Universal kit for bovine nodular skin virus, sheep pox virus and goat pox virus

Abstract

The invention belongs to the technical field of detection, and particularly relates to a universal kit for bovine nodular skin viruses, sheep pox viruses and goat pox viruses. On the basis of expressed specific recombinant p122 protein and serum to be detected as a detection sample, the double-antigen sandwich ELISA method for the bovine nodular skin virus, the sheep pox virus and the goat pox virus is established, and the method comprises the following steps: coating an antigen, sealing, adding the sample to be detected and an enzyme-labeled antigen, developing, judging a result and the like. The kit has the advantages of being good in specificity, high in sensitivity, good in repeatability, low in cost and capable of achieving batch detection, and a certain detection technology foundation can be laid for preventing and controlling the bovine nodular skin virus, the sheep pox virus and the goat pox virus.

Description

Technical Field

[0001] The invention belongs to the technical field of detection, and in particular relates to a universal test kit for bovine nodular skin virus, sheeppox virus and goatpox virus. Background Art

[0002] Lumpy skin disease (LSD), also known as bovine pimple skin disease, is a disease caused by bovine lumpy skin disease virus (LSDV). LSD is a notifiable animal disease regulated by the World Organization for Animal Health and is currently a Category II animal disease in my country. After infection with LSDV, the affected cattle will experience fever, swollen lymph nodes, and extensive nodules on the skin, mucous membranes, and organ surfaces. LSDV belongs to the Poxviridae family, the Capripoxvirus genus, which also includes goatpox virus (GTPV) and sheeppox virus (SPPV). The nucleotide sequence similarity of LSDV, GTPV, and SPPV is as high as 96%, with high antigenic similarity and serological cross-reaction. For this reason, my country clinically uses a 5-fold dose of intradermal injection of goatpox attenuated vaccine to prevent and control LSD.

[0003] The serological methods recommended by the World Organization for Animal Health (OIE) for diagnosing LSDV include virus neutralization test (VNT), immunoblot (WB) and enzyme-linked immunosorbent assay (ELISA). VNT is the gold standard for detecting antibodies to goatpoxvirus. The experimental operation requires the use of live virus and has high requirements for the laboratory operation, which is not suitable for clinical serological testing. WB is also not suitable for clinical serological testing due to its cumbersome experimental operation process. The ELISA method is widely used in clinical serological testing due to its convenience, rapidity, sensitivity, specificity and the lack of special instruments and equipment.

[0004] The application is found by analyzing the infected serum of bovine nodular skin disease and the immune serum (0dpi, 15dpi, 30dpi, 45dpi and 60dpi) of different periods, and the p122 protein serum abundance of bovine nodular skin virus is high. Further by sequence alignment, it is found that p122 protein is highly conservative in bovine nodular skin disease, sheep pox virus and goat pox virus, so p122 protein is selected as target protein to establish bovine nodular double antigen sandwich ELISA antibody detection kit, and further experimental results also show that the kit can detect sheep pox virus and goat pox virus simultaneously, therefore, the kit is a universal kit for bovine nodular skin virus, sheep pox virus and goat pox virus. In addition, the method is not limited by serum subtypes to be tested, and antibodies in different periods (early, middle and late stages) and different subtypes (IgA, IgM and IgG) serum can be detected simultaneously after immunity or infection. Because this method does not require the preparation of specific antibodies and only requires one antigen protein to complete the preparation of the kit, the kit has the characteristics of low cost, short reaction time, strong specificity and high sensitivity, and is easy to promote on a large scale. Summary of the invention

[0005] In view of the above technical problems, the present invention provides a universal double antigen sandwich ELISA method for three pathogens, LSDV, GTPV and SPPV, based on the preparation of p122 protein and using serum as the detection sample, which specifically includes the following contents:

[0006] In a first aspect, the present invention provides a use of p122 protein as a coating antigen in the preparation of a universal double antigen sandwich ELISA kit for bovine nodular skin virus, sheep pox virus and goat pox virus.

[0007] In the second aspect, the present invention provides a universal double antigen sandwich ELISA kit for bovine nodular skin virus, sheeppox virus and goatpox virus, the kit comprising: coating antigen, enzyme-labeled antigen, coating solution, diluent, ELISA plate, TBST buffer, blocking solution, color developing solution, and stop solution; the coating antigen is p122 protein, and the enzyme-labeled antigen is enzyme-labeled p122 protein.

