor (LFB) is a rapid detection tool based on capillary action. It usually takes the form of a test strip, and the sample or amplification product binds to the fixed probe or antibody when it flows on the test strip. A visible band signal is generated in the detection zone, enabling rapid detection without complex instruments and visual interpretation. It has the advantages of simple operation, intuitive results, and strong portability, and is often combined with RPA, LAMP, and other isothermal amplification technologies for pathogen detection, food safety, and clinical point-of-care testing.

[0005] The combination of isothermal nucleic acid amplification technology and CPISPR system has obvious application potential. In the early stage, the combination of isothermal nucleic acid amplification technology and CPISPR system often carries out the amplification process and the CRISPR / Cas cutting process in two systems respectively, such as SHERLO, DETECTR and HOLMES, but this undoubtedly increases the opportunity of cross contamination and aerosol contamination. The subsequent AIOD-CRISPR and STOPCovid realize the one-pot of isothermal nucleic acid amplification technology and CPISPR system, but this brings new problems, such as the Cas protein cis-cutting activity leading to the amplification product cannot accumulate rapidly, and the trans-cutting activity degrading the primer. In order to solve these problems, strategies such as physical separation of isothermal amplification system and CRISPR system, use of suboptimal protospacer adjacent motifs (PAM) sequence, and light-activated CRISPR process are proposed. The use of these methods often requires the design of reaction containers or solution components, reduces the CRISPR reaction rate, and modifies the guide RNA, resulting in additional costs and inconvenience for on-site detection.

[0006] In summary, how to provide a transgenic food detection method with simple operation, low cost, rapidness, sensitivity and visualization is one of the problems to be solved in the field of food safety and transgenic food. SUMMARY

[0007] The present application aims at the deficiencies of the prior art, and aims to provide a nucleic acid sequence combination of RPA-CRISPR / Cas12b for detecting transgenic corn plants and a nucleic acid detection method.

[0008] The present application is to react the RPA amplification process and the CRISPR / Cas12b reaction in the same container, and to minimize the mutual influence of the RPA amplification process and the CRISPR / Cas12b reaction under temperature control. The principle is as follows Figure 1As shown, in the presence of transgenic corn DBN9936 components, the RPA amplification product of the extracted genome will trigger the single-stranded transcleavage activity of Cas12b, and the fluorescence reporter (fluorescence method) or reporter (test paper method) in the CRISPR / Cas12b reaction will be cut, emitting fluorescence (fluorescence method) or making the C line of the LFB unable to form a "gold nano-nucleic acid signal probe-reporter-T line nucleic acid" sandwich structure, unable to develop color, and the T line directly binds to the gold nano-nucleic acid signal probe to develop color (test paper method). When there is no transgenic corn DBN9936 component, no RPA amplification reaction occurs, the subsequent CRISPR / Cas12b reaction cannot be activated, the fluorescence reporter cannot be cut to emit fluorescence (fluorescence method) or the reporter is combined with the gold nano-nucleic acid signal probe on the LFB based on base complementarity, and as the sensor is eluted to the upper layer, a "gold nano-nucleic acid signal probe-reporter-C line nucleic acid" sandwich structure is formed at the C line, the C line is colored, and there is no excess or only a small amount of nucleic acid signal probe combined at the T line to develop color (test paper method).

[0009] To achieve the above-mentioned purposes, the technical scheme adopted by the present application is: On the one hand, the present application provides a set of compositions for detecting corn nucleic acids, characterized in that the composition is a nucleic acid sequence combination of RPA-CRISPR / Cas12b, including an RPA primer pair and an sgRNA.

[0010] Further, the RPA primer in the composition includes an upstream primer F and a downstream primer R, wherein F is selected from any one of the nucleic acids in SEQ ID NO: 1-3; R is selected from any one of the nucleic acids in SEQ ID NO: 4-6. Preferably, F in the RPA primer is the nucleic acid shown in SEQ ID NO: 1. Further preferably, R in the RPA primer is the nucleic acid shown in SEQ ID NO: 4. Most preferably, the nucleic acid sequence of the RPA primer F is shown in SEQ ID NO: 1, and the nucleic acid sequence of the RPA primer R is shown in SEQ ID NO: 4.

[0011] Further, the sgRNA sequence in the composition is selected from any one of SEQ ID NO: 7-19.

[0012] Preferably, the sgRNA is selected from SEQ ID NO: 17.

[0013] Further, the composition further includes a Cas12b protein.

[0014] Further, the corn is genetically modified corn, preferably, the genetically modified corn is DBN9936.

[0015] The second aspect of the present application provides use of the composition of the first aspect in the preparation of a reagent for detecting corn nucleic acid.

[0016] Further, the corn is genetically modified corn, preferably, the genetically modified corn is DBN9936.

[0017] The third aspect of the present application provides a kit for detecting corn nucleic acid, characterized in that the kit comprises the composition of the first aspect.

[0018] Further, the kit further comprises reagents required by recombinase polymerase amplification (RPA); preferably, the reagents required by RPA comprise recombinase, fluorescent reporter, and magnesium ions; most preferably, the reporter is labeled with a fluorescent group and a quencher group at both ends.

