DNA virus tracer coated with protein by EDC covalent coupling method, and preparation method and application thereof

Abstract

The application provides a DNA virus tracer coated with a protein by an EDC covalent coupling method and a preparation method and application thereof. The DNA virus tracer coated with the protein of the application is composed of a core, a shell and a protein shell coated on the shell; the core is a SiO2 nanosphere with adsorbed nucleic acid on the surface, a layer of SiO2 shell is coated on the outside of the core, the surface of the shell is modified by carboxylation, and the shell is formed by covalently coupling the protein through EDC. The novel DNA virus tracer provided by the application can effectively protect the coding DNA, facilitate subsequent quantitative detection and analysis, effectively ensure that the outermost shell of the tracer is completely composed of the selected protein, and make the surface characteristics and migration characteristics of the tracer closer to the target virus.

Description

Technical Field

[0001] This invention belongs to the field of environmental protection, specifically, it relates to a DNA virus tracer that encapsulates proteins by EDC covalent coupling, its preparation method and application. Background Technology

[0002] The basic structure of a virus consists of a protein capsid and a protective nucleic acid layer (DNA / RNA) enclosing it. The properties of the viral capsid proteins play a decisive role in its transport properties. Tracers used as viral substitutes should also have surface properties as similar as possible to the target virus. Generally, they should have an outermost layer entirely of protein, with the DNA / RNA completely enclosed within the protein capsid, neither exposed nor penetrating the protein layer.

[0003] Existing DNA virus tracers involve covalently coupling SiO2 nanospheres with selected proteins and DNA. The coexistence of protein and DNA on the tracer's surface not only contradicts the surface characteristics of viruses, but more importantly, the exposed DNA is not effectively protected. This results in a tracer half-life close to that of naked DNA in this environment, but far shorter than that of the target virus. False negative results may occur due to the rapid degradation of the DNA marker. Therefore, there is an urgent need to develop a novel DNA virus tracer. Summary of the Invention

[0004] The purpose of this invention is to provide a DNA virus tracer for protein encapsulation by EDC covalent coupling, its preparation method, and its application.

[0005] Existing DNA virus tracers have the following two shortcomings: (1) the exposed DNA is not effectively protected; (2) proteins and DNA coexist on the surface of the tracer, which contradicts the surface characteristics of viruses. Using a carbodiimide crosslinking agent EDC (1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride), SiO2 nanospheres (approximately 70 nm in diameter) close to the size of the target virus are covalently coupled to the selected proteins and DNA. Figure 1 a).

[0006] The concept of this invention is as follows: A layer of DNA marker is first adsorbed onto the SiO2 core, then a SiO2 shell is coated to effectively protect the DNA. Finally, a capsid is formed on the outermost layer by covalently coupling with proteins. Figure 1 b). Specifically, first, SiO2 nanospheres ( Figure 2 a) Functionalize with TMAPS to make its surface positively charged ( Figure 2b) Then, negatively charged DNA is electrostatically adsorbed, followed by neutralization of excess negative charge with TMAPs and SiO2 growth catalyzed by TEOS to obtain a DNA-labeled SiO2 nanosphere SiO2(DNA(SiO2))( Figure 2 c). Then, different methods are used to coat the protein on the outermost layer. This invention uses the EDC covalent coupling method to carboxylate the surface of SiO2 (DNA (SiO2)) using polyacrylic acid (PAA). Figure 2 e), then using 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) as a cross-linking agent, a capsid is formed on the outermost layer of covalently coupled protein, thus obtaining [Protein(SiO2(DNA(SiO2)))]( Figure 2 f).

[0007] In order to achieve the objective of the present invention, in a first aspect, the present invention provides a protein-encapsulated DNA virus tracer, which consists of a core, a shell, and a protein capsid encapsulated in the shell.

[0008] The core is a SiO2 nanosphere with nucleic acid adsorbed on its surface. A SiO2 shell of thickness y is wrapped around the core. The shell surface is modified by carboxylation. Then, a capsid of thickness z is formed by covalently coupling proteins with EDC. Wherein, 12nm≤y≤15nm.

