Hemagglutination test for avian influenza and Newcastle disease
By setting reference hemocoagulation titer and regression test tests, the hemocoagulation titer was calculated, and the problem of multiple tests and reagent waste in the existing technology was solved, achieving efficient and accurate hemocoagulation test detection of avian influenza and Newcastle epidemics was achieved. .
Patent Information
- Application Number
- CN202110616202.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Patents(China)
- Current Assignee / Owner
- Filing Date
- 2021-06-02
- Publication Date
- 2024-12-17
- Estimated Expiration
- 2041-06-02
AI Technical Summary
The existing avian influenza and Newcastle epidemic hemagglutination test detection technology requires multiple hemagglutination tests and regression tests, resulting in inefficient diagnostic efficiency and excessive reagent use, and inaccurate results require retest verification.
The reference hemagglutination titers for the avian influenza and Newcastle epidemic hemagglutination test are 9log2 and 10log2. The number of red blood cell aggregation pores was observed through the regression test, and the hemagglutination titers were calculated using formulas, which simplified the detection process, reduced the number of tests and Reagent usage amount.
It improves the diagnostic efficiency of avian influenza and Newcastle epidemic, ensures the accuracy and consistency of results, reduces the need for repeated trial and error verification, and significantly improves the simplicity and efficiency of detection.
Smart Images
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Abstract
Description
Technical Field
[0001] The invention relates to the technical field of poultry disease detection, in particular to a hemagglutination test detection method for avian influenza and Newcastle disease. Background Art
[0002] Avian influenza and Newcastle disease are two major poultry infectious diseases that have spread around the world. Both viruses can cause severe respiratory infections in poultry, leading to high mortality rates in poultry. Outbreaks of avian influenza and Newcastle disease are related to transmission in wild birds and can cause human infections, such as conjunctivitis and influenza-like syndrome.
[0003] At present, the detection of Newcastle disease mostly adopts GBT16550-2020 Newcastle disease diagnostic technology, and the detection of avian influenza mostly adopts GB-T18936-2020 highly pathogenic avian influenza diagnostic technology. Both hemagglutination test and hemagglutination inhibition test are required in the diagnostic technology. 4-unit antigen (4HAU) is the diagnostic solution of the hemagglutination inhibition test. The preparation of 4HAU is an important part of the hemagglutination inhibition test. Its accuracy has a decisive influence on the result of the hemagglutination inhibition test. When the concentration of 4HAU is high, the HI antibody titer will decrease, and vice versa. The accuracy of the hemagglutination test directly determines the accuracy of the prepared 4HAU. In order to prevent the hemagglutination test from being inaccurate, a regression test is required to verify the result of the hemagglutination test. When the hemagglutination test is proved to be inaccurate by the regression test, it indicates that the prepared 4HAU antigen is inaccurate and the hemagglutination test needs to be repeated. Therefore, the existing detection technology often requires multiple hemagglutination tests and multiple regression tests, resulting in low diagnostic efficiency of avian influenza and Newcastle disease hemagglutination tests. Summary of the invention
[0004] The purpose of the present invention is to provide a hemagglutination test detection method for avian influenza and Newcastle disease, which overcomes the shortcomings of the above-mentioned prior art. Conventional hemagglutination test is not performed, but the reference hemagglutination titer of avian influenza hemagglutination test and / or Newcastle disease hemagglutination test is directly set, and a regression test is directly performed according to the set reference hemagglutination titer. According to the observation result of the regression test, the avian influenza hemagglutination titer or the Newcastle disease hemagglutination titer is calculated according to the avian influenza hemagglutination titer formula or the Newcastle disease hemagglutination titer formula, and the result is completely consistent with the correct avian influenza hemagglutination titer or the Newcastle disease hemagglutination titer. After a large number of tests and inspections, the avian influenza hemagglutination titer formula and the Newcastle disease hemagglutination titer formula are practical, accurate and universal. The new detection method is simple and intuitive, easy to operate, can shorten the test time, and can save the use of diagnostic reagents. Repeated tests and verifications are not required, and the diagnosis efficiency of avian influenza and Newcastle disease is greatly improved.
