Indirect ELISA detection method based on African swine fever virus D250R protein

Abstract

The invention provides an indirect ELISA antibody detection method based on African swine fever virus D250R protein, and relates to the technical field of virus detection. The method comprises the following steps: cloning a D250R protein gene into a prokaryotic expression vector pET-32a (+) to construct a recombinant plasmid pET-32a-D250R, inducing a D250R recombinant protein through IPTG (isopropyl-beta-d-thiogalactoside), purifying the recombinant protein and coating an elisa plate, and optimizing and determining the optimal conditions of indirect ELISA (Enzyme-Linked Immunosorbent Assay). On the basis of the D250R protein, serum to be detected is taken as a sample, and the established indirect ELISA antibody detection method is high in sensitivity, strong in specificity and high in coincidence rate with a commercialized kit, and provides reference for development and prevention and control of an ASF diagnosis technology.

Description

Technical Field

[0001] The present invention relates to the technical field of virus detection, and in particular to an indirect ELISA detection method based on African swine fever virus D250R protein. Background Art

[0002] African swine fever (ASF) is a highly contagious disease caused by African swine fever virus (ASFV). After acute infection, domestic pigs will experience symptoms such as high fever, anorexia, bleeding, and edema. Highly pathogenic strains can cause nearly 100% mortality in domestic pigs. ASF was first discovered in 1921 and broke out in August 2018, bringing unprecedented challenges to the prevention and control of the disease in the pig industry around the world. At present, the prevention and control of ASF is still severe, and there is still no safe and effective commercial vaccine in China. Therefore, it is particularly important to do a good job in early diagnosis of ASF and establish a rapid and effective detection method.

[0003] The indirect ELISA detection method has become the preferred diagnostic method for ASF antibody detection determined by WOAH due to its high sensitivity, simple and fast operation, and has been widely used in daily detection. African swine fever virus is a type of complex linear double-stranded DNA virus with a genome length of about 170 to 190 kb, encoding 68 structural proteins and more than 100 non-structural proteins. The functions of most viral proteins encoded by ASFV have not yet been resolved or the immunogenicity is unclear. At present, the ELISA antibody detection method mainly revolves around proteins such as p30, p10, and K205R, such as the patent documents with publication numbers CN117004652A and CN114966052A. However, the epidemic situation of ASFV is becoming increasingly complex. It is necessary to evaluate the application value of other viral proteins in ELISA antibody detection to improve the accuracy and sensitivity of the ELISA detection method, and to avoid the problems of missed detection and wrong detection to a certain extent.

[0004] The D250R protein is an ASFV-encoded hydrolase and mRNA decapping enzyme that regulates host cell transcription levels and nucleic acid substrate metabolism levels during infection, and is an important target protein for ASFV vaccine research and detection technology development. In addition, as a non-structural protein encoded by ASFV, the D250R protein is expressed in the early stages of viral replication and continues to accumulate until the late stages of infection, and has certain potential for clinical development.

[0005] Developing more indirect ELISA methods can enrich the targets of the current ELISA detection methods for African swine fever. On the other hand, as proteins such as p30 and p72 are important candidate antigens for subunit vaccines, exploring detection methods based on other target proteins is expected to be able to distinguish subunit vaccine-vaccinated pigs from wild virus-infected pigs, which has important clinical application value. At present, there are no reports on indirect ELISA antibody detection methods based on D250R protein. Summary of the invention

[0006] Based on the deficiencies in the prior art, the present invention provides an indirect ELISA detection method based on the African swine fever virus D250R protein, and establishes an indirect ELISA antibody detection method with good specificity and sensitivity using affinity-purified D250R recombinant prokaryotic protein as the coating antigen, providing a reference for the development of diagnostic technology and prevention and control of ASF.

[0007] On the one hand, the present invention provides an indirect ELISA detection kit based on African swine fever virus D250R protein, comprising:

[0008] African swine fever virus D250R protein, the amino acid sequence of which is shown in SEQ ID No. 1, can bind to a primary antibody contained in a sample to be detected during detection, wherein the primary antibody is a pig anti-African swine fever virus D250R protein antibody to be detected;

[0009] A labeled secondary antibody capable of binding to a primary antibody bound to the African swine fever virus D250R protein, and the labeled secondary antibody carries a marker for detection;

[0010] The initiator solution is used to initiate a chemical reaction between the marker on the labeled secondary antibody and the chemiluminescent compound, chromogenic substrate or fluorescent dye as a reaction object of the marker.

[0011] The antigen D250R used in the present invention is relatively unique. It is a non-structural protein and an important functional protein, while structural proteins are generally used in the prior art. On the other hand, there is no guarantee that D250R will appear in the blood of pigs that have been infected with African swine fever virus, pigs that are currently infected with African swine fever virus, or pigs that have been injected with African swine fever virus vaccine. The present invention aims to discover that pigs in the above situations produce D250R protein antibodies in their blood, and the established indirect ELISA detection method can specifically distinguish between PRV, PRRSV, PCV2, CSFV positive sera and ASFV positive sera.

[0012] Specifically, the marker on the labeled secondary antibody is peroxidase, phosphatase or luciferase.

[0013] Preferably, in the embodiment of the present invention, the peroxidase is selected as horseradish peroxidase as an example.

[0014] Preferably, the African swine fever virus D250R protein is a recombinantly expressed protein.

[0015] Specifically, the coding gene of the African swine fever virus D250R protein is cloned into an expression vector to obtain a recombinant expression plasmid, the recombinant expression plasmid is transformed into a host cell, and expressed, and the recombinantly expressed African swine fever virus D250R protein is isolated and purified to obtain.

[0016] More specifically, the method for preparing the recombinant protein comprises the following steps:

[0017] 1) Referring to the D250R gene sequence of ASFV HLJ / 2018 strain (GenBank accession number: MK333180.1), the amplification primers were designed using SnapGene software. The upstream primer sequence was 5'-GCCATGGCTGATATCATGGATACTGCCATGCAG-3', and the downstream primer sequence was 5'-TTGTCGACGGAGCTCCTAGTGCTTGTATCGTAAATAG-3'. The target fragment was amplified by PCR using pCMV-Flag-D250R template;

[0018] 2) The PCR product was identified and recovered by 1% agarose gel electrophoresis, and homologously recombined with the pET-32a(+) prokaryotic expression vector linearized by BamHI and EcoR I;

[0019] 3) The homologous recombination product was transformed and plaqued, and then the bacterial solution was PCR-selected. The positive bacterial solution was sent to Zhejiang Shangya Biotechnology Co., Ltd. for sequencing and identification. After the sequence analysis was correct, the recombinant plasmid pET-32a-D250R was extracted;

[0020] 4) The pET-32a-D250R recombinant plasmid was transformed into BL21(DE3) competent cells, and a single colony was picked to activate the culture in a test tube. After expanding to a conical flask at a ratio of 1:100, the culture was shaken at 37°C until the OD 600nm The value was 0.6-0.8, and IPTG with a final concentration of 1 mmol / L was added for induction at 37°C for 4 hours. After induction, it was ultrasonically broken, and the ultrasonic precipitate was obtained after centrifugation and resuspended with the denatured mother liquor. After resuspension and centrifugation, the supernatant was taken, filtered and fully mixed with nickel filler, and the recombinant D250R protein was obtained after affinity chromatography purification.

[0021] On the other hand, the present invention also provides an indirect ELISA detection method based on African swine fever virus D250R protein, wherein the indirect ELISA detection method is for non-disease diagnosis purposes, and the indirect ELISA detection kit based on African swine fever virus D250R protein is used.

