CN115327100A - Mycoplasma bovis plate agglutination antigen and preparation method and application thereof - Google Patents
Mycoplasma bovis plate agglutination antigen and preparation method and application thereof Download PDFInfo
- Publication number
- CN115327100A CN115327100A CN202210969928.5A CN202210969928A CN115327100A CN 115327100 A CN115327100 A CN 115327100A CN 202210969928 A CN202210969928 A CN 202210969928A CN 115327100 A CN115327100 A CN 115327100A
- Authority
- CN
- China
- Prior art keywords
- mycoplasma bovis
- strain
- antigen
- culture
- plate agglutination
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Pending
Links
Images
Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/531—Production of immunochemical test materials
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56911—Bacteria
- G01N33/56933—Mycoplasma
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6854—Immunoglobulins
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/35—Mycoplasma
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
- G01N2333/30—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Mycoplasmatales, e.g. Pleuropneumonia-like organisms [PPLO]
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2469/00—Immunoassays for the detection of microorganisms
- G01N2469/20—Detection of antibodies in sample from host which are directed against antigens from microorganisms
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Chemical & Material Sciences (AREA)
- Biomedical Technology (AREA)
- Urology & Nephrology (AREA)
- Hematology (AREA)
- Molecular Biology (AREA)
- Biotechnology (AREA)
- Biochemistry (AREA)
- Microbiology (AREA)
- Medicinal Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Food Science & Technology (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Cell Biology (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Virology (AREA)
- Tropical Medicine & Parasitology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Genetics & Genomics (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- General Engineering & Computer Science (AREA)
- Peptides Or Proteins (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
- Medicines Containing Antibodies Or Antigens For Use As Internal Diagnostic Agents (AREA)
Abstract
The invention discloses a mycoplasma bovis plate agglutination antigen and a preparation method and application thereof. The mycoplasma bovis plate agglutination antigen is prepared by using mycoplasma bovis XBY01 strain, and is prepared by subculturing the mycoplasma bovis XBY01 strain to prepare seed bacterial liquid, inoculating the seed bacterial liquid into mycoplasma bovis culture medium for amplification culture, harvesting, performing viable bacteria counting, centrifuging to obtain bacterial mud, adding 1/10 volume times of PBS containing 0.01% thimerosal for inactivation, adjusting the antigen volume according to the viable bacteria counting result, adding crystal violet solution with final concentration of 0.03% for staining and glycerol with bacterial liquid volume of 10%, mixing uniformly, and subpackaging for storage. The mycoplasma bovis plate agglutination antigen prepared by the invention has the advantages of higher sensitivity and specificity, simple operation, easy observation of results and the like, and is suitable for rapid screening and detection of mycoplasma bovis infection in pastures. The invention has simple process, low cost and good stability, and is suitable for large-scale production and application.
Description
Technical Field
The invention belongs to the technical field of veterinary diagnosis, and particularly relates to a mycoplasma bovis plate agglutination antigen and a preparation method and application thereof.
Background
The bovine mycoplasmosis is infectious disease caused by mycoplasma bovis, with symptoms of calf pneumonia, arthritis, adult cow mastitis and the like, and has high morbidity, and the infection mortality of calves can reach 20%. The bovine mycoplasmosis is ubiquitous in the world, most provinces and cities in Shandong, henan, hubei and Liaoning of China occur, and the main transmission route of the bovine mycoplasmosis is horizontal transmission, including direct contact or transmission through respiratory tract, reproductive tract and nipple. The fetus may be infected by contact with cow secretions with mycoplasma bovis during parturition, and the newborn calf may also be infected by sucking or drinking mastitis milk with mycoplasma bovis. The disease causes huge economic loss to the cattle raising industry in China every year, and seriously affects the healthy development of the cattle raising and milk industry.
Currently, registered kits which can be used for detecting bovine mycoplasmosis in China comprise: the mycoplasma bovis loop-mediated isothermal amplification detection kit and the mycoplasma bovis ELISA antibody detection kit are two varieties, but a national basic database (http:// vdts. Ivdc.org.cn:8081/cx /) of a Chinese veterinary drug information network shows that no production and wholesale records exist from 2019 to the present. Therefore, the difference between the current mycoplasma bovis detection reagent approved for production in China and the actual requirement can be judged, and the current production requirement cannot be met. Through communication with breeding enterprises and technical experts in the industry, the mycoplasma bovis detection needs a simple, quick and visual on-site detection reagent.
