CN101659993A - Molecular biology identification method for dry sea cucumbers - Google Patents
Molecular biology identification method for dry sea cucumbers Download PDFInfo
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- CN101659993A CN101659993A CN200910015703A CN200910015703A CN101659993A CN 101659993 A CN101659993 A CN 101659993A CN 200910015703 A CN200910015703 A CN 200910015703A CN 200910015703 A CN200910015703 A CN 200910015703A CN 101659993 A CN101659993 A CN 101659993A
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- stichopus japonicus
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Abstract
The invention relates to a molecular biology identification method for dry sea cucumbers, which comprises the following steps: extracting genome DNA of dry sea cucumbers by a high salt method; amplifying CO I genetic fragments by using a degenerate primer PCR; carrying out agarose gel electrophoresis detection on enzyme-digested products; and determining the result by the quantity and the length of electrophoretic bands. The method is used for identifying different varieties of sea cucumbers, has the characteristics of high accuracy and speed as well as convenience and is more accurate and reliable when compared with the traditional morphological identification.
Description
Technical field
The present invention relates to a kind of mensuration or method of inspection that comprises enzyme or microorganism, be specifically related to a kind of molecular biology identification method of dry Stichopus japonicus.
Background technology
Sea cucumber belongs to Echinodermata on taxonomy, Holothuroidea has nutritive value and economic worth, and domestic main breed variety is imitative stichopus japonicus (Apostichopus japonicus), and doing imitative stichopus japonicus is the main products that the human consumer buys.Along with the raising of people's living standard, the sea cucumber demand significantly rises, and product is identified becomes the guardian technique problem of being badly in need of solution.At present China has introduced multiple sea cucumber from external, comprise the red stichopus japonicus of introducing from Japan (Apostichopus japonicus), New Zealand sea cucumber (Australostichopus mollis), Holothuria nobilis Selenka (holothuria nobilis), yellow newborn sea cucumber (holothuriafuscogilva) etc.Difference in selling prices is bigger between the dry Stichopus japonicus not of the same race, but sea cucumber is difficult to differentiate its kind from formalness after being processed into dry product through multiple working procedure.And lack effective discrimination method, cause market circulation relatively more chaotic.
Protocols in Molecular Biology provides good terms of settlement for this problem, and CO I gene order is very conservative in living species, is widely used in the classification and the Study on Evolution of species.Utilize its nucleotide sequence difference that between different plant species, exists, use restriction enzyme that pcr amplification product is carried out enzyme and cut, can produce the nucleic acid fragment of different lengths and quantity, can tell different sorts by agarose gel electrophoresis.This method yet there are no open report in China's dry Stichopus japonicus is identified, this will identify for dry Stichopus japonicus provides scientific methods, provides technical support to standard dry Stichopus japonicus market.
Notification number: CN 101130815A, the Chinese patent literature of the day for announcing: 2008.02.27 has announced that " the PCR-RFLP discrimination method that seven kinds of oysters of The South China coastal belong to oyster " introduced a kind of molecular biology method that is used to identify oyster, and the primer sequence of the oyster polymerase chain reaction of this inventive method design is:
CO?I?L1：5’-CGGTGTTTATCAAAAACAT-3’；
CO?I?H：5’-TAAACTTCAGGGTGACCAAAAAATCA-3’.
Utilize restriction enzyme Nis I/Alu I and Nis I/Dde I that the PCR product is carried out double digestion, detect enzyme by agarose gel electrophoresis and cut product, result of determination.This invention can realize the discriminating quickly and accurately to oyster.But this discrimination method but can not be used for the discriminating of dry Stichopus japonicus.
Summary of the invention
The objective of the invention is to differentiate at existing dry Stichopus japonicus morphology inaccurate, the deficiency that the science authentication technique generally lacks provides a kind of scientific and effective, simple and efficient, molecular biology identification method of dry Stichopus japonicus accurately.
The objective of the invention is to realize by following technique means: the molecular biology identification method of dry Stichopus japonicus is characterized in that may further comprise the steps:
(1) adopt high salt method to extract dry Stichopus japonicus DNA;
(2) utilize degenerated primer pcr amplification CO I gene fragment;
(3) with restriction enzyme Csp6 I the PCR product being carried out enzyme cuts;
(4) enzyme is cut product and carry out the agarose gel electrophoresis detection, by the quantity and the length result of determination of electrophoretic band.
