CN102719541A - Method for analyzing Bacillus community structure in white spirit fermentation system - Google Patents
Method for analyzing Bacillus community structure in white spirit fermentation system Download PDFInfo
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Abstract
The invention discloses a method for analyzing the Bacillus community structure in a white spirit fermentation system, belonging to the technical field of microbial ecology. According to the invention, the diversity of a Bacillus community structure in the white spirit fermentation system is semi-quantitatively analyzed by a nested PCR-DGGE (polymerase chain reaction-denaturing gel gradient electrophoresis) method. The method comprises the following steps of: extracting the total genome DNA (deoxyribonucleic acid) in a yeast or fermented grains sample by a bead milling method; performing two steps of amplification of the Bacillus specific segment through the nested PCR; performing DGGE electrophoretic analysis; determining the microorganism species corresponding to the DNA stripe by use of a standard strain; performing glue cutting of the unknown stripe and recycling and then performing PCR again; connecting the T vector and cloning; performing DGGE electrophoresis again; performing positive cloning sequencing and performing blast comparison in the GenBank databae to obtain the microorganism information; digitally processing DGGE map by use of quantityone software; and calculating the proportion of different Bacillus strains in the total Bacillus flora according to the brightness of the stripe. The method disclosed by the invention has the advantages of simplicity, strong specificity and good repeatability, and lays foundation for further studying the importance of Bacillus in white spirit production.
Description
Technical field
In a kind of analysis liquor fermentation system
BacillusThe method of structure of community; The present invention relates to use modern molecular biology technique-polymerase chain reaction-deformation gradient gel electrophoresis (PCR-DGGE) to overcome the defective of traditional mikrobe separation method, in the fermentation system of qualitative and semi-quantitative analysis China white wine colony mikrobe
BacillusThe structure of colony is formed and the dynamic evolution rule, belongs to the microbial ecological technical field.
Background technology
China white wine production is the solid ferment process of an opening, we can say that in fact the production of liquor comprised a huge microbiotic complicated metabolic process, receives the influence of environment bigger.Along with the change of envrionment conditions, micro-flora also can change, and the flavor taste of liquor is exerted an influence.Daqu and wine unstrained spirits are important microbe carriers during liquor is produced, wherein
BacillusIt is main bacterium.Most of
BacillusThe bacterial classification that belongs to can both the synthetic starch enzyme, proteolytic enzyme, and the adverse environment condition in the liquor production is had higher resistibility, is present in from start to finish in the brewed spirit process.Therefore,
BacillusBe zymogenic bacteria and living fragrant bacterium important in the liquor fermentation system,
BacillusThe structure of colony becomes the focus of China white wine research.
Domestic scholars is mainly separated, is analyzed in the liquor fermentation system through screening, cultural method at present
BacillusSeparation obtains from the liquor fermentation system
BacillusMainly contain subtilis (
Bacilus subtilis), Bacillus circulans (
Bacillus circulans), bacillus megaterium (
Bacillus magaterium), bacillus cereus (
Bacillus cereus), Bacillus licheniformis (
Bacillus licheniformis) and bacillus amyloliquefaciens (
Bacillus amyloliquefaciens) etc.There is the researcher to find in the Maotai-flavor liquor fermenting process, some
B.licheniformisBe the critical function mikrobe that produces sauce fragrant breeze flavor, also find some
BacillusIt is the main bacteria seed that generates aspartic protease in the aromatic type high-temperature daqu.And
B.subtilisQuantity is more in the Daqu bacterium, is the important bacterium source that forms wine body aromatic substance.Special construction and specific function have been formed in the brewed spirit system
BacillusColony.
But traditional microbe research method has bigger limitation, has complex operation, and colonial morphology, biochemical characteristic be alike to cause being difficult to accurate evaluation, accuracy and poor reliability as a result, and it is harsh to be prone to omit some culture condition
BacillusShortcomings such as bacterial classification.In recent years, systems biology has proposed a viewpoint, and promptly composition of colony mikrobe and ratio all can influence the function of system in the complex system.Because isolation cultivation method can change the formation of original flora, can't analyze between the mikrobe and the mutual relationship between mikrobe and the environment, so be inappropriate for the real-time monitoring of brewed spirit process.The molecular ecology method begins to be applied in the research of liquor.
The DGGE technology is a kind of electrophoretic technique that is used to detect dna mutation that was taken the lead in proposing in 1979 by Fisher and Lerman; Its ultimate principle be since double-stranded DNA when general polyacrylamide gel electrophoresis; The dna fragmentation of different lengths can be made a distinction (migratory behaviour is decided by its molecular size and electric charge), but the dna fragmentation of same length can't make a distinction because of migratory behaviour in glue is the same.Therefore on general polyacrylamide gel basis; Added denaturing agent (urea and methane amide) gradient; Not homotactic dna fragmentation is unwinding under the corresponding denaturing agent concentration separately, and the variation of generation space configuration causes the rapid decline of electrophoretic velocity; Be stuck in its corresponding denaturing agent gradient position at last, thus can be same length but the different dna fragmentation of sequence make a distinction.DGGE is because effective biological community structure composition in the Analysis of Complex system, and more and more comes into one's own.Nowadays; Since DGGE have can detect be difficult to cultivate or can not cultured microorganism, detection speed is fast, circulation ratio is strong, the result accurately and reliably, make things convenient for advantages such as easy to operate; Be widely used in the research in a plurality of microecosystems field; Comprising soil, lake, ocean, human body and animal intestinal, leavened food or the like, is one of molecular biology method that biological community structure is formed and flora variety Dynamic Variation Analysis is main in the research complex system.
Because the preference property of pcr amplification is analyzed liquor Daqu and wine unstrained spirits with the universal primer in bacterial 16 S rDNA V3 district, tends to underestimate
BacillusVariety.For being directed against research
BacillusGroup structure, with the target gene of selecting 16S rDNA V9 district gene as DGGE.For the preference property that reduces pcr amplification, the specificity that improves amplification, adopt nest-type PRC to obtain specific fragment.The nest-type PRC ultimate principle is to utilize two cover PCR primers to carrying out the two-wheeled pcr amplification reaction.In first round amplification, first pair of primer (outer primer) is in order to produce amplified production.Second pair of primer is called nested primer, and they are combined in the inside of PCR product for the first time, and making for the second time, pcr amplified fragment is shorter than amplification for the first time.Because nest-type PRC has twice PCR amplification, thereby reduced the possibility of a plurality of target sites that increase, thereby the nest-type PRC high specificity, accuracy is high, safety is high.
Using nest-type PRC-DGGE analyzes in the China white wine fermentation system
BacillusThe flora variety have remarkable advantages, it is easy and simple to handle, detection speed is fast, good reproducibility, result accurately and reliably, directly identify kind of a level, and can detect to be difficult to cultivate and maybe can not cultivate
BacillusIn utilization this method semi-quantitative analysis liquor real attenuation system
BacillusStructure of community form and ratio, in accurate assurance liquor fermentation process
BacillusKind and dynamic evolution rule; Understand in the liquor production process Changing Pattern with flavour material, crucial mikrobe that important enzyme material is relevant in depth with quality product and the correlationship between becoming to be grouped into, instruct the improvement of production technique to have important theory and practice significance.
Summary of the invention
The present invention is intended to the defective to existing traditional mikrobe separation method, discloses a kind of nest-type PRC that uses and combines the method for denaturing gradient gel electrophoresis to come in the semi-quantitative analysis liquor fermentation system
BacillusStructure form and ratio.The technology that the present invention adopts, have quick, easy, accuracy is high, the advantage of high specificity.For confirming in the liquor fermentation system
BacillusIn structure of community composition and the liquor fermentation process
BacillusFlora dynamic evolution rule provides method for quick, in conjunction with the place an order specificity analysis of bacterial classification of laboratory culture condition, can judge its function and percentage contribution in the brewed spirit process, think that the research of functional microorganism provides reference frame in the liquor.
Technical scheme of the present invention: utilize pearl mill method to extract Daqu or wine unstrained spirits sample gene group, the amplification of nest-type PRC two-wheeled
BacillusDNA fragment specific; The dna fragmentation of the correspondence of DGGE electrophoretic separation different genera is cut glue and is reclaimed the corresponding band of superior microorganism, and pcr amplification and connection T carrier and positive colony selects once more; After the DGGE electrophoresis is compared again; Check order and identify the mikrobe information of cutting adhesive tape band correspondence, utilize quantity one software that digitized processing is carried out in the brightness of DGGE collection of illustrative plates band at last, calculate certain
BacillusTotal
BacillusThe ratio of being occupied in the flora.
With denaturing gradient gel electrophoresis in the qualitative and semi-quantitative analysis liquor fermentation system
BacillusThe method of structure of community may further comprise the steps:
(a) Daqu or the wine unstrained spirits sample of selection experiment carry out pre-treatment to sample, and extract the total genomic dna of mikrobe in the sample with pearl mill method;
(b) select
Bacillus16S rRNA V9 district gene as two pairs of primers of the target gene of DGGE design, and second take turns the forward primer of PCR 5 ' end add the GC hairpin structure;
The first round that nest-type PRC is used
BacillusThe specificity primer is:
p1-F：5’-CGATGCGTAGCCGACCTGAG-3’，
p1-R：5’-AAGGAGGTGATCCAGCCGCA-3’；
Second takes turns and is directed against
BacillusThe primer in 16S rDNA V9 district is:
p2-F：5’-ATGGCTGTCGTCAGCT-3’，
p2-R：5’-CGCCCGCCGC?GCGGCGGGCG?GGGCGGGGGC?ACGGGCGGTG?TGTAC-3’；
(c) be that template is carried out pcr amplification with the genome in the step (a);
(d) the PCR product is carried out the DGGE electrophoretic analysis under optimum condition;
(e) the dyeing back is compared with known Maker, confirms to have the corresponding kind of DNA band of identical electrophoretic mobility, the advantage band of the unknown is cut glue reclaim;
(f) the DNA band that reclaims is pcr amplification, connection T carrier again, is transformed into competent escherichia coli cell, and it is dull and stereotyped to coat the LB that is added with finite concentration Amp, selects positive colony;
(g), carry out DGGE comparison, checking once more to the bacterium colony PCR product of positive colony;
(h) will check order with reclaiming the PCR product that the DNA band has identical electrophoretic mobility, sequencing result is the blast compare of analysis in the GenBank DB;
(i) adopt quantity one software that digitized processing is carried out in the brightness of DGGE collection of illustrative plates band, the brightness that is about to every band is converted into peak area, and the peak area of all bands is added and represents
BacillusThe flora sum, certain
BacillusThe peak area of corresponding D NA band is proportion among all band peak areas, is this
BacillusTotal
BacillusShared ratio in the flora.
Sample in the step (a) can be the China white wine Daqu or the wine unstrained spirits sample of any odor type, area collection, carries out pre-treatment after sample multidraw, the mixing.
Electrophoresis apparatus is a Dcode detection in Gene Mutation system in the step (d), and deposition condition is voltage 100V, 60 ℃ of following electrophoresis 200min, and denatured gradient is 33%-43%.
Dyeing process is for using SYBR green dyeing three times, each 15min in the step (e); Unknown band is cut the method that glue reclaims: the cutting of advantage band is placed in the 1.5mL centrifuge tube of the bacterium of going out, adds an amount of sterilization ultrapure water and cleans, and adds 60 μ L sterilization ultrapure water then and in 4 ℃ of refrigerator hold over night.
The primer that pcr amplification adopts in the step (f) is identical with the primer in the step (b).
In the step (g) primer of bacterium colony PCR with step (b) in primer identical, and be the sample P CR product in positive colony daughter colony PCR product and the step (c) to be carried out again the DGGE electrophoresis compare.
Beneficial effect of the present invention: the present invention can be widely used in the brewed spirit process, comprises in system song, accumulation or the fermenting process
BacillusThe flora Changing Pattern, and compare in the liquor Daqu or wine unstrained spirits in the different process (odor type) or the place of production
BacillusThe difference that structure of community is formed is function
BacillusSeparation reference frame is provided.
Fig. 1 is among the embodiment 1
B.amyloliquefaciens,
B.oleronius,
B.firmusWith
B.cereusThe electrophoresis imaging results.
Fig. 2 is among the embodiment 1
B.subtilis,
B.licheniformisWith
B.magateriumThe electrophoresis imaging results.
Fig. 3 is embodiment 2 liquor wine unstrained spirits electrophoresis imaging results.
Embodiment
For a better understanding of the present invention, further illustrate content of the present invention below in conjunction with instance.
Will
Bacillus oleronius(CICIMB1592),
B.amyloliquefaciens(CICC20164),
B.firmus(CICIMB1543),
B.cereus(CICIMB1809),
B.subtilis(CICIMB1585),
B.licheniformis(CICIMB1540) and
B.magaterium(CICIMB1691) be inoculated in the beef extract-peptone liquid nutrient medium, spend the night in 37 ℃ of 200 commentaries on classics/min shaking culture.With the centrifugal collection thalline of bacterial culture fluid, behind the resuspended thalline of sterilized water, pearl mill appearance disruption of microorganisms cell (impacting 4min) with top speed, the centrifugal 10min of 12,000 * g collects supernatant; Add and isopyknic phenol of supernatant and chloroform organic reagent (equal-volume) mixing, the centrifugal 10min of 12,000 * g collects supernatant; Add 2 times of volume ice ethanol more than-20 ℃ of deposition 2h, 12,000 * g is centrifugal, and 10min abandons supernatant; With 70% washing with alcohol deposition, 30 μ L ultrapure water (resistivity 18.2M Ω) dissolution precipitations are used in vacuum-drying at last.The microbial total genomic dna is as template, with nested PCR method specific amplification fragment in the sample that extraction obtains.The used primer of nest-type PRC first round PCR is:
p1-F：5’-CGATGCGTA?GCCGACCTGAG-3’，
p1-R：5’-AAGGAGGTGATCCAGCCGCA-3’。
First round PCR reaction system is 25 μ L, comprises 12.5 μ L, 2 * Taq PCR MasterMix enzyme, every kind of primer 0.5 μ L (25pmol), and the sterilization ultrapure water of 10.5 μ L, the dna profiling consumption is about 10ng.First round PCR response procedures is: get into 25 circulations, 95 ℃ of 1min behind 95 ℃ of preparatory sex change 5min; 68 ℃ of 90s; 72 ℃ of 2min, last 72 ℃ are extended 10min.The PCR product detects with 1% agarose gel electrophoresis.With 100 times of first round PCR product dilutions, get 1 μ L and be second and take turns the template of PCR.
Second takes turns the used primer of PCR is:
p2-F：5’-ATGGC?TGTCGTCAGCT-3’，
p2-R：5’-CGCCCGCCGC?GCGGCGGGCG?GGGCGGGGGC?ACGGGCGGTG?TGTAC-3’。
Second to take turns the PCR reaction system identical with the first round.Second takes turns the PCR reaction conditions is: get into 25 circulations, 94 ℃ of 1min behind 94 ℃ of preparatory sex change 4min; 65 ℃ of 1min; 72 ℃ of 1min, last 72 ℃ are extended 10min.The PCR product detects with 1% agarose gel electrophoresis.
The DGGE condition is: acrylamide gel concentration is 8%, and the sex change scope is 33%-43% (100% denaturing agent is 7mol/L urea and 40% deionized formamide), and DGGE voltage is 100V, and electrophoresis temperature is 60 ℃, and electrophoresis time is 200min.The PCR applied sample amount is about 200ng.Electrophoresis finishes the back with SYBR green (adding 3 μ L SYBR green in the 30mLTAE damping fluid) dyeing 3 times, each 15min.Come the observation experiment result through gel imaging system.Experimental result is seen accompanying drawing 1 and accompanying drawing 2, and wherein swimming lane 1-4 is respectively in the accompanying drawing 1
B.amyloliquefaciens,
B.oleronius,
B.firmusWith
B.cereus, swimming lane 1-3 is respectively in the accompanying drawing 2
B.subtilis,
B.licheniformisWith
B.magateriumIn the DGGE collection of illustrative plates
BacillusEach self-forming of bacterial classification specific DNA band, and obtained good separating effect.As reference culture, carry out electrophoretic analysis simultaneously with Daqu or wine unstrained spirits sample, but Rapid identification goes out the mikrobe kind information that has the DNA band of identical electrophoretic mobility with reference culture with it.
Gather the liquor wine unstrained spirits of Shanxi fen-flavor type white spirit factory, get the wine unstrained spirits sample of fermentation 0d, 15d and 30d.During sample pretreatment, add 30mL PBS damping fluid (57.7mmol/L Na
2HPO
4, 42.3mmol/L NaH
2PO
4), vortex vibration 5min, 200g is centrifugal, and 5min discards deposition, and the centrifugal 5min of 9000g collects thalline again.Behind the resuspended thalline of PBS damping fluid, pearl mill appearance disruption of microorganisms cell (impacting 4min) with top speed, the centrifugal 10min of 12,000 * g collects supernatant; Add and isopyknic phenol of supernatant and chloroform (equal-volume) organic reagent mixing, the centrifugal 10min of 12,000 * g collects supernatant; Add 2 times of volume ice ethanol more than-20 ℃ of deposition 2h, 12,000 * g is centrifugal, and 10min abandons supernatant; With 70% washing with alcohol deposition, 30 μ L ultrapure water (resistivity 18.2M Ω) dissolution precipitations are used in vacuum-drying at last.The microbial total genomic dna is as template, with nested PCR method specific amplification fragment in the sample that extraction obtains.The used primer of nest-type PRC first round PCR:
p1-F：5’-CGATGCGTAGCCGACCTGAG-3’，
p1-R：5’-AAGG?AGGTGATCCAGCCGCA-3’。
First round PCR reaction system is 25 μ L, comprises 12.5 μ L, 2 * Taq PCR MasterMix enzyme, every kind of primer 0.5 μ L (25pmol), and the sterilization ultrapure water of 10.5 μ L, the dna profiling consumption is about 10ng.First round PCR response procedures is: get into 25 circulations, 95 ℃ of 1min behind 95 ℃ of preparatory sex change 5min; 68 ℃ of 90s; 72 ℃ of 2min, last 72 ℃ are extended 10min.The PCR product detects with 1% agarose gel electrophoresis.With 100 times of first round PCR product dilutions, get 1 μ L and be second and take turns the template of PCR.
Second takes turns the used primer of PCR is:
p2-F:5’-ATGGCTGTCGTCAGCT-3’，
p2-R：5’-CGCCCGC?CGCGCGGCGGGCGGGGCGGGGGCACGGGC?GGTGTGTAC-3’。
Second to take turns the PCR reaction system identical with the first round.
Second takes turns the PCR reaction conditions is: get into 25 circulations, 94 ℃ of 1min behind 94 ℃ of preparatory sex change 4min; 65 ℃ of 1min; 72 ℃ of 1min, last 72 ℃ are extended 10min.The PCR product detects with 1% agarose gel electrophoresis.
The DGGE condition is: acrylamide gel concentration is 8%, and the sex change scope is 33%-43% (100% denaturing agent is 7mol/L urea and 40% deionized formamide), and DGGE voltage is 100V, and electrophoresis temperature is 60 ℃, and electrophoresis time is 200min.The PCR applied sample amount is about 200ng.Electrophoresis finishes the back with SYBR green (adding 3 μ L SYBR green in the 30mLTAE damping fluid) dyeing 3 times, each 15min.Come the observation experiment result through gel imaging system.Experimental result is seen accompanying drawing 3.Swimming lane 1 is fermentation 0d sample, and swimming lane 2 is fermentation 15d sample, and swimming lane 3 is fermentation 30d sample.
Gel with after the dyeing places under the uv lamp, and the cutting of unknown band is placed in the 1.5mL centrifuge tube of the bacterium of going out, and numbering adds that an amount of sterilization ultrapure water cleans that the back adds the sterilization ultrapure water of 60 μ L and in 4 ℃ of refrigerator hold over night.Get an amount of stored sample liquid and carry out pcr amplification with p2-F/p2-R, the PCR product is cut glue with test kit reclaim as template.Brightness according to the dna fragmentation agarose gel electrophoresis band of cutting the glue recovery; The DNA and the T carrier that in centrifuge tube, add the recovery of an amount of ratio; Add 5 μ L solution I again, add the reaction mixture that the sterilization ultrapure water replenishes into 10 μ L at last, shake mixing a little.Be positioned in the metal bath, 16 ℃ of connections are spent the night.Connect product transformed into escherichia coli competent cell, coat and be added with certain density Amp LB flat board, carry out blue hickie screening, 37 ℃ of overnight cultures, positive colony of picking white carries out bacterium colony PCR.Bacterium colony PCR product is carried out DGGE analyze, choose sample as relative position according to the corresponding band of cutting glue simultaneously, eliminate false positive clone with reference to the sub-PCR product of comparison positive colony.Positive colony is sent to Shanghai gives birth to worker's order-checking, sequencing result carries out the blast compare of analysis in the GenBank DB.Comparison result is as shown in table 1.
The comparison result of table 1 positive colony
Adopt quantity one software that digitized processing is carried out in the brightness of DGGE collection of illustrative plates band, concrete steps are following: a) on the DGGE collection of illustrative plates, remove the swimming lane background, confirm and each DNA band of mark; B) all bands classification that mark is good promptly are classified as one type to the consistent band of swimming speed in the DGGE collection of illustrative plates, and manually adjust, comparison band; C) brightness (OD value) with each band is converted into peak area, and the peak area data are exported; D) peak area of all bands is added and represent
BacillusThe flora sum is with representing certain
BacillusThe band peak area ratio on all bands peak area with, can calculate this
BacillusTotal
BacillusShared ratio in the flora.Sample with the swimming lane in the accompanying drawing 2 2-fermentation 15d is an example, analyzes in the fermented wine unstrained spirits sample certain
BacillusAlways
BacillusProportion in the flora.
Main DNA stripe information in the fermentation 15d sample in the table 2 DGGE collection of illustrative plates
| The band numbering | a | b | c | d |
| The bacterium name | B.circulans | B. firmus | B.amyloliquefaciens | B.licheniformis |
| Peak area | 39.681 | 16.735 | 41.693 | 31.105 |
| Relative proportion (%) | 31 | 13 | 32 | 24 |
Claims (6)
1. with denaturing gradient gel electrophoresis in the qualitative and semi-quantitative analysis liquor fermentation system
BacillusThe method of structure of community is characterized in that may further comprise the steps:
(a) Daqu or the wine unstrained spirits sample of selection experiment carry out pre-treatment to sample, and extract the total genomic dna of mikrobe in the sample with pearl mill method;
(b) select
Bacillus16S rRNA V9 district gene as two pairs of primers of the target gene of DGGE design, and second take turns the forward primer of PCR 5 ' end add the GC hairpin structure;
The first round that nest-type PRC is used
BacillusThe specificity primer is:
p1-F：5’-CGATGCGTAGCCGACCTGAG-3’，
p1-R：5’-AAGGAGGTGATCCAGCCGCA-3’；
Second takes turns and is directed against
BacillusThe primer in 16S rDNA V9 district is:
p2-F：5’-ATGGCTGTCGTCAGCT-3’，
p2-R：5’-CGCCCGCCGC?GCGGCGGGCG?GGGCGGGGGC?ACGGGCGGTG?TGTAC-3’；
(c) be that template is carried out pcr amplification with the genome in the step (a);
(d) the PCR product is carried out the DGGE electrophoretic analysis under optimum condition;
(e) the dyeing back is compared with known Maker, confirms to have the corresponding kind of DNA band of identical electrophoretic mobility, the advantage band of the unknown is cut glue reclaim;
(f) the DNA band that reclaims is pcr amplification, connection T carrier again, is transformed into competent escherichia coli cell, and it is dull and stereotyped to coat the LB that is added with finite concentration Amp, selects positive colony;
(g), carry out DGGE comparison, checking once more to the bacterium colony PCR product of positive colony;
(h) will check order with reclaiming the PCR product that the DNA band has identical electrophoretic mobility, sequencing result is the blast compare of analysis in the GenBank DB;
(i) adopt quantity one software that digitized processing is carried out in the brightness of DGGE collection of illustrative plates band, the brightness that is about to every band is converted into peak area, and the peak area of all bands is added and represents
BacillusThe flora sum, certain
BacillusThe peak area of corresponding D NA band is proportion among all band peak areas, is this
BacillusTotal
BacillusShared ratio in the flora.
2. method according to claim 1 is characterized in that sample in the step (a) wherein can be China white wine Daqu or wine unstrained spirits sample that any odor type, area are gathered, the sample multidraw, carries out pre-treatment after mixing.
3. method according to claim 1 is characterized in that wherein the middle electrophoresis apparatus of step (d) is a Dcode detection in Gene Mutation system, and deposition condition is voltage 100V, 60 ℃ of following electrophoresis 200min, and denatured gradient is 33%-43%.
4. method according to claim 1 is characterized in that wherein the middle dyeing process of step (e) is for using SYBR green dyeing three times, each 15min; Unknown band is cut the method that glue reclaims: the cutting of advantage band is placed in the 1.5mL centrifuge tube of the bacterium of going out, adds an amount of sterilization ultrapure water and cleans, and adds 60 μ L sterilization ultrapure water then and in 4 ℃ of refrigerator hold over night.
5. method according to claim 1 is characterized in that pcr amplification adopts in the step (f) wherein primer is identical with primer in the step (b).
6. method according to claim 1; The primer that it is characterized in that bacterium colony PCR in the step (g) wherein with step (b) in primer identical, and be the sample P CR product in positive colony daughter colony PCR product and the step (c) to be carried out again the DGGE electrophoresis compare.
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102911933A (en) * | 2012-11-05 | 2013-02-06 | 四川大学 | High-quality DNA (Deoxyribonucleic Acid) extraction method of spirit brewing microorganism |
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Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101570786A (en) * | 2009-06-11 | 2009-11-04 | 江南大学 | Method for identifying structure of yeast colony of Daqu starter or fermented grain of distilled spirit by using denaturing gradient electrophoresis |
| CN102382877A (en) * | 2010-08-30 | 2012-03-21 | 中国食品发酵工业研究院 | Method for studying structural diversity of daqu bacterial community |
-
2012
- 2012-06-13 CN CN2012101936325A patent/CN102719541A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101570786A (en) * | 2009-06-11 | 2009-11-04 | 江南大学 | Method for identifying structure of yeast colony of Daqu starter or fermented grain of distilled spirit by using denaturing gradient electrophoresis |
| CN102382877A (en) * | 2010-08-30 | 2012-03-21 | 中国食品发酵工业研究院 | Method for studying structural diversity of daqu bacterial community |
Non-Patent Citations (1)
| Title |
|---|
| 高亦豹: "聚合酶链式反应-变性梯度电泳技术（PCR-DGGE）研究中国白酒大曲中微生物群落结构", 《中国优秀硕士学位论文全文数据库工程科技I辑》, vol. 2012, no. 02, 29 February 2012 (2012-02-29), pages 024 - 253 * |
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