[0008] Preferably, the coating solution is a carbonate buffer with a pH of 9.6.

[0009] Preferably, the blocking solution is TBST buffer containing 5% BSA.

[0010] Preferably, the enzyme-labeled antigen is: HRP-labeled p122 protein with a HIS tag.

[0011] In a third aspect, the present invention provides the use of the kit described in the second aspect in the detection of bovine nodular skin virus, sheeppox virus and goatpox virus for non-disease diagnosis purposes.

[0012] Preferably, the method for using the kit is:

[0013] (1) Preparation of coating antigen and enzyme-labeled antigen;

[0014] (2) Antigen coating: Use coating solution to coat p122 protein in the ELISA plate at 4°C overnight;

[0015] (3) Blocking: Wash the coated ELISA plate with TBST buffer, then add blocking solution and incubate at 37°C;

[0016] (4) Adding samples to be tested: After blocking, wash the ELISA plate with TBST buffer, add the serum samples to be tested, positive control and negative control respectively, and incubate at 37°C;

[0017] (5) Adding enzyme-labeled antigen: After incubation, wash the plate with TBST buffer, add enzyme-labeled antigen, and incubate at 37°C;

[0018] (6) Display: After incubation, the ELISA plate is washed with TBST buffer, and then a color developing solution is added, and the plate is incubated at 37°C in the dark; after incubation, a stop solution is added, and the OD value at a wavelength of 450 nm is read on an ELISA reader;

[0019] (7) Result determination: S / P>0.304 was determined as positive, and S / P<0.304 was determined as negative; where S / P=(OD 样品 -OD 阴性 Average value) / (OD 阳性 -OD 阴性 average value).

[0020] Preferably, in step (2), the amount of coating solution is 50 μL / well, and the concentration of p122 protein is 2 μg / mL.

[0021] Preferably, the dilution ratio of the serum to be tested in step (4) is 1:2.

[0022] Preferably, the dilution of the enzyme-labeled antigen in step (5) is 1:60000.

[0023] Preferably, the sealing time in step (3) is 1 hour.

[0024] Preferably, the incubation time in steps (4) and (5) is 30 minutes.

[0025] Preferably, the light-proof incubation time in step (6) is 10 minutes.

[0026] Preferably, the plate is washed three times, each time for 5 minutes.

[0027] Preferably, the method for preparing the coated antigen is:

[0028] The p122 protein was synthesized by codon optimization, and the synthetic sequence was cloned into the pET-SUMO vector to obtain the recombinant plasmid pET-SUMO-LSDV-122. The relevant recombinant plasmid construction work was entrusted to Jinkairui Biotechnology Co., Ltd.

[0029] The synthesized recombinant plasmid was transformed into the BL21 (DE3) expression strain, and after heat shock, it was evenly spread on a kanamycin-resistant plate, placed in a 37°C incubator and inverted for 12 hours, and then a single colony was picked and inoculated into 10 mL of kanamycin-resistant LB liquid culture medium for activation, and shaken at 37°C and 220 r / min until the OD value was 0.6-0.8, and IPTG with a final concentration of 0.5 mmol / L was added for induction. After culturing at 16°C and 220 r / min for 16 hours, the bacteria were collected by centrifugation; the bacteria were washed with PBS and resuspended, ultrasonically broken in an ice bath, and the supernatant was collected after centrifugation and purified through a Ni affinity chromatography pre-packed column, and the target protein was eluted with different concentrations of imidazole buffer to obtain LSDV-122 protein.

[0030] Preferably, in order to facilitate purification and increase the solubility of p122 protein, HIS tag and SUMO tag are added to the LSDV-122 protein prepared above, and SUMO protease is used for cleavage. The reaction buffer is 25mM Tris-HCl, and the ratio of SUMO protease to LSDV-122 protein is 10U:1mg. The reaction is carried out in a dialysis bag, and the enzyme digestion is carried out overnight at 4°C for 16h. After the reaction, the solution is placed in a dialysis bag for displacement and concentration, and the concentrated protein is used as the coating antigen.

[0031] Preferably, the method for preparing the enzyme-labeled antigen is: LSDV-122 protein with a His tag is subjected to HRP labeling according to the instructions of the Peroxidase Labeling Kit of Roche Company to obtain the enzyme-labeled antigen.