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

[0010] Preferably, the particle size of the DNA virus tracer is x+2y+2z, where 60nm≤x+2yb+2z≤160nm, and more 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 finely adjusted by the thickness y of the SiO2 shell and the thickness z of the protein capsid.

[0011] In this invention, the nucleic acid can be a single-stranded or double-stranded DNA with a length of 60-120 bp and without stem-loop or hairpin structures.

[0012] The protein can be a protein with properties similar to the capsid protein of the target virus, and the protein molecule has enough amino groups to ensure that the protein molecule has enough amino sites to dehydrate and condense with the carboxyl groups on the surface of carboxylated SiO2 (DNA (SiO2)) to form covalent bonds.

[0013] For example, proteins with isoelectric points close to those of the target virus surface can be selected to ensure that the surface properties are similar to those of the target virus. There may be many types of proteins that make up the capsid, but the main structural proteins play a crucial role in the surface chemistry of the target virus.

[0014] In one specific embodiment of the present invention, when the target virus is an adenovirus, the outermost layer of the DNA virus tracer can be coated with a protein with an isoelectric point close to that of the adenovirus surface, such as human α1 microglobulin / bikunin precursor (AMBP).

[0015] In practical applications, the DNA virus tracer of the present invention is used as a substitute with physical and surface chemical properties consistent with the target virus. Depending on the decay rate of the target virus, it needs to be used in conjunction with a bacteriophage, which provides the decay rate of the target virus.

[0016] Secondly, the present invention provides a method for preparing a DNA virus tracer containing protein encapsulated by EDC covalent coupling, comprising the following steps:

[0017] (1) SiO2 nanospheres are contacted with functionalized reagent TMAPS to make the surface of SiO2 nanospheres positively charged, and then nucleic acids are electrostatically adsorbed as the core.

[0018] (2) The core is brought into contact with TEOS to catalyze the growth of SiO2 and form a SiO2 shell with a thickness of y on the outside of the core, namely SiO2 (DNA (SiO2)) nanospheres, wherein 12nm≤y≤15nm;

[0019] (3) The surface of the SiO2 (DNA (SiO2)) nanospheres obtained in step (2) is carboxylated using polyacrylic acid (PAA), and then 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC) is used as a crosslinking agent to contact the carboxylated SiO2 (DNA (SiO2)) nanospheres with a protein solution to form a protein coat with a thickness of z on the surface of the SiO2 (DNA (SiO2)) nanospheres.

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

[0021] 1) Preparation of SiO2 core colloidal solution: SiO2 nanospheres were suspended in isopropanol to prepare a SiO2 nanosphere colloidal solution with a concentration of about 50 mg / mL;

[0022] 2) Preparation of positively charged TMAPS-functionalized SiO2 nanospheres: 1 mL of SiO2 nanosphere colloidal solution was ultrasonically treated to obtain a homogeneous SiO2 nanosphere colloidal solution; 10 μL of TMAPS solution was added to it, and then the mixture was stirred at 900 rpm at room temperature for 12 hours in a constant temperature oven. After washing with isopropanol, positively charged SiO2 nanospheres were obtained.

[0023] 3) Electrostatic adsorption of DNA and SiO2 shell encapsulation: First, a nucleic acid solution with a concentration of 50 μg / mL was prepared using ultrapure water. Then, 700 μL of ultrapure water, 320 μL of nucleic acid solution, and 35 μL of positively charged SiO2 nanospheres were added to a centrifuge tube, mixed, and sonicated to obtain a homogeneous solution. After centrifugation and washing with ultrapure water, the solution was sonicated to obtain a homogeneous colloidal solution. 0.5 μL of TMAPS solution was added and mixed well, followed by 0.5 μL of TEOS. The mixture was stirred at 900 rpm at room temperature for 4 hours. Then, 4 μL of TEOS was added to the system and stirred at 900 rpm at room temperature for 4 days. After washing with ultrapure water and sonicating to obtain SiO2 (DNA(SiO2)) nanospheres, the solution was obtained.