[0019] Further, the kit further comprises devices and reagents for lateral flow chromatography biosensor (LFB) detection.

[0020] Preferably, the device for LFB detection comprises a test strip pre-coated with spherical nucleic acid and labeled C-line and T-line nucleic acid; wherein the spherical nucleic acid is formed by the nucleic acid of SEQ ID NO: 26 and gold nanoparticles, the C-line labeled nucleic acid sequence is as shown in SEQ ID NO: 27, and the T-line labeled nucleic acid sequence is as shown in SEQ ID NO: 28. Preferably, the reagents required for LFB detection comprise a reporter and a buffer, and the nucleic acid sequence of the reporter is any one of the nucleic acids of SEQ ID NO: 21-25; most preferably, the nucleic acid sequence of the reporter is as shown in SEQ ID NO: 24.

[0021] Preferably, the composition and ratio of the buffer are 4×SSC + 10 mM Tris-HAc + 0.002% TritionX-100 + 2% BSA + 0.1% Tween20.

[0022] The fourth aspect of the present application provides a method for detecting corn nucleic acid, comprising the following steps: S1, extracting corn genome and performing RPA reaction in a reaction tube to which the composition of the first aspect is added; S2, performing CRISPR / Cas12b reaction after the RPA reaction in the same tube is completed; S3, detecting the RPA reaction product with end-point fluorescence value or LFB.

[0023] Further, the RPA reaction system and steps in the fluorescence value detection of S2 and S3 are as follows: (1) According to the system, add each reagent: add primer F and primer R to make the final concentration 0.2-0.3 μM, add reporter to make the final concentration 8-12 μM, add sgRNA to make the final concentration 550-650 nM, add Cas12b to make the final concentration 120-140 nM, and add magnesium acetate (MgAc) to make the final concentration 13-15 mM.

[0024] (2) The fluorescence quantitative PCR instrument is carried out at 35-40℃ for 20-40 cycles; the temperature is raised to 55-60℃ for 20-30 cycles, and the fluorescence signal is collected.

[0025] Further, the steps of the LFB detection reaction product used in S3 of the method are as follows: Add 85 μL running buffer and 15 μL RPA-CRISPR / Cas12b reaction product in the sample well, spray 1.2 μL spherical nucleic acid on the edge of the sample pad of LFB, insert the sample pad into the sample well, and wait for a few minutes to observe the results (in the method, the T line and the C line are opposite to the traditional LFB position, the red color appearing at the C line indicates that the chromatography condition of the test paper is normal; the red color appearing at the T line indicates that the result is positive, that is, the sample contains DBN9936 transgenic corn transformant components).

[0026] According to the LFB detection results, only the C line normally appears a red band, and the T line does not appear or only a light red band appears, which can be judged as no transgenic corn DBN9936 components are detected in the sample; the T line and the C line normally appear red bands, which can be judged as the transgenic corn DBN9936 components are detected in the sample.

[0027] The fifth aspect of the present application provides the use of the method of the fourth aspect in judging whether the corn sample to be tested contains transgenic corn. Further, the transgenic corn is DBN9936.

[0028] Compared with the prior art, the present application has the following advantages and beneficial effects: 1. The present application uses RPA reaction and CRISPR / Cas12b system reaction to construct real-time fluorescence detection and / or LFB detection. 2、The method solves the mutual interference of the two by sequentially performing the two-step reactions of RPA and CRISPR / Cas12b in the same reaction tube through a simple and easy-to-operate process of single change of temperature; 3、The application enables isothermal amplification and CRISPR reaction to be performed in one tube, and has good detection specificity, and the qualitative detection limit can reach 0.1 wt%.

[0029] 4、The application creatively prepares a test paper with C-line and T-line arrangement different from the traditional LFB sequence, and the T-line and C-line in the method are opposite to the positions of the traditional LFB, the appearance of red at the C-line indicates that the test paper chromatography is normal; the appearance of red at the T-line indicates that the result is positive, that is, the sample to be tested contains DBN9936 transgenic corn transformant components BRIEF DESCRIPTION OF DRAWINGS

[0030] The drawings described herein are used to provide further understanding of the embodiments of the application, constitute a part of the application, and do not constitute a limitation on the embodiments of the application. In the drawings: Figure 1 It is a technical route of a one-tube RPA-CRISPR / Cas12b combined lateral flow chromatographic sensor for on-site rapid detection of transgenic corn DBN9936.

[0031] Figure 2 It is an electrophoresis result of feasibility verification of RPA system and primer (lane M: D2000 DNA Marker; lane-: non-transgenic corn genome; lane+: transgenic corn DBN9936 genome).

[0032] Figure 3 It is sgRNA screening feasibility verification of CRISPR / Cas12b system for RPA product.

[0033] Figure 4 It is an RPA-CRISPR / Cas12b system optimization chart (A. Reporter concentration; B. Cas12b reaction temperature; C. MgAC concentration; D. Cas12b concentration; E. sgRNA concentration).

