Mycoplasma high-density culture medium as well as preparation method and application thereof

Abstract

The invention provides a mycoplasma high-density culture medium and a preparation method and application thereof, and relates to the technical field of biology, the mycoplasma high-density culture medium comprises the following components: 3-8 g / L of PPLO broth powder, 3-8 g / L of BHI broth powder, 3-8 g / L of yeast extract powder, 1.5-4 g / L of lactose, 0.3-0.7 g / L of aspartic acid, 0.03-0.08 g / L of serine, 3-6 g / L of a buffer agent, 1.5-4 g / L of DNA, 100-200 ml / L of pig serum, 0.01-0.03 g / L of phenol red, and water as a solvent. By adding aspartic acid, serine and DNA, precursor substances are provided for synthesis of mycoplasma purine and pyrimidine, and the titer of the viable count can be greatly improved. By using the mycoplasma high-density culture medium, the efficient growth of mycoplasma is ensured, and the sensitivity and the detection line of the detection method are improved.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a mycoplasma high-density culture medium and a preparation method and application thereof. Background Art

[0002] Immunization prevention is an important means to control Mycoplasma pneumoniae infection in animals. For vaccines with good immune effects, the preparation of high-quality culture media is the basis. At present, the culture media for Mycoplasma pneumoniae mainly include Hayflick's medium, modified KM2 medium, modified Thiaucourt's medium, EZH medium, etc. The culture media of Mycoplasma pneumoniae in the existing technology still have problems such as poor reproduction ability, low viable bacteria titer, and long culture time.

[0003] The viable titer of mycoplasma is generally expressed in color change units (CCU), which can roughly evaluate the viable titer of the culture. However, the difference between two adjacent titers is 10 times, and the result is not accurate enough. In order to improve the accuracy of the test results, increasing the number of repeated tests of samples will greatly increase the workload.

[0004] In view of this, the present invention is proposed. Summary of the invention

[0005] One of the purposes of the present invention is to provide a mycoplasma high-density culture medium to solve the technical problems in the prior art of poor reproduction ability of mycoplasma cultured in culture medium, long culture time, low viable bacteria titer and inaccurate viable bacteria titer detection.

[0006] A second object of the present invention is to provide a method for preparing the above-mentioned mycoplasma high-density culture medium.

[0007] A third object of the present invention is to provide a use of the above-mentioned mycoplasma high-density culture medium or the mycoplasma high-density culture medium prepared by the above-mentioned preparation method in culturing mycoplasma or preparing a product for culturing mycoplasma or detecting the number of viable bacteria or preparing a product for detecting the number of viable bacteria.

[0008] A fourth object of the present invention is to provide a product for culturing mycoplasma.

[0009] A fifth object of the present invention is to provide a method for culturing mycoplasma.

[0010] A sixth object of the present invention is to provide a method for determining the number of viable mycoplasmas for non-therapeutic or diagnostic purposes.

[0011] In order to achieve the above-mentioned purpose of the present invention, the following technical solutions are particularly adopted:

[0012] In a first aspect, the present invention provides a mycoplasma high-density culture medium, comprising the following components in concentrations: 3-8 g / L PPLO broth powder, 3-8 g / L BHI broth powder, 3-8 g / L yeast extract powder, 1.5-4 g / L lactose, 0.3-0.7 g / L aspartic acid, 0.03-0.08 g / L serine, 3-6 g / L buffer, 1.5-4 g / L DNA, 100-200 ml / L porcine serum, and 0.01-0.03 g / L phenol red, wherein the solvent is water.

[0013] Further, the composition comprises the following concentrations: 5 g / L PPLO broth powder, 5 g / L BHI broth powder, 5 g / L yeast extract powder, 2.5 g / L lactose, 0.5 g / L aspartic acid, 0.05 g / L serine, 4.76 g / L buffer, 2 g / L DNA, 100 ml / L porcine serum and 0.02 g / L phenol red, and the solvent is water.

[0014] Furthermore, it also includes 150-250 U / mL of penicillin sodium, preferably 200 U / mL of penicillin sodium.

[0015] Preferably, the pH of the mycoplasma high-density culture medium is 7.4 to 7.6;

[0016] Preferably, the buffer comprises HEPES.

[0017] In a second aspect, the present invention provides a method for preparing the above-mentioned mycoplasma high-density culture medium, comprising dissolving each component in water according to the formula amount.

