CN110551634A - separation culture method for mycoplasma capricolum goat pneumonia subspecies - Google Patents
separation culture method for mycoplasma capricolum goat pneumonia subspecies Download PDFInfo
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- CN110551634A CN110551634A CN201910833143.3A CN201910833143A CN110551634A CN 110551634 A CN110551634 A CN 110551634A CN 201910833143 A CN201910833143 A CN 201910833143A CN 110551634 A CN110551634 A CN 110551634A
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/02—Separating microorganisms from their culture media
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- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/20—Bacteria; Culture media therefor
Abstract
The invention provides a separation culture method of mycoplasma capricolum goat pneumonia subspecies, which comprises the following steps: (1) treating a disease sample to obtain a treated sample solution; (2) sample inoculation: inoculating 7-8-day-old healthy SPF (specific pathogen free) chick embryos with the treated sample liquid or diluted sample liquid obtained by diluting with sterile 0.01MpH7.2PBS, and after inoculation, placing the SPF chick embryos in an incubator to continue incubation, wherein the temperature of the incubator is 37.0-37.5 ℃, and the humidity is 50%; (3) harvesting: observing the survival condition of the chick embryos, storing the dead chick embryos after inoculation at 4 ℃ for 12-24h, and collecting the chick embryo allantoic fluid. The method can be conveniently and quickly used for the separation culture of mycoplasma capricolum subspecies pneumonia of goat, can conveniently and quickly obtain mycoplasma capricolum subspecies pneumonia antigen without animal serum, and can overcome a plurality of problems caused by the use of serum.
Description
Technical Field
The invention belongs to the technical field of veterinary biology, and particularly relates to a mycoplasma capricolum goat pneumonia subspecies separation culture method.
background
Contagious caprine pleuropneumonia, mainly caused by mycoplasma caprine and subspecies pneumonia, is an OIE official report epidemic disease. Clinically, the symptoms are fever, cough, rhinorrhea, dyspnea, etc. The disease rate and the death rate of the sheep are high in the newly epidemic area, and serious threat and economic loss are brought to the sheep raising industry.
Goat contagious pleuropneumonia is mainly prevalent in Asia and Africa at present in some countries or regions, but the pathogen isolation reports are few, mainly because of the fact that mycoplasma capriae subspecies pneumoniae has extremely high requirements on nutrition, is difficult to culture, and has high requirements on isolating pathogens. Serological positivity of the disease in most provincial sheep-raising areas in China, but isolated strains are rarely reported.
The mycoplasma capricolum goat pneumonia subspecies are very difficult to culture, the disease caused by the pathogen, namely contagious pleuropneumonia capricolum, is discovered at the earliest in 1873, and the mycoplasma capricolum goat pneumonia subspecies are separated after more than 100 years. The Chinese has the report of goat contagious pleuropneumonia in the last 20 th century, the pathogen is isolated for the first time by the Haerbin veterinary research institute in 2007, and the pathogen is isolated by the laboratory of the Lanzhou veterinary research institute in the same year. The reason that the mycoplasma capricolum subsp pneumoniae of goats is difficult to culture is that no suitable culture method or culture medium exists. At present, the most common culture medium for separating and culturing mycoplasma capricolum and subspecies pneumonia of goats is modified Thiaucurt's culture medium (MTB culture medium). Modified Hayflick's medium, modified KM2 medium, etc. were also used to culture Mycoplasma capricolum subsp pneumoniae, with culture titers inferior to that of modified Thiaucurt's medium. In the prior art, the culture medium is complex in components, contains a plurality of nutrient components such as carbon sources, nitrogen sources, proteins, inorganic salts and the like, is usually as many as 8-15 or more, is complex in preparation steps, and still has the problems of low viable bacteria titer, poor reproductive capacity and the like. In addition, the in vitro growth culture of mycoplasma capricolum goat pneumonia subspecies depends on a certain concentration of animal serum, the serum content in the culture medium in the prior art is usually between 10% and 25%, and a low-serum high-efficiency culture medium lays a good foundation for the research and development of the disease vaccine. The source, quality and batch of serum easily cause the problems of large difference between culture medium batches, high cost and the like, and in addition, the serum-containing antigen has the problems of easy increase of side reaction, influence on immune effect, biological safety and the like.
