Virus tracer with protein capsid and nucleic acid layer and preparation method and application thereof

Abstract

The present invention provides a virus tracer with a protein capsid and a nucleic acid layer, and a preparation method and application thereof. The DNA virus tracer is composed of an inner core, a nucleic acid layer, and a protein capsid wrapped outside the nucleic acid layer; the inner core is a SiO2 nanosphere with controllable particle size, and its surface is amino-modified, and a layer of DNA is covalently coupled with GA to form a nucleic acid layer, and then one or more layers of protein are covalently coupled with GA to form a protein capsid. The new DNA virus tracer provided by the present invention can effectively ensure that the outermost capsid of the tracer is completely composed of the selected protein, so that the surface characteristics and migration characteristics of the tracer can be closer to the target virus, and it can also control the type and number of layers of protein to make it close to the half-life of the target virus.

Description

Technical Field

[0001] The invention belongs to the field of environmental protection, and in particular relates to a virus tracer with a protein capsid and a nucleic acid layer, and a preparation method and application thereof. Background Art

[0002] The basic structure of a virus includes a capsid made of protein and protected nucleic acid (DNA / RNA) enclosed in the capsid. The properties of the virus's capsid protein play a decisive role in its migration properties. Tracers used as virus substitutes should also try to make the surface properties similar to those of the target virus. Generally speaking, the outermost layer should be completely protein, and the DNA / RNA should be completely wrapped in the protein capsid. It should not be exposed to the outside or pass through the protein layer.

[0003] The existing DNA virus tracer is a covalent coupling of SiO2 nanospheres with selected proteins and DNA. The protein and DNA exist simultaneously on the surface of the tracer, which is not only contrary to the surface characteristics of the virus, but more importantly, the exposed DNA is not effectively protected, making the half-life of this tracer close to the half-life of naked DNA in this environment, but much smaller than the half-life of the target virus. False negative results may occur due to the rapid decomposition of the DNA marker. Therefore, it is urgent to develop a new type of DNA virus tracer. Summary of the invention

[0004] The purpose of the present invention is to provide a virus tracer with a protein capsid and a nucleic acid layer, and a preparation method and application thereof.

[0005] The existing DNA virus tracers have the following main problems: (1) the exposed DNA is not effectively protected; (2) proteins and DNA exist simultaneously on the surface of the tracer, which is contrary to the surface characteristics of the virus. For example, the existing DNA virus tracer is a SiO2 nanosphere (a sphere with a diameter of about 70nm) close to the size of the target virus, and the carbodiimide crosslinker EDC (1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochloride) is used to covalently couple the SiO2 nanosphere with the selected protein and DNA ( Figure 1 a).

[0006] The present invention is conceived as follows: In order to solve the above problems existing in the existing DNA virus tracers, SiO2 nanospheres are amino-modified to form a core, and then covalently coupled to DNA to wrap a nucleic acid layer, and then covalently coupled to protein to form a capsid ( Figure 1 b). Specifically, firstly, SiO2 nanospheres [SiO2]( Figure 2 a) Functionalization with APTEOS (3-aminopropyltriethoxysilane) to make its surface amino-modified to form a core [NH2-SiO2] ( Figure 2b), using GA (glutaraldehyde) as a crosslinker, covalently couple DNA to the outside of the inner core NH2-SiO2 nanosphere to form a nucleic acid layer [(DNA(SiO2)]( Figure 2 c), GA was used as a cross-linking agent again to covalently couple the protein outside the DNA(SiO2) nanospheres to form a capsid, and the covalently coupled monolayer protein [Protein(DNA(SiO2))]( Figure 2 d) If GA is repeatedly covalently coupled, a multi-layered protein capsid can be formed. n (DNA(SiO2))( Figure 2 e).

[0007] In order to achieve the purpose of the present invention, in a first aspect, the present invention provides a virus tracer having a protein capsid and a nucleic acid layer, which is composed of an inner core, a nucleic acid layer and a protein capsid wrapped outside the nucleic acid layer;

[0008] The inner core is a SiO2 nanosphere (with controllable particle size), the surface of the SiO2 nanosphere is amino-modified, a layer of DNA is covalently coupled with GA (glutaraldehyde) to form a nucleic acid layer, and then n layers of protein are covalently coupled with GA (glutaraldehyde) to form a protein capsid; wherein 1≤n≤5.

