A homogeneous detection reaction reagent and detection method for African swine fever virus

Abstract

The present invention relates to a homogeneous detection reaction reagent and a detection method for African swine fever virus. The homogeneous reaction kit includes reagent A and reagent B. When the African swine fever virus in the sample is combined with specific antibodies of reagent A and reagent B to form an immune reaction conjugate, a fluorescent donor and a fluorescent receptor form a fluorescent resonance body. When the donor is excited by excitation light, the receptor emits resonance fluorescence. The intensity of the resonance fluorescence emitted by the receptor is directly related to the content of African swine fever virus in the sample. The method does not require embedding and separation, and can reduce interference factors that affect the detection accuracy in the process of removing free markers, so that the accuracy and sensitivity of the detection are improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of homogeneous immunoassay, and in particular relates to a homogeneous detection reaction reagent and a detection method for African swine fever virus. Background Art

[0002] African swine fever (ASF) is an acute, severe, and highly contagious disease of pigs caused by African swine fever virus (ASFV). ASFV is a regular icosahedron with a diameter of about 200 nanometers and is composed of multiple layers of material: a protein nucleocapsid containing a nucleoid in the center, and a lipid envelope and a protein capsid from the inside to the outside. The capsid is composed of 8280 major capsid proteins p72 and 60 pentaproteins. In addition, at least three proteins maintain the stability of the capsid structure by adhering to adjacent proteins.

[0003] Its clinical features are mainly systemic bleeding, respiratory and nervous system dysfunction, high morbidity and mortality, and great harm to the pig farming industry. At present, there is no effective vaccine or special antiviral drug that can effectively control the spread of the virus in time when the epidemic breaks out. The outbreak and prevalence are mainly controlled by culling after quarantine. Therefore, it is of great significance to establish a scientific and effective detection and diagnosis method for the prevention of African swine fever.

[0004] According to whether it is necessary to separate the immune-bound markers and the unimmune-bound free markers after the antigen-antibody reaction, the immunoassay technology is divided into homogeneous immunoassay and heterogeneous immunoassay. At present, the immunoassay technologies of gold label, enzyme label, time-resolved fluorescence and chemiluminescence all belong to heterogeneous immunoassay methods. Homogeneous immunoassay does not require multiple steps such as embedding and washing to remove unimmune-bound free markers, eliminating the cumbersome operations of embedding and separation, and more importantly, it can greatly reduce the interference factors that affect the accuracy of the test results in the process of removing unimmune-bound free markers. Therefore, in recent years, the newly established homogeneous immunoassay method based on the principle of fluorescence resonance energy transfer has attracted widespread attention for its simple, accurate operation and low cost.

[0005] Time-resolved fluoroim-munoassay (TRFIA) belongs to fluorescent immunoassay and was first proposed by Soini and Hemmila in 1979. They used rare earth ions for labeling technology, which effectively avoided the problems of spontaneous fluorescence of biological materials and easy quenching of fluorescent dyes in fluorescent immunoassay, and established a high-sensitivity immunoassay technology that is a milestone in the field of labeled immunoassay after radioimmunoassay quantitative determination.

[0006] Fluorescence Resonance Energy Transfer (FRET) is a non-radiative energy transition that resonates the donor's excited state energy to the acceptor through intermolecular electric dipole interactions, so that the acceptor emits fluorophores. If one fluorescent group (the emission spectrum of the donor) partially overlaps with another fluorescent group (the absorption spectrum of the acceptor), when the distance between the two groups meets the energy exchange conditions (generally less than 10nm), energy resonant energy transfer occurs between the donor and the acceptor, that is, when the donor is excited by the excitation light, the electrons transition from the ground state energy level to the highly excited state, and in the process of returning to the ground state, part of the excitation energy is transferred to the adjacent acceptor, causing the acceptor to emit fluorescence. The intensity of the fluorescence signal emitted by the donor and the acceptor is directly related to the amount of the donor and the acceptor that can produce resonance. This method has excellent specificity and can eliminate the background fluorescence signal generated by non-specific reactions.