[0032] Preferably, the specific steps are as follows:

[0033] (1) Add 0.3 mL of His-tagged LSDV-122 protein solution and 0.1 mL of peroxidase into an EP tube, gently pipette to mix, and incubate at 4°C overnight;

[0034] (2) Add 40 μL of triethanolamine to the mixture, mix by pipetting, then add 50 μL of sodium borohydride, mix by pipetting again, and place at 4°C for 30 min;

[0035] (3) Add 10 μL of glycine solution to the mixture and mix thoroughly by pipetting;

[0036] (4) Transfer the mixed solution into a dialysis bag and place it in dialysis solution at 4°C overnight; during this period, the dialysis solution was changed three times;

[0037] (5) Use a syringe to draw out the mixed solution from the dialysis bag and inject it into the EP tube. Add a preservation solution of equal volume to the mixed solution, gently blow and mix, and store in aliquots at -80°C.

[0038] The beneficial effects of the present invention are:

[0039] (1) High specificity: The kit provided by the present invention is used to detect bovine nodular dermatosis (LSDV) positive serum, sheep pox (SPPV) positive serum, goat pox (GTPV) positive serum, LSDV negative serum, SPPV negative serum, GTP V negative serum, Brucella (brucella) positive serum, sheep orifice (ORFV) positive serum, foot-and-mouth disease (FMDV) positive serum and bovine viral diarrhea (BVDV) positive serum as samples. The results show that only LSDV positive serum, SPPV positive serum and GTPV positive serum test results are positive, and the rest are negative. This result shows that the kit has good specificity;

[0040] (2) Good stability: Using the kit provided by the present invention, 5 replicates were set up within the group and 5 repeated tests were performed between groups for 4 LSDV-positive sera and 4 LSDV-negative sera, and the CV value (coefficient of variation) was calculated. The intra-batch repeated CV value was 1.35%-4.77%, and the inter-batch repeated CV value was 2.08%-8.82%, indicating that the method established in the present application has good stability;

[0041] (3) Strong versatility and wide detection range: The present invention analyzes bovine nodular dermatosis infected serum and immune serum at different periods (0 dpi, 15 dpi, 30 dpi, 45 dpi and 60 dpi) and finds that the kit can simultaneously detect bovine nodular dermatosis, sheeppox virus and goatpox virus. Therefore, the kit is a universal kit for bovine nodular dermatosis, sheeppox virus and goatpox virus. In addition, the method is not limited by the serum subtype to be tested and can simultaneously detect antibodies in sera at different periods (early, middle and late stages) and different subtypes (IgA, IgM and IgG) after immunity or infection.

[0042] (4) Low cost: Since the method described in the present application does not require the preparation of specific antibodies, only one antigen protein is needed to complete the preparation of the kit. Therefore, the kit is low in cost and easy to promote on a large scale.

[0043] (5) Short detection time: The use of horseradish peroxidase (HRP)-labeled LSDV P122 protein as an enzyme-labeled antigen improves the sensitivity and specificity of the test and reduces the detection time. At the same time, the coating antigen and blocking steps have been pre-processed in the kit. After getting the kit, it starts directly from adding the serum to be tested, and the incubation time of the subsequent steps is up to 30 minutes. The entire detection process is completed within 1.5 hours, which greatly shortens the detection time and improves the detection efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] Figure 1 Expression, purification and identification results of p122 protein; (A) is the eluted protein after p122 protein purification; (B) is western blot analysis using LSDV positive serum;

[0045] Figure 2 The cut-off value is determined;

[0046] Figure 3 Specificity results of the double antigen sandwich ELISA method;

[0047] Figure 4 Sensitivity results of the double antigen sandwich ELISA method. DETAILED DESCRIPTION

[0048] In order to explain the technical solution and technical purpose of the present invention, the present invention is further introduced below in combination with the accompanying drawings and specific implementation methods.

[0049] Example 1 Preparation of coated antigen and enzyme-labeled antigen

[0050] 1. Construction of recombinant plasmid

[0051] In the present application, the p122 protein was synthesized by codon optimization, and the length of the synthetic sequence was 558 bp (shown in SEQ ID NO.1). The synthetic sequence was cloned into the pET-SUMO vector to obtain the recombinant plasmid pET-SUMO-LSDV-122. The relevant recombinant plasmid construction work was entrusted to Jinkairui Biotechnology Co., Ltd.

