CN112342158B - Mycoplasma gallisepticum culture medium and preparation method thereof - Google Patents
Mycoplasma gallisepticum culture medium and preparation method thereof Download PDFInfo
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Abstract
The invention relates to a mycoplasma gallisepticum culture medium and a preparation method thereof, wherein the mycoplasma gallisepticum culture medium comprises a basic culture medium and auxiliary componentsThe basic culture medium comprises phosphate buffer solution, PPLO broth, glucose, yeast extract powder, phenol red and trehalose; the auxiliary components comprise pig serum and 80 ten thousand units/ml penicillin. The culture medium of the invention obviously improves the antigen content of the mycoplasma avian bacterial liquid, and the titer of the bacterial liquid reaches up to 10 14‑15 CCU/ml is higher than the quality standard of the prior vaccine semi-finished bacterial liquid; the method of the invention improves the antigen content of mycoplasma gallisepticum, simplifies the production process and reduces the production cost.
Description
Technical Field
The invention relates to the technical field of biology, in particular to a mycoplasma gallisepticum culture medium and a preparation method thereof.
Background
Mycoplasma Gallisepticum (MG) infection, also known as Chronic Respiratory Disease (CRD), is widespread in all poultry-raising countries of the world. After a chicken farm is infected with MG, the weak chick rate of chicks is increased, the laying rate of laying hens, the weight of broilers and the feed conversion rate are all reduced, secondary bacterial or viral infection is easy to occur, the death rate of the chicks is increased, and serious damage is caused to the development of the chicken raising industry.
MG is small in genome, and lacks of a plurality of necessary genes for coding amino acid and cofactor biosynthesis in the genome, so that energy metabolism is slow, the MG belongs to auxotrophy of amino acid, lipid and certain cofactor, and needs to obtain a plurality of nutrients from the outside to promote metabolic propagation, so that the nutrient composition of a culture medium is complex, and the culture condition is harsh. Currently, conventional media for MG isolation and MG vaccine production are developed clinically from Frey or PPLO media.
However, the existing traditional culture medium has low MG viable gradient (at 1.0X 10) 8-10 CCU/ml), long culture time (24-48 hr), low antigen protein concentration (30-50ug/ml), and no need of producing inactivated seedling with bacterial liquid (semi-finished product) for preparing seedlingThe method can be directly used, the concentration operation of the semi-finished product is heavy and complex, the loss of the concentration process is large, the protective efficacy of the finished vaccine is finally influenced, the production cost of the vaccine is increased, the serum added for vaccine production of the existing traditional culture medium is generally between 10 and 30 percent, the vaccine prepared by excessive serum undoubtedly increases the allergic stress reaction of the heterologous body to the chicken, the immune efficacy of the vaccine is finally influenced, and the production cost is increased: when the existing traditional culture medium is used for MG separation, the pathogen detection rate is low, the growth is slow (the time required for primary separation is averagely 7-12d), and diagnosis and clinical drug screening are not facilitated.
Therefore, there is an urgent need in the art for a culture medium for efficiently culturing mycoplasma gallisepticum, and further for research on diagnosis and control of MG infection.
Disclosure of Invention
Aiming at the defects of the prior art, the invention provides the mycoplasma gallisepticum culture medium and the preparation method thereof, which not only simplify the complex components of the traditional culture medium, reduce the production cost of enterprises, but also reduce the use of components such as serum and the like, and reduce the anaphylactic reaction of exogenous foreign matters to chickens. Meanwhile, the culture medium can ensure that the viable bacteria titer of the mycoplasma gallisepticum is as high as 1 x 10 14 Above, the viable bacteria titer and the separation sensitivity are far higher than those of the culture medium in the prior art, and the method is more suitable for the separation and identification of mycoplasma gallisepticum and the vaccine production.
The invention is realized by the following technical scheme:
provides a mycoplasma gallisepticum culture medium, which comprises a basic culture medium and auxiliary components, and is characterized in that: the basic culture medium comprises phosphate buffer solution, PPLO broth, glucose, yeast extract powder, phenol red and trehalose; the auxiliary components comprise pig serum and 80 ten thousand units/ml penicillin.
The basic culture medium comprises the following components in parts by weight: 1000ml of phosphate buffer solution, 15-32 g of PPLO broth, 5-10 g of glucose, 1-5 g of yeast extract powder, 1-2ml of phenol red and 1-5 g of trehalose; the concentrations of the auxiliary components are as follows: 5-10% of pig serum and 1-2ml/L of 80 ten thousand units/ml penicillin.
The phosphate buffer solution contained 5.0g of sodium chloride, 0.4g of potassium chloride, 0.2g of magnesium sulfate heptahydrate, 1.6g of disodium hydrogen phosphate dodecahydrate, and 0.2g of potassium dihydrogen phosphate.
