Rapid and effective cellar mud archaea community analysis method

An analysis method and pit mud technology are applied in the field of analysis of pit mud microbial communities to achieve the effects of rapid analysis means and improved quality and purity

Inactive Publication Date: 2013-02-06
SICHUAN UNIV
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There are certain limitations in the application of the aforementione

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  • Rapid and effective cellar mud archaea community analysis method
  • Rapid and effective cellar mud archaea community analysis method
  • Rapid and effective cellar mud archaea community analysis method

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Embodiment Construction

[0024] The present invention proposes a DNA extraction and amplification method aimed at humus pollution and archaeal special cell structure:

[0025] 1) Sample pretreatment:

[0026] (1) Weigh 5g of pit mud with different ages (1 year, 50 years, 100 years and 300 years) into a 50mL sterile centrifuge tube, add 20mL of sterile PBS (pH6.0), vortex and mix, add 5mL of 0.1mol / L aluminum sulfate solution, shake gently and mix well, centrifuge at 800r / min, collect the supernatant, wash the precipitate with PBS (pH6.0) repeatedly for 1-2 times, combine the supernatant, the process can be added according to the turbidity of the supernatant Appropriate amount of 0.1mol / L aluminum sulfate solution; centrifuge at 10000r / min for 10min, discard the supernatant.

[0027] 2) Genome DNA extraction:

[0028] ①Dissolve the precipitate obtained in 1) in 500μL DNA extraction buffer (0.1M Tris-HCl, 0.1M EDTA, 1.5M NaCl, 1%CTAB, pH8.0), add lysozyme (100mg / mL), cellulase (200mg / mL), helicase ...

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Abstract

The invention relates to an analysis method of a cellar mud archaea community, in particular to an analysis method of a highly flavored type white wine cellar mud archaea community and the characteristic of diversity. The method comprises the following specific steps of: (1) weighing a certain quantity of cellar mud, and adding a PBS (Phosphate Buffer Solution) and an aluminum sulfate solution for pretreatment; (2) adding a DNA extraction buffer solution, lysozyme, achromopeptidase and other multienzyme systems for cell wall disruption; (3) adding chloroform-phenol for extraction and impurity removal; (4) adding isopropanol to precipitate to obtain sample genome DNA; and (5) carrying out nested PCR-DGGE (Polymerase Chain Reaction-Denaturing Gradient Gel Electrophoresis) analysis by using the archaea genome DNA as a template. The PBS buffer system is provided for cellar mud microorganisms before DNA extraction, and added aluminum sulfate can efficiently remove humus; achromopeptidase capable of effectively cracking archaea cell walls is pertinently added, and the purposes of increasing the quality and the purity of the genome DNA are achieved; and true and reliable wine cellar archaea community diversity information is obtained by combining the nested PCR-DGGE fingerprint spectrum technology, and an accurate and rapid analysis means for accurately analyzing the transition rule of the highly flavored type white wine cellar mud archaea community along with the age of the cellar.

Description

technical field [0001] The invention relates to an analysis method of cellar mud microbial community, in particular to an analysis method of the archaeal bacterial community and diversity characteristics of Luzhou-flavor liquor cellar mud. Background technique [0002] With the rapid development of molecular biology technology, the culture-free method of modern molecular biology with PCR technology as the core is widely used in the evaluation of environmental microbial communities and diversity characteristics. The extraction of high-quality and high-purity genomic DNA is the first prerequisite for successful PCR amplification and analysis of environmental microbial community characteristics. However, due to the complexity of the microbial cell structure and the particularity of the sampling environment, the extracted crude DNA often has low content and low purity. Studies have shown that the contamination of environmental genomic DNA by humic substances and their analogues...

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04
Inventor 周荣清何翠容郑佳
Owner SICHUAN UNIV
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