[0034] Figure 5 It is an RPA-CRISPR / Cas12b system sensitivity detection chart (A. 15 μL RPA-CRISPR / Cas12b system sensitivity detection chart; B. DBN9936 detection standard curve drawn according to the sensitivity detection chart).

[0035] Figure 6 It is a 15 μL RPA-CRISPR / Cas12b system specificity detection chart.

[0036] Figure 7 Principle of test paper for RPA-CRISPR / Cas12b detection of transgenic corn DBN9936 (A. Principle of test paper when DBN9936 is negative; B. Principle of test paper when DBN9936 is positive).

[0037] Figure 8 Optimization chart of RPA-CRISPR / Cas12b combined test paper detection conditions (the left side of each group of charts is the actual test paper strip sample chart, and the right side is the corresponding ImageJ analysis chart, * indicates p<0.05, **** indicates p<0.001: A. Reporter species optimization, test paper strips 1-3 are 20T, 4-6 are 22T, 7-9 are 24T, 10-12 are 26T, and 13-15 are 28T. Each Reporter has three parallels; B. Optimization of spherical nucleic acid species, the DNA addition amount of test paper strips 1-3 is 7.5 μL, 4-6 is 10 μL, 7-9 is 12.5 μL, 10-12 is 15 μL, and 13-15 is 17.5 μL. Each spherical nucleic acid has three parallels; C. Reporter amount optimization, the Reporter addition amount of test paper strips 1-3 is 0 μL, 4-6 is 1 μL, 7-9 is 2 μL, 10-12 is 4 μL, 13-15 is 6 μL, 16-18 is 8 μL, 19-21 is 10 μL, 22-24 is 11 μL, 25-27 is 12 μL, and 28-30 is 13 μL. Each concentration has three parallels. Since the test paper strips 22-30 have no gray value, there is no analysis of test paper 22-30 in the right ImageJ analysis chart; D. Optimization of spherical nucleic acid dosage, the Reporter addition amount of test paper strips 1-3 is 11 μL, 4-6 is 13 μL, 7-9 is 13 μL, 10-12 is 15 μL, and 13-15 is 17 μL. The spherical nucleic acid addition amount of test paper strips 1-3 is 0.6 μL, 4-6 is 0.8 μL, 7-9 is 1.0 μL, 10-12 is 1.2 μL, and 13-15 is 1.4 μL. Each concentration has three parallels).

[0038] Figure 9 Actual sample chart of RPA-CRISPR / Cas12b test paper detection (A. Actual sample chart of test paper detection, test paper strips 1-3 are non-transgenic corn, 4-6 are DBN3369 genome 0.05 ng / μL, 7-9 are 0.5 ng / μL, 10-12 are 5 ng / μL, and 13-15 are 50 ng / μL; B. ImageJ analysis chart corresponding to actual sample detection, ** indicates p<0.01, *** indicates p<0.005). DETAILED DESCRIPTION

[0039] The following examples are used to illustrate the present application but not to limit the scope of the present application.

[0040] The experimental methods used in the following examples are conventional methods unless otherwise specified.

[0041] The materials, reagents, etc. used in the following examples can be obtained commercially unless otherwise specified.

[0042] The present application will be described in detail below with reference to the accompanying drawings by way of examples, but is not limited to the present application, and is only used as an example.

[0043] The nucleic acid sequences used in the present application and their numbers are shown in Table 1 below: Table 1 Sequences used in the present application and their numbers Example 1 Detection of RPA-CRISPR / Cas12b system mechanism of transgenic corn DBN9936 1. Agarose gel electrophoresis, the specific steps are as follows: First, 50×TAE buffer solution needs to be prepared (dissolve 242.2 g of tris base in 500 mL of water with heating and stirring, then add 500 mM of EDTA disodium solution (pH 8.0), adjust the pH to 8.0 with glacial acetic acid, and dilute to 1000 mL with water), and dilute to 1×TAE buffer solution when used. When performing agarose gel electrophoresis, first clean the gel preparation tool with distilled water, select the appropriate gel preparation plate and comb to mount it. Accurately weigh 2 g of dry agarose powder, dissolve it in a 250 mL conical flask with 100 mL of 1×TAE buffer solution to prepare a 2% agarose gel. Cover the conical flask with a breathable sealing film and place it in a microwave oven to melt, cool to room temperature, then add 5 μL of EB solution (10 mg / mL) and mix well, pour into the gel preparation plate, remove the comb after the gel solidifies, then place the gel preparation plate together with the gel into an electrophoresis tank containing an appropriate amount of 1×TAE buffer solution, remove the air in the comb holes, then take out, add 1 μL of 6×Loading Buffer to 5 μL of sample, mix well, and load into the comb hole, and add D2000 DNA Marker to the appropriate comb hole. After loading, place the gel back into the electrophoresis tank and connect the power supply, electrophorese at 120 V for 25 min, then take out the gel, and observe the position of the sample and Marker on the ultraviolet gel imaging system.

[0044] 2. The RPA reaction operation process is as follows: RPA amplification reaction uses TwistDx TwistAmp basic kit, each reaction constructs 25 μL volume as shown in Table 1, and the reagents are added in 2 times of volume in turn, and then gently mixed, 25 μL is divided, 37℃ metal bath for 40 min, 65℃ inactivation for 10 min, and the reaction is terminated.

