CN100543147C - Accurately identify the test kit and the authentication method thereof of Nian and southern catfish - Google Patents
Accurately identify the test kit and the authentication method thereof of Nian and southern catfish Download PDFInfo
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- CN100543147C CN100543147C CNB2007100783552A CN200710078355A CN100543147C CN 100543147 C CN100543147 C CN 100543147C CN B2007100783552 A CNB2007100783552 A CN B2007100783552A CN 200710078355 A CN200710078355 A CN 200710078355A CN 100543147 C CN100543147 C CN 100543147C
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Abstract
The invention provides the test kit and the detection method thereof of a kind of accurate evaluation Nian and southern catfish, this test kit comprises DNA extraction reagent, comprises reagent 1, reagent 2, reagent 3, reagent 4 and reagent 5; Polymerase chain reaction reagent comprises reaction premix reagent 6 and reagent 7; Electrophoresis and colouring reagents comprise reagent 8, reagent 9, reagent 10 and reagent 11.The present invention provides a pair of special primer NS and NA especially, utilizes this that primer is carried out pcr amplification reaction and can accurately identify Nian and southern catfish, and characteristics are accurately, in batches, to the damage of evaluation fish gently, can not cause being identified live fish death.
Description
Technical field
The present invention relates to a kind of accurately with in batches identify the test kit of Nian (Silurus asotus) and southern catfish, and the method for using this test kit evaluation Nian and southern catfish (Silurus meridionalis).
Background technology
Nian and southern catfish all belong to Nian and belong to (Silurus) fish in classification, Nian is the kind that blazons in the world, and southern catfish mainly is distributed in water systems such as China the Changjiang river, the Min River, Xiang River, and the water system that generally has southern catfish to distribute all has Nian to distribute.The individuality of Nian during adult (S.asotus) is little more a lot of than southern catfish (S.meridionalis), can distinguish them by individual size and surface.Yet in the seedling stage, generally below 250g, two kinds of catfish form fairly similars only are difficult to their right areas separately with resemblance, even if the taxonomy of fishes worker who knows a thing or two also is difficult to correctly identify them.So, be badly in need of setting up authenticate technology accurately, allow the non-specialised staff also can accurately identify two kinds of Nian.In recent years, Molecular Identification (especially PCR) technology has begun to use in the fish classification, in order accurately to identify two kinds of Nian, is necessary to set up PCR authenticate technology and development PCR identification kit.Screen a difference band by the RAPD technology at Nian and southern catfish, behind this band cloning and sequencing, this sequence compared in GenBank find not find and its height homologous sequence, explanation is a unknown nucleotide sequence, distinguishes Nian and southern catfish with this sequence for touching plate design special primer (NS and NA).
Summary of the invention
Primary and foremost purpose of the present invention is to provide the test kit of a kind of accurate evaluation Nian and southern catfish, and a pair of special primer NS and NA particularly are provided, and uses this test kit can accurately distinguish Nian and southern catfish.The present invention has set up the PCR detection technique of two kinds of Nian of accurate evaluation and has developed detection kit, to satisfy the needs that seedling is accurately identified in scientific research and the production.
Another object of the present invention is to provide above-mentioned detection kit to identify the method for two kinds of Nian.
The objective of the invention is to realize by following technical proposals:
The test kit of a kind of accurate evaluation Nian (Silurus asotus) and southern catfish (Silurus meridionalis) comprises DNA extraction reagent, polymerase chain reaction (PCR) reagent, electrophoresis and colouring reagents:
(a) DNA extraction reagent comprises
Reagent 1:DNA extracts damping fluid: 10mmol/L Tris-HCl (pH8.0), 2mmol/L EDTA (pH8.0), 0.4mol/L NaCl, 1% SDS.
Reagent 2: Proteinase K 20mg/mL.
Reagent 3: sodium-chlor 6mol/L.
Reagent 4: Virahol (analytical pure).
Reagent 5:70% ethanol.
(b) polymerase chain reaction reagent: comprise reaction premix reagent 6 and reagent 7.
Reagent 6: contain 10 times of polymerase chain reaction damping fluids and (contain MgCl
215mM), special primer is to NS and NA, dNTP (mixtures of four kinds of deoxynucleotides) and sterilization distilled water, content is respectively 2.5 μ l, 0.5-0.75 μ l (10 μ M), 0.5-0.75 μ l (10 μ M), 0.5 μ l (10 μ M each) and 19.375-20.375 μ l.