[0009] The particle size of the SiO2 nanospheres of the present invention is x, 20nm≤x≤120nm, preferably x is about 40nm.

[0010] The thickness of the nucleic acid layer is y, and the thickness of the protein capsid is z.

[0011] The particle size of the DNA virus tracer is x+2y+2z, 30nm≤x+2y+2z≤160nm; preferably, the particle size of the tracer is about 80nm. The particle size x of the SiO2 nanospheres can be controlled according to the particle size of the target virus being traced, and fine-tuned by the thickness / number of layers of the protein capsid.

[0012] In the present invention, the nucleic acid may be a single-stranded or double-stranded DNA having a length of 60-120 bp and not containing a stem-loop or hairpin structure.

[0013] The protein can be a protein with properties close to those of the capsid protein of the target virus. For example, a protein with an isoelectric point close to that of the target virus surface is selected to ensure that the surface properties are close to those of the target virus. There may be many types of proteins that make up the capsid, and the main structural proteins play a key role in the surface chemical properties of the target virus.

[0014] In a specific embodiment of the present invention, when the target virus is adenovirus, the outermost layer of the DNA virus tracer can be coated with a protein having an isoelectric point close to that of the adenovirus surface, such as bovine α s1 -Casein.

[0015] In specific applications, the DNA virus tracer of the present invention can be covalently coupled to different types of proteins multiple times through a cross-linking agent according to the decay rate of the target virus, and a protein capsid can be finally formed by controlling the number of layers of the wrapped protein, thereby simulating the half-life of the target virus in a variety of water and soil environments.

[0016] In a second aspect, the present invention provides a method for preparing a virus tracer having a protein capsid and a nucleic acid layer, comprising the following steps:

[0017] (1) contacting SiO2 nanospheres with a functionalizing agent APTEOS to amino-modify the surface of the SiO2 nanospheres to form the core, i.e., NH2-SiO2 nanospheres;

[0018] (2) using GA as a cross-linking agent, the NH2-SiO2 nanospheres are brought into contact with a DNA solution, and a nucleic acid layer with a thickness of y is formed on the outside of the inner core, i.e., DNA (SiO2) nanospheres;

[0019] (3) Using GA as a cross-linking agent, the DNA(SiO2) nanospheres obtained in step (2) are brought into contact with a protein solution, and a single-layer protein capsid is formed on the surface of the DNA(SiO2) nanospheres, i.e., Protein(DNA(SiO2)); if GA is used for repeated covalent coupling, a multi-layer protein capsid is formed, i.e., Protein n (DNA(SiO2)); where 1≤n≤5.

[0020] Furthermore, the preparation method comprises the following steps:

[0021] 1) Preparation of NH2-SiO2 nanospheres: 50 mL of ethanol and 1.4 mL of ammonia water were mixed evenly at 500 rpm, and then 2 mL of TEOS was added thereto, and the mixture was stirred continuously at 900 rpm for 24 hours; then 0.3 mL of APTEOS was added to the system, and the mixture was stirred continuously at 400 rpm for 24 hours to obtain amino-modified SiO2 nanospheres, which were washed by centrifugation three times and freeze-dried in vacuum to obtain NH2-SiO2 nanospheres; 2) Preparation of DNA solution: DNA solution was prepared with ultrapure water to obtain a DNA solution with a concentration of 0.5-4 mg / mL;

[0022] 3) Covalent coupling of DNA: NH2-SiO2 nanospheres were mixed with 0.025-10% GA in PBS buffer for 12 hours, and then purified by centrifugation washing with PBS buffer for 3 times; finally, GA-coated SiO2 nanospheres were dispersed into a DNA solution, reacted in PBS buffer for 12 hours, and then washed and resuspended with PBS buffer to obtain DNA (SiO2) nanospheres;

[0023] 4) Preparation of protein solution: Prepare a protein solution with PBS buffer to obtain a protein solution with a concentration of 2-4 mg / mL;

[0024] 5) Covalent coupling of proteins: DNA (SiO2) nanospheres were mixed with 0.025-10% GA in PBS buffer for 12 hours, and then purified by centrifugation and washing three times with PBS buffer; finally, the GA-coated DNA (SiO2) nanospheres were dispersed in a protein solution, reacted in PBS buffer for 12 hours, and then washed and resuspended with PBS buffer to obtain a virus tracer Protein (DNA (SiO2)) with a protein capsid and a nucleic acid layer; if GA is repeatedly covalently coupled, a multi-layered protein capsid can be formed, i.e., Protein n (DNA(SiO2)).

