Indirect ELISA (enzyme-linked immunosorbent assay) detection kit and detection method for goose astrovirus antibody

Abstract

The invention relates to the technical field of biological products, in particular to an indirect ELISA (Enzyme-Linked Immunosorbent Assay) detection kit and a detection method for a goose astrovirus antibody. The kit comprises an elisa plate, an elisa secondary antibody, goose astrovirus positive control serum, goose astrovirus negative control serum, a diluent, a washing solution, a developing solution and a stop solution, wherein a coating antigen of the ELISA plate is a goose astrovirus VP27 recombinant protein, the goose astrovirus is a goose astrovirus GAstV / SD / 36, and the goose astrovirus VP27 recombinant protein corresponds to an ORF2 region. The goose astrovirus GAstV / SD / 36 is used as a template, the virus antigen recognition site VP27 protein is selected as an antigen for preparing the indirect ELISA kit, the indirect ELISA detection kit of the goose astrovirus antibody is prepared, and the accuracy of the detection result of the kit can be improved.

Description

Technical Field

[0001] The invention relates to the technical field of biological products, and in particular to an indirect ELISA detection kit and a detection method for goose astrovirus antibodies. Background Art

[0002] In recent years, goose gout is caused by goose astrovirus (GoAstV) infection, and is characterized by significant urate deposition on the surface of internal organs and joint cavities. The disease mainly harms goslings under 3 weeks old, and can be transmitted horizontally and vertically, and can effectively cross the interspecies barrier to infect chickens and ducks. The infection rate of goslings is as high as 80%, and the mortality rate is as high as 50%.

[0003] GoAstV is a non-enveloped single-stranded positive-strand RNA virus with three open reading frames, namely ORF1a, ORF1b and ORF2. ORF1a and ORF1b encode non-structural polyproteins nsp1a and nsp1b, which are mainly involved in viral RNA replication, transcription and encoding of viral particles. ORF2 encodes the viral capsid protein (Cap protein for short). Under the cleavage of proteases, Cap protein is cleaved to produce three proteins: VP34, VP27 and VP25. Among them, the spike region composed of VP27 has highly infectious particles. It is the main antigen recognition site of astrovirus and the main antigen that induces the body's specific immune response.

[0004] At present, the detection of GoAstV is mainly based on pathogenic methods, including RT-PCR, fluorescent quantitative RT-PCR, etc. The serological detection method for GoAstV antibodies mainly uses serum neutralization test and agar diffusion test, and there are few ELISA detection kit products. At present, some scholars use the ORF1b gene of the new goose astrovirus to express the non-structural polyprotein nsp1b as an indirect ELISA detection kit for antigen preparation, but the nsp1b protein is not a viral antigen recognition site. There are also studies that express and obtain Cap protein as a coating antigen to prepare ELISA detection kits, but Cap protein is not the final functional protein of GoAstV and needs to be digested by trypsin into proteins such as VP34, VP27 and VP25 to play a role. There are also studies that design synthetic peptides as antigens based on the GoAstV linear B cell antigen epitope sequence. This method is costly and not suitable for promotion as a commercial ELISA kit. In addition, in most studies of goose astrovirus ELISA detection methods, the gene sequence of the recombinant protein was synthesized based on the results published in the Genebank database, and most of them were data before 2020. Compared with the current goose astrovirus gene sequence, its homology is below 97.1%, and the relationship is relatively distant.

[0005] Therefore, the existing ELISA detection kit has low detection accuracy when detecting goose astrovirus, and it is imperative to improve the accuracy of the detection results of the ELISA detection kit. Summary of the invention

[0006] In order to solve the above technical problems, the present invention provides an indirect ELISA detection kit and detection method for goose astrovirus antibodies. The kit uses the main viral antigen recognition site (VP27 protein) in the ORF2 region of goose astrovirus GAstV / SD / 36 (abbreviated as SD / 36) as the coating antigen, which can improve the accuracy of the detection results of the kit.

[0007] To this end, the present invention provides the following technical solutions:

[0008] In a first aspect, the present invention provides an indirect ELISA detection kit for goose astrovirus antibodies in an optional embodiment, comprising an enzyme-labeled plate, an enzyme-labeled secondary antibody, goose astrovirus positive control serum, goose astrovirus negative control serum, a diluent, a washing solution, a color developing solution, and a stop solution;

[0009] The coating antigen of the ELISA plate is goose astrovirus VP27 recombinant protein, the goose astrovirus is goose astrovirus GAstV / SD / 36, and the goose astrovirus VP27 recombinant protein corresponds to the ORF2 region.

