Novel duck reovirus agar diffusion antigen as well as preparation method and application thereof

Abstract

The invention belongs to the technical field of veterinary diagnosis, and particularly relates to a novel duck reovirus agar diffusion antigen as well as a preparation method and application thereof. The preparation method comprises the following steps: inoculating a novel duck reovirus GX2022 strain to an SPF (specific pathogen free) duck embryo at the age of 6-10 days, harvesting and inoculating a diseased duck embryo body within 24-144 hours, adding sterile normal saline, fully grinding, freezing and thawing (freezing at the temperature of-20 DEG C and thawing at the temperature of 25 DEG C), carrying out solid-liquid separation, collecting supernate, adding formaldehyde for inactivation, uniformly mixing the inactivated virus liquid with a freeze-drying protective additive, and freeze-drying to obtain the agar diffusion antigen. Compared with the prior art, the method has the advantages that the agar diffusion titer is as high as 1: 24, the specificity is strong, the stability is good, the titer is high, the yield is high, the reaction precipitation line is clear, the detection is convenient, the method can be used for evaluating the immune effect of the NDRV and diagnosing the infection of the NDRV, can also be used for screening NDRV antibody negative ducks, detecting exogenous virus antibodies of waterfowl live vaccines or virus seeds, performing epidemiological investigation and the like, and has a wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of veterinary diagnostic technology, specifically relating to a novel duck reovirus agar agar antigen, its preparation method, and its application. Background Technology

[0002] Novel duck reovirus disease (NDRV) is caused by a novel duck reovirus (NDRV), resulting in an acute and highly contagious disease in ducks characterized by irregular liver necrosis, splenic hemorrhage, and necrosis. In recent years, NDRV infection has shown a year-on-year upward trend, spreading to duck-farming areas nationwide and becoming a serious threat to waterfowl farming, causing significant economic losses. There are two main reasons for this: First, NDRV has a broader host spectrum, infecting chickens, ducks, and geese, leading not only to hemorrhagic necrotizing hepatitis and severe splenic necrosis but also death. Second, NDRV has a high morbidity and mortality rate, and currently lacks safe and effective vaccines and drugs for infection control and treatment. Therefore, effective disease monitoring and timely isolation and culling of infected poultry are crucial for disease control.

[0003] Currently, the main methods for determining the presence of infection in duck flocks or in vaccines and viral strains include ELISA, agar diffusion assay, and virus neutralization assay (VN), each with its own advantages and disadvantages. Among these, ELISA is the most widely studied method for NDRV antibody detection, popular for its speed, specificity, sensitivity, and ease of operation, making it suitable for large-scale duck flock testing. However, there are currently no commercially available ELISA kits, and the accuracy of ELISA kits developed by individual companies is not high. The virus neutralization assay is cumbersome and time-consuming. The agar diffusion assay (AGP) can be used to determine antibody titers and identify antigens; it is inexpensive, simple to operate, and suitable for large-scale clinical testing, and has been widely used in the detection of fowlpox virus, infectious bursal disease virus, and their antibodies. However, the establishment of an agar diffusion assay method for novel duck reoviruses has not yet been reported. Summary of the Invention

[0004] The purpose of this invention is to provide a novel duck reovirus agar amplified antigen, its preparation method, and its application. The prepared NDRV agar amplified antigen can specifically detect NDRV antibodies and has the characteristics of high sensitivity, strong specificity, and good stability.

[0005] This invention provides a novel method for preparing duck reovirus agar agar antigen, comprising the following steps: The novel duck reovirus strain GX2022 was inoculated into 6-10 day old SPF duck embryos. Diseased duck embryos within 24-144 hours of inoculation were harvested, and after being thoroughly ground with sterile physiological saline, the embryos were subjected to freeze-thaw separation. The supernatant was collected to obtain the propagated novel duck reovirus. The freezing temperature of the freeze-thaw was -20℃ and the thawing temperature was 25℃. The amplified novel duck reovirus was mixed with formaldehyde at a final concentration of 0.01~0.03 wt.%, and then mixed in a shaker at 37°C to inactivate it, thus obtaining the inactivated novel duck reovirus. The inactivated novel duck reovirus was mixed with a freeze-drying protectant and then freeze-dried to obtain the novel duck reovirus agaric antigen.

