Proximity nucleic acid probe for detecting norovirus and application thereof

Abstract

The invention belongs to the technical field of food-borne pathogenic microorganism detection, and particularly relates to an ortho-position nucleic acid probe for detecting norovirus and application of the ortho-position nucleic acid probe. The invention provides an ortho-position nucleic acid probe for identifying norovirus, the ortho-position nucleic acid probe specifically comprises a polystyrene nanoprobe and a single-chain binding protein coupling probe, and the polystyrene nanoprobe is formed by covalent binding of polystyrene microspheres, lectin and hairpin DNA. The single-chain binding protein coupling probe is formed by combining a norovirus antibody and a nucleic acid molecule through a connecting molecule. The probe is simple in synthesis condition and does not need complex purification steps. According to the present invention, a combined reaction with an activation chain, a hybridization chain reaction reagent and a catalytic self-assembly reagent is performed, and the detection sensitivity is improved through a signal cascade amplification reaction, and can achieve the minimum of 0.15 pg mL <-1 >; and moreover, the gene has excellent specificity, and the delta F'difference between the gene and pathogens of staphylococcus aureus, escherichia coli, rotavirus and astrovirus is extremely remarkable.

Description

Technical Field

[0001] The present invention belongs to the technical field of foodborne pathogenic microorganism detection, and in particular relates to an adjacent nucleic acid probe for detecting norovirus and an application thereof. Background Art

[0002] Norovirus is a major foodborne pathogen of the human gastrointestinal tract. It is a single-stranded RNA virus with clinical manifestations of vomiting, abdominal pain and diarrhea. The infection route is through direct or indirect contact with food, feces and vomitus, and the viral load in contaminated food is low, which brings certain difficulties to detection and genotype identification.

[0003] The Norovirus currently involved can be divided into GI, GII, GIII, GIV and GV according to the genotype, with GII.4 being the most common. Detection methods mainly include electron microscopy, immunological detection methods, molecular biology detection methods and biosensors. Real-time PCR has high sensitivity and specificity in detection and can be used as a standard method for detecting Norovirus in food matrices (such as shellfish), but it has extremely strict requirements for operating instruments and processes, and some require professional operation to complete. Therefore, flexible and diverse biosensor methods are designed and used for the detection of Norovirus. The combination of different biorecognition molecules and nanoprobes, and the use of intelligent detection equipment can further improve the detection throughput, sensitivity and specificity.

[0004] Proximity ligation analysis is a highly sensitive detection method based on specific probes. It was originally designed by Fredriksson et al. and applied to the detection of protein molecules. Its working principle mainly relies on the proximity probe molecules to simultaneously recognize and bind to the target substance, triggering the reaction through the interaction between nucleic acid molecules in a limited space. This reaction can be combined with other detection technologies and applied to the detection of downstream targets. Such technologies usually do not require washing or separation steps, thereby reducing the detection time of traditional immunoassays and simplifying the experimental operation process. In addition, by combining various signal amplification strategies, this technology can achieve ultra-high sensitivity detection of the object to be detected, with a sensitivity 1000 times higher than that of traditional ELISA, and significantly improved specificity compared with immune PCR technology. However, the overall design and implementation process of this method is still relatively complex, especially the design and synthesis of proximity nucleic acid probes, and complex samples may trigger some non-specific interference. This has affected the widespread application of this technology to a certain extent. Summary of the invention

[0005] The invention aims to at least solve the problem of matching application of specific probe synthesis preparation and cyclic fluorescence device in the prior art. To this end, the purpose of the present invention is to provide a set of ortho-nucleic acid probes and signal amplification reagents, which can simultaneously identify norovirus through a pair of ortho-nucleic acid probe molecules, and can be combined with nucleic acid probe reagents that can be used for hybridization chain reaction / catalytic self-assembly technology to detect norovirus through the applicant's existing cyclic fluorescence detection system.

[0006] The purpose of the first aspect of the present invention is to provide a proximity nucleic acid probe for identifying Norovirus.

[0007] The purpose of the second aspect of the present invention is to provide a reagent for detecting Norovirus.

[0008] The purpose of the third aspect of the present invention is to provide the use of the adjacent nucleic acid probe of the first aspect of the present invention and the reagent of the second aspect in the preparation of a product for detecting Norovirus.

[0009] The fourth aspect of the present invention aims to provide a product for detecting Norovirus.

