Improved liquid culture medium for direct culture and detection of mycoplasma

Abstract

The invention relates to the technical field of culture media, in particular to a liquid improved culture medium for direct culture and detection of mycoplasma, which comprises a carbon source mycoplasma culture medium and a nitrogen source mycoplasma culture medium, the carbon source mycoplasma culture medium is prepared from the following components: glucose, pig stomach digest, sodium chloride, fetal calf serum, a CMRL-1066 solution, yeast extract powder, beef extract powder, phenol red, an egg liquid substitute extract and water; the nitrogen source mycoplasma culture medium is prepared from the following components: glucose, pig stomach digest, sodium chloride, L-arginine, a CMRL-1066 solution, fetal calf serum, yeast extract powder, beef extract powder, phenol red, an egg liquid replacement extract and water; the resuscitation success rate of the primary mycoplasma is improved, the preparation difficulty is reduced, and the problem that the passage is easy to die due to the technical difficulty is solved.

Description

Technical Field

[0001] The invention relates to the technical field of culture media, and in particular to a liquid improved culture medium for direct culture and detection of mycoplasma. Background Art

[0002] With the rise of stem cell and related biopharmaceutical industries in China, the original pharmacopoeia regulations do not include mycoplasma detection for microorganisms, while mycoplasma is one of the largest sources of biopharmaceutical pollution in the world. Therefore, it is imperative to improve domestic biopharmaceutical testing. It also plays a vital role in the safety of domestic high-end stem cell therapy products. At the same time, it is also a key factor affecting the introduction of foreign mycoplasma resources into China and the establishment of China's own mycoplasma sample library to get rid of the limitation of foreign mycoplasma sample resources.

[0003] The culture medium formula currently used in China is mainly based on the knowledge system established forty or fifty years ago. Therefore, although it can be used in practical applications, it has extremely high requirements on the operator. In actual testing departments (such as obstetrics and gynecology, respiratory department, and hospital laboratory), problems such as resuscitation difficulties, cell death or limited proliferation effect often occur during use. Summary of the invention

[0004] In view of the above situation, in order to overcome the defects of the prior art, the present invention provides a liquid improved culture medium for direct culture and detection of mycoplasma.

[0005] The technical scheme adopted by the present invention is as follows: a liquid improved culture medium for direct culture and detection of mycoplasma, comprising a carbon source mycoplasma culture medium and a nitrogen source mycoplasma culture medium;

[0006] The carbon source mycoplasma culture medium comprises the following components: glucose, pig stomach digesta, sodium chloride, fetal bovine serum, CMRL-1066 solution, yeast extract powder, beef extract powder, phenol red, egg liquid substitute extract, and water;

[0007] The nitrogen source mycoplasma culture medium comprises the following components: glucose, pig stomach digesta, sodium chloride, L-arginine, CMRL-1066 solution, fetal bovine serum, yeast extract powder, beef extract powder, phenol red, egg liquid substitute extract, and water.

[0008] Furthermore, the dosage of each component in the carbon source mycoplasma culture medium is: 4.5-5.5g of glucose, 9.0-11.0g of pig stomach digesta, 2.3-2.7g of sodium chloride, 180-220ml of fetal bovine serum, 45-55ml of CMRL-1066 solution, 4.5-5.5g of yeast extract powder, 4.5-5.5g of beef extract powder, 0.015-0.025g of phenol red, 9-11ml of egg liquid substitute extract, and 630-770ml of water.

[0009] Furthermore, the dosage of each component in the nitrogen source mycoplasma culture medium is: glucose 0.9-1.1g, pig stomach digesta 9.0-11.0g, sodium chloride 2.3-2.7g, L-arginine 0.9-1.1g, CMRL-1066 solution 45-55ml, fetal bovine serum 180-220ml, yeast extract 4.5-5.5g, beef extract 4.5-5.5g, phenol red 0.015-0.025g, egg liquid substitute extract 9-11ml, water 630-770ml.

[0010] The present invention also discloses a method for preparing a liquid improved culture medium for direct culture and detection of mycoplasma, comprising the following steps:

[0011] Step 1, preparation of carbon source mycoplasma culture medium:

[0012] Measure glucose, porcine stomach digesta, sodium chloride, fetal bovine serum, CMRL-1066 solution, yeast extract powder, beef extract powder, phenol red, egg liquid substitute extract, and water according to the prescribed amount;

[0013] After mixing water, glucose, pig stomach digesta, sodium chloride, yeast extract powder, beef extract powder and phenol red uniformly, fetal bovine serum, CMRL-1066 solution and egg liquid substitute extract were added, and mixed again to obtain a carbon source mycoplasma culture medium;

[0014] The pH of the carbon source mycoplasma culture medium is adjusted to 7.6-7.8, and then sterilized for use;

[0015] Step 2, preparation of nitrogen-derived mycoplasma culture medium:

[0016] Measure glucose, porcine stomach digesta, sodium chloride, L-arginine, CMRL-1066 solution, fetal bovine serum, yeast extract powder, beef extract powder, phenol red, egg liquid substitute extract, and water according to the prescribed amount;

[0017] Mix water, glucose, pig stomach digesta, sodium chloride, L-arginine, yeast extract powder, beef extract powder and phenol red uniformly, then add fetal bovine serum, CMRL-1066 solution and egg liquid substitute extract, and mix again to obtain nitrogen source mycoplasma culture medium;

[0018] The pH of the nitrogen-based mycoplasma culture medium was adjusted to 7.4-7.6, and then sterilized for use.

[0019] The pH adjustment of the nitrogen source (arginine) mycoplasma culture medium formula is set to 7.4-7.6 instead of 7.1 mainly because the culture medium can use a specific pH value to activate arginine mycoplasma to grow in subsequent pH changes.

[0020] The beneficial effects achieved by the present invention using the above structure are as follows:

[0021] 1. Improved the success rate of primary mycoplasma recovery;

[0022] 2. Reduce the difficulty of medium preparation and improve the proliferation rate;

[0023] 3. Reduce the problem of easy death during cell propagation due to technical difficulties. DETAILED DESCRIPTION

[0024] The technical solutions in the embodiments of the present invention will be described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, rather than all the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0025] A liquid improved culture medium for direct culture and detection of mycoplasma, comprising a carbon source mycoplasma culture medium and a nitrogen source mycoplasma culture medium;

[0026] The carbon source mycoplasma culture medium comprises the following components: glucose, pig stomach digesta, sodium chloride, fetal bovine serum, CMRL-1066 solution, yeast extract powder, beef extract powder, phenol red, egg liquid substitute extract, and water;

[0027] The nitrogen source mycoplasma culture medium comprises the following components: glucose, pig stomach digesta, sodium chloride, L-arginine, CMRL-1066 solution, fetal bovine serum, yeast extract powder, beef extract powder, phenol red, egg liquid substitute extract, and water.

[0028] Example 1, a method for preparing a liquid modified culture medium for direct culture and detection of mycoplasma, comprising the following steps:

[0029] Step 1, preparation of carbon source mycoplasma culture medium:

[0030] The dosage of each component in the carbon source mycoplasma culture medium is: glucose 4.5g, pig stomach digesta 9.0g, sodium chloride 2.3g, fetal bovine serum 180ml, CMRL-1066 solution 45ml, yeast extract powder 4.5g, beef extract powder 4.5g, phenol red 0.015g, egg liquid substitute extract 9ml, water 630ml;

[0031] Measure glucose, porcine stomach digesta, sodium chloride, fetal bovine serum, CMRL-1066 solution, yeast extract powder, beef extract powder, phenol red, egg liquid substitute extract, and water according to the prescribed amount;

[0032] After mixing water, glucose, pig stomach digesta, sodium chloride, yeast extract powder, beef extract powder and phenol red uniformly, fetal bovine serum, CMRL-1066 solution and egg liquid substitute extract are added, and mixed again to obtain a mixture;

[0033] The pH of the mixture was adjusted to 7.6, and then sterilized for use;

[0034] Step 2, preparation of nitrogen-derived mycoplasma culture medium:

[0035] The dosage of each component in the carbon source mycoplasma culture medium is: glucose 0.9g, pig stomach digest 9.0g, sodium chloride 2.3g, L-arginine 0.9g, CMRL-1066 solution 45ml, fetal bovine serum 180ml, yeast extract powder 4.5g, beef extract powder 4.5g, phenol red 0.015g, egg liquid substitute extract 9ml, water 630ml;

[0036] Measure glucose, porcine stomach digesta, sodium chloride, L-arginine, CMRL-1066 solution, fetal bovine serum, yeast extract powder, beef extract powder, phenol red, egg liquid substitute extract, and water according to the prescribed amount;

[0037] After mixing water, glucose, pig stomach digesta, sodium chloride, L-arginine, yeast extract powder, beef extract powder and phenol red uniformly in a practical mixer, fetal bovine serum, CMRL-1066 solution and egg liquid substitute extract are added and mixed again to obtain a mixture;

[0038] The stirring speed in the mixer is 100-130 rpm;

[0039] The pH of the mixture was adjusted to 7.4 and then sterilized for use;

[0040] The sterilization process is sterilization by high pressure steam at 121°C for 15 minutes.

[0041] Example 2, a method for preparing a liquid modified culture medium for direct culture and detection of mycoplasma, comprising the following steps:

[0042] Step 1, preparation of carbon source mycoplasma culture medium:

[0043] The dosage of each component in the carbon source mycoplasma culture medium is: glucose 5.0g, pig stomach digesta 10.0g, sodium chloride 2.5g, fetal bovine serum 200ml, CMRL-1066 solution 50ml, yeast extract powder 5.0g, beef extract powder 5.0g, phenol red 0.02g, egg liquid substitute extract 10ml, water 700ml;

[0044] Measure glucose, porcine stomach digesta, sodium chloride, fetal bovine serum, CMRL-1066 solution, yeast extract powder, beef extract powder, phenol red, egg liquid substitute extract, and water according to the prescribed amount;

[0045] After mixing water, glucose, pig stomach digesta, sodium chloride, yeast extract powder, beef extract powder and phenol red uniformly, fetal bovine serum, CMRL-1066 solution and egg liquid substitute extract are added, and mixed again to obtain a mixture;

[0046] The pH of the mixture was adjusted to 7.7, and then sterilized for use;

[0047] The sterilization process is sterilization by high pressure steam at 121℃ for 15min;

[0048] Step 2, preparation of nitrogen-derived mycoplasma culture medium:

[0049] The dosage of each component in the carbon source mycoplasma culture medium is: glucose 1.0g, pig stomach digesta 10.0g, sodium chloride 2.5g, L-arginine 1.0g, CMRL-1066 solution 50ml, fetal bovine serum 200ml, yeast extract powder 5.0g, beef extract powder 5.0g, phenol red 0.02g, egg liquid substitute extract 10ml, water 700ml;

[0050] Measure glucose, porcine stomach digesta, sodium chloride, L-arginine, CMRL-1066 solution, fetal bovine serum, yeast extract powder, beef extract powder, phenol red, egg liquid substitute extract, and water according to the prescribed amount;

[0051] After mixing water, glucose, pig stomach digesta, sodium chloride, L-arginine, yeast extract powder, beef extract powder and phenol red uniformly, fetal bovine serum, CMRL-1066 solution and egg liquid substitute extract are added, and mixed again to obtain a mixture;

[0052] The pH of the mixture is adjusted to 7.5, and then sterilized for use;

[0053] The sterilization process is sterilization by high pressure steam at 121°C for 15 minutes.

[0054] Example 3, a method for preparing a liquid modified culture medium for direct culture and detection of mycoplasma, comprising the following steps:

[0055] Step 1, preparation of carbon source mycoplasma culture medium:

[0056] The dosage of each component in the carbon source mycoplasma culture medium is: glucose 5.5g, pig stomach digesta 11.0g, sodium chloride 2.7g, fetal bovine serum 220ml, CMRL-1066 solution 55ml, yeast extract powder 5.5g, beef extract powder 5.5g, phenol red 0.025g, egg liquid substitute extract 11ml, water 770ml;

[0057] Measure glucose, porcine stomach digesta, sodium chloride, fetal bovine serum, CMRL-1066 solution, yeast extract powder, beef extract powder, phenol red, egg liquid substitute extract, and water according to the prescribed amount;

[0058] After mixing water, glucose, pig stomach digesta, sodium chloride, yeast extract powder, beef extract powder and phenol red uniformly, fetal bovine serum, CMRL-1066 solution and egg liquid substitute extract are added, and mixed again to obtain a mixture;

[0059] The pH of the mixture was adjusted to 7.8 and then sterilized for use;

[0060] The sterilization process is sterilization by high pressure steam at 121℃ for 15min;

[0061] Step 2, preparation of nitrogen-derived mycoplasma culture medium:

[0062] The dosage of each component in the carbon source mycoplasma culture medium is: glucose 1.1g, pig stomach digest 11.0g, sodium chloride 2.7g, L-arginine 1.1g, CMRL-1066 solution 55ml, fetal bovine serum 220ml, yeast extract powder 5.5g, beef extract powder 5.5g, phenol red 0.025g, egg liquid substitute extract 11ml, water 770ml;

[0063] Measure glucose, porcine stomach digesta, sodium chloride, L-arginine, CMRL-1066 solution, fetal bovine serum, yeast extract powder, beef extract powder, phenol red, egg liquid substitute extract, and water according to the prescribed amount;

[0064] After mixing water, glucose, pig stomach digesta, sodium chloride, L-arginine, yeast extract powder, beef extract powder and phenol red uniformly, fetal bovine serum, CMRL-1066 solution and egg liquid substitute extract are added, and mixed again to obtain a mixture;

[0065] The pH of the mixture was adjusted to 7.6, and then sterilized for use;

[0066] The sterilization process is sterilization by high pressure steam at 121°C for 15 minutes.

[0067] The use process of carbon source mycoplasma culture medium:

[0068] Under sterile conditions, inoculate the sample to be tested into the prepared carbon source mycoplasma culture medium, place the culture medium under appropriate temperature (usually 35-37°C) and humidity conditions for cultivation, and regularly observe the growth of mycoplasma, such as colony morphology, color, etc.

[0069] The results are determined based on the growth and colony characteristics of mycoplasma combined with clinical information.

[0070] The use process of nitrogen source mycoplasma culture medium:

[0071] Similarly, the sample to be tested is inoculated under sterile conditions and cultured under suitable temperature and humidity conditions to observe the growth of mycoplasma, especially the growth rate and morphological changes of the colonies, to determine whether there is mycoplasma infection.

[0072] The carbon source mycoplasma culture medium and nitrogen source mycoplasma culture medium configured in Example 1, Example 2 and Example 3 are used to detect mycoplasma infection in urinary tract, gynecological infection and respiratory tract infection with high accuracy. The colony growth morphology and color after mycoplasma inoculation and culture are excellent, and the colony size is greatly increased, and the mycoplasma type can be accurately and clearly identified. At the same time, the actual content of mycoplasma in the specimen can be inferred by colony counting, and it is accurately judged whether the specimen is in a bacterial state or has reached a pathogenic concentration, providing doctors with rapid and reliable test results for formulating reasonable treatment plans.

[0073] In this solution, CMRL-1066 is added to provide all trace elements for the growth of mycoplasma and the presence of carbonate and bicarbonate ions ensures long-term pH stability without damaging the mycoplasma.

[0074] Egg liquid substitute extracts can provide the free cholesterol and growth factors needed for the growth of mycoplasma without causing great harm to mycoplasma culture due to antibiotics, heavy metals, etc. that are easily found in poultry farming.

[0075] At the same time, after comparison, the colony size obtained by the scheme in Example 2 is the largest, which is much larger than the colonies formed on the commercially available culture medium, and the color is the most obvious. Therefore, the carbon source mycoplasma culture medium and the nitrogen source mycoplasma culture medium configured in Example 2 are more accurate in detecting mycoplasma infection in the urinary tract, gynecological infection, and respiratory tract infection.

[0076] It should be noted that, in this article, the terms "comprises", "includes" or any other variations thereof are intended to cover non-exclusive inclusion, so that a process, method, article or apparatus that includes a series of elements includes not only those elements, but also other elements not explicitly listed, or also includes elements inherent to such process, method, article or apparatus.

[0077] Although embodiments of the present invention have been shown and described, it will be appreciated by those skilled in the art that various changes, modifications, substitutions and variations may be made to the embodiments without departing from the principles and spirit of the present invention, and that the scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A liquid improved culture medium for direct culture and detection of mycoplasma, characterized in that: It includes carbon source mycoplasma culture medium and nitrogen source mycoplasma culture medium; The carbon source mycoplasma culture medium comprises the following components: glucose, pig stomach digesta, sodium chloride, fetal bovine serum, CMRL-1066 solution, yeast extract powder, beef extract powder, phenol red, egg liquid substitute extract, and water; The nitrogen source mycoplasma culture medium comprises the following components: glucose, pig stomach digesta, sodium chloride, L-arginine, CMRL-1066 solution, fetal bovine serum, yeast extract powder, beef extract powder, phenol red, egg liquid substitute extract, and water.

2. The liquid improved culture medium for direct culture and detection of mycoplasma according to claim 1, characterized in that: The dosage of each component in the carbon source mycoplasma culture medium is: 4.5-5.5g of glucose, 9.0-11.0g of pig stomach digesta, 2.3-2.7g of sodium chloride, 180-220ml of fetal bovine serum, 45-55ml of CMRL-1066 solution, 4.5-5.5g of yeast extract powder, 4.5-5.5g of beef extract powder, 0.015-0.025g of phenol red, 9-11ml of egg liquid substitute extract, and 630-770ml of water.

3. The liquid improved culture medium for direct culture and detection of mycoplasma according to claim 1, characterized in that: The dosage of each component in the nitrogen source mycoplasma culture medium is: 0.9-1.1g of glucose, 9.0-11.0g of pig stomach digesta, 2.3-2.7g of sodium chloride, 0.9-1.1g of L-arginine, 45-55ml of CMRL-1066 solution, 180-220ml of fetal bovine serum, 4.5-5.5g of yeast extract powder, 4.5-5.5g of beef extract powder, 0.015-0.025g of phenol red, 9-11ml of egg liquid substitute extract, and 630-770ml of water.

4. The liquid improved culture medium for direct culture and detection of mycoplasma according to claim 2, characterized in that: The dosage of each component in the carbon source mycoplasma culture medium is: 5.0g glucose, 10.0g pig stomach digesta, 2.5g sodium chloride, 200ml fetal bovine serum, 50ml CMRL-1066 solution, 5.0g yeast extract powder, 5.0g beef extract powder, 0.02g phenol red, 10ml egg liquid substitute extract, and 700ml water.

5. The liquid improved culture medium for direct culture and detection of mycoplasma according to claim 3, characterized in that: The dosage of each component in the nitrogen source mycoplasma culture medium is: 1.0g of glucose, 10.0g of pig stomach digesta, 2.5g of sodium chloride, 1.0g of L-arginine, 50ml of CMRL-1066 solution, 200ml of fetal bovine serum, 5.0g of yeast extract powder, 5.0g of beef extract powder, 0.02g of phenol red, 10ml of egg liquid substitute extract, and 700ml of water.

6. A method for preparing a liquid improved culture medium for direct culture and detection of mycoplasma according to claims 1 to 5, characterized in that ,, including the following steps: Step 1, preparation of carbon source mycoplasma culture medium: Measure glucose, porcine stomach digesta, sodium chloride, fetal bovine serum, CMRL-1066 solution, yeast extract powder, beef extract powder, phenol red, egg liquid substitute extract, and water according to the prescribed amount; After mixing water, glucose, pig stomach digesta, sodium chloride, yeast extract powder, beef extract powder and phenol red uniformly, fetal bovine serum, CMRL-1066 solution and egg liquid substitute extract were added, and mixed again to obtain a carbon source mycoplasma culture medium; Step 2, preparation of nitrogen-derived mycoplasma culture medium: Measure glucose, porcine stomach digesta, sodium chloride, L-arginine, CMRL-1066 solution, fetal bovine serum, yeast extract powder, beef extract powder, phenol red, egg liquid substitute extract, and water according to the prescribed amount; Water was mixed with glucose, pig stomach digesta, sodium chloride, L-arginine, yeast extract powder, beef extract powder and phenol red, and then fetal bovine serum, CMRL-1066 solution and egg liquid substitute extract were added, and the mixture was mixed again to obtain nitrogen source mycoplasma culture medium.

7. The method for preparing a liquid improved culture medium for direct culture and detection of mycoplasma according to claim 6, characterized in that: In step 1, the pH of the carbon source mycoplasma culture medium is 7.6-7.

8.

8. The method for preparing a liquid improved culture medium for direct culture and detection of mycoplasma according to claim 6, characterized in that: In step 2, the pH of the nitrogen-source mycoplasma culture medium is 7.4-7.

6.

9. The method for preparing a liquid improved culture medium for direct culture and detection of mycoplasma according to claim 6, characterized in that: The carbon source mycoplasma culture medium and the nitrogen source mycoplasma culture medium are sterilized after being prepared for use.

10. The method for preparing a liquid improved culture medium for direct culture and detection of mycoplasma according to claim 6, characterized in that: In step 1 and step 2, the sterilization temperature is 121°C and the sterilization time is 15 minutes.