Detection reagent, kit and detection method for detecting AD target Micro RNA (Ribonucleic Acid) by CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) technology

Abstract

The invention belongs to the technical field of in-vitro detection, and particularly relates to a detection reagent, a kit and a detection method for detecting AD target Micro RNA (Ribonucleic Acid) by a CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) technology. According to the present invention, the AD disease detection target Micro RNA and the primer and other nucleic acid sequences are screened, the concentration of each component in the detection system is preferably selected, and the detection reagent and the kit for detecting the target Micro RNA through the CRISPR-circit technology are provided; according to the present invention, the CRISPR-circit detection system is delivered by using the liposome, and the CRISPR-circit technology and the membrane fusion technology are combined so as to achieve the in-situ detection of the exosome MicroRNA, such that the method has advantages of high sensitivity and high specificity, and can accurately and rapidly detect the target MicroRNA in the AD patient serum in situ. The invention provides a rapid and efficient method for detecting the Alzheimer's disease, and the application prospect is good.

Description

Technical Field

[0001] The present invention belongs to the field of in vitro detection technology, and specifically relates to a detection reagent, a kit and a detection method for detecting AD target Micro RNA using CRISPR technology. Background Art

[0002] Alzheimer's disease is a serious neurodegenerative disease that affects the cognition and memory of the elderly. The number of patients worldwide continues to rise, and is expected to reach 152 million in 2050. Because the pathogenesis is unknown and there is no cure, early diagnosis and intervention are crucial. However, the current neuroimaging method of measuring Aβ deposits through "carbon 11" labeling is limited by the half-life of "carbon 11", and has the disadvantages of relying on PET equipment, high price, complex operation, difficulty in accurately detecting early lesions, subjective image interpretation, and unsuitable for specific populations; although the levels of β-amyloid protein and tau protein in cerebrospinal fluid are recognized biomarkers, lumbar puncture to collect cerebrospinal fluid has high requirements for operators, and patients not only suffer great physical pain and psychological burden, but also have the risk of complications, so it is not suitable for routine screening.

[0003] Studies have shown that MicroRNA is secreted into the blood by source cells and is closely related to the development of the disease. Therefore, monitoring MicroRNA in peripheral blood is an effective solution for screening and long-term monitoring of AD patients. In AD diseases, MicroRNA-135a, MicroRNA-384, and MicroRNA-193b are significantly differentially expressed and are potential biomarkers. However, peripheral blood MicroRNA is easily degraded and difficult to be accurately detected. Exosomes (EVs, Extracellular vesicles) are small membrane vesicles whose closed-loop structure and phospholipid membrane can protect MicroRNA and make it stable.

[0004] The current method of nucleic acid amplification for detecting MicroRNA requires the lysis of EVs, which carries the risk of molecular loss. The composition and properties of liposomes are similar to those of EVs. When they are close together, they can spontaneously fuse membranes based on intermolecular forces such as electrostatic attraction, encapsulate the nucleic acid detection system, and detect MicroRNA in EVs in situ. However, PCR amplification experiments are complex, rely on precision instruments, and require repeated temperature changes, so they are not applicable; constant isothermal amplification has the problem of nonspecific amplification. However, constant isothermal amplification combined with the CRISPR system with precise recognition and efficient cutting capabilities can make the detection method simple to operate, with low requirements for personnel and equipment, high sensitivity, and good specificity. This detection method is expected to achieve large-scale screening and long-term monitoring of treatment of AD patients.

[0005] Therefore, developing a membrane fusion / CRISPR detection method to achieve in situ detection of microRNA in EVs in the peripheral blood of AD patients without the need to lyse them is a challenge in this field. Summary of the invention

[0006] In view of the above problems, the present invention provides a membrane fusion / CRISPR-circuit detection method, which aims to achieve in situ detection of MicroRNA in peripheral blood EVs of AD patients without lysis.

[0007] The present invention provides a detection reagent for detecting AD target Micro RNA using CRISPR technology, which comprises the following components: Conversion primer 0.025-25nM, Self-catalytic primer 0.025-25pM, dNTP 1-4 nM, crRNA 5-40nM, CRISPR / Cas endonuclease 20-160nM, RNA reporter 0.25-1 μM, Bst II DNA polymerase 0.625-5U / mL, T7 RNA polymerase 0.02-0.16U / μL.

[0008] Preferably, the concentration of the conversion primer is 2.5nM; and / or, the concentration of the autocatalytic primer is 0.25pM; and / or, the concentration of crRNA is 20nM; and / or, the concentration of Cas13a is 40nM; and / or, the concentration of Bst II DNA polymerase is 1.25U / mL; and / or, the concentration of T7 RNA polymerase is 0.08U / μL; and / or, the CRISPR / Cas nuclease is selected from Cas13a, Cas12a.