[0010] In the present invention, goose astrovirus GAstV / SD / 36 was deposited in the China Center for Type Culture Collection on April 22, 2024, with the deposit number being CCTCC NO: V202445.

[0011] Preferably, the goose astrovirus VP27 recombinant protein is expressed and purified in BL21 Escherichia coli competent cells using a plasmid pET-32a loaded with a gene sequence encoding the VP27 protein, wherein the expressed gene sequence is shown in SEQ ID NO. 1. The amino acid sequence of the goose astrovirus VP27 recombinant protein is shown in SEQ ID NO. 4.

[0012] Preferably, the enzyme-labeled secondary antibody is a rabbit anti-goose IgG secondary antibody labeled with horseradish peroxidase; and / or, the goose astrovirus positive control serum is the serum obtained by immunizing healthy geese with goose astrovirus VP27 recombinant protein, and the goose astrovirus negative control serum is the serum of healthy geese that are not infected with goose astrovirus GAstV / SD / 36.

[0013] Preferably, the diluent is a carbonate buffer solution, the concentration of sodium carbonate in the carbonate buffer solution is 1.59 g / L, the concentration of sodium bicarbonate is 2.93 g / L, and the pH value is 9.6; and / or, the concentration of sodium chloride in the washing solution is 80 g / L, the concentration of potassium chloride is 2 g / L, the concentration of disodium hydrogen phosphate is 14.4 g / L, the concentration of sodium dihydrogen phosphate is 2.4 g / L, the concentration of Tween-20 is 0.05%, and the balance is water. The color developing solution is TMB color developing solution; and / or, the stop solution is a 2 mol / L sulfuric acid solution.

[0014] In a second aspect, the present invention provides an indirect ELISA detection method for goose astrovirus antibodies in an optional embodiment, comprising the following steps:

[0015] S1: The VP27 protein of goose astrovirus was used as the coating antigen, mixed with the diluent and added to the wells of the ELISA plate, incubated at 4°C overnight, and washed with washing solution;

[0016] S2: Add 200 μL of 5% skim milk powder to each well of the washed ELISA plate, block at 37°C for 1 hour, discard the liquid, and wash with washing solution;

[0017] S3: Add the serum dilution mixture to be tested into each well of the ELISA plate, incubate at 37°C for 1 hour, discard the liquid, and wash with washing solution;

[0018] S4: Add 100 μL of enzyme-labeled secondary antibody to each well of the ELISA plate at a volume ratio of 1:10000, incubate at 37°C for 1 h, discard the liquid, and wash with washing solution;

[0019] S5: Add 100 μL of color development solution to each well of the ELISA plate and develop the color for 15 min in the dark;

[0020] S6: Add 100 μL of stop solution to each well of the ELISA plate to terminate the color development reaction;

[0021] S7: Use an ELISA reader to read the data at an absorbance of 450nm. When OD450 is above 0.1425, it is judged as positive;

[0022] Wherein, the dilution ratio of the serum dilution mixture to be detected is 1:10-1:50.

[0023] Preferably, in step S1, the goose astrovirus VP27 protein is prepared by the following method: using the full gene sequence of goose astrovirus GAstV / SD / 36 as a template, amplifying the VP27 gene using a primer pair, connecting it with the pET-32a plasmid to construct a recombinant plasmid, and then transforming the recombinant plasmid into BL21 Escherichia coli competent cells, adding IPTG to induce expression, and purifying the expressed recombinant protein to obtain the goose astrovirus VP27 protein.

[0024] Preferably, the sequence of the upstream primer in the primer pair is shown in SEQ ID NO.2; and / or, the sequence of the downstream primer in the primer pair is shown in SEQ ID NO.3. In step S1, the coating concentration of the goose astrovirus VP27 protein after dilution is 0.25-1 μg / mL; and / or, in step S4, the dilution ratio of the enzyme-labeled secondary antibody is 1:1000-1:5000.

[0025] The nucleotide sequence of SEQ ID NO.1 is as follows:

[0026] ATGCAGGTTACTCCCTCGCTTGTGTACAATTTCCAGGGTGAAAGGCAGAGCACTACAGAATCGTGCTCATTCCTAGTGTTTGGAATACCACAGGCAGACTCCAGGTCAAGATACAATGCAAATATAACATTCAATGTTGGCTATCGTGGAAAGACTTCAAAATCATTCACACTTGGAACACACAATTGGTGGGCTGTTATGACACTTTCACAAACAGGAGTAATTTTTGCACCACCGGCTGTGGGCACAGGGGTCTGTAATACATTGGCTACAGCCATACAACACTTAAACCCTGAGCTTGAAAAAGCAGTCCTGCGTGTGAATACCAGTACAACATCTACTGGTGGGCAGATAACGGAACTCAGGAATCGGCTCAACATCGCTGATGGGGACTATGTGATTTCAATGGGTGATCCGCAAGGAAATAGGTCAGCACTGTACTTTAGGAATTCAGACCAGAAATGGGTGTGGCTCTGGGCTGGCGACTTTAACCCTGGTGAAACTTTCCAAAGTTTTAAAATGCCAGTCCTAATTAATTGGTCAGTGTCAGATACCCAGGAACAATATAATAATAGAGTCAGGATGGTACAGTATGCTAATGCACAACAGCAGACTTTGACAGACCCTGAGGAAGATGATGATCCCCTCTCTGATGTCACTTCGCTTTTTGATCCAACAGCGGAAGATGAGACTGACTTCCACCTAGCAGTCTCGCTCAAGACCTCT。

[0027] The sequence of SEQ ID NO.2 is shown as follows:

[0028] TAAGGATCCCTAACACAGCCAAATGTG。

[0029] The sequence of SEQ ID NO.3 is shown as follows:

[0030] AAGTCGACAAGAGGTCTTGAGCGAGAC。

[0031] The amino acid sequence of SEQ ID NO.4 is shown as follows:

[0032] MQVTPSLVYNFQGERQSTTESCSFLVFGIPQADSRSRYNANITFNVGYRGKTSKSFTLGTHNWWAVMTLSQTGVIFAPPAVGTGVCNTLATAIQHLNPELEKAVLRVNTSTTSTGGQITEL RNRLNIADGDYVISMGDPQGNRSALYFRNSDQKWVWLWAGDFNPGETFQSFKMPVLINWSVSDTQEQYNNRVRMVQYANAQQQTLTDPEEDDDPLSDVTSLFDPTAEDETDFHLAVSLKTS.

[0033] Compared with the prior art, the present invention has one of the following beneficial effects:

[0034] 1. The present invention uses goose astrovirus GAstV / SD / 36 as a template, selects the virus antigen recognition site VP27 protein as an antigen for preparing an indirect ELISA kit, and prepares an indirect ELISA detection kit for goose astrovirus antibodies, which can improve the accuracy of the detection results of the kit.

[0035] 2. The indirect ELISA detection method for goose astrovirus antibodies provided by the present invention has a sensitivity of up to 1:2400, a repeatability coefficient of variation of 0.0285%-0.0345%, which is lower than 10%, and a conformity rate of 100% with neutralization detection, and has good sensitivity and stability.

[0036] 3. The indirect ELISA detection kit for goose astrovirus antibodies provided by the present invention does not cross-react with positive goose sera of GPV, TUMV, AIV, NDV, and DRV. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 is the virus phylogenetic tree in Example 1 of the present invention;

[0038] Figure 2 This is a comparison result of GoAstV nucleotide homology in Example 1 of the present invention;

[0039] Figure 3 This is a diagram showing the amplification results of the VP27 gene in Example 1 of the present invention, wherein M is DL2000 DNA Marker and 1 is VP27 gene amplification;

[0040] Figure 4 This is a diagram of the bacterial solution PCR detection result in Example 1 of the present invention, wherein M is DL2000 DNA Marker, and 1-10 are VP27 bacterial solution PCR;

[0041] Figure 5 This is a diagram showing the SDS-PAGE detection result of the VP27 protein expression in Example 1 of the present invention, wherein M is a standard molecular weight protein Marker, and 1 is a VP27 recombinant protein. DETAILED DESCRIPTION

[0042] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with specific embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0043] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.

[0044] Unless otherwise specified, the materials and reagents used in the following examples can be obtained from commercial sources.

