CN105063034A - Specific primer and method for molecular quantitative analysis of sarcandra glabra and three kinds of adulterant - Google Patents
Specific primer and method for molecular quantitative analysis of sarcandra glabra and three kinds of adulterant Download PDFInfo
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- 238000004445 quantitative analysis Methods 0.000 title abstract description 20
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- 235000010842 Sarcandra glabra Nutrition 0.000 title abstract description 7
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Abstract
The invention provides a specific primer and method for molecular quantitative analysis of sarcandra glabra and three kinds of adulterant and belongs to the field of biotechnology. An upstream specific primer SgF:5'-TGATTCTGATAGATT-TTTGAAGAATTGA-3' and a downstream primer trnL-Fb of sarcandra glabra are specifically recognized; a specific upstream primer ChF:5'-ATCTGATCACCCTTACACTTACAAGC-3 and a downstream primer trnL-Ff of chloranthus spicatus, radix chloranthi serrati and henryi hemsl are specifically recognized. A fluorogenic quantitative PCR is built through the two pairs of primers, and molecular quantitative analysis of a specific sample in mixed samples is carried out. The technology capable of analyzing sarcandra glabra and adulterant of chloranthus spicatus, henryi hemsl and radix chloranthi serrate accurately, fast, easily and quantitatively is provided, and technical supports are provided for quantitative analysis of the adulterant of related sarcandra glabra decoction pieces, chloranthus spicatus, henryi hemsl and radix chloranthi serrate.
Description
Technical field
The invention belongs to biological technical field, be specifically related to Auele Specific Primer and method that molecular method quantification analyzes Herba Pileae Scriptae and 3 kinds of adulterants, be specially a kind of discriminating Herba Pileae Scriptae
sarcandraglabra (Thunb.) Nakai, chu lan tree Chloranthusspicatus, and oneself
chloranthusserratus, with Chloranthus henryi Hemsl
chloranthushenryithe specificity labeled primers analyzed of molecular method quantification, and one utilizes this Auele Specific Primer to Herba Pileae Scriptae and chu lan tree, and 3 kinds of melange quick quantitative analytic methods such as oneself and Chloranthus henryi Hemsl.
Background technology
Herba Pileae Scriptae
sarcandraglabra (Thunb.) Nakai, be commonly called as sarcandra glaber, be generally used for treatment tinea versicolor, purpura, rheumatic arthralgia, wound.Modern study shows to have Therapeutic cancer, antitumor, anti-inflammatory, antiviral and non-specific immunity strengthens first-class pharmacological action.But wild plant germ plasm resource reduces in recent years, due to chu lan tree, Chloranthus henryi Hemsl and own profile extremely similar to Herba Pileae Scriptae, be easy to obscure mutually with Herba Pileae Scriptae.But they have different attributes and pharmaceutical use, these easy intermingle things also have corresponding hepatotoxic effect, and oneself is verified in the toxicity research of serious liver toxicity case report and animal remarkable liver toxicity, which has limited its application clinically.
Although the fresh blade of Herba Pileae Scriptae can be identified from its adulterant by the microscopical identification difference between the nonglandular hair vascular structure of vein, appurtenant and leaf, the Herba Pileae Scriptae of powder or crushing piece form and easy intermingle thing are very indiscernible by morphology and histological techniques.In the past few years, utilize chemical analysis technology as described in TLC, HPLC, MS etc. identify that Herba Pileae Scriptae and related preparations thereof had.Although these methods can supplement the limitation of morphology or histological characterization to a certain extent, but chemical fingerprint can only test section compound, limited species composition information can only be provided, the method identifying Herba Pileae Scriptae completely can not be provided to us, and be difficult to carry out quantitative analysis, particularly when the species very similar with it mix mutually.Therefore, quantitative analysis method is carried out, than just most important to monitoring quality to its related products when setting up the Product mixes such as Herba Pileae Scriptae and chu lan tree and oneself and Chloranthus henryi Hemsl.
Along with molecular biological development, DNA molecular marker as RAPD, ISSR and SSR equimolecular mark, ITS(Internaltranscribedspacer) etc. DNA bar code technology provide the method identifying reliably medicinal material.But, these molecular marking techniques based on genomic dna are easily subject to the impact of genomic dna degraded, because nuclear DNA is fewer than chloroplast DNA copy number, more easily goes to pot in finished medicinal material and cause to be differentiated by its molecular marking technique.Chloroplast DNA trnL-F region comprise trnL(UAA) intron and interval, i.e. trnL(UAA)-trnF(GAA) district is as DNA bar code, it can be easy to utilize universal primer to increase, this sequence has been proved to be and has can be used for, to the identification of various plants in kind of level, being widely used in the detection of plant prod.
