CN108486015A - A kind of culture medium and its application for Prevotella copri specificity screenings - Google Patents
A kind of culture medium and its application for Prevotella copri specificity screenings Download PDFInfo
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Abstract
The invention discloses a kind of culture medium for Prevotella copri specificity screenings and its applications, belong to microorganism field.Culture medium of the present invention is prepared convenient, it is at low cost, contain the substances such as vancomycin, kanamycins, sole carbon source and production acid indicator in culture medium, after being used cooperatively, chromogenic reaction can occur in solid culture primary surface using the Prevotella copri single bacterium colonies of the sole carbon source, the separative efficiency for improving more low-abundance Prevotella copri in flora sample is reached with this.
Description
Technical field
The present invention relates to a kind of culture medium for Prevotella copri specificity screenings and its applications, belong to micro- life
Object field.
Background technology
Prevotella copri are subordinated to general Bordetella, Bacteroidetes, are Gram-negative strict anaerobes, no matter
Grain；To oxygen and its sensitivity, can only in the environment of complete anaerobic well-grown.The polysaccharide such as metabolizable xylan, it is also metabolizable
The small molecular sugars such as hemicellulose, xylose.
Prevotella copri are one of human normal enterobacteriaceaes, but abundance is relatively low.Macro gene order-checking result is shown
Rheumatoid arthritis and psoriatic arthritis patient are compared with the P.copri containing excess in healthy population enteron aisle, and and Healthy People
Intragroup P.copri strain genes are different.Inferred according to its genome sequence, P.copri can be by secreting superoxides
Reductase and adenosine phosphate phosphosulfate reductase and cause intestinal inflammation.Experiment in vivo also confirms that P.copri can aggravate
Enteric epithelium inflammation simultaneously reduces mouse weight.
At present for the research multi-focus of Prevotella copri on its glycometabolism and immunological characteristic, about the bacterial strain
It is still uncertain with specifically contacting for rheumatoid arthritis.Also, Bu Shi sheep blood mediums are as known bacteroides vulgatus
(Bacteroidales) Effective selection means (《Infection and Immunity》, 2011,5,2012-2020), Zhi Neng great
The bacteroid kind of high abundance in the isolated enteron aisle of probability energy, and it is more difficult to get low-abundance P.copri, thus limit its depth
The scientific research entered.Therefore, how to design special media and efficiently detach P.copri, to be the research bacterium and human body
The relationship of health provides technical support, is a technical problem to be solved urgently.
Invention content
The present invention provides a kind of Prevotella copri specificity screening culture mediums, the culture medium includes that separation is trained
Base and growth medium are supported, contains vancomycin, kanamycins, sole carbon source and production acid indicator etc. in the isolation medium
Substance.
In one embodiment of the invention, the sole carbon source is xylan.
In one embodiment of the invention, the production acid indicator is bromocresol purple.
In one embodiment of the invention, the isolation medium includes following component：Sole carbon source xylan 4-
6g/L, tryptone 15-25g/L, yeast extract 4-6g/L, sodium chloride 4-6g/L, dipotassium hydrogen phosphate 0.04-0.06g/L, phosphorus
Acid dihydride potassium 0.04-0.06g/L, cysteine hydrochloride 0.5-1g/L, hemin 0.005-0.01g/L, vitamin
K10.001-0.002g/L produces acid indicator bromocresol purple 0.010-0.014g/L, Kanamycin Sulfate solution 4-5ml/L,
Vancomycin hydrochloride solution 2.0-2.5ml/L, agar 15-20g/L, surplus is sterile water, pH value 6.8-7.0.
In one embodiment of the invention, the formula of the growth medium includes following component：Brain heart infusion liquid
Body culture medium, cysteine hydrochloride 0.5-1g/L, hemin 0.005-0.01g/L, vitamin K1 0.001-
0.002g/L, surplus are sterile water.
In one embodiment of the invention, the sole carbon source in the isolation medium is xylan.
In one embodiment of the invention, the production acid indicator in the isolation medium is bromocresol purple.
Color change is 5.2 (yellow) -6.8 (purple), Prevotella copri bacterium to bromocresol purple at various ph values
Suspension pH belongs in 5.3-5.8 or so in bromocresol purple color variation range, may be used to indicate the life of Prevotella copri
Long and production acid condition.
In one embodiment of the invention, a concentration of 15-25mg/ml of the Kanamycin Sulfate solution.
In one embodiment of the invention, a concentration of 2-4mg/ml of institute's vancomycin hydrochloride solution.
The culture described above for specific isolation screening Prevotella copri is prepared the present invention also provides a kind of
The method of base：
The preparation steps of the isolation medium：
(1) it presses formula rate and weighs all components in addition to antibiotic, mixed dissolution, if Medium's PH Value is less than 6.8,
After adjusting Medium's PH Value to 6.8-7.0 with sodium hydroxide solution, sterilize 15-20 minutes at 115-121 DEG C.
(2) Kanamycin Sulfate and vancomycin hydrochloride are prepared into solution by concentration requirement, and passes through 0.22 μ respectively
M membrane filtration degermings.
(3) after the culture medium after step (1) sterilizing is cooled to 50-55 DEG C, with the solution mixing obtained by step (2).
The preparation steps of the growth medium：Culture medium powder is weighed by brain heart infusion fluid nutrient medium product requirement,
It is dissolved in distilled water, and other all components is weighed by formula rate, be uniformly mixed, sterilize at 115-121 DEG C 15-20 points
Clock.
Second object of the present invention is to provide Prevotella in a kind of application culture medium separation screening flora
The method of copri, the method are as follows：
(1) coating separation：After flora sample sterile saline gradient dilution, 80-100 μ l is taken to be coated on above-mentioned separation
Culture medium is inverted plate, 35-37 DEG C of anaerobism constant incubator culture 48-72 hours；
(2) level-one purifying culture：The single bacterium colony in picking culture medium colour changed into yellow region, on new above-mentioned isolation medium
Sectional streak after scribing line, is inverted plate, 35-37 DEG C of anaerobism constant incubator culture 48-72 hours；
(3) two level purifying culture：Picking level-one culture single bacterium colony, the sectional streak on new above-mentioned isolation medium are drawn
After line, it is inverted plate, 35-37 DEG C of anaerobism constant incubator culture 48-72 hours；
(4) three-level purifying culture：Picking two level culture single bacterium colony, is inoculated in the test tube of 5-10ml growth mediums, 35-
37 DEG C of anaerobism constant incubator cultures 24-48 hours；
(5) fungi preservation：Three-level bacteria suspension is uniformly mixed with the sterile glycerol solution of 40%-50%, keeps glycerine dense eventually
Degree is 20% or more, and mixed solution is divided in sterilized cryopreservation tube, is frozen in -80 DEG C of ultra low temperature freezers, spare.
(6) strain idenfication.
In one embodiment of the invention, the physiological saline of gradient dilution, preparation side are used in the step (1)
Method is：Sodium chloride 9g/L, cysteine hydrochloride 0.5-1g/L are weighed in proportion, and uniform dissolution uses hydroxide in distilled water
After sodium solution adjusts Medium's PH Value to 6.8-7.0, sterilize 15-20 minutes at 115-121 DEG C.
In one embodiment of the invention, the sterile glycerol solution of the 40%-50% described in the step (5),
Preparation method is：It is 1 by the volume ratio of pure glycerin and distilled water:1-1:After 1.5 mixing, cysteine hydrochloride 0.5- is added
It sterilizes 15-20 minutes at 1g/L, 115-121 DEG C.
In one embodiment of the invention, the method for strain idenfication is in the step (6)：
(6.1) three-level bacteria suspension being centrifuged 1 minute in 10000rpm, abandons supernatant, bacterium mud is resuspended with sterile water equivalent, as
Pcr template；
(6.2) PCR primer used in is 16S rRNA universal primers：27F-AGAGTTTGATCCTGGCCTCA, 1492R-
GGTTACCTTGTTACGACTT, expanding fragment length 1500-1600bp；
(6.3) PCR system is：1 μ l, 1492R primer solution of 27F primer solutions, 1 μ l, 2X Taq Mastermix Dye
0.5 μ l of 25 μ l, 22.5 μ l of sterile water and template；
(6.4) PCR system after mixing, carries out amplification reaction, and reaction condition is：95 DEG C, 5min；30 cycles：95
DEG C, 30s；55 DEG C, 30s；72 DEG C, 2min；72 DEG C, 10min；
(6.5) gained PCR product transfers to biotech firm to be sequenced, and obtained sequence results are used BLAST (http://
Www.ncbi.nlm.nih.gov/BLAST it) scans for comparing with similitude in GeneBank.
In one embodiment of the invention, the 27F primer solutions and 1492R primers described in the step (6.3)
Solution concentration is 10 μM.
Technical solution provided by the invention has the advantages that：
The present invention provides a kind of culture mediums and its application process efficiently separating screening Prevotella copri.The training
Foster base is added to the bacteroid selective antibiotic being made by certain concentration by kanamycins and vancomycin, can effectively inhibit it
The varied bacteria growing of his class；According to primary Jie Shi handbooks, most high abundance bacteroids cannot utilize xylan, this hair in enteron aisle
It is bright that low-abundance P.copri can be effectively enriched with using xylan as sole carbon source；It is additionally added bromocresol purple in culture medium simultaneously,
During screening and culturing, since P.copri can produce acid using sole carbon source and rapidly, its periphery of bacterial colonies can be due to the drop of pH value
It is low so that culture medium become yellow, cannot utilize carbon source bacteroid produce acid cause more slowly culture medium still be original purple.Therefore
Basal culture medium can go out the bacterial strain of different Sugar metabolism abilities respectively, discrimination process is simple according to the depth and speed of color change
It is easy, improve separative efficiency.The hemin and vitamin K1 being added are the growth factor needed for bacteroid, half Guang ammonia
Acid hydrochloride can effectively reduce the oxygen content in culture medium, to promote the growth of P.copri.
The culture medium of specificity screening provided by the invention is more advantageous to point compared to conventional bacteroid selective medium
From low-abundance object bacteria, the difference with miscellaneous bacteria is strengthened, improves the separative efficiency of Prevotella copri, more quickly
The easily isolated P.copri from flora sample.
Specific implementation mode
Technical scheme of the present invention is described further with reference to case study on implementation
Case study on implementation 1：The selection of carbon source in Prevotella copri special medias
It is instructed according to primary Jie Shi handbooks, high abundance bacteroid such as bacteroides uniformis (Bacteroides in enteron aisle
Uniformis), many types of bacteroid (Bacteroides thetaiotaomicron), excrement bacteroid (Bacteroides
Caccae) Ji Shi pairs bacteroid (Parabacteroides distasonis) etc. cannot or can only utilize xylan, mouse on a small quantity
Lee's sugar and mannitol.Therefore, the implementation case will screen the carbon source that can assist specific enrichment separation Prevotella copri.
The culture medium of specificity screening Prevotella copri, including isolation medium and life are used in the implementation case
Long culture medium.Wherein：
It is as follows to be separately cultured based formulas：Single carbon source (xylan, rhamnose or mannitol) 5g/L, tryptone 20g/L,
Yeast extract 5g/L, sodium chloride 5g/L, dipotassium hydrogen phosphate 0.05g/L, potassium dihydrogen phosphate 0.05g/L, cysteine hydrochloride
1g/L, bromocresol purple 0.012g/L, hemin 0.01g/L, the kanamycins sulphur of vitamin K1 0.002g/L, 20mg/ml
The vancomycin hydrochloride solution 2.5ml of acid salt solution 5ml, 3mg/ml, agar 20g, surplus is sterile water, pH value 7.0.
Grown cultures based formulas is as follows in the implementation case：Brain heart infusion fluid nutrient medium (the rich biology in Qingdao sea), half Guang
Propylhomoserin hydrochloride 1g/L, hemin 0.01g/L, vitamin K1 0.002g/L, surplus is sterile water.
The preparation steps of isolation medium in the implementation case：
(1) it presses formula rate and weighs all components in addition to antibiotic, mixed dissolution is adjusted with sodium hydroxide solution and cultivated
Base pH value sterilizes 15 minutes to after 7.0 at 121 DEG C.
(2) Kanamycin Sulfate and vancomycin hydrochloride are prepared into solution by concentration requirement, and passes through 0.22 μ respectively
M membrane filtration degermings.
(3) after the culture medium after step (1) sterilizing is cooled to 50 DEG C, with the solution mixing obtained by step (2), that is, match
The culture medium of the present invention.
The preparation steps of growth medium in the implementation case：It weighs and cultivates by brain heart infusion fluid nutrient medium product requirement
Based powders are dissolved in distilled water, and add cysteine hydrochloride 1g/L, hemin 0.01g/L, vitamin K1
0.002g/L is uniformly mixed, sterilizes 15 minutes at 121 DEG C.
Separation screening is as follows：
(1) coating separation：Healthy old men excrement is taken from Taian Shandong, 1g/L cysteine hydrochlorides are added to sterile
Physiological saline (pH7.0) gradient dilution 10-1-10-7Afterwards, each dilution gradient takes 100 μ l to be respectively coated on three kinds containing difference
The isolation medium of carbon source is inverted plate, 37 DEG C of anaerobism constant incubator cultures 48 hours；
(2) level-one purifying culture：The single bacterium colony different in isolation medium colour changed into yellow region choosing colony form respectively,
And the sectional streak on new culture medium respectively, after scribing line, it is inverted plate, 37 DEG C of anaerobism constant incubator cultures 48 are small
When；
(3) two level purifying culture：Level-one culture single bacterium colony on three kinds of culture mediums of picking respectively, divides on new culture medium
Ride after scribing line, is inverted plate, 37 DEG C of anaerobism constant incubator cultures 48 hours；
(4) three-level purifying culture：Two level culture single bacterium colony on three kinds of culture mediums of picking respectively is inoculated in 5-10ml growths
In the test tube of culture medium, 37 DEG C of anaerobism constant incubator cultures 24 hours；
(5) fungi preservation：Take 500 μ l three-levels bacteria suspensions and 500 μ l 40% it is sterile be added to 1g/L cysteine hydrochloric acid
The glycerite of salt uniformly mixes, and mixed solution is divided in sterilized cryopreservation tube, freezes in -80 DEG C of ultra low temperature freezers
It is interior, it is spare.
(6) strain idenfication：
(6.1) it takes 1ml three-levels bacteria suspension to be centrifuged 1 minute in 10000rpm, abandons supernatant, bacterium mud 1ml sterile water equivalent weights
It is outstanding, as pcr template；
(6.2) PCR primer used in is 16S rRNA universal primers：27F-AGAGTTTGATCCTGGCCTCA, 1492R-
GGTTACCTTGTTACGACTT, expanding fragment length 1500-1600bp；
(6.3) PCR system is：10 μM of 27F primer solutions 1 μ l, 10 μM of 1492R primer solutions 1 μ l, 2XTaq
0.5 μ l of 25 μ l of Mastermix Dye, 22.5 μ l of sterile water and template；
(6.4) PCR system after mixing, carries out amplification reaction, and reaction condition is：95 DEG C, 5min；30 cycles：95
DEG C, 30s；55 DEG C, 30s；72 DEG C, 2min；72 DEG C, 10min；
(6.5) gained PCR product transfers to biotech firm to be sequenced, and obtained sequence results are used BLAST (http://
Www.ncbi.nlm.nih.gov/BLAST it) scans for comparing with similitude in GeneBank.Sequencing result see the table below 1.
Note：The operation of separation screening must execute in aseptic operating platform and anaerobism work station.
The strain that culture medium of the table 1 containing different carbon source screens
The implementation case the result shows that, from 11 bacterium colonies of picking in xylan isolation medium contain P.copri bacterium
It falls 9 (accountings 81.8%), hence it is evident that better than mannitol and the screening effect of rhamnose isolation medium, wherein rhamnose separation training
Foster base is unscreened to obtain P.copri, and the P.copri clump counts accounting only 18.2% that mannitol sieves.Therefore, it is to use to select xylan
In the single carbon source of Prevotellacopri specificity screening culture mediums.
Case study on implementation 2：Specificity screening culture medium is directed to Prevotella copri separation effect in bacteroid mixed system I
The verification of rate
The culture medium of specificity screening Prevotella copri, including isolation medium and life are used in the implementation case
Long culture medium.
It is as follows to be separately cultured based formulas：Xylan 5g/L, tryptone 20g/L, yeast extract 5g/L, sodium chloride 5g/
L, dipotassium hydrogen phosphate 0.05g/L potassium dihydrogen phosphate 0.05g/L, cysteine hydrochloride 1g/L, bromocresol purple 0.012g/L, chlorination
The vancomycin salt of Kanamycin Sulfate the solution 5ml, 3mg/ml of ferroheme 0.01g, vitamin K1 0.002g, 20mg/ml
Acid salt solution 2.5ml, agar 20g/L, surplus are sterile water, pH value 6.8.
Grown cultures based formulas is as follows：Brain heart infusion fluid nutrient medium (the rich biology in Qingdao sea), cysteine hydrochloride 1g/
L, hemin 0.01g/L, vitamin K1 0.002g/L, surplus are sterile water.
Isolation medium is prepared as follows with growth medium：It is identical as case study on implementation 1.
To verify the separative efficiency that above-mentioned specificity screening culture medium is directed to Prevotella copri, in the implementation case
The control medium of use, including isolation medium and growth medium.Wherein：
Isolation medium (control medium) is prepared as follows：
(1) it is weighed by brain-heart infusion medium product formula requirement, adds distillation water dissolution, cysteine hydrochloride is added
1g/L, hemin 0.01g/L, vitamin K1 0.002g/L, agar 20g are uniformly mixed, sterilize 15 minutes at 121 DEG C.
(2) by Kanamycin Sulfate and vancomycin hydrochloride by identical in the isolation medium of specificity screening method
Concentration requirement prepares solution, and passes through 0.22 μm of membrane filtration degerming respectively.
(3) after the culture medium after step (1) sterilizing is cooled to 50 DEG C, with the solution mixing obtained by step (2), that is, match
The isolation medium of the implementation case control screening.
The growth medium preparation method for compareing screening is identical as specificity screening.
Sample preparation：The sample that the implementation case uses is bacteroid mixed liquor I, by bacteroid bacteria suspension described in table 2
Culture is mixed to after stablizing early period in the ratio in table 2.Separation screening operation must be in aseptic operating platform and anaerobism work station
Interior execution.
The composition of 2 bacteroid mixed liquor I of table
| Strain name | Clump count/cfu | Accounting % |
| Bacteroides uniformis | 4×108 | 15.38 |
| Bacteroides vulgatus | 3×108 | 11.54 |
| Bacteroides thetaiotaomicron | 4×108 | 15.38 |
| Parabacteroides distasonis | 4×108 | 15.38 |
| Bacteroides ovatus | 2×108 | 3.85 |
| Bacteroides eggerthii | 2×108 | 3.85 |
| Bacteroides xylanisolvens | 1×108 | 3.85 |
| Prevotella copri | 1×108 | 7.69 |
| Bacteroides fragilis | 2×108 | 3.85 |
| Bacteroides cellulosilyticus | 1×108 | 7.69 |
| Bacteroides dorei | 1×108 | 7.69 |
| Odoribacter splanichnicus | 1×108 | 3.85 |
| It amounts to | 2.6×109 | 100.00 |
After separation screening, 16s sequencing results are as shown in table 3.
The result that 3 specificity screening culture medium of table and control medium screen Prevotella copri
The implementation case the result shows that, P.copri bacterium colonies are unscreened to obtain using control medium, and from specificity screening culture
Contain P.copri bacterium colonies 2 (accountings 18.2%) in base in 11 bacterium colonies of picking, compared to being directed to for control medium
The separative efficiency of P.copri significantly improves.
Case study on implementation 3：Specificity screening culture medium is directed to Prevotella copri separation in bacteroid mixed system II
The verification of efficiency
The culture medium of specificity screening Prevotella copri, including isolation medium and life are used in the implementation case
Long culture medium.
It is as follows to be separately cultured based formulas：Xylan 5g, tryptone 20g, yeast extract 5g, sodium chloride 5g, phosphoric acid hydrogen
Dipotassium 0.05g/L potassium dihydrogen phosphate 0.05g, cysteine hydrochloride 1g, bromocresol purple 0.012g, hemin 0.01g, dimension
The vancomycin hydrochloride solution 2.5ml of Kanamycin Sulfate the solution 5ml, 3mg/ml of raw element K1 0.002g, 20mg/ml,
Agar 20g, surplus are sterile water, pH value 6.9.
Grown cultures based formulas is as follows：Brain heart infusion fluid nutrient medium (the rich biology in Qingdao sea), cysteine hydrochloride 1g/
L, hemin 0.01g/L, vitamin K1 0.002g/L, surplus are sterile water.
To verify the separative efficiency that above-mentioned specificity screening culture medium is directed to Prevotella copri, in the implementation case
The control medium of use, including isolation medium and growth medium.Above-mentioned specificity screening culture medium and control medium
Configuration it is identical as case study on implementation 2 as separation screening step.
Sample preparation：The sample that the implementation case uses is bacteroid mixed liquor I I, by bacteroid bacteria suspension described in table 4
Culture is mixed to after stablizing early period in the ratio in table 4.Separation screening operation must be in aseptic operating platform and anaerobism work station
Interior execution.
The composition of 4 bacteroid mixed liquor I I of table
| Strain name | Clump count/cfu | Accounting % |
| Bacteroides uniformis | 4×108 | 15.38 |
| Bacteroides thetaiotaomicron | 4×108 | 15.38 |
| Parabacteroides distasonis | 4×108 | 15.38 |
| Parabacteroides merdae | 3×108 | 11.54 |
| Bacteroides ovatus | 1×108 | 3.85 |
| Bacteroides eggerthii | 1×108 | 3.85 |
| Bacteroides xylanisolvens | 1×108 | 3.85 |
| Prevotella copri | 2×108 | 7.69 |
| Bacteroides fragilis | 1×108 | 3.85 |
| Bacteroides cellulosilyticus | 2×108 | 7.69 |
| Bacteroides dorei | 2×108 | 7.69 |
| Bacteroides salyersiae | 1×108 | 3.85 |
| It amounts to | 2.6×109 | 100.00 |
After separation screening, 16s sequencing results are as shown in table 5.
The result that 5 specificity screening culture medium of table and control medium screen Prevotella copri
The implementation case the result shows that, P.copri bacterium colonies are unscreened to obtain using control medium, and from specificity screening culture
Contain P.copri bacterium colonies 3 (accountings 27.3%) in base in 11 bacterium colonies of picking, compared to being directed to for control medium
The separative efficiency of P.copri significantly improves.
Case study on implementation 1-3 is only the preferable case study on implementation of the present invention, is not the limit of other forms made for the present invention
It is fixed.Any person skilled in the art as enlightenment or is modified as changing on an equal basis equivalent possibly also with above-mentioned technology contents
Case study on implementation.In every case be technical spirit without departing from the claims in the present invention, to the above case study on implementation made it is simple change,
Equivalent variations and remodeling, still fall within the range of the claims in the present invention protection.
Claims (10)
1. a kind of Prevotella copri specificity screening culture mediums, which is characterized in that the culture medium includes being separately cultured
Base and growth medium contain the objects such as vancomycin, kanamycins, sole carbon source and production acid indicator in the isolation medium
Matter.
2. culture medium according to claim 1, which is characterized in that the sole carbon source is xylan；The production acid indicator
For bromocresol purple.
3. culture medium according to claim 1, which is characterized in that the isolation medium includes following component：Sole carbon source
Xylan 4-6g/L, tryptone 15-25g/L, yeast extract 4-6g/L, sodium chloride 4-6g/L, dipotassium hydrogen phosphate 0.04-
0.06g/L, potassium dihydrogen phosphate 0.04-0.06g/L, cysteine hydrochloride 0.5-1g/L, hemin 0.005-0.01g/
L, vitamin K1 0.001-0.002g/L produce acid indicator bromocresol purple 0.010-0.014g/L, Kanamycin Sulfate solution
4-5ml/L, vancomycin hydrochloride solution 2.0-2.5ml/L, agar 15-20g/L, surplus is sterile water, pH value 6.8-
7.0。
4. culture medium according to claim 1, which is characterized in that the formula of the growth medium includes following component：Brain
Heart immersion liquid fluid nutrient medium, cysteine hydrochloride 0.5-1g/L, hemin 0.005-0.01g/L, vitamin K1
0.001-0.002g/L, surplus are sterile water.
5. culture medium according to claim 3, which is characterized in that a concentration of 15- of the Kanamycin Sulfate solution
25mg/ml；A concentration of 2-4mg/ml of the vancomycin hydrochloride solution.
6. a kind of preparation method of any culture mediums of claim 1-5, which is characterized in that the preparation of the isolation medium
Step：
(1) it presses formula rate and weighs all components in addition to antibiotic, mixed dissolution uses hydrogen if Medium's PH Value is less than 6.8
After sodium hydroxide solution adjusts Medium's PH Value to 6.8-7.0, sterilize 15-20 minutes at 115-121 DEG C；
(2) Kanamycin Sulfate and vancomycin hydrochloride are prepared into solution by concentration requirement, and is filtered respectively by 0.22 μm
Membrane filtration degerming；
(3) after the culture medium after step (1) sterilizing is cooled to 50-55 DEG C, with the solution mixing obtained by step (2)；
The preparation steps of the growth medium：Culture medium powder, dissolving are weighed by brain heart infusion fluid nutrient medium product requirement
Other all components are weighed in distilled water, and by formula rate, is uniformly mixed, sterilizes 15-20 minutes at 115-121 DEG C.
7. a kind of method of Prevotella copri in any culture medium separation screening flora using claim 1-5,
It is characterized in that, the method is as follows：
(1) coating separation：After flora sample sterile saline gradient dilution, 80-100 μ l is taken to be coated on above-mentioned be separately cultured
Base is inverted plate, 35-37 DEG C of anaerobism constant incubator culture 48-72 hours；
(2) level-one purifying culture：The single bacterium colony in picking culture medium colour changed into yellow region, the subregion on new above-mentioned isolation medium
Scribing line after scribing line, is inverted plate, 35-37 DEG C of anaerobism constant incubator culture 48-72 hours；
(3) two level purifying culture：Picking level-one culture single bacterium colony, the sectional streak on new above-mentioned isolation medium, scribing line knot
Shu Hou is inverted plate, 35-37 DEG C of anaerobism constant incubator culture 48-72 hours；
(4) three-level purifying culture：Picking two level culture single bacterium colony, is inoculated in the test tube of 5-10ml growth mediums, 35-37 DEG C
Anaerobism constant incubator culture 24-48 hours；
(5) fungi preservation：Three-level bacteria suspension is uniformly mixed with the sterile glycerol solution of 40%-50%, makes the final glycerol concentration be
20% or more, mixed solution is divided in sterilized cryopreservation tube, is frozen in -80 DEG C of ultra low temperature freezers, it is spare；
(6) strain idenfication.
8. method according to claim 7, which is characterized in that the physiological saline of gradient dilution is used in the step (1),
Preparation method is：Sodium chloride 9g/L, cysteine hydrochloride 0.5-1g/L are weighed in proportion, and uniform dissolution is used in distilled water
After sodium hydroxide solution adjusts Medium's PH Value to 6.8-7.0, sterilize 15-20 minutes at 115-121 DEG C.
9. method according to claim 7, which is characterized in that the sterile glycerol of the 40%-50% described in the step (5)
Solution, preparation method are：It is 1 by the volume ratio of pure glycerin and distilled water:1-1:After 1.5 mixing, cysteine hydrochloric acid is added
Salt 0.5-1g/L sterilizes 15-20 minutes at 115-121 DEG C.
10. method according to claim 7, which is characterized in that the method for strain idenfication is in the step (6)：
(6.1) three-level bacteria suspension is centrifuged 1 minute in 10000rpm, abandons supernatant, bacterium mud is resuspended with sterile water equivalent, as PCR
Template；
(6.2) PCR primer used in is 16S rRNA universal primers：27F-AGAGTTTGATCCTGGCCTCA, 1492R-
GGTTACCTTGTTACGACTT, expanding fragment length 1500-1600bp；
(6.3) PCR system is：1 μ l, 1492R primer solution of 27F primer solutions, 1 μ l, 2X Taq Mastermix Dye, 25 μ l,
0.5 μ l of 22.5 μ l of sterile water and template；
(6.4) PCR system after mixing, carries out amplification reaction, and reaction condition is：95 DEG C, 5min；30 cycles：95 DEG C,
30s；55 DEG C, 30s；72 DEG C, 2min；72 DEG C, 10min；
(6.5) gained PCR product transfer to biotech firm to be sequenced, by obtained sequence results using BLAST in GeneBank into
Row search and similitude compare.
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