[0045] Example 1

[0046] This embodiment provides a method for preparing goose astrovirus VP27 protein, comprising the following steps:

[0047] 1. Identification, isolation and sequencing of SD / 36 virus

[0048] In 2024, 67 samples of goslings suspected of suffering from gout were collected from goose farms in Shandong, Zhejiang, Hunan, Guangdong and other places. After extracting the cDNA of the samples, RT-PCR was performed using cDNA as a template and specific detection primers (upstream primer: ACTGACTGGGAAGATGG AGAAG; downstream primer: TGTAATAGTGGAACACGCAAAC). 44 samples were positive for GoAstV. The samples were separated on LMH cells, and a total of 44 viruses were isolated. After sequencing these viruses and comparing their sequence homology, it was found that 34 samples were 100% homologous and were a single virus named GAstV / SD / 36 (SD / 36 for short); 3 samples were 100% homologous and were a single virus named GAstV / SD / 4 (SD / 4 for short); the remaining viruses were named GAstV / SD / 1, GAstV / SD / 2, GAstV / SD / 3, GAstV / SD / 5, GAstV / SD / 6, GAstV / SD / 7, and GAstV / SD / 8, respectively, resulting in a total of 9 strains of goose astrovirus. The 9 strains were compared with the goose astrovirus in the NCBI gene database, and it was found that the SD / 36 virus was in the same branch as the viruses after 2021 (see Figure 1 ).

[0049] The SD / 36 virus was analyzed for homology with the GoAstV full genome sequences published by GenBank between 2015 and 2024. It was found that the SD / 36 virus had a homology of less than 97.1% with the virus before 2020 and was not in the same branch; the homology with the virus after 2021 was more than 98.5-99.0% (see Figure 2 ). SD / 36 was deposited in the China Center for Type Culture Collection on April 22, 2024, with the deposit number CCTCC NO: V202445.

[0050] The results showed that the SD / 36 virus was widely prevalent in goslings, and the virus was closely homologous to the viruses after 2021. This virus should be selected as an antigen to prepare ELISA detection kits to improve the sensitivity and specificity of the kits.

[0051] 2. Amplification of VP27 gene

[0052] VP27 protein is the main antigen recognition site of the spike region of astrovirus and can induce specific immune response. Therefore, SD / 36 strain was used as template and specific primers were used: the sequence of the upstream primer (SEQ ID NO.2) was: TAAGGATCCCT AACACAGCCAAATGTG; the sequence of the downstream primer (SEQ ID NO.3) was: AAGTCGACAAGAGGTCTTGA GCGAGAC) to amplify the VP27 gene by PCR and obtain the target fragment of 720 bp (see the results). Figure 3 ).

[0053] 3. Construction and identification of recombinant plasmids

[0054] After the pET-32a plasmid and VP27 gene were digested with restriction endonucleases BamHI and SalI, the pET-32a plasmid and VP27 gene were connected with T4 DNA ligase, and the ligation product was transformed into BL21 Escherichia coli competent cells. A single suspected positive colony was selected for PCR identification and sequencing. The results showed that all 10 single colonies selected had a single band, and the fragment size was consistent with 720bp (see the results). Figure 4 ), and the sequencing results were completely consistent with the VP27 sequence.

[0055] 4. Induced expression and identification of recombinant proteins

[0056] The recombinant plasmid pET-32a-VP27 positive bacterial solution was inoculated into LB medium at a volume ratio of 1:100, and the culture was shaken at 37°C and 220rpm. When the OD600 value of the bacterial solution was about 0.8, IPTG with a final concentration of 1mmol / L was added, and the samples were harvested after 6 hours of induction culture at 37°C. The protein expression was identified by SDS-PAGE. The results showed that the expressed protein had a specific band in the range of 25-35KDa, which was in line with the expected size (see the results). Figure 5 ).

[0057] 5. Purification of Recombinant Protein

[0058] pET-32a plasmid comes with a His tag, which is used with Ni 2+ Purify the recombinant protein by affinity chromatography. The purification steps are as follows:

[0059] 1) Cleaning: Clean the nickel column 3-5 times with 10 times the volume of membrane water;

[0060] 2) Cleaning and balancing: The nickel column is cleaned with 10 times the volume of balancing buffer to achieve the effect of balancing the nickel column;

[0061] 3) Sample loading: After the equilibrium buffer has settled, add the sample solution and allow it to slowly pass through the column and settle naturally;

[0062] 4) Washing: Add 10 times the volume of washing buffer and allow it to settle naturally;

[0063] 5) Elution: Add 5 times the volume of elution buffer, allow it to settle naturally, and collect the eluted protein solution. After purification, analyze the purified protein solution by SDS-PAGE.

[0064] Results: The purified recombinant protein had only one target band at 25-35KDa, which proved that the purification effect was good. The OD562 value of the purified recombinant protein was 0.2091 measured by BCA protein quantification kit, and the concentration of the recombinant protein was calculated to be 83.12μg / mL.