Chick embryos are commonly used to isolate newcastle disease virus, influenza virus, and the like. At present, no research report of isolated culture of mycoplasma capricolum goat pneumonia subspecies by a chick embryo culture method is seen at home and abroad.
Disclosure of Invention
In order to solve the problems in the prior art, the invention provides a separation culture method of mycoplasma capricolum and subspecies pneumoniae.
the invention provides a separation culture method of mycoplasma capricolum goat pneumonia subspecies, which comprises the following steps:
(1) Treating a disease sample to obtain a treated sample solution;
(2) Sample inoculation: inoculating the treated sample liquid or diluted sample liquid obtained by diluting with sterile 0.01M PBS (phosphate buffer solution) with pH7.2 to healthy SPF (specific pathogen free) chick embryos of 7-8 days old, and after inoculation, placing the SPF chick embryos in an incubator to continue incubation, wherein the temperature of the incubator is 37.0-37.5 ℃, and the humidity is 50%;
(3) Harvesting: observing the survival condition of the chick embryos, storing the dead chick embryos after inoculation at 4 ℃ for 12-24h, and collecting the chick embryo allantoic fluid.
Preferably, in step (1), the disease sample is fresh disease pleural fluid or alveolar lavage fluid of the dead goat.
Preferably, in the step (1), the pathological sample treatment is to filter the fresh pathological pleural fluid or alveolar lavage fluid of the diseased and dead goat through a sterile filter with a 0.45 μm filter membrane, and sterilize the filtered sample fluid.
Preferably, in the step (2), the inoculation is yolk sac inoculation, and the inoculation amount is 0.2ml for each chick embryo.
Preferably, in step (2), the healthy SPF chick embryos have the following criteria: clear blood vessel and vigorous chick embryo.
Preferably, in step (3), the dead chick embryos after 1-12 days of inoculation are stored at 4 ℃ for 12-24 h.
The invention also provides the application of the mycoplasma capricolum goat pneumonia subspecies separation culture method in mycoplasma capricolum goat pneumonia subspecies separation culture.
The invention has the beneficial effects that:
Compared with the prior art, the method does not need to prepare the mycoplasma capricolum and pneumonia subspecies culture medium of the goat with complex components and complicated steps, avoids the problem of unstable difference between gene and serum batches cultured in the prior art, and has the advantages of low cost, convenient acquisition, high separation success rate, high viable bacteria titer and the like. The method can be used for conveniently and quickly separating and culturing the mycoplasma capricolum subspecies pneumonia of the goat, can conveniently and quickly obtain the mycoplasma capricolum subspecies pneumonia antigen without animal serum, and can overcome a plurality of problems caused by serum use.
Drawings
The accompanying drawings, which are included to provide a further understanding of the invention and are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description serve to explain the principles of the invention and not to limit the invention. In the drawings:
FIG. 1 shows the result of electrophoresis of the PCR product of chick embryo allantoic fluid.
Detailed Description
The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples are commercially available unless otherwise specified.
The method for separating and culturing the mycoplasma capricolum and the subspecies pneumonia of the goat comprises the following steps:
(1) And (3) treating a disease sample: aseptically collecting fresh morbid substances of the dead goat such as pleural effusion, alveolar lavage fluid, etc., filtering with sterile filter with 0.45 μm filter membrane, and collecting filtrate. Sucking 1.0ml of filtrate, placing the filtrate in a sterile EP tube, adding 200 mul of 20 ten thousand IU penicillin, fully and uniformly mixing, placing the mixture at 4 ℃ for acting for 12-24 hours, and preserving the mixture for later use.