[0007] There are many methods for detecting African swine fever virus at present. For example, African swine fever virus enzyme-labeled detection reagents. Since the bound and free markers are separated by washing, the non-free markers are also washed away during the washing process. In addition, the washing process is prone to inconsistency, or if the cleaning is not thorough, the free markers will produce background effects, which will directly affect the sensitivity of the detection.

[0008] The principles and detection methods of the present invention are different from those of CN201711203146.6. The energy resonance of the present invention has stronger specificity, which is conducive to improving the sensitivity of detection. Summary of the invention

[0009] In order to solve the above technical problems, the present invention provides an African swine fever virus homogeneous detection reaction reagent, a kit and a detection method. The detection method uses homogeneous time-resolved fluorescence technology to detect African swine fever virus, has high sensitivity, strong specificity, and is efficient and convenient to operate. It does not require separation of free markers and non-free markers, and does not require cleaning. It is suitable for on-site detection, and the use of these methods facilitates high-throughput detection.

[0010] A homogeneous detection reagent for African swine fever virus that solves the above technical problems is characterized by comprising a reaction reagent A and a reaction reagent B,

[0011] The reaction reagent A is composed of a lanthanide cryptate compound of formula IV as a fluorescence donor and a coupled antibody molecule for capturing African swine fever virus;

[0012]

[0013] The reaction reagent B is composed of allophycocyanin as a fluorescent receptor and a coupled detection molecule; the detection molecule is an African swine fever virus antibody.

[0014] The African swine fever virus antibody is any specific antibody against African swine fever virus, including polyclonal antibodies and monoclonal antibodies; the capture molecule is the same as or different from the detection molecule.

[0015] The detection molecule in the reaction reagent B is any specific antibody against African swine fever virus, including polyclonal antibodies and monoclonal antibodies; the capture molecule is the same as or different from the detection molecule.

[0016] Preferably, the reaction reagent A is in the form of a reaction reagent A solution; wherein the capture molecule is reacted with a monoclonal antibody, and the detection molecule is reacted with another monoclonal antibody.

[0017] The reaction reagent A exists in the form of a reaction reagent A solution.

[0018] The reaction reagent B exists in the form of a reaction reagent B solution.

[0019] A homogeneous detection method for African swine fever virus of the reactant is characterized by comprising the following steps:

[0020] (1) Reagent A, test sample and reagent B need to be configured in a ratio; preferably: 1:2:1 ratio for immune reaction.

[0021] (2) Reagent A, the test sample and reagent B react simultaneously or sequentially. Preferably, reagent A and the test sample are first subjected to immune reaction and then react with reagent B.

[0022] Time-resolved fluorescence detection technology was used to detect the fluorescence detection signals at central wavelengths of 615 nm and 665 nm respectively.

[0023] In the present invention, a 0.05 mg / ml African swine fever P30 protein standard is used to dilute at least three gradient samples with a buffer solution, i.e., 10, 100, and 1000 times dilutions, and then time-resolved fluorescence detection is performed at 615 nm and 665 nm, respectively, and a standard curve is made using 665 nm / 615 nm and the concentration of the standard.

[0024] The method of the present invention detects the African swine fever virus content in domestic pigs and wild boars.

[0025] In the reaction reagent A described in the optimization scheme, the lanthanide cryptate compound shown in formula I and the capture molecule can be coupled through a streptavidin-biotin system.

[0026] The streptavidin-biotin system coupling can be specifically carried out by the following steps:

[0027] (1) labeling the lanthanide cryptate represented by formula I onto streptavidin to obtain the lanthanide cryptate represented by formula II, i.e., streptavidin;

[0028]

[0029]

[0030] (2) Conjugating the captured African swine fever virus antibody with biotin to obtain a captured antibody-biotin conjugate.

[0031] The reagent A is: the streptavidin in the lanthanide cryptate-streptavidin and the biotin in the capture antibody-biotin specifically bind to obtain the reagent A (shown in formula III).

[0032]

[0033] In a specific example of the present invention, the capture molecule is coupled using a biotin labeling kit.

[0034] In a further optimized solution, in the reaction reagent B, the allophycocyanin and the detection molecule can be coupled to the detection molecule using an allophycocyanin labeling kit.

[0035] The African swine fever virus antibody may be any antibody against African swine fever virus, including polyclonal antibodies and monoclonal antibodies.

[0036] The capture molecule and the detection molecule may be the same or different.

[0037] In the specific example of the present application, the capture molecule is a monoclonal antibody; the detection molecule is another monoclonal antibody different from the capture molecule.

[0038] The reaction reagent A is composed of a lanthanide cryptate compound as a fluorescence donor and a coupled capture molecule; the capture molecule is an African swine fever virus antibody, as shown in Formula IV:

[0039]

[0040] The reaction reagent B is composed of allophycocyanin as a fluorescent receptor and a coupled detection molecule; the detection molecule is an African swine fever virus antibody.

[0041] The present invention discloses an application of a reaction reagent for homogeneous detection of African swine fever virus. The reaction reagent is applied to homogeneous detection of African swine fever virus or to the preparation of a product for detecting African swine fever virus.

[0042] One of the applications is a kit for detecting African swine fever virus in the present invention, comprising the above reactants, namely, reaction reagent A and reaction reagent B.

[0043] In the above kit, in the specific example of the coupling of the reaction reagent A, the capture molecule is coupled using a biotin labeling kit, i.e., streptavidin-biotin system coupling, which can be performed by the following steps:

[0044] (1) labeling streptavidin onto the lanthanide cryptate represented by formula I to obtain the lanthanide cryptate / streptavidin represented by formula II;

[0045] (2) coupling biotin to the capture antibody to obtain capture antibody / biotin (Formula III);

[0046] (3) allowing the streptavidin in the lanthanide cryptate / streptavidin of the formula III to specifically bind to the biotin in the capture antibody / biotin to obtain the reagent A (formula IV).

[0047] In the specific reaction reagent B of the present invention, the allophycocyanin and the detection molecule can be specifically coupled to the detection molecule using an allophycocyanin labeling kit.

[0048] In the above-mentioned kit, the African swine fever virus antibody can be any antibody against African swine fever virus, including polyclonal antibodies and monoclonal antibodies.

[0049] The capture molecule and the detection molecule may be the same or different, in the specific examples of the present invention.

[0050] In a further optimized solution, the reaction reagent A can be present in the form of a reaction reagent A solution; the capture molecule in the reaction uses a monoclonal antibody; the detection molecule uses another monoclonal antibody. The concentration of the African swine fever monoclonal antibody in the reagent A solution can be 0.3 mg / ml, and the solvent is a neutral buffer or purified water;

[0051] The reaction reagent B may exist in the form of a reaction reagent B solution; the concentration of the African swine fever monoclonal antibody in the reaction reagent B solution may be 0.25 mg / ml, and the solvent may be a neutral buffer solution or purified water.

[0052] In a specific example of the present invention, the neutral buffer is PBS buffer.

[0053] A homogeneous detection method for African swine fever virus in the present invention comprises the following steps:

[0054] (3) Use African swine fever P30 protein (0.05 mg / ml) standard to dilute three gradient samples with PBS buffer at 10, 100, and 1000 dilutions.

[0055] (4) First, take 10 μl of reagent A and incubate with 20 μl of the test sample P30 protein for immune reaction for 10 minutes, then add 10 μl of reagent B, mix and incubate for immune reaction for 20 minutes.

[0056] (5) After the incubation, 30 μl of the mixture was taken and fluorescence was detected at 615 nm and 665 nm using a homemade homogeneous resonance time-resolved fluorescence analyzer.

[0057] In the above detection method, in the reaction reagent A, the lanthanide cryptate compound represented by formula IV and the capture molecule can be coupled via a streptavidin-biotin system.

