Transgenic poplar visual detection system and detection method

Abstract

The invention discloses a visual detection system and detection method based on transgenic poplar. The visual detection system comprises an amplification primer, Cas12a protein and crRNA, a loop allosteric G-quadruplex, a suppressor and a scanning substrate. According to the method, an LAMP reaction is utilized to amplify a poplar transgenic fragment, CRISPR-Cas12a responds to an LAMP product and cuts a suppressor, a loop allosteric G-quadruplex can be combined with a heme post-catalysis substrate TBM to generate a yellow product in the absence of the suppressor, a scanner scans a chromogenic sample, RGB numerical values are collected, picture color information is analyzed, and the detection result is obtained. And the detection result can be stored in a high-throughput and digitalized manner. According to the detection method, complex instruments such as a thermal cycler are not needed, all related equipment can be carried about, a fixed power supply is not needed, and on-site and instant detection of the transgenic poplar in the field sampling process can be achieved.

Description

Technical Field

[0001] The invention belongs to the technical field of biological detection, and in particular relates to an on-site visual detection system and a detection method for transgenic poplars. Background Art

[0002] Poplar is one of the timber species with the largest cultivated area in the mid-latitude plains in the world. The area of ​​poplar plantations in my country has reached 7 million hectares, ranking first in the world. Poplars grow fast, have strong adaptability, are widely distributed, and have strong asexual reproduction ability. They are excellent broad-leaved tree species with important economic value. However, pests and diseases and abiotic stresses such as drought, salt damage and frost damage seriously affect forestry production. These unfavorable conditions have greatly restricted the planting and promotion of poplars. Transgenic is the use of engineering technology to transfer one or several genes into an organism to obtain new traits. At present, several new poplar varieties cultivated by transgenic technology have obtained safety production certificates and have been promoted in some areas of our country. Long-term and continuous research on the safety evaluation of transgenic poplars is of great significance to the development of forest genetic improvement and breeding work and the biosafety evaluation of transgenic forests. Through testing, the potential impact of transgenic poplars on the ecological environment can be evaluated to ensure that they meet safety standards before commercial planting. At present, commonly used transgenic detection methods include PCR technology, fluorescent quantitative PCR technology, gene sequencing technology, etc. These methods require professional instruments and equipment and are generally required to be completed in the laboratory. They cannot achieve on-site and rapid detection of genetically modified samples.

[0003] Loop-mediated isothermal amplification (LAMP) technology is an efficient isothermal amplification technology with low cost. LAMP technology can achieve exponential amplification of target genes and does not rely on thermal cyclers. It is widely used in the field of gene detection. For example, LAMP technology can be combined with fluorescent quantitative PCR technology to achieve ultra-sensitive detection of target genes.

[0004] Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) and Cas proteins are widely present in bacteria and archaea, and are an adaptive immune defense system unique to prokaryotes. The system has been developed into a gene editing tool and is widely used in molecular biology related fields. Cas12a protein has trans-cleavage activity, which constitutes the CRISPR-Cas12a system with crRNA. After the system is activated, it can cut single-stranded DNA in the system. The CRISPR-Cas12a system is widely used in the construction of various biosensors including genetic testing. Biosensors based on the CRISPR-Cas12a system usually use single-stranded DNA with fluorescent labels as signal indicators. When these DNAs are cut by the CRISPR-Cas12a system, fluorescent signals are released, indicating the presence of the detection target. However, capturing these fluorescent signals requires specific equipment such as fluorometers, which limits the application of such sensors.

[0005] G-quadruplex is a nucleic acid secondary structure formed by π-π stacking of regularly guanine-rich oligonucleotides. Some specific G-quadruplexes can form DNAzymes with peroxidase activity with heme. G-quadruplex DNAzymes can catalyze chromogenic substrates such as TMB in the presence of hydrogen peroxide, causing a change in the color of the solution.

[0006] RGB stands for Red, Green, Blue and is a color model used to display images and videos. In the RGB model, colors are created by combining three basic colors of light. Each color has a value from 0 to 255, representing the intensity of that color of light. By changing these values, more than 16 million different colors can be produced. Summary of the invention

[0007] The purpose of this section is to summarize some aspects of embodiments of the present invention and briefly introduce some preferred embodiments. Some simplifications or omissions may be made in this section and the specification abstract and the invention title of this application to avoid blurring the purpose of this section, the specification abstract and the invention title, and such simplifications or omissions cannot be used to limit the scope of the present invention.

[0008] In view of the above problems and / or the problems existing in the prior art, the present invention is proposed.

[0009] Therefore, the purpose of the present invention is to overcome the deficiencies in the prior art and provide a transgenic poplar visualization detection system.

[0010] In order to solve the above technical problems, the present invention provides the following technical solutions: a transgenic poplar visualization detection system, characterized in that it includes: an amplification primer, a Cas12a protein, a crRNA, a loop region allosteric G-quadruplex, an inhibitor and a scanning substrate;

[0011] Among them, the amplification primers include F3 primers, B3 primers, FIP primers, BIP primers, Loop-F primers and Loop-B primers; the Cas12a protein and crRNA recognize the target gene and activate the trans-cleavage activity of the Cas12a protein; the loop region allosteric G-quadruplex is a deoxyribozyme with catalytic activity formed by binding to heme; the inhibitor is a trans-cleavage substrate of the LbCas12a protein, which inhibits the formation of the loop region allosteric G-quadruplex; the scanning substrate is loaded with a color developing reagent.

[0012] As a preferred embodiment of the transgenic poplar visualization detection system described in the present invention, the F3 sequence is shown in SEQ ID No.1; the B3 sequence is shown in SEQ ID No.2; the FIP sequence is shown in SEQ ID No.3; the BIP sequence is shown in SEQ ID No.4; the Loop-F sequence is shown in SEQ ID No.5; the Loop-B sequence is shown in SEQ ID No.6; the crRNA sequence is shown in SEQ ID No.7; the loop region allosteric G-quadruplex sequence is shown in SEQ ID No.8; and the inhibitor sequence is shown in SEQ ID No.9.

[0013] As a preferred solution of the transgenic poplar visualization detection system described in the present invention, the amplification primers are used to amplify the target gene fragment containing the recognition sequence of the CRISPR-Cas12a system through the LAMP reaction with the transgenic poplar genomic DNA as a template; the gene fragment can activate the trans-splicing activity of the Cas12a protein and cut the inhibitor; the cut inhibitor cannot inhibit the formation of the allosteric G-quadruplex in the loop region; the allosteric G-quadruplex in the loop region binds to heme to form a deoxyribozyme, catalyzes the oxidation of the substrate 3,3',5,5'-tetramethylbenzidine, and produces a yellow product after adding hydrochloric acid; the color developing solution is carried on a scanning substrate, a scanner is used to scan a fixed area of ​​the substrate, a digital photo is obtained, and the RGB value is read by software for analysis.

[0014] Another object of the present invention is to overcome the deficiencies in the prior art and provide a detection method for a transgenic poplar visualization detection system.

[0015] In order to solve the above technical problems, the present invention provides the following technical solutions: A method for a transgenic poplar visualization detection system, characterized in that: it includes adding poplar genomic DNA to a LAMP reaction solution and incubating to obtain a reaction solution in which the genome is amplified by LAMP;

[0016] The reaction solution in which the genome is amplified by LAMP is added to the CRISPR-Cas12a reaction solution and incubated to obtain a CRISPR-Cas12a mixed reaction solution;

[0017] The loop region allosteric G-quadruplex is mixed with a buffer to prepare a loop region allosteric G-quadruplex storage solution;

[0018] The CRISPR-Cas12a mixed reaction solution, the loop region allosteric G-quadruplex storage solution, heme, and buffer C were mixed to prepare the G-quadruplex working solution;

[0019] The G-quadruplex working liquid, TMB, hydrogen peroxide solution, and color development buffer were mixed to prepare a color development reaction solution, hydrochloric acid was added to stop the reaction, and the color change of the solution was observed. The color development reaction solution was added to a scanning substrate, and a scanner was used to scan a fixed area of ​​the substrate to obtain a digital photo, and the RGB value was read using software for analysis.

[0020] As a preferred embodiment of the method of the present invention, the preparation method of the LAMP reaction solution includes adding 1.6 μM FIP primer, 1.6 μM BIP primer, 0.4 μM Loop-F primer, 0.4 μM Loop-B primer, 0.2 μM F3 primer, 0.2 μM B3 primer, 1.4 mM dNTP and 0.32 U / μL Bst II enzyme to buffer A to obtain the LAMP reaction solution.

[0021] As a preferred embodiment of the method of the present invention, the method for preparing the CRISPR-Cas12a reaction solution comprises adding 10 nM crRNA, 30 ng / μL Cas12a protein, and 1 μM inhibitor to buffer B to obtain the CRISPR-Cas12a reaction solution.

[0022] As a preferred embodiment of the method of the present invention, the preparation method of the color development buffer comprises mixing 200 mM ammonium chloride, 100 mM disodium hydrogen phosphate, and 51 mM citric acid to obtain the color development buffer.

[0023] As a preferred embodiment of the method of the present invention, the reaction solution in which the genome is amplified by LAMP is added to the CRISPR-Cas12a reaction solution for incubation to obtain a CRISPR-Cas12a mixed reaction solution, wherein the incubation time is 10 to 60 minutes.

