Simple mycoplasma ovipneumoniae culture medium as well as preparation method and application thereof

Abstract

The invention discloses a simple mycoplasma ovipneumoniae culture medium as well as a preparation method and application thereof. The method comprises the following steps: uniformly mixing 18-24g / L of PPLO Both, 21-29g / L of yeast powder and 2-4mL / L of 1% phenol red, and then adjusting the pH value to 7.8-8.0 by using 1M NaOH; after high-pressure steam sterilization is carried out for 20 minutes at the temperature of 121 DEG C, 180-220 mL / L of pig serum, 80-120 mL / L of MEM culture medium and 0.9-1.1 mL / L of penbritin with the concentration of 50 mg / mL are added after the temperature is reduced to room temperature. The mycoplasma ovipneumoniae cultured by the culture medium disclosed by the invention can be up to 109CCU / mL at most; the preparation method of the culture medium is simpler and more convenient, the formula is simple, and auxiliary components such as glucose and sodium pyruvate do not need to be additionally added; according to the method, the culture medium is prepared by directly adding yeast powder instead of adding yeast extract in the prior art; the mycoplasma ovipneumoniae cultured by the culture medium disclosed by the invention can reach 107CCU / mL to the maximum in 24 hours, and the strain grows quickly; the culture medium does not need thallium acetate, and is more environment-friendly and harmless to human bodies.

Description

Technical Field

[0001] The invention belongs to the technical field of veterinary biological products, and particularly relates to a simple Mycoplasma ovipneumoniae culture medium and a preparation method and application thereof. Background Art

[0002] Mycoplasma ovis pneumonia has become one of the most common infectious diseases in my country's sheep industry. Mycoplasma ovis pneumonia (Mo) is the main pathogen of the disease. The existence of this disease has caused huge economic losses to the sheep industry. In my country, Hu Jingshao et al. first isolated the pathogen of the disease in Sichuan in 1982 (Hu Jingshao, Jiang Xueliang, Hu Chenglong, et al. Study on a type of hyperplastic interstitial pneumonia in sheep infected with mycoplasma [J]. Chinese Veterinary Journal, 1982, (05): 2-6.), and determined that Mo was the cause of hyperplastic interstitial pneumonia in local sheep. Mo is sensitive to physical and chemical factors and can generally be completely killed in an environment of 55°C for 5 to 15 minutes. The growth of this bacterium has high nutritional requirements and is difficult to culture in vitro. In order to conduct a thorough study on it and prevent and control Mycoplasma ovis pneumonia, it is important to increase the growth titer of Mo in the simplest way.

[0003] There are many kinds of culture media, some of which have simple ingredients but the growth titer of Mo cultured is not high (such as KM2 medium, the highest growth titer of Mo is 10 7 CCU / mL: CN201911237916.8); some have high growth titers but are more complex in composition, consuming manpower and material resources (such as the low-serum high-efficiency culture medium developed by Chen Shengli et al.: CN201611071308.0). Summary of the Invention

[0004] The present invention aims to provide a Mycoplasma ovipneumoniae culture medium with a simple formula, sufficient nutrients and the ability to allow the strain to grow rapidly; at the same time, the present invention provides a preparation method of the culture medium.

[0005] To achieve the above objectives, the present invention provides a very simple method for preparing an in vitro culture medium for Mycoplasma ovipneumoniae. The main components are: 21g / L PPLO Both, 25g / L yeast powder, and 3mL / L 1% phenol red. The culture medium is made up to 700mL with water and mixed evenly, and the pH is adjusted to 7.8-8.0 with 1M NaOH. After high-pressure steam sterilization at 121°C for 20min, 200mL / L porcine serum, 100mL / L MEM culture medium, and 1mL / L 50mg / mL ampicillin are added when the temperature drops to room temperature.

[0006] A simple preparation method for a Mycoplasma ovipneumoniae culture medium comprises the following steps: uniformly mixing 18-24 g / L of PPLO Both, 21-29 g / L of yeast powder, and 2-4 mL / L of 1% phenol red, and then adjusting the pH to 7.8-8.0 with 1M NaOH; sterilizing with high-pressure steam at 121°C for 20 min, and then adding 180-220 mL / L of porcine serum, 80-120 mL / L of MEM culture medium, and 0.9-1.1 mL / L of 50 mg / mL ampicillin after the temperature drops to room temperature.

[0007] Preferably, the concentration of PPLO Both is 21 g / L, the concentration of yeast powder is 25 g / L, and the concentration of 1% phenol red is 3 mL / L.

[0008] Preferably, the porcine serum is 200 mL / L, the MEM medium is 100 mL / L, and the 50 mg / mL ampicillin is 1 mL / L.

