Use method of DGGE (Denaturing Gradient Gel Electrophoresis) technology in detecting phytoplankton structure

Abstract

The invention discloses a use method of a DGGE (Denaturing Gradient Gel Electrophoresis) technology in detecting a phytoplankton structure. The method comprises the following steps of: filtering a phytoplankton water sample by using a bolting silk of 200 meshes; then filtering filtered seawater by using a nitrocellulose membrane with an aperture of 0.45 mum; transferring the filter membrane into an Eppendorf tube of 2ml and placing the Eppendorf tube at -20 DEG C for analyzing the diversity of the phytoplankton; collecting and washing algae cells and extracting DNA; carrying out a PCR (Polymerase Chain Reaction) and agarose electrophoresis detection; analyzing the phytoplankton structure by using the DGGE; and finally, carrying out graphic processing and data analysis. The method can accurately detect the structural composition of the phytoplankton and is beneficial to the early warning of the red tide breakout of the phytoplankton.

Description

Technical field: [0001] The invention relates to the technical field of marine phytoplankton species and / or quantity detection, in particular to a method for using DGGE technology in detecting phytoplankton community structure. Background technique: [0002] Plankton is a unique group that lives with the current in water bodies, and is easy to move passively under the action of wind and current, with no or only weak swimming ability; plankton includes two categories: phytoplankton and zooplankton. Plankton are small in size, short in life cycle, fast in reproduction, very sensitive to changes in the environment, and can respond quickly to changes in the nutritional status of water bodies. In waters with good water quality, the diversity index and uniformity index of plankton were larger; on the contrary, the species diversity of plankton decreased, and the distribution was uneven. Therefore, changes in the structural characteristics of plankton communities can be used as im...

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Problems solved by technology

However, at the same time, this technology also has defects: (1) only the main microorganisms in the environment can be detected, and the microorganisms accounting for more than 1% of the total can be detected; (2) the size of the fragments that can be separated by DGGE is between 200-700bp (3) DNA with different sequences may migrate to the same position of the gel, and sometimes the bands cannot be separated well even if the bands are recovered, re-amplified, and re-electrophoresis ; (4) There is a problem of heterogeneity between different copies of genes in some species. Even if they have only slight differences, DGGE can separate them, so a species does not only show one band on the DGGE map, which is overestimated (5) In addition, there are certain limitations in each step of the whole analysis process: some microbial species may be lost during sample collection and storage; due to the different cell wall compositions of different species, the same Different extraction methods have different extraction efficiencies for different species; PCR itself has the problem of amplification preference, and chimeras and heteroduplexes may be generated during the amplification process, which have a certain impact on the experimental results

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