CN102559686A - Deoxynivalenol nucleic acid aptamer and application thereof - Google Patents
Deoxynivalenol nucleic acid aptamer and application thereof Download PDFInfo
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- CN102559686A CN102559686A CN2011103801116A CN201110380111A CN102559686A CN 102559686 A CN102559686 A CN 102559686A CN 2011103801116 A CN2011103801116 A CN 2011103801116A CN 201110380111 A CN201110380111 A CN 201110380111A CN 102559686 A CN102559686 A CN 102559686A
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Abstract
The invention relates to a deoxynivalenol nucleic acid aptamer with combined high specificity and high affinity and application thereof. The nucleic acid aptamer can be used for detection and analysis, and separation and concentration of deoxynivalenol, and has the characteristics of high specificity, high affinity, uniform quality, high stability, short development period, low production cost and convenience for use. The invention also relates to a derivative sequence of the nucleic acid aptamer sequence; and the derivative sequence comprises a modified sequence.
Description
(1) technical field:
The invention belongs to biological technical field, relate to----SELEX technical project of utilizing Protocols in Molecular Biology and preparation a kind of with deoxynivalenol high specific and high-affinity bonded deoxynivalenol aptamer and application thereof.
(2) background technology:
(deoxynivalenol DON), claims vomitoxin again to deoxynivalenol; It is a kind of Type B trichothecene; Mainly produce, be distributed in the cereal seeds such as wheat, barley, corn more, also pollute cereal product by Fusarium graminearum and Fusarlum roseum.It occupy first of the Fusarium toxin cereal pollution rate and pollution level, not only in the head blight evolution, has vital role, and can produce toxic effect widely to people and animals; Belong to severe toxicity or medium poisonous substance; Can cause acute poisoning symptoms such as vomiting, diarrhoea, fever, also have close ties with anaemia, immunosuppression, the esophageal carcinoma, Keshan disease, in addition; It is often gone back and other mould halitoxin such as Toxins, afla pollute farm crop jointly, can influence each other behind the entering human body.Therefore the content of monitoring DON is significant.
The available multiple diverse ways of the detection of DON; Like thin-layer chromatography, enzyme linked immunological, gc, liquid chromatography, near-infrared spectrum analysis, fluorescence polarization immunoassay etc.; Wherein ELISA (ELISA) with fast, convenient, preprocessing process is simple and direct is dominant, but still there is certain problem in it.The main drawback of ELISA is monoclonal antibody or the how anti-complicated process of preparation that DON is efficient, special, and the cross reaction of the acetylize analogue of used antibody ubiquity and DON makes its value of detecting higher, is prone to false positive; Some detection method is used than sensitive 3 without hesitation; 5, the antibody surrogate DON antibody that 7-triacetyl DON immunity produces, but this carries out the acetylize processing to extract before not only requiring to analyze; And detected be 3 one AcDON, 15-AcDON and 3, the amount of 15-AcDON mixture.We can say; For DON kind toxicity small molecules; The preparation cycle of its antibody is long, production cost is high, technical difficulty is big, differences between batches are big, clone strain (cell) is difficult to difficult problems such as effectively preservations, big limitations the technological fast development of immunology detection such as enzyme-linked immunosorbent assay.Aptamer (aptamer) is a kind ofly to have the more aglucon of high specific than antibody; Even can discern monoclonal antibody the single substituting group of the undistinguishable target molecules superfine elementary errors of modifying other; It is better as a specific specificity, avidity is higher, the antibody surrogate article of quality homogeneous, good stability; Widely approved in the check and analysis Application for Field; And have the advantages that the construction cycle is short, production cost is low, easy to use, therefore, aptamer has a good application prospect in this micromolecular rapid detection of toxicity that has a complex construction analogue of DON.
(3) summary of the invention:
The object of the present invention is to provide a kind of adopt that SELEX technology screening of new generation obtains can be fit with deoxynivalenol high specific and high-affinity bonded Structure Conversion signal; And the application method of deoxynivalenol aptamer in deoxynivalenol check and analysis and sample separation enrichment be provided; Have easy, quick, economic dispatch characteristics; Compare the many advantages of fit tool that from oligonucleotide library, filter out with other combinatorial chemical libraries such as random peptide library, antibody library and phage display library.
Technical scheme of the present invention: a kind of deoxynivalenol aptamer is characterized in that being nucleotide sequence
Dna molecular shown in GCATCACTACAGTCATTACGCATCGTAGGGGGGATCGTTAAGGAAGTGCCCGGAGG CGGTATCGTGTGAAGTGCTGTCCC or its complementary nucleotides sequence.
