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High-quality DNA (Deoxyribonucleic Acid) extraction method of spirit brewing microorganism

An extraction method and high-quality technology, which is applied in the field of high-quality DNA extraction and analysis of the microbial community structure in liquor brewing, can solve the problems of unsatisfactory total DNA quality, complex micro-ecological environment, and immeasurable impact, and achieve the purity of extracted DNA High performance, wide application range and low cost

Inactive Publication Date: 2013-02-06
SICHUAN UNIV
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Problems solved by technology

However, there are the following defects in the existing technical methods: (1) the scope of application of the genomic DNA extraction method is relatively narrow (only Daqu or pit mud), and the generally applicable method is rare; (2) certain flocs are added to the pretreatment method , but the impact of flocs on microorganisms with low abundance in the microbial community is immeasurable; at the same time, because the micro-ecological environment of Daqu and pit mud is very complex, there are many interfering components, the quality of the total genome DNA obtained is not ideal

Method used

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  • High-quality DNA (Deoxyribonucleic Acid) extraction method of spirit brewing microorganism
  • High-quality DNA (Deoxyribonucleic Acid) extraction method of spirit brewing microorganism
  • High-quality DNA (Deoxyribonucleic Acid) extraction method of spirit brewing microorganism

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Embodiment

[0014] Example: Extraction, PCR amplification and DGGE of total microbial genome DNA of Daqu, glutinous rice grains and pit mud in a winery in Sichuan

[0015] ⑴ sample pretreatment

[0016] Weigh 5g Daqu, add 30mL PBS buffer (0.0557mol / LNa) 2 HPO 4 , 0.0423mol / LNaH 2 PO 4 , pH 8.0), vortexed evenly, centrifuged at 800r / min for 10min, took the supernatant, and centrifuged at 10000r / min for 10min to obtain solid colony.

[0017] ⑵ DNA extraction and detection

[0018] Resuspend the above solid bacterial mass in 500 μL DNA extraction solution (0.1M Tris-HCl, 0.1M EDTA, 1.5M NaCl, 1% CTAB, pH8.0), add 10 μL cellulase (75mg / ml), helicase (50mg / ml) and lysozyme (40mg / mL), lysed at 37°C for 1h; added 10μL 20% SDS, incubated at 65°C for 1.5h; added 10μL proteinase K (5mg / mL), 60°C water bath shaker for 1h, 4500r / Centrifuge for 10 min and collect the supernatant; extract with an equal volume of phenol:chloroform:isoamyl alcohol (25:24:1), centrifuge at 10,000 r / min at 4°C for 1...

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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 [0001] The invention relates to the technical field of liquor microbial community ecology, in particular to a high-quality DNA extraction method for liquor brewing microbial community structure analysis. Background technique [0002] Chinese liquor has a long history of brewing, and the brewing process of Chinese liquor is not the same. The brewing and production of Chinese liquor uses Daqu as the main source of microorganisms and enzymes. In actual production, the quality of Daqu is related to the yield of liquor. and wine quality. The production process of Daqu is accompanied by a series of domestication processes of microbes. The community structure of microorganisms in Daqu is directly related to the production quality of Daqu. Chinese liquor is mainly based on pit ponds and fermented grains, etc. The microorganisms in the environment, Daqu and pit mud carry out complex energy metabolism and material metabolism, and the complex metabolic process of micro...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10
Inventor 周荣清张立强郑佳
Owner SICHUAN UNIV
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