[0052] p122: acagattatgatgaaagtaataattttacagcattcgcaggttccactatatacggatatggtttaaaatcaaaaaaaaatataaaa aaaaaagtaaaattaattaatttctgtataaaaatatcaattatgacatcaatggtttcgttaattacaataacaattcttttagcatttttaataatacatgtgaattaaatcaat ttaaggaacacaaaccgtactttttaaaaaatccaaatcctactacatatagtgacgacgatactgaatctgagttaaatgtttatagatcatgtaaaggtattgtttatagcggatac tgttacacgtttaactcagaacctaaaagttttaatgatgcatacgatgattgtgaaaaaaaaatagcgaattaccatcaaataatttaatgaatgattggataagtgactacttaga tgggacgtggggcgaagacggtaacgtactttttaaagaaaaaaatcaagaacttgaagctatagatataagcgatgagatgagaagctattactgtgtaagatcttttttttaa(SEQ ID NO.1 shown).

[0053] 2. Protein Expression

[0054] The synthesized recombinant plasmid was transformed into the BL21 (DE3) expression strain, and evenly spread on the kanamycin-resistant plate after heat shock. After inverted culture in a 37°C incubator for 12 hours, a single colony was picked and inoculated in 10mL of kanamycin-resistant LB liquid medium for activation, and shaken at 37°C and 220r / min until the OD value was 0.6-0.8. IPTG with a final concentration of 0.5mmol / L was added for induction. After culture at 16°C and 220r / min for 16 hours, the bacteria were centrifuged and collected. The bacteria were washed with PBS and resuspended, ultrasonically broken in an ice bath, and the supernatant was collected after centrifugation and purified by Ni affinity chromatography pre-packed column, and the target protein was eluted with different concentrations of imidazole buffer. The target protein LSDV-122 was analyzed by SDS-PAGE and Western blot. Finally, the protein concentration was determined and the aliquots were placed in a -80°C refrigerator for storage.

[0055] LSDV-122 protein sequence: TDYDESNNFTAFAGSTIYGYGLKSKKNIKKKVKLINFCIKISIMTS MVSLITITILLAFFNNTCELNQFKEHKPYFLKNPNPTTYSDDDTESELNVYRSCKGIVYSGYC YTFNSEPKSFNDAYDDCEKKNSELPSNNLMNDWISDYLDGTWGEDGNVLFKEKNQELEAI DISDEMRSYYCVRSFF (shown in SEQ ID NO.2).

[0056] The expression and purification results of p122 protein are shown in Figure 1 As shown, the results showed that the target protein with a purity of more than 90% was successfully prepared.

[0057] 3. Preparation of coating antigen

[0058] To facilitate purification and increase protein solubility, HIS tags and SUMO tags were added to the LSDV-122 protein prepared above. In order to improve the specificity of the method, the prepared LSDV-122 protein was cleaved with SUMO protease. The reaction buffer was 25mM Tris-HCl, and the ratio of SUMO protease to LSDV-122 protein was 10U:1mg. The reaction was carried out in a dialysis bag, and the enzyme digestion was carried out at 4°C overnight for 16h. After the reaction, the solution was placed in a dialysis bag for displacement and concentration, and the concentrated protein was used as the coating antigen.

[0059] 4. Preparation of enzyme-labeled antigen

[0060] In order to shorten the time for serum diagnosis and further improve the specificity of diagnosis, the concentrated protein was labeled with HRP according to the instructions of the Roche Peroxidase Labeling Kit. The specific steps are as follows:

[0061] (1) Add 0.3 mL of antigen solution and 0.1 mL of peroxidase into an EP tube, gently pipette to mix, and incubate at 4°C overnight;

[0062] (2) Add 40 μL of triethanolamine to the mixture, mix by pipetting, then add 50 μL of sodium borohydride, mix by pipetting again, and let stand at 4°C for 30 min;

[0063] (3) Add 10 μL of glycine solution to the mixture and mix thoroughly by pipetting;

[0064] (4) Transfer the mixed solution into a dialysis bag and place it in dialysis solution at 4°C overnight; during this period, the dialysis solution was changed three times;

[0065] (5) Use a syringe to draw out the mixed solution from the dialysis bag and inject it into the EP tube. Add a preservation solution of equal volume to the mixed solution, gently blow and mix, and store in aliquots at -80°C.