[0024] As a preferred embodiment of the method of the present invention, the buffer A is prepared by 50 mM potassium chloride, 20 mM tris(hydroxymethyl)aminomethane, 10 mM ammonium sulfate, 10 mM magnesium sulfate, 0.1% Tween-20, and the pH is adjusted to 8.8 using hydrochloric acid; the buffer B is prepared by 50 mM sodium chloride, 20 mM tris(hydroxymethyl)aminomethane, 10 mM magnesium sulfate, 1 mM dithiothreitol, and the pH is 7.9; the buffer C is prepared by 300 mM ammonium chloride, 20 mM 4-hydroxyethylpiperazineethanesulfonic acid, 0.05% Triton X-100, 1% dimethyl sulfoxide, and the pH is 7.4.

[0025] As a preferred solution of the method of the present invention, the scanning substrate is fixed at a fixed position of a portable scanner for scanning, and the RGB value of the sample image is analyzed using Color Picker online software.

[0026] Beneficial effects of the present invention:

[0027] The present invention provides a transgenic poplar visualization detection system, which utilizes loop-mediated isothermal amplification reaction to amplify poplar transgenic fragments, utilizes CRISPR-Cas12a to respond to the amplified gene fragments and cut inhibitors, utilizes loop region allosteric G-quadruplex to bind heme and then catalyze substrate TBM to generate yellow products, scans color samples by scanner and analyzes RGB values, and images and digitally stores the detection results. The color images are obtained by scanner, and high-throughput detection signal acquisition and processing can be completed. The color digital photos obtained by the method, because the scanning position is fixed by a scanning substrate, the color images obtained during each scanning process are not affected by the imaging angle and the ambient light source, so that the color information of the image is analyzed using RGB values ​​more accurately; the detection method does not require complex instruments such as thermal cyclers, all the equipment involved can be carried, and no fixed power supply is required, so that on-site and real-time detection of transgenic poplars can be achieved during field sampling. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the following briefly introduces the drawings required for describing the embodiments. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without creative labor. Among them:

[0029] Figure 1 It is a detection flow chart of the present invention.

[0030] Figure 2 The present invention is an assembly and scanning process of the scanning substrate.

[0031] Figure 3 The invention discloses agarose gel electrophoresis analysis of LAMP reaction to amplify transgenic poplar gene fragments.

[0032] Figure 4 This is a polyacrylamide gel electrophoresis analysis of the transgenic poplar gene fragment activating the Cas12a trans-cleavage activity result of the present invention.

[0033] Figure 5 This is a diagram showing the results of analyzing a colorimetric sample using the ultraviolet spectrophotometer of the present invention.

[0034] Figure 6 This is a diagram showing the optimization results of the ratio of the inhibitor to the allosteric G-quadruplex in the loop region of the present invention.

[0035] Figure 7 This is a diagram showing the optimization results of the CRISPR-Cas12a shearing time of the present invention.

[0036] Figure 8 This is a graph showing the results of the present invention in detecting the wild type and six strains of transgenic Nanlin 895 poplar. DETAILED DESCRIPTION

[0037] In order to make the above-mentioned objects, features and advantages of the present invention more obvious and easy to understand, the specific implementation methods of the present invention are described in detail below in conjunction with the embodiments of the specification.

[0038] In the following description, many specific details are set forth to facilitate a full understanding of the present invention, but the present invention may also be implemented in other ways different from those described herein, and those skilled in the art may make similar generalizations without violating the connotation of the present invention. Therefore, the present invention is not limited to the specific embodiments disclosed below.

[0039] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The term "in one embodiment" that appears in different places in this specification does not necessarily refer to the same embodiment, nor does it refer to a separate or selective embodiment that is mutually exclusive with other embodiments.

[0040] The raw materials used in the examples of the present invention are all commercially available unless otherwise specified, as shown in Table 1.

[0041] Table 1

[0042]

[0043]

[0044] The nucleic acid standard is a gene fragment amplified using PCR technology, with transgenic poplar genomic DNA as a template, using 35s-F (SEQ ID No. 10), 35s-R (SEQ ID No. 11) and FidCycle high-fidelity DNA polymerase. The target gene sequence is confirmed by sequencing, and after purification, it is quantified using a nucleic acid quantifier.

[0045] Cas12a protein was obtained by prokaryotic induced expression and purification. For specific methods, see: Analytica Chimica Acta, 2023, 1283: 341950.

[0046] The wild type 895 poplar and the transgenic 895 poplar used in the present invention are from the transgenic poplar intermediate experimental field of Chenxu Forest Farm, Sihong County, Jiangsu Province.

[0047] For the source of the transgenic 895 poplar genome, see: Molecular Plant Breeding, 2020, 18(14): 4645-4656; the invention patent is an artificially synthesized cry1Ah1 gene and its application, with patent number 201810123435.3.