[0024] 4) Preparation of carboxylated SiO2 (DNA (SiO2)) nanospheres: 1 mL of SiO2 (DNA (SiO2)) nanosphere colloidal solution was added dropwise to 20 mL of 2.5 mg / mL polyacrylic acid solution. After sonication, water was added to the product, and the mixture was centrifuged and washed three times. The nanospheres were resuspended in water for the last time. Then, MES buffer was added, and the mixture was sonicated and mechanically stirred to obtain a carboxylated SiO2 (DNA (SiO2)) nanosphere colloidal solution.

[0025] 5) Preparation of protein solution: Prepare protein solution with PBS buffer to obtain a protein solution with a concentration of 2 mg / mL;

[0026] 6) Covalently coupled protein: Wash 1 mL of carboxylated SiO2 (DNA(SiO2)) nanosphere colloidal solution twice in MES buffer; after the second wash, resuspend the nanosphere precipitate in 10 mL of MES buffer and sonicate to ensure complete suspension of the nanospheres, obtaining a carboxylated SiO2 (DNA(SiO2)) nanosphere colloidal solution with a concentration of 10 mg / mL; add 10 mg / mL EDC dropwise while stirring at 18-25℃. Add 10 mL of PBS buffer and stir continuously for 15 minutes (to prevent EDC clumping); wash twice with PBS buffer, then resuspend the nanospheres in 5 mL of PBS buffer; sonicate to ensure the particles are fully suspended; add 1.85-18.5 mL of protein solution and react at 18-25℃ for 2-4 hours, stirring constantly to ensure the nanospheres and proteins are fully in contact and react; after the reaction, centrifuge and discard the supernatant, resuspend the nanosphere precipitate in 10 mL of quenching solution, and gently stir for 30 minutes. Centrifuge again and discard the supernatant, then resuspend the nanosphere precipitate in storage buffer to obtain the protein-encapsulated DNA virus tracer [Protein(SiO2(DNA(SiO2)))].

[0027] The MES buffer has a pH of 6, and the PBS buffer has a pH of 7.4.

[0028] The quenching fluid is a 1 mg / mL casein solution;

[0029] The storage buffer solution is a 10 mg / mL glycine solution.

[0030] Preferably, in step 4), after ultrasonic treatment, water is added to the product, centrifuged at 10,000 rpm for 10 minutes, and washed 3 times.

[0031] In step 3), a double-stranded DNA solution is prepared using ultrapure water. The double-stranded DNA is obtained by annealing the single-stranded DNA and its reverse complementary strand as shown in any of SEQ ID NO:1-4.

[0032] Thirdly, the present invention provides a protein-encapsulated DNA virus tracer prepared according to the method described above. Its particle size is (x+2y+2z), which can be within the range of 60-160 nm. The particle size x of the SiO2 nanospheres is controlled according to the particle size of the target virus being traced, and finely adjusted by the thickness y of the SiO2 shell and the thickness z of the protein capsid.

[0033] Fourthly, the present invention provides the application of the DNA virus tracer in the spatiotemporal tracing of non-point source pollution of pathogens in agricultural areas where organic fertilizers are applied; wherein the pathogens include viruses.

[0034] By employing the above technical solution, the present invention has at least the following advantages and beneficial effects:

[0035] (i) The novel protein-encapsulated DNA virus tracer provided by this invention can effectively protect the encoded DNA, facilitating subsequent quantitative detection and analysis.

[0036] (ii) Effectively ensure that the outermost capsid of the tracer is composed entirely of the selected protein, so that the surface and transport properties of the tracer can be more similar to the target virus. Attached Figure Description

[0037] Figure 1 A comparison diagram of protein-encapsulated DNA-encoded viral tracers of the prior art (a) and the present invention (b).

[0038] Figure 2 This is a schematic diagram illustrating the fabrication process of the DNA virus tracer containing protein encapsulated by the EDC covalent coupling method of this invention.

[0039] Figure 3 This is a conceptual diagram illustrating the spatiotemporal source tracing of pathogen non-point source pollution in an agricultural area where organic fertilizer is applied, using a protein-encapsulated DNA virus tracer, as described in a preferred embodiment of the present invention. Detailed Implementation

[0040] The following examples are used to illustrate the present invention, but are not intended to limit the scope of the 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.

