A method for preparing highly specific Salmonella pullorum plate agglutination antigen

Abstract

The invention discloses a method for preparing a highly specific Salmonella pullorum plate agglutination antigen. The Salmonella pullorum C79-1 and C79-7 strains are revived and prepared into seed liquid, inoculated with a suitable culture medium, fermented and cultured according to a specific process, inactivated and centrifuged to collect the bacteria, and then washed at 121°C for 2 hours and multiple times to remove the protein antigen components that easily cross-react with Escherichia coli or other Enterobacteriaceae bacteria commonly found in the environment. The C79-1 and C79-7 strains are adjusted to a suitable bacterial concentration, Tween-20, crystal violet and glycerol are added, and the Salmonella pullorum plate agglutination antigen is obtained by sufficient homogenization. The antigen has higher specificity and is more accurate when used for pullorum purification detection. The bacterial yield according to the fermentation process is 4 times that of ordinary fermentation and 2 times that of solid culture. No ethanol precipitation is required, and the production cost is lower. Tween-20 is added to increase the surface tension of the antigen, solve the problem of difficulty in coating evenly during use, and provide a new and better diagnostic reagent for the purification of pullorum.

Description

Technical Field

[0001] The invention relates to the technical field of veterinary diagnosis, in particular to a method for preparing a highly specific Salmonella pullorum plate agglutination antigen. Background Art

[0002] Salmonellosis, including pullorum and gallinarum, is a foodborne zoonosis primarily affecting poultry. It primarily causes septicemia and mortality in chicks and turkeys, while adult chickens remain disease-free and represent a significant source of infection. Both salmonellosis pathogens can be vertically transmitted through eggs. The disease not only severely impacts animal production but also poses a serious public health threat to humans through foodborne transmission. Currently, the prevention and control strategy for this disease in China is population decontamination. Therefore, establishing rapid and accurate diagnostic methods is crucial for its prevention, control, and elimination.

[0003] The pullorum and gallinarum plate agglutination test is currently the most commonly used method for pullorum purification. This method is simple, rapid, can be directly used for the detection of blood samples, and is suitable for clinical screening. However, the traditional pullorum and gallinarum polyvalent staining plate agglutination test antigen is prepared in accordance with the process of the "Regulations on Veterinary Biological Products of the People's Republic of China" 2000 edition. The strains used are standard C79-1 and variant C79-7 Salmonella pullorum and Gallinarum. The freeze-dried bacteria were inoculated into ordinary broth, cultured at 37°C for 18 to 24 hours, and then inoculated into ordinary agar plates, cultured at 37°C for 18 to 24 hours to obtain the first-level seeds; the first-level seeds were used to inoculate ordinary agar slants or ordinary agar small flat bottles, and cultured at 37°C for 18 to 24 hours. hours to obtain secondary seeds; the secondary seeds were made into bacterial suspensions with physiological saline or the secondary seeds could be directly streaked and inoculated into sodium thiosulfate agar flat bottles, which were cultured at 37°C for 44-48 hours, the condensed water in the flat bottles was discarded, 20-30 ml of phosphate buffered saline (0.07 mol / L, pH 7.0-7.2) containing 2% formaldehyde solution was added to each bottle, the culture was washed, and collected in a glass bottle with glass beads, the bacterial blocks were broken up by shaking, and the bottles were inactivated at 37°C for 48 hours; the bacterial solution was then filtered through a sterilized cotton gauze funnel into a large glass bottle, and about 2 times the amount of 95% ethanol was added. The solution was precipitated for more than 3 days, the supernatant was discarded, the sediment was centrifuged again, the supernatant was discarded, and the centrifugal sediment was suspended in phosphate buffered saline (0.07 mol / L, pH 7.0-7.2) containing 1% formaldehyde solution. The main disadvantages of this process are: (1) The preparation process uses solid flat bottles for culture. It takes about 1200 flat bottles to prepare 3000 bottles (15 ml / bottle) of antigen. Each flat bottle needs to be streaked with an inoculation loop to take the bacteria. It takes 6 professionals to work simultaneously for 5 hours to complete this work. It takes 4 professionals to work simultaneously for 5 hours to wash and harvest the bacteria. It takes about 1.2×10 5ml; its antigen culture efficiency is low, the risk of contamination is high, the batch stability is not ideal, and a large amount of anhydrous ethanol is required, and the production and waste liquid treatment costs are high; (2) the obtained antigen contains a large amount of K antigen and H antigen, which is easy to cross-react with Escherichia coli or other Enterobacteriaceae infections in the environment, resulting in the prepared antigen having shortcomings such as unstable sensitivity and specificity, which causes trouble in the purification process of pullorum.