[0048] Example 1

[0049] The visualization detection process of transgenic poplar in the present invention is shown in Figure 1 . Collect transgenic poplar leaves, extract poplar genomic DNA using a genomic DNA extraction kit; use loop-mediated isothermal amplification (LAMP) technology to amplify the target gene fragment, which contains the recognition sequence of the CRISPR-Cas12a system, wherein the LAMP amplification primers include FIP, BIP, Loop-F, Loop-B, F3, and B3; use LAMP to amplify the gene product to activate the trans-cleavage activity of the CRISPR-Cas12a system and cut the inhibitor, wherein the CRISPR-Cas12a system consists of crRNA and Cas12a protein; because the cut inhibitor cannot destroy the secondary structure of the allosteric G-quadruplex in the loop region, the complete allosteric G-quadruplex in the loop region binds to heme to form a catalytically active deoxyribozyme, catalyzes the TMB color development reaction, and outputs a color development signal. The oligonucleotide sequences involved in the present invention are shown in Table 2.

[0050] Table 2

[0051]

[0052]

[0053] Note: crRNA in the table is ribonucleic acid (RNA), and the rest of the sequences are deoxyribonucleic acid (DNA).

[0054] The method for detecting transgenic poplars using the above detection system comprises the following steps:

[0055] (1) Extraction of genomic DNA from poplar: About 100 mg of fresh poplar leaves were collected and genomic DNA was extracted according to the instructions of the genomic DNA extraction kit;

[0056] (2) LAMP amplification of genomic DNA fragments: 2 μL of poplar genomic DNA solution (40 ng / μL), 1 μL of 10× primer mixture (16 μM FIP, 16 μM BIP, 4 μM Loop-F, 4 μM Loop-B, 2 μM F3, 2 μM B3), 1.4 μL of dNTP (10 mM), 0.4 μL of BstII polymerase (8 U / μL) were mixed in buffer A (50 mM potassium chloride, 20 mM tris, 10 mM ammonium sulfate, 10 mM magnesium sulfate, 0.1% Tween-20, pH adjusted to 8.8 with hydrochloric acid), and the final volume was 10 μL. The LAMP reaction solution was incubated at 65°C for 30 minutes, and then incubated at 80°C for 20 minutes to terminate the reaction;

[0057] (3) Prepare a mixture of Cas12a and crRNA: Take 0.25 μL Cas12a protein (2400 ng / μL) and 1 μL crRNA (200 nM), mix in buffer B (50 mM sodium chloride, 20 mM tris(hydroxymethyl)aminomethane, 10 mM magnesium sulfate, 1 mM dithiothreitol, pH 7.9) to a final volume of 16 μL;

[0058] (4) Cutting the inhibitor by the CRISPR-Cas12a system: Add 0.2 μL of LAMP amplification product, 2 μL of inhibitor (10 μM) and 1.8 μL of deionized water to the mixture in (3) to a final volume of 20 μL, incubate at 37°C for 30 minutes, and then immediately heat to 70°C;

[0059] (5) Preparation of ring region allosteric G-quadruplex storage solution: 1 μL of ring region allosteric G-quadruplex solution (10 μM) was mixed in buffer C (300 mM ammonium chloride, 20 mM 4-hydroxyethylpiperazineethanesulfonic acid, 0.05% Triton X-100, 1% dimethyl sulfoxide, pH 7.4) to a final volume of 10 μL;

[0060] (6) Preparation of loop region allosteric G-quadruplex working solution: 2.2 μL of the prepared CRISPR-Cas12a reaction solution, 2 μL of the prepared loop region allosteric G-quadruplex storage solution, and 1 μL of heme (5 μM) were mixed in buffer C to prepare a final volume of 26 μL of G-quadruplex working solution, and incubated at 25°C for 30 minutes;

[0061] (7) Color reaction: Add 0.5 μL TMB (200 mM) and 20 μL hydrogen peroxide solution (3%) to the G-quadruplex working liquid and mix in a color development buffer to prepare a final volume of 200 μL color development reaction solution. Incubate at 25°C for 5 min. Add 200 μL hydrochloric acid (1 M) to stop the reaction and observe the color change of the solution.

[0062] (8) Obtaining standardized digital images of color samples: Add the color reaction solution to the scanning substrate, use a portable scanner to scan a fixed area of ​​the substrate, and obtain a digital photo. The image output format is JPEG format, and the image resolution is 800 DPI. The color digital photo obtained by this method is not affected by the imaging angle and the ambient light source during each scanning process because the scanning position is fixed by the scanning substrate. Scanning substrate and scanning method, see Figure 2 ;

[0063] (9) Analysis of color sample images: Use the Color Picker online software (https: / / redketchup.io / color-picker) to analyze the RGB values ​​of the sample images in the center area of ​​the scanned sample points, record the B value in the RGB value, and subtract the measured value from 255 as the sample detection reading (the lower the B value, the yellower the color); use the average reading of the 12 blank sample data plus ten times the standard deviation (Blank+10SD) as the basis for judging the positive signal. A value greater than or equal to this value is judged as positive, and a value less than this value is judged as negative.

[0064] Example 2

[0065] Figure 3 The method is to use agarose gel electrophoresis to analyze the LAMP reaction to amplify the transgenic poplar gene fragment. The method comprises the following steps:

[0066] (1) Extraction of genomic DNA from poplar: About 100 mg of fresh poplar leaves were collected and genomic DNA was extracted according to the instructions of the genomic DNA extraction kit;

[0067] (2) LAMP amplification of genomic DNA fragments: 2 μL of LAMP amplification template (40 ng / μL), 1 μL of 10× primer mixture (16 μM FIP, 16 μM BIP, 4 μM Loop-F, 4 μM Loop-B, 2 μM F3, 2 μM B3), 1.4 μL of dNTP (10 mM), 0.4 μL of BstII polymerase (8 U / μL) were mixed in buffer A (50 mM potassium chloride, 20 mM tris, 10 mM ammonium sulfate, 10 mM magnesium sulfate, 0.1% Tween-20, pH adjusted to 8.8 with hydrochloric acid), and the final volume was 10 μL. The LAMP reaction solution was incubated at 65°C for 30 minutes, and then incubated at 80°C for 20 minutes to terminate the reaction;

[0068] The templates for LAMP amplification in (2) were ionized water (blank sample), nucleic acid standards, wild-type poplar genomic DNA, and transgenic poplar genomic DNA.

[0069] (3) The LAMP products were analyzed using 1% agarose gel electrophoresis and photographed using a gel imager.

[0070] like Figure 3 As shown, no LAMP amplification bands were found in the blank sample and the wild-type poplar sample, while LAMP amplification bands were found in the nucleic acid standard sample and the transgenic poplar sample, indicating that the designed LAMP primers can specifically amplify the target fragment.

[0071] Example 3

[0072] Figure 4 The method is to use polyacrylamide gel electrophoresis to analyze the transgenic poplar gene fragment to activate the Cas12a trans-cleavage activity. It includes the following steps:

[0073] Steps (1) and (3) in this example are consistent with steps (1) and (3) in Example 1. The difference is that in step (2), the template poplar genomic DNA solution for LAMP amplification is replaced with ionized water (blank sample), nucleic acid standard, wild-type poplar genomic DNA, and transgenic poplar genomic DNA, respectively; in step (4), the inhibitor is replaced with FAM-inhibitor to facilitate gel imaging analysis.

[0074] (5) The Cas reaction solution was analyzed using 12% polyacrylamide gel electrophoresis and photographed using a gel imaging instrument.

[0075] like Figure 4As shown, neither the blank sample nor the wild-type poplar sample cleaved the FAM-repressor, while the nucleic acid standard sample and the transgenic poplar sample showed cleaved FAM-repressor, indicating that the gene fragment obtained by LAMP amplification can activate the trans-cleavage activity of Cas12a and cleave the substrate.

[0076] Example 4

[0077] Figure 5 To analyze the color samples using a UV spectrophotometer. The steps include:

[0078] Steps (1), (3) to (7) in this embodiment are similar to steps (1), (3) to (7) in Example 1.

[0079] (7) except that in step (2), the template poplar genomic DNA solution for LAMP amplification was replaced with ionized water (blank sample), nucleic acid standard, wild-type poplar genomic DNA, and transgenic poplar genomic DNA, respectively.

[0080] (8) Use a UV-visible spectrophotometer to measure the absorbance, set the maximum absorption wavelength to 450 nm, and measure the absorbance.

[0081] like Figure 5 As shown, the blank sample and the wild-type poplar sample had no obvious color and low absorbance, while the nucleic acid standard sample and the transgenic poplar sample showed obvious yellow and the absorbance increased significantly, indicating that the sample containing the gene fragment can indeed make the detection solution appear yellow and cause the absorbance of the solution to increase.

[0082] Example 5

[0083] Figure 6 To optimize the ratio of inhibitor to loop region allosteric G-quadruplex, the following steps are included:

[0084] (1) Take 0 μL, 0.1 μL, 0.2 μL, 0.6 μL, 1 μL, 1.4 μL, 1.8 μL, 2 μL, 2.2 μL, 2.4 μL, and 2.6 μL of inhibitor (1 μM) and add them to 11 centrifuge tubes, take 2 μL of loop region allosteric G-quadruplex preservation solution (1 μM) and 1 μL of heme (5 μM) and add them to the above centrifuge tubes respectively, mix in buffer C, and the final volume of each sample is 26 μL. Incubate at 25°C for 30 min.

