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DNA analysis method for zooplankton, phytoplankton and zooplankton and phytoplankton specie composition and community structures

A technology for zooplankton and community structure, which is applied in biochemical equipment and methods, and the determination/inspection of microorganisms.

Inactive Publication Date: 2015-03-25
SHANGHAI OCEAN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

As a traditional research method, microscopes are widely used in research in this field. However, there are the following problems: 1. Due to the large size difference of animal and plant samples, it is difficult to analyze them in the same sample at the same time. The proportional relationship between animals and plants is calculated, but the error generated during the conversion process is relatively large
2. Microscopic examination needs to rely on complete animal and plant samples for identification. It is often impossible to identify the residues caused by the destruction of microscopic organisms during the collection process, especially when identifying the digestive tract of aquatic organisms with plankton feeding habits. There are more bodies, which will bring great difficulties to detection microscopy methods that rely on complete individuals
3. In terms of species identification, it is necessary to determine the species through the judgment of the form. Although there are many basis and retrieval tables for species determination, it is difficult to accurately determine the species
[0003] By consulting the literature and patents, it is also found that the identification of the community structure or components of phytoplankton is relatively rare. There are two groups of eukaryotic algae and prokaryotic algae (cyanobacteria), and the general primers of eukaryotes cannot identify cyanobacteria

Method used

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  • DNA analysis method for zooplankton, phytoplankton and zooplankton and phytoplankton specie composition and community structures
  • DNA analysis method for zooplankton, phytoplankton and zooplankton and phytoplankton specie composition and community structures
  • DNA analysis method for zooplankton, phytoplankton and zooplankton and phytoplankton specie composition and community structures

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Experimental program
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Effect test

Embodiment 1

[0037] The amplification effects of zooplankton 18s rDNA primers SSU F04 and SSU R22 and phytoplankton 23s rDNA primers p23SrV f1 and p23SrV r1 were verified:

[0038] The DNA of the cultured eukaryotic phytoplankton Chlorella and the prokaryotic phytoplankton Microcystis were extracted by CTAB method, and the cultured DNA of the zooplankton Hyapha vulgaris was extracted by SDS method.

[0039] The phytoplankton 23s rDNA primers p23SrV f1 and p23SrV r1 were used to amplify the DNA of Chlorella and Microcystis. Bands 1, 2 (Chlorella) and 3, 4 (Microcystis) were obtained by gel electrophoresis, respectively. like figure 1 As shown, the amplification effect is good.

[0040] Use zooplankton 18s rDNA primers SSU F04 and SSU R22 to amplify the DNA of the flea, and gel electrophoresis to obtain bands 5 and 6, respectively, as shown in figure 2 As shown, the amplification effect is good.

Embodiment 2

[0042] Verification of the amplification effect of composite primers combined with linker primers to amplify phytoplankton, the principle is as follows image 3 shown.

[0043] The DNAs of the above-mentioned Chlorella, Microcystis, and Phyllostachys vulgaris were mixed at equal concentrations to form an equal-concentration mixed DNA sample. It can be seen from the database query that the target fragments of Chlorella 23s rDNA and Phyllostachys hyalaeum 18s rDNA are about 395bp, The target fragment of Microcystis 23s rDNA is about 420bp.

[0044] The reaction system is 25μl system: 1.8mmol / lMgCl 2 , 200ummol / l dNTPs and 1.5U DNA polymerase, 300nmol / L universal primer UT, 2nmol / L each composite primer (UT-SSU F04 and UT-SSU R22; UT-p23SrV f1 and UT-p23SrV r1), 2.5μl 10×PCR buffer, sterilized H 2 O make up to 25 μl. The temperature conditions of the reaction: pre-denaturation at 94 °C for 2 min; denaturation at 94 °C for 30 s, annealing at 56 °C for 30 s, extension at 72 °C ...

Embodiment 3

[0047] Comparison of feeding habits of silver carp and bighead carp in Qiandao Lake

[0048] Step 1: Sample collection: Take 5 Qiandao Lake silver carp and 5 bighead carp, take the contents of the foregut and mix well, take 0.1 g and store it in a -20°C refrigerator for later use.

[0049] Step 2 DNA extraction

[0050] DNA extraction from foregut contents was performed using Qiagen's Fecal Genome Extraction Kit.

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Abstract

The invention relates to a DNA analysis method for zooplankton, phytoplankton and zooplankton and phytoplankton specie composition and community structures. The method includes the following steps: (1) collecting an environmental sample and extracting DNAs of zooplanktons and phytoplanktons; (2) conducting PCR amplification on the DNAs of the zooplanktons and the phytoplanktons to obtain PCR products of the DNAs of the zooplanktons and the phytoplanktons; (3) conducting gel electrophoresis on the PCR products of the DNAs of the zooplanktons and the phytoplanktons to obtain gel with the DNAs of the zooplanktons and the phytoplanktons; (4) conducting next-generation sequencing on the gel with the DNAs of the zooplanktons and the phytoplanktons to obtain rDNA sequences of zooplankton and phytoplankton species in the environmental sample; (5) conducting specie comparison on the rDNA sequences of the zooplankton and phytoplankton species in a database, and conducting statistics and analysis on the zooplankton and phytoplankton structures. The DNA analysis method is simple, convenient, efficient, accurate and ingenious in design, and has the practical value.

Description

technical field [0001] The invention relates to the field of molecular ecology, in particular to species identification, and in particular to a DNA analysis method for zooplankton, phytoplankton and phytoplankton species composition and community structure Background technique [0002] The identification of phytoplankton and the study of community structure is an important task in water ecological investigation and environmental monitoring; at the same time, similar studies are often used in the ecological study of plankton-feeding aquatic organisms. In the traditional research, the above contents are studied by means of microscope examination. As a traditional research method, microscopy is widely used in research in this field, but there are the following problems: 1. Due to the large volume difference, it is difficult for animal and plant samples to be analyzed in the same sample at the same time, although it can be converted into The proportional relationship between an...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6844C12Q1/6869C12Q2563/185
Inventor 刘其根罗衡赵良杰胡忠军王烽竹
Owner SHANGHAI OCEAN UNIV
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