CRISPR-immune combination small molecule detection probe preparation and detection method

Abstract

The invention particularly relates to preparation of a CRISPR (clustered regularly interspaced short palindromic repeats)-immune combined small molecule detection probe as well as a related detection method and application thereof. The preparation method of the probe mainly adopts a chemical and biological coupling method, and the dsDNA-antibody probe is prepared by covalently coupling the dsDNA of which the 5'end is modified to NH2 and a monoclonal antibody by using a bifunctional cross-linking agent 4-(N-maleimide methyl) cyclohexane-1-carboxylic acid sulfonic acid group succinimide ester sodium salt (Sulfo-SMCC) and N-succinimido-S-acetylthioacetate (SATA). Secondly, according to the detection method, a dsDNA-antibody probe is taken as a bridge to combine an immune system and a CRISPR / Cas12a system, the specific recognition capability of an antibody to small molecules is utilized, signal amplification is carried out by virtue of RPA nucleic acid amplification, and fluorescence signals are generated by virtue of cis-cleavage activity and / or trans-single-chain DNA cleavage activity of CRISPR / Cas12a, so that conversion from immune reaction signals to high-intensity fluorescence signals is realized, and the detection sensitivity is high. And a CRISPR-immune combined small molecule detection system is established. The detection method has the advantages of simplicity in operation, high specificity, extremely high sensitivity, stable signal and the like.

Description

Technical Field

[0001] The present invention belongs to the field of biological detection, and specifically relates to a method for preparing a CRISPR-immune combined small molecule detection probe and related detection methods and uses thereof. Background Art

[0002] In recent years, the discovery and research of CRISPR system has provided a new method for the detection of nucleic acid targets, while the work of using CRISPR system for non-nucleic acid targets (such as small molecules) is very limited. However, in the fields of medicine, environment and food safety, the detection of small molecule markers or pollutants is very important. For example, β-agonists are a representative type of small molecule pollutants, which are often illegally used in animal breeding, can cause acute poisoning or chronic cumulative poisoning in humans, and bring long-term and inestimable impacts to the food chain and the environment. Despite the serious harm, at present, the instrumental analysis method is very mature in the detection method of banned small molecule pollutants such as β-agonists, but the disadvantage of this method is that the sample pretreatment is complicated and relies on expensive instruments and professional technicians; the unstable characteristics of the biosensor method make the method difficult to use in practical applications; the advantage of immunoassay is that it is easy to operate, time-saving, and has good stability, but the disadvantage of this method is that the sensitivity depends more on the properties of the antibody itself, and the detection performance is not enough for β-agonists that are prohibited by law. Combining the specificity and controllability of immunoassays with the ultra-high sensitivity and signal stability of the CRISPR / Cas system, establishing a CRISPR-immune combined detection system can make up for the shortcomings of current immunoassay methods.

[0003] At present, CRISPR detection of small molecules mainly uses aptamers, antibodies and bacterial allosteric transcription factors as bridges to realize the conversion of nucleic acid signals. For example, Chinese patent CN111812066A provides a detection method that uses bacterial allosteric transcription factors and CRISPR / Cas12a systems to convert small molecule signals into light signals through the cis-cleavage and trans-cleavage activities of CRISPR / Cas12a. Compared with antibodies, it is more difficult to screen suitable aptamers and bacterial allosteric transcription factors as non-nucleic acid target recognition elements. Australian patent WO2023019290A1 proposes a CRISPR / Cas12a-driven immunosensor for detecting whole Cryptosporidium oocyst cells in water samples. However, research on the detection of small molecules by CRISPR-immune combined systems is still lacking. Therefore, there is an urgent need for a new method for detecting small molecules that can integrate the CRISPR / Cas system with an immunoassay system, is simple to operate, has strong specificity, high sensitivity, and has the potential for rapid on-site detection. Summary of the invention

[0004] The technical problem to be solved by the present invention is to overcome the defects of the prior art, and provide a dsDNA-antibody probe, a composition, a small molecule detection kit and related detection methods and uses thereof that organically combine an immune response, an RPA isothermal amplification system and a CRISPR / Cas12a system. The dsDNA-antibody probe is used as a bridge to combine the immune system and the CRISPR system. On the one hand, the antibody part of the dsDNA-antibody probe is used to specifically identify small molecules, and on the other hand, the nucleic acid part of the dsDNA-antibody probe is used to achieve the purpose of signal amplification and signal output with the help of the RPA amplification system and the CRISPR / Cas12a system.

[0005] To this end, the present invention adopts the following technical solutions:

[0006] In a first aspect, the present invention provides a method for synthesizing a dsDNA-antibody probe, wherein the dsDNA-antibody probe is formed by covalently coupling dsDNA and a monoclonal antibody through a chemical biological coupling method.

[0007] Wherein, the dsDNA is arbitrary and can be subjected to RPA isothermal amplification and CRISPR system recognition activated dsDNA.

[0008] Wherein, the dsDNA is a dsDNA with -NH2 modification at the 5' end;

[0009] The monoclonal antibodies include but are not limited to salbutamol monoclonal antibodies, and may also be replaced by other small molecule monoclonal antibodies.

[0010] Among them, the chemical biological coupling method uses the bifunctional cross-linking agents 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid sulfonic acid succinimidyl ester sodium salt (Sulfo-SMCC) and N-succinimidyl-S-acetylthioacetate (SATA) to react chemically with dsDNA and antibodies respectively to form covalent bonds, see Example 1.

[0011] In a second aspect, the present invention also includes a method for constructing the dsDNA-antibody probe: dsDNA and monoclonal antibodies are covalently linked to obtain the dsDNA-antibody probe. The dsDNA-antibody probe is prepared by reacting dsDNA and antibody at a molar concentration ratio of 1:1.

[0012] In a third aspect, the present invention provides a kit for detecting small molecules, the kit comprising a recognition reagent, an amplification reagent and a signal output reagent.

[0013] Wherein, the recognition reagent comprises a dsDNA-antibody probe, wherein the dsDNA comprises an -NH2 modified portion for chemical biological coupling with the antibody, a target sequence of the RPA isothermal amplification system, a recognition site PAM of the CRISPR / Cas12a system, and a sequence complementary to the crRNA portion.

[0014] Wherein, the amplification reagent comprises the RPA isothermal amplification system, comprising target dsDNA forward and reverse primers, MgOAc, dNTPs, target dsDNA, 2×Reaction Buffer, 10×Basic E-mix, 20×Core ReactionMix and / or water;

[0015] 2×Reaction Buffer, 10×Basic E-mix, and 20×Core Reaction Mix are all reagents in the DNA isothermal rapid amplification kit of TwistDx. RPA forward and reverse primers are designed for target dsDNA.

[0016] The signal output reagent comprises the CRISPR / Cas12a system, including Cas12a enzyme, crRNA, single-stranded DNA probe (ssDNA-reporter), NEBuffer, RNase inhibitor and / or water.

[0017] Among them, the two ends of the ssDNA-reporter are conjugated with FAM reporter group and BHQ quencher group respectively.

[0018] In a fourth aspect, the present invention provides a CRISPR-immunity combined with small molecule detection method, comprising the following steps:

[0019] S1. The coating source is embedded in the ELISA plate, and the dsDNA-antibody probe is added. The antibody specifically recognizes and binds to the coating source. After washing the plate, the dsDNA-antibody probe is fixed on the ELISA plate.

[0020] S2. adding the RPA reagent to the above system to perform exponential amplification on dsDNA;

[0021] S3. Add CRISPR reagents to the above reaction system. The crRNA in CRISPR specifically recognizes the PAM site on the target dsDNA for cis-cutting, and activates the para-cutting activity of the CRISPR protein, randomly cutting the fluorescent reporter molecule to generate fluorescent signals. Use QuantStudio3 Real-time PCR or a portable UV-blue light gel cutter with a wavelength of 470nm for detection to achieve quantitative or qualitative analysis of the analyte.

[0022] Specifically, in step S1, the original coating concentration is 2.56 pg / mL, and the dilution ratio of the dsDNA-antibody probe is 1:16000, see Example 2.

[0023] Specifically, the reaction conditions of the RPA amplification reaction in step S2 were 10 μL reaction volume, which contained 0.5 μM forward primer and reverse primer, 2.0 mM dNTPs, 22.4 mM MgOAc, and the entire amplification process was completed by heating at 37°C for 25 min, see Series 4.

[0024] Specifically, the reaction conditions of the CRISPR system in step S3 are 10 μL reaction volume, including 125 nM Cas12a protein, 50 nM crRNA, 1000 nM ssDNA-reporter, and reaction time of 10 min, see series experiment 2.

[0025] The present invention has the following beneficial effects:

[0026] The present invention combines enzyme-linked immunosorbent assay, RPA amplification technology and CRISPR detection technology, combines the immune system and CRISPR / Cas12a system by synthesizing dsDNA-antibody probe as a bridge, utilizes the specific recognition ability of antibodies for small molecules, uses RPA nucleic acid amplification to amplify signals and CRISPR / Cas12a cleavage activity to generate fluorescent signals, realizes the transformation of immune signals into fluorescent signals, and establishes a CRISPR-immune combined small molecule detection system. The detection method has strong specificity and high sensitivity.

[0027] The present invention introduces a dsDNA-antibody probe, which is formed by covalently coupling dsDNA and monoclonal antibodies. On the one hand, the antibody part of the dsDNA-antibody probe is used to specifically identify small molecules, and on the other hand, the nucleic acid part of the dsDNA-antibody probe is used to achieve the purpose of signal amplification and signal output with the help of the RPA amplification system and the CRISPR / Cas12a system. The probe can simultaneously exert immune function and nucleic acid signal transduction function, making the operation simpler.

[0028] The CRISPR-immunity combined small molecule detection method described in the present invention only needs to select corresponding antibodies according to different small molecules and synthesize dsDNA-antibody probes. Other components do not need to be replaced, and the detection of different small molecules can be achieved, and its versatility is strong. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 :CRISPR-immunity combined with small molecule detection system detection scheme;

[0030] Figure 2:Diagram of target dsDNA sequence design;

[0031] Figure 3 :The coupling principle of dsDNA-antibody probe;

[0032] Figure 4 : dsDNA-antibody probe purification and characterization results, wherein Figure A is a molecular sieve chromatography purification chromatogram of dsDNA-antibody probe, Figure B is agarose nucleic acid electrophoresis results, and Figure C is SDS-PAGE results;

[0033] Figure 5 : Standard curve of antibody and dsDNA-antibody probe;

[0034] Figure 6 :Optimized fluorescence curve of CRISPR / Cas12a detection system conditions corresponding to dsDNA;

[0035] Figure 7 :The fluorescence value of the CRISPR / Cas12a detection system corresponding to the target dsDNA at 10 min of condition optimization, corresponding to the optimization of Cas12a concentration, crRNA concentration and ssDNA-reporter concentration;

[0036] Figure 8 :Sensitivity of CRISPR / Cas12a detection system to target dsDNA;

[0037] Fig. 9 : The optimization results of the RPA amplification system conditions corresponding to the target dsDNA, where Figure A is the optimization of primer concentration, Figure B is the optimization of dNTPs concentration, Figure C is the optimization of MgOAc concentration, and Figure D is the optimization of amplification time;

[0038] Fig.10 : Optimization results of coating source and dsDNA-antibody probe concentrations, wherein Figure A is the optimization of coating source concentrations, and Figure B is the optimization of dsDNA-antibody probe concentrations;

[0039] Fig.11 :Standard curve of CRISPR-immunity combined with small molecule detection system;

