Device and method for extracting total DNA of matrix-attached microorganisms

A microorganism and matrix technology, applied in the field of molecular biology, can solve the problems of tedious post-processing steps, loss of low-abundance microorganisms, easy to contaminate samples, etc., to improve typicality and representativeness, the total band is bright and clear, and overcome sampling difficult effect

Active Publication Date: 2020-06-12
NANJING UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0006] Although there have been various extraction methods developed for different researchers, these methods have their own defects in the DNA extraction of substrate-attached microorganisms in the environment, such as in-situ extraction, liquid nitrogen grinding, etc. The operation is time-consuming and labor-intensive, and multiple mortars need to be prepared. It is not easy to clean the equipment after grinding, which will easily contaminate the subsequent samples after extraction. Care must be taken during the extraction process. Do not shake to avoid DNA degradation; Bit extraction technology, one needs to completely immerse the small pieces of the matrix in the extraction solution, and the surface of the matrix must be destroyed, and the other is that the matrix itself is very easy to introduce impurities, and lysozyme is generally added to the extraction solution, causing the cell wall/membrane of microorganisms to rupture. After a large amount of biological macromolecules in the cells are dissolved, they will complex with im

Method used

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  • Device and method for extracting total DNA of matrix-attached microorganisms
  • Device and method for extracting total DNA of matrix-attached microorganisms
  • Device and method for extracting total DNA of matrix-attached microorganisms

Examples

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Embodiment 1

[0054] The device of the present invention for extracting substrate-attached total DNA of microorganisms, such as figure 1As shown, it includes a pressure-controlled flushing device 2, a negative pressure device and a collection device 8. The outlet end of the pressure-controlled flushing device 2 is provided with a flushing pipeline 3, and the pressure-controlled flushing device 2 is used to control the water outlet of the flushing pipeline 3. pressure, the negative pressure device includes a negative pressure pipeline and a negative pressure pump 9, the negative pressure pipeline includes a first negative pressure pipeline 5 and a second negative pressure pipeline 6, and the first negative pressure pipeline One end of 5 is sealed and connected with the pressure-controlled flushing device 2, and the other end is set as an open end; the flushing pipeline 3 is nested inside the first negative pressure pipeline 5, and the water outlet end of the flushing pipeline 3 is located at ...

Embodiment 2

[0077] In order to meet the needs of more accurate sampling, the open end of the first negative pressure pipeline 5 in the device of the present invention can be connected to ports of different shapes to adapt to different actual situations, such as figure 2 As shown, the open end can be connected to a tapered port to ensure that the suspension eluate sputtered out after washing under different conditions can be collected more completely.

[0078] This embodiment also provides a DNA extraction method using the device of this embodiment. The method is aimed at the super acidic circulating fluid conditions of a flue gas desulfurization and denitrification biofilm packed tower described in Chinese Patent Publication No. CN109569270B The extraction and determination of bacterial total DNA and 16S rDNA attached to the filler, and the extraction and determination of total fungal DNA and iTS at the same time, the steps are as follows:

[0079] Step 1, collection of functional flora ...

Embodiment 3

[0083] In this example, the device of Example 1 is used to extract the total DNA of fungi attached to the surface of the filler in a denitrification biofilm filler reactor, and the steps are as follows:

[0084] Step 1. Collection of functional flora samples: From the denitrification biofilm filter filled with ceramsite fillers, take about 0.5L of ceramsite fillers with good microbial growth status and place them in a 1L beaker. In this example, ceramsite The diameter of the filler is 10-30mm, and the bulk density is 300kg / m 3 , specific surface area 230~300m 2 / m 3 , Porosity 53%. Put 100mL of the prepared phosphate buffer solution of pH=7 into a water pressure container, and use a pressure of 60MPa to flush the packing to wash down the bacterial cells attached to the packing, and then enter the centrifuge tube. Remove two 50mL centrifuge tubes containing the microbial suspension from the device one after another, and centrifuge at a speed of 5,000×g for 2 minutes until th...

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Abstract

The invention discloses a device and a method for extracting total DNA of matrix-attached microorganisms, and belongs to the technical field of molecular biology, the device comprises a pressure control flushing device, a negative pressure device and a collecting device, the flushing water outlet pressure is controlled through the pressure control flushing device; suspension generated by flushingthe matrix through a flushing pipeline is collected to the collecting device through the negative pressure pipeline; the invention also discloses an extraction method of DNA. The negative pressure device is matched to collect samples; the method overcomes the problems of difficult sampling and incomplete sampling of microbial samples attached to a matrix in the prior art, significantly improves the representativeness, extraction efficiency and quality of microbial genome DNA attached to the matrix, has bright and clear strips, is basically non-degradable, and has good extraction efficiency ondifficult-to-extract microorganisms, such as fungi and the like.

Description

technical field [0001] The invention relates to the technical field of molecular biology, in particular to a device and a method for extracting the total DNA of substrate-attached microorganisms. Background technique [0002] In recent years, with the development of sequencing technology, molecular biology methods for analyzing microbial diversity and function based on total deoxyribonucleic acid (DNA) in environmental samples have been continuously improved, including fluorescent quantitative nucleic acid amplification of a specific gene Detection (qPCR) method, metagenomic technology for sequencing the total DNA of microorganisms in the environment, etc., have gradually been widely used. Metagenomic technology is recognized as the most comprehensive and accurate method for studying environmental microbial community structure and functional genes, because it has no special selectivity (such as pure culture / enrichment), and it also reduces the preference of sequencing techno...

Claims

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Application Information

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IPC IPC(8): C12M1/00C12Q1/6806
CPCC12Q1/6806
Inventor 李侃任鑫坤张徐祥许柯王庆张志超
Owner NANJING UNIV
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