[0004] Chinese patent CN113265358 A (double plate agglutination antigens of Salmonella pullorum and Mycoplasma synoviae and their preparation and application) discloses a method for preparing Salmonella pullorum C79-1 antigen as follows: (1) preparation of seed bacterial liquid: Salmonella pullorum C79-1 strains were revived and propagated in TSB liquid medium, inoculated into TSA solid plate medium, cultured at 37°C for 20 hours, washed off with Martin liquid medium, expanded and cultured, and then inoculated into a fermenter containing Martin broth medium at a volume ratio of 5.0% for fermentation; the solid plate medium was prepared by adding 1.5% purified agar powder to the liquid medium; (2) bacterial culture: after high-density fermentation at 37°C for 18 hours, the bacterial liquid was harvested, the live bacteria were counted, formaldehyde was added to a final concentration of 0.3% by volume, and the solution was inactivated at 37°C for 24 hours; (3) antigen preparation: 2 times the volume of 95% ethanol was added to the inactivated bacterial liquid, precipitated for 3 days, part of the supernatant was discarded, and the remaining 8% was removed. The supernatant was discarded after centrifugation at 000 r / min for 10 min, and the precipitate was resuspended in 1% formaldehyde saline to adjust the bacterial solution concentration to 1.0×10 10 This process uses conventional fermentation methods to culture bacteria, which can reduce the risk of contamination to a certain extent, but the bacterial yield is low and it cannot solve the high cost of using ethanol to precipitate bacteria and the unstable antigen sensitivity and specificity.

[0005] In addition, since existing culture media contain animal-derived ingredients, some culture media have high fat content or the blood lipids of the screened chickens are high, it is sometimes difficult to apply the antigen evenly when used, which affects the test results and efficiency. Summary of the Invention

[0006] The present invention aims to provide a method for preparing a highly specific Salmonella pullorum plate agglutination antigen. The process reduces production costs and contamination risks, improves diagnostic specificity, makes diagnosis more accurate, and provides a new and better diagnostic reagent for the purification of pullorum.

[0007] In order to achieve the above object, the present invention provides the following technical solutions:

[0008] A method for preparing a highly specific Salmonella pullorum plate agglutination antigen comprises the following steps:

[0009] (1) Preparation of primary seeds

[0010] Standard Salmonella pullorum strain C79-1 and variant Salmonella pullorum strain C79-7 were inoculated on ordinary agar plates for culture; colonies on the agar plates were selected and inoculated on ordinary agar slant culture medium to obtain primary seeds; the deposit numbers of strains C79-1 and C79-7 are CVCC79201 and CVCC79201, respectively;

[0011] (2) Propagation of secondary seeds

[0012] The first-level seeds are inoculated into ordinary broth medium to obtain the second-level seeds;

[0013] (3) Antigen high-density fermentation culture

[0014] The secondary seeds were fermented and cultured in a fermentation tank, the bacterial liquid was harvested, and formaldehyde with a final concentration of 0.2% was added to inactivate the solution for 12 h, and the bacterial liquid was harvested;

[0015] (4) Bacteria treatment

[0016] The bacterial solution harvested in step (3) was centrifuged at 8000 rpm for 20 min, the precipitate was collected, and resuspended in PBS (0.01 mol / L, pH 7.0-7.2), placed in a high pressure oven at 121°C for 2 h, and centrifuged at 8000 rpm for 20 min. The precipitate was collected and washed with PBS until the supernatant was clear. The bacterial solution was resuspended in PBS (0.01 mol / L, pH 7.0-7.2) containing 2% formaldehyde to adjust the bacterial solution concentration to 5 × 10 10 CFU / ml;

[0017] (5) Preparation of antigen

[0018] The bacterial suspensions of standard Salmonella pullorum C79-1 strain and variant Salmonella pullorum C79-7 strain were diluted to different concentrations, and 50ul of each suspension was used for plate agglutination test with an equal amount of standard / variant positive serum national standard containing 0.5IU. The bacterial suspension concentration that showed no less than 50% agglutination was used as the concentration for preparing the antigen.

