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Method for extracting microbial DNA in water body

An extraction method and microbial technology, applied in the field of molecular ecology, can solve the problems of low cell lysis rate, interfere with the extraction reaction, DNA loss, etc., and achieve the effects of high purity, improved elution efficiency, and single DNA band.

Inactive Publication Date: 2017-09-12
SHANGHAI ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] Due to the wide variety of water samples, complex components, and a large number of inorganic and organic compounds, especially the natural organic humic acid and heavy metals in the water environment will interfere with the extraction reaction and the subsequent PCR enzyme reaction. In addition, every crude extraction of DNA Purification steps that inevitably cause DNA loss
Therefore, it is necessary to study a convenient, fast and practical method for DNA extraction suitable for the analysis of microbial diversity in water bodies, so as to solve the problems of PCR inhibitors such as humic acid, low cell lysis rate, and serious DNA loss in DNA samples obtained by conventional methods. question

Method used

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  • Method for extracting microbial DNA in water body
  • Method for extracting microbial DNA in water body
  • Method for extracting microbial DNA in water body

Examples

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Embodiment 1

[0045] 1. Utilize the method of the present invention to extract the microbial DNA of 6 different water body samples (river water, lake water, each 2 sampling points that seawater position is different), sample is respectively taken from the Huangpu River fresh water of 2 different positions, 2 different positions The eutrophic water of Taihu Lake and seawater from the Yangtze River estuary of Chongming Island in two different locations, the extraction steps are as follows:

[0046] 1) Using the five-point sampling method, use plexiglass samplers to collect water bodies 0.5 meters below the water surface at two different locations (10-20 meters apart from the water surface), put them into sterilized bottles, store the samples at 4 degrees, and transport them back to In the laboratory, extract DNA immediately, take 50ml of water sample and filter it through a 0.22μm microporous membrane, cut the filter membrane and the filtrate into 1-2mm debris under sterile conditions, put it ...

Embodiment 2

[0069] In this example, reagent I is: 0.3M phosphate buffer solution, pH=8.0; the content of each component in reagent II is: 2wt% SDS, 60mM Tris-HCl, 200mM NaCl, 100mM EDTA, 1wt.%PVPP, pH=8.0 ; Reagent III is: 120mM aluminum sulfate; the content of each component of reagent IV is: 5M potassium acetate, 6wt.% glacial acetic acid, pH=5.0; the content of each component of reagent V is: 10M guanidine isothiocyanate, 0.6M potassium acetate The contents of each component of the reagent VI are: 10M guanidine isothiocyanate, 23mM sodium citrate; the reagent VII is: 75% ethanol.

[0070] Other operating steps are the same as in Example 1, and the DNA concentration and purity results of the extracted water body samples are shown in Table 2.

[0071] Table 2

[0072]

[0073]

[0074] Utilize the microbial DNA of 6 different water body samples (river water, lake water, the different sampling points of seawater position) that this embodiment extracts, PCR amplifies 18S rRNA gene, ...

Embodiment 3

[0076] In this example, the reagent I is: 0.25M phosphate buffer, pH=7.5; the content of each component in the reagent II is: 1.5wt% SDS, 40mM Tris-HCl, 150mM NaCl, 70mM EDTA, 0.5%PVPP, pH=8.0 ; Reagent III is: 100mM aluminum sulfate; the content of each component of reagent IV is: 3M potassium acetate, 4wt.% glacial acetic acid, pH=4.8; the content of each component of reagent V is: 8M guanidine isothiocyanate, 0.4M potassium acetate The content of each component of the reagent VI is: 8M guanidine isothiocyanate, 22mM sodium citrate; the reagent VII is: 73% ethanol.

[0077] Other operating steps are the same as in Example 1, and the results of DNA concentration and purity of the extracted water samples are shown in Table 3.

[0078] table 3

[0079] sample

[0080] Utilize the microbial DNA of 6 different water body samples (river water, lake water, the different sampling points of seawater position) that this embodiment extracts, PCR amplifies 18S rRNA gene, agar...

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Abstract

The invention relates to a method for extracting microbial DNA in a water body. The method comprises: collecting microorganisms by using a sterile microporous filtration membrane; breaking the microbial cells by using physical and chemical methods; removing humic acid and other impurities by using potassium acetate, guanidine isothiocyanate, sodium citrate and other reagents; carrying out centrifugal purification on the cell lysate a plurality of times; and extracting with ethanol and potassium acetate to obtain the purified microbial genomic DNA. According to the present invention, the extracted microbial DNA has advantages of good integrity, high purity and high yield, and has the OD260 / OD280 ratio of 1.8-2.0; after the extracted microbial DNA is subjected to agarose gel electrophoresis and staining is performed, the DNA band is clear and single, and the phenomena such as tailing and impurity band do not exist; and the extraction method has advantages of high microbial DNA yield, simple operation, low cost, no requirement of purification during the extraction process, extraction step reducing and extraction cost reducing, and is suitable for the diversity analysis of various water microorganisms.

Description

technical field [0001] The invention belongs to the field of molecular ecology, and in particular relates to a method for extracting microbial DNA in water bodies. Background technique [0002] The water body contains very rich microbial resources. The traditional pure culture technology is mainly to separate and cultivate the microorganisms through selective medium, and then carry out the classification and identification of pure bacteria. Using the traditional pure culture technology, about 0.25% of the microorganisms in the freshwater environment can be cultivated, and about 0.001-0.1% of the microorganisms in the seawater environment can be cultivated. Microorganisms cannot be analyzed, which brings great limitations to the development and utilization of microbial diversity resources. [0003] However, in recent years, various molecular biology methods (PCR, clone library, DGGE, etc.) The evaluation and analysis of population dynamics, as well as the location, expressi...

Claims

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Application Information

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IPC IPC(8): C12N15/10
CPCC12N15/1003
Inventor 李鹏唐雪明潘爱虎赵凯蒋玮王金斌武国干吕贝贝吴潇贾军伟王荣谈白蓝刘华王慧
Owner SHANGHAI ACAD OF AGRI SCI
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