CN102191309B - 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 the easily mixed kind DNA identification kit of radix bupleuri (radix bupleuri and Radix Bupeuri Scorzonerfolii.) and its and authentication method.
Background technology
Radix bupleuri is umbelliferae Bupleurum per nnial herb, and with one of large Chinese medicinal materials in short supply of root and all herbal medicine ，Shi China, price is high.Function is delivered with inner, dispersing the stagnated live-QI to relieve the stagnation of QI, and ascending spleen-QIs etc., are medicine among clinical commonly using.36 kinds of the total Bupleurum plants of China, 17 mutation, 7 modification, < < Pharmacopoeia of People's Republic of China > > (2005 editions) only stipulates that two kinds of radix bupleuri (Bupleurum chinense DC.) and Radix Bupeuri Scorzonerfolii.s (Bupleurumscorzonerifolium Willd.) are medicinal for certified products, but mixed kind on market has tens kinds.
Particularly, in recent years because resource seriously reduces, price is held ascendant trend always, and some adulterants and adulterant are occurred in a large number, has seriously confused normal market order, has endangered the healthy of patient, even causes the generation of intoxication accident.
Conventional radix bupleuri discrimination method has Medicinal Materials Characters discriminating, microscopical identification and chemical identification method.Character identification mainly refers to that the macrofeatures such as profile from radix bupleuri, size, color, epidermis characteristic, quality, cut surface character, smell judge kind.Although method is simple, the impact of experience in this method is very large, subjective.Even if carried out very detailed character description, then by other people, judged also more difficult.And normally incomplete as configuration of medicinal materials, therefore identify that difficulty is larger.
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 large 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 there is relative discriminating meaning, cannot the precise Identification true and false.
It is mainly 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 to qualitative reaction, is difficult to reach the object that quality of medicinal material is controlled.
RDNA (rDNA) sequence is because of the clock of the high conservative in its function and evolution, by more and more for the research of species taxonomy and genetic evolution relation.Internal Transcribed Spacer (ITS) sequence is present in the rrna highly repeating, and rate of evolution is fast and length is little, adds that coevolution makes this fragment very consistent between genome different units, thereby is applicable to carrying out various molecule manipulations.Because this region is subject to the impact of external environment factor less, rate of evolution is very fast, thereby in having kind, variation is little and the large characteristic that makes a variation between planting is the important molecular markers of Identification of Species and Phylogenetic Analysis, can be used for the research of biological classification, phylogeny and genetic diversity.
Though at present existing investigator explores and differentiates radix bupleuri with DNA molecular marker, does not all obtain desirable result.Major part is that radix bupleuri tender tissue (mostly being fresh or dry blade) is studied, and its medicinal part is the dry root of medicinal material that contains a large amount of secondary metabolites, and conventionally through 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 for the research of the dry root of Radix Bupleuri, and method is also unstable.
Summary of the invention
The object of the invention is to screen grasping the full gene group of radix bupleuri and Radix Bupeuri Scorzonerfolii. rDNA, 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 is to provide easy, quick, the reliable radix bupleuri DNA of detected result authentication method.
The object of the invention is to be realized by following technical scheme:
A 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: be totally 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, remaining is bi-distilled water; Described radix bupleuri positive control system: be totally 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, remaining 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 remaining 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: be totally 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, remaining is bi-distilled water; Described Radix Bupeuri Scorzonerfolii. positive control system: be totally 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, remaining 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 remaining is bi-distilled water.
A 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 and belong to DNA sequence dna together, following primer 1, primer 2:
Primer 1 is radix bupleuri primer:
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, solid phase phosphoramidite triester method;
(3) set up radix bupleuri DNA identification kit
(a) DNA extraction reagent: be totally 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: be totally 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, remaining is bi-distilled water; Described radix bupleuri positive control system: be totally 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, remaining 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 remaining 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: be totally 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, remaining is bi-distilled water; Described Radix Bupeuri Scorzonerfolii. positive control system: be totally 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, remaining 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 remaining 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 dry root of medicinal material is first except epidermis, be placed in mortar, the DNA extraction reagent 250ul that adds 65 ℃ of preheatings, in liquid nitrogen, grind, grind liquid and proceed to 65 ℃ of temperature bath 1h of centrifuge tube, 4 ℃ of centrifugal 10min of 12000rpm, supernatant liquor proceeds to centrifuge tube, add chloroform-primary isoamyl alcohol 250ul of 24: 1, the centrifugal 10min of room temperature 8000rpm, supernatant liquor proceeds to centrifuge tube, 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, the 50ulTE or the distilled water that after dry, add pH8.0, put into-20 ℃ of refrigerators standby, by the radix bupleuri identification system of test kit, 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 reaction tubes, being placed in PCR reaction instrument is undertaken by follow procedure: radix bupleuri identification system, radix bupleuri positive control system, radix bupleuri negative control system: 94 ℃ of denaturation 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 denaturation 5min, 94 ℃ of 30s, 56 ℃ of 30s of annealing temperature, 72 ℃ of 45s, 32 circulations, 72 ℃ are extended 8min, 4 ℃,
(5) result is judged
Reaction product is used 1.5-1.8% sepharose, electrophoresis on horizontal cataphoresis apparatus, and 60-70mV electrophoresis 45-60min, the DNA Marker of molecular weight 100-1200bp does reference, and 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, there is the advantages such as easy, quick.
Accompanying drawing explanation
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 radix bupleuri positive control, is 300bp; 3 is Radix Bupeuri Scorzonerfolii. positive control, is 220bp; 4 is radix bupleuri result; 5 is Radix Bupeuri Scorzonerfolii. result; 6 is radix bupleuri negative control; 7 is Radix Bupeuri Scorzonerfolii. negative control.
Embodiment
The invention will be further described to utilize embodiment below.
A DNA identification kit, it comprises:
(1) DNA extraction reagent: be totally 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: be totally 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, remaining is bi-distilled water.
2. radix bupleuri positive control system: be totally 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, remaining 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 remaining is bi-distilled water.
(3) Radix Bupeuri Scorzonerfolii. identification systems, comprise three parts:
1. Radix Bupeuri Scorzonerfolii. identification system: be totally 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, remaining is bi-distilled water.
2. Radix Bupeuri Scorzonerfolii. positive control system: be totally 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, remaining 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 remaining is bi-distilled water.
A 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 and belong to DNA sequence dna together, following primer 1, primer 2:
Primer 1 is radix bupleuri primer:
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, solid phase phosphoramidite triester method;
(3) set up radix bupleuri DNA identification kit
(a) DNA extraction reagent: be totally 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: be totally 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, remaining is bi-distilled water.
2. radix bupleuri positive control system: be totally 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, remaining 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 remaining is bi-distilled water.
(c) Radix Bupeuri Scorzonerfolii. identification systems, comprise three parts:
1. Radix Bupeuri Scorzonerfolii. identification system: be totally 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, remaining is bi-distilled water.
2. Radix Bupeuri Scorzonerfolii. positive control system: be totally 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, remaining 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 remaining 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 dry root of medicinal material first, except epidermis, is placed in mortar, adds the DNA extraction reagent 250ul of 65 ℃ of preheatings, grinds in liquid nitrogen.Grind liquid and proceed to 65 ℃ of temperature bath 1h of centrifuge tube, 4 ℃ of centrifugal 10min of 12000rpm, supernatant liquor proceeds to centrifuge tube, add chloroform-primary isoamyl alcohol 250ul of 24: 1, the centrifugal 10min of room temperature 8000rpm, supernatant liquor proceeds to centrifuge tube, 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.After dry, adding pH8.0 is 50ulTE or distilled water, puts into-20 ℃ of refrigerators standby.DNA extraction reagent and all kinds of SOLVENTS are the add-on while extracting 0.1g sample, according to actual sample amount, adjust in proportion.
Each 100 μ L of the radix bupleuri identification system of test kit, 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 are added in reaction tubes, are placed in PCR reaction instrument and are undertaken by follow procedure:
Radix bupleuri identification system, radix bupleuri positive control system, radix bupleuri negative control system: 94 ℃ of denaturation 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 denaturation 5min, 94 ℃ of 30s, 56 ℃ of 30s of annealing temperature, 72 ℃ of 45s, 32 circulations, 72 ℃ are extended 8min, 4 ℃.
(5) result is judged
Reaction product is used 1.5-1.8% sepharose, electrophoresis on horizontal cataphoresis apparatus, and 60-70mV electrophoresis 45-60min, the DNA Marker of molecular weight 100-1200bp does reference, and gel imaging analysis systematic observation is obtained a result.1.5-1.8% sepharose, market is on sale; The commercially available instrument that horizontal cataphoresis apparatus is manufactured by Beijing Liuyi Instrument Factory; The DNA Marker of molecular weight 100-1200bp is standard molecular weight, and market is on sale; 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, there is respectively positive findings in radix bupleuri and Radix Bupeuri Scorzonerfolii..
Claims (2)
1. a 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: be totally 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, remaining is bi-distilled water, described radix bupleuri positive control system: be totally 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 DNA 2-5 μ l, remaining is bi-distilled water, described radix bupleuri negative control system: total system is 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, bigleaf thorowax root, black radix bupleuri DNA were by 1: 1 mixture 2-5 μ l, and remaining is bi-distilled water, and described primer 1 is radix bupleuri primer: 5 '-ATGACCAACATTCGTAAACTC-3 ', 5 '-TCTTCATTTTAATAGGTTATA-3 ',
(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: be totally 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, remaining is bi-distilled water; Described Radix Bupeuri Scorzonerfolii. positive control system: be totally 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, Radix Bupeuri Scorzonerfolii. DNA2-5 μ l, remaining is bi-distilled water; Described Radix Bupeuri Scorzonerfolii. negative control system: total system is 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, bigleaf thorowax root, black radix bupleuri DNA were by 1: 1 mixture 2-5 μ l, and remaining is bi-distilled water, and described primer 2 is Radix Bupeuri Scorzonerfolii. primer: 5 '-AGACACATTCGTAACTC-3 ', 5 '-CTCATTTAATAGGTATA-3 '.
2. a 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 and belong to DNA sequence dna together, following primer 1, primer 2:
Primer 1 is radix bupleuri primer:
5’-ATGACCAACATTCGTAAACTC-3’
5’-TCTTCATTTTAATAGGTTATA-3’
Primer 2 is 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, solid phase phosphoramidite triester method;
(3) set up radix bupleuri DNA identification kit
(a) DNA extraction reagent: be totally 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: be totally 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, remaining is bi-distilled water; Described radix bupleuri positive control system: be totally 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, remaining 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 remaining 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: be totally 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, remaining is bi-distilled water; Described Radix Bupeuri Scorzonerfolii. positive control system: be totally 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, remaining 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 remaining 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 dry root of medicinal material is first except epidermis, be placed in mortar, the DNA extraction reagent 250 μ l that add 65 ℃ of preheatings, in liquid nitrogen, grind, grind liquid and proceed to 65 ℃ of temperature bath 1h of centrifuge tube, 4 ℃ of centrifugal 10min of 12000rpm, supernatant liquor proceeds to centrifuge tube, add chloroform-primary isoamyl alcohol 250ul of 24: 1, the centrifugal 10min of room temperature 8000rpm, supernatant liquor proceeds to centrifuge tube, 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, the 50ulTE or the distilled water that after dry, add pH8.0, put into-20 ℃ of refrigerators standby, by the radix bupleuri identification system of test kit, 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 reaction tubes, being placed in PCR reaction instrument is undertaken by follow procedure: radix bupleuri identification system, radix bupleuri positive control system, radix bupleuri negative control system: 94 ℃ of denaturation 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 denaturation 5min, 94 ℃ of 30s, 56 ℃ of 30s of annealing temperature, 72 ℃ of 45s, 32 circulations, 72 ℃ are extended 8min, 4 ℃,
(5) result is judged
Reaction product is used 1.5-1.8% sepharose, electrophoresis on horizontal cataphoresis apparatus, and 60-70mV electrophoresis 45-60min, the DNA Marker of molecular weight 100-1200bp does reference, and 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) |
| 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 |
| CN108823284A (en) * | 2018-06-21 | 2018-11-16 | 北华大学 | Rabbit testis DNA fingerprint identification kit and identification method |
| 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 |
| 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 |
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