[0045] 3, In the verification process of the application, sgRNA is prepared, and the specific steps are as follows: All sgRNAs are prepared by transcribing 13 DNAs of TSG1-13 in Table 1 by Cas12b High Yield sgRNASynthesis and Purification Kit, and the specific operation steps are as follows: Template annealing and extension: 0.5 μL Cas12b Sense Oligo (10 μM) is TSG1-13, 0.5 μL Target Antisense Oligo (10 μM), 10 μL 2 × PCR Master Mix, 9 μL Nuclease-free Water is shaken and mixed and centrifuged, denatured at 95℃ for 2 min, 5 cycles (95℃ for 15 s, 55℃ for 15 s, 72℃ for 15 s), and then cooled to 25℃ for the next step.

[0046] sgRNA transcription: 5 μL of the product of the last step, 8 μL of NTP mix, 4 μL of 5 × TranscriptMax Reaction Buffer, 2.1 μL of TranscriptMax Enzyme Mix, 0.9 μL of Nuclease-free Water are shaken and mixed and centrifuged, incubated at 37℃ for 16 h, and then the next step is performed.

[0047] Template removal: 30 μL of reaction solution (25 μL of 2 × DNase Ⅰ Buffer, 4 μL of DNase Ⅰ, 1 μL of Nuclease-free Water) is added to the transcription system, shaken and mixed and centrifuged, incubated at 37℃ for 30 min, and then the next step is performed.

[0048] sgRNA purification: The magnetic beads of the kit were equilibrated at room temperature for 30 min with shaking, 25 μL of magnetic beads were mixed with 50 μL of isopropanol and added to the product of the previous step with shaking, incubated at room temperature for 5 min, placed in a magnetic stand for 5 min to clarify the solution, remove the supernatant, add 200 μL of freshly prepared 80% ethanol for rinsing, remove the supernatant after magnetic separation for 30 s, keep on the magnetic stand after 2 rinses, open the cover and air dry for 5 min (note to avoid over-drying), take out from the magnetic stand, add 50 μL of Nuclease-free Water, mix by blowing and suction, stand for 5 min, place in the magnetic stand for 5 min, transfer the solution to a new Nuclease-free tube after the solution is clarified, dilute to 10 μM after determining the concentration, and store at -80℃.

[0049] 4、The sgRNA screening in the verification process of the application has the following specific operation steps: Mix the initial 25 μL RPA-CRISPR / Cas12b system in an ice box (dissolve the enzyme powder in 29.5 μL Primer Free Rehydration buffer, add primers F and R to a final concentration of 0.48 μM, add Reporter to a final concentration of 200 nM, add sgRNA to a final concentration of 200 nM, add Cas12b to a final concentration of 200 nM, add MgAc to a final concentration of 14 mM, add Nuclease-free Water to make up to 46 μL, and then add 2 μL of template DNA). Select sgRNA with a BIO-RAD fluorescence quantitative PCR instrument at 37℃ for 20 cycles, and at 60℃ for 20 cycles (1 min / cycle), and collect the fluorescence signal.

[0050] 5、Primer and sgRNA selection The 3 RPA forward primers (SEQ ID NO: 1-3) and 3 RPA reverse primers (SEQ ID NO: 4-6) designed in the sequence table were used for the DNA genome (50 ng / µL) extracted from transgenic corn DBN9936, and the reaction was carried out according to the RPA system in Table 1. The RPA product was detected by 2% agarose gel electrophoresis, and the results are as follows Figure 2The products of upstream primer F1 and downstream primer R1 (184 bP, lane +1) and the products of upstream primer F2 and downstream primer R1 (120 bP, lane +4) obtained corresponding bright bands, and their RPA products (lane-1 and lane-4) without bands as templates of non-transgenic corn genome. Since the products of upstream primer F1 and downstream primer R1 (184 bP) contain the sequence of the products of upstream primer F2 and downstream primer R1 (120 bP), the design of sgRNA is based on the products of upstream primer F1 and downstream primer R1 (184 bP), and the precursor DNA of 13 sgRNAs-TSG1-13 is synthesized accordingly.

[0051] The prepared sgRNAs 1-13 (SEQ ID Nos: 7-19) were tested with 50 ng / μL of negative target (non-transgenic corn genome) and positive target (transgenic corn DBN9936 genome) in the sgRNA screening method, and the results are shown in Figure 3 sgRNA9 (SEQ ID NO: 15) and sgRNA11 (SEQ ID NO: 17) had no fluorescence signal in the negative results, and had fluorescence signal in the positive results, while the fluorescence signal of sgRNA9 was generated at 37℃, and the fluorescence signal of sgRNA11 was generated after the temperature was changed to 60℃. Considering the need to inhibit the single-stranded trans-cleavage activity of Cas12b at 37℃ as much as possible, sgRNA11 (SEQ ID NO: 17) was selected for subsequent experiments, and the recognition region of sgRNA11 was not contained by the products of upstream primer F2 and downstream primer R1, so upstream primer F1 (SEQ ID NO: 1) and downstream primer R1 (SEQ ID NO: 4) were selected for subsequent experiments.

[0052] Table 1 RPA reaction system Example 2 One-pot RPA-CRISPR / Cas12b fluorescence method optimization and corresponding detection performance 15 μL RPA-CRISPR / Cas12b system optimization According to the pre-experiment design, 15 μL RPA-CRISPR / Cas12b system was prepared as follows: in an ice box, the enzyme powder in the RPA kit was dissolved in 29.5 μL Primer Free Rehydration buffer, then 5 μL CRISPR / Cas12b buffer, primers F and R with a final concentration of 0.24 μM, reporter (SEQ ID NO: 20, 5' end labeled with 6-FAM, 3' end labeled with BHQ1) with a final concentration of 10 μM, sgRNA with a final concentration of 530 nM, Cas12b with a final concentration of 130 nM, MgAc with a final concentration of 18.6 mM, and nuclease-free water were added to make up to 65 μL, and 13 μL was aliquoted and then 2 μL template DNA was added. The sgRNA selection was performed at 37°C for 40 cycles and at 58°C for 20 cycles (1 min / cycle) on a BIO-RAD fluorescence quantitative PCR instrument, and the fluorescence signal was collected. The reporter concentration (5 μM, 6.7 μM, 8.3 μM, 10.0 μM, 11.7 μM), reaction temperature (52°C, 54°C, 56°C, 58°C, 60°C), MgAc concentration (9.3 mM, 14.0 mM, 18.6 mM, 23.3 mM, 28.0 mM), Cas12b concentration (60 nM, 80 nM, 100 nM, 120 nM, 140 nM, 160 nM), and sgRNA (400 nM, 467 nM, 530 nM, 600 nM, 667 nM) were optimized in the order of optimization. After MgAc optimization, the subsequent optimization was performed according to the optimal concentration of MgAc, and the other conditions remained unchanged except for the optimized conditions. Each of the above samples was tested in triplicate on a BIO-RAD fluorescence quantitative PCR instrument at 37°C for 20 cycles and at 58°C for 20 cycles (1 min / cycle), and the fluorescence signal was collected.

[0053] The experimental results are shown in Figure 4 . First, the effect of reporter concentration on the system was investigated. When the final concentration of reporter was 14 mM, the amplification efficiency and the final value of fluorescence signal were the highest, so 14 mM was selected as the optimal reporter concentration ( Figure 4 A ); the final value of fluorescence signal was the most stable and the production efficiency was higher when the reaction temperature of Cas12b step of RPA-CRISPR / Cas12b was 58°C, so 58°C was maintained as the reaction temperature of Cas12b step for subsequent experiments ( Figure 4 B ); the final value of fluorescence signal and the fluorescence production efficiency were the highest when the MgAc concentration was 14 mM ( Figure 4C), so the MgAc concentration of 14 mM was selected for subsequent experiments; the final value of the fluorescence signal and the highest production efficiency were obtained when the Cas12b concentration was 140 nM, and considering the cost of Cas12b, the Cas12b concentration of 130 nM was selected for subsequent experiments Figure 4 D); during the sgRNA condition optimization process, the final value of the fluorescence signal and the highest production efficiency were obtained when the sgRNA concentration was 600 nM, so the sgRNA concentration of 600 nM was selected for subsequent experiments Figure 4 E).

[0054] 15 μL RPA-CRISPR / Cas12b system sensitivity detection According to the optimized 15 μL RPA-CRISPR / Cas12b system under the optimal conditions of each factor, the sensitivity detection was performed, specifically, in an ice box, after dissolving the enzyme powder with 29.5 μL Primer Free Rehydration buffer, 5 μL CRISPR / Cas12b buffer was added, primers F and R were added to a final concentration of 0.24 μM, reporter was added to a final concentration of 10 μM, sgRNA was added to a final concentration of 600 nM, Cas12b was added to a final concentration of 130 nM, MgAc was added to a final concentration of 14 mM, and Nuclease-free Water was added to 65 μL, and then 2 μL template DNA was added after 13 μL was dispensed. The template concentration gradient was diluted to 50 ng / μL, 5 ng / μL, 0.5 ng / μL, 0.05 ng / μL, and non-transgenic corn genome 50 ng / μL as a negative control, and each concentration of genome was performed in triplicate, and the fluorescence signal was collected by BIO-RAD fluorescence quantitative PCR instrument at 37°C for 20 cycles, and at 58°C for 20 cycles (1 min / cycle).

[0055] The sensitivity of the reaction system was detected, according to Figure 5 (A-B) shown, the system can detect 0.05 ng / μL (0.1%) of the target, and quantitative detection can be achieved for 100% to 0.1% of the target, and no false positive results are obtained for the negative samples, which proves the reliability of the experimental results of the system. The system can detect 0.1% of the target concentration, which meets the detection requirements of the national standard for accidental mixing of transgenic seeds.

