Amylase-loaded DNA (deoxyribonucleic acid) hydrogel as well as preparation method and application thereof

Abstract

The invention discloses amylase-loaded DNA (deoxyribonucleic acid) hydrogel as well as a preparation method and application thereof. A DNA single strand is generated through a rolling circle amplification technology to form a hydrogel scaffold, the single strand can be cross-linked with a high-affinity DNA methylation binding protein after being acted by DNA methyltransferase to finally form DNA hydrogel of a hollow net structure, amylase is added in the formation process of the DNA hydrogel, and amylase wrapping is achieved. Meanwhile, based on the preparation of the DNA hydrogel, an application of portable pathogen nucleic acid detection is established, the trans-cleavage activity of Cas12a protein is activated by a pathogen target, the structure of the DNA hydrogel can be destroyed, amylase is released to catalytically generate glucose, and pathogen detection is realized by using a personal portable glucometer. The method established by the invention provides a novel and convenient pathogen nucleic acid rapid detection method thought for the technical field of molecular diagnosis.

Description

Technical Field

[0001] The invention relates to a DNA hydrogel loaded with amylase and a preparation method and application thereof, belonging to the field of biological detection. Background Art

[0002] The design and preparation of DNA hydrogel has been one of the focuses of attention in the biomedical field in recent years. It is a hydrophilic material between solid and fluid formed by self-assembly of DNA molecules. It has high biocompatibility and a variety of designable stimulus response capabilities. It has been widely used in intelligent diagnosis and treatment fields such as drug delivery and artificial tissue.

[0003] Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) technology, referred to as CRISPR technology, some of the new discoveries have brought new opportunities to the nucleic acid detection model. CRISPR-Dx (CRISPR-based diagnostic) technology and SHERLOCK (Specific High-Sensitivity Enzymatic Reporter UnLOCKing) technology rely on Cas12a and Cas13a proteins. After pre-amplification, the trans-cleavage activity of Cas proteins is stimulated, and molecular beacons can be cut at room temperature to achieve signal amplification with high sensitivity and specificity. At present, for nucleic acid detection using CRISPR technology, the final signal collection mode still relies on microplate readers and fluorescent quantitative PCR instruments for real-time collection of fluorescent signals. This type of signal collection method greatly limits the portability of nucleic acid detection. However, the new technology of CRISPR-Dx provides new ideas for portable nucleic acid detection, making it have great application prospects in pathogen nucleic acid detection, especially POCT (point-of-care testing) detection.

[0004] There are currently a variety of CRISPR-based POCT detection methods that use various signal sensing technologies, such as electrochemical signals, fluorescence sensing, etc. to achieve detection. Among them, blood glucose meters, as a portable, easy-to-operate, and low-cost POCT platform, can be well combined with the CRISPR / Cas system for biological analysis. However, most nucleic acid detection methods that combine CRISPR technology with blood glucose meters require the synthesis of nucleic acid-protein conjugates and complex operations to achieve target-specific signal output. Summary of the invention

[0005] Purpose of the invention: The technical problem to be solved by the present invention is to provide a novel DNA hydrogel loaded with amylase and its preparation method and application.

[0006] Technical solution: To solve the above technical problems, the present invention provides a DNA hydrogel loaded with amylase, wherein the hydrogel skeleton is a single-stranded DNA, the DNA chain contains methylated cytosine bases, the single-stranded DNA is cross-linked by high-affinity DNA methylation binding proteins to form a hollow network structure, and the amylase is wrapped inside the hydrogel.

[0007] The present invention also provides a method for preparing the amylase-loaded DNA hydrogel, which is specifically as follows:

[0008] Step 1, design and synthesis of RCA template. The RCA template is specially designed to contain an intramolecular stem-loop structure with potential methylation binding sites (CpGs) on the stem sequence. The 5' end of a linear template (RCA-80) sequence is phosphorylated with T4 polynucleotide kinase, and a special circular structure, i.e. circular DNA, is formed under the action of T4 DNA ligase as a template for rolling circle nucleic acid amplification (RCA);

[0009] Step 2: The RCA reactants are modified by DNA methylation and cross-linked with tetrameric DNA methylation binding proteins. A short DNA chain (RCA-Primer) is added to the RCA circular template, and the amplified product is a long ssDNA with a repeated and regularly spaced hairpin structure. Under the catalysis of DNA methyltransferase, the CpGs sites in the hairpin structure are modified to methylated CpGs (mCpGs). The mCpGs in the amplified product cross-link DNA methylation binding proteins (SA-MBD) to form a hollow mesh structure DNA hydrogel. Amylase is added during the formation of the DNA hydrogel to achieve the encapsulation of amylase.

[0010] Wherein, the tetrameric DNA methylation binding protein in step 2 includes SA-MBD protein.

[0011] The invention completes the preparation of a novel DNA hydrogel loaded with amylase through three main steps of RCA reaction, DNA methylation modification and cross-linking of tetrameric MBD protein.

[0012] The present invention also provides the use of the amylase-loaded DNA hydrogel in preparing a pathogen nucleic acid detection reagent or a kit.

[0013] The application includes the following steps: adding the product of pathogen nucleic acid after pre-amplification to the targeted CRISPR / Cas reaction solution, DNA hydrogel and starch for reaction, and after the reaction, using a blood glucose meter for detection.

[0014] The present invention also provides a reagent or a kit for detecting pathogen nucleic acid, comprising the DNA hydrogel loaded with amylase.

[0015] The present invention also provides a method for responsively activating a CRISPR-Cas system, wherein the amylase-loaded DNA hydrogel is combined with the CRISPR-Cas system.

[0016] The present invention also provides the use of the amylase-loaded DNA hydrogel, the reagent or kit, or the CRISPR-Cas system in the detection of new coronavirus and influenza A virus.

[0017] Among them, the detection of the new coronavirus and influenza A virus includes pathogen nucleic acid detection.

[0018] The present invention also establishes a new method for portable pathogen nucleic acid detection based on the DNA hydrogel loaded with amylase. Specific principle ( Figure 1 ) is: pre-amplify a variety of pathogen nucleic acid templates through recombinase polymerase amplification technology (RPA) and convert them into DNA templates; the template activates the trans-cleavage activity of Cas12a protein through sgRNA-mediated target recognition, and the activated Cas12a protein can decompose the ssDNA scaffold of DNA hydrogel and release amylase; however, in the absence of target nucleic acid, the nuclease activity of Cas protein is dormant and will not release amylase to produce glucose, which maintains the integrity of the hydrogel. Starch is added to the CRISPR detection system, and amylase is catalyzed to produce glucose after being destroyed by Cas protein, so that the sugar concentration in the system can be detected by a portable personal blood glucose meter.

[0019] The present invention prepares a new type of DNA hydrogel loaded with amylase and combines it with the CRISPR / Cas system to successfully construct a pathogen nucleic acid detection method with a portable personal blood glucose meter as signal output, and develops a POCT platform for rapid detection of pathogen nucleic acid.

[0020] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0021] 1. The prepared new DNA hydrogel does not require the synthesis of complex nucleic acid-protein conjugates. It cross-links the high-affinity SA-MBD protein and can effectively encapsulate amylase, avoiding false positive results caused by amylase leakage;

[0022] 2. Amylase can be released under the action of the activated CRISPR / Cas system to hydrolyze starch and produce glucose readings, and a pathogen nucleic acid detection POCT platform can be established. This platform can complete highly flexible, sensitive and specific pathogen nucleic acid detection within 1 hour by coupling rapid nucleic acid amplification methods. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 It is a schematic diagram of the overall process of the present invention;

[0024] Figure 2 This is a schematic diagram of the principle of preparing a novel DNA hydrogel according to the present invention;

[0025] Figure 3 This is a diagram showing the appearance of DNA hydrogel;

[0026] Figure 4 This is a diagram showing the sensitivity verification result of the method established by the present invention to the new coronavirus target;

[0027] Figure 5 This is a diagram showing the specificity verification result of the method established by the present invention for the new coronavirus target;

[0028] Figure 6 This is a diagram showing the sensitivity validation results of the method established by the present invention for the influenza A virus target validation;

[0029] Figure 7 This is a graph showing the specific detection results of the method established by the present invention for validating influenza A virus targets. DETAILED DESCRIPTION

[0030] The technical solution of the present invention is further described below in conjunction with the accompanying drawings.

[0031] Example 1: Preparation of DNA hydrogel loaded with amylase

[0032] 1. Design of linear template: A linear template with a loop length of 80 nt (RCA-80: SEQ ID NO.6: CACGGTATTCAGTCTCTCTCTCTCTCTCTTACCGATCGCCTCTGCGATCGGTCTCTCTCTCTCTCTCTCTCAATGGTTACAGT) was synthesized by Shanghai Biotechnology Co., Ltd. Figure 2 The second step structure is shown in .

[0033] 2. Phosphorylation: Phosphorylate the 5' end of this sequence with T4 polynucleotide kinase (T4 PNK, Takara Biotechnology Co., Ltd. (Dalian)). Prepare 10μL 1×T4 PNK buffer (formula: 50mM Tris-HCl, pH8.0, 10mMMgCl2, 5mM dithiothreitol), add 1mM ATP and 10U T4 PNK to the solution, label the phosphate group at the 5' end of the linear template sequence, and the total amount of linear template input is 100pmol. Reaction conditions: Incubate at 37℃ for 30 minutes and 70℃ for 10 minutes using a thermal cycler.

[0034] 3. Circularization reaction: After phosphorylation, use T4 DNA ligase (Takara Biotechnology Co., Ltd. (Dalian)) for ligation. Specifically, add 120 pmol of short DNA chain (RCA-Primer: SEQ ID NO.7: GTTACAGTCACGGTAT) to the phosphorylated linear template. Heat the above mixture to 90°C for 30 seconds, and then slowly cool to room temperature. Add 10 μL of 1×T4 DNA ligase buffer (the buffer contains 10U T4 DNA ligase) to the product, and use a thermal cycler to incubate at 37°C for 30 minutes and 70°C for 10 minutes. Finally, the RCA template is obtained.

[0035] 4. RCA reaction: The RCA template was amplified by RCA technology, specifically: 50 μL of 1×phi29 DNA polymerase reaction buffer (formula: 50 mM Tris-HCl, pH 7.5, 10 mM MgCl2, 10 mM (NH4)2SO4, 4 mM DTT) was prepared, 10 μL of phi29 DNA polymerase (New England Biotechnology (Beijing) Co., Ltd.), 1 mM dNTPs, 1 μM RCA-Primer, 10 μM M.SssI methyltransferase (New England Biotechnology (Beijing) Co., Ltd.), 160 μM SAM (S-adenosylmethionine), 10 mM SA-MBD, 10 μg amylase (Sigma-Aldrich (Shanghai) Trading Co., Ltd.) and 10 pmol of the synthesized RCA template were added. The reaction conditions were: incubation at 37°C for 4 hours using a thermal cycler. Finally, the amylase was encapsulated to obtain the amylase-loaded DNA hydrogel. In order to verify the encapsulation effect of DNA hydrogel on amylase, the morphology of DNA hydrogel was characterized by scanning electron microscopy (see Appendix). Figure 3 The hydrogel skeleton is a single-stranded DNA, which contains methylated cytosine bases. The DNA single strands are cross-linked through a high-affinity DNA methylation binding protein to form a hollow network structure, and amylase is wrapped inside the hydrogel.

[0036] Example 2: Verification of sensitivity and specificity of amplified products of common coronavirus SARS-CoV-2 using a detection platform combining DNA hydrogel and CRISPR / Cas system

[0037] 1. Synthesis of RNA samples: Artificial RNA samples of coronavirus SARS-CoV-2 (GenBank: OQ364822.1), other popular respiratory viruses influenza A (FluA, GenBank: CY058864.1), influenza B (FluB, GenBank: CY115112.1), adenovirus (ADV, GenBank: KY511678.1), and respiratory syncytial virus (RSV, GenBank: DQ780567.1) were synthesized by T7 High Yield RNA transcription kit and used according to the manufacturer's recommendations. Clean up kit purification, synthesis steps are divided into in vitro transcription and purification.

[0038] (1) In vitro transcription:

[0039] ① The above pathogen DNA sequence was screened in the genome database and artificially synthesized by Nanjing GenScript Biotechnology Co., Ltd. and recombined into the pUC19 plasmid vector as a DNA template, wherein the DNA template contains the T7 promoter for RNA transcription (sequence in the underlined area). The nucleotide sequence of the coronavirus DNA template is shown in SEQ ID NO.1: ACTCGT TAATACGACTCACTATAGGGAG ACACATAAAAGAAATAC TTGTCACATACAATTGTTGTGATGATGATTATTTCAATAAAAAGGACTGGTATGATTTTGTAGAAAACCCAGATATATTACGCGTATACGCCAACTTAGGTGAACGTGTACGCCAAGCTTTGTTAAAAACAGTACAATTCTGTGATGCCATGCGAAATGCTGGTATTGTTGGTGTACTGACATTAGATAATCAAGATCTCAATGGTAACTGGTATGATTTCGGTGATTT CATACAAACCACGCCAGGTAGTGGAGTTCCTGTTGTAGATTCTTATTATTCATTGTTAATGCCTATATTAACCTTGACCAGGGCTTAACTGCAGAGTCACATGTTGACACTGACTTAACAAAGCCTTACATTAAGTGGGATTTGTTAAAATATGACTTCACGGAAGAGAGGTTAAAACTCTTTGACCGTTATTTTAAATATTGGGATC, the nucleotide sequence of the influenza A DNA template is as SEQ. ID NO.2 shows: ACTCGTT AATACGACTCACTATAGGGAGACTCG TTAATACGACTCACTATAGGGAGCAGTGAGCGAGGACTGCAGCGTAGACGCTTTGTCCAAAATGCCCTTAATGGGAATGGGGATCCAAATAATATGGACAGAGCAGTTAAACTGTATCGAAAGCTTAAGAGGGAGATAACATTCCATGGGGCCAAAGAAATAGCACTCAGTTATTCTGCTGGTGCACTTGCCAGTTGTATGGGACTCATATACAACAGGATGGGGGCTGTGACCACCGAATCAGCATTTGGCCTTATATGCGCAACCTGTGAACAGATTGCCGACTCCCAGCATAAGTCTCATAGGCAAATGGTAACAACAACCAATCCATTAATAAGACATGAGAACAGAATGGTTCTGGCCAGCACTACAGCTAAGGCTATGGAGCAAATGGCTGGATCGAGTGAACAAGCAGCTGAGGCCATGGAGGTTGCTAGTCAGGCCAGGCAGATGGTGCAGGCAATGAGAGCCATTGGGACTCATCCTAGCTCTAGCACTGGTCTGA, The nucleotide sequence of the DNA template for influenza B is shown in SEQ ID NO.3: ACTCGTTAATACGA CTCACTATAGGGAGACTCGTTAATACGACTCACTATAGGGAGGTACCTAAACCCTGAAAACTATTCAATGCAAGTAAAACTAGGAACGCTCTGTGCTTTATGCGAGAAACAAGCATCGCACTCGCATAGAGCCCATAGCAGAGCAGCAAGGTCTTCGGTACCTGGAGTAAGACGAGAAATGCAGATGGTTTCAGCTATGAACACAGCAAAGACAATGAATGGAATGGGAAAGGGAGAAGACGTCCAAAAACTAGCAGAAGAGCTGCAAAACAACATTGGAGTGTTGAGATCTCTAGGAGCAAGTCAAAAGAATGGAGAAGGAATTGCCAAAGATGTAATGGAAGTGCTAAAACAGAGCTCTATGGGAAATTCAGCTCTTGTGAGGAAATACTTATAATGCTCGAACCACTTCAGATTCTTTCAATTTGTTCTTTCATTTTATCAGCTCTCCATTTCATGGCTTGGACAATAGGGCATTTGAATCAAATAAAAAGAGGGGTAAACTTGAAAATACAAATAAGGAATCCAAATAAGGAGGCACTAAACAGAGAGGTGTCAATTCTGAGACACAATTACCAAAAGGAAATCCAAGCCAAAGAAACAATGAAGAAAATACTCTCTGACAACATGGAAGTATTGGGTGACCACATAGTAGTTGAAGGGCTTTCAACTGATGAGAT, The nucleotide sequence of the adenovirus DNA template is shown in SEQ ID NO. 4: GCAAA TTTGGGTCAACAAGCCATGCCTAACAGACCCAATTACATTGGATTCAGGGATAACTTTGTAGGCCTAATGTACTACAACAGTACTGGAAATATGGGAGTTTTGGCTGGCCAAGCATCACAACTGAATGCAGTGGTTGACTTGCAGGACAGAAATACTGAACTGTCATATCAGCTTTTGCTTGATTCTCTGGGAGACAGAACCAGATACTTCAGCATGTGGAATCAGGCTGTGGACAGTTACGATCCCGATGTTCGCATTATTGAA, The nucleotide sequence of the respiratory syncytial virus DNA template is as shown in SEQ ID NO.5 as shown:. ACTCGTTAATACGACTCACTATAGGGAG A TGGCTCTTAGCAAAGTCAAGTTAAATGATACATTAAATAAGGATCAGCTGCTGTCATCCAGCAAATACACTATTCAACGTAGTACAGGAGATAATATTGATACTCCCAATTATGATGTGCAAAAACACCTAAACAAACTATGTGGTATGCTATTAATCACTGAAGATGCAAATCATAAATTCACAGGGTTAATAGGTATG TTATATGCTATGTCCAGGTTAGGAAGGGAAGACACTATAAAAGATACTTAAAGATGCTGGATATCATGTTAAAGCTAATGGAGTAGATATAACAACATATCGTCAAGATATAAATGGAAAGGAAATGAAATTCGAAGTATTAACATTATCAAGCTTGACATCAGAAATACAAGTCAATATTGAGATAGAATCTAGAAAGTC.

[0040] ② Transcribe the DNA template into RNA in vitro: prepare a 20 μL system, including 2 μL 10×Transcription Buffer, 2 μL UTP Solution, 2 μL ATP Solution, 2 μL CTP Solution, 2 μL GTP Solution, 2 μL DNA template, and 2 μL T7 RNA Polymerase Mix. Mix thoroughly and centrifuge briefly to collect. Use a thermal cycler to incubate at 37°C for 16 hours to obtain the RNA product.

[0041] (2) Purification:

[0042] ① Add 100 μL of RNA binding buffer to 50 μL of RNA product, mix, then add 150 μL of 95% ethanol, mix by pipetting or flicking the test tube, do not vortex.

[0043] ② Move the mixed liquid into the centrifuge column, place the centrifuge column in the collection tube, centrifuge for 1 minute, and discard the filtrate in the collection tube.

[0044] ③Re-insert the centrifuge column into the collection tube, add 500 μL RNA elution buffer, centrifuge for 1 minute, discard the effluent, and repeat this step twice.

[0045] ④ Transfer the spin column to a 1.5 mL microcentrifuge tube without RNase and spin again for 1 minute.

[0046] ⑤ Elute with 20-30 μL of nuclease-free pure water and centrifuge for one minute. The eluted RNA can be stored at -70°C.

[0047] 2. If Figure 1 As shown, by testing a series of different concentrations (10 1 , 10 2 , 10 3 , 10 4 , 10 5 The sensitivity and specificity of the CRISPR / Cas-driven DNA hydrogel platform were evaluated using artificial in vitro transcribed coronavirus RNA samples at 100 copies / μL.

[0048] (1) The RNA sample was subjected to RPA reaction: 50 μL RPA reaction mixture contained 29.4 μL buffer A, enzyme lyophilized powder, 0.4 μM each of the forward and reverse coronavirus DNA primers RPA-ORF1-F / R, 2.5 μL buffer B, and 5 μL of coronavirus RNA target (purified coronavirus RNA product). After vortex centrifugation, incubate at 42°C for 30 minutes in a thermal cycler. Among them, RPA-ORF1-F: SEQ ID NO.8: TACGCCAAGCTTTGTTAAAAACAGTACAATTCTG; RPA-ORF1-R: SEQ ID NO.9: GGCATTAACAATGAATAATAAGAATCTACAACAGG.

[0049] The upstream and downstream primers of negative controls (FluA, FluB, ADV, RSV) are:

[0050] RPA-FluA-F: TCATATACAACAGGATGGGGGCTGTGACCACCGAA (SEQ ID NO.10)

[0051] RPA-FluA-R:CACTCGATCCAGCCATTTGCTCCATAGCCTTAGCT(SEQ ID NO.11)

[0052] RPA-FluB-F: GTCCAAAAACTAGCAGAAGAGCTGCAAAACAAC (SEQ ID NO.12)

[0053] RPA-FluB-R: AACAAATTGAAAGAATCTGAAGTGGTTCGAGCA (SEQ ID NO.13)

[0054] RPA-ADV-F: CCCAATTACATTGGATTCAGGGATAACTTTGTAGG(SEQ ID NO.14)

[0055] RPA-ADV-R: CTGATTCCACATGCTGAAGTATCTGGTTCTGTCTC (SEQ ID NO.15)

[0056] RPA-RSV-F:CTATGTGGTATGCTATTAATCACTGAAGATGC(SEQ ID NO.16)

[0057] RPA-RSV-R:GCTAATGGAGTAGATATAACAACATATCGTC(SEQ ID NO.17)

[0058] (2) The RPA amplification product was used as an activator in combination with CRISPR technology, that is, sgRNA-ORF1 (SEQ ID NO. 18: UAAUUUCUACUAAGUGUAGAUCAUACAAACCACGCCAGGUA) was mixed to perform detection on the DNA hydrogel pathogen nucleic acid detection platform.

[0059] Among them, the pathogen nucleic acid detection platform combined with DNA hydrogel and CRISPR / Cas system: 10μCRISPR / Cas system (3×NEBuffer TM r2.1, 0.15 μM LbCas12a, 0.15 μM target-specific sgRNA-ORF1), 2 μL of RPA amplification product, 10 μL of starch solution (10 mg / mL), and 10 μL of DNA hydrogel loaded with amylase were mixed and incubated at room temperature for 15 minutes. 5 μL of the mixture was taken and the glucose concentration was measured with a blood glucose meter.

[0060] The test results are as attached Figure 4 As shown, it contains 10 1 Up to 10 5 Clear glucose readings were obtained for samples containing 100 copies of the SARS-CoV-2 ORF1 gene target RNA, demonstrating the high sensitivity of the detection platform.

[0061] Using the new coronavirus target (10 3 The amplified products of other common respiratory viruses (all 10 5 The specificity of the detection platform was studied by using influenza virus A (FluA), influenza virus B (FluB), adenovirus (ADV), and respiratory syncytial virus (RSV) as negative controls.

[0062] The test results are as attached Figure 5 As shown, only the positive control showed corresponding glucose readings, while other pathogens presented negative results. These results confirmed the good specificity of the proposed platform, which was attributed to the highly specific target recognition ability of the CRISPR / Cas system.

[0063] Example 3: Design of a Cas12a1 / sgRNA system targeting influenza virus type A M gene to study the versatility of the detection platform

[0064] In this embodiment, the influenza virus type A (FluA) was verified and applied according to the reaction system and reaction conditions in Example 2.

[0065] In order to study the versatility of the proposed pathogen nucleic acid detection platform, a Cas12a1 / sgRNA system targeting the FluAM gene was designed to detect artificial RNA samples of different concentrations of influenza virus type A M gene, including 10 1 Up to 10 5 A sample containing copies of an artificial in vitro transcribed target RNA.

[0066] 1. Specific steps of RNA synthesis: The synthesis steps are divided into in vitro transcription and purification, both of which are synthesized by T7 High Yield RNA transcription kit and used according to the manufacturer's recommendations. Purify with RNA cleanup kit.

[0067] (1) In vitro transcription:

[0068] ① The DNA sequence of the pathogen influenza A virus (FluA) was screened in the genome database and artificially synthesized by Nanjing GenScript Biotech Co., Ltd. and recombined into the pUC19 plasmid vector as a DNA template, where the DNA template contains a T7 promoter.

[0069] ② Transcribe DNA into RNA in vitro: prepare a 20 μL system including 2 μL 10×Transcription Buffer, 2 μL UTP Solution, 2 μL ATP Solution, 2 μL CTP Solution, 2 μL GTP Solution, 2 μL DNA template, and 2 μL T7 RNA Polymerase Mix. Mix thoroughly and centrifuge briefly to collect. Incubate at 37°C for 16 hours in a thermal cycler.

[0070] (2) Purification:

[0071] ① RNA template is prepared by adding 100 μL of RNA binding buffer to 50 μL of RNA product, mixing and then adding 150 μL of 95% ethanol, mixing by pipetting or flicking the test tube, do not vortex.

[0072] ② Move the mixed liquid into the centrifuge column, place the centrifuge column in the collection tube, centrifuge for 1 minute, and discard the filtrate in the collection tube.

[0073] ③Insert the column back into the collection tube, add 500 μL RNA elution buffer, centrifuge for 1 minute, discard the effluent, and repeat this step twice.

[0074] ④ Transfer the spin column to a 1.5 mL microcentrifuge tube without RNase and spin again for 1 minute.

[0075] ⑤ Elute with 20-30 μL of nuclease-free pure water and centrifuge for one minute. The eluted RNA can be stored at -70°C.

[0076] 2. Perform RPA reaction on RNA sample: 50μL RPA reaction mixture contains 29.4μL buffer A, enzyme lyophilized powder, 0.4μM each of forward and reverse coronavirus DNA primers ORF1-F / R, 2.5μL buffer B, and 5μL coronavirus RNA target. After vortex centrifugation, incubate at 42°C for 30 minutes in a thermal cycler. Among them, RPA-ORF1-F: SEQ ID NO.8: TACGCCAAGCTTTGTTAAAAACAGTACAATTCTG; RPA-ORF1-R: SEQ ID NO.9: GGCATTAACAATGAATAATAAGAATCTACAACAGG.

[0077] The upstream and downstream primers of negative controls (FluA, FluB, ADV, RSV) are:

[0078] RPA-FluA-F: TCATATACAACAGGATGGGGGCTGTGACCACCGAA (SEQ ID NO.10)

[0079] RPA-FluA-R:CACTCGATCCAGCCATTTGCTCCATAGCCTTAGCT(SEQ ID NO.11)

[0080] RPA-FluB-F: GTCCAAAAACTAGCAGAAGAGCTGCAAAACAAC (SEQ ID NO.12)

[0081] RPA-FluB-R: AACAAATTGAAAGAATCTGAAGTGGTTCGAGCA (SEQ ID NO.13)

[0082] RPA-ADV-F: CCCAATTACATTGGATTCAGGGATAACTTTGTAGG(SEQ ID NO.14)

[0083] RPA-ADV-R: CTGATTCCACATGCTGAAGTATCTGGTTCTGTCTC (SEQ ID NO.15)

[0084] RPA-RSV-F:CTATGTGGTATGCTATTAATCACTGAAGATGC(SEQ ID NO.16)

[0085] RPA-RSV-R:GCTAATGGAGTAGATATAACAACATATCGTC(SEQ ID NO.17)

[0086] 3. The amplified product of RPA is used as an activator and combined with CRISPR to detect the DNA hydrogel pathogen nucleic acid detection platform. The preparation method of this pathogen nucleic acid detection platform is as follows: 10 μL CRISPR / Cas system (3×NEBuffer TM r2.1, 0.15μM LbCas12a, 0.15μM target-specific sgRNA-M: SEQ ID NO.19: UAAUUUCUACUAAGUGUAGAUCCGACUCCCAGCAUAAGUCU), 2μL of RPA amplification product, 10μL of starch solution (10mg / mL), and 10μL of DNA hydrogel were mixed and incubated at room temperature for 15 minutes. 5μL of the mixture was taken and the glucose concentration was measured with a blood glucose meter. The test results are shown in the attached Figure 6 As shown, it contains 10 1 Up to 10 5 Clear glucose readings were obtained for samples containing 100 copies of influenza A virus M gene target RNA, demonstrating the high sensitivity of the detection platform.

[0087] Using synthetic influenza virus A as a target (10 3 The amplified products of novel coronavirus (CoV-2), influenza B virus (FluB), adenovirus (ADV) and respiratory syncytial virus (RSV) (all 10 5 The test results are shown in the attached Figure 7As shown, only the positive control showed corresponding glucose readings, while other pathogens presented negative results. These results confirmed the good specificity of the proposed platform, which was attributed to the highly specific target recognition ability of the CRISPR / Cas system.

[0088] The experimental results show that the proposed detection platform can be used for FluA detection with high sensitivity and specificity. The versatility of the detection platform has been verified. The platform we proposed can be applied to other pathogen nucleic acid detection by simply replacing the corresponding amplification products and sgRNA of the CRISPR / Cas system.

Claims

1. A DNA hydrogel loaded with amylase, characterized in that: The hydrogel skeleton is a single-stranded DNA that contains methylated cytosine bases. The single-stranded DNA is cross-linked through high-affinity DNA methylation binding proteins to form a hollow network structure, and amylase is wrapped inside the hydrogel.

2. A method for preparing the amylase-loaded DNA hydrogel according to claim 1, characterized in that: The following steps are involved: (1) Phosphorylate the 5' end of a linear template sequence using T4 polynucleotide kinase and form circular DNA under the action of T4 DNA ligase; (2) The circular DNA described in step (1) is used as an RCA template for amplification, S-adenosylmethionine is used as a methyl donor, and methyl groups are selectively added to the cytosine of the two CG nucleotides in the amplified product, so that the CG sequence position is located on the stem of the stem-loop structure. The amplified product is bound to the tetrameric DNA methylation binding protein after the action of DNA methyltransferase, and finally forms a DNA hydrogel with a spatial network structure. Amylase is added during the formation of the DNA hydrogel to achieve the encapsulation of the amylase, thereby obtaining the DNA hydrogel loaded with amylase.

3. The method according to claim 2, characterized in that: The tetrameric DNA methylation binding protein in step (2) includes SA-MBD protein.

4. Use of the amylase-loaded DNA hydrogel according to claim 1 in preparing a pathogen nucleic acid detection reagent or kit.

5. The application according to claim 4, characterized in that: The following steps are involved: The product of pre-amplification of pathogen nucleic acid was added to the targeted CRISPR / Cas reaction solution, DNA hydrogel and starch for reaction. After the reaction, it was tested using a blood glucose meter.

6. A reagent or kit for detecting pathogen nucleic acid, characterized in that: The invention comprises the DNA hydrogel loaded with amylase as claimed in claim 1.

7. A method for response activation of a CRISPR-Cas system, characterized in that The amylase-loaded DNA hydrogel according to claim 1 is combined with the CRISPR-Cas system.

8. Use of the amylase-loaded DNA hydrogel according to claim 1, the reagent or kit according to claim 6, or the CRISPR-Cas system according to claim 7 in the detection of new coronavirus and influenza A virus.

9. The use according to claim 8, characterized in that: The novel coronavirus and influenza A virus detection includes pathogen nucleic acid detection.

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