Method for total DNA extraction and diversity analysis of ship ballast water microorganisms

A technology for ship ballast water and microorganisms, which is applied in the field of environmental microbial molecular ecology to achieve the effects of simple operation, simple and convenient method, and avoiding DNA loss.

Active Publication Date: 2015-09-30
SHANGHAI OCEAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, there is no relevant report on the use of PCR-DGGE technology to study the microbial diversity of ship ballast water at home and abroad.

Method used

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  • Method for total DNA extraction and diversity analysis of ship ballast water microorganisms
  • Method for total DNA extraction and diversity analysis of ship ballast water microorganisms
  • Method for total DNA extraction and diversity analysis of ship ballast water microorganisms

Examples

Experimental program
Comparison scheme
Effect test

Embodiment

[0053] Select a ship, take samples, and take ballast water from ballast tanks in different spatial positions of the ship, analyze the bacterial population structure and determine the dominant bacterial group as follows

[0054] 1) Extraction and detection of microbial total DNA in ballast water:

[0055] Use water collectors to collect ballast water in different ballast tanks of ships, collect 2L samples in sterilized blue-necked glass bottles, and store them at 4°C; take ballast water samples and filter them through 3 μm and 0.22 μm microporous membranes (filter membranes) Prior sterilization treatment) After two-stage filtration, discard the 3 μm microporous filter membrane, and the ballast water microorganisms are on the 0.22 μm microporous filter membrane; use sterilized surgical forceps to transfer the 0.22 μm filter membrane to a 1.5mL centrifuge tube, Immediately freeze in a -80°C refrigerator for 4 hours; take out the centrifuge tube with the microbial filter membrane ...

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Abstract

The invention discloses a method for total DNA extraction and diversity analysis of ship ballast water microorganisms. The method comprises the steps that according to the characteristics of ship ballast water, two-level filtering and refrigerating fixation is performed by using millipore filtration membranes of 3 micrometers and 0.22 micrometer, and adhering of the microorganisms to the filtration membranes is reinforced; the microorganisms are processed by using cetyltrimethyl ammonium bromide (CTAB) to enable the total DNA purity of the obtained microorganisms to meet the requirements of directly performing subsequent molecular biology study; 16SrDNA amplification and denaturing gradient gel electrophoresis (DGGE) can be performed on extracted DNA fragments, and the diversity degree of ship ballast water microorganism populations can be judged after digitalization analysis is performed on electrophoretogram; the varieties of dominant bacterial populations of the ballast water is obtained by performing rubber tapping recovering, clone sequencing and BLAST sequence alignment on special bands, and therefore the structual components of the microorganism populations are determined. According to the method, a basis is laid for studying the diversity and ecological function of the microorganisms in the ship ballast water and sediment.

Description

technical field [0001] The invention belongs to the field of molecular ecology of environmental microorganisms, and in particular relates to a method for extracting and analyzing the total DNA of microorganisms in ballast water of ships. Background technique [0002] The ship's ballast water is the seawater that is ballasted into the ship's ballast tank when it sets sail in order to maintain the stability of the ship's hull when it is empty, and is discharged from the ballast tank when it arrives at the port for loading. The discharge of ballast water and sediment is the main way for marine organisms to spread between the port of departure and the port of destination, and has been identified by the World Environmental Protection Fund (GEF) as one of the four major threats to the ocean. [0003] When the ship arrives at the port for loading, the ballast water will be unloaded, and the ballast tank will be cleaned at the same time. Due to the special structure of the ballast t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10C12Q1/68C12Q1/04
Inventor 吴惠仙肖南燕薛俊增王琼张拿慧
Owner SHANGHAI OCEAN UNIV
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