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DNA Marker for detecting coal geology microbe archaea species, and preparation method and application thereof

A microbial and archaeal technology, applied in biochemical equipment and methods, microbial determination/inspection, DNA/RNA fragments, etc., can solve the problem of no DNAMarker research report, etc., to reduce experimental time, sharp bands, Highly reproducible effect

Active Publication Date: 2017-09-05
SHANXI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

But so far, there is no research report on DNAMarker in PCR-DGGE detection of microbial flora in coal geological environment

Method used

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  • DNA Marker for detecting coal geology microbe archaea species, and preparation method and application thereof
  • DNA Marker for detecting coal geology microbe archaea species, and preparation method and application thereof
  • DNA Marker for detecting coal geology microbe archaea species, and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0048] 1. Extract the genomic DNA from the pre-enrichment culture medium of different enrichment culture methods, and select 2 sets of primers for PCR amplification;

[0049] 1.1 For the extraction steps of genomic DNA, see patent document CN2016103727233, and the verification map of genomic DNA 0.7% agarose gel is attached figure 1 shown;

[0050] 1.2 For the first PCR amplification, the obtained PCR amplification products were purified and recovered with the agarose gel DNA recovery kit of Zhongke Ruitai (Beijing) Biotechnology Co., Ltd., stored at -20°C for later use, and the PCR reaction was carried out in PTC-200 , run on Bio-Rad, USAPCR instrument;

[0051] 1.2.1 PCR reaction system:

[0052]

[0053]

[0054] 1.2.2 The PCR reaction program is: 95°C pre-denaturation for 5min, 95°C for 45s, 55°C for 30s, 72°C for 60s, 34 cycles, 72°C for 10min, 10°C for ever,

[0055] 1.2.3 The primer sequence is as follows:

[0056] 0357f: CCCTACGGGGCGCAGCAG;

[0057] 0691r: G...

Embodiment 2

[0114] 1. Extract the genomic DNA from the water samples produced from high-rank coal seams in Qinshui Basin, Shanxi and the early and mid-term enrichment culture solution of Zhaotong lignite in Yunnan, and select 2 sets of primers for PCR amplification. Genome extraction and PCR amplification refer to specific implementation plan 1 1.1, 1.2, 1.3 in.

[0115] 2. DGGE analysis:

[0116] 2.1 Select the concentration of polyacrylamide gel to be 8%, and the range of gel denaturation is 40% to 60%. Take 18ml each of 40% and 60% gel, add 50μL of TEMED and 40μL of 10% ammonium persulfate respectively, and make gradient mixture The gel can be solidified at room temperature in summer, and can be solidified in a 37°C incubator in winter. If it is solidified at room temperature, the gel time should be at least 3 hours;

[0117] 2.2 After the gel is completely solidified, pull out the comb, install the whole board on the DGGE bracket, put the DGGE bracket with the board into the electrop...

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Abstract

The invention relates to a special specie DNA Marker for detecting coal geology microbe archaea in the denaturing gel gradient electrophoresis (DGGE) molecule detection of coal geology environment microbe florae, and belongs to the field of molecular microbial ecology. The DNA Marker for detecting coal geology microbe archaea species is composed of a DNA fragment a with the nucleotide sequence represented by SEQ. ID. NO.1, a DNA fragment b with the nucleotide sequence represented by SEQ. ID. NO.2, a DNA fragment c with the nucleotide sequence represented by SEQ. ID. NO.3, a DNA fragment d with the nucleotide sequence represented by SEQ. ID. NO.4, a DNA fragment e with the nucleotide sequence represented by SEQ. ID. NO.5, a DNA fragment f with the nucleotide sequence represented by SEQ. ID. NO.6, a DNA fragment g with the nucleotide sequence represented by SEQ. ID. NO.7, and a DNA fragment h with the nucleotide sequence represented by SEQ. ID. NO.8.

Description

technical field [0001] The invention relates to a species DNA Marker specially used for archaea in coal geological environment for denaturing gradient gel electrophoresis (DGGE) molecular detection of microbial flora in coal geological environment, belonging to the field of microbial molecular ecology. Background technique [0002] Microorganisms in the coal geological environment refer to the microorganisms detected in coal seam output water samples or coal samples, or the microorganisms detected after enrichment and culture of the above two, which are mainly divided into two categories: bacteria and archaea. The archaea in the coal geological environment mainly refer to the methanogenic archaea. Methanogenic archaea are widely distributed, from soil to lake sediments, from land to ocean, from sub-zero low temperature environments to high temperature environments above 100 °C, and are found in most anoxic environments on the earth. They play an important role in the earth's...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04C12N15/11C12N15/70
CPCC12N15/70C12Q1/689C12Q2600/166
Inventor 杨秀清陈彦梅王保玉袁宗本吴瑞薇
Owner SHANXI UNIV
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