A kit for detecting porcine reproductive and respiratory syndrome virus (PRRSV) and its preparation method.

Abstract

This invention relates to a kit for detecting porcine reproductive and respiratory syndrome virus (PRRSV) and its preparation method, belonging to the field of virus detection technology. Specifically, the preparation steps include: S1, extracting and purifying antigens from the virus using a composite precipitant (polyethylene glycol, ammonium sulfate, and modified cellulose nanocrystals); S2, coating the purified antigens onto a microplate to form a solid-phase antigen; S3, preparing and aseptically storing the solid-phase antigen, enzyme label, diluent, buffer, chromogenic agent, and stop solution to obtain the kit for detecting PRRSV. The raw materials used in this preparation method, such as cellulose nanocrystals, polyethylene glycol, and ammonium sulfate, are all environmentally friendly materials, and no harmful substances are generated during the preparation process, conforming to the concept of green chemistry. It features improved precipitation efficiency, enhanced antigen stability, optimized precipitation conditions, improved purification quality, and environmental friendliness.

Description

Technical Field

[0001] This application relates to the field of virus detection technology, and more specifically, to a kit for detecting porcine reproductive and respiratory syndrome virus (PRRSV) and a method for preparing the same. Background Technology

[0002] Porcine reproductive and respiratory syndrome (PRRS), also known as blue ear disease, is a global infectious disease caused by porcine reproductive and respiratory virus (PRRSV). This disease causes enormous economic losses to the pig farming industry and severely impacts the health and productivity of pig herds. Therefore, establishing a rapid, accurate, and sensitive method for detecting PRRSV is of great significance for disease prevention, control, and treatment.

[0003] Currently, methods for detecting porcine reproductive and respiratory syndrome virus (PRRSV) mainly include virus isolation and nucleic acid detection. However, these methods have certain limitations in terms of accuracy, sensitivity, and ease of operation. Virus isolation methods are time-consuming, complex, and require high-level laboratory conditions; while nucleic acid detection methods are highly sensitive, they can be affected by interference from other nucleic acid substances in the sample and require highly skilled operators. Existing sandwich antibody-antigen kits still have some problems when detecting PRRSV, such as insufficient accuracy and unstable sensitivity. This is mainly because factors such as antibody quality, antigen purity, and buffer formulation in the kit have a significant impact on the detection results.

[0004] Based on the above statements, this application provides a kit for detecting porcine reproductive and respiratory syndrome virus (PRRSV) and a method for preparing the same. Summary of the Invention

[0005] To address the problems mentioned in the background art, this application provides a kit for detecting porcine reproductive and respiratory syndrome virus (PRRSV) and a method for preparing the same.

[0006] This application provides a kit for detecting porcine reproductive and respiratory syndrome virus (PRRSV) and its preparation method, employing the following technical solution:

[0007] A method for preparing a kit for detecting porcine reproductive and respiratory syndrome virus (PRRSV) includes the following preparation steps:

[0008] S1. Preparation and purification of antigen: The antigen was extracted from porcine reproductive and respiratory syndrome virus (PRRSV) and purified using a compound precipitant to obtain the viral antigen.

[0009] S2, Coating of the reaction plate: The viral antigen obtained in step S1 is coated on a microplate to obtain porcine reproductive and respiratory syndrome virus solid-phase antigen.

[0010] S3. Reagent preparation: Sterilely store porcine reproductive and respiratory syndrome virus (PRRSV) solid-phase antigen, enzyme label, sample diluent, buffer, chromogenic agent and stop solution to obtain a kit for detecting PRRSV.

[0011] The composite precipitant is composed of ammonium sulfate and modified cellulose nanocrystals.

[0012] Furthermore, the modified cellulose nanocrystals are prepared by the following steps:

[0013] Carboxylated cellulose nanocrystals and polyethylene glycol were mixed at a mass ratio of 1g:(5-10)g, 2-3mL of stannous octoate was added, and the mixture was stirred at a speed of 30-90rpm. The system temperature was raised to 80-90℃ under an inert atmosphere and heated for 4-6 hours. Then anhydrous ethanol was added, the system was centrifuged, and dialyzed for 48-72 hours to obtain modified cellulose nanocrystals.