[0006] Preferably, the novel duck reovirus GX2022 strain is diluted 1000 times and then inoculated, with an inoculation volume of 0.2 mL dilution / embryo.

[0007] Preferably, dead or diseased duck embryos are harvested within 24-144 hours after inoculation.

[0008] Preferably, the freeze-drying protectant is a sucrose solution containing 5-10 wt.% gelatin.

[0009] Preferably, the concentration of sucrose in the sucrose solution is 10~40 wt.%.

[0010] Preferably, the volume ratio of the inactivated novel duck reovirus to the freeze-drying protectant is 7-10:1.

[0011] This invention provides a novel duck reovirus agarose antigen prepared by the preparation method described in the above technical solution.

[0012] This invention provides the application of the novel duck reovirus agar agar antigen described in the above technical solution in one or more of the following: (1) Preparation of novel duck reovirus antibody detection products; (2) Preparation of a novel product for evaluating the immunogenicity of duck reovirus; (3) Prepare novel diagnostic products for duck reovirus infection; (4) Screening for novel duck reovirus antibody-negative ducks; (5) Prepare detection products for exogenous virus antibodies against live waterfowl vaccines and / or strains; (6) Epidemiological investigation.

[0013] Preferably, the product includes products for agar diffusion testing.

[0014] This invention provides a method for detecting novel duck reovirus antibodies for non-diagnostic purposes, comprising the following steps: (1) Prepare agarose gel with a concentration of 1% using PBS buffer with a concentration of 0.01 mol / L and a pH of 7.2, and make agarose plates by punching holes. (2) Add the novel duck reovirus agar amplified antigen described in the above technical solution to the central well, and add the test sample, positive control and negative control to the surrounding wells of the central well in sequence; (3) Incubate the agar plate in a humidified chamber at 37°C for 24 hours, and then place it at 25°C for 24-72 hours. Observe the precipitation line. When a precipitation line is formed between the test sample and the antigen well, and coincides with the end of the precipitation line of the positive control, the test sample is judged as positive. When there is no precipitation line between the test sample and the antigen well, and the end of the precipitation line of the positive control is bent toward the test sample well, the test sample is judged as weakly positive. When no precipitation line is formed between the test sample and the antigen well, and the precipitation line of the positive control points toward the test sample well, the test sample is judged as negative.

[0015] Beneficial effects: This invention involves inoculating 6-10 day old SPF duck embryos with a novel duck reovirus strain GX2022. Diseased duck embryos within 24-144 hours of inoculation are harvested, thoroughly ground with sterile physiological saline, and then subjected to freeze-thaw cycles (-20°C to 25°C) for solid-liquid separation. The supernatant is collected, inactivated with formaldehyde, and then mixed evenly with a freeze-drying protectant before freeze-drying to obtain agar plate antigen with a titer as high as 1:2. 4 It can be stored for a long time below -15℃, and has high specificity, good stability, high potency, high yield, and clear reaction precipitation line, making it convenient for detection. It can be used not only for evaluating the immunization effect of NDRV and diagnosing NDRV infection, but also for screening NDRV antibody-negative ducks, detecting exogenous virus antibodies in live vaccines or strains of waterfowl, and conducting epidemiological surveys, etc., and has broad application prospects.

[0016] Furthermore, the NDRV antibody agar diffusion assay method established based on the agar diffusion antigen prepared in this invention has a high positive detection rate. Compared with existing ELISA detection technology, the antibody negative and positive concordance rate is 100%. It has the characteristics of low experimental operation requirements and low detection cost, and is particularly suitable for large-scale clinical NDRV antibody detection. Attached Figure Description

[0017] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the accompanying drawings used in the embodiments will be briefly described below.