[0065] 6. Preparation of positive control serum and negative control serum

[0066] Forty healthy goslings aged 7 days were selected and randomly divided into two groups, A and B, with 20 in each group. The purified VP27 protein was mixed and emulsified with Freund's complete adjuvant (Sigma-Aldrich (Shanghai) Trading Co., Ltd., catalog number: F5881) in a volume ratio of 1:1, 100 μg / goose, and the goslings in group A were first immunized by multiple subcutaneous injections in the neck. Group B was used as a negative control group and was not treated. 14 days after immunization, the purified VP27 protein was mixed and emulsified with Freund's incomplete adjuvant (Sigma-Aldrich (Shanghai) Trading Co., Ltd., catalog number: F5506) in a volume ratio of 1:1, 100 μg / goose, and the goslings in group A were secondarily immunized by multiple subcutaneous injections in the neck. Group B was used as a negative control group and was not treated. 21 days after immunization, blood was collected from the two groups of geese to separate serum, and the neutralization titer was determined. The positive and negative serum identified were stored at -20°C for later use.

[0067] Example 2

[0068] This embodiment provides a method for determining the optimal antigen coating amount, optimal serum dilution, optimal serum reaction conditions, optimal secondary antibody dilution, optimal reaction conditions and indirect ELISA critical value of an indirect ELISA detection kit for goose astrovirus antibodies.

[0069] 1. Determination of the optimal coating amount of antigen

[0070] The recombinant protein was diluted to 0.0625, 0.125, 0.25, 0.5, and 1 μg / mL with carbonate buffer (Na2CO31.59 g / L, NaHCO32.93 g / L, PH=9.6), and 100 μL was added to each well. After overnight coating at 4°C, the liquid was discarded, the cells were washed 5 times with PBST, and patted dry. 200 μL of 5% skim milk powder was added to each well, and the cells were blocked at 37°C for 1 h. The liquid was discarded, the cells were washed 5 times with PBST, and patted dry. 100 μL of 5% skim milk powder was added to each well at a ratio of 1:200. GoAstV positive serum, with negative serum as control, incubated at 37°C for 1h; discarded the liquid, washed 5 times with PBST, patted dry, added 100μL rabbit anti-goose IgG-HRP to each well at a ratio of 1:10000, incubated at 37°C for 1h; discarded the liquid, washed 5 times with PBST, added 100μL TMB colorimetric solution to each well, color developed for 15min in dark, added 100μL stop solution to each well, measured OD450 value, calculated P / N value, selected the group with the largest P / N value, and determined the optimal antigen coating concentration. The results are shown in Table 1:

[0071] Table 1 Optimal antigen coating amount test

[0072]

[0073] It can be seen from Table 1 that when the antigen coating concentration is 0.25 μg / mL, the maximum P / N value is 4.6640. Therefore, 0.25 μg / mL is the optimal concentration for antigen coating.

[0074] 2. Determination of the optimal serum dilution

[0075] The positive and negative sera to be tested were diluted at a ratio of 1:10, 1:50, 1:00, 1:200, 1:400, and 1:800, respectively, and the test was carried out according to the optimal coating concentration of the antigen. The other conditions remained unchanged, the OD450 value was measured, the P / N value was calculated, and the group with the largest P / N value was selected to determine the optimal dilution of the serum to be tested. The results are shown in Table 2:

[0076] Table 2 Results of optimal serum dilution

[0077] Serum dilution + - P / N 1:10 2.4091 0.2985 8.0718 1:50 0.8216 0.0814 10.0995 1:100 0.4337 0.0593 7.3136 1:200 0.2282 0.0520 4.3875 1:400 0.1462 0.0523 2.7971 1:800 0.09755 0.04925 1.9807

[0078] It can be seen from Table 2 that when the dilution of the serum to be tested is 1:50, the maximum P / N value is . Therefore, the optimal dilution of the serum to be tested is 1:50.

[0079] 3. Determination of the optimal serum reaction conditions

[0080] According to the above optimized antigen concentration and serum dilution ratio, the serum to be tested was reacted at room temperature and 37°C for 30 min, 45 min, 60 min, and 90 min, respectively, and ELISA reaction was performed. The group with the largest P / N value was selected to determine the optimal reaction conditions for the serum to be tested. The results are shown in Table 3:

[0081] Table 3 Serum optimal reaction time and temperature test

[0082] Serum incubation time + - P / N 30min 0.8236 0.1324 1.9437 45min 0.2922 0.1616 1.8084 60min 0.3444 0.2033 1.6938 90min 0.3829 0.2275 1.6834 Serum incubation temperature + - P / N Room temperature (25℃) 0.288 0.1384 2.0804 37℃ 0.2954 0.1387 2.1307

[0083] It can be seen from Table 3 that when the serum to be tested is incubated at 37°C for 30 minutes, the P / N value is the largest.