[0014] Furthermore, in step S1, the viral antigen is specifically prepared by the following steps:

[0015] S11. Porcine reproductive and respiratory virus (PRRSV) cell culture harvest medium was obtained by culturing Marc-145 cells with PRRSV JXA1-R strain.

[0016] S12. Centrifuge the porcine reproductive and respiratory syndrome virus (PRRSV) cell culture harvest broth, collect the supernatant, add a composite precipitant to the supernatant, and filter the supernatant; after filtration, concentrate the virus clarification solution using a tangential flow membrane with a pore size of 30-100 kDa to obtain the virus concentrate.

[0017] S13. Add modified PBS buffer to the virus concentrate, mix well, and repeat the concentration step and the step of adding modified PBS buffer 3-5 times; then purify the system by chromatography using a composite packed column to obtain the virus antigen.

[0018] Furthermore, in step S1, the viral antigen is specifically prepared by the following steps:

[0019] S11. Porcine reproductive and respiratory syndrome virus (PRRSV) cell culture harvest broth was obtained by inoculating Marc-145 cells with PRRSV JXA1-R strain. The inoculum size was set at 5-10%, and the specific culture conditions were: pH 5.0-7.0, automatic ammonia addition, dissolved oxygen 30%, and stirring speed 300 rpm. After 20 h of fermentation, 50% (volume fraction) glycerol was added at a rate of 20 g / (L·h). After 30 h of fermentation, the addition was stopped, and methanol was added at a rate of 1.5 g / (L·h) for 16 hours to induce fermentation. After that, fermentation was stopped, and the PRRSV cell culture harvest broth was obtained.

[0020] S12. Centrifuge the porcine reproductive and respiratory syndrome virus (PRRSV) cell culture harvest medium, collect the supernatant, add a composite precipitant to the supernatant, and then filter the supernatant using a 0.45μm filter cartridge; after filtration, concentrate the virus clarification solution using a tangential flow membrane with a pore size of 30-100 kDa, and set the peristaltic pump output flow rate to 20 L / min during the concentration process to obtain the virus concentrate;

[0021] S13. Add modified PBS buffer to the virus concentrate, with a volume ratio of virus concentrate to modified PBS buffer of 1:(5-10). After mixing, repeat the concentration step and the step of adding modified PBS buffer 3-5 times. Then, purify the system by chromatography using a composite packing column, setting the sample loading volume to 1-5 column volumes to obtain the virus antigen.

[0022] Furthermore, in step S3, the volume ratio of porcine reproductive and respiratory syndrome virus solid antigen, enzyme marker, sample diluent, buffer, chromogenic agent and stop solution is 1:(10-15):50:40:(5-10):(5-10).

[0023] Furthermore, the enzyme marker is horseradish peroxidase-labeled anti-pig IgG.

[0024] Furthermore, the modified PBS buffer comprises the following components: based on PBS buffer, it further contains 8.0 g / L NaCl, 0.2 g / L KCl, 3.58 g / L Na2HPO4·12H2O, 0.24 g / L KH2PO4, and the pH value of the modified PBS buffer is 5.0-7.0.

[0025] Furthermore, in step S2, the porcine reproductive and respiratory syndrome virus (PRRSV) solid-phase antigen is specifically prepared by the following steps:

[0026] The viral antigen obtained in step S1 is diluted at a ratio of 1:100-1000 and then added to each well of a microplate. The microplate is then incubated at 37°C for 30-60 minutes. After washing the microplate, it is allowed to air dry at room temperature to obtain the porcine reproductive and respiratory syndrome virus (PRRSV) solid-phase antigen.