[0018] Figure 1 The results of the NDRV agar amplification antigen titer determination in Example 5; Figure 2 The results are the NDRV agar antigen specificity detection results in Example 5. Detailed Implementation

[0019] This invention provides a novel method for preparing duck reovirus agar agar antigen, comprising the following steps: The novel duck reovirus strain GX2022 was inoculated into 6-10 day old SPF duck embryos. Diseased duck embryos within 24-144 hours of inoculation were harvested, and after being thoroughly ground with sterile physiological saline, the embryos were subjected to freeze-thaw separation. The supernatant was collected to obtain the propagated novel duck reovirus. The freezing temperature of the freeze-thaw was -20℃ and the thawing temperature was 25℃. The amplified novel duck reovirus was mixed with formaldehyde at a final concentration of 0.01~0.03 wt.%, and then mixed in a shaker at 37°C to inactivate it, thus obtaining the inactivated novel duck reovirus. The inactivated novel duck reovirus was mixed with a freeze-drying protectant and then freeze-dried to obtain the novel duck reovirus agaric antigen.

[0020] This invention involves inoculating 6-10 day old SPF duck embryos with a novel duck reovirus strain GX2022. In one embodiment, the novel duck reovirus strain GX2022 is diluted 1000-5000 times before inoculation, with an inoculation volume of 0.1-0.3 mL diluent / embryo; in another embodiment, the novel duck reovirus strain GX2022 is diluted 1000 times before inoculation, with an inoculation volume of 0.2 mL diluent / embryo. In one embodiment, the SPF duck embryos described in this invention are 6-8 days old.

[0021] Following inoculation, this invention harvests dead or diseased duck embryos within 24-144 hours of inoculation. As one implementation method, this invention harvests dead or diseased duck embryos within 24-120 hours of inoculation. The timing of harvesting in this invention eliminates duck embryo mortality caused by non-specific factors and aligns with the characteristics of viral replication, ensuring that the harvested virus content reaches its peak.

[0022] After obtaining the diseased duck embryos, the present invention adds sterile physiological saline to the diseased duck embryos, grinds them thoroughly, performs freeze-thaw separation, collects the supernatant, and obtains the novel duck reovirus after propagation; the freezing temperature of the freeze-thaw is -20℃ and the thawing temperature is 25℃.

[0023] In one embodiment, the mass-to-volume ratio of the diseased duck embryo to sterile physiological saline is 1g:2-4mL; in another embodiment, the mass-to-volume ratio is 1g:3mL. In one embodiment, the freeze-thaw cycle is 2-4 times; in another embodiment, it is 3 times. In another embodiment, the solid-liquid separation method includes centrifugation. In one embodiment, the centrifugation speed is 6000-10000 rpm; in another embodiment, it is 8000 rpm. In one embodiment, the centrifugation time is 20-40 min; in another embodiment, it is 30 min.

[0024] After obtaining the amplified novel duck reovirus, the present invention inactivates the amplified novel duck reovirus by mixing it with formaldehyde at a final concentration of 0.01~0.03 wt.%, thereby obtaining an inactivated novel duck reovirus. As one embodiment, the present invention inactivates the amplified novel duck reovirus by mixing it with formaldehyde at a final concentration of 0.01%.

[0025] In one embodiment, the inactivation temperature of the present invention is 35~38℃; in another embodiment, the inactivation temperature of the present invention is 37℃. In one embodiment, the inactivation time of the present invention is 16~28h; in another embodiment, the inactivation time of the present invention is 24h.

[0026] After obtaining the inactivated novel duck reovirus, this invention mixes the inactivated novel duck reovirus with a lyophilization protectant and freeze-dries it to obtain the novel duck reovirus agar agar antigen. As one embodiment, the lyophilization protectant of this invention is a sucrose solution containing 5-10 wt.% gelatin. As another embodiment, the sucrose solution of this invention is a 10-40 wt% sucrose solution. The lyophilization protectant specified in this invention requires a smaller dosage compared to lyophilization protectants prepared in other proportions, reducing the influence of other components on the virus while still providing protection and not reducing the virus titer.

[0027] In one embodiment, the volume ratio of the inactivated novel duck reovirus to the freeze-drying protectant is 7-10:1; in another embodiment, the volume ratio of the inactivated novel duck reovirus to the freeze-drying protectant is 8:1.

[0028] This invention provides a novel duck reovirus agarose antigen prepared by the preparation method described in the above technical solution.

[0029] The novel duck reovirus agar agar agar agar agar agar agar titer prepared by this invention is as high as 1:2.4 It can be stored for a long time below -15℃, and has high specificity, good stability, high potency, and high yield. It is convenient to detect and can be used not only for evaluating the immunization effect of NDRV and diagnosing NDRV infection, but also for screening NDRV antibody-negative ducks, detecting exogenous virus antibodies in live vaccines or strains of waterfowl, and conducting epidemiological surveys. It has broad application prospects.

[0030] In view of the above advantages, the application of the novel duck reovirus agar agar antigen described in this invention in one or more of the following is also within the scope of protection of this invention: (1) preparing novel duck reovirus antibody detection products; (2) preparing novel duck reovirus immunization effect evaluation products; (3) preparing novel duck reovirus infection diagnosis products; (4) screening novel duck reovirus antibody-negative ducks; (5) preparing exogenous virus antibody detection products for live waterfowl vaccines and / or strains; (6) epidemiological investigation.

[0031] As one embodiment, the product of the present invention includes a product for agar diffusion testing. This invention provides a method for detecting novel duck reovirus antibodies for non-diagnostic purposes, comprising the following steps: (1) Prepare agarose gel with a concentration of 1% using PBS buffer with a concentration of 0.01 mol / L and a pH of 7.2, and make agarose plates by punching holes. (2) Add the novel duck reovirus agar amplified antigen described in the above technical solution to the central well, and add the test sample, positive control and negative control to the surrounding wells of the central well in sequence; (3) Incubate the agar plate in a humidified chamber at 37°C for 24 hours, and then place it at 25°C for 24-72 hours. Observe the precipitation line. When a precipitation line is formed between the test sample and the antigen well, and coincides with the end of the precipitation line of the positive control, the test sample is judged as positive. When there is no precipitation line between the test sample and the antigen well, and the end of the precipitation line of the positive control is bent toward the test sample well, the test sample is judged as weakly positive. When no precipitation line is formed between the test sample and the antigen well, and the precipitation line of the positive control points toward the test sample well, the test sample is judged as negative.

[0032] In one embodiment, the 1% agarose gel of the present invention contains 6-10 wt.% sodium chloride; in another embodiment, the 1% agarose gel of the present invention contains 8 wt.% sodium chloride. In one embodiment, the thickness of the agarose gel in the agar diffusion plate of the present invention is 2.0-4.0 mm; in another embodiment, the thickness of the agarose gel in the agar diffusion plate of the present invention is 3.0 mm. In one embodiment, the pore diameters of the central hole and the surrounding holes of the present invention are 3.0-4.0 mm; in another embodiment, the pore diameters of the central hole and the surrounding holes of the present invention are 3.0 mm. In one embodiment, the pore distance from the central hole to the surrounding holes of the present invention is 3.0 mm. In one embodiment, the addition amount of the central hole and the surrounding holes of the present invention is 25 μL each.

[0033] The detection method provided by this invention has a high positive detection rate. When measuring chicken serum samples immunized with inactivated antigen of novel duck reovirus, the antibody positive rate is 100%. Compared with the ELISA detection method, the antibody negative and positive concordance rate is 100%. It also has the characteristics of low experimental operation requirements and low detection cost, and is particularly suitable for large-scale clinical detection of novel duck reovirus antibodies.

[0034] To further illustrate the present invention, the following detailed description, in conjunction with the accompanying drawings and embodiments, describes a novel duck reovirus agar agar antigen, its preparation method, and its application, but these descriptions should not be construed as limiting the scope of protection of the present invention.

[0035] Example 1 Preparation of NDRV agar agar antigen 1. NDRV inoculation of SPF duck embryos and harvesting NDRV-GX2022 strain (published in the literature: Kong Dongni, Deng Yong, Chen Mengjiao, et al. Isolation, identification and cell adaptation study of a novel duck reovirus [J]. Progress in Animal Medicine, 2025(5). DOI:10.3969 / j.issn.1007-5038.2025.05.007.) was diluted 1000 times and yolk sacs were inoculated into 6-8 day old SPF duck embryos, 0.2 mL / embryo. The embryos were incubated in a 37℃ incubator and candled twice daily. Chicken embryos that died within 24 hours were discarded, and diseased duck embryos that died within 24-120 hours were harvested, weighed, and 3 times the volume of sterile physiological saline were added. After thorough grinding, the embryos were subjected to repeated freeze-thaw cycles at -20℃ and 25℃ three times, centrifuged at 8000 rpm / min for 30 minutes, and the supernatant was collected as NDRV.

[0036] 2. Inactivation of NDRV agar antigen The harvested NDRV was added to a formaldehyde solution with a final concentration of 0.01% and inactivated in a shaker at 37°C for 24 hours. After inactivation, small samples of NDRV were inoculated into SPF duck embryos and passaged blindly for two generations for inactivation testing. If no cytopathic effect was observed in any generation of chicken embryo hepatocellular carcinoma cells (LMH cells), inactivation was considered complete; otherwise, inactivation was incomplete and required repeat inactivation. The results showed that inactivation was complete after 24 hours.

[0037] 3. Stability of NDRV agar agar antigen Inactivated NDRV was mixed with a protective agent (a 10 wt.% sucrose solution containing 5 wt.% gelatin) at a volume ratio of 8:1. After thorough mixing, the mixture was freeze-dried using a freeze-drying technique (the technique was described in the literature "Establishment of Purification and Freeze-Drying Process for Freeze-Dried Inactivated Rabies Vaccine", by Cui Songqi et al., published in the journal "Animal Husbandry" in April 2022), to obtain NDRV agar-agar antigen. The prepared NDRV agar-agar antigen freeze-dried product was stored at -20℃, and the NDRV agar-agar antigen titer was determined at different storage times. The results are shown in Table 1.

[0038] Table 1. Results of NDRV agar-agar antigen potency assay at different storage times (-20℃)

[0039] As can be seen from Table 1, the NDRV agar-agar antigen prepared in this invention does not decrease in potency even after being stored at -20℃ for 30 months.

[0040] Example 2 Establishment of an agar amplification assay for NDRV antibody detection 1. Preparation of agar plates: Add 1.0g of high-quality agar powder or agarose and 8.0g of sodium chloride to 100mL of PBS (0.01M, pH 7.2), heat in a water bath to melt, and after cooling slightly (60~65℃), pour into a petri dish or glass slide (thickness about 3.0mm). After the agar solidifies and cools to room temperature, store in a refrigerator at 2~8℃ for later use.

[0041] 2. Punching holes: Use a hexagonal hole punch to punch holes in the petri dish or glass slide. The hole diameter is 3.0 mm and the hole spacing is 3.0 mm. Remove the agar from the holes.

[0042] 3. Sealing the bottom: Gently heat the bottom of the agar plate with an alcohol lamp until the agar at the bottom is slightly melted. Alternatively, a small amount of melted 1% agar or agarose can be added to the bottom of the agar well where it contacts the plate.

[0043] 4. Sample addition: Depending on the purpose of the experiment, the sample addition method is as follows: (1) Clinical testing: Add antigen (approximately 25 μl) to the central well and serum (approximately 25 μl) to the peripheral well.

[0044] (2) Determination of egg yolk antibody or serum titer: Add antigen (about 25 μl) to the central well and add egg yolk antibody or serum (about 25 μl) to be tested, serially diluted with PBS (0.01M pH7.2) to the peripheral wells.

[0045] All of the above tests should include both positive and negative controls.

[0046] 5. Reaction: Place the agar plate in a humidified chamber and incubate at 37°C for 24 hours. Then, place it at room temperature for another 24-72 hours and observe the precipitation line.

[0047] 6. Judgment: A test is considered successful only if a clear precipitation line appears between the antigen well and the positive control serum, and no precipitation line appears between the antigen well and the negative control serum. Specific criteria are as follows: If a precipitation line forms between the test serum and the antigen well, and coincides with the end of the precipitation line of the positive control serum, the test serum is judged as positive. If there is no precipitation line between the test serum and the antigen well, but the end of the precipitation line of the positive serum bends towards the test serum well, it is judged as weakly positive. Weakly positive samples should be retested; if it remains weakly positive, it is judged as positive; otherwise, it is negative. If no precipitation line forms between the test serum and the antigen well, and the precipitation line of the positive control serum points towards the test serum well, it is judged as negative.

[0048] Example 3 NDRV agar amplification antigen is used for the determination of NDRV antibody agar amplification titer. 1. Preparation of positive control serum and negative control serum NDRV virus was inactivated by adding formaldehyde to a final concentration of 0.01 wt.% and shaking at 37°C for 24 hours to obtain NDRV inactivated antigen. Using the obtained NDRV inactivated antigen as an immunogen, 30 60-day-old SPF chickens were immunized by intramuscular injection (2.00 mL / chick). A booster immunization was administered subcutaneously 14 days later (2.00 mL / chick). Blood was collected 21 days later to separate serum. Blood was aseptically collected when the neutralizing antibody titer was ≥1:32. Five control chickens were also included. Positive and negative control sera were prepared by collecting serum from positive and control chickens, respectively.

[0049] 2. Using the NDRV agar agar antigen prepared in Example 1, the agar agar titer of NDRV antibody was detected according to the method in Example 2. Specific method: The prepared NDRV agar agar antigen was diluted 4-fold and added to the central well of an agar plate, filling the well completely without overflowing. The serum to be tested was serially diluted 2-fold and added sequentially to the surrounding wells, filling each well completely without overflowing. Positive and negative control sera were added to the surrounding wells. The agar plate was placed in a humidified chamber at 37°C for 24 hours, and then placed at room temperature for 24–72 hours. The agar agar antibody titer was then read. These sera were simultaneously tested by ELISA according to Chinese Patent CN118501444A, and the results are shown in Table 2.

[0050] Table 2 Results of NDRV antibody detection by ELISA and the agar amplification assay of the present invention

[0051] As can be seen from Table 2, the results obtained by the two methods have a 100% agreement rate.

[0052] Example 4 NDRV antigen is used for the detection of exogenous viruses in avian viral live vaccines. Six key live waterfowl vaccines produced by domestic manufacturers were selected. NDRV antibody detection was performed using the NDRV agar agar antigen prepared in Example 1, following the method in Example 2. Furthermore, the chicken test method was used to test the exogenous NDRV virus in the following live waterfowl vaccines according to the current "Veterinary Pharmacopoeia of the People's Republic of China," Part III. A PBS group was set up as a negative control, and an NDRV-infected group as a positive control. The results are shown in Table 3.

[0053] Table 3 Table 3 Results of vaccine detection by ELISA and the agar diffusion assay method of this invention.

[0054] As shown in Table 3, the results of the ELISA test and the agar amplification test using the present invention are consistent. All selected live waterfowl vaccines are free of NDRV contamination. The negative control is NDRV negative and the positive control is NDRV positive.

[0055] Example 5 1. NDRV antigen titer (sensitivity) determination The NDRV-positive serum prepared in Example 3 was used as an intermediate well. The NDRV agar-agar antigen prepared in Example 1 was serially diluted 2-fold, and PBS was used as a negative control (+). The original NDRV antigen was used as a positive control (+), and the antigen titer was determined. The results showed that the titer of the agar-agar antigen was 1:16 ( Figure 1 ).

[0056] 2. NDRV antigen-specific detection The NDRV-positive serum prepared in Example 3 was used as an intermediate well. The NDRV agar agar antigen prepared in Example 1 was serially diluted 2-fold. Sample 1 was the avian infectious bursal disease virus agar agar antigen standard, sample 2 was the fowlpox virus agar agar antigen standard, and sample 3 was the avian Marek's disease virus agar agar antigen standard. The NDRV agar agar antigen prepared in Example 1 was used as sample 4 and the positive control (+), and PBS was used as the negative control (-) for antigen specificity testing. The results showed that only sample 4 and the positive control (+) reacted with the NDRV-positive serum and produced a precipitation line. Samples 1-3 did not produce a precipitation line. Figure 2 ).

[0057] As can be seen from the above, the NDRV agar agar antigen prepared by this invention has the characteristics of high titer, strong specificity, and good stability, and has a high positive detection rate for NDRV antibodies, making it suitable for the detection of NDRV antibodies. Furthermore, the preparation method provided by this invention is scientifically sound, low in cost, and simple to operate.

[0058] Although the above embodiments have provided a detailed description of the present invention, they are only some embodiments of the present invention, and not all embodiments. People can obtain other embodiments based on these embodiments without creative effort, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for preparing a novel duck reovirus agar agar antigen, characterized in that, Includes the following steps: The novel duck reovirus strain GX2022 was inoculated into 6-10 day old SPF duck embryos. The embryos of dead and diseased ducks within 24-144 hours after inoculation were harvested, and after being thoroughly ground with sterile physiological saline, the mixture was subjected to freeze-thaw separation. The supernatant was collected to obtain the propagated novel duck reovirus. The freezing temperature of the freeze-thaw was -20℃ and the thawing temperature was 25℃. The amplified novel duck reovirus was mixed with formaldehyde at a final concentration of 0.01~0.03 wt.%, and then mixed in a shaker at 37°C to inactivate it, thus obtaining the inactivated novel duck reovirus. The inactivated novel duck reovirus was mixed with a freeze-drying protectant and then freeze-dried to obtain the novel duck reovirus agaric antigen.

2. The preparation method according to claim 1, characterized in that, The novel duck reovirus strain GX2022 was diluted 1000 times and then inoculated at a dose of 0.2 mL dilution per embryo.

3. The preparation method according to claim 1, characterized in that, Harvest diseased or dead duck embryos within 24-144 hours after inoculation.

4. The preparation method according to claim 1, characterized in that, The freeze-drying protectant is a sucrose solution containing 5-10 wt.% gelatin.

5. The preparation method according to claim 4, characterized in that, The concentration of sucrose in the sucrose solution is 10~40 wt.%.

6. The preparation method according to any one of claims 1 to 5, characterized in that, The volume ratio of the inactivated novel duck reovirus to the freeze-dried protectant is 7-10:

1.

7. The novel duck reovirus agaroplastin antigen obtained by the preparation method according to any one of claims 1 to 6.

8. The use of the novel duck reovirus agar agar antigen of claim 7 in one or more of the following: (1) Preparation of novel duck reovirus antibody detection products; (2) Preparation of a novel product for evaluating the immunogenicity of duck reovirus; (3) Prepare novel diagnostic products for duck reovirus infection; (4) Screening for novel duck reovirus antibody-negative ducks; (5) Prepare detection products for exogenous virus antibodies against live waterfowl vaccines and / or strains; (6) Epidemiological investigation.

9. The application according to claim 8, characterized in that, The products include those used in agar diffusion tests.

10. A method for detecting novel duck reovirus antibodies for non-diagnostic purposes, characterized in that, Includes the following steps: (1) Prepare agarose gel with a concentration of 1% using PBS buffer with a concentration of 0.01 mol / L and a pH of 7.2, and make agarose plates by punching holes. (2) Add the novel duck reovirus agar amplified antigen as described in claim 7 to the central well, and add the test sample, positive control and negative control to the surrounding wells of the central well in sequence; (3) Incubate the agar plate in a humidified chamber at 37°C for 24 hours, and then place it at 25°C for 24-72 hours. Observe the precipitation line. When a precipitation line is formed between the test sample and the antigen well, and coincides with the end of the precipitation line of the positive control, the test sample is judged as positive. When there is no precipitation line between the test sample and the antigen well, and the end of the precipitation line of the positive control is bent toward the test sample well, the test sample is judged as weakly positive. When no precipitation line is formed between the test sample and the antigen well, and the precipitation line of the positive control points toward the test sample well, the test sample is judged as negative.

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