[0018] In a third aspect, the present invention provides the use of the above-mentioned mycoplasma high-density culture medium or the mycoplasma high-density culture medium prepared by the above-mentioned preparation method in culturing mycoplasma or preparing a product for culturing mycoplasma or detecting the number of viable bacteria or preparing a product for detecting the number of viable bacteria;

[0019] Preferably, the mycoplasma comprises at least one of Mycoplasma ovis, Mycoplasma hyopneumoniae, Mycoplasma bovis, Mycoplasma gallisepticum, Mycoplasma capricolum, Mycoplasma mycoides subspecies capricolum, or Mycoplasma arginini.

[0020] In a fourth aspect, the present invention provides a product for culturing mycoplasma, comprising the mycoplasma high-density culture medium described above or the mycoplasma high-density culture medium prepared by the above-mentioned preparation method;

[0021] Preferably, the product comprises a reagent or a kit.

[0022] In a fifth aspect, the present invention provides a method for culturing mycoplasma, using the above-mentioned mycoplasma high-density culture medium, the mycoplasma high-density culture medium prepared by the above-mentioned preparation method, or the above-mentioned product to cultivate mycoplasma.

[0023] Further, it includes mycoplasma recovery culture and / or mycoplasma subculture;

[0024] Preferably, the mycoplasma resuscitation culture comprises inoculating the stored mycoplasma into a mycoplasma high-density culture medium for resuscitation culture, and the volume ratio of the mycoplasma to the mycoplasma high-density culture medium is 1:5-50;

[0025] Preferably, the mycoplasma subculture comprises inoculating the revived mycoplasma into a mycoplasma high-density culture medium at an inoculation rate of 2-20% for subculture.

[0026] Preferably, the resuscitation culture conditions include culturing at 37°C until the pH is 6.6-6.8;

[0027] Preferably, the subculture conditions include culturing at 37° C. for 12 to 28 hours.

[0028] In a sixth aspect, the present invention provides a method for determining the number of viable mycoplasmas for non-therapeutic or diagnostic purposes, comprising culturing several groups of mycoplasma high-density culture media containing mycoplasmas until color change occurs, counting the number of color changes in each dilution culture medium, and calculating the half color change unit; and / or,

[0029] When all wells have changed color, record the last well that changes color to get the color change unit;

[0030] The concentration of the mycoplasma changes in a gradient;

[0031] The mycoplasma high-density culture medium is the mycoplasma high-density culture medium mentioned above or the mycoplasma high-density culture medium prepared by the above-mentioned preparation method.

[0032] Furthermore, the concentration of the mycoplasma is varied in a gradient including a dilution of 10 -1 Up to 10 -12 Mycoplasma concentration.

[0033] The present invention provides a mycoplasma high-density culture medium, which uses PPLO broth powder, BHI broth powder and lactose as basic components to provide carbon source, nitrogen source and energy for mycoplasma; yeast extract powder provides nutrients such as nitrogen source, microorganisms and trace elements; buffer provides a stable pH range; by adding pig serum, cholesterol, saturated fatty acids and unsaturated fatty acids required for mycoplasma growth are provided; by adding phenol red as a pH indicator, the growth status of mycoplasma is judged by the color change of the culture solution; by adding aspartic acid, serine and DNA, precursor substances are provided for the synthesis of mycoplasma purine and pyrimidine, especially for the nucleic acid synthesis of mycoplasma, which can greatly improve the titer of viable bacteria. On the other hand, a mycoplasma viable bacteria determination method for non-treatment or diagnosis purposes is provided, which ensures the efficient growth of mycoplasma by using the above-mentioned mycoplasma high-density culture medium, improves the titer of viable bacteria, improves the sensitivity and detection line of the detection method, and enables low-concentration mycoplasma to be accurately detected. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] In order to more clearly illustrate the specific implementation methods of the present invention or the technical solutions in the prior art, the drawings required for use in the specific implementation methods or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some implementation methods of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0035] Figure 1 The growth curve of Mycoplasma ovis cultured in the Mycoplasma ovis high-density culture medium and culture medium with different components before optimization provided by the present invention;

[0036] Figure 2 The growth curve of Mycoplasma ovis cultured in the high-density culture medium of Mycoplasma ovis provided by the present invention and the culture medium in the prior art;

[0037] Figure 3 The growth curve of Mycoplasma ovis cultured in the Mycoplasma ovis high-density culture medium and the culture medium before the optimization of the component addition amount provided by the present invention;

[0038] Figure 4 The growth curve of Mycoplasma ovis cultured in a Mycoplasma ovis high-density culture medium in a reactor provided by the present invention;

[0039] Figure 5 The growth curve of F3 generation Mycoplasma ovis cultured in the Mycoplasma ovis high-density culture medium with different serum types provided by the present invention;

[0040] Figure 6 The growth curve of F4 generation Mycoplasma ovis cultured in the Mycoplasma ovis high-density culture medium with different serum types provided by the present invention;

