CN106526168A - Nucleotide aptamer probe kit used for quick detection of Doxycycline residue and application thereof - Google Patents
Nucleotide aptamer probe kit used for quick detection of Doxycycline residue and application thereof Download PDFInfo
- Publication number
- CN106526168A CN106526168A CN201610989401.3A CN201610989401A CN106526168A CN 106526168 A CN106526168 A CN 106526168A CN 201610989401 A CN201610989401 A CN 201610989401A CN 106526168 A CN106526168 A CN 106526168A
- Authority
- CN
- China
- Prior art keywords
- doxycycline
- aptamers
- probe
- aptamer
- quick detection
- 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
-
- 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
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54326—Magnetic particles
-
- 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
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54313—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals the carrier being characterised by its particulate form
- G01N33/54346—Nanoparticles
-
- 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
- G01N33/94—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors
- G01N33/9446—Antibacterials
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2446/00—Magnetic particle immunoreagent carriers
- G01N2446/20—Magnetic particle immunoreagent carriers the magnetic material being present in the particle core
Landscapes
- Health & Medical Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Engineering & Computer Science (AREA)
- Immunology (AREA)
- Molecular Biology (AREA)
- Chemical & Material Sciences (AREA)
- Biomedical Technology (AREA)
- Hematology (AREA)
- Urology & Nephrology (AREA)
- Food Science & Technology (AREA)
- Biochemistry (AREA)
- Cell Biology (AREA)
- Biotechnology (AREA)
- Medicinal Chemistry (AREA)
- Physics & Mathematics (AREA)
- Analytical Chemistry (AREA)
- Microbiology (AREA)
- General Health & Medical Sciences (AREA)
- General Physics & Mathematics (AREA)
- Pathology (AREA)
- Nanotechnology (AREA)
- Bioinformatics & Cheminformatics (AREA)
- Pharmacology & Pharmacy (AREA)
- Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
Abstract
Provided is a nucleotide aptamer probe kit used for quick detection of Doxycycline residue and application thereof. The Doxycycline nucleic acid aptamer nucleic acid probe adopts a nano-magnetic sphere as a core, the outer surface of the probe is bonded with a Doxycycline nucleic acid aptamer chain; wherein, the Doxycycline nucleic acid aptamer chain (Doxycycline nucleic acid aptamer artificial sequence) is 5'- GTA CGG AAT TCGCTA GCC GGG CGG CAG GCC ACG GCT GGG GTT GGT CCC ACT GCG GAT CCG AGC TCC ACG TG-3'. The kit aptamer probe is a novel recognition element of Doxycycline, and has the advantages of being good in stability, high in sensitivity, low in cost, easy to prepare and modify and high in specificity of marks.
Description
Technical field
The present invention relates to the screening technique and its application to doxycycline specific recognition DNA aptamers, and in particular to raw
Thing technology and food antibiotic detection field.
Background technology
Doxycycline (Doxycycline) is tetracycline antibiotics (Tetracyclines), is commonly used to treatment dynamic
Thing infectious disease, is also act as feed additive, with prevention disease and promotion growth of animal.In real work, due to arbitrarily adding
Big using dosage and not in accordance with the off-drug period, causes medicine to remain in animal tissue.Tetracycline medication extended residual is in animal
In tissue, cause fastbacteria to be propagated between the mankind, seriously damage health.Many countries and international organization are dynamic by setting up
The effective detection method of physical property food veterinary drug residue, controls medicament residue problem.Such as, China and European Union to Dox in animal
Remain MRL (MRL) to make explicit provisions in tissue：The MRL of muscle, liver and kidney respectively 100 μ g/kg,
300 μ g/kg and 600 μ g/kg.In order to control residue of veterinary drug, human health is ensured, it is necessary to set up a kind of fast and effectively residual
Stay detection method to remain which to be monitored.The method of the detection doxycycline (OTC) of report mainly includes efficient liquid phase recently
Chromatography, fluorescence method, mass spectrography and other methods etc..But these methods generally relatively time-consuming and price is relatively expensive,
Also have higher requirement to experimenter, it is higher to the pre-processing requirements of sample and be difficult promote.Immunoassay is using anti-
The original antibody specific binding various materials of reaction detection, food antibiotics leftover detection field it is relatively broad apply.Enzyme
The efficient catalytic reaction of the immunoreation of antigen and antibody and enzyme is organically combined by linked immunosorbent assay (ELISA), is effectively protected
High selectivity and high sensitivity in card analysis, what report was most is immune analysis method.Existing some companies American-European at present give birth to
ELISA kit product of the product for antibiotics leftover detection, but expensive, the limitednumber of enzyme labelled antibody, Zhi Nengjian
Survey the common antibiotic remainss of a few species.As doxycycline is micromolecular compound, itself have immunogenicity, need by
Which could produce specific antibody as the antigenic determinant stimulating animal of carrier after be connected such as protein with biomacromolecule, give birth to
Produce and there is more restriction with application, including：Antigen is coupled synthesis, the screening of high-titer antibody；Result from the animals such as Mus, rabbit
Heterologous antibody is possible to produce nonspecific reaction in use；The preparation cost of antibody is high, wastes time and energy, monoclonal cell strain
It is not easy to maintain；Unstable properties are detected between criticizing in batch.These all greatly limit immune analysis method in how western ring
Application in element detection.
