CN105274244B - A kind of method and its special primer pair for identifying Rhizoma Acori Calami - Google Patents
A kind of method and its special primer pair for identifying Rhizoma Acori Calami Download PDFInfo
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Abstract
The invention discloses a kind of methods and its special primer pair for identifying Rhizoma Acori Calami.Primer pair provided by the present invention is made of primer ZCP CP19s and primer ZCP CP19a, and primer ZCP CP19s and primer ZCP CP19a are single strand dna, and nucleotide sequence is followed successively by sequence 1 and sequence 2 in sequence table.PCR amplification is carried out to the genomic DNA of sample to be tested using primer ZCP CP19s and primer ZCP CP19a, if it is possible to realize effectively amplification, then contain in the sample to be tested or candidate contains Rhizoma Acori Calami；If can not achieve effective amplification, do not contained in the sample to be tested or candidate does not contain Rhizoma Acori Calami.It is demonstrated experimentally that using identification Rhizoma Acori Calami provided by the invention method can quick easy identification Rhizoma Acori Calami, to ensure the safety of Rhizoma Acori Calami clinical application, with great promotion prospect and application value.
Description
Technical field
The invention belongs to biological technical fields, and in particular to a kind of method and its special primer pair for identifying Rhizoma Acori Calami.
Background technology
Rhizoma Acori Calami (Rhizoma Acori calami) is the conventional crude drugs of Tibetan, is aroid Rhizoma Acori Calami
The dry rhizome of (Acorus calamus L.), has the function of warm stomach and anti-inflammatory analgetic, for mend stomach sun, cure mainly indigestion,
Food stagnates, diphtheria and anthrax etc..Rhizoma Acori Calami also known as calamus or rhizoma calami are distributed in each provinces and regions in China, in world wide
Inside it is distributed in the temperate zone and subtropical zone of north and south two hemispheres.Rhizoma Acori Calami has a variety of chemical compositions and pharmacological action, has and kills
Worm, antibacterial, calm, antidepression, anti-inflammatory, antitumor and hypoglycemic etc. pharmacological actions.
Grass-leaved sweetflag (Acortw tatarinowii Schott) falls within Araeceae, is that the Ji Yuan of grass-leaved sweetflag medicinal material plants
Object.Though there is similarity in the effect of it and Rhizoma Acori Calami, also different, the two is different medicinal materials, necessary on clinical application
Differentiated, it is impossible to it is mixed or alternative, therefore the two is differentiated and distinguished when medicinal material is bought with clinical practice.Hide Chang
The methods of Pu medicinal material Main Basiss formalness, microscopic features, thin-layered chromatography, ultraviolet spectra Absorption Characteristics and liquid chromatogram into
The discriminating of the mixed adulterant of row.The resolution ratio that these methods have is not high, and some method complexity are time-consuming, are unfavorable for promoting, there is an urgent need to build
Vertical quick and easy true and false product identification method, to ensure the safety of Rhizoma Acori Calami clinical application.
The content of the invention
How quick, the easy identification Rhizoma Acori Calami of the technical problems to be solved by the invention.
To solve the above problems, present invention firstly provides a kind of for identifying the primer pair of Rhizoma Acori Calami.
The primer pair provided by the present invention for being used to identify Rhizoma Acori Calami, can be by primer ZCP-CP19s and primer ZCP-CP19a
Composition, the primer ZCP-CP19s and the primer ZCP-CP19a are single strand dna, and nucleotide sequence is followed successively by sequence
Sequence 1 and sequence 2 in table.
It is above-mentioned for identifying in the primer pair of Rhizoma Acori Calami, the primer ZCP-CP19s and the primer ZCP-CP19a's rubs
You are than that can be 1:1.
