A Mycoplasma laidlawii culture medium, a culture and detection method thereof, and a verification test method for the sterilization performance of a membrane filter

Abstract

The present invention discloses a L-form acholeplasma medium, a culture and detection method thereof, and a verification test method for the sterilization performance of a membrane filter. The medium comprises Composition I, an osmotic pressure regulator, an acid-base indicator, and a pH regulator; Composition I is used to provide the carbon source, nitrogen source, growth factors, vitamins, and other nutrients required for the growth of L-form acholeplasma, including a carbon source that can be fermented by L-form acholeplasma and converted into an acidic substance, and horse serum, and the volume fraction of horse serum in the medium is 5% - 10%; the pH of the medium is 7.5 - 7.8. The medium provided by the present invention is a medium dedicated to the culture and detection of L-form acholeplasma. Applying it to the culture and detection of L-form acholeplasma, and the verification of the mycoplasma removal ability of the filter membrane, etc., can improve the viable bacteria titer of L-form acholeplasma production, reduce the detection difficulty, reduce the medium cost, strain cultivation cost, and detection cost, and improve work efficiency.

Description

Technical Field

[0001] The present invention relates to the technical fields of bacterial culture media, microbial detection, and performance testing of porous materials (such as filter membranes), and particularly relates to a L-form acholeplasma medium, a culture and detection method thereof, and a verification test method for the sterilization performance of a membrane filter. Background Art

[0002] L-form acholeplasma belongs to one of the mycoplasmas and is one of the most common mycoplasma flora that contaminate cell cultures. Other common mycoplasmas in cell cultures include Mycoplasma pneumoniae and Mycoplasma orale. Mycoplasma is a prokaryotic microorganism that lives independently with a size between bacteria and viruses. It exists widely in humans and animals. It has only a cell membrane and no cell wall. It is not easily infected by β-lactam and is not stained by Gram stain. The monomer presents a polymorphic state (showing various shapes, from coccus-shaped to rod-shaped, to filaments), with different sizes, ranging from 0.2 μm to 0.3 μm or smaller. L-form acholeplasma is small in size and has been found to be able to pass through a 0.2 μm filter membrane but will be retained by a 0.1 μm filter. Therefore, foreign manufacturers have proposed using L-form acholeplasma as a model mycoplasma for testing the bacterial retention ability of 0.1 μm sterilization-grade membrane filters and using the plate counting method for detection. In addition, no one else has proposed using L-form acholeplasma as a model for verifying the mycoplasma removal ability of membrane filters. Moreover, compared with common and more concerned mycoplasmas such as Mycoplasma pneumoniae and Mycoplasma orale, there is little research on L-form acholeplasma, especially its culture and detection methods, because L-form acholeplasma is not pathogenic. Therefore, there is currently no dedicated culture medium for culturing and detecting L-form acholeplasma on the market, and the culture media and methods used to culture and detect other mycoplasmas (such as Mycoplasma pneumoniae and Mycoplasma orale) are not applicable or not very suitable for culturing L-form acholeplasma and further realizing detection. Therefore, at the present stage, when verifying the mycoplasma removal ability of membrane filters, the culture medium formula and its preparation method for culture and detection are provided by the American Type Culture Collection (ATCC). However, the inventors of the present application have found that this culture medium has defects such as a low viable cell count during laboratory actual application, difficult accurate reading, high detection cost, high environmental requirements, and a long growth cycle. Therefore, there is a greater need for a culture medium with lower culture and detection difficulty.

[0003] Upon retrieval, it was found that US Patent US 8361741 also discloses a medium for Acholeplasma laidlawii. Although the Acholeplasma laidlawii cultured can reach a relatively high titer, additional agar still needs to be added for titer detection and the plate counting method is used. Inevitably, the plate counting method has problems such as high consumption (2 - 3 petri dishes are required for 1 dilution, and about 15 mL of medium is needed for one petri dish), difficult accurate reading, resulting in high counting and detection costs (the colonies of Acholeplasma laidlawii are invisible to the naked eye and an inverted microscope is needed), high requirements for the detection environment, and a long growth cycle.

[0004] In addition, the medium formula for Acholeplasma laidlawii and its culture method published by ATCC require a high serum content of 20%, and the titer of Acholeplasma laidlawii cultured is relatively low, generally at 1×10 7 ~1×10 8 CCU / mL. According to the requirements of ASTM F838 - 20 standard, when testing the mycoplasma retention ability of membrane filters, the concentration of Acholeplasma laidlawii in the challenge liquid needs to meet at least 10 7 CCU per square centimeter of the membrane surface. A large amount is used during the test, and the stock solution contains too much horse serum, which is likely to cause blockage of the membrane filter during the verification of mycoplasma retention ability.

[0005] The currently commonly used method for detecting the titer of mycoplasma retained by membrane filters is the plate counting method. For example, the method for detecting the titer of mycoplasma used to rate the mycoplasma retention ability of membrane filters by Pall, Millipore, Sartorius and other companies jointly published in the PDA Journal of Pharmaceutical Science and Technology is the plate counting method. The method for detecting the titer of Acholeplasma laidlawii in the sterilizing grade filter disclosed in US Patent US8361741 is also the plate counting method. The detection result of the plate counting method is relatively accurate, but because its reading is difficult (the colonies are invisible to the naked eye), it has high requirements for the culture environment and a relatively long detection cycle. Therefore, it is somewhat difficult for ordinary laboratories to obtain stable detection results. At the same time, the medium cost of Acholeplasma laidlawii is relatively high, and the reagent usage of the plate counting method is large, resulting in high detection costs. Summary of the Invention

[0006] In view of the above-mentioned disadvantages of the prior art, the purpose of the present invention is to provide a L-form acholeplasma medium, a culture and detection method thereof, and a verification test method for the sterilization performance of a membrane filter. This medium is a special medium for the culture and detection of L-form acholeplasma, which is applied to the culture and detection of L-form acholeplasma, as well as the verification of the mycoplasma removal ability of the filter membrane. It can improve the viable cell titer of L-form acholeplasma production, reduce the detection difficulty, and at the same time reduce the cost of the medium itself and the cost of strain cultivation, thereby further reducing the detection cost. It can also shorten the culture time and improve work efficiency.