Reagent 7:Taq enzyme (5 unit/μ l).
(c) electrophoresis and colouring reagents comprise reagent 8, reagent 9, reagent 10 and reagent 11.
Reagent 8: be 50 times of TAE (electrophoretic buffer), contain trihydroxy methyl aminomethane-acetate and ethylenediamine tetraacetic acid (EDTA), concentration is respectively 0.04M and 0.001M; (using 50 times of distilled water dilutings during actual the use).
Reagent 9: be gel loading buffer, contain w/v and be 40% sucrose and 0.25% tetrabromophenol sulfonphthalein and the blue or green FF of 0.25% dimethylbenzene;
Reagent 10: electrophoresis level agarose.
Reagent 11: ethidium bromide 0.5 μ g/ μ l.
In order to realize the present invention better, described special primer NS and NA sequence are as follows:
Special primer NS refers to 5 ' AACTGTAGTCCACGAACAACATCTT3 ',
Special primer NA refers to 5 ' CTAAACTTTCTTACGGCTTCTCCC3 '.
The preferred group of described polymerase chain reaction reagent becomes: reagent 6:2.5 μ l 10 * PCR Buffer, 0.5 μ l NS, 0.5 μ l NA, 0.5 μ ldNTPs (10mM each), 20.375 μ l ddH
2O; Reagent 7:0.125 μ l Taq enzyme.
Adopt above-mentioned test kit to identify the method for Nian and southern catfish, may further comprise the steps:
(1) gets fin 50-100mg, shred, add 400 μ l reagent 1;
(2) add 10 μ l reagent 2,55-65 ℃ of water-bath 2h dissolves fully to particle;
(3) add 300 μ l reagent 3, vibration mixing 30 seconds, the centrifugal 30min of 10,000 * g gets supernatant to new centrifuge tube then;
(4) add and previous step (the 3rd step) the isopyknic reagent 4 of institute's supernatant liquor of getting, place 1h for-20 ℃, the centrifugal 15min of 10,000 * g then, supernatant discarded;
(5) with 5 washing precipitations of 1ml reagent, the centrifugal 5min of 10,000 * g, control is done in the air, and the dissolving of sterilization distilled water is DNA extraction liquid;
(6) pcr amplification: get the template of 0.5-1 μ l DNA as pcr amplification; Getting 23.875-24.375 μ l reagent 6 mixes with 0.125 μ l reagent 7; On thermal cycler, unwind 3-5 minute by 94 ℃; Use 94 ℃ of sex change 20-30 seconds then, in 60-62 ℃ of renaturation 30-40 second, 72 ℃ are extended 45-90 second, so circulate 30-35 time, are incubated 8-12 minute at 72 ℃ at last, and the purpose that will increase specific fragment is increased to 10
8More than the mol.
(7) electrophoresis and color developing detection: get the sample after 5-10 μ l increases, mix with 1-2 μ l reagent 9 (gel loading buffers), electrophoresis (50 times of conduct work of reagent 8 usefulness distilled water dilutings electrophoretic buffer) in the sepharose (add 10ml in the 0.1g reagent 10 and diluted 50 times reagent 8) of 1.0% (W/V), color developing detection under UV-light.If the band of a treaty 640 nucleotide pairs (bp) illustrates that this fish is Nian; If the band of a treaty 420 nucleotide pairs (bp) illustrates that this fish is a southern catfish.
Description of drawings
Fig. 1 is for identifying that Nian and southern catfish test kit are to the identification experiments of two kinds of fishes figure as a result.
M:DNA marker wherein, 1: be Nian characteristic band (about 640bp) 2: be southern catfish characteristic band (about 420bp).
Embodiment
Following embodiment is further to explanation of the present invention, but embodiments of the present invention are not limited thereto.
Embodiment 1
Can use 100 times the evaluation Nian and the test kit of southern catfish, its composition is composed as follows:
(a) DNA extraction reagent comprises
1,40 milliliter of reagent, its composition is: 10mmol/L Tris-HCl, pH8.0; 2mmol/L EDTA, pH8.0;
0.4mol/L?NaCl，1％?SDS；
2,0.8 milliliters of reagent, its composition is: Proteinase K, 20mg/mL;
3,30 milliliters of reagent, its composition is: sodium-chlor, 6mol/L;
Reagent 4:30ml Virahol (analytical pure);
Reagent 5:100ml 70% ethanol.
(b) polymerase chain reaction reagent: comprise reaction premix reagent 6 and reagent 7;
Reagent 6, contain 100 parts of following moietys (numeral in the bracket is 1 part a volume, and 100 parts total amount is 2437.5 μ l): 10 times of polymerase chain reaction damping fluids (contain 15mM MgCl
2) (2.5 μ l), special primer 10 μ M NS (0.5 μ l) and 10 μ M NA (0.5 μ l) (asking biotech company's (giving birth to worker, handsome company or Dalian Takara company as Shanghai) according to synthetic get final product of sequence (NS:5 ' AACTGTAGTCCACGAACAACATCTT 3 ', NA:5 ' CTAAACTTTCTTACGGCTTCTCCC 3 ')), the mixture (0.5 μ l (10 μ M each)) of four kinds of deoxynucleotides and the distilled water (20.375 μ l) of sterilizing;
Reagent 7,130 μ l recommend to use precious biotechnology (Dalian) the product Taq of company limited enzyme, 5U/ μ l; (c) electrophoresis and colouring reagents comprise reagent 8, reagent 9, reagent 10 and reagent 11;
8,100 milliliters of reagent are 50 times of electrophoretic buffers, contain trihydroxy methyl aminomethane-acetate and ethylenediamine tetraacetic acid (EDTA), and concentration is respectively 0.04M and 0.001M; (using 50 times of distilled water dilutings during actual the use)
9,0.5 milliliters of reagent are gel loading buffer, contain w/v and be 40% sucrose and 0.25% tetrabromophenol sulfonphthalein and the blue or green FF of dimethylbenzene;
Reagent 10,10 gram electrophoresis level agaroses; (press the W/V configuration with the reagent 8 of 50 times of dilutions during use: 1.0% agarose)
Reagent 11,300 μ l ethidium bromides (0.5 μ g/ μ l).(adding 1 μ g ethidium bromide according to every milliliter of agarose during use)
Use operation steps:
1, gets fin 100mg, shred, add 400 μ l reagent 1;
2, add 2,55 ℃ of water-bath 2h of 10 μ l reagent, to solution, do not have particulate matter;
3, add 300 μ l reagent 3, vibration mixing 30 seconds, the centrifugal 30min of 10,000 * g gets supernatant to new centrifuge tube then;
4, the isopyknic reagent 4 of adding and previous step (the 3rd step) institute's supernatant liquor of getting is placed 1h for-20 ℃, the centrifugal 15min of 10,000 * g then, supernatant discarded;
5, with 5 washing precipitations of 1ml reagent, the centrifugal 5min of 10,000 * g, control is done in the air, and the dissolving of sterilization distilled water is DNA extraction liquid;
6, pcr amplification: get the template of 0.5 μ lDNA as pcr amplification; Getting 24.375 μ l reagent 6 mixes with 0.125 μ l reagent 7; On thermal cycler, unwind 3 minutes by 94 ℃; Use 94 ℃ of sex change 30 seconds then, in 62 ℃ of 30 seconds of renaturation, 72 ℃ were extended for 45 seconds, so circulated 30 times, at last 72 ℃ of insulations 8 minutes.
7, electrophoresis and color developing detection: get the sample after 10 μ l increase, mix with 2 μ l reagent 9 (gel loading buffer), electrophoresis (50 times of conduct work of reagent 8 usefulness distilled water dilutings electrophoretic buffer) in the sepharose (add 10ml in the 0.1g reagent 10 and diluted 50 times reagent 8) of 1.0% (W/V), color developing detection under UV-light.If the band of a treaty 640 nucleotide pairs (bp) illustrates that this fish is Nian; If the band of a treaty 420 nucleotide pairs (bp) illustrates that this fish is a southern catfish.
