CN102443585A - Zearalenone nucleic acid aptamer and application thereof - Google Patents
Zearalenone nucleic acid aptamer and application thereof Download PDFInfo
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- CN102443585A CN102443585A CN2011103803431A CN201110380343A CN102443585A CN 102443585 A CN102443585 A CN 102443585A CN 2011103803431 A CN2011103803431 A CN 2011103803431A CN 201110380343 A CN201110380343 A CN 201110380343A CN 102443585 A CN102443585 A CN 102443585A
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Abstract
The invention relates to a nucleic acid aptamer combined with high specificity and high affinity of zearalenone and application of the nucleic acid aptamer. The nucleic acid aptamer can be used for detection, analysis, separation and concentration of the zearalenone and has the characteristics of favorable specificity, high affinity, uniformity in quality, favorable stability, short development period, low production cost and convenience for use. The invention also relates to a derivation sequence of a nucleic acid aptamer sequence, and the derivation 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 zearalenone high specific and high-affinity bonded zearalenone aptamer and application thereof.
(2) background technology:
Zearalenone (zearalenone; ZEN) claim the F-2 toxin again; Being a kind estrogen-like mycotoxins that is mainly produced by sickle-like bacteria, is 2,4-RALs compounds; From the mouldy corn that has polluted sickle-like bacteria, separated at first obtaining by people such as Stob, in extensively being present in cereal crop such as the corn that goes mouldy, Chinese sorghum, wheat, rice and suckling in 1962.Research shows; ZEN has very strong genotoxicity and teratogenesis; When 1nmol/L～10nmol/L concentration, can stimulate transcribing of ERs, cause animal miscarriage, stillborn foetus, return reproduction unusual phenomenoies such as feelings, can also reduce the feed consumption rate of domestic animal; Cause growing decline, immunosuppression, breeding difficulty have become second killer of pig industry now; Simultaneously, it is the possibility induced tumor also, and damage cns, blood system and immunity system are to the health existence harm greatly of humans and animals.
Because of polluting the financial loss of bringing, ZEN more and more receives the attention of national governments; Most of at present country has all made very strict regulation to the ZEN content in the middle of food, cereal, the feed, therefore cereal, feed, food etc. is carried out the ZEN content detection and just seems very important.ZEN detection method relatively more commonly used at present comprises HPLC, vapor-phase chromatography, thin layer chromatography, performance liquid chromatography-mass spectrometry method, microorganism detection method and immunological detection etc.But methods such as performance liquid chromatography have advantages such as accuracy height, the strong microdetermination of susceptibility, but detection efficiency is low, and are also very high to the requirement of instrument; Thin layer chromatography is simple to operate, and cost is low, need not expensive instrument, but its complex steps, sensitivity is not good enough.Immunological detection measure ZEN have high specificity, highly sensitive, cost is low, need not large-scale expensive instrument, be suitable for the advantages such as rapid detection of extensive sample, more and more receives the welcome of check unit of basic unit.Yet for this toxicity small molecules of ZEN, 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 its immunology detection.Aptamer (aptamer) 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 the micromolecular rapid detection of this toxicity of ZEN.
(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 zearalenone high specific and high-affinity bonded Structure Conversion signal, and the application method of zearalenone aptamer in zearalenone check and analysis and sample separation enrichment is 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 zearalenone aptamer is characterized in that being nucleotide sequence
Dna molecular shown in GCATCACTACAGTCATTACGCATCGTGGGGATGGGAGGTTGTTTACGCAGGAGATG TTAATCGTGTGAAGTGCTGTCCC or its complementary nucleotides sequence.
The verivate of said zearalenone aptamer has the identical function purposes.
The verivate of said zearalenone aptamer is that nucleotide sequence carries out amination or sulfhydrylation or isotropic substance modification.
The verivate of said zearalenone aptamer is that nucleotide sequence combines vitamin H or enzyme or digoxin or fluorescent substance or nano luminescent material.
A kind of application of zearalenone aptamer is characterized in that the zearalenone aptamer is used for the check and analysis of zearalenone, 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. zearalenone 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: 200 OliGreen 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.
ZEN detection kit: detection sensitivity: 0.95ng/ml, repeatability: CV＜6%
With corn homogenate methanol extract is sample dilution in 1: 50, and the recovery is seen table:
A kind of application of zearalenone aptamer is characterized in that the zearalenone aptamer is used for may further comprise the steps from sample separation, enrichment zearalenone:
(1) coupling there is the magnetic particle of zearalenone aptamer and treats the isolating solution thorough mixing that contains zearalenone;
(2) with magnetic separator enrichment magnetic particle, and clean, have the magnetic particle of aptamer to dissociate from coupling zearalenone then, obtain the zearalenone single-component.
A kind of application of complementary sequence of zearalenone aptamer is characterized in that being applied to may further comprise the steps in the zearalenone check and analysis:
1. zearalenone 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 zearalenone 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 zearalenone aptamer of the present invention:
1, fluorescence quantitative PCR method is estimated the activity of zearalenone aptamer:
Thorough washing assembling (realizing through its complementary sequence) has the magnetic particle of zearalenone aptamer, and the zearalenone 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 zearalenone aptamer has the specificity affinity to zearalenone.
2, dye method is estimated the activity of zearalenone aptamer:
Thorough washing assembling (realizing through its complementary sequence) has the magnetic particle of zearalenone aptamer, and the zearalenone 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 zearalenone aptamer has the specificity affinity to zearalenone.
3, through the activity rating of the zearalenone aptamer of base group modification:
According to the active method of fluorescence quantitative PCR method evaluation zearalenone aptamer, carry out the zearalenone aptamer of amination zearalenone aptamer, sulfhydrylation zearalenone aptamer, isotropic substance zearalenone aptamer, biotin labeled zearalenone aptamer, HRP mark, the zearalenone aptamer of AP mark, the zearalenone aptamer of digoxigenin labeled, the zearalenone aptamer and the nano luminescent material Y of TAMRA mark respectively with fluorescence quantitative PCR method
2SiO
5: the activity rating of the zearalenone aptamer of Eu mark.
The result:
It is thus clear that the zearalenone aptamer of the zearalenone aptamer of amination zearalenone aptamer, sulfhydrylation zearalenone aptamer, isotropic substance zearalenone aptamer, biotin labeled zearalenone aptamer, HRP mark, the zearalenone aptamer of AP mark, digoxigenin labeled, the zearalenone aptamer and the nano luminescent material Y of TAMRA mark
2SiO
5: the zearalenone aptamer of Eu mark all has the specificity affinity to zearalenone.
Advantage of the present invention is: have easy, quick, economic dispatch characteristics; Compare with other combinatorial chemical libraries such as random peptide library, antibody library and phage display library; The many advantages of fit tool that from oligonucleotide library, filter out: 1) itself be oligonucleotide; 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.Therefore, the aptamer technology has a good application prospect, particularly in the check and analysis field.
(4) embodiment:
Embodiment: a kind of zearalenone aptamer is characterized in that being nucleotide sequence
Dna molecular shown in GCATCACTACAGTCATTACGCATCGTGGGGATGGGAGGTTGTTTACGCAGGAGATG TTAATCGTGTGAAGTGCTGTCCC 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, and two ends are fixed sequence program, and the centre is the stochastic sequence of 35 bases:
5 ' GCATCACTACAGTCATTACGCATCG-(N35)-ATCGTGTGAAGTGCTGTCCC 3 ', storage capacity is 10
14More than; Primer 1:5 ' GCATCACTACAGTCATTACGCA-3 '; Primer 2: 5 ' GGGACAGCACTTCACACGAT 3 ', primer 3:5 ' GCGTAATGACAAAAAAAAAAACAT-C6-NH23 ', primer 4:5 ' Biotin-GGGACAGCACTTCACACGAT 3 '.Zearalenone 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 zearalenone aptamer:
Initial library is coupled on the magnetic particle through complementary sequence, is that anti-sieve element, zearalenone serve as that the screening molecule screens with solvent.In the presence of zearalenone, 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 zearalenone.
A kind of application of zearalenone aptamer is characterized in that the zearalenone aptamer is used for the check and analysis of zearalenone, 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. zearalenone 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.
ZEN detection kit: detection sensitivity: 0.95ng/ml, repeatability: CV＜6%
With corn homogenate methanol extract is sample dilution in 1: 50, and the recovery is seen table:
A kind of application of zearalenone aptamer is characterized in that the zearalenone aptamer is used for may further comprise the steps from sample separation, enrichment zearalenone:
Coupling there is the magnetic particle of zearalenone aptamer and treats the isolating solution thorough mixing that contains zearalenone; With magnetic separator enrichment magnetic particle; And clean, there is the magnetic particle of aptamer to dissociate from coupling zearalenone then, obtain the zearalenone single-component.
