CN102911933A - High-quality DNA (Deoxyribonucleic Acid) extraction method of spirit brewing microorganism - Google Patents
High-quality DNA (Deoxyribonucleic Acid) extraction method of spirit brewing microorganism Download PDFInfo
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- CN102911933A CN102911933A CN2012104352142A CN201210435214A CN102911933A CN 102911933 A CN102911933 A CN 102911933A CN 2012104352142 A CN2012104352142 A CN 2012104352142A CN 201210435214 A CN201210435214 A CN 201210435214A CN 102911933 A CN102911933 A CN 102911933A
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Abstract
The invention relates to a high-quality DNA (Deoxyribonucleic Acid) extraction method of a spirit brewing microorganism. The high-quality DNA extraction method is characterized by comprising the following steps of: (1) performing simple pre-treatment on a sample to obtain thallus mixture; (2) adding DNA extract, various enzyme solution, SDS (Sodium Dodecyl Sulfonate), protease and the like into the mixture to fully release DNA in thallus; (3) adding phenol: chloroform: isoamyl alcohol and chloroform: isoamyl alcohol to extract miscellaneous protein so as to purify the DNA; and (4) settling the DNA by the isoamyl alcohol at a low temperature and washing with absolute ethyl alcohol to obtain high-quality microbial genome total DNA. The high-quality DNA extraction method has the advantages of capability of realizing that all used reagents are the conventional reagents, low cost, wide application range, easiness in operation, mild conditions, high purity of extracted DNA, capability of performing subsequent PCR (Polymerase Chain Reaction) and DGGE (Denaturing Gradient Gel Electrophoresis) analysis without purifying the genome DAN and the like.
Description
Technical field
The present invention relates to liquor microflora ecotechnology field, specifically a kind of high quality DNA extracting method for the brewed spirit structural analysis of microbial community.
Background technology
China white wine has the long history of brewageing, and the making method of China white wine is not quite similar, brewageing of China white wine produced with the main source of Daqu as microorganism and enzyme, and in actual production, the quality of spirit quality is related to the quality of the yield of liquor and the wine of liquor.Be accompanied by these those long domestication processes that disappear of a series of microorganisms in the production process of Daqu, the structure of community of microorganism is directly connected to the quality of production of Daqu in the middle of the Daqu.China white wine mainly with Jiao Chi and wine unstrained spirits etc. as production basis, microorganism in the middle of environment, Daqu and the cellar for storing things mud is carrying out complicated energy metabolism and substance metabolism, the metabolic process of microorganism complexity also is the forming process of liquor flavor precursor substance, group's difference and the transition of microorganism in the Daqu that the research different process is produced, same Daqu yeast making process and the cellar for storing things mud have certain directive significance for the production of Daqu and liquor.Technology related to the present invention has DNA extraction method and DNA extraction test kit etc., be 201210107413.0 to have put down in writing the pre-treating process that a kind of Daqu wine cellar mud microorganism total DNA extracts just like number of patent application, may further comprise the steps: (1) takes by weighing an amount of cellar for storing things mud, put into the centrifuge tube of sterilization, add sterile saline, fully vibration is centrifugal, abandoning supernatant; With method repeated washing sample 1 time; (2) add dehydrated alcohol to above-mentioned sample, fully vibration is centrifugal, abandoning supernatant; With method repeated washing sample 1 time; (3) add aseptic ultrapure water, mix, the vibration washing sample, centrifugal, abandoning supernatant; The cellar for storing things mud sample that (4) will fully wash wraps up the centrifuge tube mouth with the dual-layer sterilization gauze ,-70 ℃ of freezing 30min, and cryogenic vacuum is drained to powdery, is stored in-20 ℃ of refrigerators, is used for