CN102191309A - Bupleurum DNA (deoxyribonucleic acid) identifying kit and identifying method - Google Patents
Bupleurum DNA (deoxyribonucleic acid) identifying kit and identifying method Download PDFInfo
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Abstract
The invention relates to the technical field of traditional Chinese medicine detection, in particular to a bupleurum DNA (deoxyribonucleic acid) identifying kit and identifying method. The identifying kit comprises a DNA extraction reagent, a bupleurum identifying system and a bupleurum scorzonerifolium willd identifying system. The bupleurum DNA identifying method comprises the steps of: designing two pairs of specific oligonucleotide primers of the bupleurum ribosomal DNA, artificially synthesizing two pairs of primers, establishing a bupleurum DNA identifying kit, determining a reaction process, judging the result, and the like. The bupleurum DNA identifying kit and identifying method can be used for identifying and detecting traditional Chinese medicines, the bupleurum DNA identifying kit can accurately identify the specificity of bupleurum and can simultaneously identify the bupleurum, the bupleurum scorzonerifolium willd and confusing species thereof, and the identifying method has the advantages of simplicity, quickness and the like.
Description
Technical field
The present invention relates to use biotechnology to identify that radix bupleuri (radix bupleuri and Radix Bupeuri Scorzonerfolii.) and its easily mix kind DNA identification kit and authentication method.
Background technology
Radix bupleuri is umbelliferae Bupleurum per nnial herb, with root and all herbal medicine, is one of large Chinese medicinal materials in short supply of China, and price is high.Function deliver and in, dispersing the stagnated live-QI to relieve the stagnation of QI, ascending spleen-QI etc., be clinical commonly used among medicine.36 kinds of the total Bupleurum plants of China, 17 mutation, 7 modification, the Pharmacopoeia of the People's Republic of China (2005 editions) only stipulates that radix bupleuri (Bupleurum chinense DC.) and Radix Bupeuri Scorzonerfolii. (Bupleurumscorzonerifolium Willd.) are that certified products is medicinal for two kinds, but the kind of using with on the market has tens kinds.
Particularly, make some pseudo-product and adulterant occur in a large number, seriously confused normal market order, endangered patient's body health, even caused the generation of intoxication accident in recent years because resource seriously reduces, and price is held ascendant trend always.
Radix bupleuri discrimination method commonly used has medicinal material character identification, microscopical identification and chemical differential method.Character identification is meant that mainly macrofeatures such as profile from radix bupleuri, size, color, epidermis feature, quality, cut surface character, smell judge kind.Though method is simple, the influence of experience in this method is very big, subjective.Even carried out very detailed character description, judge also difficult again by other people.And normally incomplete as configuration of medicinal materials, therefore identify that difficulty is bigger.
At present existing scholar has worked out radix bupleuri tissue and powder key, for the microscopical identification of radix bupleuri provides certain foundation, but the powder of Bupleurum plant does not have too big difference, the quantity of the having or not of the thickness of the shape of fiber, length, wall, starch small grain, pitted vessel etc. just, only have relative discriminating meaning, can't accurately identify the true and false.
It mainly is the qualitative reaction of saikoside a, d that chemistry is differentiated, but the kind of most of radix bupleuri all contains saikoside, and characteristic is not strong, thereby only saikoside a, d is carried out qualitative reaction, is difficult to reach the purpose of quality of medicinal material control.
RDNA (rDNA) sequence has been used for the research of species classification and genetic evolution relation more and more because of the clock of high conservative on its function and evolution.Ribosomal gene transcribed spacer (ITS) sequence is present in the height multiple rrna, and rate of evolution is fast and length is little, adds that coevolution makes this fragment very consistent between the genome different units, thereby is fit to carry out various molecule manipulations.Because this zone is subjected to the influence of external environment factor less, rate of evolution is very fast, thereby the little and big characteristic of variation between planting of variation in having kind, be that kind is identified and the important molecular markers of Phylogenetic Analysis, can be used for classification, phylogeny and the Genetic Diversity of biology.
Though at present existing investigator explores and differentiates radix bupleuri with dna molecular marker, does not all obtain ideal results.Major part is that radix bupleuri tender tissue (mostly being fresh or dry blade) is studied, and its medicinal part is the dried root of medicinal material that contains a large amount of secondary metabolites, and usually through the processing and the process of preparing Chinese medicine, achievement in research lacks practicality; And part does not obtain the distinctive molecule marker of radix bupleuri on the one hand at the research of the dried root of radix bupleuri medicinal material, and method is also unstable.
Summary of the invention
The objective of the invention is the full gene group of grasping radix bupleuri and Radix Bupeuri Scorzonerfolii. rDNA is screened, the specificity that can accurately differentiate radix bupleuri is provided, can identify the identification kit of radix bupleuri, Radix Bupeuri Scorzonerfolii. and its easily mixed kind simultaneously; Another object of the present invention provides easy, quick, the reliable radix bupleuri DNA of detected result authentication method.
The objective of the invention is to realize by following technical scheme:
A kind of radix bupleuri DNA identification kit is characterized in that it comprises:
(1) DNA extraction reagent: total system 1000 μ l, 2%CTAB, 100mmol/L Tris-HCl, 30mmol/L EDTA, 2.5mmol/L NaCI, 3%PVP-40;
(2) radix bupleuri identification systems, comprise radix bupleuri identification system, radix bupleuri positive control system and radix bupleuri negative control system, described radix bupleuri identification system: totally be 100 μ l, contain damping fluid 10 μ l, 12.5mM dNTP 4-10 μ l, 0.1mM " primer 1 " 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; Described radix bupleuri positive control system: totally be 100 μ l, contain damping fluid 10 μ l, 12.5mM dNTP 4-10 μ l, 0.1mM " primer 1 " 1.5-4.0 μ l, Taq archaeal dna polymerase 2-10 μ l, radix bupleuri DNA 2-5 μ l, surplus is bi-distilled water; Described radix bupleuri negative control system: total system is 100 μ l, contains damping fluid 10 μ l, 12.5mM dNTP 4-10 μ l, " 0.1mM primer 1 " 1.5-4.0 μ l, Taq archaeal dna polymerase 2-10 μ l, bigleaf thorowax root, black radix bupleuri DNA were by 1: 1 mixture 2-5 μ l, and surplus is bi-distilled water;
(3) Radix Bupeuri Scorzonerfolii. identification systems, comprise Radix Bupeuri Scorzonerfolii. identification system, Radix Bupeuri Scorzonerfolii. positive control system and Radix Bupeuri Scorzonerfolii. negative control system, described Radix Bupeuri Scorzonerfolii. identification system: totally be 100 μ l, contain damping fluid 10 μ l, 12.5mM dNTP 4-10 μ l, 0.1mM " primer 2 " 1.5-4.0 μ l, TaqDNA polysaccharase 2-10 μ l, sample DNA 2-5 μ l to be checked, surplus is bi-distilled water; Described Radix Bupeuri Scorzonerfolii. positive control system: totally be 100 μ l, contain damping fluid 10 μ l, 12.5mM dNTP 4-10 μ l, 0.1mM " primer 2 " 1.5-4.0 μ l, Taq archaeal dna polymerase 2-10 μ l, Radix Bupeuri Scorzonerfolii. DNA 2-5 μ l, surplus is bi-distilled water; Described Radix Bupeuri Scorzonerfolii. negative control system: total system is 100 μ l, contains damping fluid 10 μ l, 12.5mM dNTP 4-10 μ l, " 0.1mM primer 2 " 1.5-4.0 μ l, TaqDNA polysaccharase 2-10 μ l, bigleaf thorowax root, black radix bupleuri DNA were by 1: 1 mixture 2-5 μ l, and surplus is bi-distilled water.
A kind of radix bupleuri DNA authentication method is characterized in that it comprises following steps:
(1) two pairs of special Oligonucleolide primers of design radix bupleuri rDNA require only to contain radix bupleuri and Radix Bupeuri Scorzonerfolii. dna sequence dna, do not contain to belong to dna sequence dna together, following primer 1, primer 2:
5’-ATGACCAACATTCGTAAACTC-3’
5’-TCTTCATTTTAATAGGTTATA-3’
5’-AGACACATTCGTAACTC-3’
5’-CTCATTTAATAGGTATA-3’
(2) two pairs of special Oligonucleolide primers of synthetic radix bupleuri rDNA adopt ABI3900 high-throughput synthesizer, the solid phase phosphoramidite triester method;
(3) set up radix bupleuri DNA identification kit
(a) DNA extraction reagent: totally be 1000 μ l, 2%CTAB, 100mmol/L Tris-HCl, 30mmol/L EDTA, 2.5mmol/L NaCI, 3%PVP-40;
(b) radix bupleuri identification systems, comprise radix bupleuri identification system, radix bupleuri positive control system and radix bupleuri negative control system, described radix bupleuri identification system: totally be 100 μ l, contain damping fluid 10 μ l, 12.5mM dNTP 4-10 μ l, 0.1mM " primer 1 " 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; Described radix bupleuri positive control system: totally be 100 μ l, contain damping fluid 10 μ l, 12.5mM dNTP 4-10 μ l, 0.1mM " primer 1 " 1.5-4.0 μ l, Taq archaeal dna polymerase 2-10 μ l, radix bupleuri DNA 2-5 μ l, surplus is bi-distilled water; Described radix bupleuri negative control system: total system is 100 μ l, contains damping fluid 10 μ l, 12.5mM dNTP 4-10 μ l, " 0.1mM primer 1 " 1.5-4.0 μ l, Taq archaeal dna polymerase 2-10 μ l, bigleaf thorowax root, black radix bupleuri DNA were by 1: 1 mixture 2-5 μ l, and surplus is bi-distilled water;
(C) Radix Bupeuri Scorzonerfolii. identification systems, comprise Radix Bupeuri Scorzonerfolii. identification system, Radix Bupeuri Scorzonerfolii. positive control system and Radix Bupeuri Scorzonerfolii. negative control system, described Radix Bupeuri Scorzonerfolii. identification system: totally be 100 μ l, contain damping fluid 10 μ l, 12.5mM dNTP 4-10 μ l, 0.1mM " primer 2 " 1.5-4.0 μ l, TaqDNA polysaccharase 2-10 μ l, sample DNA 2-5 μ l to be checked, surplus is bi-distilled water; Described Radix Bupeuri Scorzonerfolii. positive control system: totally be 100 μ l, contain damping fluid 10 μ l, 12.5mM dNTP 4-10 μ l, 0.1mM " primer 2 " 1.5-4.0 μ l, Taq archaeal dna polymerase 2-10 μ l, Radix Bupeuri Scorzonerfolii. DNA 2-5 μ l, surplus is bi-distilled water; Described Radix Bupeuri Scorzonerfolii. negative control system: total system is 100 μ l, contains damping fluid 10 μ l, 12.5mM dNTP 4-10 μ l, " 0.1mM primer 2 " 1.5-4.0 μ l, TaqDNA polysaccharase 2-10 μ l, bigleaf thorowax root, black radix bupleuri DNA were by 1: 1 mixture 2-5 μ l, and surplus is bi-distilled water;
(4) design response procedures
All sample standard deviation to be checked DNA extraction agent treated: get sample 0.1g to be checked, clean, dry up, shred, the dried root of medicinal material removes epidermis earlier, place mortar, the DNA extraction reagent 250ul that adds 65 ℃ of preheatings, in liquid nitrogen, grind, grind liquid and change 65 ℃ of temperature baths of centrifuge tube 1h over to, 4 ℃ of centrifugal 10min of 12000rpm, supernatant liquor changes centrifuge tube over to, add chloroform-primary isoamyl alcohol 250ul of 24: 1, the centrifugal 10min of room temperature 8000rpm, supernatant liquor changes centrifuge tube over to, the Virahol 200ul that adds-20 ℃ of precoolings,-20 ℃ of freezing 1h, 4 ℃ of centrifugal 5min of 12000rpm, abandon supernatant liquor, dry, adding 70% ethanol 200ul cleans, the centrifugal 3min of room temperature 12000rpm, discard ethanol, add 50ulTE or the distilled water of pH8.0 after the drying, it is standby to put into-20 ℃ of refrigerators, radix bupleuri identification system with test kit, the Radix Bupeuri Scorzonerfolii. identification system, radix bupleuri positive control system, Radix Bupeuri Scorzonerfolii. positive control system, each 100 μ L of radix bupleuri negative control system and Radix Bupeuri Scorzonerfolii. negative control system add in the reaction tubes, place PCR reaction instrument to be undertaken: the radix bupleuri identification system by follow procedure, radix bupleuri positive control system, radix bupleuri negative control system: 94 ℃ of pre-sex change 5min, 94 ℃ of 30s, 58 ℃ of 30s of annealing temperature, 72 ℃ of 45s, 32 circulations, 72 ℃ are extended 8min, 4 ℃; Radix Bupeuri Scorzonerfolii. identification system, Radix Bupeuri Scorzonerfolii. positive control system, Radix Bupeuri Scorzonerfolii. negative control system: 94 ℃ of pre-sex change 5min, 94 ℃ of 30s, 56 ℃ of 30s of annealing temperature, 72 ℃ of 45s, 32 circulations, 72 ℃ are extended 8min, 4 ℃;
(5) result judges
Reaction product is used the 1.5-1.8% sepharose, electrophoresis on the horizontal strip electrophoresis instrument, and 60-70mV electrophoresis 45-60min, the DNA Marker of molecular weight 100-1200bp does reference, and the gel imaging analysis systematic observation is obtained a result.
