CN107541521A - A kind of authentication method of radix coniti coreani DNA bar code and radix coniti coreani based on big data - Google Patents
A kind of authentication method of radix coniti coreani DNA bar code and radix coniti coreani based on big data Download PDFInfo
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Abstract
The invention discloses a kind of radix coniti coreani DNA bar code based on big data and the authentication method of radix coniti coreani, belong to molecular identification technical field.Pass through the Aconitum distribution of medicinal plants area that takes a broad survey, and multiple place samplings are carried out in species distribution area as far as possible, build the bar code large database concept of the category medicinal plant ITS sequence, then by it was found that the peculiar informative site of the distinctive ITS sequence of radix coniti coreani, further obtains its distinctive DNA bar code：The DNA bar code is the sequence for being 629bp by the primer sequence SEQ ID NO.1 and SEQ ID NO.2 overall lengths for expanding to obtain, and 89bp is A, and 443bp is A, and 447bp is that A, 491bp and 492bp are AT, and 499bp is T, and 577bp is C.The Rapid identification of medicinal plant radix coniti coreani will be greatly facilitated using the DNA bar code.
Description
Technical field
The present invention relates to molecular identification technical field, more particularly to a kind of radix coniti coreani DNA bar code based on big data,
And the method using DNA bar code identification radix coniti coreani.
Background technology
Radix coniti coreani (Aconitum coreanum (H.L é veill é) Rapaics), also known as close monkshood, rhizoma typhonii, mountain loudspeaker
Flower and yellow black garland etc., are Ranunculaceae Aconitum perennial herb medicinal plant, are that Jilin Province particularly Changbaishan area is genuine
Medicinal material.Its root tuber Korean aconite roof is used as medicine, and has the effect of wind-dispelling, eliminating dampness, resolving sputum analgesic.Aconitum plant has more than 200 to plant in China,
The platymiscium belongs to for poisonous plant simultaneously, the phenomenon generally existing of homonym or synonym in Aconitum medicinal material use.
Country's report Aconitum medicine poisoning is of common occurrence, and various aconitum plants are not easy to distinguish according to traditional form or Microscopic Identification method
Know.
DNA bar code technology (DNA Barcoding) is by dividing the DNA sequence dna of a standard target gene
Analysis, so as to quickly and accurately carry out the technology of species identification.In recent years, DNA bar code technology had proven to a row
Effective bioassay means, can not only make strong supplement to conventional identification method, and because it is more objective, accurate
Really, break through the transition to experience in the past to rely on, can help to identify species, find novel species etc..
At present, in the correlation molecule identification research of Aconitum medicinal plant, sample takes when species identification storehouse structure be present
Sample deficiency, the problem of failing to consider to there may be the variant sites similarities and differences between Different Individual between different plant species or in same species, lead
The poor reliability of cause identification, accuracy rate are low.
The content of the invention
In view of this, it is an object of the invention to provide a kind of radix coniti coreani DNA bar code based on big data, and utilize
The method that the DNA bar code identifies radix coniti coreani, improve the reliability and accuracy rate of qualification result.
Technical scheme is as follows：
The present invention provides a kind of radix coniti coreani DNA bar code based on big data, and the DNA bar code is by primer sequence
The sequence that the overall length that SEQ ID NO.1 and SEQ ID NO.2 expand to obtain is 629bp, 89bp is A, and 443bp is A, the
447bp is that A, 491bp and 492bp are AT, and 499bp is T, and 577bp is C.
Preferably, the sequence of the DNA bar code is as shown in SEQ ID NO.3.
The present invention also provides a kind of radix coniti coreani authentication method based on big data DNA bar code, comprises the following steps：
(1) sample to be tested DNA is extracted；
(2) enter performing PCR amplification using DNA bar code primer, obtain 629bp amplified production, the DNA bar code primer
Sequence is SEQ ID NO.1 and SEQ ID NO.2；
(3) amplified production is sequenced, the sequence of radix coniti coreani species discriminating is carried out according to following specific position
Row aspect ratio pair：89bp is A, and 443bp is A, and 447bp is that A, 491bp and 492bp are AT, and 499bp is T, the
577bp is C；
If amplified production meets above-mentioned condition, the sample to be tested is radix coniti coreani.
