Aptamer test strip for transduction and amplification of CHA and CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) combined mediated signal

Abstract

The invention belongs to the field of pesticide detection, and discloses a CHA and CRISPR combined mediated signal transduction and amplification aptamer test strip, which comprises the steps of test strip assembly and signal probe preparation, release of an initiation sequence after a target is combined with an aptamer to trigger CHA reaction to generate a pre-amplification signal, activation of a CRISPR / Cas12a system to cut a reporter molecule, and detection of the CHA and CRISPR combined mediated signal transduction and amplification. Therefore, transduction from an adapter signal to an immune signal and secondary amplification are realized, and Turn on type signal output is realized. In a specificity test, the method has no cross reaction on the structure and functional analogues of procymidone; and after being stored at room temperature for 28 days, the signal stability is kept at 84.61% or above. In actual vegetable sample detection, the recovery rate is 94.00%-104.20%. The trace detection capability of the research is obviously improved, and the Turn on type signal expression form is beneficial to visual interpretation of a detection result, so that a new strategy and reference are provided for development of an aptamer test strip of a small molecule target.

Description

Technical Field

[0001] The present invention relates to an aptamer test strip jointly mediated by CHA and CRISPR for signal transduction and amplification, and belongs to the field of pesticide detection. Background Art

[0002] In recent years, test strips have become an important tool for on-site detection of small molecule targets such as pesticides due to their advantages of portability and low cost. Since small molecule targets have small molecular weights, generally only 1-2 epitopes, and due to the influence of steric hindrance, test strips for small molecule targets often adopt a competitive model, and the signal presentation form is the "Turn off" type. However, traditional "Turn off" type test strips have insufficient sensitivity for trace detection and are prone to the phenomenon of "false positive". In contrast, the detection signal of the "Turn on" type signal output mode gradually increases with the increase of the target concentration, and the signal presentation is intuitive, which is suitable for visual interpretation of detection results.

[0003] As an easily modified and highly specific biorecognition element, aptamers are used to construct signal probes to capture targets and transmit signals in the technology. In particular, some researchers have tried to combine aptamer signals with other rapid detection methods to achieve the conversion of single signals to various sensing signal types such as colorimetry and electrochemistry. Among them, the CRISPR / Cas12a system, as a powerful gene editing tool, not only has high-specificity target recognition ability, but also can activate its non-specific ssDNA cleavage activity to achieve signal transduction and amplification. The research team of Professor Zhenlin Xu from South China University of Technology used 6 enterotoxin genes of Bacillus cereus as detection targets, and with the help of the RPA reaction and the CRISPR / Cas12a system, converted the target gene signal in the sample into the fluorescence signal of an immunochromatographic strip. This method can quickly and simply detect Bacillus cereus within 60 minutes, and the LOD reaches 10 -4 ng / μL. After the CRISPR / Cas12a system is activated, it cleaves the ssDNA reporter molecule to achieve signal transduction. Therefore, combining the CRISPR / Cas12a system with aptamers is expected to achieve the conversion of the "Turn off" type signal of small molecule targets to the "Turn on" type; at the same time, with the secondary amplification of the CRISPR / Cas12a system, the detection sensitivity can be significantly improved.

[0004] In addition, to achieve trace detection, some researchers have attempted to combine aptamers with nucleic acid amplification technologies to enhance sensitivity by pre-amplifying aptamer signals. Among them, the CHA reaction is an enzyme-free isothermal amplification technology with advantages such as simple operation, high cost-effectiveness, and high sensitivity. The team of Wang Lianhui from Nanjing University of Posts and Telecommunications applied the CHA amplification strategy to the imaging of adenosine triphosphate in living cells, with an LOD of 0.21 μM, which is 83.9 times higher than the aptamer enzyme-catalyzed amplification strategy and 142.9 times higher than the method without amplification strategy. Similarly, the research group of Qian Ning from Ningbo University used the CHA reaction in a microchip electrophoresis platform to amplify aptamer signals. Under the optimal amplification conditions, the signal generated by this platform is 300 times higher than that of the platform without CHA amplification. More importantly, the CHA product is dsDNA, which can directly activate the cleavage property of Cas12a as a target. Summary of the Invention

[0005] The object of the present invention is to propose an aptamer test strip jointly mediated by CHA and CRISPR for signal transduction and amplification, providing reference and technical support for improving the detection sensitivity of aptamer test strips.