extracting cellar for storing things mud microorganism total DNA.Be 201010266385.8 to have put down in writing the method that relates to a kind of studying structural diversity of daqu bacterial community just like number of patent application, key step is as follows: 1): directly extract the Daqu genomic dna; 2): selecting bacteria universal primer, the DNA fragment specific among the amplification bacterial ribosome DNA; 3): DGGE electrophoretic separation PCR product; 4): cut glue and reclaim band corresponding to microorganism in the DGGE finger printing; 5): PCR again, product is connected to the T carrier, the positive colony checking is also carried out in blue hickie screening; 6): order-checking obtains the kind information of the corresponding microorganism of DGGE band.Be 201210107413.0 to have put down in writing the method that microorganism total DNA in a kind of liquor Daqu extracts just like number of patent application, it is characterized in that before extracting DNA, the liquor Daqu being carried out pre-treatment, pre-treatment comprises the steps: to get Daqu, add pretreatment liquid: the phosphoric acid buffer that contains 0.05-0.2W/V%PVPP (cross-linking polyethylene pyrrolidone) and 3-5W/V% twen-80 or soil temperature-60, pH8.0, wherein the weightmeasurement ratio of Daqu and pretreatment liquid is 3-8: 25, and jolting; Supersound process; Leave standstill rear low-speed centrifugal; Get supernatant liquor, high speed centrifugation; Remove the phosphoric acid buffer that the supernatant liquor precipitation adds 0.05-0.2W/V%PVPP (cross-linking polyethylene pyrrolidone); Break up rear high speed centrifugation, remove supernatant liquor, obtain sample.But, there is following defective in the existing technological method: the scope that the ⑴ genome DNA extracting method is used narrower (only being Daqu or cellar for storing things mud), and blanket method is rarely seen; ⑵ added certain throw out in the pre-treating process, but throw out is inestimable on the impact of the lower microorganism of abundance in the microflora; Simultaneously, because the micro-ecological environment of Daqu and cellar for storing things mud is very complicated, have a lot of interference components to exist, the quality of the genome DNA of acquisition is unsatisfactory.Therefore, extensive, the simple effective and stable method that can from Daqu, poor unstrained spirits and cellar for storing things mud, extract high quality DNA of a kind of scope of application in the urgent need to.
Summary of the invention
The purpose of this invention is to provide extensive, the simple effective and stable method that can from Daqu, poor unstrained spirits and cellar for storing things mud, extract high quality DNA of a kind of scope of application.Its concrete steps are as follows:
⑴ sample pretreatment
Take by weighing a certain amount of Daqu, add 30mL PBS damping fluid (0.0557mol/L Na
2HPO
4, 0.0423mol/L NaH
2PO
4, pH8.0), vortex is even, and the centrifugal 10min of 800r/min gets supernatant liquor, and the centrifugal 10min of 10000r/min gets solid-state cenobium.
⑵ DNA extraction and detection
Above-mentioned solid-state cenobium is resuspended in 500 μ L DNA extraction liquid (0.1M Tris-HCl, 0.1M EDTA, 1.5M NaCl, 1%CTAB, pH8.0) in, add 10 μ L cellulases (75mg/mL), helicase (50mg/mL) and N,O-Diacetylmuramidase (40mg/mL), 37 ℃ of cracking 1h; Add 10 μ L 20%SDS, 65 ℃ of insulation 1.5h; Add 10 μ L Proteinase Ks (5mg/mL), 60 ℃ of shaking bath 1h, the centrifugal 10min of 4500r/min collects supernatant liquor; Use equal-volume phenol: chloroform: primary isoamyl alcohol (25:24:1) extracting, 4 ℃ of centrifugal 10min of 10000r/min collect supernatant liquor; Use the equal-volume chloroform: primary isoamyl alcohol (24:1) extracting, the centrifugal 10min of 10000r/min gets supernatant liquor and moves in the clean pipe; The Virahol that adds 0.6 times of volume precooling ,-20 ℃ of lower placements more than the 2h, 4 ℃ of centrifugal 20min of 13000r/min remove supernatant liquor; Absolute ethanol washing with precooling precipitates vacuum lyophilization 2-3 time.DNA is dissolved in 100 μ L TE(10mM Tris-HCl, 1 mM EDTA, pH8.0) in the solution, be stored in-20 ℃ of refrigerators, the validity of PCR-DGGE Detection and Extraction high quality DNA.
