Primer and probe for identifying temperature-sensitive type of coral symbiotic photosynthetic algae and identifying method

A temperature-sensitive, photosynthetic algae technology, applied in the direction of DNA / RNA fragments, recombinant DNA technology, etc., can solve the problems of coral symbiotic photosynthetic algae without uniform standards and difficult morphological identification

Inactive Publication Date: 2016-03-30
THIRD INST OF OCEANOGRAPHY STATE OCEANIC ADMINISTATION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no uniform standard for the identification of coral symbiotic photosynthetic algae in the world, and it is difficult to carry out accurate identification in form

Method used

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  • Primer and probe for identifying temperature-sensitive type of coral symbiotic photosynthetic algae and identifying method
  • Primer and probe for identifying temperature-sensitive type of coral symbiotic photosynthetic algae and identifying method
  • Primer and probe for identifying temperature-sensitive type of coral symbiotic photosynthetic algae and identifying method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Embodiment 1: Separation and cultivation of test algal species

[0040] Collect the helmet coral tissue samples in the sea area of ​​China. The coral tissue is scraped or cut with a scalpel blade, and the extraction buffer (artificial seawater filtered by a 0.45um microporous membrane and 5mM EDTA, 40mg / L ampicillin and 30mg / L Kanamycin), remove the coral tissue from the surface of the skeleton, collect the washing fluid, and shake vigorously for about 3 minutes to destroy the mucus. Centrifuge the above-mentioned host extract at 5000g for 5min, collect the precipitate, suspend and mix with extraction buffer, centrifuge the obtained slurry at 2000g for 5min, discard the liquid, resuspend the precipitate with buffer, and mix. Repeat 2-4 times until the extracted symbiotic algae are pure and save for future use. Take another drop of the suspension solution for microscopic examination (10×10), if there are impurities, repeat the above steps,

[0041] Until the extracted ...

Embodiment 2

[0043] Embodiment 2: the extraction of DNA of algal species

[0044] The algal cells of Example 1 are collected by centrifugation, that is, the algal cells are placed in a 10ml centrifuge tube, 4°C, 13,000rpm, and centrifuged for 15min. After the supernatant is removed, the precipitated bacteria are placed in a sterile ultra-clean work Blow dry. Then, use the CTAB method to extract the genomic DNA of the algae; first, transfer the excess algae collected by centrifugation to a 1.5mleppendorf tube; add 600ul of CTAB extract, place it in a water bath at 65°C for 1 hour, and shake it several times during the period. In order to fully break up the algae cells. Next, remove the centrifuge tube from the water bath, add the DNA extraction solution and an equal volume of phenol, chloroform, and isoamyl alcohol (volume ratio 25:24:1) mixed solution, vortex vigorously for 5 min, and collect the supernatant by centrifugation. Finally, add an equal volume of chloroform / isoamyl alcohol (v...

Embodiment 3

[0045] Embodiment 3: Taqman fluorescent quantitative PCR detection

[0046] Design real-time quantitative PCR primers and Taqman probe combination (FAM-CladeC) according to the specific sequence of temperature-sensitive CladeC of coral symbiotic photosynthetic algae, wherein FAM-CladeC includes one of the following primer-probe sets:

[0047] FAM-CladeC Group 1:

[0048] Primer: FAM-CladeC-F1: 5'-GCTGAACACGGACCCAT-3'SEQ ID NO: 2;

[0049] FAM-CladeC-R1: 5'-GGATGCTTGCGGTGAGATA-3' SEQ ID NO: 3;

[0050] Probe: FAM-CladeC-P1: 5'-AAGGGTTGGTGAGTGTGCATCTGT-3'; SEQ ID NO: 4;

[0051] The second group of FAM-CladeC:

[0052] Primer: FAM-CladeC-F2: 5'-GTGAGTCTGACTTCATGCTAGAG-3'SEQ ID NO: 5;

[0053] FAM-CladeC-R2: 5'-GCTTTGCGCGCTGTTATT-3'SEQ ID NO: 6;

[0054] Probe: FAM-CladeC-P2: 5'-AGCATGAGCAGCGTCACTCAAGTA-3'; SEQ ID NO: 7;

[0055] The third group of FAM-CladeC:

[0056] Primer: FAM-CladeC-F3: 5'-CCACGAAGGTAGAAACCTGAA-3'SEQ ID NO: 8;

[0057] FAM-CladeC-R3: 5'-CCTTGCGTTCTTA...

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Abstract

The invention discloses a primer and a probe for identifying the temperature-sensitive type of coral symbiotic photosynthetic algae and an identifying method. The primer is SEQ ID NO: 2-3, and the probe is SEQ ID NO: 4, or the primer and the probe are SEQ ID NO: 5-7 respectively, or the primer and the probe are SEQ ID NO: 8-10 respectively, or the primer and the probe are SEQ ID NO: 11-13 respectively, or the primer and the probe are SEQ ID NO: 14-16, or the primer and the probe are SEQ ID NO: 17-19, and the 5' end and the 3' end of the probe are marked through FAM-Clade fluorescent dye and a TAMRA quenching group. Qualitative and quantitative identification of the temperature-sensitive type of the coral symbiotic photosynthetic algae can be performed.

Description

technical field [0001] The invention relates to the field of biological technology, in particular to a primer, a probe and an identification method for identifying the temperature-sensitive type of coral symbiotic photosynthetic algae. Background technique [0002] Coral reefs are of great ecological and economic value and are estimated to be home to one-third of marine life and responsible for more than 25% of ocean productivity. However, due to global warming in recent years, seawater temperature rise, seawater acidification, and marine environment pollution have caused reef-building corals to face a series of major threats. my country's coral reefs are mainly distributed in the South China Sea, mainly atolls, and coastal reefs are distributed along the coasts of Hainan Island, Guangdong Province and Taiwan Island. Due to the relatively backward establishment of protected areas and protection measures for coral reefs in my country, coupled with serious human-induced damag...

Claims

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Application Information

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IPC IPC(8): C12Q1/68C12N15/11
CPCC12Q1/6895C12Q1/6851
Inventor 林镇跃陈明谅陈建明
Owner THIRD INST OF OCEANOGRAPHY STATE OCEANIC ADMINISTATION
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