CN107619879A - A kind of primer pair and its application for being used to identify Dendrobidium huoshanness and dendrobium candidum - Google Patents
A kind of primer pair and its application for being used to identify Dendrobidium huoshanness and dendrobium candidum Download PDFInfo
- Publication number
- CN107619879A CN107619879A CN201711111212.7A CN201711111212A CN107619879A CN 107619879 A CN107619879 A CN 107619879A CN 201711111212 A CN201711111212 A CN 201711111212A CN 107619879 A CN107619879 A CN 107619879A
- Authority
- CN
- China
- Prior art keywords
- primer
- single strand
- dendrobidium huoshanness
- seq
- dendrobium candidum
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Withdrawn
Links
Landscapes
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a kind of primer pair for being used to identify Dendrobidium huoshanness and dendrobium candidum, it is made up of primer SEQ ID No.1 single strand dnas and primer SEQ ID No.2 single strand dnas, the SEQ ID No.1 single strand dnas are the single strand dna in sequence 1 in sequence table, and the SEQ ID No.2 single strand dnas are the single strand dna in sequence 2 in sequence table.The present invention passes through specific primer pair, including primer SEQ ID No.1 single strand dnas and primer SEQ ID No.2 single strand dnas, quick, the accurate discriminating between Dendrobidium huoshanness and dendrobium candidum is realized using rapid PCR methods, differentiates for Dendrobidium huoshanness and provides new method.
Description
Technical field
The present invention relates to technical field of molecular biology, more particularly to a kind of it is used to identifying Dendrobidium huoshanness and dendrobium candidum
Primer pair and its application.
Background technology
Dendrobidium huoshanness (Dendrobium huoshanense) is the distinctive plant in Dabie Mountains, Anhui Province area, and is country
The rare Chinese herbal medicine of focused protection, it is under the jurisdiction of orchid family (Orchidaceae) Dendrobium (Dendrobium).Dendrobium candidum
(Dendrobium catenatum) belongs to sibling species together for Dendrobidium huoshanness, and both morphological features are more close, after maple bucket is made
Both not easily pass through profile and differentiated, cause commercially dendrobium candidum turn into one of adulterant of Dendrobidium huoshanness.Due to suddenly
The mountain stem of noble dendrobium is differentiated relatively difficult with dendrobium candidum by profile, there is certain limitation using traditional authentication method.Therefore, need badly
A kind of fast and accurately authentication method is found, to ensure the accuracy of Dendrobidium huoshanness Med Mat Appreciation.Li Dan, Li Zhenjian etc., are delivered
Identification based on ITS sequence stem of noble dendrobium material and phylogenetic analysis, gardening journal, 2012,39 (8):1539~1550, utilize
DNA bar code ITS sequence identified Dendrobidium huoshanness and its nearly edge species, but this method needs to be sequenced, take compared with
It is long, and large-scale instrument is used, it is unfavorable for realizing quick, Site Detection.
The content of the invention
Based on technical problem existing for background technology, the present invention proposes a kind of for identifying Dendrobidium huoshanness and dendrobium candidum
Primer pair and its application.
A kind of primer pair SH-CP25 for being used to identify Dendrobidium huoshanness and dendrobium candidum proposed by the present invention, the primer pair
It is made up of primer SEQ ID No.1 single strand dnas and primer SEQ ID No.2 single strand dnas, the SEQ ID No.1
Single strand dna is the single strand dna in sequence 1 in sequence table, and the SEQ ID No.2 single strand dnas are sequence table
Single strand dna in middle sequence 2；
Wherein, sequence 1 is SH-CP25s：5’-TTCGTGGGATGGGGAGC-3’；
Sequence 2 is SH-CP25a：5’-TGGACATCCGAGCCCTA-3’.
Present invention also offers a kind of kit for being used to identify Dendrobidium huoshanness and dendrobium candidum, the kit includes drawing
Thing is to, dNTP and archaeal dna polymerase, and the primer pair is by primer SEQ ID No.1 single strand dnas and primer SEQ ID No.2
Single strand dna forms.
Further, the molal quantity that two single strand dnas of primer pair are formed in the kit is identical.
