A kit for detecting porcine epidemic diarrhea and its preparation method

Abstract

The present invention relates to a kit for detecting porcine epidemic diarrhea and a preparation method thereof, belonging to the technical field of virus detection, and specifically comprising the following steps: after extracting the antigen, treating it with a virus purification agent containing polyethylene glycol, nuclease and concanavalin, and then obtaining a virus antigen suspension with a composite dissociation solution. The suspension is coated on a microporous reaction plate to form a solid-phase antigen. Subsequently, the porcine epidemic diarrhea virus solid-phase antigen, enzyme marker, sample diluent, Tris buffer, color developer and stop solution are aseptically stored to prepare a kit for detecting porcine epidemic diarrhea. The present invention provides a novel kit and a preparation method thereof, which can not only effectively purify porcine epidemic diarrhea virus, but also significantly reduce the risk of protein precipitation, while overcoming nucleic acid interference and improving purification efficiency.

Description

Technical Field

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

[0002] As a common infectious disease, rapid and accurate detection of porcine epidemic diarrhea is crucial for epidemic prevention and control. At present, Elisa kits are an effective means of detecting porcine epidemic diarrhea virus. During the preparation of the kit, the purity of the virus directly determines the detection effect of the kit. Therefore, how to efficiently purify the virus becomes the key to preparing high-quality kits.

[0003] Concanavalin is a material that can be used to purify porcine epidemic diarrhea virus. However, during the purification process, the protein is easily exposed to the air-water interface or the liquid-solid interface through operations such as stirring and filtering, which changes the spatial structure of the protein and forms precipitation. As a result, the total amount of protein used to purify the virus decreases, the purification material cannot be fully utilized, and the purification effect decreases. In addition, during the virus purification process, a large amount of nucleic acid will be released when cells or bacteria are lysed, resulting in an increase in the viscosity of the system and affecting the purification efficiency. And the combined use of nucleases will increase the probability of abnormal protein precipitation, resulting in a decrease in the purification effect.

[0004] Based on the above statements, the present application provides a kit for detecting porcine epidemic diarrhea 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 epidemic diarrhea and a preparation method thereof, which specifically includes the following steps: after extracting the antigen, it is treated with a virus purification agent containing polyethylene glycol, nuclease and concanavalin, and then a virus antigen suspension is obtained with a composite dissociation solution. The suspension is coated on a microporous reaction plate to form a solid-phase antigen. Subsequently, the porcine epidemic diarrhea virus solid-phase antigen, enzyme marker, sample diluent, Tris buffer, color developer and stop solution are aseptically stored to prepare a kit for detecting porcine epidemic diarrhea. The present invention provides a new type of kit and a preparation method thereof, which can not only effectively purify porcine epidemic diarrhea virus, but also significantly reduce the risk of protein precipitation, while overcoming nucleic acid interference and improving purification efficiency.

[0006] A method for preparing a kit for detecting porcine epidemic diarrhea comprises the following preparation steps: S1. Preparation and purification of antigens: extract the antigens, treat them with a virus purification agent, and then centrifuge them with a composite dissociation solution to obtain a virus antigen suspension; Among them, the virus purification agent is composed of polyethylene glycol, nuclease and concanavalin; S2, reaction plate coating: coating the virus antigen suspension obtained in step S1 on a microporous reaction plate to obtain a porcine epidemic diarrhea virus solid phase antigen; S3. Reagent preparation: Sterilely store the porcine epidemic diarrhea virus solid phase antigen, enzyme marker, sample diluent, Tris buffer, color developer and stop solution to obtain a kit for detecting porcine epidemic diarrhea.

[0007] Furthermore, in step S1, the viral antigen suspension is prepared by the following steps: S11. Select piglets with typical clinical symptoms of diarrhea, take samples and place them in a sterilized collection tube container, add physiological saline at a mass volume ratio of 1 g:10 mL, stir evenly and then perform ultrasonic disruption on the system; S12, the virus purification agent is mixed with the system after ultrasonic fragmentation in S11 at a mass ratio of (1-3): 100 at a rate of 30-90 rpm for 20-30 minutes at room temperature, and then centrifuged, the supernatant is discarded, and the precipitate is added to the dissociation solution for centrifugal dissociation at a centrifugal rate of 6000-8000 rpm, and the dissociation time is 10-30 minutes. The virus antigen suspension is obtained.

[0008] Furthermore, in step S11, the specific parameters of the ultrasonic crushing treatment are: The ultrasonic crushing treatment temperature is 0-4°C, the time is 10-30 minutes, ultrasonication is performed for 1-5 seconds and then the ultrasonication is continued for 1-5 seconds with an interval of 2-7 seconds; the ultrasonication power is 200-600W.

[0009] Furthermore, in step S1, the concentration of polyethylene glycol in the virus purification agent is 8-10 mg / mL, the amount of nuclease used is 10-20 U / mL, and the amount of concanavalin A is 50-100 ng / mL.

[0010] Furthermore, the molecular weight of the polyethylene glycol is 2000-4000.

[0011] Furthermore, in step S12, the dissociation solution is a Tirs-HCl buffer solution containing methyl-α-D-pyranoside and NaCl, and the pH value is 6.8-7.2.

[0012] Furthermore, in step S12, the dissociation solution is a 0.01 mol / L Tirs-HCl buffer solution containing 0.2 mol / L methyl-α-D-pyranoside and 50 mmol / L NaCl, and the pH value is 6.8-7.2.

[0013] Furthermore, the specific operations in step S2 are: The viral antigen suspension obtained in step S1 is diluted with PBS buffer and then added to each well of the microplate. The microplate is then incubated, washed, and dried to obtain the solid phase antigen.

[0014] Furthermore, the volume ratio of porcine epidemic diarrhea virus solid phase antigen, enzyme marker, sample diluent, Tris buffer, color developer and stop solution is 1:(10-15):50:40:(5-10):(5-10).

[0015] In summary, this application has the following beneficial effects: In the technical solution of the present invention, polyethylene glycol, nuclease and concanavalin are used as components of the virus purifier. Polyethylene glycol, as a macromolecular protective agent, can prevent the aggregation of concanavalin and nuclease in the system by providing steric hindrance, and can reduce the inactivation or precipitation caused by the interaction between protein molecules in the mixed system. Nuclease can efficiently degrade nucleic acid impurities in virus samples and completely degrade them into oligonucleotides, which helps to remove nucleic acids entangled on the surface of viruses and inclusion bodies, thereby improving the purity and safety of the virus. At the same time, polyethylene glycol can interact with water molecules on the surface of nuclease and concanavalin to maintain the hydration state of the protein, reduce its denaturation probability, and improve the stability of each component in the system. DETAILED DESCRIPTION

[0016] 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 described embodiments 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 creative work are within the scope of protection of the present invention.

[0017] The specific implementation methods of this application involve: The nuclease used was Benzonase.

[0018] The enzyme marker is anti-pig IgG labeled with horseradish peroxidase.

[0019] The color developer is tetramethylbenzidine.

[0020] The stop solution is sulfuric acid.

[0021] Example 1 The present application provides a method for preparing a kit for detecting porcine epidemic diarrhea, comprising the following preparation steps: S1. Preparation and purification of antigens: extract the antigens, treat them with a virus purification agent, and then use a composite dissociation solution for centrifugation to obtain a virus antigen suspension. The specific operation is as follows: S11. Select piglets with typical clinical symptoms of diarrhea, take samples and place them in a sterilized collection tube container, add physiological saline at a mass volume ratio of 1 g:10 mL, stir evenly and then perform ultrasonic disruption on the system; The specific parameters of the ultrasonic treatment are as follows: the ultrasonic treatment temperature is 0-4°C, ultrasonic treatment is continued for 1 second after 1 second of ultrasonic treatment, and the total ultrasonic treatment time is 10 minutes; the ultrasonic power is 200W.

[0022] S12. The virus purification agent was mixed with the system after ultrasonic disruption in S11 at a mass ratio of 1:100 at a rate of 30 rpm for 20 minutes at room temperature, followed by centrifugation, the supernatant was discarded, and the precipitate was added to the dissociation solution for centrifugal dissociation at a centrifugal rate of 6000 rpm and a dissociation time of 10 minutes to obtain a virus antigen suspension.

[0023] The virus purifying agent is composed of polyethylene glycol, nuclease and concanavalin A protein. The concentration of polyethylene glycol in the virus purifying agent is 8 mg / mL, the amount of nuclease used is 10 U / mL, and the amount of concanavalin A protein used is 50 ng / mL. The molecular weight of polyethylene glycol is 2000; The dissociation solution is a 0.01 mol / L Tirs-HCl buffer solution containing 0.2 mol / L methyl-α-D-pyranoside and 50 mmol / L NaCl, and the pH value is 6.8.

[0024] S2, reaction plate coating: coating the virus antigen suspension obtained in step S1 on a microporous reaction plate to obtain a porcine epidemic diarrhea virus solid phase antigen; The viral antigen suspension obtained in step S1 was diluted with PBS buffer at a volume ratio of 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 solid phase antigen. S3. Reagent preparation: Sterile storage of porcine epidemic diarrhea virus solid phase antigen, enzyme marker, sample diluent, Tris buffer, colorimetric agent and stop solution to obtain a kit for detecting porcine epidemic diarrhea; Among them, the volume ratio of porcine epidemic diarrhea virus solid phase antigen, enzyme marker, sample diluent, Tris buffer, color developer and stop solution is 1:10:50:40:5:5.

[0025] Example 2 The present application provides a method for preparing a kit for detecting porcine epidemic diarrhea, comprising the following preparation steps: S1. Preparation and purification of antigens: extract the antigens, treat them with a virus purification agent, and then use a composite dissociation solution for centrifugation to obtain a virus antigen suspension. The specific operation is as follows: S11. Select piglets with typical clinical symptoms of diarrhea, take samples and place them in a sterilized collection tube container, add physiological saline at a mass volume ratio of 1 g:10 mL, stir evenly and then perform ultrasonic disruption on the system; The specific parameters of the ultrasonic treatment are as follows: the ultrasonic treatment temperature is 0°C, ultrasonic treatment is continued for 3 seconds after 3 seconds of ultrasonic treatment and then for 5 seconds, the total ultrasonic treatment time is 20 minutes, and the ultrasonic power is 400W.

[0026] S12. The virus purification agent was mixed with the system after ultrasonic disruption in S11 at a mass ratio of 2:100 at a rate of 60 rpm for 25 minutes at room temperature, followed by centrifugation. The supernatant was discarded and the precipitate was added to the dissociation solution for centrifugal dissociation at a centrifugal rate of 7000 rpm and a dissociation time of 20 minutes to obtain a virus antigen suspension.

[0027] The virus purifying agent is composed of polyethylene glycol, nuclease and concanavalin A protein. The concentration of polyethylene glycol in the virus purifying agent is 9 mg / mL, the amount of nuclease used is 15 U / mL, and the amount of concanavalin A protein used is 80 ng / mL. The molecular weight of polyethylene glycol is 3000; The dissociation solution is a 0.01 mol / L Tirs-HCl buffer solution containing 0.2 mol / L methyl-α-D-pyranoside and 50 mmol / L NaCl, and the pH value is 7.0.

[0028] S2, reaction plate coating: coating the virus antigen suspension obtained in step S1 on a microporous reaction plate to obtain a porcine epidemic diarrhea virus solid phase antigen; The viral antigen suspension obtained in step S1 was diluted with PBS buffer at a volume ratio of 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 solid phase antigen. S3. Reagent preparation: Sterile storage of porcine epidemic diarrhea virus solid phase antigen, enzyme marker, sample diluent, Tris buffer, colorimetric agent and stop solution to obtain a kit for detecting porcine epidemic diarrhea; Among them, the volume ratio of porcine epidemic diarrhea virus solid phase antigen, enzyme marker, sample diluent, Tris buffer, color developer and stop solution is 1:12:50:40:7:8.

[0029] Example 3 The present application provides a method for preparing a kit for detecting porcine epidemic diarrhea, comprising the following preparation steps: S1. Preparation and purification of antigens: extract the antigens, treat them with a virus purification agent, and then use a composite dissociation solution for centrifugation to obtain a virus antigen suspension. The specific operation is as follows: S11. Select piglets with typical clinical symptoms of diarrhea, take samples and place them in a sterilized collection tube container, add physiological saline at a mass volume ratio of 1 g:10 mL, stir evenly and then perform ultrasonic disruption on the system; The specific parameters of the ultrasonic treatment are as follows: the ultrasonic treatment temperature is 4°C, ultrasonic treatment is continued for 5 seconds after 5 seconds of ultrasonic treatment and then for 7 seconds, the total ultrasonic treatment time is 30 minutes, and the ultrasonic power is 600W.

[0030] S12. The virus purification agent was mixed with the system after ultrasonic disruption in S11 at a mass ratio of 3:100 at a rate of 90 rpm for 30 minutes at room temperature, followed by centrifugation, the supernatant was discarded, and the precipitate was added to the dissociation solution for centrifugal dissociation at a centrifugal rate of 8000 rpm and a dissociation time of 30 minutes to obtain a virus antigen suspension.

[0031] The virus purifying agent is composed of polyethylene glycol, nuclease and concanavalin A protein. The concentration of polyethylene glycol in the virus purifying agent is 10 mg / mL, the amount of nuclease used is 20 U / mL, and the amount of concanavalin A protein used is 100 ng / mL. The molecular weight of polyethylene glycol is 4000; The dissociation solution is a 0.01 mol / L Tirs-HCl buffer solution containing 0.2 mol / L methyl-α-D-pyranoside and 50 mmol / L NaCl, and the pH value is 7.2.

[0032] S2, reaction plate coating: coating the virus antigen suspension obtained in step S1 on a microporous reaction plate to obtain a porcine epidemic diarrhea virus solid phase antigen; The viral antigen suspension obtained in step S1 was diluted with PBS buffer at a volume ratio of 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 solid phase antigen. S3. Reagent preparation: Sterile storage of porcine epidemic diarrhea virus solid phase antigen, enzyme marker, sample diluent, Tris buffer, colorimetric agent and stop solution to obtain a kit for detecting porcine epidemic diarrhea; Among them, the volume ratio of porcine epidemic diarrhea virus solid phase antigen, enzyme marker, sample diluent, Tris buffer, color developer and stop solution is 1:15:50:40:10:10.

[0033] Comparative Example 1 The difference between this comparative example and Example 1 is that the molecular weight of the polyethylene glycol used in the virus purification agent in this comparative example is 200.

[0034] Comparative Example 2 The difference between this comparative example and Example 1 is that the molecular weight of the polyethylene glycol used in the virus purification agent in this comparative example is 8000.

[0035] Comparative Example 3 The difference between this comparative example and Example 1 is that the virus purification agent in this comparative example consists of nuclease and concanavalin A protein, the amount of nuclease used in the virus purification agent is 20 U / mL, and the amount of concanavalin A protein used is 100 ng / mL.

[0036] Comparative Example 4 The difference between this comparative example and Example 1 is that the virus purification agent in this comparative example is composed of polyethylene glycol and concanavalin, the concentration of polyethylene glycol in the virus purification agent is 10 mg / mL, the amount of concanavalin is 100 ng / mL, and the molecular weight of polyethylene glycol is 4000.

[0037] Performance Testing The kits for detecting porcine epidemic diarrhea prepared in Examples 1-3 and Comparative Examples 1-4 of the present application were tested.

[0038] Accuracy test method: 300 serum samples of pigs known to be infected with porcine epidemic diarrhea virus were selected, and 300 serum samples of pigs not infected with porcine epidemic diarrhea virus were selected as controls. The serum samples were diluted 1:1 with diluent and added to 50 μL in a microplate, with 3 replicate wells for each sample. Then incubate at 37°C for 1 hour, then wash the well plate with washing solution to remove unbound antibodies and impurities, then add enzyme markers and incubate under the same conditions, wash the plate again, add color substrate, and observe color changes. After adding the stop solution, use an ELISA reader to read the absorbance value at 450nm, and determine the presence or absence of antibodies in the sample based on the comparison of the OD value with the critical value (CUT OFF).

[0039] Sensitivity test method: Prepare porcine epidemic diarrhea virus antibody samples with known concentrations ranging from 0.1-1.0 pg / mL, and use the same detection method as above to test the lowest antibody concentration value.

[0040] The specific performance test results are shown in Table 1: Table 1

[0041] From the results of Comparative Examples 1-3 in Table 1 above, it can be seen that the use of a polyethylene glycol component with an appropriate molecular weight can well improve the stability of concanavalin, improve its virus purification effect, and improve the accuracy and sensitivity of the kit. From the results in Comparative Example 4, it can be seen that the use of nuclease can efficiently degrade nucleic acid impurities in virus samples and improve the detection effect of the kit.

[0042] In the description of the specification, the description with reference to the terms "one embodiment", "example", "specific example", etc. means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described can be combined in any one or more embodiments or examples in a suitable manner.

[0043] 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 specific embodiments described or replace them in a similar manner. As long as they do not deviate from the invention or exceed the scope defined by the claims, they shall all fall within the protection scope of the present invention.

Claims

1. A method for preparing a kit for detecting porcine epidemic diarrhea, characterized in that: The method comprises the following preparation steps: S1. Preparation and purification of antigens: extract the antigens, treat them with a virus purification agent, and then centrifuge them with a composite dissociation solution to obtain a virus antigen suspension; Among them, the virus purification agent is composed of polyethylene glycol, nuclease and concanavalin; S2, reaction plate coating: coating the virus antigen suspension obtained in step S1 on a microporous reaction plate to obtain a porcine epidemic diarrhea virus solid phase antigen; S3. Reagent preparation: Sterilely store the porcine epidemic diarrhea virus solid phase antigen, enzyme marker, sample diluent, Tris buffer, color developer and stop solution to obtain a kit for detecting porcine epidemic diarrhea.

2. The method for preparing a kit for detecting porcine epidemic diarrhea according to claim 1, characterized in that: In step S1, the viral antigen suspension is specifically prepared by the following steps: S11. Select piglets with typical clinical symptoms of diarrhea, take samples and place them in a sterilized collection tube container, add physiological saline, stir evenly, and then perform ultrasonic disruption on the system; S12, mixing the virus purification agent with the system after ultrasonic disruption in S11 at room temperature for 20-30 minutes, then centrifuging, discarding the supernatant, adding the precipitate to the dissociation solution for centrifugal dissociation, the centrifugal speed is 6000-8000rpm, the dissociation time is 10-30 minutes, and the virus antigen suspension is obtained.

3. The method for preparing a kit for detecting porcine epidemic diarrhea according to claim 2, characterized in that: In step S11, the specific parameters of the ultrasonic crushing treatment are: The ultrasonic crushing treatment temperature is 0-4°C, ultrasonication is performed for 1-5 seconds and then continued for 1-5 seconds at an interval of 2-7 seconds, and the total ultrasonication time is 10-30 minutes; the ultrasonication power is 200-600W.

4. The method for preparing a kit for detecting porcine epidemic diarrhea according to claim 2, characterized in that: In step S1, the concentration of polyethylene glycol in the virus purification agent is 8-10 mg / mL, the amount of nuclease used is 10-20 U / mL, and the amount of concanavalin A is 50-100 ng / mL.

5. The method for preparing a kit for detecting porcine epidemic diarrhea according to claim 4, characterized in that: The molecular weight of the polyethylene glycol is 2000-4000.

6. The method for preparing a kit for detecting porcine epidemic diarrhea according to claim 2, characterized in that: In step S12, the dissociation solution is a Tirs-HCl buffer solution containing methyl-α-D-pyranoside and NaCl, and the pH value is 6.8-7.

2.

7. The method for preparing a kit for detecting porcine epidemic diarrhea according to claim 1, characterized in that: The specific operations in step S2 are: The viral antigen suspension obtained in step S1 is diluted with PBS buffer and then added to each well of the microplate. The microplate is then incubated, washed, and dried to obtain the solid phase antigen.

8. The method for preparing a kit for detecting porcine epidemic diarrhea according to claim 1, characterized in that: The volume ratio of porcine epidemic diarrhea virus solid phase antigen, enzyme marker, sample diluent, Tris buffer, color developer and stop solution is 1:(10-15):50:40:(5-10):(5-10).

9. A kit prepared by the method for preparing a kit for detecting porcine epidemic diarrhea according to any one of claims 1 to 8.

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