[0022] The indirect ELISA detection method comprises the following steps:

[0023] (1) coating the African swine fever virus D250R recombinant protein onto an ELISA plate, washing, blocking, and then washing again;

[0024] (2) adding the sample to be tested for incubation, and washing after incubation;

[0025] (3) adding the labeled secondary antibody for incubation, and washing after incubation;

[0026] (4) Add the initiator solution for detection.

[0027] The specific method of indirect ELISA detection includes the following steps:

[0028] 1) Coating: The recombinant protein was purified using the above method, and 50 mmol / L Tris-HCl was used as the coating solution to dilute the D250R protein concentration to 1.0 μg / mL for coating the ELISA plate at 37°C for 1 h;

[0029] 2) Washing: discard the coating solution in the ELISA plate, add PBST washing solution (0.01 mol / L phosphate buffer containing 0.05% Tween-20) 200 μL / well, wash 4 times;

[0030] 3) Blocking: discard the washing solution in the ELISA plate, add 1% BSA 100 μL / well, and block at 37°C for 1 hour;

[0031] 4) Washing: discard the coating solution in the ELISA plate, add 200 μL / well of PBST washing solution, and wash 4 times;

[0032] 5) Serum incubation: discard the washing solution in the ELISA plate, dilute the serum to be tested 1:200 with 5% skim milk, add 100 μL / well to the ELISA plate, and incubate at 37°C for 1 hour;

[0033] 6) Washing: discard the primary antibody dilution in the ELISA plate, add 200 μL / well of PBST washing solution, and wash 4 times;

[0034] 7) Secondary antibody incubation: discard the washing solution in the ELISA plate, dilute HRP-labeled goat anti-swine IgG 1:5000 with 5% skim milk, add 100 μL / well to the ELISA plate, and incubate at room temperature for 1 hour;

[0035] 8) Washing: discard the secondary antibody dilution in the ELISA plate, add 400 μL / well of PBST washing solution, and wash 4 times;

[0036] 9) Color development: discard the washing solution in the ELISA plate, add 100 μL / well of TMB substrate color development solution, and develop color at 37°C for 20 min;

[0037] 10) Termination: Add 50 μL of 2 mol / L H 2 SO 4 Terminate the reaction;

[0038] Result interpretation: Use an ELISA plate reader to read the OD 450nm value, when the OD of the sample to be tested 450nm When the value is less than 0.3589, it is considered negative; when OD 450nm A value ≥ 0.3589 was considered positive.

[0039] The established indirect ELISA detection method can distinguish between PRV, PRRSV, PCV2, CSFV positive sera and ASFV positive sera, with good specificity; the reading of positive serum at 1:3200 dilution is still greater than the critical value, with high sensitivity; the maximum coefficient of variation of intra-batch repeated tests is 6.67%, and the maximum coefficient of variation of inter-batch repeated tests is 9.24%, with good repeatability; 163 sera were tested simultaneously with the commercial test kit, and the compliance rate reached 98.8%.

[0040] The indirect ELISA detection method is not for disease diagnosis. It is mainly based on the detection and analysis of biomarkers, and is intended to assess the overall health status and lifestyle adaptability of an individual, or for laboratory research, rather than for disease diagnosis, prevention or treatment. This method provides individuals with personalized health advice and lifestyle guidance through quantitative detection of specific biomarkers in the blood, combined with advanced data analysis methods.

[0041] Preferably, the sample to be tested is pig serum, whole blood or plasma, and the sample to be tested comes from a pig that has been infected with African swine fever virus, a pig that is currently infected with African swine fever virus, or a pig that has been injected with an African swine fever virus vaccine.

[0042] In some embodiments of the present invention, the marker on the labeled secondary antibody is horseradish peroxidase. In step (4), the initiator solution is TMB substrate color development solution. After color development, a stop solution is added to terminate the reaction. Then, the detection is performed and the OD value of the ELISA plate is read using an ELISA reader. 450nm value, when the OD of the sample to be tested 450nm When the value is less than 0.3589, it is considered negative; when OD 450nm A value ≥ 0.3589 was considered positive.

