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Soil microbial diversity analysis method

A technology of diversity analysis and analysis method, applied in the field of analysis of microbial diversity in soil, can solve the problems of radioactive contamination, time-consuming and laborious, unfavorable operation, etc., and achieve the effect of reducing radioactive contamination, good repeatability and good separation

Inactive Publication Date: 2015-08-12
TAIZHOU UNIV +1
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Although the conventional DGGE method is a well-recognized method that can obtain relatively accurate detection results, this method needs to run the gel, which is time-consuming and laborious, and the detection process takes a long time, and the formazan used in the denaturing gel Amide and acrylamide are toxic, and in order to improve the sensitivity, it is necessary to use isotope-labeled primers, there is radioactive contamination, and it is not conducive to practical operation

Method used

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  • Soil microbial diversity analysis method
  • Soil microbial diversity analysis method
  • Soil microbial diversity analysis method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0031] The analysis method of bacterial diversity in the soil in the present embodiment is as follows:

[0032] Soil Collection:

[0033] The soil in this example is from a broccoli field in Linhai City, Zhejiang Province. In fact, there is no special requirement for the specific location of the soil, and general soil can be used; specifically, the litter on the surface of the soil is removed first, Use a ring knife to collect 0-20cm of soil, bring it back to the laboratory, and complete DNA extraction within 48 hours;

[0034] Soil microbial total DNA extraction and quantification:

[0035] Soil microbial genomic DNA was extracted using FastPrep Company's Soil Microorganism Genomic DNA Extraction Kit (FastDNA Spin Kit for Soil), and then the Nanodrop 2000 micro-volume ultraviolet spectrophotometer was used to determine the DNA concentration and quality, so that A 260 / A 280The ratio was controlled between 1.6-2.0, and then the DNA was diluted to 20ng / μl with sterile water ...

Embodiment 2

[0051] The analysis method of fungal diversity in the soil in the present embodiment is as follows:

[0052] Soil Collection:

[0053] The soil in this example is the soil from an abandoned wasteland in Dongguan, Guangdong Province. In fact, there is no special requirement for the specific collection site of the soil, and general soil can be used. Specifically, the litter on the surface of the soil is removed first, and the environmental protection method is used. Collect 0-20cm of soil with a knife, bring it back to the laboratory, and complete DNA extraction within 48 hours;

[0054] Soil microbial total DNA extraction and quantification:

[0055] Soil microbial genome DNA was extracted using FastPrep's Soil Microorganism Genome Extraction Kit (FastDNA Spin Kit for Soil), and then the Nanodrop 2000 micro-volume UV spectrophotometer was used to determine the DNA concentration and quality, so that A 260 / A 280 The ratio was controlled between 1.6-2.0, and then the DNA was d...

Embodiment 3

[0078] The specific analysis method of this example is the same as that of Example 1, and will not be repeated here. The only difference is that the denaturation temperature of the WAVE nucleotide fragment analyzer is set to 55°C or 57°C for specific implementation one by one.

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Abstract

The invention relates to a soil microbial diversity analysis method, belonging to the technical field of soil microbial analysis. In order to solve the problems of heavy pollution and long time consumption, the soil microbial diversity analysis method is provided. The soil microbial diversity analysis method includes microbial genome DNA extraction of acquired soil to obtain a genome DNA extract; detection of the genome DNA extract to determine the soil genomic DNA concentration and purity; PCR amplification of DNA extracted from the soil to obtain a PCR amplification product; and microbial diversity analysis of the PCR amplification product by use of denaturing high Performance liquid chromatography. According to the method, primer marking is not needed, glue leaking using toxic substance formamide and acrylamide is not needed, the radioactive pollution is reduced, and the soil microbial diversity analysis method has the effects of less pollution, high degree of automation, short detection time, high sensitivity, and good detection strip separation effect.

Description

technical field [0001] The invention relates to a method for analyzing microbial diversity in soil, and belongs to the technical field of soil microbial analysis. Background technique [0002] At present, the research on soil microbial diversity is mainly by means of molecular biology, which directly obtains the DNA fragments of microorganisms from the soil for analysis, so that the analysis of environmental microbial diversity becomes a method as comprehensive as possible. In recent years, many methods have been reported on the analysis of microbial diversity in soil. However, the main focus is on the extraction and analysis of the total DNA of the bacterial community in the soil, and less attention has been paid to the extraction and analysis of the total DNA of the fungal community in the soil. Denaturing gradient gel electrophoresis (DGGE) analysis is commonly used in the study of soil bacterial and fungal diversity. Such as the Chinese patent application (publication ...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12Q1/04
Inventor 李钧敏金则新陈露茜
Owner TAIZHOU UNIV
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