Kit for detecting porcine reproductive and respiratory syndrome virus and preparation method thereof

Abstract

The invention relates to a kit for detecting a porcine reproductive and respiratory syndrome virus and a preparation method thereof, and belongs to the technical field of virus detection.The preparation method specifically comprises the following preparation steps that S1, a composite precipitator (polyethylene glycol, ammonium sulfate and modified cellulose nanocrystals) is used for extracting and purifying an antigen from the virus; s2, coating a microporous reaction plate with the purified antigen to form a solid-phase antigen; and S3, preparing and aseptically storing the solid-phase antigen, the enzyme marker, a diluent, a buffer solution, a color developing agent and a stop solution to prepare the kit for detecting the porcine reproductive and respiratory syndrome virus. The raw materials such as the cellulose nanocrystal, the polyethylene glycol and the ammonium sulfate used in the preparation method are environment-friendly materials, harmful substances are not generated in the preparation process, and the concept of green chemistry is met. The method has the characteristics of improvement of precipitation efficiency, enhancement of antigen stability, optimization of precipitation conditions, improvement of purification quality, environmental friendliness and the like.

Description

Technical Field

[0001] The present application relates to the technical field of virus detection, and more specifically, to a kit for detecting porcine blue ear virus and a preparation method thereof. Background Art

[0002] Porcine reproductive and respiratory syndrome (PRRS), also known as porcine reproductive and respiratory syndrome (PRRS), is a global infectious disease caused by the porcine reproductive and respiratory syndrome virus (PRRSV). The disease causes significant economic losses to the swine industry, severely impacting the health and productivity of pigs. Therefore, developing a rapid, accurate, and sensitive method for detecting PRRSV is crucial for disease prevention, control, and treatment.

[0003] Currently, the methods used to detect 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 laboratory conditions. Although nucleic acid detection methods are highly sensitive, they are subject to interference from other nucleic acid substances in the sample and require high operating skills from the experimenter. Existing sandwich antibody-antigen test kits still have some problems when detecting porcine reproductive and respiratory syndrome virus (PRRSV), such as insufficient accuracy and unstable sensitivity. This is mainly due to the fact that factors such as the antibody quality, antigen purity, and buffer formulation in the test kit have a significant impact on the test results.

[0004] Based on the above statements, the present application provides a kit for detecting porcine reproductive and respiratory syndrome virus and a preparation method thereof. Summary of the Invention

[0005] In order to solve the problems raised in the background technology, the present application provides a kit for detecting porcine blue ear virus and a preparation method thereof.

[0006] The present application provides a kit for detecting porcine reproductive and respiratory syndrome virus and a preparation method thereof, which adopts the following technical solutions: A method for preparing a kit for detecting porcine reproductive and respiratory syndrome virus (PRRSV) comprises the following steps: S1. Preparation and purification of antigens: extracting antigens from porcine blue ear virus and purifying them using a composite precipitant to obtain viral antigens; S2, reaction plate coating: The viral antigen obtained in step S1 is coated on a microporous reaction plate to obtain a porcine reproductive and respiratory syndrome virus solid phase antigen; S3. Reagent preparation: Sterilely store the porcine reproductive and respiratory syndrome virus solid phase antigen, enzyme marker, sample diluent, buffer, color developing agent, and stop solution to obtain a kit for detecting porcine reproductive and respiratory syndrome virus; Wherein, the composite precipitant consists of ammonium sulfate and modified cellulose nanocrystals.

[0007] Furthermore, the modified cellulose nanocrystals are prepared by the following steps: Carboxylated cellulose nanocrystals and polyethylene glycol were mixed in a mass ratio of 1 g:(5-10) g, 2-3 mL of stannous octoate was added, and the mixture was stirred at a rate of 30-90 rpm. The system temperature was raised to 80-90°C under an inert atmosphere. After heating for 4-6 hours, anhydrous ethanol was added, the system was centrifuged, and dialyzed for 48-72 hours to obtain modified cellulose nanocrystals.

[0008] Furthermore, in step S1, the viral antigen is specifically prepared by the following steps: S11, using Marc-145 cells inoculated with PRRSV JXA1-R strain to obtain porcine reproductive and respiratory syndrome virus cell culture harvest fluid; S12, centrifuging the porcine blue ear virus cell culture harvest, collecting the supernatant, adding a composite precipitant to the supernatant, and filtering the supernatant; after filtration, concentrating the virus clarified liquid using a tangential flow membrane, wherein the pore size of the tangential flow membrane is 30-100KDa, to obtain a virus concentrate; S13. Add modified PBS buffer to the virus concentrate, mix well, and repeat the concentration step and the addition of modified PBS buffer step 3-5 times; then purify the system by chromatography using a composite filler column to obtain the virus antigen.

[0009] Furthermore, in step S1, the viral antigen is specifically prepared by the following steps: S11. PRRS virus cell culture harvest fluid was obtained by inoculating Marc-145 cells with the 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 feed, 30% dissolved oxygen, and a stirring rate of 300 rpm. After 20 hours of fermentation, 50% (volume fraction) glycerol was added at a rate of 20 g / L·h. This addition was stopped after 30 hours of fermentation. Methanol was added at a rate of 1.5 g / L·h for 16 hours of induction, and then fermentation was stopped to obtain the PRRS virus cell culture harvest fluid. S12, centrifuging the porcine blue ear virus cell culture harvest, collecting the supernatant, adding a composite precipitant to the supernatant, and then filtering the supernatant using a 0.45 μm filter element; after filtration, concentrating the virus clarified liquid using a tangential flow membrane, wherein the pore size of the tangential flow membrane is 30-100 KDa, and setting the peristaltic pump flow output flow rate to 20 L / min during the concentration process to obtain a virus concentrate; 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 addition of modified PBS buffer step 3-5 times; then, purify the system by chromatography using a composite filler column, setting the sample volume to 1-5 column volumes, to obtain the viral antigen.

[0010] Furthermore, in step S3, the volume ratio of the porcine reproductive and respiratory syndrome virus solid phase antigen, enzyme marker, sample diluent, buffer, color developer and stop solution is 1:(10-15):50:40:(5-10):(5-10).

[0011] Furthermore, the enzyme marker is horseradish peroxidase-labeled anti-pig IgG.

[0012] Furthermore, the improved 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, and 0.24 g / L KH2PO4. The pH value of the improved PBS buffer is 5.0-7.0.

[0013] Furthermore, in step S2, the porcine reproductive and respiratory syndrome virus solid phase antigen is specifically prepared by the following steps: The viral antigen obtained in step S1 is diluted at 1:100-1000 and then added to each well of the microplate. The microplate is then incubated at 37°C for 30-60 minutes, and then the well plate is washed and naturally dried at room temperature to obtain the porcine blue ear virus solid phase antigen.

[0014] In summary, this application has the following beneficial effects: In the technical solution of the present invention, the extraction and purification steps of the antigen are optimized, and by controlling the cell culture conditions and the use of a composite precipitant, the viral antigen is effectively purified, impurities are removed, and the purity and quality of the antigen are improved. The surface of the carboxylated cellulose nanocrystal carries a carboxyl group, which can undergo an esterification reaction and hydrogen bond with the hydroxyl group in the polyethylene glycol molecule, thereby achieving the modification of the cellulose nanocrystal. By regulating the hydrophilicity and charge properties of the surface of the modified cellulose nanocrystal, it is improved to more effectively bind to impurities and unnecessary components in the antigen. In addition, the introduction of polyethylene glycol into the system can reduce the binding between the cellulose nanocrystal and the antigen, thereby protecting the activity of the antigen. Ammonium sulfate, as a salting-out agent, can affect the solubility of the protein by reducing the ionic strength of the solution. The compounding of modified cellulose nanocrystals and ammonium sulfate can further enhance the precipitation effect and improve the purity of the antigen.

[0015] By using inorganic salt components in different proportions, the salt ion concentration in the PBS buffer is regulated, the adsorption between the virus and the container wall is reduced, and the osmotic pressure and ionic strength of the buffer are maintained, thereby facilitating the separation and purification of the virus and helping to maintain the stability and activity of the virus particles.

[0016] This preparation method uses environmentally friendly raw materials such as cellulose nanocrystals, polyethylene glycol, and ammonium sulfate. No harmful substances are produced during the preparation process, aligning with the concept of green chemistry. It has the advantages of improving precipitation efficiency, enhancing antigen stability, optimizing precipitation conditions, improving purification quality, and being environmentally friendly. DETAILED DESCRIPTION

[0017] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the embodiments described are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts shall fall within the scope of protection of the present invention.

[0018] In the chromatographic purification process of the composite filler column described in the following examples and comparative examples, an anion exchange chromatography medium DEAE-Sepharose Fast Flow was selected, and the medium specification was 1.6 cm × 10 cm; the chromatography purification sample load was set to 2 column volumes, and the sample load flow rate was 1 mL / min; a 0-1 M NaCl linear gradient (gradient volume 2 column volumes) was used, and the flow rate was 1 mL / min.

[0019] The final concentration of the enzyme marker in the kit product in the following examples and comparative examples is 0.3 μg / mL.

[0020] Example 1 A method for preparing a kit for detecting porcine reproductive and respiratory syndrome virus (PRRSV) comprises the following steps: S1. Preparation and purification of antigens: Extract antigens from porcine blue ear virus and purify them using a composite precipitant to obtain viral antigens. The specific operations are as follows: S11. PRRSV cell culture harvest fluid was obtained by inoculating Marc-145 cells with the PRRSV JXA1-R strain. The inoculum size was set at 5%, and the specific culture conditions were: pH 5.0, automated ammonia feed, 30% dissolved oxygen, and a stirring rate of 300 rpm. After 20 hours of fermentation, 50% (volume fraction) glycerol was added at a rate of 20 g / L·h. This addition was stopped after 30 hours of fermentation. Methanol was added at a rate of 1.5 g / L·h for 16 hours of induction, and then fermentation was stopped to obtain the PRRSV cell culture harvest fluid. S12, centrifuging the porcine blue ear virus cell culture harvest, collecting the supernatant, adding a composite precipitant to the supernatant, and then filtering the supernatant using a 0.45 μm filter element; after filtration, concentrating the virus clarified liquid using a tangential flow membrane, wherein the pore size of the tangential flow membrane is 30 KDa, and setting the peristaltic pump flow output flow rate to 20 L / min during the concentration process to obtain a virus concentrate; The composite precipitant is composed of ammonium sulfate and modified cellulose nanocrystals mixed in a mass ratio of 1:1, and the modified cellulose nanocrystals are prepared by the following steps: Carboxylated cellulose nanocrystals and polyethylene glycol-200 were mixed in a mass ratio of 1 g:5 g, 2 mL of stannous octoate was added, and the mixture was stirred at 30 rpm. The temperature of the system was raised to 80°C under an inert atmosphere. After heating for 5 hours, 20 mL of anhydrous ethanol was added, and the system was centrifuged at 10,000 rpm and dialyzed for 48 hours at a cutoff of 1,000 Da to obtain modified cellulose nanocrystals. S13, adding modified PBS buffer to the virus concentrate, with a volume ratio of virus concentrate to modified PBS buffer of 1:5, mixing, and repeating the concentration step and the addition of modified PBS buffer step three times; then, the system was purified by chromatography using a composite filler column, with the sample volume set to 2 column volumes, to obtain the viral antigen; 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, and KH2PO4 0.24 g / L. The pH value of the modified PBS buffer is 5.0.

[0021] S2. Reaction plate coating: The viral antigen obtained in step S1 is coated on a microporous reaction plate to obtain a PRRS virus solid phase antigen. The specific operation is as follows: The viral antigen obtained in step S1 was diluted 1:500 and then added to each well of the microplate. The microplate was then incubated at 37°C for 30 minutes, and then the well plate was washed and naturally dried at room temperature to obtain the porcine blue ear virus solid phase antigen.

[0022] S3. Reagent preparation: Sterilely store the porcine reproductive and respiratory syndrome virus solid phase antigen, enzyme marker, sample diluent, PBS buffer, colorimetric agent, and stop solution to obtain a kit for detecting porcine reproductive and respiratory syndrome virus; Among them, the volume ratio of porcine blue ear virus solid phase antigen, enzyme marker, sample diluent, buffer, color developer and stop solution is 1:10:50:40:5:5; the enzyme marker is horseradish peroxidase-labeled anti-swine IgG.

[0023] Example 2 A method for preparing a kit for detecting porcine reproductive and respiratory syndrome virus (PRRSV) comprises the following steps: S1. Preparation and purification of antigens: Extract antigens from porcine blue ear virus and purify them using a composite precipitant to obtain viral antigens. The specific operations are as follows: S11. PRRSV cell culture harvest fluid was obtained by inoculating Marc-145 cells with the PRRSV JXA1-R strain. The inoculum size was set at 8%, and the specific culture conditions were: pH 6.5, automated ammonia feed, 30% dissolved oxygen, and a stirring rate of 300 rpm. After 20 hours of fermentation, 50% (volume fraction) glycerol was added at a rate of 20 g / L·h. This addition was stopped after 30 hours of fermentation. Methanol was added at a rate of 1.5 g / L·h for 16 hours of induction, and then fermentation was stopped to obtain the PRRSV cell culture harvest fluid. S12. Centrifuge the porcine blue ear virus cell culture harvest, collect the supernatant, add a composite precipitant to the supernatant, and then filter the supernatant using a 0.45 μm filter element; after filtration, concentrate the virus clarified liquid using a tangential flow membrane, wherein the pore size of the tangential flow membrane is 70 KDa, and the peristaltic pump output flow rate is set to 20 L / min during the concentration process to obtain a virus concentrate; the composite precipitant is composed of ammonium sulfate and modified cellulose nanocrystals mixed in a mass ratio of 1:1, and the modified cellulose nanocrystals are prepared by the following steps: Carboxylated cellulose nanocrystals and polyethylene glycol-200 were mixed in a mass ratio of 1 g:7 g, 2 mL of stannous octoate was added, and the mixture was stirred at 60 rpm. The temperature of the system was raised to 85°C under an inert atmosphere. After heating for 5 hours, 20 mL of anhydrous ethanol was added, and the system was centrifuged at 10,000 rpm and dialyzed for 60 hours at a cutoff of 1,000 Da to obtain modified cellulose nanocrystals. S13, adding modified PBS buffer to the virus concentrate, with a volume ratio of virus concentrate to modified PBS buffer of 1:7, mixing, and repeating the concentration step and the addition of modified PBS buffer step 5 times; then the system was purified by chromatography using a composite filler column, and the sample volume was set to 3 column volumes to obtain the viral antigen; 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, and KH2PO4 0.24 g / L. The pH value of the modified PBS buffer is 6.5.

[0024] S2. Reaction plate coating: The viral antigen obtained in step S1 is coated on a microporous reaction plate to obtain a PRRS virus solid phase antigen. The specific operation is as follows: The viral antigen obtained in step S1 was diluted 1:500 and then added to each well of the microplate. The microplate was then incubated at 37°C for 45 minutes, and then the well plate was washed and naturally dried at room temperature to obtain the porcine blue ear virus solid phase antigen.

[0025] S3. Reagent preparation: Sterilely store the porcine reproductive and respiratory syndrome virus solid phase antigen, enzyme marker, sample diluent, PBS buffer, colorimetric agent, and stop solution to obtain a kit for detecting porcine reproductive and respiratory syndrome virus; Among them, the volume ratio of porcine blue ear virus solid phase antigen, enzyme marker, sample diluent, buffer, color developer and stop solution is 1:12:50:40:7:7; the enzyme marker is horseradish peroxidase-labeled anti-swine IgG.

[0026] Example 3 A method for preparing a kit for detecting porcine reproductive and respiratory syndrome virus (PRRSV) comprises the following steps: S1. Preparation and purification of antigens: Extract antigens from porcine blue ear virus and purify them using a composite precipitant to obtain viral antigens. The specific operations are as follows: S11. PRRSV cell culture harvest fluid was obtained by inoculating Marc-145 cells with the PRRSV JXA1-R strain. The inoculum size was set at 10%, and the specific culture conditions were: pH 7.0, automatic ammonia feed, 30% dissolved oxygen, and a stirring rate of 300 rpm. After 20 hours of fermentation, 50% (volume fraction) glycerol was added at a rate of 20 g / L·h. This addition was stopped after 30 hours of fermentation. Methanol was added at a rate of 1.5 g / L·h for 16 hours of induction, and then fermentation was stopped to obtain the PRRSV cell culture harvest fluid. S12. Centrifuge the porcine blue ear virus cell culture harvest, collect the supernatant, add a composite precipitant to the supernatant, and then filter the supernatant using a 0.45 μm filter element; after filtration, concentrate the virus clarified liquid using a tangential flow membrane, wherein the pore size of the tangential flow membrane is 90 KDa, and the peristaltic pump output flow rate is set to 20 L / min during the concentration process to obtain a virus concentrate; the composite precipitant is composed of ammonium sulfate and modified cellulose nanocrystals mixed in a mass ratio of 1:1, and the modified cellulose nanocrystals are prepared by the following steps: Carboxylated cellulose nanocrystals and polyethylene glycol-200 were mixed in a mass ratio of 1 g:10 g, 3 mL of stannous octoate was added, and the mixture was stirred at 90 rpm. The temperature of the system was raised to 90°C under an inert atmosphere. After heating for 5 hours, 20 mL of anhydrous ethanol was added, and the system was centrifuged at 10,000 rpm and dialyzed for 72 hours at a cutoff of 1,000 Da to obtain modified cellulose nanocrystals. S13, adding modified PBS buffer to the virus concentrate, with a volume ratio of virus concentrate to modified PBS buffer of 1:10, mixing, and repeating the concentration step and the addition of modified PBS buffer step 5 times; then, the system was purified by chromatography using a composite filler column, and the sample volume was set to 5 column volumes to obtain the viral antigen; 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, and KH2PO4 0.24 g / L. The pH value of the modified PBS buffer is 7.0.

[0027] S2. Reaction plate coating: The viral antigen obtained in step S1 is coated on a microporous reaction plate to obtain a PRRS virus solid phase antigen. The specific operation is as follows: The viral antigen obtained in step S1 was diluted 1:500 and then added to each well of the microplate. The microplate was then incubated at 37°C for 60 minutes, and then the well plate was washed and naturally dried at room temperature to obtain the porcine blue ear virus solid phase antigen.

[0028] S3. Reagent preparation: Sterilely store the porcine reproductive and respiratory syndrome virus solid phase antigen, enzyme marker, sample diluent, PBS buffer, colorimetric agent, and stop solution to obtain a kit for detecting porcine reproductive and respiratory syndrome virus; Among them, the volume ratio of porcine blue ear virus solid phase antigen, enzyme marker, sample diluent, buffer, color developer and stop solution is 1:15:50:40:10:10; the enzyme marker is horseradish peroxidase-labeled anti-swine IgG.

[0029] Comparative Example 1 The difference between this comparative example and Example 1 is that polyethylene glycol-200, ammonium sulfate and carboxylated cellulose nanocrystals are mixed in a mass ratio of 6:1:5 to prepare a composite precipitant.

[0030] Comparative Example 2 The difference between this comparative example and Example 1 is that polyethylene glycol-200 and carboxylated cellulose nanocrystals are mixed in a mass ratio of 1:1 to prepare a composite precipitant.

[0031] Comparative Example 3 This comparative example uses PBS buffer instead of modified PBS buffer.

[0032] Performance Testing The kits prepared in Examples 1-3 and Comparative Examples 1-2 of the present application were tested.

[0033] Accuracy test: 300 serum samples from pigs known to be infected with porcine reproductive and respiratory syndrome virus (PRRSV) and 300 serum samples from uninfected pigs were selected as test subjects. Samples were added, incubated, plates were washed, enzymes were added, and the color development results were recorded after termination in a sterile environment to determine the detection accuracy of the kit.

[0034] Sensitivity test: The lowest antibody concentration that produces a detectable signal was determined within a concentration range of 0.1-1.0 pg / mL. Specific performance test results are shown in Table 1 below.

[0035] Table 1 As shown in Table 1 above, the comprehensive performance of the kit samples prepared in Examples 1-3 of the present application is significantly better than that of the samples prepared in Comparative Examples 1-3, that is, within the technical solution defined in the present application, the comprehensive performance of the porcine blue ear virus detection kit prepared is excellent. As shown in the results of Comparative Example 1, the combination of polyethylene glycol and cellulose nanocrystals improves the hydrophilicity and charge properties of the cellulose nanocrystal surface, promotes its combination with impurities in the antigen, improves the precipitation effect of the composite precipitant on impurities, and improves the sensitivity of the kit. As shown in the results of Comparative Example 2, the use of modified polyethylene glycol and ammonium sulfate in combination can reduce the ionic strength in the system, regulate the solubility of the protein, and further enhance the precipitation effect. As shown in the results of Comparative Example 3, the optimization of the component concentration in the buffer can maintain the osmotic pressure and assist the regulation of the ionic strength by ammonium sulfate in the composite precipitant, which helps to maintain the stability of the virus particles and facilitates the separation and purification of the virus.

[0036] Throughout the specification, references to terms such as "one embodiment," "example," or "specific example" indicate that the specific features, structures, materials, or characteristics described in conjunction with that embodiment or example are included in at least one embodiment or example of the present invention. In this specification, schematic representations of these 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 any one or more embodiments or examples.

[0037] The above contents are merely examples and explanations of the present invention. Those skilled in the art may make various modifications or additions to the described specific embodiments or replace them in similar ways. As long as they do not deviate from the invention or exceed the scope defined by the claims, they should all fall within the scope of protection of the present invention.

Claims

1. A method for preparing a kit for detecting porcine reproductive and respiratory syndrome virus, characterized in that: The method comprises the following preparation steps: S1. Preparation and purification of antigens: extracting antigens from porcine blue ear virus and purifying them using a composite precipitant to obtain viral antigens; S2, reaction plate coating: The viral antigen obtained in step S1 is coated on a microporous reaction plate to obtain a porcine reproductive and respiratory syndrome virus solid phase antigen; S3. Reagent preparation: Sterilely store the porcine reproductive and respiratory syndrome virus solid phase antigen, enzyme marker, sample diluent, buffer, color developing agent, and stop solution to obtain a kit for detecting porcine reproductive and respiratory syndrome virus; Wherein, the composite precipitant consists of ammonium sulfate and modified cellulose nanocrystals.

2. The method for preparing a kit for detecting porcine reproductive and respiratory syndrome virus according to claim 1, wherein In step S1, the viral antigen is specifically prepared by the following steps: S11, using Marc-145 cells inoculated with PRRSV JXA1-R strain to obtain porcine reproductive and respiratory syndrome virus cell culture harvest fluid; S12, centrifuging the porcine blue ear virus cell culture harvest, collecting the supernatant, adding a composite precipitant to the supernatant, and filtering the supernatant; after filtration, concentrating the virus clarified liquid using a tangential flow membrane, wherein the pore size of the tangential flow membrane is 30-100KDa, to obtain a virus concentrate; S13. Add modified PBS buffer to the virus concentrate, mix well, and repeat the concentration step and the addition of modified PBS buffer step 3-5 times; then purify the system by chromatography using a composite filler column to obtain the virus antigen.

3. The method for preparing a kit for detecting porcine reproductive and respiratory syndrome virus according to claim 1, wherein In step S2, the porcine blue ear virus solid phase antigen is specifically prepared by the following steps: The viral antigen obtained in step S1 is diluted at 1:100-1000 and then added to each well of the microplate. The microplate is then incubated for 30-60 minutes, and then the well plate is washed and naturally dried at room temperature to obtain the porcine blue ear virus solid phase antigen.

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

5. The method for preparing a kit for detecting porcine reproductive and respiratory syndrome virus according to claim 1, wherein In step S3, the enzyme marker is horseradish peroxidase-labeled anti-pig IgG.

6. The method for preparing a kit for detecting porcine reproductive and respiratory syndrome virus according to claim 2, wherein: In step S13, the modified PBS buffer comprises the following components: based on PBS buffer, further containing 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.

7. The method for preparing a kit for detecting porcine reproductive and respiratory syndrome virus according to claim 1, wherein: The modified cellulose nanocrystals are prepared by the following steps: Carboxylated cellulose nanocrystals and polyethylene glycol were mixed in a mass ratio of 1 g:(5-10) g, 2-3 mL of stannous octoate was added, and the mixture was stirred at a rate of 30-90 rpm. The system temperature was raised to 80-90°C under an inert atmosphere. After heating for 4-6 hours, anhydrous ethanol was added, the system was centrifuged, and dialyzed for 48-72 hours to obtain modified cellulose nanocrystals.

8. A kit for detecting porcine reproductive and respiratory syndrome virus prepared by the preparation method according to any one of claims 1 to 7.

Images