The step that the described high salt method of step (1) is extracted DNA is:
1. get dry Stichopus japonicus 100-200mg, in Milli-Q water, soaked 5-6 hour grind into powder in liquid nitrogen;
2. be transferred in the 50ml centrifuge tube, add 10ml and extract damping fluid, it consists of 50mM Tris, 100mM NaCl, 150mM EDTA, 1%SDS, 0.30mg/ml Proteinase K, 0.1% mercaptoethanol, 8.0,65 ℃ of digestion of pH 3h;
3. slowly add 3ml 6mol/L NaCl, saltout and remove unnecessary albumen and polysaccharide, centrifugal 10 minutes of 4 ℃ of 10000rpm collect supernatant liquor;
4. add the equal-volume ratio and be 25: 24: 1 phenol, chloroform, primary isoamyl alcohol, centrifugal 10 minutes of 15000rpm collects supernatant liquor;
5. the primary isoamyl alcohol that adds 0.6 times of volume of precooling ,-20 ℃ of precipitation 1-2h are after room temperature is placed 15min, centrifugal 3 minutes of 15000rpm removes supernatant, collects the DNA precipitation, with concentration is 70% washing with alcohol precipitation, places to blow in 5-10 minute at super clean bench and dries, and is dissolved in the 200ul TE damping fluid.
The described PCR of step (2), promptly the polymerase chain reaction comprises the following steps:
1. the system of polymerase chain reaction is 25 μ l, and the consumption of all ingredients is respectively:
10 * PCR damping fluid, 2.5 μ l
MgCl
2?25mM 1.6μl
dNTP?2.5mM 2.0μl
COIf?15μM 1μl
COIr?15μM 1μl
Taq enzyme 5U/ μ l 0.125 μ l
The DNA 1 μ l that extract on top
PCR level water 15.775 μ l
2. the polymerase chain enzyme reaction is carried out on the PCR instrument, and reaction conditions is:
94 ℃ of pre-sex change 5 minutes, then by 35 circulating reactions of following conditioned response:
94 ℃ of sex change 45 seconds
50 ℃ of renaturation 45 seconds
72 ℃ were extended 1 minute
After the loop ends, last 72 ℃ were extended 10 minutes.
The endonuclease reaction system of the described Csp6I of step (3) is 20 μ l, and the consumption of all ingredients is respectively:
PCR product 12 μ l
10 * enzyme cutting buffering liquid, 2 μ l
Csp6I(10U/μl) 1μl
Deionized water 5 μ l
With above all ingredients mixing, to put in the 0.2ml PCR pipe, 37 ℃ of temperature were bathed 4 hours, and flick 2-3 time the centre.
The described agarose gel electrophoresis of step (4) detects step: get 8 μ l enzymes and cut product, with 2.5% sepharose under 120V, electrophoresis 1-2 hour, add the DNA standard simultaneously, the taking-up gel is put under the UV-light and is observed electrophoretic band, with the contrast of DNA standard, by the quantity and the length result of determination of electrophoretic band.
The employed primer sequence of the described pcr amplification of step (2) is respectively:
COIf：ATAATGATAGGAGG[A/G]TTTGG
COIr：GCTCGTGT[A/G]TCTAC[A/G]TCCAT。
The standard band spectrum that the electrophoretic band enzyme that the described agarose gel electrophoresis of step (4) detects is cut the result is:
Blue or green stichopus japonicus (Apostichopus japonicus) 386bp+306bp
Red stichopus japonicus (Apostichopus japonicus) 692bp
New Zealand sea cucumber (Australostichopus mollis) 539bp+153bp
Holothuria nobilis Selenka (Holothuria nobilis) 306bp+288bp+98bp
Yellow newborn sea cucumber (Holothuria fuscogilva) 594bp+98bp.
Compared with prior art the molecular biology identification method of this dry Stichopus japonicus has following tangible advantage:
1, the molecular biology identification method of this dry Stichopus japonicus improves traditional DNA extraction method, has effectively removed the impurity in the dry Stichopus japonicus, can access high purity, the DNA of high yield.
2, the molecular biology identification method of this dry Stichopus japonicus can be identified different sea cucumber species, have accurately, rapidly, characteristics easily, can finish in common 1 day, compare the method for CO I complete sequence determination and not only can save time but also can reduce expense.
3, the molecular biology identification method of this dry Stichopus japonicus is lower to the plant and instrument requirement of using, and differentiates more accurately and reliably than traditional morphology.
4, the molecular biology identification method of this dry Stichopus japonicus fundamentally solves the difficult problem that current sea cucumber kind can't accurately be identified, has filled up the industry blank, and is significant.
