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Method for quantitatively analyzing clostridia, lactobacilli, bacilli and methanobacteria in pit mud

A Bacillus, quantitative analysis technology, applied in the field of bioengineering, can solve the problems of time-consuming and laborious, lack of stability and reliability of detection results, and achieve the effect of strengthening monitoring, simple operation, and simplifying detection procedures

Active Publication Date: 2014-06-04
LUZHOU PINCHUANG TECH CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, the quantitative detection of microorganisms in pit mud still adopts the method of screening of selective medium and separation and counting of pure culture, which is easily affected by external environmental factors, strain characteristics, etc., and the detection results lack stability and reliability, and are time-consuming and laborious.

Method used

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  • Method for quantitatively analyzing clostridia, lactobacilli, bacilli and methanobacteria in pit mud
  • Method for quantitatively analyzing clostridia, lactobacilli, bacilli and methanobacteria in pit mud
  • Method for quantitatively analyzing clostridia, lactobacilli, bacilli and methanobacteria in pit mud

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] The preparation of embodiment 1 standard curve

[0038] 1. Synthesize specific primers for Clostridium, lactic acid bacteria, Bacillus and methanogens, and specifically amplify the screened Clostridium, lactic acid bacteria, Bacillus and methanogens

[0039] Clostridium-specific primer sequence (5′→3′):

[0040] Clost-F: AAAGGRAGATTAATACCGCATAA;

[0041] Clost-R: TTCTTCCTAATCTCTACGCA.

[0042] Lactic acid bacteria specific primer sequence (5'→3'):

[0043] Lacto-F: AGAACACCAGTGGCGAAGG;

[0044] Lacto-R: CAGGCGGAGTGCTTAATGC.

[0045] Bacillus-specific primer sequence (5′→3′):

[0046] Bacil-F: ATGGCTGTCGTCAGCT;

[0047] Bacil-R: ACGGGCGGTGTGTAC.

[0048] Methanogen-specific primer sequence (5′→3′):

[0049] Metha-F: GGTGGTGTMGGATTCACACARTAYGCWACAGC;

[0050] Metha-R: TTCATTGCRTAGTTWGGRTAGTT.

[0051] R in the above primers is A or G; W is A or T; M is A or C; Y is T or C.

[0052] 2. Screening of Clostridium, Lactobacillus, Bacillus and Methanogen in pit mud and...

Embodiment 2

[0090] The preparation of embodiment 2 standard samples

[0091] 1. Collection of pit mud samples

[0092] Pit mud samples were collected (100-year-old, 200-year-old, and 300-year-old pit mud were collected respectively). If DNA cannot be extracted in time after sample collection, it should be stored in a -80°C refrigerator immediately.

[0093] 2. Extraction method of microbial total DNA in pit mud

[0094] Weigh 1g of pit mud sample into a 50mL centrifuge tube, add 5mL of PBS buffer solution pH7.64 and 8-9 sterilized glass beads to the centrifuge tube, vortex for 5min, centrifuge at 200×g for 5min, and absorb the supernatant. Add 5mL PBS buffer solution to the precipitate and repeat washing twice, combine the three supernatants (operated on ice), centrifuge at 12000rpm for 5min, and take the precipitate. Add 1mL CTAB extract (2%CTAB, 5mol / L NaCl, 1mol / LTris-HCl(pH8.0), 0.5mol / L EDTA) and 20μL mercaptoethanol to the centrifuge tube, shake at 65°C for 30min , add 5 μL of pr...

Embodiment 3

[0096] Quantitative analysis of Clostridium, lactic acid bacteria, bacillus and methane bacteria in the pit mud microbial community of embodiment 3

[0097] Quantification of Clostridium, Lactobacillus, Bacillus and Methanogen in the pit mud sample: using the total DNA of the pit mud obtained in Example 2, and specific primers for Clostridium, Lactobacillus, Bacillus and Methanogen in Example 1. Daqu fluorescent quantitative PCR analysis was performed according to the fluorescent quantitative PCR reaction system and PCR program in Example 1.

[0098] Quantification of Clostridia in pit mud samples: according to the linear equation y=-0.3303x+13.137(R 2 =0.9978) to calculate the copy number of Clostridia in pit mud samples of different ages, so as to obtain the change trend of Clostridium biomass in different pit ages (such as figure 1 shown).

[0099] Quantification of lactic acid bacteria in the pit mud sample: according to the linear equation y=-0.3411x+14.07(R 2 =0.9978)...

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Abstract

The invention belongs to the technical field of bioengineering, and in particular relates to a method for quantitatively analyzing clostridia, lactobacilli, bacilli and methanobacteria in pit mud. The technical problem to be solved by the invention is to provide a new choice for analyzing the clostridia, lactobacilli, bacilli and methanobacteria in the pit mud. The technical scheme of the invention is as follows: the method for quantitatively analyzing the clostridia, lactobacilli, bacilli and methanobacteria in the pit mud comprises the steps of a, preparation of a standard curve; b, extraction of genome DNA; c, amplification; d, quantitative analysis. The method disclosed by the invention can be used for analyzing the change trend of the clostridia, lactobacilli, bacilli and methanobacteria in the pit mud.

Description

technical field [0001] The invention belongs to the technical field of bioengineering, and in particular relates to a quantitative analysis method for clostridium, lactic acid bacteria, bacillus and methane bacteria in pit mud. Background technique [0002] The cellar is a wine-making container that integrates various reactions such as saccharification, alcoholization, and esterification in the production process of liquor. As the saying goes, "good wine comes out of old cellars". In the practice of liquor production, it is often found that the fermented grains near the bottom and wall of the cellar are rich in aroma, and the root cause is the metabolism of functional bacteria related to aroma in the pit mud. effect. The pit itself is the carrier of various microorganisms. With the long-term continuous fermentation, the functional microorganisms in the pit mud are continuously domesticated and enriched, and finally form its unique microbial community. Therefore, the type a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12Q1/06C12R1/145C12R1/07
CPCC12Q1/06C12Q1/6851C12Q1/686C12Q2545/114C12Q2531/113
Inventor 许正宏史劲松陆震鸣肖辰王松涛
Owner LUZHOU PINCHUANG TECH CO LTD
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