[0040] Fig.12 : Standard curve of SAL in pig urine samples. DETAILED DESCRIPTION

[0041] The present invention is further described in detail below in conjunction with the accompanying drawings and specific examples of the specification. The examples are only used to explain the present invention and are not used to limit the scope of the present invention. The test methods used in the following examples are conventional methods unless otherwise specified; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0042] pass Figure 1 The principle of the CRISPR-immune combined small molecule detection method of the present invention is briefly described as follows: the coating source is fixed on the ELISA plate, and the dsDNA-antibody probe is added and incubated with the analyte at the same time. The analyte and the coating source compete for the antibody at the same time, and then the excess probe and drug are washed away, and then the CRISPR / Cas12a detection system is directly added to the ELISA well. More analytes will bring stronger competitive ability, resulting in fewer dsDNA-antibody probes bound to the coating source, and the number of Cas12a finally activated is smaller, and the signal value generated is lower, and a standard curve with a negative correlation between the concentration of the analyte and the fluorescence intensity is established.

[0043] According to the above principle, it is necessary to design for dsDNA. Since the specific recognition of Cas12a will be affected by the steric effect, it is necessary to consider whether the sequence characteristics of the target dsDNA will affect the specific binding of Cas12a to the target dsDNA in the design of the target dsDNA. Therefore, the target dsDNA sequence consists of four parts (such as Figure 2 As shown in the figure): (1) a linker arm connected to the antibody, with a length of 5-10bp; (2) the sequence contains the PAM site "TTTV" and a target sequence complementary to crRNA; (3) provides sufficient binding space for the Cas12a enzyme / crRNA complex; (4) RPA primer recognition sequence. The role of the linker arm is to increase the distance between the antibody and the CRISPR / Cas12a recognition site to prevent Cas12a from being unable to recognize the target DNA due to steric hindrance. At the same time, it also provides sufficient distance between dsDNA and the antibody to prevent the target dsDNA in the dsDNA-antibody probe from blocking the specific binding site of the antibody.

[0044] The following examples all use salbutamol (SAL) in β-agonists as the detection object to establish a CRISPR-immunity combined small molecule detection method. The antibody is a salbutamol monoclonal antibody, and the dsDNA is a double-stranded DNA with a length of 219bp, a sequence containing the PAM site "TTTV" and conforming to the above four parts. The dsDNA-antibody probe synthesis method described in the present invention can be applied to any monoclonal antibody, not limited to the salbutamol monoclonal antibody.

[0045] Example 1. Synthesis of dsDNA-antibody probe

[0046] Principle of dsDNA-antibody probe preparation Figure 2As shown. First, the dsDNA modified with -NH2 at the 5' end forms a covalent amide bond with the n-hydroxysuccinimide (NHS) ester at one end of the bifunctional crosslinker 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid sulfosuccinimide ester sodium salt (Sulfo-SMCC); the NHS ester at one end of N-succinimidyl-S-acetylthioacetate (SATA) reacts with the -NH2 on the lysine residue of the antibody, and then hydroxylamine deprotects the thiol group to expose the thiol group; the maleimide group at the other end of Sulfo-SMCC reacts with the exposed thiol group at pH = 6.5-7.5 to form a stable thioether bond. The specific steps are as follows:

[0047] Considering the possible losses caused by subsequent purification and concentration, the initial amount of antibody coupling should be at least 1 mg. Here, 9 mg of antibody is taken as an example. The amount of each reagent can be increased or decreased in the same proportion according to actual needs.

[0048] (1) Dialyze 9 mg of antibody (60 nmol) in phosphate buffered saline (PBS) solution at pH 7.4 at 4°C for 48 hours to displace the primary amine groups that may be contained in the buffer in which the antibody is stored, preventing nonspecific reactions during the subsequent coupling process, and adjusting the pH value of the antibody background solution to 7.4, which is conducive to the reaction between the antibody and SATA;

[0049] (2) SATA-modified antibodies: Take SATA solution (600 nmol) prepared with 10 times the molar amount of antibody and add it to the dialyzed antibody, mix well, and react at room temperature for 30 min to obtain SATA-modified antibodies; the SATA-modified antibodies are concentrated and purified by ultrafiltration tubes to remove excess SATA reagents; the ultrafiltration tubes are centrifuged at 6000 r / min and 4°C. During the concentration and purification process, the ultrafiltration tubes need to be taken out every 10 minutes of centrifugation, and the liquid is applied to the membranes on both sides with a pipette. The solution is repeatedly blown and aspirated several times to prevent protein precipitation caused by excessive concentration, until 200 μL of the solution is left, and finally the liquid volume is supplemented to 900 μL with PBS;

[0050] (3) Sulfo-SMCC modification of target dsDNA: Take 600 nmol of Sulfo-SMCC solution prepared with 10 times the molar amount of DNA and add it to the prepared 60 nmol target dsDNA, mix well, and react at room temperature for 30 min to obtain SATA-modified dsDNA; the SATA-modified dsDNA is concentrated and cleaned according to step (2), and finally the solution volume is supplemented to 900 μL with PBS and then temporarily stored at -20°C;

[0051] (4) Weigh 1.74 g of hydroxylamine hydrochloride and 0.365 g of disodium EDTA and dissolve them in 40 mL of PBS solution. Adjust the pH of the solution to 7.4 with sodium hydroxide solution to prepare a hydroxylamine hydrochloride solution. Take 100 μL of the prepared hydroxylamine hydrochloride solution and add it to the concentrated and impurity-free antibody, mix well, and react at room temperature for 2 hours.

[0052] (5) Add the Sulfo-SMCC-modified target dsDNA stored in step (3) to ensure that the molar concentration ratio of target dsDNA to antibody is 1:1, mix well, and react at room temperature for 1 hour;

[0053] (6) Concentrate using an ultrafiltration tube and remove excess reaction reagents in the process. The operation is the same as step (2) of concentration and impurity removal until the solution is 250 μL, then take out the liquid and add PBS solution to 1 mL to obtain the initial product of dsDNA-antibody probe;

[0054] (7) Using molecular sieves to separate and purify the initial product of the dsDNA-antibody probe to obtain a purified dsDNA-antibody probe.

[0055] During the coupling process, there are uncoupled target dsDNA and antibodies, which need to be purified to prevent them from interfering with the subsequent quantitative system. The purification method uses a one-step molecular sieve chromatography method based on the difference in molecular weight between the dsDNA-antibody probe and its target dsDNA. The molecular sieve chromatography method uses a Superdex 200 pre-packed column to separate the dsDNA-antibody and the uncoupled target dsDNA based on the difference in molecular weight between them.

[0056] Among them, the molecular weight of monoclonal antibodies is generally around 150kDa, and the dsDNA used in this example is 219bp, with a molecular weight of approximately 130kDa. The dsDNA-antibody probe (280kDa) has a large difference in molecular weight from the uncoupled target dsDNA and antibodies, so it can be separated using a molecular sieve. The first peak to emerge is the dsDNA-antibody probe, and the subsequent peaks are the uncoupled target dsDNA and antibodies. Since the molecular weight difference between the latter two is 20kDa, the gap is small, so they flow out together within the same retention time. The results of molecular sieve chromatography purification of the dsDNA-antibody probe are shown in the figure. Figure 4 As shown in Figure A.

[0057] The results of nucleic acid electrophoresis and SDS-PAGE identification of the purified dsDNA-antibody probe are as follows: Figure 4 As shown in Figures B and C. Since the antibody in the dsDNA-antibody probe cannot move on the protein gel due to the tethering of dsDNA, the probe will be at a higher position than the antibody. Figure 4Lanes 4 and 5 of Figures B and C correspond to a mixture of dsDNA-antibody probe and unconjugated antibody and target dsDNA, respectively, wherein the probe band is single, indicating that a pure dsDNA-antibody probe was obtained.

[0058] Experiment 1. Performance comparison of dsDNA-antibody probe before and after coupling

[0059] The ic-ELISA method was used to identify the activity of the antibody before coupling and the purified dsDNA-antibody probe. After the chessboard titration method, the appropriate antigen-antibody concentration combination was found and the standard curve was established. The specific steps are as follows:

[0060] (1) Coating: Dilute the salbutamol (SAL) coating source to 1 μg / mL with coating buffer, add 100 μL / well to the ELISA plate, and incubate in a 37°C water bath for 12 hours.

[0061] (2) Blocking: Take out the ELISA plate, wash it twice using an automatic plate washer and pat it dry. Add 120 μL of 6% skimmed milk powder blocking solution and block it for 3 h. Shake off the blocking solution in the plate and dry it at 37°C for 1 h. The 6% skimmed milk powder blocking solution is prepared by dissolving 6 g of skimmed milk powder in 100 mL of phosphate-buffered saline (PBST).

[0062] (3) Sample addition: In the chessboard titration method, the antibody with an initial concentration of 1 μg / mL was first diluted with PBST to 7 concentrations (0.5 μg / mL, 0.25 μg / mL, 0.13 μg / mL, 62.5 ng / mL, 31.25 ng / mL, 15.63 ng / mL, 7.81 ng / mL); 50 μL PBST was added to the odd-numbered wells of the blocked ELISA plate, and 50 μL PBST-diluted salbutamol (1 μg / mL) was added to the even-numbered wells. Then, 50 μL of the antibody with the same dilution factor was added to each row, and the blank control (NTC) in the last row was set to 50 μL PBST; during the calibration curve establishment, the salbutamol with an initial concentration of 10 μg / mL was first diluted with PBST to 7 concentrations (1000 ng / mL, 100 ng / mL, 10 ng / mL, 1 ng / mL, 0.1 ng / mL, 0.01 ng / mL, 0.001 ng / mL); add 50 μL of the drug with the same dilution factor to each row of the sealed ELISA plate, set the last row as a blank with 50 μL PBST, then add 0.25 μg / mL antibody to each well, incubate at 37℃ for 40 min, wash the plate 5 times, and pat dry;

[0063] (4) Add secondary antibody: dilute HRP enzyme-labeled goat anti-mouse 5000 times with PBST, add 100 μL to each well, incubate at 37°C for 30 min, wash the plate 5 times, and pat dry;

[0064] (5) Color development: Add 100 μL of color development solution to each well and incubate at 37°C for 10 min.

[0065] (6) Termination: Add 50 μL of stop solution to each well to terminate the reaction and read the absorbance of each well at 450 nm on a multifunctional microplate reader.

[0066] The results are as follows Figure 4 As shown, SAL antibody IC 50 1.49 ng / mL, LOD 0.18 ng / mL; dsDNA-antibody probe IC 50 The IC of dsDNA-antibody probe was 1.83 ng / mL and the LOD was 0.2 ng / mL. 50 The LOD value did not change much compared with that before coupling, and the performance of the antibody was basically unaffected. Therefore, the dsDNA-antibody probe can be used for the subsequent establishment of CRISPR-immune combined small molecule detection methods.

[0067] Experiment 2. Optimization of CRISPR / Cas12a detection system conditions

[0068] Before establishing the CRISPR-immune combined small molecule detection system, the corresponding CRISPR / Cas12a detection system was optimized for the target dsDNA. Based on the preparation of the target dsDNA and the corresponding crRNA, a preliminary system was first used: 50 nM Cas12a, 150 nM crRNA, 200 nM ssDNA-reporter (FAM-TTTTTT-BHQ), 100 nM DNA, 2 μL NEBuffer 2.1, and sterilized ultrapure water was added to 20 μL.

