CN105886512A - A group of oligonucleotide aptamers for identifying clenbuterol hydrochloride, salbutamol and ractopamine with high specificity - Google Patents
A group of oligonucleotide aptamers for identifying clenbuterol hydrochloride, salbutamol and ractopamine with high specificity Download PDFInfo
- Publication number
- CN105886512A CN105886512A CN201610128853.2A CN201610128853A CN105886512A CN 105886512 A CN105886512 A CN 105886512A CN 201610128853 A CN201610128853 A CN 201610128853A CN 105886512 A CN105886512 A CN 105886512A
- Authority
- CN
- China
- Prior art keywords
- oligonucleotide
- apt
- ractopamine
- aptamer
- oligonucleotide aptamer
- 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.)
- Granted
Links
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/115—Aptamers, i.e. nucleic acids binding a target molecule specifically and with high affinity without hybridising therewith ; Nucleic acids binding to non-nucleic acids, e.g. aptamers
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/10—Type of nucleic acid
- C12N2310/16—Aptamers
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Biomedical Technology (AREA)
- Chemical & Material Sciences (AREA)
- Genetics & Genomics (AREA)
- Molecular Biology (AREA)
- Immunology (AREA)
- Biotechnology (AREA)
- Hematology (AREA)
- Organic Chemistry (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Microbiology (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- General Engineering & Computer Science (AREA)
- Biochemistry (AREA)
- General Health & Medical Sciences (AREA)
- Urology & Nephrology (AREA)
- Physics & Mathematics (AREA)
- Cell Biology (AREA)
- Biophysics (AREA)
- Plant Pathology (AREA)
- Food Science & Technology (AREA)
- Medicinal Chemistry (AREA)
- Analytical Chemistry (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
The invention provides a group of oligonucleotide aptamers Apt-1, Apt-2 and Apt-3 which are capable of simultaneously identifying clenbuterol hydrochloride and salbutamol, an oligonucleotide aptamer CLB-2 which is capable of identifying the clenbuterol hydrochloride with high specificity, an oligonucleotide aptamer SAL-5 which is capable of identifying the salbutamol with high specificity and two oligonucleotide aptamers RAC-5 and RAC-6 which are capable of identifying ractopamine with high specificity. Through an SELEX (Systematic Evolution of Ligands by Exponential Enrichment) technology based on Fe3O4 magnetic nanoparticle separation, a random oligonucleotide library is immobilized on avidin-enveloped magnetic nanoparticles by virtue of a complementary chain of a biotinylation marker, and the oligonucleotide aptamers, which are high in specific affinity, are finally obtained by conducting screening by 16 turns. The aptamers are broad in application prospect; and the aptamers, by virtue of marker function genes or fluorescent dyes, are applicable to detection of the clenbuterol hydrochloride, the salbutamol and the ractopamine in food; therefore, a new choice is provided for existing detection methods which depend on antibodies.
Description
Technical field
The present invention relates to biological technical field of food safety, be related specifically to utilize SELEX technology (the part system of index concentration
System evolution technology) screen the oligonucleotide aptamer of one group of identification clenobuterol hydrochloride, the few core of one group of identification albuterol respectively
Thuja acid aptamer and the oligonucleotide aptamer of one group of identification Ractopamine, for based on thin in oligonucleotide aptamer detection food
The application of meat essence provides scientific basis and theoretical basis.
Background technology
Clenbuterol hydrochloride is as the receptor,β activator of a kind of synthetic, it is possible to reduce Animal fat content, increases lean meat
Rate, simultaneously facilitates growth of animal, reduces feedstuff consumption, thus is used for husbandry sector.But clenbuterol hydrochloride metabolism in animal body
Slowly, enter human body body accumulation through meat products, make human body produce dizzy, weak, cardiopalmus, the poisoning symptom such as numb limbs and tense tendons,
The health of the serious harm mankind.On market, the main clenbuterol hydrochloride molecule used includes: clenobuterol hydrochloride (CLB), albuterol
And Ractopamine (RAC) (SAL).Clenobuterol hydrochloride was synthesized by American scientist first in 1964, clinically
Having the effect of expansion bronchus, to preventing and treating bronchial asthma, chronic bronchitis, the pulmonary disease such as emphysema has good controlling
Therapeutic effect.It is found to have promotion muscle development and fat-splitting effect later, is therefore added in the feedstuff of herding rush in a large number
Enter growth of animal.Owing to the illegal clenobuterol hydrochloride that adds by strict supervision and effectively hits, albuterol and Ractopamine
Gradually rise as a kind of novel " clenbuterol hydrochloride ", become the succedaneum of clenobuterol hydrochloride, also raised animal by people's abuse
In material.Clenobuterol hydrochloride, albuterol, Ractopamine metabolism in animal body is relatively slow, and remains in animal body in a large number,
Then pass through in meat products enters human body and accumulate, causing human body poisoning even dead.Therefore, China and all over the world its other country
Family launches respectively laws and regulations and forbids that clenbuterol hydrochloride uses in feedstuff and Production of Livestock and Poultry.
At present, clenobuterol hydrochloride, albuterol, Ractopamine assay method mainly has and is divided into instrumental method and immunology
Method.Instrumental method instrumental method includes high performance liquid chromatography (HPLC), gas chromatography (GC), liquid chromatography-mass spectrography
Combination (LC MS), gas chromatography-mass spectrography (GC-MS), micelle capillary electric chromatogram (CE), thin layer chromatography (TLC)
Deng, these methods have been successfully used for the detection of clenbuterol hydrochloride.Although instrumental method has favorable reproducibility, detection limit is low,
Highly sensitive advantage, but detection equipment is costly, and sample treatment is complex, is extremely difficult to Site Detection purpose.Immunity
Method relies on antigen and the detection method of antibody specificity combination, and the method has simple to operate, highly sensitive, high specificity,
Without advantages such as large-scale instruments.But, due to clenobuterol hydrochloride, albuterol, Ractopamine is all small haptens,
Do not possess immunogenicity, could stimulating animal secretion resist after the macromolecular carrier protein binding such as itself and BSA need to being prepared complete antigen
Body, the therefore the most loaded down with trivial details but also cost intensive of antibody preparation, the antibody prepared also easily is affected by environmental factorss such as temperature.
Oligonucleotide aptamer is to be screened from the random oligonucleotide strand library of external synthesis by SELEX technology and obtain,
Can be with the high special one section of affine shorter single-stranded DNA sequence of specific structure and target.Nowadays, oligonucleotide aptamer
Have been widely used for such as target detection, enzyme level, the various field such as regulation and drug delivery.Oligonucleotide aptamer
Comparing antibody exhibits and go out great advantage, except having higher pathoklisis, oligonucleotide aptamer screens the most in vitro
Carrying out, the screening cycle is short, synthesizes convenient and low cost, and some functional groups of easy labelling and reporter molecules, with time variation and renaturation
Reversible and speed is fast, stablizes, little by environmental influence, can preserve for a long time.Along with SELEX technology constantly improve into
Step, has filtered out the aptamer of various target, such as small-molecule substance (organic dyestuff, metal, medicine, aminoacid, core
Thuja acid and peptide etc.), protein (including enzyme, antibody, the gene regulation factor, and lectin), tumor cell, virus
With pathogenic bacterium etc..But there is no at present about clenobuterol hydrochloride, albuterol, prepared by the oligonucleotide aptamer of Ractopamine
The research report of method.The present invention is with illegal additive clenobuterol hydrochloride common in food or feedstuff, albuterol, Rec
Dopamine is target, and with secure the Fe of random oligonucleotide library3O4Magnetic nanoparticle is hatched, and screening obtains one group of knowledge
The oligonucleotide aptamer of other clenobuterol hydrochloride, the oligonucleotide aptamer of one group of identification albuterol and one group of identification Rec are many
The oligonucleotide aptamer of bar amine, the oligonucleotide aptamer of preparation has stability height, synthesizes convenient, easy mark function group
And reporter molecules, the quick detection of clenbuterol hydrochloride in food and feedstuff will be widely used in.
Summary of the invention
It is an object of the invention to provide the oligonucleotide aptamer of one group of identification clenobuterol hydrochloride, one group of identification albuterol
Oligonucleotide aptamer and the oligonucleotide aptamer of one group of identification Ractopamine, for exploitation clenbuterol hydrochloride novel separation and concentration or
Analyze detection instrument and establish good basis.
A kind of method that it is a further object of the present invention to provide oligonucleotide aptamer preparing clenbuterol hydrochloride, it can facilitate, accurately
Ground obtains and clenobuterol hydrochloride, albuterol, and the high special affine oligonucleotide aptamer of Ractopamine, effect is notable.
The inventive method utilizes based on Fe3O4The SELEX technology that magnetic nanoparticle separates, passes through random oligonucleotide library
The complementary strand of biotinylation labelling is fixed on Avidin and is coated Fe3O4Magnetic nanoparticle.With clenobuterol hydrochloride, albuterol,
Ractopamine is hatched with fixing library as target.After the 16 SELEX repeated screenings taken turns, enriched library is carried out gram
Grand order-checking, and analyze affinity and the specificity representing sequence, the final oligonucleotide obtaining one group of identification clenobuterol hydrochloride is fitted
Gamete, the oligonucleotide aptamer of one group of identification albuterol and the oligonucleotide aptamer of one group of identification Ractopamine.
Accompanying drawing explanation
Fig. 1 is based on Fe3O4The schematic diagram of the SELEX technology that magnetic nanoparticle separates.
Fig. 2 is sequence Apt1, Apt2, Apt-3, CLB-2, SAL-5, the simulation secondary structure figure of RAC-5, RAC-6.
Fig. 3 is sequence Apt1, Apt2, Apt-3, CLB-2, SAL-5, the saturated knot of RAC-5, RAC-6 oligonucleotide aptamers
Close curve chart.
Fig. 4 is sequence Apt1, Apt2, Apt-3, CLB-2, SAL-5, the specificity of RAC-5, RAC-6 oligonucleotide aptamers
Result of the test.
Detailed description of the invention:
Below in conjunction with Figure of description and embodiment, the present invention is further illustrated, but is not to limit the present invention.
Embodiment 1: clenobuterol hydrochloride, albuterol, the SELEX screening of the specific binding oligonucleotide aptamer of Ractopamine
1, iii vitro chemical synthesis initial random oligonucleotide (ssDNA) library and primer are (by American I ntegrated DNA Technologies
Company completes), sequence is as follows:
5 '-AGCAGCACAGAGGTCAGATG-N40-CCTATGCGTGCTACCGTGAA-3 ' (40N represent 40 with
Machine nucleotide)；
Forward primer:: 5 '-AGCAGCACAGAGGTCAGATG-3 '
5 ' phosphorylation downstream primer: 5 '-P-TTCACGGTAGCACGCATAGG-3 '
Biotinylated library complementary short-chain P1:5 '-Bio-AGCACGCATAGG-3 '
By ssDNA pool and primer all with TE buffer become 100 μMs of stock solutions be stored in-20 DEG C standby.
2, ssDNA pool fixing and hatching
First round screening reaction system is that 600 μ L, 1nmol ssDNA library and short chain P1 add BB with 1:2 mole ratio
Buffer (50mM Tris-HCl, 5mM KCl, 100mM NaCl, 1mM MgCl2, pH 7.4) in after mix homogeneously,
95 DEG C of heating 10min, transfer to the complementary 3h of hybridization at 37 DEG C.Then by 600 μ g parents after Complementary hybridization chain and cleaning
Magnetic bead coated with element, at 37 DEG C, reacts 6h under 130rpm, specific binding by Avidin and biotin, makes ssDNA
Library is fixed on magnetic bead.The immobilized magnetic bead of ssDNA uses BB buffer to be cleaned multiple times, to remove non-specific binding
ssDNA.600 μ L ssDNA immobilized magnetic bead solution with mix target (clenobuterol hydrochloride, albuterol, Rec DOPA
Amine, initial concentration is respectively for 0.1mM) at 37 DEG C, hatch 2h, the ssDNA affine with target specificity dissociates from magnetic bead
Get off.Under additional the action of a magnetic field, the ssDNA that specificity is affine and target are stayed in supernatant, and carry out PCR as template
Amplification.
3, prepared by PCR amplification and ssDNA strand
The supernatant of negative control group and experimental group incubation system is carried out PCR amplification, 50 μ L PCR amplification system as template
For: 5 μ L templates, 1 μ L forward primer (10 μm ol/L), 1 μ L phosphorylation reverse primer (10 μm ol/L), 1 μ L dNTPmix
(5mmol/L), 0.5 μ L Taq archaeal dna polymerase (5U/ μ L), 5 μ L 10 × PCR amplification buffers, 36.5 μ L ultra-pure waters.
Thermal circulation parameters is: 94 DEG C of degeneration 5min, then 94 DEG C of degeneration 30s, 56 DEG C of annealing 30s, and 72 DEG C extend 30s,
Carrying out 20 and take turns circulation, then 72 DEG C extend 2min, last 4 DEG C of coolings.PCR primer uses 8% polyacrylamide gel
Electrophoretic separation, uses Gelred dyeing to be placed on Bio-Rad gel imaging instrument and takes pictures, verify PCR primer dsDNA size
Whether is 80bp, band is the most single.PCR primer uses PCR primer Purification Kit.Use ND-1000 micro-
Amount ultraviolet-uisible spectrophotometer measures purified pcr product concentration, calculates Lambda exonuclease addition, it is ensured that
Lambda exonuclease can excise the reverse DNA chain that 5 ' end phosphate groups are modified completely, to obtain the list of next round screening
Chain DNA.The enzyme cutting buffering liquid of Lambda exonuclease and 1/10 volume is added at 37 DEG C in purified pcr product
Endonuclease reaction 1h, at 75 DEG C, enzyme denaturing 10min terminates reaction.Digestion products uses 8% denaturing polyacrylamide gel (containing 7M
Carbamide) electrophoresis separates 20min under 250V, and the gel 15min that dyes in Gelred dyeing liquor is placed on Bio-Rad gel
Under imager, imaging is taken pictures, and determines that enzyme action is the most complete, and single stranded product stripe size is the most correct.Digestion products is transferred to 1.5
In the centrifuge tube of ml and add isopyknic phenol: chloroform: isoamyl alcohol (V:V:V=25:24:1), mix homogeneously 30s is in vain
Color emulsion liquid, 4 DEG C, 2000rpm is centrifuged 5min, and 4 DEG C, 8000rpm is centrifuged 1min, the supernatant is carefully taken out
Move in centrifuge tube.Add equal-volume chloroform: isoamyl alcohol (V:V=24:1), mixing, under above-mentioned identical centrifugal condition centrifugal also
Collect supernatant.Supernatant adds the NaAC of 1/10 volume 3M, fully adds 2 times of volume dehydrated alcohol after mixing,
-20 DEG C of precipitates overnight are placed after mixing.Solution 4 DEG C after precipitation, 14000rpm is centrifuged after 15min removes supernatant and adds 200 μ L
70% ethanol, turns upside down and cleans precipitation, then at 4 DEG C, 14000rpm is centrifuged 15min, goes supernatant visible white solid to sink
Form sediment, be placed in 50 DEG C of baking ovens dried addition 200 μ L TE buffer solution and use ND-1000 trace UV, visible light to divide
The concentration of light photometric determination purification ssDNA.
4, Cycle Screening
Second takes turns to the 11st to take turns and screens according to first round method, and screening reaction system is 300 μ L.For increasing screening pressure,
Improve the affinity of screening oligonucleotide aptamer, add amount 100pmol in ssDNA library in fixed system, and along with screening
The increase of wheel number is gradually decremented to 20pmol, and the target mixture concentration adding incubation system is gradually decremented to 1 μM by 0.1mM,
Incubation time is gradually decremented to 1h by 2h.For improve oligonucleotide aptamer specificity, from third round start every one take turns into
Row is the most counter to be sieved, and immobilization magnetic bead in ssDNA library is initially charged anti-sieve material adrenalin hydrochloride, dopamine hydrochloride, weight winestone
Acid norepinephrine, isoproterenol sulfate, Avidin hatch combination with target after hatching cleaning again.Simultaneously from the 12nd
Take turns to 16 and take turns, ssDNA library individually with clenobuterol hydrochloride, albuterol, Ractopamine is hatched, the most additionally
Two kinds of molecules add anti-sieve nest system, are respectively directed to clenobuterol hydrochloride, albuterol and Ractopamine with acquisition specific binding
Oligonucleotide aptamer.
5, cloning and sequencing and sequence analysis
Screen 16 take turns after, clenobuterol hydrochloride, Ractopamine, the PCR of three kinds of target sievings of albuterol will be respectively directed to
Product is served Hai Sheng work Bioisystech Co., Ltd and is carried out determined dna sequence.40 sequences of acquisition are measured for every kind of target,
DNAMAN and RNA Structure 4.6 software is used to analyze homology information and the secondary structure of 40 sequences respectively.Knot
Close software analysis result, for target clenobuterol hydrochloride, sequence is divided into 8 families, from each family, selects 1 structure
Stable, the candidate oligonucleotide aptamer totally 8 that free energy level is relatively low.For target albuterol, sequence is divided into 10 families,
1 Stability Analysis of Structures is selected, the candidate oligonucleotide aptamer totally 10 that free energy level is relatively low from each family.For target Lay
Sequence is divided into 9 families by gram dopamine, selects 1 Stability Analysis of Structures, the oligonucleotide that free energy level is relatively low from each family
Aptamer totally 9.Found that Apt-1, the candidate that Apt-2, Apt-3 simultaneously appear in clenobuterol hydrochloride and albuterol is few
In nucleotide aptamer sequence.By the candidate oligonucleotide aptamer selected by Shanghai Sangon Biological Engineering Technology And Service Co., Ltd
Synthesis 5 ' end flag F AM sequence, for affinity and specificity analyses.
6, the affinity of three kinds of clenbuterol hydrochloride oligonucleotide aptamers and specificity analyses
6.1 affinity analyses
There is based on graphene oxide oxidation the characteristic of absorption single stranded DNA, construct oligonucleotide aptamer affinity authentication
Method.By the clenbuterol hydrochloride target (1 μM) of fixed concentration respectively with corresponding a series of variable concentrations (10,25,50,75,
100,150,200nM) candidate oligonucleotide aptamer is hatched, and cumulative volume is 300 μ L, and 37 DEG C of lucifuges hatch 2h,
And replace target as negative control group using BB buffer.Hatch add after combination the GO absorption of optimum amount ratio not with target
In conjunction with aptamer, after centrifugally operated use F-7000 fluorescent spectrophotometer measuring supernatant 490nm excite lower 520nm to launch
Fluorescence intensity, three parallel repetitions of Setup Experiments, experiment use lucifuge process.With experimental group relative to the fluorescence of negative control group
Intensity is as vertical coordinate, using aptamer concentration as abscissa, uses GraphPad Prism 5.0 software to carry out nonlinear regression plan
Add up to dissociation constant K calculating aptamerdIt is worth, and drafting combines saturation curve, thus obtain energy and clenobuterol hydrochloride, husky butylamine
Alcohol, the preferable oligonucleotide aptamer of Ractopamine affinity, the oligonucleotide aptamer that i.e. dissociation constant is relatively low.(being shown in Table 1).
Oligonucleotide aptamer dissociation constant K of table 1. high-affinitydValue
Fig. 3 is sequence Apt1, Apt2, Apt-3, CLB-2, SAL-5, RAC-5 and RAC-6 is bent with the saturated combination of corresponding target
Line chart.
6.2 specificity analyses
According to the analysis result of 5.1, it is thus achieved that preferable Apt-1, Apt-2, Apt-3 and CLB-2 affine with clenobuterol hydrochloride
Sequence, preferable Apt-1, Apt-2, Apt-3 and SAL-5 sequence affine with albuterol, with Ractopamine affinity relatively
Good RAC-5 and RAC-6 sequence, wherein clenobuterol hydrochloride and albuterol are all had by Apt-1, Apt-2, Apt-3
High-affinity.Therefore, by oligonucleotide aptamer Apt-1, Apt-2, Apt-3, CLB-2, SAL-5, RAC-5, RAC-6
Carry out specificity analyses.Specificity experiments analyzes clenobuterol hydrochloride, Ractopamine, the oligonucleotide aptamer of albuterol
To counter sieve material (adrenalin hydrochloride, dopamine hydrochloride, noradrenaline bitartrate, isoproterenol sulfate,
Avidin) adhesion.The oligonucleotide aptamer of anti-sieve material and 200nM hatches 2h 37 DEG C of lucifuges, and delays with BB
Rushing liquid replaces target as negative control group.The GO adding optimum amount ratio after hatching combination adsorbs the few core not being combined with target
Thuja acid aptamer, uses F-7000 fluorescent spectrophotometer measuring supernatant 490nm to excite lower 520nm to launch after centrifugally operated
Fluorescence intensity, three parallel repetitions of Setup Experiments, experiment uses lucifuge to process.Result display Apt-1, Apt-2, Apt-3,
CLB-2 combines clenobuterol hydrochloride, Apt-1, Apt-2, Apt-3, and SAL-5 combines albuterol, RAC-5 and RAC-6
All being better than other anti-materials that sieves in conjunction with Ractopamine ability, specific test result is as shown in Figure 3.Therefore, by based on Fe3O4
The SELEX technology that magnetic nanoparticle separates is prepared one group of oligonucleotide simultaneously identifying clenobuterol hydrochloride and albuterol and is fitted
Gamete Apt-1, Apt-2, Apt-3, the oligonucleotide aptamer CLB-2 of a high specific identification clenobuterol hydrochloride, one
Oligonucleotide adaptation SAL-5 of bar high specific recognition albuterol and the oligonucleotide of two high specific recognition Ractopamines are adaptive
Sub-RAC-5 and RAC-6, novel separation and concentration or analysis detection instrument for exploitation clenbuterol hydrochloride establish good basis.For food or
Clenobuterol hydrochloride in feedstuff, albuterol, the detection of Ractopamine provides important foundation.
The present invention includes but not limited to above example, every any equivalent carried out under the spirit and principles in the present invention or
Locally this enters, and all will be regarded as within protection scope of the present invention.
Claims (3)
1. one group can identify clenobuterol hydrochloride and husky butylamine oligonucleotide aptamer Apt-1, Apt-2 and Apt-3 simultaneously, one
The oligonucleotide aptamer CLB-2 of energy high specific identification clenobuterol hydrochloride, the widow of an energy high specific identification albuterol
Nucleotide aptamer SAL-5, oligonucleotide aptamer RAC-5 and RAC-6 of two energy high specific recognition Ractopamines, its
It is characterised by sequence 1 in oligonucleotide aptamer sequence such as sequence table, the sequence shown in 2,3,4,5,6,7.
2. as described in claim 1 oligonucleotide aptamer, it is characterised in that its 5 ' end or 3 ' ends can carry out FITC,
Amino, biotin or thiol chemistry are modified.
3. oligonucleotide aptamer separation and concentration and analysis detection hydrochloric acid gram human relations in food or feedstuff as described in claim 1
Special sieve, albuterol and the application of Ractopamine.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610128853.2A CN105886512B (en) | 2016-03-07 | 2016-03-07 | Oligonucleotide aptamer group for high-specificity recognition of clenbuterol hydrochloride, salbutamol and ractopamine |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610128853.2A CN105886512B (en) | 2016-03-07 | 2016-03-07 | Oligonucleotide aptamer group for high-specificity recognition of clenbuterol hydrochloride, salbutamol and ractopamine |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN105886512A true CN105886512A (en) | 2016-08-24 |
| CN105886512B CN105886512B (en) | 2020-06-09 |
Family
ID=57014184
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610128853.2A Active CN105886512B (en) | 2016-03-07 | 2016-03-07 | Oligonucleotide aptamer group for high-specificity recognition of clenbuterol hydrochloride, salbutamol and ractopamine |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN105886512B (en) |
Cited By (9)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108508199A (en) * | 2018-06-25 | 2018-09-07 | 沭阳康源泰博生物科技有限公司 | A kind of salbutamol Sample pretreatment kit based on immunomagnetic beads |
| CN108760706A (en) * | 2018-06-08 | 2018-11-06 | 农业部环境保护科研监测所 | A kind of method of quick screening low cadmium-accumulation rice varieties |
| CN109628458A (en) * | 2019-02-19 | 2019-04-16 | 江南大学 | A kind of the oligonucleotides aptamers and its application of specific recognition histamine |
| CN109813774A (en) * | 2019-03-14 | 2019-05-28 | 青岛科技大学 | A kind of method of photic electrochemical sensor detection clenbuterol hydrochloride ractopamine |
| CN109810980A (en) * | 2019-02-02 | 2019-05-28 | 江南大学 | A kind of aptamer of specific recognition tyrasamine and its application |
| CN109897856A (en) * | 2019-03-13 | 2019-06-18 | 江南大学 | The oligonucleotides aptamers of one group-specific identification spermine |
| CN109975561A (en) * | 2019-04-25 | 2019-07-05 | 河北医科大学 | A method of the super sensitivity detection dopamine based on aptamer |
| CN110618265A (en) * | 2019-09-12 | 2019-12-27 | 广东省药品检验所（广东省药品质量研究所、广东省口岸药品检验所） | Visual detection method of salbutamol |
| CN111172166A (en) * | 2020-02-15 | 2020-05-19 | 江南大学 | nucleic acid aptamer for specifically recognizing β -lactoglobulin and application thereof |
Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103343126A (en) * | 2013-07-19 | 2013-10-09 | 暨南大学 | Ractopamine aptamer and electrochemical biosensor for detecting same |
| CN104109677A (en) * | 2014-07-30 | 2014-10-22 | 暨南大学 | Clenbuterol aptamer and electrochemical biosensor of aptamer for detecting clenbuterol |
| CN104407128A (en) * | 2014-11-28 | 2015-03-11 | 中国农业科学院农业质量标准与检测技术研究所 | Aptamer-based ractopamine visual detection kit |
| CN104593374A (en) * | 2015-03-02 | 2015-05-06 | 江南大学 | Oligonucleotide aptamer for specifically identifying patulin |
| CN105505939A (en) * | 2015-12-30 | 2016-04-20 | 中国人民解放军第二军医大学 | High-affinity aptamer capable of being specifically combined with gonyautoxin 1 (GTX1) and gonyautoxin 4 (GRX4) and application thereof |
-
2016
- 2016-03-07 CN CN201610128853.2A patent/CN105886512B/en active Active
Patent Citations (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103343126A (en) * | 2013-07-19 | 2013-10-09 | 暨南大学 | Ractopamine aptamer and electrochemical biosensor for detecting same |
| CN104109677A (en) * | 2014-07-30 | 2014-10-22 | 暨南大学 | Clenbuterol aptamer and electrochemical biosensor of aptamer for detecting clenbuterol |
| CN104407128A (en) * | 2014-11-28 | 2015-03-11 | 中国农业科学院农业质量标准与检测技术研究所 | Aptamer-based ractopamine visual detection kit |
| CN104593374A (en) * | 2015-03-02 | 2015-05-06 | 江南大学 | Oligonucleotide aptamer for specifically identifying patulin |
| CN105505939A (en) * | 2015-12-30 | 2016-04-20 | 中国人民解放军第二军医大学 | High-affinity aptamer capable of being specifically combined with gonyautoxin 1 (GTX1) and gonyautoxin 4 (GRX4) and application thereof |
Non-Patent Citations (6)
| Title |
|---|
| DAN CHEN ET AL.: "A novel aptasensor for electrochemical detection of ractopamine,clenbuterol,salbutamol,phenylethanolamine and procaterol", 《BIOSENSORS AND BIOELECTRONICS》 * |
| SHUNXIANG GAO ET AL.: "Gonyautoxin1/4 aptamers with high-affinity and high-specificity: From efficient selection to aptasensor application", 《BIOSENSORS AND BIOELECTRONICS》 * |
| 周妮 等: "莱克多巴胺核酸适配体电化学生物传感器的研制", 《中国生物工程杂志》 * |
| 巩文慧: "基于文库固定化的瘦肉精适配子筛选及应用研究", 《中国优秀硕士学位论文全文数据库工程科技I辑》 * |
| 杨菲 等: "基于适配体复合膜电化学传感器测定莱克多巴胺的研究", 《化学试剂》 * |
| 杨菲: "莱克多巴胺适配体识别速测技术研究", 《中国优秀硕士学位论文全文数据库工程科技I辑》 * |
Cited By (15)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN108760706A (en) * | 2018-06-08 | 2018-11-06 | 农业部环境保护科研监测所 | A kind of method of quick screening low cadmium-accumulation rice varieties |
| CN108760706B (en) * | 2018-06-08 | 2021-08-06 | 农业部环境保护科研监测所 | Method for rapidly screening rice varieties with low cadmium accumulation |
| CN108508199A (en) * | 2018-06-25 | 2018-09-07 | 沭阳康源泰博生物科技有限公司 | A kind of salbutamol Sample pretreatment kit based on immunomagnetic beads |
| CN109810980B (en) * | 2019-02-02 | 2022-09-16 | 江南大学 | Nucleic acid aptamer for specifically recognizing tyramine and application thereof |
| CN109810980A (en) * | 2019-02-02 | 2019-05-28 | 江南大学 | A kind of aptamer of specific recognition tyrasamine and its application |
| CN109628458A (en) * | 2019-02-19 | 2019-04-16 | 江南大学 | A kind of the oligonucleotides aptamers and its application of specific recognition histamine |
| CN109628458B (en) * | 2019-02-19 | 2021-11-12 | 江南大学 | Oligonucleotide aptamer capable of specifically recognizing histamine and application thereof |
| CN109897856A (en) * | 2019-03-13 | 2019-06-18 | 江南大学 | The oligonucleotides aptamers of one group-specific identification spermine |
| CN109897856B (en) * | 2019-03-13 | 2022-08-12 | 江南大学 | Oligonucleotide aptamer group capable of specifically recognizing spermine |
| CN109813774B (en) * | 2019-03-14 | 2021-02-19 | 青岛科技大学 | Method for detecting clenbuterol by photo-induced electrochemical sensor |
| CN109813774A (en) * | 2019-03-14 | 2019-05-28 | 青岛科技大学 | A kind of method of photic electrochemical sensor detection clenbuterol hydrochloride ractopamine |
| CN109975561A (en) * | 2019-04-25 | 2019-07-05 | 河北医科大学 | A method of the super sensitivity detection dopamine based on aptamer |
| CN110618265A (en) * | 2019-09-12 | 2019-12-27 | 广东省药品检验所（广东省药品质量研究所、广东省口岸药品检验所） | Visual detection method of salbutamol |
| CN111172166A (en) * | 2020-02-15 | 2020-05-19 | 江南大学 | nucleic acid aptamer for specifically recognizing β -lactoglobulin and application thereof |
| CN111172166B (en) * | 2020-02-15 | 2021-10-15 | 江南大学 | Nucleic acid aptamer for specifically recognizing beta-lactoglobulin and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN105886512B (en) | 2020-06-09 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN105886512A (en) | A group of oligonucleotide aptamers for identifying clenbuterol hydrochloride, salbutamol and ractopamine with high specificity | |
| CN106834295B (en) | Broad-spectrum nucleic acid aptamer for specifically recognizing bacterial lipopolysaccharide and directional screening method thereof | |
| Ashley et al. | Selection of bovine catalase aptamers using non‐SELEX | |
| CN104630230B (en) | The aptamer of one group of specific recognition okadaic acid | |
| CN107058329B (en) | Aptamer specifically binding to Newcastle disease virus and screening method and application thereof | |
| CN108613862B (en) | Double-target synchronous aptamer screening method based on capillary electrophoresis | |
| Duan et al. | Simultaneous coupled with Separate SELEX for heterocyclic biogenic amine-specific aptamers screening and their application in establishment of an effective aptasensor | |
| CN107858357B (en) | Bovine respiratory syncytial G protein specific aptamer | |
| CN112143732B (en) | ssDNA aptamer for specifically recognizing N-cadherin, and screening method and application thereof | |
| CN102952848B (en) | A kind of method detecting non-coding tiny RNA | |
| CN109971843A (en) | A kind of sequencing approach of unicellular transcript profile | |
| CN109576272A (en) | A kind of DKK-1 aptamer and its application | |
| CN108866060A (en) | A kind of aptamer, kit and detection method specifically binding shell-fish arginine kinase | |
| CN105541990B (en) | Aptamers K15 of skate angiogenesis inhibitor 1 and screening method and application thereof | |
| CN102719549A (en) | Method for selecting goose carcass trait microsatellite molecular marker | |
| CN112813071B (en) | Aptamer sequence for specifically recognizing ribavirin and application thereof | |
| CN115558706A (en) | DNA nano cage fluorescent probe for in-situ detection of miRNA in exosome and application thereof | |
| CN109897856B (en) | Oligonucleotide aptamer group capable of specifically recognizing spermine | |
| CN110257489A (en) | A kind of detection technique system of 30 Multiple-allele SNP sites based on the sequencing of two generations | |
| CN109810980A (en) | A kind of aptamer of specific recognition tyrasamine and its application | |
| CN105506150B (en) | A kind of preparation method of EMSA methods and its probe and the probe | |
| CN113462693A (en) | Application of ssDNA aptamer in identification of iridovirus infected cells of micropterus salmoides | |
| CN109371031A (en) | A kind of screening technique specifically binding bovine serum albumin(BSA) aptamer | |
| CN114621958B (en) | Single-stranded DNA aptamer sequence for specifically recognizing ATP and application thereof | |
| CN103131722A (en) | Swine gene expression muscle creatine kinase (MCK)-diacylglycerol acyltrabsferase 1(DGAT1) carrier and preparation method thereof |
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 |