It is above-mentioned for identify Rhizoma Acori Calami primer pair application, be following a)-f) in it is any：
A) prepare to detect or aid in the kit for detecting Rhizoma Acori Calami；
B) detect or aid in whether to contain in detection sample to be tested or candidate contains Rhizoma Acori Calami；
C) prepare to identify or aid in the kit for identifying Rhizoma Acori Calami；
D) identify or aid in identify sample to be tested whether be or candidate is Rhizoma Acori Calami；
E) prepare for identify or aid in identify commercially available Rhizoma Acori Calami true or false kit；
F) identify or aid in the true or false of the commercially available Rhizoma Acori Calami of identification.
The present invention also provides for identifying the kit of Rhizoma Acori Calami.
The kit provided by the present invention for being used to identify Rhizoma Acori Calami contains the above-mentioned primer pair for being used to identify Rhizoma Acori Calami.
The present invention also protects the preparation method of the above-mentioned kit for being used to identify Rhizoma Acori Calami, it may include is used to identify by above-mentioned
The step of each primer of the primer pair of Rhizoma Acori Calami is individually packed.
The application of the above-mentioned kit for being used to identify Rhizoma Acori Calami falls within protection scope of the present invention.It is above-mentioned to be hidden for identifying
The application of the kit of calamus can be following b1) or b2) or b3)：
B1) detect or aid in whether to contain in detection sample to be tested or candidate contains Rhizoma Acori Calami；
B2) identify or aid in identify sample to be tested whether be or candidate is Rhizoma Acori Calami；
B3) identify or aid in the true or false of the commercially available Rhizoma Acori Calami of identification.
The method that whether sample to be tested contains or candidate contains Rhizoma Acori Calami is detected the present invention also provides a kind of, it may include such as
Lower step：The total DNA of sample to be tested is extracted, PCR amplification is carried out using the above-mentioned primer pair for being used to identify Rhizoma Acori Calami, if it is possible to
It realizes effectively amplification, then contains in the sample to be tested or candidate contains Rhizoma Acori Calami；It is described if can not achieve effective amplification
It is not contained in sample to be tested or candidate does not contain Rhizoma Acori Calami.The sample to be tested is planted for the Ji Yuan of Chinese medicine or, the Chinese medicine
Object or, be derived from the Chinese medicine Original plant tissue or organ.
The present invention also provides it is a kind of identify sample to be tested whether be or method that candidate is Rhizoma Acori Calami, it may include following step
Suddenly：The genomic DNA of sample to be tested is extracted, PCR amplification is carried out using the above-mentioned primer pair for being used to identify Rhizoma Acori Calami, if it is possible to
Realize effectively amplification, then the sample to be tested is or candidate is Rhizoma Acori Calami；It is described to treat test sample if can not achieve effective amplification
Product are not or candidate is not Rhizoma Acori Calami.The sample to be tested is the Original plant of Chinese medicine or, the Chinese medicine or, is derived from institute
State the tissue or organ of the Original plant of Chinese medicine.
The present invention also provides a kind of methods for the true or false for identifying commercially available Rhizoma Acori Calami, it may include following steps：Extract city
The genomic DNA of Rhizoma Acori Calami is sold, PCR amplification is carried out using the above-mentioned primer pair for being used to identify Rhizoma Acori Calami, if it is possible to which realization has
Effect amplification, then the commercially available Rhizoma Acori Calami is or candidate is genuine piece；If can not achieve effective amplification, the commercially available Rhizoma Acori Calami is
Or candidate is adulterant.
In any of the above-described the method, described " can realize effective amplification " is to contain 150-200bp in pcr amplification product
DNA fragmentation.
In any of the above-described the method, described " can not achieve effective amplification " is not contain 150- in pcr amplification product
The DNA fragmentation of 200bp.
The DNA fragmentation of the 150-200bp can be the DNA fragmentation of 173bp.
The DNA fragmentation of the 173bp concretely DNA fragmentation in sequence table shown in sequence 3.
In the above method, when carrying out the PCR amplification, concretely 62 DEG C of the annealing temperature of use.
In the above method, when carrying out the PCR amplification, the amplification program of use concretely 95 DEG C of 5min；95℃
30s, 62 DEG C of 20s, 25 Xun Huans；72℃ 10min.