The verivate of said deoxynivalenol aptamer has the identical function purposes.
The verivate of said deoxynivalenol aptamer is that nucleotide sequence carries out amination or sulfhydrylation or isotropic substance modification.
The verivate of said deoxynivalenol aptamer is that nucleotide sequence combines vitamin H or enzyme or digoxin or fluorescent substance or nano luminescent material.
A kind of application of deoxynivalenol aptamer is characterized in that the deoxynivalenol aptamer is used for the check and analysis of deoxynivalenol, and it may further comprise the steps:
(1) the competition plate is prepared: 50 μ g/ml arm molecule aminopropyltriethoxywerene werene or poly-lysine 100 μ l encapsulate in 4 ℃ and spend the night, linking agent time phenylene diisothiocyanic acid salt or LUTARALDEHYDE activation 6h, 200-2000pmol/ml NH
2The 100 μ l couplings of-primer, 0.2mol/L thanomin or glycocoll sealing 2h, sodium borohydride reduction, the 1%BSA sealing, PBS washing back is subsequent use;
(2) detect step:
1. deoxynivalenol aptamer 5-10pmol, 94 ℃ of 3min, 5min sex change on ice;
2. with 0-100pmol target mixing, TV 100 μ l add in the entering plate incubated at room 30min;
3. draw 50 μ l and add in the fluorescent plate, add 1: 200OliGreen 100 μ l, effect 5min;
4. measure fluorescent value (Ex:485nm, Em:525nm).
Typical curve: through 6 revision tests, being ordinate zou with fluorescence numerical value, is X-coordinate with the normal concentration, does typical curve.
Sensitivity calculations: through 6 revision tests, the target content of detection is 0ng/ml ± 3*STDV.
Repeatability: standard specimen replication 5 times, calculate it and measure plastisied dispersion CV value.
The recovery: with the dilution of corn methanol extract is sample for 50 times, adds basic, normal, high concentration standard, and replication 3 times calculates its recovery.
DON detection kit: detection sensitivity: 0.13ng/ml, repeatability: CV＜7%
With corn homogenate methanol extract is sample dilution in 1: 50, and the recovery is seen table:
A kind of application of deoxynivalenol aptamer is characterized in that the deoxynivalenol aptamer is used for from sample separation, enrichment deoxynivalenol, and it may further comprise the steps:
(1) coupling there is the magnetic particle of deoxynivalenol aptamer and treats the isolating solution thorough mixing that contains deoxynivalenol;
(2) with magnetic separator enrichment magnetic particle, and clean, have the magnetic particle of aptamer to dissociate from coupling deoxynivalenol then, obtain the deoxynivalenol single-component.
A kind of application of complementary sequence of deoxynivalenol aptamer is characterized in that being applied in the deoxynivalenol check and analysis, and it may further comprise the steps:
1. deoxynivalenol detects chromatograph test strip, and its substruction is formed and comprised: supporting pad, Radioactive colloidal gold pad, nitrocellulose filter, absorbent pad etc.;
2. contain aggregates of nanoparticles (through biotin labeling) the 6 μ l that aptamer and its complementary sequence are assembled on the Radioactive colloidal gold pad, contain the detection line of 1-10mg/ml Streptavidin 2 μ l on the nitrocellulose filter;
When 3. detecting, test strip baffle end is inserted in the solution of testing sample, about 20s, the complete moistening web plate of liquid also gets into film; Take out paper slip, be placed on and let liquid continue to flow on the platform, if do not have target in the liquid to be measured, aggregate is bigger; Can not get into nitrocellulose filter with flow, not develop the color, if there is the target deoxynivalenol to exist in the liquid to be measured; The aggregate that can cause assembling dissociates and forms the dispersive nano particle, and the dispersive nano particle gets into cellulose membrane with flow, and Streptavidin combines on the biotin labeling thing on the nano particle and the film; Showing red, is positive result, and colour developing degree and the proportional relation of target concentration.
The characteristic evaluation and test of a kind of deoxynivalenol aptamer of the present invention:
1, fluorescence quantitative PCR method is estimated the activity of deoxynivalenol aptamer:
Thorough washing assembling (realizing through its complementary sequence) has the magnetic particle of deoxynivalenol aptamer, and the deoxynivalenol with final concentration 1 μ M is at war with then, room temperature reaction 30min.With the competing reaction product is that template is carried out the quantitative fluorescent PCR reaction; Adopt the test kit Platinum SYBR Green qPCR SuperMix-UDG of Invitrogen company; Contain template 2 μ l, primer P1, each 20pmol of P2 in the 20 μ l systems, 65 ℃ of annealing temperatures.Be provided with simultaneously with the control group of solvent methanol as the competition thing.
The result:
It is thus clear that the deoxynivalenol aptamer has the specificity affinity to deoxynivalenol.
2, dye method is estimated the activity of deoxynivalenol aptamer:
Thorough washing assembling (realizing through its complementary sequence) has the magnetic particle of deoxynivalenol aptamer, and the deoxynivalenol with final concentration 1 μ M is at war with then, room temperature reaction 30min.After the Oligreen dyestuff diluted by 1: 200 with binding buffer liquid, equal proportion added in the competing reaction liquid, and room temperature lucifuge reaction 2-5min uses the 480nm optical excitation, measures the emission light at 520nm place.Be provided with simultaneously with control group and the binding buffer liquid background control group of methyl alcohol as the competition thing.
The background control group does not detect emission light as a result, and the experimental group fluorescence intensity is significantly higher than the methyl alcohol control group, and visible deoxynivalenol aptamer has the specificity affinity to deoxynivalenol.
3, through the activity rating of the deoxynivalenol aptamer of base group modification:
According to the active method of fluorescence quantitative PCR method evaluation deoxynivalenol aptamer, carry out the deoxynivalenol aptamer of amination deoxynivalenol aptamer, sulfhydrylation deoxynivalenol aptamer, isotropic substance deoxynivalenol aptamer, biotin labeled deoxynivalenol aptamer, HRP mark, the deoxynivalenol aptamer of AP mark, the deoxynivalenol aptamer of digoxigenin labeled, the deoxynivalenol aptamer and the nano luminescent material Y of TAMRA mark respectively with fluorescence quantitative PCR method
2SiO
5: the activity rating of the deoxynivalenol aptamer of Eu mark.
The result:
It is thus clear that amination deoxynivalenol aptamer; Sulfhydrylation deoxynivalenol aptamer; Isotropic substance deoxynivalenol aptamer; Biotin labeled deoxynivalenol aptamer; The deoxynivalenol aptamer of HRP mark; The deoxynivalenol aptamer of AP mark; The deoxynivalenol aptamer of digoxigenin labeled; The deoxynivalenol aptamer of TAMRA mark; And nano luminescent material Y
2SiO
5: the deoxynivalenol aptamer of Eu mark all has the specificity affinity to deoxynivalenol.
Advantage of the present invention is: itself be oligonucleotide 1), molecular weight is less, can chemosynthesis, practice thrift cost; 2) have affinity and the specificity higher than antibody; 3) be convenient to mark and can be at different sites mark selectively; 4) repeatability and good stability, and be easy to preserve, promptly insensitive with violent condition to high temperature; 5) the aptamer technology has a good application prospect, particularly in the check and analysis field.
(4) embodiment:
Embodiment: a kind of deoxynivalenol aptamer is characterized in that being the dna molecular shown in nucleotide sequence GCATCACTACAGTCATTACGCATCGTAGGGGGGATCGTTAAGGAAGTGCCCGGAGG CGGTATCGTGTGAAGTGCTGTCCC or its complementary nucleotides sequence.
The single stranded DNA that is used for SELEX among the present invention is synthetic by Takara company with hangar and primer; Two ends are fixed sequence program; The centre is the stochastic sequence of 35 bases: 5 ' GCATCACTACAGTCATTACGCATCG-(N35)-ATCGTGTGAAGTGCTGTCCC 3 '; Storage capacity is more than 1014, primer l:5 ' GCATCACTACAGTCATTACGCA-3 ', primer 2: 5 ' GGGACAGCACTTCACACGAT 3 '; Primer 3:5 ' GCGTAATGACAAAAAAAAAAACAT-C6-NH2 3 ', primer 4:5 ' Biotin-GGGACAGCACTTCACACGAT 3 '.Deoxynivalenol is available from ALEXIS company, and magnetic bead is purchased the company in invitrogen, and the purified reagent of oligonucleotide is purchased the company in Qiagen, and PCR test kit and T carrier are purchased the company in Pu Luomaige (Promega).
A kind of screening preparation of deoxynivalenol aptamer:
Initial library is coupled on the magnetic particle through complementary sequence, is that anti-sieve element, deoxynivalenol serve as that the screening molecule screens with solvent.In the presence of deoxynivalenol, there is the oligonucleotide sequence of binding ability to dissociate with it, behind pcr amplification from the magnetic particle; Adopt affine separation and purification method to prepare secondary library; Repeat to screen 8 and take turns, cloning and sequencing, the mono-clonal aptamer of acquisition deoxynivalenol.
A kind of application of deoxynivalenol aptamer is characterized in that the deoxynivalenol aptamer is used for the check and analysis of deoxynivalenol, and it may further comprise the steps:
(1) the competition plate is prepared: and poly-lysine (50 μ g/ml, CBS) 100 μ l encapsulate in 4 ℃ and spend the night LUTARALDEHYDE (2.5%; PBS) activation 6h, NH2-primer (2000pmol/ml, PBS) 100 μ l couplings; Thanomin (0.2mol/L, pH8.0) sealing 2h, sodium borohydride reduction; BSA (1%, PBS) sealing, PBS washing back is subsequent use;
(2) detect step:
1. deoxynivalenol aptamer 5-10pmol, 94 ℃ of 3min, 5min sex change on ice; 2. with target (0-100pmol) mixing, TV 100 μ l add in the entering plate incubated at room 30min; 3. draw 50 μ l and add in the fluorescent plate, add 100 μ l OliGreen (1: 200, TAE), effect 5min; 4. measure fluorescent value (Ex:485nm, Em:525nm).
Typical curve: through 6 revision tests, being ordinate zou with fluorescence numerical value, is X-coordinate with the normal concentration, does typical curve.
Sensitivity calculations: through 6 revision tests, the target content of detection is 0ng/ml ± 3*STDV.
Repeatability: standard specimen replication 5 times, calculate it and measure plastisied dispersion CV value.
The recovery: with the dilution of corn methanol extract is sample for 50 times, adds basic, normal, high concentration standard, and replication 3 times calculates its recovery.
DON detection kit: detection sensitivity: 0.13ng/ml, repeatability: CV＜7%
With corn homogenate methanol extract is sample dilution in 1: 50, and the recovery is seen table:
A kind of application of deoxynivalenol aptamer is characterized in that the deoxynivalenol aptamer is used for from sample separation, enrichment deoxynivalenol, and it may further comprise the steps:
Coupling there is the magnetic particle of deoxynivalenol aptamer and treats the isolating solution thorough mixing that contains deoxynivalenol; With magnetic separator enrichment magnetic particle; And clean; There is the magnetic particle of aptamer to dissociate from coupling deoxynivalenol then, obtains the deoxynivalenol single-component.
A kind of application of complementary sequence of deoxynivalenol aptamer is characterized in that being applied in the deoxynivalenol check and analysis, and it may further comprise the steps:
1. deoxynivalenol detects chromatograph test strip, and its substruction is formed and comprised: supporting pad, Radioactive colloidal gold pad, nitrocellulose filter, absorbent pad etc.;
2. contain aggregates of nanoparticles (through biotin labeling) the 6 μ l that aptamer and its complementary sequence are assembled on the Radioactive colloidal gold pad, contain the detection line of 10mg/ml Streptavidin 2 μ l on the nitrocellulose filter;
When 3. detecting, test strip baffle end is inserted in the solution of testing sample, about 20s, the complete moistening web plate of liquid also gets into film; Take out paper slip, be placed on and let liquid continue to flow on the platform, if do not have target in the liquid to be measured, aggregate is bigger; Can not get into nitrocellulose filter with flow, not develop the color, if there is the target deoxynivalenol to exist in the liquid to be measured; The aggregate that can cause assembling dissociates and forms the dispersive nano particle, and the dispersive nano particle gets into cellulose membrane with flow, and Streptavidin combines on the biotin labeling thing on the nano particle and the film; Showing red, is positive result, and colour developing degree and the proportional relation of target concentration.
Sequence table
SEQUENCE?LISTING
< 110>China National Academy Nanotechnology & Engineering
< 120>deoxynivalenol aptamer and application thereof
<130>
<140>
<141>
<160>1
<170>PatentIn?version?3.4
<210>1
<211>80
<212>DNA
< 213>artificial sequence
<400>1
gcatcactac?agtcattacg?catcgtaggg?gggatcgtta?aggaagtgcc?cggaggcggt
60
atcgtgtgaa gtgctgtccc
80
Claims (6)
1. a deoxynivalenol aptamer is characterized in that being nucleotide sequence
Dna molecular shown in GCATCACTACAGTCATTACGCATCGTAGGGGGGATCGTTAAGGAAGTGCCCGGAGG CGGTATCGTGTGAAGTGCTGTCCC or its complementary nucleotide sequence.
2. according to the said a kind of deoxynivalenol aptamer of claim 1, the verivate that it is characterized in that having with said deoxynivalenol aptamer the identical function purposes is that nucleotide sequence carries out amination or sulfhydrylation or isotropic substance modification.
3. according to the said a kind of deoxynivalenol aptamer of claim 1, the verivate that it is characterized in that having with said deoxynivalenol aptamer the identical function purposes is that nucleotide sequence combines vitamin H or enzyme or digoxin or fluorescent substance or nano luminescent material.
4. the application of the said deoxynivalenol aptamer of claim 1 is characterized in that being used for the check and analysis of deoxynivalenol.
5. the application of the said deoxynivalenol aptamer of claim 1 is characterized in that being used for from sample separation, enrichment deoxynivalenol.
6. the application of claim 2 or 3 said deoxynivalenol nucleic acid aptamer derivative is characterized in that being applied in deoxynivalenol check and analysis and the separation and concentration.
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Cited By (8)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105695473A (en) * | 2016-03-09 | 2016-06-22 | 湖南科技大学 | Detection method of fungaltoxin DON (deoxynivalenol) and detection kit |
| CN106645344A (en) * | 2016-11-08 | 2017-05-10 | 湖南科技大学 | Preparation method and application of deoxynivalenol (DON) electrochemical sensor |
| CN107262074A (en) * | 2017-01-23 | 2017-10-20 | 北京美正生物科技有限公司 | A kind of deoxynivalenol aptamers affinity column and its production and use |
| CN107807034A (en) * | 2017-10-31 | 2018-03-16 | 北京农业质量标准与检测技术研究中心 | A kind of vomitoxin aptamer affinity column and preparation method and application |
| CN109833648A (en) * | 2019-01-15 | 2019-06-04 | 北京农业质量标准与检测技术研究中心 | Vomitoxin and its derivative aptamers affinity column and the preparation method and application thereof |
| CN110885827A (en) * | 2019-11-26 | 2020-03-17 | 昆明理工大学 | Aptamer specifically bound with vomitoxin, preparation method and application |
| CN110923238A (en) * | 2019-11-26 | 2020-03-27 | 昆明理工大学 | Aptamer specifically bound with vomitoxin, preparation method and application |
| CN113774063A (en) * | 2021-08-30 | 2021-12-10 | 北京农业质量标准与检测技术研究中心 | Vomitoxin specific aptamer and application |
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Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN105695473A (en) * | 2016-03-09 | 2016-06-22 | 湖南科技大学 | Detection method of fungaltoxin DON (deoxynivalenol) and detection kit |
| CN105695473B (en) * | 2016-03-09 | 2020-02-21 | 湖南科技大学 | Detection method and detection kit for mycotoxin deoxynivalenol |
| CN106645344A (en) * | 2016-11-08 | 2017-05-10 | 湖南科技大学 | Preparation method and application of deoxynivalenol (DON) electrochemical sensor |
| CN106645344B (en) * | 2016-11-08 | 2018-07-17 | 湖南科技大学 | A kind of preparation method and applications of deoxynivalenol electrochemical sensor |
| CN107262074A (en) * | 2017-01-23 | 2017-10-20 | 北京美正生物科技有限公司 | A kind of deoxynivalenol aptamers affinity column and its production and use |
| CN107807034A (en) * | 2017-10-31 | 2018-03-16 | 北京农业质量标准与检测技术研究中心 | A kind of vomitoxin aptamer affinity column and preparation method and application |
| CN109833648A (en) * | 2019-01-15 | 2019-06-04 | 北京农业质量标准与检测技术研究中心 | Vomitoxin and its derivative aptamers affinity column and the preparation method and application thereof |
| CN109833648B (en) * | 2019-01-15 | 2021-02-26 | 北京农业质量标准与检测技术研究中心 | Vomitoxin and derivative aptamer affinity column thereof, and preparation method and application thereof |
| CN110885827A (en) * | 2019-11-26 | 2020-03-17 | 昆明理工大学 | Aptamer specifically bound with vomitoxin, preparation method and application |
| CN110923238A (en) * | 2019-11-26 | 2020-03-27 | 昆明理工大学 | Aptamer specifically bound with vomitoxin, preparation method and application |
| CN113774063A (en) * | 2021-08-30 | 2021-12-10 | 北京农业质量标准与检测技术研究中心 | Vomitoxin specific aptamer and application |
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