Preferably, the amplification system of the PCR is 25 μ L, and component proportioning is as follows：10 μm of each 1.0 μ of the forward and reverse primers of ol/L
The μ L of 10 × PCRbuffer of L, 25mmol/L, 2.5 μ L, 2.5mM dNTP, 2.5 μ L, 5unit/ μ L Taq archaeal dna polymerases 0.25,
50~100ng/L template DNAs 1.0,16.75 μ L of μ L, ddH2O.
It is further preferred that the amplification condition of the PCR is：94 DEG C of pre-degeneration 2min, 1 circulation；94 DEG C denaturation 30s, 52
DEG C annealing 1min, 72 DEG C extension 2min, 35 circulation；72 DEG C of extension 7min.
Preferably, it is of the present invention sequencing use two-way sequencing, the primer sequence such as SEQ ID NO.1 of the sequencing and
Shown in SEQ ID NO.2.
It is further preferred that the reaction system of the sequencing is 5 μ L：Wherein ddH2The μ L of O 3,10 μm of forward and reverse primers of ol/L
Each 0.25 μ L, the μ L of 0.5 μ L, PCR purified product of sequencing reaction mixture 1.
It is furthermore preferred that the reaction condition of the sequencing is：96 DEG C of pre-degenerations 10s, 50 DEG C of annealing 5s, 60 DEG C of extension 4min,
33 circulations.
Compared with prior art, the present invention has advantages below：
The present invention is carried out multiple places in species distribution area and adopted by the Aconitum distribution of medicinal plants area that takes a broad survey
Sample, the bar code large database concept of the category medicinal plant ITS sequence is built, then by it was found that the ITS sequence of radix coniti coreani
Peculiar informative site, further obtain its distinctive DNA bar code.
Compared to conventional morphology or method for identifying molecules, the present invention is to be carried out extensively to Aconitum medicinal plant first
On the basis of general sampling, radix coniti coreani is identified by the use of ITS sequence as DNA bar code, is expanded by entering performing PCR to ITS sequence
And sequencing, qualification result is directly obtained, detection process is quick, and as a result accuracy is high.This method will greatly facilitate medicinal plant
The Rapid identification of radix coniti coreani.
Brief description of the drawings
Fig. 1 is the pcr amplification product collection of illustrative plates of the sample segment used in structure Aconitum medicinal plant, wherein, positioned at the right
" M " of position is GeneRuler 100bp Plus DNA Ladder；
Fig. 2 is the sample segment aligned sequences result used in Aconitum medicinal plant.
Embodiment
The present invention is by the Aconitum distribution of medicinal plants area that takes a broad survey, and progress is more as far as possible in species distribution area
Individual place sampling, the bar code large database concept of the category medicinal plant ITS sequence is built, then by it was found that radix coniti coreani is special
Some peculiar informative sites of ITS sequence, further obtain its distinctive DNA bar code.
The radix coniti coreani DNA bar code of the present invention is to expand to obtain by primer sequence SEQ ID NO.1 and SEQ ID NO.2
Overall length be 629bp sequence, in the sequence, specific recognition site is：89bp is A, and 443bp is A, 447bp
It is AT for A, 491bp and 492bp, 499bp is T, and 577bp is C.The amplimer of the present invention is using ITS sequences
The universal primer of row, expand in obtained 629bp sequence, chrysanthemum can be identified according to the base species of above-mentioned specific position
The rhizome of Chinese monkshood.
Because the radix coniti coreani DNA bar code of the present invention is obtained based on the Aconitum medicinal herbs obtained as far as possible more
Arrive, it is contemplated that the problem of variant sites similarities and differences between Different Individual are there may be between different plant species or in same species, is one
Radix coniti coreani DNA bar code of the kind based on big data.Can be quick and precisely using the above-mentioned radix coniti coreani DNA bar code of the present invention
Radix coniti coreani is identified, overcome in the prior art identify poor reliability the defects of.
The present invention to the DNA of sample to be tested by extracting, by the way that amplified production is sequenced, by amplified production
Sequence is compared with the specific recognition sites in radix coniti coreani DNA bar code of the present invention, directly obtains qualification result.Detected
Journey is quick, and as a result accuracy is high, the Rapid identification available for medicinal plant radix coniti coreani.
In the present invention, the DNA extraction method of sample to be tested uses method well-known to those skilled in the art.In the present invention
In specific embodiment, the STb gene of sample to be tested is extracted using 4 × CTAB methods of improvement.The present invention is to DNA sources in sample to be tested
It is not particularly limited, can is leaf, root or the stem of sample to be tested.
Sequence used is ITS universal sequence when PCR is expanded and amplimer is sequenced in the present invention, specifically
For：
ITS4(SEQ ID NO.1)：5'-TCCTCCGCTTATTGATATGC-3',
ITS5(SEQ ID NO.2)：5'-GGAAGTAAAAGTCGTAACAAGG-3'.
In the present invention, PCR amplification method and the method that amplified production is sequenced are ripe using those skilled in the art
The method known.
Present invention preferably employs 25 μ L PCR amplification system, component proportioning is：10 μm of each 1.0 μ L of the forward and reverse primers of ol/L,
μ L, the 5unit/ μ L Taq DNA polymerase of 10 × PCR of 25mmol/L buffer, 2.5 μ L, 2.5mM dNTP 2.5
0.25 μ L, 50~100ng/L template DNA 1.0 μ L, ddH2O 16.75μL.The present invention does not have to the reagent source in amplification system
There is a particular determination, agents useful for same can use Commercial reagents well-known to those skilled in the art in amplification system.
PCR amplification condition is：94 DEG C of pre-degeneration 2min, 1 circulation；94 DEG C of denaturation 30s, 52 DEG C of annealing 1min, 72 DEG C are prolonged
Stretch 2min, 35 circulations；72 DEG C of extension 7min.The sample DNA sequence of all standby screenings under above-mentioned amplification system and amplification condition
Can Successful amplification.Those skilled in the art can be carried out on the basis of above-mentioned technical proposal to amplification system and amplification condition
Appropriate Reasonable adjustment, such as change the volume, the concentration of component, the temperature and time of adjustment amplification of amplification system, belong to this
The protection domain of invention.Preferred pair amplified production of the present invention is purified.Using purifying side well-known to those skilled in the art
Method is carried out.If amplification obtains 629bp amplified production, sample to be tested is possible to as radix coniti coreani, is passed through to be sequenced and is compared
Whether specific recognition site is that radix coniti coreani is further identified to sample to be tested.
The present invention is not particularly limited to the mode of sequencing, using sequence measurement well-known to those skilled in the art, such as
Unidirectional sequencing or two-way sequencing.In the specific embodiment of the invention, it is preferred to use two-way sequencing.
5 μ L sequencing reaction system is preferably used in the present invention：ddH2O 3 μ L, 10 μm of each 0.25 μ of the forward and reverse primers of ol/L
L, the μ L of 0.5 μ L, PCR purified product of sequencing reaction mixture 1.Wherein sequencing agents useful for same can use well known in the art
Commercial reagents.The reaction condition of sequencing is preferably：96 DEG C of pre-degeneration 10s, 50 DEG C of annealing 5s, 60 DEG C of extension 4min, 33 circulate.
It is appropriate reasonable that those skilled in the art can be carried out on the basis of above-mentioned technical proposal to sequencing reaction system and sequencing condition
Adjustment, such as change the volume, the concentration of component, the temperature and time of adjustment sequencing of reaction system, belong to the guarantor of the present invention
Protect scope.After the completion of reaction, product is settled, purified, after thermal denaturation, upper machine is sequenced.The present invention to sedimentation, it is pure
Change, thermal denaturation and sequencing procedure are not particularly limited, using the method known to skilled person.
Sequencing result is compared with the radix coniti coreani DNA bar code of the present invention, test sample is treated if following condition is met
This is radix coniti coreani：89bp is A, and 443bp is A, and 447bp is that A, 491bp and 492bp are AT, and 499bp is T,
577bp is C.
The present invention can be to it using universal primer and the operable PCR amplifications of those skilled in the art and sequence measurement
Rapid identification is carried out, detection process is quick, result accuracy is high.
For the object, technical solutions and advantages of the present invention are more clearly understood, the present invention is entered with reference to embodiment
Row detailed description, but they can not be interpreted as limiting the scope of the present invention.
Embodiment 1：
1st, the collection and preservation of Aconitum medicinal plant sample
The holding items of consulting literatures and domestic major specimen museum, field is formulated according to the sampling request of DNA bar code and adopted
Sample strategy, each species gather multiple individuals (1-13) of separate sources in its distributed area, all individual leaves as far as possible
Piece is preserved with silica dehydrator.Aconitum more than 70 is obtained altogether plants (containing mutation) 206 parts blade material, the sequence of remainder sample
Information downloads (totally 23) from NCBI gene pools.Wherein, the sample of radix coniti coreani is from Heilongjiang Province, Jilin Province and Liaoning Province
5 places, cover the main distributed area of the species, and each sampling point respectively gathers 3 parts of leaf samples.The bill of materials and sample source are shown in
Table 1.
The Aconitum medicinal plant sample of table 1 and source
2nd, Genome DNA extraction
The STb gene of above-mentioned aconitum plant blade is extracted using 4 × CTAB methods of improvement, is comprised the following steps that：
(1) mortar is cleaned and be dried for standby in baking oven, alcohol calcination mortar is used before extracting STb gene, pestle, spoon is to go out
Bacterium.
(2) choose clean blade to be placed in mortar, with liquid nitrogen coolant after moisture content completely loss, firmly grind material
Material is allowed to be in powdered, in then 2ml that ground material is transferred to precooling centrifuge tube.
(3) 4 × CTAB extract solutions and 2 μ l beta -mercaptoethanols (2%V/V) of 1ml preheatings are added in 2ml centrifuge tube,
Material is thoroughly dispersed in extract solution, warm bath about 1.5 hours in 65 DEG C of water-baths, during which shake up 3~5 times.
(4) material of warm bath is taken out to be placed at room temperature, to be cooled to after room temperature and adds isometric chloroform-isoamyl alcohol
(volume ratio 24:1) solution, shake up 5~10 minutes, then centrifuged 5 minutes with 10000~12000 revs/min.
(5) supernatant (about 700~800 μ l) is transferred in a new centrifuge tube to (paying attention to should not be miscellaneous in drawing
Matter sucks up), add isometric chloroform-isoamyl alcohol (volume ratio 24:1), shake up 5~10 minutes, then with 10000~
12000 revs/min centrifuge 5~10 minutes.
(6) supernatant (about 450~600 μ l) is transferred in a new centrifuge tube, adds the isopropanol of 70% volume, sunk
DNA is dropped, is gently overturned 2~3 times, it is seen that more than 30 minutes are stood in white flock precipitate, room temperature or 4 DEG C of refrigerators, then 10000
~12000 leave the heart 5~10 minutes, abandon supernatant.
(7) respectively washed 2 times with 200 μ l 70% ethanol and absolute ethyl alcohol, supernatant is abandoned after 20~30s of brief centrifugation, then
Centrifuge tube is placed in 37 DEG C of baking ovens (or at room temperature) ethanol is volatilized.Add 30~50 μ l TE solution and 1 after STb gene drying
~2 μ l RNase A are digested 2~3 hours in 37 DEG C of baking ovens with ribalgilase (RNaseA), then in -20 DEG C (or 4 DEG C)
Saved backup in refrigerator.
3rd, pcr amplification reaction
DNA concentration is detected with ultraviolet specrophotometer (UV-VIS spectrophotometer (TU-1800)), last dilute
Release standby to 50~100ng/ μ L.
RDNA ITS sequence is expanded using following primer：
ITS4(SEQ ID NO.1)：5'-TCCTCCGCTTATTGATATGC-3',
ITS5(SEQ ID NO.2)：5'-GGAAGTAAAAGTCGTAACAAGG-3'.
PCR reaction systems are 25 μ L, and component proportioning is as follows：Forward and reverse primer (10 μm of ol/L) each 1.0 μ L, 10 ×
PCRbuffer(Mg2+Plus, 25mmol/L) 2.5 2.5 μ L, Taq DNA polymerase (5unit/ μ of μ L, dNTP (2.5mM)
L) 0.25 μ L, the μ L of template DNA 1.0 (50~100ng/ μ L), ddH2O16.75μL。
The primer of all standby screenings can Successful amplification under following amplification condition：94 DEG C of pre-degeneration 2min, 1 circulation；94
DEG C denaturation 30s, 52 DEG C annealing 1min, 72 DEG C extension 2min, 35 circulation；72 DEG C of extension 7min, 4 DEG C of preservations after end.
Amplified production is purified, method illustrates operation by kit (giving birth to work Sangon in Shanghai).Use 1.5% fine jade
Sepharose electrophoresis detection PCR primer, is as a result proved, the Aconitum sample primer of all standby screenings is equal under amplification condition of the present invention
Can Successful amplification.Sample segment electrophoresis pattern is referring to Fig. 1.
4th, amplified production is sequenced
Sequencing reaction reagent uses the PRISM Dye Terminator Cycle Sequencing of ABI companies
ReactionKit, two-way sequencing.
Sequencing reaction system is 5 μ L：Wherein ddH2The μ L of O 3, sequencing each 0.25 μ L of upstream and downstream primer (10 μm of ol/L), sequencing
The μ L of reactant mixture (mix, Big Dye v3.1) 0.5 μ L, PCR purified product 1.
Amplimer sequence in the sequence synchronization rapid 3 of sequencing primer, i.e. SEQ ID NO.1 and SEQ ID NO.2.
Sequencing reaction condition is：96 DEG C of pre-degeneration 10s, 50 DEG C of annealing 5s, 60 DEG C of extension 4min, 33 circulate；After end
Add 20 μ L sedimentation agent (absolute ethyl alcohols：3M sodium acetate=20：1), 4 DEG C of sedimentations are stayed overnight.Sedimentation products add after purification through 70% alcohol
20μL ddH2Loading after O is denatured 2 minutes under the conditions of 95 DEG C, is sequenced on the automatic sequencers of ABI 3730.
5th, data analysis
Sequencing result is spliced and compared respectively with software SeqMan (DNAstar) and BioEdit (ver 7.0.0)
Right, the insertion and deletion in matrix is replaced with "-".The ITS sequence matrix of Aconitum medicinal plant is built, obtains ITS sequence data
Storehouse.
6th, the sequence signature of sequence alignment and radix coniti coreani
The result of sequence alignment is：Whole matrix overall length 640bp, by 229 Sequence compositions.Wherein, 23 are downloaded from NCBI
Bar, the present invention obtain 206, and sequence adheres to 72 kinds of Aconitum (containing mutation) common plant of being used as medicine separately.In a matrix, radix coniti coreani institute
The ITS sequence recognition site for having sample is：It is A in 92bp bases, 206bp and 207bp positions is lack, 452bp and 456bp positions
A is set to, remaining species is G；500-501bp has AT insertions, remaining species sample site deletion；508bp positions are T, remaining
Species sample is G；587bp positions are C, and remaining is T.
If only seeing the sequence of single species, the radix coniti coreani extension increasing sequence of all separate sources is consistent, with primer I TS4 and
The amplified production sequence that ITS5 expands to obtain is as shown in SEQ ID NO.3, and the overall length of the sequence is 629bp, and 89bp is base
A, 443bp A, 447bp A, 491bp and 492bp are AT, 499bp T, 577bp C.
The specific information site that radix coniti coreani is obtained according to the present invention can be used for as the DNA bar code of radix coniti coreani
The identification of radix coniti coreani.The authentication method of the present invention is merely with general versatility primer, by PCR amplifications and sequence measurement,
Rapid identification can be carried out to radix coniti coreani by above-mentioned 7 specific recognition sites.
Embodiment 2
The radix coniti coreani being had no result without flower and other Aconitum plant leafs gathered using medicinal material growing area and field is material
Material, DNA extractions are carried out using the method for embodiment 1, PCR amplifications and sequencing, qualification result are shown, are planted in known radix coniti coreani
The 89bp that thing obtains ITS sequence is base A, and 443bp A, 447bp A, 491bp and 492bp are AT, 499bp T,
577bp is C, and other unknown aconitum plants have no above-mentioned specific position, it is possible thereby to identify radix coniti coreani sample.
Described above is only the preferred embodiment of the present invention, it is noted that for the ordinary skill people of the art
For member, under the premise without departing from the principles of the invention, some improvements and modifications can also be made, these improvements and modifications also should
It is considered as protection scope of the present invention.
Sequence table
<110>Kunming Inst. of Botany, Chinese Academy of Sciences
<120>A kind of authentication method of radix coniti coreani DNA bar code and radix coniti coreani based on big data
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 20
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 1
tcctccgctt attgatatgc 20
<210> 2
<211> 22
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 2
ggaagtaaaa gtcgtaacaa gg 22
<210> 3
<211> 629
<212> DNA
<213> Aconitum coreanum
<400> 3
cgaaacctgc ccagcagagc gacccgcgaa caagtgaaaa caaaaccgga cggaccgaag 60
aggggcgcat gcccccgatc gcccgcccat cggaccgcga cctcttctgc gaccgcactg 120
atttgtgggt ggaggggtgg gttgttgagt ccgcacaaaa ccaaaaaccg gcgcgacagg 180
cgccaaggaa atcttagcgg aaagagggct tccccgttcg cggaggcagt cttcagaatc 240
cgatactcaa acgactctcg gcaacggata tctcggctct tgcatcgatg aagaacgtag 300
cgaaatgcga tacttggtgt gaattgcaga atcccgtgaa ccatcgagtc tttgaacgca 360
agttgcgccc gaggccatta ggtcgagggc acgtctgcct gggcgtcaca cacagcgtcg 420
caccccgtca accacgttgt cgaggaacgg agattggccc cccgggcccc tgcgggcacg 480
gtcggcacaa atatgttttt ccccggcggc gagcgtcgcg gtcagcggtg gttgtatttc 540
tcatcctcca aagacatcaa gacgcgtcgt cctcgtcgca tgttgggaca catcgacccc 600
acgaagtcgc tttgcgcgac attcaccct 629
Claims (8)
1. a kind of radix coniti coreani DNA bar code based on big data, it is characterised in that the DNA bar code is by primer sequence
The sequence that the overall length that SEQ ID NO.1 and SEQ ID NO.2 expand to obtain is 629bp, 89bp is A, and 443bp is A, the
447bp is that A, 491bp and 492bp are AT, and 499bp is T, and 577bp is C.
2. DNA bar code according to claim 1, it is characterised in that the sequence of the DNA bar code such as SEQ ID
Shown in NO.3.
3. a kind of radix coniti coreani authentication method based on big data DNA bar code, it is characterised in that comprise the following steps：
(1) sample to be tested DNA is extracted；
(2) enter performing PCR amplification using DNA bar code primer, obtain 629bp amplified production, the DNA bar code primer sequence
For SEQ ID NO.1 and SEQ ID NO.2；
(3) amplified production is sequenced, the sequence that radix coniti coreani species discriminating is carried out according to following specific position is special
Sign compares：89bp is A, and 443bp is A, and 447bp is that A, 491bp and 492bp are AT, and 499bp is T, the
577bp is C；
If amplified production meets above-mentioned condition, the sample to be tested is radix coniti coreani.
4. according to the method for claim 3, it is characterised in that the amplification system of the PCR is 25 μ L, and component is with such as
Under：Each μ L of 1.0 μ, 10 × PCR of L, 25mmol/L buffer solutions, 2.5 μ L, 2.5mM dNTP 2.5 of 10 μm of forward and reverse primers of ol/L,
0.25 μ L, 50~100ng/L template DNA of 5unit/ μ L Taq archaeal dna polymerases 1.0 μ L, ddH2O 16.75μL。
5. according to the method for claim 4, it is characterised in that the amplification condition of the PCR is：94 DEG C of pre-degeneration 2min, 1
Circulation；94 DEG C of denaturation 30s, 52 DEG C of annealing 1min, 72 DEG C of extension 2min, 35 circulate；72 DEG C of extension 7min.
6. according to the method for claim 3, it is characterised in that the sequencing is two-way sequencing, the primer sequence of the sequencing
Row are as shown in SEQ ID NO.1 and SEQ ID NO.2.
7. according to the method for claim 6, it is characterised in that the reaction system of the sequencing is 5 μ L：Wherein ddH2O 3μ
L, 10 μm of each 0.25 μ L of the forward and reverse primers of ol/L, the μ L of 0.5 μ L, PCR purified product of sequencing reaction mixture 1.
8. according to the method for claim 7, it is characterised in that the reaction condition of the sequencing is：96 DEG C of pre-degeneration 10s,
50 DEG C of annealing 5s, 60 DEG C of extension 4min, 33 circulate.
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| CN112980987A (en) * | 2021-03-15 | 2021-06-18 | 中国医学科学院药用植物研究所 | Aconitum molecular identity card and application thereof |
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