Isolated culture method of Mycoplasma capricolum subsp.Capripneumoniae (Mccp)

A technology for separating and culturing mycoplasma, which is applied in the field of separating and culturing Mycoplasma capricolum subsp. capricolum, can solve the problems that have not yet been seen, and achieves the effects of avoiding unstable differences between batches, high separation success rate, and high titer of viable bacteria.

Pending Publication Date: 2019-12-10
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

At present, there is no research report on the isolation and cultivation of Mycoplasma goat pneumoniae subspecies by chicken embryo culture method at home and abroad

Method used

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  • Isolated culture method of Mycoplasma capricolum subsp.Capripneumoniae (Mccp)
  • Isolated culture method of Mycoplasma capricolum subsp.Capripneumoniae (Mccp)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] The method for isolating Mycoplasma capricoides goat pneumonia subspecies in pleural effusion samples is as follows:

[0028] (1) Treatment of disease samples: aseptically collect pleural effusion of fresh disease materials from goats with Mycoplasma goati subsp. Draw 1.0ml of the filtered sample solution into a sterile EP tube, add 200μl of 200,000 IU penicillin, mix well, place at 4°C for 24 hours, and store for later use.

[0029] (2) Sample inoculation: Dilute the processed sample solution 10 times with sterile 0.01M pH7.2 PBS to prepare 10- 1 Diluted sample solution, the sample treatment solution, 10 -1 The yolk sac of the diluted sample solution was inoculated with 7-day-old healthy SPF chicken embryos (with clear blood vessels and vigorous vitality), and each chicken embryo was inoculated with 0.2ml, and the sample processing stock solution, 10 -1 Two 7-day-old healthy SPF chicken embryos were inoculated with each dilution sample treatment solution. After the ...

Embodiment 2

[0034] The method for isolating Mycoplasma capricosum subsp. goat pneumonia in alveolar lavage fluid samples is as follows:

[0035] (1) Treatment of disease samples: aseptically collect alveolar lavage fluid from dead goats with Mycoplasma capricoides subsp. Draw 1.0ml of the filtered sample solution into a sterile EP tube, add 200μl of 200,000 IU penicillin, mix well, place at 4°C for 24 hours, and store for later use.

[0036] (2) Sample inoculation: Dilute the processed sample solution 10 times with sterile 0.01M pH7.2 PBS to prepare 10 -1 Diluted sample solution, the sample treatment solution, 10 -1 The yolk sac of the diluted sample solution was inoculated with 7-day-old healthy SPF chicken embryos (with clear blood vessels and vigorous vitality), and each chicken embryo was inoculated with 0.2ml, and the sample processing stock solution, 10 -1 Two 7-day-old healthy SPF chicken embryos were inoculated with each dilution sample treatment solution. After the sample inoc...

Embodiment 3

[0042] The method of the present invention is compared with the culture medium of the prior art on the separation of Mycoplasma capricoides goat pneumonia subspecies.

[0043] Under the same conditions, use the method of the present invention and the prior art (improved Thiaucourt's culture medium) to separate the disease material (Mycoplasma capricosum subspecies capricornus pneumonia PCR positive). The specific method is as follows: Aseptically collect and process 4 parts of pleural effusion or alveolar lavage fluid of goats that died of the disease, filter them with a sterile filter with a filter membrane of 0.45 μm, and store the filtrate for future use. Draw 1.0ml filter sample liquid and carry out separation and cultivation according to the method of the present invention; Get 0.5ml and inoculate into 4.5ml improved Thiaucourt's substratum in addition, cultivate in 37 ℃ of incubators, judge according to substratum color change and PCR identification result Mycoplasma goat...

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Abstract

The invention provides an isolated culture method of Mycoplasma capricolum subsp.Capripneumoniae (Mccp). The method comprises the following steps: (1), treatment of a pathological material: a treatedsample solution is obtained; (2), sample inoculation: the treated sample solution is diluted by 0.01 M of sterile PBS (phosphate buffer solution) with pH of 7.2, 7-8-day-old healthy SPF (specific pathogen free) chick embryos are inoculated with the diluted sample solution, and after the inoculation, the SPF chick embryos are put in an incubator and incubated continuously, wherein the temperature of the incubator is 37.0-37.5 DEG C and the humidity is 50%; and (3), harvesting: the survival condition of the chick embryos is observed, dead chick embryos after the inoculation are preserved at 4 DEG C for 12-24 h, and chick embryo allantoic fluid is collected. The method can be applied to isolated culture of the Mccp conveniently and rapidly, a Mccp antigen containing no animal serum can be obtained conveniently and rapidly, and various problems caused by application of serum can be solved.

Description

technical field [0001] The invention belongs to the technical field of veterinary biology, and in particular relates to a method for isolating and cultivating Mycoplasma capricum subsp. goat pneumonia. Background technique [0002] Goat infectious pleuropneumonia, mainly caused by Mycoplasma capricosum subspecies goat pneumonia, is an OIE legally notifiable disease. Clinically, the main manifestations are fever, cough, runny nose, and dyspnea. In the new epidemic areas, it often has the characteristics of high morbidity and high mortality, which brings serious threats and economic losses to the sheep industry. [0003] Goat infectious pleuropneumonia is currently prevalent in some countries or regions in Asia and Africa, but there are few reports on the isolation of the pathogen, mainly because the subspecies of Mycoplasma capricoides goat pneumonia has extremely high nutritional requirements and is difficult to cultivate. In addition, the isolation of disease materials req...

Claims

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Application Information

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IPC IPC(8): C12N1/02C12N1/20C12R1/35
CPCC12N1/02C12N1/20
Inventor 陈胜利储岳峰郝华芳颜新敏袁婷张晓亮刘永生
Owner LANZHOU INST OF VETERINARY SCI CHINESE ACAD OF AGRI SCI
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