[0007] To achieve the above and other related purposes, the present invention provides a L-form acholeplasma medium in the form of a liquid medium, which includes Composition I, an osmotic pressure regulator, an acid-base indicator, and a pH regulator;

[0008] Composition I is used to provide the carbon source, nitrogen source, growth factors, vitamins, and other nutrients required for the growth of L-form acholeplasma. Composition I includes a carbon source that can be fermented by L-form acholeplasma and converted into an acidic substance, and serum;

[0009] The serum is selected from at least one of horse serum, chicken serum, and fetal bovine serum, and the volume fraction of the serum in the medium is 5% - 10%;

[0010] The pH of the medium is 7.5 - 7.8.

[0011] Further, in the medium, the mass concentration of the carbon source that can be fermented by L-form acholeplasma and converted into an acidic substance is 10 - 15 g / L.

[0012] Further, Composition I also includes yeast extract powder, and the mass concentration of the yeast extract powder in the medium is 2 - 15 g / L.

[0013] Further, in the medium, the mass ratio of the total mass of the components in Composition I except serum to the mass of the osmotic pressure regulator and the acid-base indicator is 37 - 60:2 - 5:0.05 - 0.1.

[0014] Further, in the medium, the total mass concentration of the components in Composition I except serum is 37 - 60 g / L, the mass concentration of the osmotic pressure regulator is 2 - 5 g / L, and the mass concentration of the acid-base indicator is 0.05 - 0.1 g / L.

[0015] Further, Composition I also includes beef heart extract powder and yeast extract powder, and in the medium, the mass ratio of the carbon source that can be fermented by L-form acholeplasma and converted into an acidic substance to the beef heart extract powder and yeast extract powder is 10 - 15:25 - 30:2 - 15.

[0016] Furthermore, the carbon source that can be fermented by Acholeplasma laidlawii and converted into acidic substances is selected from glucose.

[0017] Furthermore, the nitrogen source is selected from yeast extract.

[0018] Furthermore, the composition 1 further includes beef heart extract and / or yeast extract.

[0019] Furthermore, the osmotic pressure regulator is selected from inorganic salts. The inorganic salts include metal ions and acid radical ions. The metal ions are selected from sodium ion Na + and / or potassium ion K + , and the acid radical ions are selected from chloride ion Cl - .

[0020] Furthermore, the pH regulator is selected from sodium hydroxide and / or potassium hydroxide.

[0021] Furthermore, the acid-base indicator is selected from phenol red and / or neutral red.

[0022] Furthermore, the composition 1 includes the following components: glucose, beef heart powder, yeast powder, horse serum.

[0023] Furthermore, the culture medium includes the following components: glucose, beef heart powder, yeast powder, horse serum, sodium chloride, phenol red, and the mass ratio of glucose, beef heart powder, yeast powder, sodium chloride to phenol red is 10-15:25-30:2-15:2-5:0.05-0.1.

[0024] Furthermore, the preparation method of the culture medium includes:

[0025] Dissolve the components in the composition 1 except for the serum and the osmotic pressure regulator, and then sterilize to obtain a sterilized solution;

[0026] After the sterilized solution is cooled, add serum and acid-base indicator in a sterile environment, mix well, and then add pH regulator to adjust the pH to 7.5-7.8 to obtain the culture medium.

[0027] The present invention also provides a method for culturing and detecting Acholeplasma laidlawii, including: culturing a sample to be tested and / or Acholeplasma laidlawii with the above-mentioned culture medium to obtain a harvested liquid, and detecting Acholeplasma laidlawii in the harvested liquid.

[0028] Furthermore, the sample to be tested or Acholeplasma laidlawii is cultured with the above-mentioned culture medium in a carbon dioxide environment with a concentration other than 5%. That is, it is cultured in a non-carbon dioxide environment.

[0029] Furthermore, the culture time does not exceed 24 hours.

[0030] Further, the culture temperature is 34 - 38°C.

[0031] Further, detecting Acholeplasma laidlawii in the harvested fluid includes:

[0032] Judging whether Acholeplasma laidlawii exists in the sample to be tested by observing the color of the harvested fluid;

[0033] And / or, detecting the titer of Acholeplasma laidlawii in the harvested fluid.

[0034] The present invention also provides a method for verifying the sterilization performance of a membrane filter, including: culturing Acholeplasma laidlawii according to the method described above to obtain a harvested fluid, and using the harvested fluid to test the filter membrane to detect the retention ability of the filter membrane for Acholeplasma laidlawii.

[0035] Further, culturing Acholeplasma laidlawii according to the method described above to obtain a harvested fluid with a viable cell concentration of 10 10-11 CCU / mL.

[0036] Further, diluting the harvested fluid and using it as a challenge fluid to test the filter membrane.

[0037] Further, the filter membrane is a filter membrane with a pore size of 0.1 micrometer.

