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Analysis method for detecting diversity of soil microorganism

A technology of soil microorganisms and analysis methods, applied in the field of analysis of soil microbial diversity, can solve the problems of difficult detection, low DNA recovery rate, loss of DNA, etc.

Inactive Publication Date: 2012-04-04
SHANGHAI HAIDI GARDENING
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  • Abstract
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  • Application Information

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Problems solved by technology

Therefore, the DNA initially extracted by most methods must be recovered and purified. However, part of the DNA will be lost more or less during the recovery process, especially the recovery rate of large fragments of DNA is relatively low. Some people use dialysis bags to recover and purify. It can only reach 65.34%, and a part of DNA is still lost. It is difficult to detect in the follow-up detection of microbial species that actually exist in small quantities, so it cannot truly reflect the soil micro-ecological structure

Method used

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  • Analysis method for detecting diversity of soil microorganism
  • Analysis method for detecting diversity of soil microorganism
  • Analysis method for detecting diversity of soil microorganism

Examples

Experimental program
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Embodiment 1

[0027] Extraction of soil DNA: Weigh 0.5g soil sample of medicinal plant rhizosphere soil (its pH value is 4.30) in Luoji County, Sichuan Province in a 2ml centrifuge tube, add 1.3ml extract (0.1mol Tris, 0.1mol EDTA, 0.1 mol phosphate buffer, 1.5mol NaCl, 1% CTAB, pH8.0) vortexed and mixed, then placed in a -80°C refrigerator for 30 minutes (or liquid nitrogen quick-frozen for 5 minutes), took it out and thawed it in a 65% water bath, and repeated freezing and thawing 3 times Add 100 μL of proteinase K (1 mg / ml), shake at 37°C for 30 minutes, add 200 μL of 20% SDS, bathe in water at 65°C for 2 hours (during every 15 to 20 minutes, gently invert and mix well), centrifuge at room temperature for 10 minutes at 6000 r / min , collect the supernatant, try not to get the impurities between the supernatant and the soil sediment; add 0.75ml extract solution and 50μL 20% SDS to the sediment, vortex 10s in 65℃ water bath for 10min, centrifuge at room temperature 6000r / min for 10min, and c...

Embodiment 2

[0035] The soil sample in the present embodiment is taken from the saline soil of Lop Nur Lake in Xinjiang, and its pH is 6.96, the extraction of its soil DNA, the detection of soil DNA concentration and purity, the PCR reaction of soil DNA, the denaturing gradient gel electrophoresis of PCR reaction product ( DGGE) analysis method is the same as embodiment 1.

Embodiment 3

[0037] The soil sample in this example is taken from the rhizosphere soil of Populus euphratica on the Tarim River bank in Xinjiang, and its pH is 8.64. Gel electrophoresis (DGGE) analysis method is the same as that in Example 1.

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Abstract

The invention relates to a biological analysis method, especially to an analysis method for detecting the diversity of soil microorganism. The method comprises the following steps of: (1) carrying out DNA extraction in soil; (2) detecting DNA concentration and purity in soil by using the DNA extract obtained from Step (1); (3) carrying out PCR specific amplification on the extracted DNA from soil; and (4) carrying out denaturing gradient gel electrophoresis analysis on the PCR reaction product. By the adoption of the method for detection and analysis of various soils, the result is consistent with the real state of microorganisms as there is no loss of the extracted DNA solution during the treatment process. Therefore, the invention provides a perfect solution for the research of the soil microorganism state.

Description

technical field [0001] The invention relates to a biological analysis method, in particular to an analysis method for detecting soil microbial diversity. Background technique [0002] At present, the research on soil microorganisms is mainly by means of molecular biology, and the metagenomic DNA of microorganisms is obtained directly from the soil for analysis. This strategy makes the analysis of environmental microbial diversity as comprehensive as possible. In recent years, there have been many reports on soil metagenomic DNA extraction methods. However, it mainly focuses on the extraction of the total DNA of the bacterial community in the soil, and less attention is paid to the extraction of the total DNA of the fungal community in the soil and its extraction effect. The samples from the environment contain very complex components, especially the humic acids in the soil cannot be removed during the extraction process, which directly affects the subsequent PCR amplificati...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04
Inventor 丁国才
Owner SHANGHAI HAIDI GARDENING
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