A preparation method of the mycoplasma gallisepticum culture medium comprises the following steps:
step one, preparing a basic culture medium: preparing a basic culture medium according to the formula of the components of the basic culture medium formula;
step two, adjusting the pH: adjusting the pH of the basic culture medium to 7.5-7.8 by using 10mol/LNaOH solution;
step three, sterilization: sterilizing the basic culture medium at 116 deg.C for 15-30 min;
step four, adding auxiliary components: adding auxiliary components such as serum and penicillin under aseptic condition, wherein the serum is added according to 5-10% of the total culture medium, and the penicillin is added according to the final concentration of 800U/ml;
and step five, adjusting the pH value of the mycoplasma gallisepticum culture medium to 7.5-7.8 by using 10mol/LNaOH solution, and storing at the temperature of 2-8 ℃.
An application of the mycoplasma gallisepticum culture medium in preparation of mycoplasma gallisepticum vaccines.
The invention has the beneficial effects that:
according to the invention, a phosphate buffer system is added into the culture medium, so that the pH stability of the culture medium is improved, mycoplasma can grow under the optimal condition, and the antigen yield is improved.
Secondly, the yeast extract in the CM culture medium is replaced by convenient, stable and efficient yeast extract powder, so that the production process is simplified, the labor is saved, and the product stability is improved. The antifungal thallium acetate was removed from the CM medium.
Thirdly, trehalose is added into the culture medium, so that the lag phase time is shortened, the passage time is shortened from 22 +/-1 h to about 18 +/-1 h, the production efficiency is improved, the titer of viable bacteria is improved, and the titer of bacteria liquid is as high as 10 14-15 CCU/ml is higher than the quality standard of the prior vaccine semi-finished bacterial liquid.
Detailed Description
In order to clearly explain the technical features of the present solution, the present solution is explained by the following detailed description.
A mycoplasma gallisepticum culture medium comprises a basic culture medium and auxiliary components, wherein the basic culture medium comprises phosphate buffer solution, PPLO broth, glucose, yeast extract powder, phenol red and trehalose; the auxiliary components comprise pig serum and 80 ten thousand units/ml penicillin.
The basic culture medium comprises the following components in parts by weight: 1000ml of phosphate buffer solution, 15-32 g of PPLO broth, 5-10 g of glucose, 1-5 g of yeast extract powder, 1-2ml of phenol red and 1-5 g of trehalose; the concentrations of the auxiliary components are as follows: 5-10% of pig serum and 1-2ml/L of 80 ten thousand units/ml penicillin.
The phosphate buffer solution contained 5.0g of sodium chloride, 0.4g of potassium chloride, 0.2g of magnesium sulfate heptahydrate, 1.6g of disodium hydrogen phosphate dodecahydrate, and 0.2g of potassium dihydrogen phosphate.
A preparation method of the mycoplasma gallisepticum culture medium comprises the following steps:
step one, preparing a basic culture medium: preparing a basic culture medium according to the formula of the components of the basic culture medium formula;
step two, adjusting the pH: adjusting the pH of the basic culture medium to 7.5-7.8 by using 10mol/LNaOH solution;
step three, sterilization: sterilizing the basic culture medium at 116 deg.C for 15-30 min;
step four, adding auxiliary components: adding auxiliary components such as serum and penicillin under aseptic condition, wherein the serum is added according to 5-10% of the total culture medium, and the penicillin is added according to the final concentration of 800U/ml;
and step five, adjusting the pH value of the mycoplasma gallisepticum culture medium to 7.5-7.8 by using 10mol/LNaOH solution, and storing at the temperature of 2-8 ℃.
The mycoplasma gallisepticum culture medium can be applied to preparation of mycoplasma gallisepticum vaccines.
Example 2
1. Culture of mycoplasma gallisepticum bacterial liquid
Mycoplasma gallisepticum (CR strain) production seeds were inoculated at a volume ratio (V: V) of 10% into CM liquid medium, modified Frey medium and the medium of example 1, mixed well, and then subjected to static culture at 37 ℃ to harvest when the color of the medium turned yellow and the pH decreased from 7.6 to 6.4. Passage 3 was followed in the same manner and passage time was recorded.
2. Viable bacterial titer (CCU) assay
26 small test tubes (outer diameter (mm) × length (mm) 12: 100) are taken for each sample, 1.8ml of culture medium is filled in each tube, 0.2ml of culture sample needing to be tested for viable bacterial titer is added in the 1 st test tube, the mixture is uniformly mixed on a micro-rotary oscillator, 0.2ml of the mixture is sucked and added in the 2 nd test tube, the serial dilution is carried out to the 12 th tube in the way, the 13 th test tube is not added with the sample as a negative control, and the test is repeated for 2 times. The test tube is placed in a 37 ℃ incubator for static culture, the test tube is observed for 1 time at a certain time period (9:00-10:00) every day, the color change of the culture medium is mainly observed, the culture medium added with the sample is compared with a control group, the continuous observation is carried out for 7 days, and finally the dilution of the test tube with the color change is the CCU titer of the culture, and the test is carried out for 3 times in total.
3. Results
3.1 color Change Unit measurement results
The results of 3 growth tests CCU (color change units) of 3 media inoculated with the Mycoplasma gallisepticum CR strain are shown in the following Table:
determination results of 3 growth tests CCU of MG CR strain inoculated with 3 culture media:
5.2 passage time results
From the results of measurement of viable cell titer by the culture of Mycoplasma gallisepticum CR strain using the medium of the present invention, CM medium and modified Frey medium, 3 generations of the medium of Mycoplasma gallisepticum of the present invention were cultured under the same test conditionsTest results CCU at 10 14 -10 15 CCU/ml; and CM and modified Frey Medium 3 subculture CCU at 10 7.0-9.0 CCU/ml. The 3-generation culture time of the mycoplasma gallisepticum culture medium is 17-18 h; and the CM and the improved Frey culture medium have the culture time of 22-26h, which shows that the mycoplasma gallisepticum culture medium has the characteristics of capability of ensuring that the mycoplasma gallisepticum is at a higher growth speed and high viable bacteria titer.
In conclusion, the invention improves the culture medium of mycoplasma gallisepticum in the prior art through a series of improvement of culture modes, obtains a new culture medium formula, and simultaneously provides the preparation method of the culture medium. The method of the invention improves the antigen content of mycoplasma gallisepticum, simplifies the production process and reduces the production cost.
Of course, the above description is not limited to the above examples, and the undescribed technical features of the present invention can be implemented by or using the prior art, and will not be described herein again; the above embodiments are merely for illustrating the technical solutions of the present invention and not for limiting the present invention, and the present invention has been described in detail with reference to the preferred embodiments, and those skilled in the art should understand that changes, modifications, additions or substitutions which are made by those skilled in the art within the spirit of the present invention are also within the scope of the claims of the present invention.
Claims (3)
1. A mycoplasma gallisepticum culture medium consists of a basic culture medium and auxiliary components, and is characterized in that: the basic culture medium consists of phosphate buffer solution, PPLO broth, glucose, yeast extract powder, phenol red and trehalose; the auxiliary components consist of pig serum and 80 ten thousand units/ml penicillin; the basic culture medium comprises the following components in parts by weight: 1000ml of phosphate buffer solution, 15-32 g of PPLO broth, 5-10 g of glucose, 1-5 g of yeast extract powder, 1-2ml of phenol red and 1-5 g of trehalose; the concentrations of the auxiliary components are as follows: 5-10% of pig serum and 1-2ml/L of 80 ten thousand units/ml penicillin; wherein: the phosphate buffer solution contained 5.0g of sodium chloride, 0.4g of potassium chloride, 0.2g of magnesium sulfate heptahydrate, 1.6g of disodium hydrogen phosphate dodecahydrate, and 0.2g of potassium dihydrogen phosphate.
2. A method for preparing the Mycoplasma gallisepticum culture medium of claim 1, comprising: the method comprises the following steps:
step one, preparing a basic culture medium: preparing a basic culture medium according to the basic culture medium formula;
step two, adjusting the pH: adjusting the pH of the basic culture medium to 7.5-7.8 by using 10mol/LNaOH solution;
step three, sterilization: sterilizing the basic culture medium at 116 deg.C for 15-30 min;
step four, adding auxiliary components: adding serum and penicillin under aseptic condition, wherein the serum is added according to 5-10% of the total culture medium, and the penicillin is added according to the final concentration of 800U/ml;
and step five, adjusting the pH value of the mycoplasma gallisepticum culture medium to 7.5-7.8 by using 10mol/LNaOH solution, and storing at the temperature of 2-8 ℃.
3. Use of a mycoplasma gallisepticum culture medium according to claim 1 for the preparation of a mycoplasma gallisepticum vaccine.
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| CN102168073B (en) * | 2010-11-30 | 2013-03-20 | 瑞普（保定）生物药业有限公司 | Method for improving proliferation capability of mycoplasma gallisepticum in vaccine production |
| CN103074246B (en) * | 2012-08-31 | 2015-09-02 | 南京天邦生物科技有限公司 | A kind of low serum high-efficient culture chicken virus mycoplasma low virulent strain substratum and preparation method thereof |
| CN103060221A (en) * | 2012-08-31 | 2013-04-24 | 南京天邦生物科技有限公司 | Culture medium adopting continuous enrichment culture for mycoplasma gaujseptium and preparation method |
| CN106282300B (en) * | 2015-05-26 | 2019-12-24 | 临沂大学 | Mycoplasma gallisepticum detection method |
| CN106967651A (en) * | 2017-05-18 | 2017-07-21 | 隋兆峰 | A kind of chicken virus mycoplasma culture medium and preparation method thereof |
| CN108949606B (en) * | 2017-06-29 | 2021-08-27 | 兆丰华生物科技(南京)有限公司 | High-density fermentation culture process for mycoplasma gallisepticum |
| CN109913396B (en) * | 2019-04-22 | 2023-01-06 | 华中农业大学 | Liquid culture medium and method for separating and culturing mycoplasma gallisepticum by using same |
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