Disclosure of Invention
Aiming at the defects in the prior art, the invention provides a mycoplasma bovis plate agglutination antigen and a preparation method and application thereof. The plate agglutination antigen developed and developed by the mycoplasma bovis XBY01 strain and the control serum thereof are selected for the rapid detection of the mycoplasma bovis antibody in the bovine serum, and the method is favorable for solving the problems in the current mycoplasma bovis infection detection and prevention and control in China.
In order to achieve the purpose of the invention, the invention adopts the technical scheme that:
provides a mycoplasma bovis plate agglutination antigen prepared by using mycoplasma bovis XBY01 strain, and the concentration of viable bacteria before the inactivation of the strain of the mycoplasma bovis XBY01 strain is 1.0 x 10 11 CCU/mL。
The preparation method of the mycoplasma bovis plate agglutination antigen is provided, and comprises the following steps:
(1) The mycoplasma bovis XBY01 strain is subjected to passage to prepare a basic seed solution;
(2) Inoculating the basic seed solution into a mycoplasma bovis culture medium for amplification culture to obtain a production seed solution;
(3) Carrying out fermentation culture on the production seed liquid, and carrying out CCU counting and sterile inspection on the bacterial liquid;
(4) Adding PBS containing thimerosal into the fermentation culture bacterial liquid for resuspension and inactivation, adjusting the bacterial liquid concentration according to the CCU counting result, adding crystal violet solution for dyeing and glycerin for preparing bovine mycoplasma plate agglutination antigen, mixing uniformly, and subpackaging for storage.
Further, the preparation method of the mycoplasma bovis plate agglutination antigen specifically comprises the following steps:
(1) Preparing a basic seed solution: taking mycoplasma bovis XBY01 strain freeze-dried strain, starting the strain, inoculating the strain to a mycoplasma bovis culture medium according to the amount of 10%, culturing the strain at 37 ℃ for 18-36 h, taking the strain as primary basic seeds after harvesting, inoculating the primary basic seed solution for passage for 1 time according to the proportion of 10%, and freezing and storing the strain at-80 ℃ after harvesting and subpackaging as secondary basic seeds;
(2) Preparation of production seed liquid: inoculating the second-level basic seeds into a production mycoplasma bovis culture medium according to a proportion of 10%, placing the culture medium in a constant-temperature shaking table at 37 ℃, performing shake culture at 120rpm for 18-36 hours until the culture medium turns yellow, and performing enlarged culture to obtain a production seed solution;
(3) Fermentation culture: adopting a bioreactor to culture a mycoplasma bovis XBY01 strain in a large scale, firstly sterilizing the bioreactor, aseptically adding a mycoplasma bovis culture medium, inoculating a production seed solution according to the proportion of 1;
(4) Antigen preparation: adding 0.2-0.4% sodium carboxymethylcellulose into the bacterial liquid, standing at 2-8 deg.C for 7-10 days, centrifuging bacterial liquid at 8000rpm for 10min, re-suspending the precipitate with PBS containing 0.01% thimerosal, and adjusting the concentration of bacterial liquid to 1.1 × 10 11 CCU/mL, and inactivating at 37 ℃ for 3 hours; repeatedly stirring uniformly, adding 3% crystal violet solution to a final concentration of 0.03% for dyeing, continuously stirring for 20 minutes, adding 10% glycerol of bacterial liquid volume, continuously stirring for 10 minutes to obtain the mycoplasma bovis plate agglutination antigen, subpackaging, and storing at 2-8 ℃.
Provides a mycoplasma bovis plate agglutination antigen, and an application in mycoplasma bovis infection detection.
The invention has the beneficial effects that:
1. compared with the mycoplasma bovis loop-mediated isothermal amplification detection kit and the mycoplasma bovis ELISA antibody detection kit which are registered at home at present, the mycoplasma bovis flat agglutination antigen provided by the application has the characteristics of simplicity and intuition in use, and can be used by a common feeder according to a specification and the result judgment.
2. The mycoplasma bovis flat agglutination antigen provided by the application has low production cost and detection cost, and is beneficial to large-scale production, popularization and application.