[0069] First, the reaction time in the CRISPR / Cas12a detection system was optimized. The configured CRISPR / Cas12a detection system was placed in a QuantStudio3 Real-time PCR instrument and incubated at 37°C to continuously collect fluorescence signals. The results are as follows: Figure 5 As shown, the fluorescence basically reached saturation after 10 minutes of reaction.

[0070] In the CRISPR / Cas12a system, Cas12a only exhibits trans-cleavage activity when Cas12a, crRNA and target dsDNA form a ternary complex, and can non-specifically cut the ssDNA-reporter probe to release fluorescent signals. Therefore, the concentrations of cas12a, crRNA and ssDNA-reporter are the main factors affecting the detection performance. The concentrations of Cas12a protein, crRNA and ssDNA-reporter were further optimized according to the reaction time of 10 min, and the optimized CRISPR / Cas12a detection system was obtained, specifically Cas12a protein concentration of 125nM, crRNA concentration of 50nM, ssDNA-reporter concentration of 1000nM, 100 nM DNA, 2 μL NEBuffer 2.1, and sterilized ultrapure water was supplemented to 20μL, as shown in Table 1 and Figure 6 shown.

[0071] Table 1: CRISPR / Cas12a detection system optimization parameters

[0072]

[0073] Later, due to actual cost reasons, the CRISPR system was reduced to a 10μL system, which did not affect the test results.

[0074] Experiment series 3. Performance of CRISPR / Cas12a detection system

[0075] The established CRISPR / Cas12a detection system was tested by adding different concentrations of target dsDNA to test its detection effect. The target dsDNA concentration was diluted to 10 1 ng / μL, 10 0 ng / μL, 10 -1 ng / μL, 10 -2 ng / μL, 10 -3 ng / μL, the optimized CRISPR / Cas12a detection system (obtained from series 2) was used to test the minimum recognition concentration of target dsDNA. A blank control was set up in each group. The blank control system was the same as that of the experimental group, and sterilized ultrapure water was used instead of the DNA template. Each group of experiments was set up in triplicate. The recognition performance of target dsDNA was compared, and this was used as a screening condition for universal dsDNA-antibody probes and as a reference for the concentration of template DNA added during RPA amplification.

[0076] The results are as follows Figure 7 As shown, when the target dsDNA added to the system is 10 1ng / μL, the CRISPR / Cas12a detection system produced green fluorescence visible to the naked eye. At the same time, statistical analysis of the fluorescence value and the blank control group showed a significant difference, which could achieve a better recognition effect.

[0077] The amount of DNA template used in the subsequent experiment was 10 1 ng / μL.

[0078] Experiment 4. Optimization of RPA amplification system conditions

[0079] First, refer to the instructions of the RPA amplification kit to prepare an RPA reaction system: 0.5 μM forward and reverse primers, 14 mM MgOAc, 0.8 mM dNTPs, 10 1 ng / μL DNA template, 5 μL 2×Reaction Buffer, 1 μL 10×Basic E-mix and 0.5 μL 20×Core Reaction Mix, and add sterile ultrapure water to 10 μL. In this study, the primer concentration, dNTPs concentration, MgOAc concentration and reaction time in the RPA reaction system were optimized. The optimized RPA reaction system was 0.5 μM for the forward and reverse primers, 2 mM for the dNTPs concentration, 22.4 mM for the MgOAc concentration, and 10 1 ng / μL DNA template, 5μL 2× Reaction Buffer, 1μL 10× Basic E-mix and 0.5μL 20× Core Reaction Mix, add sterile ultrapure water to 10μL, reaction time 25min, as shown in Table 2 and Figure 8 shown.

[0080] Table 2 Optimized parameters of RPA amplification system

[0081]

[0082] The optimization results of the RPA reaction system were obtained by adding the optimized CRISPR / Cas12a detection system after the RPA reaction, placing it in the QuantStudio3 Real-time PCR instrument for incubation at 37°C for 10 min, and then collecting the fluorescence signal. Then a portable blue light gel cutting instrument with a wavelength of 470 nm was used for naked eye observation.

[0083] Example 2. Establishment of CRISPR-immunity combined with small molecule detection method

[0084] This method was established based on the principle of CRISPR-immune combined with small molecule detection method and combined with multiple series of experimental analysis and optimization in this case.

[0085] Since this method includes RPA amplification, false positives are very likely to occur. The reason for this may be that the open reaction site, such as the enzyme-labeled well, is easily contaminated by aerosols during the RPA amplification reaction, resulting in false positives. Therefore, the reaction site is transferred from the open enzyme-labeled well to the closed EP tube (after testing, the EP tube can also undergo immune reactions). Therefore, the implementation steps of the CRISPR-immune combined small molecule detection system finally constructed by the present invention are as follows:

[0086] It should be noted that this detection method is divided into three stages, which are used to achieve the purpose of determining the working concentration of antigen and antibody, establishing a standard curve, and detecting real samples. Each stage implements steps 1) to 7), the difference is that in the first stage, step A is implemented in step 3), in the second stage, step B is implemented in step 3), and in the third stage, step C is implemented in step 3).

[0087] 1) Coating: Weigh 1.59 g Na2CO3 and 2.93 g NaHCO3 and dissolve them in 1 L of grade 3 water to prepare coating buffer. Use the coating buffer to dilute the SAL coating to 2.56 pg / mL (the original coating concentration is obtained from series 5), add 10 μL / tube into the EP tube, and incubate in a 37°C constant temperature water bath for 3 hours;

[0088] 2) Blocking: After taking out the EP tube, manually shake off the coating solution in the tube; add 200 μL of phosphate-buffered saline (PBST) to the EP tube, manually shake off the solution in the tube, and wash the EP tube twice with the PBST; add 20 μL of 6% skimmed milk powder blocking solution (prepared by dissolving 6 g skimmed milk powder in 100 mL PBST), incubate in a 37°C constant temperature water bath for 1 hour, manually shake off the blocking solution in the EP tube, and dry at 37°C for 1 hour;

[0089] 3) Adding reagents: Step A, step B and step C in step (3) are respectively implemented in the three-stage experiment of the detection method, as follows:

[0090] A. Use PBST to dilute 43.2 mg / mL dsDNA-antibody probe into 7 concentrations (1000, 2000, 4000, 8000, 16000, 32000 and 64000 times). The initial concentration of dsDNA-antibody probe measured by micro-spectrophotometer is 43.2 mg / mL. Since dsDNA-antibody probe contains dsDNA, it will interfere with the accurate determination of its absorbance at 280 nm. Therefore, this reference value is taken. 50 μL PBST and 50 μL diluted dsDNA-antibody probe are added to the sealed EP tube. A blank control (NTC) of 100 μL PBST is set in the last row.

