CN101434990B - Mink heart DNA detection kit and identification method - Google Patents
Mink heart DNA detection kit and identification method Download PDFInfo
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- CN101434990B CN101434990B CN2008100516433A CN200810051643A CN101434990B CN 101434990 B CN101434990 B CN 101434990B CN 2008100516433 A CN2008100516433 A CN 2008100516433A CN 200810051643 A CN200810051643 A CN 200810051643A CN 101434990 B CN101434990 B CN 101434990B
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Abstract
The invention relates to the Chinese medicine detection technology field and belongs to a mink heart DNA detection kit, comprising buffer solution, 12.5mM dNTP, one type of 0.1mM primer 1 or 2, Taq DNA polymerase, sample DNA to be detected, and the identification reaction system of double-distilled water; containing buffer solution, 12.5mM dNTP, one type of 0.1mM primer 1 or 2, Taq DNA polymerase, mink heart DNA, and the positive control reaction system of the double-distilled water; including buffer solution, 12.5mM dNTP, one type of 0.1mM primer 1 or 2, Taq DNA polymerase, mixture of chicken heart, duck heart, goose heart and rabbit heart DNA according to 1:1, and the negative control reaction system of the double-distilled water. The mink heart DNA identification method comprises steps such as designing mink heart mitochondrial DNA two-pair specific oligonucleotide primer, artificially synthetizing mink heart mitochondrial DNA two-pair specific oligonucleotide primer, fixing reaction procedures and result judgment, and the like; the method can simultaneously and accurately differ the specificity of various animal hearts which are easily mixed with the mink heat; and the identification method has the advantages of simplicity, rapidness, and reliable detection result, etc.
Description
Technical field
The present invention relates to Chinese medicine detection technique field, more particularly, is a kind of mink heart DNA detection kit and authentication method.
Background technology
The ermine heart belongs to thin precious medicinal material, is the main ingredient in China's Chinese patent medicine-Li heart ball.Sharp heart ball has the effect of heart tonifying and tranquilizing, can treat diseases such as rheumatic heart disease, tachycardia, arrhythmia and heart failure.At present, still the legal discrimination method that does not have the ermine heart in the national standard, do not see the composition Study of the ermine heart and the bibliographical information of discriminating both at home and abroad yet, existing Chinese medicinal materials discrimination method can't be distinguished the ermine heart and the heart that easily mixes animal, so brought very big difficulty for the quality control of the ermine heart and preparation thereof, be difficult to satisfy evaluation to the Chinese medicinal materials ermine heart.
Summary of the invention
The inventor follows the carrier organism Heredity theory of Mitochondrial DNA (mtDNA) as genetic information, it is moderate that mitochondrial cytochrome b (cyt b) gene has rate of evolution, the feature of classification, phylogeny and the genetic diversity of genetic diversity and biology in being applied to plant, between kind.The objective of the invention is the full gene group of ermine heart Mitochondrial DNA is screened, a kind of specificity that can accurately differentiate the ermine heart and heart, the duck heart, the goose heart and the rabbit heart is provided, mink heart DNA detection kit easy to use, and provide that it is easy, quick, the reliable authentication method of detected result.
The objective of the invention is to realize by following technical scheme:
A kind of mink heart DNA detection kit is characterized in that, it comprises:
(a) identification reaction system: totally be 100 μ L, wherein contain damping fluid 10 μ L, 12.5mMdNTP 4-10 μ L, 0.1mM a kind of 1.5-4.0 μ L in primer 1 and the primer 2, Taq archaeal dna polymerase 2-10 μ L, sample DNA 2-5 μ L to be checked, surplus is bi-distilled water;
(b) positive control reaction system: totally be 100 μ L, wherein contain damping fluid 10 μ L, 12.5mM dNTP4-10 μ L, 0.1mM a kind of 1.5-4.0 μ L in primer 1 and the primer 2, Taq archaeal dna polymerase 2-10 μ L, mink heart DNA 2-5 μ L, surplus is bi-distilled water;
(c) negative control reaction system: total system is 100 μ L, wherein contain damping fluid 10 μ L, 12.5mM dNTP 4-10 μ L, 0.1mM a kind of 1.5-4.0 μ L in primer 1 and the primer 2, TaqDNA polysaccharase 2-10 μ L, heart, the duck heart, the goose heart and rabbit heart DNA mixed 2-5 μ L by 1: 1, and surplus is bi-distilled water.
The mink heart DNA authentication method is characterized in that, it comprises following step:
(1) design ermine heart Mitochondrial DNA two pairs of special Oligonucleolide primers contain the mink heart DNA sequence, a kind of in following primer 1 and the primer 2:
5’ATGACCAACATTCGTAAAACTC 3’
5’CAGATTCACTCTACAAGACT 3’
5’GGAACCAGTCTTGTAGAGTG 3’
5’TCTTCATTTTAATAGGTTAT 3’
(2) two pairs of special Oligonucleolide primers of synthetic ermine heart Mitochondrial DNA adopt ABI3900 high-throughput synthesizer, and solid phase phosphoramidite three esters are synthetic;
(3) determine response procedures
Respectively with (a) identification reaction system of mink heart DNA detection kit, (b) positive control reaction system and (c) each 100 μ L of total system of negative control reaction system add in reaction tubes, place PCR reaction instrument to be undertaken by follow procedure:
Primer 1:94 ℃ of pre-sex change 3-7min, 94 ℃ of 30s, the 58 ℃ of 30s that anneal, 72 ℃ of 1min-30s, 40 circulations, 72 ℃ are extended 5-8min, 4 ℃;
Primer 2: 94 ℃ of pre-sex change 3-7min, 94 ℃ of 1min-30s, the 57 ℃ of 45s that anneal, 72 ℃ of 1min-30s, 35 circulations, 72 ℃ are extended 5-8min, 4 ℃;
(4) result judges
Reaction product is used the 1.5-1.8% sepharose, electrophoresis on DYY-III type horizontal strip electrophoresis instrument, and 60-70mV electrophoresis 45min-1h, the DNA Marker of molecular weight 100-1500bp does reference, and gel imaging analysis systematic observation and analysis are obtained a result.
Ermine heart identification kit of the present invention is the species specificity that utilizes ermine heart Mitochondrial DNA and cytochrome b to have, and can accurately distinguish the specificity of the multiple animal hearts that easily mixes with the ermine heart simultaneously; Advantage such as that authentication method has is easy, quick, detected result is reliable.
Description of drawings
Fig. 1 detects the ermine heart for primer 1 and heart, the duck heart, the goose heart and rabbit hearty cord are really schemed.
Fig. 2 detects the ermine heart for primer 2 and heart, the duck heart, the goose heart and rabbit hearty cord are really schemed.
Among Fig. 1: 1 negative result, 2 is standard molecular weight, 3 positive contrasts, is 430bp, 4 is sample results to be checked;
Among Fig. 2: 5 positive contrasts, be 580bp, 6 is standard molecular weight, and 7 is sample results to be checked, 8 negative results.
Embodiment
Utilize embodiment that the present invention is further described below.
A kind of mink heart DNA detection kit, it comprises:
(a) identification reaction system: totally be 100 μ L, wherein contain damping fluid 10 μ L, 12.5mMdNTP 4-10 μ L, 0.1mM a kind of 1.5-4.0 μ L in primer 1 and the primer 2, Taq archaeal dna polymerase 2-10 μ L, sample DNA 2-5 μ L to be checked surplusly mends to 100 μ L for bi-distilled water;
(b) positive control reaction system: totally be 100 μ L, wherein contain damping fluid 10 μ L, 12.5mM dNTP 4-10 μ L, 0.1mM a kind of 1.5-4.0 μ L in primer 1 and the primer 2, Taq archaeal dna polymerase 2-10 μ L, mink heart DNA 2-5 μ L surplusly mends to 100 μ L for bi-distilled water;
(c) negative control reaction system: totally be 100 μ L, wherein contain damping fluid 10 μ L, 12.5mM dNTP 4-10 μ L, 0.1mM a kind of 1.5-4.0 μ L in primer 1 and the primer 2, Taq archaeal dna polymerase 2-10 μ L, heart, the duck heart, the goose heart and rabbit heart DNA be by the mixture 2-5 μ L that mix to constitute at 1: 1, surplus for the bi-distilled water benefit to 100 μ L.
The used damping fluid of identification reaction system, positive control reaction system and negative control reaction system is an available reagent, and damping fluid contains 500mM KCl, 100mM Tri s-HCl (PH8.4), 15mM MgCl
2, market is on sale; 12.5mM dNTP is the 12.5mM deoxynucleoside triphosphate, market is on sale; The Taq archaeal dna polymerase, market is on sale.
The mink heart DNA authentication method, it comprises following step:
(1) design ermine heart Mitochondrial DNA two pairs of special Oligonucleolide primers require only to contain the mink heart DNA sequence, do not contain heart, the duck heart, the goose heart and rabbit heart dna sequence dna, a kind of in following primer 1 and the primer 2:
5’ATGACCAACATTCGTAAAACTC 3’
5’CAGATTCACTCTACAAGACT 3’
5’GGAACCAGTCTTGTAGAGTG 3’
5’TCTTCATTTTAATAGGTTAT 3’
(2) two pairs of special Oligonucleolide primers of synthetic ermine heart Mitochondrial DNA adopt ABI3900 high-throughput synthesizer, and solid phase phosphoramidite three esters are synthetic.Solid phase phosphoramidite three esters are synthetic by Shanghai bio-engineering corporation, for market on sale.
(3) determine response procedures
Respectively with (a) identification reaction system of mink heart DNA detection kit, (b) positive control reaction system and (c) each 100 μ L of total system of negative control reaction system add in the 500 μ L reaction tubess, place PCR reaction instrument to be undertaken by follow procedure:
Primer 1:94 ℃ of pre-sex change 3-7min, 94 ℃ of 30s, the 58 ℃ of 30s that anneal, 72 ℃ of 1min-30s, 40 circulations, 72 ℃ are extended 5-8min, 4 ℃;
Primer 2: 94 ℃ of pre-sex change 3-7min, 94 ℃ of 1min-30s, the 57 ℃ of 45s that anneal, 72 ℃ of 1min-30s, 35 circulations, 72 ℃ are extended 5-8min, 4 ℃;
(4) result judges
Reaction product is used the 1.5-1.8% sepharose, electrophoresis on DYY-III type horizontal strip electrophoresis instrument, and 60-70mV electrophoresis 45min-1h, the DNA Marker of molecular weight 100-1500bp does reference, and gel imaging analysis systematic observation and analysis are obtained a result.
The 1.5-1.8% sepharose is that market DYY-III type on sale horizontal strip electrophoresis instrument is that Beijing Liuyi Instrument Factory makes; The DNA Marker of molecular weight 100-1500bp is a standard molecular weight, and market is on sale; The gel imaging analysis system is that market is on sale.
With reference to Fig. 1,, have only the ermine heart positive findings to occur for primer 1 detects the ermine heart and heart, the duck heart, the goose heart and rabbit hearty cord fruit.
For primer 2 detects the ermine heart and heart, the duck heart, the goose heart and rabbit hearty cord fruit, have only the ermine heart positive findings to occur with reference to Fig. 2.
Claims (2)
1. mink heart DNA detection kit is characterized in that it comprises:
(a) identification reaction system: totally be 100 μ L, wherein contain damping fluid 10 μ L, 12.5mMdNTP 4-10 μ L, 0.1mM 5 ' ATGACCAACATTCGTAAAACTC 3 ' of primer 1 and 5 ' CAGATTCACTCTACAAGACT 3 ' are 1.5-4.0 μ L, Taq archaeal dna polymerase 2-10 μ L, sample DNA 2-5 μ L to be checked, surplus is bi-distilled water;
(b) positive control reaction system: totally be 100 μ L, wherein contain damping fluid 10 μ L, 12.5mM dNTP4-10 μ L, 0.1mM 5 ' ATGACCAACATTCGTAAAACTC 3 ' of primer 1 and 5 ' CAGATTCACTCTACAAGACT 3 ' are 1.5-4.0 μ L, Taq archaeal dna polymerase 2-10 μ L, mink heart DNA 2-5 μ L, surplus is bi-distilled water;
(c) negative control reaction system: total system is 100 μ L, wherein contain damping fluid 10 μ L, 12.5dNTP 4-10 μ L, 0.1mM 5 ' ATGACCAACATTCGTAAAACTC 3 ' of primer 1 and 5 ' CAGATTCACTCTACAAGACT3 ' are 1.5-4.0 μ L, Taq archaeal dna polymerase 2-10 μ L, heart, the duck heart, the goose heart and rabbit heart DNA mixed 2-5 μ L by 1: 1, and surplus is bi-distilled water.
