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Chaetuceros sp. detecting probe and method

A technology for detecting probes and Chaetoceros sp. It is applied in the determination/inspection of microorganisms, biochemical equipment and methods, DNA/RNA fragments, etc. It can solve the problem of lack of research and achieve the effect of small sample size.

Inactive Publication Date: 2006-05-17
OCEAN UNIV OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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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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  • Chaetuceros sp. detecting probe and method
  • Chaetuceros sp. detecting probe and method
  • Chaetuceros sp. detecting probe and method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] Embodiment 1. The cultivation of Chaetoceros and the preparation of its DNA sample

[0018] Chaetoceros 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 medium used is conventional F / 2 medium, and the culture conditions are: light / dark cycle of 12h / 12h, light intensity of 4000Lx, and culture temperature of 22°C-25°C.

[0019] Use a 0.45 μm microporous membrane or centrifuge at 10,000 r / min to collect Chaetoceros cells in logarithmic growth phase, wash with 200 μl TE buffer (10 mmol / L Tris-HCl pH8.0, 1 mmol / L EDTA pH8.0) for about 20mg of algae, add 2 times volume of extraction buffer (3% (w / v) CTAB, 1% (w / v) sarkosyl, 20mmol / L EDTA, 1.4mol / L NaCl, 0.1 mol / L Tris-HCl pH8.0, 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 at 4°C for 3 minu...

Embodiment 2

[0021] Design and synthesis of embodiment 2.C.sp primer1 and C.sp primer2

[0022] By comparing with the sequences of the corresponding regions of all known other eukaryotes and prokaryotes in Genbank, it is found that the sequence shown in SEQ ID NO.1 is very different from the corresponding sequences of other organisms. Based on this sequence, two nucleic acid molecular probes as shown in SEQ ID NO.2 and SEQ ID NO.3 were designed. 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 Chaetoceros 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. gracilis, C. minimum, Skeletonema costatum, Gymnodinium sp., G. mikimotoi, spiny Pseudonitzschia pungens, Nitzschia closterium, Thalassiosira nordenskioeldii, Navicula membranacea, Melosira sp., isolated from Dapeng Bay Alexandrium tamarens from Dalian Bay and Heterosigma akashiwo isolated from Dalian Bay have repeatedly picked single algal cells under a microscope to obtain pure cultures. These algae were cultured and the DNAs of these algae and Chaetoceros were extracted by the method described in Example 1.

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

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Abstract

The present invention belongs to the field of molecular biology technology for detecting environment microbe, and discloses Chaetuceros sp. ribosome DNA and transcription unit spacer nucleic acid sequence as well as oligonucleotide probe designed based on the sequence. Using the nucleic acid molecular probe as primer for PCR reaction and judging the PCR products in the corresponding size can detect Chaetuceros sp. accurately and quickly. The present invention also discloses the fluorescent quantitative PCR technology with these probes for fast and accurate detection and counting of Chaetuceros sp.

Description

technical field [0001] The invention belongs to the technical field of detecting micro-organisms in marine environment by means of molecular biology methods. Specifically, it relates to a nucleotide sequence that can be used to detect Chaetoceros, a nucleic acid molecular probe designed based on the sequence, and a method for using the molecular probe to detect Chaetoceros. Background technique [0002] Chaetcoers is the most common and important type of phytoplankton, and it is also a very important type of diatoms in my country's coastal waters; it is distributed in the vast sea areas of tropical, subtropical, temperate and even frigid regions, and is found in various sea areas of my country. distributed. At present, there are about 175 species of Chaetoceros recorded in the world, and about 50 species have been found in the coastal waters of my country. Among these chaetoceros, some are important red tide organisms, which can proliferate in large quantities to form red t...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68C12N15/11
Inventor 于志刚李荣秀何闪英米铁柱蔡青松甄毓
Owner OCEAN UNIV OF CHINA
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