CN105018614B - Radix Paeoniae Alba kind detection method - Google Patents
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Abstract
The present invention relates to a kind of Radix Paeoniae Alba kind detection methods, belong to plant variety authentication technique field.Identify model method includes the following steps: establishing: taking the Radix Paeoniae Alba of different cultivars, its DNA is extracted respectively, then the DNA extracted is subjected to polymerase chain reaction amplification with primer, obtain amplified production, and the amplified production is separated by electrophoresis, the DNA electrophoretogram of different cultivars Radix Paeoniae Alba is obtained, Radix Paeoniae Alba Variety identification model is established with the spectrogram information of the electrophoretogram；Variety identification: taking Radix Paeoniae Alba to be measured, obtains its DNA electrophoretogram according to the method described above, and the spectrogram information of the Radix Paeoniae Alba electrophoretogram to be measured is substituted into above-mentioned identification model and is analyzed, and judgement obtains the kind of the Radix Paeoniae Alba.This method provides effective reliable identification of means for the differentiation of Radix Paeoniae Alba kind.There is huge advantage relative to traditional form identification, in the research and application of white Peony Root, to solve the problems, such as that kind confusion and kind superiority and inferiority provide a kind of new means.
Description
Technical field
The present invention relates to a kind of plant variety discrimination methods, more particularly to a kind of Radix Paeoniae Alba kind detection method.
Background technique
ISSR (inter-simple sequence repeat) is that Zietkeiwitcz grew up equal to 1994
A kind of molecular marking technique on the basis of microsatellite.The basic principle is that being primer with the microsatellite DNA of anchoring, i.e., in SSR sequence
The end 3' or the end 5' of column add 2-4 random nucleotide, and in PCR reaction, anchor primer can cause specific site to be annealed, and cause
DNA fragmentation carries out PCR amplification between the less big repetitive sequence in the interval complementary with anchor primer.The area inter SSR expanded
Multiple bands in domain are differentiated by polyacrylamide gel electrophoresis, and expanding bands of a spectrum is mostly dominant performance.
The Germplasm resources diversity for (being free of mineral drug) studies have shown that Chinese medicine according to modern molecular biology be by
Caused by its gene pleiomorphism as a result, and gene pleiomorphism can be detected in DNA molecular marker technical level, this be than
The genetic marker more representative of its variation type is detected on form, tissue and cellular level.The polymorphism that ISSR is disclosed is higher, can
Obtain the information times over RAPD (Random Amplification Polymorphic DNA, randomly amplified polymorphic DNA)
Amount, accuracy almost can be with RFLP (Restriction Fragment Length Polymorphism, Restriction Fragment Length
Polymorphism) it compares favourably.
Radix Paeoniae Alba (Radix Paeoniae), alias Chinese herbaceous peony, perennial herb are Ranunculaceae (Ranunculaceae) plant
The peeling dry root of Chinese herbaceous peony (Paeonia lactifloraPall), property slight bitter, bitter acid.Its main component is Paeoniflorin, Chinese herbaceous peony
Medicine lactone glycoside, oxygen Paeoniflorin, benzoylpaeoniflorin are generally called total glucosides of paeony.It is more with anti-inflammatory, antiviral, spasmolysis and analgesia, protect liver etc.
Kind pharmacological activity.Radix Paeoniae Alba is in the history of existing more than 3900 years of the cultivation in China, and since its pharmacological activity is strong, clinical application is extensive, white
Chinese herbaceous peony is for a long time just by the primary raw material as plurality of Chinese and new drug.Since most of cultivation white Peony Root merely through drawing in short term
Kind and domestication, in traditional Radix Paeoniae Alba genunie medicinal materials there may be it is some by for many years selection but non-Economical Purification place it is excellent
Non-defective unit kind.The Radix Paeoniae Alba in China always has a three great tradition kinds, i.e. producing region Hangzhoupro for being the ground such as Herb paeony root, the Zhejiang Dongyang of Hui nationality
The river Radix Paeoniae Alba on the ground such as Radix Paeoniae Alba and Sichuan Zhong Jiang.In addition also there is Radix Paeoniae Alba output on Shandong, Shaanxi and Jiangsu and other places.Genuine Radix Paeoniae Alba is treated
It imitates, good quality, becomes the optimal selection of commodity Radix Paeoniae Alba.
