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Probe and method for detecting red naked dinoflagellate

A technology of Gymnodinium red and detection probe is applied in the field of detection of Gymnodinium red, which can solve the problems of lack of research, and achieve the effects of good species specificity and small sample volume.

Inactive Publication Date: 2005-11-23
OCEAN UNIV OF CHINA
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  • Abstract
  • Description
  • Claims
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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 red naked dinoflagellate
  • Probe and method for detecting red naked dinoflagellate
  • Probe and method for detecting red naked dinoflagellate

Examples

Experimental program
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Embodiment 1

[0016] Embodiment 1. the cultivation of Gymnodinosa red and the preparation of DNA sample thereof:

[0017] The Gymnodinosa red used in the present invention is isolated from the natural seawater sample of the East my country Sea. 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 4000Lx, culture temperature 22°C-25°C.

[0018] Use a 0.45 μm microporous membrane or centrifuge at 1000 r / min to collect the algae cells in the logarithmic growth phase, and use 200 μl of TE buffer (10 mmol / L Tris-HCl (tris-hydroxymethylaminomethane hydrochloride) pH8. 0, 1mmol / L EDTA (ethylenediaminetetraacetic acid) (pH8.0) to wash about 20mg of algae, add 2 times the volume of extraction buffer (3% (w / v) CTAB (ten Hexaalkyl-trimethyl-ammonium bromide), 1% (w / v) sarkosyl (sodium lauryl sarcosinate), 20mmol / L EDTA, 1.4mol / L NaCl, 0.1mol...

Embodiment 2

[0019] Design and synthesis of embodiment 2.G.sanguinea primer1 and G.sanguinea primer2:

[0020] By comparing with the sequences of the corresponding regions of all known other eukaryotes and prokaryotes in Genbank, it is found that the sequences shown in Sequence Table 1 are very different from the corresponding sequences of other organisms. For this reason, the inventors use this sequence Two nucleic acid molecular probes of G.sanguinea primer1 and G.sanguinea primer2 were designed on the basis. According to the nucleotide composition and arrangement of the two probes or their complementary sequences, the nucleotide sequences described in Sequence List 2 and Sequence List 3 can be obtained on a commercial DNA synthesizer.

Embodiment 3

[0021] Embodiment 3. detects red dinoflagellate with routine PCR method:

[0022] In this embodiment, several strains of microalgae 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, Thalassiosira nordenskioeldii, Pseudonitzschiapungens, Nitzschia closterium, Navicula membraneacea, Melosira sp., Skeletonema costatum, Thalassiosirasp. ), Prorocentrem minimun isolated from Bohai Bay, Alexandrium tamarens isolated from Dapeng Bay, and Heterosigmaakashiwo isolated from Dalian Bay. Single algal cells were picked to obtain pure cultures. These algae were cultured and the DNAs of these algae and Gymnodinium rubrum were extracted according to the method described in Example 1.

[0023] 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 des...

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Abstract

The present invention discloses the oligonucleotide probe based on the design of red gymnodinia ribosome DNA (rDNA) nucleic acid sequence, belonging to the technical field of environment microbe inspection by means of molecular biological method. This nucleic acid probe is used as a pair of guide objects to carry out PCR reaction for accurate and fast inspection of red gymnodinia through predicting whether there is PCR product of corresponding size. The present invention also discloses a fast and accurate inspection and quantitative counting method of red gymnodinia by fluorescent quantitative PCR technology with the probe.

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 Gymnodinosa rubrum, a nucleic acid molecular probe designed based on the sequence, and a method for using the molecular probe to detect Gymnodinium rubrum. Background technique [0002] Gymnodinium sanguinea belongs to the genus Gymnodinium dinoflagellates, which can form a large-scale red tide, causing a large number of deaths of cultured scallops, sea cucumbers, and abalones, resulting in huge economic losses. Among the more than 40 species of red tide organisms recorded in Jiaozhou Bay, Gymnodinosa is one of the main species of red tide algae. Therefore, it is of great significance to detect Gymnodinosa in a timely, accurate and rapid manner, and to keep abreast of its dynamic changes in the ocean. The traditional identification method, th...

Claims

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Application Information

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