[0038] As described above, the Acholeplasma laidlawii culture medium, detection method and its application in filter membrane detection of the present invention have the following beneficial effects:

[0039] 1. In the culture medium of the present invention, Composition I serves as the supply of the main carbon source, nitrogen source, growth factors, vitamins and other nutrients for the growth of Acholeplasma laidlawii; an acid-base indicator is added to the culture medium, and the acid-base indicator changes color through the change of pH, so that the result can be directly judged by the naked eye without the need to observe with an inverted microscope; an osmotic pressure regulator is introduced to maintain the osmotic pressure of Acholeplasma laidlawii and promote the growth and metabolism of Acholeplasma laidlawii; the pH of the culture medium is controlled at 7.5 - 7.8 by a pH regulator to provide a suitable pH environment for the normal growth and metabolism of Acholeplasma laidlawii.

[0040] 2. To reduce the impact of serum on the mycoplasma retention capacity test of the filtration membrane, the present invention reduces the serum concentration in the culture medium from 20% to less than 10%. At the same time, due to the addition of excellent carbon sources such as glucose, the demand for serum can be reduced. Compared with the culture media with known formulations on the market, although the present invention uses less serum, the viable bacteria titer produced is higher. And during the mycoplasma retention capacity test of the filtration membrane, the harvested liquid is diluted and used as the challenge liquid, which can minimize the impact of serum on the test, while reducing the cost of the culture medium itself and the cost of cultivating strains, thereby reducing the detection cost.

[0041] 3. To reduce the impact of the color of yeast extract powder on the observation of CCU results, the present invention controls the yeast extract powder in the culture medium to be below 15 g / L. At the same time, due to the addition of excellent carbon sources such as glucose, the demand for nitrogen sources such as yeast extract powder can be reduced.

[0042] 4. Using the culture medium of the present invention can significantly increase the concentration of viable bacteria cultivated. When using the culture medium with a known formulation on the market, the viable bacteria concentration can only reach 10 7-8 CCU / mL, while the present invention can reach 10 10-11 CCU / mL.

[0043] 5. The culture medium of the present invention, compared with the culture media with known formulations on the market, shortens the culture time from 24 - 48 h to within 24 h, effectively shortening the culture time required for detection and improving work efficiency.

[0044] 6. While the culture medium of the present invention is used as the culture solution for Acholeplasma laidlawii, it can also be used as the culture medium for detection. And it has a lower price than traditional culture media, a simpler required culture environment, does not need to be cultured in a 5% carbon dioxide environment, and also has advantages such as a shorter detection period (results can be obtained in 4 days), stable detection conditions, and easy result reading. Description of the Drawings

[0045] Figure 1 It shows the culture solution obtained by culturing the harvested liquid obtained by culturing with the culture medium of Example 1 (parallel group 1) in Example 3 of the present invention for 18 h, diluting it with different gradients, and then continuing to culture for 24 h.

[0046] Figure 2 It shows the culture solution obtained by culturing the harvested liquid obtained by culturing with the culture medium of Example 1 (parallel group 1) in Example 3 of the present invention for 18 h, diluting it with different gradients, and then continuing to culture for 96 h.

[0047] Figure 3 It shows the culture solution obtained by culturing the harvested liquid obtained by culturing with the culture medium of Comparative Example 1 in Example 3 of the present invention for 48 h, diluting it with different gradients, and then continuing to culture for 24 h.

[0048] Figure 4 It shows the culture solution obtained by continuing to culture the harvested solution obtained by culturing with the culture medium of Comparative Example 1 for 48 h in Example 3 of the present invention after different gradient dilutions for 96 h.

[0049] Figures 1-4 Among them, 1-9:10 -1 ~10 -9 Dilution, NC: Negative control (only containing the culture medium of Example 1).

[0050] Figure 5 It shows the detection graph of the challenge solution obtained by diluting the harvested solution obtained by culturing with the culture medium of Example 1 (parallel group 1) in Example 3 of the present invention for 18 h by 10 -1 ~10 -9 Dilution. Among them, 1-9:10 -1 ~10 -9 Dilution detection results, NC: Blank control (only containing the harvested solution obtained by culturing with the culture medium of Example 1 for 18 h);

[0051] Figure 6 It shows the colony photo observed under the microscope when the titer is detected by the plate counting method in Example 3 of the present invention. Detailed implementation mode

[0052] The following specific examples illustrate the implementation modes of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific implementation modes, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0053] In the present invention, unless otherwise specified, the term "a plurality of" means two or more.

[0054] The character " / " indicates that the objects before and after are in an "or" relationship. For example, A / B means: A or B.

[0055] The term "and / or" is a description of the associated relationship of an object, indicating that three relationships can exist. For example, A and / or B means: A or B, or, the three relationships of A and B.

[0056] An embodiment of the present invention provides a L-form mycoplasma-free medium in the form of a liquid medium, including Composition I, an osmotic pressure regulator, an acid-base indicator, and a pH regulator;

[0057] The first composition is used to provide the carbon source, nitrogen source, growth factors, vitamins and other nutrients required for the growth of Acholeplasma laidlawii. The first composition includes a carbon source that can be fermented by Acholeplasma laidlawii and converted into an acidic substance, and serum;

[0058] The serum is selected from at least one of horse serum, chicken serum and fetal bovine serum, and the volume fraction of the serum in the medium is 5% - 10%;

[0059] The pH of the medium is 7.5 - 7.8.

[0060] In the medium provided by the present invention, the first composition serves as the supply of the main carbon source, nitrogen source, growth factors, vitamins and other nutrients for the growth of Acholeplasma laidlawii; an acid-base indicator is added to the medium, and the color of the acid-base indicator changes due to the change of pH, so that the result can be directly judged by the naked eye without the need to observe with an inverted microscope; the osmotic pressure regulator can maintain the osmotic pressure of Acholeplasma laidlawii and promote the growth and metabolism of Acholeplasma laidlawii; the pH of the medium is controlled at 7.5 - 7.8 by a pH regulator to provide a suitable pH environment for the normal growth and metabolism of Acholeplasma laidlawii.