[0041] Experimental reagents: SiO2 nanospheres (40 nm), isopropanol (purity >99.8%), trimethyl[3-(trimethoxysilyl)propyl]chloronitrogen (TMAPS; 50% (wt / wt) dissolved in methanol), double-stranded DNA obtained by annealing the single strands and their reverse complementary strands from Table 1, ultrapure water, tetraethoxysilane (TEOS, purity ≥99.0%), polyacrylic acid solution (PAA, 2.5 mg / mL), MES buffer (pH 6.0), PBS buffer (pH 7.4). Quenching solution: 1 mg / mL casein solution. Storage buffer: 10 mg / mL glycine solution. 1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC, 10 mg / mL).

[0042] SiO2 nanospheres were purchased from nanoComposix (https: / / nanocomposix.com / collections / solid-silica-sp heres / products / nanoxact-silica-nanospheres?variant=15906837954649). Human α1 microglobulin / bikunin precursor (AMBP) was purchased from Creative Biomart, USA (https: / / www.creativebiomart.net / product / NormalSearch?q=AMBP) (purity >98%). Deoxyribonuclease I (DNase I) was purchased from Thermo Fisher Scientific (https: / / www.thermofisher.cn / order / catalog / product / EN0521).

[0043] Table 1 DNA sequences (SEQ ID NO: 1-4)

[0044]

[0045] Note: Bold underlined primers indicate forward primers, and bold italicized primers indicate reverse primers.

[0046] Example 1: Preparation method of DNA viral tracer containing protein encapsulated by EDC covalent coupling

[0047] In this embodiment, adenovirus with double-stranded DNA as the nucleic acid is used as the target virus to prepare a protein-encapsulated DNA virus tracer.

[0048] 1. Preparation of SiO2 core colloidal solution: Uniform SiO2 nanospheres with a particle size of 40 nm were suspended in isopropanol to prepare a SiO2 nanosphere colloidal solution with a concentration of approximately 50 mg / mL. Figure 2 a).

[0049] 2. Preparation of positively charged TMAPS-functionalized SiO2 nanospheres: The prepared SiO2 nanosphere colloidal solution was mixed thoroughly, and 1 mL was added to a 2 mL microcentrifuge tube. The mixture was sonicated for 10 minutes to obtain a homogeneous SiO2 nanosphere colloidal solution. Then, 10 μL of TMAPS solution was added, and the mixture was stirred at 900 rpm at room temperature for 12 hours in a constant temperature incubator. After two centrifugal washes with isopropanol, positively charged SiO2 nanospheres were obtained. Figure 2 b).

[0050] 3. Electrostatic Adsorption of DNA and SiO2 Shell Encapsulation: A 50 μg / mL double-stranded T5 DNA solution (SEQ ID NO: 1) was prepared using ultrapure water. 700 μL of ultrapure water, 320 μL of DNA solution, and 35 μL of positively charged SiO2 nanospheres were added to a centrifuge tube, mixed thoroughly, and sonicated for 2 minutes to obtain a relatively homogeneous solution. After washing twice with ultrapure water by centrifugation, the solution was sonicated again to obtain a homogeneous colloidal solution. Then, 0.5 μL of 50% TMAPS solution was added, mixed thoroughly, and then 0.5 μL of TEOS was added. The mixture was stirred at 900 rpm at room temperature for 4 hours, and then 4 μL of TEOS was added. The mixture was stirred at 900 rpm at room temperature for another 4 days. After washing twice with ultrapure water and sonicating to mix, a SiO2 (DNA(SiO2)) colloidal solution was obtained. Figure 2 c).

[0051] 4. To ensure uniform particle size of SiO2 (DNA (SiO2)), SiO2 (DNA (SiO2)) colloidal solution was freeze-dried under vacuum to obtain SiO2 (DNA (SiO2)) nanospheres. In a micro grinder, 1g of zirconium beads were added to 1mg of SiO2 (DNA (SiO2)) nanospheres and the mixture was ground for 2-8 minutes with zirconium beads of 0.1mm diameter. Then, the mixture was centrifuged at 21000g and washed 3 times to remove the supernatant and add ultrapure water.