[0084] 4. Determination of the optimal dilution of secondary antibodies and optimal reaction conditions

[0085] The HRP-labeled rabbit anti-goose IgG secondary antibody was diluted 1:1000, 1:2500, 1:5000, and 1:10000, and incubated at room temperature and 37°C for 30 min, 45 min, 60 min, and 90 min, respectively. ELISA was performed under the optimized reaction conditions, and the group with the largest P / N value was selected to determine the optimal working concentration and reaction time of the enzyme-labeled secondary antibody. The results are shown in Tables 4 and 5.

[0086] Table 4 Experiment on the optimal dilution of secondary antibody

[0087] Secondary antibody dilution + - P / N 1:1000 1.1189 0.7201 1.5538 1:2500 0.5796 0.3622 1.6001 1:5000 0.3235 0.1983 1.6318 1:10000 0.1766 0.1225 1.4422

[0088] Table 5 Experiments on the optimal reaction conditions of secondary antibodies

[0089]

[0090] It can be seen from Tables 4 and 5 that when the enzyme-labeled secondary antibody dilution ratio is 1:5000 and incubated at 37°C for 90 min, the P / N value is the largest.

[0091] 5. Determination of indirect ELISA cut-off value

[0092] According to the optimized reaction conditions, 20 negative sera were tested, and the mean value (x) and standard deviation (SD) of OD450 were calculated. According to statistical principles, when the OD450 of the serum sample to be tested is ≥x+3×SD, it is judged as positive, OD450≤x+2×SD is judged as negative, and those between the two are judged as suspicious. According to the results, the mean value (x) of OD450 is calculated to be 0.0754, and the variance (SD) is 0.223, so when the OD450 of the serum to be tested is ≥0.1425, it is judged as positive.

[0093] Example 3

[0094] This example provides a sensitivity, specificity and repeatability test of an indirect ELISA detection kit for goose astrovirus antibodies.

[0095] 1. Indirect ELISA sensitivity test

[0096] The GoAstV positive serum was diluted 1:50, 1:100, 1:200, 1:400, 1:800, 1:1600, 1:2400, and 1:3200 times, respectively, and tested using the established ELISA method to analyze the sensitivity of the method. The results are shown in Table 6:

[0097] Table 6 Sensitivity test

[0098]

[0099]

[0100] It can be seen from Table 6 that when the positive serum was diluted 1:2400, the OD450 value of the indirect ELISA test was 0.1753, which was a positive reaction. This shows that the maximum serum dilution factor that can be detected by the ELISA test is 1:2400.

[0101] 2. Indirect ELISA specificity test

[0102] The known GoAstV positive serum and negative serum, GPV, TUMV, AIV, NDV, and DRV positive goose serum were selected respectively, and the established indirect ELISA method was used to detect whether there was a cross reaction with the established method. The results are shown in Table 7:

[0103] Table 7 Specificity test

[0104] serum OD450 GoAst positive serum 0.8632 GoAst negative serum 0.1001 GPV 0.0544 TUMV 0.0523 AIV 0.0509 NDV 0.0491 DRV 0.0515

[0105] It can be seen from Table 7 that only the GoAstV positive serum is positive, and the OD450 of the other sample serum is lower than 0.167 (OD value is calculated according to the method of Example 2), showing negative. This shows that the established indirect ELISA method has no cross reaction with these sera and has good specificity.

[0106] 3. Indirect ELISA repeatability test

[0107] Five positive serum samples were taken for testing at four different time points, and the test results were statistically analyzed and the coefficient of variation was calculated. The results are shown in Table 8:

[0108] Table 8 Repeatability test

[0109]

[0110] It can be seen from Table 8 that the coefficient of variation was between 0.0285% and 0.0345% when tested at four different time points, indicating that the indirect ELISA method has good stability.

[0111] Example 4

[0112] This embodiment provides an indirect ELISA detection method for goose astrovirus antibodies, comprising the following steps:

[0113] S1: The VP27 protein of goose astrovirus was used as the coating antigen, mixed with the diluent (concentration of 0.25 μg / mL), added to the wells of the ELISA plate, incubated at 4°C overnight, and washed with washing solution;

[0114] S2: Add 200 μL of 5% skim milk powder to each well of the washed ELISA plate, block at 37°C for 1 hour, discard the liquid, and wash with washing solution;

[0115] S3: Add the serum dilution mixture to be tested (dilution ratio is 1:50) to each well of the ELISA plate, incubate at 37°C for 1 hour, discard the liquid, and wash with washing solution;

[0116] S4: Add 100 μL of enzyme-labeled secondary antibody (dilution ratio 1:5000) to each well of the ELISA plate at a volume ratio of 1:10000, incubate at 37°C for 1 h, discard the liquid, and wash with washing solution;

[0117] S5: Add 100 μL of color development solution to each well of the ELISA plate and develop the color for 15 min in the dark;

[0118] S6: Add 100 μL of stop solution to each well of the ELISA plate to terminate the color development reaction;

[0119] S7: Use an ELISA reader to read the data at an absorbance of 450nm. When OD450 is above 0.1425, it is judged as positive.

[0120] 80 goose serum samples collected from slaughterhouses and goose breeding farms were randomly divided into two large portions, each containing 40 goose serum samples. The first large portion was tested according to the above steps, and the second large portion was tested according to the virus neutralization test. The test results are shown in Table 9:

[0121] Table 9 Clinical sample test results

[0122] Neutralization test Indirect ELISA Positive 35 35 Negative 5 5 total 40 40

[0123] It can be seen from Table 9 that the indirect ELISA method detected 35 positive sera and 5 negative sera; the virus neutralization test detected 35 positive sera and 5 negative sera; the total coincidence rate of the two was 100% (Table 10), and the two had a high coincidence rate and consistency, indicating that the indirect ELISA method has a more accurate detection result.

[0124] Example 5

[0125] This example provides an indirect ELISA test kit for goose astrovirus antibodies and three commercially available test kits for accuracy in detecting goose astrovirus, comprising the following steps:

[0126] Three indirect ELISA test kits for goose astrovirus antibodies were purchased from three biotechnology companies, named Kit A, Kit B, and Kit C. The antigen of Kit A is the non-structural polyprotein nsp1a encoded by the ORF1a gene, the antigen of Kit B is the non-structural polyprotein nsp1b encoded by the ORF1b gene, and the antigen of Kit C is the viral capsid protein Cap protein encoded by the ORF2 gene. A total of 20 strains of goose astrovirus were selected from the laboratory virus library, including 2 strains in 2018, 3 strains in 2020, 5 strains in 2022, 6 strains in 2023, and 4 strains in 2024. The viruses were prepared into inactivated vaccines and inoculated into geese by intramuscular injection at a dose of 0.5 ml / feather; immunization was performed once every 14 days for 3 times. After immunization for 3 times, blood was collected on the 21st day to collect serum. Serum antibodies were detected using 4 indirect ELISA test kits and neutralization tests, and the test results are shown in Table 10:

[0127] Table 10 Serum sample test results

[0128]

[0129] It can be seen from Table 10 that for 20 positive serum tests, the indirect ELISA method of the present invention detected 19 positives and 1 negative, and the negative serum was an immune serum prepared with the strain of 2018; Kit A detected 16 positives and 4 negatives, of which the negative serum was an immune serum prepared with the strain of 2022-2024; Kit B detected 15 positives and 5 negatives, of which the negative serum was an immune serum prepared with the strain of 2022-2024; Kit C detected 17 positives and 3 negatives, of which the negative serum was an immune serum prepared with the strain of 2023-2024; Neutralization test detected 19 positive sera and 1 negative serum. The indirect ELISA method test results of the present invention are consistent with the neutralization test results at a rate of 100%. The detection rates of kits A, B, and C for positive serum are 80%, 75%, and 85%, respectively, and they are mainly targeted at strains before 2020. The detection rate for strains after 2022 has dropped significantly. It can be seen that the indirect ELISA detection kit for goose astrovirus antibodies provided by the present invention has greatly improved the accuracy of detecting goose astrovirus compared to conventional indirect ELISA detection kits on the market.

[0130] Although the principles of the present invention are described in detail above in conjunction with the preferred embodiments of the present invention, those skilled in the art should understand that the above embodiments are merely explanations of the exemplary implementations of the present invention, and are not intended to limit the scope of the present invention. The details in the embodiments do not constitute limitations on the scope of the present invention, and any obvious changes such as equivalent transformations, simple replacements, etc. based on the technical solution of the present invention, without departing from the spirit and scope of the present invention, fall within the protection scope of the present invention.