[0009] Preferably, the conversion primer and the autocatalytic primer combination comprises at least one of the following combinations: The nucleotide sequence of the conversion primer is SEQ ID NO. 2, and the nucleotide sequence of the autocatalytic primer is SEQ ID NO. 3; Or, the nucleotide sequences of the conversion primer and the autocatalytic primer are both SEQ ID NO. 6; Alternatively, the nucleotide sequences of the conversion primer and the autocatalytic primer are both SEQ ID NO. 8.

[0010] Preferably, the nucleotide sequence of crRNA is SEQ ID NO. 4; and / or, the nucleotide sequence of the RNA reporter molecule is UUUUUU.

[0011] Preferably, both ends of the RNA reporter molecule are modified with a fluorescent group and a fluorescent quenching group respectively; And / or, the detection reagent further comprises a recombinant RNase inhibitor at a concentration of 5-10 U / μL; And / or, the detection reagent also includes a buffer with a volume fraction of 10-40%, the components of the buffer include 10-40 mM Tris-HCl, 20-100 mM KCl, 5-20 mM magnesium sulfate, 0.05-0.5% mass volume ratio of PEG-8000, 0.5-2 mM dithiothreitol, 0.5-2 mg / mI BSA, and the pH of Tris-HCl is 7-8.5.

[0012] The present invention provides use of a detection reagent for detecting AD target Micro RNA using any of the above-mentioned CRISPR technologies in the preparation of a MicroRNA detection kit in the serum of patients with Alzheimer's disease or cancer.

[0013] The present invention provides a kit for detecting AD target Micro RNA using CRISPR technology, comprising any of the above-mentioned detection reagents for detecting AD target Micro RNA using CRISPR technology, or a lipid sphere containing any of the above-mentioned detection reagents for detecting AD target Micro RNA using CRISPR technology; In the lipid sphere containing the detection reagent for detecting AD target Micro RNA using CRISPR technology, the usage ratio of the detection reagent for detecting AD target Micro RNA using CRISPR technology to the lipid sphere is 1-2 mL: 23-46 mg.

[0014] Preferably, the lipid sphere containing the detection reagent for detecting AD target Micro RNA using CRISPR technology is prepared according to the following steps: mixing the detection reagent for detecting AD target Micro RNA using CRISPR technology with the lipid sphere.

[0015] Preferably, the lipid sphere is prepared according to the following steps: mixing cationic lipid molecules, amphiphilic phospholipid molecules and cholesterol, rotary evaporating to obtain a lipid film, and then incubating and extruding in an aqueous solution to obtain a lipid film; And / or, the mixing is incubated at 37° C. for 2-4 h.

[0016] The present invention provides a kit for detecting AD target Micro RNA using any of the CRISPR technologies described above for non-diagnostic purposes, which comprises the following steps: Take the test solution, mix it with the CRISPR technology detection kit for AD target Micro RNA, incubate it at 37°C for 15-45 min, and detect the fluorescence signal.

[0017] The present invention provides a detection reagent, a kit and a detection method for detecting AD target Micro RNA using CRISPR technology. The present invention screens the nucleic acid sequences such as the detection target Micro RNA and its primers for AD diseases, optimizes the concentration of each component in the detection system, and provides a detection reagent and a kit for detecting the target Micro RNA using CRISPR-circuit technology. The present invention utilizes liposomes to deliver the CRISPR-circuit detection system, combines the CRISPR-circuit technology with the membrane fusion technology, and realizes in situ detection of exosome MicroRNA, which has the advantages of high sensitivity and high specificity, and can accurately and quickly detect the target MicroRNA in the serum of AD patients in situ. By synergistically coupling the molecular circuit with the CRISPR-Cas13a system, it is used for the exponential amplification and fluorescence detection of MicroRNA, and at the same time significantly simplifies and speeds up the analysis workflow (only 30 minutes). The liposome delivery method avoids possible RNA degradation or cross contamination in conventional detection methods, and improves the sensitivity of MicroRNA detection. The method does not require expensive equipment and time-consuming steps, can be used for convenient on-site diagnosis, and has a wide range of applications and can be innovated across fields, such as medical diagnosis, gene editing verification, and environmental / food safety monitoring. The present invention provides a fast and efficient method for diagnosing Alzheimer's disease, and has good application prospects.

[0018] Obviously, according to the above contents of the present invention, in accordance with common technical knowledge and customary means in the art, without departing from the above basic technical ideas of the present invention, other various forms of modification, replacement or change may be made.

[0019] The above contents of the present invention are further described in detail below through specific implementation methods in the form of embodiments. However, this should not be understood as the scope of the above subject matter of the present invention being limited to the following examples. All technologies realized based on the above contents of the present invention belong to the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a diagram showing the verification results of the detection system of Experimental Example 1.

[0021] Figure 2 The results of the detection system of Experimental Example 1 for detecting MicroRNA-135a, MicroRNA-193b, and MicroRNA-384 are shown.

[0022] Figure 3 It is a screening result graph of the concentration of each component in the detection system of Experimental Example 1; Figure A is a screening result graph of the conversion primer Cp concentration, and the Cp concentration experimental group is compared, ***p<0.001; Figure B is a screening result graph of the autocatalytic primer Ap concentration, and the Ap concentration experimental group is compared, ***p<0.001; Figure C is a screening result graph of the T7 RNA polymerase concentration, and the T7 RNA polymerase concentration experimental group is compared, ***p<0.001; Figure D is a screening result graph of the Bst II DNA polymerase concentration, and the Bst II DNA polymerase concentration experimental group is compared, ***p<0.001; Figure E is a screening result graph of the Cas13a concentration, and the Cas13a concentration experimental group is compared, ***p<0.001; Figure F is a screening result graph of the crRNA concentration, and the crRNA concentration experimental group is compared, ***p<0.001.

[0023] Figure 4 The results of Western blot analysis were used to determine the protein contents of CD9, CD63, and CD81 in cells and exosomes.

[0024] Figure 5 This is a biological transmission electron microscopy image of exosomes.

[0025] Figure 6 This is a biological transmission electron microscopy image of the fusion of exosomes and lipid globules.

[0026] Figure 7 These are the experimental results of lipid spheres, exosomes and their fused microspheres measured by photodynamic scattering instrument; Lipo represents liposomes, Exo represents exosomes, and Lipo+Exo represents fused microspheres; Figure A is the diameter change diagram, and Figure B is the zeta potential result diagram.

[0027] Figure 8 This is a graph showing the results of the detection kit in Example 1 detecting different concentrations of extracellular vesicles in the serum of Alzheimer's patients.

[0028] Fig. 9 This is a graph showing the results of the detection kit of Example 1 detecting different MicroRNA exosomes. DETAILED DESCRIPTION

[0029] In the following examples and experimental examples, reagents and raw materials not specifically described are all commercially available products.

[0030] Example 1 A membrane fusion / CRISPR-circuit detection kit and detection method 1. Kit composition and preparation method The kit of this example is prepared by encapsulating the detection system in Table 1 in liposome microspheres.

[0031] (1) The detection system in Table 1.

[0032] Table 1 Detection system Notes: The buffer contained 20 mM Tris-HCl (pH 7.9), 50 mM KCl, 10 mM magnesium sulfate, 0.1% (W / V) PEG-8000, 1 mM DTT (Dithiothreitol (DTT), a small molecule organic reducing agent), and 1 mg / mI BSA. Tris-HCl (pH 7.9), KCl, MgSO4, and PEG 8000 were purchased from Sigma-Aldrich, and DTT and BSA were purchased from TAKARA Biomedical Technology Co., Ltd. (Kusatsu, Japan).

[0033] In other embodiments, in the above system, the concentration of Cp is adjusted within the range of 0.5-500 nM, the concentration of Ap is adjusted within the range of 0.5-500 nM, the concentration of T7 RNA polymerase is adjusted within the range of 0.5-4 U / mL, the concentration of Bst II DNA polymerase is adjusted within the range of 0.05-0.4 U / μL, the concentration of Cas13a is adjusted within the range of 100-800 nM, and the concentration of crRNA is adjusted within the range of 50-400 nM.

[0034] (2) Nucleic acid sequence The nucleic acid sequences involved in this example are shown in Table 2.

[0035] Table 2 Nucleotide sequences The nucleotide sequence of RNA Reporter is UUUUUU, its 5' end is modified with 6-FAM fluorescent group, and its 3' end is modified with BHO fluorescent quenching group.