[0066] At the same time, the applicant compared the effects of HIS-tagged proteins and non-HIS-tagged proteins on HRP labeling. The results showed that HIS-tagged proteins can improve the HRP labeling efficiency. Therefore, HIS-tagged proteins were selected for HRP labeling.

[0067] LSDV-122 protein sequence with HIS tag: TDYDESNNFTAFAGSTIYGYGLKSKKNIKKKVKLI NFCIKISIMTSMVSLITITILLAFFNNTCELNQFKEHKPYFLKNPNPTTYSDDDTESELNVYRS CKGIVYSGYCYTFNSEPKSFNDAYDDCEKKNSELPSNNLMNDWISDYLDGTWGEDGNVLF KEKNQELEAIDISDEMRSYYCVRSFFGGGSHHHHHH (shown in SEQ ID NO. 3).

[0068] Example 2 Optimization and establishment of double antigen sandwich ELISA method

[0069] 1. Basic steps of double antigen sandwich ELISA method

[0070] (1) Antigen coating: Use coating solution to coat 122 protein, the coating solution volume is: 50 μL / well, 4°C overnight;

[0071] (2) Blocking: After antigen coating, the ELISA plate was blocked: the plate was first washed with TBST buffer (3 times, 5 min each time), and then a blocking solution (TBST buffer containing 5% BSA) was added and incubated at 37°C for 1 h.

[0072] (3) Adding samples to be tested and enzyme-labeled antigens: After blocking, wash the plate with TBST buffer (wash 3 times, 5 minutes each time), add 50 μL of serum samples to be tested, positive controls, and negative controls respectively, and incubate at 37°C for 30 minutes. After incubation, wash the plate with TBST buffer (wash 3 times, 5 minutes each time), add 50 μL of enzyme-labeled antigen working solution to each well, and incubate at 37°C for 30 minutes.

[0073] (4) Color development: After incubation, wash the plate with TBST buffer (3 times, 5 min each time), then add 50 μL of color development solution and incubate in a 37°C incubator in the dark for 10 min. Add 50 μL of stop solution to each well and read the OD value at 450 nm on a microplate reader.

[0074] The coating solution is carbonate buffer, formula: 1.59g sodium carbonate, 2.93g sodium bicarbonate, adjust pH to 9.6 and add deionized water to 1L;

[0075] TBST buffer solution formula: 8 g sodium chloride, 0.2 g potassium chloride and 0.3 g tris(hydroxymethyl)aminomethane, adjust pH to 7.4, add 1 ml Tween 20 and add distilled water to make up to 1 L.

[0076] 2. Method Optimization

[0077] 2.1 Optimal concentration of coated antigen and optimal dilution of serum to be tested

[0078] The coating antigen prepared in Example 1 was coated with the coating solution by matrix titration method, and the coating antigen concentration was diluted to 8 μg / mL, 6 μg / mL, 3 μg / mL, 2 μg / mL and 1 μg / mL, respectively, and 50 μL was added to each well of a 96-well ELISA plate;

[0079] The standard positive serum and negative serum were diluted at 1:1, 1:2, 1:4, and 1:8, respectively;

[0080] The other steps are the same as those shown in 1 above.

[0081] P / N value (positive control serum OD 450 Mean / negative control serum OD 450 The serum dilution multiple and the antigen coating concentration when the mean (average) is greater than 2.1 and the maximum are the optimal serum dilution and optimal coating concentration.

[0082] The experimental results are shown in Table 1 below. It was finally determined that the optimal coating antigen concentration was 2 μg / mL and the optimal dilution of the serum to be tested was 1:2.

[0083] Table 1 Matrix titration results of different coating concentrations of antigen and different dilutions of serum

[0084]

[0085] 2.2 Determination of coating fluid type

[0086] Based on the optimal antigen concentration (2 μg / ml) and the optimal serum dilution (1:2) determined in 2.1 above, the coating solution type was further determined, and carbonate buffer, NaHCO3, and phosphate buffer were used as coating solutions to dilute the antigen to 2 μg / ml, and the optimal coating solution type was when the P / N value was greater than 2.1 and the maximum. The other steps were the same as in 1 above.