[0091] B. The salbutamol drug with an initial concentration of 6 ng / mL was diluted with PBST to 7 concentrations (1 ng / mL, 0.17 ng / mL, 0.027 ng / mL, 0.0046 ng / mL, 0.7 pg / mL, 0.13 pg / mL, 0.021 pg / mL), and 50 μL of the diluted salbutamol drug and 50 μL of the dsDNA-antibody probe with a dilution ratio of 1:16000 (obtained from series 5) were added to the sealed EP tube. A blank control of 50 μL PBST and 50 μL dsDNA-antibody probe was set up in the last row.

[0092] C. Using pig urine as the real sample, first dilute the pig urine sample with 5 times the volume of PBST. This dilution factor can basically eliminate the matrix interference caused by the pig urine sample (obtained from series experiment 7). Then, add 50 μL of the diluted pig urine sample to the sealed EP tube, and then add 50 μL of the dsDNA-antibody probe diluted 16,000 times (obtained from series experiment 5);

[0093] 4) After adding the reagent, incubate at 37°C for 40 min, and manually shake off the solution in the tube; add 200 μL of sterile ultrapure water to the EP tube, then manually shake off the liquid in the tube, and repeatedly wash the EP tube five times with sterile ultrapure water; then use sterile ultrapure water to wash the EP tube to fully wash away the unbound probes and inorganic salts in the tube, reducing the impact on the subsequent RPA and CRISPR systems;

[0094] 5) RPA amplification: add 10 μL of the optimal RPA reaction system, including 0.5 μM forward and reverse primer concentrations, 2 mM dNTPs concentration, 22.4 mM MgOAc concentration, 5 μL 2 × Reaction Buffer, 1 μL 10 × BasicE-mix and 0.5 μL 20 × Core Reaction Mix, add sterile ultrapure water to 10 μL (obtained from series 4), and incubate at 37°C for 25 minutes;

[0095] 6) CRISPR / Cas12a detection system: Add 10 μL of the optimal CRISPR / Cas12a detection system, specifically Cas12a protein concentration of 125nM, crRNA concentration of 50nM, ssDNA-reporter concentration of 1000nM, 2 μL NEBuffer2.1, add sterile ultrapure water to 20 μL (obtained from series 2), incubate at 37°C for 10 minutes, inactivate at 98°C for 2 minutes, and then use QuantStudio3Real-time PCR instrument to collect fluorescence signals;

[0096] 7) The collected fluorescence signals were processed using Origin 2021 software. The optimal coating concentration (i.e., antigen working concentration) was finally determined to be 2.56 pg / mL, and the dsDNA-antibody probe dilution ratio (i.e., antibody working concentration) was 1:16000; the standard curve IC of the CRISPR-immunity combined small molecule detection system 10 0.64 pg / mL, IC 50 It was 11.08 pg / mL, and the detection range was 1.83~66.97 pg / mL; as well as the detection of pig urine samples.

[0097] The CRISPR-immunity combined small molecule detection method described in this technical solution integrates the specificity of immunoassay, the convenience of RPA isothermal amplification, and the ultra-high sensitivity and signal stability of the CRISPR / Cas system. 10 Improved by 280 times, IC 50 The sensitivity has been significantly improved by 134 times. At the same time, this method is universal. It only needs to replace the salbutamol monoclonal antibody with the corresponding small molecule monoclonal antibody to achieve high-sensitivity detection of different small molecules, making up for the shortcomings of the existing immune method in detecting legally prohibited drugs.

[0098] Experiment 5. Optimization results of coating source and dsDNA-antibody probe concentration

[0099] According to the method of Example 2, the concentration of the coating source and the concentration of the dsDNA-antibody probe were optimized by single factor for the purpose of determining the working concentration of the antigen and antibody (the initial concentrations of the two were 2.2 mg / mL and 43.2 mg / mL, respectively. Since the latter contained dsDNA, it would interfere with the accurate determination of its absorbance at 280 nm, so the concentration could only be used as a reference. The dilution ratio was used to describe the experimental results later). The coating source concentration conditions were 0.16 pg / mL, 0.64 pg / mL, 2.56 pg / mL, 10.24 pg / mL, and 40.96 pg / mL, respectively. The dilution multiples of the dsDNA-antibody probe were 1000, 2000, 4000, 8000, 16000, 32000, and 64000 times, respectively. The best coating source / probe concentration combination was found. The optimization results are as follows. Fig. 9 shown.

[0100] From the results, it can be seen that as the concentrations of coating source and dsDNA-antibody probe continue to decrease, the fluorescence intensity continues to decrease. Since lower coating source and dsDNA-antibody probe concentrations can promote competitive reactions and thus show better performance, a coating source / probe concentration combination with lower signal intensity should be selected. From the results, it was found that when the antibody concentration remained unchanged (dilution ratio was 1:1000), the fluorescence intensity decreased significantly when the coating source concentration decreased from 2.56 pg / mL to 0.64 pg / mL. According to the fluorescence intensity generated under the latter condition, it will not provide enough signal range for the calibration curve of the joint system. At the same time, in order to prevent the signal from being too low and causing large errors in the establishment of the subsequent joint system, the coating source concentration (i.e., antigen working concentration) was selected as 2.56 pg / mL. The coating original concentration was used to optimize the dsDNA-antibody probe concentration. When the dsDNA-antibody probe dilution ratio increased from 1:16000 to 1:32000, the fluorescence intensity also decreased significantly and remained basically unchanged afterwards. Based on the selection basis of the above analysis, the dsDNA-antibody probe dilution ratio (i.e., antibody working concentration) was 1:16000.

[0101] Experiment 6. Establishment of standard curve for CRISPR-immunity combined with small molecule detection system

[0102] According to series of experiments 5, the coating original concentration of 2.56 pg / mL and the dsDNA-antibody probe dilution factor of 1:16000 were selected as the optimal antigen-antibody working concentrations, and the standard curve of the CRISPR-immunity combined with small molecule detection system was established according to the method of Example 2.

[0103] Data processing and standard curve drawing: The B / B0 value is used as the ordinate, and the drug concentration is used as the abscissa, where B is the average absorbance of three parallel wells, and B0 is the blank average. The standard curve of ic-ELISA is obtained by fitting with Origin 2021 software.

[0104] The standard curve of CRISPR-immunity combined with small molecule detection system is as follows Fig.10 As shown, the IC10 is 0.64 pg / mL, the IC50 is 11.08 pg / mL, and the detection range is 1.83~66.97 pg / mL.

[0105] Experiment series 7. Detection of pig urine samples using CRISPR-immunity combined with small molecule detection system

[0106] Before testing the actual pig urine sample, it is necessary to explore the matrix effect of the sample. Adding an appropriate amount of PBST solution to eliminate the matrix effect can improve the accuracy of the system. The pre-treated pig urine sample was diluted 2 times, 5 times, and 10 times with PBST solution, and used to dilute the SAL standard respectively. The standard curve was established according to the operating steps of the combined system and compared with the standard curve established by diluting the SAL standard with PBST.

[0107] The results are as follows Fig.10 As shown in the figure, when the pig urine sample was diluted 5 times, the fitted curve basically overlapped with the standard curve obtained by using PBST as the diluent and the IC50 value (11.36 pg / mL) was the closest, indicating that this dilution factor can basically eliminate the matrix interference caused by the pig urine sample.

[0108] In order to evaluate the practical performance of the method, after testing the detection capability of the instrument, salbutamol standards (200, 1000, 5000 pg / mL) were first added to the pig urine samples, and the spiked samples were diluted 20 times with the pig urine samples (SAL concentrations were 10, 50, 250 pg / mL). The spiked samples were determined using the established CRISPR-immune combined detection system, and the spiked samples were confirmed using the LC-MS / MS method. The accuracy and precision of the system were evaluated based on the sample spike recovery rate and coefficient of variation (difference between parallel group data). Three parallel experiments were set up for each group. The recovery rate was calculated using formula (1) and the coefficient of variation was calculated using formula (2).

[0109] Recovery rate (%) = (measured value - blank value) * 100% / added value (1)

[0110] Coefficient of variation (%) = standard deviation * 100% / mean value (2)

[0111] Table 3 SAL recovery experiment in pig urine samples (n=3)

[0112]

[0113] The results showed (as shown in Table 3) that the recovery rate of SAL by the combined detection system established in the present invention was between 90.9% and 134.0%, and the coefficient of variation was between 1.1% and 7.8%, which was basically consistent with the results of the LC-MS / MS method (recovery rate 103.2% to 118.4%, coefficient of variation 1.7% to 10.2%). The above results indicate that the CRISPR-immunity combined small molecule detection system established in the present invention has high accuracy and precision, and can be used for the actual detection of SAL residues in pig urine.

[0114] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention rather than to limit the protection scope of the present invention. For ordinary technicians in this field, other different forms of changes or modifications can be made based on the above descriptions and ideas. It is not necessary and impossible to list all the implementation methods here. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the claims of the present invention.

Claims

1. A dsDNA-antibody probe, comprising an antibody and a target dsDNA, characterized in that: The antibody is any monoclonal antibody, the dsDNA is a target dsDNA with a length of 100-300 bp and a PAM site "TTTV" in the sequence, and the two are synthesized into a dsDNA-antibody probe by a maleimide-thiol coupling method, wherein the target dsDNA sequence consists of four parts, including: a linker with a length of 5-10 bp connected to the antibody, a target sequence containing a PAM site "TTTV" and complementary to crRNA in the sequence, providing sufficient binding space for the Cas12a enzyme / crRNA complex, and an RPA primer recognition sequence.

2. The dsDNA-antibody probe according to claim 1, characterized in that The dsDNA is 219 bp in length.

3. The method for preparing a dsDNA-antibody probe according to claim 1 or 2, characterized in that: The following steps are involved: (1) dialyzing 60 nmol of the antibody in phosphate buffered saline (PBS) at pH 7.4 at 4°C for 48 hours to obtain dialyzed antibody; (2) Antibody modified with N-succinimidyl-S-acetylthioacetate (SATA): Add 600 nmol of the prepared SATA solution to the dialyzed antibody, mix well, and react at room temperature for 30 minutes to obtain the SATA-modified antibody. Then transfer the SATA-modified antibody to an ultrafiltration tube, and repeatedly centrifuge the ultrafiltration tube to concentrate and remove impurities until the solution is 200 μL, and then add phosphate buffered saline to the liquid volume to 900 μL. (3) Modification of target dsDNA with bifunctional crosslinker 4-(N-maleimidomethyl)cyclohexane-1-carboxylic acid sulfonic acid succinimidyl ester sodium salt (Sulfo-SMCC): Add 600 nmol of the prepared Sulfo-SMCC solution to the prepared 60 nmol of the target dsDNA, mix well, and react at room temperature for 30 minutes to obtain SATA-modified dsDNA. Transfer the SATA-modified dsDNA to an ultrafiltration tube, centrifuge the ultrafiltration tube repeatedly, concentrate and remove impurities from the SATA-modified dsDNA, add phosphate buffered saline solution to the solution volume to 900 μL, and then temporarily store it at -20°C. (4) Add 100 μL of hydroxylamine hydrochloride solution to the concentrated and impurity-free antibody, mix well, and react at room temperature for 2 hours; (5) Add the Sulfo-SMCC-modified target dsDNA stored in step (3), wherein the molar concentration ratio of the Sulfo-SMCC-modified target dsDNA to the SATA-modified antibody is 1:1, mix well, and react at room temperature for 1 hour; (6) Concentrating and removing impurities in a centrifugal ultrafiltration tube until 250 μL of the solution remains, then taking out the liquid and replenishing the solution to 1 mL with PBS to obtain the initial product of the dsDNA-antibody probe; (7) Using molecular sieves to separate and purify the initial product of the dsDNA-antibody probe to obtain a purified dsDNA-antibody probe.

4. The method for preparing a dsDNA-antibody probe according to claim 3, characterized in that: The concentration and impurity removal process of step (2), step (3) and step (6) comprises: centrifuging the ultrafiltration tube at 6000 r / min and 4°C, taking out the ultrafiltration tube after every 10 minutes of centrifugation, and using a pipette to apply the liquid to the membranes on both sides, and repeatedly blowing and sucking 3-5 times.

5. The method for preparing a dsDNA-antibody probe according to claim 4, characterized in that: The hydroxylamine hydrochloride solution comprises the following preparation steps: a) Weigh 1.74 g of hydroxylamine hydrochloride and 0.365 g of disodium ethylenediaminetetraacetic acid (EDTA) and add them to 40 mL of PBS solution, mix well, and obtain a mixed solution; b) adding sodium hydroxide solution to the mixed solution to adjust the pH value of the mixed solution to 7.4, thereby obtaining a hydroxylamine hydrochloride solution.

6. The method for preparing a dsDNA-antibody probe according to claim 5, characterized in that: The antibody is a salbutamol monoclonal antibody, and the dsDNA is a double-stranded DNA with a length of 219 bp.

7. A CRISPR-immunity combined with small molecule detection method, characterized in that: The detection method is divided into three stages, each of which includes steps 1)-7), which respectively correspond to step A, step B and step C in step 3), specifically: 1) Coating: Dilute the salbutamol coating source to 2.56 pg / mL with coating buffer, add 10 μL / tube into a microcentrifuge tube (EP tube), and incubate in a 37°C constant temperature water bath for 3 hours; 2) Blocking: After taking out the EP tube, manually shake off the coating solution in the tube; add 200 μL of phosphate-buffered saline (PBST) to the EP tube, manually shake off the solution in the tube, and repeat the PBST to wash the EP tube twice; then add 20 μL of 6% skimmed milk powder blocking solution to the EP tube, place the EP tube in a 37°C constant temperature water bath and incubate for 1 hour, manually shake off the blocking solution in the EP tube, and dry at 37°C for 1 hour; 3) Add reagents: A. Use PBST to dilute the dsDNA-antibody probe, add 50μL PBST and 50μL diluted dsDNA-antibody probe to the sealed EP tube, and set a blank control (NTC) of 100μL PBST in the last row; B. Dilute the salbutamol drug with an initial concentration of 6 ng / mL with PBST, add 50 μL of the diluted salbutamol drug and 50 μL of the dsDNA-antibody probe with a dilution ratio of 1:16000 to the sealed EP tube, and set a blank control of 50 μL PBST and 50 μL dsDNA-antibody probe in the last row; C. Add 5 times the volume of PBST to dilute the pig urine sample, then add 50 μL of the diluted pig urine sample to the sealed EP tube, and then add 50 μL of the dsDNA-antibody probe diluted 16,000 times; 4) After adding the reagent, incubate the EP tube at 37°C for 40 minutes, manually spin dry the solution in the tube, add 200 μL of sterile ultrapure water to the EP tube and then spin dry the liquid in the tube manually, and repeatedly wash the EP tube with sterile ultrapure water for 5 times; 5) RPA amplification: add 10 μL RPA reaction system, add sterile ultrapure water to 10 μL, and incubate at 37°C for 25 min; 6) CRISPR / Cas12a detection system: add 10 μL RISPR / Cas12a detection system, add sterile ultrapure water to 20 μL, incubate at 37°C for 10 minutes, inactivate at 98°C for 2 minutes, and then use the instrument to collect the fluorescence signal; 7) Use software to process the collected fluorescence signals to obtain detection data.

8. The CRISPR-immunity combined with small molecule detection method according to claim 7, characterized in that: The RPA reaction system described in step 7) is as follows: forward and reverse primer concentrations are 0.5 μM each, dNTPs concentration is 2 mM, MgOAc concentration is 22.4 mM, 5 μL 2×Reaction Buffer, 1 μL 10×Basic E-mix and 0.5 μL 20×Core Reaction Mix.

9. The CRISPR-immunity combined with small molecule detection method according to claim 8, characterized in that: The CRISPR / Cas12a detection system described in step 8) is specifically Cas12a protein concentration 125nM, crRNA concentration 50nM, ssDNA-reporter concentration 1000nM, and 2μL NEBuffer 2.

1.

10. The CRISPR-immunity combined with small molecule detection method according to claim 9, characterized in that: In the step 3) step A, the dsDNA-antibody probe is diluted into 7 concentrations, namely 1000, 2000, 4000, 8000, 16000, 32000 and 64000 times; in step B, the salbutamol drug is diluted into 7 concentrations, namely 1 ng / mL, 0.17 ng / mL, 0.027 ng / mL, 0.0046 ng / mL, 0.7 pg / mL, 0.13 pg / mL and 0.021 pg / mL.

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