Detection method for biological community structures of ocean brown tide

A detection method and technology of biological communities, which can be used in the determination/inspection of microorganisms, biochemical equipment and methods, etc., and can solve problems such as difficult practical operation, difficult to distinguish, and small individuals.

Inactive Publication Date: 2015-09-30
LIAONING OCEAN & FISHERIES SCI RES INST
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Problems solved by technology

[0003] Traditionally, the identification and detection of plankton mainly rely on the microscope to observe the morphological differences for classification. However, this method has certain limitations in practical applications: first, plankton, especially micro and pico plankton, are small in size, Difficult to observe, difficult to operate, conventional microscopic examination cannot achieve species identification; secondly, this method relies on experienced taxonomic researchers, requires solid taxonomic skills, and is time-consuming and laborious, not suitable for large-scale sample research; Finally, the morphological differences of many plankton are not obvious, and it is difficult to distinguish and easy to cause human error
Because brown tide species are pico organisms smaller than 3 μm, it is impossible to identify its species by general methods such as microscope identification. There are many pico-organisms that may have brown tides, and DNA sequencing technology is used to detect the 18S rDNA of pico-organisms in the target waters, and the obtained DNA fragment sequences are input into GenBank for retrieval and comparison, and the Latin and Chinese names of corresponding species are searched. , and then can identify the species of brown tide organisms, there is no proprietary primer sequence designed for brown tide organisms, and no 18S rDNA sequencing comparison method is used in the analysis of brown tide biological community structure. The present invention uses this While expanding the method, specially designed and verified the primer sequences designed for brown tide organisms, and used the probability of clone sequencing to count the parameters such as the dominance degree, community diversity index, and evenness index of various pico organisms
[0012] 2010.11.24 published invention patent "a detection method of phytoplankton community structure" (application number: 201010184810) is a detection method using denaturing gradient gel electrophoresis (DGGE) technology to study the detection method of phytoplankton community structure, but this method cannot identify specific Species, can only count parameters such as biodiversity based on electrophoretic strips, but cannot know the name of dominant species and its degree of dominance, etc.

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  • Detection method for biological community structures of ocean brown tide
  • Detection method for biological community structures of ocean brown tide
  • Detection method for biological community structures of ocean brown tide

Examples

Experimental program
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Effect test

Embodiment 1

[0049] The detection method of marine brown tide biological community structure includes the following steps a to e:

[0050] a. Sample collection and pretreatment

[0051] Collect 2-5L of surface seawater, first filter it through a 200-mesh sieve, and then use a nitrocellulose membrane with a pore size of 0.22 μm to collect pico, micro and small plankton, transfer the filter membrane to a 10mL sterile centrifuge tube, and place in- Freeze at 20°C or -80°C for analysis of pico-eukaryotes.

[0052] b. Extraction of DNA

[0053] (1) Lyse algae cells: transfer the above filter membrane into a 1.5mL sterilized centrifuge tube, cut it into pieces with sterilized scissors, add 500 μL CTAB (cetyltrimethylammonium bromide method) buffer, 1 μL mercapto Mix ethanol and 5 μL proteinase K evenly, and place in an air bath at 55°C for 1-2 hours until the algae liquid is transparent;

[0054] (3) Extraction: Add 300 μL saturated phenol, 300 μL CI (chloroform: isoamyl alcohol = 24:1) and g...

Embodiment 2

[0080] At present, the amplification primers used in the 18S rDNA eukaryotic molecular identification technology are general primers for eukaryotic organisms. In order to detect the species of brown tide organisms, we designed and verified the exclusive primers for brown tide algae.

[0081] 1. Primer design

[0082] 179 algal 18S rRNA sequences were downloaded from the NCBI database, including 11 phyla, 23 classes, and 179 species. The software MEGA4 was used to perform multiple sequence alignment on 179 sequences, and the comparison results are shown in figure 1 .

[0083] According to the comparison results, the gene regions F(V4)632-653, R(V4)1054-1076, F(V9), 1385-1405, and R(V9)1737-1759 were selected, and their sequences are shown in Table 2.

[0084] Table 2. Algae 18S rRNA variable region and its base sequence of the present invention

[0085]

[0086] Use the software DNAMAN to detect the complementarity of the two primers, and FASTPCR to evaluate the propertie...

Embodiment 3

[0143] From June to July 2013, the seawater in Dalian's exchange island culture ponds and near-shore waters was golden yellow. The brown tide warning analysis was carried out in this sea area by the method described in Example 1. After taking 1L surface seawater in the target sea area, the sample After collection and pretreatment, lyse the algae cells, extract their DNA, perform PCR using V4-F / R, build a library with the kit, and use the Illumina MiSeq sequencing platform PE300 sequencing solution to carry out sequencing on the machine according to Example 2. Data information, obtain OTUs information and further obtain information such as algae species and abundance. Finally, statistical analysis was performed according to the method of step e in Example 1, and the test results are shown in Table 7-9. In this embodiment, three parallels were performed for each test sample, and the standard error of the test result of the first dominant species was within ±0.04.

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Abstract

The invention discloses a detection method for the biological community structures of an ocean brown tide. According to the detection method, miniature organism 18S rDNA in a water area environment is detected by virtue of a metagenomics high-throughput sequencing technology, the obtained OUTs and species annotations are combined, and then diversity, uniformity, abundance and dominance index analysis is carried out on the OUTs, thus judging the dominance degrees of organisms in the ocean brown tide; statistics and judgment for the dominance evolvement rules of dominant species in brown tide outbreaks are promoted. The embodiment of the invention designs and verifies primer sequences designed with regard to the organisms in the brown tide, and a verification for the actual brown tide outbreak cases proves that the method disclosed by the invention is capable of authenticating the biological community structures of the ocean brown tide in ocean water, providing a technical support for brown tide monitoring operation, and providing a technical support for avoiding environment risks for healthy mariculture.

Description

technical field [0001] The invention relates to the technical field of detection of marine pico-eukaryotic species, in particular to a detection method of marine brown tide biological community structure. Background technique [0002] Research overview of brown tide at home and abroad: brown tide is a harmful algal bloom caused by microscopic algae, which can also be understood as a red tide of picoplankton, which is a brown tide. , The duration is longer. American research institutions named this harmful algal bloom "brown tide" to distinguish it from the general "red tide", and it was adopted by relevant international agencies (Cosper EM, et al., 1990). In recent years, brown tides have occurred frequently in the Bohai Sea of ​​China, and the main species of disaster is Aureococcus anophagefferens, which belongs to microalgae. This microalgae is extremely harmful to the aquaculture industry, causing shellfish to stagnate or even die. China is the third country to experi...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
CPCC12Q1/6888
Inventor 宋伦刘卫东王年斌韩家波孙明宋广军吴金浩张玉凤王昆王召会马志强李楠宋永刚李爱田金杨爽赵海勃刘桂英胡超魁
Owner LIAONING OCEAN & FISHERIES SCI RES INST
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