[0006] To achieve the above object, the technical solution adopted by the present invention is as follows: An aptamer test strip jointly mediated by CHA and CRISPR for signal transduction and amplification, which is prepared by the following method:

[0007] 1. An aptamer test strip jointly mediated by CHA and CRISPR for signal transduction and amplification, characterized in that it includes the following steps:

[0008] (1) Design a procymidone probe sequence, a hairpin primer, a capture probe, and a quality control probe containing an initiation sequence respectively;

[0009] (2) Enrich the sample in the sample pool and trigger the CHA reaction;

[0010] (3) Construct a CRISPR / Cas12a system;

[0011] (4) Assemble an anti-6-FAM immunochromatographic test strip;

[0012] In the step (1), the sequence of the procymidone probe is 5'-CAAAATCTACCTACTCACACTATAAGGGAATTCGTCGACGTAGCGAACGCATGGGCCGGCCGCGGCGCATGCGTCGACCTG-3', the capture short chain is 5'-Biotin-GTTCGCTACGTCGACGAATTCCCTTATAGTGT-3', the sequence of the hairpin primer 1 is 5'-ATAGTATAGTGTGAGTAGGTAGATTTTGCCATGTGTAGACAAAATCTACCTACTC-3', the sequence of the hairpin primer 2 is 5'-AGATTTTGTCTACACATGGCAAAATCTACCTACTCCCATGTGTAGA-3', the sequence of the reporter molecule is 5'-FAM-TTTATTT-Biotin-3', and the sequence of the crRNA is 5'-UAAUUUCUACUAAGUGUAGAUUCUACACAUGGCAAAAUCUA-3'.

[0013] Furthermore, the sample pool preparation method in step (2) is as follows: pre-coating streptavidin into each well of the detachable ELISA plate, incubating overnight at 4°C, washing three times, mixing the capture short chain modified with Biotin and the aptamer probe with procymidone and performing renaturation treatment, adding the formed hybrid nucleic acid complex into the ELISA well, incubating at 37°C, and washing the plate for standby use.

[0014] Furthermore, the triggering method for amplifying the CHA nucleic acid signal in step (2) is: subjecting the sample solution containing the procymidone aptamer probe, the hairpin primer 1, and the hairpin primer 2 to renaturation treatment respectively, and incubating them at room temperature to form a CHA amplification product.

[0015] Furthermore, the construction method of the CRISPR / Cas12a system in step (3) is as follows: Cas12a protein and crRNA are incubated at 37°C for 15 min to form a Cas12a / crRNA complex, CHA amplification product and reporter molecule are added, and the mixture is incubated at 37°C.

[0016] Furthermore, the preparation method of the signal probe AuNPs-Ab in step (4) is: using 0.1 M K2CO3 to adjust the pH of AuNPs to 6.5, adding a rabbit polyclonal antibody (Ab) that recognizes 6-FAM, and standing at 37°C for 30 min to form the signal probe AuNPs-Ab.

[0017] Further, the assembly method of the test strip in step (4) is as follows: paste the NC membrane at the middle position of the bottom plate, spray streptavidin and goat anti-rabbit secondary antibody solution with a certain concentration onto the C-line and T-line positions of the NC membrane area in sequence, dry the bottom plate, soak the sample pad in the immunological blocking solution for 10 min, then dry it at 37°C, assemble the test strip in the order of "sample pad, NC membrane, absorbent pad", with an overlap of 1-2 mm between every two adjacent components, and cut it with a guillotine according to a width of 3.8 mm / strip, thus obtaining the anti-6-FAM immunological test strip.

[0018] An aptamer test strip jointly mediated by CHA and CRISPR for signal transduction and amplification prepared based on the above method, the aptamer test strip is strip-shaped, the sample addition position is at one end of the sample pad, and the detection area is located in the NC membrane area, and it is characterized in that: from one end of the sample pad to one end of the absorbent pad, there are a C-line and a T-line in sequence.

[0019] The above-mentioned aptamer test strip jointly mediated by CHA and CRISPR for signal transduction and amplification is applied to the rapid detection of procymidone in agricultural products.

[0020] Further, the detection sensitivity of the procymidone is ≥0.015 ng / mL.

[0021] The present invention has the following beneficial effects:

[0022] The present invention designs a strategy jointly mediated by CHA and CRISPR for signal transduction and amplification, realizes the conversion of aptamer signal to immunochromatographic test strip and "Turn on" type signal output, and the detection limit is significantly reduced to 0.015 ng / mL. The present invention has good specificity, performs excellently in vegetable sample matrix, and has good room temperature storage stability, providing a new paradigm for the on-site rapid detection of small molecule targets. Description of the Drawings

[0023] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the following drawings are only some embodiments of the present invention, and for those of ordinary skill in the art, other drawings can be obtained according to these drawings without creative efforts.

[0024] Figure 1 It is the design drawing of the aptamer test strip of the present invention.

[0025] Figure 2 It is the test characterization drawing of the effective triggering of CHA of the present invention, the feasibility of the CRISPR / Cas12a system, and the anti-6-FAM immunological test strip.

[0026] Figure 3 It is the optimization of the process parameters of the aptamer test strip of the present invention.

[0027] Figure 4 It is the detection calibration curve graph of the aptamer test strip of the present invention.

[0028] Figure 5 It is the graph of the specificity and stability results of the aptamer test strip of the present invention. Detailed implementation manners

[0029] Next, in combination with the embodiments of the present invention, the technical solutions of the present invention will be clearly and completely described. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all of the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0030] Example 1: Preparation of sample pool

[0031] Add 100 μL of streptavidin to each well of a detachable microplate and incubate overnight at 4 °C to allow streptavidin to adsorb to the polystyrene well walls. Wash the plate three times with PBST (135 mM NaCl, 2.7 mM KCl, 1.5 mM KH2PO4, 8 mM Na2HPO4, 0.5% Tween-20, pH 7.4). Mix the capture short chain and the procymidone aptamer probe in a molar ratio of 1:1.2 and perform denaturation-renaturation treatment: at 95 °C for 10 min, at 60 °C for 1 min, at 4 °C for 5 min, with a cooling rate of 0.1 °C / s. Add 100 μL of the hybrid nucleic acid complex cDNA-APTpro to each well, incubate at 37 °C, then wash the plate three times with PBST and store at 4 °C for later use.

[0032] Example 2: CHA nucleic acid signal amplification reaction

[0033] The sample solution containing the procymidone aptamer probe, hairpin primer 1, and hairpin primer 2 are respectively subjected to denaturation-renaturation treatment: at 95 °C for 10 min, at 60 °C for 1 min, at 4 °C for 5 min, with a cooling rate of 0.1 °C / s. Mix 1 μL of 10 μM hairpin primer 1 and 10 μM hairpin primer 2 in a certain proportion, add 1 μL of the sample solution containing the procymidone aptamer probe, and make up the total volume of the system to 10 μL. Incubate at room temperature, and the formed CHA amplification product is the target of the CRISPR / Cas12a system.

[0034] Example 3: Construction of CRISPR / Cas12a system

[0035] Add 2 μL of NEBufferTM In r2.1 (10×), 2 μL of 1 μM Cas12a protein and 1 μM crRNA were added simultaneously, and the reaction system was made up to 17 μL with diethyl pyrocarbonate-treated water. It was incubated at 37 °C for 15 min to form the Cas12a / crRNA complex. Then, 2 μL of the CHA amplification product and 1 μL of the reporter molecule were added, and the final volume was 20 μL, and it was incubated at 37 °C.

[0036] Example 4: Preparation of the signal probe AuNPs-Ab

[0037] 0.1 M K2CO3 was added dropwise to 5 mL of AuNPs to adjust the pH to 6.5. Rabbit polyclonal antibody (Ab) recognizing 6-FAM was added thereto, and it was shaken while adding. It was left standing at 37 °C for 30 min to form the signal probe AuNPs-Ab. 10% BSA was added dropwise until the BSA content in the system was 1%. It was left standing at 37 °C for 30 min to allow BSA to fully block the non-specific sites on the surface of AuNPs. The mixed solution was centrifuged at 8000 rpm at 4 °C for 10 min to remove the precipitate. The supernatant was then centrifuged at 13000 rpm at 4 °C for 30 min. The supernatant was washed twice and resuspended with 500 μL of 0.2 M boric acid buffer containing 0.2% PEG20000.

[0038] Example 5: Assembly of the test strip

[0039] First, the NC membrane was pasted at the middle position of the bottom plate. Streptavidin and goat anti-rabbit secondary antibody solution were respectively sprayed onto the C line and T line positions of the NC membrane area. The bottom plate was placed flat in an oven at 37 °C and dried for 2 h. At the same time, the sample pad was soaked in an immunological blocking solution (20 mM NaH2PO4·2H2O, 80 mM Na2HPO4·12H2O, 0.5% BSA, 1% PVP-K30, 1% Tween-20, 0.25% ethylenediaminetetraacetic acid) for 10 min. After soaking, it was taken out and dried at 37 °C for 2 h. The test strip was assembled in the order of "sample pad, NC membrane, absorbent pad", with an overlap of 1 - 2 mm between every two adjacent components. After it was completely and firmly bonded, it was cut with a cutting machine at a width of 3.8 mm / strip to obtain the anti-6-FAM immunological test strip.

[0040] Example 6: Detection procedure

[0041] 20 μL of the signal enrichment probe was added to 80 μL of the sample solution. The test strip was inserted into the sample pool for chromatography and incubation. The color development of the strip was observed to qualitatively analyze the sample. The picture data of the blank group and the experimental group were simultaneously obtained using a smartphone, and quantitative analysis was performed using ImageJ software.