Aptamer is the external synthetic of class Jing developed in recent years and the single stranded oligonucleotide that filters out,
Can efficiently, specifically combine various biological target molecules, aptamer appears as chemical-biological educational circles and biomedicine
Boundary provides a kind of new research platform.Aptamer have self stability it is good, prepare synthesis it is relatively easy, quick, easy
The advantages of acquisition, easy functional modification and labelling, therefore, using flexibly extensively in biosensor design.In recent years, it was based on
The biosensor research of aptamer is greatly paid close attention to by people.
At present using the report that nucleic acid aptamer is the antibiotic detection in food of identification molecular application, detection method
More based on electrochemica biological sensor.For traditional large-scale instrument, the present invention can not realize that field quick detection, pre-treatment are numerous
It is trivial, and immune analysis method need to prepare the shortcoming of antibody, establish the fluoroscopic examination based on the identification of Beads enrichment-aptamers
Method, can be used for quick, the high-sensitivity detection of doxycycline remnant in food.
The content of the invention
The present invention screens the single stranded DNA aptamers sequence that can be combined with doxycycline high-affinity, establishes and is based on
The fluorescence detection method of Beads enrichment-aptamers identification, can be used for quick, the Gao Ling of doxycycline remnant in animal derived food
Sensitivity is detected.
The object of the invention：It is that a kind of construction method of the aptamers for determining doxycycline is provided, in view of doxycycline point
The construction featuress of son, the method for devising the preferred doxycycline of magnetic bead SELEX technologies of improvement, by the combination based on SELEX
Chemical technology, screening obtain specifically binding the DNA aptamers of doxycycline.Aptamers are passed through into biotin-avidin system
System is fixed on nanometer magnetic bead surface, the aptamers complementary strand thereof with FAM labellings, detects with launch wavelength in different exciting
Fluorescent marker on complementary strand.When target molecule is added, aptamer and target molecule specifically bind, and cause to be complementary to miscellaneous
The dissociation of the fluorescent label DNA short chain of friendship, causes the change of fluorescence intensity to carry out doxycycline in quantitative analyses sample.
The nucleotide aptamers probe reagent box of the quick detection doxycycline remnant that the present invention is provided, including following reagent
For doxycycline aptamer nucleic probe, PBS, dissociation solution；It is characterized in that：The doxycycline nucleic acid adaptation
With nanoscale magnetic bead as kernel, outer surface is bonded with doxycycline aptamer chain to body nucleic probe；Wherein, doxycycline nucleic acid
Aptamers chain (doxycycline aptamer artificial sequence) is：5’-GTA CGG AAT TCG CTA GCC GGG CGG CAG
GCC ACG GCT GGG GTT GGT CCC ACT GCG GAT CCG AGC TCC ACG TG-3 ' are referring specifically to adnexa：《Core
Nucleotide sequence table》.
During the dissociation solution is 900mL water, 8g NaCl, 0.37g KCl, 0.135g Na2HPO4.2H2O, 1g glucoses,
5g HEPES, dissolving, NaOH adjust PH to 7.05, are settled to 1000ml.
The preparation method of the aptamers nucleic probe is：Fe is prepared using chemical coprecipitation3O4Nano-particle, then
Amidized nano material is coupled with Avidin using glutaraldehyde method；Doxycycline aptamers are modified in nanometer magnetic bead table
Face, complementary hybridization chain marked fluorophor respectively at 5 ' ends with doxycycline aptamers, by complementary with aptamers, constitute
Fluorescently-labeled aptamers nucleic probe.