[0010] In order to achieve the above-mentioned purpose of the present invention, the technical solution adopted by the present invention is:

[0011] The first aspect of the present invention provides an adjacent nucleic acid probe for identifying norovirus, comprising a polystyrene nanoprobe and a single-stranded binding protein-coupled probe.

[0012] In some embodiments of the present invention, the polystyrene nanoprobe comprises a Norovirus recognition molecule, a nucleic acid molecule A and polystyrene microspheres.

[0013] In some embodiments of the present invention, the sequence of the nucleic acid molecule A is shown in SEQ ID NO: 1. The nucleic acid molecule A can form a hairpin structure.

[0014] In some embodiments of the present invention, the Norovirus recognition molecule and nucleic acid molecule A are coupled to the surface of polystyrene microspheres.

[0015] In some embodiments of the present invention, the Norovirus recognition molecule in the polystyrene nanoprobe includes at least one of a lectin, a nucleic acid aptamer, and an antibody.

[0016] In some embodiments of the present invention, the norovirus recognition molecule is a lectin.

[0017] In some embodiments of the present invention, the lectin includes concanavalin A (ConA), lentil agglutinin (LCA), pea agglutinin (PSA), aurantiacus aurantiacus agglutinin (AAL), urea oleifera agglutinin I (UEA-I), wheat germ agglutinin (WGA), tomato agglutinin (LEL), potato agglutinin (STL), Datura stramonium agglutinin (DSL), succinylated wheat germ agglutinin (Succinylated At least one of WGA, Griffonia suffruticosa lectin II (GSL-II), PHA-E, PHA-L, Ricinus communis lectin I (RCA-I), Erythrina indica lectin (ECL), Jacalin, Peanut agglutinin (PNA), Dolichos biflorus lectin (DBA), Soybean agglutinin (SBA), Vicia villosum lectin (VVL), Griffonia suffruticosa lectin I (GSL-I), and Sambucus nigra lectin (SNL); preferably Concanavalina lectin A (ConA).

[0018] In some embodiments of the present invention, the polystyrene microspheres are carboxylated.

[0019] In some embodiments of the present invention, the diameter of the polystyrene microspheres is 50 to 200 nm.

[0020] In some embodiments of the present invention, the Norovirus recognition molecule and nucleic acid molecule A are coupled to the surface of polystyrene microspheres.

[0021] In some embodiments of the present invention, the coupling method is selected from one of streptavidin-biotin and EDC / NHS; preferably, EDC / NHS.

[0022] In some embodiments of the present invention, the single-stranded binding protein-coupled probe includes a Norovirus antibody, a linker molecule, and a nucleic acid molecule B.

[0023] In some embodiments of the present invention, the Norovirus antibody is linked to the single-chain binding protein via a linker molecule.

[0024] In some embodiments of the present invention, the sequence of the nucleic acid molecule A is shown in SEQ ID NO: 2. The nucleic acid molecule B can form a hairpin structure, and has a specific single-stranded DNA connection domain (5'-AAGTATTACCAGAAA-3') at the 5' end.

[0025] In some embodiments of the present invention, the 5' end of the nucleic acid molecule A has an amino modification.

[0026] In some embodiments of the present invention, the norovirus antibody is a commercial antibody Rabbit Anti-Norovirus Capsid protein VP1 antibody (Beijing Bioson Biotechnology Co., Ltd., catalog number: bs-19320R). The specific selection of the antibody does not affect the implementation of the present invention, and other commercial norovirus antibodies in the field or other public antibodies that can bind to norovirus do not constitute a limitation of the present invention.

[0027] In some embodiments of the present invention, the Norovirus antibody is linked to the single-chain binding protein via a linker molecule.

[0028] In some embodiments of the present invention, the linking molecule is an AND Linker or a click chemistry reaction; preferably an AND Linker.

[0029] AND Linker is a bifunctional fusion protein having an Fc linker domain and a single-stranded DNA linker domain, wherein the Fc linker domain can bind to the Fab region of the target antibody, and the single-stranded DNA linker domain binds to the target nucleic acid molecule.

[0030] The second aspect of the present invention provides a reagent for detecting Norovirus, comprising the proximity nucleic acid probe described in the first aspect of the present invention.

[0031] In some embodiments of the present invention, the reagents further include cascade signal amplification reagents; the cascade signal amplification reagents include activation chains, hybridization chain reaction reagents, and catalytic self-assembly reagents.