Embodiment
The present invention is described in further detail below in conjunction with embodiment: the molecular biology identification method of dry Stichopus japonicus may further comprise the steps:
(1) adopt high salt method to extract dry Stichopus japonicus DNA, concrete extraction step is:
1. get dry Stichopus japonicus 100-200mg, in Milli-Q water, soaked 5-6 hour grind into powder in liquid nitrogen;
2. be transferred in the 50ml centrifuge tube, add 10ml and extract damping fluid, it consists of 50mM Tris, 100mM NaCl, 150mM EDTA, 1%SDS, 0.30mg/ml Proteinase K, 0.1% mercaptoethanol, 8.0,65 ℃ of digestion of pH 3h;
3. slowly add 3ml 6mol/L NaCl, saltout and remove unnecessary albumen and polysaccharide, centrifugal 10 minutes of 4 ℃ of 10000rpm collect supernatant liquor;
4. add the equal-volume ratio and be 25: 24: 1 phenol, chloroform, primary isoamyl alcohol, centrifugal 10 minutes of 15000rpm collects supernatant liquor;
5. the primary isoamyl alcohol that adds 0.6 times of volume of precooling ,-20 ℃ of precipitation 1-2h are after room temperature is placed 15min, centrifugal 3 minutes of 15000rpm removes supernatant, collects the DNA precipitation, with concentration is 70% washing with alcohol precipitation, places to blow in 5-10 minute at super clean bench and dries, and is dissolved in the 200ul TE damping fluid.
Adopt high salt method to extract DNA and can effectively remove albumen and the polysaccharide that dry Stichopus japonicus contains, obtain the higher genomic dna of purity.
(2) utilize degenerated primer pcr amplification CO I gene fragment, specifically comprise the following steps:
1. the system of polymerase chain reaction is 25 μ l, and the consumption of all ingredients is respectively:
10 * PCR damping fluid, 2.5 μ l
MgCl
2?25mM 1.6μl
dNTP?2.5mM 2.0μl
COIf?15μM 1μl
COIr?15μM 1μl
Taq enzyme 5U/ μ l 0.125 μ l
The DNA 1 μ l that extract on top
PCR level water 15.775 μ l
2. the polymerase chain enzyme reaction is carried out on the PCR instrument, and reaction conditions is:
94 ℃ of pre-sex change 5 minutes, then by 35 circulating reactions of following conditioned response:
94 ℃ of sex change 45 seconds
50 ℃ of renaturation 45 seconds
72 ℃ were extended 1 minute
After the loop ends, last 72 ℃ were extended 10 minutes.
3. the employed primer sequence of pcr amplification is respectively:
COIf：ATAATGATAGGAGG[A/G]TTTGG
COIr：GCTCGTGT[A/G]TCTAC[A/G]TCCAT。
(3) with restriction enzyme Csp6I the PCR product being carried out enzyme cuts; Described Csp6I endonuclease reaction system is 20 μ l, and the consumption of all ingredients is respectively:
PCR product 12 μ l
10 * enzyme cutting buffering liquid, 2 μ l
Csp6I?10U/μl 1μl
Deionized water 5 μ l
With above all ingredients mixing, to put in the 0.2ml PCR pipe, 37 ℃ of temperature were bathed 4 hours, and flick 2-3 time the centre.
(4) enzyme is cut product and carry out the agarose gel electrophoresis detection, get 8 μ l enzymes and cut product, with 2.5% sepharose (containing 0.5% ethidium bromide) under 120V, electrophoresis 1-2 hour, add the DNA standard simultaneously, the taking-up gel is put under the UV-light and is observed electrophoretic band, with the contrast of DNA standard, judges the result who differentiates by the quantity and the length of electrophoretic band.The standard band spectrum that sea cucumber PCR product enzyme not of the same race is cut rear electrophoresis is respectively:
Blue or green stichopus japonicus (Apostichopus japonicus) 386bp+306bp
Red stichopus japonicus (Apostichopus japonicus) 692bp
New Zealand sea cucumber (Australostichopus mollis) 539bp+153bp
Holothuria nobilis Selenka (Holothuria nobilis) 306bp+288bp+98bp
Yellow newborn sea cucumber (Holothuria fuscogilva) 594bp+98bp.
The molecular biology identification method of this dried sea-cucumber can identify different sea cucumber species, has accurately, rapidly, just Prompt characteristics are differentiated more accurately and reliably than traditional morphology.