Recently as the fast development of Market of Chinese Materia Medica, new Radix Paeoniae Alba planting base is continuously emerged.Radix Paeoniae Alba always with Bo,
Hangzhoupro, river be it is genuine, according to the research of Hu Shilin etc., singly with paeoniflorin content in, Bo Chinese herbaceous peony, river Chinese herbaceous peony are better than Hangzhoupro Chinese herbaceous peony, simultaneously
He has found the Radix Paeoniae Alba of Shandong Cao County production although being unknown, and appearance is similar with the above road local specialties, and paeoniflorin content is up to
1.95%, it prompts to be likely to become emerging Genuine producing area at this.White Peony Root source is other than part is from planting base, largely
Crude drug is still from the purchase of casual household.Therefore, the lack of standardization of working condition causes white Peony Root quality irregular；Simultaneously because
Long-term dispersion plantation, the structure of Radix Paeoniae Alba blastogenesis at present, source are not very clear that some comes from source area, and some is not from
It is of the same period to introduce a fine variety product, have plenty of not of the same race with ground, therefore former plant genetic structure has and largely makes a variation, may between kind
There are comparable hybrid UV curings.It is necessary to be strict in terms of source and Radix Paeoniae Alba germ plasm resource, plantation specification, white Peony Root is got clear
Genuineness, establish variety and quality standard, instruct GAP (Good Agriculturing Practice, Producing medicinal herbs quality
Management regulation) standardized production.
It and is the kind confusion for solving Radix Paeoniae Alba plantation to the genetic analysis of genunie medicinal materials provenance, kind scientific verification and label
Problem and the basis for examining quality present on raw material market are the key that guarantee genuine white Peony Root genuineness.Road
Ground Radix Paeoniae Alba and non-genuineness Radix Paeoniae Alba mode of appearance, quality texture are all closely similar, are difficult to accurately distinguish with conventional method
Ground Radix Paeoniae Alba and non-genuineness Radix Paeoniae Alba.
Summary of the invention
Based on this, it is necessary to which, in view of the above-mentioned problems, providing a kind of Radix Paeoniae Alba kind detection method, this method is with Radix Paeoniae Alba gene
It based on polymorphism, is detected in DNA molecular marker technical level, the kind of Radix Paeoniae Alba is identified, from gene level area
Divide genuineness Radix Paeoniae Alba and non-genuineness Radix Paeoniae Alba.
A kind of Radix Paeoniae Alba kind detection method, comprising the following steps:
It establishes and identifies model: taking the Radix Paeoniae Alba of different cultivars, extract its DNA respectively, the DNA primer that then will be extracted
Polymerase chain reaction amplification is carried out, amplified production is obtained, and the amplified production is separated by electrophoresis, it is white to obtain different cultivars
The DNA electrophoretogram of Chinese herbaceous peony establishes Radix Paeoniae Alba Variety identification model with the spectrogram information of the electrophoretogram；
Variety identification: taking Radix Paeoniae Alba to be measured, obtains its DNA electrophoretogram according to the method described above, by the Radix Paeoniae Alba electrophoretogram to be measured
Spectrogram information is substituted into above-mentioned identification model and is analyzed, and judgement obtains the kind of the Radix Paeoniae Alba；
The primer includes at least one of following primer:
Primer 1:AGAGAGAGAGAGAGAGT；
Primer 2: AGAGAGAGAGAGAGAGC；
Primer 3:AGAGAGAGAGAGAGAGG；
Primer 4:GAGAGAGAGAGAGAGAC；
Primer 5:CACACACACACACACAA；
Primer 6:CACACACACACACACAG；
Primer 7:ACACACACACACACACT；
Primer 8:ACACACACACACACACC；
Primer 9:AGAGAGAGAGAGAGAGYT；
Primer 10:AGAGAGAGAGAGAGAGYA；
Primer 11:GAGAGAGAGAGAGAGAYT；
Primer 12:GAGAGAGAGAGAGAGAYG；
Primer 13:CACACACACACACACARC；
Wherein: R is A or T, and Y is C or G.
