Human mycoplasma pneumoniae culture medium, mycoplasma culture method and application

Abstract

The invention provides a human mycoplasma pneumoniae culture medium, a mycoplasma culture method and application, and relates to the technical field of microbial materials. The human mycoplasma pneumoniae culture medium comprises a basic culture medium, fetal calf serum, ampicillin sodium and an additive, and the additive comprises the following components based on the total volume of the human mycoplasma pneumoniae culture medium: 0.8-1.6 mg / L of glutamic acid, 0.4-1.2 mg / L of cysteine, 0.6-1.4 mg / L of glutathione, 50-150 mM / L of sodium pyruvate and 50-200 mL / L of a yeast extract. And the pH value of the human mycoplasma pneumoniae culture medium after sterilization is 7.6-8.0. According to the invention, the growth and reproduction speed of the mycoplasma pneumoniae is accelerated through the synergistic effect of the specific additive composition and content and the whole culture medium, a large number of mycoplasma pneumoniae wet thalli can be obtained in a short time, the survival rate of the human mycoplasma pneumoniae is obviously improved, and the method can be widely applied to large-scale production of the human mycoplasma pneumoniae natural antigen.

Description

Technical Field

[0001] This invention relates to the field of microbial culture technology, and in particular to a human-derived Mycoplasma pneumoniae culture medium, a method for culturing Mycoplasma, and its applications. Background Technology

[0002] Mycoplasma pneumoniae (Mp) is a minimal prokaryotic pathogenic microorganism intermediate between viruses and bacteria. Lacking many important biosynthetic pathways, Mp has high nutritional requirements for its culture medium. Animal serum is added during cultivation to provide cholesterol and other long-chain fatty acids that the mycoplasma cannot synthesize. Yeast extract, tissue extract, and coenzymes are also required for its growth. Mp lacks a cell wall, making it sensitive to its culture environment. It reproduces slowly, primarily through binary fission, with a reproduction cycle of 3-4 hours. Initially isolated colonies are small and strawberry-shaped; after repeated passages on solid media, it takes 1-4 weeks or even longer to develop the typical "fried egg" colonies. Laboratory methods for detecting Mp include pathogen isolation and culture and morphological detection, antigen detection, serological methods, and molecular biological methods. Mycoplasma pneumoniae has a long growth cycle, and pathogen isolation and culture are time-consuming, which is not conducive to laboratory diagnosis. While various PCR and gene probes for molecular diagnostics have high sensitivity and specificity, samples are prone to contamination and require sophisticated equipment, making them difficult to promote widely. Therefore, there is an urgent need for a simple, rapid, and high-survival-rate method for culturing human Mycoplasma pneumoniae.

[0003] Existing technology CN 113637613A discloses a culture medium for human Mycoplasma pneumoniae, its preparation method, and its application. The culture medium is prepared by mixing basal culture medium, culture medium additives, fetal bovine serum, and sodium penicillin at a volume ratio of 800 ml:(0.5~1.5) ml:(180~220) ml:(2.0~3.0) ml. The basal culture medium includes tryptone, soybean peptone, bacterial peptone, sodium pyruvate, beef extract, yeast extract, sodium chloride, L-cysteine, glucose, and phenol red. The culture medium additives contain glutathione and vitamin C. Using this culture medium to culture human Mycoplasma pneumoniae can accelerate its growth and reproduction, shortening the culture time to 40 hours. However, the operation steps of this culture medium are complex, and each 1 L of culture medium can produce only 0.2 g of wet cells, which cannot guarantee the survival rate while increasing the growth rate.

[0004] Therefore, there is a need for a culture medium and culture method that are easy to operate and can improve growth rate and survival rate. Summary of the Invention

[0005] This invention addresses the current deficiency of lacking a culture medium that can effectively improve the growth rate and survival rate of human Mycoplasma pneumoniae. It provides a culture medium for human Mycoplasma pneumoniae that significantly improves the growth rate and survival rate of human Mycoplasma pneumoniae culture through the synergistic effect of its components, while also increasing the yield of wet cells.

[0006] Another objective of this invention is to provide a human-derived Mycoplasma pneumoniae culture medium for the cultivation of mycoplasma.

[0007] Another object of the present invention is to provide a method for culturing human Mycoplasma pneumoniae.

[0008] In a first aspect, the present invention protects a human Mycoplasma pneumoniae culture medium, comprising a basal culture medium and fetal bovine serum, characterized in that it further comprises ampicillin sodium and additives, wherein, based on the total volume of the human Mycoplasma pneumoniae culture medium, the additives comprise the following components: Glutamic acid 0.8~1.6 mg / L, cysteine ​​0.4~1.2 mg / L, glutathione 0.6~1.4 mg / L, sodium pyruvate 50~150 mM / L, yeast extract 50~200 mL / L; The pH value of the human mycoplasma pneumoniae culture medium after sterilization is 7.6~8.0.

[0009] According to the human mycoplasma pneumoniae culture medium protected by the present invention, preferably, the mass concentration ratio of cysteine ​​and glutathione in the additive is 1:1~2.

[0010] According to the human mycoplasma pneumoniae culture medium protected by the present invention, preferably, the additive comprises the following components: Glutamic acid 1.0~1.2 mg / L, cysteine ​​0.8~1.2 mg / L, glutathione 0.6~1.0 mg / L, sodium pyruvate 100~150 mM / L, yeast extract 100~200 mL / L.

[0011] According to the human mycoplasma pneumoniae culture medium protected by the present invention, preferably, the amount of fetal bovine serum in each 1000 mL human mycoplasma pneumoniae culture medium is 100-200 mL.

[0012] According to the human mycoplasma pneumoniae culture medium protected by the present invention, preferably, the amount of ampicillin sodium in each 1000 mL of human mycoplasma pneumoniae culture medium is 500,000 to 1,000,000 units.

[0013] According to the human mycoplasma pneumoniae culture medium protected by the present invention, preferably, the volume ratio of the basal culture medium, fetal bovine serum, ampicillin sodium and additives is 600~900mL:(100~200)mL:(1~2)mL:(1~202)mL.

[0014] Secondly, the present invention also specifically protects the application of a human mycoplasma pneumoniae culture medium in mycoplasma culture.

[0015] Thirdly, the present invention also specifically protects the application of a human mycoplasma pneumoniae culture medium in the preparation of human mycoplasma pneumoniae molecular diagnostic antigens.

[0016] Fourthly, the present invention also specifically protects a method for culturing mycoplasma, comprising the following steps: inoculating human mycoplasma pneumoniae into the human mycoplasma pneumoniae culture medium and culturing it in a carbon dioxide environment, preferably with an inoculation ratio of 1:9 to 11.

[0017] According to the mycoplasma culture method protected by the present invention, preferably, wet bacterial cells are collected at a pH of 6.3 to 7.2, and more preferably at a pH of 6.7 to 6.9.

[0018] Beneficial effects: This invention provides a culture medium for human Mycoplasma pneumoniae. Through the synergistic effect of specific additive composition and content with the overall culture medium, the growth and reproduction rate of Mycoplasma pneumoniae is accelerated. A large number of wet Mycoplasma pneumoniae cells can be obtained in a short time, shortening the culture time of Mycoplasma pneumoniae from 7-14 days to 96 hours. It also significantly improves the survival rate of human Mycoplasma pneumoniae. Each 1L of culture medium can produce nearly 0.7g of wet cells, which can be widely used for the mass production of natural antigens of human Mycoplasma pneumoniae. Detailed Implementation

[0019] The following examples are for illustrative purposes only and are not intended to limit the scope of the invention. Where specific techniques or conditions are not specified in the examples, they should be performed according to the techniques or conditions described in the literature in this field, or according to the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased from legitimate channels.

[0020] In a specific embodiment, the present invention provides a human Mycoplasma pneumoniae culture medium, comprising a basal culture medium, fetal bovine serum, and further comprising ampicillin sodium and additives. Based on the total volume of the human Mycoplasma pneumoniae culture medium, the additives comprise the following components: Glutamic acid 0.8~1.6 mg / L, cysteine ​​0.4~1.2 mg / L, glutathione 0.6~1.4 mg / L, sodium pyruvate 50~150 mM / L, yeast extract 50~200 mL / L.

[0021] The pH value of the human mycoplasma pneumoniae culture medium after sterilization is 7.6~8.0.

[0022] This pH value is the most suitable pH value for the growth of Mycoplasma pneumoniae, which can ensure the rapid proliferation of Mycoplasma pneumoniae and improve its survival rate. The synergistic effect has a significant effect on increasing the yield of Mycoplasma pneumoniae culture.

