CRISPR (clustered regularly interspaced short palindromic repeats)-based homogeneous chemiluminescence kit as well as preparation method and use method thereof

Abstract

The invention discloses a CRISPR (clustered regularly interspaced short palindromic repeats)-based homogeneous chemiluminescence kit as well as a preparation method and a use method thereof, and belongs to the technical field of nucleic acid detection. The kit comprises a Cas enzyme, crRNA, a chemiluminescence reporter molecule, a free radical scavenger and hydrogen peroxide. The chemiluminescent reporter molecule comprises a reporter molecule modified with a chemical group for coupling, one end of the reporter molecule is connected with a peroxidase-like catalytic activity center for reacting with peroxide to generate active free radicals, and the other end of the reporter molecule is connected with a luminescent substrate for reacting with the active free radicals to generate luminescence. According to the kit, a multifunctional microplate reader can be used for collecting chemiluminescence signals for quantifying the concentration of target nucleic acid. The kit is simple in operation steps and short in detection time, does not need light source excitation, and avoids self-luminous interference of a biological sample matrix, so that the kit has more excellent sensitivity and detection dynamic range.

Description

Technical Field

[0001] The present invention belongs to the technical field of nucleic acid detection, and particularly relates to a homogeneous chemiluminescence kit based on CRISPR technology, and a preparation method and a usage method thereof. Background Technique

[0002] The CRISPR (Clustered regularly interspaced short palindromic repeats) detection technology is a highly sensitive and highly specific molecular diagnostic tool based on the CRISPR-Cas system, and is widely used in the fields of gene editing and nucleic acid detection. Its core principle is to use CRISPR-associated (Cas) proteins (such as Cas9, Cas12, Cas13, Cas14) to specifically recognize and cleave target DNA or RNA sequences under the guidance of guide RNA (crRNA). For example, while cleaving the target nucleic acid, Cas12 and Cas13 can also non-specifically cleave (trans-cleavage activity) surrounding single-stranded DNA or RNA reporter molecules, and this property is used to develop highly sensitive fluorescence or lateral flow detection methods, such as SHERLOCK (based on Cas13) and DETECTR (based on Cas12). Compared with traditional PCR amplification, the CRISPR technology has a shorter detection time, gets rid of complex instruments (PCR requires a temperature-varying system) and professional operators, so it has great potential and application prospects in the field of molecular diagnosis.

[0003] Traditional CRISPR reporter molecules adopt the principle of fluorescence resonance energy transfer (FRET), that is, a BHQ fluorescence quenching group is connected to the 5' of the reporter molecule nucleic acid strand, and a fluorescent group FAM is connected to the 3'. After the Cas-crRNA complex binds to the target sequence, the trans-cleavage activity is activated, cleaving the reporter molecule, the distance between the fluorescence quenching group and the fluorescent group becomes larger, and the fluorescence recovers. The fluorescence intensity is proportional to the concentration of the target sequence to be detected. However, fluorescence needs to be excited by a light source, there is self-luminescence interference, the detection sensitivity of the existing methods cannot meet the clinical needs, and the dynamic range of this method is relatively narrow. Therefore, increasing the sensitivity of the CRISPR detection method is a key problem to be solved.

[0004] The principle of homogeneous chemiluminescence technology is that the change in the spatial distance between the catalytic center and the luminescent substrate causes the change in the intensity of the chemiluminescence signal, so as to quantitatively detect the target molecule to be detected. When the spatial distance between the catalytic center and the luminescent substrate is close, the chemiluminescence signal is strong; when the distance between the two becomes far, the chemiluminescence signal weakens.

[0005] Based on this principle, the present invention modifies catalytic centers and luminescent substrates at both ends of the reported nucleic acid strand. The target nucleic acid sequence activates the Cas enzyme to cleave the reporter molecule, causing a change in spatial distance, thereby changing the chemiluminescence signal, and thus quantifying the target nucleic acid sequence. Compared with fluorescence, the chemiluminescence signal is triggered by a chemical reaction, without external light interference, avoiding photobleaching and interference from biological sample matrices. Moreover, the sensitivity of chemiluminescence is generally 10 1 -10 2 times higher than that of fluorescence and has a wider linear range. Therefore, the chemiluminescence excitation method of the present invention has the characteristics of being more economical, more efficient, and having more excellent detection performance. Summary of the Invention

[0006] Aiming at the deficiencies of traditional CRISPR detection technologies based on fluorescent reporter molecules, the present invention designs and provides a homogeneous chemiluminescence kit based on CRISPR technology, its preparation method and usage method. The method of the present invention uses chemiluminescence as the signal output method, with advantages such as no need for external light excitation, no need for washing steps, low instrument requirements, and high sensitivity.