[0027] In summary, this application has the following beneficial effects:

[0028] In this invention, the extraction and purification steps of the antigen are optimized. By controlling cell culture conditions and the use of a composite precipitant, viral antigens are effectively purified, impurities are removed, and the purity and quality of the antigen are improved. Carboxylated cellulose nanocrystals have carboxyl groups on their surface, which can undergo esterification and hydrogen bonding with the hydroxyl groups in polyethylene glycol molecules, thereby modifying the cellulose nanocrystals. By controlling the hydrophilicity and charge properties of the modified cellulose nanocrystal surface, their binding with impurities and unwanted components in the antigen is improved. Furthermore, the introduction of polyethylene glycol into the system can reduce the binding between cellulose nanocrystals and antigens, protecting the activity of the antigen. Ammonium sulfate, as a salting-out agent, can affect the solubility of proteins by reducing the ionic strength of the solution. The combined use of modified cellulose nanocrystals and ammonium sulfate can further enhance the precipitation effect and improve the purity of the antigen.

[0029] By using inorganic salt components in different proportions to adjust the salt ion concentration in the PBS buffer, the adsorption between the virus and the container wall can be reduced, and the osmotic pressure and ionic strength of the buffer can be maintained, thereby facilitating the separation and purification of the virus and helping to maintain the stability and activity of the virus particles.

[0030] The raw materials used in this preparation method, such as cellulose nanocrystals, polyethylene glycol, and ammonium sulfate, are all environmentally friendly materials, and no harmful substances are generated during the preparation process, which aligns with the principles of green chemistry. It features improved precipitation efficiency, enhanced antigen stability, optimized precipitation conditions, improved purification quality, and environmental friendliness. Detailed Implementation

[0031] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0032] In the following examples and comparative examples, during the chromatographic purification process using the composite packed column described, the anion exchange chromatography medium DEAE-Sepharose Fast Flow was selected, with a medium size of 1.6cm × 10cm; the chromatographic purification sample loading volume was set to 2 column volumes, and the sample loading flow rate was 1mL / min; a 0-1 M NaCl linear gradient (gradient volume 2 times column volume) was used, with a flow rate of 1mL / min.

[0033] In the following examples and comparative examples, the final concentration of the enzyme marker in the kit product was 0.3 μg / mL.

[0034] Example 1

[0035] A method for preparing a kit for detecting porcine reproductive and respiratory syndrome virus (PRRSV) includes the following preparation steps:

[0036] S1. Preparation and purification of antigen: The antigen was extracted from porcine reproductive and respiratory syndrome virus (PRRSV) and purified using a compound precipitant to obtain the viral antigen. The specific steps were as follows:

[0037] S11. Porcine reproductive and respiratory syndrome virus (PRRSV) cell culture harvest broth was obtained by culturing Marc-145 cells with PRRSV JXA1-R strain. The inoculum size was set at 5%, and the specific culture conditions were: pH 5.0, automatic ammonia addition, dissolved oxygen 30%, and stirring speed 300 rpm. After 20 h of fermentation, 50% (volume fraction) glycerol was added at a rate of 20 g / (L·h). After 30 h of fermentation, the addition was stopped, and methanol was added at a rate of 1.5 g / (L·h) for 16 hours to induce fermentation. After that, fermentation was stopped, and the porcine reproductive and respiratory syndrome virus (PRRSV) cell culture harvest broth was obtained.

[0038] S12. Centrifuge the porcine reproductive and respiratory syndrome virus (PRRSV) cell culture harvest medium, collect the supernatant, add a composite precipitant to the supernatant, and then filter the supernatant using a 0.45μm filter cartridge; after filtration, concentrate the virus clarification solution using a tangential flow membrane with a pore size of 30 kDa, and set the peristaltic pump output flow rate to 20 L / min during the concentration process to obtain the virus concentrate;

[0039] The composite precipitant is composed of ammonium sulfate and modified cellulose nanocrystals mixed in a 1:1 mass ratio, and the modified cellulose nanocrystals are prepared by the following steps:

[0040] Carboxylated cellulose nanocrystals and polyethylene glycol-200 were mixed at a mass ratio of 1g:5g, 2mL of stannous octoate was added, the mixture was stirred at 30rpm, and the system temperature was raised to 80℃ under an inert atmosphere. After heating for 5 hours, 20mL of anhydrous ethanol was added, the system was centrifuged at 10000rpm, and the dialysis treatment was set to 1000Da for 48 hours to obtain modified cellulose nanocrystals.