[0052] 5. The size and structure of the ground and washed SiO2 (DNA(SiO2)) were observed using transmission electron microscopy (TEM), and its zeta potential (ζ) and hydrodynamic diameter were measured using a Zetasizer Nano ZS. The particle size of SiO2 (DNA(SiO2)) was approximately 65 nm.

[0053] 6. To ensure that the DNA is completely encapsulated in SiO2 (DNA(SiO2)) nanospheres and effectively protected, the uniformly ground SiO2 (DNA(SiO2)) nanospheres were gently mixed in 0.2 U / μL DNase I solution and incubated at 37°C for 1 hour to digest any DNA fragments that were not completely encapsulated on the surface, resulting in SiO2 (DNA(SiO2)) nanospheres with completely protected DNA. Figure 2 d).

[0054] 7. The protein is coated on the outermost layer using the EDC covalent coupling method:

[0055] (1) Preparation of carboxylated SiO2 (DNA(SiO2)) nanospheres: 20 mL of PAA solution (2.5 mg / mL) was added to a 50 mL three-necked flask, followed by sonication and stirring with a polytetrafluoroethylene stir bar. 1 mL of the SiO2 (DNA(SiO2)) colloidal solution prepared in step 6 was added dropwise to the well-stirred PAA solution, and the mixture was sonicated for 30 minutes. The product was then centrifuged at 10,000 rpm for 10 minutes, washed three times with ultrapure water, and finally resuspended and dispersed the nanospheres with ultrapure water to obtain 100 mg / mL carboxylated SiO2 (DNA(SiO2)) nanospheres. Figure 2 e); (2) Preparation of protein solution: Prepare human α1 microglobulin / bikunin precursor (AMBP) solution in PBS buffer (pH 7.4) to obtain a protein solution with a concentration of 2 mg / mL.

[0056] (3) Covalent coupling with proteins: Wash 1 mL (100 mg / mL) of carboxylated SiO2 (DNA(SiO2)) nanospheres twice in 10 mL of MES buffer. After the second wash, resuspend the particles in 10 mL of MES buffer and sonicate to ensure complete suspension of the nanospheres, obtaining a 10 mg / mL carboxylated SiO2 (DNA(SiO2)) nanosphere colloidal solution. While mixing, add 10 mL of EDC (10 mg / mL) dropwise to prevent EDC clumping. Stir continuously at room temperature (18-25℃) for 15 minutes. Wash twice with PBS buffer, then resuspend in 5 mL of PBS buffer and sonicate to ensure complete suspension of the particles. Add 1.85-18.5 mL of protein solution and react at room temperature for 2-4 hours, stirring continuously to ensure sufficient contact and reaction between the nanospheres and proteins. Centrifuge, discard the supernatant, and resuspend in 10 mL of quenching solution, stirring gently for 30 minutes. After centrifugation and discarding the supernatant, resuspend in storage buffer to obtain the protein-encapsulated DNA virus tracer [Protein(SiO2(DNA(SiO2)))]( Figure 2 f), store at 4℃ for later use.

[0057] Protein selection: The outermost layer is coated with a protein whose isoelectric point is close to that of the adenovirus surface, and the protein molecule has a sufficient number of amino groups to ensure that there are enough amino sites to form covalent bonds through dehydration condensation with the carboxyl groups on the surface of the carboxylated SiO2 (DNA(SiO2)) nanospheres. In this embodiment, human α1 microglobulin / bikunin precursor (GenBank: P02760) was selected.

[0058] 8. The size and structure of the DNA virus tracer [Protein(SiO2(DNA(SiO2))] were observed using transmission electron microscopy (TEM), and its zeta potential (ζ) and hydrodynamic diameter were measured using a Zetasizer Nano ZS. The protein-encapsulated DNA virus tracer [Protein(SiO2(DNA(SiO2))] prepared in this example has a particle size of approximately 80 nm, consistent with the 70-90 nm of the target adenovirus. Figure 2 f).

[0059] The DNA half-life in the tracer of this invention is consistent with the DNA half-life in the SiO2(DNA(SiO2)) tracer, and it can be stored at room temperature for more than 116 years. Figure 1 The half-life of the tracer is similar to that of naked DNA, generally less than one day under normal soil and water conditions.

[0060] 9. Quantitative detection of DNA virus tracers

[0061] The DNA virus tracer [Protein(SiO2(DNA(SiO2))] was sequentially immersed in pepsin solution (pH 2.0, simulating gastric juice) and trypsin solution (pH 6.8, simulating intestinal juice) for 2 hours and 12 hours, respectively, to simulate the protein digestion process and remove the outermost protein coat. It was then immersed in NH4F etching solution until clear and free of precipitate, ensuring complete dissolution of silica. The released DNA was then purified using a QIAquick PCR purification kit. Finally, qPCR was performed using the TaqMan probe method, and the DNA copy number concentration of the sample was obtained by comparison with a standard curve.

[0062] 10. The uniformly sized SiO2 (DNA(SiO2)) nanospheres obtained at the end of step 6, with completely preserved DNA. Figure 2 d) Determine the correspondence between the number of tracers and the number of encapsulated DNA copies: The number of tracers is calculated from the total mass of the SiO2 (DNA(SiO2)) nanospheres, the density of SiO2, and the particle size of SiO2 (DNA(SiO2)) measured in step 5. The number of encapsulated DNA copies is obtained by the detection method described in step 9. Thus, the relationship between the number of tracers and the number of encapsulated DNA copies can be established, i.e., how many double-stranded DNAs are encapsulated on average in each SiO2 (DNA(SiO2)) nanosphere.

[0063] Example 2: Application of Protein-Encapsulated DNA Virus Tracers

[0064] Existing DNA virus tracers cannot accurately deduce the migration and transformation process of the target virus from the tracer's migration and transformation process in soil and water media, making them difficult to apply in the field where pathogenic viruses cannot be introduced and only virus tracers can be used. This embodiment uses a DNA virus tracer prepared in Example 1, whose physical filtration and chemical adsorption properties in soil are similar to those of the target virus but whose half-life is much longer, in combination with a bacteriophage as a substitute for the target virus. This allows for the deduction of the pathogen's migration and transformation process, thereby enabling spatiotemporal tracing of non-point source pollution of pathogens in agricultural areas where organic fertilizers are applied. For example, when the target virus is an adenovirus, the corresponding bacteriophage could be crAssphage bacteriophage.

[0065] 1. A scenario of virus infiltration and filtration after organic fertilizer application in unsaturated soil is simulated by adding water with a certain head to an unsaturated vertical soil column. A saturated horizontal soil column simulates the migration of the virus into the drainage ditch after entering the saturated soil. To avoid mutual interference between the tracer and the target virus in the soil, such as occupying adsorption sites, parallel soil column experiments will be conducted. Several identical soil columns will be filled, and each time only one tracer (DNA virus tracer or bacteriophage) or target virus will be added along with the conservative tracer KBr (used to reverse soil hydrodynamic parameters). The recovery rate and breakthrough curve obtained from the experiment will be combined with a soil pollutant migration and transformation model to obtain functions that infer the filtration parameters, adsorption-desorption rates, and decay rates of the target virus from the DNA virus tracer and the phage decay rate in unsaturated and saturated soils under different conditions.

[0066] 2. Water and sediment from several representative ditch sections at different irrigation periods were collected, and a 5-meter-long, 1-meter-high, and 0.6-meter-wide flume was used to simulate the drainage conditions of different ditch sections at different irrigation periods. All tracers (DNA virus tracers and bacteriophages), the target virus, and the conserved tracer KBr (used for reverse hydrodynamic parameters) were added to the flume. The concentration change curves of each tracer and the target virus in the overlying water at different migration distances were obtained from the experiment. Combined with the convection and dispersion equations that consider pollutant decomposition and adsorption / desorption on particulate matter, sedimentation of suspended particulate matter in water, and scouring and sedimentation of bottom sediment, a function was obtained to infer the convection and dispersion parameters, adsorption / desorption rates, and decay rates of the target virus using the DNA virus tracer convection and dispersion parameters and adsorption / desorption rates, and the phage decay rate under different irrigation periods and ditch sections.

