CN109554499A - A kind of primer sets, the detection method of fritillaria and kit - Google Patents
A kind of primer sets, the detection method of fritillaria and kit Download PDFInfo
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- CN109554499A CN109554499A CN201811571473.1A CN201811571473A CN109554499A CN 109554499 A CN109554499 A CN 109554499A CN 201811571473 A CN201811571473 A CN 201811571473A CN 109554499 A CN109554499 A CN 109554499A
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- 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
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Abstract
The invention discloses a kind of primer sets, the detection method of fritillaria and kits；The detection method of fritillaria includes: PCR amplification step；Electrophoresis step；: there is the segment of 310bp in authentication step in electrophoretic band, then contain bulbus fritillariae cirrhosae in sample to be tested；Occur the segment of 310bp and 730bp in electrophoretic band, then contains fritillaria thunbergii in sample to be tested；Occur the segment of 260bp in electrophoretic band, then contains Siberian fritillary bulb in sample to be tested；Occur the segment of 330bp and 200bp in electrophoretic band, then contains fritillary bulb in sample to be tested；Kit includes PCR amplification system；PCR amplification system includes primer sets, 10 × Taq DNA polymerase buffer liquid, 10mM dNTPs and Taq archaeal dna polymerase；The detection method passes through the difference of PCR product electrophoretic band, it can be seen that the fritillaria kind of sample.
Description
Technical field
The present invention relates to a kind of primer sets, the detection method of fritillaria and kits, belong to molecular Biological Detection technology neck
Domain.
Background technique
Fritillaria is Liliaceae herbaceos perennial, as a kind of clearing heat and moistening lung, the good medicine of preventing phlegm from forming and stopping coughing, applicating history
Source is for a long time.There are about more than 20 of medical value in the Fritillaria Linn in China, is distributed mainly on Sichuan, Xinjiang, Gansu, Zhejiang
Etc. ground.Because of its place of production difference, the medical value between each strain of fritillaria has certain difference, wherein with the medicinal of bulbus fritillariae cirrhosae
It is worth highest.According to 2015 editions " Chinese Pharmacopoeia ", bulbus fritillariae cirrhosae shares six kinds of Ji Yuan, i.e. bulbus fritillariae cirrhosae, Fritillaria unibracteata, Gansu shellfish at present
Mother, Bulbus Fritillariae cirrhosae, taipei fritillary bulb and Wa Bu fritillaria.Bulbus fritillariae cirrhosae often occurs using it in the market because price is more expensive and resource is limited
The phenomenon that adulterating such as fritillary bulb and fritillaria thunbergii, it is different from certified products bulbus fritillariae cirrhosae drug effect to mix adulterant for its strain fritillaria, once it uses with
It then will affect drug safety.Therefore a kind of more rapidly accurate molecular biology method is developed, for examining in Chinese materia medica preparation
Detection containing specific fritillaria kind very it is necessary to.
Summary of the invention
For overcome the deficiencies in the prior art, the first purpose of this invention is to provide a kind of primer sets, the primer sets
For expanding the ITS1 gene order of fritillaria.
Second object of the present invention is to provide a kind of detection method of fritillaria, which passes through PCR product electricity
The difference of swimming band, it can be seen that the fritillaria kind of sample.
Third object of the present invention is to provide a kind of kit, and kit includes primer sets, carries out the kind of fritillaria
Detection.
Realize that the first purpose of this invention can reach by adopting the following technical scheme that: a kind of primer sets, including with
It is at least a pair of in lower primer pair 1, primer pair 2 and primer pair 3:
Primer pair 1:
ITS1-F1:5 '-CGTAACAAGGTTTCCGTAGGTGAA-3 '；
ITS1-R1:5 '-GCTACGTTCTTCATCGAT-3 '；
Primer pair 2:
ITS1-F2:5 '-CGCGATCGCCTCCAAGCG-3 '；
ITS1-R1:5 '-GCTACGTTCTTCATCGAT-3 '；
Primer pair 3:
ZB1-F1:5 '-ACAGTACTGGGGGAGAAGTC-3 '；
ZB1-R1:5 '-CAAATGGAGGGTATGTGTCG-3 '.
Further, primer sets include primer pair 1, primer pair 2 and primer pair 3.