[0087] Carbonate buffer solution formula: 1.59 g sodium carbonate, 2.93 g sodium bicarbonate, adjust pH to 9.6 and add deionized water to make up to 1 L.

[0088] Phosphate buffer formula: 8g NaCl, 0.2g KCl, 1.44g Na2HPO4 and 0.24g KH2PO4, adjust the pH to 7.4 and add deionized water to 1L.

[0089] NaHCO3 buffer recipe: 4.2005 g NaHCO3, adjust pH to 9.6 and add deionized water to 1 L.

[0090] From the results in Table 2, it can be seen that when the coating solution is carbonate buffer, the P / N value is the largest, which meets the established conditions. Therefore, this coating solution is the best coating solution.

[0091] Table 2 Detection results of different coating fluid types

[0092]

[0093] 2.3 Confirmation of coating conditions

[0094] Based on the optimal antigen coating concentration, serum dilution multiple and coating solution type described in 2.1-2.2 above, other experimental conditions were fixed (same as in 1 above), and the diluted antigen coating solution was incubated at 37°C for 1h, 37°C for 2h and 4°C overnight, and the OD was read according to the conventional operation steps of antigen sandwich ELISA. 450 The P / N values ​​of each group were compared, and the condition corresponding to the maximum P / N value was selected as the optimal coating condition. The optimal coating condition was determined to be 4°C overnight.

[0095] 2.4 Determination of blocking solution type

[0096] Based on the optimal conditions determined in 2.1-2.3 above, the blocking solution type was adjusted, and 3% BSA, 5% BSA, 8% BSA, 3% skim milk powder, 5% skim milk powder and 8% skim milk powder were used as blocking solutions to carry out the blocking test and dilute the serum samples. The other steps were the same as in 1 above; the optimal blocking solution type was taken when the P / N value was greater than 2.1 and the maximum. The experimental results are shown in Table 3 below, and the results show that 5% BSA is the optimal blocking solution type.

[0097] Table 3 Test results of different blocking liquid types

[0098]

[0099] 2.5 Determination of closure conditions

[0100] Based on the optimal reaction conditions determined in 2.1-2.4 above, only the blocking conditions were adjusted: four different blocking conditions were set at 4°C for 6h, 4°C overnight (16-18h), 37°C for 1h, and 37°C for 2h. The other steps were the same as in 1 above. The optimal blocking condition was determined to be 37°C for 1h when the P / N value was greater than 2.1 and was the maximum.

[0101] 2.6 Determination of the optimal reaction time of the serum to be tested

[0102] Based on the optimal reaction conditions determined in 2.1-2.5 above, only the incubation time of the serum to be tested was adjusted, and four time points of 15min, 30min, 60min and 90min were set respectively. The optimal reaction time was taken when the P / N value was greater than 2.1 and was the maximum. Finally, the incubation time was determined to be 30min.

[0103] 2.7 Optimal working concentration of enzyme-labeled antigen

[0104] Based on the optimal reaction conditions determined in 2.1-2.6 above, only the enzyme-labeled antigen dilution was adjusted to 1:10000, 1:20000, 1:40000, 1:60000 and 1:80000, respectively. The optimal enzyme-labeled antigen dilution was taken when the P / N value was greater than 2.1 and was the largest.

[0105] The experimental results are shown in Table 4 below, which show that 1:60000 is the optimal dilution of the enzyme-labeled antigen.

[0106] Table 4 Detection results of different enzyme-labeled antigen dilutions

[0107]

[0108] 2.8 Optimal working time of enzyme-labeled antigen

[0109] Based on the optimal reaction conditions determined in 2.1-2.7 above, only the enzyme-labeled antigen incubation time was adjusted, and three time points of 15 min, 30 min and 60 min were set respectively. The optimal reaction time was taken when the P / N value was greater than 2.1 and was the maximum. Finally, the incubation time was determined to be 30 min.

[0110] 2.9 Determination of the optimal color development time

[0111] Based on the optimal reaction conditions determined in 2.1-2.8 above, only the color development time was adjusted, and four time points of 5min, 10min, 15min and 20min were set respectively. The optimal reaction time was taken when the P / N value was greater than 2.1 and was the maximum. Finally, the incubation time was determined to be 10min.