Embodiment 2
1, gets fin 50mg, shred, add 400 μ l reagent 1;
2, add 2,60 ℃ of about 2h of water-bath of 10 μ l reagent, to solution, do not have particulate matter;
3, add 300 μ l reagent 3, vibration mixing 30 seconds, the centrifugal 30min of 10,000 * g gets supernatant to new centrifuge tube then;
4, the isopyknic reagent 4 of adding and previous step (the 3rd step) institute's supernatant liquor of getting is placed 1h for-20 ℃, the centrifugal 15min of 10,000 * g then, supernatant discarded;
5, with 5 washing precipitations of 1ml reagent, the centrifugal 5min of 10,000 * g, control is done in the air, and the dissolving of sterilization distilled water is DNA extraction liquid;
6, pcr amplification: get the template of 1 μ l DNA as pcr amplification; Getting 23.875 μ l reagent 6 mixes with 0.125 reagent 7; On thermal cycler, unwind 3 minutes by 94 ℃; Use 94 ℃ of sex change 30 seconds then, in 60 ℃ of 30 seconds of renaturation, 72 ℃ were extended for 45 seconds, so circulated 30 times, at last 72 ℃ of insulations 8 minutes.
7, electrophoresis and color developing detection: get the sample after 10 μ l increase, mix with 2 μ l reagent 9 (gel loading buffer), electrophoresis (50 times of conduct work of reagent 8 usefulness distilled water dilutings electrophoretic buffer) in the sepharose (add 10ml in the 0.1g reagent 10 and diluted 50 times reagent 8) of 1.0% (W/V), color developing detection under UV-light.If the band of a treaty 640 nucleotide pairs (bp) illustrates that this fish is Nian; If the band of a treaty 420 nucleotide pairs (bp) illustrates that this fish is a southern catfish, detected result is seen Fig. 1.
Claims (3)
1, the test kit of a kind of accurate evaluation Nian and southern catfish comprises DNA extraction reagent, polymerase chain reaction reagent, electrophoresis and colouring reagents:
(a) DNA extraction reagent comprises
Reagent 1:DNA extracts damping fluid: 10mmol/L Tris-HCl, pH8.0; 2mmol/L EDTA, pH8.0; 0.4mol/L NaCl, 1% SDS;
Reagent 2: Proteinase K 20mg/mL;
Reagent 3: sodium-chlor 6mol/L;
Reagent 4: Virahol;
Reagent 5:70% ethanol;
(b) polymerase chain reaction reagent: comprise reaction premix reagent 6 and reagent 7;
Reagent 6: contain 10 times of polymerase chain reaction damping fluids, wherein contain MgCl
215mM, 2.5 μ l; Special primer NS0.5-0.75 μ l, 10 μ M, NA 0.5-0.75 μ l, 10 μ M; The mixture 0.5 μ l of four kinds of deoxynucleotides, every kind 10 μ M; Sterilization distilled water 19.375-20.375 μ l;
Reagent 7:Taq enzyme, 5U/ μ l;
Described special primer NS and NA are as follows:
Special primer NS refers to 5 ' AACTGTAGTCCACGAACAACATCTT3 ';
Special primer NA refers to 5 ' CTAAACTTTCTTACGGCTTCTCCC 3 ';
(c) electrophoresis and colouring reagents comprise reagent 8, reagent 9, reagent 10 and reagent 11;
Reagent 8: be 50 times of electrophoretic buffers, contain trihydroxy methyl aminomethane-acetate and ethylenediamine tetraacetic acid (EDTA), concentration is respectively 0.04M and 0.001M;
Reagent 9: be gel loading buffer, contain w/v and be 40% sucrose and 0.25% tetrabromophenol sulfonphthalein and the blue or green FF of 0.25% dimethylbenzene;
Reagent 10: electrophoresis level agarose;
Reagent 11: ethidium bromide, 0.5 μ g/ μ l.
2, the test kit of accurate evaluation Nian according to claim 1 and southern catfish is characterized in that, the consisting of of described polymerase chain reaction reagent:
Reagent 6:2.5 μ l 10 * PCR Buffer, 0.5 μ l NS, 0.5 μ l NA, every kind of 10mM of 0.5 μ l dNTPs, 20.375 μ lddH
2O;
Reagent 7:0.125 μ l Taq enzyme.
3, adopt the described test kit of each claim of claim 1-2 to identify the method for Nian and southern catfish, it is characterized in that comprising the steps:
(1) gets fin 50-100mg, shred, add 400 μ l reagent 1;
(2) add 10 μ l reagent 2,55-65 ℃ of water-bath 2h dissolves fully to particle;
(3) add 300 μ l reagent 3, vibration mixing 30 seconds, the centrifugal 30min of 10,000 * g gets supernatant to new centrifuge tube then;
(4) add the isopyknic reagent 4 of (3) step institute's supernatant liquor of getting, place 1h for-20 ℃, the centrifugal 15min of 10,000 * g then, supernatant discarded;
(5) with 5 washing precipitations of 1ml reagent, the centrifugal 5min of 10,000 * g, control is done in the air, and the dissolving of sterilization distilled water is DNA extraction liquid;
(6) pcr amplification: get the template of 0.5-1 μ l DNA as pcr amplification; Getting 23.875-24.375 μ l reagent 6 mixes with 0.125 μ l reagent 7; On thermal cycler, unwind 3-5 minute by 94 ℃; Use 94 ℃ of sex change 20-30 seconds then, in 60-62 ℃ of renaturation 30-40 second, 72 ℃ are extended 45-90 second, so circulate 30-35 time, are incubated 8-12 minute at 72 ℃ at last, and the purpose that will increase specific fragment is increased to 10
8More than the mol;
(7) electrophoresis and color developing detection: get the sample after the 5-10 μ l amplification, mix with 1-2 μ l reagent 9, electrophoresis in the sepharose of 1.0%W/V, with 50 times of reagent 8 usefulness distilled water dilutings as the electrophoretic buffer of working, color developing detection under UV-light; If the band of a treaty 640 nucleotide pairs illustrates that this fish is Nian; If the band of a treaty 420 nucleotide pairs illustrates that this fish is a southern catfish.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CNB2007100783552A CN100543147C (en) | 2007-03-30 | 2007-03-30 | Accurately identify the test kit and the authentication method thereof of Nian and southern catfish |
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| CNB2007100783552A CN100543147C (en) | 2007-03-30 | 2007-03-30 | Accurately identify the test kit and the authentication method thereof of Nian and southern catfish |
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| CN100543147C true CN100543147C (en) | 2009-09-23 |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102161988A (en) * | 2011-01-21 | 2011-08-24 | 中国科学院海洋研究所 | Method for simply, conveniently and quickly extracting trace total deoxyribonucleic acid (DNA) of single roes and fries |
-
2007
- 2007-03-30 CN CNB2007100783552A patent/CN100543147C/en not_active Expired - Fee Related
Non-Patent Citations (6)
| Title |
|---|
| 三种鲇遗传多样性的RAPD分析. 王朝明，邹桂伟，郑蓓蓓，罗相忠，潘光碧.淡水渔业，第35卷第4期. 2005 |
| 三种鲇遗传多样性的RAPD分析. 王朝明，邹桂伟，郑蓓蓓，罗相忠，潘光碧.淡水渔业，第35卷第4期. 2005 * |
| 大口鲇、鲇及其杂种F1种质遗传标记研究. 王朝明.华中农业大学硕士论文，第3期. 2006 |
| 大口鲇、鲇及其杂种F1种质遗传标记研究. 王朝明.华中农业大学硕士论文，第3期. 2006 * |
| 长江流域南方鲇（Silurus meridionalis）和鲇（Silurusasotus）分子遗传标记的建立. 李学英，王大忠，廖吉文.遵义医学院学报，第24卷第3期. 2001 |
| 长江流域南方鲇（Silurus meridionalis）和鲇（Silurusasotus）分子遗传标记的建立. 李学英，王大忠，廖吉文.遵义医学院学报，第24卷第3期. 2001 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN102161988A (en) * | 2011-01-21 | 2011-08-24 | 中国科学院海洋研究所 | Method for simply, conveniently and quickly extracting trace total deoxyribonucleic acid (DNA) of single roes and fries |
| CN102161988B (en) * | 2011-01-21 | 2013-10-23 | 中国科学院海洋研究所 | Method for simply, conveniently and quickly extracting trace total deoxyribonucleic acid (DNA) of single roes and fries |
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| CN101024858A (en) | 2007-08-29 |
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