Realize that second object of the present invention can reach by adopting the following technical scheme that: a kind of detection side of fritillaria
Method, comprising:
PCR amplification step: sample to be tested target fragment is obtained by PCR amplification；Using drawing as described above in PCR amplification
Object group；
Electrophoresis step: the product that PCR amplification step is obtained carries out gel electrophoresis；
: there is the segment of 310bp in authentication step in electrophoretic band, then contain bulbus fritillariae cirrhosae in sample to be tested；In electrophoretic band
There is the segment of 310bp and 730bp, then contains fritillaria thunbergii in sample to be tested；Occur the segment of 260bp in electrophoretic band, then to
Contain Siberian fritillary bulb in sample；Occur the segment of 330bp and 200bp in electrophoretic band, then contains fritillary bulb in sample to be tested.
Further, detection method further includes gDNA extraction step: extracting gDNA from sample, and carries out nucleic acid quantification inspection
It surveys, gDNA is then used for PCR amplification.
Further, in PCR amplification step, pcr amplification reaction system includes: template DNA；Primer sets；10×Taq DNA
Polymerase buffer；10mM dNTPs；Taq archaeal dna polymerase.
Further, in PCR amplification step, pcr amplification reaction system are as follows:
Further, in PCR amplification step, pcr amplification reaction program are as follows: 95 DEG C of initial denaturation 2min；95 DEG C of denaturation 30s,
52 DEG C of annealing 30s, 72 DEG C of extension 30s, 32 recycle；72 DEG C of final extension 10min.
Further, in electrophoresis step, preparation 2% Ago-Gel of mass concentration progress electrophoresis, deposition condition 120V,
30min。
Realize that third object of the present invention can reach by adopting the following technical scheme that: a kind of kit, including PCR
Amplification system；PCR amplification system include primer sets as described above, 10 × Taq DNA polymerase buffer liquid, 10mM dNTPs and
Taq archaeal dna polymerase.
Further, kit further includes that gDNA extracts system.
Compared with prior art, the beneficial effects of the present invention are:
1, the invention belongs on industry technology for the first time by bulbus fritillariae cirrhosae, fritillary bulb, fritillaria thunbergii and the ITS1 of Siberian fritillary bulb sequence into
Row compares, and designs corresponding PCR amplification primer sets, detection method, carries out the cultivar identification of fritillaria；
2, detection method step is simple, by the difference of PCR product electrophoretic band, it can be seen that the fritillaria product of sample
Kind；
3, kit of the invention includes the reagent and material for realizing the detection of fritillaria kind, convenient to use.
Detailed description of the invention
Fig. 1 is that bulbus fritillariae cirrhosae, fritillary bulb, Siberian fritillary bulb and fritillaria thunbergii ITS1 genetic fragment compare；
Fig. 2 is the electrophoretogram of embodiment 3.
Specific embodiment
In the following, being described further in conjunction with attached drawing and specific embodiment to the present invention:
Design principle of the invention is as follows: using the homology and otherness of variety classes fritillaria gene order, design is drawn
Object group including but not limited to carries out PCR amplification, by right using the genomic DNA (gDNA) of fritillaria bulb or powder as template
The difference of the agarose gel electrophoresis band of PCR product, come judge test sample type bulbus fritillariae cirrhosae ITS1 sequence
(GenBank:KX669649.1) as shown in SEQ ID NO.1；The ITS1 sequence (GenBank:KT861550.1) of fritillary bulb is such as
Shown in SEQ ID NO.2；The ITS1 sequence (GenBank:KP712003.1) of fritillaria thunbergii is as shown in SEQ ID NO.3；Siberian fritillary bulb
ITS1 sequence (GenBank:HQ010405.1) as shown in SEQ ID NO.4.As shown in Figure 1, bulbus fritillariae cirrhosae (CB), fritillary bulb
(PB), Siberian fritillary bulb (YB) and fritillaria thunbergii (ZB) ITS1 genetic fragment comparison result, wherein having and only bulbus fritillariae cirrhosae ITS1 gene piece
There are a Sma I restriction enzyme sites (CCCGGG) in section.
