CN105274243B - A kind of method and its special primer pair of identification tulip bulb - Google Patents
A kind of method and its special primer pair of identification tulip bulb Download PDFInfo
- Publication number
- CN105274243B CN105274243B CN201510815696.8A CN201510815696A CN105274243B CN 105274243 B CN105274243 B CN 105274243B CN 201510815696 A CN201510815696 A CN 201510815696A CN 105274243 B CN105274243 B CN 105274243B
- Authority
- CN
- China
- Prior art keywords
- tulip bulb
- tested
- sample
- candidate
- primer
- 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.)
- Active
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
- C12Q1/6888—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms
- C12Q1/6895—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes for detection or identification of organisms for plants, fungi or algae
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q2600/00—Oligonucleotides characterized by their use
- C12Q2600/13—Plant traits
Landscapes
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Analytical Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Organic Chemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Health & Medical Sciences (AREA)
- Biotechnology (AREA)
- Zoology (AREA)
- Wood Science & Technology (AREA)
- Immunology (AREA)
- Mycology (AREA)
- Microbiology (AREA)
- Molecular Biology (AREA)
- Botany (AREA)
- Biophysics (AREA)
- Physics & Mathematics (AREA)
- Biochemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- General Health & Medical Sciences (AREA)
- Genetics & Genomics (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention discloses a kind of methods and its special primer pair of identification tulip bulb.Primer pair provided by the present invention is made of primer LYB CP20s and primer LYB CP20a, and primer LYB CP20s and primer LYB CP20a 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 LYB CP20s and primer LYB CP20a, if it is possible to realize effectively amplification, then contain in the sample to be tested or candidate contains tulip bulb；If can not achieve effective amplification, do not contained in the sample to be tested or candidate without containing tulip bulb.It is demonstrated experimentally that identification tulip bulb that can be quick and easy using the method for identification tulip bulb provided by the invention, to ensure the safety of tulip bulb clinical application, with great promotion prospect and application value.
Description
Technical field
The invention belongs to biotechnologies, and in particular to a kind of method and its special primer pair of identification tulip bulb.
Background technology
Tulip bulb (Bulbus Tulipae) removes for Liliaceae (Liliaceae) plant Tulipa edulis (Amana edulis)
Dry bulb after film quality skin and villus has effects that detoxicating and resolving a mass, promoting circulation of blood stagnation resolvation, cure mainly abscess of throat, scrofula, ulcer,
Sore swells and the postpartum stasis of blood are stagnant etc..Recent study shows that tulip bulb has significant effect for the treatment of mammary gland disease, therefore leads
Cause the increase of tulip bulb demand.But tulip bulb is mainly derived from wild, and wild resource is gradually deficient, results in going out for adulterant
Now with it is mixed, hamper the accuracy of clinical application.In order to ensure safety and the validity of clinical application, need to tulip bulb
And adulterant is differentiated.
Tulip bulb and its adulterant Main Basiss formalness, microscopic features, thin-layered chromatography, ultraviolet spectra Absorption Characteristics
Differentiated with the methods of liquid chromatogram.The resolution ratio having in these methods is not high, and some method complexity are time-consuming, are unfavorable for pushing away
Extensively, therefore there is an urgent need to establish the method for quick, easy identification tulip bulb, to ensure the safety of tulip bulb clinical application.
Invention content
The technical problems to be solved by the invention how identification tulip bulb quickly, easy.
To solve the above problems, present invention firstly provides a kind of primer pairs for identifying tulip bulb or Tulipa edulis.
Primer pair provided by the present invention for identifying tulip bulb or Tulipa edulis, can be by primer LYB-CP20s and primer
LYB-CP20a is formed, and each primer is single strand dna, and nucleotide sequence is followed successively by sequence 1 and sequence 2 in sequence table.
In the above-mentioned primer pair for identifying tulip bulb or Tulipa edulis, the primer LYB-CP20s and primer LYB-CP20a
Molar ratio can be 1:1.