The present invention establishes above-mentioned Radix Paeoniae Alba kind detection method, relative to tradition based on the molecular labeling of DNA level
Morphological Identification has huge advantage, in the research and application of white Peony Root, to solve the problems, such as that kind confusion and kind superiority and inferiority mention
A kind of new means have been supplied, have also provided technical support for the work of introducing a fine variety and cultivate of Radix Paeoniae Alba.And this method have it is fast and convenient,
The characteristics of only needing a small amount of sample can be realized, it is possible to authenticate the kind of Radix Paeoniae Alba seedling, so as to select to plant from gene level
Kind guarantees the quality of medicinal material from source.
Also, the present invention obtains above-mentioned ISSR primer by screening, carries out PCR amplification, obtained product energy with the primer
The difference and classification for reflecting Radix Paeoniae Alba kind, in this way detect Radix Paeoniae Alba kind, have classification accurate, and method is stable and again
The good advantage of renaturation.
The primer is made of primer 1- primer 13 in one of the embodiments,.Select above-mentioned all primers as one
A composite entity, obtained pcr amplification product have the characteristics that electrophoretic image abundant information is various and stable, best embody white
The difference and classification of Chinese herbaceous peony kind.
The reaction system of the polymerase chain reaction amplification in one of the embodiments, are as follows: the concentration of DNA profiling is
30~80ng/25 μ L, the concentration of archaeal dna polymerase are 1.0~2.0U/25 μ L, and the concentration of magnesium ion is 1.5~2.5m mol/L,
The concentration of dNTPs is 180~270 μm of ol/L, and the concentration of every kind of primer is 0.2~0.5 μm of ol/L.Above-mentioned reaction system has
Preferable expanding effect, can be stablized and the amplified production of high-quality a large amount, and subsequent separation and identification are conducive to.
The response procedures of the polymerase chain reaction amplification in one of the embodiments, are as follows: 93~98 DEG C first changes
Property 3-5 minutes, 93~98 DEG C denaturation 40-50 seconds, 51~58 DEG C annealing 40-50 seconds, then 70~75 DEG C extension 80-100 seconds,
30-40 circulation；Last 70~75 DEG C of extensions 8-12 minutes.Above-mentioned response procedures have preferable expanding effect, can obtain
The amplified production of stable and high-quality a large amount is conducive to subsequent separation and identification.
The spectrogram information obtains by the following method in one of the embodiments: to reproducible on electrophoretogram and
Clearly electrophoretic band counts band, has band to be denoted as 1, no band is denoted as 0, obtains 0,1 matrix data, which is
Spectrogram information.
In one of the embodiments, according to the spectrogram information, the similarity factor between the Radix Paeoniae Alba of each kind is calculated, and
Clustering is carried out, the identification model of different cultivars Radix Paeoniae Alba is obtained.
The method for extracting Radix Paeoniae Alba DNA in one of the embodiments, is as follows: Radix Paeoniae Alba being placed in the mortar of pre-cooling, is added
Liquid nitrogen grinding is transferred quickly in centrifuge tube at fine powder；The cetyl trimethylammonium bromide buffering of 60-70 DEG C of preheating is added
Liquid, 60-70 DEG C water-bath 20-60 minutes；It is cooled to room temperature, the chloroform that volume ratio is 20-28:1 is added: isoamyl mixed alkoxide solution is mixed
Even, 10000-14000rpm is centrifuged 10-20 minutes, takes supernatant, and NaAc and dehydrated alcohol, 10000- after mixing is added
14000rpm is centrifuged 3-7min, abandons supernatant, obtains DNA precipitating；The ethanol solution washing DNA precipitating of 60-80% is added, and will
The DNA be deposited in room temperature dry to get.
The condition of the electrophoretic separation in one of the embodiments, are as follows: carry out electrophoresis with agarose gel electrophoresis method, electricity
Swimming voltage is 120~180V, and electrophoresis time is 0.5h~1h.
Compared with prior art, the invention has the following advantages:
Radix Paeoniae Alba kind detection method of the invention, establishes Variety identification model according to the Radix Paeoniae Alba DNA of known kind first, then
The DNA information of Radix Paeoniae Alba to be measured is brought into the identification model, the inspection of Radix Paeoniae Alba kind is carried out based on the molecular labeling of DNA level
It surveys, provides effective reliable identification of means for the differentiation of genuineness Radix Paeoniae Alba and non-genuineness Radix Paeoniae Alba.It reflects relative to traditional form
Surely there is huge advantage, in the research and application of white Peony Root, to solve the problems, such as that kind confusion and kind superiority and inferiority provide one
The new means of kind, also provide technical support for the work of introducing a fine variety and cultivate of Radix Paeoniae Alba.And this method is with fast and convenient, it is only necessary to
The characteristics of a small amount of sample can be realized, it is possible to authenticate the kind of Radix Paeoniae Alba seedling, so as to select varieties of plant from gene level, from
Guarantee the quality of medicinal material on source.
Also, the present invention also passes through many experiments and filters out most representative primer, and suitable experiment condition, mentions
The high accuracy of the detection method, stability and repeatability.
Detailed description of the invention
Fig. 1 is the cluster arborescence of reference substance Radix Paeoniae Alba in embodiment 1；
Fig. 2 is the cluster arborescence of reference substance and product to be tested Radix Paeoniae Alba in embodiment 1.
Specific embodiment
The present invention is described further explains with attached drawing with reference to embodiments, but does not cause to the present invention any
Limitation.
Laboratory apparatus and reagent in following embodiment is as follows:
Instrument: nucleic acid-protein analyzer (Implen Nanophotometer), PCR amplification instrument (BIO-RAD), at a high speed from
Scheming (Eppendorf 5418), ultra low temperature freezer (Thermo 902-ULTS), electrophoresis apparatus, electrophoresis tank (BIO-RAD), gel
Imaging system (Bio-Rad).
Reagent: Taq archaeal dna polymerase (Promega, 5U/ μ L), dNTP (Promega, 10mM), DNAmarker (new east station of Guangzhou
Contain Biotechnology Co., Ltd), 10 × Loading Buffer (Takara), agarose (Biowest), Golden View (north
Jing Boling section is Biotechnology Co., Ltd).
Embodiment 1
A kind of Radix Paeoniae Alba kind detection method, comprising the following steps:
One, it establishes and identifies model.
1, the Radix Paeoniae Alba reference substance of different cultivars is taken, the reference substance type of excavation is 4 years raw sexual propagation Radix Paeoniae Albas and 4 years raw nothings
Sexual reproduction Radix Paeoniae Alba.
Sexual propagation Radix Paeoniae Alba plants sample by the inner planting base of Hu Qiao company 18, is the kind introduced from Shandong, and altogether 5
, it is respectively labeled as base 1, base 2, base 3, base 4, base 5.
Vegetative propagation Radix Paeoniae Alba is the sample of 18 inner casual household's planting sites, the Bozhou land race Radix Paeoniae Alba of bud head breeding, altogether 5
, it is respectively labeled as No. 1, No. 2, No. 3, No. 4, No. 5.
2, the Radix Paeoniae Alba genomic DNA of above-mentioned known kind is extracted.
0.2g Radix Paeoniae Alba seedling tender leaf is weighed respectively to be placed in the mortar of pre-cooling, and liquid nitrogen grinding is added into fine powder, shifts rapidly
Into 2mL centrifuge tube；The 800 μ L of CTAB (cetyl trimethylammonium bromide) buffer of 65 DEG C of preheatings, 65 DEG C of water-baths 30 are added
Minute；It is cooled to room temperature, the chloroform of 800 μ L is added: isoamyl alcohol (24:1) mixed solution simultaneously mixes well, 12000rpm centrifugation 15
Minute；Take supernatant into a 1.5mL centrifuge tube, be added 50 μ L NaAc, 1mL dehydrated alcohols, mix well, 12000rpm from
Heart 5min abandons supernatant；The ethanol solution that 1mL concentration expressed in percentage by volume is 70% is added and washs DNA precipitating, and DNA is deposited in room
Temperature is dried, and 100 μ L TE are added after drying and sufficiently dissolve, save backup in -20 DEG C, with 1% agarose gel electrophoresis and ultraviolet point
The quality and concentration of light photometry detection genomic DNA.
3, above extracted genomic DNA is subjected to polymerase chain reaction with following primer respectively.
Primer sequence table
Primer 1:AGAGAGAGAGAGAGAGT
Primer 2: AGAGAGAGAGAGAGAGC
Primer 3:AGAGAGAGAGAGAGAGG
Primer 4:GAGAGAGAGAGAGAGAC
Primer 5:CACACACACACACACAA
Primer 6:CACACACACACACACAG
Primer 7:ACACACACACACACACT
Primer 8:ACACACACACACACACC
Primer 9:AGAGAGAGAGAGAGAGYT
Primer 10:AGAGAGAGAGAGAGAGYA
Primer 11:GAGAGAGAGAGAGAGAYT
Primer 12:GAGAGAGAGAGAGAGAYG
Primer 13:CACACACACACACACARC
Note: R represents A or T, and Y represents C or G
Wherein: polymerase chain reaction volume is 25 μ L.Reaction system ingredient are as follows: 2.5 μ L 10 × buffer buffers
(i.e. DNA gel sample loading buffer), 2.0 μ L concentration are the MgCl of 25mmol/L2, 0.5 μ L concentration is the dNTPs of 10mmol/L,
0.75 μ L concentration is the primer of 10 μm of ol/L, and DNA profiling 50ng obtained above, 0.3 μ L concentration is the archaeal dna polymerase of 5U/ μ L,
Residual volume supplies 25 μ L with ultrapure water.
Reaction cycle parameter are as follows: 95 DEG C, 5 minutes；Subsequent 95 DEG C, 45 seconds；51~58 DEG C of annealing, 45 seconds；72 DEG C, 90 seconds,
35 circulations；Last 72 DEG C of extensions, 10 minutes.
4, polymerase chain reaction product is separated by electrophoresis respectively with agarose gel electrophoresis
1% agarose 200mL is configured with 1 × TAE buffer, shakes up and dissolves, boiled with micro-wave oven to clear.In heat
8.3 μ L of Golden View is added in glue to shake up, after naturally cool to 50 DEG C or so, encapsulating is in the electrophoresis tank for being plugged sample comb
Making sheet.After gel solidifies completely, comb is carefully taken out.Gel is put into electrophoresis tank, cathode is leaned in well side, is added appropriate
TAE working buffer solution, make its liquid level not have glue surface about 1mm deep just.10 × loading buffer is mixed with pipettor
Sample DNA and molecular weight standard (Marker) from loading wells be respectively perpendicular be added loading wells in.It is correct to connect after sample-adding
Electrophoresis tank and power supply, after electrophoretic voltage 180V, electrophoresis 0.5h to indicator bromophenol blue away from bottom end about 2cm when take out gel slab,
It is analyzed in gel analysis system, photograph deposit.
5, Radix Paeoniae Alba Variety identification model is established.
To reproducible and band, clearly electrophoretic band is counted, and every DNA bands of a spectrum as a unit, have band to be denoted as
1, no band is denoted as 0, obtains 0,1 matrix data, with 1,0 Input matrix NTSys software, calculates the similar system between each sample
Number, as shown in table 1 below, and using clustering between each kind of UPGMA algorithm progress, as shown in Figure 1, building clusters arborescence,
Obtain Radix Paeoniae Alba Variety identification model.
Similarity factor known to table 1 between kind Radix Paeoniae Alba
Two, Variety identification
1, following table 2 sample to be tested is taken.
2 Radix Paeoniae Alba sampling situations table of table
2, it according to the method described above, extracts and obtains its DNA electrophoretogram, according to above-mentioned electrophoretic band method of counting, obtain 0,1 square
Battle array data, calculate the similarity factor between each sample, as shown in table 3 below, and using cluster point between each kind of UPGMA algorithm progress
Analysis, as shown in Fig. 2, building cluster arborescence, seedling sample to be measured gather in clustering figure with which kind of reference substance for a major class,
I.e. it is believed that sample to be tested and this kind of reference substance are the Radix Paeoniae Alba of same kind.
Similarity factor between 3 each sample Radix Paeoniae Alba of table
Three, testing result.
By clustering Fig. 1 it is found that No. 1-base in introduced variety Radix Paeoniae Alba reference substance base 5 is gathered for a major class, Bozhou sheet
Ground kind Radix Paeoniae Alba reference substance No. 1-No. 5 gathers for a major class, consistent with sampling situations.
By clustering Fig. 2 it is found that sample SN8, SN9, SN10 (i.e. three samples of ten rivers acquisition) and reference substance base 1
Number, base 2 and base 3 are gathered for a major class, be introduced variety Radix Paeoniae Alba；Sample SN1, SN2, SN3, SN4, SN5, SN6 and SN7
(three samples of i.e. 18 inner acquisitions, three of 19 inner acquisitions and ten rivers acquisition a sample) with reference substance 1, No. 3,
No. 4 are gathered for a major class, are Bozhou Radix Paeoniae Alba land race.
From the above results, the kind of identification Radix Paeoniae Alba seedling that can be simple and fast using method of the invention, solves
Radix Paeoniae Alba seedling is difficult to the problem of differential variety from appearance, provides important breeding technology branch for the large-scale plantation of Radix Paeoniae Alba
It holds.
Embodiment 2
A kind of Radix Paeoniae Alba kind detection method, it is substantially similar to the method for embodiment 1, the difference is that:
Amplified production in order to obtain, used polymerase chain reaction system ingredient are as follows:
Polymerase chain reaction volume is 25 μ L.Reaction system ingredient are as follows: 2.5 μ L 10 × buffer buffer (i.e. DNA
Gel loading buffer), 1.0 μ L concentration are the MgCl of 25mmol/L2, 0.3 μ L concentration is the dNTPs of 10mmol/L, and 0.5 μ L is dense
Degree is the primer of 10 μm of ol/L, and DNA profiling 20ng obtained above, 0.1 μ L concentration is the archaeal dna polymerase of 5U/ μ L, residual volume
It is supplied with ultrapure water.
Amplified production obtained above is separated by electrophoresis with agarose gel electrophoresis, compared with Example 1, electrophoresis
Band is few, and fuzzy, it is difficult to carry out the calculating of subsequent clustering and similarity factor.
Embodiment 3
A kind of Radix Paeoniae Alba kind detection method, it is substantially similar to the method for embodiment 1, the difference is that:
Amplified production in order to obtain, used polymerase chain reaction system ingredient are as follows:
Polymerase chain reaction volume is 25 μ L.Reaction system ingredient are as follows: 2.5 μ L 10 × buffer buffer (i.e. DNA
Gel loading buffer), 2.5 μ L concentration are the MgCl of 25mmol/L2, 0.5 μ L concentration is the dNTPs, 1.25 μ L of 10mmol/L
Concentration is the primer of 10 μm of ol/L, and DNA profiling 50ng obtained above, 0.1 μ L concentration is the archaeal dna polymerase of 5U/ μ L, residual body
Product is supplied with ultrapure water.
Amplified production obtained above is separated by electrophoresis with agarose gel electrophoresis, compared with Example 1, electrophoresis
Band is fuzzy, it is more difficult to carry out the calculating of subsequent clustering and similarity factor.
Embodiment 4
A kind of Radix Paeoniae Alba kind detection method, it is substantially similar to the method for embodiment 1, the difference is that:
Amplified production in order to obtain, used polymerase chain reaction system ingredient are as follows:
Polymerase chain reaction volume is 25 μ L.Reaction system ingredient are as follows: (i.e. DNA is solidifying for 2.5 μ L10 × buffer buffers
Glue sample loading buffer), 2.5 μ L concentration are the MgCl of 25mmol/L2, 0.75 μ L concentration is the dNTPs of 10mmol/L, and 0.75 μ L is dense
Degree is the primer of 10 μm of ol/L, and DNA profiling 20ng obtained above, 0.3 μ L concentration is the archaeal dna polymerase of 5U/ μ L, residual volume
It is supplied with ultrapure water.
Amplified production obtained above is separated by electrophoresis with agarose gel electrophoresis, compared with Example 1, electrophoresis
Band is fuzzy, it is more difficult to carry out the calculating of subsequent clustering and similarity factor.
Comparative example 1
A kind of Radix Paeoniae Alba kind detection method, it is substantially similar to the method for embodiment 1, the difference is that:
The primer of use are as follows:
PR1:TATATATATATATATAC
PR2:CTCTCTCTCTCTCTCTG
PR5:GTGTGTGTGTGTGTGTC
After carrying out polymerase chain reaction using the above primer, with agarose gel electrophoresis respectively to polymerase chain reaction
Product is answered to be separated by electrophoresis, the result is that the calculating of subsequent clustering and similarity factor can not be carried out without electrophoretic band.
Comparative example 2
A kind of Radix Paeoniae Alba kind detection method, it is substantially similar to the method for embodiment 1, the difference is that:
The primer of use are as follows:
PR3:ACACACACACACACACT
PR4:TGTGTGTGTGTGTGTGG
After carrying out polymerase chain reaction using the above primer, with agarose gel electrophoresis respectively to polymerase chain reaction
Product is answered to be separated by electrophoresis, the result is that band can not carry out subsequent without polymorphism, i.e. the electrophoretic band indistinction of all samples
Clustering and similarity factor calculating.
Each technical characteristic of embodiment described above can be combined arbitrarily, for simplicity of description, not to above-mentioned reality
It applies all possible combination of each technical characteristic in example to be all described, as long as however, the combination of these technical characteristics is not deposited
In contradiction, all should be considered as described in this specification.
The embodiments described above only express several embodiments of the present invention, and the description thereof is more specific and detailed, but simultaneously
It cannot therefore be construed as limiting the scope of the patent.It should be pointed out that coming for those of ordinary skill in the art
It says, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to protection of the invention
Range.Therefore, the scope of protection of the patent of the invention shall be subject to the appended claims.
Claims (5)
1. a kind of Radix Paeoniae Alba kind detection method, which comprises the following steps:
It establishes and identifies model: taking the Radix Paeoniae Alba of different cultivars, extract its DNA respectively, then carry out the DNA extracted with primer
Polymerase chain reaction amplification obtains amplified production, and the amplified production is separated by electrophoresis, and obtains different cultivars Radix Paeoniae Alba
DNA electrophoretogram establishes Radix Paeoniae Alba Variety identification model with the spectrogram information of the electrophoretogram；
Variety identification: taking Radix Paeoniae Alba to be measured, obtains its DNA electrophoretogram according to the method described above, by the spectrogram of the Radix Paeoniae Alba electrophoretogram to be measured
Information is substituted into above-mentioned identification model and is analyzed, and judgement obtains the kind of the Radix Paeoniae Alba；
The primer is made of primer 1- primer 13:
Primer 1:AGAGAGAGAGAGAGAGT；
Primer 2: AGAGAGAGAGAGAGAGC；
Primer 3:AGAGAGAGAGAGAGAGG；
Primer 4:GAGAGAGAGAGAGAGAC；
Primer 5:CACACACACACACACAA；
Primer 6:CACACACACACACACAG；
Primer 7:ACACACACACACACACT；
Primer 8:ACACACACACACACACC；
Primer 9:AGAGAGAGAGAGAGAGYT；
Primer 10:AGAGAGAGAGAGAGAGYA；
Primer 11:GAGAGAGAGAGAGAGAYT；
Primer 12:GAGAGAGAGAGAGAGAYG；
Primer 13:CACACACACACACACARC；
Wherein: R is A or T, and Y is C or G；
The spectrogram information obtains by the following method: to reproducible and band on electrophoretogram, clearly electrophoretic band is counted,
There is band to be denoted as 1, no band is denoted as 0, obtains 0,1 matrix data, which is spectrogram information；
According to the spectrogram information, the similarity factor between the Radix Paeoniae Alba of each kind is calculated, and carries out clustering, obtains different product
The identification model of kind Radix Paeoniae Alba.
2. Radix Paeoniae Alba kind detection method according to claim 1, which is characterized in that the polymerase chain reaction amplification
Reaction system are as follows: the concentration of DNA profiling is 30~80ng/25 μ L, and the concentration of archaeal dna polymerase is 1.0~2.0U/25 μ L, magnesium from
The concentration of son is 1.5~2.5mmol/L, and the concentration of dNTPs is 180~270 μm of ol/L, the concentration of every kind of primer is 0.2~
0.5μmol/L。
3. Radix Paeoniae Alba kind detection method according to claim 2, which is characterized in that the polymerase chain reaction amplification
Response procedures are as follows: 93~98 DEG C first denaturation 3-5 minutes, 93~98 DEG C denaturation 40-50 seconds, 51~58 DEG C annealing 40-50 seconds, so
70~75 DEG C extension 80-100 seconds afterwards, 30-40 circulation；Last 70~75 DEG C of extensions 8-12 minutes.
4. Radix Paeoniae Alba kind detection method according to claim 1, which is characterized in that the method for extracting Radix Paeoniae Alba DNA is as follows: will
Radix Paeoniae Alba is placed in the mortar of pre-cooling, and liquid nitrogen grinding is added into fine powder, is transferred quickly in centrifuge tube；60-70 DEG C of preheating is added
Cetyl trimethylammonium bromide buffer, 60-70 DEG C water-bath 20-60 minutes；It is cooled to room temperature, addition volume ratio is 20-28:
1 chloroform: isoamyl mixed alkoxide solution mixes, and 10000-14000rpm is centrifuged 10-20 minutes, takes supernatant, and NaAc and nothing is added
Water-ethanol, 10000-14000rpm is centrifuged 3-7min after mixing, abandons supernatant, obtains DNA precipitating；Add the ethyl alcohol of 60-80%
Solution wash DNA precipitating, and by the DNA be deposited in room temperature dry to get.
5. Radix Paeoniae Alba kind detection method according to claim 1, which is characterized in that the condition of the electrophoretic separation are as follows: use
Agarose gel electrophoresis method carries out electrophoresis, and electrophoretic voltage is 120~180V, and electrophoresis time is 0.5h~1h.
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| 利用DNA ISSR分子标记技术对芍药属植物栽培品种的分类鉴定方法;索志立;《生物技术通报》;20081231;第2008年卷(第增刊期);109-112 * |
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