(2) And (2) sample inoculation, namely diluting the processed sample liquid by 10 times of sterile PBS (0.01 MpH7.2) to prepare a sample liquid with a dilution of 10 -1, inoculating the sample treatment liquid and the sample liquid yolk sac with a dilution of 10 -1 to healthy SPF (specific pathogen free) chick embryos with the age of 7-8 days (the blood vessels are clear and the chick embryos are vigorous), inoculating 0.2ml of each chick embryo, respectively inoculating 2-3 healthy SPF chick embryos with the age of 7-8 days to the sample treatment stock solution and the sample treatment liquid with a dilution of 10 -1, and after the sample inoculation is finished, continuously incubating the chick embryos in an incubator for 12 days, wherein the temperature of the incubator is 37.0-37.5 ℃ and the humidity is 50%.
(3) Harvesting: observing the survival condition of chick embryos every day, preserving the chick embryos which die 1-12 days after inoculation at 4 ℃ for 12-24h, harvesting chick embryo allantoic fluid, placing the chick embryo allantoic fluid in a sterile test tube or an EP tube, and marking.
Example 1
The method for separating mycoplasma capricolum and pneumonia subspecies in the pleural effusion sample comprises the following steps:
(1) And (3) treating a disease sample: aseptically collecting fresh pathological breast water of the dead goat after the mycoplasma capricolum pneumonia subspecies of the goat, filtering by using an aseptic filter with a filter membrane of 0.45 mu m, and storing the filtrate for later use. Sucking 1.0ml of the filtered sample liquid, placing the filtered sample liquid in a sterile EP tube, adding 200 mul of 20 ten thousand IU penicillin, fully and uniformly mixing, placing the mixture at 4 ℃ for 24 hours, and preserving the mixture for later use.
(2) and (2) sample inoculation, namely diluting the processed sample liquid by 10 times of sterile 0.01M PBS (phosphate buffer solution) with pH7.2 to prepare 10- 1 diluted sample liquid, inoculating the sample treatment liquid and the 10 -1 diluted sample liquid egg yolk sac into 7-day-old healthy SPF (specific pathogen free) chick embryos (with clear blood vessels and vigorous chick embryo activity), inoculating 0.2ml of each chick embryo, respectively inoculating 2 7-day-old healthy SPF chick embryos with the sample treatment stock solution and the 10 -1 diluted sample treatment liquid, and after the sample inoculation is finished, continuously incubating the chick embryos in an incubator for 12 days, wherein the incubator is 37.0-37.5 ℃ in temperature and 50% in humidity.
(3) Harvesting: observing the survival condition of the chick embryos every day, preserving the chick embryos which die 1-12 days after inoculation at 4 ℃ for 20h, harvesting the chick embryo allantoic fluid, placing the chick embryo allantoic fluid in a sterile test tube or an EP tube, and marking.
(4) taking part of chick embryo allantoic fluid to perform PCR identification and viable bacteria titer determination on mycoplasma capricolum subspecies pneumonia of goat, performing PCR identification on an upstream primer 5'-ATCATTTTTAATCCCTTCAAG-3' and a downstream primer 5'-TACTATGAGTAATTATAATATATGCAA-3', performing PCR system on a PCRmix 12.5 mu L, an upstream primer 1 mu L, a downstream primer 1 mu L, DNA 2 mu L, a ddH 2 O8.5 mu L and a 25 mu L system, and performing PCR reaction under the conditions of 95 ℃ for 5min, 35 cycles (95 ℃ for 30s, 47 ℃ for 30s and 72 ℃ for 25s), 72 ℃ for 10min and 4 ℃ for storing a target fragment of 316 bp.
The result shows that the chick embryos start to die after the breast water sample is inoculated for 1 day, the allantoic fluid of the dead chick embryos is collected, the mycoplasma culture medium is inoculated, the color of the culture medium turns yellow, and the titer of viable bacteria reaches 10 8 CCU/ml, the PCR identification of the goat mycoplasma goat pneumonia subspecies is carried out on the allantoic fluid, the PCR reaction is positive, and the result is shown in figure 1.