The advantage that the present invention has is that agents useful for same is conventional reagent, and the DNA purity of with low cost, applied widely, simple to operate, mild condition, extraction is higher, need not that genomic dna is carried out purifying and can carry out the advantages such as follow-up pcr amplification and DGGE analysis.
Description of drawings
Fig. 1 is Daqu, poor unstrained spirits and cellar for storing things mud microbe genome DNA electrophoretogram.Swimming lane M is Marker(λ DNA/HindIII); Swimming lane 1,2 is the Daqu sample; Swimming lane 3,4 is respectively poor unstrained spirits and cellar for storing things mud sample.
Fig. 2 is Daqu, poor unstrained spirits and mud microbial bacterial genomic dna 16S rDNA V3 district, cellar for storing things amplification electrophoretogram.Swimming lane M is Marker(D2000); Swimming lane 1,2 is the Daqu sample; Swimming lane 3,4 is respectively poor unstrained spirits and cellar for storing things mud sample.
Fig. 3 is Daqu, poor unstrained spirits and cellar for storing things mud fungal genomic DNA 18S rDNA amplification electrophoretogram.Swimming lane 1,2 is the Daqu sample; Swimming lane 3,4 is respectively poor unstrained spirits and cellar for storing things mud sample.
Fig. 4 is Daqu, poor unstrained spirits and cellar for storing things mud bacterial genomes DNA 16S rDNA V3 district amplified production DGGE collection of illustrative plates.Swimming lane 1,2 is the Daqu sample; Swimming lane 3,4 is respectively poor unstrained spirits and cellar for storing things mud sample.
Fig. 5 is Daqu, poor unstrained spirits and cellar for storing things mud fungal genomic DNA 18S rDNA amplified production DGGE collection of illustrative plates.Swimming lane 1,2 is the Daqu sample; Swimming lane 3,4 is respectively poor unstrained spirits and cellar for storing things mud sample.
Embodiment
Embodiment: extraction, pcr amplification and the DGGE of Sichuan brewery Daqu, poor unstrained spirits and the total DNA of cellar for storing things mud microbial genome
⑴ sample pretreatment
Take by weighing the 5g Daqu, add 30mL PBS damping fluid (0.0557mol/LNa
2HPO
4, 0.0423mol/LNaH
2PO
4, pH8.0), vortex is even, and the centrifugal 10min of 800r/min gets supernatant liquor, and the centrifugal 10min of 10000r/min gets solid-state cenobium.
⑵ DNA extraction and detection
Above-mentioned solid-state cenobium is resuspended in 500 μ L DNA extraction liquid (0.1M Tris-HCl, 0.1M EDTA, 1.5M NaCl, 1%CTAB, pH8.0) in, add 10 μ L cellulases (75mg/ml), helicase (50mg/ml) and N,O-Diacetylmuramidase (40mg/mL), 37 ℃ of cracking 1h; Add 10 μ L 20%SDS, 65 ℃ of insulation 1.5h; Add 10 μ L Proteinase Ks (5mg/mL), 60 ℃ of shaking bath 1h, the centrifugal 10min of 4500r/min collects supernatant liquor; Use equal-volume phenol: chloroform: primary isoamyl alcohol (25:24:1) extracting, 4 ℃ of centrifugal 10min of 10000r/min collect supernatant liquor; Use the equal-volume chloroform: primary isoamyl alcohol (24:1) extracting, the centrifugal 10min of 10000r/min gets supernatant liquor and moves in the clean pipe; The Virahol that adds 0.6 times of volume precooling ,-20 ℃ of lower placements more than the 2h, 4 ℃ of centrifugal 20min of 13000r/min remove supernatant liquor; Absolute ethanol washing with precooling precipitates vacuum freezedrying 2-3 time.DNA is dissolved in 100 μ L TE(10mM Tris-HCl, 1 mM EDTA, pH8.0) in the solution, be stored in-20 ℃ of refrigerators, the validity of PCR-DGGE Detection and Extraction high quality DNA.
Claims (2)
1. high quality DNA extracting method that is used for the brewed spirit structural analysis of microbial community is characterized in that may further comprise the steps:
⑴ sample pretreatment
Take by weighing a certain amount of Daqu, add 30mL PBS damping fluid (0.0557mol/L Na
2HPO
4, 0.0423mol/L NaH
2PO
4, pH8.0), vortex is even, and the centrifugal 10min of 800r/min gets supernatant liquor, and the centrifugal 10min of 10000r/min gets solid-state cenobium.
⑵ DNA extraction and detection
Above-mentioned solid-state cenobium is resuspended in 500 μ L DNA extraction liquid (0.1M Tris-HCl, 0.1M EDTA, 1.5M NaCl, 1%CTAB, pH8.0) in, add 10 μ L cellulases (75mg/mL), helicase (50mg/mL) and N,O-Diacetylmuramidase (40mg/mL), 37 ℃ of cracking 1h; Add 10 μ L 20%SDS, 65 ℃ of insulation 1.5h; Add 10 μ L Proteinase Ks (5mg/mL), 60 ℃ of shaking bath 1h, the centrifugal 10min of 4500r/min collects supernatant liquor; Use equal-volume phenol: chloroform: primary isoamyl alcohol (25:24:1) extracting, 4 ℃ of centrifugal 10min of 10000r/min collect supernatant liquor; Use the equal-volume chloroform: primary isoamyl alcohol (24:1) extracting, the centrifugal 10min of 10000r/min gets supernatant liquor and moves in the clean pipe; The Virahol that adds 0.6 times of volume precooling ,-20 ℃ of lower placements more than the 2h, 4 ℃ of centrifugal 20min of 13000r/min remove supernatant liquor; Absolute ethanol washing with precooling precipitates vacuum lyophilization 2-3 time.DNA is dissolved in the 100 μ L TE solution, is stored in-20 ℃ of refrigerators, the validity of PCR-DGGE Detection and Extraction high quality DNA.
2. the extracting method of described a kind of Daqu and cellar for storing things mud microbial total genomic dna according to claim 1 is characterized in that described step 2) TE solution is 10mM Tris-HCl, 1 mM EDTA, pH8.0.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104178482A (en) * | 2014-09-11 | 2014-12-03 | 泸州品创科技有限公司 | Method for extracting total microbial DNA from strong-flavor yeasts |
| CN104630203A (en) * | 2013-11-13 | 2015-05-20 | 深圳华大基因研究院 | Method for preparing insect intestinal flora DNA (Deoxyribonucleic Acid) |
| CN105002165A (en) * | 2015-08-17 | 2015-10-28 | 河北衡水老白干酒业股份有限公司 | Method for improving extraction quality of total DNA of koji kaoliang spirit fermented grains through pretreatment |
| CN108559771A (en) * | 2018-04-04 | 2018-09-21 | 贵州省产品质量监督检验院 | The multifarious detection method of microbe colony during a kind of brewed spirit |
| CN110305862A (en) * | 2019-08-19 | 2019-10-08 | 郑州轻工业学院 | A method of extracting total serum IgE from Luzhou-flavor liquo fermented grain |
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Cited By (6)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN104630203A (en) * | 2013-11-13 | 2015-05-20 | 深圳华大基因研究院 | Method for preparing insect intestinal flora DNA (Deoxyribonucleic Acid) |
| CN104178482A (en) * | 2014-09-11 | 2014-12-03 | 泸州品创科技有限公司 | Method for extracting total microbial DNA from strong-flavor yeasts |
| CN105002165A (en) * | 2015-08-17 | 2015-10-28 | 河北衡水老白干酒业股份有限公司 | Method for improving extraction quality of total DNA of koji kaoliang spirit fermented grains through pretreatment |
| CN108559771A (en) * | 2018-04-04 | 2018-09-21 | 贵州省产品质量监督检验院 | The multifarious detection method of microbe colony during a kind of brewed spirit |
| CN110305862A (en) * | 2019-08-19 | 2019-10-08 | 郑州轻工业学院 | A method of extracting total serum IgE from Luzhou-flavor liquo fermented grain |
| CN110305862B (en) * | 2019-08-19 | 2023-05-09 | 郑州轻工业学院 | Method for extracting total RNA from fermented grains of Luzhou-flavor liquor |
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Application publication date: 20130206 |