[0056] 15 μL RPA-CRISPR / Cas12b system specificity detection According to the optimized 15 μL each factor under the optimal condition of RPA-CRISPR / Cas12b system for specific detection, specifically, in the ice box, after dissolving the enzyme powder with 29.5 μL Primer Free Rehydration buffer, 5 μL CRISPR / Cas12b buffer was added, the final concentration of primer F and primer R was 0.24 μM, the final concentration of reporter was 10 μM, the final concentration of sgRNA was 600 nM, the final concentration of Cas12b was 130 nM, the final concentration of MgAc was 14 mM, the final concentration of Nuclease-free Water was 65 μL, 13 μL was divided, and 2 μL template DNA was added. As a sample for specific detection, mixed transgenic corn (MRZ604, NK603, 10.2.2), mixed transgenic soybean genome (GTS40-3-2, ZH10-6, DBN9004), mixed transgenic corn, soybean and rape genome (MON88902, RF3, GT73), non-transgenic corn genome and DBN9936 genome mixed into non-transgenic corn genome to a final concentration of 10% and 1%, above samples each three parallel to BIO-RAD fluorescence quantitative PCR instrument 37℃ for 20 cycles, 58℃ for 20 cycles (1 min / cycle), and the fluorescence signal was collected.

[0057] The transgenic corn mixture, transgenic soybean mixture and transgenic corn, soybean and rape mixture were detected according to Figure 6 The results showed that, except for DBN9936 sample, no fluorescence signal was generated in other samples, proving the specificity of the method. When DBN9936 was mixed with non-transgenic corn genome, the detection efficiency decreased, which was speculated to be due to the influence of other genomes reducing the detection efficiency, but 1% concentration of target and non-transgenic corn genome mixture could still be detected.

[0058] Example 3 One-pot RPA-CRISPR / Cas12b test paper method optimization and corresponding detection performance The principle of test paper method is shown in Figure 7 (A-B), which is summarized as: The application combines the RPA-CRISPR / Cas12b one-pot reaction product with LFB to detect transgenic corn DBN9936. In the absence of transgenic corn DBN9936 components, the RPA amplification reaction does not occur, and the subsequent CRISPR / Cas12b reaction cannot be activated, and the reporter is combined with the gold nano-nucleic acid signal probe on the LFB based on base complementarity, and then the sensor is eluted upward, and a "gold nano-nucleic acid signal probe-reporter-C line nucleic acid" sandwich structure is formed at the C line, so that the C line is colored. At this time, there is no excess gold nano-probe on the T line to be combined and colored. In the presence of transgenic corn DBN9936 components, the RPA amplification product of the extracted genome will trigger the single-stranded transcleavage activity of Cas12b, and the reporter will be cut in the CRISPR / Cas12b reaction, so that the "gold nano-nucleic acid signal probe-reporter-C line nucleic acid" sandwich structure cannot be formed at the C line of the LFB, and the C line cannot be colored. The T line is directly combined with the gold nano-nucleic acid signal probe and colored. In actual detection, the C line will not be completely colorless, because the reporter will not be completely cut, so the color change of the T line will be used to determine whether the target exists.

[0059] 1. Construction of test strip and corresponding detection operation steps Spherical nucleic acid preparation method: 100 mL of pure water is added to a glass flask containing a stirring magnet and heated to boiling, 26 μL of 1 mM chloroauric acid solution is added, and 10 mL of 38.8 mM sodium citrate solution is added to prepare 13 nm AuNPs. The solution is kept boiling for 15-20 min, and then cooled to room temperature. 200 μL of AuNPs solution is taken in a 1.5 mL centrifuge tube, and centrifuged at 13500 rpm in a centrifuge for 10 min, and the supernatant is removed as much as possible. 10 μL of 100 μM DNA (C8A, SEQ ID NO: 26) is added and mixed uniformly, and evaporated in a 90°C metal bath. 1350 μL of pure water is added, and centrifuged at 13500 rpm in a centrifuge for 15 min. The supernatant is removed as much as possible, and pure water is added to a volume of 25 μL. The spherical nucleic acid preparation is complete. Change the amount of added DNA to 5 μL, 7.5 μL, 10 μL, 12.5 μL, 15 μL, and 17.5 μL, respectively, and prepare the spherical nucleic acid according to the above method.

[0060] Test strip construction and assembly: nucleic acid AC8 (SEQ ID NO: 27) and C8T (SEQ ID NO: 28) were used as C-line and T-line respectively, the nucleic acid was dissolved to 100 μM after centrifugation at 12000 rpm for 15 min, and incubated with equal volume of 1 mg / mL streptavidin dissolved in 1xPBS buffer at 4°C overnight. The T-line solution was loaded into three-dimensional jetting platform jet 1, fixed at a position 1.1 cm away from the lower edge of the NC membrane, and the C-line solution was loaded into three-dimensional jetting platform jet 2, fixed at a position 1.6 cm away from the lower edge of the NC membrane, so that the distance between the T-line jet and the C-line jet was 5.0 mm. The NC membrane was stuck to the PVC bottom plate, and the line speed was set to 1.0 μL / cm, and the line solution was uniformly sprayed on the NC membrane. After the sprayed NC membrane was dried at 37°C for 3 h, the water absorption pad was stuck to the back plate close to the upper edge of the back plate, and carefully smoothed; the binding pad was stuck to the appropriate position of the back plate, and carefully smoothed; the sample pad was stuck to the back plate close to the lower edge of the back plate, and carefully smoothed. The test paper strip was cut into 3.5 mm wide test paper strips using a programmable cutter, and the cut test paper was placed in a packaging bag containing a desiccant, and stored at room temperature for standby.

