CN101845483A - Method for identifying Karenia mikimotoi - Google Patents
Method for identifying Karenia mikimotoi Download PDFInfo
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- CN101845483A CN101845483A CN200910048127A CN200910048127A CN101845483A CN 101845483 A CN101845483 A CN 101845483A CN 200910048127 A CN200910048127 A CN 200910048127A CN 200910048127 A CN200910048127 A CN 200910048127A CN 101845483 A CN101845483 A CN 101845483A
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- karenia mikimotoi
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Abstract
The invention discloses a method for identifying Karenia mikimotoi, relating to identification of algous specific moleculars and aiming to solve the technical problem of providing specific primers for polymerase chain reaction identification of Karenia mikimotoi. The method is characterized by utilizing Primer 5.0 software to design specific identification primers Ki 1F3 and Ki 1B3 for Karenia mikimotoi ribosome transcribed spacers ITS by algae culture and total DNA extraction, wherein the primer sequence of the Ki 1F3 is CAACTTGAATTCATTGTGAACC, and the primer sequence of the Ki1B3 is ATGTGAATCAAACATGTGGT. The agarose gel electrophoresis test shows that the genome DNA of the Karenia mikimotoi has amplification bands after PCR amplification of the genome DNA. The method is used for preventing and forecasting red tides.
Description
Technical field
The present invention relates to identify for red tide prevention prediction provides the algae of reference.
Background technology
Be used at present differentiate that the primer of red tide algae is generally universal primer, as utilize rrna 5.8S rDNA, 18S rDNA, the 28S rDNA of little algae, the designed primer of the relative conservative region of gene order in ITS district, this primer can be applicable to eukaryote or Pyrrophyta algae, scope is bigger, comparatively convenient as first evaluation, but because its specificity is not strong, can't disposablely navigate to concrete algae kind, therefore be difficult to reach the requirement that the red tide algae is identified.And adopt universal primer after polymerase chain reaction (PCR) experiment, and obtain the product sequence and need pass through gene sequencing, can clear and definite its affiliated kind after the analysis.This procedure step is more, consuming time, expend a large amount of expenses, and can't be useful in and lack in the order-checking condition.Therefore, the stronger primer of exploitation specific aim is current research focus, consult the up-to-date research report of delivering, existing expert utilizes gene orders such as rrna 18S rDNA, chloroplast(id) rbcl to design the primer probe of differentiating single class algae kind, disposable pcr amplification just can be identified and put in place, need not order-checking, shortened qualification cycle greatly, saved a large amount of expenses simultaneously.
Summary of the invention
The problem that the present invention need solve provides the triumphant human relations algae of Michaelis polymerase chain reaction and identifies Auele Specific Primer.
The present invention is characterized in that utilizing Primer 5.0 softwares that the triumphant human relations algae of Michaelis (Karenia mikimotoi) rrna transcribed spacer (ITS district) has been designed specificity primers designed Ki1F3 and Ki1B3 by algal species cultivation, total DNA extraction,
Primer sequence is Ki1F3:CAACTTGAATTCATTGTGAACC;
Ki1B3：ATGTGAATCAAACATGTGGT；
Genomic dna is behind pcr amplification, and by the agarose gel electrophoresis check, it is the triumphant human relations algae of Michaelis that amplified band is arranged.
The present invention is specifically designed to and identifies the triumphant human relations algae of Michaelis, then is negative to other algae kinds, behind pcr amplification, need not gene sequencing, directly by the agarose gel electrophoresis check, just can be defined as the triumphant human relations algae of Michaelis.This invention is simple to operate, is particularly suitable for operating in the common laboratory with regular-PCR instrument.
Embodiment
The present invention's experiment is carried out according to the following steps:
(1) algal species cultivation: research object is the triumphant human relations algae of Michaelis (Karenia mikimotoi).The contrast algae has the different cap algae of ring-type (Heterocapsa circularisquama), Alexandrium tamarense (Alexandrium tamarense), chain Alexander algae (A.catenella), Alexandrium mimutum Halim by using (A.minutum), ocean Prorocentrum (Prorocentrum micans), Riamb's Prorocentrum micans (Prorocentrum lima).Substratum adopts natural sea-water, and according to the f/2 formulated that does not contain silicate, wherein nitrogen, phosphorus concentration add in addition by requirement of experiment.Substratum is standby behind 121 ℃ of high pressure steam sterilization 20min.Culture temperature is (25 ± 1) ℃, is light source with the fluorescent lamp, and intensity of illumination is 100lx, and Light To Dark Ratio is 12h: 12h.
(2) total DNA extraction: centrifugal collection is in frustule exponential phase of growth, extracts genome DNA with modified CTAB method, phenol, chloroform extracting and purifying.
(3) design of primers: according to the ITS gene order (AF183473) of the triumphant human relations algae of Michaelis among the GenBank, contrast does not belong to together, multiple other algae kinds ITS region sequence of section, order, guiding principle, look for the characteristic gene order, utilize 5.0 designs of Primer software, filter out many to primer, by the PCR experiment, determine that best primer is Ki1F3:CAACTTGAATTCATTGTGAACC and Ki 1B3:ATGTGAATCAAACATGTGGT.
(4) pcr amplification: adopt the design primer that triumphant human relations algae of Michaelis and multiple contrast algae kind genomic dna are carried out pcr amplification, reaction conditions is: carry out 30 circulations behind 95 ℃ of pre-sex change 5min, each circulation comprises 94 ℃ of sex change 45s, 58 ℃ of annealing 45s, 72 ℃ are extended 45s, extend 10min, 4 ℃ of preservations in 72 ℃ at last.
(5) electrophoresis check: the PCR product is electrophoresis on 1.5% sepharose, and gel imaging system is taken pictures, and whether the check slice result has specificity.The result has only the triumphant human relations algae of Michaelis that amplified band is arranged as can be known, and other algae are all less than amplification.
Claims (3)
1. method for identifying Karenia mikimotoi by algal species cultivation, total DNA extraction, is characterized in that utilizing Primer 5.0 softwares that the triumphant human relations algae of Michaelis (Karenia mikimotoi) rrna transcribed spacer ITS district has been designed specificity primers designed Ki1F3 and Ki1B3,
Primer sequence is Ki 1F3:CAACTTGAATTCATTGTGAACC;
Ki1B3：ATGTGAATCAAACATGTGGT；
Genomic dna is behind pcr amplification, and by the agarose gel electrophoresis check, the person is the triumphant human relations algae of Michaelis the amplified band.
2. method for identifying Karenia mikimotoi according to claim 1, it is characterized in that DNA carries out pcr amplification, reaction conditions is: carry out 30 circulations behind 95 ℃ of pre-sex change 5min, each circulation comprises 94 ℃ of sex change 45s, 58 ℃ of annealing 45s, 72 ℃ are extended 45s, extend 10min, 4 ℃ of preservations in 72 ℃ at last.
3. method for identifying Karenia mikimotoi according to claim 1 and 2 is characterized in that PCR product electrophoresis on 1.5% sepharose detects specific amplification.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200910048127XA CN101845483B (en) | 2009-03-24 | 2009-03-24 | Method for identifying Karenia mikimotoi |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN200910048127XA CN101845483B (en) | 2009-03-24 | 2009-03-24 | Method for identifying Karenia mikimotoi |
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| Publication Number | Publication Date |
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| CN101845483A true CN101845483A (en) | 2010-09-29 |
| CN101845483B CN101845483B (en) | 2012-05-30 |
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| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN200910048127XA Expired - Fee Related CN101845483B (en) | 2009-03-24 | 2009-03-24 | Method for identifying Karenia mikimotoi |
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101975814A (en) * | 2010-10-11 | 2011-02-16 | 国家海洋局第一海洋研究所 | Method for detecting potential genetic toxicity of organic pollutant in seawater |
| JP2015146774A (en) * | 2014-02-06 | 2015-08-20 | 国立大学法人愛媛大学 | Primer sets for detecting red tide, and methods for detecting red tide |
| CN112795689A (en) * | 2021-02-09 | 2021-05-14 | 中国科学院海洋研究所 | Method for rapidly detecting multiple species in Kareniaceae |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1699599A (en) * | 2005-04-08 | 2005-11-23 | 中国海洋大学 | Probe and method for detecting heterosigma akashiwo |
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2009
- 2009-03-24 CN CN200910048127XA patent/CN101845483B/en not_active Expired - Fee Related
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101975814A (en) * | 2010-10-11 | 2011-02-16 | 国家海洋局第一海洋研究所 | Method for detecting potential genetic toxicity of organic pollutant in seawater |
| CN101975814B (en) * | 2010-10-11 | 2012-12-05 | 国家海洋局第一海洋研究所 | Method for detecting potential genetic toxicity of organic pollutant in seawater |
| JP2015146774A (en) * | 2014-02-06 | 2015-08-20 | 国立大学法人愛媛大学 | Primer sets for detecting red tide, and methods for detecting red tide |
| CN112795689A (en) * | 2021-02-09 | 2021-05-14 | 中国科学院海洋研究所 | Method for rapidly detecting multiple species in Kareniaceae |
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| Publication number | Publication date |
|---|---|
| CN101845483B (en) | 2012-05-30 |
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