Example 2
The method for separating mycoplasma capricolum and goat pneumonia subspecies from alveolar lavage fluid samples comprises the following steps:
(1) And (3) treating a disease sample: and (3) aseptically collecting alveolar lavage fluid of the goat mycoplasma capricolum subsp pneumoniae morbid and dead goat, filtering the alveolar lavage fluid by using a sterile filter with a 0.45-micron filter membrane, and storing the filtrate for later use. Sucking 1.0ml of the filtered sample liquid, placing the filtered sample liquid in a sterile EP tube, adding 200 mul of 20 ten thousand IU penicillin, fully and uniformly mixing, placing the mixture at 4 ℃ for 24 hours, and preserving the mixture for later use.
(2) And (2) sample inoculation, namely diluting the processed sample liquid by 10 times of sterile 0.01M PBS (phosphate buffer solution) with pH7.2 to prepare 10 -1 diluted sample liquid, inoculating the sample treatment liquid and 10 -1 diluted sample liquid egg yolk sac into 7-day-old healthy SPF (specific pathogen free) chick embryos (with clear blood vessels and vigorous chick embryo activity), inoculating 0.2ml of each chick embryo, respectively inoculating 2 7-day-old healthy SPF chick embryos with the sample treatment stock solution and the 10 -1 diluted sample treatment liquid, and after the sample inoculation is finished, continuously incubating the chick embryos in an incubator for 12 days, wherein the incubator is 37.0-37.5 ℃ in temperature and 50% in humidity.
(3) Harvesting: observing the survival condition of the chick embryos every day, and preserving the chick embryos which die 1-12 days after inoculation at 4 ℃ for 20 h. The chick embryo allantoic fluid is harvested and placed in a sterile test tube or an EP tube and labeled.
(4) Taking part of chick embryo allantoic fluid to perform PCR identification and viable bacteria titer determination on mycoplasma capricolum subspecies pneumonia of goat, performing PCR identification on an upstream primer 5'-ATCATTTTTAATCCCTTCAAG-3' and a downstream primer 5'-TACTATGAGTAATTATAATATATGCAA-3', performing PCR system on a PCRmix 12.5 mu L, an upstream primer 1 mu L, a downstream primer 1 mu L, DNA 2 mu L, a ddH 2 O8.5 mu L and a 25 mu L system, and performing PCR reaction under the conditions of 95 ℃ for 5min, 35 cycles (95 ℃ for 30s, 47 ℃ for 30s and 72 ℃ for 25s), 72 ℃ for 10min and 4 ℃ for storing a target fragment of 316 bp.
The result shows that the alveolar lavage fluid sample is inoculated with chicken embryos, the dead chicken embryos are collected and stored at 4 ℃ for later use, the allantoic fluid of the dead chicken embryos is collected, mycoplasma culture medium (improved Thiauport's culture medium) is inoculated, the color of the culture medium turns yellow, the viable bacteria titer reaches 10 9 CCU/ml, PCR identification is carried out on the allantoic fluid for mycoplasma capricolum and goat pneumonia subspecies, the PCR reaction is positive, and the result is shown in figure 1.
FIG. 1 shows the result of electrophoresis of the PCR product of chick embryo allantoic fluid. Wherein, M is DL5000 DNAmarker, 1 is a breast water sample and collects a chick embryo allantoic fluid PCR product, 2 is an alveolar lavage fluid sample and collects a chick embryo allantoic fluid PCR product, 3 is a positive control, and 4 is a negative control.
Example 3
Compared with the prior art, the method of the invention separates the mycoplasma capricolum subspecies pneumonia of the goat.
under the same conditions, the method of the invention is used for separating the pathological material (mycoplasma capricolum and goat pneumonia subspecies PCR positive) from the prior art (improved Thiaucorn's culture medium). The specific method comprises the following steps: 4 parts of pleural fluid or alveolar lavage fluid of the diseased and dead goats are collected and treated in an aseptic manner, filtered by a sterile filter with a 0.45-micron filter membrane, and the filtrate is stored for later use. Sucking 1.0ml of filtered sample liquid to perform separation culture according to the method of the invention; and inoculating 0.5ml of the modified Thiaucotrt's culture medium to 4.5ml of the modified Thiaucotrt's culture medium, culturing in an incubator at 37 ℃, and judging the separation condition of the mycoplasma capricolum and the subspecies pneumonia of the goat according to the color change of the culture medium and the PCR identification result. The results are shown in Table 1.
Wherein, the formula of the improved Thiaucourt's culture medium is as follows: PPLO broth (21g/L), sodium pyruvate (2g/L), glucose (2g/L), 25% yeast extract (100mL/L), 1% phenol red (2.5mL/L), inactivated horse serum (200mL/L), penicillin (20 ten thousand IU/L), deionized water (700mL/L), and adjusting the pH to 7.2-7.6 with 1mol/L NaOH.
TABLE 1
The results show that the method can completely separate the mycoplasma capricolum pneumonia subspecies from the diseased material with the separation rate of 100 percent (4/4), and the improved Thiaucourt's culture medium can also completely separate the mycoplasma capricolum pneumonia subspecies from 4 diseased materials.
For pleural fluid disease materials, the titer of the viable bacteria of the separated bacteria reaches 10 7 -10 8 CCU/ml, which is higher than the titer (10 6 -10 7 CCU/ml) of the viable bacteria of the separated bacteria of the conventional improved Thiaucourt's culture medium, for lavage fluid disease materials, the titer of the viable bacteria of the separated bacteria of the method reaches 10 9 CCU/ml, which is obviously higher than the titer (10 7 -10 8 CCU/ml) of the viable bacteria of the separated bacteria of the conventional improved Thiaucourt's culture medium.
Finally, it should be noted that: although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that changes may be made in the embodiments and/or equivalents thereof without departing from the spirit and scope of the invention. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention.
Claims (7)
1. A mycoplasma capricolum goat pneumonia subspecies separation culture method is characterized in that: the method comprises the following steps:
(1) Treating a disease sample to obtain a treated sample solution;
(2) Sample inoculation: inoculating the treated sample liquid or diluted sample liquid obtained by diluting with sterile 0.01M PBS (phosphate buffer solution) with pH7.2 to healthy SPF (specific pathogen free) chick embryos of 7-8 days old, and after inoculation, placing the SPF chick embryos in an incubator to continue incubation, wherein the temperature of the incubator is 37.0-37.5 ℃, and the humidity is 50%;
(3) harvesting: observing the survival condition of the chick embryos, storing the dead chick embryos after inoculation at 4 ℃ for 12-24h, and collecting the chick embryo allantoic fluid.
2. The method of claim 1, wherein: in the step (1), the disease sample is fresh disease pleural effusion or alveolar lavage fluid of the diseased and dead goat.
3. The method of claim 2, wherein: in the step (1), the pathological sample treatment is to filter fresh pathological pleural fluid or alveolar lavage fluid of the diseased and dead goats through a sterile filter with a 0.45-micron filter membrane and sterilize the filtered sample fluid.
4. The method of claim 1, wherein: in the step (2), the inoculation is yolk sac inoculation, and the inoculation amount is 0.2ml for each chick embryo.
5. The method of claim 1, wherein: in the step (2), the standard of the healthy SPF chick embryo is as follows: clear blood vessel and vigorous chick embryo.
6. The method of claim 1, wherein: in the step (3), the dead chick embryos inoculated for 1 to 12 days are stored at 4 ℃ for 12 to 24 hours.
7. The use of the isolated culture method of mycoplasma capricolum subsp pneumoniae of any one of claims 1-6 in isolated culture of mycoplasma capricolum subsp pneumoniae.
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