The nucleotide aptamers probe reagent box of described quick detection doxycycline remnant is more in detection poultry meat apoplexy due to endogenous wind
Application in western ring element residual.
In the present invention, magnetic nano-particle and aptamers used are Chongqing biological medicine and apparatus research center laboratory
Synthesis.
Wherein, above-mentioned probe is to modify doxycycline aptamers in nanometer magnetic bead surface, the fluorescently-labeled adaptation of difference
Body complementary strand thereof, after adding target molecule, aptamer is specifically bound with target molecule, causes to be complementary to the glimmering of hybridization
The dissociation of signal DNA short chains, causes fluorescence intensity change, establishing criteria curve to try to achieve the content of doxycycline in sample.
Above-mentioned nanometer magnetic bead is to prepare Fe using chemical coprecipitation3O4Nano-particle, then using glutaraldehyde method by ammonia
The nano material of base is coupled with Avidin.
Biotin has been modified at the 5 ' ends of the doxycycline aptamer apt 1, by biotin-avidin system
System, while be fixed on the nanometer magnetic bead surface of Avidin modification.
Complementary hybridization chain marked fluorophor respectively at 5 ' ends with doxycycline aptamers, by mutual with aptamers
Mend, constitute fluorescent labeling nucleic probe.
A kind of nucleotide aptamers probe reagent box of quick detection doxycycline remnant, including following reagent：Above-mentioned institute
The doxycycline aptamer nucleic probe stated, PBS, dissociation solution.
The nucleotide aptamers probe reagent box of quick detection doxycycline remnant described above, the dissociation solution is
In 900mL water, 8g NaCl, 0.37g KCl, 0.135g Na2HPO4.2H2O, 1g glucoses, 5g HEPES, dissolving, NaOH are adjusted
PH is settled to 1000ml to 7.05.
The preparation method of the nucleotide aptamers probe reagent box of quick detection doxycycline remnant described above, adopts
Chemical coprecipitation prepares Fe3O4Then amidized nano material is coupled with Avidin by nano-particle using glutaraldehyde method.
Doxycycline aptamers are modified in nanometer magnetic bead surface, complementary hybridization chain is in 5 ' end difference labellings with doxycycline aptamers
Fluorophor, by complementary with aptamers, constitutes fluorescently-labeled aptamers nucleic probe.
Specially：1) in 60mL ethylene glycol, add 1, the 6- hexamethylene diamines of 13g, 4.0g anhydrous sodium acetates and 2.0g six to be hydrated
Ferric chloride (FeCl3 6H2O), 50 DEG C of stirrings, obtains colloid solution, transfers the solution into 100mL band teflon-lineds
In reactor, 198 DEG C of reaction 6h；Room temperature is cooled down, and abandons supernatant liquid, and deionized water goes out lower floor's solid matter, and Magneto separate is collected；
Respectively deionized water and washing with alcohol 2 times, 50 DEG C are dried 5-10h；
2) quantitatively weigh nano material to be suspended in 10mM PBS, ultrasonic disperse 15min, add glutaraldehyde solution so as to
Final concentration of volume ratio 5%；Then shaken at room temperature 2h, Magneto separate, abandon supernatant, and 10mM PBS are washed three times, remove unnecessary penta
Dialdehyde；10mM PBS are subsequently adding, ultrasonic disperse adds a certain amount of Avidin, shaken at room temperature 12h, Magneto separate to abandon supernatant,
10mM PBSs three times；
3) nano material that Avidin is modified is suspended in into ultrasonic disperse in 10mM PBS, adds certain density nucleic acid to fit
Ligand solution, shaken at room temperature 4h, Magneto separate abandon supernatant, and 10mM PBSs three times are resuspended in removing unconjugated aptamers
In PBS.
Test kit described above is in the application in detection doxycycline remnant；For adding after target molecule, nucleic acid is fitted
Part and target molecule specifically bind, and cause the dissociation of fluorescent label DNA short chain for being complementary to hybridize, cause fluorescence intensity to become
Change, establishing criteria curve tries to achieve the content of doxycycline in sample.In the detectable animal derived food of the method, doxycycline is residual
Stay.
The method have the benefit that：The test kit aptamers probe is the new recognition component of doxycycline,
With having good stability, sensitivity height, low cost, easily prepare, the advantage of the easy high specific of modification and labelling.
Specific embodiment
The preparation of the nucleotide aptamers probe reagent box of 1 quick detection doxycycline remnant of embodiment
1. the preparation of amination nanometer magnetic bead
1, the 6- hexamethylene diamines of 13g, six water of 4.0g anhydrous sodium acetates (CH3COONa) and 2.0g is added in 60mL ethylene glycol
Ferric chloride (FeCl3 6H2O) is closed, 50 DEG C of stirrings obtain colloid solution, transfer the solution into 100mL band polytetrafluoroethyllining linings
Reactor in, 198 DEG C reaction 6h.Room temperature is cooled down, and abandons supernatant liquid, and deionized water goes out lower floor's solid matter, and Magneto separate is received
Collection.Respectively deionized water and washing with alcohol 2 times, 50 DEG C are dried 5-10h.
2. Avidin is coupled amidized nanometer magnetic bead
Quantitatively weigh nano material to be suspended in PBS (10mM), ultrasonic disperse 15min, add glutaraldehyde solution so as to eventually
Concentration is 5% (v/v).Then shaken at room temperature 2h, Magneto separate, abandon supernatant, and 10mM PBS are washed three times, remove unnecessary penta 2
Aldehyde.The PBS of 10mM is subsequently adding, ultrasonic disperse adds a certain amount of Avidin, shaken at room temperature 12h, Magneto separate to abandon supernatant,
10mM PBSs three times.The suction surveyed before and after Avidin combines magnetic nanoparticle at 280nm using ultraviolet spectrophotometer
Light value.
3. the nanometer magnetic bead that the aptamers connection Avidin of biotin modification is modified
The nano material that Avidin is modified is suspended in into ultrasonic disperse in 10mM PBS, certain density nucleic acid adaptation is added
Body (doxycycline aptamer artificial sequence in described in nucleotides sequence list) solution, shaken at room temperature 4h, Magneto separate are abandoned
Clearly, 10mM PBSs three times are resuspended in PBS with removing unconjugated aptamers.
4. aptamers and complementary strand thereof
By the complementary strand of certain density FAM labellings, in adding above-mentioned suspension, 37 DEG C of lucifuges are incubated 1h, and Magneto separate is abandoned
Supernatant, PBS wash three times to remove free complementary strands, are resuspended in PBS, using spectrofluorophotometer, respectively at
Under 495nm, 588nm excitation wavelength and 520nm, 608nm launch wavelength, its fluorescent value is surveyed.
5. sample-adding and detection
The nano material complex of step 3 is dissolved in dissociation solution (in 900mL water, 8g NaCl, 0.37g KCl,
0.135g Na2HPO4.2H2O, 1g glucose, 5g HEPES, dissolving, NaOH adjust PH to 7.05, are settled to 1000mL), add
Testing sample or mark product solution, 45 DEG C of incubation lucifuge reaction 40-60min, Magneto separate abandon supernatant, and PBS washs three times and removes dissociation
Under complementary strand, be resuspended in PBS, detect fluorescent value under similarity condition.
6. the drafting of standard curve
Doxycycline standard substance are detected by aforesaid operations step, set up standard curve.Standard concentration is followed successively by
0th, 2.5,5,10,20,40,80 μ g/L, are placed in quartz colorimetric utensil, long in Ex495nm excitation wavelengths and an Em520nm ejected wave
Under, each experiment is repeated 5 times, and surveys its fluorescent value.It is B that standard concentration is fluorescent value corresponding to 0ng/mL0, variable concentrations standard
The corresponding fluorescent value of product is B, and the meansigma methodss of the standard substance fluorescent value for being obtained are divided by B0The light absorption value (suppression ratio) of standard.With not
It is abscissa with standard concentration, with suppression ratio B/B0Map for vertical coordinate, draw standard curve.
7. eat medicine prison sample to determine
Any tissue of the healthy animal without antibacterials is chosen, the blank tissue Jing after screening is shredded, refiner is used
4000r/min homogenizing 5min, weigh tissue homogenizing thing 1.0g in 50mL centrifuge tubes, add 3% trichloroacetic acid (meat 4mL, liver
9mL), vortex is mixed, and overturns vibration 20min, room temperature 10000r/min centrifugation 10min.Take 200 μ L of supernatant to be centrifuged in 1.5mL
Guan Zhong, adds 20 μ L of 1mol/L sodium hydroxide solutions, vortex to mix, adds sample buffer (180 μ L of meat, 380 μ L of liver) to mix,
5min is centrifuged with 10000r/min, supernatant is used as sample solution.Muscle dilution gfactor is 10, and liver dilution gfactor is 30.It is accurate
The appropriate standard substance storing solution of doxycycline is really measured, doxycycline standard substance storing solution is dissolved in into PBS, pig muscle, Hepar Sus domestica 3 respectively
Plant in substrate, final concentration of 0,2.5,5,10,20,40,80 μ g/L, curve is drawn according to the operational approach of step 6, tested
As a result comparative analysiss.
The application of the nucleotide aptamers probe reagent box of 2 quick detection doxycycline remnant of embodiment
1. the foundation of standard curve
Method in application invention content determines doxycycline standard substance, sets up standard curve.It is respectively configured doxycycline
Series standard concentration be 0,0,2.5,5,10,20,40,80 μ g/L, with various criterion product concentration as abscissa, with suppression ratio B/
B0For vertical coordinate, standard curve is drawn, regression equation is set up.Standard curve is in typical S types, and Regression Equations are y=-
43.76x+94.95, R2=0.99, its IC50For 10.7 μ g/L, lowest detection line is 1.79 μ g/L, detection method standard curve
Detection range is 1.8～80 μ g/L.
2. tissue sample matrix effect test
By doxycycline standard working solution respectively with 3 kinds of samples such as PBS, pig liver sample treatment liquid, muscular tissue treatment fluids
Product substrate carries out series concentration dilution, under optimum reaction condition, effect experiment numerical value of the measured standard substance in each substrate.
The method is tested to a series of concentration doxycycline in matrix sample, and acquired results draw standard curve, its linear relationship
98% is all higher than, gained substrate PBS, pig muscle, the IC of liver50Respectively 11.7 μ g/L, 14.5 μ g/kg, 16.3 μ g/kg.Knot
Although fruit explanation bio-matrix difference has certain impact, its IC to the light absorption value of standard substance50Change is little, illustrates different samples
Treatment fluid affects little to the sensitivity and testing result of method, and the processing method of sample is feasible.
3. detection method lowest detectable limit in the tissue
The detection method is used, standard curve is made.The fluorescent value of 20 parts of blank tissues (pig muscle and liver) is determined respectively,
Suppression ratio calculating is carried out, regression equation is substituted into, computation organization's sample detection line, by testing doxycycline in pig muscle and Hepar Sus domestica
Detection line in dirty sample is respectively 1.6 μ g/kg, 1.7 μ g/kg.
4. organize TIANZHU XINGNAO Capsul
Test animal tissue, the response rate of liver sample between 83.5%～98.6%, average 91.2%；Muscle samples
The response rate between 84.2%～98.7%, average 93.7%.The average variation within batch coefficient of pig liver and Swine muscle sample
Respectively 6.1%, 5.3%；The average interassay coefficient of variation of pig liver and Swine muscle sample is respectively 6.8%, 8.2%.It is flat
Criticize interior interassay coefficient of variation and be respectively less than 9%.Seen by the result of batch interior interassay coefficient of variation and the response rate, test result indicate that should
The repeatability of method, degree of accuracy, accuracy are relatively good, can meet food medicine prison on-site verification needs.
SEQUENCE LISTING
<110>Chongqing Normal University
<120>A kind of nucleotide aptamers probe reagent box of quick detection doxycycline remnant and its application
<130> 2016
<160> 1
<170> PatentIn version 3.3
<210> 1
<211> 71
<212> DNA
<213>Doxycycline aptamer artificial sequence
<400> 1
gtacggaatt cgctagccgg gcggcaggcc acggctgggg ttggtcccac tgcggatccg 60
agctccacgt g 71
Claims (4)
1. a kind of nucleotide aptamers probe reagent box of quick detection doxycycline remnant, is doxycycline including following reagent
Aptamer nucleic probe, PBS, dissociation solution；It is characterized in that：The doxycycline aptamer nucleic probe
With nanoscale magnetic bead as kernel, outer surface is bonded with doxycycline aptamer chain；Wherein, doxycycline aptamer chain
Nucleotides sequence is classified as：5’- GTA CGG AAT TCG CTA GCC GGG CGG CAG GCC ACG GCT GGG GTT GGT
CCC ACT GCG GAT CCG AGC TCC ACG TG-3’。
2. the nucleotide aptamers probe reagent box of quick detection doxycycline remnant according to claim 2, its feature
It is：During the dissociation solution is 900mL water, 8g NaCl, 0.37 g KCl, 0.135 g Na2HPO4.2H2O, 1g glucoses,
5g HEPES, dissolving, NaOH adjust PH to 7.05, are settled to 1000 ml.
3. the nucleotide aptamers probe reagent box of a kind of quick detection doxycycline remnant, it is characterised in that：The aptamers
The preparation method of nucleic probe is：Fe is prepared using chemical coprecipitation3O4Nano-particle, then using glutaraldehyde method by amino
The nano material of change is coupled with Avidin；Doxycycline aptamers are modified in nanometer magnetic bead surface, with doxycycline aptamers
Complementary hybridization chain marked fluorophor respectively at 5 ' ends, by complementary with aptamers, constitute fluorescently-labeled aptamers core
Acid probe.
4. the nucleotide aptamers probe reagent box of the quick detection doxycycline remnant described in any one of claim 1-3 is in inspection
The application surveyed in poultry meat apoplexy due to endogenous wind doxycycline remnant.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610989401.3A CN106526168B (en) | 2016-11-10 | 2016-11-10 | A kind of nucleotide aptamers probe reagent box of quick detection doxycycline remnant and its application |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201610989401.3A CN106526168B (en) | 2016-11-10 | 2016-11-10 | A kind of nucleotide aptamers probe reagent box of quick detection doxycycline remnant and its application |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN106526168A true CN106526168A (en) | 2017-03-22 |
| CN106526168B CN106526168B (en) | 2018-08-24 |
Family
ID=58350826
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201610989401.3A Active CN106526168B (en) | 2016-11-10 | 2016-11-10 | A kind of nucleotide aptamers probe reagent box of quick detection doxycycline remnant and its application |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN106526168B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111398576A (en) * | 2020-05-15 | 2020-07-10 | 西华大学 | Kit, probe and method for rapidly and sensitively detecting ofloxacin |
Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009041776A2 (en) * | 2007-09-27 | 2009-04-02 | Korea University Industrial & Academic Collaboration Foundation | Dna aptamer binding to oxytetracycline with specificity and production method thereof |
| KR20090103100A (en) * | 2008-03-27 | 2009-10-01 | 고려대학교 산학협력단 | Twenty one Single-stranded DNA aptamers having high affinity to tetracycline and it's analogues with high specificity and production method thereof |
| CN101639477A (en) * | 2009-08-20 | 2010-02-03 | 华中农业大学 | ELISA detection method for doxycycline remnant, kit and application |
| CN103852460A (en) * | 2014-03-25 | 2014-06-11 | 新疆农垦科学院 | Method for detecting multi-residues of antibiotics by magnetic nano fluorescence sensor based on aptamer |
| CN104360077A (en) * | 2014-11-25 | 2015-02-18 | 重庆市科学技术研究院 | Aptamer nucleic acid probe kit for detecting doxycycline residue as well as preparation method and application thereof |
| CN104745589A (en) * | 2015-03-11 | 2015-07-01 | 河北大学 | Screening method and application of nucleic acid aptamers for specifically recognizing streptomycin |
| CN105044003A (en) * | 2015-07-09 | 2015-11-11 | 惠州出入境检验检疫局检验检疫综合技术中心 | Nano-sensor for colorimetric/fluorescence dual-mode rapid detection of antibiotics and antibiotic detection method |
| CN105911042A (en) * | 2016-06-21 | 2016-08-31 | 吉林大学 | Unmarked aptamer fluorescence sensor for detecting tetracycline |
-
2016
- 2016-11-10 CN CN201610989401.3A patent/CN106526168B/en active Active
Patent Citations (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2009041776A2 (en) * | 2007-09-27 | 2009-04-02 | Korea University Industrial & Academic Collaboration Foundation | Dna aptamer binding to oxytetracycline with specificity and production method thereof |
| KR20090103100A (en) * | 2008-03-27 | 2009-10-01 | 고려대학교 산학협력단 | Twenty one Single-stranded DNA aptamers having high affinity to tetracycline and it's analogues with high specificity and production method thereof |
| CN101639477A (en) * | 2009-08-20 | 2010-02-03 | 华中农业大学 | ELISA detection method for doxycycline remnant, kit and application |
| CN103852460A (en) * | 2014-03-25 | 2014-06-11 | 新疆农垦科学院 | Method for detecting multi-residues of antibiotics by magnetic nano fluorescence sensor based on aptamer |
| CN104360077A (en) * | 2014-11-25 | 2015-02-18 | 重庆市科学技术研究院 | Aptamer nucleic acid probe kit for detecting doxycycline residue as well as preparation method and application thereof |
| CN104745589A (en) * | 2015-03-11 | 2015-07-01 | 河北大学 | Screening method and application of nucleic acid aptamers for specifically recognizing streptomycin |
| CN105044003A (en) * | 2015-07-09 | 2015-11-11 | 惠州出入境检验检疫局检验检疫综合技术中心 | Nano-sensor for colorimetric/fluorescence dual-mode rapid detection of antibiotics and antibiotic detection method |
| CN105911042A (en) * | 2016-06-21 | 2016-08-31 | 吉林大学 | Unmarked aptamer fluorescence sensor for detecting tetracycline |
Non-Patent Citations (1)
| Title |
|---|
| 张璇 等: "基于酶联适配体的四环素检测方法研究", 《现代食品科技》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN111398576A (en) * | 2020-05-15 | 2020-07-10 | 西华大学 | Kit, probe and method for rapidly and sensitively detecting ofloxacin |
Also Published As
| Publication number | Publication date |
|---|---|
| CN106526168B (en) | 2018-08-24 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN104360077A (en) | Aptamer nucleic acid probe kit for detecting doxycycline residue as well as preparation method and application thereof | |
| CN103852460B (en) | Based on the method that how residual the magnetic nano fluorescent sensor detection of antibiotics of aptamers is | |
| CN104897652B (en) | One step homogeneous chemistry luminescence method carries out the method and particulate used of small molecule detection | |
| CN102023147B (en) | Method for detecting ochratoxin A by magnetic separation of adapter-functionalized magnetic nano material and marking of up-conversion fluorescent nano material | |
| Wang et al. | Simultaneous detection of Staphylococcus aureus and Salmonella typhimurium using multicolor time-resolved fluorescence nanoparticles as labels | |
| JP2011527745A (en) | Method for producing homogeneous plastic adherent aptamer magnetic fluorescent beads (Plastic-adherentaptamer-magneticbead-fluorphore) and other sandwich assays | |
| CN104502585A (en) | Nano-sensor for detecting antibiotics and preparation method and application of nano-sensor | |
| EP2118653A2 (en) | Materials and methods for efficient and accurate detection of analytes | |
| CN102703453A (en) | DNA aptamer specifically recognizing streptomycin and application of DNA aptamer | |
| AU2019101761A4 (en) | An aptamer affinity column of Alternariol and preparation method and application thereof | |
| CN107389919A (en) | A kind of label-free fluorescence aptamer sensor and its preparation method and application | |
| CN103898203B (en) | The detection method of Cryptosporidum parvum and detection kit | |
| CN105675877B (en) | It is a kind of that the method that the identification of Magneto separate aptamers detects two kinds of pathogenic bacteria simultaneously is marked based on double-colored time-resolved fluorescence | |
| Munir et al. | A spellbinding interplay between biological barcoding and nanotechnology | |
| CN113444773B (en) | Method and kit for detecting tick pathogen nucleic acid based on liquid chip | |
| CN103698526A (en) | Rapid detection method and kit for mercury poisoning based on magnetic separation and quantum dot marking | |
| CN109593764B (en) | Aptamer biosensor for rapidly detecting saxitoxin and preparation method thereof | |
| CN107084951B (en) | Method for detecting zearalenone based on time-resolved fluorescence labeling-aptamer | |
| CN106526168B (en) | A kind of nucleotide aptamers probe reagent box of quick detection doxycycline remnant and its application | |
| CN107656059B (en) | A kind of Fluorescent detector and its preparation method and application for p53 albumen | |
| CN1527940A (en) | Protein chip for diagnosis allergy and detection method for allergen and antibody | |
| AU2006226844A1 (en) | Elucidation of high affinity, high specificity oligonucleotides and derivatized oligonucleotide sequences for target recognition | |
| KR102196281B1 (en) | DNA aptamer binding to edifenphos with specificity and Uses thereof | |
| KR101342080B1 (en) | Nucleic Acid Aptamer Capable of Specifically Binding to Tetracyclines Compound and Use thereof | |
| KR100930974B1 (en) | DNA aptamer binding to Retinol Binding Protein 4 with specificity and production method thereof |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |