Non-nucleic acid target rapid detection method based on magnetic separation-CRISPR signal amplification, kit and application

Abstract

The invention provides a non-nucleic acid target rapid detection method based on magnetic separation-CRISPR signal amplification, a kit and application, and belongs to the technical field of detection. The method specifically comprises the following steps: S1, preparing an antibody modified magnetic nanoparticle solution; s2, modifying the antigen / antigen analogue and single-stranded DNA to the surface of a gold nanoparticle to obtain a gold nanoparticle compound solution; s3, preparing the magnetic nanoparticle solution, the gold nanoparticle compound solution and the non-nucleic acid target sample solution to be detected into an MNPs-Ab compound solution; s4, mixing a Cas12a protein solution, a new England buffer solution, a crRNA solution, an RNA enzyme inhibitor solution and a fluorescent probe solution to obtain a system complex solution; and S5, carrying out mixed reaction on the MNPs-Ab compound solution and a system compound solution, and finally carrying out fluorescence detection. The method is high in sensitivity and short in detection time, and has the capability of quantitatively detecting non-nucleic acid targets, so that the method has a good development prospect.

Description

Technical Field

[0001] The present invention belongs to the field of detection technology, and specifically relates to a method, a kit and an application of rapid detection of non-nucleic acid targets based on magnetic separation-CRISPR signal amplification. Background Art

[0002] Residues of non-nucleic acid targets such as antibiotics, toxins, and pesticides seriously endanger human health. Therefore, rapid detection of non-nucleic acid targets such as antibiotics is crucial to improving public safety. At present, the commonly used detection methods for such substances mainly include high performance liquid chromatography (HPLC), liquid chromatography-tandem mass spectrometry (HPLC-MS / MS), gas chromatography (GC), and enzyme-linked immunosorbent assay (ELISA). However, these technologies usually require a large amount of organic solvents, lengthy operating procedures, and high detection costs. Therefore, it is crucial to develop new rapid detection methods.

[0003] The CRISPR / Cas system is an acquired immune system present in bacteria and archaea. The CRISPR technology developed based on this is a star tool in the field of gene editing. In recent years, due to its convenient operation, high detection sensitivity and strong specificity, CRISPR technology has attracted more and more attention in the field of detection. Among them, the CRISPR / Cas12a system is favored due to its high specificity and cutting efficiency. In the presence of the target, the CRISPR / Cas12a system can be activated, so that any single-stranded DNA (ssDNA) can be non-specifically cut. This feature provides great potential for the development of new rapid detection methods. At present, rapid detection methods based on the CRISPR / Cas12a system have been widely developed. However, CRISPR / Cas12a can only be activated by nucleic acid targets. Therefore, the CRISPR / Cas12a system is still challenging in the rapid detection of non-nucleic acid targets.

[0004] At present, the non-nucleic acid target detection method based on the CRISPR / Cas12a system mainly introduces functional DNA such as aptamers as a medium to establish a quantitative relationship between target recognition and CRISPR / Cas12a cleavage activity. However, due to the recognition range of functional nucleic acids, such methods are still limited to the detection of a small number of non-nucleic acid targets. Therefore, it is urgent to develop a more universal CRISPR / Cas rapid detection method. Summary of the invention

[0005] In view of this, the purpose of the present invention is to provide a method, kit and application of rapid detection of non-nucleic acid targets based on magnetic separation-CRISPR signal amplification. The method of the present invention expands the application of the CRISPR / Cas12a system to non-nucleic acid small molecule detection by preparing a gold nanoparticle-antigen / antigen analog-single-stranded DNA (AuNPs-Ag-ssDNA) complex that can both competitively bind to the antibody site of the target to be detected and activate the cutting activity of the CRISPR / Cas12a system. The present invention has high sensitivity, short detection time, and the ability to quantitatively detect non-nucleic acid targets, so it has good development prospects.

[0006] In order to achieve the above object, the present invention provides the following technical solutions: In a first aspect, the present invention provides a method for rapid detection of non-nucleic acid targets based on magnetic separation-CRISPR signal amplification, characterized in that it comprises the following steps: S1. modifying the surface of carboxylated magnetic nanoparticles with antibodies that can specifically recognize the non-nucleic acid target to be detected to obtain an antibody-modified magnetic nanoparticle solution, i.e., MNPs-Ab solution; S2. modifying the antigen / antigen analog and single-stranded DNA (ssDNA) corresponding to the non-nucleic acid target onto the surface of the gold nanoparticles to obtain a gold nanoparticle complex solution, i.e., AuNPs-Ag-ssDNA solution; S3. The magnetic nanoparticle solution, the gold nanoparticle complex solution, and the non-nucleic acid target sample solution to be detected are mixed in a phosphate buffer solution and subjected to a shake reaction at room temperature, followed by magnetic separation to obtain a MNPs-Ab complex that captures the non-nucleic acid target to be detected and AuNPs-Ag-ssDNA on the surface; then, after washing with PBS, the complex is resuspended in DEPC water to obtain a MNPs-Ab complex solution that captures the non-nucleic acid target to be detected and AuNPs-Ag-ssDNA on the surface, i.e., a MNPs-Ab-AuNPs-Ag-ssDNA solution; S4. Mix the Cas12a protein solution, New England buffer (NEBuffer r2.1), crRNA solution, RNase inhibitor solution and fluorescent probe solution to obtain a CRISPR / Cas12a system complex solution; S5. The MNPs-Ab-AuNPs-Ag-ssDNA solution is mixed with the CRISPR / Cas12a system complex solution for reaction; the fluorescence value at the corresponding emission wavelength is measured under the excitation wavelength of the fluorescent group of the fluorescent probe, and then the standard curve of the relationship between concentration and fluorescence value is substituted to obtain the concentration of the non-nucleic acid target to be measured in the sample solution.

[0007] In the present invention, the inventors innovatively utilized the target to be detected and AuNPs-Ag-ssDNA to competitively bind to the antibody binding sites on the surface of MNPs-Ab, and utilized AuNPs-Ag-ssDNA bound to the surface of MNPs-Ab to activate the cleavage activity of the CRISPR / Cas12a system, thereby converting the target recognition information into the cleavage activity of the CRISPR / Cas12a system, thereby realizing the quantitative detection of non-nucleic acid targets and broadening the detection application scope of the CRISPR / Cas12a system.

[0008] Preferably, the non-nucleic acid targets to be detected that can be specifically identified include but are not limited to enrofloxacin, levofloxacin, and ciprofloxacin.

[0009] Preferably, the mass ratio between the antibody and the carboxylated magnetic nanoparticles in S1 is 1:50; The particle size of the magnetic nanoparticles is 50-500 nm.

[0010] Preferably, the volume ratio of the gold nanoparticles, antigen / antigen analogues, and ssDNA in S2 is 2:1:1; The particle size of the gold nanoparticles is 10-30 nm.

[0011] Preferably, the length of the single-stranded DNA in S2 is 20-30 nt and is complementary to the crRNA base in S4; The 5' or 3' end of the ssDNA is modified with a thiol group.

[0012] Preferably, the volume ratio of the magnetic nanoparticle solution, the gold nanoparticle complex solution, and the non-nucleic acid target sample solution to be detected in S3 is 3:1:13.

[0013] Preferably, the pH of the phosphate buffer in S3 is 5-8; and the number of washing times is 2-4 times.

[0014] Preferably, the volume ratio of the Cas12a protein solution, the New England buffer (NEBuffer r2.1), the crRNA solution, the RNase inhibitor solution, and the fluorescent probe solution in the S4 is 0.1:5:1.25:0.25:2.

[0015] Preferably, the volume ratio of the MNPs-Ab complex solution to the CRISPR / Cas12a system complex solution in S5 is 30:20.

[0016] In a second aspect, the present invention provides a kit for rapid detection of non-nucleic acid targets, comprising the magnetic nanoparticle solution, gold nanoparticle complex solution, and CRISPR / Cas12a system complex solution described above.

[0017] In a third aspect, the present invention provides use of the above kit in detecting antibiotics, toxins or pesticides.

[0018] Contains at least the following beneficial technical effects: (1) The detection method of the present invention expands the application of the CRISPR / Cas12a system to the detection of non-nucleic acid small molecules by competitively binding the non-nucleic acid target to be detected and AuNPs-Ag-ssDNA to the antibody sites on the surface of MNPs-Ab and the CRISPR / Cas12a non-specific cleavage fluorescent probe.

[0019] (2) The method described in the present invention creatively introduces AuNPs-Ag-ssDNA complexes that can both competitively bind to the antibody sites of the target and activate the cleavage activity of the CRISPR / Cas12a system, thereby converting the target recognition behavior into the cleavage activity of CRISPR / Cas12a, constructing a fluorescent biosensor method, and applying CRISPR / Cas12a to the detection of non-nucleic acid small molecules.

[0020] (3) The method of the present invention fully utilizes the intrinsic enzyme catalytic amplification effect of the CRISPR / Cas12a system. Compared with the existing HPLC, ELISA and CRISPR detection technologies, it significantly improves the detection sensitivity and the detection limit is 0.86 ng / mL.

[0021] (4) The method of the present invention does not require large-scale, sophisticated instruments and can quickly achieve quantitative detection, thus lowering the detection threshold. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 This is a schematic diagram of the principle of detecting enrofloxacin in the present invention.

[0023] Figure 2 This is a verification diagram for the preparation of MNPs-Ab of the present invention.

[0024] Figure 3 This is a verification diagram of the preparation of AuNPs-ENR-ssDNA of the present invention.

[0025] Figure 4 This is a standard curve diagram for detecting enrofloxacin in the present invention.

[0026] Figure 5 This is the detection specificity of enrofloxacin detected by the method of the present invention. DETAILED DESCRIPTION

[0027] The present invention provides a method for rapid detection of non-nucleic acid targets based on magnetic separation-CRISPR signal amplification, comprising the following steps: S1. modifying the surface of carboxylated magnetic nanoparticles with an antibody that can specifically recognize the non-nucleic acid target to be detected to obtain an antibody-modified magnetic nanoparticle solution; S2. modifying the antigen / antigen analog and single-stranded DNA corresponding to the non-nucleic acid target onto the surface of the gold nanoparticles to obtain a gold nanoparticle complex solution; S3. Mix the magnetic nanoparticle solution, the gold nanoparticle complex solution, and the non-nucleic acid target sample solution to be detected in a phosphate buffer and shake at room temperature for reaction before magnetic separation; then wash with PBS and resuspend in DEPC water to obtain a MNPs-Ab complex solution; S4. Mix the Cas12a protein solution, New England buffer, crRNA solution, RNase inhibitor solution and fluorescent probe solution to obtain a CRISPR / Cas12a system complex solution; S5. The MNPs-Ab complex solution is mixed with the CRISPR / Cas12a system complex solution for reaction; the fluorescence value at the corresponding emission wavelength is measured under the excitation wavelength of the fluorescent group of the fluorescent probe, and then the standard curve of the relationship between concentration and fluorescence value is substituted to obtain the concentration of the non-nucleic acid target to be measured in the sample solution.

[0028] In the present invention, S1 specifically includes the following steps: 1.1) Prepare a 2-(N-morpholino)ethanesulfonic acid (MES) solution with a pH of 5-7 and containing 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), wherein the concentration of EDC is 5-50 mmol / L; 1.2) Prepare a MES solution with a pH of 5-7 containing sodium N-hydroxysuccinimide sulfonate (sulfo-NHS), wherein the concentration of sulfo-NHS is 5-50 mmol / L; 1.3) mixing the MES solution containing EDC in step 1.1) and the MES solution containing sulfo-NHS in step 1.2), wherein the ratio of EDC solution to sulfo-NHS solution is 1:1 / V:V, to obtain a MES mixed solution containing EDC and sulfo-NHS; 1.4) 20 parts of carboxylated magnetic beads with a concentration of 1-50 mg / mL are ultrasonically dispersed and dissolved in 380 parts of MES buffer. After magnetic separation, the particles are repeatedly washed 2-4 times with MES buffer. The washed particles are resuspended in 400 parts of the MES mixed solution containing EDC and sulfo-NHS obtained in step 1.3), shaken at room temperature, magnetically separated, and the particles are repeatedly washed 2-4 times with borate buffer (BB). The washed particles are then dispersed in 400 parts of BB buffer to obtain a carboxyl-activated magnetic nanoparticle mixture; 1.5) adding 4 portions of antibody solution with a concentration of 0.1-5 mg / mL to the carboxyl-activated magnetic nanoparticle mixture obtained in step 1.4), and shaking the mixture at room temperature for at least 60 min to obtain an antibody-modified magnetic nanoparticle dispersion, i.e., MNPs-Ab solution; 1.6) Add 400 parts of 1% to 5% bovine serum albumin solution to the MNPs-Ab solution obtained in step 1.5), shake and react at room temperature, wash repeatedly 2 to 4 times with PBS at pH 5 to 8, and finally disperse in 100 parts of PBS buffer to obtain a bovine serum albumin-blocked MNPs-Ab solution, which is stored at 4°C for future use.

[0029] In the present invention, S2 specifically includes the following steps: 2.1) Prepare ssDNA solution with a concentration of 5-100 μmol / L and antigen / antigen analog solution with a concentration of 0.1-1 mg / mL; 2.2) AuNPs were prepared by citric acid reduction method. After the reaction, the precipitate was centrifuged and washed 2 to 4 times with deionized water at pH 6 to 8. The washed precipitate was then ultrasonically dispersed in 100 parts of deionized water at pH 6 to 8 to obtain a 10 to 30 nm AuNPs solution. 2.3) 100 parts of the AuNPs solution obtained in step 2.2), 50 parts of the antigen / antigen analog solution obtained in step 2.1) and 50 parts of the ssDNA solution were mixed, shaken at room temperature, centrifuged to obtain a precipitate, and the precipitate was repeatedly washed 2 to 4 times with deionized water at a pH of 6 to 8, and then dispersed in 200 parts of deionized water at a pH of 6 to 8 to obtain a gold nanoparticle composite solution, i.e., a gold nanoparticle-antigen / antigen analog-single-stranded DNA (AuNPs-Ag-ssDNA) solution, which was stored at 4°C for future use.

[0030] In the present invention, the specific steps in S3 are: mixing 30 parts of MNPs-Ab solution with a concentration of 0.5-1 mg / mL, 10 parts of AuNPs-Ag-ssDNA solution, 30 parts of PBS solution (pH 5-8, containing 0.05% Tween-20 and 0.1% BSA) and 130 parts of the sample solution to be tested, shaking the solution at room temperature, magnetically separating the particles after the reaction, washing the particles 2-4 times with PBS buffer with a pH of 5-8, and resuspending the particles in 35 parts of DEPC water to obtain an MNPs-Ab complex with a surface captured target and AuNPs-Ag-ssDNA, i.e., a MNPs-Ab-AuNPs-Ag-ssDNA solution.

[0031] In the present invention, the specific steps in S4 are: 0.1 parts of a Cas12a protein solution with a concentration of 10-500 nmol / L, 5 parts of (10×) New England buffer (NEBuffer r2.1), 1.25 parts of a crRNA solution with a concentration of 1-500 nmol / L, 0.25 parts of an RNase inhibitor solution with a concentration of 1-100 U, and 2 parts of a fluorescent probe solution with a concentration of 1-500 nmol / L are mixed to obtain a CRISPR / Cas12a system composite solution.

[0032] In the present invention, the specific steps in S5 are: after mixing 30 parts of MNPs-Ab-AuNPs-Ag-ssDNA solution with 20 parts of CRISPR / Cas12a system composite solution, reacting at 25-40°C for at least 20 min, measuring the fluorescence value at the emission wavelength of the fluorescent group under the excitation wavelength of the fluorescent group in the fluorescent probe, substituting the fluorescence value into the standard curve to obtain the concentration of the non-nucleic acid target to be measured.

[0033] In the present invention, the process of establishing the standard curve is specifically as follows: using PBS with a pH of 5 to 8 as the base liquid, preparing non-nucleic acid target standard solutions to be tested with different concentration gradients (wherein the concentration gradient is selected from 0.001 to 1000 ng / mL); treating each group of standard solutions with different target concentrations to obtain the fluorescence value corresponding to the corresponding concentration of each group, and performing linear fitting with the logarithmic value of the concentration of the standard solution as the abscissa and the corresponding fluorescence value as the ordinate to obtain a standard curve.

[0034] In the present invention, in the fluorescent probe solution, the fluorescent probe includes a fluorescent group, a quenching group and single-stranded DNA, the 5' end of the fluorescent probe is modified with a fluorescent group, the 3' end of the fluorescent probe is modified with a quenching group, and the number of nucleotide bases of the single-stranded DNA is 3-10 nt; in the crRNA solution, the nucleotide sequence of the crRNA is complementary to the ssDNA base in AuNPs-Ag-ssDNA.

[0035] In the present invention, the fluorescent probe is represented by: 5'-fluorescent group-single-stranded DNA-quencher group-3'.

[0036] In the present invention, the fluorescent group includes one or more fluorescent groups such as 5 / 6 carboxyfluorescein (FAM), cyanine dye (Cy5), tetrachloro-6-carboxyfluorescein (TET), hexachloro-6-methylfluorescein (HEX) and carboxy-X-rhodamine (ROX), and the quenching group includes one or more quenching groups such as BHQ-1, BHQ-2, BHQ-3 and TAMRA.

[0037] Various exemplary embodiments of the present invention will now be described in detail. This detailed description should not be considered as limiting the present invention, but should be understood as a more detailed description of certain aspects, features, and embodiments of the present invention.

[0038] It should be understood that the terms described in the present invention are only for describing special embodiments and are not intended to limit the present invention. In addition, for the numerical range in the present invention, it should be understood that each intermediate value between the upper and lower limits of the scope is also specifically disclosed. Each smaller range between the intermediate value in any stated value or stated range and any other stated value or intermediate value in the described range is also included in the present invention. The upper and lower limits of these smaller ranges can be independently included or excluded in the scope.

[0039] Unless otherwise indicated, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art. Although the present invention describes only preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials associated with the documents. In the event of a conflict with any incorporated document, the content of this specification shall prevail.

[0040] It will be apparent to those skilled in the art that various modifications and variations may be made to the specific embodiments of the present invention description without departing from the scope or spirit of the present invention. Other embodiments derived from the present invention description will be apparent to those skilled in the art. The present application description and examples are exemplary only.

[0041] The words “include,” “including,” “have,” “contain,” etc. used in this document are open-ended terms, meaning including but not limited to.

[0042] The "room temperature" and "normal temperature" mentioned in the present invention are all calculated as 25±2°C unless otherwise specified.

[0043] The raw materials used in the following examples of the present invention are all commercially available unless otherwise specified; the ssDNA in the examples of the present invention was synthesized by Shengong Bioengineering.

[0044] Example 1 1) preparing an enrofloxacin monoclonal antibody modified immunomagnetic nanoparticle (MNPs-Ab) solution; 1.1) Prepare 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) solution with a concentration of 20 mmol / L; 1.2) Preparation N- Hydroxysuccinimide sulfonic acid sodium salt (sulfo-NHS) solution, concentration 30 mmol / L; 1.3) After ultrasonication, the magnetic beads with a concentration of 10 mg / mL were mixed with 380 μL 2-( N -morpholine) ethanesulfonic acid (MES, containing 0.01% Tween-20) buffer was ultrasonically washed, and redissolved in a mixed solution of 400 μL of the EDC solution obtained in step 1.1) and the sulfo-NHS solution obtained in step 1.2) (EDC solution: sulfo-NHS solution = 1:1 / V:V), after shaking at room temperature for 30 min, it was repeatedly washed 3 times with borate buffer (BB, containing 0.01% Tween-20), and continued to be dispersed in 400 μL BB (containing 0.01% Tween-20) to obtain a dispersion of magnetic nanoparticles after carboxyl activation; In this embodiment, the average particle size of the carboxylated magnetic beads is 150 nm; 1.4) Add 4 μL of 1 mg / mL enrofloxacin monoclonal antibody solution to the carboxyl-activated magnetic nanoparticle dispersion obtained in step 1.3), and shake at room temperature to obtain an enrofloxacin monoclonal antibody-modified immunomagnetic nanoparticle (MNPs-Ab) solution; 1.5) Add 400 μL of 2% bovine serum albumin solution to the MNPs-Ab obtained in step 1.4), shake and react at room temperature, wash three times with PBS (pH 7, containing 0.01% Tween-20), and then disperse in 100 μL PBS (pH 7, containing 0.01% Tween-20) to obtain a bovine serum albumin-blocked MNPs-Ab solution, which is stored at 4 °C for later use.

[0045] Figure 2 This is a verification diagram for the preparation of MNPs-Ab. In order to confirm the presence of antibodies on the surface of MNPs, rabbit anti-mouse antibodies (HRP-IgG) labeled with horseradish peroxidase (HRP) were used. HRP-IgG can be captured by MNPs-Ab and catalyze the decomposition of H2O2 to produce hydroxyl radicals (·OH). These free radicals can oxidize 3,3',5,5'-tetramethylbenzidine (TMB) to produce blue oxidized TMB. After adding sulfuric acid stop solution, it has a maximum absorption peak at 450 nm. Figure 2 As shown in the figure, the MNPs-Ab sample has a larger absorption value at 450 nm. In contrast, the absorption peak of unmodified MNPs is smaller, indicating that MNPs-Ab is successfully prepared.

[0046] 2) Design and synthesize a single-stranded DNA (ssDNA) that can activate the cutting activity of the CRISPR / Cas12a system. The sequence of the ssDNA is 5'-TTTTCTCTGGATGGTATGCCCGG-3', which can be specifically recognized by the crRNA in the CRISPR / Cas12a system and activate the trans-cutting activity of the CRISPR / Cas12a system.

[0047] 3) Gold nanoparticles were modified simultaneously with bovine serum albumin-enrofloxacin complex (BSA-ENR) and single-stranded DNA (ssDNA) to obtain a gold nanoparticle complex solution.

[0048] 3.1) Prepare 10 μmol / L ssDNA aqueous solution; 3.2) Prepare 0.6 mg / mL BSA-ENR solution; 3.3) Prepare phosphate-buffered saline (PBS) buffer, pH 6, containing 0.05% Tween-20 and 0.1% BSA; 3.4) Use citric acid reduction method to prepare 55 mL of gold nanoparticle (AuNPs) solution with 0.0565 mg sodium citrate and 0.0197 mg chloroauric acid. Take 100 μL of AuNPs solution, wash it once with deionized water at pH 7, and then redisperse it in 100 μL deionized water (pH 7). Then add 50 μL of ssDNA solution obtained in step 3.1) and 50 μL of BSA-ENR solution obtained in step 3.2), shake and react at room temperature for 30 min, centrifuge and wash the precipitate with deionized water (pH 7) for 3 times, finally disperse it in 200 μL deionized water (pH 7) to obtain AuNPs-ENR-ssDNA solution, and store it at 4 ℃ for later use.

[0049] Figure 3 This is a verification diagram of the preparation of AuNPs-ENR-ssDNA. When the AuNPs surface is not modified with protein, after adding NaCl, its surface charge is neutralized, AuNPs aggregate, and the characteristic absorption peak around 517 nm disappears; when its surface is modified with BSA-ENR and ssDNA, it can maintain good dispersibility in a high salt environment. Figure 3 It can be seen that the unmodified AuNPs are severely aggregated and have no characteristic absorption peak, while AuNPs-ENR-ssDNA can resist the high salt environment and still has an obvious absorption peak around 520 nm, which proves the successful preparation of AuNPs-ENR-ssDNA.

[0050] 4) Enrofloxacin detection, details are as follows: 4.1) Prepare CRISPR / Cas12a system complex solution; 4.1.1) Mix 200 nmol / L Cas12a protein, 10×NEBuffer r2.1, 250 nmol / L crRNA, 10 U RNase inhibitor and 400 nmol / L fluorescent probe to obtain a CRISPR / Cas12a system complex solution, and store it in the dark at 4°C for later use.

[0051] 4.2) Establishing a standard curve model between fluorescence value and logarithmic value of enrofloxacin concentration; 4.2.1) Prepare a solution of enrofloxacin at a concentration of 0.5-100 ng / mL in phosphate buffered saline (PBS, pH 6, containing 0.05% Tween-20 and 0.1% BSA); 4.2.2) 30 μL of the MNPs-Ab solution obtained in step 1), 10 μL of the AuNPs-ENR-ssDNA solution obtained in step 3), 30 μL of the phosphate-free saline (PBS) buffer (pH 6, containing 0.05% Tween-20 and 0.1% BSA) obtained in step 3.3) and 130 μL of the enrofloxacin solution obtained in step 4.2.1) were mixed and reacted at room temperature for 30 min. After the reaction, the magnetically separated particles were washed three times with PBS (containing 0.05% Tween-20 and 0.1% BSA) at pH 6 and resuspended in 35 μL of DEPC water to obtain a mixed solution of the MNPs-Ab complex (MNPs-Ab-AuNPs-ENR-ssDNA) with enrofloxacin and AuNPs-ENR-ssDNA captured on the surface; 4.2.3) Mix 30 μL of the mixed solution containing MNPs-Ab-AuNPs-ENR-ssDNA obtained in step 4.2.2) with 20 μL of the CRISPR / Cas12a system complex solution obtained in step 4.1), react at 37°C for 30 min, measure the fluorescence value at 520 nm at an excitation wavelength of 492 nm, and establish a standard curve model between the fluorescence value and the logarithmic value of the enrofloxacin concentration.

[0052] In this embodiment, the fluorescent probe uses a single-stranded DNA with a FAM fluorescent group and a BHQ-1 quenching group modified at both ends, and the sequence of the fluorescent probe is 5'-FAM-TTATT-BHQ1-3'. The higher the concentration of enrofloxacin in the sample, the less AuNPs-ENR-ssDNA bound to the surface of MNPs, and the lower the activity of the CRISPR / Cas12a system activated. Since only a small amount of the FAM fluorescent group in the fluorescent probe is far away from the BHQ-1 quenching group, the resulting fluorescence intensity is weak.

[0053] This example uses 7 known different enrofloxacin concentrations and sub-examples to test the results as shown in the following table: The relationship curve was established with the logarithmic value of enrofloxacin concentration as the abscissa and the fluorescence value at 520 nm as the ordinate. It can be obtained that the linear detection range of this method is 0.5~100 ng / mL, and the linear regression equation is: y = -973.29 x+ 2790.7( R 2 =0.9597), and the detection limit was 0.86 ng / mL ( Figure 4 ), and thus a standard curve model between enrofloxacin concentration and fluorescence value was established.

[0054] It can be seen from the above embodiments that the present invention expands the application of the CRISPR / Cas12a system to non-nucleic acid small molecule detection by introducing AuNPs-ENR-ssDNA complexes that can both competitively bind to antibody sites with the target to be detected and activate the cleavage activity of the CRISPR / Cas12a system.

[0055] Therefore, the magnetic nanoparticle solution, gold nanoparticle complex solution, and CRISPR / Cas12a system complex solution in this embodiment can be prepared into a kit.

[0056] Example 2 This example uses the kit obtained in Example 1 to detect the concentration of enrofloxacin in the pool water spiked sample. The specific detection process and results are as follows: 4.3) Detecting the concentration of enrofloxacin in the pool water spiked samples; 4.3.1) After the pool water was filtered through a 0.22 μm filter membrane, 100 mmol / L PBS, 0.05% Tween-20 and 0.1% BSA were added to obtain a pool water composite solution; 4.3.2) Add enrofloxacin standard sample to the pool water composite solution in step 4.3.1), and the enrofloxacin spiked concentrations are 1 ng / mL, 25 ng / mL and 50 ng / mL respectively; 4.3.3) Obtain the fluorescence value of the measured sample according to steps 4.2.2) to 4.2.3) in Example 1, and calculate the actual concentration of enrofloxacin contained in the pool water spiked sample.

[0057] This example involves 3 levels of enrofloxacin spike concentrations and performs sub-examples (n=3). The test results are shown in the following table: The recoveries of the three pool water spiked samples of this method ranged from 80.95% to 119.19%, which can meet the needs of enrofloxacin residue detection in actual samples.

[0058] Example 3 This example uses the kit obtained in Example 1 to investigate the detection specificity of the method of the present invention: 4.4) Investigate the detection specificity of the method; 4.4.1) Prepare ciprofloxacin (CIP), chloramphenicol (CAP), doxycycline (DOXC), oxytetracycline (OTC), sulfamethoxazole (SMZ), and enrofloxacin (ENR) at a concentration of 10 ng / mL in PBS (pH 6, containing 0.05% Tween-20 and 0.1% BSA); 4.4.2) Obtain the fluorescence value of the measured sample according to steps 4.2.2) to 4.2.3).

[0059] Figure 5 This is a bar graph showing the specific effects of the method of the present invention on different antibiotics. The method has no effect on the fluorescence of CAP, DOXC, OTC, and SMZ, but produces obvious fluorescence changes on enrofloxacin and its metabolite CIP.

[0060] CIP is the main metabolite of enrofloxacin in animals. Given the structural similarities between ENR and CIP, some cross-reactivity may be exhibited. Since the limit value for enrofloxacin in my country's national standard "GB 31650-2019 National Food Safety Standard Maximum Residue Limits of Veterinary Drugs in Food" is the sum of CIP and enrofloxacin, the cross-reactivity of this method to CIP does not affect its practical application.

[0061] The invention has high sensitivity, can quickly detect enrofloxacin qualitatively / semi-quantitatively, and has good development prospects; it also has the ability to detect other analogs.

[0062] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A method for rapid detection of non-nucleic acid targets based on magnetic separation-CRISPR signal amplification, characterized in that: The following steps are involved: S1. modifying the surface of carboxylated magnetic nanoparticles with an antibody that can specifically recognize the non-nucleic acid target to be detected to obtain an antibody-modified magnetic nanoparticle solution; S2. modifying the antigen / antigen analog and single-stranded DNA corresponding to the non-nucleic acid target onto the surface of the gold nanoparticles to obtain a gold nanoparticle complex solution; S3. Mix the magnetic nanoparticle solution, the gold nanoparticle complex solution, and the non-nucleic acid target sample solution to be detected in a phosphate buffer and shake at room temperature for reaction before magnetic separation; then wash with PBS and resuspend in DEPC water to obtain a MNPs-Ab complex solution; S4. Mix the Cas12a protein solution, New England buffer, crRNA solution, RNase inhibitor solution and fluorescent probe solution to obtain a CRISPR / Cas12a system complex solution; S5. The MNPs-Ab complex solution is mixed with the CRISPR / Cas12a system complex solution for reaction; the fluorescence value at the corresponding emission wavelength is measured under the excitation wavelength of the fluorescent group of the fluorescent probe, and then the standard curve of the relationship between concentration and fluorescence value is substituted to obtain the concentration of the non-nucleic acid target to be measured in the sample solution.

2. The method for rapid detection of non-nucleic acid targets according to claim 1, characterized in that: The non-nucleic acid targets that can specifically identify the target include but are not limited to enrofloxacin, levofloxacin, and ciprofloxacin.

3. The method for rapid detection of non-nucleic acid targets according to claim 1, characterized in that: The mass ratio between the antibody and the carboxylated magnetic nanoparticles in S1 is 1:50; The particle size of the magnetic nanoparticles is 50-500 nm.

4. The method for rapid detection of non-nucleic acid targets according to claim 1, characterized in that: The volume ratio of the gold nanoparticles, antigen / antigen analogs, and ssDNA in S2 is 2:1:1; The particle size of the gold nanoparticles is 10-30 nm.

5. The method for rapid detection of non-nucleic acid targets according to claim 1, characterized in that: The length of the single-stranded DNA in S2 is 20-30 nt and is complementary to the crRNA base in S4; The 5' or 3' end of the single-stranded DNA is modified with a thiol group.

6. The method for rapid detection of non-nucleic acid targets according to claim 1, characterized in that: The volume ratio of the magnetic nanoparticle solution, the gold nanoparticle complex solution, and the non-nucleic acid target sample solution to be detected in S3 is 3:1:

13.

7. The method for rapid detection of non-nucleic acid targets according to claim 1, characterized in that: The volume ratio of the Cas12a protein solution, the New England buffer, the crRNA solution, the RNase inhibitor solution, and the fluorescent probe solution in the S4 is 0.1:5:1.25:0.25:

2.

8. The method for rapid detection of non-nucleic acid targets according to claim 1, characterized in that: The volume ratio of the MNPs-Ab complex solution to the CRISPR / Cas12a system complex solution in S5 is 30:

20.

9. A kit for rapid detection of non-nucleic acid targets, characterized in that: It comprises the magnetic nanoparticle solution, the gold nanoparticle complex solution and the CRISPR / Cas12a system complex solution in the non-nucleic acid target rapid detection method according to any one of claims 1 to 8.

10. Use of the kit according to claim 9 in detecting antibiotics, toxins or pesticides.

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