[0043] By applying the above technical solution, the present invention has the following technical effects:

[0044] The present invention clones the ASFV D250R protein gene into the pET-32a(+) prokaryotic expression vector to obtain a recombinant expression plasmid pET-32a-D250R, and obtains a high-purity recombinant His-D250R protein by inducing expression through IPTG and denaturing and purifying. The recombinant protein is used as an antigen to coat an ELISA plate, and an indirect ELISA antibody detection method based on the D250R protein with high sensitivity, strong specificity, good repeatability, and high consistency with the detection rate of commercial kits is established, enriching the detection targets of the African swine fever ELISA method, and providing support for the further development of the African swine fever diagnosis method. BRIEF DESCRIPTION OF THE DRAWINGS

[0045] Figure 1 It is the PCR amplification product of D250R gene, where M: Marker; 1·D250R gene fragment;

[0046] Figure 2 SDS-PAGE (A) and Western blot (B) analysis of His-D250R recombinant protein before and after induction and ultrasound, where M: Marker; 1: bacterial protein before IPTG induction; 2: bacterial protein after IPTG induction; 3: supernatant after ultrasound; 4: precipitate after ultrasound;

[0047] Figure 3 SDS-PAGE (A) and Western blot (B) identification of recombinant protein after purification, where M: Marker; 1: flow-through after binding to nickel filler; 2: Buffer A eluent; 3·Buffer B eluent; 4: Buffer C eluent; 5-7: Buffer D eluent;

[0048] Figure 4 It is used to identify the reactivity of the recombinant protein with ASFV positive serum, where M: Marker; 1: pre-induction bacterial protein; 2: post-induction bacterial protein; 3 and 4: purified His-D250R recombinant protein;

[0049] Figure 5 To optimize the indirect ELISA reaction conditions, A: Determination of coating temperature and time; B: Determination of serum incubation conditions; C: Determination of secondary antibody dilution ratio; D: Determination of secondary antibody incubation conditions; E: Determination of substrate reaction conditions;

[0050] Figure 6 This is a test of the indirect ELISA method, in which A: critical value determination; B: sensitivity test; C: specificity test. DETAILED DESCRIPTION

[0051] The technical solution of the present invention is further described in detail below in conjunction with the accompanying drawings and implementation modes.

[0052] Example 1 Construction of D250R prokaryotic expression plasmid

[0053] 1) Referring to the D250R gene sequence of ASFV HLJ / 2018 strain (GenBank accession number: MK333180.1) as shown in SEQID No. 2, the amplification primers were designed using SnapGene software. The upstream primer sequence was 5′-gccatggctgatatcatggatactgccatgcag-3′, and the downstream primer sequence was 5′-ttgtcgacggagctcctagtgcttgtatcgtaaatag-3′. The target fragment was amplified by PCR using pCMV-Flag-D250R template. The PCR reaction system was 1 μL each of the upstream and downstream primers, 12.5 μL of KODOne enzyme, 1 μL of template, and ddH 2 The PCR amplification conditions were 94°C for 2 min, 98°C for 10 s, 54°C for 5 s, 68°C for 15 s, 30 cycles, and 68°C for 5 min.

[0054] 2) The PCR products were identified by electrophoresis on 1% agarose gel at 180V for 15 min. Figure 1 The target band of about 750 bp was successfully amplified and the band was recovered by gel cutting. The pET-32a(+) prokaryotic expression vector was linearized by BamH I and EcoR I. The enzyme digestion system was: 2 μL each of BamH I and EcoR I, 5 μL of 10×QuickCut Buffer, 1 μg of pET-32a(+) plasmid, ddH 2 O to 25 μL. Enzyme digestion conditions were 37°C for 2 h.

[0055] 3) The target gene fragment recovered from rubber tapping was homologously recombined with the linearized vector. The homologous recombination system was 30 ng of gene fragment, 240 ng of linearized vector, 2× Buffer 5 μL, and ddH 2 O The total system was supplemented to 10 μL. The homologous recombination condition was 50°C for 15 min. The homologous recombination product was transformed into E. coli DH5α competent cells and evenly spread on ampicillin selective LB solid plates and inverted at 37°C for 18 hours. A single colony was picked and the picked colony was expanded and cultured with ampicillin selective LB liquid medium at 37°C and 200 rpm. The culture liquid was PCR-treated with specific primers used for the amplified fragment, and the PCR product was identified by agarose gel. The PCR-positive bacterial solution was selected and sent to Zhejiang Shangya Biotechnology Co., Ltd. for sequencing and identification. After the sequence analysis was correct, the recombinant plasmid pET-32a-D250R was extracted.

[0056] Example 2 Inducible expression and solubility analysis of recombinant protein

[0057] 1) Induced expression of recombinant protein: The pET-32a-D250R recombinant plasmid was transformed into E. coli BL21 (DE3) competent cells and evenly spread on ampicillin selective LB solid plates and inverted cultured at 37°C for 18 h. A single colony was picked and amplified using ampicillin selective LB liquid medium at 37°C with shaking at 200 rpm. 600nm When the pH value was 0.6, IPTG was added at a final concentration of 1 mmol / L and induced at 37°C for 4 h. 1 mL of the bacterial solution before and after induction was taken respectively, and the bacteria were collected by centrifugation at 3000×g for 5 min. 50 μL of strong lysis buffer (5% SDS+1% Triton X-100) was added for lysis at room temperature, and 4× protein loading buffer was added and heated at 100°C for 10 min.

[0058] 2) Recombinant protein expression identification and solubility analysis: 100 mL of induced bacterial solution was centrifuged at 5000×g for 10 min, the supernatant was discarded, and the solution was resuspended in 15 mL PBS and ultrasonically disrupted in an ice bath. The ultrasonic program was as follows: ultrasonic for 3 seconds, 4 seconds interval, 50% power, each ultrasonic disruption for 10 minutes, 5-10 minutes interval, and ultrasonication for a total of 3 times. After the ultrasonication, the bacterial solution was centrifuged at 4°C and 12000×g for 10 minutes to separate the insoluble components. The supernatant after separation was added with 4× protein loading buffer and then incubated at 100°C for 10 minutes. The precipitate was added with 50 μL of strong lysis buffer (5% SDS+1% Triton X-100) and lysed at room temperature, and then added with 4× protein loading buffer and incubated at 100°C for 10 minutes. The bacterial solution samples before and after induction and the supernatant precipitate samples after ultrasonication were identified by SDS-PAGE, and Western blot was performed using His rabbit antibody as the primary antibody and HRP-labeled goat anti-rabbit IgG as the secondary antibody. The results are shown in Figure 2 , indicating that the His-D250R recombinant protein was successfully induced and the protein was mainly distributed in the inclusion bodies.

[0059] Example 3 Affinity purification and identification of recombinant proteins

[0060] 1) The precipitate separated by centrifugation after ultrasound in Example 2 was resuspended in the denatured mother solution, and after resuspension, the insoluble components were removed by centrifugation at 12000×g for 10 min at 4° C. The supernatant was filtered through a 0.45 μm filter and combined with 2 mL Ni-NTA agarose at 4° C. overnight.

[0061] 2) Move the bound recombinant protein and Ni-NTA agarose mixture to the empty column of the elution column, and let the liquid flow out by gravity. Then prepare the reagent according to the instructions of Ni-NTA agarose (purchased from QIAGEN, catalog number: 30210). The formula is shown in Table 1. Use different reagents (Buffer A, Buffer B, Buffer C, Buffer D) to wash the impurities and elute the target protein. After adding 4× protein loading buffer, the obtained washing and elution solutions were placed in a 100℃ metal bath for 10 minutes, and identified by SDS-PAGE and Western blot. His rabbit antibody was used as the primary antibody and HRP-labeled goat anti-rabbit IgG was used as the secondary antibody for Western blot identification. The results are shown in Figure 3 , indicating that the His-D250R recombinant protein was mainly in the Buffer D eluted purification product, with an elution concentration of 0.64 mg / mL and a single band.

[0062] Table 1 Protein purification reagent formula

[0063]

[0064] At the same time, the antigenicity of His-D250R protein was analyzed using ASFV positive serum as primary antibody and HRP-labeled goat anti-swine IgG as secondary antibody. Figure 4 , showing that the recombinant protein can be recognized by ASFV positive serum, and the purified protein has a single band and is specific compared with before and after induction, indicating that the His-D250R recombinant protein has good reactivity with antibodies.

[0065] Example 4 Establishment of indirect ELISA antibody detection method based on D250R protein

[0066] 1) Determination of the optimal coating concentration and the dilution multiple of the serum to be tested

[0067] The recombinant D250R protein was diluted with 50 mmol / L Tris-HCl at five different working concentrations of 0.5 μg / mL, 1.0 μg / mL, 2.0 μg / mL, 4.0 μg / mL, and 6.0 μg / mL by the chessboard method. At the same time, the serum to be tested was diluted at 1:50, 1:100, 1:200, 1:400, 1:600, 1:800, and 1:1200. The OD 450 The antigen coating concentration when the nm value reading was >2 and the P / N value was the largest was taken as the optimal antigen coating concentration. As shown in Table 2, 1.0 μg / mL was determined to be the optimal antigen coating concentration, and the serum dilution factor was 1:200.

[0068] Table 2 Determination of the optimal coating concentration and the dilution multiple of the serum to be tested

[0069]

[0070] 2) Determination of optimal coating conditions

[0071] According to the determined coating concentration and the dilution multiple of the serum to be tested, four coating temperature and time schemes were selected for optimization: 37℃1h, 37℃2h, 4℃12h and 4℃16h. The data analysis is as follows: Figure 5 A. The results showed that when coating was performed at 37°C for 1 hour, the P / N value was the largest, so the optimal coating condition was 37°C for 1 hour.

[0072] 3) Determination of optimal sealing conditions

[0073] According to the determined conditions, the experiment was conducted, and the blocking time was optimized as 37°C 0.5h, 37°C 1h, 37°C 2h, and room temperature (the room temperature in this embodiment is 25°C) 2h. The blocking solution was optimized as 5% skim milk, 1% BSA, and 3% BSA. The data analysis is shown in Table 3. The results show that when 1% BSA is selected as the blocking solution and blocked at 37°C for 1h, the P / N value is the largest, so the optimal blocking condition is 1% BSA 37°C for 1h.

[0074] Table 3 Determination of optimal sealing conditions

[0075]

[0076] 4) Determination of the optimal incubation temperature and time for the serum to be tested

[0077] According to the determined conditions, the experiment was carried out and six schemes were selected for optimization: 37℃ 0.5h, 37℃ 1h, 37℃ 1.5h, room temperature 0.5h, room temperature 1h, and room temperature 1.5h. The data analysis was as follows: Figure 5 B. The results showed that the P / N value was the largest when the serum was incubated at 37°C for 1 hour.

[0078] 5) Determination of the optimal secondary antibody dilution ratio and incubation conditions

[0079] According to the determined conditions, the experiment was carried out and the dilution ratio of the secondary antibody was optimized according to 6 dilution ratios: 1:1000, 1:2500, 1:5000, 1:7500, 1:10000, and 1:15000. The data analysis is as follows: Figure 5 C. The results showed that the P / N value was the largest when the secondary antibody dilution ratio was 1:5000. Then, six schemes were optimized: 37℃0.5h, 37℃1h, 37℃2h, room temperature 0.5h, room temperature 1h, and room temperature 2h. The data analysis is shown in the figure. Figure 5 D. The results show that when the sample is blocked at room temperature, the negative readings are significantly reduced, while the positive readings are not reduced much. Therefore, when the sample is incubated at room temperature for 1 hour, the P / N value is the largest.

[0080] 6) Determination of optimal color development temperature and time

[0081] According to the determined conditions, the experiment was carried out, and the time and temperature combinations of 37℃5min, 37℃7min, 37℃10min, 37℃15min, 37℃20min, 37℃30min and room temperature 5min, room temperature 7min, room temperature 10min, room temperature 15min, room temperature 20min, room temperature 30min were optimized. The data analysis was as follows: Figure 5 E. The results showed that the P / N value was the highest when the color was developed at 37°C for 20 minutes.

[0082] Example 6 Experiment based on the indirect ELISA antibody detection method of D250R protein

[0083] 1) Determination of critical value

[0084] The ELISA method based on the optimal reaction conditions was used to test 24 randomly selected ASFV negative sera and determine their OD 450nm According to the statistical analysis, the results are as follows Figure 6 A, average reading of 24 sera is 0.1701, and the standard deviation (SD) is 0.0629. The calculated critical value is 0.3589. Therefore, the judgment criteria of the indirect ELISA detection method of D250R protein established are: when OD 450nm When the value is less than 0.3589, it is considered negative; when OD 450nm A value ≥ 0.3589 was considered positive.

[0085] 2) Sensitivity test

[0086] The positive serum was diluted two-fold from 1:100 to 1:12800, and the OD was determined according to the established indirect ELISA method. 450nm , with OD 450nm The maximum dilution factor greater than the critical value is taken as the maximum dilution factor of the serum to be tested. Figure 6 B. When the dilution ratio is 1:3200, the reading is greater than the critical value, and when the dilution ratio is greater than 1:3200, the reading is less than the critical value, indicating that the sensitivity of the optimized ELISA method reaches 1:3200, which has good sensitivity.

[0087] 3) Specificity test

[0088] Based on the optimized D250R protein indirect ELISA method, ASFV negative and positive sera were used as controls to detect PRV, PRRSV, PCV2, and CSFV infected positive sera, and their OD values ​​were read. 450nmThe value is compared with the established critical value to determine whether it is positive or negative. Figure 6 C, showing that the readings of the four sera were all below the critical value, indicating that the indirect ELISA method optimized based on the D250R protein had good specificity and could distinguish the above four sera from ASFV-positive sera.

[0089] 4) Repeatability test

[0090] Take three ASFV positive and negative sera respectively, and set three replicate wells for each serum based on the optimized indirect ELISA method of D250R protein, and read the OD 450nm The values ​​were tested for intra-batch repeatability, and the mean and standard deviation of the readings of each serum duplicate well were calculated. The coefficient of variation was calculated according to statistical principles = (standard deviation / mean value) × 100%. The results are shown in Table 4, showing that the maximum value of the coefficient of variation was 6.67%, indicating that the intra-batch repeatability was good.

[0091] In addition, 3 ASFV positive and negative sera were taken, and 2 replicate wells were set for each serum. Different experimenters conducted four independent replicate tests at different times and read their OD 450nm The values ​​were tested repeatedly between batches, and the average values ​​of the two replicate wells in each test were calculated. The average values ​​and standard deviations of three independent tests of the same serum were calculated in turn, and the coefficient of variation was calculated according to statistical principles. The results are shown in Table 5, showing that the highest coefficient of variation was 9.24%, indicating that the reproducibility between batches was good.

[0092] Table 4 Results of intra-batch repeatability test

[0093] Sample No. average value Standard Deviation Coefficient of variation % 1 1.511 0.057 3.796 2 0.890 0.029 3.206 3 1.018 0.068 6.669 4 0.155 0.007 4.396 5 0.048 0.001 1.701 6 0.085 0.005 5.524

[0094] Table 5 Results of inter-batch repeatability test

[0095] Sample No. average value Standard Deviation Coefficient of variation % 1 1.925 0.076 3.938 2 2.450 O.158 6.437 3 1.649 0.089 5.425 4 0.217 0.009 3.976 5 0.059 0.005 8.030 6 0.104 0.010 9.235

[0096] 5) Compliance test

[0097] The indirect ELISA method established in this study and the ASF commercial ELISA detection kit were used to detect 160 negative sera and 3 standard positive sera with different dilution multiples. The results are shown in Table 6.

[0098] Table 6 Compliance test results

[0099]

[0100] Among them: "+" indicates positive, and "-" indicates negative.

[0101] The results showed that the commercial kit detected 3 positive samples and 160 negative samples; the ELISA method established in this study detected 5 positive samples and 158 negative samples. Two negative sera were mistakenly detected as positive, and the rest were consistent with the test results of the kit, indicating that the consistency rate of this method with the commercial kit reached 98.8% (Table 6).

Claims

1. An indirect ELISA detection kit based on African swine fever virus D250R protein, characterized in that: include: African swine fever virus D250R protein, the amino acid sequence of which is shown in SEQ ID No. 1, can bind to a primary antibody contained in a sample to be detected during detection, wherein the primary antibody is a pig anti-African swine fever virus D250R protein antibody to be detected; A labeled secondary antibody capable of binding to a primary antibody bound to the African swine fever virus D250R protein, and the labeled secondary antibody carries a marker for detection; The initiator solution is used to initiate a chemical reaction between the marker on the labeled secondary antibody and the chemiluminescent compound, chromogenic substrate or fluorescent dye as a reaction object of the marker.

2. The indirect ELISA detection kit based on African swine fever virus D250R protein according to claim 1, characterized in that: The marker on the labeled secondary antibody is peroxidase, phosphatase or luciferase.

3. The indirect ELISA detection kit based on African swine fever virus D250R protein according to claim 2, characterized in that: The peroxidase is horseradish peroxidase.

4. The indirect ELISA detection kit based on African swine fever virus D250R protein according to claim 1, characterized in that: The African swine fever virus D250R protein is a recombinant expression protein.

5. The indirect ELISA detection kit based on African swine fever virus D250R protein according to claim 4, characterized in that: The coding gene of the African swine fever virus D250R protein is cloned into an expression vector to obtain a recombinant expression plasmid, the recombinant expression plasmid is transformed into a host cell, and the expression is performed, and the recombinantly expressed African swine fever virus D250R protein is obtained by separation and purification.

6. An indirect ELISA detection method based on African swine fever virus D250R protein, the indirect ELISA detection method is for non-disease diagnosis purposes, characterized in that: Using the indirect ELISA detection kit based on African swine fever virus D250R protein according to any one of claims 1 to 5, The indirect ELISA detection method comprises the following steps: (1) coating the African swine fever virus D250R protein onto an ELISA plate, washing, blocking, and then washing again; (2) adding the sample to be tested for incubation, and washing after incubation; (3) adding the labeled secondary antibody for incubation, and washing after incubation; (4) Add the initiator solution for detection.

7. The indirect ELISA detection method based on African swine fever virus D250R protein according to claim 6, characterized in that: The samples to be tested are pig serum, whole blood or plasma, and come from pigs that have been infected with African swine fever virus, pigs that are currently infected with African swine fever virus, or pigs that have been vaccinated against African swine fever virus.

8. The indirect ELISA detection method based on African swine fever virus D250R protein according to claim 6, characterized in that: The marker on the labeled secondary antibody is horseradish peroxidase. In step (4), the initiator solution is TMB substrate colorimetric solution. After color development, a stop solution is added to terminate the reaction. Then, the detection is performed and the OD value of the ELISA plate is read using an ELISA reader. 450nm value, when the OD of the sample to be tested 450nm When the value is less than 0.3589, it is considered negative; when OD 450nm A value ≥ 0.3589 was considered positive.

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