CN106834467A - A kind of DNA bar code method for identifying molecules of earthworm - Google Patents
A kind of DNA bar code method for identifying molecules of earthworm Download PDFInfo
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Abstract
The invention discloses the DNA bar code method for identifying molecules and its CO I sequence of a kind of earthworm, PCR (PCR) is carried out using the method amplify sample CO I genes, PCR primer is delivered to biological order-checking company after agarose gel is verified and is sequenced, by sequencing result by check and correction, sequence assembly by hand, and compare with open sequence, if with the homology of the gene order SEQ ID NO.1 NO.4 more than 99%, you can judge that described test serum is LUMBRICUS or Shanghai earthworm source.The present invention uses reliable DNA molecular identification technology, and Morphological Identification is carried out without multi-product kind over the ground, can fast and accurately identify《Pharmacopoeia of People's Republic of China》The earthworm kind of regulation and non-medicinal material earthworm kind, can further discriminate between LUMBRICUS and Shanghai earthworm kind in addition, enhance accuracy and reliability, it is ensured that earthworm security being used as medicine as medicinal material.
Description
The application is the divisional application of the patent of invention to Patent No. 201410162028.5.The applying date of original application is
On 04 21st, 2014, Application No. 201410162028.5, invention and created name was a kind of DNA bar code molecule mirror of earthworm
Determine method.
Technical field
The present invention relates to species identification field, and in particular to the DNA bar code method for identifying molecules of earthworm.
Background technology
Earthworm, i.e. earthworm, cold in nature, taste is micro- salty, is traditional Chinese medicine, and《Pharmacopoeia of People's Republic of China》Version in 2010
Only Annelida Ju Yin sections animal Pheretima aspergillum (the Pheretima aspergillum recorded under (one) " earthworm " item
(E.Perrier)), popular Pheretimatschiliensis (Pheretima vulgaris Chen), power simulation
(Pheretima.guillelmi (Michaelsen)) or comb the region between the heart and the diaphragm hair earthworm (Pheretima pectinifera Michaelsen)
Hirudo leech.Former is practised and claims " LUMBRICUS ", practises title " Shanghai earthworm " for three kinds afterwards.Earthworm exists《Sheng Nong's herbal classic》It is listed in low-grades,
Have functions that heat-clearing arresting convulsion, dredging collateral, relieving asthma, diuresis.It is used for hyperpyrexia, coma, twitch, joint pain, lung after normal frying
The diseases such as heat panting is coughed, oliguria oedema, hypertension.Li Shizhen (1518-1593 A.D.) exists《Compendium of Materia Medica》Referred to as there is clearing and activating the channels and collaterals, promoting blood circulation and removing blood stasis, prevention
Treatment cardiovascular and cerebrovascular disease effect.Modern study shows that earthworm contains various pharmacological components, and pharmacological action is almost related to people
Body each system, mainly have antithrombotic, thrombolysis and relieving asthma, anti-arrhythmia, step-down, immune enhancing, antiulcer, antipyretic, anti-inflammatory,
Many pharmacological actions such as analgesia, immunological regulation, wound healing, calmness, antitumor, clinical practice is widely.
The identification of multi-product kind over the ground is the basis being used as medicine, and the identification of current Chinese medicine earthworm uses live body discriminating mostly
Method, its formalness of Main Basiss and internal structure.But because the most formalness of animal medicinal material is similar, tissue is special
Levy without specificity, and by after place of production roughing and process of preparing, it is difficult to find out species shape and color in itself, micro- spy
Levying also has different degrees of destruction, causes LUMBRICUS to judge not relatively difficult.Therefore, need badly and seek a kind of new method, with more
Mend the defect of conventional sorting methods.
DNA bar code technology (DNA Barcoding) is divided by the DNA sequence dna to a standard genes of interest
Analysis, so as to quickly and accurately carry out the technology of species identification.DNA barcode the most frequently used in animal at present are cell colors
No. 1 partial sequence of gene (COI) of plain C oxidizing ferment.In recent years, DNA bar code technology have proved to be one it is effective
Bioassay means, can not only make strong supplement to conventional identification method, and because it is more objective, accurate, break through
Experience is depended on unduly in the past, can be helped identify species, be found novel species and the evolution pass of hidden kind, reconstruction species and higher level taxa
System etc..With DNA bar code technology, can be very good fast and effectively to identify the kind of LUMBRICUS and Shanghai earthworm.
The present invention provides a kind of DNA bar code method for identifying molecules of earthworm, without morphological observation, can quick and precisely reflect
It is fixed《Pharmacopoeia of People's Republic of China》The earthworm kind of regulation and non-medicinal material earthworm kind, it is ensured that the security of Chinese herb.