[0041] S13. Add modified PBS buffer to the virus concentrate. The volume ratio of virus concentrate to modified PBS buffer is 1:5. After mixing, repeat the concentration step and the step of adding modified PBS buffer 3 times. Then, purify the system by chromatography using a composite packing column. Set the sample loading volume to 2 column volumes to obtain the virus antigen.

[0042] The modified PBS buffer contains the following components: based on PBS buffer, it also contains 8.0 g / L NaCl, 0.2 g / L KCl, 3.58 g / L Na2HPO4·12H2O, 0.24 g / L KH2PO4, and has a pH of 5.0.

[0043] S2. Reaction Plate Coating: The viral antigen obtained in step S1 is coated onto a microplate to obtain porcine reproductive and respiratory syndrome virus (PRRSV) solid-phase antigen. The specific operation is as follows:

[0044] The viral antigen obtained in step S1 was diluted at a ratio of 1:500 and then added to each well of a microplate. The microplate was then incubated at 37°C for 30 minutes. After washing the microplate, it was allowed to air dry at room temperature to obtain the porcine reproductive and respiratory syndrome virus (PRRSV) solid-phase antigen.

[0045] S3. Reagent preparation: Sterilely store porcine reproductive and respiratory syndrome virus (PRRSV) solid-phase antigen, enzyme label, sample diluent, PBS buffer, chromogenic agent and stop solution to obtain a kit for detecting PRRSV.

[0046] The volume ratio of porcine reproductive and respiratory syndrome virus (PRRSV) solid-phase antigen, enzyme marker, sample diluent, buffer, chromogenic agent, and stop solution was 1:10:50:40:5:5; the enzyme marker was horseradish peroxidase-labeled anti-porcine IgG.

[0047] Example 2

[0048] A method for preparing a kit for detecting porcine reproductive and respiratory syndrome virus (PRRSV) includes the following preparation steps:

[0049] S1. Preparation and purification of antigen: The antigen was extracted from porcine reproductive and respiratory syndrome virus (PRRSV) and purified using a compound precipitant to obtain the viral antigen. The specific steps were as follows:

[0050] S11. Porcine reproductive and respiratory syndrome virus (PRRSV) cell culture harvest broth was obtained by culturing Marc-145 cells with PRRSV JXA1-R strain. The inoculum size was set at 8%, and the specific culture conditions were: pH 6.5, automatic ammonia addition, dissolved oxygen 30%, and stirring speed 300 rpm. After 20 h of fermentation, 50% (volume fraction) glycerol was added at a rate of 20 g / (L·h). After 30 h of fermentation, the addition was stopped, and methanol was added at a rate of 1.5 g / (L·h) for 16 hours to induce fermentation. After that, fermentation was stopped, and the porcine reproductive and respiratory syndrome virus (PRRSV) cell culture harvest broth was obtained.

[0051] S12. Centrifuge the porcine reproductive and respiratory syndrome virus (PRRSV) cell culture harvest medium, collect the supernatant, add a composite precipitant to the supernatant, and then filter the supernatant using a 0.45 μm filter cartridge; after filtration, concentrate the virus concentrate using a tangential flow membrane with a pore size of 70 kDa, and set the peristaltic pump output flow rate to 20 L / min during the concentration process to obtain a concentrated virus solution; the composite precipitant is composed of ammonium sulfate and modified cellulose nanocrystals mixed in a 1:1 mass ratio, and the modified cellulose nanocrystals are prepared by the following steps:

[0052] Carboxylated cellulose nanocrystals and polyethylene glycol-200 were mixed at a mass ratio of 1g:7g, 2mL of stannous octoate was added, the mixture was stirred at 60rpm, and the system temperature was raised to 85℃ under an inert atmosphere. After heating for 5 hours, 20mL of anhydrous ethanol was added, the system was centrifuged at 10000rpm, and the system was dialyzed at a cutoff of 1000Da for 60 hours to obtain modified cellulose nanocrystals.