[0112] 2.10 Determination of cut-off value (positive judgment value)

[0113] According to the above optimal reaction conditions, 109 negative sera and 57 positive sera were tested, the S / P value was calculated, the ROC curve was generated, and the cut-off value was determined accordingly. Figure 2 As shown, the critical value of this method is 0.304; result judgment: when S / P<0.304, it is judged as negative; when S / P>0.304, it is judged as positive.

[0114] 3. Establishment of double antigen sandwich ELISA method

[0115] According to the above conditions optimization, a double antigen sandwich ELISA method was established. The specific steps are as follows:

[0116] (1) Coating: 2 μg / mL of LSDV-122 protein (prepared in Example 1) was coated with a coating solution, the volume of which was 50 μL / well, at 4°C overnight; the coating solution was a carbonate buffer (1.59 g of sodium carbonate, 2.93 g of sodium bicarbonate, adjusted to pH = 9.6 and fixed to 1 L with deionized water).

[0117] (2) Blocking: After antigen coating, the ELISA plate was blocked: the plate was first washed with TBST buffer (3 times, 5 min each time), and then a blocking solution (TBST buffer containing 5% BSA) was added and incubated at 37°C for 1 h.

[0118] TBST buffer solution formula: 8 g sodium chloride, 0.2 g potassium chloride and 0.3 g tris(hydroxymethyl)aminomethane, adjust pH to 7.4, add 1 ml Tween 20 and add distilled water to make up to 1 L.

[0119] (3) Adding samples to be tested: After blocking, wash the ELISA plate with TBST buffer (3 times, 5 min each time). After washing, add 50 μL of serum samples to be tested, positive control, and negative control, respectively, and incubate at 37°C for 30 min.

[0120] (4) Adding enzyme-labeled antigen: After incubation, the ELISA plate was washed with TBST buffer (3 times, 5 min each time), and 50 μL of the enzyme-labeled antigen (prepared in Example 1) working solution was added to each well, and the plate was incubated in a 37° C. incubator for 30 min.

[0121] (5) Color development: After incubation, wash the plate with TBST buffer (3 times, 5 min each time), then add 50 μL of color development solution and incubate in a 37°C incubator in the dark for 10 min. Add 50 μL of stop solution to each well and read the OD value at 450 nm on a microplate reader.

[0122] (6) Result determination: When S / P>0.304, it is determined as positive, and when S / P<0.304, it is determined as negative. S / P=(OD 样品 -OD 阴性 Average value) / (OD 阳性 -OD 阴性 average value).

[0123] Example 3 Validation of the Double Antigen Sandwich ELISA Method

[0124] 1. Specificity detection

[0125] Referring to the method described in Part 3 of Example 2, bovine nodular dermatitis (LSDV) positive serum, sheep pox (SPPV) positive serum, goat pox (GTPV) positive serum, LSDV negative serum, SPPV negative serum, GTPV negative serum, Brucella (brucella) positive serum, sheep orifice (ORFV) positive serum, foot-and-mouth disease (FMDV) positive serum and bovine viral diarrhea (BVDV) positive serum were used as samples for detection to evaluate the specificity of the detection method of the present application.

[0126] The results are as follows Figure 3 As shown, except for LSDV-positive serum, SPPV-positive serum and GTPV-positive serum, the rest were negative. This result indicates that the detection method provided in the present application is a universal detection method for LSDV, SPPV and GTPV, and it has good specificity and accuracy.

[0127] 2. Sensitivity testing

[0128] Referring to the method described in Part 3 of Example 2, LSDV, SPPV and GTPV positive sera were diluted at 1:2, 1:4, 1:8, 1:16, 1:32, 1:64 and 1:128, respectively, for testing to evaluate the sensitivity of the method.

[0129] The results are as follows Figure 4 As shown, the sensitivity of LSDV and GTPV was 1:32, and the sensitivity of SPPV was 1:16, which showed that the method had good sensitivity to all three viruses.

[0130] 3. Repeatability test

[0131] Referring to the method described in Part 3 of Example 2, 4 LSDV-positive sera and 4 LSDV-negative sera were set up with 5 replicate wells within the group and 5 repeated tests between groups, and the CV value (coefficient of variation) was calculated to determine the stability of the method.

[0132] The results are shown in Table 5. From the data in Table 5, it can be seen that the intra-batch repeat CV value is 1.35%-4.77%, and the inter-batch repeat CV value is 2.08%-8.82%. This result shows that the method established in this application has good stability.

[0133] Table 5 Repeatability test of double antigen sandwich ELISA method

[0134]

[0135] 4. Sample testing

[0136] To further evaluate the performance of the kit, 115 clinical samples were tested with reference to the method described in Part 3 of Example 2 and the Lanyan Biotech Bovine Nodule Antibody Detection Kit.

[0137] The results are shown in Table 6. When 115 clinical samples were tested, it was found that: the method described in this application detected 66 positive samples and 49 negative samples; the bovine nodule antibody detection kit of Lanyan Biology detected 65 positive samples and 50 negative samples; among them, the method described in this application and the bovine nodule antibody detection kit of Lanyan Biology simultaneously detected 62 positive samples and 46 negative samples. That is, the detection compliance rate of the method described in this application and the bovine nodule antibody detection kit of Lanyan Biology for bovine nodular skin disease clinical samples is 93.9%. This result shows that the method described in this application has a good effect in the detection of clinical samples of bovine nodular skin disease.

[0138] Table 6 Evaluation of clinical samples by double antigen sandwich ELISA method and commercial kit

[0139]

[0140] The above shows and describes the basic principles and main features of the present invention and the advantages of the present invention. It should be understood by those skilled in the art that the present invention is not limited by the above embodiments, and the above embodiments and descriptions are only for explaining the principles of the present invention. Without departing from the spirit and scope of the present invention, the present invention may have various changes and improvements, and these changes and improvements all fall within the scope of the present invention to be protected.

Claims

1. Application of p122 protein as coating antigen in the preparation of universal double antigen sandwich ELISA kit for bovine nodular skin virus, sheep pox virus and goat pox virus.

2. A universal double antigen sandwich ELISA kit for bovine nodular skin virus, sheep pox virus and goat pox virus, characterized in that: The kit comprises: coating antigen, enzyme-labeled antigen, coating solution, diluent, enzyme-labeled plate, TBS T buffer, blocking solution, color developing solution and stop solution; the coating antigen is p122 protein, and the enzyme-labeled antigen is enzyme-labeled p122 protein.

3. The kit according to claim 2, characterized in that The coating solution is a carbonate buffer solution with a pH of 9.

6.

4. The kit according to claim 2, characterized in that The blocking solution is TBST buffer containing 5% BSA.

5. The kit according to claim 2, characterized in that The enzyme-labeled antigen is: HRP-labeled p122 protein with a HIS tag.

6. Use of the kit according to any one of claims 1 to 5 in the detection of bovine nodular skin virus, sheeppox virus and goatpox virus for non-disease diagnosis purposes.

7. The use according to claim 6, characterized in that The method of using the kit is as follows: (1) Preparation of coating antigen and enzyme-labeled antigen; (2) Antigen coating: Use coating solution to coat p122 protein in the ELISA plate at 4°C overnight; (3) Blocking: Wash the coated ELISA plate with TBST buffer, then add blocking solution and incubate at 37°C; (4) Adding samples to be tested: After blocking, wash the ELISA plate with TBST buffer, add the serum samples to be tested, positive control and negative control respectively, and incubate at 37°C; (5) Adding enzyme-labeled antigen: After incubation, wash the plate with TBST buffer, add enzyme-labeled antigen, and incubate at 37°C; (6) Display: After incubation, the ELISA plate is washed with TBST buffer, and then a color developing solution is added, and the plate is incubated at 37°C in the dark; after incubation, a stop solution is added, and the OD value at a wavelength of 450 nm is read on an ELISA reader; (7) Result determination: S / P>0.304 was determined as positive, and S / P<0.304 was determined as negative; where S / P=(OD 样品 -OD 阴性 Average value) / (OD 阳性 -OD 阴性 average value).

8. The use according to claim 7, characterized in that In step (2), the amount of coating solution used is 50 μL / well, and the concentration of p122 protein is 2 μg / mL.

9. The use according to claim 7, characterized in that The dilution of the serum to be tested in step (4) is 1:2; the dilution of the enzyme-labeled antigen in step (5) is 1:60000.

10. The use according to claim 7, characterized in that The blocking time in step (3) is 1 hour; the incubation time in steps (4) and (5) is 30 minutes; and the light-proof incubation time in step (6) is 10 minutes.

Images