[0067] When migrating in soil, the surface chemistry of both the viral tracer and the target virus directly affects their adsorption. When migrating in drainage ditches, both the viral tracer and the target virus also adsorb and desorb onto silt and suspended particles in the water; this adsorption and desorption process is also closely related to their surface chemistry. Therefore, ensuring that the surface of the viral tracer is completely encapsulated by proteins, just like the target virus, is crucial.

[0068] 3. Different crops require different amounts of organic fertilizer and irrigation water, and different soil textures also have varying filtration effects on pathogens. For example... 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 this invention is encoded with different DNA sequences to mark the farmland and time of application, and together with bacteriophages, it is applied with organic fertilizer at different times and in different farmlands. Figure 3The DNA virus tracers applied to four farmlands (each encapsulating one of the four different DNA sequences listed in Table 1) were used to sample nearby groundwater wells and drainage ditches. The concentration changes of the DNA virus tracers and bacteriophages in groundwater and surface water were detected. Then, using the functions derived from the soil column and water tank experiments and simulations in the first two steps—which jointly describe the migration and transformation process of the target virus using DNA virus tracers and bacteriophages—the time and concentration of the target virus entering groundwater and drainage ditches, as well as the time and concentration of its migration over different distances in the drainage ditches, were estimated. This allowed for the spatiotemporal tracing of pathogen non-point source pollution in agricultural areas where organic fertilizer was applied, identifying farmlands and time periods prone to pathogen non-point source pollution.

[0069] Although the present invention has been described in detail above with general descriptions and specific embodiments, modifications or improvements can be made to it, which will be obvious to those skilled in the art. Therefore, all such modifications or improvements made without departing from the spirit of the present invention fall within the scope of protection claimed by the present invention.

Claims

1. A protein-coated DNA virus tracer characterized in that, The DNA virus tracer is composed of a core, a shell and a protein shell wrapped in the shell; The core is a SiO2 nanosphere adsorbing nucleic acid on the surface, and a SiO2 shell with a thickness of y is wrapped on the outside of the core, the surface of the shell is carboxylated, and then the protein is covalently coupled by EDC to form a shell with a thickness of z; wherein 12 nm≤y≤15 nm; The particle size of the SiO2 nanosphere is x, and 20 nm≤x≤120 nm; The particle size of the DNA virus tracer is x+2y+2z, and 60 nm≤x+2y+2z≤160 nm; The nucleic acid is single-stranded or double-stranded DNA with a length of 60-120 bp and without stem loop and hairpin structure; The protein is a protein with a similar isoelectric point to the surface of the capsid protein of the target virus.

2. A preparation method of a DNA virus tracer wrapped with protein by EDC covalent coupling method, characterized in that it comprises the following steps: (1) Contacting SiO2 nanospheres with functional reagent TMAPS to make the surface of the SiO2 nanospheres positively charged, and then electrostatically adsorbing nucleic acid as a core; (2) Contacting the core with TEOS to catalyze the growth of SiO2 and form a SiO2 shell with a thickness of y on the outside of the core, i.e. SiO2(DNA(SiO2)) nanospheres, wherein 12 nm≤y≤15 nm; (3) Carboxylating the surface of the SiO2(DNA(SiO2)) nanospheres obtained in step (2) by using polyacrylic acid, and then contacting the carboxylated SiO2(DNA(SiO2)) nanospheres with a protein solution by using EDC as a crosslinking agent to form a protein shell with a thickness of z on the surface of the SiO2(DNA(SiO2)) nanospheres; The particle size of the SiO2 nanosphere is x, and 20 nm≤x≤120 nm; The nucleic acid is single-stranded or double-stranded DNA with a length of 60-120 bp and without stem loop and hairpin structure; The protein is a protein with a similar isoelectric point to the surface of the capsid protein of the target virus.

