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Mycoplasma synoviae culture medium and preparation method thereof

A technology of mycoplasma synovialum and culture medium, which is applied in the field of mycoplasma synovialum culture medium and its preparation, can solve problems such as unfavorable diagnosis and clinical drug screening, affecting the protective efficacy of finished vaccines, and heavy concentration operations of semi-finished products, and achieves improved The effect of harvesting the total amount of antigen, improving product stability, and increasing the rate of material transfer

Active Publication Date: 2021-02-26
山东滨州沃华生物工程有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the existing traditional culture medium MS has a low titer of live bacteria (at 1.0×10 8-10 CCU / ml), the culture time is 24-48h long, the antigenic protein concentration is low (30-50ug / ml), and the bacterial liquid (semi-finished product) used for seedling production needs to be concentrated when producing inactivated seedlings, and the concentration operation of the semi-finished product is heavy, complicated and easy to operate. Pollution, large losses in the concentration process, ultimately affect the protective efficacy of the finished vaccine, and further increase the production cost of the vaccine. The serum added to the existing traditional culture medium is generally between 10-30%. Excessive serum preparation The vaccine will undoubtedly increase the allergic stress response of the heterologous body to the chicken, which will eventually affect the immune efficacy of the vaccine and increase the production cost
When the existing traditional medium is used for MS isolation, the pathogen detection rate is low and the growth is slow (the average time required for the initial isolation is 7-12 days), which is not conducive to diagnosis and clinical drug screening

Method used

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  • Mycoplasma synoviae culture medium and preparation method thereof
  • Mycoplasma synoviae culture medium and preparation method thereof
  • Mycoplasma synoviae culture medium and preparation method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] A culture medium for Mycoplasma gallisanum bursa, comprising basal medium and auxiliary components, the basal medium includes phosphate buffer, glucose, hydrolyzed milk protein, coenzyme I, arginine hydrochloride, MEM, yeast extract powder, 1 % phenol red and trehalose; auxiliary components include porcine serum and penicillin.

[0024] The weight of each component in the above basal medium is: 800-1000ml of phosphate buffer, 5-18g of glucose, 3-9g of hydrolyzed milk protein, 0.5-1.5g of coenzyme I, 5-15ml of arginine hydrochloride, L-cysteine ​​hydrochloride 0.5 ~ 1.5g, MEM 2 ~ 5g, yeast extract powder 1-5g, 1% phenol red, trehalose 1-5g; the concentration of each component in the above auxiliary components is: pig serum 5 -10% and 800,000 units / ml penicillin 1-2ml / L.

[0025] As an implementation of this example, in the culture medium: 900ml of phosphate buffer, 15g of glucose, 6g of hydrolyzed milk protein, 1g of coenzyme I, 10ml of arginine hydrochloride, 1g of L-c...

Embodiment 2

[0034] Take the chickens with obvious joint swelling in the clinically affected chicken flock, a total of 5 chicken farms, 6 chickens in each chicken farm. Separation is carried out by the MS medium, the improved Frey medium, and the improved CM medium in Example 1 of the present invention, respectively.

[0035] Take the trachea / cleft palate swab and joint fluid of each chicken and dissolve them in 5ml of MS medium, shake and mix well, use a 0.45um filter to sterilize, take 1ml of each and inoculate into the three kinds of medium, and let it stand at 37°C After 2 days of culture, if the color does not change, the first generation is blindly passed according to 10% of the inoculum size; if the second generation of blind passage does not change color after 10 days, it is negative; if the color of the medium changes, it is identified by PCR and sent for sequencing identification.

[0036] The separation results of the sensitivity of the three media to MS are as follows:

[0037...

Embodiment 3

[0040] 1. Culture of Mycoplasma gallinarum bursa

[0041] The culture medium selected in this experiment is Mycoplasma gallinarum WVU1853 strain, which was isolated and preserved clinically. Z1 seeds were inoculated in the improved CM medium, the improved Frey medium, and the MS medium of Example 1 according to the volume ratio (V:V) of 10%. After fully mixing, they were shaken and cultured at 100r / min at 37°C. When the color of the culture solution Turn yellow and harvest when the pH drops from 7.6 to 6.8. Passage for 3 generations in the same way, record the passage time and measure the discoloration unit and protein concentration of the 3rd generation.

[0042] 2. Viable bacterial titer (CCU) determination method

[0043]Take 28 small test tubes for each sample (outer diameter (mm) × length (mm) is 12:100), each tube is filled with 1.8ml of culture medium, and 0.2ml of the culture that needs to measure the titer of viable bacteria is added to the first test tube Mix the ...

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Abstract

The invention relates to a mycoplasma synoviae culture medium and a preparation method thereof. The culture medium comprises a basal culture medium and auxiliary components. The basal culture medium comprises a phosphate buffer solution, glucose, lactoalbumin hydrolysate, coenzyme I, arginine hydrochloride, L-cysteine hydrochloride, MEM, yeast extract powder, 1% phenol red, and trehalose. The auxiliary components comprise pig serum and penicillin. According to the culture medium, the antigen content of mycoplasma synoviae bacterial liquid is remarkably increased, the bacterial liquid titer reaches up to 1012.0-13.0 CCU / ml. Meanwhile, the separation rate of the MS wild strains is remarkably improved and is as high as 93%. According to the preparation method of the culture medium, the antigen content of mycoplasma synoviae is increased, the production process is simplified, and the production cost is reduced.

Description

technical field [0001] The invention relates to the technical field of biological culture, in particular to a culture medium for Mycoplasma gallisanum bursae and a preparation method thereof. Background technique [0002] Chicken synovial mycoplasma disease, also known as chicken infectious synovitis, is a joint exudative synovial fluid of chickens, ducks, geese, quails, pigeons, etc. caused by Mycoplasmasynoviae (MS) Acute or chronic infectious diseases characterized by capsulitis and tenosynovitis are generally not easy to observe and mainly manifest as subclinical symptoms. Mixed infection of MS with Newcastle disease virus, chicken infectious bronchial virus, Escherichia coli, etc. will aggravate the condition. MS belongs to the order Molliculata, and Mycoplasma synovia is different from other mycoplasmas. It was first proposed by Olson et al. in 1956 and designated as an independent species. Infection of chickens can lead to obvious lameness, stunted growth and carcas...

Claims

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Application Information

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IPC IPC(8): C12N1/20C12R1/35
CPCC12N1/20Y02A50/30
Inventor 朱杰翟庆贺杨灵芝耿兴良吴洪才李香云
Owner 山东滨州沃华生物工程有限公司
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