[0032] In some embodiments of the present invention, the nucleic acid sequence of the activation chain is shown in SEQ ID NO:3.

[0033] In some embodiments of the present invention, the hybridization chain reaction reagent includes four hairpin nucleic acids H1, H2, H3 and H4.

[0034] In some embodiments of the present invention, the nucleic acid sequences of H1, H2, H3 and H4 are shown in SEQ ID NOs: 4 to 7, respectively.

[0035] In some embodiments of the present invention, the catalytic self-assembly reagent includes two hairpin nucleic acids H5 and H6.

[0036] In some embodiments of the present invention, the nucleic acid sequences of H5 and H6 are shown in SEQ ID NOs: 8 to 9, respectively.

[0037] In some embodiments of the present invention, the two ends of the hairpin nucleic acid H3 are modified with a quenching group and a fluorescent group, respectively.

[0038] In some embodiments of the present invention, the fluorescent group is selected from any one of FAM, CY3, CY5, HTX, ROX, VIC, TAMRA and HEX; preferably, it is CY3.

[0039] In some embodiments of the present invention, the quenching group is selected from any one of BHQ1, BHQ2 and BHQ3; preferably, it is BHQ-2.

[0040] In the second aspect of the present invention, under the condition of the presence of norovirus, the polystyrene nanoprobe and the single-stranded protein-coupled probe are triggered in the adjacent position and generate the Activator / P-1 / P-2 composite structure under the condition of the presence of the activation chain (Activator), and the oligonucleotide chain released by the structure can be used as the starter chain I of the hybridization chain reaction (HCR) and the catalytic self-assembly (CHA) reaction. HCR amplification is completed by four hairpin structures (H1, H2, H3 and H4), and the end of H3 is modified with Cy3 and BHQ-2 molecules respectively. When in the hairpin structure, since Cy3 and BHQ-2 are close to each other, the fluorescence is in a quenched state due to the effect of fluorescence resonance energy transfer. When the starter chain I triggers the HCR hybridization reaction and opens the hairpin structure of H3, the fluorescence is restored. A specific hybridization reaction will occur between the hairpin DNAs, triggering signal amplification. This amplification process forms a long repetitive double-stranded DNA (H1-H2-H3-H4)n through continuous opening and hybridization of hairpin DNA. The repeating unit exposes the T-terminal nucleic acid fragments that can induce CHA to form a long double-stranded H5-H6 structure. The end of this structure contains the initiator chain I that triggers HCR amplification, thus promoting the continuous generation of the initiator chain, thereby ensuring the cyclic exponential amplification of the reaction.

[0041] The third aspect of the present invention provides the use of the proximity nucleic acid probe of the first aspect of the present invention and the reagent of the second aspect in the preparation of a product for detecting Norovirus.

[0042] The fourth aspect of the present invention provides a kit for detecting norovirus, comprising the reagent described in the second aspect of the present invention.

[0043] In some embodiments of the present invention, the kit further comprises a buffer.

[0044] The fifth aspect of the present invention provides a method for using the kit according to the fourth aspect of the present invention, comprising the following steps:

[0045] 1) mixing the polystyrene nanoprobe, the single-stranded binding protein-coupled probe and the sample to be tested and incubating;

[0046] 2) adding activation chain, hybridization chain reaction reagent, catalytic self-assembly reagent, mixing, and incubating;

[0047] 3) Detect the fluorescence signal.

[0048] In some embodiments of the present invention, the incubation time is 15 to 60 minutes, and the incubation temperature is 25 to 40°C.

[0049] In some embodiments of the present invention, the mass ratio of the polystyrene nanoprobe to the single-stranded binding protein-coupled probe is (5-20):1.

[0050] The beneficial effects of the present invention are:

[0051] The present invention provides an adjacent nucleic acid probe for identifying norovirus, including a single-stranded protein-coupled probe and a polystyrene nanoprobe. The probe has simple synthesis conditions and does not require complex purification steps. The probe can react with an activation chain, a hybridization chain reaction reagent, and a catalytic self-assembly reagent to improve the sensitivity of detection through a signal cascade amplification reaction, and the sensitivity can be as low as 0.15 pg mL -1 It also has excellent specificity, with significant differences in ΔF′ between pathogens such as Staphylococcus aureus, Escherichia coli, rotavirus, and astrovirus. BRIEF DESCRIPTION OF THE DRAWINGS

[0052] Figure 1 To detect Norovirus in food based on the principle of HCR / CHA cyclic amplification triggered by nucleic acid proximity ligation technology; (A) Preparation of Norovirus recognition probe ConA@PS@P-1; (B) Preparation of Rabbit Anti-Norovirus Capsid proteinVP1antibody@AND linker@P-2 recognition probe; (C) Norovirus proximity recognition and HCR / CHA cyclic amplification detection process.