[0061] Preparation method of loading buffer: the preparation method of loading buffer is 4xSSC, 10 mM Tris-Hac, 0.002% Trition X-100, 2% BSA, 0.1% Tween 20, and the pH is adjusted to 7.4 with HAc, and attention should be paid not to shake violently to prevent BSA from failing.

[0062] 2. Test strip condition optimization experiment (1) Reporter type: 0.8 μL of spherical nucleic acid probe prepared with 10 μL DNA was added to the sample pad of each test strip along the pre-coating, and the preparation method was as shown above, so that it did not penetrate into the binding pad, and was dried with a hair dryer until the spherical nucleic acid changed from red to black. In the enzyme-labeled plate, 13 μL of 10 μM Reporter was added in turn, and the Reporter types were 20T (SEQ ID NO: 21), 22T (SEQ ID NO: 22), 24T (SEQ ID NO: 23), 26T (SEQ ID NO: 24), and 28T (SEQ ID NO: 25), and 83 μL of loading buffer was added. Each Reporter was done in triplicate. The dried test strip was inserted into the enzyme-labeled plate, and waited for 10 min until the color of the T-line and C-line remained stable, and the C-line was plotted with ImageJ. The experimental results are shown in Figure 8A, 26T (SEQ ID NO: 24) is the optimal condition because its peak area on the C line is the highest, which proves that the ligation effect of the Reporter is the best. In the process of detecting actual samples, 26T is selected as the Reporter to add to the reaction system.

[0063] Spherical nucleic acid species: 0.8 μL of spherical nucleic acid probes were pre-coated on the sample pad of each test strip, and the preparation method is shown above. The amount of DNA was 7.5 μL, 10 μL, 12.5 μL, 15 μL, and 17.5 μL, respectively, so that it did not penetrate the conjugate pad. It was dried with a hair dryer until the spherical nucleic acid changed from red to black. Each spherical nucleic acid probe was made in triplicate. 13 μL of 10 μM 24T Reporter was added to the enzyme-labeled plate, and 83 μL of loading buffer was added. The dried test strip was inserted into the enzyme-labeled plate, and the color of the T line and the C line was allowed to remain stable for 10 min. The C line was analyzed by ImageJ. The experimental results are shown in Figure 8 B, the test strip with 10 μL of DNA has the highest peak area, and the increase in the amount of DNA has little effect on the results. In addition, the DNA end is modified with a thiol group, which is more expensive. Therefore, the spherical nucleic acid prepared with 10 μL of DNA is selected as the final probe.

[0064] Reporter amount: 0.8 μL of spherical nucleic acid probes were pre-coated on the sample pad of each test strip, and the preparation method is shown above. 10 μM 24T Reporter was added to the enzyme-labeled plate, and the volume was 0, 1, 2, 4, 6, 8, 10, 11, 12, 13 μL, respectively. The loading buffer was added to 100 μL, and each concentration was made in triplicate. The dried test strip was inserted into the enzyme-labeled plate, and the color of the T line was allowed to remain stable for 10 min. The T line was analyzed by ImageJ. The experimental results are shown in Figure 8 C, after determining the Reporter species and the spherical nucleic acid species, the quantitative ability of the test strip was preliminarily determined. The peak area decreased significantly with the decrease in the amount of Reporter.

[0065] Optimization of the amount of spherical nucleic acid probes: On the sample pad of each test strip, 0.6 μL, 0.8 μL, 1.0 μL, 1.2 μL, and 1.4 μL of spherical nucleic acid probes prepared from 10 μL of DNA were respectively pre-coated, and the preparation method is shown above. The spherical nucleic acid probes were blown dry with a hair dryer until the spherical nucleic acid probes changed from red to black. In the enzyme-labeled plate, 10 μM 24TReporter was added in the volume of 11, 13, 13, 15, and 17 μL, respectively, and the volume was made up to 100 μL with the loading buffer. Three parallel samples were prepared for each concentration. The dried test strips were inserted into the enzyme-labeled plate, and the color of the T line and the C line was allowed to stabilize for 10 min. The C line was plotted using ImageJ. The experimental results are shown in Figure 8 D, 1.2 μL of spherical nucleic acid probes was finally selected, and 1.5 μL of 100 μM Reporter was added to the system. Because 15 μL of 10 μM Reporter can completely capture the spherical nucleic acid probes on the C line, and the signal intensity of 1.2 μL of spherical nucleic acid probes is appropriate, and the amplification efficiency of 1.5 μL of Reporter is the highest, and the final value of the fluorescence signal is the highest.