[0041] Figure 7 The growth curve of F3 generation Mycoplasma ovis cultured in the Mycoplasma ovis high-density culture medium with different serum contents provided by the present invention;

[0042] Figure 8 The growth curve of F4 generation Mycoplasma ovis cultured in the Mycoplasma ovis high-density culture medium with different serum contents provided by the present invention;

[0043] Fig. 9 A comparison chart of CCU determination results of different live bacteria detection methods provided by the present invention;

[0044] Fig.10 CCU of different culture media provided by the present invention 50 Comparison chart of measurement results. DETAILED DESCRIPTION

[0045] Unless otherwise defined herein, scientific and technical terms used in conjunction with the present invention shall have the meanings commonly understood by those of ordinary skill in the art. The meaning and scope of the terms should be clear, however, in the case of any potential ambiguity, the definitions provided herein take precedence over any dictionary or external definitions. In this application, unless otherwise stated, the use of "or" means "and / or". In addition, the use of the term "including" and other forms is non-limiting.

[0046] The methods and techniques of the present invention are generally performed according to conventional methods well known in the art and as described in various general and more specific references that are cited and discussed throughout the present specification unless otherwise indicated.

[0047] The invention provides a mycoplasma high-density culture medium, comprising the following components in concentrations: 3-8 g / L PPLO broth powder, 3-8 g / L BHI broth powder, 3-8 g / L yeast extract powder, 1.5-4 g / L lactose, 0.3-0.7 g / L aspartic acid, 0.03-0.08 g / L serine, 3-6 g / L buffer, 1.5-4 g / L DNA, 100-200 ml / L porcine serum and 0.01-0.03 g / L phenol red, wherein the solvent is water.

[0048] PPLO broth powder, BHI broth powder and lactose are used as basic ingredients to provide carbon source, nitrogen source and energy for mycoplasma; yeast extract powder provides nitrogen source, microorganisms and trace elements and other nutrients; buffer provides a stable pH range; by adding porcine serum, cholesterol, saturated fatty acids and unsaturated fatty acids required for the growth of mycoplasma are provided; by adding phenol red as a pH indicator, the growth status of mycoplasma can be judged by the color change of the culture solution; by adding aspartic acid, serine and DNA, precursor substances are provided for the synthesis of mycoplasma purine and pyrimidine, especially providing a material basis for the nucleic acid synthesis of mycoplasma, which can greatly increase the titer of viable bacteria.

[0049] In order to further improve the titer of the viable count of the culture medium, in some specific embodiments, the culture medium includes components of the following concentrations: 5 g / L PPLO broth powder, 5 g / L BHI broth powder, 5 g / L yeast extract powder, 2.5 g / L lactose, 0.5 g / L aspartic acid, 0.05 g / L serine, 4.76 g / L buffer, 2 g / L DNA, 100 ml / L porcine serum and 0.02 g / L phenol red, and the solvent is water.

[0050] In some specific embodiments, 150-250 U / mL of sodium penicillin is also included. It inhibits the growth of miscellaneous bacteria, has no inhibition on the growth of mycoplasma, and can reduce the risk of bacterial contamination during the culture process. Experiments have shown that 200 U / mL of sodium penicillin is preferred for the best effect.

[0051] In some specific embodiments, the pH of the mycoplasma high-density culture medium is 7.4-7.6.

[0052] In some specific embodiments, the buffer comprises HEPES.

[0053] According to another aspect of the present invention, there is also provided a method for preparing the above-mentioned mycoplasma high-density culture medium, comprising dissolving each component in water according to the formula amount.

[0054] In some specific embodiments, the pH value is adjusted to 7.4-7.6 by sodium hydroxide solution after mixing.

[0055] According to another aspect of the present invention, there is also provided a use of the above-mentioned mycoplasma high-density culture medium or the mycoplasma high-density culture medium prepared by the above-mentioned preparation method in culturing mycoplasma or preparing a product for culturing mycoplasma or detecting the number of viable bacteria or preparing a product for detecting the number of viable bacteria.

[0056] The present invention studies the application of the above culture medium in Mycoplasma ovis, Mycoplasma capricolum, Mycoplasma hyopneumoniae and Mycoplasma bovis, wherein Mycoplasma ovis is the pathogen of interstitial pneumonia in sheep and goats, which can cause respiratory diseases. For other animal mycoplasmas that cause respiratory diseases, the types of nutrients required during the culture and proliferation process are similar, and the only difference is the special nutrients such as cholesterol, saturated fatty acids and unsaturated fatty acids, nucleic acid precursors, etc. The culture medium provided by the present invention can meet the needs of different mycoplasma species for special nutrients by adding pig serum rich in cholesterol, saturated fatty acids and unsaturated fatty acids, aspartic acid, serine and DNA. In some specific embodiments, the mycoplasma includes at least one of Mycoplasma ovis, Mycoplasma hyopneumoniae, Mycoplasma bovis, Mycoplasma gallisepticum, Mycoplasma capricolum, Mycoplasma mycoides subspecies capricolum, or Mycoplasma arginine.