[0053] Figure 2 The SEM characterization results of ConA@PS@P-1; A is the electron micrograph of carboxylated polystyrene microspheres (particle size is about 200nm); B is the electron micrograph of polystyrene microsphere probes modified with lectin A alone; C is the electron micrograph of nucleic acid P-1 alone; D is the electron micrograph of both lectin A and nucleic acid P-1 chains modified simultaneously.

[0054] Figure 3 Schematic diagram of Norovirus antibody conjugation to ssDNA and SDS-PAGE (10%, w / w) electrophoresis characterization; (A) Schematic diagram of ANDlinker bifunctional fusion protein conjugation to antibody and ssDNA; (B) SDS-PAGE protein electrophoresis characterization of Rabbit Anti-Norovirus Capsidprotein VP1 antibody@AND linker@P-2.

[0055] Figure 4 Schematic diagram of the structure of the proximity nucleic acid probe and the amplification process; (A) The lectin-modified probe P-1 and the antibody-modified P-2 simultaneously recognize norovirus, triggering the confined proximity of P-1 and P-2; (B) The activating oligonucleotide Activator binds to the circular part of P-1; (C) The site released after P-1 binds to the circular site of P-2 serves as the starting activator for the HCR reaction; (D and E) The HCR amplification is initiated by four hairpin structures H1, H2, H3 and H4, and double-stranded DNA nanowires are generated through sequential cross-hybridization reactions between HCR hairpins; (F) CHA amplification triggers cross-hybridization between H5 and H6, releasing the starting activator I for the new HCR reaction.

[0056] Figure 5 Time series fluorescence spectra (A) and standard curves (B) of proximity ligation analysis based on ConA@PS@P-1 probe and Rabbit Anti-Norovirus Capsid proteinVP1antibody@AND linker@P-2 probe for the detection of Norovirus GII.

[0057] Figure 6 Cyclic fluorescence spectra of norovirus GII in oyster-1 (A) and oyster-2 (B) based on NPLA-HCA. Lines 1, 2, and 3 represent blank, actual sample, and standard sample (10 pg g -1 and 0.5 pg g -1 )'s cyclic fluorescence spectrum.

[0058] Figure 7 NPLA-HCA is used in the selective test of norovirus GII detection (the concentration is 0.5 pg mL -1 ). A is the time series fluorescence spectra of different analytes; B is the ratio ΔF′ results of different analytes. DETAILED DESCRIPTION

[0059] The following will be combined with the embodiments to clearly and completely describe the concept of the present invention and the technical effects produced, so as to fully understand the purpose, characteristics and effects of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, other embodiments obtained by those skilled in the art without creative work are all within the scope of protection of the present invention.

[0060] Terminology Note:

[0061] ConA@PS@P-1 represents polystyrene microsphere probe;

[0062] Rabbit Anti-Norovirus Capsid protein VP1 antibody@AND linker@P-2 indicates a single-stranded binding protein probe;

[0063] EDC is 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride;

[0064] NHS is N-hydroxysuccinimide.

[0065] Cy3 is Cyanine 3.

[0066] BHQ-2 stands for Black Hole Quencher-2.

[0067] Table 1 Sequence information

[0068]

[0069]

[0070] in Figure 1 It is a schematic diagram of the process of the present invention.

[0071] Example 1 Synthesis of polystyrene nanoprobe ConA@PS@P-1

[0072] 1) Add 2.0 mg of carboxylated polystyrene microspheres (PS) into a 1.5 mL centrifuge tube and add 500 μL of MEST (10 mmol·L -1 Morpholineethanesulfonic acid (MES), 0.05% (w / w) Tween-20) was vortexed for 30 seconds and centrifuged at 8000 rpm for 10 minutes to remove the supernatant. This step was repeated twice.

[0073] 2) Resuspend the washed material in 850 μL MES, 100 μL EDC (5 mg mL -1 ) and 50 μL NHS (5 mg mL -1 The reaction solution was incubated at 37°C for 60 min and then washed with MEST buffer. The supernatant was removed after centrifugation at 8000 rpm for 10 min. This step was repeated three times.

[0074] 3) Add 100 μg ConA (concanavalin A, purchased from Shanghai Shenggong Biotechnology Co., Ltd.) and 1 μmol·L -1 , 20 μL 5'-terminal amino-modified P-1 chain (Note: denatured at 95°C for 5 min before use, then cooled to room temperature before use, SEQ ID NO: 2), add 0.01 mol·L -1PBS buffer was made up to 1 mL. The reaction mixture was also placed in a 37°C constant temperature shaker for 1 h. After the reaction was completed, the supernatant was removed after centrifugation at 9000 rpm for 10 min to remove unbound ConA and P-1 chain substances, and then washed 3 times with 1 mL PBST.

[0075] 4) The prepared ConA@PS@P-1 probe was resuspended in 1 mL 0.01 mol·L-1 PBS buffer (containing 0.02% NaN3, 0.5% BSA) and stored at 4°C for future use.

[0076] The same method was used to prepare polystyrene microsphere probes modified with lectin A alone and polystyrene microsphere probes modified with nucleic acid P-1 alone. The carboxylated polystyrene microspheres, polystyrene microsphere probes modified with lectin A alone, polystyrene microsphere probes modified with nucleic acid P-1 alone, and ConA@PS@P-1 probes were subjected to SEM characterization experiments. The results are shown in Figure 2 shown.

[0077] Example 2 Preparation of single-stranded protein-coupled probe Rabbit Anti-Norovirus Capsid protein VP1antibody@AND linker@P-2

[0078] 1) P-2 nucleic acid chain (SEQ ID NO: 2) was treated with 0.01 mol·L -1 Dissolve in PBS buffer to 100 μmol·L -1 , denature at 95°C for 5 min, and then cool to room temperature.

[0079] 2) Absorb 100 μmol·L -1 2 μL of P-2 nucleic acid chain was added to an enzyme-free sterilized centrifuge tube, followed by the addition of 1 mg mL -1 ANDlinker 1.8 μL (B&K Technology Co., Ltd., China) and ligation buffer (100 mmol·L -1 HEPES, 300mmol·L -1 NaCl, 2mmol·L -1 MgCl2,2mmol·L -1 MnCl2), and finally dd H2O was added to obtain a final reaction volume of 20 μL. After mixing, the mixture was reacted at 37°C for 30 min.

[0080] 3) After the reaction is complete, draw 12 μL of 1 mg·mL -1Rabbit Anti-Norovirus Capsid proteinVP1 antibody (Beijing Bioson Biotechnology Co., Ltd., Catalog No.: bs-19320R) was added, mixed and reacted at 37°C for 30 minutes.

[0081] 4) After the above reaction is completed, the reaction solution is separated and purified using a SpinOUT GT-1200 column:

[0082] a. The sample loading volume is controlled at 20% of the resin bed volume;

[0083] b. Mark one side of the column and ensure that the mark faces outward during all centrifugation procedures.

[0084] c. Remove the SpinOUT column and centrifuge at 1000 g for 1 min to ensure that the resin bed is tightly packed.

[0085] d. Remove the sealing caps at both ends and place them in a collection centrifuge tube;

[0086] e. Centrifuge at 1000 g for 2 min to remove the retained buffer;

[0087] f. Accurately pipette 200 μL of the reaction solution into the centrifugal separation column, and centrifuge at 1000 g for 4 minutes to collect the Rabbit Anti-Norovirus Capsid protein VP1 antibody@AND linker@P-2 protein-nucleic acid complex at the bottom.

[0088] The end of the P-2 nucleic acid chain used in the probe contains a 5'-AAGTATTACCAGAAA-3' (SEQ ID NO: 10) fragment, which can bind to the specific binding domain of AND linker (react with the 5'-phosphate group after cleavage). The Fc binding domain in the ANDlinker protein can specifically connect to the Fc functional region of the antibody. AND linker also contains a special nucleic acid binding site protein. Therefore, modifying AAGTATTACCAGAAA at the 5' end of the P-2 chain can achieve coupling between P-2 and AND linker, and finally form an antibody-single-stranded DNA complex probe with a stoichiometric ratio of 1:1. This one-step coupling method can achieve rapid and efficient biological coupling of antibodies and nucleic acids, while maintaining the activity of the biological probe.

[0089] The schematic diagram of the Norovirus antibody conjugation to ssDNA in this example is as follows Figure 3 As shown in A.

[0090] Figure 3B in the middle is SDS-PAGE (10%, w / w) electrophoresis characterization. From right to left, lane 4 is the protein Maker (standard range: 18.4kDa-116kDa), lane 3 is AND LINKER, lane 2 is the Rabbit Anti-Norovirus Capsidprotein VP1 antibody@AND linker@P-2 complex, and lane 1 is the Rabbit anti-Novovius VP1antibody. Multiple protein bands with different molecular weights can be observed in the Rabbit Anti-Norovirus Capsid protein VP1 antibody@AND linker@P-2 column, and the migration distance is shorter than that of lanes 1 and 3, indicating that the molecular weight is relatively large, indirectly indicating that the Rabbit Anti-Norovirus Capsid protein VP1 antibody@AND linker@P-2 complex was successfully prepared.

[0091] Example 3 Cascade signal amplification

[0092] In order to improve the reaction limit, this example proposes a hybridization chain reaction (HCR) and catalytic self-assembly (CHA) cascade signal amplification strategy (abbreviated as NPLA-HCA) of proximity ligation technology. The details are as follows:

[0093] When norovirus is present, ConA@PS@P-1 and Rabbit Anti-Norovirus Capsidprotein VP1antibody@AND linker@P-2 are adjacently triggered and generate an Activator / P-1 / P-2 composite structure in the presence of an activator chain. The Activator chain releases the stem and loop sequences of P-1 by hybridization and pairing with the P-1 chain. At this time, due to its close spatial position to P-2, it can quickly activate the opening of the P-2 hairpin structure. The terminal oligonucleotide chain released by the hybridization of P-1 and P-2 can be used as the subsequent initiator activator I for triggering HCR and CHA. HCR amplification is jointly completed by four hairpin structures (H1, H2, H3 and H4), in which the cleavage fragments of the CHA-catalyzed hairpin self-assembly triggering site (T) are grafted on the 5' end of H2 and the 3' end of H4, respectively. This design ensures that these hairpin structures are in a stable state of secondary structure in the absence of initiator activators. The generation of the starter activation chain is triggered by the adjacent nucleic acid reaction, and a long double-stranded DNA nanowire is generated by the sequential cross-hybridization reaction between the HCR hairpins, and a large number of new CHA trigger sites are generated to activate CHA. CHA catalysis is designed by two hairpins (sequence reference H5 and H6 in Table 1), and its function is to use T to trigger the cross-hybridization between H5 and H6, thereby releasing a new HCR initial activator I, which in turn activates HCR amplification again. Therefore, the continuous generation of the starter chain can be promoted, thereby ensuring the cyclic exponential amplification of the reaction.

[0094] The schematic diagram of the amplification process is as follows Figure 4 shown.

[0095] Example 4 Norovirus Detection Effect Test

[0096] This embodiment provides a method for detecting norovirus by triggering HCR / CHA cyclic amplification reaction using nucleic acid proximity ligation technology, and the specific steps are as follows:

[0097] 1) First, all DNA chains were dissolved in ultrapure water to prepare 50 μmol·L -1 The stock solution was stored at -20°C for later use. During use, the DNA strands were dissolved in 1.0 mol·L -1 NaCl, 50mmol·L -1 MgCl2, 10mmol·L -1 In order to ensure that the DNA chain can be correctly folded into a stable hairpin structure, it is annealed in the reaction buffer, which is first heated at 95°C for 5 minutes and then kept at 25°C for 2 hours.

[0098] 2) 1 μg·mL -1 ConA@PS@P-1 (40 μL) and inactivated Norovirus-like particles at different concentrations (0, 0.5, 1, 2, 5, 10, 20, 50 pg / mL) were mixed (20 μL), and then freshly prepared 0.1 μg mL -1 Rabbit Anti-Norovirus Capsid protein VP1 antibody@AND linker@P-2 (40μL) probe was incubated and reacted at 37°C for 15 minutes.

[0099] 3) Then, the NPLA-HCA hairpin mixture (H1-H6 concentration 200nmol·L -1 , added volume 20μL), Activator chain (20nmol·L -1 , added in an amount of 20 μL) and mixed with the reaction system of step (2), the final reaction volume was 220 μL, and the reaction was carried out at 37°C for 12 min.

[0100] 4) The above reaction unit was injected into the online cyclic fluorescence system through the injection valve to collect the signal of the NPLA-HCA process, which lasted for 20 minutes at 25°C. The excitation wavelength was 550nm and the emission wavelength was 570nm, and the instrument was set to time scan mode. The final result was solved using the relative fluorescence ratio:

[0101] ΔF ′ =(F n -F1) / F0 (1)

[0102] Among them, F n is the fluorescence intensity of the sample passing through the detection cell for the Nth time during the reciprocating cycle (n is taken as 5 during the experiment), F1 is the fluorescence intensity of the sample when it first enters the detection area, and F0 is the fluorescence intensity corresponding to the blank sample.

[0103] The results are as follows Figure 5 As shown in the figure, the time fluorescence sequence of Rabbit Anti-Norovirus Capsidprotein VP1antibody@AND linker@P-2 / Norovirus GII / ConA@PS@P-1 and HCR / CHA triggered at different Norovirus GII concentrations. As the concentration of the analyte increases, the ratio of the signal intensity ΔF′ between the initial cycle and the end of the reaction increases, which is consistent with the principle of NPLA-HCA constructed by this circulation system. -1 Within the concentration range of norovirus GII, ΔF′ showed a linear relationship with the concentration of norovirus GII (R 2=0.9985), the linear equation is ΔF′=1.84C′+4.37(C′=log2C), and the detection limit (S / N=3) is 0.15pg·mL -1 .

[0104] Example 5 Norovirus detection effect test in food

[0105] 1) Take 1.00g of oyster samples (two inactivated samples provided by the CDC, numbered 1 and 2) and mix them with 9mL of Hank's Balanced Salt Solution (1×) to dilute the norovirus sample at a volume ratio of 1:9 (w / w). Then, vortex vigorously and centrifuge the suspension at 7000g for 10min at 4°C. Then carefully extract the supernatant, dilute to 1mL and store at -80°C for later use.

[0106] 2) Prepare a series of gradient concentrations of Norovirus GII-like particles, with the injection concentration range of 0.2 pg mL -1 -200pgmL -1 , respectively with 1 μg mL -1 ConA@PS@P-1 (40 μL) and Rabbit Anti-Norovirus Capsidprotein VP1antibody@AND linker@P-2 (40 μL) were mixed and incubated at 37°C for 15 min.

[0107] 3) Add NPLA-HCA hairpin mixture (H1-H6 concentration 200nmol·L -1 , added volume 20μL) and Activator chain (20nmol·L -1 , added in an amount of 20 μL), after complete mixing, the samples were injected into the cyclic fluorescence detection system respectively. The control sequence flow of the solenoid valve and the peristaltic pump is shown in Table 2. After the injection is completed, all modules are started to run the instructions uniformly to complete the batch acquisition of the fluorescence sequence. It was carried out at an excitation wavelength of 550 nm and an emission wavelength of 570 nm. The instrument was set to time scan mode with an acquisition time of 12 min. The final result was solved using the relative fluorescence ratio, and the concentration of norovirus in the sample was solved by the equation. Finally, the cyclic fluorescence spectrum of the actual sample oyster was detected as shown in the figure Figure 6 Norovirus was detected in two oyster samples, with the positive samples containing 12.67 pg·g -1 and 0.68 pg·g -1 The relative errors of the above two positive sample results were 2.6% and 10.5%, indicating that the NPLA-HCA detection method combined with the CAFA detection device established in this study has high accuracy.

[0108] Table 2 Control operation sequence of online cycle fluorescence device

[0109]

[0110]

[0111] Example 5 Specificity Test

[0112] 1) First, all DNA chains were dissolved in ultrapure water to prepare 50 μmol·L -1 The stock solution was stored at -20°C for later use. During use, the DNA strands were dissolved in 1.0 mol·L -1 NaCl, 50mmol·L -1 MgCl2,10mmol·L -1 In order to ensure that the DNA chain can be correctly folded into a stable hairpin structure, it is annealed in the reaction buffer, which is first heated at 95°C for 5 minutes and then kept at 25°C for 2 hours.

[0113] 2) Norovirus GII, a mixture containing norovirus GII (rotavirus and astrovirus), Staphylococcus aureus, Escherichia coli, rotavirus, and astrovirus (all at a concentration of 0.5 pg mL -1 ) and other samples (20 μL each) were added with 1 μg mL -1 ConA@PS@P-1 (40 μL) was mixed (20 μL), and then freshly prepared 0.1 μg mL -1 Rabbit Anti-Norovirus Capsid protein VP1 antibody@AND linker@P-2(40μL)probes were incubated together and reacted at 37℃ for 15min.

[0114] 3) Then, the NPLA-HCA hairpin mixture (H1-H6 concentration 200nmol·L -1 , added volume 20μL), Activator chain (20nmol·L -1 , added in an amount of 20 μL) and mixed with the reaction system of step (2), the final reaction volume was 220 μL, and the reaction was carried out at 37°C for 12 min.

[0115] 4) The above reaction unit was injected into the online cyclic fluorescence system through the injection valve to collect the signal of the NPLA-HCA process, which lasted for 20 minutes at 25° C. The excitation wavelength was 550 nm and the emission wavelength was 570 nm, and the instrument was set to time scan mode.

[0116] The results of ΔF′ are as follows Figure 7 As shown in the figure, there was no significant difference between Norovirus GII and the mixture containing Norovirus GII (with rotavirus and astrovirus added) through Tukey test, while the ΔF′ difference between the pathogens of Staphylococcus aureus, Escherichia coli, rotavirus and astrovirus was extremely significant (p<0.001), further indicating that the established NPLA-HCA method has good selectivity for Norovirus GII, and Rabbit Anti-Norovirus Capsid protein VP1antibody has a high selectivity through single-stranded DNA-antibody Fc-terminal binding protein.

Claims

1. A proximity nucleic acid probe for identifying norovirus, characterized in that: These include polystyrene nanoprobes and single-stranded binding protein-coupled probes; The polystyrene nanoprobe comprises a norovirus recognition molecule, a nucleic acid molecule A and polystyrene microspheres; The sequence of the nucleic acid molecule A is shown in SEQ ID NO: 1; The norovirus recognition molecule and nucleic acid molecule A are coupled to the surface of the polystyrene microspheres; The single-stranded protein-coupled probe comprises a Norovirus antibody, a linker molecule, and a nucleic acid molecule B; The norovirus antibody is connected to the single-chain binding protein via a linker molecule; The sequence of the nucleic acid molecule B is shown in SEQ ID NO:

2.

2. The proximity nucleic acid probe according to claim 1, characterized in that: The norovirus recognition molecule in the polystyrene nanoprobe includes at least one of a lectin, a nucleic acid aptamer, and an antibody.

3. The proximity nucleic acid probe according to claim 1, characterized in that: The coupling method is selected from one of streptavidin-biotin and EDC / NHS.

4. The proximity nucleic acid probe according to claim 1, characterized in that: The connecting molecule in the single-stranded binding protein coupled probe is AND Linker.

5. A reagent for detecting norovirus, characterized in that: The method comprises the proximity nucleic acid probe according to any one of claims 1 to 4.

6. The reagent according to claim 5, characterized in that: The reagents include cascade signal amplification reagents; The cascade signal amplification reagents include activation chains, hybridization chain reaction reagents, and catalytic self-assembly reagents; The nucleic acid sequence of the activation chain is shown in SEQ ID NO: 3; The hybridization chain reaction reagent includes four hairpin nucleic acids H1, H2, H3 and H4; The nucleic acid sequences of H1, H2, H3 and H4 are shown in SEQ ID NOs: 4 to 7, respectively; The catalytic self-assembly reagent includes two hairpin nucleic acids H5 and H6; The nucleic acid sequences of H5 and H6 are shown in SEQ ID NOs: 8 to 9, respectively.

7. The reagent according to claim 6, characterized in that: The two ends of the hairpin nucleic acid H3 are modified with a quenching group and a fluorescent group respectively.

8. Use of the adjacent nucleic acid probe according to any one of claims 1 to 4 and / or the reagent according to any one of claims 5 to 7 in the preparation of a product for detecting norovirus.

9. A kit for detecting norovirus, comprising the reagent according to any one of claims 5 to 7.

10. The kit according to claim 9, characterized in that: The kit includes a buffer.

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