Claims

1. An indirect ELISA detection kit for goose astrovirus antibodies, characterized in that: It includes an enzyme-labeled plate, an enzyme-labeled secondary antibody, a goose astrovirus positive control serum, a goose astrovirus negative control serum, a diluent, a washing solution, a color developing solution and a stop solution; The coating antigen of the ELISA plate is goose astrovirus VP27 recombinant protein, the goose astrovirus is goose astrovirus GAstV / SD / 36, and the goose astrovirus VP27 recombinant protein corresponds to the ORF2 region.

2. The indirect ELISA detection kit for goose astrovirus antibodies according to claim 1, characterized in that: The goose astrovirus VP27 recombinant protein is expressed and purified in BL21 Escherichia coli competent cells using a plasmid pET-32a loaded with a gene sequence encoding the VP27 protein, wherein the expressed gene sequence is shown in SEQ ID NO.

1.

3. The indirect ELISA detection kit for goose astrovirus antibodies according to claim 1, characterized in that: The enzyme-labeled secondary antibody is a rabbit anti-goose IgG secondary antibody labeled with horseradish peroxidase; and / or, The goose astrovirus positive control serum is the serum obtained by immunizing healthy geese with goose astrovirus VP27 recombinant protein, and the goose astrovirus negative control serum is the serum of healthy geese that are not infected with goose astrovirus GAstV / SD / 36.

4. The indirect ELISA detection kit for goose astrovirus antibodies according to claim 1, characterized in that: The diluent is a carbonate buffer solution, the concentration of sodium carbonate in the carbonate buffer solution is 1.59 g / L, the concentration of sodium bicarbonate is 2.93 g / L, and the pH value is 9.6; and / or, The washing liquid has a sodium chloride concentration of 80 g / L, a potassium chloride concentration of 2 g / L, a disodium hydrogen phosphate concentration of 14.4 g / L, a sodium dihydrogen phosphate concentration of 2.4 g / L, a Tween-20 concentration of 0.05%, and the remainder being water.

5. The indirect ELISA detection kit for goose astrovirus antibodies according to claim 1, characterized in that: The color developing solution is TMB color developing solution; and / or, The stop solution is a 2 mol / L sulfuric acid solution.

6. An indirect ELISA method for detecting goose astrovirus antibodies, characterized in that: The following steps are involved: S1: The VP27 protein of goose astrovirus was used as the coating antigen, mixed with the diluent and added to the wells of the ELISA plate, incubated at 4°C overnight, and washed with washing solution; S2: Add 200 μL of 5% skim milk powder to each well of the washed ELISA plate, block at 37°C for 1 hour, discard the liquid, and wash with washing solution; S3: Add the serum dilution mixture to be tested into each well of the ELISA plate, incubate at 37°C for 1 hour, discard the liquid, and wash with washing solution; S4: Add 100 μL of enzyme-labeled secondary antibody to each well of the ELISA plate at a volume ratio of 1:10000, incubate at 37°C for 1 h, discard the liquid, and wash with washing solution; S5: Add 100 μL of color development solution to each well of the ELISA plate and develop the color for 15 min in the dark; S6: Add 100 μL of stop solution to each well of the ELISA plate to terminate the color development reaction; S7: Use an ELISA reader to read the data at an absorbance of 450nm. When OD450 is above 0.1425, it is judged as positive; Wherein, the dilution ratio of the serum dilution mixture to be detected is 1:10-1:

50.

7. The indirect ELISA detection method for goose astrovirus antibodies according to claim 6, characterized in that: In step S1, the goose astrovirus VP27 protein is prepared by the goose astrovirus method as follows: The full gene sequence of goose astrovirus GAstV / SD / 36 was used as a template, the VP27 gene was amplified using a primer pair, and the gene was connected with a pET-32a plasmid to construct a recombinant plasmid. The recombinant plasmid was then transformed into BL21 Escherichia coli competent cells, IPTG was added to induce expression, and the expressed recombinant protein was purified to obtain the goose astrovirus VP27 protein.

8. The indirect ELISA detection method for goose astrovirus antibodies according to claim 7, characterized in that: The sequence of the upstream primer in the primer pair is shown in SEQ ID NO.2; and / or, The sequence of the downstream primer in the primer pair is shown as SEQ ID NO.

3.

9. The indirect ELISA detection method for goose astrovirus antibodies according to claim 6, characterized in that: In step S1, the coating concentration of the goose astrovirus VP27 protein after dilution is 0.25-1 μg / mL; and / or, In step S4, the dilution ratio of the enzyme-labeled secondary antibody is 1:1000-1:5000.

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