[0061] The serum concentration will affect the culture of Acholeplasma laidlawii. To reduce the influence of serum on the test of the mycoplasma interception ability of the filter membrane, the present invention reduces the serum concentration in the medium from 20% to less than 10%; at the same time, because excellent carbon sources such as glucose are added, the demand for serum can be reduced. Compared with the media with known formulations on the market, although the present invention uses less serum, the viable bacteria titer produced is higher, and during the test of the mycoplasma interception ability of the filter membrane, the harvested liquid is diluted and used as the challenge liquid, which can minimize the influence of serum on the test, while reducing the cost of the medium itself and the cost of cultivating strains, thereby reducing the detection cost.

[0062] In some embodiments, in the medium, the mass concentration of the carbon source that can be fermented by Acholeplasma laidlawii and converted into an acidic substance is 10 - 15 g / L. The concentration of carbon sources such as glucose will affect the titer detection. The present invention finds that controlling the concentration within the above range can shorten the culture time required for detection and obtain a more obvious color change situation.

[0063] In some embodiments, the first composition further includes yeast extract powder, and the mass concentration of the yeast extract powder in the medium is 2 - 15 g / L. The present invention reduces the amount of yeast extract powder used to reduce the influence of the color of the yeast extract powder on the observation of the detection result; at the same time, because excellent carbon sources such as glucose are added, the demand for nitrogen sources such as yeast extract powder can be reduced.

[0064] In some embodiments, in the culture medium, the mass ratio of the total mass of the components in Composition I other than serum to the osmotic pressure regulator and the acid-base indicator is 37-60:2-5:0.05-0.1.

[0065] Further, in some embodiments, in the culture medium, the total mass concentration of the components in Composition I other than serum is 37-60 g / L, the mass concentration of the osmotic pressure regulator is 2-5 g / L, and the mass concentration of the acid-base indicator is 0.05-0.1 g / L.

[0066] In some embodiments, Composition I further includes beef heart infusion powder and yeast extract powder, and in the culture medium, the mass ratio of the carbon source that can be fermented by Acholeplasma laidlawii and converted into acidic substances to the beef heart infusion powder and yeast extract powder is 10-15:25-30:2-15.

[0067] Further, in some embodiments, in the culture medium, the mass concentrations of the carbon source that can be fermented by Acholeplasma laidlawii and converted into acidic substances, the beef heart infusion powder, and the yeast extract powder are 10-15 g / L, 25-30 g / L, and 2-15 g / L, respectively.

[0068] In some embodiments, the carbon source that can be fermented by Acholeplasma laidlawii and converted into acidic substances is glucose.

[0069] In some embodiments, the nitrogen source is yeast extract.

[0070] In some embodiments, Composition I further includes beef heart extract and / or yeast extract. Among them, the beef heart extract is preferably fresh beef heart extract, such as beef heart infusion powder, which is rich in microbial growth promoting factors and can provide vitamin B complex required for the growth of Acholeplasma laidlawii. The yeast extract can be, for example, yeast extract powder and / or yeast extract paste. The yeast extract is a biological culture medium product rich in nutrients such as protein, amino acids, peptides, polypeptides, nucleic acids, vitamins, and trace elements, which is made from high-protein baker's yeast or brewer's yeast through processes such as autolysis, enzymatic hydrolysis, concentration, and drying. Yeast extract powder is the powder product of yeast extract, and yeast extract paste is the paste product of yeast extract.

[0071] In some embodiments, the osmotic pressure regulator includes but is not limited to inorganic salts. The inorganic salts include metal ions and acid radical ions. The metal ions include but are not limited to sodium ion Na + 、potassium ion K + etc., and the acid radical ions include but are not limited to chloride ion Cl - etc. Exemplarily, the osmotic pressure regulator includes but is not limited to sodium chloride, potassium chloride, etc.

[0072] In some embodiments, the pH regulator includes but is not limited to sodium hydroxide, potassium hydroxide, etc.

[0073] In some embodiments, the acid-base indicator includes but is not limited to phenol red, neutral red, etc. The pH color change range of phenol red is 6.8 - 8.0. It is red under neutral conditions, yellow under acidic conditions, and purple under alkaline conditions; the pH color change range of neutral red is 6.8 - 8.0. It is red under acidic conditions and yellow under alkaline conditions. Therefore, it is recommended to use them as acid-base indicators and add them to the culture medium of the present invention, and the pH value range of the culture medium can be judged by visual observation. Currently, in laboratories, culture media added with phenol red are mostly used for culturing cells, so phenol red is more recommended. When preparing the culture medium, it is recommended to first prepare the acid-base indicator into a solution with a mass concentration of 0.5% - 1%, and then add it.

[0074] In some embodiments, Composition I includes the following components: glucose, beef heart infusion powder, yeast extract powder, horse serum. The growth conditions of horse serum, chicken serum, and fetal bovine serum are similar. However, in the market, the price of chicken serum is about 2 times that of horse serum, and the price of fetal bovine serum is at least 3 times that of horse serum. Therefore, choosing horse serum is beneficial for cost control.

[0075] Further, in some embodiments, in the culture medium, the mass concentrations of the glucose, beef heart infusion powder, and yeast extract powder are 10 - 15 g / L, 25 - 30 g / L, and 2 - 15 g / L respectively.

[0076] In some embodiments, the culture medium includes the following components: glucose, beef heart infusion powder, yeast extract powder, horse serum, sodium chloride, phenol red, and the mass ratio of glucose, beef heart infusion powder, yeast extract powder, sodium chloride to phenol red is 10 - 15:25 - 30:2 - 15:2 - 5:0.05 - 0.1.

[0077] Further, in some embodiments, the culture medium includes the following components in the following amounts: 10 - 15 g / L glucose, 25 - 30 g / L beef heart infusion powder, 2 - 15 g / L yeast extract powder, 50 - 100 mL / L horse serum, 2 - 5 g / L sodium chloride, 0.05 - 0.1 g / L phenol red.

[0078] In some embodiments, the culture medium uses water as a solvent, and the water includes but is not limited to ultrapure water, deionized water, etc.

[0079] In some embodiments, the present invention also provides a preparation method of the above-mentioned L-form mycoplasma-free medium, including:

[0080] Dissolve the components of Composition I except for the serum and the osmotic pressure regulator, and then sterilize to obtain a sterilized solution;

[0081] After the sterilizing solution is cooled, serum and an acid-base indicator are added in a sterile environment. After mixing, a pH regulator is added to adjust the pH to 7.5 - 7.8, obtaining the medium.

[0082] In some embodiments, the components of Composition 1 except serum and the osmotic pressure regulator are dissolved by heating and stirring.

[0083] In some embodiments, the sterilization temperature is 110 - 121 °C, such as 110, 112, 114, 116, 118, 120 °C, etc.

[0084] In some embodiments, the sterilization time is 10 - 15 min.

[0085] Another embodiment of the present invention provides a method for culturing and detecting Acholeplasma laidlawii, including: culturing a sample to be tested and / or Acholeplasma laidlawii using the above-mentioned medium to obtain a harvest solution, and detecting Acholeplasma laidlawii in the harvest solution.

[0086] In some embodiments, the sample to be tested or Acholeplasma laidlawii is cultured using the medium in an environment without 5% carbon dioxide, that is, cultured in a common biochemical incubator.

[0087] In some embodiments, the culturing time does not exceed 24 hours, such as 15, 16, 17, 18, 19, 20, 21, 22, 23, 24 hours, etc.

[0088] In some embodiments, the culturing temperature is 34 - 38 °C, such as 34, 35, 36, 37, 38 °C, etc.

[0089] In some embodiments, detecting Acholeplasma laidlawii in the harvest solution includes:

[0090] Judging whether Acholeplasma laidlawii exists in the sample to be tested by observing the color of the harvest solution;

[0091] and / or detecting the titer of Acholeplasma laidlawii in the harvest solution.

[0092] In some embodiments, it is judged whether Acholeplasma laidlawii exists in the sample to be tested by visually observing the color of the obtained culture solution. Using the medium of the present invention, the result can be read by the naked eye without the need to observe with an inverted microscope, which is simple and fast.

[0093] In some embodiments, the detection method for the titer of Acholeplasma laidlawii in the obtained culture solution includes but is not limited to the plate counting method (plate colony counting method), polymerase chain reaction (PCR) method, fluorescence staining detection method, etc.

[0094] In some embodiments, the calculation methods for the titer of Acholeplasma laidlawii in the obtained culture medium include, but are not limited to, the color change unit (CCU) method, the Karber method, the Reed-Muench method (also known as the two-point method), etc.

[0095] The present invention does not impose special requirements on the detection and calculation methods for the titer of Acholeplasma laidlawii, and other existing bacterial titer detection and calculation methods can also be used.

[0096] Another embodiment of the present invention provides a method for verifying the sterilization performance of a membrane filter, including: culturing Acholeplasma laidlawii according to the method described above to obtain a harvest solution, and using the harvest solution to test the filter membrane to detect the retention ability of the filter membrane for Acholeplasma laidlawii.

[0097] In some embodiments, Acholeplasma laidlawii is cultured according to the method described above to obtain a harvest solution with a viable cell concentration of 10 10-11 CCU / mL.

[0098] In some embodiments, the harvest solution is diluted and used as a challenge solution to test the filter membrane.

[0099] Further, in some embodiments, the dilution factor is 10 to 1000 times, such as 10, 20, 50, 100, 200, 500, 1000 times, etc.

[0100] In some embodiments, the filter membrane is a filter membrane with a pore size of 0.1 micrometers, such as a polyethersulfone (PES) membrane, a polyvinylidene fluoride (PVDF) membrane, a nylon membrane, a polytetrafluoroethylene (PTFE) microporous membrane, etc.

[0101] The following specific examples are given to illustrate the present invention in detail. It should also be understood that the following examples are only used to specifically illustrate the present invention and cannot be construed as limiting the protection scope of the present invention. Some non-essential improvements and adjustments made by those skilled in the art based on the above content of the present invention fall within the protection scope of the present invention. The specific process parameters and the like in the following examples are also only an example within a suitable range, that is, those skilled in the art can make selections within a suitable range through the description herein, rather than being limited to the specific values in the following examples.

[0102] The Acholeplasma laidlawii strain used in the following examples is ATCC 23206, which is a mycoplasma standard strain specifically cultured by ATCC for membrane filter detection.

[0103] Example 1

[0104] In this example, the formula and preparation process of the L-form Mycoplasma medium are as follows:

[0105] Take 10 g of glucose, 25 g of beef heart infusion powder, 5 g of yeast extract powder, 100 mL of horse serum, 2.5 g of sodium chloride, 10 mL of 0.5% phenol red solution (prepared by dissolving 0.5 g of powdered phenol red in 100 mL of ultrapure water) and 890 mL of ultrapure water, and prepare the medium according to the following steps.

[0106] (1) Add glucose, beef heart infusion powder, yeast extract powder and sodium chloride to ultrapure water, place it on an electric furnace, heat and stir until completely dissolved to obtain a mixed solution;

[0107] (2) Sterilize the mixed solution obtained in step (1) at 116 °C for 10 - 15 min to obtain a sterilized solution;

[0108] (3) After the sterilized solution obtained in step (2) is cooled, add horse serum and phenol red solution in a sterile environment, mix well, and adjust the pH to 7.5 - 7.8 with 1 mol / L sodium hydroxide solution to obtain the medium.

[0109] Example 2

[0110] Screening of the concentrations of each component in the L-form Mycoplasma medium

[0111] According to the preparation method of Example 1, prepare media containing different concentrations of horse serum, glucose and yeast extract powder according to Tables 1 - 3, cultivate according to the following method, observe the results and detect the titer to analyze and compare the influence on the cultivation of L-form Mycoplasma.

[0112] The method for culturing L-form Mycoplasma and detecting the titer (CCU) is as follows:

[0113] Use ultrapure water to rinse to ensure that the inside of the vial is clean and free of impurities that may affect the growth of L-form Mycoplasma. Then sterilize it using a dry heat sterilizer or a moist heat sterilizer, but the moist heat sterilizer needs to be dried. The vial has a volume of 5 mL.

[0114] The total volume of the culture solution used in this example is 2 mL. Add 0.9 mL of the medium to a centrifuge tube, take 0.1 mL of the test sample and add it to the centrifuge tube, mix well and perform 10-fold serial dilutions. After mixing the diluted samples evenly, take 0.1 mL and add it to a vial, then add 1.9 mL of the medium to the vial, mix gently, place it in a 37 °C biochemical incubator for 4 days to observe the results, and calculate the titer of L-form Mycoplasma using the Karber method.

[0115] Table 1. Influence of different concentrations of horse serum on the cultivation of L-form Mycoplasma

[0116] Table 2. Effects of different glucose concentrations on CCU detection

[0117] Glucose concentration (g / L) 5 10 15 20 Time required for detection and culture 6d 4d 6d 6d Color change +++ +++ ++ ++

[0118] Table 3. Effects of different concentrations of yeast extract on CCU detection

[0119] Yeast extract concentration (g / L) 2 5 10 15 Time required for detection and culture 5d 4d 4d 4d Color change +++ +++ ++ +

[0120] Note: “+” indicates obvious color change. The more “+” there are, the more obvious the color change is.

[0121] As shown in Table 1, the concentration of horse serum has a positive effect on the proliferation of Acholesterolabraxene, but when the concentration reaches 100mL / L, the titer of Acholesterolabraxene does not increase significantly. Therefore, considering the production cost and the impact on the membrane filter sterilization performance verification test, 100mL / L concentration of horse serum is a better choice.

[0122] As can be seen from Table 2, in addition to being the carbon source for the culture medium of Acholesterola reinhardtii, glucose also serves as a substance that produces acid after fermentation and causes pH changes. Too low a concentration of glucose (such as 5 g / L) will increase the time it takes for Acholesterola reinhardtii to proliferate in the vial and turn phenol red. Too high a concentration of glucose (such as 15 or 20 g / L) will affect the pH change of the culture medium, resulting in unclear color changes. Therefore, a usage of 10 g / L is a better choice.

[0123] As shown in Table 3, yeast extract powder provides a high-quality nitrogen source for A. reinhardtii. Increasing the usage can promote the proliferation of A. reinhardtii. However, it appears dark brown when dissolved in ultrapure water. The higher the concentration, the darker the color, which masks the color change of phenol red indicator. As shown in Table 3, the usage concentration of 5 g / L can achieve the best effect in providing nutrition and color observation.

[0124] Comparative Example 1

[0125] Liquid culture medium was prepared according to the formula and method of Leibniz cholesterogen-free medium provided by ATCC Medium 243. The specific process is as follows:

[0126] Take 25g of heart infusion broth and 100mL of 15% yeast extract solution (prepared by dissolving 150g of yeast extract in 1000mL of deionized water) in 800mL of deionized water, and sterilize at 121℃ for 15 minutes; after sterilization, add 200mL of heat-inactivated (56℃ for 30 minutes) horse serum under a sterile environment, mix well, and adjust the pH to 7.4±0.2 with 1mol / L sodium hydroxide solution, shake gently, and set aside.

[0127] Example 3

[0128] 1. Cultivation and harvesting of Acholeplasma laidlawii bacterial liquid

[0129] The culture media prepared in Example 1 and Comparative Example 1 were respectively used for inoculation at a ratio of 1:10 of the bacterial liquid to the culture medium to culture Acholeplasma laidlawii. Among them, the culture medium of Example 1 was cultured in a biochemical incubator at 37°C, while the culture medium of Comparative Example 1 needed to be cultured in a 5% carbon dioxide incubator at 37°C.

[0130] Samples were taken at 24 h and 48 h respectively in Comparative Example 1, and measured using a UV spectrophotometer at OD 500 The OD 500 value at 24 h was 0.234, and the OD 500 value at 48 h was 0.613. Two parallel groups were set up in Example 1, and both were harvested when the color of the culture medium was orange (pH about 6.8), and the culture time was 18 h.

[0131] 2. Detection of Acholeplasma laidlawii titer (CCU)

[0132] The sample taken at 24 h when cultured with the culture medium of Comparative Example 1, the harvested liquid obtained after culturing for 48 h, and the harvested liquid obtained after culturing for 18 h with the culture medium of Example 1 were diluted 10-fold with the culture medium of Example 1. 0.1 mL of the diluted liquid from each group was respectively added to a vial containing only 1.9 mL of the culture medium of Example 1, and 2 mL of the culture medium of Example 1 was set as a negative control. They were cultured in an incubator at 37°C for 4 days, and the color change was observed. The CCU titer was calculated using the Karber method for the vials with color change, and the results are shown in the following table.

[0133] Table 4. Harvesting / sampling time and titer (CCU) detection results of samples / harvested liquids obtained by culturing with different culture media

[0134]

[0135]

[0136] As shown in Table 4, when comparing the culture effects of the culture media prepared in Example 1 and Comparative Example 1 under the same test conditions, it can be found that: from the perspective of the harvesting time, the culture medium of Example 1 can be harvested within 24 h, while the titer of the culture medium of Comparative Example 1 did not reach the highest point at 24 h, so the harvesting time is preferably controlled at 24 h to 48 h, and the specific harvesting time needs to be evaluated by means of the absorbance value. From the perspective of the culture concentration of the bacterial liquid, the titer of the harvested liquid obtained with the culture medium of Comparative Example 1 was 10 7-8 CCU / mL, while the titer of the harvested liquid obtained with the culture medium of Example 1 was 1010-11 CCU / mL. It can be seen that by using the culture medium of the present invention, a harvest solution with a titer of 10 10-11 CCU / mL can be obtained within 24 hours, which indicates that compared with the culture media with known formulations on the market, the present invention can not only significantly increase the culture concentration of live bacteria, but also significantly shorten the culture time.

[0137] 3. Application research of the culture medium of the present invention as a culture medium for detection

[0138] The harvest solution obtained by culturing with the culture medium of Example 1 (parallel group 1) for 18 hours was diluted at dilutions of 10 -1 to 10 -9 . 0.1 mL of the diluted solution at each dilution was respectively added to vials containing only 1.9 mL of the culture medium of Example 1, and cultured in a biochemical incubator at 37°C for 24 hours and 96 hours to obtain the culture solutions corresponding to 1-9 in Figure 1 and Figure 2 , and the color change was observed.

[0139] The harvest solution obtained by culturing with the culture medium of Comparative Example 1 for 48 hours was diluted at dilutions of 10 -1 to 10 -9 . 0.1 mL of the diluted solution at each dilution was respectively added to vials containing only 1.9 mL of the culture medium of Example 1, and cultured in a carbon dioxide incubator at 37°C for 24 hours and 96 hours to obtain the culture solutions corresponding to 1-9 in Figure 3 and Figure 4 , and the color change was observed.

[0140] 2 mL of the culture medium of Example 1 was set as a negative control (NC).

[0141] According to Figures 1-4 the following conclusions can be drawn: From the application effect of the culture medium of the present invention as a culture medium for CCU titer detection, the CCU titer detection time is significantly shortened. For the culture media for mycoplasma detection on the market (such as the culture medium of Comparative Example 1), the detection time mostly requires about 7 days, while the present invention can obtain the detection result in 4 days, and no color change occurs in the vials with higher dilutions after extending the culture time. At the same time, in terms of result evaluation, the color difference change using the culture medium of the present invention is obvious, and the result can be visually judged by the naked eye with little subjective influence.

[0142] 4. Membrane filter retention effect test

[0143] To simulate the retention test of the membrane filter, the harvest fluid obtained by culturing in the medium of Example 1 (parallel group 1) for 18 h and the harvest fluid obtained by culturing in the medium of Comparative Example 1 for 48 h were selected. A flux test was carried out on a PES filter membrane with a pore size of 0.1 μm (which can intercept Acholeplasma laidlawii) at 0.1 MPa. According to the requirements of ASTM F838-20 standard, the titer of Acholeplasma laidlawii in the challenge fluid should be no less than 10 7.50 CCU / mL. Therefore, the harvest fluid obtained by culturing in the medium of Comparative Example 1 needs to use the undiluted solution as the challenge fluid, and the harvest fluid obtained by culturing in the medium of Example 1 can be diluted with PBS buffer at a ratio of 1:100 and used as the challenge fluid. The simulated retention test situation is shown in Table 5.

[0144] Table 5. Simulated retention test situation of harvest fluids obtained by culturing with different media

[0145] Culture medium Control Example 1 Example 1 (Parallel Group 1) Challenge liquid volume 200 mL 200 mL Harvest liquid dosage 200 mL 2 mL Initial flow rate 17 mL / min 25 mL / min Total filtration time 14 min 10 min

[0146] As can be seen from the above, the cultivation of Acholeplasma laidlawii using the medium of Example 1 requires less serum and has a higher harvested titer. Therefore, when used as a test verification for a sterile-grade membrane filter, it can be diluted 100 - 1000 times. The membrane filter is less affected by serum, medium and their metabolites during this process, so the flux test results are better.

[0147] 5. Comparison of the retention test effects of membrane filters

[0148] According to the requirements of ASTM F838-20 standard, the harvest fluid obtained by culturing in the medium of Example 1 (parallel group 1) for 18 h and the harvest fluid obtained by culturing in the medium of Comparative Example 1 for 48 h were selected, and diluted with PBS to a challenge fluid with a titer not less than 10 7.50 CCU / mL (or not less than 1.0×10 8 CFU / mL). A retention test was carried out on a single-layer filter membrane (PES filter membrane with a pore size of 0.2 μm (which can absolutely allow Acholeplasma laidlawii to pass through)) and two different batches of double-layer filter membranes (the upper layer is a PES filter membrane with a pore size of 0.2 μm (which can absolutely allow Acholeplasma laidlawii to pass through), and the lower layer is a PES filter membrane with a pore size of 0.1 μm (which can intercept Acholeplasma laidlawii)) at 0.1 MPa. The CCU method and the plate counting method were used to detect the titers of the challenge fluid before retention and the filtrate sample after retention respectively, and the log reduction value (LRV) was calculated according to the following formula. The results are shown in Table 6.

[0149]

[0150] Figure 5Shown is the test chart of the challenge solution obtained by diluting the harvest solution cultured in the medium of Example 1 (parallel group 1) for 18 h with a dilution factor of 10 -1 ~10 -9 times.

[0151] As Figure 5 shown, during the culture process, as Mycoplasma acholeplasma laidlawii proliferated and metabolized, the medium at different dilution factors gradually changed from dark red to orange-yellow, and the medium color was clear and transparent. The detection period was short (results could be obtained in 4 days), the detection situation was stable, and the results were easy to read, etc.

[0152] Figure 6 Shown is the colony photo observed under a microscope when the harvest solution cultured in the medium of Comparative Example 1 for 48 h was subjected to titer detection by the plate counting method.

[0153] As Figure 6 shown, under a 40-fold (40×) microscope, typical fried-egg-shaped colonies could be seen; at the same time, it could also be seen that since the colonies were of different sizes, it was difficult to read.

[0154] As shown in Table 6, by comparing the detection results obtained based on two different media and two different titer detection methods, it was found that the LRV deviation between the CCU method and the plate counting method was within 0.5 log. This indicates that the harvest solution obtained by culturing Mycoplasma acholeplasma laidlawii with the medium of the present invention can be used as a challenge solution for detecting the mycoplasma retention ability of membrane filters after dilution, and the titer of Mycoplasma acholeplasma laidlawii in the filtrate can be determined by titer detection methods such as the CCU method to verify the mycoplasma removal ability of the membrane filter.

[0155] Table 6. Detection results obtained by using different media and different titer detection methods

[0156]

[0157] The above embodiments are only illustrative of the principles and effects of the present invention, and are not intended to limit the present invention. Any person familiar with this technology can modify or change the above embodiments without departing from the spirit and scope of the present invention. Therefore, all equivalent modifications or changes made by those with ordinary knowledge in the technical field without departing from the spirit and technical idea disclosed by the present invention should still be covered by the claims of the present invention.

Claims

1. A liquid-form L-form mycoplasma medium, characterized in that, The L-form mycoplasma medium consists of Composition I, an osmotic pressure regulator, an acid-base indicator, and a pH regulator; Composition I is used to provide the carbon source, nitrogen source, growth factors, vitamins, and other nutrients required for the growth of L-form mycoplasma. Composition I consists of glucose, beef heart infusion powder, yeast extract powder, and serum; In the medium, the mass concentrations of glucose, beef heart infusion powder, yeast extract powder, osmotic pressure regulator, and acid-base indicator are 10 g / L, 25 - 30 g / L, 5 g / L, 2 - 5 g / L, and 0.05 - 0.1 g / L respectively; The serum is selected from at least one of horse serum, chicken serum, and fetal bovine serum, and the volume fraction of the serum in the medium is 10%; The pH of the medium is 7.5 - 7.

8.

2. The L-form mycoplasma medium according to claim 1, wherein: The osmotic pressure regulator is selected from inorganic salts; and / or, the pH regulator is selected from sodium hydroxide and / or potassium hydroxide; and / or, the acid-base indicator is selected from phenol red and / or neutral red.

3. The L-form acholeplasma culture medium according to claim 1 or 2, characterized in that: Composition I consists of the following components: glucose, beef heart infusion powder, yeast extract powder, horse serum.

4. The L-form acholeplasma culture medium according to claim 3, characterized in that: The medium consists of the following components: glucose, beef heart infusion powder, yeast extract powder, horse serum, sodium chloride, phenol red, and the mass ratio of glucose, beef heart infusion powder, yeast extract powder, sodium chloride, and phenol red is 10:25 - 30:5:2 - 5:0.05 - 0.

1.

5. The L-form acholeplasma medium according to claim 1, characterized in that: The preparation method of the medium includes: Dissolve the components of Composition I except serum and the osmotic pressure regulator, and then sterilize to obtain a sterilized solution; After the sterilized solution is cooled, add serum and acid-base indicator in a sterile environment, mix well, and then add the pH regulator to adjust the pH to 7.5 - 7.8 to obtain the medium.

6. A method for culturing and detecting Acholeplasma laidlawii, characterized in that, including: Culturing a test sample and / or L-form mycoplasma in an environment without 5% carbon dioxide using the medium according to any one of claims 1 - 5 and obtaining a harvest solution, and the culturing time does not exceed 24 hours; Detecting the L-form mycoplasma in the harvest solution, including: judging whether there is L-form mycoplasma in the test sample by observing the color of the harvest solution; and / or, detecting the titer of L-form mycoplasma in the harvest solution.

7. The L-form mycoplasma culture and detection method according to claim 6, characterized in that: The culturing temperature is 34 - 38°C.

8. A method for verifying the sterilization performance of a membrane filter, characterized in that, including: Cultivate Acholeplasma laidlawii by the method according to any one of claims 6 to 7 and obtain a harvest solution with a viable cell concentration of 10 10-11 CCU / mL. Dilute the said harvest solution and use it as a challenge solution to test the filter membrane, and detect the retention ability of the filter membrane for Acholeplasma laidlawii.

9. The method for verifying the sterilization performance of the membrane filter according to claim 8, wherein: The filter membrane is a filter membrane with a pore size of 0.1 micrometers.

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