3. The mycoplasma bovis plate agglutination antigen provided by the application can be stored for more than 3 years under the storage condition of 2-8 ℃, can be stored for more than 2 months at room temperature, and is convenient to transport and store.
Drawings
FIG. 1 is a schematic diagram showing the results of the reaction between the Mycoplasma bovis plate agglutination antigen and the test bovine serum of example 4, wherein the results are, from left to right, weakly positive (++), positively (++++), strongly positive (++++++), and negatively (-) respectively.
Detailed Description
The following description of the embodiments of the present invention is provided to facilitate the understanding of the present invention by those skilled in the art, but it should be understood that the present invention is not limited to the scope of the embodiments, and it will be apparent to those skilled in the art that various changes may be made without departing from the spirit and scope of the invention as defined and defined in the appended claims, and all matters produced by the invention using the inventive concept are protected.
Example 1
Preparation of mycoplasma bovis plate agglutination antigen:
(1) Preparing a basic seed solution: taking freeze-dried strain of mycoplasma bovis XBY01 separated, identified and stored by Beijing animal veterinary institute of Chinese academy of agricultural sciences, starting the strain, inoculating the strain to a mycoplasma bovis culture medium according to the amount of 10%, culturing the strain at 37 ℃ for 24-36 hours, reducing the pH of the culture medium to about 6.8, using the culture medium as primary basic seed after harvesting, inoculating the primary basic seed solution according to the proportion of 10% for passage for 1 time, and freezing and storing the primary basic seed solution at-80 ℃ after harvesting and subpackaging to serve as secondary basic seed.
(2) Preparation of production seed liquid: and (3) taking the second-level basic seeds, inoculating the second-level basic seeds into a production mycoplasma bovis culture medium according to a proportion of 10%, placing the culture medium in a constant-temperature shaking table at 37 ℃, performing shake culture for 18-36 hours at 120rpm until the culture medium turns yellow, reducing the pH value of the culture medium to about 6.8, and performing enlarged culture to obtain a production seed solution.
(3) Fermentation culture: adopting a bioreactor to culture a mycoplasma bovis XBY01 strain in a large scale, firstly sterilizing the bioreactor, aseptically adding a mycoplasma bovis culture medium, inoculating a produced seed solution according to the proportion of 1. And keeping 260mL of the serum, and freezing and storing the serum at the temperature below-15 ℃ for preparing positive serum.
(4) Antigen preparation: adding 0.3% sodium carboxymethylcellulose into the bacterial liquid, standing at 5 deg.C for 8 days, centrifuging at 8000rpm for 10min, re-suspending the precipitate with PBS containing 0.01% merthiolate, and adjusting the bacterial liquid concentration to 1.1 × 10 11 CCU/mL, and inactivating the mixture at 37 ℃ for 3 hours; repeatedly stirring uniformly, adding 3% crystal violet solution to a final concentration of 0.03% for dyeing, continuously stirring for 20 minutes, adding 10% glycerol of bacterial liquid volume, continuously stirring for 10 minutes to obtain the mycoplasma bovis plate agglutination antigen, subpackaging, and storing at 2-8 ℃.
Example 2
And (3) inspecting a semi-finished product (inspecting fermentation culture liquid):
[ sterility test ] the test was carried out according to appendix 3306 of the "pharmacopoeia of Chinese beasts" (2020 edition, three parts), namely, the mycoplasma bovis fermentation broth of example 1 was tested, 1mL was inoculated into a vial containing 50mL of TG medium, after static culture at 37 ℃ for 3 days, cultures were taken from each vial and inoculated into 1 TG tubule, 1 GA tubule and 2 TSB tubules, respectively, 0.2mL was inoculated into each vial, 1 of the TSB tubules was cultured in a biochemical incubator at 25 ℃, the remaining 3 were cultured at 37 ℃ and no bacterial growth was observed after 7 days.
[ CCU count ] 14 small test tubes containing 1.8mL of mycoplasma liquid culture medium were labeled 1, 2, 3 \823012, and 2 were blank controls. Adding 0.2mL of mycoplasma bovis fermentation culture liquid obtained in example 1 into a No. 1 small tube, uniformly mixing, sucking 0.2mL of mycoplasma bovis fermentation culture liquid from the No. 1 small tube, adding the mycoplasma bovis fermentation culture liquid into the No. 2 small tube, and repeating the steps until the mycoplasma bovis fermentation culture liquid is diluted 10 times to 10 times -12 (tube No. 12). Mixing the above 12 small tubes with2 control tubes were placed in a 37 ℃ incubator and incubated for 10 days. Observing the color change condition of each small tube, keeping the color of the control tube unchanged, and obtaining the highest dilution of the test tube with the color change, namely the CCU titer of the mycoplasma bovis fermentation culture solution. In this example, the CCU titer of the M.bovis Zymobacter fermentation broth should not be less than 1.0X 10 10 CCU/mL。
Example 3
Preparation of mycoplasma bovis control negative and positive sera:
selecting 4 healthy calves of 5-6 months old, detecting a serum antibody result by an ELISA kit to be negative, collecting 100 mL/head of bovine blood, and extracting serum; after 14 days, blood is collected again by 100 mL/head, serum is extracted, and after mixing, split charging and freeze drying are carried out to obtain the control negative serum.
Preparation of immune antigen: thawing 200mL of mycoplasma bovis XBY01 strain culture, centrifuging at 8000rpm for 30min, taking precipitate, re-suspending with 25mL of PBS (pH 7.4), adding 0.01% thimerosal, inactivating at 37 ℃ for 4h, adding 5mL of sterilized alumina gel adjuvant, fully mixing to obtain the mycoplasma bovis immunizing antigen, and storing at 2-8 ℃.
The healthy calves are subjected to intramuscular injection of immune mycoplasma bovis immune antigen at a rate of 2 mL/head 14 days after the 2 nd blood sampling, are subjected to boosting immunization once at the same dose 14 days after the first immunization, are subjected to boosting immunization once again at 28 days after the first immunization, and are subjected to thawing of the remaining 60mL of bacterial liquid for nasal cavity spray immunization at a rate of 10 mL/head. After the second boosting immunization, the blood is collected for titer measurement, 30 mu L of the blood is taken after 1.
Collecting 100 mL/head of qualified bovine blood of serum antibody, and extracting serum; the immunization is strengthened once again 14 days after blood collection; collecting blood again 14 days after immunization to determine antibody titer, collecting 100 mL/head of qualified bovine blood of serum antibody, and extracting serum; the positive serum can be continuously obtained by circularly collecting blood. Mixing the serum with qualified antibody titer, centrifuging, filtering for sterilization, packaging, and lyophilizing to obtain control positive serum.
Example 4
The use method of the bovine mycoplasma plate agglutination antigen and the control negative and positive serum comprises the following steps:
and taking out the mycoplasma bovis plate agglutination antigen, recovering to room temperature, and performing plate agglutination test on bovine serum to be detected to detect mycoplasma bovis antibody in the bovine serum. Specifically, 1 drop (about 30. Mu.L/drop) of the plate agglutination antigen is dropped on the reaction plate, then an equal amount of the serum to be detected is dropped, the mixture is fully mixed, the liquid surface with the diameter of about 1.5-2 cm is coated by a gun head, and the negative and positive control samples are also detected by using the method. The reaction plate was gently shaken and the results were determined within 2 minutes. Positive when 50% (++) or more agglutination occurs; negative in the absence of agglutination; between the two is a suspicious reaction. Positive controls should have agglutination that is "+ +" or above "+ +", and negative controls should have no agglutination.
TABLE 1 interpretation standards for serum plate agglutination assays
FIG. 1 shows the results of M.bovis plate agglutination.
Example 5
And (3) testing bovine mycoplasma plate agglutination antigen and control negative and positive serum:
1. the test of the bovine mycoplasma plate agglutination antigen comprises the following test items:
1.1 the product is a purple uniform suspension, after standing for a long time, the thallus is weighed down, the upper part is clear, and no agglomerations appear after shaking.
1.2 sterility testing
The method is carried out according to appendix 3306 of the Chinese veterinary pharmacopoeia (2020 edition, three parts), namely, randomly taking 5 bottles of mycoplasma bovis plate agglutination antigen for examination, respectively taking 1mL of the antigen to inoculate into a small bottle containing 50mL of TG culture medium, after static culture for 3 days at 37 ℃, respectively taking the culture from each bottle to inoculate 1 TG tubule, 1 GA tubule and 2 TSB tubules, each of which is inoculated with 0.2mL, placing 1 of the TSB tubules in a biochemical incubator at 25 ℃, placing the other 3 of the TSB tubules in static culture at 37 ℃, and observing after 7 days that no bacteria grow.
1.3 potency test
After the lyophilized anti-mycoplasma bovis positive serum was diluted with PBS 1.
1.4 nonspecific test antigen was taken and subjected to plate agglutination test with negative serum, PBS and physiological saline, respectively, and no agglutination should occur.
2. And (4) testing the negative control serum and the positive control serum, wherein the test items comprise the following contents.
2.1 the product is a purple uniform suspension, after standing for a long time, the thallus is weighed down, the upper part is clear, and no agglomerations appear after shaking.
2.2 sterility test
Sterility test was performed according to appendix 3306 of the pharmacopoeia of beasts of China (2020 edition, three parts) without bacterial growth.
2.3 Titers test
Diluting the positive control serum with PBS (1); negative control sera did not agglutinate.
Example 6
Clinical application of mycoplasma bovis plate agglutination antigen
The plate agglutination antigen prepared by the method detects 150 bovine serum samples provided by a certain pasture in Shandong province, and the pasture is suspected of having pneumonia symptoms of calves and mycoplasma bovis infection in cattle herds. Since no domestic mycoplasma bovis detection kit is sold, the comparative test was performed using a mycoplasma bovis ELISA detection kit produced by IDEXX corporation, usa, and the comparative test was performed with the mycoplasma bovis plate agglutination antigen prepared in example 1. The result shows that the detection result of the ELISA detection kit is that 30 parts of mycoplasma bovis antibody are positive, the detection result of the mycoplasma bovis plate agglutination antigen provided by the application is 34 parts of positive, all ELISA result positive samples are covered, and the coverage rate is 100%, so that the mycoplasma bovis plate agglutination antigen prepared by the method can be used for detecting mycoplasma bovis serum samples. The method has the characteristics of simplicity, rapidness and intuition in use, and can be used by a common feeder according to the instruction, and the result judgment is carried out.
It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential attributes thereof. The present embodiments are therefore to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein. Any reference sign in a claim should not be construed as limiting the claim concerned.
Furthermore, it should be understood that although the present specification describes embodiments, not every embodiment includes only a single embodiment, and such description is for clarity purposes only, and it is to be understood that all embodiments may be combined as appropriate by one of ordinary skill in the art to form other embodiments as will be apparent to those of skill in the art from the description herein.
Claims (4)
1. A Mycoplasma bovis plate agglutination antigen characterized by being prepared from a Mycoplasma bovis XBY01 strain, wherein the Mycoplasma bovis plate agglutination antigen contains the Mycoplasma bovis XBY01 strain at a concentration of 1.0X 10 before inactivation 11 CCU/mL。
2. A method of preparing the mycoplasma bovis plate agglutination antigen according to claim 1, comprising the steps of:
(1) The mycoplasma bovis XBY01 strain is subjected to passage to prepare a basic seed solution;
(2) Inoculating the basic seed solution into a mycoplasma bovis culture medium for amplification culture to obtain a production seed solution;
(3) Carrying out fermentation culture on the production seed liquid, and carrying out CCU counting and sterile inspection on the bacterial liquid;
(4) Adding thimerosal-containing PBS into the fermentation culture bacterial liquid for resuspension and inactivation, adjusting the bacterial liquid concentration according to the CCU counting result, adding crystal violet solution for dyeing and glycerin for preparing the bovine mycoplasma plate agglutination antigen, mixing uniformly, and subpackaging for storage.
3. The method for preparing a mycoplasma bovis plate agglutination antigen according to claim 2, specifically comprising the steps of:
(1) Preparing a basic seed solution: taking mycoplasma bovis XBY01 strain freeze-dried strain, starting the strain, inoculating the strain to a mycoplasma bovis culture medium according to the amount of 10%, culturing the strain at 37 ℃ for 18-36 h, taking the strain as primary basic seeds after harvesting, inoculating the primary basic seed solution for passage for 1 time according to the proportion of 10%, and freezing and storing the strain at-80 ℃ after harvesting and subpackaging as secondary basic seeds;
(2) Preparation of production seed liquid: inoculating the second-level basic seeds into a production mycoplasma bovis culture medium according to a proportion of 10%, placing the culture medium in a constant-temperature shaking table at 37 ℃, performing shake culture at 120rpm for 18-36 hours until the culture medium turns yellow, and performing enlarged culture to obtain a production seed solution;
(3) Fermentation culture: adopting a bioreactor to culture mycoplasma bovis XBY01 strain in a large scale, sterilizing the bioreactor, aseptically adding a mycoplasma bovis culture medium, inoculating a seed solution according to the proportion of 1;
(4) Antigen preparation: adding 0.2-0.4% sodium carboxymethylcellulose into the bacterial liquid, standing at 2-8 deg.C for 7-10 days, centrifuging bacterial liquid at 8000rpm for 10min, re-suspending the precipitate with PBS containing 0.01% thimerosal, and adjusting the concentration of bacterial liquid to 1.1 × 10 11 CCU/mL, put at 37 ℃ to killThe mixture is alive for 3 hours; repeatedly stirring uniformly, adding 3% crystal violet solution to a final concentration of 0.03% for dyeing, continuously stirring for 20 minutes, adding 10% of glycerol with the volume of bacterium solution, continuously stirring for 10 minutes to obtain the mycoplasma bovis plate agglutination antigen, subpackaging, and storing at 2-8 ℃.
4. Use of the mycoplasma bovis plate agglutination antigen according to claim 1 in the detection of mycoplasma bovis infection.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210969928.5A CN115327100A (en) | 2022-08-12 | 2022-08-12 | Mycoplasma bovis plate agglutination antigen and preparation method and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202210969928.5A CN115327100A (en) | 2022-08-12 | 2022-08-12 | Mycoplasma bovis plate agglutination antigen and preparation method and application thereof |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN115327100A true CN115327100A (en) | 2022-11-11 |
Family
ID=83924532
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202210969928.5A Pending CN115327100A (en) | 2022-08-12 | 2022-08-12 | Mycoplasma bovis plate agglutination antigen and preparation method and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN115327100A (en) |
Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| RU2202797C2 (en) * | 2001-03-02 | 2003-04-20 | Федеральное государственное учреждение Всероссийский научно-исследовательский институт защиты животных | Kit for detecting antibodies to the agent of poultry respiratory mycoplasmosis |
| CN104645324A (en) * | 2015-01-27 | 2015-05-27 | 新疆天康畜牧生物技术股份有限公司 | Application of swine enzootic hyopneumoniae vaccine strain |
| CN104789500A (en) * | 2015-04-03 | 2015-07-22 | 江苏省家禽科学研究所 | Pullorum staining agglutination antigen as well as preparation method and application thereof |
| CN105816868A (en) * | 2016-03-21 | 2016-08-03 | 青岛易邦生物工程有限公司 | Inactivated vaccine for chicken bursa synovialis mycoplasma |
| CN111707822A (en) * | 2020-08-20 | 2020-09-25 | 兆丰华生物科技(南京)有限公司 | Mycoplasma gallisepticum antibody detection reagent and preparation method and application thereof |
| CN113265358A (en) * | 2021-05-25 | 2021-08-17 | 山东省滨州畜牧兽医研究院 | Salmonella pullorum and mycoplasma synoviae double plate agglutination antigen and preparation method and application thereof |
-
2022
- 2022-08-12 CN CN202210969928.5A patent/CN115327100A/en active Pending
Patent Citations (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| RU2202797C2 (en) * | 2001-03-02 | 2003-04-20 | Федеральное государственное учреждение Всероссийский научно-исследовательский институт защиты животных | Kit for detecting antibodies to the agent of poultry respiratory mycoplasmosis |
| CN104645324A (en) * | 2015-01-27 | 2015-05-27 | 新疆天康畜牧生物技术股份有限公司 | Application of swine enzootic hyopneumoniae vaccine strain |
| CN104789500A (en) * | 2015-04-03 | 2015-07-22 | 江苏省家禽科学研究所 | Pullorum staining agglutination antigen as well as preparation method and application thereof |
| CN105816868A (en) * | 2016-03-21 | 2016-08-03 | 青岛易邦生物工程有限公司 | Inactivated vaccine for chicken bursa synovialis mycoplasma |
| CN111707822A (en) * | 2020-08-20 | 2020-09-25 | 兆丰华生物科技(南京)有限公司 | Mycoplasma gallisepticum antibody detection reagent and preparation method and application thereof |
| CN113265358A (en) * | 2021-05-25 | 2021-08-17 | 山东省滨州畜牧兽医研究院 | Salmonella pullorum and mycoplasma synoviae double plate agglutination antigen and preparation method and application thereof |
Non-Patent Citations (7)
| Title |
|---|
| G.G.阿尔顿 等: "《布氏菌病实验室技术》", 31 October 1980 * |
| QINGCHUN SHEN等: "Complete Genome Sequence of Mycoplasma bovis Strain XBY01, Isolated from Henan Province, China", 《MICROBIOLOGY RESOURCE ANNOUNCEMENTS》 * |
| ZULFEKAR ALI等: "Seroprevalence of Mycoplasma gallisepticum antibody by ELISA and serum plate agglutination test of laying chicken", 《VETERINARY WORLD》 * |
| 王天宇 等: "牛支原体病流行病学及其诊断技术研究进展", 《畜牧与兽医》 * |
| 王明俊 等: "《兽医生物制品学》", 31 July 1997, 中国农业出版社 * |
| 王玙 等: "甘肃陇东南规模养牛场常发二三类牛病的血清流行病学调查", 《中国兽医杂志》 * |
| 耿仁浩 等: "细胞和病毒活疫苗中支原体污染的PCR检测方法建立与应用", 《中国农业科学》 * |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN108904796B (en) | Rabbit hemorrhagic disease virus baculovirus vector, pasteurella multocida disease bivalent inactivated vaccine and preparation method thereof | |
| US20220003763A1 (en) | Inert Carrier Salmonella and Potential Use Thereof | |
| WO2020233147A1 (en) | Inert carrier escherichia coli and potential use thereof | |
| CN113265358B (en) | Salmonella pullorum and mycoplasma synoviae double plate agglutination antigen and preparation method and application thereof | |
| GB2600848A (en) | Inert carrier indirect agglutination test detection system and application thereof | |
| CN111548996A (en) | Poultry cancer cell serum-free suspension cell line and establishment method and application thereof | |
| CN111514288A (en) | Preparation method of canine distemper and canine parvo bivalent inactivated vaccine | |
| CN109913404A (en) | The preparation method of infections chicken cloacal bursa virus live vaccine | |
| CN104258386B (en) | A kind of mink viral enteritis inactivated vaccine-canine distemper live vaccine combination | |
| CN109097340B (en) | Avian adenovirus, quadruple vaccine and preparation method thereof | |
| CN117844686A (en) | Separation, identification and application of avibacterium paragallinarum serum A strain with cross protection effect | |
| CN113957007A (en) | Inactivated vaccine for mycoplasma synoviae | |
| CN103864931B (en) | A kind of preparation of pseudoabies standard positive serum and freeze-drying store method thereof | |
| CN115327100A (en) | Mycoplasma bovis plate agglutination antigen and preparation method and application thereof | |
| CN113376387B (en) | Pigeon Newcastle disease VI b subtype positive serum standard substance and preparation method thereof | |
| CN112618706B (en) | Triple vaccine for salmonella, riemerella anatipestifer and escherichia coli disease | |
| CN113046409B (en) | Selective chromogenic medium for swine erysipelas bacillus and swine pasteurella multocida bacterial colonies | |
| CN112402599B (en) | Canine distemper and parvovirus disease bivalent inactivated vaccine for dogs and preparation method thereof | |
| CN110448690B (en) | Chicken infectious rhinitis (A type + B type + C type), and avicularia nose bacterium (A type) bigeminy inactivated vaccine | |
| CN110007077B (en) | Porcine epidemic diarrhea virus agar-agar and detection method for antibody detection by using same | |
| CN106367399B (en) | A method of pig parvoviral disease vaccine is produced using full suspension technology | |
| CN115097146A (en) | Mycoplasma ovipneumoniae flat agglutination antigen and preparation method and application thereof | |
| CN106511990A (en) | A concentration process and preparing process for a freeze-dried brucellosis live vaccine for veterinary use | |
| CN109280648B (en) | Method for preparing mink parvoviral enteritis antigen-protein complex, antigen-protein complex and application of antigen-protein complex | |
| CN106290861B (en) | A kind of detection method of exogenous avian leukosis virus |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination |