Method for high-efficiency extraction of extracellular DNA in sediments

A sediment and high-efficiency technology, applied in the fields of environmental science and bioengineering, to achieve the effect of strong practicability and high degree of reliability

Inactive Publication Date: 2017-03-08
TIANJIN UNIV
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  • Abstract
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  • Application Information

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Problems solved by technology

[0004] The purpose of the present invention is to provide a method for the extraction of microbial extracellular DNA in

Method used

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  • Method for high-efficiency extraction of extracellular DNA in sediments
  • Method for high-efficiency extraction of extracellular DNA in sediments
  • Method for high-efficiency extraction of extracellular DNA in sediments

Examples

Experimental program
Comparison scheme
Effect test

Example Embodiment

[0026] Example 1: Extraction of extracellular DNA from sediment samples;

[0027] Step 1: Construction of extracellular DNA internal standard gene;

[0028] Section 1.1: Construction of cloning vector of extracellular DNA internal standard gene;

[0029] The CESA9 gene in Arabidopsis thaliana was amplified by PCR and then gelatinized and recovered, 0.5-4μl PCR was gently mixed with 1μlpEASY-T1 cloning vector, and reacted at room temperature for 5 minutes. After the reaction, the centrifuge tube was placed on ice;

[0030] Section 1.2: Amplification of internal standard genes in extracellular DNA

[0031] Add the ligation product to 50μl of competent E.coli DH5α cells, heat shock in a 42℃ water bath for 30 seconds, and immediately place on ice for 2 minutes; add 250μl of LB medium equilibrated to room temperature, incubate at 200rpm and 37℃ for 1 hour; take 8μl 500mM IPTG and 40μl 20mg / ml X-gal were mixed, spread evenly on the prepared plate, and placed in a 37°C incubator for 30 minute...

Example Embodiment

[0046] Example 2: PCR testing the extraction efficiency of microbial extracellular DNA in sediments;

[0047] (1) Target gene primer sequence

[0048]

[0049] (2) Qualitative PCR reaction program

[0050]

[0051] (3) Fluorescent real-time quantitative PCR reaction

[0052]

[0053] (4) Qualitative PCR reaction system

[0054]

[0055] (5) Quantitative PCR reaction system

[0056]

[0057] Table 2: Detection of extraction efficiency of microbial extracellular DNA from sediment samples

[0058]

[0059] Using the method described in Example 2, the extraction efficiency of the microbial extracellular DNA in the sediment samples can be all higher than 44%, and the highest can reach 57%. Therefore, the present invention is suitable for extracting microbial extracellular DNA in sediment samples. In addition, the application scope of the present invention not only includes sediments, but also has high DNA quality and extraction efficiency for soil and other media.

[0060]

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Abstract

Provided is a method for high-efficiency extraction of extracellular DNA in sediments. The method includes construction of an extracellular DNA internal standard gene (CESA9), extraction of sediment sample extracellular DNA, and quantification of the internal standard gene. The internal standard gene CESA9 used for calculating the DNA extraction efficiency in the method is a section of a cellulose synthase gene in arabidopsis thaliana and cannot be detected in to-be-extracted DNA environmental samples. The method can separate and purify the extracellular DNA in the environmental samples. Through measurement by an ultraviolet spectrophotometer, the OD260/OD280 of the extracted DNA can reach 1.57, the OD260/OD230 can reach 1.78, subsequent molecular operation can be carried out, and the practicability is relatively high. Through analysis of the extraction efficiency of the DNA, the extraction rate of the extracellular DNA in the sediments by the method can reach 57%, and the method is indicated to have relatively high reliability degree.

Description

technical field [0001] The invention relates to a simple extraction method for microbial extracellular DNA in environmental samples, especially in sediment samples. The method uses biotechnology means to process sediment samples, and belongs to the technical fields of environmental science and bioengineering. Background technique [0002] The widespread existence and spread of antibiotic resistance genes in the natural environment has caused huge environmental and health risks. It is necessary to accurately detect the number of resistance genes in the natural environment. Antibiotic resistance genes in the environment are usually located on some mobile genetic elements, such as plasmids, integrons, transposons, etc. These resistance genes can spread and spread through the horizontal transfer of mobile genetic elements. Antibiotic resistance genes located inside bacteria can often enter other bacteria through conjugation and transduction, while genes located outside bacteria ...

Claims

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Application Information

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IPC IPC(8): C12N15/10C12Q1/68
CPCC12N15/1003C12Q1/6851C12Q2531/113C12Q2545/101
Inventor 谭璐毛大庆徐艳罗义
Owner TIANJIN UNIV
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