It is above-mentioned for identify tulip bulb or Tulipa edulis primer pair application, be following a1)-a12) and in it is any：
A1 the kit for detecting or assisting detection tulip bulb) is prepared；
A2 it) detects or assists whether to contain in detection sample to be tested or candidate contains tulip bulb；
A3 the kit for identifying or assisting identification tulip bulb) is prepared；
A4) identify or assist to identify sample to be tested whether be or candidate is tulip bulb；
A5 the kit for identifying or assisting to identify the true or false of commercially available tulip bulb) is prepared；
A6) identify or assist the true or false of the commercially available tulip bulb of identification；
A7 the kit for detecting or assisting detection Tulipa edulis) is prepared；
A8 it) detects or assists whether to contain in detection sample to be tested or candidate contains Tulipa edulis；
A9 the kit for identifying or assisting identification Tulipa edulis) is prepared；
A10) identify or assist to identify sample to be tested whether be or candidate is Tulipa edulis；
A11 the kit for identifying or assisting to identify the true or false of commercially available Tulipa edulis) is prepared；
A12) identify or assist the true or false of the commercially available Tulipa edulis of identification.
The present invention also provides the kits for identifying tulip bulb or Tulipa edulis.It is provided by the present invention to be used to identify light
The kit of arrowhead or Tulipa edulis contains the above-mentioned primer pair for identifying tulip bulb or Tulipa edulis.
The present invention also protects the above-mentioned preparation method for identifying the kit of tulip bulb or Tulipa edulis, it may include will be above-mentioned
For identifying the step of each primer of the primer pair of tulip bulb or Tulipa edulis is individually packed.
It is above-mentioned to be used to identify that the application of the kit of tulip bulb or Tulipa edulis also belongs to protection scope of the present invention.Above-mentioned use
In the kit for identifying tulip bulb or Tulipa edulis application be following b1) b2) or b3)：
B1 it) detects or assists whether to contain in detection sample to be tested or candidate contains tulip bulb or Tulipa edulis；
B2) identify or assist to identify sample to be tested whether be or candidate is tulip bulb or Tulipa edulis；
B3) identify or assist the true or false of the commercially available tulip bulb of identification or Tulipa edulis.
Whether contain the present invention also provides a kind of detection sample to be tested or the candidate method containing tulip bulb, it may include such as
Lower step：The total DNA for extracting sample to be tested is carried out using any of the above-described primer pair for identifying tulip bulb or Tulipa edulis
PCR amplification, if it is possible to realize effectively amplification, then contain in the sample to be tested or candidate contains tulip bulb；If cannot be real
Existing effect amplification does not contain in the sample to be tested then or candidate does not contain tulip bulb.The sample to be tested can be Chinese medicine,
Or, the Original plant of the Chinese medicine, or, being derived from the tissue or organ of the Original plant of the Chinese medicine.
The present invention also provides it is a kind of identify sample to be tested whether be or method that candidate is tulip bulb, it may include following step
Suddenly：The genomic DNA for extracting sample to be tested is carried out using any of the above-described primer pair for identifying tulip bulb or Tulipa edulis
PCR amplification, if it is possible to realize effectively amplification, then the sample to be tested is or candidate is tulip bulb；If can not achieve effectively
Amplification, then the sample to be tested is not or candidate is not tulip bulb.The sample to be tested can be Chinese medicine, or, the Chinese medicine
Original plant, or, being derived from the tissue or organ of the Original plant of the Chinese medicine.
The present invention also provides a kind of methods of the true or false of the commercially available tulip bulb of identification, it may include following steps：Extract city
The genomic DNA for selling tulip bulb carries out PCR expansions using any of the above-described primer pair for identifying tulip bulb or Tulipa edulis
Increase, if it is possible to realize effectively amplification, then the commercially available tulip bulb is or candidate is genuine piece；If can not achieve effective amplification,
Then the commercially available tulip bulb is or candidate is adulterant.
The present invention also provides a kind of methods of the true or false of the commercially available Tulipa edulis of identification, it may include following steps：Extract city
The genomic DNA for selling Tulipa edulis carries out PCR expansions using any of the above-described primer pair for identifying tulip bulb or Tulipa edulis
Increase, if it is possible to realize effectively amplification, then the commercially available Tulipa edulis is or candidate is genuine piece；If can not achieve effective amplification,
Then the commercially available Tulipa edulis is or candidate is adulterant.
In any of the above-described the method, described " can realize effective amplification " can be to contain in the pcr amplification product
The DNA fragmentation of 400-500bp.
In any of the above-described the method, described " can not achieve effective amplification " can be not contained in the pcr amplification product
The DNA fragmentation of 400-500bp.
The DNA fragmentation of the 400-500bp can be the DNA fragmentation of 432bp.