[0053] S13. Add modified PBS buffer to the virus concentrate. The volume ratio of virus concentrate to modified PBS buffer is 1:7. After mixing, repeat the concentration step and the step of adding modified PBS buffer 5 times. Then, purify the system by chromatography using a composite packing column. Set the sample loading volume to 3 column volumes to obtain the virus antigen.

[0054] The modified PBS buffer contains the following components: based on PBS buffer, it also contains 8.0 g / L NaCl, 0.2 g / L KCl, 3.58 g / L Na2HPO4·12H2O, 0.24 g / L KH2PO4, and has a pH of 6.5.

[0055] S2. Reaction Plate Coating: The viral antigen obtained in step S1 is coated onto a microplate to obtain porcine reproductive and respiratory syndrome virus (PRRSV) solid-phase antigen. The specific operation is as follows:

[0056] The viral antigen obtained in step S1 was diluted at a ratio of 1:500 and then added to each well of a microplate. The microplate was then incubated at 37°C for 45 minutes. After washing the microplate, it was allowed to air dry at room temperature to obtain the porcine reproductive and respiratory syndrome virus (PRRSV) solid-phase antigen.

[0057] S3. Reagent preparation: Sterilely store porcine reproductive and respiratory syndrome virus (PRRSV) solid-phase antigen, enzyme label, sample diluent, PBS buffer, chromogenic agent and stop solution to obtain a kit for detecting PRRSV.

[0058] The volume ratio of porcine reproductive and respiratory syndrome virus (PRRSV) solid-phase antigen, enzyme marker, sample diluent, buffer, chromogenic agent, and stop solution was 1:12:50:40:7:7; the enzyme marker was horseradish peroxidase-labeled anti-porcine IgG.

[0059] Example 3

[0060] A method for preparing a kit for detecting porcine reproductive and respiratory syndrome virus (PRRSV) includes the following preparation steps:

[0061] S1. Preparation and purification of antigen: The antigen was extracted from porcine reproductive and respiratory syndrome virus (PRRSV) and purified using a compound precipitant to obtain the viral antigen. The specific steps were as follows:

[0062] S11. Porcine reproductive and respiratory syndrome virus (PRRSV) cell culture harvest broth was obtained by culturing Marc-145 cells with PRRSV JXA1-R strain. The inoculum size was set at 10%, and the specific culture conditions were: pH 7.0, automatic ammonia addition, dissolved oxygen 30%, and stirring speed 300 rpm. After 20 h of fermentation, 50% (volume fraction) glycerol was added at a rate of 20 g / (L·h). After 30 h of fermentation, the addition was stopped, and methanol was added at a rate of 1.5 g / (L·h) for 16 hours to induce fermentation. After that, fermentation was stopped, and the PRRSV cell culture harvest broth was obtained.

[0063] S12. Centrifuge the porcine reproductive and respiratory syndrome virus (PRRSV) cell culture harvest medium, collect the supernatant, add a composite precipitant to the supernatant, and then filter the supernatant using a 0.45 μm filter cartridge; after filtration, concentrate the virus concentrate using a tangential flow membrane with a pore size of 90 kDa, and set the peristaltic pump output flow rate to 20 L / min during the concentration process to obtain a concentrated virus solution; the composite precipitant is composed of ammonium sulfate and modified cellulose nanocrystals mixed in a 1:1 mass ratio, and the modified cellulose nanocrystals are prepared by the following steps:

[0064] Carboxylated cellulose nanocrystals and polyethylene glycol-200 were mixed at a mass ratio of 1g:10g, 3mL of stannous octoate was added, the mixture was stirred at 90rpm, and the system temperature was raised to 90℃ under an inert atmosphere. After heating for 5 hours, 20mL of anhydrous ethanol was added, the system was centrifuged at 10000rpm, and the dialysis treatment was set to 1000Da for 72 hours to obtain modified cellulose nanocrystals.

[0065] S13. Add modified PBS buffer to the virus concentrate. The volume ratio of virus concentrate to modified PBS buffer is 1:10. After mixing, repeat the concentration step and the step of adding modified PBS buffer 5 times. Then, purify the system by chromatography using a composite packing column. Set the sample loading volume to 5 column volumes to obtain the virus antigen.