Primer pair 1 amplifies the segment of 310bp from bulbus fritillariae cirrhosae gDNA, and the piece of 330bp is amplified from fritillary bulb gDNA
Section, the segment of 310bp is amplified from fritillaria thunbergii gDNA, the segment of 260bp is amplified from Siberian fritillary bulb gDNA；
Primer pair 2 amplifies the segment of 200bp from fritillary bulb gDNA；
Primer pair 3 only amplifies the segment of 730bp, fragment sequence SEQ ID NO.5 from fritillaria thunbergii gDNA.
Embodiment 1:
The gDNA for carrying out sample is extracted, and prepares bulbus fritillariae cirrhosae, fritillary bulb, Siberian fritillary bulb and fritillaria thunbergii sample；
Reagent: lysate (L1), DNA extract (E1), buffer (W1), rinsing liquid (W2), (TE is molten for DNA eluent
Liquid)；
Steps are as follows:
1) fritillaria flesh tissue or dry weight tissue are taken, liquid nitrogen is added and sufficiently mills；
2) ground powder is quickly transferred to be pre-loaded in the centrifuge tube of 65 DEG C of preheating lysate L1 (before experiment
Mercaptoethanol is first added in the L1 of preheating, makes its final concentration of mass percent 0.1%), after being mixed by inversion rapidly, by centrifuge tube
65 DEG C of water-bath 20min are placed on, reverse centrifuge tube is during water-bath to mix sample for several times；
3) chloroform is added, mixes well, 12,000rpm (~13,400 × g) are centrifuged 5min；
4) upper strata aqueous phase obtained by step 3) is transferred in new centrifuge tube, extract E1 is added, mixes well；
5) liquid of step 4) is transferred in adsorption column, 12,000rpm (~13,400 × g) are centrifuged 30s, discard waste liquid；
6) buffer W1 (please first check whether before use and dehydrated alcohol has been added) is added in adsorption column, 12,000rpm (~
13,400 × g) centrifugation 30s, outwells waste liquid, adsorption column is put into collecting pipe；
7) rinsing liquid W2 (please first check whether before use and dehydrated alcohol has been added) is added into adsorption column, 12,000rpm
(~13,400 × g) is centrifuged 30s, outwells waste liquid, adsorption column is put into collecting pipe；
8) repetitive operation step 7)；
9) adsorption column is put back in collecting pipe, 12,000rpm (~13,400 × g) are centrifuged 2min, outwell waste liquid；It will absorption
Column, which is placed in, to be placed at room temperature for several minutes, thoroughly to dry rinsing liquid W2 remaining in adsorbent material；
The purpose of this step is to remove rinsing liquid remaining in adsorption column, and the residual of ethyl alcohol will affect subsequent in rinsing liquid
Enzyme reaction (digestion, PCR etc.) experiment；
10) adsorption column is transferred in clean centrifuge tube, DNA eluent TE is vacantly added dropwise to the intermediate position of adsorbed film,
It is placed at room temperature for 2-5min, 12,000rpm (~13,400 × g) are centrifuged 2min, solution is collected into centrifuge tube；
11) Nanodrop or Qubit carries out nucleic acid quantification detection.
Embodiment 2:
The gDNA that embodiment 1 is obtained carries out PCR amplification, and amplification system is as follows:
Pcr amplification reaction program are as follows: 95 DEG C of initial denaturation 2min；95 DEG C of denaturation 30s, 52 DEG C of annealing 30s, 72 DEG C of extension 30s,
32 circulations；72 DEG C of final extension 10min；4 DEG C of preservations；
The primer pair used are as follows:
Primer pair 1:
ITS1-F1:5 '-CGTAACAAGGTTTCCGTAGGTGAA-3 '；
ITS1-R1:5 '-GCTACGTTCTTCATCGAT-3 '；
Primer pair 2:
ITS1-F2:5 '-CGCGATCGCCTCCAAGCG-3 '；
ITS1-R1:5 '-GCTACGTTCTTCATCGAT-3 '；
Primer pair 3:
ZB1-F1:5 '-ACAGTACTGGGGGAGAAGTC-3 '；
ZB1-R1:5 '-CAAATGGAGGGTATGTGTCG-3 '.
Obtain sample to be tested ITS1 sequence fragment pcr amplification product.
Embodiment 3:
Electrophoresis and authentication step:
It prepares 2% Ago-Gel of mass concentration and carries out electrophoresis, deposition condition 120V, 30min；
For electrophoretogram as shown in Fig. 2, CBS swimming lane is that only bulbus fritillariae cirrhosae, PB swimming lane are fritillary bulb, YB swimming lane is Siberian fritillary bulb, ZB
Swimming lane is fritillaria thunbergii.It can learn: occur the segment of 310bp in electrophoretic band, then contain bulbus fritillariae cirrhosae in sample to be tested；Electrophoresis strip
There is the segment of 310bp and 730bp in band, then contains fritillaria thunbergii in sample to be tested；Occur the segment of 260bp in electrophoretic band,
Then contain Siberian fritillary bulb in sample to be tested；Occur the segment of 330bp and 200bp in electrophoretic band, then contains flat shellfish in sample to be tested
It is female.
Embodiment 4:
A kind of kit, including gDNA extract system and PCR amplification system；
It includes lysate, DNA extract, buffer, rinsing liquid, DNA eluent that wherein gDNA, which extracts system, be may also include
DNA adsorption column and casing；
PCR amplification system includes primer sets, 10 × Taq DNA polymerase buffer liquid, 10mM dNTPs and Taq DNA polymerization
Enzyme and nuclease-free water.
The kit is used to detect the kind of fritillaria, realizes the detecting step of embodiment 1-3.
For those skilled in the art, it can make other each according to the above description of the technical scheme and ideas
Kind is corresponding to be changed and deforms, and all these change and deform the protection model that all should belong to the claims in the present invention
Within enclosing.
SEQUENCE LISTING
<110>Kang Mei Hua Da gene technology Co., Ltd
<120>a kind of primer sets, the detection method of fritillaria and kit
<130> 2018
<160> 5
<170> PatentIn version 3.3
<210> 1
<211> 788
<212> DNA
<213>bulbus fritillariae cirrhosae
<400> 1
caggtcgcga gcggttcgcc gcctgtgacg tcacgagaag tccactgaac cttatcattt 60
agaggaagga gaagtcgtaa caaggtttcc gtaggtgaac ctgcggaagg atcattgtcg 120
agaaacgacc gagagaccgc gaactcgtaa acggatgaca ccgtgtcggg cgggcactat 180
gcccgccctg cccgggacct cgcatcgtgc ctctccgggc acgatttgcg ggggacggac 240
gaaaccccgg cgcggcctgc gccaaggaac atatgacagg acggacgcac gtcaacgcct 300
ctgcggcggg gcggcgttcg ctctctatcc atacgactct cggcaacgga tatctcggct 360
ctcgcatcga tgaagaacgt agcgaaatgc gatacttggt gtgaattgca gaatcccgtg 420
aaccatcgag tctttgaacg caagttgcgc ccgaggcctt tcggccgagg gcacgcctgc 480
ctgggcgtca cgccttgttt cgctccgtgc ccaatgaccc ttcgggtgcg gtcacggatg 540
cggagattgg ccccccgtgc ctcgtgtgcg gcgggcttaa gcgcgggctg tcggcgtcgg 600
gatgggcacg acgagtggtg gacggagcac cagcaggatg ttgtggcccc ctgtcgcctt 660
aaggggctca agagacccga aacgggcgag ccgtgctccg tacgaggagg gcgtgccgtc 720
tcgcaatgcg accccaggtc aggcggggac acccgctgag tttaagcata tcaataagcg 780
gaggagaa 788
<210> 2
<211> 611
<212> DNA
<213>fritillary bulb
<400> 2
tcgagaaccg accgakagac cgcgaacccg taaacggatg acaccgtgtc gggcgggcac 60
tatgcccgcc ctgcccggga cctcgcatcg tgyctctccg ggcacgattt gcgggggacg 120
gacgaaaccc cggcgcggcc tgcgccaagg aacatatgac aggacggacg cacgtcaacg 180
cctctgcggc ggggcgacgt tcgctctcta tccatacgac tctcggcaac ggatatctcg 240
gctctcgcat cgatgaagaa cgtagcgaaa tgcgatactt ggtgtgaatt gcagaatccc 300
gtgaaccatc gagtctttga acgcaagttg cgcccgaggc ctttcggccg agggcacgcc 360
tgcctgggcg tcacgccttg tttcgctccg tgcccaatga cccttcgggt gcggtcacgg 420
atgcggagat tggccccccg tgcctcgtgt gcggcgggct taagcgcggg ctgtcggcgt 480
cgggatgggc acgacgagtg gtggacggag caccagcagg atgttgtggc ccccygtcgc 540
cttaaggggc tcaagagacc cggaccgggc gagccgtgct ccgtacgagg agggcgtgcc 600
gtctcgcaat g 611
<210> 3
<211> 608
<212> DNA
<213>fritillaria thunbergii
<400> 3
tcgagaaccg accgagagac cgcgaacccg taaacggatg acaccgtgtc gggcgggcac 60
tatgcccgcc ctgctcggga cctcgcgtcg tgtctccgga cacgatttgc gggggacgga 120
cgaaaccccg gcgcggcctg cgccaaggaa catatgacag gacggacgca cgccaacgcc 180
tctgcggcgg ggcgacgttc gctctctatc catacgactc tcggcaacgg atatctcggc 240
tctcgcatcg atgaagaacg tagcgaaatg cgatacttgg tgtgaattgc agaatcccgt 300
gaaccatcga gtctttgaac gcaagttgcg cccgaggcct ttcggccgag ggcacgcctg 360
cctgggcgtc acgccttgtt tcgctccgtg cccaacgccc cttcgggggc ggtcacggat 420
gcggagattg gccccccgtg cctcgtgtgc ggcgggctta agcgcgggct gtcggcgccg 480
ggatgggcac gacgagtggt ggacggagca ccagcaggat gtcgtggccc cccgtcgcct 540
taaggggccc aagagacccg gaccgggcga gccgtgctcc gtacgaggag ggcgtgccgt 600
ctcgcaat 608
<210> 4
<211> 715
<212> DNA
<213>Siberian fritillary bulb
<400> 4
tttccgtagg gtgaacctgc ggaaggatca ttgtcgagaa ctgaccgaga gaccgcgaac 60
ccgtaaacgg atgacaccgt gtcgggcggg cactgtgctc gccctgctcg ggacctcgca 120
ccgtgttcgc gatcgcctca gggcgctccg gacacggttt gcgggggacg gacgaaaccc 180
cggcgcggcc tgcgccaagg aacatatgac aggacggacg cacgtcaacg cctctgcggc 240
ggggcgacgt tcgatctcta tccatacgac tctcggcaac ggatatctcg gctctcgcat 300
cgatgaagaa cgtagcgaaa tgcgatactt ggtgtgaatt gcagaatccc gtgaaccatc 360
gagtctttga acgcaagttg cgcccgaggc ctttcggccg agggcacgcc tgcctgggcg 420
tcacgccttg tttcgctccg tgcccattgc cccttcgggg ggcggtcacg gatgcggaga 480
ctggccctcc gtgcctcgtg tgcggcgggc ttaagcgcgg gctgtcggcg ccgggatggg 540
cacgacgagt ggtggacgga gcaccagcag gatgtcgtgg ccccccgtcg cctcaagggg 600
ctcaagagac ccggaccagg cgagccgtgc tccgtacgag gagggcgtgc cctctcgcaa 660
tgcgacccca ggtcaagcgg ggacacccgc tgagttttaa gcatatcaat aagcg 715
<210> 5
<211> 745
<212> DNA
<213>fritillaria thunbergii
<400> 5
cgacgaagaa cggacagtac tgggggagaa gtcaaatact gttttatctg cctgaaggct 60
gcttcgcact cttccgtcca ttcaaactct ctgcttcctc atagcatcgc tgtaaaagtt 120
tttatcttgt ccgatgatct tgatatgaag cggtaaaggg ctgctattct tccactcagt 180
acctgtatgt ctttcactgt tttggggctt tccatcccca gtatcgattt tgtttgcgct 240
ggatctgcgc tgattctctt cttgtgcact atgtgcccta agaacttacc taaagctact 300
ccaaatatgc atttttttgg attcaatttc agggagaaga tgcgtaatcg gtcgaaggcc 360
tcctctaggt tctggatatg atcctccttt ttagaacttt ttaccagtaa atcatcaatt 420
tatgtgccta ctgttttccc tagtagccct tgaaagattc tggtaatcaa ccgttgaaaa 480
gttgctcctg cattcttaag tccaaagggc atcaccgtgt agcaatataa ccctaaggga 540
gtagtgaaac tggtgtgttc ctcatcttgc agatttaact tgatttgatt atagcctgaa 600
caggcatcca aaaaagatat cctctcgtga cccaccagtg agtccaccaa ctggcatatc 660
cgtggtagcg ggaaactgtc cttgggacag gctttattta gatctgtgta gtcgacacat 720
accctccatt tgccgttctt cgtcg 745
Claims (10)
1. a kind of primer sets, which is characterized in that including at least a pair of in following primer pair 1, primer pair 2 and primer pair 3:
Primer pair 1:
ITS1-F1:5 '-CGTAACAAGGTTTCCGTAGGTGAA-3 '；
ITS1-R1:5 '-GCTACGTTCTTCATCGAT-3 '；
Primer pair 2:
ITS1-F2:5 '-CGCGATCGCCTCCAAGCG-3 '；
ITS1-R1:5 '-GCTACGTTCTTCATCGAT-3 '；
Primer pair 3:
ZB1-F1:5 '-ACAGTACTGGGGGAGAAGTC-3 '；
ZB1-R1:5 '-CAAATGGAGGGTATGTGTCG-3 '.
2. primer sets as described in claim 1, which is characterized in that the primer sets include primer pair 1, primer pair 2 and primer
To 3.
3. a kind of detection method of fritillaria, characterized by comprising:
PCR amplification step: sample to be tested target fragment is obtained by PCR amplification；Using as described in claim 1 in PCR amplification
Primer sets；
Electrophoresis step: the product that PCR amplification step is obtained carries out gel electrophoresis；
: there is the segment of 310bp in authentication step in electrophoretic band, then contain bulbus fritillariae cirrhosae in sample to be tested；Occur in electrophoretic band
The segment of 310bp and 730bp then contains fritillaria thunbergii in sample to be tested；Occurs the segment of 260bp in electrophoretic band, then to test sample
Contain Siberian fritillary bulb in product；Occur the segment of 330bp and 200bp in electrophoretic band, then contains fritillary bulb in sample to be tested.
4. the detection method of fritillaria as claimed in claim 3, which is characterized in that the detection method further includes that gDNA extracts step
It is rapid: to extract gDNA from sample, and carry out nucleic acid quantification detection, gDN A is then used for PCR amplification.
5. the detection method of fritillaria as claimed in claim 3, which is characterized in that in the PCR amplification step, PCR amplification is anti-
Answering system includes: template DNA；Primer sets；10 × Taq DNA polymerase buffer liquid；10mM dNTPs；Taq archaeal dna polymerase.
6. the detection method of fritillaria as claimed in claim 3, which is characterized in that in the PCR amplification step, PCR amplification is anti-
Answer system are as follows:
Nuclease-free water is settled to 50 μ L.
7. the detection method of fritillaria as claimed in claim 3, which is characterized in that in the PCR amplification step, PCR amplification is anti-
Answer program are as follows: 95 DEG C of initial denaturation 2min；95 DEG C of denaturation 30s, 52 DEG C of annealing 30s, 72 DEG C of extension 30s, 32 recycle；72 DEG C final
Extend 10min.
8. the detection method of fritillaria as claimed in claim 3, which is characterized in that in the electrophoresis step, prepare mass concentration
2% Ago-Gel carries out electrophoresis, deposition condition 120V, 30min.
9. a kind of kit, which is characterized in that including PCR amplification system；The PCR amplification system includes such as claim 1 institute
The primer sets stated, 10 × Taq DNA polymerase buffer liquid, 10mM dNTPs and Taq archaeal dna polymerase.
10. kit as claimed in claim 9, which is characterized in that the kit further includes that gDNA extracts system.
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Citations (7)
| Publication number | Priority date | Publication date | Assignee | Title |
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| US6569625B1 (en) * | 1999-07-14 | 2003-05-27 | The Hong Kong University Of Science & Technology | Fritillaria species identification |
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