The DNA fragmentation of the 432bp concretely DNA fragmentation shown in sequence 3 in sequence table.
In any of the above-described the method, when carrying out the PCR amplification, concretely 55 DEG C of the annealing temperature of use.
In any of the above-described the method, when carrying out the PCR amplification, the amplification program of use concretely 95 DEG C of 5min；
95 DEG C of 30s, 55 DEG C of 30s, 25 cycles；72℃10min.
The Chinese medicine can be tulip bulb and/or hair arrowhead and/or pleionebulbocodioides rolfe and/or tinosporae.
The Original plant of the Chinese medicine can be Tulipa edulis (Amana edulis), Cremastra appendiculata (Cremastra
Appendiculata (D.Don) Makino), pleione bulbocodioides (Pleione bulbocodioides) or Pleione yunnanensis
(Pleione yunnanensis)。
It being capable of quick and simplicity identification light using the method and its special primer pair of identification tulip bulb provided by the invention
Arrowhead has great promotion prospect and application value to ensure the safety of tulip bulb clinical application.
Description of the drawings
Fig. 1 is the phylogenetic tree of tulip bulb 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 implementation mode
The present invention is further described in detail With reference to embodiment, the embodiment provided is only for explaining
The bright present invention, the range being not intended to be limiting of the invention.
Experimental method in following embodiments is unless otherwise specified conventional method.
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.
Tulip bulb (Original plant is Tulipa edulis (Amana edulis)) in the present embodiment is purchased from Quanjiao, Anhui Province county, and hair is kind
(Original plant is for mushroom (Original plant is Cremastra appendiculata (Cremastra appendiculata (D.Don) Makino)), pleionebulbocodioides rolfe
Pleione bulbocodioides (Pleione bulbocodioides) or Pleione yunnanensis (Pleione yunnanensis)) and tinosporae purchase
From in Hui nationality's medicinal material market.Each Chinese medicine meets related under each medicinal material item of text of Chinese Pharmacopoeia (version in 2010)
Regulation, by identification, every Chinese medicine material object is consistent with title, and quality complies with standard.
The kit and its application method of embodiment 1, preparation for identifying tulip bulb
One, the design and synthesis of the primer pair for identifying tulip bulb
The ITS sequence of tulip bulb (Original plant is Tulipa edulis (Amana edulis)) and adulterant is searched from GenBank,
Refer to table 1.
The ITS sequence quantity and GenBank accession number of 1. tulip bulb of table and adulterant
Use respectively 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 water pepper (Polygonum hydropiper)
(GenBank accession number is JF977851 and JF977850) is outer group, and interior group includes then tulip bulb 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 MrModeltest version2.3 softwares according to AIC (Akaike Information
Criterion) the optimality model of test stone selection data, selected optimality model is (GTR+G).Markovian Meng Teka
Lip river method (Markov Chains Monte Carlo, MCMC) is set as four chains and ran for 1000000 generations.In order to determine it
Convergent, MCMC are separately operable twice.In every 100 generation, extracts a sample, forms 20002 samples altogether.It learns by analysis,
Whole service reaches steady after 20000 generations, in this way, remaining sample number is 19602 in total, with remaining sample reconstructing system tree
And estimate its posterior probability values.When building MP trees, setting bootstrapping number of repetition bootstrap nreps are 1000 times, using opening
Hairdo searching analysis bootstrapping duplicate data collection, heuristic search are set as, by gradually additive process generates initial tree at random, repeating 10
It is secondary, using TBR branch exchanges.
The phylogenetic tree built by BI methods and MP methods shows that the genealogical tree of 2 kinds of methods structure is completely the same (Fig. 1).
Posterior probability (posterior probability, PP) value and the MP trees of BI trees are indicated at the node of Tu1Zhong Ge pedigrees branch
Support of booting (Bootstrap, BS).The result shows that the haplotype of tulip bulb all sequences forms monosystem (PP=1.00, BS
=90).
Under the premise of it is monosystem group to determine tulip bulb, all ITS sequences are carried out pair with software Clustal X 1.81
Position sequence and compare, finds differential fragment, designed and synthesized according to the distinctive variant sites of tulip bulb and to be identified for tulip bulb
A large amount of PCR primers 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 best is obtained eventually：
Primer LYB-CP20s：5 '-GAGAAGTCGTAACAAGGTTTCCGTAG-3 ' (sequence 1 in sequence table)；
Primer LYB-CP20a：5 '-AGGCAGGCGTGCCCGCA-3 ' (sequence 2 in sequence table).
