Method for separating mycoplasma ovipneumoniae

A technology for Mycoplasma pneumoniae and sheep, which is applied in the field of veterinary biotechnology research, can solve the problems of complex composition, batch differences of culture medium, poor reproductive ability, etc., and achieves the effects of high degree of cleanliness, high separation rate and simple operation.

Pending Publication Date: 2019-11-15
LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the prior art, the composition of the culture medium for Mycoplasma pneumoniae in ovis is complicated, and in addition to containing many nutritional components such as carbon source, nitrogen source, protein, inorganic salt, etc., it generally needs to add animal serum ranging from 10% to 20%
The preparation steps of the prior art culture medium are cumbersome, and often need to prepare as many as 8 to 9 kinds of reagents. In addition, it is often necessary to prepare reagents such as fresh yeast extrac

Method used

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  • Method for separating mycoplasma ovipneumoniae
  • Method for separating mycoplasma ovipneumoniae
  • Method for separating mycoplasma ovipneumoniae

Examples

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Embodiment 1

[0023] A method for separating Mycoplasma pneumoniae of the present invention, the steps are as follows:

[0024] (1) Sampling and sample processing: goat nasal swabs were collected with sterile cotton swabs. Add 3.0ml of sterilized 0.01M pH7.2 PBS to the nasal swab sample, shake fully with a shaker for 1min, squeeze the cotton swab clean with sterilized tweezers, discard the cotton swab, and make up with sterilized 0.01M pH7.2 PBS 3.5ml. The above-mentioned sample treatment solution was filtered and sterilized through a sterile filter with a filter membrane pore size of 0.45 μm, and the mycoplasma contained in the sample could pass through the filter. Take 1.0ml of the filtrate, add 200μl of penicillin with a final concentration of 200,000 units, and place it at 4°C for 15 hours to form the inoculum.

[0025] (2) Inoculation of chicken embryos: Inoculate 7-day-old healthy SPF chicken embryos with the processed sample inoculum solution, discard the fertilized eggs, dead embr...

Embodiment 2

[0031] The inventive method compares with prior art culture medium:

[0032] Under the same conditions, use the method provided by the present invention and the improved Thiaucourt's medium in the prior art to separate the PCR-positive nasal swab samples of Mycoplasma ovis pneumoniae. The specific method is as follows: add 3.0ml of sterilized 0.01M pH7.2 PBS to the goat nose swab collected by a sterile cotton swab, fully shake it with an oscillator for about 1min, squeeze the cotton swab with sterilized tweezers, discard the cotton swab, and use Sterile 0.01M pH 7.2 PBS made up to 3.5ml. The samples are processed according to the method of the invention, and chicken embryos are inoculated for isolating Mycoplasma pneumoniae of sheep. Another 0.5ml was inoculated into 4.5ml modified Thiaucourt's medium, 10 -1 ~10 -3 The culture medium was diluted two-fold, cultured in a 37°C incubator, and the color change of the culture medium was observed and identified by PCR to determine...

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Abstract

The invention provides a method for separating mycoplasma ovipneumoniae, which comprises the following steps: (1) collecting a sample and treating the sample to obtain a sample inoculation solution; (2) inoculating chick embryos; (3) separating the mycoplasma ovipneumoniae: collecting dead chick embryos after inoculation and/or non-dead chick embryos after 9 days, taking chick embryo allantoic fluid to carry out viable bacteria detection on the mycoplasma ovipneumoniae, and if the chick embryo allantoic fluid is collected to be positive, storing the material, if the chick embryo allantoic fluid of the first generation is negative, the collected chick embryo allantoic fluid is blindly transmitted to the chick embryo by the same method, and if the chick embryo allantoic fluid is still negative after three generations of blind transmission, determining the sample to be negative for mycoplasma ovipneumoniae, and discharging the material. The method disclosed by the invention is simple to operate, does not need to prepare a mycoplasma ovipneumoniae culture medium with complex components, and avoids the problem that in the prior art, high-pressure sterilization or a high-capacity filteris often needed in preparation of an isolation culture medium; and according to the method, the mycoplasma ovipneumoniae without animal serum can be obtained.

Description

technical field [0001] The invention belongs to the technical field of veterinary biotechnology research, and in particular relates to a method for isolating ovine mycoplasma pneumoniae. Background technique [0002] Mycoplasma ovis pneumoniae is a microorganism that can cause respiratory disease in sheep. Mycoplasma ovis can infect both sheep and goats. The pathogen can also be isolated from the nasal cavity of healthy sheep. The pathogen has a global distribution. The pathogen is currently prevalent in my country and poses a serious threat to the sheep industry. [0003] Mycoplasma ovis pneumoniae is relatively delicate, and its isolation and culture require special media. The improved Thiaucourt's medium is a commonly used isolation medium for Mycoplasma ovis pneumoniae. The effect of culturing Mycoplasma ovis pneumoniae is higher than that of other media such as the improved KM2 medium, and the effect is better. In the prior art, the composition of the culture mediu...

Claims

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Application Information

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IPC IPC(8): C12N1/20C12N1/02C12R1/35
CPCC12N1/02C12N1/20
Inventor 郝华芳陈胜利储岳峰袁婷颜新敏刘永生
Owner LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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