[0025] Wherein, the pH of the PBS buffer is 7.2-7.4.

[0026] In step 2), ultrapure water is used to prepare a single-stranded DNA solution, wherein the nucleotide sequence of the single-stranded DNA is shown in any one of SEQ ID NOs: 1-4.

[0027] In a third aspect, the present invention provides a virus tracer having a protein capsid and a nucleic acid layer prepared according to the method. Its particle size is x+2y+2z, which can be controlled by the particle size x of the SiO2 nanospheres in the range of 30-160nm according to the particle size of the target virus being traced, and fine-tuned by the thickness / number of layers of the protein capsid.

[0028] In a fourth aspect, the present invention provides the use of the DNA virus tracer in the spatiotemporal tracing of pathogen non-point source pollution in agricultural areas where organic fertilizers are applied; wherein the pathogens include viruses.

[0029] By means of the above technical solution, the present invention has at least the following advantages and beneficial effects:

[0030] (i) The novel protein-coated DNA virus tracer provided by the present invention can achieve effective protection of the encoding DNA, facilitating subsequent quantitative detection and analysis.

[0031] (ii) Effectively ensure that the outermost capsid of the tracer is completely composed of the selected protein, so that the surface characteristics and migration characteristics of the tracer can be closer to the target virus.

[0032] (III) According to the decay rate of the target virus, different types and numbers of protein capsids are covalently coupled multiple times through cross-linking agents to simulate the half-life of the target virus in various water and soil environments. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 Comparative diagram of protein-encapsulated DNA-encoded viral tracers of the prior art (a) and the present invention (b).

[0034] Figure 2 The figure is a schematic diagram of the production process of the virus tracer with a protein capsid and a nucleic acid layer according to the present invention.

[0035] Figure 3 This is a conceptual diagram of the spatiotemporal tracing of pathogen non-point source pollution in agricultural areas where organic fertilizers are applied by a virus tracer having a protein capsid and a nucleic acid layer in a preferred embodiment of the present invention. DETAILED DESCRIPTION

[0036] The present invention provides a method for preparing a virus tracer having a protein capsid and a nucleic acid layer, comprising the following steps:

[0037] (1) contacting SiO2 nanospheres with a functionalizing agent APTEOS to amino-modify the surface of the SiO2 nanospheres to form the core, i.e., NH2-SiO2 nanospheres;

[0038] (2) using GA as a cross-linking agent, the inner core, i.e., the NH2-SiO2 nanosphere, is brought into contact with a DNA solution, thereby forming a nucleic acid layer with a thickness of y on the outer side of the inner core, i.e., the DNA (SiO2) nanosphere;

[0039] (3) Using GA as a cross-linking agent, the DNA(SiO2) nanospheres obtained in step (2) are brought into contact with a protein solution, thereby forming a single-layer protein capsid on the surface of the DNA(SiO2) nanospheres, i.e., Protein(DNA(SiO2)); if GA is used for repeated covalent coupling, a multi-layer protein capsid can be formed, i.e., Protein n (DNA(SiO2)).

[0040] Furthermore, the preparation method comprises the following steps:

[0041] 1) Preparation of inner core NH2-SiO2 nanospheres: 50 mL of ethanol and 1.4 mL of ammonia water were mixed uniformly at 500 rpm, and then 2 mL of TEOS was added thereto, and the mixture was stirred continuously at 900 rpm for 24 hours; then 0.3 mL of APTEOS was added thereto, and the mixture was stirred continuously at 400 rpm for 24 hours to obtain amino-modified SiO2 nanospheres, which were washed by centrifugation three times and freeze-dried in vacuum to obtain NH2-SiO2 nanospheres;

[0042] 2) Preparation of DNA solution: Prepare a DNA solution with ultrapure water to obtain a DNA solution with a concentration of 0.5-4 mg / mL;