[0005] The technical solution adopted by the present invention to solve the technical problem is:
[0006] A hemagglutination test method for detecting avian influenza and Newcastle disease comprises the following steps:
[0007] (1) Set the reference hemagglutination titer of the avian influenza hemagglutination test to 9 log 2 , set the reference hemagglutination titer of Newcastle disease hemagglutination test to 10log 2 ;
[0008] (2) According to the "Avian Influenza, Newcastle Disease 4HAU Antigen Preparation Table", the reference hemagglutination titer is 9log 2 Avian influenza 4HAU antigen, or prepare a reference hemagglutination titer of 10log 2 The Newcastle disease 4HAU antigen was thoroughly shaken and the regression test was performed to observe the number of red blood cell agglutination holes;
[0009] (3) Avian influenza hemagglutination titer formula: According to the regression test observation results of step (2), when the number of hemagglutination holes of red blood cells is more than 2 by X, the avian influenza hemagglutination titer = (9 + X) log 2 When the number of hemagglutination holes of red blood cells is less than 2 by X, the hemagglutination titer of avian influenza is (9-X)log 2 , where X is 0, 1, 2;
[0010] (4) Newcastle disease hemagglutination titer formula: According to the regression test observation results of step (2), when the number of red blood cell agglutination holes is more than 2 by X, Newcastle disease hemagglutination titer = (10 + X) log 2 When the number of red blood cell agglutination holes is less than 2, the Newcastle disease hemagglutination titer = (10-X) log 2 , where X is 0, 1, or 2.
[0011] Preferably, the regression test in step (2) comprises the following steps:
[0012] ① Take a 96-well V-shaped microplate, use a micropipette to add 0.025 mL of PBS to wells 1 to 4, make 4 parallel samples, and change the dropper;
[0013] ②Pipette 0.025mL of the mixed antigen of 4HAU and add it to the first well, mix well, pipette 0.025mL from the first well and add it to the second well, mix well and pipette 0.025mL and add it to the third well, and so on to dilute it to the fourth well, pipette 0.025mL from the fourth well and discard it, and change the dripper;
[0014] ③ Add 0.025 mL of 1% chicken red blood cell suspension or 0.75% chicken aldehyde red blood cell suspension to each well;
[0015] ④ Mix by oscillation, let stand at room temperature of 20℃-25℃ for 20 minutes, then observe the results. Tilt the reaction plate, observe and record the number of red blood cell agglutination holes.
[0016] Preferably, in step ①, PBS is a pH 7.2 phosphate buffer, and its preparation method comprises the following steps: weigh 8.0 g of sodium chloride, 0.2 g of potassium chloride, 1.44 g of disodium hydrogen phosphate, and 0.24 g of sodium dihydrogen phosphate, add distilled water to 1000 mL, dissolve the above ingredients in sequence, adjust the pH to 7.2 with hydrochloric acid, package, and sterilize at 121°C for 15 min.
[0017] Preferably, the preparation method of 1% chicken red blood cell suspension in step ③ comprises the following steps: using Aldrich solution as an anticoagulant, collecting anticoagulated blood from at least three SPF roosters or non-immune chickens without avian influenza and Newcastle disease antibodies, placing the blood in a centrifuge tube, adding 3-4 times the volume of PBS to mix, centrifuging at 2000r / min for 5min-10min, removing plasma and white blood cell layer, repeating the above process, washing repeatedly 3-4 times, washing plasma and white blood cells, centrifuging at 2000r / min for 10min, finally drawing packed red blood cells and making a suspension with PBS with a volume fraction of 1%, and storing at 4°C for standby use.
[0018] Preferably, the preparation method of Aldrich solution comprises the following steps: weighing 2.05 g of glucose, 0.8 g of sodium citrate, 0.055 g of citric acid, and 0.42 g of sodium chloride, adding distilled water to 100 mL, adjusting the pH value to 6.1 after dissipating the heat to dissolve, sterilizing by high pressure at 69 kPa for 15 min, and storing at 4°C for later use.