2. a mink heart DNA authentication method is characterized in that, it comprises following step:
(1) a pair of special Oligonucleolide primers of design ermine heart Mitochondrial DNA, contain the mink heart DNA sequence, following primer 1:
5’ATGACCAACATTCGTAAAACTC?3’
5’CAGATTCACTCTACAAGACT 3’
(2) a pair of special Oligonucleolide primers of synthetic ermine heart Mitochondrial DNA adopts ABI3900 high-throughput synthesizer, and solid phase phosphoramidite three esters are synthetic;
(3) determine response procedures
Respectively with (a) identification reaction system of the described mink heart DNA detection kit of claim 1, (b) positive control reaction system and (c) each 100 μ L of total system of negative control reaction system add in reaction tubes, place PCR reaction instrument to be undertaken by follow procedure:
Primer 1:94 ℃ of pre-sex change 3-7min, 94 ℃ of 30s, the 58 ℃ of 30s that anneal, 72 ℃ of 1min-30s, 40 circulations, 72 ℃ are extended 5-8min, 4 ℃;
(4) result judges
Reaction product is used the 1.5-1.8% sepharose, electrophoresis on DYY-III type horizontal strip electrophoresis instrument, and 60-70mV electrophoresis 45min-1h, the DNA Marker of molecular weight 100-1500bp does reference, and gel imaging analysis systematic observation and analysis are obtained a result.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2008100516433A CN101434990B (en) | 2008-12-15 | 2008-12-15 | Mink heart DNA detection kit and identification method |
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN2008100516433A CN101434990B (en) | 2008-12-15 | 2008-12-15 | Mink heart DNA detection kit and identification method |
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| Publication Number | Publication Date |
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| CN101434990A CN101434990A (en) | 2009-05-20 |
| CN101434990B true CN101434990B (en) | 2011-03-23 |
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Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101792816B (en) * | 2010-04-20 | 2012-01-11 | 东北林业大学 | Primer for amplifying marten species cytochrome b gene and method for identifying sable and pine marten |
| CN102643912B (en) * | 2012-04-11 | 2014-01-08 | 中国农业科学院特产研究所 | Amplification primer for detecting mink derived ingredients |
| CN104032008B (en) * | 2014-06-12 | 2016-04-13 | 中国动物疫病预防控制中心 | The PCR primer pair of qualification or assistant identification recoon dog tissue and/or organ and application thereof |
| CN105296477B (en) * | 2015-11-20 | 2018-10-30 | 华中农业大学 | Mink in mink source Components identification and animal product, rabbit, dog ingredient multiple PCR detection kit |
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2008
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Non-Patent Citations (4)
| Title |
|---|
| 张丽华 等.<水貂心肌线粒体mtDNA鉴定及RFLP特征>.<遗传学进步与人口健康高峰论坛论文集>.2007,全文. |
| 张丽华 等.<貂组织线粒体DNA及细胞色素b鉴定及特征>.<吉林大学学报>.2008,第34卷(第5期),全文. |
| 张丽华等.<水貂心肌线粒体mtDNA鉴定及RFLP特征>.<遗传学进步与人口健康高峰论坛论文集>.2007,全文. * |
| 张丽华等.<貂组织线粒体DNA及细胞色素b鉴定及特征>.<吉林大学学报>.2008,第34卷(第5期),全文. * |
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Granted publication date: 20110323 Termination date: 20121215 |