Any of the above-described Chinese medicine can be Rhizoma Acori Calami and/or grass-leaved sweetflag and/or acorus graminens soland and/or purple bergenia herb.
The Original plant can be Rhizoma Acori Calami (Acorus calamus L.), grass-leaved sweetflag (Acortw tatarinowii
Schott), anemone altaica (Anemone altaica Fisch.ex C.A.Mey.) or purple bergenia herb (Bergenia
purpurascens(Hook.f.et Thoms.)Engl.)。
It being capable of quick easy identification Tibetan using the method and its special primer pair of identification Rhizoma Acori Calami provided by the invention
Calamus to ensure the safety of Rhizoma Acori Calami clinical application, has great promotion prospect and application value.
Description of the drawings
Fig. 1 is the phylogenetic tree of Rhizoma Acori Calami and adulterant.
Fig. 2 is universal primer to PCR amplification gel electrophoresis figure.
Fig. 3 is the specific detection of Specific PCR primers pair.
Fig. 4 is the sensitivity technique of Specific PCR primers pair.
Specific embodiment
The present invention is further described in detail With reference to embodiment, the embodiment provided is only for explaining
The bright present invention, the scope being not intended to be limiting of the invention.
Experimental method in following embodiments is conventional method unless otherwise specified.
The materials, reagents and the like used in the following examples is commercially available unless otherwise specified.
Quantitative test in following embodiment, is respectively provided with three repeated experiments, and results are averaged.
Rhizoma Acori Calami (Original plant is Rhizoma Acori Calami (Acorus calamus L.)) in the present embodiment is purchased from Jiangsu Province Suqian
City, grass-leaved sweetflag (Original plant is grass-leaved sweetflag (Acortw tatarinowii Schott)), acorus graminens soland (Original plant be Ah
Your safe pasqueflower (Anemone altaica Fisch.ex C.A.Mey.)) and purple bergenia herb (Original plant is purple bergenia herb
(Bergenia purpurascens (Hook.f.et Thoms.) Engl.)) it is purchased from Hui nationality's medicinal material market.In each
Medicinal material meets the pertinent regulations under each medicinal material item of text of Chinese Pharmacopoeia (version in 2010), passes through identification, every Chinese medicine
Material object is consistent with title, and quality complies with standard.
Embodiment 1 prepares to identify the kit and its application method of Rhizoma Acori Calami
First, for identifying the design of the primer pair of Rhizoma Acori Calami and synthesis
The ITS sequence of Rhizoma Acori Calami and adulterant is searched from GenBank, refers to table 1.
1. Rhizoma Acori Calami of table and the ITS sequence quantity of adulterant and GenBank accession number
Respectively with Bayesian Method (Bayesian inference, BI) and parsimony principle (maximum parsimony, MP) into
Row Phylogenetic Analysis, structure two kinds of genealogical trees of BI and MP are (with 2 ITS sequences of smartweed (Polygonum orientale)
(GenBank accession number is EU196898 and JX144668) is outer group, and interior group then includes the Rhizoma Acori Calami and adulterant shown in table 1
ITS sequence).Wherein BI trees MrBayes version 3.1.2 software buildings, MP trees PAUP*version 4.0beta
10 software buildings.When building BI trees, with 2.3 softwares of MrModeltest version according to AIC (Akaike Information
Criterion) the optimality model of test stone selection data, selected optimality model is (GTR+I).Markovian Meng Teka
Lip river method (Markov Chains Monte Carlo, MCMC) is arranged to four chains and ran for 1100000 generations.In order to determine it
Convergent, MCMC are separately operable twice.In every 100 generation, extracts a sample, forms 22002 samples altogether.It learns by analysis,
Whole service reaches steady after 50000 generations, in this way, remaining sample number is 21002 in total, with remaining sample reconstructing system tree
And estimate its posterior probability values.When building MP trees, bootstrapping number of repetition bootstrap nreps are set for 1000 times, using opening
Hairdo searching analysis bootstrapping repeated data collection, heuristic search are arranged to, by progressively additive process generates initial tree at random, repeat 10
It is secondary, using TBR branch exchanges.