Claims

1. An aptamer test strip for CHA and CRISPR combined-mediated signal transduction and amplification, characterized in that, The steps are as follows: (1) Design a procymidone probe sequence, a hairpin primer, a capture probe, and a quality control probe containing an initiation sequence respectively; (2) Prepare a sample pool to enrich the sample and trigger the CHA reaction; (3) Construct a CRISPR / Cas12a system; (4) Assemble an anti-6-FAM immunostrip; In the step (1), the procymidone probe sequence is 5’-CAAAATCTACCTACTCACACTATAAGGGAATTCGTCGACGTAGCGAACGCATGGGCCGGCCGCGGCGCATGCGTCGACCTG-3’, the capture short chain is 5’-Biotin-GTTCGCTACGTCGACGAATTCCCTTATAGTGT-3’, the sequence of the hairpin primer 1 is 5’-ATAGTATAGTGTGAGTAGGTAGATTTTGCCATGTGTAGACAAAATCTACCTACTC-3’, the sequence of the hairpin primer 2 is 5’-AGATTTTGTCTACACATGGCAAAATCTACCTACTCCCATGTGTAGA-3’, the sequence of the reporter molecule is 5’-FAM-TTTATTT-Biotin-3’, and the sequence of the crRNA is 5’-UAAUUUCUACUAAGUGUAGAUUCUACACAUGGCAAAAUCUA-3’.

2. The aptamer test strip for jointly mediating signal transduction and amplification by CHA and CRISPR according to claim 1, characterized in that, In the step (2), the preparation method of the sample pool is as follows: streptavidin is pre-coated in each well of a detachable enzyme-linked immunosorbent assay (ELISA) plate overnight at 4 °C. After washing three times, the capture short chain modified with Biotin is mixed with the procymidone aptamer probe and subjected to denaturation-renaturation treatment. The formed hybrid nucleic acid complex is added to the ELISA wells and incubated at 37 °C, and then the plate is washed for standby.

3. The aptamer test strip for jointly mediating signal transduction and amplification by CHA and CRISPR according to claim 1, characterized in that, In the step (2), the triggering method for CHA nucleic acid signal amplification is as follows: the sample solution containing the procymidone aptamer probe, the hairpin primer 1, and the hairpin primer 2 are respectively subjected to denaturation-renaturation treatment and incubated at room temperature to form CHA amplification products.

4. An aptamer test strip for CHA and CRISPR combined-mediated signal transduction and amplification according to claim 1, characterized in that, In the step (3), the construction method of the CRISPR / Cas12a system is as follows: the Cas12a protein and the crRNA are incubated at 37 °C for 15 min to form a Cas12a / crRNA complex, and then the CHA amplification products and the reporter molecule are added and incubated at 37 °C.

5. The aptamer test strip for jointly mediating signal transduction and amplification by CHA and CRISPR according to claim 1, wherein In the step (4), the preparation method of the signal probe AuNPs-Ab is as follows: the pH of AuNPs is adjusted to 6.5 with 0.1 M K2CO3, and a rabbit polyclonal antibody (Ab) recognizing 6-FAM is added and allowed to stand at 37 °C for 30 min to form the signal probe AuNPs-Ab.

6. The aptamer test strip for jointly mediating signal transduction and amplification by CHA and CRISPR according to claim 1, wherein The assembly method of the test strip in step (4) is as follows: paste the NC membrane at the middle position of the bottom plate, spray streptavidin and goat anti-rabbit secondary antibody solution with a certain concentration onto the C-line and T-line positions of the NC membrane area in sequence, dry the bottom plate, soak the sample pad in the immune blocking solution for 10 min, then dry it at 37 °C, assemble the test strip in the order of "sample pad, NC membrane, absorbent pad", with an overlap of 1-2 mm between every two adjacent components, and cut it with a cutting machine at a width of 3.8 mm / strip to obtain the 6-FAM immunoassay test strip.

7. An aptamer test strip for CHA and CRISPR combined mediated signal transduction and amplification prepared by the method according to any one of claims 1-6, wherein the aptamer test strip is strip-shaped, the sample addition position is at one end of the sample pad, and the detection area is located in the NC membrane area, and is characterized in that: From one end of the sample pad to the other end of the absorbent pad, there are the C-line and the T-line in sequence.

8. The method for using an aptamer test strip for jointly mediating signal transduction and amplification by CHA and CRISPR according to claim 7, characterized in that: Mix 20 μL of the signal enrichment probe with 80 μL of the sample solution, insert the aptamer test strip into the sample pool for chromatography and incubation, and observe the color development of the strip to qualitatively and quantitatively analyze the sample.

9. Application of an aptamer test strip for signal transduction and amplification jointly mediated by CHA and CRISPR as claimed in claim 8 in the rapid detection of procymidone in agricultural products.

10. The application according to claim 9, characterized in that: The detection sensitivity of the procymidone is ≥ 0.015 ng / mL.

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