[0019] Preferably, the preparation method of the 0.75% chicken aldehyded red blood cell suspension in step ③ comprises the following steps: washing fresh red blood cells with 0.1mol / L PBS solution for 5 times, centrifuging at 3000r / min for 4min each time, washing with 10 times the compressed volume of red blood cells with PBS solution for the last time, and centrifuging at 3000r / min for 10min, and removing the supernatant; adding 36% formaldehyde solution cooled to 4°C according to 8 times the amount of deposited red blood cells, mixing evenly, placing at 4°C for 24h, and shaking gently during the process; after taking out, adding 2 parts of 30% formaldehyde solution cooled to 4°C to 1 part of the deposited red blood cells, mixing evenly, and placing at 4°C for 24h, and then washing with PBS solution for 5 times to prepare 0.75% aldehyded red blood cell solution, adding sodium azide with a final concentration of 0.1% at 4°C for standby use.
[0020] The beneficial effects of the present invention are as follows: compared with the prior art, the hemagglutination test detection method for avian influenza and Newcastle disease of the present invention has the following advantages: the diagnostic technology of avian influenza and Newcastle disease is improved, and the conventional hemagglutination test is not performed, but the reference hemagglutination titer of the avian influenza hemagglutination test and the Newcastle disease hemagglutination test is directly set to 9log 2 and 10log 2According to the set reference hemagglutination titer, a regression test is directly performed and the number of hemagglutination holes of red blood cells is observed. According to the regression test, the accurate hemagglutination titer of avian influenza or Newcastle disease is calculated according to the hemagglutination titer formula of avian influenza or Newcastle disease. The new hemagglutination test detection method of the present invention does not need to repeatedly perform hemagglutination test and regression test verification, which greatly improves the diagnostic efficiency of hemagglutination test of avian influenza and Newcastle disease. DETAILED DESCRIPTION
[0021] Set the hemagglutination titer of avian influenza and Newcastle disease to 9log respectively 2 , 10log 2 A regression test was performed, and the detection method of calculating the hemagglutination titer by the hemagglutination titer formula was used in the avian influenza and Newcastle disease hemagglutination test detections in Example 2 and Example 4, and compared with the highly pathogenic avian influenza diagnostic technology (GB / T 18936-2020) and Newcastle disease diagnostic technology (GB / T 16550-2020) respectively to test the practicality and accuracy of the new detection method.
[0022] Example 1 Reagent Preparation
[0023] The preparation of 1% chicken red blood cell suspension comprises the following steps: using Aldrich solution as an anticoagulant, collecting anticoagulated blood from at least three SPF roosters or non-immune chickens without avian influenza and Newcastle disease antibodies, putting the blood into a centrifuge tube, adding 3-4 times the volume of PBS to mix, centrifuging at 2000r / min for 5min-10min, removing the plasma and white blood cell layer, repeating the above process, washing three times, washing the plasma and white blood cells, centrifuging at 2000r / min for 10min, finally absorbing the packed red blood cells and using PBS to prepare a suspension with a volume fraction of 1%, and storing at 4°C for standby use.
[0024] The preparation method of Alder's solution includes the following steps: weighing 2.05 g of glucose, 0.8 g of sodium citrate, 0.055 g of citric acid, and 0.42 g of sodium chloride, adding distilled water to 100 mL, adjusting the pH value to 6.1 after dissipating the heat to dissolve, sterilizing under high pressure at 69 kPa for 15 min, and storing at 4°C for later use.
[0025] The preparation of 0.75% chicken aldehyded red blood cell suspension comprises the following steps: fresh red blood cells are fully washed with 0.1mol / L PBS solution for 5 times, centrifuged at 3000r / min for 4min each time, washed with PBS solution of 10 times the compressed volume of red blood cells for the last time, centrifuged at 3000r / min for 10min, and the supernatant is removed; 36% formaldehyde solution cooled to 4°C is added according to 8 times the amount of sedimented red blood cells, mixed evenly, and placed at 4°C for 24h with continuous gentle shaking; after taking out, 2 parts of 30% formaldehyde solution cooled to 4°C are added to 1 part of sedimented red blood cells, mixed evenly, and placed at 4°C for 24h, and then washed with PBS solution for 5 times to prepare 0.75% aldehyded red blood cell solution, and sodium azide with a final concentration of 0.1% is added to the solution and placed at 4°C for standby use.
[0026] The "Avian Influenza, Newcastle Disease 4HAU Antigen Preparation Table" is shown in Table 1:
[0027]
[0028] Example 2 A hemagglutination test method for detecting avian influenza
[0029] The avian influenza virus antigen used in the experiment of Example 2 is the avian influenza virus H7N9 (RE-2 strain) hemagglutination inhibition test antigen produced by Harbin Guosheng Biotechnology Co., Ltd., with production batch number 20190415.
[0030] 1. Material preparation
[0031] (1) 96-well V-shaped microplate, micropipette (with dropper), and disposable pipette trough.
[0032] (2) 1% chicken red blood cell suspension or 0.75% chicken aldehyde red blood cell suspension.
[0033] (3) pH 7.2, 0.01 mol / L PBS.
[0034] (4) Avian influenza virus H7N9 (RE-2 strain) hemagglutination inhibition test antigen.
[0035] 2. Operation method
[0036] (1) Set the reference hemagglutination titer of the avian influenza hemagglutination test to 9 log 2 ;
[0037] (2) According to the "Avian Influenza, Newcastle Disease 4HAU Antigen Preparation Table", the reference hemagglutination titer is 9log 2 The avian influenza 4HAU antigen was fully shaken and a regression test was performed to observe the number of red blood cell agglutination holes; specifically:
[0038] ① Add 0.025 mL of PBS to wells 1 to 4 of a 96-well V-shaped microplate, make 4 parallel samples, and change the dripper.
[0039] ②Pipette 0.025mL of the antigen solution mixed with 4HAU into the first well and mix well.
[0040] ③ Pipette 0.025mL from the first well and add it to the second well. After mixing, pipette 0.025mL and add it to the third well. Repeat this process to dilute the solution to the fourth well. Pipette 0.025mL from the fourth well and discard it. Then change the dripper.
[0041] ④ Add 0.025 mL of 1% chicken red blood cell suspension to each well (shake the 1% chicken red blood cell suspension thoroughly before adding).
[0042] ⑤ Mix by oscillation, let stand at room temperature (20℃-25℃) for 20 minutes and observe the results.
[0043] Results of regression test: The number of erythrocyte agglutination holes observed was 1;
[0044] (3) Avian influenza hemagglutination titer formula: According to the regression test observation results of step (2), when the number of hemagglutination holes of red blood cells is less than 2 by 1, the avian influenza hemagglutination titer = (9-1)log 2 =8log 2 ;
[0045] Test results: Example 1 Detection of avian influenza virus H7N9 (RE-2 strain) Hemagglutination inhibition test antigen hemagglutination titer is 8log 2 .
[0046] Example 3 Highly Pathogenic Avian Influenza Diagnostic Technology (GB / T 18936-2020) Detection Method
[0047] Example 3 is a control test of Example 2. The avian influenza virus antigen used in the test is the avian influenza virus H7N9 (RE-2 strain) hemagglutination inhibition test antigen produced by Harbin Guosheng Biotechnology Co., Ltd., with production batch number 20190415.
[0048] 1. Material preparation
[0049] (1) 96-well V-shaped microplate, micropipette (with dropper), and disposable pipette trough.
[0050] (2) 1% chicken red blood cell suspension.
[0051] (3) pH 7.2, 0.01 mol / L PBS.
[0052] (4) Hemagglutination inhibition test antigen and standard positive serum and negative serum of avian influenza virus H7N9 (RE-2 strain).
[0053] 2. Operation method
[0054] 2.1 Hemagglutination (HA) test (micro method)
[0055] (1) Add 0.025 mL of PBS to wells 1 to 12 of the microplate and change the dripper.
[0056] (2) Pipette 0.025 mL of avian influenza virus H7N9 (RE-2 strain) hemagglutination inhibition test antigen solution into well 1 and mix well.
[0057] (3) Pipette 0.025 mL of virus antigen solution from well 1 and add it to well 2. After mixing, pipette 0.025 mL and add it to well 3. Repeat this process until the solution is diluted to well 11. Pipette 0.025 mL from well 11 and discard it, then replace the dripper.
[0058] (4) Add 0.025 mL PBS to each well.
[0059] (5) Add 0.025 mL of 1% chicken red blood cell suspension to each well (shake the 1% chicken red blood cell suspension thoroughly before adding).
[0060] (6) Vortex to mix, let stand at room temperature (20°C-25°C) for 40 min, and observe the results (if the ambient temperature is too high, place it at 4°C). The red blood cells in the control well will sink to the bottom of the well in a distinct button shape.
[0061] (7) Result determination: Tilt the reaction plate and observe whether the red blood cells flow in a teardrop shape. The highest dilution of the antigen or virus that completely hemagglutinates (does not flow) represents one hemagglutination unit (HAU).
[0062] Preliminary results of the hemagglutination (HA) test: The hemagglutination titer of the avian influenza virus H7N9 (RE-2 strain) hemagglutination inhibition test antigen was 8log2.
[0063] According to the "4HAU Antigen Preparation Table for Avian Influenza and Newcastle Disease", 4HAU antigen with a hemagglutination titer of 8log2 was prepared for regression test.
[0064] 2.2 Regression test
[0065] ① Add 0.025 mL of PBS to wells 1 to 4 of a 96-well V-shaped microplate, make 4 parallel samples, and change the dripper.
[0066] ②Pipette 0.025mL of the antigen solution mixed with 4HAU into the first well and mix well.
[0067] ③ Pipette 0.025mL from the first well and add it to the second well. After mixing, pipette 0.025mL and add it to the third well. Repeat this process to dilute the solution to the fourth well. Pipette 0.025mL from the fourth well and discard it. Then change the dripper.
[0068] ④ Add 0.025 mL of 1% chicken red blood cell suspension to each well (shake the 1% chicken red blood cell suspension thoroughly before adding).
[0069] ⑤ Mix by oscillation, let stand at room temperature (20℃-25℃) for 20 minutes and observe the results.
[0070] Regression test results: Red blood cell agglutination was observed in 2 wells, and the test was successful.
[0071] Final result of hemagglutination (HA) test: The hemagglutination titer of the hemagglutination inhibition test antigen of avian influenza virus H7N9 (RE-2 strain) detected by the highly pathogenic avian influenza diagnostic technology (GB / T 18936-2020) test method is 8log 2 .
[0072] By comparing the test results of Example 2 and Example 3, it can be seen that the detection method of Example 2 is accurate in detecting the hemagglutination titer of the hemagglutination inhibition test antigen of avian influenza virus H7N9 (RE-2 strain).
[0073] Example 4 A hemagglutination test method for Newcastle disease
[0074] The Newcastle disease virus antigen used in the test is the Newcastle disease hemagglutination inhibition test antigen produced by Qingdao Lijian Diagnostic Technology Development Center, with production batch number 010131901.
[0075] 1. Material preparation
[0076] (1) 96-well V-shaped microplate, micropipette (with dropper), and disposable pipette trough.
[0077] (2) 1% chicken red blood cell suspension or 0.75% chicken aldehyde red blood cell suspension.
[0078] (3) pH 7.2, 0.01 mol / L PBS.
[0079] (4) Newcastle disease hemagglutination inhibition test antigen (produced by Qingdao Lijian Diagnostic Technology Development Center, production batch number 010131901)
[0080] 2. Operation method
[0081] (1) Set the reference hemagglutination titer of Newcastle disease hemagglutination test to 10log 2 ;
[0082] (2) According to the "Avian Influenza, Newcastle Disease 4HAU Antigen Preparation Table", prepare the reference hemagglutination titer of 10log 2 The Newcastle disease 4HAU antigen was shaken thoroughly, and the regression test was performed to observe the number of red blood cell agglutination holes, specifically:
[0083] ① Add 0.025 mL of PBS to wells 1 to 4 of a 96-well V-shaped microplate, make 4 parallel samples, and change the dripper.
[0084] ②Pipette 0.025mL of the antigen solution mixed with 4HAU into the first well and mix well.
[0085] ③ Pipette 0.025mL from the first well and add it to the second well. After mixing, pipette 0.025mL and add it to the third well. Repeat this process to dilute the solution to the fourth well. Pipette 0.025mL from the fourth well and discard it. Then change the dripper.
[0086] ④ Add 0.025 mL of 1% chicken red blood cell suspension to each well (shake the 1% chicken red blood cell suspension thoroughly before adding).
[0087] ⑤ Mix by oscillation, let stand at room temperature (20℃-25℃) for 20 minutes and observe the results.
[0088] Results of regression test: The number of erythrocyte agglutination holes observed was 3;
[0089] (4) Newcastle disease hemagglutination titer formula: According to the regression test observation results of step (2), when the number of red blood cell agglutination holes is 1 more than 2, Newcastle disease hemagglutination titer = (10+1)log 2 =11log 2 .
[0090] Test results: The hemagglutination titer of Newcastle disease hemagglutination inhibition test antigen (produced by Qingdao Lijian Diagnostic Technology Development Center, production batch number 010131901) detected by the new detection method was 11log2.
[0091] Example 5 Newcastle disease diagnostic technology (GB / T 16550-2020) detection method
[0092] Example 5 is a control test of Example 4. The Newcastle disease virus antigen used in the test is the Newcastle disease hemagglutination inhibition test antigen produced by Qingdao Lijian Diagnostic Technology Development Center, with production batch number 010131901.
[0093] 1. Materials and reagents
[0094] (1) Equipment: ordinary balance, analytical balance, ordinary centrifuge, micro oscillator, boiling sterilizer, refrigerator, autoclave, micropipette, dropper, 96-well V-shaped microplate, disposable pipette trough.
[0095] (2) pH 7.2 phosphate buffered saline (PBS).
[0096] (3) 1% chicken red blood cell suspension.
[0097] (4) Newcastle disease hemagglutination inhibition test antigen (produced by Qingdao Lijian Diagnostic Technology Development Center, production batch number 010131901)
[0098] 2. Operation method
[0099] 2.1 Hemagglutination test operation process
[0100] (1) Take a 96-well V-shaped microplate and add 0.025 mL of PBS to each well from well 1 to well 12 using a micropipette;
[0101] (2) Pipette 0.025 mL of Newcastle disease hemagglutination inhibition test antigen into well 1 and pipette 3-5 times to mix thoroughly;
[0102] (3) 0.025 mL of the mixed Newcastle disease hemagglutination inhibition test antigen was drawn from well 1 and added to well 2. After mixing, 0.025 mL was drawn and added to well 3. Serial dilutions were performed in sequence to well 11. Finally, 0.025 mL was drawn from well 11 and discarded. Well 12 was set as PBS control.
[0103] (4) Add 0.025 mL of PBS to each well;
[0104] (5) Add 0.025 mL of 1% chicken red blood cell suspension to each well;
[0105] (6) Mix the reaction mixture by oscillation, and let it stand at room temperature (20°C-25°C) for 40 min before observing the results. If the ambient temperature is too high, let it stand at 4°C for 60 min. The result is determined when the red blood cells in the PBS control well sink to the bottom of the well in a distinct button shape.
[0106] Result judgment:
[0107] When the PBS control well shows the correct result, tilt the reaction plate to observe whether the red blood cells are completely agglutinated. The maximum dilution of the virus that completely agglutinates is the hemagglutination titer of the antigen. The highest dilution of the virus that completely agglutinates is 1 hemagglutination unit (HAU).
[0108] Preliminary results of hemagglutination (HA) test: Newcastle disease hemagglutination inhibition test antigen hemagglutination titer is 11log 2 .
[0109] According to the "Avian Influenza, Newcastle Disease 4HAU Antigen Preparation Table", the hemagglutination titer is 11log 2 The 4HAU antigen was used for regression test.
[0110] 2.2 Regression test
[0111] ① Add 0.025 mL of PBS to wells 1 to 4 of a 96-well V-shaped microplate, make 4 parallel samples, and change the dripper.
[0112] ②Pipette 0.025mL of the antigen solution mixed with 4HAU into the first well and mix well.
[0113] ③ Pipette 0.025mL from the first well and add it to the second well. After mixing, pipette 0.025mL and add it to the third well. Repeat this process to dilute the solution to the fourth well. Pipette 0.025mL from the fourth well and discard it. Then change the dripper.
[0114] ④ Add 0.025 mL of 1% chicken red blood cell suspension to each well (shake the 1% chicken red blood cell suspension thoroughly before adding).
[0115] ⑤ Mix by oscillation, let stand at room temperature (20℃-25℃) for 20 minutes and observe the results.
[0116] Regression test results: Red blood cell agglutination was observed in 2 wells, and the test was successful.
[0117] Final result of hemagglutination (HA) test: The hemagglutination titer of Newcastle disease hemagglutination inhibition test antigen (produced by Qingdao Lijian Diagnostic Technology Development Center, production batch number 010131901) detected by Newcastle disease diagnostic technology (GB / T 16550-2020) is 11log 2 .
[0118] By comparing the test results of Example 4 and Example 5, it is found that the detection method of Example 4 is accurate in detecting the hemagglutination titer of the Newcastle disease hemagglutination inhibition test antigen produced by Qingdao Lijian Diagnostic Technology Development Center.
[0119] In summary, the hemagglutination titer of the avian influenza hemagglutination inhibition test antigen detected by the method of Example 2 is completely consistent with the hemagglutination titer of the avian influenza hemagglutination inhibition test antigen detected by the highly pathogenic avian influenza diagnostic technology (GB / T 18936-2020); the hemagglutination titer of the Newcastle disease hemagglutination inhibition test antigen detected by the method of Example 4 is completely consistent with the hemagglutination titer of the Newcastle disease hemagglutination inhibition test antigen detected by the Newcastle disease diagnostic technology (GB / T 16550-2020).
[0120] After Examples 2-5, the test personnel were replaced and Newcastle disease hemagglutination inhibition test antigens from different manufacturers were used; avian influenza hemagglutination inhibition test antigens from different manufacturers, different subtypes and strains were used to carry out comparison tests of the detection method proposed in the present invention with Newcastle disease diagnostic technology (GB / T 16550-2020) and highly pathogenic avian influenza diagnostic technology (GB / T 18936-2020), respectively. The avian influenza hemagglutination test case is shown in Table 2, and the Newcastle disease hemagglutination test case is shown in Table 3.
[0121] Table 2 Cases of avian influenza hemagglutination test
[0122]
[0123] Table 3 Newcastle disease hemagglutination test cases
[0124]
[0125]
[0126] After experimental verification of the test antigens in Table 2 and Table 3, the method proposed in the present invention is completely consistent with the hemagglutination titer of the avian influenza hemagglutination inhibition test antigen detected by the highly pathogenic avian influenza diagnostic technology (GB / T 18936-2020), and the method proposed in the present invention is completely consistent with the hemagglutination titer of the Newcastle disease hemagglutination inhibition test antigen detected by the Newcastle disease diagnostic technology (GB / T 16550-2020).
[0127] The above-mentioned specific implementations are only specific cases of the present invention. The patent protection scope of the present invention includes but is not limited to the product form and style of the above-mentioned specific implementations. Any appropriate changes or modifications made to them by ordinary technicians in the technical field that conform to the claims of the present invention shall fall within the patent protection scope of the present invention.
Claims
1. A hemagglutination test method for detecting avian influenza and Newcastle disease, characterized in that: The steps include: (1) The reference hemagglutination titer of the avian influenza hemagglutination test was set to 9 log2, and the reference hemagglutination titer of the Newcastle disease hemagglutination test was set to 10 log2; (2) According to the "Avian Influenza and Newcastle Disease 4HAU Antigen Preparation Table", prepare an avian influenza 4HAU antigen with a reference hemagglutination titer of 9log2, or prepare a Newcastle disease 4HAU antigen with a reference hemagglutination titer of 10log2, shake well, perform a regression test, and observe the number of hemagglutination holes of red blood cells; (3) Formula for avian influenza hemagglutination titer: According to the regression test observation results of step (2), when the number of hemagglutination holes of red blood cells is X more than 2, the avian influenza hemagglutination titer = (9 + X) log2; when the number of hemagglutination holes of red blood cells is X less than 2, the avian influenza hemagglutination titer = (9 - X) log2, where X is 0, 1, or 2; (4) Newcastle disease hemagglutination titer formula: According to the regression test observation results of step (2), when the number of red blood cell agglutination holes is X more than 2, Newcastle disease hemagglutination titer = (10 + X) log2, when the number of red blood cell agglutination holes is X less than 2, Newcastle disease hemagglutination titer = (10 - X) log2, where X is 0, 1, or 2; The regression test in step (2) comprises the following steps: ① Take a 96-well V-shaped microplate, use a micropipette to add 0.025 mL of PBS to wells 1 to 4, make 4 parallel samples, and change the dropper; ②Pipette 0.025mL of the mixed antigen of 4HAU and add it to the first well, mix well, pipette 0.025mL from the first well and add it to the second well, mix well and pipette 0.025mL and add it to the third well, and so on to dilute it to the fourth well, pipette 0.025mL from the fourth well and discard it, and change the dripper; ③ Add 0.025 mL of 1% chicken red blood cell suspension or 0.75% chicken aldehyde red blood cell suspension to each well; ④ Mix by oscillation, let stand at room temperature of 20℃-25℃ for 20 minutes, then observe the results. Tilt the reaction plate, observe and record the number of red blood cell agglutination wells.
2. The hemagglutination test detection method for avian influenza and Newcastle disease according to claim 1, characterized in that: In step ①, PBS is a pH 7.2 phosphate buffer, and its preparation method includes the following steps: weigh 8.0 g of sodium chloride, 0.2 g of potassium chloride, 1.44 g of disodium hydrogen phosphate, and 0.24 g of sodium dihydrogen phosphate, add distilled water to 1000 mL, dissolve the above ingredients in sequence, adjust the pH to 7.2 with hydrochloric acid, package, and sterilize at 121°C for 15 min.
3. The hemagglutination test detection method for avian influenza and Newcastle disease according to claim 1, characterized in that: The preparation method of 1% chicken red blood cell suspension in step 3. comprises the following steps: using Aldrich solution as anticoagulant, collecting anticoagulated blood of at least three SPF roosters or non-immune chickens without avian influenza and Newcastle disease antibodies, putting into a centrifuge tube, adding 3-4 times of volume of PBS to mix, centrifuging at 2000r / min for 5min-10min, removing plasma and white blood cell layer, repeating the above process, washing repeatedly 3-4 times, cleaning plasma and white blood cells, centrifuging at 2000r / min for 10min, finally absorbing the packed red blood cells and making them into a 1% suspension by volume with PBS, and storing them at 4 ℃ for standby use.
4. The hemagglutination test detection method for avian influenza and Newcastle disease according to claim 1, characterized in that: The preparation method of the 0.75% chicken aldehyded red blood cell suspension in step ③ comprises the following steps: washing fresh red blood cells with 0.1mol / L PBS solution for 5 times, centrifuging at 3000r / min for 4min each time, washing with 10 times the PBS solution of the compressed volume of red blood cells for the last time, centrifuging at 3000r / min for 10min, and removing the supernatant; adding 36% formaldehyde solution cooled to 4°C according to 8 times the amount of deposited red blood cells, mixing evenly, placing at 4°C for 24h, and shaking gently during the mixing; after taking out, adding 2 parts of 30% formaldehyde solution cooled to 4°C according to 1 part of deposited red blood cells, mixing evenly, placing at 4°C for 24h, and then washing with PBS solution for 5 times to prepare 0.75% aldehyded red blood cell solution, adding sodium azide with a final concentration of 0.1% at 4°C for standby use.
5. The hemagglutination test detection method for avian influenza and Newcastle disease according to claim 3, characterized in that: The preparation method of the Aldrich solution comprises the following steps: weighing 2.05 g of glucose, 0.8 g of sodium citrate, 0.055 g of citric acid, and 0.42 g of sodium chloride, adding distilled water to 100 mL, adjusting the pH value to 6.1 after dissipating the heat to dissolve, sterilizing under high pressure at 69 kPa for 15 min, and storing at 4° C. for later use.