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Probe and method for detecting heterosigma akashiwo

A technology for the detection of Akashio algae and detection probes, which is applied in the field of molecular biology detection of microorganisms in the marine environment, can solve the problems of lack of research and achieve good species specificity and small sample volume

Inactive Publication Date: 2005-11-23
OCEAN UNIV OF CHINA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, at present, there is no research on this algae at home and abroad.

Method used

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  • Probe and method for detecting heterosigma akashiwo
  • Probe and method for detecting heterosigma akashiwo
  • Probe and method for detecting heterosigma akashiwo

Examples

Experimental program
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Embodiment 1

[0016] Example 1. Preparation of nucleotide sequences for detection of Akashiwa algae

[0017] The algae used in the present invention are isolated from natural seawater samples of Dalian Bay. Pure cultures are obtained by repeatedly picking individual algal cells under a microscope. The culture medium used is conventional F / 2 culture medium, culture conditions: light / dark cycle 12h / 12h, light intensity 4000 Lx, culture temperature 22°C-25°C.

[0018] Use a 0.45 μm microporous membrane or centrifuge at 10,000 r / min to collect the algae cells in the logarithmic growth phase of Akashiwa algae, and use TE buffer (10mmol / L Tris-HCl (Dihydroxymethylaminomethane hydrochloric acid) pH8.0 , 1mmol / LEDTA (ethylenediaminetetraacetic acid) pH8.0) to wash about 20mg of algae, add 2 times the volume of extraction buffer (3% (w / v) CTAB (hexadecane base-trimethyl-ammonium bromide), 1% (w / v) sarkosyl (sodium lauryl sarcosinate), 20mmol / L EDTA, 1.4mol / L NaCl, 0.1mol / LTris-HCl pH8.0 , 1% (v / v...

Embodiment 2

[0020] Design and synthesis of embodiment 2.H.akashiwo primer1 and H.akashiwo primer2

[0021] 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 the sequence, two nucleic acid molecular probes of H.akashiwo primer1 and H.akashiwo primer2 were designed. According to the nucleotide composition and arrangement of the two probes or their complementary sequences, the nucleotide sequences described in SEQ ID NO.2 and SEQ ID NO.3 can be obtained on a commercial DNA synthesizer.

Embodiment 3

[0022] Example 3. Detection of Akashiwa algae by conventional PCR method

[0023] In this embodiment, several strains of microalgae in the algae bank of this laboratory were selected as reference algae, and they were: Chaetoceros curvisetus (Chaetoceros curvisetus), Chaetoceros curvisetus, C. C. gracile, C. minimum, Gymnodinium sp., G. mikimotoi, Thalassiosira nordenskioeldii, Pseudonitzschia pungens, Nitzschia closterium, Navicula membranacea, Melosira sp., Skeletonemacostatum, isolated from Tamar in Mirs Bay Alexandrium tamarens, they all obtain pure cultures by repeatedly picking single algal cells under a microscope. These algae were cultured and the DNAs of these algae and I. akashiwo algae were extracted according to the method described in Example 1.

[0024] Get 1 μ l of DNA extracts of various algae, carry out PCR according to the PCR reaction system described in Example 1, with the nucleus described in SEQ ID NO.2 (H.akashiwo primer1) and SEQ ID NO.3 (H.akashiwo pr...

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Abstract

The present invention belongs to the field of environment microbe inspection technology with molecular biological method. The invention discloses red tide heterosigma akashiwo alga ribosome DNA(rDNA)and the nucleic acid sequence of interval area of transcription unit (ITS) as well as the oligonucleotide probe based on the design of this sequence. This nucleic acid probe is used as a pair of guide object to carry out PCR reaction. Accurate and fast inspection of red tide heterosigma akashiwo alga can be realized through predicting whether there is the PCR product of corresponding size. The present invention also discloses a fast and accurate inspection quantitative counting method of red tide heterosigma akashiwo alga by fluorescent quantitative PCR technology with the probe.

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 I. akashiwo, a nucleic acid molecular probe designed based on the sequence, and a method for using the molecular probe to detect I. akashiwo. Background technique [0002] Heterosigma akashiwo is a harmful algae widely distributed in the coastal waters of the world. It is also a common red tide organism in my country's coastal waters. It has been reported to cause red tides many times. Therefore, timely, accurate and rapid detection of I. akashiwo is of great significance. [0003] Taxonomically, I. akashiwo algae belongs to the genus Syndrome of Chrysophyta. Although it is not very difficult to distinguish Heterophyta akashiwo morphologically, this method is cumbersome, time-consuming and labor-intensive, and the workload is huge when the sample...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/68
Inventor 于志刚李荣秀甄毓何闪英蔡青松米铁柱
Owner OCEAN UNIV OF CHINA
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