[0019] During the fermentation in step (3), the pH value is controlled at 7.0-8.0 using aqueous ammonia.

[0020] In step (3), after fermentation for 12 hours, 40% glucose is added at a ratio of 5% of the volume of the fermentation liquid, and glucose is then added once every 4 hours.

[0021] In the step (3), the secondary seeds are inoculated at a ratio of 2% of the total amount of the fermentation liquid and fermented in a fermenter containing a common broth medium at 37° C. During the fermentation, the dissolved oxygen is controlled to be greater than 40%, and the pH value is controlled at 7.0 to 8.0 with ammonia water. After fermentation for 12 hours, 40% glucose is added at a ratio of 5% of the volume of the fermentation liquid, and then 40% glucose is added once every 4 hours at a ratio of 5% of the volume of the fermentation liquid. When the pH value begins to rise, formaldehyde with a final concentration of 0.2% is added for inactivation for 12 hours, and the bacterial liquid is harvested.

[0022] The final concentration of crystal violet in the antigen prepared in step (5) is 3%, the final concentration of glycerol is 10%, and the final concentration of Tween-20 is 1%.

[0023] In step (5), crystal violet and PBS are first placed in a homogenizer and homogenized at 7000 rpm for 3-4 minutes, glycerol and Tween-20 are added, and homogenization is continued for 4 minutes. Finally, bacterial solution is added, homogenized at 2000 rpm for 2 minutes, and quantitatively packaged to prepare antigens.

[0024] The present invention has the following beneficial effects: (1) The bacterial body treatment adopts the high-pressure sterilization method to remove the K antigen and H antigen that are easy to cross-react with Escherichia coli or other Enterobacteriaceae bacteria in the environment. Since the purification of Salmonella enterica serotypes O9 and O12 is mainly targeted at the Salmonella group, the removal of K antigen and H antigen does not affect the purification, thereby improving the specificity and accuracy of diagnosis; (2) No ethanol is used in the bacterial body treatment, which reduces the use of organic reagents, reduces production costs and waste liquid treatment costs; (3) The bacterial body production adopts a specific high-density fermentation process, by adding glucose to supplement the carbon source required for bacterial metabolism, using ammonia water instead of NaOH to adjust the pH value, and reducing the salt content of the culture medium, the antigen yield can reach 2.7×10 10 CFU / ml, the yield is more than 4 times that of conventional fermentation and 2 times that of solid culture; (4) Tween-20 component is added to the antigen configuration, which has been verified to have no effect on the test results, but increases the surface tension of the antigen, solving the impact of high oil content in the culture medium or high chicken blood lipids on the test results. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The results of hyperlipidemic serum test with and without Tween-20 antigen added;

[0026] Figure 2 It is the specific test result;

[0027] Figure 3 It is the criterion for judging the results of the plate agglutination test for Salmonella pullorum and Galli typhoid. DETAILED DESCRIPTION

[0028] In order to facilitate understanding by those skilled in the art, the present invention will be further described below with reference to the examples, which are not intended to limit the present invention.

[0029] 1. Antigen preparation process for Salmonella pullorum agglutination test

[0030] (1) Preparation of primary seeds

[0031] Open freeze-dried CVCC79201 (strain C79-1) and CVCC79201 (strain C79-7), dissolve each in physiological saline, and streak onto standard agar plates. Incubate at 37°C for 18-24 hours, and observe bacterial morphology and purity. Select smooth, moist, slightly raised, translucent, round colonies on the standard agar plates, inoculate several standard agar slant plates, and incubate at 37°C for 24 hours. After purity testing, serological testing, specificity testing, and biochemical characterization, those that meet the requirements are designated as first-class seed.

[0032] (2) Propagation of secondary seeds

[0033] Take the ordinary agar slant culture (first-level seed) to inoculate 500ml of ordinary broth medium, culture at 37℃ for 12h, and use it as the second-level seed after passing the purity test.

[0034] (3) Antigen high-density fermentation culture

[0035] Ordinary broth medium powder was prepared in a fermenter according to the instructions and sterilized by high pressure. Secondary seeds were inoculated at a ratio of 2% of the total fermentation liquid and cultured at 37°C. During the fermentation, the dissolved oxygen was controlled to be >40%, and the pH value was controlled at 7.0-8.0 with ammonia water. After fermentation for 12 hours, 40% glucose was added at a ratio of 5% of the fermentation liquid volume, and glucose was added once every 4 hours. When the pH value began to rise during fermentation, formaldehyde with a final concentration of 0.2% was added to inactivate the culture for 12 hours, and the bacterial liquid was harvested.

[0036] Results: Fermentation is completed, the OD of bacteria 600nm It can reach 24, that is, the number of live bacteria before inactivation is about 2.7×10 10 CFU / ml.

[0037] (4) Bacteria treatment

[0038] The bacterial solution in step (3) was centrifuged at 8000 rpm for 20 min, the precipitate was collected, and resuspended with PBS (0.01 mol / L, pH 7.0-7.2), placed in a high pressure oven at 121°C for 2 h, and centrifuged at 8000 rpm for 20 min. The precipitate was collected and washed three times with PBS until the supernatant was clear. The bacterial solution was resuspended with PBS containing 2% formaldehyde (0.01 mol / L, pH 7.0-7.2), and the collected bacterial solution was filtered through 8 layers of gauze and collected in a large glass bottle. The solution was filtered through a No. 2 sulfuric acid filter and adjusted to a bacterial solution concentration of 5 × 10 10 CFU / ml.

[0039] (5) Preparation of antigen

[0040] The bacterial suspensions of standard Salmonella pullorum C79-1 strain and variant Salmonella pullorum C79-7 strain were diluted to 1.0×10 10 CFU / ml, 1.2×10 10 CFU / ml, 1.5×10 10 CFU / ml, 1.7×10 10 CFU / ml, 2.0×10 10 There are 5 different concentrations of CFU / ml. 50ul of each concentration was used for plate agglutination test with an equal amount of standard / variant positive serum national standard containing 0.5IU. The concentration of bacterial solution that showed no less than 50% agglutination was used as the concentration of prepared antigen.

[0041] Adjust the concentrations of the standard Salmonella pullorum strain C79-1 and the variant Salmonella pullorum strain C79-7 to the respective bacterial suspensions determined above in 1000 ml, with a final concentration of 3% crystal violet, 10% glycerol, and 1% Tween-20. Make up to 1000 ml with PBS (0.07 mol / L, pH 7.0-7.2) containing 1% formaldehyde. Homogenize the crystal violet and PBS in a homogenizer at 7000 rpm for 3-4 minutes. Add glycerol and Tween-20 and continue homogenizing for another 4 minutes. Finally, add the bacterial suspension and homogenize at 2000 rpm for 2 minutes. Quantitatively aliquot the suspension to prepare the antigen.

[0042] Results: The final concentration of the standard antigen was 1.5×10 10 CFU / ml, the final concentration of variant antigen was 1.2×10 10 CFU / ml.

[0043] Adding Tween-20 to the antigen preparation facilitates mixing of serum and antigen during use, resulting in more accurate results. Testing of 15 samples revealed consistent results, but the addition of Tween-20 facilitated visualization and reduced testing time.

[0044] Hyperlipidemic serum was tested with and without Tween-20 antigen, and the results were as follows: Figure 1 As shown, the left side shows the test result of serum without Tween-20, and the antigen and serum are unevenly mixed and coated. The right side shows the test result of antigen prepared by the new process, and the serum and antigen are easy to mix and coat, and the test results are easier to observe.

[0045] 2. Testing the prepared Salmonella pullorum plate agglutination test antigen

[0046] 1. Sterility test

[0047] It shall be carried out in accordance with the appendix of the current Chinese Veterinary Pharmacopoeia.

[0048] 2. Potency determination

[0049] A plate agglutination test was performed using 50ul of antigen and an equal volume of 10IU / ml of standard and variant positive serum national standards. The test was preliminarily judged to be qualified when 50% agglutination occurred within 2 minutes.

[0050] 3. Specificity test

[0051] The antigens prepared by the process of the present invention (hereinafter referred to as the antigens prepared by the process of the present invention) and the antigens prepared in accordance with the 2000 edition of the "Regulations on Veterinary Biological Products of the People's Republic of China" (hereinafter referred to as the original process antigens) were tested using the iELISA method on 120 serum samples of grandparent chickens, negative serum for pullorum, and positive serum for Escherichia coli collected from clinical sites.

[0052] Table 1 Specificity test results

[0053]

[0054] The results showed that the original process antigen detected 42 samples of Salmonella pullorum and Gallistyphus positive, the antigen of this process detected 35 samples of Salmonella pullorum and Gallistyphus positive, and the iELISA method detected 36 samples of Salmonella pullorum and Gallistyphus positive. The detection results of the antigen of this process and the iELISA based on O antigen were basically consistent; the detection results of the antigen of this process, the original process antigen and iELISA for pullorum-negative serum were all negative; the detection result of the antigen of this process for chicken Escherichia coli-positive serum was negative, while the detection result of the antigen of the original process was weakly positive. Figure 2The left side of the middle is the result of the antigen of this process detecting Escherichia coli positive serum, which is judged to be negative. The right side is the result of the antigen of the original process detecting Escherichia coli positive serum, which is judged to be positive. This shows that the antigen of the original process will cross-react with Escherichia coli when detecting Salmonella Gallinarum Pullorum, resulting in false positives. The antigen of this process is not interfered by Escherichia coli. The antigen prepared by this process has better specificity and is more accurate when used for pullorum purification detection.

[0055] Application and judgment

[0056] This antigen is used to detect Salmonella pullorum in chickens in chicken farms. The test object is serum or freshly collected blood. 30ul of antigen is thoroughly mixed with 30ul of the sample to be tested. The result is determined within 2 minutes. The test is considered positive if more than 50% agglutination is observed. Figure 3 From left to right in the middle, they are ++++, +++, ++, +, -, among which, ++++: large agglutination occurs quickly, the antigen is 100% agglutinated, and the bottom solution is clear;

[0057] +++: Obvious agglutination occurs, 75% to 99% of the antigen is agglutinated, and the base solution is slightly turbid;

[0058] ++: Significant small agglutinated particles appear, 50% to 74% of the antigen is agglutinated, and the bottom liquid is turbid;

[0059] +: A small amount of agglutinated particles appear, 25% to 49% of the antigen is agglutinated, and the bottom solution is turbid;

[0060] -: No agglomerated particles.

[0061] 4. Comparison of high-density fermentation process, conventional fermentation and solid culture

[0062] 1. Solid culture

[0063] Inoculate the freeze-dried strains of Salmonella pullorum C79-1 and C79-7 into ordinary broth, incubate at 37°C for 18-24 hours, then inoculate ordinary agar plates and incubate at 37°C for 18-24 hours to obtain primary seeds; use the primary seeds to inoculate ordinary agar flasks and incubate at 37°C for 18-24 hours to obtain secondary seeds; use physiological saline to make a bacterial suspension of the secondary seeds, or directly streak the secondary seeds into sodium thiosulfate agar flasks and incubate at 37°C for 44-48 hours.

[0064] 2. Conventional fermentation

[0065] Salmonella pullorum C79-1 and C79-7 strains were resuscitated and propagated in TSB liquid medium, then inoculated into TSA solid plate medium. After incubation at 37°C for 20 hours, the bacterial lawn was washed off with Martin liquid medium, expanded, and then inoculated into a fermenter containing Martin broth medium at a volume ratio of 5.0% for fermentation. After fermentation at 37°C for 18 hours, the bacterial liquid was harvested.

[0066] 3. High-density fermentation

[0067] Ordinary broth medium powder was prepared in a fermenter according to the instructions and sterilized by high pressure. Secondary seeds were inoculated at a ratio of 2% of the total fermentation liquid and cultured at 37°C. During the fermentation, the dissolved oxygen was controlled to be >40%, and the pH value was controlled at 7.0-8.0 with ammonia water. After fermentation for 12 hours, 40% glucose was added at a ratio of 5% of the fermentation liquid volume, and glucose was added once every 4 hours. When the pH value began to rise during fermentation, formaldehyde with a final concentration of 0.2% was added to inactivate the culture for 12 hours, and the bacterial liquid was harvested.

[0068] 4. Comparison of bacterial yields in different processes

[0069] The bacterial yield is calculated based on the yield of culturing the C79-1 strain in 10,000 mL of culture medium. The bacterial yield is shown in the table below (the cost of solid culture medium includes the preparation and post-processing of dry powder culture medium and flat bottles; the cost of liquid culture medium includes the cost of dry culture medium powder, preparation, and tank preparation). The bacterial production adopts the high-density fermentation culture process described in this application. By adding glucose to supplement the carbon source required for bacterial metabolism, using ammonia water instead of NaOH to adjust the pH value and reduce the salt content of the culture medium, the antigen yield, i.e., the antigen concentration, can reach 2.7×10 10 CFU / ml, the yield was 4 times that of conventional fermentation and 2 times that of solid culture. The specific results are shown in Table 2.

[0070] Table 2 Comparison of yield and cost of antigen production using different culture methods

[0071]

[0072] The present invention improves the production process of the original pullorum and gallinarum polyvalent staining plate agglutination test antigen, thereby reducing production costs and pollution risks, and improving the specificity and sensitivity of diagnosis, making the diagnosis more accurate. It does not require ethanol precipitation, thereby reducing costs and environmental pressures. Tween-20 is added to increase the surface tension of the antigen, solving the problem of difficulty in uniform coating during use, thereby providing a new and better diagnostic reagent for the purification of pullorum.

[0073] The above embodiments are merely preferred embodiments for the purpose of fully illustrating the present invention, and the scope of protection of the present invention is not limited thereto. Equivalent substitutions or modifications made by those skilled in the art based on the present invention are within the scope of protection of the present invention. The scope of protection of the present invention shall be subject to the claims.

Claims

1. A method for preparing a highly specific Salmonella pullorum plate agglutination antigen, characterized in that: The following steps are involved: (1) Preparation of primary seeds Standard Salmonella pullorum strain C79-1 and variant Salmonella pullorum strain C79-7 were inoculated on ordinary agar plates for culture; colonies on the agar plates were selected and inoculated on ordinary agar slant culture medium to obtain primary seeds; the deposit numbers of strains C79-1 and C79-7 are CVCC79201 and CVCC79201, respectively; (2) Propagation of secondary seeds The first-level seeds are inoculated into ordinary broth culture medium to obtain the second-level seeds; (3) Antigen high-density fermentation culture The secondary seeds were fermented and cultured in a fermentation tank, the bacterial liquid was harvested, and formaldehyde with a final concentration of 0.2% was added to inactivate the solution for 12 h, and the bacterial liquid was harvested; (4) Bacteria treatment The bacterial solution harvested in step (3) was centrifuged at 8000 rpm for 20 min, the precipitate was collected, and resuspended with 0.01 mol / L PBS, pH 7.0-7.

2. The solution was placed in a high pressure oven at 121°C for 2 h, centrifuged at 8000 rpm for 20 min, the precipitate was collected, and washed with 0.01 mol / L PBS, pH 7.0-7.2 until the supernatant was clear. The bacterial solution was resuspended with 0.01 mol / L PBS, pH 7.0-7.2 containing 2% formaldehyde, and the bacterial solution concentration was adjusted to 5 × 10 10 CFU / ml; (5) Preparation of antigen The bacterial suspensions of the standard Salmonella pullorum strain C79-1 and the variant Salmonella pullorum strain C79-7 were diluted to different concentrations, and 50 μl of each suspension was used for plate agglutination test with an equal amount of the national standard of standard / variant positive serum containing 0.5 IU. The concentration of the bacterial suspension that showed no less than 50% agglutination was used as the concentration for preparing the antigen. In the step (3), the secondary seeds are inoculated at a ratio of 2% of the total volume of the fermentation liquid and fermented in a fermenter containing a common broth medium at 37° C. During the fermentation, the dissolved oxygen is controlled to be greater than 40%, and the pH value is controlled at 7.0 to 8.0 with ammonia water. After fermentation for 12 hours, 40% glucose is added at a ratio of 5% of the volume of the fermentation liquid, and glucose is added once every 4 hours. When the pH value begins to rise during fermentation, formaldehyde with a final concentration of 0.2% is added for inactivation for 12 hours, and the bacterial liquid is harvested.

2. The method for preparing a highly specific Salmonella pullorum plate agglutination antigen according to claim 1, wherein: The final concentration of crystal violet in the antigen prepared in step (5) is 3%, the final concentration of glycerol is 10%, and the final concentration of Tween-20 is 1%.

3. The method for preparing a highly specific Salmonella pullorum plate agglutination antigen according to claim 2, wherein: In step (5), crystal violet and PBS are first placed in a homogenizer and homogenized at 7000 rpm for 3-4 minutes, glycerol and Tween-20 are added, and homogenization is continued for 4 minutes. Finally, bacterial solution is added, homogenized at 2000 rpm for 2 minutes, and quantitatively packaged to prepare antigens.

Images