[0043] 3) Covalent coupling of DNA: at room temperature, the inner core NH2-SiO2 nanospheres were mixed with 0.025-10% GA in PBS buffer for 12 hours, so that the aldehyde group (-CHO) of GA and the amino group (-NH2) of the inner core were fully reacted on the surface of the NH2-SiO2 nanospheres, and then centrifuged and washed three times with PBS buffer for purification; finally, the GA-coated SiO2 nanospheres were dispersed in a DNA solution, reacted in PBS buffer for 12 hours, and then washed and resuspended with PBS buffer to obtain DNA (SiO2) nanospheres;

[0044] 4) Preparation of protein solution: Prepare a protein solution with PBS buffer to obtain a protein solution with a concentration of 2-4 mg / mL;

[0045] 5) Covalent coupling of proteins: DNA (SiO2) nanospheres were mixed with 0.025-10% GA in PBS buffer at room temperature for 12 hours to allow the aldehyde group (-CHO) of GA and the amino group (-NH2) of DNA to fully react on the surface of the DNA (SiO2) nanospheres, and then purified by centrifugation washing with PBS buffer for 3 times; finally, the GA-coated DNA (SiO2) nanospheres were dispersed in a protein solution, reacted in PBS buffer for 12 hours, and then washed and resuspended with PBS buffer to obtain a protein-coated DNA virus tracer [Protein (DNA (SiO2))]; if GA is repeatedly covalently coupled, a multilayer protein capsid can be formed, i.e., Protein n (DNA(SiO2)).

[0046] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art, and the raw materials used are all commercially available products.

[0047] Experimental reagents: ammonia water (25%), tetraethoxysilane (TEOS, GC ≥ 99.9%), ethanol (purity ≥ 99.0%), single-stranded DNA using the single-stranded DNA in Table 1, double-stranded DNA obtained by annealing the single-stranded DNA in Table 1 and its reverse complementary strand, ultrapure water, 3-aminopropyltriethoxysilane (APTEOS, purity ≥ 99.0%), glutaraldehyde (GA, 50% aqueous solution), PBS buffer (pH 7.2).

[0048] Cow α s1 -Casein (Bovine milkα s1-casein) was purchased from Sigma-Aldrich, USA (https: / / www.sigmaaldrich.com / US / en / search / bovine-milk-casein?focus=products&page=1&perpage=30&sort=relevance&term=bovine%20milk%20casein&type=product). α1-acid glycoprotein (α1-aci d-glycoprotein) was purchased from Sigma-Aldrich, USA (https: / / www.sigmaaldrich.cn / CN / zh / product / sigma / g9885).

[0049] Table 1 DNA sequences (SEQ ID NO: 1-4)

[0050]

[0051] Note: The bold underline indicates the forward primer, and the italic underline indicates the reverse primer.

[0052] Example 1 Preparation method of virus tracer having nucleic acid layer and single-layer protein capsid

[0053] In this example, adenovirus was used as the target virus to prepare a protein-coated DNA virus tracer.

[0054] 1. Preparation of inner core NH2-SiO2 nanospheres: 50 mL of ethanol and 1.4 mL of ammonia water were mixed evenly under stirring at 500 rpm, and then 2 mL of TEOS was added thereto, and stirring was continued at 900 rpm for 24 hours; then 0.3 mL of APTEOS was added thereto, and stirring was continued at 400 rpm for 24 hours to obtain amino-modified SiO2 nanospheres, which were washed by centrifugation three times and freeze-dried in vacuum to obtain NH2-SiO2 nanospheres.

[0055] 2. Nucleic acid layer expansion: Prepare a 0.5 mg / mL single-stranded DAP3 DNA solution (SEQ ID NO: 4) with ultrapure water. Mix the core NH2-SiO2 nanospheres with 10% GA in PBS buffer for 12 hours at room temperature, so that the aldehyde group (-CHO) of GA and the amino group (-NH2) of the core react fully on the surface of the NH2-SiO2 nanospheres, and then centrifuge and wash with PBS buffer for 3 times for purification; finally, disperse the GA-wrapped SiO2 nanospheres into the DNA solution, react in PBS buffer for 12 hours, and then wash and resuspend with PBS buffer to obtain DNA (SiO2) nanospheres.