[0066] The modified PBS buffer contains the following components: based on PBS buffer, it also contains 8.0 g / L NaCl, 0.2 g / L KCl, 3.58 g / L Na2HPO4·12H2O, 0.24 g / L KH2PO4, and has a pH of 7.0.

[0067] S2. Reaction Plate Coating: The viral antigen obtained in step S1 is coated onto a microplate to obtain porcine reproductive and respiratory syndrome virus (PRRSV) solid-phase antigen. The specific operation is as follows:

[0068] The viral antigen obtained in step S1 was diluted at a ratio of 1:500 and then added to each well of a microplate. The microplate was then incubated at 37°C for 60 minutes. After washing the microplate, it was allowed to air dry at room temperature to obtain the porcine reproductive and respiratory syndrome virus (PRRSV) solid-phase antigen.

[0069] S3. Reagent preparation: Sterilely store porcine reproductive and respiratory syndrome virus (PRRSV) solid-phase antigen, enzyme label, sample diluent, PBS buffer, chromogenic agent and stop solution to obtain a kit for detecting PRRSV.

[0070] The volume ratio of porcine reproductive and respiratory syndrome virus (PRRSV) solid-phase antigen, enzyme marker, sample diluent, buffer, chromogenic agent, and stop solution was 1:15:50:40:10:10; the enzyme marker was horseradish peroxidase-labeled anti-porcine IgG.

[0071] Comparative Example 1

[0072] The difference between this comparative example and Example 1 is that a composite precipitant was prepared by mixing polyethylene glycol-200, ammonium sulfate and carboxylated cellulose nanocrystals in a mass ratio of 6:1:5.

[0073] Comparative Example 2

[0074] The difference between this comparative example and Example 1 is that a composite precipitant was prepared by mixing polyethylene glycol-200 and carboxylated cellulose nanocrystals in a 1:1 mass ratio.

[0075] Comparative Example 3

[0076] This comparative example uses PBS buffer instead of modified PBS buffer.

[0077] Performance testing

[0078] The kits prepared in Examples 1-3 and Comparative Examples 1-2 of this application were tested.

[0079] Accuracy test: 300 serum samples from pigs known to be infected with porcine reproductive and respiratory syndrome virus (PRRSV) and 300 serum samples from pigs not infected with PRRSV were selected as test subjects. The sample addition, incubation, plate washing, enzyme addition, and termination were performed under sterile conditions, and the color development results were recorded to determine the detection accuracy of the kit.

[0080] Sensitivity test: The minimum antibody concentration required to produce a detectable signal was determined within the concentration range of 0.1-1.0 pg / mL. The specific performance test results are shown in Table 1 below.

[0081] Table 1

[0082]

[0083] As shown in Table 1 above, the overall performance of the kit samples prepared in Examples 1-3 of this application is significantly better than that of the samples prepared in Comparative Examples 1-3. That is, within the technical scope defined in this application, the porcine reproductive and respiratory syndrome virus (PRRSV) detection kit exhibits excellent overall performance. The results in Comparative Example 1 show that the combination of polyethylene glycol and cellulose nanocrystals improves the hydrophilicity and charge properties of the cellulose nanocrystal surface, promoting its binding with impurities in the antigen, improving the precipitation effect of the composite precipitant on impurities, and increasing the sensitivity of the kit. The results in Comparative Example 2 show that using modified polyethylene glycol and ammonium sulfate in combination can reduce the ionic strength in the system, regulate protein solubility, and further enhance the precipitation effect. The results in Comparative Example 3 show that optimizing the component concentration in the buffer solution can maintain osmotic pressure and assist in the regulation of ionic strength by ammonium sulfate in the composite precipitant, helping to maintain the stability of virus particles and facilitating virus isolation and purification.

[0084] In the description of this specification, the references to terms such as "an embodiment," "example," "specific example," etc., indicate that a specific feature, structure, material, or characteristic described in connection with that embodiment or example is included in at least one embodiment or example of the present invention. In this specification, the illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0085] The above description is merely an example and illustration of the present invention. Those skilled in the art can make various modifications or additions to the specific embodiments described, or use similar methods to replace them, as long as they do not deviate from the invention or exceed the scope defined in the claims, all of which should fall within the protection scope of the present invention.