[0058] In a specific example of the present invention, the capture molecule is coupled using a biotin labeling kit.

[0059] In the reaction reagent B, the allophycocyanin and the detection molecule can be specifically coupled to the detection molecule using an allophycocyanin labeling kit.

[0060] The African swine fever virus antibody described in the detection method may be any antibody against African swine fever virus, including polyclonal antibodies and monoclonal antibodies.

[0061] The capture molecule and the detection molecule may be the same or different; in a specific example of the present invention, the capture molecule is a monoclonal antibody; the detection molecule is a monoclonal antibody that is not from the same source as the capture molecule.

[0062] The detection method of the present invention uses homogeneous time-resolved fluorescence technology to detect African swine fever virus, which has high sensitivity, strong specificity, efficient and convenient operation, no need to separate free markers and non-free markers, no need for cleaning, and is suitable for on-site detection. The use of these methods facilitates high-throughput detection.

[0063] The homogeneous kit of the present invention includes reagent A and reagent B. When the African swine fever virus in the sample combines with the specific antibodies of reagent A and reagent B to form an immune reaction conjugate, the fluorescent donor and the fluorescent acceptor form a fluorescent resonance body, and the donor is excited by the excitation light, and the acceptor emits resonance fluorescence. The present invention is based on the spectral characteristics of the cryptate compound formula 1 ( Figure 2 ) and allophycocyanin, using 340nm light excitation to receive two fluorescence signals at 615nm and 665nm. The resonance fluorescence intensity emitted by the receptor is directly related to the African swine fever virus content in the sample. The method of the present invention does not require embedding and separation, can reduce the interference factors that affect the accuracy of detection in the process of removing free markers, and improve the accuracy and sensitivity of detection.

[0064] The 615nm and 665nm fluorescence signals described in the present invention are only related to the amount of African swine fever virus in the sample and the antigen-antibody immune reaction binding of reagent A and reagent B, which can eliminate the interference of other fluorescent backgrounds and improve the detection sensitivity. BRIEF DESCRIPTION OF THE DRAWINGS

[0065] Figure 1 Schematic diagram of the working principle of fluorescence resonance energy transfer (FRET);

[0066] Figure 2 The fluorescence spectrum of the lanthanide cryptate compound shown in the formula in the embodiment of the present invention is

[0067] (Where A is the excitation spectrum and B is the emission spectrum). DETAILED DESCRIPTION

[0068] The homogeneous time-resolved fluorescence technique used in the present invention combines fluorescence resonance energy transfer (FRET) and time-resolved technology (TR), and is a method for detecting an analyte in a homogeneous system.

[0069] like Figure 1 As shown, in the present invention, the reaction reagent A and the reaction reagent B are respectively an energy donor and a lanthanide cryptate. Different from the existing homogeneous immunological detection method based on lanthanide elements, the present invention uses the lanthanide cryptate shown in formula I as the lanthanide cryptate in the reaction reagent A for African swine fever virus. The fluorescence spectrum of the lanthanide cryptate shown in formula I is shown in Figure 2 shown.

[0070] The streptavidin labeling step refers to Sun Yanwen's "Immuno-labeled Energy Acceptors of Cryptid Rare Earth Chelators", in which the energy donor is a long-lived fluorescence, the acceptor fluorescence is a short-lived fluorescence, and the emission light of the fluorescence donor is converted into the excitation light of the fluorescence acceptor through resonance. When used, the above two reagents are incubated with the sample to be tested, and through the antigen-antibody immune complex reaction, the distance between the immune donor and the acceptor is very close, which meets the conditions for resonance energy transfer.

[0071] The streptavidin labeling steps refer to Sun Yanwen's "Research on Immunolabeling Technology of Cryptate Rare Earth Chelators" [D]. Jilin University, 2018. First, activate the lanthanide cryptate and streptavidin in turn, mix the activated substances in equal proportions, and after sufficient reaction, put them into the Filtration tube and centrifuge them at 12000×g for 10 minutes to separate the target product.

[0072] Example 1

[0073] A homogeneous detection reagent for African swine fever virus, comprising a reaction reagent A and a reaction reagent B,

[0074] The reaction reagent A is composed of a lanthanide cryptate compound of formula I as a fluorescence donor and a coupled capture molecule;

[0075] The capture molecule is an African swine fever virus antibody;

[0076]

[0077] The reaction reagent B is composed of allophycocyanin as a fluorescent receptor and a coupled detection molecule; the detection molecule is an African swine fever virus antibody.

[0078] In the reaction reagent A, the lanthanide cryptate compound shown in formula I and the capture molecule are coupled via a streptavidin-biotin system.

[0079] The streptavidin-biotin system coupling can be carried out specifically through the following steps:

[0080] (1) labeling the lanthanide cryptate represented by formula I onto streptavidin to obtain the lanthanide cryptate represented by formula II, i.e., streptavidin;

[0081]

[0082] (2) coupling the captured African swine fever virus antibody with biotin to obtain a captured antibody-biotin conjugate of formula III;

[0083]

[0084] (3) The streptavidin in the lanthanide cryptate-streptavidin specifically binds to the biotin in the capture antibody-biotin to obtain the reagent A, as shown in Formula IV,

[0085]

[0086] In reaction reagent B, allophycocyanin and detection molecule are coupled to the detection molecule using an allophycocyanin labeling kit.

[0087] The African swine fever virus antibody can be any antibody against the African swine fever virus, including polyclonal antibodies and monoclonal antibodies; the capture molecule is the same as or different from the detection molecule.

[0088] Reagent A is composed of a lanthanide cryptate as a fluorescence donor and a coupled capture molecule, as shown in Formula IV; the capture molecule is an African swine fever virus antibody:

[0089] Example 2

[0090] The African swine fever virus homogeneous detection kit includes the reagents in the above Example 1, namely, reaction reagent A and reaction reagent B.

[0091] Example 3

[0092] The homogeneous detection kit for African swine fever virus comprises the reagents in the above embodiment 1, namely, reaction reagent A and reaction reagent B. Reaction reagent A is present in the form of reaction reagent A solution; wherein the capture molecule is reacted with a monoclonal antibody, and the detection molecule is reacted with another monoclonal antibody; and the reaction reagent B can be present in the form of reaction reagent B solution.

[0093] The concentration of African swine fever monoclonal antibody in reagent A solution can be 0.3 mg / ml, and the solvent is neutral buffer or purified water; the concentration of African swine fever monoclonal antibody in the reaction reagent B solution can be 0.25 mg / ml, and the solvent is neutral buffer or purified water; in the optimization scheme, the neutral buffer is PBS buffer.

[0094] Example 3

[0095] Follow the instructions of the biotin labeling kit to couple the capture antibody. The steps are as follows:

[0096] (1) Place 1 mg of the capture antibody to be labeled in a Filtration tube and add the corresponding volume of Labeling Buffer to a total volume of 0.5 mL. Centrifuge at 12,000 × g for 10 min.

[0097] (2) Dissolving NH2-Reactive-Biotin: Add 30 μL DMF to the NH2-Reactive-Biotin bottle and let it stand for 10 min until it is fully dissolved.

[0098] (3) Immediately add 13.3 μL NH2-Reactive-Biotin and an appropriate amount of Labeling Buffer to the above Filtration tube to make the final concentration of the antibody 2 mg / mL, and gently pipette to mix. Place in a 37°C incubator and incubate in the dark for 30 min.

[0099] (4) Centrifuge at 12,000 × g for 10 min.

[0100] (5) Add an appropriate amount of Labeling Buffer to the above Filtration tube to make the final volume 0.5 mL, gently pipette to mix, and centrifuge at 12,000×g for 10 min. Repeat the operation once.

[0101] (6) Add 0.2 mL of Labeling Buffer to the Filtration tube and pipette gently. Invert the filter cartridge and place it in another centrifuge tube. Centrifuge at 6,000 × g for 10 min.

[0102] (7) Collect the solution in the centrifuge tube, which is the biotin-labeled capture antibody.

[0103] The capture molecule was coupled by the streptavidin-biotin system according to the following steps: the lanthanide cryptate chelator labeled with streptavidin and the capture antibody coupled with biotin were uniformly mixed at a concentration ratio of 2:1, and reacted at 37°C for 20 minutes to obtain reaction reagent A.

[0104] The allophycocyanin labeling kit was purchased from Frdbio, and its catalog number is ARL0023k-1;

[0105] The detection molecule is another monoclonal antibody against African swine fever virus, which recognizes a different site from the capture antibody.

[0106] Example 4

[0107] Follow the instructions of the Allophycocyanin Labeling Kit to couple the detection molecule, as follows:

[0108] 1. Activation of APC protein and modification of detection molecules

[0109] 1. Modification of detection molecules:

[0110] (1) Antibody modification reagent (purchased from Frdbio, catalog number ARL0023k-1); dissolved in ddH2O to prepare a 2 mg / ml antibody modification reagent solution.

[0111] (2) Take the antibody to be labeled (purity>90%), adjust the concentration to above 5 mg / ml, add 5 μl of antibody modification reagent solution per mg of antibody, mix gently, and react at room temperature with stirring for 60-90 min.

[0112] (3) Add labeling buffer to the inner core of the ultrafiltration tube, centrifuge the ultrafiltration tube multiple times to replace the solution, remove excess antibody modification reagent, and collect the liquid in the inner core of the ultrafiltration tube as the modified antibody, and adjust the concentration to 3 mg / ml.

[0113] 2. Activation of APC protein

[0114] (1) Centrifuge the APC protein suspension at 12,000 rpm at 4°C for 5 min and carefully remove the supernatant to minimize residual liquid.

[0115] (2) The bottom precipitate was redissolved with labeling buffer, centrifuged at 12,000 rpm for 5 min at 4°C, and the supernatant blue solution was taken as the APC protein solution;

[0116] (3) APC protein activation reagent (purchased from Frdbio, catalog number ARL0023k-1) was dissolved in ddH2O to prepare a 5 mg / ml solution, and 100 μl of APC protein was added to the APC protein solution at a ratio of 1 mg APC protein per 100 μl of APC protein. The mixture was reacted at room temperature in the dark for 2 hours.

[0117] (4) After the reaction is completed, the solution is centrifuged through an ultrafiltration tube 4 times, each time at 120,000 rpm for 5 min, and replaced with labeling buffer to remove free APC protein activation reagent. The solution in the filter element is the activated APC protein.

[0118] 2. Activated APC protein coupled with antibody:

[0119] (1) The concentration of activated APC protein was adjusted to 5 mg / ml;

[0120] (2) The modified antibody and activated APC protein were mixed at a mass ratio of 1:2 (2 mg of activated APC protein for every mg of modified antibody) and reacted at room temperature in the dark for 2 hours;

[0121] (3) Add 200ul DMSO to the blocking reagent tube to prepare a 10mg / ml blocking reagent solution; add 3.4ul per milligram of antibody to the reaction product of step 2) to block the unreacted active groups;

[0122] (4) The labeled antibodies are packaged and stored at 4°C for future use, or appropriate protective agents are added and stored at -20°C for future use.

[0123] The pH of the PBS buffer used in the following examples is 7.4, and its preparation process is as follows: 4.06 g Na2HPO4·12H2O, 0.27 g KH2PO4, 0.20 g KCl, added to 1000 mL purified water to dissolve.

[0124] In the following examples, a homemade homogeneous time-resolved fluorescence spectrometer was used to measure the time-resolved fluorescence at 615 nm and 665 nm at room temperature, and the area was calculated as the measurement signal.

[0125] Example 5 African swine fever virus detection

[0126] Follow the steps below to test for African swine fever virus:

[0127] Using PBS as solvent, take 10ul of the three African swine fever virus P30 gradient samples of 10, 100, and 1000 diluted African swine fever virus standard solutions, mix with 20ul reagent A and 10ul reagent B, and incubate at 37°C for 30min.

[0128] After incubation, a homemade homogeneous resonance fluorescence immunoassay was used to detect the fluorescence signals of the receptor at 615nm and 665nm. Due to resonance, the donor fluorescence transferred part of the fluorescence energy to the receptor, which weakened the donor fluorescence somewhat (very weak, regarded as a stable value). The receptor fluorescence increased due to the increase in the conjugate and the resonance energy, and the receptor fluorescence increased accordingly. After 665nm / 615nn calculation, a standard curve of African swine fever virus P30 concentration and fluorescence was prepared. The African swine fever virus P30 concentration is proportional to the content of African swine fever virus. According to the developed standard curve of African swine fever virus P30 concentration and fluorescence and the detection method, the content of African swine fever virus in domestic pigs and wild boars can be detected.

[0129] The detection operation of this method is simple and the specificity is good. The reagent components of the kit are reagent A, reagent B and buffer (or diluent). There is no need to wash and separate the detection samples, which is very conducive to achieving high-throughput detection.

[0130] The fluorescence value obtained by detection shows that: as the number of detected samples increases, the above implementation / test examples are only examples for clear explanation, and are not limitations on the implementation methods. For ordinary technicians in the relevant field, other different forms of changes or modifications can be made on the basis of the above description. It is not necessary and impossible to list all the implementation methods here. The obvious changes or modifications derived from this are still within the protection scope of the invention.

Claims

1. A homogeneous detection reagent for African swine fever virus, characterized in that: It includes reaction reagent A and reaction reagent B, The reaction reagent A is composed of a lanthanide cryptate compound shown in formula IV as a fluorescence donor and a coupled capture African swine fever virus antibody molecule, and the reaction reagent A includes a capture molecule; The reaction reagent B is composed of allophycocyanin as a fluorescent receptor and a coupled detection molecule; the detection molecule is an African swine fever virus antibody; the reaction reagent A exists in the form of a reaction reagent A solution, and the reaction reagent B exists in the form of a reaction reagent B solution.

2. The African swine fever virus homogeneous detection reagent according to claim 1, characterized in that: The African swine fever virus antibody is any specific antibody against African swine fever virus, including polyclonal antibodies and monoclonal antibodies; the capture molecule is the same as or different from the detection molecule.

3. The African swine fever virus homogeneous detection reagent according to claim 1, characterized in that: The detection molecule in the reaction reagent B is any specific antibody against African swine fever virus, including polyclonal antibodies and monoclonal antibodies; the capture molecule is the same as or different from the detection molecule.

4. The homogeneous detection reagent for African swine fever virus according to claim 1, characterized in that: The reaction reagent A is in the form of a reaction reagent A solution; wherein the capture molecule uses a monoclonal antibody, and the detection molecule uses another monoclonal antibody.

5. A homogeneous detection method for African swine fever virus for non-diagnostic purposes using the detection reagent of claim 1, characterized in that: The steps include: Reagent A, test sample and reagent B need to be configured in a ratio; 1:2:1 ratio for immune reaction; Among them, the time-resolved fluorescence detection technology is used to detect the fluorescence detection signals of the central wavelengths of 615nm and 665nm respectively; Use 0.05 mg / ml African swine fever P30 protein standard to dilute at least three gradient samples with buffer, i.e. 10, 100, and 1000 times dilution, and then perform time-resolved fluorescence detection at 615 nm and 665 nm, respectively, and use 665 nm / 615 nm and the concentration of the standard to make a standard curve.

6. According to the method of claim 5, the African swine fever virus content in domestic pigs and wild boars is detected.

7. The method according to claim 5, characterized in that: Reagent A, the test sample and reagent B react simultaneously or sequentially. Reagent A and the test sample first undergo an immune reaction and then react with reagent B.

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