[0007] To achieve the above technical objectives, the present invention adopts the following technical solutions: On the one hand, the present invention provides a homogeneous chemiluminescence kit based on CRISPR, which comprises a Cas enzyme, a crRNA, a chemiluminescent reporter molecule, a radical scavenger, and hydrogen peroxide; The Cas enzyme ensures the specificity of the detection. The Cas enzyme binds to the crRNA to form a Cas-crRNA complex, which is used to identify the target nucleic acid sequence and initiate non-specific cleavage activity, where the crRNA can be complementary paired with the target nucleic acid sequence; The chemiluminescent reporter molecule comprises a reporter molecule modified with a chemical group for coupling. A peroxidase-like catalytic activity center is connected to one end of the reporter molecule for reacting with peroxide to generate active free radicals, and a luminescent substrate is connected to the other end for reacting with the active free radicals to produce luminescence; The radical scavenger is used to improve the signal-to-noise ratio of the detection system; The hydrogen peroxide is a peroxidase-like catalytic substrate and serves as a chemiluminescence trigger; The CRISPR technology is one of the Cas12, Cas13, and Cas14 detection systems.

[0008] In the homogeneous chemiluminescence kit based on CRISPR, the chemical group is at least one of epoxy group, chloromethyl group, mercapto group, amino group, hydroxyl group, maleimide group, sulfonic acid group, carboxyl group, aldehyde group, biotin, dibenzocyclooctyne, or N-hydroxysuccinimide.

[0009] The described homogeneous chemiluminescence kit based on CRISPR, wherein the reporter molecule is at least one of single-stranded DNA, single-stranded RNA, DNA molecular beacon structure, DNA nanoflower structure, DNA hydrogel or peptide nucleic acid complex; The reporter molecule contains a characteristic base sequence for trans-cleavage by Cas enzyme.

[0010] The described homogeneous chemiluminescence kit based on CRISPR, wherein the peroxidase-like catalytic activity center is at least one of natural peroxidase, metal complex, metal nanoparticle, metal oxide nanoparticle, carbon-based nanomaterial, heme and its derivatives, amino acid or peptide, metal-organic framework, polymer composite, molybdenum compound and its derivatives; Preferably, the metal complex is at least one of iron complex, manganese complex or cobalt complex; Preferably, the metal nanoparticle is at least one of gold nanoparticle, silver nanoparticle or platinum nanoparticle; Preferably, the metal oxide nanoparticle is magnetite nanoparticle or cerium dioxide nanoparticle; Preferably, the carbon-based nanomaterial is carbon nanotube or graphene; The luminescent substrate is at least one of luminol and its analogs, lucigenin, peroxyoxalate, fluorescein, imidazole compounds, polycyclic aromatic hydrocarbons, acridine compounds, luciferase substrates, chlorophyll derivatives, quantum dots, carbon dots or nano-fluorescent rare earth complexes.

[0011] The described homogeneous chemiluminescence kit based on CRISPR, wherein the radical scavenger is at least one of vitamins, glutathione, tert-butanol, superoxide dismutase, tryptophan, phenolic compounds, uric acid, bilirubin, creatinine or glucuronic acid; Preferably, the vitamin is ascorbic acid and its derivatives, vitamin E or vitamin A.

[0012] In a second aspect, the present invention provides a preparation method of the homogeneous chemiluminescence kit based on CRISPR according to any one of the above, comprising the following steps: (1) Connecting the peroxidase-like catalytic activity center and the luminescent substrate to both sides of the reporter molecule modified with chemical groups respectively by chemical coupling method to obtain a chemiluminescent reporter molecule; (2) Formulating Cas enzyme, crRNA, radical scavenger and hydrogen peroxide to obtain the kit.

[0013] In the described preparation method, the chemical coupling method is at least one of diazo method, glutaraldehyde method, glutaric anhydride method, carbodiimide method, 1,3-dipolar cycloaddition reaction or biotin-streptavidin method.

[0014] In a third aspect, the present invention provides a method for using a homogeneous chemiluminescence kit based on CRISPR according to any one of the above, comprising the following steps: (i) Co-incubating Cas enzyme and crRNA to form a Cas-crRNA complex; (ii) Adding the Cas-crRNA complex and the chemiluminescent reporter molecule to the sample to be tested for co-incubation reaction; When the target nucleic acid sequence exists in the sample to be tested, the target nucleic acid sequence is complementary paired with the crRNA sequence, activating the activity of the Cas enzyme, cleaving the chemiluminescent reporter molecule, making the spatial distance between the catalytic center and the luminescent substrate larger, and the chemiluminescence intensity weakens; when the target nucleic acid sequence does not exist in the sample to be tested, the chemiluminescent reporter molecule is not cleaved, the spatial distance between the catalytic center and the luminescent substrate remains unchanged, and the chemiluminescence intensity remains unchanged; (iii) After the co-incubation reaction is completed, adding an aqueous solution of a radical scavenger; (iv) Diluting hydrogen peroxide with a carbonate buffer solution, and injecting the diluted hydrogen peroxide using a multi-functional microplate reader with a syringe to trigger the chemiluminescent signal, and the chemiluminescent signal intensity is inversely proportional to the concentration of the nucleic acid to be tested.

[0015] In the described method for use, the concentration of the aqueous solution of the radical scavenger is 0.005 - 10 mM; The concentration of the diluted hydrogen peroxide is 1 - 100 mM; The molar ratio of the crRNA to the Cas enzyme is (1 - 2):1; The conditions for the co-incubation reaction in steps (i) and (ii) are: temperature 25 - 37°C, time 10 - 60 minutes; The volume ratio of the Cas-crRNA complex, the chemiluminescent reporter molecule, the aqueous solution of the radical scavenger to the diluted hydrogen peroxide is (30 - 45):5:(1 - 10):(20 - 500).

[0016] In a fourth aspect, the present invention provides the application of the homogeneous chemiluminescence kit according to any one of the above in the detection and quantification of target genes in samples; The sample is blood, plasma, serum, urine, semen, saliva, sweat, cell culture, tissue extract, solvent, swab sample, seawater, industrial water sample, food sample, environmental sample, plant material, eukaryotic cell, bacterium, plasmid, virus, fungus or a cell from prokaryotes; Preferably, the blood is anticoagulated blood; Preferably, the environmental sample is soil or water.

[0017] Compared with the prior art, the present invention has the following beneficial effects: The kit of the present invention can use a multifunctional microplate reader to collect chemiluminescence signals for quantifying the concentration of target nucleic acids. Compared with traditional PCR quantification techniques, the operation steps of this kit are simple and the detection time is short; compared with existing methods based on fluorescent reporter molecules, this method does not require light source excitation, avoiding interference from the autofluorescence of biological samples, and thus has better sensitivity and detection dynamic range. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 is a schematic diagram of the homogeneous chemiluminescence kit based on the CRISPR technology of the present invention; Figure 2 is the standard curve of the homogeneous chemiluminescence kit for detecting HPV16; Figure 3 is the result of detecting chemiluminescence signals in Example 3; Figure 4 is the standard curve of the homogeneous chemiluminescence kit for detecting BRCA-1; Figure 5 is the standard curve of the homogeneous chemiluminescence kit for detecting ORF1ab in SARS-CoV-2. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0019] In order to make the present invention more clear and understandable, the following further describes the present invention in detail in conjunction with embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0020] It should be noted that the term "target nucleic acid sequence" as used in the present invention refers to a mixture to be tested containing or suspected of containing a target nucleic acid sequence. The sample to be tested can be diluted with a diluent according to needs before use.

[0021] Example 1: Preparation of the Kit of the Present Invention The working principle of the homogeneous chemiluminescence kit based on the CRISPR technology of the present invention is as Figure 1As shown, the kit includes: Cas enzyme, crRNA, chemiluminescent reporter molecule, ascorbic acid, hydrogen peroxide; wherein the Cas enzyme can ensure the specificity of detection, bind to the crRNA complementary to the target nucleic acid strand to form a Cas-crRNA complex, which is used to identify the target nucleic acid strand and initiate non-specific cleavage activity; the chemiluminescent reporter molecule is the substrate cleaved by the activated Cas-crRNA complex, wherein one end of the reporter molecule is connected to the catalytic center of peroxidase-like enzyme, which can react with peroxide to generate active free radicals, and the other end is connected to the luminescent substrate, which reacts with the active free radicals to produce luminescence; the radical scavenger is used to improve the signal-to-noise ratio of the detection system; hydrogen peroxide is used for the peroxidase-like enzyme to catalyze the substrate and serves as a chemiluminescence trigger.

[0022] The chemiluminescent reporter molecule contains a reporter molecule, a catalytic center of peroxidase-like enzyme and a luminescent substrate. Among them, the catalytic center of peroxidase-like enzyme is a G-quadruplex with hemin as the catalytic center; the luminescent substrate is a luminol analogue ABEI. The G-quadruplex is a DNA sequence that can form a tetrahedral structure in the presence of potassium ions.

[0023] Hemin can firmly bind to the G-quadruplex, and then form a catalytic center with peroxidase-like enzyme activity. ABEI is a luminol analogue with an amino group. Under alkaline conditions, it can be oxidized to the excited state by active free radicals and emit blue light (425 nm) when returning to the ground state. This system uses the Cas12a trans-cleavage sequence (TTATT) n Design a single-stranded reporter molecule, modify dibenzocyclooctyne (DBCO) at its 5' end, and the 3' end is a G-quadruplex DNA sequence. Connect ABEI to the reporter molecule with N-hydroxysuccinimide-polyethylene glycol-azide (NHS-PEG4-N3) as the linker. The modified chemiluminescent reporter molecule can emit strong blue light under the trigger of hydrogen peroxide. When the analyte is present, Cas12a-crRNA is activated and cleaves the chemiluminescent reporter molecule, and the chemiluminescence signal intensity decreases.

[0024] Among them, the molecular formula of ABEI is shown in the following formula (Ⅰ):

[0025] Formula (Ⅰ).

[0026] The specific implementation steps for preparing the above chemiluminescent reporter molecule are as follows: Dissolve the report single-stranded DNA (ssDNA) powder in DEPC-treated water to a concentration of 100 μM. Take 10 μL of 100 μM of the reported ssDNA and mix it with 190 μL of boric acid buffer (50 mM, pH 8.5) containing 10 mM KCl. Subsequently, heat the mixture at 95 °C for 5 minutes and then anneal it to room temperature to form a G-quadruplex structure. Then, incubate 100 μL of 10 μM hemin with the reported ssDNA solution at room temperature for 40 minutes. To conjugate ABEI to ssDNA, the amino group on ABEI is linked to NHS-PEG4-N3, and the N3 group reacts with the DBCO located at the 5'-end of ssDNA. Specifically, add 7.2 μL of 2 mg / mL ABEI and 2 μL of 2 mg / mL NHS-PEG4-N3 to the ssDNA solution that has formed a G-quadruplex and incubate at 37 °C for 1 hour. Finally, dialyze the functionalized ssDNA in DEPC water to remove unreacted free molecules.

[0027] Example 2: (1) Using the homogeneous chemiluminescence kit prepared in Example 1 above, perform the CRISPR reaction to test the analyte: Take HPV16 as the model analyte and dilute it to gradient concentrations of 10 aM, 100 aM, 1 fM, 10 fM, 100 fM, 1 pM, 10 pM, 100 pM, 500 pM, 1 nM, 10 nM. The CRISPR reaction is carried out in a 96-well white microplate. Incubate 20 μL of Cas12a (100 nM) and 10 μL of crRNA (300 nM) at 37 °C for 10 minutes. Then, further incubate 5 μL of the chemiluminescent reporter molecule (1.5 μM), 5 μL of the analyte with the Cas12a-crRNA complex at 37 °C for 30 minutes. Subsequently, add 8 μL of ascorbic acid AA (0.005 mM) and 50 μL of 5 mM hydrogen peroxide to the mixture in sequence, and collect the chemiluminescent signal to quantify the target HPV16. The reaction buffer is 10 mM Tris-HCl, pH 7.9 (containing 15 mM MgCl2, 100 μg / mL BSA, 50 mM NaCl). The nucleic acid sequences involved are shown in Table 1 below.

[0028] Table 1 Nucleic acid sequences in Examples 1 and 2

[0029] (2) Plotting the standard curve for the detection of HPV16: Taking the obtained chemiluminescence intensity as the ordinate and the HPV16 concentration (pM) as the abscissa, a standard curve was plotted, and a non-linear equation was calculated. The non-linear standard curve is as shown in the following formula (1): Formula (1); Non-linear fitting R² = 0.9946, and the standard curve is as Figure 2 shown. The lowest detection limit of this method was defined as the concentration when the chemiluminescence signal was inhibited by 90%. The detection limit calculated by this method was 41.6 fM.

[0030] Example 3: Using the homogeneous chemiluminescence kit prepared in Example 1 above, a CRISPR reaction was carried out. The samples were cervical swab samples from clinical women, a total of 17 (7 negative samples and 10 positive samples): (1) The cervical swab samples were centrifuged in a centrifuge (13000 rpm, 10 minutes) to collect suspended cells, and then the cells were lysed using a commercial DNA extraction kit to release the HPV virus; (2) Using a commercial RPA amplification kit, the samples were amplified for 10 minutes according to the kit instructions; (3) 20 μL of Cas12a (100 nM) and 10 μL of crRNA (300 nM) were incubated at 37°C for 10 minutes. Then, 5 μL of the chemiluminescence reporter molecule (1.5 μM), 10 μL of the amplified sample were further incubated with the Cas12a-crRNA complex at 37°C for 30 minutes. Subsequently, 8 μL of ascorbic acid (0.005 mM) and 50 μL of 5 mM hydrogen peroxide were added to the mixture in sequence, and the chemiluminescence signal was collected to determine the negative / positive of the sample (positive samples had low signals and negative samples had high signals). The reaction buffer was 10 mM Tris-HCl, pH 7.9 (containing 15 mM MgCl2, 100 μg / mL BSA, 50 mM NaCl).

[0031] (4) As Figure 3 shown, the critical value for distinguishing negative and positive in this method was determined according to the detection signal (the dotted line signal value in the figure was 16171 a.u.).

[0032] Example 4: The working principle of the homogeneous chemiluminescence kit based on the CRISPR technology of the present invention is as Figure 1As shown in the figure, the kit includes: Cas enzyme, crRNA, chemiluminescent reporter molecule, ascorbic acid radical scavenger, hydrogen peroxide; among them, the Cas enzyme can ensure the specificity of detection, bind to the crRNA complementary to the target nucleic acid strand to form a Cas-crRNA complex, which is used to identify the target nucleic acid strand and initiate non-specific cleavage activity; the chemiluminescent reporter molecule is the substrate cleaved by the activated Cas-crRNA complex, where one end of the reporter molecule is connected to the catalytic active center of peroxidase-like enzyme, which can react with peroxide to generate active free radicals, and the other end is connected to the luminescent substrate, which reacts with the active free radicals to produce luminescence; the radical scavenger is used to improve the signal-to-noise ratio of the detection system; hydrogen peroxide is used for the peroxidase-like enzyme to catalyze the substrate and act as a chemiluminescence trigger agent.

[0033] The chemiluminescent reporter molecule contains a reporter molecule ssDNA, a peroxidase-like enzyme catalytic active center, and a luminescent substrate. Among them, the peroxidase-like enzyme catalytic active center is horseradish peroxidase (HRP); the luminescent substrate is a nano-fluorescent rare earth complex (a europium (Eu) complex embedded in carboxylated (-COOH) polystyrene). The 5' end of the reporter molecule ssDNA is modified with an amino group (-NH2), and the 3' end is modified with biotin. Streptavidin-conjugated HRP (HPP-SA) binds to biotin, thereby modifying the peroxidase catalytic active center on the reporter molecule. The luminescent substrate is connected to the reporter molecule by the carbodiimide method. The modified chemiluminescent reporter molecule can emit strong red light (615 nm) under the trigger of hydrogen peroxide. When the analyte is present, Cas12a-crRNA is activated and cleaves the chemiluminescent reporter molecule, and the chemiluminescence signal intensity weakens.

[0034] The preparation process of the above chemiluminescent reporter molecule is specifically as follows: (1) Preparation of polystyrene microspheres embedded with EuⅢ (EuPS) The swelling method is used to prepare EuPS, and the specific steps are as follows: First, dissolve 5 mg of Eu³⁺ in 3 mL of propylene glycol methyl ether solvent, and preheat the solution to 80°C. Then, add 1 mL of carboxylated polystyrene microspheres (particle size 100 nm, concentration 100 mg / mL) to it, and maintain the reaction for 30 minutes. Subsequently, immediately stop the heating operation and cool the solution to room temperature. Then, wash the synthesized EuPS three times with deionized water and perform centrifugation at 13500 rpm for 15 minutes to separate the product. Subsequently, redisperse the obtained residue in 4 mL of deionized water. Finally, store the prepared EuPS solution in a dark environment at 4°C for subsequent use.

[0035] (2) Preparation of chemiluminescent reporter molecule Dissolve the reported ssDNA powder in DEPC-treated water to a concentration of 100 μM. Connect EuPS to ssDNA using the one-step EDC method. Pipette 10 μL of EuPS and dissolve it in 1 mL of PBS buffer (0.01 M, pH = 7.4), add 10 μM ssDNA, and perform electrostatic adsorption for 30 min. Subsequently, add 5 μg of EDC and stir for 30 min, repeating this process 3 times; then add 100 μL of mgBSA (10 mg / mL), perform electrostatic adsorption for 30 min, and then add 5 μg of EDC and react for 30 min; add 5 μg of EDC again and react for 30 min, then centrifuge, and redissolve the precipitate in 100 μL of the reconstitution solution. The composition of the reconstitution solution: 25% sucrose, 0.1% sodium azide, 1% BSA, and 1% Tween 20 in 0.01 M PBS buffer (pH = 7.4). Finally, add 280 μL of HRP-SA with a concentration of 100 μg / mL to the reaction solution and react at room temperature for 30 minutes.

[0036] Example 5: (1) Use the homogeneous chemiluminescence kit prepared in Example 3 above to perform the CRISPR reaction and test the test substance: Take the breast cancer susceptibility gene (BRCA-1) as the model test substance and dilute it to gradient concentrations of 10 aM, 100 aM, 1 fM, 10 fM, 100 fM, 1 pM, 10 pM, 100 pM, 500 pM, 1 nM, and 10 nM. The CRISPR reaction is carried out in a 96-well white microplate. Incubate 30 μL of Cas12a (100 nM) and 15 μL of crRNA (300 nM) at 37 °C for 30 minutes. Then, further incubate 5 μL of the chemiluminescent reporter molecule (1.5 μM), 5 μL of the test substance, and the Cas12a-crRNA complex at 37 °C for 30 minutes. Subsequently, sequentially add 5 μL of ascorbic acid AA (0.01 mM) and 50 μL of 20 mM hydrogen peroxide to the mixture, and collect the chemiluminescent signal to quantify the target BRCA-1. The reaction buffer is commercial 10 mM Tris-HCl, pH 7.9 (containing 15 mM MgCl2, 100 μg / mL BSA, 50 mM NaCl). The nucleic acid sequences involved in Examples 4 and 5 are shown in Table 2 below: Table 2 Nucleic acid sequences in Examples 4 and 5

[0037] (2) Plot the standard curve for detecting BRCA-1 Taking the obtained chemiluminescence intensity as the ordinate and the BRCA-1 concentration (pM) as the abscissa, a standard curve was plotted, and a non-linear equation was calculated. The non-linear standard curve is shown in the following formula (2): Formula (2); Non-linear fitting R² = 0.9971, and the standard curve is as Figure 4 shown. The lowest detection limit of this method was defined as the concentration when the signal was at 90%, and the detection limit calculated by this method was 18.6 fM.

[0038] Example 6: The working principle of the homogeneous chemiluminescence kit based on the CRISPR technology of the present invention is as Figure 1 shown. The kit includes: Cas enzyme, crRNA, chemiluminescent reporter molecule, ascorbic acid free radical scavenger, hydrogen peroxide; wherein the Cas enzyme can ensure the specificity of detection, and binds with the crRNA complementary to the target nucleic acid strand to form a Cas-crRNA complex, which is used to identify the target nucleic acid strand and initiate non-specific cleavage activity; the chemiluminescent reporter molecule is the substrate cleaved by the activated Cas-crRNA complex, one end of its reporter molecule is connected with the catalytic activity center of peroxidase-like enzyme, which can react with peroxide to generate active free radicals, and the other end is connected with a luminescent substrate, which reacts with the active free radicals to produce luminescence; the free radical scavenger is used to improve the signal-to-noise ratio of the detection system; hydrogen peroxide is used for the peroxidase-like enzyme to catalyze the substrate and serves as a chemiluminescence trigger.

[0039] The chemiluminescent reporter molecule contains a reporter molecule, a peroxidase-like enzyme catalytic activity center and a luminescent substrate. Among them, AuPtCo nanoparticles serve as the peroxidase-like enzyme catalytic activity center; acridinium ester serves as the luminescent substrate. AuPtCo nanomaterials have peroxidase-like enzyme activity, and Au on the surface of the nanomaterials can be connected with the reporter molecule through Au-S bonds. Acridinium ester has a variety of commercially mature modifiers. In this case, acridinium ester with NHS group is selected, which can be coupled with the amino-modified reporter molecule. The modified reporter molecule can produce a strong chemiluminescent signal under the trigger of hydrogen peroxide. When the analyte is present, Cas13a-crRNA is activated and cleaves the chemiluminescent reporter molecule, and the chemiluminescent signal intensity decreases. Among them, the molecular formula of acridinium ester is shown in the following formula (Ⅱ):

[0040] Formula (Ⅱ).

[0041] The preparation process of the above chemiluminescent reporter molecule is specifically as follows: (1) Synthesis of AuPtCo nanoparticles First, dissolve 30 mg of Pluronic F-127 in 4 mL of deionized water and sonicate it until completely dissolved. Subsequently, add 0.5 mL of chloroauric acid (HAuCl4, 10 mM), 2 mL of chloroplatinic acid (H2PtCl6, 10 mM), and 2 mL of cobalt chloride (CoCl2, 10 mM) in sequence. At this time, the solution turns light yellow. Then, add 2 mL of citric acid (100 mM), slowly heat the mixed solution to 65 °C, and continuously stir for 5 hours. Centrifuge at 12,000 rpm for 10 minutes, wash three times with ultrapure water, and redissolve it in water for standby.

[0042] (2) Preparation of chemiluminescent reporter molecule Dissolve the SH-RNA-NH2 single-stranded powder in DEPC-treated water to a concentration of 100 μM. Take 5 μL of 1M tris(2-carboxyethyl)phosphine (TCEP) and react with 200 μL of 15 μM thiol-modified RNA at room temperature for 30 minutes. Then, mix 200 μL of the reduced SH-RNA-NH2 with 700 μL of AuPtCo nanoparticles, add 100 μL of citric acid (0.1M, pH 3.0), and react at room temperature for 3 minutes. Then, adjust the pH to 8.2, add 5 μL of 100 μM acridinium ester, and react at 37 °C for 1 hour. After the reaction, dialyze in RNase-free ultrapure water to remove free small molecules.

[0043] Example 7: (1) Use the homogeneous chemiluminescent kit prepared in Example 5 above to perform the CRISPR reaction and test the sample to be detected: Using the ORF1ab RNA in SARS-CoV-2 as the pattern analyte, and diluting it to gradient concentrations of 10 aM, 100 aM, 1 fM, 10 fM, 100 fM, 1 pM, 10 pM, 100 pM, 500 pM, 1 nM, and 10 nM. The CRISPR reaction was carried out in a 96-well white microplate. Incubate 20 μL of Cas13a (100 nM) and 10 μL of crRNA (300 nM) at 37 °C for 20 minutes. Then, further incubate 5 μL of the chemiluminescent reporter molecule (1.5 μM), 5 μL of the analyte with the Cas13a-crRNA complex at 37 °C for 30 minutes. Subsequently, add 10 μL of ascorbic acid AA (0.01 mM) and 75 μL of 50 mM hydrogen peroxide to the mixture in sequence, and collect the chemiluminescent signal to quantify the target ORF1ab RNA. The reaction buffer was commercial 10 mM Tris-HCl, pH 7.9 (containing 15 mM MgCl2, 100 μg / mL BSA, 50 mM NaCl). The nucleic acid sequences involved in Examples 6 and 7 are shown in Table 3 below: Table 3 Nucleic Acid Sequences in Examples 6 and 7

[0044] (2) Plotting the standard curve for detecting SARS-CoV-2 Taking the obtained chemiluminescent intensity as the ordinate and the ORF1ab concentration (pM) as the abscissa to plot the standard curve and calculate the non-linear equation. The non-linear standard curve is shown in the following formula (3):

[0045] Formula (3); Non-linear fitting R² = 0.9794, and the standard curve is as Figure 5 shown. The lowest detection limit of this method was defined as the concentration when the signal was at 90%, and the detection limit calculated by this method was 18.3 fM.

[0046] Those of ordinary skill in the art can understand that the above embodiments are specific examples for implementing the present application. In actual applications, various changes can be made in form and details without departing from the spirit and scope of the present application. Any person skilled in the art can make their own changes and modifications without departing from the spirit and scope of the present application. Therefore, the protection scope of the present application should be subject to the scope defined by the claims.

Claims

1. A CRISPR-based homogeneous chemiluminescence kit, characterized in that, The kit contains a Cas enzyme, crRNA, a chemiluminescent reporter molecule, a radical scavenger, and hydrogen peroxide; The Cas enzyme binds to crRNA to form a Cas-crRNA complex, which is used to recognize the target nucleic acid sequence and initiate non-specific cleavage activity, where crRNA can be complementary paired with the target nucleic acid sequence; The chemiluminescent reporter molecule contains a reporter molecule modified with a chemical group for coupling. At one end of the reporter molecule, a peroxidase-like catalytic activity center is connected, which is used to react with peroxide to generate reactive radicals, and at the other end, a luminescent substrate is connected, which is used to react with the reactive radicals to produce luminescence.

2. The homogeneous chemiluminescence kit based on CRISPR according to claim 1, wherein, The chemical group is at least one of epoxy group, chloromethyl group, mercapto group, amino group, hydroxyl group, maleimide group, sulfonic acid group, carboxyl group, aldehyde group, biotin, dibenzocyclooctyne or N-hydroxysuccinimide.

3. The homogeneous chemiluminescence kit based on CRISPR according to claim 1, wherein The reporter molecule is at least one of single-stranded DNA, single-stranded RNA, DNA molecular beacon structure, DNA nanoflower structure, DNA hydrogel or peptide nucleic acid complex; The reporter molecule contains a characteristic base sequence for trans-cleavage by the Cas enzyme.

4. A CRISPR-based homogeneous chemiluminescence kit according to claim 1, wherein, The peroxidase-like catalytic activity center is at least one of natural peroxidase, metal complex, metal nanoparticle, metal oxide nanoparticle, carbon-based nanomaterial, heme and its derivatives, amino acid or peptide, metal-organic framework, polymer composite material, molybdenum compound and its derivatives; Preferably, the metal complex is at least one of iron complex, manganese complex or cobalt complex; Preferably, the metal nanoparticle is at least one of gold nanoparticle, silver nanoparticle or platinum nanoparticle; Preferably, the metal oxide nanoparticle is magnetite nanoparticle or cerium dioxide nanoparticle; Preferably, the carbon-based nanomaterial is carbon nanotube or graphene; The luminescent substrate is at least one of luminol and its analogs, lucigenin, peroxyoxalate, fluorescein, imidazole compounds, polycyclic aromatic hydrocarbons, acridine compounds, luciferase substrates, chlorophyll derivatives, quantum dots, carbon dots or nano-fluorescent rare earth complexes; 5. A CRISPR-based homogeneous chemiluminescence kit according to claim 1, characterized in that, The radical scavenger is at least one of vitamins, glutathione, tert-butanol, superoxide dismutase, tryptophan, phenolic compounds, uric acid, bilirubin, creatinine or glucuronic acid; Preferably, the vitamin is ascorbic acid and its derivatives, vitamin E or vitamin A.

6. The preparation method of a CRISPR-based homogeneous chemiluminescence kit according to any one of claims 1-5, characterized in that, It includes the following steps: (1) Use a chemical coupling method to connect a peroxidase-like catalytic activity center and a luminescent substrate on both sides of a reporter molecule modified with a chemical group respectively to obtain a chemiluminescent reporter molecule; (2) Prepare a Cas enzyme, crRNA, a radical scavenger and hydrogen peroxide for use.

7. The preparation method according to claim 6, characterized in that, The chemical coupling method is at least one of diazo method, glutaraldehyde method, glutaric anhydride method, carbodiimide method, 1,3-dipolar cycloaddition reaction or biotin-streptavidin method.

8. The method for using a CRISPR-based homogeneous chemiluminescence kit according to any one of claims 1-5, characterized in that, It includes the following steps: (ⅰ) Co-incubate the Cas enzyme and crRNA to form a Cas-crRNA complex; (ii) Add the Cas-crRNA complex and the chemiluminescent reporter molecule to the sample to be tested for co-incubation reaction; When the target nucleic acid sequence exists in the sample to be tested, the target nucleic acid sequence is complementary paired with the crRNA sequence, activating the Cas enzyme activity, cleaving the chemiluminescent reporter molecule, making the spatial distance between the catalytic center and the luminescent substrate larger, and the chemiluminescent intensity weakens; when the target nucleic acid sequence does not exist in the sample to be tested, the chemiluminescent reporter molecule is not cleaved, the spatial distance between the catalytic center and the luminescent substrate remains unchanged, and the chemiluminescent intensity remains unchanged; (iii) After the co-incubation reaction is completed, add an aqueous solution of a radical scavenger; (iv) Dilute hydrogen peroxide with carbonate buffer solution, and use a multi-functional microplate reader with a syringe to inject the diluted hydrogen peroxide to trigger the chemiluminescent signal, and the chemiluminescent signal intensity is inversely proportional to the concentration of the nucleic acid to be tested.

9. The method of use according to claim 8, characterized in that, The concentration of the aqueous solution of the radical scavenger is 0.005 - 10 mM; The concentration of the diluted hydrogen peroxide is 1 - 100 mM; The molar ratio of the crRNA to the Cas enzyme is (1 - 2):1; The conditions for the co-incubation reaction in steps (i) and (ii) are: temperature 25 - 37 °C, time 10 - 60 minutes; The volume ratio of the Cas-crRNA complex, the chemiluminescent reporter molecule, the aqueous solution of the radical scavenger to the diluted hydrogen peroxide is (30 - 45):5:(1 - 10):(20 - 500).

10. The application of the CRISPR-based homogeneous chemiluminescent kit according to any one of claims 1 - 5 in the detection and quantification of target genes in a sample; The sample is blood, plasma, serum, urine, semen, saliva, sweat, cell culture, tissue extract, solvent, swab sample, seawater, industrial water sample, food sample, environmental sample, plant material, eukaryotic cell, bacterium, plasmid, virus, fungus or a cell from a prokaryote; Preferably, the blood is anticoagulated blood; Preferably, the environmental sample is soil or water.

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