Radix bupleuri identification kit of the present invention is compared with existing radix bupleuri authentication method with authentication method, the present invention is utilizing radix bupleuri DNA to have on the basis of species specificity, invent test kit and the authentication method of distinguishing radix bupleuri, Radix Bupeuri Scorzonerfolii. and its easily mixed kind simultaneously, had advantages such as easy, quick.
Description of drawings
Accompanying drawing is that test kit detects radix bupleuri, Radix Bupeuri Scorzonerfolii. and bigleaf thorowax root, black radix bupleuri result, and positive findings appears respectively in radix bupleuri and Radix Bupeuri Scorzonerfolii..
Wherein: 1 is standard molecular weight; 2 is the radix bupleuri positive control, is 300bp; 3 is the Radix Bupeuri Scorzonerfolii. positive control, is 220bp; 4 is the radix bupleuri result; 5 is the Radix Bupeuri Scorzonerfolii. result; 6 is the radix bupleuri negative control; 7 is the Radix Bupeuri Scorzonerfolii. negative control.
Embodiment
The invention will be further described to utilize embodiment below.
A kind of radix bupleuri DNA identification kit, it comprises:
(1) DNA extraction reagent: totally be 1000 μ l, 2%CTAB, 100mmol/L Tris-HCl, 30mmol/L EDTA, 2.5mmol/L NaCI, 3%PVP-40.
(2) radix bupleuri identification systems comprise three parts:
1. radix bupleuri identification system: totally be 100 μ l, contain damping fluid 10 μ l, 12.5mM dNTP 4-10 μ l, 0.1mM " primer 1 " 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.
2. radix bupleuri positive control system: totally be 100 μ l, contain damping fluid 10 μ l, 12.5mM dNTP4-10 μ l, 0.1mM " primer 1 " 1.5-4.0 μ l, Taq archaeal dna polymerase 2-10 μ l, radix bupleuri DNA2-5 μ l, surplus is bi-distilled water.
3. radix bupleuri negative control system: total system is 100 μ l, contains damping fluid 10 μ l, 12.5mMdNTP 4-10 μ l, " 0.1mM primer 1 " 1.5-4.0 μ l, Taq archaeal dna polymerase 2-10 μ l, bigleaf thorowax root, black radix bupleuri DNA were by 1: 1 mixture 2-5 μ l, and surplus is bi-distilled water.
(3) Radix Bupeuri Scorzonerfolii. identification systems comprise three parts:
1. Radix Bupeuri Scorzonerfolii. identification system: totally be 100 μ l, contain damping fluid 10 μ l, 12.5mM dNTP4-10 μ l, 0.1mM " primer 2 " 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.
2. Radix Bupeuri Scorzonerfolii. positive control system: totally be 100 μ l, contain damping fluid 10 μ l, 12.5mMdNTP 4-10 μ l, 0.1mM " primer 2 " 1.5-4.0 μ l, Taq archaeal dna polymerase 2-10 μ l, Radix Bupeuri Scorzonerfolii. DNA 2-5 μ l, surplus is bi-distilled water.
3. Radix Bupeuri Scorzonerfolii. negative control system: total system is 100 μ l, contains damping fluid 10 μ l, 12.5mM dNTP 4-10 μ l, " 0.1mM primer 2 " 1.5-4.0 μ l, Taq archaeal dna polymerase 2-10 μ l, bigleaf thorowax root, black radix bupleuri DNA were by 1: 1 mixture 2-5 μ l, and surplus is bi-distilled water.
A kind of radix bupleuri DNA authentication method, it comprises following steps:
(1) two pairs of special Oligonucleolide primers of design radix bupleuri rDNA require only to contain radix bupleuri and Radix Bupeuri Scorzonerfolii. dna sequence dna, do not contain to belong to dna sequence dna together, following primer 1, primer 2:
5’-ATGACCAACATTCGTAAACTC-3’
5’-TCTTCATTTTAATAGGTTATA-3’
5’-AGACACATTCGTAACTC-3’
5’-CTCATTTAATAGGTATA-3’
(2) two pairs of special Oligonucleolide primers of synthetic radix bupleuri rDNA adopt ABI3900 high-throughput synthesizer, the solid phase phosphoramidite triester method;
(3) set up radix bupleuri DNA identification kit
(a) DNA extraction reagent: totally be 1000 μ l, 2%CTAB, 100mmol/L Tris-HCl, 30mmol/L EDTA, 2.5mmol/L NaCI, 3%PVP-40.
(b) radix bupleuri identification systems comprise three parts:
1. radix bupleuri identification system: totally be 100 μ l, contain damping fluid 10 μ l, 12.5mM dNTP 4-10 μ l, 0.1mM " primer 1 " 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.
2. radix bupleuri positive control system: totally be 100 μ l, contain damping fluid 10 μ l, 12.5mM dNTP4-10 μ l, 0.1mM " primer 1 " 1.5-4.0 μ l, Taq archaeal dna polymerase 2-10 μ l, radix bupleuri DNA2-5 μ l, surplus is bi-distilled water.
3. radix bupleuri negative control system: total system is 100 μ l, contains damping fluid 10 μ l, 12.5mMdNTP 4-10 μ l, " 0.1mM primer 1 " 1.5-4.0 μ l, Taq archaeal dna polymerase 2-10 μ l, bigleaf thorowax root, black radix bupleuri DNA were by 1: 1 mixture 2-5 μ l, and surplus is bi-distilled water.
(c) Radix Bupeuri Scorzonerfolii. identification systems comprise three parts:
1. Radix Bupeuri Scorzonerfolii. identification system: totally be 100 μ l, contain damping fluid 10 μ l, 12.5mM dNTP4-10 μ l, 0.1mM " primer 2 " 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.
2. Radix Bupeuri Scorzonerfolii. positive control system: totally be 100 μ l, contain damping fluid 10 μ l, 12.5mMdNTP 4-10 μ l, 0.1mM " primer 2 " 1.5-4.0 μ l, Taq archaeal dna polymerase 2-10 μ l, Radix Bupeuri Scorzonerfolii. DNA 2-5 μ l, surplus is bi-distilled water.
3. Radix Bupeuri Scorzonerfolii. negative control system: total system is 100 μ l, contains damping fluid 10 μ l, 12.5mM dNTP 4-10 μ l, " 0.1mM primer 2 " 1.5-4.0 μ l, Taq archaeal dna polymerase 2-10 μ l, bigleaf thorowax root, black radix bupleuri DNA were by 1: 1 mixture 2-5 μ l, and surplus is bi-distilled water.
(4) design response procedures
All sample standard deviation to be checked DNA extraction agent treated: get sample 0.1g to be checked, clean, dry up, shred, the dried root of medicinal material removes epidermis earlier, places mortar, adds the DNA extraction reagent 250ul of 65 ℃ of preheatings, grinds in liquid nitrogen.Grind liquid and change 65 ℃ of temperature baths of centrifuge tube 1h over to, 4 ℃ of centrifugal 10min of 12000rpm, supernatant liquor changes centrifuge tube over to, add chloroform-primary isoamyl alcohol 250ul of 24: 1, the centrifugal 10min of room temperature 8000rpm, supernatant liquor changes centrifuge tube over to, the Virahol 200ul that adds-20 ℃ of precoolings ,-20 ℃ of freezing 1h.4 ℃ of centrifugal 5min of 12000rpm abandon supernatant liquor, dry, and add 70% ethanol 200ul and clean, and the centrifugal 3min of room temperature 12000rpm discards ethanol.Adding pH8.0 after the drying is 50ulTE or distilled water, and it is standby to put into-20 ℃ of refrigerators.DNA extraction reagent and all kinds of SOLVENTS are the add-on when extracting the 0.1g sample, adjust in proportion according to the actual sample amount.
Each 100 μ L of radix bupleuri identification system, Radix Bupeuri Scorzonerfolii. identification system, radix bupleuri positive control system, Radix Bupeuri Scorzonerfolii. positive control system, radix bupleuri negative control system and Radix Bupeuri Scorzonerfolii. negative control system of test kit are added in the reaction tubes, place PCR reaction instrument to be undertaken by follow procedure:
Radix bupleuri identification system, radix bupleuri positive control system, radix bupleuri negative control system: 94 ℃ of pre-sex change 5min, 94 ℃ of 30s, 58 ℃ of 30s of annealing temperature, 72 ℃ of 45s, 32 circulations, 72 ℃ are extended 8min, 4 ℃.Radix Bupeuri Scorzonerfolii. identification system, Radix Bupeuri Scorzonerfolii. positive control system, Radix Bupeuri Scorzonerfolii. negative control system: 94 ℃ of pre-sex change 5min, 94 ℃ of 30s, 56 ℃ of 30s of annealing temperature, 72 ℃ of 45s, 32 circulations, 72 ℃ are extended 8min, 4 ℃.
(5) result judges
Reaction product is used the 1.5-1.8% sepharose, electrophoresis on the horizontal strip electrophoresis instrument, and 60-70mV electrophoresis 45-60min, the DNA Marker of molecular weight 100-1200bp does reference, and the gel imaging analysis systematic observation is obtained a result.The 1.5-1.8% sepharose, market is on sale; The commercially available instrument that the horizontal strip electrophoresis instrument is made by Beijing Liuyi Instrument Factory; The DNA Marker of molecular weight 100-1200bp is a standard molecular weight, and market is on sale; The gel imaging analysis system, market is on sale.
With reference to accompanying drawing, for test kit detects radix bupleuri and Radix Bupeuri Scorzonerfolii. result schematic diagram, positive findings appears respectively in radix bupleuri and Radix Bupeuri Scorzonerfolii..
Claims (2)
1. radix bupleuri DNA identification kit is characterized in that it comprises:
(1) DNA extraction reagent: total system 1000 μ l, 2%CTAB, 100mmol/L Tris-HCl, 30mmol/L EDTA, 2.5mmol/L NaCI, 3%PVP-40;
(2) radix bupleuri identification systems, comprise radix bupleuri identification system, radix bupleuri positive control system and radix bupleuri negative control system, described radix bupleuri identification system: totally be 100 μ l, contain damping fluid 10 μ l, 12.5mMdNTP 4-10 μ l, 0.1mM " primer 1 " 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; Described radix bupleuri positive control system: totally be 100 μ l, contain damping fluid 10 μ l, 12.5mM dNTP 4-10 μ l, 0.1mM " primer 1 " 1.5-4.0 μ l, Taq archaeal dna polymerase 2-10 μ l, radix bupleuri DNA 2-5 μ l, surplus is bi-distilled water; Described radix bupleuri negative control system: total system is 100 μ l, contains damping fluid 10 μ l, 12.5mM dNTP 4-10 μ l, " 0.1mM primer 1 " 1.5-4.0 μ l, Taq archaeal dna polymerase 2-10 μ l, bigleaf thorowax root, black radix bupleuri DNA were by 1: 1 mixture 2-5 μ l, and surplus is bi-distilled water;
(3) Radix Bupeuri Scorzonerfolii. identification systems, comprise Radix Bupeuri Scorzonerfolii. identification system, Radix Bupeuri Scorzonerfolii. positive control system and Radix Bupeuri Scorzonerfolii. negative control system, described Radix Bupeuri Scorzonerfolii. identification system: totally be 100 μ l, contain damping fluid 10 μ l, 12.5mM dNTP 4-10 μ l, 0.1mM " primer 2 " 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; Described Radix Bupeuri Scorzonerfolii. positive control system: totally be 100 μ l, contain damping fluid 10 μ l, 12.5mM dNTP 4-10 μ l, 0.1mM " primer 2 " 1.5-4.0 μ l, Taq archaeal dna polymerase 2-10 μ l, Radix Bupeuri Scorzonerfolii. DNA 2-5 μ l, surplus is bi-distilled water; Described Radix Bupeuri Scorzonerfolii. negative control system: total system is 100 μ l, contains damping fluid 10 μ l, 12.5mM dNTP 4-10 μ l, " 0.1mM primer 2 " 1.5-4.0 μ l, Taq archaeal dna polymerase 2-10 μ l, bigleaf thorowax root, black radix bupleuri DNA were by 1: 1 mixture 2-5 μ l, and surplus is bi-distilled water.
2. radix bupleuri DNA authentication method is characterized in that it comprises following steps:
(1) two pairs of special Oligonucleolide primers of design radix bupleuri rDNA require only to contain radix bupleuri and Radix Bupeuri Scorzonerfolii. dna sequence dna, do not contain to belong to dna sequence dna together, following primer 1, primer 2:
Primer 1 is the radix bupleuri primer:
5’-ATGACCAACATTCGTAAACTC-3’
5’-TCTTCATTTTAATAGGTTATA-3’
Primer 2 is the Radix Bupeuri Scorzonerfolii. primer:
5’-AGACACATTCGTAACTC-3’
5’-CTCATTTAATAGGTATA-3’
(2) two pairs of special Oligonucleolide primers of synthetic radix bupleuri rDNA adopt ABI3900 high-throughput synthesizer, the solid phase phosphoramidite triester method;
(3) set up radix bupleuri DNA identification kit
(a) DNA extraction reagent: totally be 1000 μ l, 2%CTAB, 100mmol/L Tris-HCl, 30mmol/L EDTA, 2.5mmol/L NaCI, 3%PVP-40;
(b) radix bupleuri identification systems, comprise radix bupleuri identification system, radix bupleuri positive control system and radix bupleuri negative control system, described radix bupleuri identification system: totally be 100 μ l, contain damping fluid 10 μ l, 12.5mMdNTP 4-10 μ l, 0.1mM " primer 1 " 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; Described radix bupleuri positive control system: totally be 100 μ l, contain damping fluid 10 μ l, 12.5mM dNTP 4-10 μ l, 0.1mM " primer 1 " 1.5-4.0 μ l, Taq archaeal dna polymerase 2-10 μ l, radix bupleuri DNA 2-5 μ l, surplus is bi-distilled water; Described radix bupleuri negative control system: total system is 100 μ l, contains damping fluid 10 μ l, 12.5mM dNTP 4-10 μ l, " 0.1mM primer 1 " 1.5-4.0 μ l, Taq archaeal dna polymerase 2-10 μ l, bigleaf thorowax root, black radix bupleuri DNA were by 1: 1 mixture 2-5 μ l, and surplus is bi-distilled water;
(C) Radix Bupeuri Scorzonerfolii. identification systems, comprise Radix Bupeuri Scorzonerfolii. identification system, Radix Bupeuri Scorzonerfolii. positive control system and Radix Bupeuri Scorzonerfolii. negative control system, described Radix Bupeuri Scorzonerfolii. identification system: totally be 100 μ l, contain damping fluid 10 μ l, 12.5mM dNTP4-10 μ l, 0.1mM " primer 2 " 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; Described Radix Bupeuri Scorzonerfolii. positive control system: totally be 100 μ l, contain damping fluid 10 μ l, 12.5mM dNTP 4-10 μ l, 0.1mM " primer 2 " 1.5-4.0 μ l, Taq archaeal dna polymerase 2-10 μ l, Radix Bupeuri Scorzonerfolii. DNA 2-5 μ l, surplus is bi-distilled water; Described Radix Bupeuri Scorzonerfolii. negative control system: total system is 100 μ l, contains damping fluid 10 μ l, 12.5mM dNTP 4-10 μ l, " 0.1mM primer 2 " 1.5-4.0 μ l, Taq archaeal dna polymerase 2-10 μ l, bigleaf thorowax root, black radix bupleuri DNA were by 1: 1 mixture 2-5 μ l, and surplus is bi-distilled water;
(4) design response procedures
All sample standard deviation to be checked DNA extraction agent treated: get sample 0.1g to be checked, clean, dry up, shred, the dried root of medicinal material removes epidermis earlier, place mortar, the DNA extraction reagent 250ul that adds 65 ℃ of preheatings, in liquid nitrogen, grind, grind liquid and change 65 ℃ of temperature baths of centrifuge tube 1h over to, 4 ℃ of centrifugal 10min of 12000rpm, supernatant liquor changes centrifuge tube over to, add chloroform-primary isoamyl alcohol 250ul of 24: 1, the centrifugal 10min of room temperature 8000rpm, supernatant liquor changes centrifuge tube over to, the Virahol 200ul that adds-20 ℃ of precoolings,-20 ℃ of freezing 1h, 4 ℃ of centrifugal 5min of 12000rpm, abandon supernatant liquor, dry, adding 70% ethanol 200ul cleans, the centrifugal 3min of room temperature 12000rpm, discard ethanol, add 50ulTE or the distilled water of pH8.0 after the drying, it is standby to put into-20 ℃ of refrigerators, radix bupleuri identification system with test kit, the Radix Bupeuri Scorzonerfolii. identification system, radix bupleuri positive control system, Radix Bupeuri Scorzonerfolii. positive control system, each 100 μ L of radix bupleuri negative control system and Radix Bupeuri Scorzonerfolii. negative control system add in the reaction tubes, place PCR reaction instrument to be undertaken: the radix bupleuri identification system by follow procedure, radix bupleuri positive control system, radix bupleuri negative control system: 94 ℃ of pre-sex change 5min, 94 ℃ of 30s, 58 ℃ of 30s of annealing temperature, 72 ℃ of 45s, 32 circulations, 72 ℃ are extended 8min, 4 ℃; Radix Bupeuri Scorzonerfolii. identification system, Radix Bupeuri Scorzonerfolii. positive control system, Radix Bupeuri Scorzonerfolii. negative control system: 94 ℃ of pre-sex change 5min, 94 ℃ of 30s, 56 ℃ of 30s of annealing temperature, 72 ℃ of 45s, 32 circulations, 72 ℃ are extended 8min, 4 ℃;
(5) result judges
Reaction product is used the 1.5-1.8% sepharose, electrophoresis on the horizontal strip electrophoresis instrument, and 60-70mV electrophoresis 45-60min, the DNA Marker of molecular weight 100-1200bp does reference, and the gel imaging analysis systematic observation is obtained a result.
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| CN103571956A (en) * | 2013-11-01 | 2014-02-12 | 山西大学 | Method for detecting bupleurum chinense and bupleurum scorzonerifolium by utilizing specific primer PCR (polymerase chain reaction) |
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| CN105238855A (en) * | 2015-09-18 | 2016-01-13 | 北华大学 | Kit and method for identifying cornu cervi DNA |
| CN107326069A (en) * | 2017-06-07 | 2017-11-07 | 苏州市李良济健康产业有限公司 | A kind of primer pair and its application for being used to identify radix bupleuri |
| CN114438248A (en) * | 2022-02-21 | 2022-05-06 | 广州白云山光华制药股份有限公司 | Method for identifying bupleurum falcatum and/or bupleurum stenophyllum based on SCAR molecular marker |
| CN114457183A (en) * | 2022-02-21 | 2022-05-10 | 广州白云山光华制药股份有限公司 | SCAR molecular marker, specific primer pair and method for identifying Xikangchui |
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| CN103911364A (en) * | 2013-01-07 | 2014-07-09 | 中国中医科学院中药研究所 | Special herb PCR reaction strengthening agent |
| CN103571956A (en) * | 2013-11-01 | 2014-02-12 | 山西大学 | Method for detecting bupleurum chinense and bupleurum scorzonerifolium by utilizing specific primer PCR (polymerase chain reaction) |
| CN105238855A (en) * | 2015-09-18 | 2016-01-13 | 北华大学 | Kit and method for identifying cornu cervi DNA |
| CN107326069A (en) * | 2017-06-07 | 2017-11-07 | 苏州市李良济健康产业有限公司 | A kind of primer pair and its application for being used to identify radix bupleuri |
| CN114438248A (en) * | 2022-02-21 | 2022-05-06 | 广州白云山光华制药股份有限公司 | Method for identifying bupleurum falcatum and/or bupleurum stenophyllum based on SCAR molecular marker |
| CN114457183A (en) * | 2022-02-21 | 2022-05-10 | 广州白云山光华制药股份有限公司 | SCAR molecular marker, specific primer pair and method for identifying Xikangchui |
| CN114438248B (en) * | 2022-02-21 | 2024-04-16 | 广州白云山光华制药股份有限公司 | Method for identifying bupleurum chinense and/or bupleurum tenuifolia based on SCAR molecular marker |
| CN114457183B (en) * | 2022-02-21 | 2024-04-16 | 广州白云山光华制药股份有限公司 | SCAR molecular marker for identifying Western Kang Chaihu, specific primer pair and method |
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