[0085] (2) To the samples in (1), 0.5 μL TMB (200 mM) and 20 μL hydrogen peroxide solution (3%) were added and mixed in a color development buffer to make a final volume of 200 μL for each sample. The mixture was incubated at 25°C for 5 minutes, and 200 μL hydrochloric acid (1 M) was added to stop the reaction.

[0086] (3) Use a UV-visible spectrophotometer to measure the absorbance, set the maximum absorption wavelength to 450 nm, and measure the absorbance.

[0087] like Figure 6 As shown, when the ratio of the inhibitor to the allosteric G-quadruplex in the loop region is 1:1, the absorbance reaches the lowest platform, indicating that the allosteric G-quadruplex structure in the loop region is completely destroyed by the inhibitor and cannot be combined with heme to form a DNAzyme-catalyzed TMB color development reaction. In order to ensure that the inhibitor fully inhibits the allosteric G-quadruplex, the present invention selects a ratio of the inhibitor to the allosteric G-quadruplex in the loop region of 1.1:1.

[0088] Example 6

[0089] Figure 7 Optimization of CRISPR-Cas12a shearing time. The steps include:

[0090] (1) LAMP amplification of genomic DNA fragments: 2 μL of LAMP amplification template (40 ng / μL), 1 μL of 10× primer mixture (16 μM FIP, 16 μM BIP, 4 μM Loop-F, 4 μM Loop-B, 2 μM F3, 2 μM B3), 1.4 μL of dNTP (10 mM), 0.4 μL of BstII polymerase (8 U / μL) were mixed in buffer A (50 mM potassium chloride, 20 mM tris, 10 mM ammonium sulfate, 10 mM magnesium sulfate, 0.1% Tween-20, pH adjusted to 8.8 with hydrochloric acid), and the final volume was 10 μL. The LAMP reaction solution was incubated at 65°C for 30 minutes, and then incubated at 80°C for 20 minutes to terminate the reaction;

[0091] Among them, the amplification templates are deionized water (negative sample) and nucleic acid standards (positive sample).

[0092] (2) Prepare a mixture of Cas12a and crRNA: Take 0.25 μL Cas12a protein (2400 ng / μL) and 1 μL crRNA (200 nM), mix in buffer B (50 mM sodium chloride, 20 mM tris(hydroxymethyl)aminomethane, 10 mM magnesium sulfate, 1 mM dithiothreitol, pH 7.9) to a final volume of 16 μL;

[0093] (3) Cutting the inhibitor by the CRISPR-Cas12a system: Add 0.2 μL of LAMP amplification product, 2 μL of inhibitor (10 μM) and 1.8 μL of deionized water to the mixture in (3) to a final volume of 20 μL, incubate at 37°C for 10 to 60 minutes, and then immediately heat to 70°C;

[0094] (4) Preparation of ring region allosteric G-quadruplex storage solution: 1 μL of ring region allosteric G-quadruplex solution (10 μM) was mixed in buffer C (300 mM ammonium chloride, 20 mM 4-hydroxyethylpiperazineethanesulfonic acid, 0.05% Triton X-100, 1% dimethyl sulfoxide, pH 7.4) to a final volume of 10 μL;

[0095] (5) Preparation of loop region allosteric G-quadruplex working solution: 2.2 μL of the prepared CRISPR-Cas12a reaction solution, 2 μL of the prepared loop region allosteric G-quadruplex storage solution, and 1 μL of heme (5 μM) were mixed in buffer C to prepare a final volume of 26 μL of G-quadruplex working solution, and incubated at 25°C for 30 minutes;

[0096] (6) Color reaction: Add 0.5 μL TMB (200 mM) and 20 μL hydrogen peroxide solution (3%) to the G-quadruplex working liquid and mix in a color development buffer to prepare a final volume of 200 μL color development reaction solution. Incubate at 25°C for 5 min, add 200 μL hydrochloric acid (1 M) to stop the reaction, and observe the color change of the solution.

[0097] (7) Use a UV-visible spectrophotometer to measure the absorbance, set the maximum absorption wavelength to 450 nm, and measure the absorbance.

[0098] like Figure 7 As shown in the figure, negative samples showed low absorbance values ​​at different CRISPR-Cas12a shearing times, indicating that there was no target gene fragment in the sample. For positive samples, the absorbance value increased with time at different CRISPR-Cas12a shearing times, and reached the maximum value at 30 minutes. Therefore, the CRISPR-Cas12a shearing time was selected as 30 minutes.

[0099] Example 7

[0100] Table 3 is the detection sensitivity analysis of the present invention. It includes the following steps:

[0101] (1) LAMP amplification of genomic DNA fragments: 2 μL of nucleic acid standard, 1 μL of 10× primer mixture (16 μM FIP, 16 μM BIP, 4 μM Loop-F, 4 μM Loop-B, 2 μM F3, 2 μM B3), 1.4 μL of dNTP (10 mM), 0.4 μL of Bst II polymerase (8 U / μL) were mixed in buffer A (50 mM potassium chloride, 20 mM tris, 10 mM ammonium sulfate, 10 mM magnesium sulfate, 0.1% Tween-20, pH adjusted to 8.8 using hydrochloric acid), and the final volume was 10 μL. The LAMP reaction solution was incubated at 65°C for 30 minutes, and then incubated at 80°C for 20 minutes to terminate the reaction.

[0102] Steps (2) to (6) of this embodiment are consistent with steps (3) to (7) in embodiment 1.

[0103] Sensitivity analysis uses the false negative rate in the detection process of samples containing a specific number of target gene copies as a reference. If the false negative rate is less than 5%, it proves that this method can stably detect samples containing the target gene copy number. As shown in Table 3, when the amplification template is 2 copies of the nucleic acid standard, the false negative rate of the detection is 27%; when the amplification template is 5 copies of the nucleic acid standard, the false negative rate of the detection is 9%; when the amplification template is 10 copies of the nucleic acid standard, the false negative rate of the detection is 0. The above results show that the detection method of the present invention can achieve a false negative rate of 0 and a positive rate of 100% in the detection process of 30 samples when the target gene is 10 copies, and the detection performance is stable.

[0104] Table 3 Detection sensitivity analysis

[0105]

[0106] Example 8

[0107] Figure 8 The method is used to detect the color patterns of wild-type and six strains of transgenic Nanlin 895 poplar. The steps are as follows:

[0108] (1) Extraction of genomic DNA from poplars: About 100 mg of fresh poplar leaves were collected and genomic DNA was extracted according to the instructions of the genomic DNA extraction kit. The poplar leaves were collected from six strains of wild-type Nanlin 895 poplar and transgenic Nanlin 895 poplar (strains A to F).

[0109] Steps (2) to (8) of this embodiment are consistent with steps (2) to (8) of embodiment 1.

[0110] like Figure 8 As shown, all blank samples and wild-type Nanlin 895 poplar samples had no obvious color, while six strains of transgenic Nanlin 895 poplar (strains A to F) showed a yellow color that was clearly discernible to the naked eye.

[0111] Table 4 shows the RGB analysis results of the color development images of the wild type and six strains of transgenic Nanlin 895 poplar. Figure 8 As shown in Table 4, all blank samples and wild-type Nanlin 895 poplar samples were judged as negative samples, while six strains of transgenic Nanlin 895 poplar (strains A to F) were judged as positive.

[0112] Table 4 RGB analysis results of color images of wild type and six strains of transgenic Nanlin 895 poplar

[0113]

[0114]

[0115] Comparative Example 1

[0116] Table 5 is a comparison of the results of this method and the qPCR method. It includes the following steps:

[0117] (1) Fresh leaves of wild-type 895 poplar and six strains (A to F) of transgenic Nanlin 895 poplar were collected and genomic DNA was extracted according to the instructions of the genomic DNA extraction kit. The genomic DNA samples of the same poplar leaf were divided into two parallel parts;

[0118] Steps (2) to (9) of this comparative example are consistent with steps (2) to (9) in Example 1;

[0119] (10) Parallel samples of genomic DNA from poplar leaves in (1) were taken and tested using the qPCR method. The primers were qPCR-F (SEQ ID No. 12) and qPCR-B (SEQ ID No. 13). A Ct value less than or equal to 36 was considered positive, and a Ct value greater than 36 was considered negative.

[0120] As shown in Table 5, for all samples, the detection conclusions obtained by this method were consistent with those of the qPCR method.

[0121] Table 5 Comparison of the detection results of this method and qPCR method

[0122]

[0123]

[0124] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit it. Although the present invention has been described in detail with reference to the preferred embodiments, those skilled in the art should understand that the technical solutions of the present invention can be modified or replaced by equivalents without departing from the spirit and scope of the technical solutions of the present invention, which should all be included in the scope of the present invention.

Claims

1. A transgenic poplar visualization detection system, characterized in that: include, Amplification primers, Cas12a protein, crRNA, loop region allosteric G-quadruplex, inhibitor and scanning substrate; Among them, the amplification primers include F3 primers, B3 primers, FIP primers, BIP primers, Loop-F primers and Loop-B primers; the Cas12a protein and crRNA recognize the target gene and activate the trans-cleavage activity of the Cas12a protein; the loop region allosteric G-quadruplex is a deoxyribozyme with catalytic activity formed by binding to heme; the inhibitor is a trans-cleavage substrate of the LbCas12a protein, which inhibits the formation of the loop region allosteric G-quadruplex; the scanning substrate is loaded with a color developing reagent.