[0066] (4) Detection of actual samples by test strips After the final reaction conditions of the test strips were determined, the RPA-CRISPR / Cas12b system was applied to the detection of DBN9936 actual samples, and Figure 9 A-B results show that this method can completely realize the qualitative detection of DBN9936, and the T line peak area of 0.1% positive samples and non-transgenic corn has a significant difference. When only Reporter is used, quantitative detection can be achieved, but after the actual sample is subjected to the RPA-CRISPR / Cas12b system, there is no significant gradient change between 0.1% and 100% positive samples from the graph. At present, quantitative detection has not been achieved. It is speculated that the length of the Reporter has increased compared with the fluorescence method, thereby affecting the final cutting efficiency of RPA-CRISPR / Cas12b, and there is a risk of connection with the C line even if the Reporter is cut once due to its excessive length. At the same time, in the negative sample, although there is a significant difference, the spherical nucleic acid cannot be completely captured by the C line, which is speculated to be due to the weak trans-cleavage activity of CRISPR / Cas12b under negative conditions. Therefore, in the subsequent research, the amount and length of the Reporter should also be adjusted to achieve the effect of actual on-site quantitative detection.

[0067] While the application has been described in detail and with reference to specific embodiments thereof, it will be apparent to one skilled in the art that various modifications or changes can be made therein without departing from the spirit and scope thereof. Accordingly, it is intended that all such modifications and changes be included within the scope of the application as claimed.

Claims

1. A set of compositions for detecting maize nucleic acids, characterized in that, The composition is a combination of RPA-CRISPR / Cas12b nucleic acid sequences, including RPA primer pairs and sgRNA. The RPA primer pairs in the composition include upstream primer F and downstream primer R, wherein F is selected from any nucleic acid in SEQ ID NO:1-3; R is selected from any nucleic acid in SEQ ID NO:4-6; and the sgRNA sequence in the composition is selected from any one of SEQ ID NO:7-19.

2. The composition according to claim 1, characterized in that, The nucleic acid sequence of the upstream primer F in the RPA primer pair is shown in SEQ ID NO:1, the nucleic acid sequence of the downstream primer R is shown in SEQ ID NO:4, and the nucleic acid sequence of the sgRNA is shown in SEQ ID NO:

17.

3. The use of the composition according to claim 1 or 2 in the preparation of reagents for detecting maize nucleic acid samples; characterized in that, The corn in question is genetically modified corn.

4. A kit for detecting maize nucleic acid, characterized in that, The kit comprises the composition of claim 1 or 2.

5. The reagent kit according to claim 4, characterized in that, The kit also includes Cas12b protein, reagents required for recombinase polymerization amplification of RPA; the reagents required for RPA include recombinase, fluorescent reporter, and magnesium ions.

6. The reagent kit according to claim 4, characterized in that, The kit also includes a device and reagents for lateral chromatography biosensor (LFB) detection. The LFB detection device includes a test strip pre-coated with spherical nucleic acid and nucleic acid labeled with C-lines and T-lines. The spherical nucleic acid is formed by combining the nucleic acid shown in SEQ ID NO:26 with gold nanoparticles. The nucleic acid sequence labeled with C-lines is shown in SEQ ID NO:27, and the nucleic acid sequence labeled with T-lines is shown in SEQ ID NO:

28. The reagents required for LFB detection include a reporter and a buffer solution. The nucleic acid sequence of the reporter is any one of the nucleic acids shown in SEQ ID NO:21-25.

7. A method for detecting nucleic acids in corn, characterized in that, The method includes the following steps: S1. Extract maize genome and perform RPA reaction in a reaction tube containing the composition of claim 1 or 2; S2. After the RPA reaction in the same tube is completed, the temperature is increased to carry out the CRISPR / Cas12b reaction; S3. Detect the reaction products of RPA reaction and CRISPR / Cas12b system using endpoint fluorescence value or LFB.

8. The method according to claim 7, characterized in that, When using endpoint fluorescence values ​​for product detection, steps S1-S3 are performed as follows: (1) Add the following reagents according to the system: add primer F and primer R to make the final concentration 0.2~0.3 μM, add reporter to make the final concentration 8~12 μM, add sgRNA to make the final concentration 550~650 nM, add Cas12b to make the final concentration 120~140 nM, and add magnesium acetate (MgAc) to make the final concentration 13~15 mM; (2) Perform 20-40 cycles at 35-40℃ using a real-time PCR instrument; then perform 20-30 cycles at 55-60℃ and collect fluorescence signals.

9. The method according to claim 7, characterized in that, When using LFB for product detection, step S3 is performed as follows: Add running buffer and RPA-CRISPR / Cas12b reaction product to the sample well, spray spherical nucleic acid onto the edge of the LFB sample pad, insert the test strip sample pad into the sample well, wait a few minutes, and the results can be observed. The T-line and C-line are positioned opposite to those of the traditional LFB. A red line at the C-line indicates normal chromatographic activity of the test strip, while a red line at the T-line indicates a positive result.

10. The application of the method according to any one of claims 7 to 9 in determining whether a corn sample to be tested contains genetically modified corn, characterized in that, The genetically modified corn in question is DBN9936.

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