The phylogenetic tree built by BI methods and MP methods shows that the genealogical tree that 2 kinds of methods are built is completely the same (Fig. 1).
Posterior probability (posterior probability, the PP) value of BI trees and MP trees are indicated at the node of Tu1Zhong Ge pedigrees branch
Support of booting (Bootstrap, BS).The result shows that the haplotype of Rhizoma Acori Calami all sequences forms monosystem (PP=0.91, BS
=100).
On the premise of definite Rhizoma Acori Calami is monosystem group, all ITS sequences are aligned with software Clustal X1.81
It sorts and compares, find differential fragment, designed and synthesized according to the distinctive variant sites of Rhizoma Acori Calami for the big of Rhizoma Acori Calami identification
Measure PCR primer pair.
To a large amount of PCR primers of acquisition to being screened successively, compliance test result, specificity verification and sensitivity verification, most
It is as follows that a pair of primer pair specific, that sensitivity is optimal is obtained eventually：
Primer ZCP-CP19s：5 '-CGGATGCGGATGTTGGC-3 ' (sequence 1 in sequence table)；
Primer ZCP-CP19a：5 '-GGTGATGGTGGGGTTCAT-3 ' (sequence 2 in sequence table).
Primer ZCP-CP19s and primer ZCP-CP19a is single strand dna.
The length of theoretical pcr amplification product is 173bp.
2nd, prepare to identify the kit of Rhizoma Acori Calami
For identifying that the kit of Rhizoma Acori Calami is by ZCP-CP19a points of the primer ZCP-CP19s of step 1 synthesis and primer
After not packing not individually, with the 10 × PCR Buffer, Taq DNA polymerase, MgCl individually packed2(25mM) and
DNTPs etc. is packaged in same kit.
3rd, identify sample to be tested in whether the foundation of the method containing Rhizoma Acori Calami
Using Rhizoma Acori Calami whether is contained in the kit identification sample to be tested of step 2, step is as follows：
1st, PCR amplification
Extract the genomic DNA of sample to be tested and using it as template, it is anti-to carry out PCR amplification using the kit of step 2
Should, obtain pcr amplification product.
Reaction system：Genomic DNA (6-50) ng, 10 × PCR Buffer, 2.5 μ L, the Taq DNA of sample to be tested
polymerase(5U/μL)0.5μL、MgCl2(25mM) 1.5 μ L, 0.5 μ L of dNTPs (10mM), primer ZCP-CP19s and ZCP-
Each 10pmol of CP19a are mended with deionized water to 25 μ L.
Response procedures：95℃ 5min；95 DEG C of 30s, 62 DEG C of 20s, totally 25 cycle；72℃ 10min.
2nd, pcr amplification product detects
Pcr amplification product is detected using agarose gel electrophoresis method, it is specific as follows：
5 μ L pcr amplification products are taken, using 2% Ago-Gel, electrophoresis 30min, gel imaging under 80-90V of voltage
It observes and takes pictures under system.Result judgement is carried out according to the size of purpose band：If it is about containing size in pcr amplification product
The purpose band (sequence 3 of sequence table) of 173bp, then contain in the sample to be tested or candidate contain Rhizoma Acori Calami；If PCR amplification
The purpose band (sequence 3 of sequence table) that size is about 173bp is not contained in product, then does not contain or waits in the sample to be tested
Choosing does not contain Rhizoma Acori Calami.
The specificity for being used to identify the kit of Rhizoma Acori Calami prepared by embodiment 2, embodiment 1
In triplicate, the step of repetition is as follows every time for experiment：
1st, about 50mg is taken to dry sample to be tested (Rhizoma Acori Calami, grass-leaved sweetflag, acorus graminens soland or purple bergenia herb), base is extracted with CTAB methods
Because of a group DNA, the concentration of the genomic DNA of sample to be tested is between 30ng/ μ l-50ng/ μ l.
2nd, the genomic DNA of the sample to be tested extracted using step 1 is template, with artificial synthesized ZCP-TY4s：5’-
TCGGCAACGGATATCTAG-3 ' and ZCP-TY4a：5 '-TTGCGTTCAAAGACTCG-3 ' are universal primer, carry out PCR expansions
Increase, detect the genomic DNA quality of sample to be tested.
Reaction total system be 25 μ L, 0.5 μ L of genomic DNA, 10 × PCR Buffer, 2.5 μ L including sample to be tested,
Taq DNA polymerase(5U/μL)0.5μL、MgCl2(25mM) 1.5 μ L, 0.5 μ L of dNTPs (10mM), primer ZCP-TY4s
With each 10pmol of ZCP-TY4a, mended with deionized water to 25 μ L.
Response procedures：95℃ 5min；95 DEG C of 30s, 62 DEG C of 20s, totally 25 cycle；72℃ 10min.
According to above-mentioned steps, Rhizoma Acori Calami genomic DNA is extracted, and the genomic DNA of sample to be tested is replaced in equal volume
Rhizoma Acori Calami genomic DNA, other steps are constant, carry out pcr amplification reaction, as positive control.
According to above-mentioned steps, the genomic DNA of sample to be tested is replaced with to isometric sterilizing ultra-pure water, other steps are equal
It is constant, pcr amplification reaction is carried out, as blank control.
Experimental result see Fig. 2 (swimming lane M for DNA molecular standard (DNA Marker, be followed successively by 600 from top to bottom, 500,
400th, 300,200 and 100bp), swimming lane 1 be using Rhizoma Acori Calami genomic DNA as template, swimming lane 2 be using grass-leaved sweetflag genomic DNA as
Template, swimming lane 3 are using acorus graminens soland genomic DNA as template, and swimming lane 4 is using purple bergenia herb genomic DNA as template, and swimming lane 5 is
Using the ultra-pure water that sterilizes as template).The result shows that positive control and sample to be tested can realize that effectively amplification, electrophoresis showed contain
The purpose band that size is about 173bp；Blank control can not achieve effective amplification, and it is about 173bp that electrophoresis showed, which does not contain size,
Purpose band.The purpose band that size is about 173bp is recycled into sequencing, sequence is as shown in sequence 3 in sequence table.It can be seen that it treats
The genomic DNA quality of test sample sheet is satisfied by requiring.
3rd, the genomic DNA of the sample to be tested extracted using step 1 is template, using the primer of 1 step 1 of embodiment synthesis
The primer pair of ZCP-CP19s and primer ZCP-CP19a compositions, carries out PCR amplification.Specific reaction system and response procedures are the same as real
Apply 1 step 31 of example.Experiment is set using negative control of the ultra-pure water as template that sterilize simultaneously.After reaction, according to embodiment 1
The method of step 32 carries out result judgement to sample to be tested.
Experimental result see Fig. 3 (swimming lane M for DNA molecular standard (DNA Marker, be followed successively by 600 from top to bottom, 500,
400th, 300,200 and 100bp), swimming lane 1 be using Rhizoma Acori Calami genomic DNA as template, swimming lane 2 be using grass-leaved sweetflag genomic DNA as
Template, swimming lane 3 are using acorus graminens soland genomic DNA as template, and swimming lane 4 is using purple bergenia herb genomic DNA as template, and swimming lane 5 is
Using the ultra-pure water that sterilizes as template).The result shows that only Rhizoma Acori Calami can amplify the purpose band that size is about 173bp.By size
The purpose band recycling sequencing of about 173bp, sequence is as shown in sequence 3 in sequence table.This shows being used for for the preparation of embodiment 1
Identify that the kit of Rhizoma Acori Calami being capable of precise Identification Rhizoma Acori Calami.
The sensitivity for being used to identify the kit of Rhizoma Acori Calami prepared by embodiment 3, embodiment 1
In triplicate, the step of repetition is as follows every time for experiment：
1st, with the genomic DNA of CTAB methods extraction Rhizoma Acori Calami, it is named as DNA1.The concentration of DNA is 105.8ng/ μ in DNA1
L。
2nd, 1mL DNA1 are drawn and add in abundant mixing in the test tube filled in 1mL sterilizing ultra-pure waters, obtain DNA2；With such
Pushing into DNA3, DNA4, DNA5, DNA6 and DNA7, after dilution the DNA concentration of each gradient be respectively 52.9ng/ μ L,
26.45ng/ μ L, 13.23ng/ μ L, 6.61ng/ μ L, 3.31ng/ μ L and 1.65ng/ μ L.
3rd, the DNA1 extracted using step 1 is template, the primer ZCP-CP19s and primer that are synthesized with 1 step 1 of embodiment
ZCP-CP19a is primer, carries out PCR amplification, obtains pcr amplification product.Specific response procedures are the same as 1 step 31 of embodiment.
Reaction total system is 25 μ L, including 0.5 μ L of DNA1,10 × PCR Buffer, 2.5 μ L, Taq DNA
polymerase(5U/μL)0.5μL、MgCl2(25mM) 1.5 μ L, 0.5 μ L of dNTPs (10mM), primer LYB-TY1s and LYB-
Each 10pmol of TY1a, deionized water are mended to 25 μ L.
According to the method described above, DNA1 is replaced with respectively DNA2, DNA3, DNA4, DNA5, DNA6 prepared by step 2 and
DNA7, other steps are constant, obtain corresponding pcr amplification product.
After reaction, result judgement is carried out according to the method for 1 step 32 of embodiment.
Experimental result is shown in that ((DNA Marker, are followed successively by swimming lane M the picture left above in Fig. 4 from top to bottom for DNA molecular standard
600th, 500,400,300,200 and 100bp), swimming lane 1 be using DNA1 as template, swimming lane 2 be using DNA2 as template, swimming lane 3 be with
DNA3 is template, and swimming lane 4 is using DNA4 as template, and swimming lane 5 is using DNA5 as template, and swimming lane 6 is the swimming lane 7 using DNA6 as template
For using DNA7 as template).It is obtained greatly the result shows that carrying out PCR amplification as template using DNA1, DNA2, DNA3, DNA4 or DNA5
The small about purpose band of 173bp, and it is about 173bp to carry out PCR amplification cannot obtain size as template using DNA6 and DNA7
Purpose band.Show minimum detection of the kit to Rhizoma Acori Calami genomic DNA provided by the present invention for identifying Rhizoma Acori Calami
It is limited to 3.31ng.
According to the method for above-mentioned steps 1 to 3, Rhizoma Acori Calami is replaced with to grass-leaved sweetflag, acorus graminens soland and purple bergenia herb respectively, it is other
Step is constant, obtains corresponding pcr amplification product.(swimming lane M is DNA molecular mark to the top right plot that experimental result is shown in successively in Fig. 4
Accurate (DNA Marker are followed successively by 600,500,400,300,200 and 100bp from top to bottom), the grass-leaved sweetflag into swimming lane 14 of swimming lane 8
Genomic DNA concentration be followed successively by 105.8ng/ μ L, 52.9ng/ μ L, 26.45ng/ μ L, 13.23ng/ μ L, 6.61ng/ μ L,
3.31ng/ μ L and 1.65ng/ μ L), lower-left figure in Fig. 4 (swimming lane M for DNA molecular standard (DNA Marker, from top to bottom according to
Secondary is 600,500,400,300,200 and 100bp), the concentration of the genomic DNA of acorus graminens soland into swimming lane 21 of swimming lane 15 is successively
For 105.8ng/ μ L, 52.9ng/ μ L, 26.45ng/ μ L, 13.23ng/ μ L, 6.61ng/ μ L, 3.31ng/ μ L and 1.65ng/ μ L)
With in Fig. 4 bottom-right graph (swimming lane M for DNA molecular standard (DNA Marker, be followed successively by 600 from top to bottom, 500,400,300,
200 and 100bp), the concentration of the genomic DNA of purple bergenia herb into swimming lane 28 of swimming lane 22 is followed successively by 105.8ng/ μ L, 52.9ng/ μ
L, 26.45ng/ μ L, 13.23ng/ μ L, 6.61ng/ μ L, 3.31ng/ μ L and 1.65ng/ μ L).The result shows that it cannot obtain big
The small about purpose band of 173bp.
Claims (11)
1. for identifying the primer pair of Rhizoma Acori Calami, it is made of primer ZCP-CP19s and primer ZCP-CP19a, each primer is
Single strand dna, nucleotide sequence are followed successively by sequence 1 and sequence 2 in sequence table.
2. the application of primer pair described in claim 1 is following a）-f）In it is any：
a）It prepares to detect or aid in the kit for detecting Rhizoma Acori Calami；
b）Whether contain in detection or auxiliary detection sample to be tested or candidate contains Rhizoma Acori Calami；
c）It prepares to identify or aid in the kit for identifying Rhizoma Acori Calami；
d）Identify or aid in identify sample to be tested whether be or candidate is Rhizoma Acori Calami；
e）Prepare for identify or aid in identify commercially available Rhizoma Acori Calami true or false kit；
f）Identification or auxiliary identify the true or false of commercially available Rhizoma Acori Calami.
3. the kit containing primer pair described in claim 1；The kit is used to identifying or aiding in identification Rhizoma Acori Calami.
4. the preparation method of the kit described in claim 3, including by primer ZCP-CP19s described in claim 1 and institute
State the step of primer ZCP-CP19a is individually packed.
5. the application of kit described in claim 3 is following b1）Or b2）Or b3）：
b1）Whether contain in detection or auxiliary detection sample to be tested or candidate contains Rhizoma Acori Calami；
b2）Identify or aid in identify sample to be tested whether be or candidate is Rhizoma Acori Calami；
b3）Identification or auxiliary identify the true or false of commercially available Rhizoma Acori Calami.
6. a kind of detect the method that whether sample to be tested contains or candidate contains Rhizoma Acori Calami, include the following steps：
The total DNA of sample to be tested is extracted, PCR amplification is carried out using primer pair described in claim 1, if it is possible to realize and effectively expand
Increase, then contain in the sample to be tested or candidate contains Rhizoma Acori Calami；If can not achieve effective amplification, in the sample to be tested
It does not contain or candidate does not contain Rhizoma Acori Calami.
Identify whether sample to be tested is or method that candidate is Rhizoma Acori Calami to include the following steps 7. a kind of：
The genomic DNA of sample to be tested is extracted, PCR amplification is carried out using primer pair described in claim 1, if it is possible to which realization has
Effect amplification, then the sample to be tested is or candidate is Rhizoma Acori Calami；If can not achieve effective amplification, the sample to be tested is not
Or candidate is not Rhizoma Acori Calami.
8. method as claimed in claims 6 or 7, it is characterised in that：The sample to be tested is Chinese medicine or, the Chinese medicine
Original plant or, be derived from the Chinese medicine Original plant tissue or organ.
9. method as claimed in claims 6 or 7, it is characterised in that：
Described " can realize effective amplification " is the DNA fragmentation containing 173bp in pcr amplification product；
Described " can not achieve effective amplification " is the DNA fragmentation that 173bp is not contained in pcr amplification product.
10. a kind of method for the true or false for identifying commercially available Rhizoma Acori Calami, includes the following steps：
The genomic DNA of commercially available Rhizoma Acori Calami is extracted, PCR amplification is carried out using primer pair described in claim 1, if it is possible to realize
Effectively amplification, then the commercially available Rhizoma Acori Calami is or candidate is genuine piece；If can not achieve effective amplification, the commercially available Rhizoma Acori Calami
For or candidate be adulterant.
11. method as claimed in claim 10, it is characterised in that：
Described " can realize effective amplification " is the DNA fragmentation containing 173bp in pcr amplification product；
Described " can not achieve effective amplification " is the DNA fragmentation that 173bp is not contained in pcr amplification product.
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| 西藏栽培与野生藏菖蒲的性状和粉末鉴别分析;肖剑琴等;《中国林副特产》;20090430(第2期);全文 * |
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