CN108866205A - Identify the specific primer of hiruto based on molecular biology method - Google Patents

Identify the specific primer of hiruto based on molecular biology method Download PDF

Info

Publication number
CN108866205A
CN108866205A CN201810824002.0A CN201810824002A CN108866205A CN 108866205 A CN108866205 A CN 108866205A CN 201810824002 A CN201810824002 A CN 201810824002A CN 108866205 A CN108866205 A CN 108866205A
Authority
CN
China
Prior art keywords
hiruto
primer
sample
molecular biology
identify
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.)
Pending
Application number
CN201810824002.0A
Other languages
Chinese (zh)
Inventor
郑健
刘杰
过立农
昝珂
马双成
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
National Institutes for Food and Drug Control
Original Assignee
National Institutes for Food and Drug Control
Priority date (The priority date 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 date listed.)
Filing date
Publication date
Application filed by National Institutes for Food and Drug Control filed Critical National Institutes for Food and Drug Control
Priority to CN201810824002.0A priority Critical patent/CN108866205A/en
Publication of CN108866205A publication Critical patent/CN108866205A/en
Pending legal-status Critical Current

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
    • 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
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]

Abstract

The invention discloses the specific primer for identifying hiruto based on molecular biology method, upstream primer is：5'-TCTGGCTCTTAGGCTTCG-3', downstream primer are：5'-GTTGTCTTCCTGGCTGTT-3'.Quickly identify the method for hiruto based on the primer amplified, key step is as follows：（1）The extraction of total DNA, the genomic DNA extracted are carried out to sample to be tested；（2）The genomic DNA extracted using step (1) carries out polymerase chain reaction amplification as template, using upstream primer described in claim 1, downstream primer；（3）To step（2）Gained reaction product carries out agarose gel electrophoresis detection and gel imaging, realizes and identifies to the specificity of hiruto, provides reference for the molecular biological variety identification method of hiruto.

Description

Identify the specific primer of hiruto based on molecular biology method
Technical field
The invention belongs to technical field of molecular biology, and in particular to one kind identifies hiruto based on molecular biology method Specific primer.
Background technique
Hiruto (Poecilobdella manillensis) is commonly called as phnom penh leech, the quasi- doctor leech of alias horse Buddhist nun, chemistry at Divide mainly there are many fatty acid, amino acid and microelement etc., effective component is mainly hirudin, Bufrudin A, Bufrudin B, in addition to this hiruto also contains some other bioactive ingredients, such as Anticoagulant peptide, fibrinoclase, hyalomitome Sour enzyme, vasodilator, platelet aggregation inhibitory activity ingredient, bacteriostatic active ingredients etc..The pharmacological action of hiruto mainly has anti- Platelet aggregation and blood coagulation resisting function, inhibition thrombosis effect, effect for reducing fat, antitumor action promote angiogenesis effect, Effective component, pharmacological action, mechanism of action and the molecular biology related data of comprehensive hiruto are further development and utilization Hiruto is laid a good foundation, and hiruto occurs as a kind of newtype drug, is prevention and cure of cardiovascular disease, and in particular hyperlipidemia is suffered from Person brings dawn.
The active constituent and drug activity of different leech kinds often have differences, and are with eurysome golden thread leech and hiruto Example, the active constituent of the two has very big difference, and hiruto has more preferably drug effect than the eurysome golden thread leech being widely used at present Activity.Hiruto is also known as phnom penh leech, and the Phnom Penh of body two sides is its identification mark, but hiruto is after death and its dry product gold Side disappears, and is not easy to differentiate, identify, leech kind is miscellaneous more in the market, wherein it is no lack of some import leech medicinal material kinds, how will be luxuriant and rich with fragrance Ox leech distinguishes with other kinds, most important for reasonable employment hiruto.
Hiruto was indexed to by Yunnan Province in 2013《Yunnan Province's Chinese medicine standard》In, the strong medicine of Guangxi Zhuang Autonomous Region Hiruto has also been included in volume two, has pushed the standardized process of hiruto medicinal material.Currently, for the identification side of hiruto kind Method mainly has vertical flat plate sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), gene order and molecule Evolutionary analysis method etc..
Currently, relying primarily on character identification and molecular biology identification means, character to the identification method of hiruto medicinal material Identification is very high to the professional requirement of appraiser, and different concocting methods, condition of storage can all give the identification of hiruto medicinal material Larger difficulty is brought, current molecular biology identification means are the method for DNA bar code, need to be by the amplified production of universal primer It is sequenced, and sequencing sequence is compared, taken a long time, and required operator to be familiar with molecular biosciences profession and know Know and be familiar with the use of professional software.
Summary of the invention
The technical problem to be solved by the present invention is to provide one kind in view of the deficiency of the prior art and be based on dividing Sub- biological method identifies the specific primer of hiruto, so effective that distinguish hiruto and other leech samples, for phenanthrene The molecular biological variety identification method of ox leech provides reference.
The present invention be solve the problems, such as it is set forth above used by technical solution be：
Identify the specific primer of hiruto based on molecular biology method, which is characterized in that upstream primer is：5'- TCTGGCTCTTAGGCTTCG-3', downstream primer are：5'-GTTGTCTTCCTGGCTGTT-3'.
Above-mentioned primer is quickly identifying the application in hiruto.
The present invention also provides a kind of method for quickly identifying hiruto using above-mentioned primer, key step is as follows：
(1) extraction of total DNA, the genomic DNA extracted are carried out to sample to be tested；
(2) genomic DNA extracted using step (1) carries out polymerase using above-mentioned upstream primer, downstream primer as template Chain reaction (PCR) amplification；
(3) agarose gel electrophoresis detection and gel imaging are carried out to reaction product obtained by step (2), realized to hiruto Specificity identify.
According to the above scheme, in step (3), specific identification is carried out to hiruto according to the result of gel imaging, effective Under the premise of extracting sample to be tested DNA profiling, when observing single bright electrophoretic band, then identify that sample to be tested is hiruto； When not observing single bright electrophoretic band, then identify that sample to be tested is not hiruto.
Compared with prior art, the beneficial effects of the invention are as follows：
It hiruto and its is made a living more than close kind with sucking blood, identify by universal primer in the prior art and be easy by phenanthrene Ox leech sucks the interference of blood, influences sequencing result, and it is long that the period needed is sequenced, and the analysis of sequencing sequence needs to have There is professional knowledge and understands professional software.The present invention provides the specific primer of a pair of of hiruto, is reached by specific amplification The purpose of hiruto and other kinds is distinguished, it is intuitive whether band comparison can be generated by the result of observation gel electrophoresis Qualification result is embodied, while reducing the time of sequencing and sequencing result comparison analysis.The present invention passes through hiruto specificity Primer identification hiruto reduces the operating time of medicine inspection personnel, compresses Check-Out Time, improves checkability, while reducing theory For the profession limitation of reviewer.
Detailed description of the invention
Fig. 1 is the NJ tree established based on COI sequence.
Fig. 2 is specific amplification electrophoresis detection result figure, wherein M：Maker(DL2000)；K：Negative control (H2O)；1 ~20：Number SZ-1~SZ-20 sample.
Fig. 3 is the electrophoresis detection slice result of number SZ-10, SZ-14, SZ-18 hiruto sample under different annealing temperature, It is successively from left to right wherein number SZ-10, SZ-14, SZ-18 at each temperature.
Specific embodiment
For a better understanding of the present invention, below with reference to the embodiment content that the present invention is furture elucidated, but the present invention is not It is limited only to the following examples.
In following embodiments, involved key instrument and reagent are as follows：(ThernoFisher is public for ABI Veriti PCR instrument Department), EPS-301 electrophoresis apparatus (Amersham company), GelDoc XR+ full automatic gel imaging system (BIORAD company)；Animal Blood tissues genome extraction kit DNeasy Blood&Tissue Kit (article No. 69504, QIAGEN company), 2 × Taq Master Mix buffer (article No. GK8006, GENEray company), (article No. 41003, Biotium are public by DNA dyestuff GelRed Department), agarose (Biowest company), trishydroxymethylaminomethane Tris-base (Sigma company), general COI primer 5'- The Shanghai TAAGAGGTTTACCACCGTTAT-3' and 5'-GTAGATGAAG TAGTTGACCCAA-3'(JaRa bioengineering is limited Company).
In following embodiments, leech fresh sample is respectively collected from Guangdong Province, Yunnan Province, Guangxi Zhuang Autonomous Region, medicinal material Dry product is collected from Anguo, Bozhou, the peaceful medicinal material market in Guangzhou.Wherein sample SZ-4, SZ-7, SZ-8, SZ-10, SZ-14, SZ- 17, SZ-20 is accredited as hiruto Poecilobdella by National Institute for Food and Drugs Control Zheng Jian researcher Manillensis, sample message are shown in Table 1.
1 leech sample message table of table
In following embodiments, sequencing peak figure using CodonCode Aligner 3.7.1 software (CodonCode Co., USA) check and correction splicing removes primer section；All sequences are utilized into 5.0 software of MEGA (molecular evolutionary Genetics analysis) it analyses and compares；Phylogenetic tree is established using NJ tree (Neighbor-Joining Tree) method, is led to It crosses bootstrap (1000 repetitions) and rate inspection is supported to each branch.
Embodiment 1
Identify a pair of of specific primer of hiruto based on molecular biology specific amplification, upstream primer is：5'- TCTGGCTCTTAGGCTTCG-3', downstream primer are：5'-GTTGTCTTCCTGGCTGTT-3', by Shanghai JaRa bioengineering Co., Ltd's synthesis.
Quickly identify the method for hiruto using above-mentioned primer, sample to be tested selects number SZ-1~SZ-20 in table 1 respectively Leech sample carries out parallel laboratory test, identifies whether number SZ-1~SZ-20 leech sample is hiruto；Specific step is as follows：
(1) extraction of total DNA：20~30mg of sample to be tested is taken, dry product is crushed with MM400 ball milling instrument, fresh sample hand Art shreds, and carries out DNA extraction using the DNeasy Blood&Tissue Kit genome extraction kit of QIAGEN company；
(2) polymerase chain reaction：20 μ L of PCR reaction system includes 2 × Taq Master Mix buffer, 10 μ L, up and down Swim each 0.4 μ L of .5 μm of ol/L of primer 2,1 μ L of DNA profiling 50ng, the 8.2 μ L of distilled water of sterilizing.PCR reaction condition is：95 DEG C pre- It is denaturalized 4min；95 DEG C of denaturation 30s, 45~55 DEG C of annealing 30s, 72 DEG C of extension 1min are recycled 35 times；72 DEG C of extension 7min.
(3) agarose gel electrophoresis detection and gel imaging are carried out to reaction product obtained by step (2).50 × TAE electrophoresis The preparation of buffer：Trishydroxymethylaminomethane 242g, glacial acetic acid 57.1mL, EDTA-Na22H2O 37.2g are taken, is added suitable Deionized water is measured, dissolution is sufficiently stirred, is settled to 1000mL, as storage liquid, dilute when gel electrophoresis 50 times into work Liquid.The concentration 2% (mass volume ratio) of Ago-Gel, configuration Ago-Gel 40mL be added 3 μ L of DNA dyestuff GelRed and 3 μ L of reaction product obtained by step (2), electrophoresis detection voltage are 3~5V/cm, electrophoresis time 30min.Detected through gel electrophoresis result As shown in Fig. 2, wherein number SZ-1, SZ-4, SZ-8, SZ-10, SZ-14, SZ-17, SZ-18, SZ-19, SZ-20 totally 9 samples Product observe single bright band, are accredited as hiruto；The sample to be tested of other numbers does not observe single bright band, identification For non-hiruto.
Verifying：By universal primer COI (5'-TAAGAGGTTTACCACCGTTAT-3' and 5'- GTAGATGAAGTAGTTGACCCAA-3' amplified production) carries out detected through gel electrophoresis, 20 crowdes of sample (number SZ-1~SZ-20 Sample) electrophoresis detection result be illustrated as single bright band, by the amplified production of 20 batches of COI primers send Invitrogen (match Mo Feishier Science and Technology Ltd.) it is sequenced.
The sequencing sequence of leech sample COI sequence uses CodonCode Aligner 3.7.1 software (CodonCode Co., USA) check and correction splicing, primer section is removed, the COI sequence length for obtaining 20 batches of leech samples is 656bp.By all sequences It is analysed and compared using 5.0 software of MEGA (molecular evolutionary genetics analysis).Using NJ tree (Neighbor-Joining Tree) method establishes phylogenetic tree, by bootstrap (1 000 repetitions) to each branch into Row supporting rate is examined, and it is as shown in Figure 1 to obtain the NJ tree established based on COI sequence.SZ-4,SZ-8,SZ-10,SZ-14,SZ-17, Totally 6 samples are identified as hiruto, SZ-1, SZ-4, SZ-8, SZ-10, SZ-14, SZ-17, SZ-18, SZ- in Fig. 1 to SZ-20 19, totally 9 samples gather SZ-20 is one, and COI sequence is completely the same, and sequence results are： ACTCTATATTTGATTCTAGGGGCTTGGGCAGCTATATTAGGCTCCTCTATAAGAACTATTATTCGAATTGAGTTATC TCAACCAGGTAGGTTTCTTGGGGATGATCAACTTTATAATTCTTTAATTACTGCACATGGACTTATTATAATTTTTT TTATAGTAATACCTATTTTAATCGGTGGGTTTGGTAATTGACTTTTACCGTTAATAATTGGTGCCCCAGATATGGCT TTTCCACGATTAAATAATTTTAGGTTTTGATTATTACCACCTTCATTAACTATATTAGTAAGATCATCAATAATTGA ATCCGGTGTTGGTACAGGATGGACTATTTATCCACCATTAGCTGATAGAGTTTCTCACTCAGGACCTTGTGTAGATA TAGCTATCTTTTCATTGCATATAGCTGGTGCATCATCTATTTTAGGTTCTTTAAATTTTATTTCTACTATTATTAAT ATGCGAACTAATGGTATAAGTAATGAACGAGTTCCATTATTTGTTTGATCTGTTGTAATTACTACTATCTTATTATT ACTTTCATTACCTGTATTAGCAGCAGCTATTACAATGTTATTAACTGATCGTAATTTAAATACTTCATTTTTTGATC CAATGGGTGGTGGAGATCCAGTATTATTTCAACACTTATT, thus may determine that this 9 samples are hiruto, other 11 There are base differences with hiruto sample for the COI sequence of a sample, and wherein the base difference between SZ-2 and hiruto is minimum, are 63 bases, thus may determine that other 11 samples are non-hiruto kind.The conclusion is consistent with the present embodiment 1 as a result,.
Embodiment 2
The investigation of annealing temperature has been carried out to the specific primer in embodiment 1, has investigated 45 DEG C, 47 DEG C, 49 DEG C, 51 altogether DEG C, 53 DEG C, 55 DEG C of totally 6 annealing temperatures, according to investigate as a result, determine 49 DEG C for 7 specific amplification hiruto sequence of primer Optimum annealing temperature.Specific step is as follows：
(1) extraction of total DNA：Three batches of samples for randomly selecting number SZ-10, SZ-14, SZ-18, take sample to be tested 20~ 30mg, dry product are crushed with MM400 ball milling instrument, and fresh sample is shredded with operating scissors, utilize the DNeasy Blood& of QIAGEN company Tissue Kit genome extraction kit carries out DNA extraction；
(2) polymerase chain reaction：20 μ L of PCR reaction system includes 2 × Taq Master Mix buffer, 10 μ L, up and down Swim each 0.4 μ L of 5 μm of ol/L of primer 2, DNA profiling 50ng/ μ L1 μ L, the 8.2 μ L of distilled water of sterilizing.PCR reaction condition is：95℃ Initial denaturation 4min；95 DEG C of denaturation 30s, (45 DEG C, 47 DEG C, 49 DEG C, 51 DEG C, 53 DEG C, 55 DEG C) annealing 30s, 72 DEG C of extension 1min are followed Ring 35 times；72 DEG C of extension 7min.
(3) agarose gel electrophoresis detection and gel imaging are carried out to reaction product obtained by step (2).50 × TAE electrophoresis The preparation of buffer：Take trishydroxymethylaminomethane 242g, glacial acetic acid 57.1mL, EDTA-Na2·2H2O 37.2g is added appropriate Deionized water is sufficiently stirred dissolution, is settled to 1000mL, as storage liquid, dilute when gel electrophoresis 50 times into working solution. 3 μ L of DNA dyestuff GelRed, electrophoresis is added in the concentration 2% (mass volume ratio) of Ago-Gel, configuration Ago-Gel 40mL Detection voltage is 3~5V/cm, electrophoresis time 30min.
Wherein, 3 batches of hiruto samples of number SZ-10, SZ-14, SZ-18 for randomly selecting are respectively in 45 DEG C, 47 DEG C, 49 DEG C, 51 DEG C, 53 DEG C, the amplified production detected through gel electrophoresis result under 55 DEG C of six annealing temperatures is as shown in figure 3, in annealing temperature When being 49 DEG C, band is most obvious in electrophoresis detection result, comprehensively considers, and determines that 49 DEG C are the best of hiruto specific primer 7 Annealing temperature.
The above is only a preferred embodiment of the present invention, it is noted that come for those of ordinary skill in the art It says, without departing from the concept of the premise of the invention, several modifications and variations can also be made, these belong to of the invention Protection scope.
110 > National Institute for Food and Drugs Control of <
120 > of < identifies the specific primer of hiruto based on molecular biology method
160 > 5 of <
<210>1
<211>18
<212>DNA
<213>artificial sequence
<400>1
tctggctctt aggcttcg 18
<210>2
<211>18
<212>DNA
<213>artificial sequence
<400>2
gttgtcttcc tggctgtt 18
<210>3
<211>21
<212>DNA
<213>artificial sequence
<400>3
taagaggttt accaccgtta t 21
<210>4
<211>22
<212>DNA
<213>artificial sequence
<400>4
gtagatgaag tagttgaccc aa 22
<210>5
<211>656
<212>DNA
<213>Hiruto
<400>5
actctatatt tgattctagg ggcttgggca gctatattag gctcctctat aagaactatt 60
attcgaattg agttatctca accaggtagg tttcttgggg atgatcaact ttataattct 120
ttaattactg cacatggact tattataatt ttttttatag taatacctat tttaatcggt 180
gggtttggta attgactttt accgttaata attggtgccc cagatatggc ttttccacga 240
ttaaataatt ttaggttttg attattacca ccttcattaa ctatattagt aagatcatca 300
ataattgaat ccggtgttgg tacaggatgg actatttatc caccattagc tgatagagtt 360
tctcactcag gaccttgtgt agatatagct atcttttcat tgcatatagc tggtgcatca 420
tctattttag gttctttaaa ttttatttct actattatta atatgcgaac taatggtata 480
agtaatgaac gagttccatt atttgtttga tatgttgtaa ttactactat cttattatta 540
ctttcattac ctgtattagc agcagctatt acaatgttat taactgatcg taatttaaat 600
acttcatttt ttgatccaat gggtggtgga gatccagtat tatttcaaca cttatt 656

Claims (4)

1. identifying the specific primer of hiruto based on molecular biology method, which is characterized in that upstream primer is：5'- TCTGGCTCTTAGGCTTCG-3', downstream primer are：5'-GTTGTCTTCCTGGCTGTT-3'.
2. specific primer described in claim 1 identifies the application in hiruto reagent in preparation.
3. quickly identifying the method for hiruto based on molecular biology specific amplification, which is characterized in that key step is as follows：
（1）Effective extraction of total DNA, the genomic DNA extracted are carried out to sample to be tested；
（2）The genomic DNA extracted using step (1) is carried out as template using upstream primer described in claim 1, downstream primer Polymerase chain reaction amplification；
（3）To step（2）Gained reaction product carries out agarose gel electrophoresis detection and gel imaging, realizes the spy to hiruto The opposite sex identifies.
4. quickly identifying the method for hiruto based on molecular biology specific amplification according to claim 3, feature exists In step（3）In, specific identification is carried out to hiruto according to the result of gel imaging, when observing single bright band, then Identify that sample to be tested is hiruto；When not observing single bright band, then identify that sample to be tested is not hiruto.
CN201810824002.0A 2018-07-25 2018-07-25 Identify the specific primer of hiruto based on molecular biology method Pending CN108866205A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN201810824002.0A CN108866205A (en) 2018-07-25 2018-07-25 Identify the specific primer of hiruto based on molecular biology method

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN201810824002.0A CN108866205A (en) 2018-07-25 2018-07-25 Identify the specific primer of hiruto based on molecular biology method

Publications (1)

Publication Number Publication Date
CN108866205A true CN108866205A (en) 2018-11-23

Family

ID=64305158

Family Applications (1)

Application Number Title Priority Date Filing Date
CN201810824002.0A Pending CN108866205A (en) 2018-07-25 2018-07-25 Identify the specific primer of hiruto based on molecular biology method

Country Status (1)

Country Link
CN (1) CN108866205A (en)

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109504782A (en) * 2018-11-30 2019-03-22 中国食品药品检定研究院 A pair of of specificity identifies the primer of eurysome golden thread leech
CN109680071A (en) * 2018-11-30 2019-04-26 中国食品药品检定研究院 Identify the primer set and method of leech kind
CN109762908A (en) * 2018-11-30 2019-05-17 岛津企业管理（中国）有限公司 Identify hiruto specific primer to and method, application

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002066628A2 (en) * 2001-02-20 2002-08-29 Aventis Pharma Deutschland Gmbh Supersecretable peptides, processes for their production, and parallel improvement of the secreted form of one or more other polypeptides
CN103848914A (en) * 2012-11-29 2014-06-11 河北以岭医药研究院有限公司 Bufrudin polypeptide with anticoagulant activity and preparation method and purpose thereof
CN105506103A (en) * 2015-12-28 2016-04-20 天津中医药大学 Mitochondria genome amplified universal primer mixture as well as design and amplification method thereof
CN107937566A (en) * 2017-12-29 2018-04-20 江苏大学 The specific primer and its identification method of one group of identification leech

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2002066628A2 (en) * 2001-02-20 2002-08-29 Aventis Pharma Deutschland Gmbh Supersecretable peptides, processes for their production, and parallel improvement of the secreted form of one or more other polypeptides
CN103848914A (en) * 2012-11-29 2014-06-11 河北以岭医药研究院有限公司 Bufrudin polypeptide with anticoagulant activity and preparation method and purpose thereof
CN105506103A (en) * 2015-12-28 2016-04-20 天津中医药大学 Mitochondria genome amplified universal primer mixture as well as design and amplification method thereof
CN107937566A (en) * 2017-12-29 2018-04-20 江苏大学 The specific primer and its identification method of one group of identification leech

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
GENBANK: "X72785.1", 《NCBI》 *
GENBANK: "X72786.1", 《NCBI》 *
谭恩光等: "广东菲牛蛭水蛭素基因的克隆和序列测定 ", 《中山医科大学学报》 *
黎渊弘: "广西菲牛蛭水蛭素基因cDNA的克隆与序列分析 ", 《中国生化药物杂志》 *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109504782A (en) * 2018-11-30 2019-03-22 中国食品药品检定研究院 A pair of of specificity identifies the primer of eurysome golden thread leech
CN109680071A (en) * 2018-11-30 2019-04-26 中国食品药品检定研究院 Identify the primer set and method of leech kind
CN109762908A (en) * 2018-11-30 2019-05-17 岛津企业管理（中国）有限公司 Identify hiruto specific primer to and method, application
CN109680071B (en) * 2018-11-30 2022-04-05 中国食品药品检定研究院 Complete set of primers and method for identifying leech variety

Similar Documents

Publication Publication Date Title
CN106947838A (en) African swine fever virus nonstructural gene real-time fluorescence LAMP detection primer group, kit and detection method
CN108866205A (en) Identify the specific primer of hiruto based on molecular biology method
CN104498599B (en) One group of microsporidian molecule universal detector primer and kit thereof
CN110066878B (en) DNA molecular identification method of earthworm medicinal material in Naoxintong preparation
CN107988376A (en) A kind of application of relevant SNP marker of the colour of skin in black-bone chicken Genotyping
CN110567951A (en) Apple stem groove virus visual detection system based on CRISPR-Cas12a technology and detection method thereof
CN116814812A (en) Primer probe set for identifying scorpion of Buthus martensii Karsch and real-time fluorescence PCR identification method
CN109136389A (en) A kind of DNA bar code standard detection segment CO II, kit and its application method identifying American cockroach
CN106282373A (en) A kind of Herba Dendrobii and the contrast authentication method of Henan Herba Dendrobii
CN105543373B (en) Glycyrrhiza Uralensis, glycyrrhiza glabra, swollen fruit Radix and its Hybrid rapid molecular identification method
CN105603067A (en) Specific primer pair for rapid identification of alien invasive Pomacea in China based on multiple PCR (Polymerase Chain Reaction) and application of specific primer pair
WO2023231532A1 (en) Snp site combination for identifying variety of lonicera japonica thunb., primer combination, and method for identifying variety of lonicera japonica thunb.
CN112322768A (en) Method for diagnosing hippophae rhamnoides branch wilt and rapidly detecting RPA (resilient root antigen) of pathogenic bacteria
CN109762919B (en) Method for rapidly identifying raspberry and various confuses thereof
CN103451312A (en) Method for quickly identifying scorpion medicinal materials in field of molecular biology
CN110541035A (en) Sinocyclocheilus sinensis DNA barcode sequence and application thereof
CN112359135B (en) Indel labeled primer of fritillaria taipaiensis and application thereof
CN110885898B (en) Molecular specific marker primer and method for identifying amaranthus rugosus and 2 common confused products
CN107630014A (en) A kind of authentication method of Aconitum transsectum DNA bar code and Aconitum transsectum based on big data
CN113005218A (en) LAMP (loop-mediated isothermal amplification) detection primer, kit and detection method for fusarium solani
CN111440891A (en) DNA barcode composition for identifying origin of cordyceps sinensis and application thereof
CN105200050B (en) Differentiate the molecular specificity labeled primers and method of cordate houttuynia and Chinese gymnotheca herb
CN108998560A (en) Identify SNP marker, ApoE gene method and the application of rhizoma corydalis
CN108998512A (en) The hands-free kit and detection method for CYP2C19 genetic polymorphism detection taken
NL2029315B1 (en) Method for identifying cynomorium songaricum rupr

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
RJ01 Rejection of invention patent application after publication

Application publication date: 20181123

RJ01 Rejection of invention patent application after publication