Claims

1. A method for preparing a reagent kit for detecting porcine reproductive and respiratory syndrome virus (PRRSV), characterized in that, The preparation steps include the following: S1. Preparation and purification of antigen: The antigen was extracted from porcine reproductive and respiratory syndrome virus (PRRSV) and purified using a compound precipitant to obtain the viral antigen. S2, Coating of the reaction plate: The viral antigen obtained in step S1 is coated on a microplate to obtain porcine reproductive and respiratory syndrome virus solid-phase antigen. S3. Reagent preparation: Sterilely store porcine reproductive and respiratory syndrome virus (PRRSV) solid-phase antigen, enzyme label, sample diluent, buffer, chromogenic agent and stop solution to obtain a kit for detecting PRRSV. The composite precipitant is composed of ammonium sulfate and modified cellulose nanocrystals; the modified cellulose nanocrystals are prepared by the following steps: Carboxylated cellulose nanocrystals and polyethylene glycol were mixed at a mass ratio of 1g:(5-10)g, 2-3mL of stannous octoate was added, and the mixture was stirred at a speed of 30-90rpm. The system temperature was raised to 80-90℃ under an inert atmosphere and heated for 4-6 hours. Then anhydrous ethanol was added, the system was centrifuged, and dialyzed for 48-72 hours to obtain modified cellulose nanocrystals.

2. The method for preparing a reagent kit for detecting porcine reproductive and respiratory syndrome virus (PRRSV) according to claim 1, characterized in that, In step S1, the viral antigen is specifically prepared through the following steps: S11. Porcine reproductive and respiratory virus (PRRSV) cell culture harvest medium was obtained by culturing Marc-145 cells with PRRSV JXA1-R strain. S12. Centrifuge the porcine reproductive and respiratory syndrome virus (PRRSV) cell culture harvest broth, collect the supernatant, add a composite precipitant to the supernatant, and filter the supernatant; after filtration, concentrate the virus clarification solution using a tangential flow membrane with a pore size of 30-100 kDa to obtain the virus concentrate. S13. Add modified PBS buffer to the virus concentrate, mix well, and repeat the concentration step and the step of adding modified PBS buffer 3-5 times; then purify the system by chromatography using a composite packed column to obtain the viral antigen; wherein, in step S13, the modified PBS buffer includes the following components: based on PBS buffer, it also contains NaCl 8.0 g / L, KCl 0.2 g / L, Na2HPO4·12H2O 3.58 g / L, KH2PO4 0.24 g / L, and the pH value of the modified PBS buffer is 5.0-7.

0.

3. The method for preparing a reagent kit for detecting porcine reproductive and respiratory syndrome virus (PRRSV) according to claim 1, characterized in that, In step S2, the porcine reproductive and respiratory syndrome virus (PRRSV) solid-phase antigen is specifically prepared by the following steps: The viral antigen obtained in step S1 is diluted at a ratio of 1:100-1000 and then added to each well of a microplate. The microplate is then incubated for 30-60 minutes, washed, and allowed to air dry at room temperature to obtain the porcine reproductive and respiratory syndrome virus solid-phase antigen.

4. The method for preparing a reagent kit for detecting porcine reproductive and respiratory syndrome virus (PRRSV) according to claim 1, characterized in that, In step S3, the volume ratio of porcine reproductive and respiratory syndrome virus (PRRSV) solid antigen, enzyme marker, sample diluent, buffer, chromogenic agent, and stop solution is 1:(10-15):50:40:(5-10):(5-10).

5. A method for preparing a reagent kit for detecting porcine reproductive and respiratory syndrome virus (PRRSV) according to claim 1, characterized in that, In step S3, the enzyme marker is horseradish peroxidase-labeled anti-pig IgG.

6. A kit for detecting porcine reproductive and respiratory syndrome virus (PRRSV) prepared by the method described in any one of claims 1-5.

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