This seminar also finds in early-stage Study, and for drying or through finished medicinal material, the authentication technique based on chloroplast DNA trnL-F region is more more stable effectively than the molecular marking technique based on nuclear DNA.This research is to Herba Pileae Scriptae, chu lan tree, Chloranthus henryi Hemsl and and oneself trnL-F region sequence check order, analyze its sequential structure and feature, find single nucleotide polymorphism (singlenucleotidepolymorphismSNP) molecule marker, design identifies Herba Pileae Scriptae respectively, chu lan tree, Chloranthus henryi Hemsl and and oneself specific recognition primer, set up multiple PCR technique, the specificity of primers designed, on this basis, utilize these special primers pair, build real-time fluorescence quantitative PCR, can simple quantitative analysis Herba Pileae Scriptae and chu lan tree fast, Chloranthus henryi Hemsl and and the method for oneself 3 kinds of adulterants.The present invention is that Herba Pileae Scriptae and 3 molecular method quantification analyses obscuring kind provide technical support, carry out quantitative analysis to the relevant prepared slices of Chinese crude drugs to have very important significance, retrieval has no Herba Pileae Scriptae and chu lan tree, Chloranthus henryi Hemsl and and the application for a patent for invention of molecular method quantification of 3 kinds of adulterants such as own.
Summary of the invention
Main purpose of the present invention is to provide one can be accurate, fast, simple quantitative analysis Herba Pileae Scriptae and chu lan tree, Chloranthus henryi Hemsl and and the technology of oneself 3 kinds of adulterants, for the melange quantitative analysis of relevant Herba Pileae Scriptae medicine materical crude slice and these 3 kinds of adulterants provides technical support.
For achieving the above object, the present invention adopts following technical scheme:
Molecular method quantification analyze Herba Pileae Scriptae and 3 kinds of adulterants Auele Specific Primer, upstream specific primer the SgF:5 '-TGATTCTGATAGATT-TTTGAAGAATTGA-3 ' of specific recognition Herba Pileae Scriptae and downstream primer trnL-Fb:5 '-GGGACTTGAACCCTCACGATT-3 '; Simultaneously special upstream primer the ChF:5 '-ATCTGATCACCCTTACACTTACAAGC-3 ' of specific recognition chu lan tree and oneself and Chloranthus henryi Hemsl and downstream primer trnL-Ff:5 '-ATTTGAACTGGTGACACGAG-3 '.
Molecular method quantification analyzes the method for Herba Pileae Scriptae and 3 kinds of adulterants, builds quantitative fluorescent PCR and carries out 40 circulations, reaction system
As follows: total amount is 20 μ L, the DNA of 10ng is extracted as amplification masterplate, 10 μ L2 × SYBRGreenReal-timePCRMasterMix (TaKaRa), 0.2 μM of upstream specific primer from plant, 0.2 μM of downstream universal primer, all the other are sterilized water; Response procedures is as follows: plant at real-time quantitative PCR instrument (ABI7900), 50 DEG C of denaturations 2 minutes, 95 DEG C of denaturations 10 minutes, at 95 DEG C of sex change 15s, and primer annealing 58 DEG C of 15s and extend 30s, 40 circulations at 72 DEG C.
Concrete grammar is as follows:
(1) design specific recognition primer, and verify its specificity
According to the Herba Pileae Scriptae of order-checking experiment in early stage gained, chu lan tree, and trnL-F gene order that is own and Chloranthus henryi Hemsl, analyze its special SNP site, design upstream specific primer the SgF(5 '-TGATTCTGATAGATTTTTGAAGAATTGA-3 ' of Herba Pileae Scriptae respectively), design can simultaneously specific recognition chu lan tree, and special upstream primer ChF(5 '-ATCTGATCACCCTTACACTTACAAGC-3 ' that is own and Chloranthus henryi Hemsl) and downstream universal primer trnL-Ff(5 '-ATTTGAACTGGTGACACGAG-3 '), build multiplex PCR system: total amount is 20 μ L, get the DNA that extracts in 10ng plant as masterplate, add 1 μ L1 × TransStartTopTaqDNA polymeric enzyme reaction liquid, 2 μ L10 × TransStartTopTaqBuffer, dNTP2.5mM, two specific primer SgF of 0.2 μM of downstream primer trnL-Ff and upstream, each 0.1 μM of ChF, all the other are sterilized water.Build multiplex PCR system, response procedures is as follows: 95 DEG C of denaturations 2 minutes, at 95 DEG C of sex change 30s, primer annealing 30s and 72 DEG C of downward-extensions 2 minutes at 50 DEG C, carry out 35 circulations, last circulates in 72 DEG C and extends 7 minutes, to guarantee complete extension product.Only have Herba Pileae Scriptae to achieve 575bp, chu lan tree and oneself and Chloranthus henryi Hemsl generate specific 216bp, and biased sample then produces 2 band such as 575bp and 216bp simultaneously.Confirm the specificity of primer.
(2) quantitative analysis method:
Upstream specific primer the SgF(5 '-TGATTCTGATAGATT-TTTGAAGAATTGA-3 ' of Herba Pileae Scriptae) with downstream primer trnL-Fb(5 '-GGGACTTGAACCCTCACGATT-3 ') form the primer pair of specific recognition Herba Pileae Scriptae, specific recognition chu lan tree simultaneously, and special upstream primer ChF(5 '-ATCTGATCACCCTTACACTTACAAGC-3 ' that is own and Chloranthus henryi Hemsl) and downstream primer trnL-Ff(5 '-ATTTGAACTGGTGACACGAG-3 ') form specific recognition chu lan tree, and the primer pair of 3 kinds of samples such as oneself and Chloranthus henryi Hemsl, build real-time quantitative PCR system respectively.Build quantitative fluorescent PCR and carry out 40 circulations, reaction system is as follows: total amount is 20 μ L, the DNA of 10ng is extracted as amplification masterplate from plant, 10 μ L2 × SYBRGreenReal-timePCRMasterMix (TaKaRa), 0.2 μM of upstream specific primer and 0.2 μM of downstream universal primer, all the other are sterilized water; Response procedures is as follows: in real-time quantitative PCR instrument (ABI7900), 50 DEG C of denaturations 2 minutes, 95 DEG C of denaturations 10 minutes; At 95 DEG C of sex change 15s, primer annealing 58 DEG C of 15s and 72 DEG C extend 30s, 40 circulations.Primer SgF and trnL-Fb is used for the quantitative analysis of Herba Pileae Scriptae in mixing prod; Primer ChF and trnL-Ff is used for the quantitative analysis of the Chloranthus (chu lan tree and oneself and Chloranthus henryi Hemsl) in mixing prod.The concentration of all primers is 0.2 μM.Real time quantitative PCR method is used for quantitative analysis method can by relative quantitative analysis method R=2
-△ △ Ctmethod is derived, due in same DNA sample with its STb gene for template increases, the corresponding Ct(total of biased sample total masterplate DNA) value is constant, the Ct(total namely in a specific biased sample) value is constant.Then the ratio of hotchpotch quantity can pass through this formulae discovery in same sample:
Ct(total)) be the Ct value of total mixing sample, the Ct value that Ct (Sg) is primer SgF, trnL-Fb, namely represent the Ct value of Herba Pileae Scriptae content, the Ct value that Ct (Ch) is primer trnL-Ff, ChF, namely represent the Ct value of three kinds of adulterant content.
The invention has the advantages that:
Owing to only carrying out quantitative analysis according to some activity characteristic component content, be subject to the impact of the external conditions such as plant growth environment, be difficult to carry out accurate quantitative analysis.Present method carries out molecular method quantification based on the content of hereditary material DNA, is not subject to the impact of the externalitiess such as the place of production of medicinal material, relative to stoichiometric method, more accurately, also more simple and convenient.
Because chloroplast DNA is more than the copy number of genomic dna, more stable in product after drying and processing, present method utilizes chloroplast DNA as the detected object of quantitative analysis, Billy as detected object, carries out molecular method quantification to the related products of processing more reliable and stable with genomic dna.
Present method can carry out molecular method quantification analysis to Herba Pileae Scriptae and 3 kinds of common adulterants simultaneously, accurate quantitative analysis 3 kinds of adulterants there is ratio, efficiency is higher, more convenient.
Accompanying drawing explanation
Fig. 1 utilizes primer trnL-Ff, the product gel electrophoresis (M:2logDNAladder of SgF, ChF multiplex PCR; 1-8: Herba Pileae Scriptae; 9-11: and oneself; 12-13: Chloranthus henryi Hemsl; 14-15: Herba Pileae Scriptae and own mixture; 16: Herba Pileae Scriptae, Chloranthus henryi Hemsl mixture).
Ct/x graphic representation (the R of Fig. 2 pair of primers SgF, trnL-Fb
2=0.9989).
Ct/x graphic representation (the R of Fig. 3 pair of primers ChF, trnL-Ff
2=0.9982).
Embodiment
instrument
PCR instrument (Eppendorf, model 5332), electrophoresis system (Beijing Liuyi Instrument Factory, model DYY-12), cryogenic freezing whizzer (Eppendorf, model 5810R), gel image analyser (BIO-RADChemiDocXRS), micropipet (Eppendorf).
reagent
2 × CTAB extracting solution, 1 × TAE damping fluid, agarose (Promega company), ethidium bromide (Fluka company), TransStart TopTaqDNAPolymerase (Beijing Quan Shi King Company), three chlorine first alkane ﹑ are domestic analytical pure without water second alcohol ﹑ Virahol.
material
This research experiment sample is identified by Fujian University of Traditional Chinese Medicine Wei Yicong lecturer and the sample picking up from Different sources collected: 13 kinds of Herba Pileae Scriptaes, four kinds of chu lan trees, four kinds of Chloranthus henryi Hemsl and three kinds and own sample collect from Chinese Different sources.Five kinds of Herba Pileae Scriptae sample powder and three kinds of chu lan tree sample powder are collected from local market and are got.
extract:
(1) get the fresh Herba Pileae Scriptae of 5g and Chloranthus plant leaf shreds, be placed in the mortar through sterilization, add 0.5gPVP powder, then add liquid nitrogen and grind rapidly, by powder collection in sack, be placed in-20 DEG C of preservations, long-time preservation is then placed in-80 DEG C;
(2) get the preheating in 65 DEG C of water of 10mLCTAB solution, then add 0.2mL (200 μ L) mercaptoethanol (mercaptoethanol: CTAB solution=1:50) in the CTAB of preheating;
(3) the powder 0.2g got in step (1) manages (centrifuge tube) in 2mLEP, adds rapidly the CTAB solution (adding during sample low temperature) containing mercaptoethanol of 1ml preheating, vibrates and within 2 minutes, put it into 65 DEG C of water-baths, water-bath about 1 hour; (vibration in every 20 minutes once, can suitably be vibrated several times more)
(4) in pipe, add 600 μ L chloroform-isoamyl alcohol mixed solutions (chloroform: primary isoamyl alcohol volume ratio=24:1, must operate at stink cupboard), shake up 5 minutes, then be placed on 15-20 DEG C with 12000rpm centrifugal 10 minutes;
(5) get 600 μ L supernatant liquors, then add 1.2mL(2 doubly) dehydrated alcohol of precooling or 360 μ L(0.6 doubly) Virahol, be placed in-20 DEG C of precipitations about 3 hours or spend the night;
(6) take out EP pipe under 4 DEG C of conditions with 12000rmp centrifugal 10 minutes, remove supernatant liquor, be inverted in thieving paper number minute;
(7) add 700 μ L70% ethanol again, turn upside down for several times, washing precipitation, after under 4 DEG C of conditions with 12000rmp centrifugal 10 minutes, remove supernatant liquor, repeat, be finally placed in super clean bench and dry up; (noting: can not dry up excessively)
(8) in EP pipe, add 30-50 μ L deionized water (ddH2O) or 1 × TE solution, place 3 hours or spend the night, make it fully dissolve in 4 DEG C, be difficult to if hand dissolve, available mixing machine mixes, and short its dissolves;
(9) can detect it whether containing genomic dna with sepharose, after carry out purifying.
primer specificity discriminance analysis
According to the Herba Pileae Scriptae of order-checking experiment in early stage gained, chu lan tree, and trnL-F gene order that is own and Chloranthus henryi Hemsl, analyze its special SNP site, design Herba Pileae Scriptae respectively, and upstream specific primer SgF(5 '-TGATTCTGATAGATTTTTGAAGAATTGA-3 ' that is own and Chloranthus henryi Hemsl), ChF(5 '-ATCTGATCACCCTTACACTTACAAGC-3 ') respectively with downstream universal primer trnL-Ff(5 '-ATTTGAACTGGTGACACGAG-3 ') build multiplex PCR system: total amount is 20 μ L, get the DNA that extracts in 10ng plant as template, add 1 μ L1 × TransStartTopTaqDNA polymeric enzyme reaction liquid (TransGen biotechnology), 2 μ L10 × TransStartTopTaqBuffer, dNTP2.5mM, two specific primer SgF of 0.2 μM of downstream primer trnL-Ff and 0.1 μM upstream, ChF, all the other are sterilized water.Build multiplex PCR system, response procedures is as follows: 95 DEG C of denaturations 2 minutes, 95 DEG C of 30s sex change, extend 2 minutes, carry out 35 circulations at the primer annealing 30s of 50 DEG C with at 72 DEG C.Last circulates in 72 DEG C and extends 7 minutes, to guarantee complete extension product.Only have Herba Pileae Scriptae to achieve 575bp, and oneself and Chloranthus henryi Hemsl generate specific 216bp, biased sample then produces 2 band such as 575bp and 216bp simultaneously, sees Fig. 1.
auele Specific Primer is utilized to carry out molecular method quantification analysis
For determining whether this specific primer is suitable for the molecular method quantification analysis of biased sample, we have carried out PCR in real time detection with the Herba Pileae Scriptae of gradient dilution and the mixture D NA sample of easy intermingle thing thereof.Tested sample DNA carries out continuous ten times of dilutions (1/10/100/1000/10000 times of dilution), is be incorporated as 2 μ L sample DNAs in 20 μ L reactive systems as masterplate in total amount.Relative to Ct value structure curve of specific DNA, result shows, the Ct value of 2 pairs of special primers and the logarithm of starting template exist linear relationship, and R
2>0.99, illustrates that these 2 pairs of primers of design can carry out quantitative assay to unknown sample, and therefore, designed real-time fluorescence quantitative PCR is the method for effectively this biased sample being carried out to molecular method quantification.As shown in (Fig. 2) and (Fig. 3), the Ct value of pair of primers SgF, trnL-Fb amplification and the logarithmic value of sample relative quantity form regression curve:
Y=-3.0885log(X)+39.015,R
2=0.9989。
Equally, the Ct value of pair of primers ChF and trnL-Ff amplification and the logarithmic value of sample relative quantity form regression curve:
Y=-3.5698log(X)+37.004,R
2=0.9982。
In this research, the Ct value of 2 pairs of primers of this mixed sample and 4 weaker concns (1/10/100/1000 times is diluted) of DNA profiling all there is linear relationship, therefore, can by accurate quantitative analysis in this dilution intervals.The content ratio of chloroplast DNA in the easy intermingle thing of same sample of Herba Pileae Scriptae and adulterant calculates by the method, namely 3 kinds obscure sample can by following formulae discovery relative to the relative content of Herba Pileae Scriptae sample:
.
In this research, easy intermingle thing chloroplast DNA content is 12.94 times higher than Herba Pileae Scriptae.These results show, PCR in real time is the ratio of Herba Pileae Scriptae and the chloroplast DNA content of its 3 kinds of adulterants in an enough responsive and biased sample of method assessment accurately, and carries out according to this content the ratio assessing Herba Pileae Scriptae and easy intermingle thing biased sample thereof.Because specific mixing sample Chloroplast DNA degradation rate relative to Herba Pileae Scriptae and easily intermingle thing be identical, therefore this method effectively can carry out molecular method quantification analysis to the biased sample of Herba Pileae Scriptae and easy intermingle thing thereof.
Table 1Ct value draws the relative populations of sample
The foregoing is only preferred embodiment of the present invention, all equalizations done according to the present patent application the scope of the claims change and modify, and all should belong to covering scope of the present invention.
SEQUENCELISTING
<110> Fujian University of Traditional Chinese Medicine
<120> molecular method quantification analyze Herba Pileae Scriptae and 3 kinds of adulterants Auele Specific Primer and method
<130>4
<160>4
<170>PatentInversion3.3
<210>1
<211>28
<212>DNA
<213> artificial sequence
<400>1
tgattctgatagatttttgaagaattga
28
<210>2
<211>21
<212>DNA
<213> artificial sequence
<400>2
gggacttgaaccctcacgatt
21
<210>3
<211>26
<212>DNA
<213> artificial sequence
<400>3
atctgatcacccttacacttacaagc
26
<210>4
<211>20
<212>DNA
<213> artificial sequence
<400>4
atttgaactggtgacacgag
20
Claims (2)
1. molecular method quantification analyze Herba Pileae Scriptae and 3 kinds of adulterants Auele Specific Primer, it is characterized in that: specific recognition chu lan tree and upstream specific primer ChF:5 '-ATCTGATCACCCTTACACTTACAAGC-3 ' that is own and Chloranthus henryi Hemsl 3 kinds of adulterants simultaneously.
2. one kind as claimed in claim 1 primer be used for the method that molecular method quantification analyzes Herba Pileae Scriptae and 3 kinds of adulterants, it is characterized in that: build quantitative fluorescent PCR and carry out 40 circulations, reaction system is as follows: total amount is 20 μ L, the DNA of 10ng is extracted as amplification masterplate from plant, 10 μ L2 × SYBRGreenReal-timePCRMasterMix, 0.2 μM of upstream specific primer, 0.2 μM of downstream universal primer, all the other are sterilized water; Response procedures is as follows: in real-time quantitative PCR instrument, 50 DEG C of denaturations 2 minutes, 95 DEG C of denaturations 10 minutes; 95 DEG C of sex change 15s, primer annealing 58 DEG C of 15s and 72 DEG C extend 30s, 40 circulations.
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| CN201510514887.0A CN105063034B (en) | 2015-08-21 | 2015-08-21 | Molecular method quantification analyzes the specific primer and method of Chloranthus glaber and 3 kinds of adulterants |
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| CN201510514887.0A CN105063034B (en) | 2015-08-21 | 2015-08-21 | Molecular method quantification analyzes the specific primer and method of Chloranthus glaber and 3 kinds of adulterants |
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| CN105063034A true CN105063034A (en) | 2015-11-18 |
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116004897A (en) * | 2022-12-16 | 2023-04-25 | 广西壮族自治区中国科学院广西植物研究所 | Method for identifying different species in chloranthus |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105087564A (en) * | 2015-08-21 | 2015-11-25 | 福建中医药大学 | Specific molecular marking primer and method for identifying sarcandra glabra and three adulterants |
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2015
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Patent Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105087564A (en) * | 2015-08-21 | 2015-11-25 | 福建中医药大学 | Specific molecular marking primer and method for identifying sarcandra glabra and three adulterants |
Non-Patent Citations (5)
| Title |
|---|
| HONG-ZHI KONG ET AL.: "Phylogeny of Chloranthus (Chloranthaceae) Based on Nuclear Ribosomal ITS and Plastid trnL-F sequence data", 《AMERICAN JOURNAL OF BOTANY》 * |
| WEI Y ET AL.: "Molecular authentication and quantitative analysis of Sarcandra glabra and adulterated chloranthus products using SNP markers.", 《MITOCHONDRIAL DNA A DNA MAPP SEQ ANAL.》 * |
| 蒋超 等: "基于双向位点特异性PCR 的金银花真伪鉴别方法研究", 《中国中药杂志》 * |
| 魏艺聪 等: "不同居群草珊瑚的ITS序列分析及模式识别研究", 《中草药》 * |
| 魏艺聪 等: "草珊瑚与金粟兰叶片双位点特异性PCR鉴别方法研究", 《中国中药杂志》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN116004897A (en) * | 2022-12-16 | 2023-04-25 | 广西壮族自治区中国科学院广西植物研究所 | Method for identifying different species in chloranthus |
| CN116004897B (en) * | 2022-12-16 | 2023-07-07 | 广西壮族自治区中国科学院广西植物研究所 | Method for identifying different species in chloranthus |
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| CN105063034B (en) | 2018-04-10 |
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Effective date of registration: 20231201 Address after: 355399 No.1 Xingye Road, Biopharmaceutical Park, Chengjiao Township, Zherong County, Ningde City, Fujian Province Patentee after: Fujian Longxiangu Biopharmaceutical Co.,Ltd. Address before: 350003 No. 54, 282 Gulou District, Fujian, Fuzhou Patentee before: FUJIAN University OF TRADITIONAL CHINESE MEDICINE |
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