2. The transgenic poplar visualization detection system according to claim 1, characterized in that: The F3 primer sequence is shown in SEQ ID No. 1; the B3 primer sequence is shown in SEQ ID No. 2; The FIP primer sequence is shown in SEQ ID No.3; the BIP primer sequence is shown in SEQ ID No.4; the Loop-F primer sequence is shown in SEQ ID No.5; the Loop-B primer sequence is shown in SEQ ID No.6; the crRNA sequence is shown in SEQ ID No.7; the loop region allosteric G-quadruplex sequence is shown in SEQ ID No.8; and the inhibitor sequence is shown in SEQ ID No.

9.

3. The transgenic poplar visualization detection system according to claim 1 or 2, characterized in that: The amplification primers are used to amplify a target gene fragment containing a recognition sequence of the CRISPR-Cas12a system by a LAMP reaction with transgenic poplar genomic DNA as a template; the gene fragment can activate the trans-scission activity of the Cas12a protein and cut the inhibitor; the cut inhibitor cannot inhibit the formation of the ring region allosteric G-quadruplex; the ring region allosteric G-quadruplex combines with heme to form a deoxyribozyme, catalyzes the oxidation of the substrate 3,3',5,5'-tetramethylbenzidine, and generates a yellow product after adding hydrochloric acid; the color developing solution is carried on a scanning substrate, a scanner is used to scan a fixed area of ​​the substrate, a digital photo is obtained, and the RGB value is read by software for analysis.

4. A method for a transgenic poplar visualization detection system, characterized in that: The method comprises adding poplar genomic DNA to a LAMP reaction solution and incubating the solution to obtain a reaction solution in which the genome is amplified by LAMP; The reaction solution in which the genome is amplified by LAMP is added to the CRISPR-Cas12a reaction solution and incubated to obtain a CRISPR-Cas12a mixed reaction solution; The loop region allosteric G-quadruplex is mixed with a buffer to prepare a loop region allosteric G-quadruplex storage solution; The CRISPR-Cas12a mixed reaction solution, the loop region allosteric G-quadruplex storage solution, heme, and buffer C were mixed to prepare the G-quadruplex working solution; The G-quadruplex working liquid, TMB, hydrogen peroxide solution, and color development buffer were mixed to prepare a color development reaction solution, hydrochloric acid was added to stop the reaction, and the color change of the solution was observed. The color development reaction solution was added to a scanning substrate, and a scanner was used to scan a fixed area of ​​the substrate to obtain a digital photo, and the RGB value was read using software for analysis.

5. The method according to claim 4, characterized in that: The preparation method of the LAMP reaction solution comprises adding 1.6 μM FIP primer, 1.6 μM BIP primer, 0.4 μM Loop-F primer, 0.4 μM Loop-B primer, 0.2 μM F3 primer, 0.2 μM B3 primer, 1.4 mM dNTP and 0.32 U / μL BstII enzyme into buffer A to obtain the LAMP reaction solution.

6. The method according to claim 4, characterized in that: The preparation method of the CRISPR-Cas12a reaction solution includes adding 10nM crRNA, 30ng / μL Cas12a protein, and 1μM inhibitor to buffer B to obtain the CRISPR-Cas12a reaction solution.

7. The method according to claim 4, characterized in that: The preparation method of the color development buffer comprises mixing 200 mM ammonium chloride, 100 mM disodium hydrogen phosphate, and 51 mM citric acid to obtain the color development buffer.

8. The method according to claim 4, characterized in that: The reaction solution in which the genome is amplified by LAMP is added to the CRISPR-Cas12a reaction solution for incubation to obtain a CRISPR-Cas12a mixed reaction solution, wherein the incubation time is 10 to 60 minutes.

9. The detection method according to claim 4, characterized in that: The preparation method of the buffer A is 50mM potassium chloride, 20mM tris(hydroxymethylaminomethane), 10mM ammonium sulfate, 10mM magnesium sulfate, 0.1% Tween-20, and the pH is adjusted to 8.8 using hydrochloric acid; the preparation method of the buffer B is 50mM sodium chloride, 20mM tris(hydroxymethylaminomethane), 10mM magnesium sulfate, 1mM dithiothreitol, and the pH is 7.9; the preparation method of the buffer C is 300mM ammonium chloride, 20mM 4-hydroxyethylpiperazineethanesulfonic acid, 0.05% Triton X-100, 1% dimethyl sulfoxide, and the pH is 7.

4.

10. The detection method according to claim 4, characterized in that: The scanning substrate is fixed at a fixed position of a portable scanner for scanning, and the RGB values ​​of the sample image are analyzed using Color Picker online software.

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