The content of the invention
The present invention overcomes the defect for existing in Morphological Identification earthworm method at present, improves the accurate of earthworm qualification result
Degree, is a kind of easily operated, sensitivity authentication method high.Technical scheme is as follows：
First from the earthworm sample extraction genomic DNA of collection, PCR (PCR) is carried out using pair of primers
One section of CO I gene of sample is amplified, amplified production is sequenced, then carry out sequence analysis comparison, set up LUMBRICUS and Shanghai earthworm
Sequence criteria database, on the basis of relatively database D NA, by analyzing the product result of PCR, root
According to the difference of CO I gene sequences, so as to reach the purpose of quick, precise Identification earthworm species.What the present invention was designed is used for《In
Magnificent people's republic's pharmacopeia》The earthworm kind of regulation and non-medicinal material earthworm cultivar identification method, using following steps：
1st, earthworm genomic DNA is extracted：Routinely animal DNA extracting method or blood/cell/tissue genomic DNA
Extracts kit carries out DNA extractions, and the DNA concentration of sample is diluted into 0.1 μ g/ μ l-2 μ g/ μ l with the deionized water for sterilizing.
2nd, amplification of DNA fragments, carries out PCR, i.e., expanded with special primer, and primer pair sequence is
LCO1490：5′-GGTCAACAAATCATAAAGATATTGG-3′；
HCO2198：5′-TAAACTTCAGGGTGACCAAAAAATCA-3′；
Amplification program is 94 DEG C of predegenerations 1 minute, and 45 DEG C are annealed 1.5 minutes, and 72 DEG C extend 1.5 minutes, and 5 circulate 94 DEG C
Denaturation 1 minute, 50 DEG C are annealed 1.5 minutes, and 72 DEG C extend 1 minute, and 35 72 DEG C of circulations extend 5 minutes.
3rd, PCR primer is entered into row agarose gel electrophoresis analysis, PCR fragment size is detected using DNAmarker.
4th, the judgement of qualification result：If there is the band of obvious clearly 709bp sizes, and without miscellaneous band, then can send biology
Sequencing company is sequenced.
5th, sequencing result is carried out into manual check and correction, sequence assembly, if with the gene order SEQ ID NO.1-SEQ
ID NO.4, if with any sequence homology more than 99%, you can judge that described test serum is above-mentioned kind.
Wherein, described earthworm DNA bar code method for identifying molecules uses blood/cell/tissue genomic DNA and carries
Take kit.
The primer：
Forward primer is 5 '-GGTCAACAAATCATAAAGATATTGG-3 ',
Reverse primer is 5 '-TAAACTTCAGGGTGACCAAAAAATCA-3 '.
The archaeal dna polymerase for being used is pfu high-fidelity enzymes.
Described electrophoresis is the agarose gel electrophoresis of 1%-2%.
Described stripe size needs to be detected with DNA maker.
Described DNA sequence dna splicing software includes CodonCode Aligner, Sequencher, Genious, DNA
The softwares such as star.
Compared with traditional Morphological Identification method, the gene order that the present invention is obtained is advantageously implemented《The Chinese people are total to
With state's pharmacopeia》The identification of the earthworm kind of regulation and non-medicinal material earthworm kind.
Specific embodiment
With reference to specific embodiment, the present invention will be further described：
1st, the collection of earthworm sample and preservation：The sample of collection is from Guangxi, Shanghai and other places.The sample of harvest is through 10% wine
Smart anaesthetic treatment about 20 minutes, then put to death and preserve within alcohol-pickled 15 minutes through 75%.
2nd, the pre-treatment of test specimen：Taking-up is stored in the earthworm sample in 75% alcohol, removes internal organ, it is to avoid internal organ content
The pollution of thing, remainder is cleaned 3 times with distilled water immersion, washes away alcohol, is taken afterbody about 20mg dissecting scissors and is inserted 2ml EP
Pipe, adds magnetic bead, and concussion is crushed.56 DEG C of Proteinase K warm bath 1 hour, is completely dissolved to tissue.Add 350 μ l chloroforms：Isoamyl
Alcohol (24: 1) is mixed, and 10,000 × g room temperatures are centrifuged 5 minutes, in careful absorption supernatant to new 1.5mL EP pipes.
3rd, prepared by DNA profiling：Using the blood/cell/tissue genome DNA extracting reagent kit DNA of Tiangeng biotech firm
Extracted, and the DNA concentration of sample is diluted to 0.5 μ g/ μ l with the deionized water for sterilizing.
4th, primer synthesis：The present embodiment the primer is as follows：
- the GGTCAACAAATCATAAAGATATTGG-3 ' of forward primer 5 '；
- the TAAACTTCAGGGTGACCAAAAAATCA-3 ' of reverse primer 5 '.
5th, PCR expand the present embodiment PCR reaction systems it is as follows
PCR reaction systems are 25 μ l：ddH2O 8.5uL, 2 × Taq PCR Master Mix 12.5uL, forward primer/
Reverse primer (2.5umol) each 1uL, DNA profiling 2uL 2 × Taq PCR Master Mix contain Taq archaeal dna polymerases,
dNTPs、MgCl2, reaction buffer, the reinforcing agent of PCR reactions and optimization agent and stabilizer, concentration is 2 ×, and without dyestuff；
Amplification program：Reaction condition is 94 DEG C of predegenerations 1 minute, and 45 DEG C are annealed 1.5 minutes, and 72 DEG C extend 1.5 minutes, 5 circulations 94
DEG C denaturation 1 minute, 50 DEG C are annealed 1.5 minutes, and 72 DEG C extend 1 minute, and 35 72 DEG C of circulations extend 5 minutes 6, agarose electrophoresis test
Card PCR results agarose electrophoresis display sample amplification is good, and the clear free from admixture of band can directly send biological order-checking company to be surveyed
Sequence.
6th, sequencing result sequence assembly：By sequencing result CodonCodeAligner biosoftwares, sequence is imported, will
By sequence assembling after primer shearing, then compare with SEQ ID NO.1-4 respectively, H1 samples are same with SEQ ID NO.1
Source rate 100%, is LUMBRICUS, and Pheretima aspergillum belongs to《Pharmacopoeia of People's Republic of China》The earthworm kind of regulation；H2, H3 sequence
It is the popular Pheretimatschiliensis of Shanghai earthworm with SEQ ID NO.2 homologies 100%, belongs to《Pharmacopoeia of People's Republic of China》The ground of regulation
Multi-product kind；H5, H6, H7 sample and SEQ ID NO.3 homologies 100%, are Shanghai earthworm power simulation, are belonged to《The Chinese people
Republic's pharmacopeia》The earthworm kind of regulation；H8, H9 with SEQ ID NO.4 homologies 100%, be Shanghai earthworm comb the region between the heart and the diaphragm hair earthworm, category
In《Pharmacopoeia of People's Republic of China》The earthworm kind of regulation.
Embodiment set forth in the present invention is not limitation of the invention, described in above-described embodiment and specification only
Be preference of the invention, any those skilled in the art according to conventional meanses it is contemplated that or fine setting changes and improvements,
Each fall within protection scope of the present invention.
Claims (11)
1. the DNA bar code method for identifying molecules of a kind of earthworm, it is characterised in that carry out Morphological Identification without multi-product kind over the ground,
Medicinal earthworm kind can fast and accurately be identified.
2. method according to claim 1, it is characterised in that identification LUMBRICUS and Shanghai earthworm kind can be distinguished.
3. method according to claim 1, it is characterised in that identification Shanghai earthworm, including popular Pheretimatschiliensis, prestige can be distinguished
Honest and clean Pheretimatschiliensis and comb the region between the heart and the diaphragm hair earthworm.
4. method according to claim 1 is DNA bar code method for identifying molecules, and its step mainly includes：
(1) the separation and Extraction genomic DNA from earthworm sample tissue to be measured；
(2) genomic DNA extracted with step (1) is template pair of primers, by PCR amplification；
(3) then take appropriate step (2) PCR amplification go out DNA product agarose electrophoresis separate identification amplification
Primer size, send biological order-checking company to be sequenced；
(4) sequencing result is spliced, the homology with the gene order SEQ ID NO.1-SEQ ID NO.4 is compared
Right, homology is more than 99%, you can judge identification earthworm kind.
5. primer according to claim 4, it is characterised in that the sequence of primed DNA is：
LCO1490：5’-GGTCAACAAATCATAAAGATATTGG-3’；
HCO2198：5’-TAAACTTCAGGGTGACCAAAAAATCA-3’.
6. PCR according to claim 4, it is characterised in that amplification condition is 94 DEG C of predegenerations 1 minute, 45
DEG C annealing 1.5 minutes, 72 DEG C extend 1.5 minutes, 5 circulation；94 DEG C are denatured 1 minute, and 50 DEG C are annealed 1.5 minutes, and 72 DEG C extend 1
Minute, 35 circulations；72 DEG C extend 5 minutes.
7. amplified production according to claim 4, it is characterised in that amplified fragments are 709bp, use 1%-2% agarose electrophoresis
Detection.
8. earthworm DNA bar code standard detection gene order according to claim 4, it is characterised in that the bar code mark
Quasi- detection gene is COI genes, with LUMBRICUS Pheretima aspergillum gene order SEQ ID NO.1 as described below：
AACACTATACTTCATTTTAGGAATTTGAGCTGGAATAATTGGAGCAGGTATAAGACTCCTTATTCGAATTGAA
CTAAGACAACCGGGCTCATTCCTGGGCAGAGATCAACTATATAACACAATTGTCACTGCTCATGCATTCTTAATAAT
TTTCTTTCTAGTTATACCAGTATTCATTGGAGGATTTGGAAATTGACTTCTTCCTCTAATACTCGGAACGCCAGATA
TAGCATTCCCACGACTTAATAATATAAGATTTTGACTTTTACCACCCTCTCTAATTCTATTAGTATCTTCTGCCGCT
GTGGAGAAAGGAGCAGGAACCGGATGAACAGTATATCCACCCCTAGCAAGAAATATTGCGCATGCTGGACCATCTGT
GGACCTCGCAATTTTTTCCCTTCACTTAGCGGGGGCATCATCTATCCTTGGAGCTATCAACTTTATTACCACAGTAA
TTAATATACGTTGATCAGGTCTACGACTAGAACGAATTCCACTATTTGTATGAGCAGTAGTAATTACTGTAGTCCTA
CTACTTCTATCCCTACCAGTACTCGCGGGGGCTATTACAATACTTCTAACAGATCGAAACCTAAATACATCTTTCTT
CGACCCAGCCGGTGGAGGAGATCCAATTCTATATCAACATCTATTC。
9. earthworm DNA bar code standard detection gene order according to claim 4, it is characterised in that the bar code mark
Quasi- detection gene is COI genes, with the popular Pheretimatschiliensis gene order SEQ ID NO.2 of Shanghai earthworm as described below：
GAATAGGTGTTGATATAAAATAGGGTCTCCCCCGCCAGCGGGGTCAAAGAAGGAGGTATTGAGATTACGGTCT
GTTAATAGTATTGTAATGGCCCCAGCAAGCACTGGAAGGGATAGTAGGAGAAGTACTACGGTGATTACTACTGCTCA
TACGAATAATGGAATTCGTTCTAGTCGTAGACCGGATCATCGCATATTAATTACTGTGGTAATAAAGTTAATAGCCC
CTAGAATTGAGGATGCACCAGCAAGGTGTAAAGAGAAAATCGCTAGGTCTACTGATGGTCCCGCATGTGCAATATTT
CTAGCAAGTGGGGGGTAAACTGTCCATCCTGTTCCGGCCCCCTTCTCCACAGCAGCAGAAGACACTAATAGAATGAG
AGATGGAGGTAGAAGTCAAAATCTCATGTTATTGAGTCGTGGAAAAGCTATGTCTGGTGTTCCAAGCATTAATGGGA
GAAGTCAGTTTCCAAATCCTCCAATAAATACTGGTATTACTAGAAAAAAAATTATTAAAAAAGCATGAGCTGTAACG
ATTGTATTATATAGTTGATCTCTGCCAAGAAATGAGCCTGGCTGTCTTAACTCAATTCGGATTAGTAATCTTATTCC
AGCTCCAATTATTCCGGCTCAGATACCTAGAATGAAATATAGGGTT。
10. earthworm DNA bar code standard detection gene order according to claim 4, it is characterised in that the bar code
Standard detection gene is COI genes, with Shanghai earthworm power simulation gene order SEQ ID NO.3 as described below：
GAATAGGTGTTGATATAAAATAGGGTCTCCCCCGCCAGCGGGGTCAAAGAAGGAGGTATTGAGATTACGGTCT
GTTAATAGTATTGTAATGGCCCCAGCAAGTACTGGAAGGGATAGTAGGAGAAGTACTACGGTGATTACCACTGCTCA
TACGAATAATGGAATTCGTTCTAGTCGTAGCCCGGATCATCGCATATTAATTACTGTGGTAATAAAGTTAATAGCCC
CTAGAATTGAGGATGCACCAGCAAGGTGTAAAGAGAAAATCGCCAGGTCTACTGATGGTCCCGCATGTGCAATATTT
CTAGCAAGTGGGGGGTAAACTGTTCACCCTGTTCCGGCCCCCTTCTCTACAGCAGCAGAAGACACTAATAGAATGAG
AGATGGAGGTAGAAGTCAAAATCTTATGTTATTGAGTCGTGGAAAAGCTATGTCTGGTGTTCCAAGCATTAATGGGA
GAAGCCAGTTTCCAAATCCCCCAATAAATACTGGTATTACTAGAAAAAAAATTATTAAAAAAGCATGAGCTGTAACG
ATTGTATTATATAGTTGATCTCTGCCAAGAAATGAGCCCGGCTGCCTTAATTCAATTCGGATTAGTAATCTTATTCC
AGCTCCAATTATTCCGGCTCAAATACCTAGAATGAAATATAGTGTT。
11. earthworm DNA bar code standard detection gene orders according to claim 4, it is characterised in that the bar code
Standard detection gene is COI genes, with Shanghai earthworm comb the region between the heart and the diaphragm hair earthworm gene order SEQ ID NO.4 as described below：
GAATAGATGTTGATATAGAATTGGATCTCCTCCACCGGCTGGGTCGAAGAAAGATGTATTTAGGTTTCGATCT
GTTAGAAGTATTGTAATAGCCCCCGCGAGTACTGGTAGGGATAGAAGTAGTAGGACTACAGTAATTACTACTGCTCA
TACAAATAGTGGAATTCGTTCTAGTCGTAGACCTGATCAACGTATATTAATTACTGTGGTAATAAAGTTGATAGCTC
CAAGGATAGATGATGCCCCCGCTAAGTGAAGGGAAAAAATTGCGAGGTCCACAGATGGTCCAGCATGCGCAATATTT
CTTGCTAGGGGTGGATATACTGTTCATCCGGTTCCTGCTCCTTTCTCCACAGCGGCAGAAGATACTAATAGAATTAG
AGAGGGTGGTAAAAGTCAAAATCTTATATTATTAAGTCGTGGGAATGCTATATCTGGCGTTCCGAGTATTAGAGGAA
GAAGTCAATTTCCAAATCCTCCAATGAATACTGGTATAACTAGAAAGAAAATTATTAAGAATGCATGAGCAGTGACA
ATTGTGTTATATAGTTGATCTCTGCCCAGGAATGAGCCCGGTTGTCTTAGTTCAATTCGAATAAGGAGTCTTATACC
TGCTCCAATTATTCCAGCTCAAATTCCTAAAATGAAGTATAGTGTT。
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| CN114277156A (en) * | 2021-12-21 | 2022-04-05 | 华润三九医药股份有限公司 | Specific primer pair for identifying Pheretima aspergillum formula granules and application thereof |
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| CN105112525A (en) * | 2015-08-27 | 2015-12-02 | 中国医学科学院药用植物研究所 | Method and PCR (polymerase chain reaction) reagent kit for identifying DNA (deoxyribonucleic acid) barcodes of animal medicinal materials |
| CN106591426B (en) * | 2016-03-02 | 2020-04-03 | 西南林业大学 | COI gene standard complete sequence and molecular identification method of great brow apes |
| CN107779512A (en) * | 2016-08-24 | 2018-03-09 | 周亚伟 | A kind of PCR method for identifying earthworm |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1588049A (en) * | 2004-09-27 | 2005-03-02 | 上海中药创新研究中心 | Method for producing Chinese medicine fingerprint atlas |
| CN103451312A (en) * | 2013-09-25 | 2013-12-18 | 张龙霏 | Method for quickly identifying scorpion medicinal materials in field of molecular biology |
-
2014
- 2014-04-21 CN CN201710072861.4A patent/CN106834467A/en active Pending
- 2014-04-21 CN CN201410162028.5A patent/CN103898234A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN1588049A (en) * | 2004-09-27 | 2005-03-02 | 上海中药创新研究中心 | Method for producing Chinese medicine fingerprint atlas |
| CN103451312A (en) * | 2013-09-25 | 2013-12-18 | 张龙霏 | Method for quickly identifying scorpion medicinal materials in field of molecular biology |
Non-Patent Citations (2)
| Title |
|---|
| 吕国庆: ""动物性中药材地龙DNA条形码的初步研究"", 《中国优秀硕士学位论文全文数据库 医药卫生科技辑》 * |
| 吕国庆等: ""动物性中药材地龙 DNA 条形码初步研究"", 《广东农业科学》 * |
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| CN114277156A (en) * | 2021-12-21 | 2022-04-05 | 华润三九医药股份有限公司 | Specific primer pair for identifying Pheretima aspergillum formula granules and application thereof |
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