A kind of application of complementary sequence of zearalenone aptamer is characterized in that being applied to may further comprise the steps in the zearalenone check and analysis:
1. zearalenone 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 zearalenone 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.
Claims (6)
1. a zearalenone aptamer is characterized in that being the dna molecular shown in nucleotide sequence GCATCACTACAGTCATTACGCATCGTGGGGATGGGAGGTTGTTTACGCAGGAGATG TTAATCGTGTGAAGTGCTGTCCC or its complementary nucleotides sequence.
2. according to the said a kind of zearalenone aptamer of claim 1, the verivate that it is characterized in that having with said zearalenone 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 zearalenone aptamer of claim 1, the verivate that it is characterized in that having with said zearalenone 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 zearalenone aptamer of claim 1 is characterized in that being used for the check and analysis of zearalenone.
5. the application of the said zearalenone aptamer of claim 1 is characterized in that being used for from sample separation, enrichment zearalenone.
6. the application of claim 2 or 3 said zearalenone nucleic acid aptamer derivative is characterized in that being applied in zearalenone check and analysis and the separation and concentration.
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| CN104844692A (en) * | 2015-04-13 | 2015-08-19 | 南昌大学 | Dodecapeptide capable of binding zearalenone, and applications thereof |
| CN105543345A (en) * | 2015-12-17 | 2016-05-04 | 湖南科技大学 | Method and kit for detecting zearalenone |
| CN105713966A (en) * | 2016-01-24 | 2016-06-29 | 湖南科技大学 | Method for rapidly detecting zearalenone |
| CN107084951A (en) * | 2017-04-11 | 2017-08-22 | 江南大学 | A kind of method that aptamers recognition detection zearalenone is marked based on time-resolved fluorescence |
| CN110261363A (en) * | 2019-08-06 | 2019-09-20 | 青岛农业大学 | A method of the sensor detects zearalenone to biosensor, the preparation method of detection zearalenone with use |
| CN112114134A (en) * | 2019-06-19 | 2020-12-22 | 爱科来株式会社 | Target substance detection method, target substance detection kit, and target substance detection system |
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Cited By (13)
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| CN104788543B (en) * | 2015-04-13 | 2018-03-02 | 南昌大学 | A kind of zearalenone antibody analog and its application based on polypeptide |
| CN104804070A (en) * | 2015-04-13 | 2015-07-29 | 南昌大学 | Polypeptide molecule capable of being specifically bound with ZEN (Zearalenone) and application of polypeptide molecule |
| CN104844692A (en) * | 2015-04-13 | 2015-08-19 | 南昌大学 | Dodecapeptide capable of binding zearalenone, and applications thereof |
| CN104788543A (en) * | 2015-04-13 | 2015-07-22 | 南昌大学 | Polypeptide-based zearalenone antibody mimics and application thereof |
| CN104844692B (en) * | 2015-04-13 | 2018-05-11 | 南昌大学 | A kind of dodecapeptide and its application with reference to zearalenone |
| CN104804070B (en) * | 2015-04-13 | 2018-03-02 | 南昌大学 | Peptide molecule and its application of zearalenone can be specifically bound |
| CN105543345A (en) * | 2015-12-17 | 2016-05-04 | 湖南科技大学 | Method and kit for detecting zearalenone |
| CN105713966A (en) * | 2016-01-24 | 2016-06-29 | 湖南科技大学 | Method for rapidly detecting zearalenone |
| CN105713966B (en) * | 2016-01-24 | 2020-08-11 | 湖南科技大学 | Method for rapidly detecting zearalenone |
| CN107084951A (en) * | 2017-04-11 | 2017-08-22 | 江南大学 | A kind of method that aptamers recognition detection zearalenone is marked based on time-resolved fluorescence |
| CN112114134A (en) * | 2019-06-19 | 2020-12-22 | 爱科来株式会社 | Target substance detection method, target substance detection kit, and target substance detection system |
| CN112114134B (en) * | 2019-06-19 | 2024-02-02 | 爱科来株式会社 | Target substance detection method, target substance detection kit, and target substance detection system |
| CN110261363A (en) * | 2019-08-06 | 2019-09-20 | 青岛农业大学 | A method of the sensor detects zearalenone to biosensor, the preparation method of detection zearalenone with use |
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Application publication date: 20120509 |