The present invention provide it is a kind of identification whether the method containing Dendrobidium huoshanness, comprise the following steps：
1. from testing sample extract genomic DNA be used as template, using primer SEQ ID No.1 single strand dnas with
The primer SH-CP25s and SH-CP25a of primer SEQ ID No.2 single strand dnas composition PCR in PCR reaction solutions and PCR instrument
Amplification；
2. PCR primer detects：PCR primer is detected using agarose gel electrophoresis method, identified according to the size of purpose band
Whether contain Dendrobidium huoshanness in testing sample.
Further, the PCR reaction solutions include following reagent：2% template DNA, 4%10pmol sense primers, 4%pmol
Anti-sense primer, 10%10 × buffer buffer solutions, 6% concentration are 25mM MgCl2The aqueous solution, 2% concentration are 10mM's
DNTPs, 2%5U/ μ L Taq archaeal dna polymerases, surplus is aseptic double-distilled water.
Further, the Dendrobidium huoshanness or be dendrobium candidum.
Compared with the prior art, the present invention has the beneficial effect that：
1st, in the present invention, by specific primer to (primer SEQ ID No.1 single strand dnas and primer SEQ ID
No.2 single strand dnas), quick, the accurate discriminating between Dendrobidium huoshanness and dendrobium candidum is realized using rapid PCR methods,
Differentiate for Dendrobidium huoshanness and provide new method.
2nd, compared with traditional form authentication method, this method does not need morphological taxonomy basis and realizes Rapid identification suddenly
The mountain stem of noble dendrobium and dendrobium candidum, solve the problems, such as from Morphological Identification difficulty.
3rd, technology is versatile, less demanding to instrument and equipment, the easy popularization and application in real work, and operating process
Simply, take shorter, high sensitivity and relatively stablize.
Brief description of the drawings
Fig. 1 is the phylogenetic tree of Dendrobidium huoshanness, dendrobium candidum and its sibling species；
Fig. 2 is the gel electrophoresis figure of universal primer amplification；
Fig. 3 is the gel electrophoresis figure that Specific PCR primers expand to SH-CP25；
Fig. 4 is the gel electrophoresis figure that Specific PCR primers expand to SH-CP25 under various concentrations DNA profiling；
Embodiment
Below in conjunction with the accompanying drawings the present invention is made with specific embodiment further to explain.
It is the bootstrapping support of posterior probability PP and the MP tree of BI trees above pedigree branch node in accompanying drawing Fig. 1 of the present invention
BS, it is PP values on the left side of oblique line, the right of oblique line is BS values；
M in Fig. 2：DNA molecular standard (DNA Marker, is followed successively by 2000,1000,750,500,250 and from top to bottom
100bp), 1- Dendrobidium huoshanness, 2- dendrobium candidums, 3- blank controls；
M in Fig. 3：DNA molecular standard (DNA Marker, is followed successively by 2000,1000,750,500,250 and from top to bottom
100bp), 1- expands result, the 2- Specific PCR primers of 1 Dendrobidium huoshanness sample with Specific PCR primers to SH-CP25
Result, the 3- blank controls of 1 dendrobium candidum are expanded to SH-CP25；
M in Fig. 4：DNA molecular standard (DNA Marker, is followed successively by 2000,1000,750,500,250 and from top to bottom
100bp), 1-7 be respectively Dendrobidium huoshanness DNA profiling concentration be followed successively by 210ng/ μ l, 105ng/ μ l, 52ng/ μ l, 26ng/ μ l,
13ng/ μ l, 7ng/ μ l and 3ng/ μ l, 8-14 be respectively dendrobium candidum DNA profiling concentration be followed successively by 210ng/ μ l, 105ng/ μ l,
52ng/ μ l, 26ng/ μ l, 13ng/ μ l, 7ng/ μ l and 3ng/ μ l.
First, for the design and synthesis of the primer pair for identifying Dendrobidium huoshanness
Searched from GenBank or carry out that the ITS sequence for obtaining Dendrobidium huoshanness and adulterant is sequenced by this research, referred to down
Table 1.Wherein, this research sequencing gained haplotype sequence, including Hap5, Hap6 and Hap7.Sequence 4 is referred in sequence table to sequence
Shown in 6, wherein, Hap5 corresponds to sequence 4, and Hap6 corresponds to sequence 5, and Hap7 corresponds to sequence 6.
The ITS sequence of the Dendrobidium huoshanness of table 1, dendrobium candidum and part sibling species
Entered respectively with Bayesian Method (Bayesian inference, BI) and parsimony principle (maximum parsimony, MP)
Row Phylogenetic Analysis, build two kinds of genealogical trees of BI and MP.During constructing system tree, with rhombic lip stem of noble dendrobium Dendrobium
Leptocladum 2 ITS sequences (GenBank accession number is respectively AF521612 and EU840697) are used as outer group.Wherein BI
Tree uses MrBayes version 3.1.2 software buildings, the MP trees software buildings of PAUP*version 4.0beta 10.Structure
During BI trees, examined with the softwares of MrModeltest version 2.3 according to AIC (Akaike Information Criterion)
The optimality model of standard selection data, selected optimality model are (SYM+G).Markovian monte carlo method (Markov
Chains Monte Carlo, MCMC) it is arranged to four chains and ran for 500000 generations.In order to determine its convergence situation, MCMC divides
Do not run twice.In every 100 generation, extracts a sample, forms 10002 samples altogether.Learn by analysis, whole service is 20000
Reach steady after generation, so, remaining sample number is 9602 altogether, with remaining sample reconstructing system tree and estimates its posterior probability
Value.When building MP trees, it is 1000 times to set bootstrapping number of repetition bootstrap nreps, is analyzed and booted using heuristic search
Duplicate data collection, heuristic search are arranged to, by progressively additive process produces initial tree at random, be repeated 10 times, hand over using TBR branches
Change.The phylogenetic tree built by BI methods and MP methods shows that the genealogical tree trunk of 2 kinds of method structures is consistent (Fig. 1).In Fig. 1
Posterior probability (posterior probability, PP) value and the bootstrapping of MP trees of BI trees are indicated at the node of each pedigree branch
Support (Bootstrap, BS).As a result show, the haplotype of Dendrobidium huoshanness all sequences forms monosystem (PP=1.00, BS=
96)。
On the premise of it is determined that Dendrobidium huoshanness is monosystem group, with software Clustal X 1.81 to all ITS sequences in table 1
Carry out aligning sequence and compare, find differential fragment, design Dendrobidium huoshanness detection Specific PCR primers are as follows：
Sequence 1 is SH-CP25s：5’-TTCGTGGGATGGGGAGC-3’；
Sequence 2 is SH-CP25a：5’-TGGACATCCGAGCCCTA-3’；
Theoretical PCR primer length is 262bp (sequence 3).
The primer pair of primer SH-CP25s single strand dnas and primer SH-CP25a single strand dnas composition is applied to mirror
Determine Dendrobidium huoshanness and dendrobium candidum technical field.
2nd, for the assembling for the kit for identifying Dendrobidium huoshanness
After the primer SH-CP25s and SH-CP25a of step 1 design synthesis are individually packed, with individually packing
DNTP, archaeal dna polymerase, 10 × buffer buffer solutions etc. be packaged in same kit, that is, obtain the present invention and be used to identify
The kit of Dendrobidium huoshanness.
3rd, identify testing sample in whether the method containing Dendrobidium huoshanness
Identify in testing sample whether contain Dendrobidium huoshanness according to the method comprised the following steps using kit：
1st, PCR is expanded
From testing sample extract genomic DNA be used as template, using step 1 design synthesis primer SH-CP25s with
SH-CP25a (sequence 1 and sequence 2) proceeds as follows PCR amplifications：PCR reacts the μ L of cumulative volume 25, including following reagent：0.5μ
L template DNAs, 1.0 μ L sense primers (10pmol), 1.0 μ L anti-sense primers (10pmol), 2.5 μ L 10 × buffer buffer solutions,
1.5 μ L concentration are the 25mM MgCl2 aqueous solution, and 0.5 μ L concentration is 10mM dNTPs, 0.5 μ L Taq archaeal dna polymerases (5U/ μ
L), aseptic double-distilled water complements to 25 μ L.
After PCR reaction solutions have been prepared, gently concussion is mixed, and PCR pipe is put into PCR instrument, enters performing PCR amplification, specific anti-
Answer condition as follows：95℃5min；95 DEG C of 30s, 60 DEG C of 30s, 72 DEG C of 30s, 45 circulations；72℃10min.
2nd, PCR primer detects
PCR primer is detected using agarose gel electrophoresis method, it is specific as follows：
5 μ L amplified productions are taken, using 2% Ago-Gel, electrophoresis 30 minutes under voltage 80-90V, gel imaging system
Observe and take pictures under system.According to the size of purpose band, determine whether contain Dendrobidium huoshanness in testing sample as follows：
If obtaining the purpose band (sequence 3) that size is about 262bp, contain Dendrobidium huoshanness in the testing sample；If there is no
Size is 262bp purpose band (sequence 3), then does not contain Dendrobidium huoshanness in the testing sample.
4th, the specificity for identifying Dendrobidium huoshanness using kit is analyzed
Testing sample：Dendrobidium huoshanness (picking up from Huoshan) and dendrobium candidum (picking up from Huoshan).In meeting
The diagnostic characteristics of state's flora and pertinent literature describes.By identification, the material object of each sample is consistent with title, and quality meets mark
It is accurate.
1st, genomic DNA is extracted from testing sample
About 50mg drying samples (can also use 100mg fresh samples to carry out extracting genome DNA certainly) are taken respectively, are used
CTAB methods extract STb gene.It is specific as follows：
Ground being placed in without the drying medicinal material to go mouldy in pulverizer, cross 40 mesh sieves.Powder is transferred to the micro- of 2.0mL
Measure in centrifuge tube, add the sterilized CTAB extract solutions of 900 μ L (formula：2% (2g/100ml) CTAB, 100mmol/L Tris-
HCl pH=8.0,20mmol/L EDTA, 1.4mol/L NaCl), 0.02g PVP 40000,10 μ L beta -mercaptoethanols it is abundant
Vibration mixes, 65 DEG C of water-bath 1.5h-2h, during which jog 2-3 times.Taken out after end and be cooled to room temperature, 900 μ L chloroforms of addition-different
Amylalcohol (volume ratio 24：1), fully vibration mixes, 12000g centrifugations 10min.Supernatant is taken, adds isometric chloroform-isoamyl alcohol (body
Product ratio 24：1), fully vibration mixes, 12000g centrifugations 10min.Take supernatant, add the aqueous isopropanol of 2/3 volume precooling, -20
DEG C place more than 0.5h.To take out, 12000g centrifugation 10min, abandon supernatant, precipitation is washed twice with 70% (volume fraction) ethanol,
37 DEG C volatilize ethanol, are dissolved with appropriate aqua sterilisa, -20 DEG C of preservations.
The genomic DNA quality of the Dendrobidium huoshanness of extraction and dendrobium candidum above is detected with universal primer TY1s and TY1a.
TY1s：5’-GATGGATGAACCCTCAAATC-3’；
TY1a：5’-GGCAGCCAACGAGAAGA-3’.
Reaction total system is 25 μ L, including the μ L (5-50ng) of DNA profiling 0.5, and the μ L (10pmol) of sense primer 1, downstream is drawn
1.5 0.5 μ L, the Taq DNA of μ L, dNTPs (10mM) of μ L, MgCl2 (25mM) of thing 1 μ L (10pmol), 10 × PCR Buffer 2.5
Polymerase (5U/ μ L) 0.5 μ L, ddH2O complement to 25 μ L.Universal primer PCR reaction condition is：95 DEG C of 5min, 35 circulations are (every
Individual circulation includes 95 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 30s), 72 DEG C of 10min.
As a result find that all samples can amplify DNA bands (accompanying drawing 2).
2nd, PCR is expanded
The genomic DNA extracted from testing sample is template, using primer pair (primer SH-CP25s and SH-CP25a)
Enter performing PCR amplification.Specific reaction system and reaction condition are the same as the PCR amplification conditions in embodiment step 3.Experiment is set simultaneously
Put by template of distilled water and be used as negative control.Experiment is in triplicate.
After reaction terminates, the method detected according to the PCR primer in embodiment step 3 is identified testing sample.
As a result as shown in Figure 3, as can be seen from the figure：
Dendrobidium huoshanness and dendrobium candidum are examined using the primer pair (primer SH-CP25s and SH-CP25a) of the present invention
Survey, Dendrobidium huoshanness can amplify the purpose band that clip size is about 262bp.And dendrobium candidum does not amplify purpose band.
This shows that the reaction system can accurately identify Dendrobidium huoshanness with dendrobium candidum.The purpose bar for being about 262bp by size
Band recovery sequencing, its sequence are just being sequence 3 in sequence table.
5th, the sensitivity analysis of Dendrobidium huoshanness is identified using kit
Testing sample：Dendrobidium huoshanness (is picked up from Huoshan).By identification, sample object is consistent with title, quality symbol
Standardization.
1st, genomic DNA is extracted from testing sample
About 50mg drying samples (can also use 100mg fresh samples to carry out extracting genome DNA certainly) are taken respectively, are used
CTAB methods extract STb gene.Concrete operations are referring to the step 1 of embodiment 2.
2nd, PCR is expanded
The genomic DNA extracted from Dendrobidium huoshanness is subjected to doubling dilution, obtaining genomic DNA concentration is respectively
210ng/ μ l, 105ng/ μ l, 52ng/ μ l, 26ng/ μ l, 13ng/ μ l, 7ng/ μ l and 3ng/ μ l serial dilutions, it is dilute with series
It is respectively template to release liquid, and the primer pair (primer SH-CP25s and SH-CP25a) designed using the step 1 of embodiment 1 is entered performing PCR and expanded
Increase.Specific reaction system and reaction condition are the same as PCR amplification conditions in embodiment step 3.Experiment simultaneously set using distilled water as
Template is as negative control.Experiment is in triplicate.
After reaction terminates, testing sample is identified according to the PCR primer detection method in embodiment step 3.
As a result as shown in Figure 4, as can be seen from the figure：
Dendrobidium huoshanness is detected using the primer pair (primer SH-CP25s and SH-CP25a) of the present invention, when template is dense
Spend still to be able to detect the purpose band that size is about 262bp during 52ng/ μ l.The purpose band that size is about 262bp is returned
Sequencing is received, its sequence is just being sequence 3 in sequence table.
The foregoing is only a preferred embodiment of the present invention, but protection scope of the present invention be not limited thereto,
Any one skilled in the art the invention discloses technical scope in, technique according to the invention scheme and its
Inventive concept is subject to equivalent substitution or change, should all be included within the scope of the present invention.
Sequence table
<110>Western Anhui University
<120>A kind of primer pair and its application for being used to identify Dendrobidium huoshanness and dendrobium candidum
<141> 2017-11-13
<160> 6
<170> SIPOSequenceListing 1.0
<210> 1
<211> 17
<212> DNA
<213>Artificial sequence (Synthetic sequence)
<400> 1
ttcgtgggat ggggagc 17
<210> 2
<211> 17
<212> DNA
<213>Artificial sequence (Synthetic sequence)
<400> 2
tggacatccg agcccta 17
<210> 3
<211> 262
<212> DNA
<213>Dendrobidium huoshanness (Dendrobium huoshanense)
<400> 3
ttcgtgggat ggggagctgt cgcacgccat attgattgac acgactctcg gcaatggata 60
tctcggctct cgcatcgatg aagagcgcag cgaaatgcga tatgtggtgc gaattgcaga 120
atcccgcgaa ccatcgagtc tttgaacgca agttgcgcct gaggccaacc ggctgagggc 180
acgtccgcct gggcgtcaag cattttatca ctccgtgcct actctcccat ccatggatgt 240
gttgctaggg ctcggatgtc ca 262
<210> 4
<211> 629
<212> DNA
<213>The Henan stem of noble dendrobium (Dendrobium henanense)
<400> 4
aaacacaacg agcgattttg tgaacctgta aaaataagcg gtggctcttg ctgctgcgat 60
aaaatccacc cgagtcatcg cctcatcccc tctttggggt ggggacgtga tgaaggatgg 120
atgaaccctc aaatcggcgc agcgtagcgc caagggaatc ttgaaacaca agcctataaa 180
tgggttttgt gggatggggt gttgtcgcac gccatattga ttgacacgac tctcggcaat 240
ggatatctcg gctctcgcat cgatgaagag cgcagcgaaa tgcgatatgt ggtgcgaatt 300
gcagaatccc gcgaaccatc gagtctttga acgcaagttg cgcctgaggc caactggctg 360
agggcacgtc cgcctgggcg tcaagcattt tatcactctg tgcctagtct cccatccatg 420
gatgtgttgc caaggctcgg atgtgcacgg tggctcgtcg tgccccttgg tgcggcgggc 480
tgaagggctg gtcatcttct cgttggctgc caacaataag gggtggatta aataaggcct 540
atgctattgt gtcaagcgcg cctgagagat ggtcatactt ttctatgtga tcccaattca 600
tgcgttgatc catggatggc gtatcgaat 629
<210> 5
<211> 629
<212> DNA
<213>The Henan stem of noble dendrobium (Dendrobium henanense)
<400> 5
aaacacaacg agcgattttg tgaacctgta aaaataagcg gtggctcttg ctgctgcgat 60
aaaatccacc cgagtcatcg cctcatcccc tctttggggt ggggacgtga tgaaggatgg 120
atgaaccctc aaatcggcgc agcgtagcgc caagggaatc ttgaaacaca agcctataaa 180
tgggttttgt gggatggggt gttgtcgcac gccatattga ttgacacgac tctcggcaat 240
ggatatctcg gctctcgcat cgatgaagag cgcagcgaaa tgcgatatgt ggtgcgaatt 300
gcagaatccc gcgaaccatc gagtctttga acgcaagttg cgcctgaggc caactggctg 360
agggcacgtc cgcctgggcg tcaagcattt tatcactctg tgcctagtct cccatccatg 420
gatgtgttgc caaggctcgg atgtgcacgg tggctcgtcg tgccccttgg tgcggcgggc 480
tgaagggcgg gtcatcttct cgttggctgc caacaataag gggtggatta aataaggcct 540
atgctattgt gtcaagcgcg cctgagagat ggtcatactt ttctatgtga tcccaattca 600
tgcgttgatc catggatggc gtatcgaat 629
<210> 6
<211> 628
<212> DNA
<213>Dendrobidium huoshanness (Dendrobium huoshanense)
<400> 6
aaacacaacg agcgattttg tgaacctgta aaaataagcg gtggctcttg ctgctgcgat 60
aaaatccacc cgagtcatcg cctcatcccc tctttggggt ggggacgtga tgaaggatgg 120
atgaaccctc aaatcggcgc agcgttgcgc caagggaatc ttgaagcaca agcccataaa 180
tgggtttcgt gggatggggt gctgtcgcac gccatattga ttgacacgac tctcggcaat 240
ggatatctcg gctctcgcat cgatgaagag cgcagcgaaa tgcgatatgt ggtgcgaatt 300
gcagaatccc gcgaaccatc gagtctttga acgcaagttg cgcctgaggc caaccggctg 360
agggcacgtc cgcctgggcg tcaagcattt tatcactccg tgcctactct cccatccatg 420
gatgtgttgc taaggctcgg atgtgcacgg tggctcgtcg tgccccttgg tgcggcgggc 480
tgaagggcgg gtcatcttct cgttggctgc caacaataag gggtggatta aataaggcct 540
atgctattgt gtcaagcgcg cccgagagat ggtcatactt tttaggtgat cccaattcat 600
gcgttgatcc atggatggcg tatcgaat 628
Claims (7)
1. a kind of primer pair for being used to identify Dendrobidium huoshanness and dendrobium candidum, it is characterised in that the primer pair is by primer SEQ
ID No.1 single strand dnas and primer SEQ ID No.2 single strand dnas composition, the SEQ ID No.1 single strand dnas
For the single strand dna in sequence in sequence table 1, the SEQ ID No.2 single strand dnas is in sequences 2 in sequence table
Single strand dna；
Wherein, sequence 1 is SH-CP25s：5’-TTCGTGGGATGGGGAGC-3’；
Sequence 2 is SH-CP25a：5’-TGGACATCCGAGCCCTA-3’.
A kind of 2. kit for being used to identify Dendrobidium huoshanness and dendrobium candidum, it is characterised in that：The kit include primer pair,
DNTP and archaeal dna polymerase, the primer pair are single-stranded by primer SEQ ID No.1 single strand dnas and primer SEQ ID No.2
DNA molecular forms.
3. the kit according to claim 2 for being used to identify Dendrobidium huoshanness and dendrobium candidum, it is characterised in that：The examination
The molal quantity that two single strand dnas of primer pair are formed in agent box is identical.
A kind of 4. application for being used to identify Dendrobidium huoshanness and dendrobium candidum primer pair described in claim 1, it is characterised in that institute
The primer pair for stating primer SEQ ID No.1 single strand dnas and primer SEQ ID No.2 single strand dnas composition is applied to mirror
Determine Dendrobidium huoshanness and dendrobium candidum.
5. it is a kind of identification whether the method containing Dendrobidium huoshanness, it is characterised in that the authentication method comprises the following steps：
1. genomic DNA is extracted from testing sample as template, using primer SEQ ID No.1 single strand dnas and primer
The primer SH-CP25s and SH-CP25a of SEQ ID No.2 single strand dnas composition PCR in PCR reaction solutions and PCR instrument expand
Increase；
2. PCR primer detects：PCR primer is detected using agarose gel electrophoresis method, identified according to the size of purpose band to be measured
Whether contain Dendrobidium huoshanness in sample.
6. it is according to claim 5 identification whether the method containing Dendrobidium huoshanness, it is characterised in that：The PCR reaction solutions
Including following reagent：2% template DNA, 4% 10 pmol sense primers, 4% pmol anti-sense primers, 10% 10 × buffer bufferings
Liquid, 6% concentration are 25 mM MgCl2The aqueous solution, 2% concentration are 10 mM dNTPs, and 2% 5U/ μ L Taq archaeal dna polymerases are remaining
Measure as aseptic double-distilled water.
7. it is according to claim 5 identification whether the method containing Dendrobidium huoshanness, it is characterised in that：The Dendrobidium huoshanness or
For dendrobium candidum.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711111212.7A CN107619879A (en) | 2017-11-13 | 2017-11-13 | A kind of primer pair and its application for being used to identify Dendrobidium huoshanness and dendrobium candidum |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201711111212.7A CN107619879A (en) | 2017-11-13 | 2017-11-13 | A kind of primer pair and its application for being used to identify Dendrobidium huoshanness and dendrobium candidum |
Publications (1)
| Publication Number | Publication Date |
|---|---|
| CN107619879A true CN107619879A (en) | 2018-01-23 |
Family
ID=61098914
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201711111212.7A Withdrawn CN107619879A (en) | 2017-11-13 | 2017-11-13 | A kind of primer pair and its application for being used to identify Dendrobidium huoshanness and dendrobium candidum |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN107619879A (en) |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110878376A (en) * | 2019-12-31 | 2020-03-13 | 安徽师范大学 | SSR molecular marker primer for identifying dendrobium huoshanense and application thereof |
| CN112725511A (en) * | 2021-02-22 | 2021-04-30 | 拱北海关技术中心 | Micro-drop digital PCR (polymerase chain reaction) primer, probe, kit and method for quantitatively detecting dendrobium officinale |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106119408A (en) * | 2016-09-23 | 2016-11-16 | 皖西学院 | A kind of authentication method of Herba Dendrobii |
| CN106282373A (en) * | 2016-09-23 | 2017-01-04 | 皖西学院 | A kind of Herba Dendrobii and the contrast authentication method of Henan Herba Dendrobii |
-
2017
- 2017-11-13 CN CN201711111212.7A patent/CN107619879A/en not_active Withdrawn
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN106119408A (en) * | 2016-09-23 | 2016-11-16 | 皖西学院 | A kind of authentication method of Herba Dendrobii |
| CN106282373A (en) * | 2016-09-23 | 2017-01-04 | 皖西学院 | A kind of Herba Dendrobii and the contrast authentication method of Henan Herba Dendrobii |
Non-Patent Citations (1)
| Title |
|---|
| CHIN-TUNG WU等: "Internal Transcribed Spacer Sequence Based Identification and Phylogenic Relationship of Herba Dendrobii", 《JOURNAL OF FOOD AND DRUG ANALYSIS》 * |
Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110878376A (en) * | 2019-12-31 | 2020-03-13 | 安徽师范大学 | SSR molecular marker primer for identifying dendrobium huoshanense and application thereof |
| CN110878376B (en) * | 2019-12-31 | 2022-11-22 | 安徽师范大学 | SSR molecular marker primer for identifying dendrobium huoshanense and application thereof |
| CN112725511A (en) * | 2021-02-22 | 2021-04-30 | 拱北海关技术中心 | Micro-drop digital PCR (polymerase chain reaction) primer, probe, kit and method for quantitatively detecting dendrobium officinale |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN103898235B (en) | A kind of DNA bar code method for identifying molecules of Hirudo | |
| Setaro et al. | Sebacinales form ectendomycorrhizas with Cavendishia nobilis, a member of the Andean clade of Ericaceae, in the mountain rain forest of southern Ecuador | |
| CN103173532B (en) | Method for identifying honeysuckle and lonicera confuse and application of same | |
| CN106282373A (en) | A kind of Herba Dendrobii and the contrast authentication method of Henan Herba Dendrobii | |
| CN103468709B (en) | Chloroplast genome of dendrobium huoshanense and germplasm identification method | |
| CN106754886A (en) | Based on the method that transcription sequencing obtains black fruit fructus lycii SSR primers | |
| CN107586867A (en) | Thin shell mountain pecan Peach cultivars Pawnee characteristic sequence, labeled primer and authentication method | |
| CN106119408A (en) | A kind of authentication method of Herba Dendrobii | |
| CN107557434A (en) | Thin shell mountain pecan Peach cultivars Van Deman characteristic sequence, labeled primer and authentication method | |
| CN110184393A (en) | A kind of primer sets, purposes, kit and its detection method detecting African swine fever virus | |
| CN107619879A (en) | A kind of primer pair and its application for being used to identify Dendrobidium huoshanness and dendrobium candidum | |
| CN106434944A (en) | Application of SNP molecular marker closely linked to aphid resistance gene of prunus persica | |
| CN105219882A (en) | Mycobacterium avium-intracellulare compound group detects by primer and probe and its detection method | |
| CN106701943A (en) | Persimmon tree colletotrichum gloeosporioide SSR (Simple Sequence Repeat) primer pair developed based on sibling species genome and application of primer pair | |
| CN104232782A (en) | PCR (polymerase chain reaction) primer for detecting tobacco soil-borne fungal pathogens as well as application and method of PCR primer | |
| CN106350598B (en) | For the PCR primer pair of Citrus Huanglongbing pathogen early warning, kit and method | |
| CN105256052B (en) | A kind of kit and its detection method for Legionella quick detection and parting | |
| CN106148560A (en) | A kind of comparison authentication method for Herba Dendrobii with weight lip Herba Dendrobii | |
| CN105200130B (en) | The bar code identification method and its primer special of Michigan clavibacter difference subspecies | |
| CN107164525A (en) | A kind of DNA for differentiating 6 kinds of Pterocarpus timber combines bar code and its discrimination method and application | |
| CN103834735A (en) | Nucleotide sequence and molecular probe for identifying dendrobium officinale and applications of molecular probe | |
| CN106282372A (en) | Herba Dendrobii and the comparison authentication method of Dendrobium loddigesii | |
| CN103436612A (en) | PCR-FRLP quick detecting method of common sturgeons | |
| CN106434645A (en) | ITS (internal transcribed spacer) sequence of dalbergia odorifera and method for identifying dalbergia odorifera by ITS sequence | |
| CN106811506A (en) | A kind of method for detecting Botryosphaeria berengeriana f. sp and its relative populations |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| WW01 | Invention patent application withdrawn after publication |
Application publication date: 20180123 |
|
| WW01 | Invention patent application withdrawn after publication |