Claims (7)
1, the molecular biology identification method of dry Stichopus japonicus is characterized in that may further comprise the steps:
(1) adopt high salt method to extract dry Stichopus japonicus DNA;
(2) utilize degenerated primer pcr amplification CO I gene fragment;
(3) with restriction enzyme Csp6 I the PCR product being carried out enzyme cuts;
(4) enzyme is cut product and carry out the agarose gel electrophoresis detection, by the quantity and the length result of determination of electrophoretic band.
2, the molecular biology identification method of dry Stichopus japonicus according to claim 1 is characterized in that: the step that the described high salt method of step (1) is extracted DNA is:
1. get dry Stichopus japonicus 100-200mg, in Milli-Q water, soaked 5-6 hour grind into powder in liquid nitrogen;
2. be transferred in the 50ml centrifuge tube, add 10ml and extract damping fluid, it consists of 50mM Tris, 100mM NaCl, 150mM EDTA, 1%SDS, 0.30mg/ml Proteinase K, 0.1% mercaptoethanol, 8.0,65 ℃ of digestion of pH 3h;
3. slowly add 3ml 6mol/L NaCl, saltout and remove unnecessary albumen and polysaccharide, centrifugal 10 minutes of 4 ℃ of 10000rpm collect supernatant liquor;
4. add the equal-volume ratio and be 25: 24: 1 phenol, chloroform, primary isoamyl alcohol, centrifugal 10 minutes of 15000rpm collects supernatant liquor;
5. the primary isoamyl alcohol that adds 0.6 times of volume of precooling ,-20 ℃ of precipitation 1-2h are after room temperature is placed 15min, centrifugal 3 minutes of 15000rpm removes supernatant, collects the DNA precipitation, with concentration is 70% washing with alcohol precipitation, places to blow in 5-10 minute at super clean bench and dries, and is dissolved in the 200ul TE damping fluid.
3, the molecular biology identification method of dry Stichopus japonicus according to claim 1 is characterized in that: the described PCR of step (2), promptly the polymerase chain reaction comprises the following steps:
1. the system of polymerase chain reaction is 25 μ l, and the consumption of all ingredients is respectively:
10 * PCR damping fluid, 2.5 μ l
MgCl
2?25mM 1.6μl
dNTP 2.5mM 2.0μl
COIf 15μM 1μl
COIr 15μM 1μl
Taq enzyme 5U/ μ l 0.125 μ l
The DNA 1 μ l that extract on top
PCR level water 15.775 μ l
2. the polymerase chain enzyme reaction is carried out on the PCR instrument, and reaction conditions is:
94 ℃ of pre-sex change 5 minutes, then by 35 circulating reactions of following conditioned response:
94 ℃ of sex change 45 seconds
50 ℃ of renaturation 45 seconds
72 ℃ were extended 1 minute
After the loop ends, last 72 ℃ were extended 10 minutes.
4, the molecular biology identification method of dry Stichopus japonicus according to claim 1 is characterized in that: the endonuclease reaction system of the described Csp6 I of step (3) is 20 μ l, and the consumption of all ingredients is respectively:
PCR product 12 μ l
10 * enzyme cutting buffering liquid, 2 μ l
Csp6?I(10U/μl) 1μl
Deionized water 5 μ l
With above all ingredients mixing, to put in the 0.2ml PCR pipe, 37 ℃ of temperature were bathed 4 hours, and flick 2-3 time the centre.
5, the molecular biology identification method of dry Stichopus japonicus according to claim 1, it is characterized in that: the described agarose gel electrophoresis of step (4) detects step and is: get 8 μ l enzymes and cut product, with 2.5% sepharose under 120V, electrophoresis 1-2 hour, add the DNA standard simultaneously, the taking-up gel is put under the UV-light and is observed electrophoretic band, with the contrast of DNA standard, by the quantity and the length result of determination of electrophoretic band.
6, according to the molecular biology identification method of claim 1 or 3 described dry Stichopus japonicuss, it is characterized in that: the employed primer sequence of the described pcr amplification of step (2) is respectively:
COIf：ATAATGATAGGAGG[A/G]TTTGG
COIr：GCTCGTGT[A/G]TCTAC[A/G]TCCAT。
7, the molecular biology identification method of dry Stichopus japonicus according to claim 1 or 5 is characterized in that: the standard band spectrum that the electrophoretic band enzyme that the described agarose gel electrophoresis of step (4) detects is cut the result is:
Blue or green stichopus japonicus (Apostichopusjaponicus) 386bp+306bp
Red stichopus japonicus (Apostichopusjaponicus) 692bp
New Zealand sea cucumber (Australostichopus mollis) 539bp+153bp
Holothuria nobilis Selenka (Holothuria nobilis) 306bp+288bp+98bp
Yellow newborn sea cucumber (Holothuria fuscogilva) 594bp+98bp.
