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

A technology for ship ballast water and diversity analysis, applied in the field of environmental microbial molecular ecology, to achieve the effects of avoiding DNA loss, saving time, and simple and convenient methods

Active Publication Date: 2017-09-12
SHANGHAI OCEAN UNIV
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  • 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 extraction and diversity analysis of microbial total DNA in ship ballast water
  • Method for extraction and diversity analysis of microbial total DNA in ship ballast water
  • Method for extraction and diversity analysis of microbial total DNA in ship ballast water

Examples

Experimental program
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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 extracting and analyzing the total DNA of microorganisms in ship's ballast water; according to the characteristics of ship's ballast water, the present invention uses two-stage filtration and freeze-fixation of 3 μm and 0.22 μm microporous membranes to strengthen the For the attachment of microorganisms on the filter membrane, the purity of the total DNA of microorganisms obtained by treating with cetyltrimethylammonium bromide (CTAB) meets the needs of direct subsequent molecular biology research, and the extracted DNA fragments can be amplified for 16SrDNA And denaturing gradient gel electrophoresis (DGGE). After digital analysis of the electrophoresis graph, the diversity of microbial populations in the ship’s ballast water can be judged. The specific bands are cut and recovered by gel cutting, cloned and sequenced, and BLAST sequence comparison is performed to obtain the ballast water. The types of water-dominant bacterial flora can be determined to determine the structure and composition of microbial communities, which will lay a foundation for future research on microbial diversity and ecological functions in ship ballast water and sediments.

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 Patents(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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