CN110938560A - Rapid and efficient mycoplasma hyopneumoniae culture medium and preparation method thereof - Google Patents
Rapid and efficient mycoplasma hyopneumoniae culture medium and preparation method thereof Download PDFInfo
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Abstract
The invention provides a rapid and efficient mycoplasma hyopneumoniae enrichment medium and a preparation method thereof, belonging to the technical field of veterinary biology. The culture medium comprises I, II and III parts; the main components of the feed comprise DMEM, hydrolyzed milk protein, HEPES, yeast extract, PPLO, BHI, arginine, glutamine, sodium pyruvate, DNA, acidified pig serum and the like; the culture medium is rapid, efficient and stable when culturing the mycoplasma hyopneumoniae, the mycoplasma hyopneumoniae seed liquid is inoculated into a 100ml culture medium system according to the ratio of 1: 13 in a laboratory, and the CCU of the mycoplasma hyopneumoniae can reach 10 within 40-48 hours11Per ml; the culture medium not only improves the culture titer of the mycoplasma hyopneumoniae, but also improves the growth rate of the mycoplasma hyopneumoniae, and when the culture medium is used for producing the antigen for the vaccine, the production benefit is improved, the production cost is reduced, and the economic benefit is obvious.
Description
Technical Field
The invention belongs to the technical field of veterinary biology, and particularly relates to a culture medium capable of quickly and efficiently culturing mycoplasma hyopneumoniae and a preparation method thereof.
Background
Mycoplasma hyopneumoniae mainly causes chronic respiratory diseases of pigs, is often characterized by secondary infection, can aggravate clinical symptoms of other infectious diseases, causes serious metabolic dysfunction and dysfunction of pigs, and is one of the most main infectious diseases causing economic loss in the pig industry. Generally, comprehensive prevention and treatment measures are mainly taken against the disease, including ideal breeding environment and use of medicines and vaccines; the ideal environment mainly relates to proper feeding density, feeding mode, temperature, ventilation and the like; in addition, although the drugs such as antibiotics have good effects on clinical diseases, large side effects and adverse consequences exist, and drug resistance, drug residues, meat product badness and the like are challenges; therefore, vaccine prevention will continue to become the main means for disease prevention in the future.
At present, in the preparation of mycoplasma hyopneumoniae vaccine antigens, a plurality of mycoplasma hyopneumoniae culture media are used; an inactivated cell culture medium with pig lung tissue, a pig lung buried block cell-free culture medium, a KM2 culture medium invented by Jiangsu agricultural academy of sciences, a Saimer Feisher (Thermo fisher) commercial Mycoplasma culture medium (OXID brand), and the like.
The traditional culture medium or commercial culture medium and laboratory culture medium have the problems of slow growth, low concentration, long culture time, high cost and the like, and restrict the production of mycoplasma hyopneumoniae antigens and finally influence the immune effect of vaccines.
Disclosure of Invention
In order to solve the above problems, the present invention provides a novel culture medium for mycoplasma hyopneumoniae.
The technical scheme of the invention comprises the following steps:
the mycoplasma hyopneumoniae basal medium dry powder is characterized in that: it comprises the following 3 parts:
part I:
and II, part:
42-60 parts by weight of PPLO powder,
40-60 parts of BHI powder;
part III:
when the weight parts are g, the molar parts are nmol.
The culture medium dry powder is characterized by comprising the following 3 parts:
part I:
and II, part:
42 parts by weight of PPLO powder,
40 parts of BHI powder;
part III:
a preparation method of a mycoplasma hyopneumoniae basal culture medium comprises the steps of taking part I, part II and part III of the culture medium dry powder according to the proportion, dissolving the parts in water, adjusting the pH value, respectively preparing 4400-;
preferably, the agent for adjusting the pH is a NaOH and HCl solution;
when parts by weight are g, parts by volume are ml.
Further, the culture medium dry powder is prepared by taking part I, part II and part III according to the proportion, dissolving the parts I, II and III in water, adjusting the pH value, respectively preparing 4900, 3000 and 100 parts by volume of aqueous solutions with the pH value of 7.4-7.6, and then mixing the aqueous solutions;
when parts by weight are g, parts by volume are ml.
Furthermore, a pH indicator is added into the aqueous solution corresponding to the part I; preferably, the pH indicator is phenol red;
and/or, antibiotics are added into the aqueous solution corresponding to the part I; preferably, the antibiotic is penicillin.
The mycoplasma hyopneumoniae basal medium prepared by any one of the methods.
The mycoplasma hyopneumoniae enrichment medium comprises the basic medium and acidified pig serum; the volume ratio of the basic culture medium to the acidified pig serum is (4-6) to 1.
The preparation method of the acidified pig serum comprises the following steps:
1) the pig serum is inactivated at 56 deg.C,
2) adjusting the pH value to 4.3-4.5 for acidification,
3) removing the precipitate to obtain a supernatant,
4) and adjusting the pH value to 7-8 to obtain the product.
In the mycoplasma hyopneumoniae enrichment medium,
the volume ratio of the basic culture medium to the acidified pig serum is 5: 1;
and/or in the preparation method of the acidified pig serum, the acidification duration of the step 2) is 4-24 h;
and/or, in the preparation method of the acidified pig serum, the temperature of the step 2) is 4 ℃.
The application of the mycoplasma hyopneumoniae basic culture medium or the mycoplasma hyopneumoniae enrichment culture medium in culturing mycoplasma hyopneumoniae is disclosed.
The rapid and efficient mycoplasma hyopneumoniae culture medium is optimized based on comprehensive analysis and application of knowledge of inorganic and organic chemistry, biochemistry, microbiology and the like; analyzing, combining and screening various nutrient components of the culture medium, such as osmotic pressure, pH value, a buffer system, a carbon source, a nitrogen source, vitamins, cholesterol, inorganic salt and the like; a culture medium for efficiently and rapidly culturing mycoplasma hyopneumoniae is developed; the method can obviously increase the culture titer of the mycoplasma hyopneumoniae and also has the characteristic of short culture time, and the advantages are relatively comprehensive. In the basic culture medium, besides further enriching basic nitrogen source and carbon source nutrient components, substances such as BHI, yeast extract and the like for increasing cholesterol and vitamins are innovatively added, and acid treatment pig serum with less harmful substances and a milder HEPES buffer system are introduced; as a result, the viable bacteria titer of mycoplasma in the developed culture medium reaches 1011To the power, the culture time is also shortened to 36-48h (30ml culture system). Therefore, the culture medium provided by the invention obviously improves the antigen yield and production efficiency of the mycoplasma hyopneumoniae, and makes a further contribution to preventing the mycoplasma hyopneumoniae disease.
Obviously, many modifications, substitutions, and variations are possible in light of the above teachings of the invention, without departing from the basic technical spirit of the invention, as defined by the following claims.
The present invention will be described in further detail with reference to the following examples. This should not be understood as limiting the scope of the above-described subject matter of the present invention to the following examples. All the technologies realized based on the above contents of the present invention belong to the scope of the present invention.
Detailed Description
EXAMPLE 1 preparation of the culture Medium of the present invention
Preparing a basic culture medium:
medium part I:
preparation of medium part I: dissolving the above components (1) - (6) in a certain amount of distilled water, adjusting pH to 7.6 with 5M NaOH, adding distilled water to constant volume of 490ml, filtering, sterilizing, and keeping at-20 deg.C.
Medium part II:
(8) PPLO powder 4.2g
(9) BHI powder 4.0g
(10) 300ml of distilled water
Preparation of medium part II: dissolving the components (8) - (9) in a certain amount of water one by one, adjusting the pH to 7.6 by using 5M NaOH, and then fixing the volume to 300ml by using distilled water; storing at 110 deg.C under high pressure for 15min and 4 deg.C.
Medium part III:
preparation of medium part III: dissolving the components (11) - (14) in a certain amount of distilled water one by one, adjusting pH to 7.6 with 40% fuming HCl, adding distilled water to a constant volume of 10ml, filtering for sterilization, and storing at-20 deg.C for later use.
The culture medium I, II and the part III are mixed according to the volume ratio of 49: 30: 1 to obtain 800ml of basal medium.
The preparation method of the acidified pig serum comprises the following steps:
1) inactivating 160ml of pig serum in 56 ℃ water bath for 30 min;
2) adjusting pH of pig serum to 4.3 with 40% HCl, and standing at 4 deg.C for 4-24 hr.
3) The precipitate was centrifuged at 2000rpm for 15min and filtered through filter paper.
4) Adjusting pH to 7.0 with 5M NaOH, filtering for sterilization, and storing at-20 deg.C for use.
The pig serum is subjected to the steps 1) to 4) to obtain 160ml of acidified pig serum.
Mixing 800ml of basic culture medium with 160ml of acidified pig serum to obtain a complete culture medium, subpackaging, and storing at-70 ℃ for later use.
EXAMPLE 2 preparation of the culture Medium of the present invention
Preparing a basic culture medium:
medium part I:
preparation of medium part I: dissolving the above components (1) - (6) in a certain amount of distilled water, adjusting pH to 7.6 with 5M NaOH, adding distilled water to constant volume of 490ml, filtering, sterilizing, and keeping at-20 deg.C.
Medium part II:
(8) PPLO powder 6g
(9) BHI powder 6g
(10) Distilled water to constant volume of 300ml
Preparation of medium part II: dissolving the components (8) - (9) in a certain amount of water one by one, adjusting the pH to 7.6 by using 5M NaOH, and then fixing the volume to 300ml by using distilled water; storing at 110 deg.C under high pressure for 15min and 4 deg.C.
Medium part III:
preparation of medium part III: dissolving the components (11) - (14) in a certain amount of distilled water one by one, adjusting pH to 7.6 with 40% fuming HCl, adding distilled water to 6.25ml, filtering for sterilization, and storing at-20 deg.C for use.
The culture medium I, II and the part III are mixed according to the volume ratio of 49: 30: 1 to obtain 800ml of basal medium.
The preparation method of the acidified pig serum comprises the following steps:
1) inactivating 100ml of pig serum in 56 deg.C water bath for 30 min;
2) adjusting pH of pig serum to 4.3 with 40% HCl, and standing at 4 deg.C for 4-24 hr.
3) The precipitate was centrifuged at 2000rpm for 15min and filtered through filter paper.
4) Adjusting pH to 7.0 with 5M NaOH, filtering for sterilization, and storing at-20 deg.C for use.
The pig serum is subjected to the steps 1) to 4) to obtain 100ml of acidified pig serum.
Mixing 500ml of basic culture medium with 100ml of acidified pig serum to obtain complete culture medium, subpackaging, and storing at-70 ℃ for later use.
EXAMPLE 3 preparation of the culture Medium of the present invention
Preparing a basic culture medium:
medium part I:
preparation of medium part I: dissolving the above components (1) - (6) in a certain amount of distilled water, adjusting pH to 7.6 with 5M NaOH, adding distilled water to constant volume of 490ml, filtering, sterilizing, and keeping at-20 deg.C.
Medium part II:
(8) PPLO powder 4.2g
(9) BHI powder 4.0g
(10) 300ml of distilled water
Preparation of medium part II: dissolving the components (8) - (9) in a certain amount of water one by one, adjusting the pH to 7.6 by using 5M NaOH, and then fixing the volume to 300ml by using distilled water; storing at 110 deg.C under high pressure for 15min and 4 deg.C.
Medium part III:
preparation of medium part III: dissolving the components (11) - (14) in a certain amount of distilled water one by one, adjusting pH to 7.6 with 40% fuming HCl, adding distilled water to a constant volume of 10ml, filtering for sterilization, and storing at-20 deg.C for later use.
The culture medium I, II and the part III are mixed according to the volume ratio of 44: 35: 1 to obtain 800ml of basal medium.
The preparation method of the acidified pig serum comprises the following steps:
1) inactivating 133.3ml of pig serum in 56 ℃ water bath for 30 min;
2) adjusting pH of pig serum to 4.3 with 40% HCl, and standing at 4 deg.C for 4-24 hr.
3) The precipitate was centrifuged at 2000rpm for 15min and filtered through filter paper.
4) Adjusting pH to 7.0 with 5M NaOH, filtering for sterilization, and storing at-20 deg.C for use.
The pig serum is subjected to the steps 1) to 4) to obtain 133.3ml of acidified pig serum.
Mixing 800ml of basic culture medium with 133.3ml of acidified pig serum to obtain a complete culture medium, subpackaging, and storing at-70 ℃ for later use.
EXAMPLE 4 preparation of the culture Medium of the present invention
Preparing a basic culture medium:
medium part I:
preparation of medium part I: dissolving the above components (1) - (6) in a certain amount of distilled water, adjusting pH to 7.6 with 5M NaOH, adding distilled water to constant volume of 490ml, filtering, sterilizing, and keeping at-20 deg.C.
Medium part II:
(8) PPLO powder 4.2g
(9) BHI powder 4.0g
(10) 300ml of distilled water
Preparation of medium part II: dissolving the components (8) - (9) in a certain amount of water one by one, adjusting the pH to 7.6 by using 5M NaOH, and then fixing the volume to 300ml by using distilled water; storing at 110 deg.C under high pressure for 15min and 4 deg.C.
Medium part III:
preparation of medium part III: dissolving the components (11) - (14) in a certain amount of distilled water one by one, adjusting pH to 7.6 with 40% fuming HCl, adding distilled water to a constant volume of 10ml, filtering for sterilization, and storing at-20 deg.C for later use.
The culture medium I, II and the part III are mixed according to the volume ratio of 54: 25: 1 to obtain 800ml of basal medium.
The preparation method of the acidified pig serum comprises the following steps:
1) inactivating 200ml of pig serum in 56 ℃ water bath for 30 min;
2) adjusting pH of pig serum to 4.3 with 40% HCl, and standing at 4 deg.C for 4-24 hr.
3) The precipitate was centrifuged at 2000rpm for 15min and filtered through filter paper.
4) Adjusting pH to 7.0 with 5M NaOH, filtering for sterilization, and storing at-20 deg.C for use.
The pig serum is subjected to the steps 1) to 4) to obtain 200ml of acidified pig serum.
Mixing 800ml of basic culture medium with 200ml of acidified pig serum to obtain a complete culture medium, subpackaging, and storing at-70 ℃ for later use.
Experimental example 1 culture and detection of Mycoplasma hyopneumoniae
A comparative culture test was conducted on Mycoplasma hyopneumoniae by using the medium of the present invention, KM2 medium and a commercial culture medium (mycoplasm broth).
The preparation of the medium of the present invention was the same as in example 1.
The culture and detection steps are as follows:
1) mycoplasma hyopneumoniae strains and culture conditions
After the mycoplasma hyopneumoniae strain SA stored in a laboratory is taken out from a refrigerator at the temperature of-70 ℃ and dissolved, the mycoplasma hyopneumoniae strain SA is respectively inoculated in a fast and efficient culture medium, a KM2 culture medium and a commercial mycoplasma culture medium (mycoplasm brothbase) at the temperature of 37 ℃ for recovery culture, and the culture systems are respectively 2 ml; after the color of each culture medium turns yellow, transferring two generations of rejuvenation respectively in the same inoculation mode; after rejuvenation, the respective culture broth was inoculated at 8% (V/V) into respective 20ml of culture medium for amplification culture, and when the culture medium became red and yellow (ph changed from 7.6 to 6.5), the culture was taken out.
2) Determination of viable bacteria titer (CCU) of Mycoplasma hyopneumoniae
(1) A96-well cell culture plate and a certain amount of rapid and efficient mycoplasma hyopneumoniae culture medium are prepared.
(2) The medium was added to a 96-well plate with a calandria gun, 180ul was added per well, 11 dilution gradients and 1 negative control were made, and 3 parallel groups were made for each mycoplasma hyopneumoniae bacterial solution.
(3) And sucking 20ul of each of the cultured 3 corresponding mycoplasma hyopneumoniae bacterial solutions, adding the sucked 20ul of each mycoplasma hyopneumoniae bacterial solution into the 1 st row of holes, uniformly mixing, and discarding the gun heads.
(4) Diluting from the first row of holes to the 11 th hole in a continuous gradient of 10 times (after diluting 1 hole each time, discarding the gun head and replacing with a new gun head to suck liquid, adding the liquid into the next hole and mixing uniformly)
3) BCA method (bicinchoninic acid) for measuring mycoprotein concentration of Mycoplasma hyopneumoniae
a. Preparation of mycoprotein of Mycoplasma hyopneumoniae
(1) 10ml of each of the 3 kinds of culture medium liquid is taken, centrifuged for 30 minutes at 8000, and then the supernatant is discarded.
(2) Resuspend 3 bacteria with equal volume of phosphate buffer, 8000 rotate to centrifuge for 30 minutes, then discard the supernatant.
(3) Repeat 2 steps 2 times.
(4) The mycoplasma cells were resuspended in an equal volume of distilled water.
(5) The bacteria are cracked by ultrasonic waves, and the parameters are set as (400W, working for 3 seconds, interval of 5 seconds and lasting for 30 minutes), so that the bacteria lysate is obtained.
b. Drawing of standard curve
(1) 7 PCR tubes were taken and 60ul ddH2O (in 3 parallel groups) was added to each tube, starting with tube 2.
(2) Pipette 120ul of BSA standard protein solution into the 1 st PCR tube.
(3) Then 60u BSA is sucked from the 1 st PCR tube and added into the 2 nd tube for multiple dilution, and so on.
(4) Then 20ul of BSA solution at each concentration was transferred to a 96-well plate and each gradient was repeated 3 times.
(5) 160ul of AB working solution was added to each standard protein well and mixed by shaking.
(6) Cover the lid and incubate at 37 ℃ for 30 min.
(7) Absorbance was measured at 562 nm.
(8) And drawing a standard curve of protein quantity and absorbance.
c. Determination of the mycoprotein content
20ul of lysate of the bacteria is absorbed and uniformly mixed with 160ul of AB working solution, after incubation for 30min at 37 ℃, the absorbance at 562nm is measured, and the protein concentration of the bacteria is obtained according to a standard curve.
4) Results
(1) The results of 3 growth tests CCU (color change Unit) of 3 kinds of culture media inoculated with Mycoplasma hyopneumoniae and the BCA method for determining the average value of the corresponding mycoprotein concentrations are shown in Table 1.
TABLE 1 results of determination of average mycoprotein concentration by means of CCU assay and BCA assay of Mycoplasma hyopneumoniae SA strains inoculated with 3 media for 3 growth tests
From the results of measurement of viable cell titer (CCU) of Mycoplasma hyopneumoniae (SA strain) cultured with the medium of the present invention, KM2 medium and Mycoplasma nutrition BROTH (MYCOPLASMA BROTH BASE (OXID)), it was found that the medium of the present invention (ADH) andthe KM2 culture medium has basically consistent culture time and can reach the maximum value (CCU) of the growth of the respective culture medium, but the culture medium (ADH) of the invention is at least 100 times higher than the viable titer (CCU) of the mycoplasma hyopneumoniae cultured by the KM2 culture medium; the content of mycoprotein determined by the BCA method shows that the culture medium is 2.57 times of that of the KM2 culture medium; mycoplasma nutrient broth (Mycoplas bacterium BASE (OXID)) is a commercial medium purchased from Siemens Feishil Scientific, and mycoplasma cultured in this medium cannot be compared with those in 2 cases, regardless of viable bacterial titer (CCU) or mycoprotein content. In addition, the viable titer (CCU) measured in the culture medium (ADH) was 5X 1010Taking into account the errors of the measurement method and according to the comparison of the mycoprotein content with the values described in the foreign literature (mycoprotein content 0.3mg/ml corresponds to a CCU of 1011) Therefore, the test result also can conclude that the titer (CCU) of the viable mycoplasma hyopneumoniae bacteria cultured by the culture medium (ADH) can reach 1011。
In conclusion, the mycoplasma hyopneumoniae enrichment culture medium disclosed by the invention has the characteristics of high culture efficiency, high growth speed and the like when used for culturing mycoplasma hyopneumoniae, and has a good application prospect.
Claims (10)
1. The mycoplasma hyopneumoniae basal medium dry powder is characterized in that: it comprises the following 3 parts:
part I:
and II, part:
42-60 parts by weight of PPLO powder,
40-60 parts of BHI powder;
part III:
when the weight parts are g, the molar parts are nmol.
2. The dry media powder of claim 1, comprising the following 3 parts:
part I:
and II, part:
42 parts by weight of PPLO powder,
40 parts of BHI powder;
part III:
3. a preparation method of a mycoplasma hyopneumoniae basal culture medium is characterized in that part I, part II and part III are taken according to the proportion of claim 1 or 2 and respectively dissolved in water, the pH value is adjusted to prepare 4400-;
preferably, the agent for adjusting the pH is a NaOH or HCl solution;
when parts by weight are g, parts by volume are ml.
4. The preparation method of the mycoplasma hyopneumoniae basal medium according to claim 3, wherein the preparation method comprises the steps of dissolving the part I, the part II and the part III in water respectively according to the proportion of claim 1 or 2, adjusting the pH value to prepare 4900, 3000 and 100 parts by volume of aqueous solutions with the pH value of 7.4-7.6, and mixing the aqueous solutions to obtain the mycoplasma hyopneumoniae basal medium;
when parts by weight are g, parts by volume are ml.
5. The method for preparing a basic culture medium for Mycoplasma hyopneumoniae according to claim 3 or 4, wherein:
a pH indicator is added into the corresponding aqueous solution of the part I; preferably, the pH indicator is phenol red;
and/or, antibiotics are added into the aqueous solution corresponding to the part I; preferably, the antibiotic is penicillin.
6. A basic culture medium for Mycoplasma hyopneumoniae prepared by the method of any one of claims 3 to 5.
7. A mycoplasma hyopneumoniae enrichment medium comprising the basal medium of claim 6, and acidified swine serum; the volume ratio of the basic culture medium to the acidified pig serum is (4-6) to 1;
the preparation method of the acidified pig serum comprises the following steps:
1) the pig serum is inactivated at 56 deg.C,
2) adjusting the pH value to 4.3-4.5 for acidification,
3) removing the precipitate to obtain a supernatant,
4) and adjusting the pH value to 7-8 to obtain the product.
8. The mycoplasma hyopneumoniae enrichment medium of claim 7, wherein:
the volume ratio of the basic culture medium to the acidified pig serum is 5: 1.
9. The mycoplasma hyopneumoniae enrichment medium of claim 7 or 8, wherein: in the step 2) of the preparation method of the acidified pig serum, the acidification duration is 4-24 hours; and/or, the acidification temperature is 4 ℃.
10. Use of the mycoplasma hyopneumoniae basal medium or the mycoplasma hyopneumoniae enrichment medium of any one of claims 6-9 for culturing mycoplasma hyopneumoniae.
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| CN110938560B (en) | 2023-08-18 |
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