[0036] (3) Preparation of lipid spheres 10 mg (2,3-dioleoyl-propyl)-trimethylammonium-chloride) DOTAP, 10 mg (2-oleoyl-1-palmitin glycero-3-phosphocholine) POPC and 3 mg cholesterol were dissolved in 1 mL chloroform, and the chloroform was removed by rotary evaporation to form a lipid film at the bottom of a round-bottom flask. The film was hydrated in 10 mL DPBS (37°C, 1 hour), oscillated on a vortex for 1 minute, and then extruded 21 times with a 0.1 μm polycarbonate membrane to form a lipid microsphere solution. Then 1 mL of the detection system was added, mixed thoroughly, placed in a water bath, and incubated at 37°C for 2 hours. Finally, centrifuged (6000 rpm, 5 minutes), washed 10 times with DPBS, resuspended in 1 mL DPBS, and stored at 4°C.

[0037] 2. Detection Method (1) Detection of MicroRNA-135a Take 1 μL of the test solution, mix it with the detection system in Table 1, incubate it at 37°C for 15 min, and detect the fluorescence signal.

[0038] (2) Detection of MicroRNA-135a in peripheral blood EVs Take the blood to be tested, obtain the EVs solution to be tested by differential centrifugation, take 1 μL of the EVs solution to be tested, mix it with 20 μL of the lipid sphere solution of the load detection system prepared in this example, incubate at 37° C. for 30 min, and detect the fluorescence signal.

[0039] Example 2 A membrane fusion / CRISPR-circuit detection kit and detection method The kit was prepared according to the method of Example 1, except that the primer sequences were different and the MicroRNA to be detected was different.

[0040] The primers and the nucleic acid sequence of MicroRNA-384 detected in the detection system of this example are shown in Table 3.

[0041] Table 3 Nucleic acid sequences of MicroRNA-384 and its primers Example 3 A membrane fusion / CRISPR-circuit detection kit and detection method The kit was prepared according to the method of Example 1, except that the primer sequences were different and the MicroRNA to be detected was different.

[0042] The primers and nucleic acid sequences of MicroRNA-193b detected in the detection system of this embodiment are shown in Table 4.

[0043] Table 4 Nucleic acid sequences of MicroRNA-193b and its primers Example 4 Use of a membrane fusion / CRISPR-circuit detection kit for non-diagnostic purposes In the biological sample library, the membrane fusion / CRISPR-circuit detection kit prepared by the present invention is used to detect the expression level of MicroRNA-135a in the blood samples of Alzheimer's patients in the biological sample library, and evaluate whether the quality and storage conditions of the samples are good for quality control purposes.

[0044] The technical solution of the present invention is further illustrated by experiments below.

[0045] Experimental Example 1 Verification of CRISPR-circuit detection system and optimization of conditions 1. Experimental Methods (1) Testing system The detection system of this experimental example is shown in Table 5: Table 5 Detection system of Experimental Example 1 Notes: The buffer contained 20 mM Tris-HCl (pH 7.9), 50 mM KCl, 10 mM magnesium sulfate, 0.1% (W / V) PEG-8000, 1 mM DTT (Dithiothreitol (DTT), a small molecule organic reducing agent), and 1 mg / mI BSA. Tris-HCl (pH 7.9), KCl, MgSO4, and PEG 8000 were purchased from Sigma-Aldrich, and DTT and BSA were purchased from TAKARA Biomedical Technology Co., Ltd. (Kusatsu, Japan).

[0046] (2) Verification of the testing system The validation experiment was divided into 7 groups. After adding each component, the mixture was incubated at 37°C for 15 min and the fluorescence signal was detected. Among them, the converter primer (Cp) used the sequence of MicroRNA-135a-Cp, and the autocatalytic primer (Ap) used the sequence of MicroRNA-135a-Ap. The following are the components added to each group: ① According to the detection system, add Cp, Ap, Bst II DNA polymerase, T7 RNA polymerase, 1 μL 10 3fMMicroRNA-135a, dNTP, crRNA, Buffer, Cas13a, RNA Reporter, recombinant RNase inhibitor; ② According to the detection system, add Cp, Ap, Bst II DNA polymerase, T7 RNA polymerase, dNTP, crRNA, Buffer, Cas13a, RNA Reporter, and recombinant RNase inhibitor; ③ According to the detection system, add Cp, Ap, Bst II DNA polymerase, 1 μL 10 3 fM MicroRNA-135a, dNTP, crRNA, Buffer, Cas13a, RNA Reporter, recombinant RNase inhibitor; ④ According to the detection system, add Cp, Ap, T7 RNA polymerase, 1 μL 10 3 fM MicroRNA-135a, dNTP, crRNA, Buffer, Cas13a, RNA Reporter, recombinant RNase inhibitor; ⑤ According to the detection system, add Cp, Bst II DNA polymerase, T7 RNA polymerase, 1 μL 10 3 fMMicroRNA-135a, dNTP, crRNA, Buffer, Cas13a, RNA Reporter, recombinant RNase inhibitor; ⑥ According to the detection system, add Ap, Bst II DNA polymerase, T7 RNA polymerase, 1 μL 10 3 fMMicroRNA-135a, dNTP, crRNA, Buffer, Cas13a, RNA Reporter, recombinant RNase inhibitor; ⑦ According to the detection system, add Bst II DNA polymerase, T7 RNA polymerase, 1 μL 10 3 fM MicroRNA-135a, dNTP, crRNA, Buffer, Cas13a, RNA Reporter, recombinant RNase inhibitor.