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Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101942510A (en) * | 2010-07-15 | 2011-01-12 | 中国海洋大学 | Multiple PCR method for rapidly identifying four varieties of sea cucumbers |
| CN102146375A (en) * | 2011-03-09 | 2011-08-10 | 南开大学 | Method for massively extracting earthworm genome DNA |
| CN102721730A (en) * | 2012-06-21 | 2012-10-10 | 大连海洋大学 | Method for obtaining two-dimensional electrophoresis patterns of proteins of gonads of stichopus japonicus by two-dimensional electrophoresis technology |
| CN102851380A (en) * | 2012-09-20 | 2013-01-02 | 中国水产科学研究院黄海水产研究所 | Method utilizing degenerate primer to detect diversity of bacteria catalase in seawater |
| CN102911934A (en) * | 2012-11-06 | 2013-02-06 | 大连工业大学 | DNA (Deoxyribonucleic Acid) extraction method of sea cucumber body surface bacteria |
| CN102994493A (en) * | 2012-11-06 | 2013-03-27 | 中国科学院南海海洋研究所 | Method for extracting total DNA (deoxyribonucleic acid) from body wall of sea cucumber |
| CN103014175A (en) * | 2013-01-22 | 2013-04-03 | 中国海洋大学 | PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) method for identifying seven sea cucumber species |
| CN106636072A (en) * | 2017-01-22 | 2017-05-10 | 中国医学科学院药用植物研究所 | General DNA extraction method for identification of animal traditional Chinese medicine molecules and kit |
| CN109652408A (en) * | 2019-02-28 | 2019-04-19 | 广东美立康生物科技有限公司 | The method for saving rapidly extracting high concentration DNA in sample from micro alcoholic solution |
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Cited By (13)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101942510A (en) * | 2010-07-15 | 2011-01-12 | 中国海洋大学 | Multiple PCR method for rapidly identifying four varieties of sea cucumbers |
| CN102146375A (en) * | 2011-03-09 | 2011-08-10 | 南开大学 | Method for massively extracting earthworm genome DNA |
| CN102146375B (en) * | 2011-03-09 | 2012-11-14 | 南开大学 | Method for massively extracting earthworm genome DNA |
| CN102721730A (en) * | 2012-06-21 | 2012-10-10 | 大连海洋大学 | Method for obtaining two-dimensional electrophoresis patterns of proteins of gonads of stichopus japonicus by two-dimensional electrophoresis technology |
| CN102851380B (en) * | 2012-09-20 | 2014-07-09 | 中国水产科学研究院黄海水产研究所 | Method utilizing degenerate primer to detect diversity of bacteria catalase in seawater |
| CN102851380A (en) * | 2012-09-20 | 2013-01-02 | 中国水产科学研究院黄海水产研究所 | Method utilizing degenerate primer to detect diversity of bacteria catalase in seawater |
| CN102911934A (en) * | 2012-11-06 | 2013-02-06 | 大连工业大学 | DNA (Deoxyribonucleic Acid) extraction method of sea cucumber body surface bacteria |
| CN102994493A (en) * | 2012-11-06 | 2013-03-27 | 中国科学院南海海洋研究所 | Method for extracting total DNA (deoxyribonucleic acid) from body wall of sea cucumber |
| CN102911934B (en) * | 2012-11-06 | 2014-06-25 | 大连工业大学 | DNA (Deoxyribonucleic Acid) extraction method of sea cucumber body surface bacteria |
| CN103014175A (en) * | 2013-01-22 | 2013-04-03 | 中国海洋大学 | PCR-RFLP (polymerase chain reaction-restriction fragment length polymorphism) method for identifying seven sea cucumber species |
| CN106636072A (en) * | 2017-01-22 | 2017-05-10 | 中国医学科学院药用植物研究所 | General DNA extraction method for identification of animal traditional Chinese medicine molecules and kit |
| CN106636072B (en) * | 2017-01-22 | 2020-06-16 | 中国医学科学院药用植物研究所 | General DNA extraction method and kit for animal traditional Chinese medicine molecular identification |
| CN109652408A (en) * | 2019-02-28 | 2019-04-19 | 广东美立康生物科技有限公司 | The method for saving rapidly extracting high concentration DNA in sample from micro alcoholic solution |
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Application publication date: 20100303 |