3. The method of claim 2, wherein, The following steps are included: 1) Preparation of SiO2 core colloidal solution: SiO2 nanospheres are suspended in isopropyl alcohol to prepare a SiO2 nanosphere colloidal solution with a concentration of 50 mg / mL; 2) Preparation of TMAPS functionalized SiO2 nanospheres with positively charged surface: 1 mL of SiO2 nanosphere colloidal solution is treated by ultrasonic to obtain a uniform SiO2 nanosphere colloidal solution; 10 µL of TMAPS solution is added, and then stirred at a speed of 900 rpm in a thermostat at room temperature for 12 hours; after washing with isopropyl alcohol, SiO2 nanospheres with positively charged surface are obtained; 3) Electrostatic adsorption of DNA and SiO2 shell encapsulation: first, prepare a nucleic acid solution with a concentration of 50 μg / mL using ultrapure water; then add 700 μL of ultrapure water, 320 μL of nucleic acid solution and 35 μL of SiO2 nanoballs with positive surface charges into a centrifuge tube, mix and ultrasonic treatment to obtain a uniform solution; after ultrasonic treatment, centrifugal washing with ultrapure water to obtain a uniform colloidal solution; add 0.5 μL of TMAPS solution, mix, then add 0.5 μL of TEOS, stir at a speed of 900 rpm at room temperature for 4 hours; then add 4 μL of TEOS to the system, stir at a speed of 900 rpm at room temperature for 4 days; after washing with ultrapure water and mixing by ultrasonic treatment, SiO2(DNA(SiO2)) nanoball colloidal solution is obtained; 4) Preparation of carboxylated SiO2(DNA(SiO2)) nanoballs: add 1 mL of SiO2(DNA(SiO2)) nanoball colloidal solution to 20 mL of 2.5 mg / mL polyacrylic acid solution, add water to the product after ultrasonic treatment, centrifugal washing 3 times, and finally resuspend the nanoballs with water, then add MES buffer solution, ultrasonic cleaning, mechanical stirring to obtain carboxylated SiO2(DNA(SiO2)) nanoball colloidal solution; 5) Preparation of protein solution: prepare a protein solution with PBS buffer to obtain a protein solution with a concentration of 2 mg / mL; 6) Covalent coupling of protein: wash 1 mL of carboxylated SiO2(DNA(SiO2)) nanoball colloidal solution in MES buffer solution for 2 times; after the second washing, resuspend the nanoballs in 10 mL of MES buffer solution to obtain a carboxylated SiO2(DNA(SiO2)) nanoball colloidal solution with a concentration of 10 mg / mL; at a temperature of 18-25°C, add 10 mg / mL of EDC 10 mL dropwise while stirring, and continue stirring for 15 minutes; wash with PBS buffer for 2 times, then resuspend the nanoballs in 5 mL of PBS buffer; add 1.85-18.5 mL of protein solution, react at a temperature of 18-25°C for 2-4 hours, continuously stirring during the reaction, after the reaction, centrifugal discard the supernatant, resuspend the nanoball precipitate in 10 mL of quenching solution, stir for 30 minutes, then centrifugal discard the supernatant, resuspend the nanoball precipitate in storage buffer, and obtain the protein-coated DNA virus tracer; wherein the pH of the MES buffer solution is 6, and the pH of the PBS buffer solution is 7.4; the quenching solution is 1 mg / mL casein solution; the storage buffer is 10 mg / mL glycine solution.

4. The method of claim 3, wherein, After ultrasonic treatment in step 4), add water to the product, centrifugal at 10000 rpm for 10 minutes, and wash 3 times.

5. The method according to claim 3 or 4, characterized in that, In step 3), prepare a double-stranded DNA solution using ultrapure water, and the double-stranded DNA is obtained by annealing a single-stranded DNA shown in any one of SEQ ID NO: 1-4 and its reverse complementary strand.

6. The protein-coated DNA viral tracer prepared according to the method of any one of claims 2-5.

7. The DNA viral tracer of claim 6, wherein, The particle size thereof is x+2y+2z, 60 nm≤x+2y+2z≤160 nm.

8. Use of the DNA viral tracer of claim 1, 6 or 7 in the temporal tracing of the source of pathogen pollution in an agricultural area where organic fertilizer is applied. wherein The pathogen is a virus.

Images