[0047] (3) Validation of MicroRNA Detection Four detection systems were constructed, namely MicroRNA-135a group, MicroRNA-193b group, MicroRNA-384 group and negative control group. The nucleic acid sequence of Cp in MicroRNA-135a group is SEQ ID NO. 2, and the nucleic acid sequence of Ap is SEQ ID NO. 3; the nucleic acid sequences of Cp and Ap in MicroRNA-193b group are SEQ ID NO. 8; the nucleic acid sequences of Cp and Ap in MicroRNA-384 group are SEQ ID NO. 6; the nucleic acid sequence of Cp in the negative control group is SEQ ID NO. 2, and the nucleic acid sequence of Ap is SEQ ID NO. 3.

[0048] According to the detection system in Table 5, 1 μL of MicroRNA-135a, MicroRNA-193b, MicroRNA-384 and distilled water were added to the four detection systems respectively, incubated at 37°C for 15 min, and the fluorescence signal was detected.

[0049] (4) Optimization of components in the detection system The condition optimization experiment was divided into 6 groups. According to the detection system in Table 5, the concentrations of Cp, Ap, T7 RNA polymerase, Bst II DNA polymerase, Cas13a, and crRNA were optimized respectively. The Cp and Ap of the detection system were MicroRNA-135a-Cp and MicroRNA-135a-Ap. The target gene detected was 1 μL 10 3 MicroRNA-135a of fM.

[0050] The following are the experimental methods for each group: ① Test the fluorescence intensity of the target gene at the same concentration under the same experimental conditions when the Cp concentrations are 0.5 nM, 5 nM, 50 nM, and 500 nM respectively.

[0051] ② When the Ap concentrations were 0.5 pM, 5 pM, 50 pM, and 500 pM, respectively, the fluorescence intensity of the target gene at the same concentration under the same experimental conditions.

[0052] ③ Test the fluorescence intensity of the target gene at the same concentration under the same experimental conditions when the concentration of T7 RNA polymerase is 0.5 U / μL, 1 U / μL, 2 U / μL, and 4 U / μL respectively.

[0053] ④ Test the fluorescence intensity of the target gene at the same concentration under the same experimental conditions when the concentration of Bst II DNA polymerase is 0.05 U / μL, 0.1 U / μL, 0.2 U / μL, and 0.4 U / μL respectively.

[0054] ⑤ When the concentration of Cas13a was tested at 100 nM, 200 nM, 400 nM, and 800 nM, the fluorescence intensity of the target gene at the same concentration under the same experimental conditions.

[0055] ⑥ When the crRNA concentrations were tested at 50 nM, 100 nM, 200 nM, and 400 nM, respectively, the fluorescence intensity of the target gene at the same concentration under the same experimental conditions.

[0056] 2. Experimental Results (1) Verification of the testing system The verification results of the test system are as follows: Figure 1 As shown: Only when all components in the detection system are present, can an obvious fluorescent signal be detected, indicating that in the detection system of Micro RNA, Cp, Ap, Bst II DNA polymerase, and T7 RNA polymerase are indispensable. When the target to be tested is not added to the detection system, there is no obvious fluorescent signal, indicating that the detection system has a low background signal. When Ap is not added to the detection system, a certain fluorescent signal is detected, but the signal intensity is weak, indicating that the detection system can be used to detect Micro RNA-135a, and the addition of Ap produces the effect of signal amplification. Therefore, the detection system of this experimental example has been verified to be used for detecting Micro RNA-135a, and produces the effect of signal amplification.

[0057] (2) Validation of MicroRNA Detection Verification results are as follows Figure 2 As shown: Compared with the negative control group, the detection system of this experimental example successfully amplified and detected MicroRNA-135a, MicroRNA-193b, and MicroRNA-384.

[0058] (3) Optimization of components in the detection system Filter results such as Figure 3 As shown: Under each screening experimental condition, the enhancement of fluorescence signal was detected. In particular, when the Cp concentration was 50 nM, the Ap concentration was 5 pM, the T7 RNA polymerase concentration was 2 U / μL, the Bst II DNA polymerase concentration was 0.1 U / μL, the Cas13a concentration was 200 nM, and the crRNA concentration was 200 nM, the fluorescence signal enhancement effect was most obvious.

[0059] The above experiments show that the detection system of the present invention has a low background signal, and Cp, Ap, Bst II DNA polymerase, and T7 RNA polymerase are indispensable; when the primers are changed, MicroRNA-135a, MicroRNA-193b, and MicroRNA-384 can be detected accordingly. The preferred conditions of the detection system are: the optimal Cp concentration is 50 nM, the optimal Ap concentration is 5 pM, the optimal T7 RNA polymerase concentration is 2 U / μL, the optimal Bst II DNA polymerase concentration is 0.1 U / μL, the optimal Cas13a concentration is 200nM, and the optimal crRNA concentration is 200 nM.

[0060] Experimental Example 2 Characterization of exosome-lipid sphere fusion 1. Experimental Methods (1) Cell culture Human neuroblastoma (SH-SY5Y cells) and human neural stem cells (hNSCs) were cultured in DMEM containing 10% FBS (without extracellular vesicles), 100 units / mL penicillin, and 100 units / mL streptomycin at 37°C and 5% CO2. To prepare FBS without extracellular vesicles, FBS was ultracentrifuged at 110,000g for 10 h and then filtered with a 0.22 μm filter membrane (Thermo Fisher Scientific, Shanghai) and stored at -20°C.

[0061] (2) Extraction of exosomes ① Collect the cell culture medium and centrifuge at 3000g for 20 minutes to remove cells and large cell debris.

[0062] ② Centrifuge the cell culture medium obtained in the above step at 11000g for 30 minutes to remove small particle cell debris and collect the supernatant.

[0063] ③ Ultracentrifuge the supernatant at 110,000 g for 140 minutes to obtain exosomes (also known as extracellular vesicles).

[0064] ④ The obtained cell exosomes were dissolved in Dulbecco's phosphate buffered saline (DPBS) (purchased from Shanghai Thermo Fisher Scientific Co., Ltd.) and washed by ultracentrifugation at 110,000 g for 140 minutes. Finally, the cell exosomes were resuspended in DPBS and stored at -80°C for further use.

[0065] (3) Fusion of exosomes and lipid globules 10 mg (2,3-dioleoyl-propyl)-trimethylammonium-chloride) DOTAP, 10 mg (2-oleoyl-1-palmitin-glycero-3-phosphocholine) POPC and 3 mg cholesterol were dissolved in 1 mL chloroform, and the chloroform was removed by rotary evaporation to form a lipid film at the bottom of a round-bottom flask. The film was hydrated in 10 mL DPBS (37°C, 1 hour), shaken on a vortexer for 1 minute, and then extruded 21 times with a 0.1 μm polycarbonate membrane to form lipid microspheres.

[0066] The lipid spheres and exosomes were incubated at 37°C for 15 min to characterize the fusion of exosomes and lipid spheres.

[0067] (3) Western blot experiment of exosomes ② Western blot analysis of exosome markers (CD9, CD63, CD81): SH-SY5Y cells and exosome sediments were mixed with 200 μL RIPA lysis buffer (Solebo Biotechnology Co., Ltd., Beijing), and the CD9, CD63, and CD81 protein concentrations were determined using the BCA protein detection kit (Solebo Biotechnology Co., Ltd., Beijing), and then denatured at 95°C for 5 min. The loading amount of cells was obtained by the internal reference protein (GAPDH), and the samples were loaded with the specified protein amount to determine whether CD9, CD63, and CD81 were enriched in exosomes compared with cells.

[0068] (4) Transmission electron microscopy characterization of cell exosomes and fusion microspheres The morphology of exosomes and exosome-lipid sphere fusion was characterized by biological transmission electron microscopy (TEM, JEM 1400 Plus, JEOL). The samples were first fixed with 2.5% glutaraldehyde and washed with PBS buffer, then secondarily fixed with 1% osmium acid, washed with PBS buffer, and then dehydrated with ethanol solution. Subsequently, the samples were infiltrated with acetone and epoxy resin overnight, embedded with epoxy resin, and sliced ​​with an ultramicrotome (Leica EM UC7). The slices were placed on copper grids, stained with uranyl acetate, and imaged under a transmission electron microscope at 100 kV.

[0069] (5) Dynamic light scattering characterization of exosomes and fusion microspheres Dynamic light scattering (DLS) was used to monitor the changes in hydrodynamic diameter during the fusion process of lipid microspheres and exosomes, and the zeta potential of lipid microspheres, exosomes and fused vesicles was measured.

[0070] 2. Experimental Results (1) Western blot characterization results of cell exosomes Western blot results Figure 4 As shown: Compared with cells, CD9, CD63, and CD81 proteins are enriched in exosomes, indicating that exosomes were effectively extracted by the method of this experimental example.

[0071] (2) Transmission electron microscopy results Transmission electron microscopy results of exosomes Figure 5 As shown in Figure 2, exosomes are nano-sized spheres. The transmission electron microscopy results of exosome fusion with lipid spheres are shown in Figure 2. Figure 6 As shown in the figure, it can be seen that the two microspheres are in contact and partially fused, indicating that exosomes and lipid spheres can fuse.

[0072] (3) DLS characterization results DLS results are as follows Figure 7 As shown: The particle size of lipid globules and exosomes is about 200nm. When the two are fused, the particle size increases significantly. As time goes by, the size of the fused exosomes and lipid globules gradually increases, indicating that the lipid globules and exosomes have fused, and as the fusion time increases, the degree of fusion increases. Zeta potential further proves the fusion of lipid globules and exosomes.

[0073] The above results show that exosomes were extracted from cells in this experimental example, and it was proved that exosomes can fuse with lipid globules.

[0074] Experimental Example 3 Performance Verification of Membrane Fusion / CRISPR-circuit Detection Method The membrane fusion / CRISPR-circuit detection kit and its detection method used in this experimental example are as described in Example 1.

[0075] 1. Sensitivity analysis The concentration of the membrane fusion / CRISPR-circuit detection kit and its detection method was 10 -1 ——10 4 fM of serum exosomes from Alzheimer's patients, and a negative control was set up at the same time, and distilled water was added to the negative control group.

[0076] Among them, the concentration of exosomes is measured by the concentration of Micro 135a in the exosomes. The preparation method of exosomes from the serum of Alzheimer's patients with different concentrations is: after collecting the exosomes in the serum of Alzheimer's patients, the concentration of the contained MicroRNA is detected by the PCR method, and after dilution, the exosome solution with the target MicroRNA concentration is obtained.

[0077] Test results such as Figure 8 As shown in Figure 2, as the concentration of exosomes increases, the fluorescence signal increases. The sensitivity is 10 1fM, the detection method of Example 1 of the present invention has high sensitivity.

[0078] 2. Specificity Analysis The membrane fusion / CRISPR-circuit detection kit and its detection method were used to detect different types of exosomes. The different types of exosomes were exosomes isolated from SH-SY5Y cells and hNSCs cells.

[0079] Test results such as Fig. 9 As shown, the fluorescence signal of the exosomes of SH-SY5Y cells is more obvious than that of the exosomes of hNSCs cells, indicating that the detection method of Example 1 of the present invention is specific.

[0080] 3. Clinical Sample Testing Sera from 10 AD patients and 10 healthy subjects were collected and tested using the membrane fusion / CRISPR-circuit detection system and detection method of Examples 1-3, respectively.

[0081] The test results are shown in Table 6. When MicroRNA-135a is used as the detection target in Example 1, the false negative result is 10% and the false positive result is 10%; when MicroRNA-384 is used as the detection target in Example 2, the false negative result is 20% and the false positive result is 20%; when MicroRNA-193b is used as the detection target in Example 3, the false negative result is 50% and the false positive result is 20%. Therefore, the present invention preferably uses MicroRNA-135a as the detection target.

[0082] Table 6 Clinical sample test results The above results show that the membrane fusion / CRISPR-circuit detection system and detection method of this embodiment have high sensitivity and high specificity, can meet the requirements of actual detection, and the preferred detection target is MicroRNA-135a.

[0083] Through the above embodiments and experimental examples, it can be seen that the present invention provides a detection system, kit and detection method for in situ detection of MicroRNA in exosomes by combining membrane fusion technology and CRISPR-circuit technology. The present invention screens the detection targets and primers of AD diseases, optimizes the concentrations of each component in the detection system, and uses liposomes to deliver the CRISPR-circuit detection system, thereby realizing high sensitivity, high specificity and high accuracy detection of MicroRNA in exosomes of AD patients. By synergistically coupling the molecular circuit with the CRISPR-Cas13a system, it is used for exponential amplification and fluorescence detection of MicroRNA, while significantly simplifying and speeding up the analysis workflow (only 30 minutes). The liposome delivery method avoids possible RNA degradation or cross-contamination in conventional detection methods, and improves the sensitivity of MicroRNA detection. The method does not require expensive equipment and time-consuming steps, can be used for convenient on-site diagnosis, and the method is widely used and can be innovated across fields, such as medical diagnosis, gene editing verification and environmental / food safety monitoring. The present invention provides a fast and efficient method for diagnosing Alzheimer's disease, and has good application prospects.

Claims

1. A detection reagent for detecting AD target Micro RNA using CRISPR technology, characterized in that: It includes the following components: Conversion primer 0.025-25nM, Self-catalytic primer 0.025-25pM, dNTP 1-4 nM, crRNA 5-40nM, CRISPR / Cas endonuclease 20-160nM, RNA reporter 0.25-1 μM, Bst II DNA polymerase 0.625-5U / mL, T7 RNA polymerase 0.02-0.16U / μL.

2. The detection reagent for detecting AD target Micro RNA by CRISPR technology according to claim 1, characterized in that: The concentration of the conversion primer is 2.5nM; and / or, the concentration of the autocatalytic primer is 0.25pM; and / or, the concentration of crRNA is 20nM; and / or, the concentration of Cas13a is 40nM; and / or, the concentration of Bst II DNA polymerase is 1.25U / mL; and / or, the concentration of T7RNA polymerase is 0.08U / μL; and / or, the CRISPR / Cas nuclease is selected from Cas13a, Cas12a.

3. The detection reagent for detecting AD target Micro RNA by CRISPR technology according to claim 1, characterized in that: The conversion primer and the autocatalytic primer combination comprises at least one of the following combinations: The nucleotide sequence of the conversion primer is SEQ ID NO. 2, and the nucleotide sequence of the autocatalytic primer is SEQ ID NO. 3; Or, the nucleotide sequences of the conversion primer and the autocatalytic primer are both SEQ ID NO. 6; Alternatively, the nucleotide sequences of the conversion primer and the autocatalytic primer are both SEQ ID NO.

8.

4. The detection reagent for detecting AD target Micro RNA by CRISPR technology according to claim 1, characterized in that: The nucleotide sequence of crRNA is SEQ ID NO. 4; and / or, the nucleotide sequence of RNA reporter molecule is UUUUUU.

5. The detection reagent for detecting AD target Micro RNA by CRISPR technology according to claim 1, characterized in that: The two ends of the RNA reporter molecule are modified with a fluorescent group and a fluorescent quenching group respectively; And / or, the detection reagent further comprises a recombinant RNase inhibitor at a concentration of 5-10 U / μL; And / or, the detection reagent also includes a buffer with a volume fraction of 10-40%, the components of the buffer include 10-40mM Tris-HCl, 20-100mM KCl, 5-20mM magnesium sulfate, 0.05-0.5% mass volume ratio of PEG-8000, 0.5-2mM dithiothreitol, 0.5-2mg / mI BSA, and the pH of Tris-HCl is 7-8.

5.

6. Use of the detection reagent for detecting AD target Micro RNA using CRISPR technology as described in any one of claims 1-5 in the preparation of a kit for detecting MicroRNA in the serum of patients with Alzheimer's disease or cancer.

7. A kit for detecting AD target Micro RNA using CRISPR technology, characterized in that: A detection reagent for detecting AD target Micro RNA using CRISPR technology according to any one of claims 1 to 5, or a lipid sphere containing a detection reagent for detecting AD target Micro RNA using CRISPR technology according to any one of claims 1 to 5; In the lipid sphere containing the detection reagent for detecting AD target Micro RNA using CRISPR technology, the usage ratio of the detection reagent for detecting AD target Micro RNA using CRISPR technology to the lipid sphere is 1-2 mL: 23-46 mg.

8. The kit for detecting AD target Micro RNA using CRISPR technology according to claim 7, characterized in that: The lipid sphere containing the detection reagent for detecting AD target Micro RNA using CRISPR technology is prepared according to the following steps: the detection reagent for detecting AD target Micro RNA using CRISPR technology is mixed with the lipid sphere.

9. The kit for detecting AD target Micro RNA using CRISPR technology according to claim 8, characterized in that: The lipid sphere is prepared according to the following steps: cationic lipid molecules, amphiphilic phospholipid molecules and cholesterol are mixed, rotary evaporated to obtain a lipid film, and then incubated in an aqueous solution and extruded to obtain a lipid film; And / or, the mixing is incubated at 37° C. for 2-4 h.

10. A detection method for non-diagnostic purposes using a kit for detecting AD target Micro RNA using the CRISPR technology described in any one of claims 7 to 9, characterized in that: The steps include: Take the test solution, mix it with the CRISPR technology detection kit for AD target Micro RNA, incubate it at 37°C for 15-45 minutes, and detect the fluorescence signal.

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