[0009] A simple Mycoplasma ovipneumoniae culture medium is prepared by the above method.

[0010] Application of the simplified Mycoplasma ovipneumoniae culture medium in culturing Mycoplasma ovipneumoniae.

[0011] The simplified Mycoplasma ovipneumoniae culture medium is used in preparing Mycoplasma ovipneumoniae vaccine antigens.

[0012] Compared with the prior art, the present invention has the following beneficial effects:

[0013] 1. Compared with the existing culture medium KM2, the amount of Mycoplasma ovipneumoniae cultured is 10 7 CCU / mL. The culture medium of the present invention can culture Mycoplasma ovipneumoniae up to 10 9 CCU / mL;

[0014] 2. Compared with most existing Mycoplasma ovipneumoniae culture media, the preparation method of the culture medium of the present invention is simpler and has a simpler formula, without the need to add auxiliary ingredients such as glucose and sodium pyruvate;

[0015] 3. The present invention chooses to directly add yeast powder instead of the traditional method of adding yeast extract to prepare the culture medium;

[0016] 4. The culture medium of the present invention can produce up to 10% Mycoplasma ovipneumoniae in just 24 hours. 7 CCU / mL, the strain grows rapidly;

[0017] 5. The culture medium of the present invention does not require the addition of thallium acetate, is more environmentally friendly, and is harmless to the human body. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1The present invention is a schematic diagram of a method for preparing a simple Mycoplasma ovipneumoniae culture medium.

[0019] Figure 2 This is the growth of Mycoplasma ovipneumoniae in the culture medium of Example 1 of the present invention. DETAILED DESCRIPTION

[0020] Example 1

[0021] Figure 1 The present invention is a schematic diagram of a method for preparing a simple Mycoplasma ovipneumoniae culture medium.

[0022] Preparation of simplified Mycoplasma ovipneumoniae culture medium:

[0023] The chemical reagents used in the following examples are all conventional reagents and are commercially available.

[0024] Prepare 1000 mL of Mycoplasma ovipneumoniae culture medium:

[0025] (1) PPLO Both 21 g (purchased from BD Biosciences, USA);

[0026] (2) Yeast powder 25 g (yeast powder purchased from OXOID, UK);

[0027] (3) 1M NaOH (NaOH powder was purchased from Tianjin Damao Chemical Reagent Factory. 1M NaOH can be prepared by dissolving an appropriate amount of powder in deionized water)

[0028] (4) 1% phenol red 3 mL (phenol red powder was purchased from Beijing Solebow Technology Co., Ltd. 1% phenol red solution needs to be prepared in advance. The preparation process is as follows: weigh 1 g of phenol red powder in a mortar and pestle, add 1 M sodium hydroxide solution dropwise, and continue grinding until the phenol red powder is completely dissolved. Aspirate the dissolved phenol red into a 100 mL volumetric flask and then make up to 100 mL with deionized water);

[0029] (5) 200 mL of sterile porcine serum (purchased from Zhengzhou Pingrui Biotechnology Co., Ltd.)

[0030] (6)MEM 100 mL (purchased from GIBCO, USA)

[0031] (7) 1 mL of 50 mg / mL ampicillin sodium (purchased from Beijing Solebow Technology Co., Ltd., 50 mg / mL ampicillin sodium needs to be prepared in advance. The preparation process is as follows: weigh 0.5 g of ampicillin sodium powder, add 10 mL of deionized water to completely dissolve it, filter it through a 0.22 μm filter, and then package and store it at -20°C for use).

[0032] Mix (1), (2), and (4), add deionized water to fully dissolve, and adjust the pH to 7.8-8.0 with 1M NaOH. Sterilize at 121°C for 20 minutes. After the temperature drops to room temperature, add (5), (6), and (7). This completes the preparation of the culture medium.

[0033] Example 2

[0034] Preparation of simplified Mycoplasma ovipneumoniae culture medium:

[0035] All chemical reagents used are conventional reagents and can be purchased commercially.

[0036] Prepare 1000 mL of Mycoplasma ovipneumoniae culture medium:

[0037] (1)PPLO Both 18g;

[0038] (2) Yeast powder 21g;

[0039] (3) 1M NaOH;

[0040] (4) 1% phenol red 2 mL;

[0041] (5) 180 mL of sterile porcine serum;

[0042] (6) MEM medium 80 mL;

[0043] (7) 0.9 mL of 50 mg / mL ampicillin sodium.

[0044] Mix (1), (2), and (4), add deionized water to fully dissolve, and adjust the pH to 7.8-8.0 with 1M NaOH. Sterilize at 121°C for 20 minutes. After the temperature drops to room temperature, add (5), (6), and (7). This completes the preparation of the culture medium.

[0045] Example 3

[0046] Preparation of simplified Mycoplasma ovipneumoniae culture medium:

[0047] Prepare 1000 mL of Mycoplasma ovipneumoniae culture medium:

[0048] (1)PPLO Both 24g;

[0049] (2) Yeast powder 29g;

[0050] (3) 1M NaOH;

[0051] (4) 1% phenol red 4 mL;

[0052] (5) 220 mL of sterile porcine serum

[0053] (6) MEM culture medium 120 mL

[0054] (7) 1.1 mL of 50 mg / mL ampicillin sodium.

[0055] Mix (1), (2), and (4), add deionized water to fully dissolve, and adjust the pH to 7.8-8.0 with 1M NaOH. Sterilize at 121°C for 20 minutes. After the temperature drops to room temperature, add (5), (6), and (7). This completes the preparation of the culture medium.

[0056] Experimental example

[0057] Detection method:

[0058] Viable bacterial titer (CCU) determination: Take 12 centrifuge tubes, add 900 μL of the culture medium obtained in Example 1 to each tube, add 100 μL of well-grown Mycoplasma ovipneumoniae culture to the first tube, mix thoroughly with a shaker, replace the pipette tip, draw 100 μL from the first tube and add it to the second tube, and dilute tenfold to the eleventh tube in sequence. Discard 100 μL of the culture medium from the eleventh tube to obtain the dilution of the culture medium of 10, 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 100, 200, 300, 400, 500, 600, 700, 800, 900, 1000, 1000, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10000, 10000, 20000, 30000, 40000, 50000, 60000, 80000, 90000, 100000, 200000, 3 ... -1 -10 -11 The twelfth tube is the corresponding culture medium control. Set up three replicates. Place the centrifuge tube in a 37°C constant temperature incubator and incubate statically. Observe the color change daily for two weeks. The highest dilution that produces the color change is the viable bacterial titer of the culture, expressed in color change units (CCU). Repeat the experiment three times.

[0059] result:

[0060] The results of three tests showed that the culture medium of Example 1 of the present invention was used to culture Mycoplasma ovipneumoniae, and the CCU determination results of the three tests were all 10 9 CCU / mL.

[0061] The growth of Mycoplasma ovipneumoniae in the culture medium of Example 1 of the present invention is as follows: Figure 2 shown.

[0062] 900 μL of the culture medium obtained in Example 2 and Example 3 were added to each tube respectively, and the remaining experimental steps were the same as above.

[0063] result:

[0064] The results of three tests showed that the culture medium of Examples 2 and 3 of the present invention was used to culture Mycoplasma ovipneumoniae, and the CCU determination results of the three tests were all 10 8 CCU / mL.

[0065] The inventors measured the strain growth titers of the three examples mentioned above and found that Example 1 had a better effect than the other two examples; the highest growth titer of Example 1 was one order of magnitude higher than that of the other two examples.

[0066] The above specific embodiments do not limit the scope of protection of the present invention. Those skilled in the art will appreciate that various modifications, combinations, sub-combinations, and substitutions may occur depending on design requirements and other factors. Any modifications, equivalent substitutions, and improvements made within the spirit and principles of the present invention are intended to be included within the scope of protection of the present invention.

Claims

1. A method for preparing a simple Mycoplasma ovipneumoniae culture medium, characterized in that: The following steps are involved: Mix 18-24 g / L of PPLOBoth, 21-29 g / L of yeast extract, and 2-4 mL / L of 1% phenol red, then adjust the pH to 7.8-8.0 with 1 M NaOH. After high-pressure steam sterilization, wait until the temperature drops to room temperature and add 180-220 mL / L of porcine serum, 80-120 mL / L of MEM medium, and 0.9-1.1 mL / L of 50 mg / mL ampicillin.

2. The preparation method according to claim 1, characterized in that Autoclave at 121°C for 20 min.

3. The preparation method according to claim 1, characterized in that The PPLO Both was 21 g / L, the yeast powder was 25 g / L, and the 1% phenol red was 3 mL / L.

4. The preparation method according to claim 1 or 3, characterized in that The porcine serum was 200 mL / L, the MEM culture medium was 100 mL / L, and the 50 mg / mL ampicillin was 1 mL / L.

5. A simplified culture medium for Mycoplasma ovipneumoniae, characterized in that: Prepared by the method according to any one of claims 1 to 4.

6. Use of the simplified Mycoplasma ovipneumoniae culture medium according to claim 5 in culturing Mycoplasma ovipneumoniae.

7. Use of the simplified Mycoplasma ovipneumoniae culture medium according to claim 5 in preparing Mycoplasma ovipneumoniae vaccine antigens.

Images