[0023] It should be noted that: The synergistic mechanism of the additive components in the human mycoplasma pneumoniae culture medium of the present invention is as follows: The yeast extract added in this invention provides basic nutrients for the growth of *Mycoplasma pneumoniae*; pyruvate promotes energy metabolism in *Mycoplasma pneumoniae*; glutamate supplements carbon and nitrogen for its growth; and cysteine / glutathione reduces oxidative damage to *Mycoplasma pneumoniae*. With the combined addition of these components, cysteine ​​and glutathione form an antioxidant cycle to maintain a reducing environment; glutamate, through transamination, combines with pyruvate to generate alanine, balancing carbon and nitrogen metabolism; and the yeast extract provides coenzymes and growth factors, enhancing the utilization efficiency of glutamate / cysteine. These synergistic effects provide multidimensional support for *Mycoplasma pneumoniae* culture, significantly improving the growth rate and survival rate of human *Mycoplasma pneumoniae*, while also increasing the yield of wet cells.

[0024] The human-derived Mycoplasma pneumoniae culture medium provided by this invention has a sterilized pH value of 7.6-8.0, which is a suitable pH value for the growth of Mycoplasma pneumoniae. This pH value can ensure the rapid proliferation of Mycoplasma pneumoniae and improve its survival rate. The synergistic effect can significantly increase the yield of Mycoplasma pneumoniae culture.

[0025] In some specific embodiments, the concentration of glutamic acid in the culture medium additive mentioned in this invention can be, for example, a point value or any range of values ​​such as 0.8 mg / L, 1.0 mg / L, 1.2 mg / L, 1.4 mg / L, 1.6 mg / L.

[0026] In the culture medium additives mentioned in this invention, the concentration of cysteine ​​can be, for example, a point value of 0.4 mg / L, 0.6 mg / L, 0.8 mg / L, 1.0 mg / L, 1.2 mg / L, or any range thereof; In the culture medium additives mentioned in this invention, the concentration of glutathione can be, for example, a point value of 0.6 mg / L, 0.8 mg / L, 1.0 mg / L, 1.2 mg / L, 1.4 mg / L, or any range thereof. In the culture medium additives mentioned in this invention, the concentration of sodium pyruvate can be, for example, a point value of 50 mM / L, 80 mM / L, 100 mM / L, 120 mM / L, 150 mM / L, or any range of values. In the culture medium additives mentioned in this invention, the concentration of yeast extract can be, for example, a point value or any range of values ​​such as 50 mL / L, 80 mL / L, 100 mL / L, 120 mL / L, 150 mL / L, 180 mL / L, or 200 mL / L.

[0027] Mycoplasma lacks a cell wall and readily generates ROS (reactive oxygen species) during its metabolism, making it extremely sensitive to oxidative damage. In some specific embodiments, to fully synergistically combine cysteine ​​and glutathione to form a core antioxidant barrier, the mass concentration ratio of cysteine ​​to glutathione in the additives mentioned in this invention is preferably controlled at 1:1 to 2. For example, it can be specific values ​​such as 1:1, 1:1.2, 1:1.5, 1:2, or any range thereof.

[0028] In some specific embodiments, in order to better promote the rapid growth of mycoplasma culture, improve the survival rate of human mycoplasma pneumoniae culture, and increase the amount of wet bacterial cells that can be collected per 1 L of culture medium, the additives mentioned in this invention include the following components: Glutamic acid 1.0~1.2 mg / L, cysteine ​​0.8~1.2 mg / L, glutathione 0.6~1.0 mg / L, sodium pyruvate 100~150 mM / L, yeast extract 100~200 mL / L.

[0029] In some specific embodiments, the amount of fetal bovine serum in each 1000 mL of human mycoplasma pneumoniae culture medium mentioned in this invention is 100-200 mL, for example, it can be a point value of 100 mL, 120 mL, 140 mL, 160 mL, 180 mL, 200 mL, or any range of values.

[0030] In some specific embodiments, the amount of ampicillin sodium in each 1000 mL of human mycoplasma pneumoniae culture medium mentioned in this invention is 500,000 to 1,000,000 units, for example, it can be a point value of 500,000 units, 600,000 units, 700,000 units, 800,000 units, 900,000 units, 1,000,000 units, or any range of values.

[0031] In some specific embodiments, the volume ratio of basal culture medium, fetal bovine serum, ampicillin sodium and additives in the human mycoplasma pneumoniae culture medium mentioned in this invention is 600~900mL:(100~200)mL:(1~2)mL:(1~202)mL.

[0032] In some specific embodiments, the basal culture medium mentioned in this invention, calculated based on a total volume of 1000 mL, includes the following components: 500mL of pig stomach digestive fluid, 500mL of beef extract (1:2), 5.0g of yeast extract, 2.5g of sodium chloride, 5.0g of glucose, and 0.02g of phenol red.

[0033] In a specific embodiment, the present invention also provides the application of human mycoplasma pneumoniae culture medium in mycoplasma culture.

[0034] The human mycoplasma pneumoniae culture medium provided by this invention can promote the rapid growth and reproduction of human mycoplasma pneumoniae, shortening the culture time from 7-14 days to 96 hours. It has a high mycoplasma survival rate and can obtain a large number of wet mycoplasma pneumoniae cells in a short time. It solves the problems of slow growth and low survival rate of human mycoplasma pneumoniae in existing mycoplasma pneumoniae culture media. It can be widely used in the field of mycoplasma culture, especially human mycoplasma pneumoniae culture.

[0035] In a specific embodiment, this aspect also provides the application of human mycoplasma pneumoniae culture medium in the preparation of human mycoplasma pneumoniae molecular diagnostic antigens.

[0036] Using the Mycoplasma pneumoniae culture medium provided by this invention as a raw material, the natural antigen of Mycoplasma pneumoniae can be obtained through four steps: centrifugation collection, crushing treatment, crude product acquisition, and pure product acquisition. This ensures the removal of impurity proteins, and the antigen activity can be detected by chromatography. This method can be widely used in the preparation process of molecular diagnostic antigens for Mycoplasma pneumoniae.

[0037] This application method is simple to follow and can be mass-produced. It also has good specificity and sensitivity for Mycoplasma pneumoniae samples, thereby further improving the clinical detection sensitivity and specificity of Mycoplasma pneumoniae antigen in disease diagnosis and providing patients with accurate treatment plans.

[0038] In some specific application implementations, the preparation of Mycoplasma pneumoniae antigen can be carried out using the following methods: Take the inactivated human Mycoplasma pneumoniae antigen culture medium, centrifuge at 4°C to obtain wet Mycoplasma pneumoniae cells, wash with mycoplasma washing solution, centrifuge at 4°C, discard the supernatant, repeat washing to obtain the precipitate, which is Mycoplasma pneumoniae; The obtained Mycoplasma pneumoniae was placed in lysis buffer, mixed, sonicated, centrifuged at 4°C, and the supernatant was collected, which is the natural antigen of Mycoplasma pneumoniae antibody.

[0039] Performance validation of Mycoplasma pneumoniae antigen detection Utilizing chromatographic detection performance: The kit employs colloidal gold immunochromatography and an indirect method to detect Mycoplasma pneumoniae antibodies in samples, specifically: The natural antigen of Mycoplasma pneumoniae was coated on a nitrocellulose membrane to prepare a colloidal gold-labeled immunochromatographic detection kit, which consists of the following components: (1) a nitrocellulose membrane coated with the natural antigen of Mycoplasma pneumoniae; (2) a colloidal gold-labeled mouse anti-human IgM antibody; (3) a sample diluent; and (4) negative and positive control sera. Specificity = detection of true negative / (detection of true negative + detection of false positive); sensitivity = detection of true positive / (detection of true positive + detection of false negative).

[0040] The results of the natural antigen of Mycoplasma pneumoniae MP in this invention were highly consistent with those of the positive control.

[0041] In a specific embodiment, the present invention also provides a method for mycoplasma culture, comprising the following steps: Human mycoplasma pneumoniae was inoculated into the human mycoplasma pneumoniae culture medium provided by the present invention and cultured in a carbon dioxide environment.

[0042] In some specific exemplary embodiments, the inoculation ratio of human Mycoplasma pneumoniae mentioned in this invention can be, for example, 1:9 to 11, such as 1:9, 1:10, 1:11, etc.

[0043] The inoculation ratio is the ratio of seed liquid volume to culture medium volume.

[0044] The concentration of environmental carbon dioxide in the carbon dioxide environment mentioned in this invention can range from 4% to 6%, for example, it can be 5% (volume concentration).

[0045] In some specific embodiments, in order to cultivate and collect more wet bacterial cells, the mycoplasma culture method mentioned in this invention further includes the following steps: collecting wet bacterial cells at a pH of 6.3 to 7.2, preferably at a pH of 6.7 to 6.9.

[0046] In some specific exemplary embodiments, wet bacterial cells can be collected at pH values ​​of 6.3, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, etc.

[0047] Example 1 A culture medium for human Mycoplasma pneumoniae includes a basal culture medium, fetal bovine serum, ampicillin sodium, and additives. The contents of each component of the additives, based on the total volume of the human Mycoplasma pneumoniae culture medium, are as follows: Glutamic acid 1.2 mg / L, cysteine ​​0.8 mg / L, glutathione 1.0 mg / L, sodium pyruvate 100 mM / L, yeast extract 200 mL / L. The amount of fetal bovine serum in every 1000 mL of human mycoplasma pneumoniae culture medium is 200 mL; The amount of ampicillin sodium in each 1000 mL of human Mycoplasma pneumoniae culture medium is 800,000 units. The volume ratio of basal culture medium, fetal bovine serum, ampicillin sodium, and additives was 600 mL: 200 mL: 1 mL: 201 mL (1 mL of glutamic acid, cysteine, glutathione, and sodium pyruvate, and 200 mL of yeast extract).

[0048] The pH value of the human mycoplasma pneumoniae culture medium after sterilization was adjusted to 8.0 through acid-base regulation.

[0049] Example 2 A human-derived Mycoplasma pneumoniae culture medium is basically the same as that in Example 1, except that the content of each component of the additives is as follows, based on the total volume of the human Mycoplasma pneumoniae culture medium: Glutamic acid 0.8 mg / L, cysteine ​​0.8 mg / L, glutathione 1.0 mg / L, sodium pyruvate 150 mM / L, yeast extract 50 mL / L.

[0050] Example 3 A human-derived Mycoplasma pneumoniae culture medium is basically the same as that in Example 1, except that the content of each component of the additives is as follows, based on the total volume of the human Mycoplasma pneumoniae culture medium: Glutamic acid 0.8 mg / L, cysteine ​​0.4 mg / L, glutathione 0.6 mg / L, sodium pyruvate 150 mM / L, yeast extract 200 mL / L.

[0051] The pH value of the human mycoplasma pneumoniae culture medium after sterilization was adjusted to 7.4 through acid-base regulation.

[0052] Example 4 A human-derived Mycoplasma pneumoniae culture medium is basically the same as that in Example 1, except that the content of each component of the additives is as follows, based on the total volume of the human Mycoplasma pneumoniae culture medium: Glutamic acid 1.6 mg / L, cysteine ​​1.2 mg / L, glutathione 1.4 mg / L, sodium pyruvate 50 mM / L, yeast extract 100 mL / L.

[0053] Example 5 A human mycoplasma pneumoniae culture medium is basically the same as that in Example 1, except that the amount of ampicillin sodium in each 1000 mL of human mycoplasma pneumoniae culture medium is 500,000 units.

[0054] Example 6 A culture medium for human Mycoplasma pneumoniae is basically the same as that in Example 1, except that the volume ratio of basal culture medium, fetal bovine serum, ampicillin sodium and additives is 850 mL: 100 mL: 1 mL: 51 mL (1 mL of glutamic acid, cysteine, glutathione and sodium pyruvate, and 50 mL of yeast extract).

[0055] Example 7 A method for culturing human Mycoplasma pneumoniae includes the following steps: The culture method described above involves inoculating human mycoplasma pneumoniae into the human mycoplasma pneumoniae culture medium of Examples 1-6 at a ratio of 1:10 and culturing it in an environment with a concentration of 5% carbon dioxide.

[0056] pH was monitored in real time for human Mycoplasma pneumoniae cultures, and wet cells were collected at a pH of 6.8.

[0057] Example 8 A method for culturing human Mycoplasma pneumoniae includes the following steps: The culture method described above involves inoculating human mycoplasma pneumoniae into the human mycoplasma pneumoniae culture medium of Example 1 at a ratio of 1:10 and culturing it in an environment with a carbon dioxide concentration of 5%.

[0058] The pH of human Mycoplasma pneumoniae cultures was monitored in real time, and wet cells were collected at pH values ​​of 6.3, 6.5, 6.6, 6.7, 6.9, 7.0, 7.1, 7.2 and 7.4.

[0059] The specific wet cell weights are shown in Table 1.

[0060] Table 1 pH value 7.4 7.2 7.1 7.0 6.9 6.8 6.7 6.6 6.5 6.3 Collect the wet bacterial cells by weight (g). 0.251 0.325 0.356 0.544 0.675 0.685 0.655 0.526 0.425 0.415 Comparative Example 1 A method for culturing human mycoplasma pneumoniae is basically the same as that in Example 7, except that the culture medium does not contain additives, the amount of basic culture medium is increased, and the overall amount of culture medium is kept consistent.

[0061] Comparative Example 2 A method for culturing human Mycoplasma pneumoniae, using commercially available human Mycoplasma pneumoniae culture medium (Qingdao Haibo Biotechnology, Mycoplasma broth culture medium), and culturing Mycoplasma according to the method in Example 7.

[0062] Result detection Culture time determination: The time it took for the culture medium to change color under the same conditions in each group is shown in Table 2.

[0063] Table 2 culture medium Time of color change Commercially available culture media 216h Additive-free culture medium 168h Culture media of Examples 1-6 96h The collection of wet bacterial cells in different embodiments is shown in Table 3 below: Table 3. Weight of wet cells collected at pH 6.8 for each example and comparative example Serial Number Collect the wet bacterial cells by weight (g). Culture endpoint CFU / mL Example 1 0.685 2.4*10^7 Example 2 0.389 1.0*10^7 Example 3 0.457 1.3*10^7 Example 4 0.486 1.4*10^7 Example 5 0.673 2.3*10^7 Example 6 0.403 1.1*10^7 Comparative Example 1 0.323 9.5*10^7 Comparative Example 2 0.216 8.1*10^6 The CFU length of Mycoplasma pneumoniae in this invention is calculated and detected using the following method: The sample to be tested is diluted a series of times (e.g., 10-fold dilution) and inoculated onto a solid culture medium (the composition of the solid culture medium is approximately the same as in Example 1, with the addition of agarose to solidify the medium). After a period of incubation (generally 10-14 days), the characteristic "fried egg-like" colonies are observed and counted under a microscope. By calculating the colony count, the viable bacterial count in the original sample can be determined.

[0064] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them; although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some of the technical features; and these modifications or substitutions do not cause the essence of the corresponding technical solutions to deviate from the spirit and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A culture medium for human Mycoplasma pneumoniae, comprising a basal culture medium and fetal bovine serum, characterized in that, It also includes ampicillin sodium and additives, which, based on the total volume of the human Mycoplasma pneumoniae culture medium, comprise the following components: Glutamic acid 0.8~1.6 mg / L, cysteine ​​0.4~1.2 mg / L, glutathione 0.6~1.4 mg / L, sodium pyruvate 50~150 mM / L, yeast extract 50~200 mL / L; The pH value of the human mycoplasma pneumoniae culture medium after sterilization is 7.6~8.

0.

2. The human-derived Mycoplasma pneumoniae culture medium according to claim 1, characterized in that, The mass concentration ratio of cysteine ​​to glutathione in the additive is 1:1~2.

3. The human mycoplasma pneumoniae culture medium according to claim 1 or 2, characterized in that, The additive comprises the following components: Glutamic acid 1.0~1.2 mg / L, cysteine ​​0.8~1.2 mg / L, glutathione 0.6~1.0 mg / L, sodium pyruvate 100~150 mM / L, yeast extract 100~200 mL / L.

4. The human-derived Mycoplasma pneumoniae culture medium according to any one of claims 1 to 3, characterized in that, The amount of fetal bovine serum in every 1000 mL of human mycoplasma pneumoniae culture medium is 100-200 mL.

5. The human mycoplasma pneumoniae culture medium according to any one of claims 1 to 3, characterized in that, The amount of ampicillin sodium in every 1000 mL of human mycoplasma pneumoniae culture medium is 500,000 to 1,000,000 units.

6. The human mycoplasma pneumoniae culture medium according to any one of claims 1 to 5, characterized in that, The volume ratio of the basal culture medium, fetal bovine serum, ampicillin sodium, and additives is (600~900) mL : (100~200) mL : (1~2) mL : (1~202) mL.

7. The application of the human mycoplasma pneumoniae culture medium according to any one of claims 1 to 6 in mycoplasma culture.

8. The use of the human mycoplasma pneumoniae culture medium according to any one of claims 1 to 6 in the preparation of human mycoplasma pneumoniae molecular diagnostic antigen.

9. A method for culturing mycoplasma, characterized in that, Includes the following steps: Human mycoplasma pneumoniae is inoculated into the culture medium for human mycoplasma pneumoniae as described in any one of claims 1 to 6 and cultured in a carbon dioxide environment, preferably at an inoculation ratio of 1:9 to 11.

10. The mycoplasma culture method according to claim 9, characterized in that, Wet cells were collected at a pH of 6.3-7.2, preferably at a pH of 6.7-6.9.