Primer LYB-CP20s and primer LYB-CP20a is single strand dna.
The length of theoretical pcr amplification product is 432bp.
Two, the kit for identifying tulip bulb is prepared
For identifying that the kit of tulip bulb is the primer LYB-CP20s for synthesizing step 1 and LYB-CP20a points of primer
After not packing not individually, with the 10 × PCR Buffer, Taq DNA polymerase, MgCl individually packed2(25mM) and
DNTPs etc. is packaged in the same kit.
Three, identify sample to be tested in whether the foundation of the method containing tulip bulb
Using whether tulip bulb is contained in the kit identification sample to be tested of step 2, steps are as follows：
1, PCR amplification
It extracts the genomic DNA of sample to be tested and using it as template, it is anti-that PCR amplification is carried out using the kit of step 2
It answers, obtains 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 LYB-CP20s and LYB-
Each 10pmol of CP20a are mended with deionized water to 25 μ L.
Response procedures：95℃5min；95 DEG C of 30s, 55 DEG C of 30s, totally 25 recycle；72℃10min.
2, 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 at 80-90V of voltage
It observes and takes pictures under system.Result judgement is carried out according to the size of purpose band：If being about containing size in pcr amplification product
The purpose band (sequence 3 of sequence table) of 432bp, then contain in the sample to be tested or candidate contain tulip bulb；If PCR amplification
The purpose band (sequence 3 of sequence table) for being about 432bp without containing size in product, then do not contain or wait in the sample to be tested
Choosing does not contain tulip bulb.
The specificity of kit for identifying tulip bulb prepared by embodiment 2, embodiment 1
In triplicate, the step of repetition is as follows every time for experiment：
1, it takes about 50mg to dry sample to be tested (tulip bulb, hair arrowhead, pleionebulbocodioides rolfe or tinosporae), gene is extracted with CTAB methods
Group DNA, the concentration of the genomic DNA of sample to be tested is between 30ng/ μ l-50ng/ μ l.
2, the genomic DNA of the sample to be tested extracted using step 1 is template, with artificial synthesized LYB-TY1s：5’-
AACGGATATCTCGGCTCT-3 ' and LYB-TY1a：5 '-GCAACTTGCGTTCAAAA-3 ' are universal primer, carry out PCR expansions
Increase, detects the genomic DNA quality of sample to be tested.
Reaction total system be 25 μ L, including 0.5 μ L of genomic DNA of sample to be tested, 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
With each 10pmol of LYB-TY1a, deionized water is mended to 25 μ L.
Response procedures：95℃5min；95 DEG C of 30s, 55 DEG C of 30s, 72 DEG C of 30s, totally 25 recycle；72℃10min.
According to above-mentioned steps, tulip bulb genomic DNA is extracted, and the genomic DNA of sample to be tested is replaced in equal volume
Tulip bulb 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 is shown in Fig. 2 (swimming lane M be DNA molecular standard (DNA Marker, be followed successively by 600 from top to bottom, 500,
400,300,200 and 100bp), swimming lane 1 is using tulip bulb genomic DNA as template, and swimming lane 2 is to be with hair arrowhead genomic DNA
Template, swimming lane 3 be using pleionebulbocodioides rolfe genomic DNA as template, swimming lane 4 be using tinosporae genomic DNA as template, swimming lane 5 be with
Sterilizing ultra-pure water is template).The result shows that positive control and sample to be tested can realize that effectively amplification, electrophoresis showed contain greatly
The small about purpose band of 432bp；Blank control can not achieve effective amplification, and electrophoresis showed is about 432bp's without containing size
Purpose band.The purpose band that size is about 432bp is recycled and is sequenced, in sequence such as sequence table shown in sequence 3.It can be seen that be measured
The genomic DNA quality of sample is satisfied by requirement.
3, 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 LYB-CP20s and primer LYB-CP20a compositions, carry out PCR amplification.Specific reaction system and response procedures are the same as real
Apply 1 step 31 of example.It tests while being arranged using the ultra-pure water that sterilizes as the negative control of template.After reaction, according to embodiment 1
The method of step 32 carries out result judgement to sample to be tested.
Experimental result is shown in Fig. 3 (swimming lane M be DNA molecular standard (DNA Marker, be followed successively by 600 from top to bottom, 500,
400,300,200 and 100bp), swimming lane 1 is using tulip bulb genomic DNA as template, and swimming lane 2 is to be with hair arrowhead genomic DNA
Template, swimming lane 3 be using pleionebulbocodioides rolfe genomic DNA as template, swimming lane 4 be using tinosporae genomic DNA as template, swimming lane 5 be with
Sterilizing ultra-pure water is template).The result shows that only tulip bulb can amplify the purpose band that size is about 432bp.About by size
It is sequenced for the purpose band recycling of 432bp, in sequence such as sequence table shown in sequence 3.This show prepared by embodiment 1 for reflecting
The kit for determining tulip bulb being capable of precise Identification tulip bulb.
The sensitivity of kit for identifying tulip bulb prepared by embodiment 3, embodiment 1
In triplicate, the step of repetition is as follows every time for experiment：
1, the genomic DNA that tulip bulb is extracted with CTAB methods, is named as DNA1.A concentration of 43.2ng/ μ of DNA in DNA1
L。
2, absorption 1mL DNA1 are added in the test tube filled in 1mL sterilizing ultra-pure waters and mix well, and obtain DNA2；With such
Pushing at DNA3, DNA4, DNA5, DNA6 and DNA7, after dilution the DNA concentration of each gradient be respectively 21.6ng/ μ L,
10.8ng/ μ L, 5.4ng/ μ L, 2.7ng/ μ L, 1.35ng/ μ L and 0.675ng/ μ L.
3, the DNA1 extracted using step 1 is template, the primer LYB-CP20s and primer that are synthesized with 1 step 1 of embodiment
LYB-CP20a 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 DNA10.5 μ L, 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 the picture left above in Fig. 4, (swimming lane M is that (DNA Marker, are followed successively by DNA molecular standard from top to bottom
600,500,400,300,200 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 obtains size the result shows that carrying out PCR amplification as template using DNA1, DNA2 or DNA3 and is about
The purpose band of 432bp, and size cannot be obtained as template progress PCR amplification using DNA4, DNA5, DNA6 and DNA7 and be about
The purpose band of 432bp.Show that kit provided by the invention is limited to 5.4ng to the minimum detection of tulip bulb genomic DNA.
According to the method for above-mentioned steps 1 to 3, tulip bulb is replaced with to a mao arrowhead, pleionebulbocodioides rolfe and tinosporae, Qi Tabu respectively
It is rapid constant, obtain corresponding pcr amplification product.(swimming lane M is DNA molecular standard to the top right plot that experimental result is shown in successively in Fig. 4
(DNA Marker are followed successively by 600,500,400,300,200 and 100bp from top to bottom), hair arrowhead in swimming lane 8 to swimming lane 14
The concentration of genomic DNA is followed successively by 43.2ng/ μ L, 21.6ng/ μ L, 10.8ng/ μ L, 5.4ng/ μ L, 2.7ng/ μ L, 1.35ng/ μ
L and 0.675ng/ μ L), lower-left figure in Fig. 4 (swimming lane M be 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 pleionebulbocodioides rolfe is followed successively by 43.2ng/ μ in swimming lane 15 to swimming lane 21
L, 21.6ng/ μ L, 10.8ng/ μ L, 5.4ng/ μ L, 2.7ng/ μ L, 1.35ng/ μ L and 0.675ng/ μ L) and Fig. 4 in bottom-right graph
(swimming lane M is DNA molecular standard (DNA Marker are followed successively by 600,500,400,300,200 and 100bp from top to bottom), swimming lane
22 are followed successively by 43.2ng/ μ L, 21.6ng/ μ L, 10.8ng/ μ L, 5.4ng/ to the concentration of the genomic DNA of tinosporae in swimming lane 28
μ L, 2.7ng/ μ L, 1.35ng/ μ L and 0.675ng/ μ L).The result shows that cannot obtain the purpose item that size is about 432bp
Band.
Claims (10)
1. the primer pair for identifying tulip bulb or Tulipa edulis, is made of, each item primer LYB-CP20s and primer LYB-CP20a
Primer is single strand dna, and nucleotide sequence is followed successively by sequence 1 and sequence 2 in sequence table.
2. the application of primer pair described in claim 1 is following a1）-a12）In it is any：
a1）Prepare the kit for detecting or assisting detection tulip bulb；
a2）Whether contain in detection or auxiliary detection sample to be tested or candidate contains tulip bulb；
a3）Prepare the kit for identifying or assisting identification tulip bulb；
a4）Identify or assist to identify sample to be tested whether be or candidate is tulip bulb；
a5）Prepare the kit for identifying or assisting to identify the true or false of commercially available tulip bulb；
a6）Identification or auxiliary identify the true or false of commercially available tulip bulb；
a7）Prepare the kit for detecting or assisting detection Tulipa edulis；
a8）Whether contain in detection or auxiliary detection sample to be tested or candidate contains Tulipa edulis；
a9）Prepare the kit for identifying or assisting identification Tulipa edulis；
a10）Identify or assist to identify sample to be tested whether be or candidate is Tulipa edulis；
a11）Prepare the kit for identifying or assisting to identify the true or false of commercially available Tulipa edulis；
a12）Identification or auxiliary identify the true or false of commercially available Tulipa edulis.
3. the kit containing primer pair described in claim 1, the kit is for identifying tulip bulb or Tulipa edulis.
4. the preparation method of the kit described in claim 3, including by primer LYB-CP20s described in claim 1 and institute
State the step of primer LYB-CP20a 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 tulip bulb or Tulipa edulis；
b2）Identify or assist to identify sample to be tested whether be or candidate is tulip bulb or Tulipa edulis；
b3）Identification or auxiliary identify the true or false of commercially available tulip bulb or Tulipa edulis.
6. whether a kind of detection sample to be tested contains or the candidate method containing tulip bulb, include the following steps：
The total DNA for extracting sample to be tested carries out PCR amplification using primer pair described in claim 1, if it is possible to realize and effectively expand
Increase, then contains in the sample to be tested or candidate contains tulip bulb；If can not achieve effective amplification, in the sample to be tested
It does not contain or candidate without containing tulip bulb；
Described " can realize effective amplification " is the DNA fragmentation containing 432bp in the pcr amplification product；
Described " can not achieve effective amplification " is the DNA fragmentation that 432bp is not contained in the pcr amplification product.
Identifying whether sample to be tested is or method that candidate is tulip bulb to include the following steps 7. a kind of：
The genomic DNA for extracting sample to be tested carries out PCR amplification, if it is possible to which realization has using primer pair described in claim 1
Effect amplification, then the sample to be tested is or candidate is tulip bulb；If can not achieve effective amplification, the sample to be tested is not
Or candidate is not tulip bulb；
Described " can realize effective amplification " is the DNA fragmentation containing 432bp in the pcr amplification product；
Described " can not achieve effective amplification " is the DNA fragmentation that 432bp is not contained in the pcr amplification product.
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, being derived from the tissue or organ of the Original plant of the Chinese medicine.
9. a kind of method of the true or false of the commercially available tulip bulb of identification, includes the following steps：
The genomic DNA for extracting commercially available tulip bulb carries out PCR amplification, if it is possible to realize using primer pair described in claim 1
Effectively amplification, then the commercially available tulip bulb is or candidate is genuine piece；If can not achieve effective amplification, the commercially available tulip bulb
For or candidate be adulterant；
Described " can realize effective amplification " is the DNA fragmentation containing 432bp in the pcr amplification product；
Described " can not achieve effective amplification " is the DNA fragmentation that 432bp is not contained in the pcr amplification product.
10. a kind of method of the true or false of the commercially available Tulipa edulis of identification, includes the following steps：
The genomic DNA for extracting commercially available Tulipa edulis carries out PCR amplification, if it is possible to realize using primer pair described in claim 1
Effectively amplification, then the commercially available Tulipa edulis is or candidate is genuine piece；If can not achieve effective amplification, the commercially available Tulipa edulis
For or candidate be adulterant；
Described " can realize effective amplification " is the DNA fragmentation containing 432bp in the pcr amplification product；
Described " can not achieve effective amplification " is the DNA fragmentation that 432bp is not contained in the pcr amplification product.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510815696.8A CN105274243B (en) | 2015-11-23 | 2015-11-23 | A kind of method and its special primer pair of identification tulip bulb |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510815696.8A CN105274243B (en) | 2015-11-23 | 2015-11-23 | A kind of method and its special primer pair of identification tulip bulb |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN105274243A CN105274243A (en) | 2016-01-27 |
| CN105274243B true CN105274243B (en) | 2018-09-07 |
Family
ID=55144068
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510815696.8A Active CN105274243B (en) | 2015-11-23 | 2015-11-23 | A kind of method and its special primer pair of identification tulip bulb |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105274243B (en) |
Families Citing this family (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111500762B (en) * | 2020-05-26 | 2022-06-24 | 广西壮族自治区农业科学院 | Sagittaria trifolia SSR primer group and application thereof |
-
2015
- 2015-11-23 CN CN201510815696.8A patent/CN105274243B/en active Active
Non-Patent Citations (3)
| Title |
|---|
| AP-PCR技术在中药山慈菇鉴定上的应用;阮小丽等;《中国自然资源学会全国第四届天然药物资源学术研讨会论文集》;20000814;120 * |
| Species identification of the medicinal plant Tulipa edulis (Liliaceae) by DNA barcode marker;Hong-liang Ma等;《Biochemical Systematics and Ecology》;20140524;第55卷;362-368 * |
| 山慈菇与光慈菇的鉴别及正确使用;童静玲等;《海峡药学》;20111231;第23卷(第2期);28-29 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN105274243A (en) | 2016-01-27 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| Gao et al. | Evolution of Oryza chloroplast genomes promoted adaptation to diverse ecological habitats | |
| Mochon et al. | Serological profiling of a Candida albicans protein microarray reveals permanent host-pathogen interplay and stage-specific responses during candidemia | |
| Koetschan et al. | Internal transcribed spacer 1 secondary structure analysis reveals a common core throughout the anaerobic fungi (Neocallimastigomycota) | |
| White et al. | CloVR-ITS: automated internal transcribed spacer amplicon sequence analysis pipeline for the characterization of fungal microbiota | |
| Appelt et al. | Polyphasic analysis of a middle ages coprolite microbiota, Belgium | |
| CN105567789B (en) | The PCR primer pair composition and its application of identification or auxiliary identification sturgeon germplasm | |
| CN103255220A (en) | Tortoise shell DNA detection kit and identification method | |
| CN107475403A (en) | The analysis method of the method for detection Circulating tumor DNA, kit and its sequencing result from peripheral blood dissociative DNA | |
| CN103074433A (en) | Turtle shell DNA detection kit and identification method | |
| CN106119408A (en) | A kind of authentication method of Herba Dendrobii | |
| CN104946730A (en) | PCR (Polymerase Chain Reaction) specific primers and detection method for authenticating antlers | |
| CN105603106B (en) | Identification peach kernel and amygdalate PCR method and kit based on ITS sequence site | |
| CN106282373A (en) | A kind of Herba Dendrobii and the contrast authentication method of Henan Herba Dendrobii | |
| Gadelhaq et al. | Molecular characterization of Eimeria species naturally infecting Egyptian Baldi Chickens | |
| Wang et al. | Revision of three camaenid and one bradybaenid species (Gastropoda, Stylommatophora) from China based on morphological and molecular data, with description of a new bradybaenid subspecies from Inner Mongolia, China | |
| Li et al. | Application of molecular genetics method for differentiating Martes zibellina L. heart from its adulterants in traditional Chinese medicine based on mitochondrial cytochrome b gene | |
| CN107164471B (en) | Molecular identification method for rapidly identifying truth of beauveria bassiana in traditional Chinese medicine stiff silkworm | |
| CN105274243B (en) | A kind of method and its special primer pair of identification tulip bulb | |
| CN104531713A (en) | Multiple PCR primers and method for constructing human TCBR library based on high-throughput sequencing | |
| CN101875980B (en) | Kit and method for detecting Macrobrachium rosenbergii Nodavirus | |
| Mufti et al. | Genetic characterization of Fasciola samples from bovine hosts in Pakistan by sequences of ribosomal internal transcribed spacer regions. | |
| Omori et al. | Complete nucleotide sequences of the mitochondrial genomes of two avian malaria protozoa, Plasmodium gallinaceum and Plasmodium juxtanucleare | |
| CN104946729A (en) | PCR (Polymerase Chain Reaction) specific primers and detection method for authenticating tortoise shell | |
| CN105176988A (en) | Method for identifying traditional Chinese medical material corn cervi pantotrichum based on PCR-RFLP | |
| CN105039584A (en) | American ginseng DNA detection reagent box and identification method |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |