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Detection probe of Thalassiosira rotula Meunier and detection method

A technology for detecting probes and thalassina, which is applied in the determination/inspection of microorganisms, biochemical equipment and methods, etc., which can solve the problems of lack of research and achieve the effect of good species specificity and small sample amount

Inactive Publication Date: 2011-08-31
OCEAN UNIV OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, there is currently no research on this aspect of the algae at home and abroad.

Method used

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  • Detection probe of Thalassiosira rotula Meunier and detection method
  • Detection probe of Thalassiosira rotula Meunier and detection method
  • Detection probe of Thalassiosira rotula Meunier and detection method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] Example 1. Preparation of Nucleotide Sequences for Detection of Thalassiosira

[0018] Thalassiosira used in the present invention is isolated from natural seawater samples of Jiaozhou Bay. Pure cultures are obtained by repeatedly picking individual algal cells under a microscope. The culture medium used is conventional F / 2 culture medium, and the culture conditions are: the light / dark cycle is 12h / 12h, the light intensity is 4000Lx, and the culture temperature is 22°C-25°C.

[0019] Use a 0.45 μm microporous membrane or centrifuge at 10,000 r / min to collect Thalassiosira cells in the logarithmic growth phase, wash with TE buffer (10mmol / L Tris-HCl pH8.0, 1mmol / L EDTA pH8.0) Under the algae, add 2 times the volume of extraction buffer (3% (w / v) CTAB, 1% (w / v) sarkosyl, 20mmol / LEDTA, 1.4mol / L NaCl, 0.1mol / L Tris-HCl pH 8.0, 1% (v / v) 2-mercaptoethanol), treated at 55°C for 1 hour, and mixed by inverting every 10 minutes; take it out and let it stand in the refrigerator...

Embodiment 2

[0021] Design and synthesis of embodiment 2.T.rotula primer1 and T.rotula primer2

[0022] By comparing with the sequences of the corresponding regions of all other eukaryotes and prokaryotes known in Genbank, it is found that the sequence shown in SEQ ID NO.1 in the sequence table is very different from the corresponding sequences of other organisms. Based on this sequence, the inventor designed two nucleic acid molecular probes as described in SEQ ID NO.2 and SEQ ID NO.3 in the sequence listing. According to the nucleotide composition and arrangement of the two probes or their complementary sequences, the above nucleotide sequence can be obtained on a commercial DNA synthesizer.

Embodiment 3

[0023] Example 3. Detection of Thalassiosira by conventional PCR method

[0024] In this embodiment, several strains of dominant phytoplankton in the algae bank of this laboratory are selected as reference algae, and they are: chaetoceros curvisetus (Chaetoceros curvisetus), chaetoceros curvisetus (C. C. gracile, C. minimum, Gymnodinium sp., G. mikimotoi, Pseudonitzschia pungens, new Nitzschia closterium, Navicula membraneacea, Melosira sp., Skeletonema costatum, Prorocentrem minimun isolated from Bohai Bay, isolated from Alexandrium tamarens from Dapeng Bay and Heterosigma akashiwo from Dalian Bay were isolated by repeatedly picking individual algal cells under a microscope to obtain pure cultures. These algae were cultivated and the DNAs of these algae and Thalassiosira tolurum were extracted according to the method described in Example 1.

[0025] Get 1 μ l of DNA extracts of various algae, carry out PCR by the PCR reaction system described in Example 1, with the nucleoti...

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Abstract

The invention belongs to using molecular biology method to detect environment technique field. It discloses oligonucleotide probe designed according to rDNA. The nucleic acid molecule probe practices PCR reaction for a pair of primer to detect round sea chain algae. It also discloses detection and fixed quantity counting round sea chain algae method by fluorescence fixed quantity RCR technique.

Description

technical field [0001] The invention belongs to the technical field of detecting marine environment microorganisms by means of molecular biology methods. Specifically, it relates to a nucleotide sequence that can be used to detect Thalassiosira tonula, a nucleic acid molecular probe designed on the basis of the sequence, and a method for using the molecular probe to detect Thalassiosira torula. Background technique [0002] Thalassiosira rotula is a kind of phytoplankton of eurythermal and euryalline that exists widely in the world. It is also one of the common dominant species of diatoms in my country's coastal waters. The value-added speed of Thalassiosira discus is extremely fast, and it can produce some short-chain aldehydes. These aldehydes can inhibit the development of Copepoda eggs. When red tides occur, they can affect the population structure of Copepods. The inhibition of pod reproduction is one of the research hotspots in marine ecology. Therefore, it is of gre...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68
Inventor 于志刚李荣秀何闪英米铁柱蔡青松甄毓
Owner OCEAN UNIV OF CHINA