[0057] According to another aspect of the present invention, a product for culturing mycoplasma is also provided;

[0058] Specifically, the product includes a reagent or a kit.

[0059] According to another aspect of the present invention, a method for culturing mycoplasma is also provided, using the above-mentioned mycoplasma high-density culture medium, the mycoplasma high-density culture medium prepared by the above-mentioned preparation method, or the above-mentioned product base to culture mycoplasma.

[0060] In some specific embodiments, the method includes mycoplasma resuscitation culture, mycoplasma subculture, or mycoplasma resuscitation culture and mycoplasma subculture, which can be selected according to needs.

[0061] Among them, resuscitation culture includes restoring the activity of mycoplasma that has been frozen or stored for a long time. Subculture includes increasing the number of mycoplasma, improving its sensitivity and detection line in subsequent tests, and enabling low concentrations of mycoplasma to be accurately detected.

[0062] In some specific embodiments, the mycoplasma resuscitation culture comprises inoculating the stored mycoplasma into a mycoplasma high-density culture medium for resuscitation culture, and the volume ratio of the mycoplasma to the mycoplasma high-density culture medium is 1:5-50.

[0063] Experiments have shown that when the pH is 6.6-6.8 at 37°C, the F1 or F2 generation is reached and the recovery is completed. In some specific embodiments, the recovery culture conditions include culturing at 37°C until the pH is 6.6-6.8.

[0064] In some specific embodiments, the mycoplasma subculture comprises inoculating the revived mycoplasma into a mycoplasma high-density culture medium at an inoculation rate of 2-20% for subculture.

[0065] Experiments have shown that the viable titer of mycoplasma can reach 10 after continued culture for 12 to 28 hours. 10.00 CCU 50 / ml or more, up to 10 11.30 CCU 50 In some specific embodiments, the subculture conditions include culturing at 37° C. for 12 to 28 hours.

[0066] According to another aspect of the present invention, a method for determining the number of viable mycoplasma bacteria for non-therapeutic or diagnostic purposes is provided, comprising culturing several groups of mycoplasma high-density culture media containing mycoplasma until color change occurs, counting the number of color changes at each dilution, and calculating the half color change unit (CCU). 50 );

[0067] and / or, recording the last well that changes color when all wells change color to obtain a color change unit;

[0068] The concentration of the mycoplasma changes in a gradient;

[0069] The mycoplasma high-density culture medium is the mycoplasma high-density culture medium mentioned above or the mycoplasma high-density culture medium prepared by the above-mentioned preparation method.

[0070] This method ensures the efficient growth of mycoplasma by using the above-mentioned mycoplasma high-density culture medium, improves the viable bacterial count titer, improves the sensitivity and detection line of the detection method, and enables low concentrations of mycoplasma to be accurately detected. It can not only determine CCU, but also can be used to determine CCU 50 .

[0071] Among them, the half color change unit (CCU) was calculated according to the Reed and Muench method. 50 ), refer to the results of CCU, and do not consider the inoculation amount when calculating the results to ensure CCU 50 The result is at the same level as that of CCU. The calculation formula of half color change unit is as follows:

[0072]

[0073] LogCCU 50 = logarithm of the dilution above 50% color change + distance ratio x logarithm of the dilution factor.

[0074] The mycoplasma viable count determination for non-therapeutic or non-diagnostic purposes includes the mycoplasma viable count determination for the purpose of monitoring epidemiology, studying the growth pattern of mycoplasma, the pathogenic matrix and its interaction with the host, or quality control.

[0075] In some specific embodiments, the method can also be performed in conjunction with a 96-well deep-well plate, which facilitates the setting of multiple replicates and improves the accuracy of the test results; and compared with a 96-well cell plate, the total volume of the system is increased to avoid dry wells.

[0076] In some specific embodiments, the concentration of the mycoplasma is varied in a gradient, including a dilution of 10 -1 Up to 10 -12 Mycoplasma concentration.

[0077] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments. Obviously, the described embodiments are part of the embodiments of the present invention, rather than all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0078] Reagents: The manufacturer information of each reagent is shown in Table 1.

[0079] Table 1

[0080] Reagents factory PPLO Bouillon Powder Becton, Dickinson and Company BHI Broth Powder Becton, Dickinson and Company Yeast extract powder (YEAST) UK OXOID Company lactose Beijing Solarbio Technology Co., Ltd. Aspartic acid Sigma-Aldrich Serine Sigma-Aldrich HEPES Beijing Solarbio Technology Co., Ltd. Calf thymus DNA Sigma-Aldrich Phenol red Sigma-Aldrich Penicillin Sodium for Injection CSPC Sinopharm (Shijiazhuang) Pharmaceutical Co., Ltd. Pig serum (inactivated) Vick Biotech Co., Ltd.

[0081] 1. Mycoplasma High Density Culture Medium

[0082] The detailed reagent amounts for preparing 1 L of the mycoplasma high-density culture medium of each embodiment and comparative example are shown in Table 2.

[0083] Table 2

[0084]

[0085]

[0086] Dissolve and mix the reagents according to the formula in Table 1, make up to 1 L with ultrapure water, and adjust the pH to 7.4-7.6 with 1 M NaOH.

[0087] Comparative Example 5

[0088] Improved EZH medium: PPLO broth powder (17.5 g / L), hydrolyzed milk protein (2.5 g / L), yeast extract powder (5 g / L), glucose (2 g / L), sodium pyruvate (2 g / L), MEM (1 g / L), penicillin (200 U / ml), 4 g / L phenol red (2.5 ml / L), inactivated horse serum (200 ml). After mixing, adjust the pH to 7.4-7.6 with 1 M NaOH, sterilize through 0.22 micron filter membrane and aliquot for use.

[0089] Comparative Example 6

[0090] Improved Hayflick's medium: PPLO broth powder (21g / L), glucose (1g / L), 250ml / L yeast extract (10ml / L), penicillin (200U / ml), 4g / L phenol red (2.5ml / L), sodium pyruvate (1g / L), inactivated horse serum (200ml). After mixing, adjust the pH to 7.4-7.6 with 1M NaOH, sterilize through 0.22μm filter membrane and aliquot for use.

[0091] Comparative Example 7

[0092] Improved KM2 medium: 1% MEM (500ml / L), 1.7% hydrolyzed milk protein 10x Hank's solution (30ml / L), inactivated horse serum (200ml / L), 25% yeast extract (20ml / L), 5% thallium acetate (4ml / L), penicillin (200U / ml), glucose (4g / L), sodium pyruvate (2g / L), 0.4% phenol red (4ml / L), pH adjusted to 7.4-7.6 with 1mol / L NaOH. Sterilize through 0.22 micron filter and prepare for use.

[0093] Comparative Example 8

[0094] Modified Thiaucourt's medium: Bacto PPLO broth (17.5 g / L), inactivated horse serum (250 ml / L), yeast extract (100 ml / L), 50% glucose (4 ml / L), 5% thallium ester (4 ml / L), penicillin (200 U / mL), 25% sodium pyruvate (8 ml / L), 0.4% phenol red (4 ml / L), pH adjusted to 7.4-7.6 with 1 mol / L NaOH. Sterilize through 0.22 micron filter and prepare for use.

[0095] Comparative Example 9

[0096] The difference from Example 1 is that the added amount of aspartic acid is 0.25 g.

[0097] Comparative Example 10

[0098] The difference from Example 1 is that the added amount of aspartic acid is 0.8 g.

[0099] Comparative Example 11

[0100] The difference from Example 1 is that the added amount of serine is 0.02 g.

[0101] Comparative Example 12

[0102] The difference from Example 1 is that the amount of serine added is 0.09 g.

[0103] Comparative Example 13

[0104] The difference from Example 1 is that the amount of DNA added is 1.3 g.

[0105] Comparative Example 14

[0106] The difference from Example 1 is that the amount of DNA added is 4.5 g.

[0107] 2. Mycoplasma culture and viable bacteria count detection

[0108] The culture media of Examples 1 to 3 and Comparative Examples 1 to 14 were respectively used to culture Mycoplasma ovis, wherein the Mycoplasma ovis was purchased from the China Veterinary Drug Administration and stored frozen.

[0109] 2.1 Recovery culture of Mycoplasma ovis

[0110] Mycoplasma ovis was inoculated into the culture medium at a ratio of 1:20 for recovery culture, and the culture was placed at 37°C until the pH was 6.6-6.8, and the culture was harvested and recorded as F1 and F2.

[0111] 2.2 Subculture

[0112] The revived Mycoplasma ovis (F2 generation) was inoculated into the culture medium at a ratio of 2% for subculture. In order to observe the growth of Mycoplasma ovis under different culture medium treatments, samples were taken at 4h, 8h, 12h, 16h, 20h, 24h, 28h, 32h, 36h, 40h, 44h, and 48h to detect the number of viable bacteria and determine the growth curve. The experiment was repeated 3 times.

[0113] 2.3 Detection of viable bacteria count

[0114] Use a 96-well deep-well plate, add 900 μl of culture medium to each well, add 100 μl of the test bacterial solution to the first column, mix well, pipette 100 μl and add to the next column, and repeat to 12 wells, discard 100 μl, that is, the dilution of the test bacterial solution increases from 10 to 12. -1 Up to 10 -12 Each sample was replicated 8 times. The deep-well plate was incubated at 37°C and the color change was recorded daily.

[0115] Count the number of tubes with different dilutions of culture medium that change color and calculate the half color change unit (CCU). 50 ). When all wells have changed color, record the last well that changes color as the value of the viable bacterial color change unit (CCU).

[0116] 2.4 The results of the growth curves of different culture media were consistent in the three repeated tests. Figure 1 to Figure 3 shown.

[0117] Depend on Figure 1 to Figure 3It can be seen that under the same culture conditions, the number of viable bacteria in Examples 1 to 3 is higher than that in each comparative example, among which the number of viable bacteria in Example 1 is the highest, which can reach 10 9.88～11.00 CCU 50 / ml.

[0118] Compared with Example 1, the number of viable bacteria in the culture medium of Comparative Example 1 can reach 10 9.00～9.81 CCU 50 / ml; the number of viable bacteria in the culture medium of comparative example 2 can reach 10 in the stable period 8.52～9.50 CCU 50 / ml; the number of viable bacteria in the culture medium of comparative example 3 can reach 10 in the stable period 8.62～9.43 CCU 50 / ml; the number of viable bacteria in the culture medium of comparative example 4 can reach 10 in the stable period 8.06～8.68 CCU 50 / ml; It can be seen that removing any one of aspartic acid, serine and DNA will reduce the number of viable bacteria in the culture medium during the stable period and reduce the culture effect. Among them, the culture medium with all three components removed has the worst effect.

[0119] Compared with Example 1, the stable period of viable bacteria count in the improved EZH medium of Comparative Example 5 is 16h to 28h, which can reach 10 8.33～8.67 CCU 50 / ml; The viable bacterial count of Hayflick's medium in Comparative Example 6 is stable during 12h to 28h, and can reach 10 8.00 ～8.33 CCU 50 / ml; the stable period of viable bacteria count in the improved KM2 medium of comparative example 7 is 20h to 28h, which can reach 10 8.00 ～8.33 CCU 50 / ml; the trend of the early growth curve of Mycoplasma ovis cultured in the modified Thiaucourt's medium of comparative example 8 is basically the same, and the number of viable bacteria is stable during 20h to 32h, which can reach 10 8.10～8.67 CCU 50 / ml; the culture medium of Example 1 has a stable period of 12h to 24h in the number of viable Mycoplasma ovis, which can reach 10 9.88～11.00 CCU 50 / ml, the viable bacterial count titer is significantly higher than that of the prior art culture medium, and the time to reach a high viable bacterial count is short. That is, the results show that the sheep mycoplasma culture medium of the present invention has the characteristics of rapid growth and high viable bacterial titer.

[0120] Compared with Example 1, the number of viable bacteria in the culture medium of Comparative Example 9 can reach 10 in the stable period. 9.12～9.95 CCU 50 / ml; the number of viable bacteria in the culture medium of comparative example 10 can reach 10 in the stable period9.53～10.30 CCU 50 / ml; the number of viable bacteria in the culture medium of comparative example 11 can reach 10 in the stable period 9.06～9.90 CCU 50 / ml; the number of viable bacteria in the culture medium of comparative example 12 can reach 10 in the stable period 9.40～10.20 CCU 50 / ml; the viable bacterial count of the culture medium in comparative example 13 can reach 10 in the stable period 9.16～9.96 CCU 50 / ml; the number of viable bacteria in the culture medium of comparative example 14 can reach 10 in the stable period 9.40～10.43 CCU 50 / ml. It can be seen that the culture efficiency of the culture medium is not dependent on the content of aspartic acid, serine and DNA. When the content is too high or too low, the culture efficiency will be affected and the optimal viable titer cannot be achieved.

[0121] 3. Cultivation of Mycoplasma ovis in a reactor

[0122] Using the high-density culture medium for Mycoplasma ovis provided in Example 1, Mycoplasma ovis was inoculated into the high-density culture medium for Mycoplasma ovis at a ratio of 1:20 for recovery culture, and cultured at 37°C until the pH was 6.6-6.8. After harvesting, it was inoculated into a 3L reactor filled with Mycoplasma ovis high-density culture medium at a ratio of 2% for subculture, and the conditions were set at 37°C, 100 rpm, and 1M NaOH was used to control the pH to 7.0. Samples were taken after 4h, 8h, 12h, 16h, 20h, 24h, and 28h of culture, and the number of viable bacteria was detected according to the method in step 2.3. The specific results are as follows: Figure 4 shown.

[0123] Depend on Figure 4 It can be seen that the viable bacterial titer of the medium of the present invention can be maintained at a high level after amplification of the culture, and can be maintained at a high level within 12h to 28h. 10.00 CCU 50 / ml or more, up to 10 11.30 CCU 50 / ml.

[0124] 4. Serum Optimization

[0125] 4.1 Serum type screening

[0126] 1) Preparation of culture medium: All reagents except serum reagent in Example 1 were selected as the basic formula, and horse serum and pig serum were selected and combined with the basic formula to prepare culture medium with a serum content of 20%.

[0127] 2) Verification of culture medium: Use the above culture medium at a ratio of 1:20 to revive Mycoplasma ovis, culture at 37°C until pH 6.6-6.8, and harvest, record as F1 and F2 respectively; and pass down at a ratio of 2%, record as F3 and F4. Take samples of F3 and F4 generations cultured for 4h, 8h, 12h, 16h, 20h, 24h, 28h, 32h, 36h, 40h, 44h, 48h, respectively, to test the number of viable bacteria to determine the culture effect of different sera. The results are as follows Figure 5 and Figure 6 As shown, it can be seen that the culture effect of pig serum is slightly better than that of horse serum.

[0128] 4.2 Screening of serum content

[0129] 1) Preparation of culture medium: pig serum was selected and combined with the basic formula to prepare culture medium with serum content of 20%, 15% and 10% respectively.

[0130] 2) Verification of culture medium: Use the above culture medium at a ratio of 1:20 to revive Mycoplasma ovis, culture at 37°C until pH 6.6-6.8, and harvest, respectively, and record as F1, F2, F3, and F4. Take samples of F3 and F4 generations cultured for 4h, 8h, 12h, 16h, 20h, 24h, 28h, 32h, 36h, 40h, 44h, and 48h to test the number of viable bacteria to determine the culture effect of different sera. The results are as follows Figure 7 and Figure 8 As shown, it can be known that the culture effects of pig serum contents of 20%, 15% and 10% are equivalent, and the serum content of the culture medium of the present invention is selected to be 10%, that is, compared with the existing culture medium, the serum cost of the culture medium of the present invention can be reduced by at least 1 time.

[0131] 5. Screening of viable bacteria count detection methods

[0132] In order to further illustrate the live bacteria detection method of step 2.3, the F2 bacterial solution, F3 bacterial solution and F4 bacterial solution harvested at different time points in 4.2 were selected as the test bacterial solutions. The sheep mycoplasma high-density culture medium provided in Example 1 was selected, and the number of live bacteria in the test bacterial solution was detected according to the live bacteria detection method provided in step 2.3, which was recorded as the experimental group. At the same time, a control group 1 was set up. The difference from the experimental group was that a 96-well cell plate was used instead of a 96-well deep-well plate, and 180 μl of culture medium + 20 μl of the test bacterial solution was added to each well; a control group 2 was set up. The difference from the experimental group was that the culture solution was diluted 10 times to 10 using a 2ml EP tube according to the conventional method. -12 ; Control group 3, different from the experimental group, selected comparative example 5 (modified EZH medium) as the culture medium for the tested bacterial solution. Calculate CCU and CCU respectively 50 value.

[0133] CCU test results Fig. 9 As shown, compared with the control group 1, the experimental group used a 900μl+100μl system and did not have dry holes, while the 96-well cell plate (180μl+20μl) had dry holes, which caused a false negative test result; compared with the control group 2, the number of live bacteria detected by the experimental group using a 2ml deep-well plate was closer to that of the control group 2, and the EP tube in the control group 2 was a commonly used measurement method in the prior art, and its results can be regarded as close to the true value. Therefore, the experimental group can truly reflect the number of live bacteria. At the same time, the gun operation increased the number of repetitions, reduced the workload, and improved work efficiency. However, the modified EZH culture medium of comparative example 5 used in the control group 3 was not included in the statistics after being processed using the CCU calculation method because the results tended to extreme values ​​and were far away from the true value. Fig. 9 . CCU of F3 bacterial suspension 50 The results of the test are as follows Fig.10 As shown, the extreme values ​​in the experimental group detection process use CCU 50 The result after the calculation method is closer to the true value, while the control group 3 cannot achieve the detection effect of the experimental group due to the use of the culture medium of comparative example 5. It can be seen that even if the deep-well plate is used for determination, not any culture medium can achieve the detection effect of the experimental group. The application of the deep-well plate and the culture medium in the detection method has a synergistic effect. Fig. 9 and Fig.10 It can be seen that the high-density culture medium for Mycoplasma ovis provided by the present invention can be applied not only to the CCU assay, but also to the CCU 50 Determination method.

[0134] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or replace some or all of the technical features therein with equivalents. However, these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A mycoplasma high-density culture medium, characterized in that: The invention comprises components with the following concentrations: 3-8 g / L PPLO broth powder, 3-8 g / L BHI broth powder, 3-8 g / L yeast extract powder, 1.5-4 g / L lactose, 0.3-0.7 g / L aspartic acid, 0.03-0.08 g / L serine, 3-6 g / L buffer, 1.5-4 g / L DNA, 100-200 ml / L pig serum and 0.01-0.03 g / L phenol red, and the solvent is water.

2. The mycoplasma high-density culture medium according to claim 1, characterized in that The invention comprises components with the following concentrations: 5 g / L PPLO broth powder, 5 g / L BHI broth powder, 5 g / L yeast extract powder, 2.5 g / L lactose, 0.5 g / L aspartic acid, 0.05 g / L serine, 4.76 g / L buffer, 2 g / L DNA, 100 ml / L porcine serum and 0.02 g / L phenol red, and the solvent is water.

3. The mycoplasma high-density culture medium according to claim 1 or 2, characterized in that: It also includes 150-250 U / mL of sodium penicillin, preferably 200 U / mL of sodium penicillin; Preferably, the pH of the mycoplasma high-density culture medium is 7.4 to 7.6; Preferably, the buffer comprises HEPES.

4. The method for preparing a mycoplasma high-density culture medium according to any one of claims 1 to 3, characterized in that: The method comprises dissolving each component into water according to the formula amount.

5. Use of the mycoplasma high-density culture medium according to any one of claims 1 to 3 or the mycoplasma high-density culture medium prepared by the preparation method according to claim 4 in culturing mycoplasma or preparing a product for culturing mycoplasma or detecting the number of viable bacteria or preparing a product for detecting the number of viable bacteria; Preferably, the mycoplasma comprises at least one of Mycoplasma ovis, Mycoplasma hyopneumoniae, Mycoplasma bovis, Mycoplasma gallisepticum, Mycoplasma capricolum, Mycoplasma mycoides subspecies capricolum, or Mycoplasma arginini.

6. A product for culturing mycoplasma, characterized in that: A mycoplasma high-density culture medium comprising the mycoplasma high-density culture medium according to any one of claims 1 to 3 or the mycoplasma high-density culture medium prepared by the preparation method according to claim 4; Preferably, the product comprises a reagent or a kit.

7. A method for culturing mycoplasma, characterized in that: Mycoplasma is cultured using the mycoplasma high-density culture medium according to any one of claims 1 to 3, the mycoplasma high-density culture medium prepared by the preparation method according to claim 4, or the product according to claim 6.

8. The method according to claim 7, characterized in that Including mycoplasma recovery culture and / or mycoplasma subculture; Preferably, the mycoplasma resuscitation culture comprises inoculating the stored mycoplasma into a mycoplasma high-density culture medium for resuscitation culture, and the volume ratio of the mycoplasma to the mycoplasma high-density culture medium is 1:5-50; Preferably, the mycoplasma subculture comprises inoculating the revived mycoplasma into a mycoplasma high-density culture medium at an inoculation rate of 2 to 20% for subculture; Preferably, the resuscitation culture conditions include culturing at 37°C until the pH is 6.6-6.8; Preferably, the subculture conditions include culturing at 37° C. for 12 to 28 hours.

9. A method for determining the number of viable mycoplasmas for non-therapeutic or diagnostic purposes, characterized in that: The method comprises culturing several groups of mycoplasma high-density culture media containing mycoplasma until color change occurs, counting the number of color changes in each dilution culture media, and calculating the half color change unit; and / or, When all wells have changed color, record the last well that changes color to get the color change unit; The concentration of the mycoplasma changes in a gradient; The mycoplasma high-density culture medium is the mycoplasma high-density culture medium according to any one of claims 1 to 3 or the mycoplasma high-density culture medium prepared by the preparation method according to claim 4.

10. The method for determining the number of viable mycoplasmas according to claim 9, characterized in that: The concentration of mycoplasma was varied in a gradient including dilution from 10 -1 Up to 10 -12 Mycoplasma concentration.

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