CN109295170A - A kind of method of based on PCR-RFLP technical appraisement wide dragon - Google Patents
A kind of method of based on PCR-RFLP technical appraisement wide dragon Download PDFInfo
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- CN109295170A CN109295170A CN201811116589.6A CN201811116589A CN109295170A CN 109295170 A CN109295170 A CN 109295170A CN 201811116589 A CN201811116589 A CN 201811116589A CN 109295170 A CN109295170 A CN 109295170A
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- 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
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- C12Q1/6813—Hybridisation assays
- C12Q1/6827—Hybridisation assays for detection of mutation or polymorphism
- C12Q1/683—Hybridisation assays for detection of mutation or polymorphism involving restriction enzymes, e.g. restriction fragment length polymorphism [RFLP]
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Abstract
The invention discloses a kind of methods of based on PCR-RFLP technical appraisement wide dragon.The present invention carries out PCR amplification using the universal primer of COI gene, obtain mitochondrial cytochrome oxidizing ferment (COI) genetic fragment of each earthworm kind, and sequence, comparison are carried out to it, restriction endonuclease map analysis is carried out to different cultivars earthworm COI segment complete sequence by DNAMAN software, it finally screens a kind of restriction endonuclease RsaI and digestion is carried out to PCR product, wide dragon and other kinds can be identified as a result, passing through restriction enzyme mapping with the digestion of 2% Ago-Gel detection PCR product.Operation of the present invention is simple and reliable, can provide new technological means for the identification of wide dragon commodity.
Description
Technical field:
The invention belongs to Chinese material medicine resource identification technology fields, and in particular to a kind of based on PCR-RFLP technical appraisement is broadly
The method of dragon.
Background technique:
Wide dragon is the hirudo leech of Ju Yin section animal Pheretima aspergillum (Pheretima aspergillum), and main product is in Guangdong
Guangxi, is the Animal Medicine material of Chinese tradition, and wide dragon class preparation is extensive in clinical application.Pharmacological action that there are many wide dragons,
Mainly there is decompression, relievings asthma, is antipyretic and anti-inflammatory, antithrombotic, antitumor etc..Pheretima has thousands of years medication history in folks of china,
It is recorded according to Chinese Pharmacopoeia, pheretima includes wide dragon and Shanghai pheretima, and wherein it is best in quality to be considered industry for the quality of wide dragon.In
The earthworm kind of state is more, and form size is different due to breed difference, and after being processed into Chinese medicine, the discrimination for increasing kind is difficult
Degree, its drug effect difference of different cultivars are big.Through investigating, wide dragon commodity adulterant is more in Chinese Medicinal Materials Markets, has seriously affected Chinese medicine
The stability and safety of material medication, need to establish new identification technology, are accurately finished the identification of pheretima commodity.
Polymerase chain reaction restriction fragment length polymorphism analysis method (PCR-RFLP) is using round pcr as base
Plinth, restriction enzyme have specific recognition to amplified production sequence, and digestion is cut into different size segment, gene-amplification purpose sequence
The restriction endonuclease map of column is different, to generate the DNA band of different length, is reflected with this for Chinese medicine commodity
It is fixed.PCR-RFLP is widely used in Materia Medica Identification, including ginseng, Herba Epimedii, bulbus fritillariae cirrhosae, fleece-flower root etc..Chinese Pharmacopoeia
It records, part medicinal material has used PCR-RFLP technology to carry out authenticity.But it is related to utilize PCR-RFLP technical appraisement earthworm product
Kind, which has no, to be had been reported that.The present invention is applied in the wide dragon Commodity Identification of Market of Chinese Materia Medica using PCR-RFLP technology, and market is reduced
Adulterant is mixed into, and is provided safeguard for Chinese medicine quality.
Summary of the invention:
The object of the present invention is to provide a kind of methods of based on PCR-RFLP technical appraisement wide dragon that operation is simple and reliable.
The method of based on PCR-RFLP technical appraisement wide dragon of the invention, comprising the following steps: extract earthworm sample to be measured
The genomic DNA of product is as template, with the universal primer HCO2198:5 '-of COI gene
TAAACTTCAGGGTGACCAAAAAATCA-3 ' and LCO1490:5 '-GGTCAACAAATCATAAAGATATTGG-3 ', which is used as, to be drawn
Object carries out PCR amplification, and PCR product is carried out digestion with restriction enzyme RsaI after purification, carries out electrophoresis inspection to digestion products
It surveys, if there are 3 segments, earthworm sample to be measured is wide dragon.
It is preferred that the reaction system of the PCR amplification is 25 μ L, including 1 μ L, 10 × PCR Buffer of DNA profiling, 2.5 μ
L, 25mmol/L MgCl22 μ L, 10mmol/L dNTPs 0.5 μ L, Taq DNA polymerase 1U's, 10 μm of ol/L
HCO2198 and each 0.5 μ L of LCO1490 primer, surplus is water；Response procedures are as follows: 95 DEG C of pre- change 4min；95 DEG C of denaturation 45s, 55 DEG C
Anneal 45s, 72 DEG C of extension 45s, 35 circulations；72 DEG C of extension 10min.
The endonuclease reaction system of the digestion is 20 μ L, 82 μ L of μ L, 10 × Buffer of PCR product including purifying,
0.1% BSA, 0.5 μ L of 2 μ L, restriction enzyme RsaI, surplus is water；Reaction condition are as follows: 37 DEG C of reaction 2h, add 10 ×
Reaction was completed by 2 μ L of Loading Buffer.
The present invention carries out PCR amplification using COI gene universal primer, obtains the mitochondrial cytochrome oxygen of each earthworm kind
Change enzyme (COI) genetic fragment, and carry out sequence, comparison to it, by DNAMAN software to different cultivars earthworm COI segment
Complete sequence carries out restriction endonuclease map analysis, finally screens a kind of restriction endonuclease RsaI and produces to PCR
Object carries out digestion, detects (containing EB) digestion of PCR product as a result, can identify by restriction enzyme mapping with 2% Ago-Gel
Wide dragon and other kinds.Operation of the present invention is simple and reliable, can provide new technological means for the identification of wide dragon commodity.
Detailed description of the invention:
Fig. 1 is PCR product COI fragment electrophoretic figure, from left to right, M:DL2000DNA Marker, 1: wide dragon sample 1,
2: wide dragon sample 2,3: wide dragon sample 3,4: Eisenia Foetida sample 1,5: Eisenia Foetida sample 2,6: the far blind earthworm of dark holes
Sample 1,7: the far blind earthworm sample 2,8 of dark holes: power simulation sample 1,9: power simulation sample 2,10: big chamber earthworm sample 1,
11: big chamber earthworm sample 2, N: negative control.
Fig. 2 is restriction enzyme site analysis chart of the restriction enzyme RsaI to different earthworm kind COI segments, restriction enzyme
Enzyme RsaI identifies that base sequence site is GT/AC, and wherein wide dragon COI segment has 2 RsaI enzyme recognition sites, and compatriot loves victory
The far blind earthworm of earthworm, dark holes and power simulation COI segment only have 1 RsaI enzyme recognition site, and big chamber earthworm COI segment does not have then
RsaI enzyme recognition site.
Fig. 3 is digestion products electrophoresis detection figure, from left to right, M:DL2000DNA Marker, 1: wide dragon sample 1,2:
Wide dragon sample 2,3: wide dragon sample 3,4: Eisenia Foetida sample 1,5: Eisenia Foetida sample 2,6: the far blind earthworm sample of dark holes
Product 1,7: the far blind earthworm sample 2,8 of dark holes: power simulation sample 1,9: power simulation sample 2,10: big chamber earthworm sample 1,11:
Big chamber earthworm sample 2, N: negative control.
Specific embodiment:
Below with reference to embodiment, the present invention is further illustrated, is not limitation of the present invention.
Embodiment 1:
1, the extraction of DNA:
Take different cultivars earthworm (wide dragon, Eisenia Foetida (Eisenia foetida), the far blind earthworm (Amynthas of dark holes
Obscurito porus), power simulation (Pheretima guillelmi) and big chamber earthworm (Metaphire magna)) group
30mg is knitted, DNA extraction is carried out with animal tissue's DNA extraction kit (Tiangeng), obtains genomic DNA as template.
2, COI fragment amplification, purifying and sequencing:
With COI gene universal primer HCO2198:5 '-TAAACTTCAGGGTGACCAAAAAATCA-3 ' (such as SEQ ID
Shown in NO.1) and LCO1490:5 '-GGTCAACAAATCATAAAGATATTGG-3 ' (as shown in SEQ ID NO.2) progress PCR
PCR reaction system is established in amplification, and it includes 1 μ L, 10 × PCR Buffer of DNA profiling that PCR reaction carries out in 25 μ L systems
2.5 μ L, MgCl2(25mmol/L) 2 μ L, dNTPs (10mmol/L) 0.5 μ L, Taq DNA polymerase (2.5U/ μ L) 0.4 μ
L, HCO2198 and LCO1490 primer (10 μm of ol/L) each 0.5 μ L adds sterilizing ultrapure water to 25 μ L；Response procedures are as follows: 95 DEG C pre-
Become 4min；95 DEG C of denaturation 45s, 55 DEG C of annealing 45s, 72 DEG C of extension 45s, 35 recycle；72 DEG C of extension 10min, 4 DEG C of preservations；PCR
Product is purified using PCR product purification kit (Tiangeng), the PCR product (i.e. COI segment) purified, sequencing
(Fig. 1) is completed by Shanghai Invitrogen Corp..The nucleotide sequence of wide dragon COI segment is as shown in SEQ ID NO.3.
3, alignment and restriction enzyme site analysis:
According to sequencing result, using DNAMAN software, the comparison of COI fragment sequence and COI piece are carried out to different cultivars earthworm
Section complete sequence restriction endonuclease map analysis has selected restriction enzyme RsaI according to the determination of the difference of restriction enzyme mapping
It carries out digestion identification (Fig. 2).
4, endonuclease reaction:
Endonuclease reaction system is 20 μ L, 82 μ L of μ L, 10 × Buffer of PCR product (i.e. COI segment) including purifying,
2 μ L, 10U/ μ L restriction enzyme RsaI of 0.1%BSA, 0.5 μ L adds sterilizing ultrapure water to add 10 to 20 μ L, 37 DEG C of reaction 2h
Reaction was completed by 2 μ L of × Loading Buffer.Thus digestion products are obtained.
5, endonuclease reaction product detection:
The Ago-Gel that compound concentration is 2% carries out electrophoresis detection to digestion products；Through restriction enzyme RsaI enzyme
After cutting, it is about 3 segments of 305bp, 209bp, 156bp that wide dragon COI segment, which generates size, and Eisenia Foetida COI segment generates
Size is about 2 segments of 548bp, 122bp；It is about 2 segments of 507bp, 155bp that the far blind earthworm COI segment of dark holes, which generates size,；
It is about 2 segments of 515bp, 143bp that power simulation COI segment, which generates size,；Big chamber earthworm COI segment does not have restricted inscribe
Enzyme RsaI recognition site, therefore band is still about 1 segment (Fig. 3) of 659bp.
Sequence table
<110>Guangdong Province's living resources Applied Research Laboratory
<120>a kind of method of based on PCR-RFLP technical appraisement wide dragon
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 26
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 1
taaacttcag ggtgaccaaa aaatca 26
<210> 2
<211> 25
<212> DNA
<213>artificial sequence (Artificial Sequence)
<400> 2
ggtcaacaaa tcataaagat attgg 25
<210> 3
<211> 670
<212> DNA
<213>Pheretima aspergillum (Pheretima aspergillum)
<400> 3
tctaggattt gagccggata attggagccg gaataagact tcttattcgt attgaattaa 60
gacaacctgg atccttcctt ggaagagatc agctatacaa cacaattgta acagcacacg 120
catttctaat aattttcttt ctagtaatgc cagtatttat tggtgggttt ggaaactgac 180
tgctcccact tatactagga acccccgaca tagcattccc acgtctaaat aacataagat 240
tttgactttt gccaccatcc ttaattctat tagtaaggtc tgcggctgtt gaaaagggag 300
ccggtaccgg atggacagtt tacccccctt tagcaagaaa catagcacat gcgggcccct 360
ctgtagacct tgcaattttc tcactacatt tagcgggtgc ctcatcaatt ttaggtgcca 420
ttaactttat cactacagta attaacatgc gatgatcggg gctacgctta gaacgaattc 480
cactatttgt ttgagccgta gtaattactg tagtacttct actattgtcg cttcccgtat 540
tagccggtgc tattactata ttactaacag accgaaatct aaatacatcc ttctttgacc 600
ccgctggagg tggcgaccca attctatatc aacatctatt ctgatttttt ggtcacctgg 660
gaaagtttaa 670
Claims (3)
1. a kind of method of based on PCR-RFLP technical appraisement wide dragon, which comprises the following steps: extract earthworm to be measured
The genomic DNA of earthworm sample is as template, with the universal primer HCO2198:5 '-of COI gene
TAAACTTCAGGGTGACCAAAAAATCA-3 ' and LCO1490:5 '-GGTCAACAAATCATAAAGATATTGG-3 ', which is used as, to be drawn
Object carries out PCR amplification, and PCR product is carried out digestion with restriction enzyme RsaI after purification, carries out electrophoresis inspection to digestion products
It surveys, if there are 3 segments, earthworm sample to be measured is wide dragon.
2. the method for based on PCR-RFLP technical appraisement wide dragon according to claim 1, which is characterized in that described
The reaction system of PCR amplification is 25 μ L, including 1 μ L, 10 × PCR Buffer of DNA profiling, 2.5 μ L, 25mmol/L MgCl2 2μ
0.5 μ L, Taq DNA polymerase 1U of L, 10mmol/L dNTPs, HCO2198 and the LCO1490 primer of 10 μm of ol/L are each
0.5 μ L, surplus are water；Response procedures are as follows: 95 DEG C of pre- change 4min；95 DEG C of denaturation 45s, 55 DEG C of annealing 45s, 72 DEG C of extension 45s, 35
A circulation；72 DEG C of extension 10min.
3. the method for based on PCR-RFLP technical appraisement wide dragon according to claim 1, which is characterized in that the enzyme
The endonuclease reaction system cut is 20 μ L, 822 μ L of μ L, 0.1%BSA of μ L, 10 × Buffer of PCR product including purifying, limitation
0.5 μ L of property restriction endonuclease RsaI, surplus is water；Reaction condition are as follows: 37 DEG C of reaction 2h add 10 × Loading Buffer, 2 μ L to tie
Shu Fanying.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110066878A (en) * | 2019-03-20 | 2019-07-30 | 中山大学 | The DNA molecular discrimination method of pheretima medicinal material in a kind of cerebral ischemic preparation |
| CN110923331A (en) * | 2019-12-03 | 2020-03-27 | 牡丹江友搏药业有限责任公司 | Primer pair and application thereof in identification of limnodrilus |
| CN110951891A (en) * | 2019-11-15 | 2020-04-03 | 牡丹江友搏药业有限责任公司 | Primer composition and application thereof in identification of limnodrilus |
| CN112522423A (en) * | 2020-12-15 | 2021-03-19 | 南京林业大学 | Fuworm microsatellite molecular marker and polymorphism primer and application thereof |
| CN113512591A (en) * | 2020-12-21 | 2021-10-19 | 中国农业科学院蔬菜花卉研究所 | Method and kit capable of identifying 3 fruit flies simultaneously |
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| CN1197116A (en) * | 1997-01-03 | 1998-10-28 | 王骏 | Applications of DNA internally-cut enzyme segment polymorphism in discriminating Chinese medicinal crop |
| CN103898234A (en) * | 2014-04-21 | 2014-07-02 | 牡丹江友搏药业股份有限公司 | Method for identifying DNA bar code molecule of earthworm |
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2018
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Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1197116A (en) * | 1997-01-03 | 1998-10-28 | 王骏 | Applications of DNA internally-cut enzyme segment polymorphism in discriminating Chinese medicinal crop |
| CN103898234A (en) * | 2014-04-21 | 2014-07-02 | 牡丹江友搏药业股份有限公司 | Method for identifying DNA bar code molecule of earthworm |
| CN106834467A (en) * | 2014-04-21 | 2017-06-13 | 牡丹江友搏药业有限责任公司 | A kind of DNA bar code method for identifying molecules of earthworm |
Cited By (7)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110066878A (en) * | 2019-03-20 | 2019-07-30 | 中山大学 | The DNA molecular discrimination method of pheretima medicinal material in a kind of cerebral ischemic preparation |
| CN110951891A (en) * | 2019-11-15 | 2020-04-03 | 牡丹江友搏药业有限责任公司 | Primer composition and application thereof in identification of limnodrilus |
| CN110923331A (en) * | 2019-12-03 | 2020-03-27 | 牡丹江友搏药业有限责任公司 | Primer pair and application thereof in identification of limnodrilus |
| CN110923331B (en) * | 2019-12-03 | 2023-05-02 | 牡丹江友搏药业有限责任公司 | Primer pair and application thereof in identification of limnodrilus |
| CN112522423A (en) * | 2020-12-15 | 2021-03-19 | 南京林业大学 | Fuworm microsatellite molecular marker and polymorphism primer and application thereof |
| CN112522423B (en) * | 2020-12-15 | 2023-07-18 | 南京林业大学 | Molecular marker of lumbrolumbricus microsatellite and polymorphic primer and application thereof |
| CN113512591A (en) * | 2020-12-21 | 2021-10-19 | 中国农业科学院蔬菜花卉研究所 | Method and kit capable of identifying 3 fruit flies simultaneously |
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Address after: 510260 No. 105 West Xingang Road, Guangzhou, Guangdong, Haizhuqu District Applicant after: Institute of zoology, Guangdong Academy of Sciences Address before: 510260 No. 105 West Xingang Road, Guangzhou, Guangdong, Haizhuqu District Applicant before: Guangdong Institute of Applied Biological Resources |
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