[0056] 3. The size and structure of DNA(SiO2) nanospheres were observed using transmission electron microscopy (TEM), and their zeta potential (ζ) and hydrodynamic diameter were determined using ZetasizerNano ZS.

[0057] 4. Monolayer protein expansion: Prepare bovine α-protein in PBS buffer (pH 7.2). s1 -casein solution to obtain a protein solution with a concentration of 2 mg / mL. At room temperature, DNA(SiO2) nanospheres were mixed with 10% GA in PBS buffer for 12 hours to allow the aldehyde group (-CHO) of GA and the amino group (-NH2) of DNA to fully react on the surface of the DNA(SiO2) nanospheres, and then centrifuged and washed three times with PBS buffer for purification; finally, the GA-coated DNA(SiO2) nanospheres were dispersed in the protein solution, reacted in PBS buffer for 12 hours, and then washed and resuspended with PBS buffer to obtain the protein-coated DNA virus tracer [Protein(DNA(SiO2))].

[0058] Protein selection: The outermost layer is wrapped with a protein close to the isoelectric point of the adenovirus surface, and the protein molecule has enough amino groups to ensure that the protein molecule has enough amino sites to react with the DNA (SiO2) nanospheres wrapped with the aldehyde crosslinking agent GA. s1 -Casein (GenBank: P02662).

[0059] 5. The size and structure of the DNA virus tracer [Protein(DNA(SiO2))] were observed using a transmission electron microscope (TEM), and its Zeta potential (ζ) and hydrodynamic diameter were measured using a Zetasizer Nano ZS. The protein-coated DNA virus tracer [Protein(DNA(SiO2))] prepared in this example had a particle size of about 80 nm, which was consistent with the target virus adenovirus 70-90 nm.

[0060] 6. Quantitative detection of DNA virus tracers

[0061] The DNA virus tracer [Protein (DNA (SiO2))] was soaked in a pepsin solution (simulated gastric fluid) at pH 2.0 and a trypsin solution (simulated intestinal fluid) at pH 6.8 for 2 hours and 12 hours respectively to simulate the protein digestion process to remove the outermost protein shell. Then soaked in NH4F etching solution until clear and transparent without precipitation to ensure that the silica is completely dissolved. The released DNA was then purified using the QIAquick PCR purification kit. After that, the Taqman probe method was used for qPCR detection, and the DNA copy number concentration of the sample was obtained by comparison with the standard curve.

[0062] 7. DNA (SiO2) nanospheres obtained at the end of step 6 ( Figure 2 d) Determine the correspondence between the number of tracers and the number of DNA copies: The number of tracers is calculated from the total mass of the DNA (SiO2) nanospheres, the density of SiO2 and DNA, and the particle size of the DNA (SiO2) measured in step 5. The number of DNA copies is obtained by the detection method described in step 6. The relationship between the number of tracers and the number of DNA copies can be established, that is, how many single-stranded DNAs are encapsulated in each tracer on average.

[0063] Example 2 Preparation method of virus tracer having nucleic acid layer and double-layer protein capsid

[0064] In this example, adenovirus was used as the target virus to prepare a protein-coated DNA virus tracer.

[0065] 1. Preparation of inner core NH2-SiO2 nanospheres: 50 mL of ethanol and 1.4 mL of ammonia water were mixed evenly under stirring at 500 rpm, and then 2 mL of TEOS was added thereto, and stirring was continued at 900 rpm for 24 hours; then 0.3 mL of APTEOS was added thereto, and stirring was continued at 400 rpm for 24 hours to obtain amino-modified SiO2 nanospheres, which were washed by centrifugation three times and freeze-dried in vacuum to obtain NH2-SiO2 nanospheres.

[0066] 2. Nucleic acid layer expansion: Prepare a 0.5 mg / mL single-stranded GM2 DNA solution (SEQ ID NO: 1) with ultrapure water. Mix the core NH2-SiO2 nanospheres with 10% GA in PBS buffer for 12 hours at room temperature, so that the aldehyde group (-CHO) of GA and the amino group (-NH2) of the core react fully on the surface of the NH2-SiO2 nanospheres, and then centrifuge and wash with PBS buffer for 3 times for purification; finally, disperse the GA-wrapped SiO2 nanospheres into the DNA solution, react in PBS buffer for 12 hours, and then wash and resuspend with PBS buffer to obtain DNA (SiO2) nanospheres.

[0067] 3. The size and structure of DNA(SiO2) nanospheres were observed using transmission electron microscopy (TEM), and their zeta potential (ζ) and hydrodynamic diameter were determined using ZetasizerNano ZS.

[0068] 4. Monolayer protein expansion: Prepare bovine α-protein in PBS buffer (pH 7.2). s1 -casein solution to obtain a protein solution with a concentration of 2 mg / mL. At room temperature, DNA (SiO2) nanospheres were mixed with 10% GA in PBS buffer for 12 hours to allow the aldehyde group (-CHO) of GA and the amino group (-NH2) of DNA to fully react on the surface of the DNA (SiO2) nanospheres, and then centrifuged and washed 3 times with PBS buffer for purification; finally, the GA-coated DNA (SiO2) nanospheres were dispersed in the protein solution, reacted in PBS buffer for 12 hours, and then washed and resuspended with PBS buffer to obtain the protein-coated DNA virus tracer [Bovinemilkα s1 -casein(DNA(SiO2))].

[0069] 5. Multi-layer protein layer expansion: Prepare α1-acid glycoprotein solution in PBS buffer (pH 7.2) to obtain a protein solution with a concentration of 2 mg / mL. s1 -casein(DNA(SiO2)) nanospheres were mixed with 10% GA in PBS buffer for 12 h, so that the aldehyde group (-CHO) of GA would react with bovine α s1 -Amino group (-NH2) of casein in Bovine milkα s1 -casein(DNA(SiO2)) nanospheres were fully reacted on the surface, and then purified by centrifugation and washing three times with PBS buffer; finally, the Bovine milkα s1 -casein(DNA(SiO2)) nanospheres were dispersed into α1-acid glycoprotein solution, reacted in PBS buffer for 12 hours, and then washed and resuspended in PBS buffer to obtain a double-layer protein-coated DNA virus tracer [α1-acid-glycoprotein(Bovine milkα s1 -casein(DNA(SiO2)))].

[0070] Protein selection: The outermost layer is wrapped with a protein close to the isoelectric point of the adenovirus surface, and the protein molecule has enough amino groups to ensure that the protein molecule has enough amino sites, and the Bovinemilkα wrapped with the aldehyde crosslinker GA s1-casein(DNA(SiO2)) nanospheres were used for the reaction. In this example, α1-acid glycoprotein (GenBank: P02763) was selected.

[0071] Two or more protein layers can better control the half-life of the tracer, allowing it to better simulate the half-life of the target virus in water and soil environments.

[0072] Example 3 Application of a virus tracer having a protein capsid and a nucleic acid layer

[0073] Existing DNA virus tracers cannot accurately infer the migration and transformation process of the target virus from the migration and transformation process of the tracer in the water and soil medium, and are difficult to truly apply in the field where pathogenic viruses cannot be used and only virus tracers can be used. In this embodiment, the DNA virus tracer prepared in Example 1, which has physical filtration and chemical adsorption properties and half-life in the soil close to those of the target virus, is used as a substitute for the target virus to trace the migration and transformation process of the pathogen, thereby realizing the spatiotemporal tracing of pathogen non-point source pollution in agricultural areas where organic fertilizers are applied. For example, when the target virus is adenovirus.

[0074] 1. Use water infiltration with a certain head on an unsaturated vertical soil column to simulate the infiltration and filtration of viruses after applying organic fertilizers in the unsaturated zone soil, and use a saturated horizontal soil column to simulate the process of viruses migrating to the drainage ditch after entering the saturated zone soil. In order to avoid the mutual influence of tracers and target viruses in the soil, such as occupying adsorption sites, a parallel soil column experiment is planned, that is, a number of completely identical soil columns are filled, and each time only the conservative tracer KBr (used to reverse the soil hydrodynamic parameters) is put into the DNA virus tracer or target virus. The recovery rate and penetration curve obtained in the experiment are combined with the simulation of the pollutant migration and transformation model in the soil to obtain the filtration parameters, adsorption and desorption rates, and decay rates of DNA virus tracers in unsaturated zone soils and saturated zone soils under different conditions. The function of the filtration parameters, adsorption and desorption rates, and decay rates of the target virus is inferred.

[0075] 2. Take water and bottom mud from several representative ditch sections at different irrigation periods, and use a 5-meter-long, 1-meter-high, and 0.6-meter-wide flume to simulate the drainage conditions of different ditch sections at different irrigation periods. All tracers (DNA virus tracers and bacteriophages), target viruses, and conservative tracer KBr (used to reverse the hydrodynamic parameters) are put into the flume together. The concentration change curves of each tracer and target virus in the overlying water at different migration distances obtained in the experiment are combined with the convection-diffusion equation that considers the decomposition of pollutants and adsorption and desorption on particles, the sedimentation of suspended particles in water, and the scouring and siltation of water on the bottom mud. The convection-diffusion parameters, adsorption-desorption rate, and decay rate of DNA virus tracers are used to reverse the convection-diffusion parameters, adsorption-desorption rate, and decay rate of the target virus under different irrigation periods and different ditch sections.

[0076] When migrating in the soil, the surface chemical properties of the virus tracer and the target virus directly affect their adsorption in the soil. When migrating in the drain, the virus tracer and the target virus will also adsorb and desorb on the sediment and suspended particles in the water. This adsorption and desorption process is also closely related to their surface chemical properties. Therefore, it is particularly important to ensure that the surface of the virus tracer is completely coated with proteins like the target virus.

[0077] 3. The amount of organic fertilizer and irrigation water required to grow different crops is different, and soils of different textures have different filtering effects on pathogens. Figure 3 As shown, based on the soil column and water tank experiments and simulations in the first two steps, the DNA virus tracer of the present invention is encoded with different DNA sequences to mark the farmland and time of investment, and the organic fertilizer is invested at different times and in different farmlands ( Figure 3 The DNA virus tracers put into the four farmlands in the plots were encapsulated with the four different DNA sequences in Table 1, and samples were collected from nearby groundwater wells and drainage ditches to detect the concentration changes of DNA virus tracers in groundwater and surface water. Then, the function of using DNA virus tracers to describe the migration and transformation process of the target virus obtained by the soil column and water tank experiments and simulations in the first two steps was used to estimate the time and concentration of the target virus entering the groundwater and drainage ditches, as well as the time and concentration of migrating different distances in the drainage ditches, to conduct spatiotemporal tracing of pathogen non-point source pollution in the agricultural area where organic fertilizers are applied, and to trace the farmlands and time periods that are prone to pathogen non-point source pollution.

[0078] Although the present invention has been described in detail above with general descriptions and specific embodiments, it is obvious to those skilled in the art that some modifications or improvements can be made based on the present invention. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection claimed by the present invention.

Claims

1. A DNA virus tracer having a protein capsid and a nucleic acid layer, characterized in that: It consists of an inner core, a nucleic acid layer, and a protein capsid wrapped around the nucleic acid layer. The inner core is a SiO2 nanosphere, the surface of the SiO2 nanosphere is amino-modified, a layer of DNA is covalently coupled with GA to form a nucleic acid layer, and then n layers of protein are covalently coupled with GA to form a protein capsid; wherein 1≤n≤5; The particle size of the SiO2 nanosphere is x, 20 nm≤x≤120 nm; The thickness of the nucleic acid layer is y, and the thickness of the protein capsid is z; The particle size of the DNA virus tracer is x+2y+2z, 30 nm≤x+2y+2z≤160 nm; The nucleic acid is a single-stranded or double-stranded DNA with a length of 60-120 bp and without a stem-loop or hairpin structure; The protein is a protein that is close in nature to the capsid protein of the target virus; The GA is glutaraldehyde; The method for preparing the virus tracer comprises the following steps: (1) The SiO2 nanospheres are contacted with the functionalization reagent APTEOS to amino-modify the surface of the SiO2 nanospheres to serve as the core, i.e., NH2-SiO2 nanospheres; (2) using GA as a cross-linking agent, the NH2-SiO2 nanospheres are brought into contact with a DNA solution, thereby forming a nucleic acid layer with a thickness of y on the outside of the inner core, i.e., DNA (SiO2) nanospheres; (3) Using GA as a cross-linking agent, the DNA(SiO2) nanospheres obtained in step (2) are brought into contact with a protein solution, and a single-layer protein capsid is formed on the surface of the DNA(SiO2) nanospheres, i.e., Protein(DNA(SiO2)); if GA is repeatedly covalently coupled, a multi-layer protein capsid is formed, i.e., the virus tracer Protein n (DNA(SiO2)); where 1≤n≤5.

2. The method for preparing the DNA virus tracer having a protein capsid and a nucleic acid layer according to claim 1, characterized in that: The following steps are involved: (1) The SiO2 nanospheres are contacted with the functionalization reagent APTEOS to amino-modify the surface of the SiO2 nanospheres to serve as the core, i.e., NH2-SiO2 nanospheres; (2) using GA as a cross-linking agent, the NH2-SiO2 nanospheres are brought into contact with a DNA solution, thereby forming a nucleic acid layer with a thickness of y on the outside of the inner core, i.e., DNA (SiO2) nanospheres; (3) Using GA as a cross-linking agent, the DNA(SiO2) nanospheres obtained in step (2) are brought into contact with a protein solution, and a single-layer protein capsid is formed on the surface of the DNA(SiO2) nanospheres, i.e., Protein(DNA(SiO2)); if GA is repeatedly covalently coupled, a multi-layer protein capsid is formed, i.e., the virus tracer Protein n (DNA(SiO2)); where 1≤n≤5; The particle size of the SiO2 nanosphere is x, 20 nm≤x≤120 nm; The thickness of the protein capsid is z; The nucleic acid is a single-stranded or double-stranded DNA with a length of 60-120 bp and without a stem-loop or hairpin structure; The protein is a protein that is close in nature to the capsid protein of the target virus; The GA is glutaraldehyde.

3. The method according to claim 2, characterized in that The following steps are involved: 1) Preparation of NH2-SiO2 nanospheres: 50 mL of ethanol and 1.4 mL of ammonia water were mixed evenly at 500 rpm, and then 2 mL of TEOS was added thereto, and the mixture was stirred continuously at 900 rpm for 24 hours; then 0.3 mL of APTEOS was added to the system, and the mixture was stirred continuously at 400 rpm for 24 hours to obtain amino-modified SiO2 nanospheres, which were washed by centrifugation three times and freeze-dried in vacuum to obtain NH2-SiO2 nanospheres; 2) Preparation of DNA solution: Prepare DNA solution with ultrapure water to obtain a DNA solution with a concentration of 0.5-4 mg / mL; 3) Covalent coupling of DNA: NH2-SiO2 nanospheres were mixed with 0.025-10% GA in PBS buffer for 12 hours, and then purified by centrifugation washing with PBS buffer for 3 times; finally, GA-coated SiO2 nanospheres were dispersed into DNA solution, reacted in PBS buffer for 12 hours, and then washed and resuspended with PBS buffer to obtain DNA (SiO2) nanospheres; 4) Preparation of protein solution: Prepare a protein solution with PBS buffer to obtain a protein solution with a concentration of 2-4 mg / mL; 5) Covalent coupling of proteins: DNA (SiO2) nanospheres were mixed with 0.025-10% GA in PBS buffer for 12 hours, and then purified by centrifugation and washing three times with PBS buffer; finally, the GA-coated DNA (SiO2) nanospheres were dispersed in a protein solution, reacted in PBS buffer for 12 hours, and then washed and resuspended with PBS buffer to obtain a viral tracer Protein (DNA (SiO2)) with a protein capsid and a nucleic acid layer; if GA is repeatedly covalently coupled, a multi-layered protein capsid can be formed, i.e., Protein n (DNA(SiO2)); Wherein, the pH of the PBS buffer is 7.2-7.

4.

4. The method according to claim 2 or 3, characterized in that: In step 2), ultrapure water is used to prepare a single-stranded DNA solution, wherein the nucleotide sequence of the single-stranded DNA is shown in any one of SEQ ID NOs: 1-4.

5. A DNA virus tracer having a protein capsid and a nucleic acid layer prepared according to the method of any one of claims 2 to 4.

6. The DNA virus tracer according to claim 5, characterized in that Its particle size is x+2y+2z, 30 nm≤x+2y+2z≤160 nm.

7. Use of the DNA virus tracer according to claim 1, 5 or 6 in the spatiotemporal tracing of pathogen non-point source pollution in agricultural areas where organic fertilizers are applied; in, The pathogen is a virus.

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