CN112375762B - Carbaryl aptamer, aptamer derivative and application thereof - Google Patents

Carbaryl aptamer, aptamer derivative and application thereof Download PDF

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CN112375762B
CN112375762B CN202011229380.8A CN202011229380A CN112375762B CN 112375762 B CN112375762 B CN 112375762B CN 202011229380 A CN202011229380 A CN 202011229380A CN 112375762 B CN112375762 B CN 112375762B
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刘媛
郭文斐
杨高建
何农跃
邓燕
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Abstract

The invention discloses a carbaryl aptamer, a aptamer derivative and application thereof, wherein the sequence of the carbaryl aptamer is a DNA sequence shown in SEQ ID NO.1 or a nucleic acid sequence coupled with a fluorescent group, a quenching group, biotin, a nano material and the like on the basis of the DNA sequence or a single-stranded DNA molecule with the same specific recognition function obtained by deleting, adding or replacing a base on any one of the carbaryl aptamer sequences; the carbaryl aptamer derivative is a derivative of any one of the carbaryl aptamers with the same function, which is obtained by amination, sulfhydrylation or isotopic modification of the carbaryl aptamers; the carbaryl aptamer or the derivative thereof can be used for preparing carbaryl recognition probes or products for detecting/assisting to detect carbaryl, and the carbaryl aptamer or the derivative thereof is easy to synthesize and modify, easy to store, high in affinity, strong in specificity and the like for target substance carbaryl, and wide in application.

Description

Carbaryl aptamer, aptamer derivative and application thereof
Technical Field
The invention belongs to the technical field of molecular biology, and particularly relates to a carbaryl aptamer, a aptamer derivative and application thereof.
Background
Carbaryl (Carbaryl) is a carbamate pesticide, and is widely used in agricultural production due to its good pesticidal effect and low cost. However, carbaryl is a neurotoxin which inhibits the inhibitory action of acetylcholinesterase (AChE) and causes accumulation of acetylcholine in tissues, and extensive use of carbaryl pesticides in agriculture may cause accumulation in vegetables, fruits or water, further causing harm to human health and the environment. Therefore, the development of a reliable, sensitive and convenient carbaryl detection method has important significance not only in environmental research but also in food research and agriculture.
At present, the sevin detection method widely applied to food and environmental samples is an instrumental analysis method, such as liquid chromatography-tandem mass spectrometry, high performance liquid chromatography-mass spectrometry and gas chromatography-mass spectrometry. Although sensitive and reliable, these instrumental methods limit rapid detection of carbaryl in the field due to expensive equipment and professional operating requirements. In addition, acetylcholinesterase biosensors have been widely used for the detection of carbamates and organophosphates based on the principle of inhibiting specific enzyme activities, however, these AChE biosensors involve other materials such as quantum dots, sol-gels, films, and nanomaterials to improve detection sensitivity, making the preparation of these biosensors very complicated.
An aptamer (aptamer) is a nucleic acid molecule with a specific structure and function obtained by in vitro screening techniques (SELEX), and can bind to a target substance with high specificity and high selectivity. Compared with the traditional recognition molecules, the aptamer has the advantages of high affinity, strong specificity, good biocompatibility, easy synthesis and modification and the like, and is widely concerned in the fields of biomedicine, food safety and the like.
Therefore, screening of the aptamer capable of specifically recognizing carbaryl is beneficial to overcoming the defect that the conventional AChE sensor detects carbaryl.
Disclosure of Invention
The technical problem to be solved by the present invention is to provide a carbaryl aptamer, a derivative of the aptamer and an application thereof, aiming at the defects of the prior art.
In order to achieve the technical purpose, the technical scheme adopted by the invention is as follows: a carbaryl aptamer having a nucleotide sequence (SEQ ID No.1) of:
5’-ATTGGCACTCCACGCATAGGGTTATGTTAATAAGATGAACCCACGTTCCAGATGGCATCGCCTATGCGTGCTACCGTGAA-3’。
further, a fluorescent group, a quenching group, biotin, a nano material, a sulfhydryl group, an amino group or an enzyme is coupled on the nucleotide sequence of the carbaryl aptamer.
The invention also provides a carbaryl aptamer, which is a single-stranded DNA molecule which is obtained by deleting, adding or replacing nucleotides of any one carbaryl aptamer and has the same specific recognition function with the carbaryl aptamer.
The invention also provides a carbaryl aptamer derivative, which is a derivative of any one carbaryl aptamer, wherein the carbaryl aptamer derivative is aminated, sulfhydrylated or isotopically modified to obtain the aptamer with the same function as the carbaryl aptamer.
The invention also provides an application of any carbaryl aptamer or carbaryl aptamer derivative in carbaryl detection.
The invention also provides an application of the carbaryl aptamer or carbaryl aptamer derivative in preparation of a product for detecting or assisting in detecting carbaryl, wherein the product comprises a carbaryl recognition probe, and the carbaryl recognition probe is the carbaryl aptamer or carbaryl aptamer derivative.
The invention also provides a carbaryl aptamer fluorescent sensor which is prepared by mixing a carbaryl recognition probe and a carbaryl quenching probe in the Du's phosphate buffer solution, wherein the working concentrations of the carbaryl recognition probe and the carbaryl quenching probe are respectively 100nM and 300nM, and the nucleotide sequence of the carbaryl recognition probe is as follows: 5 '-FAM-CTCAGTCGCTAGGGTTATGTTAATAAGATGAACCCACGTTCCAGATGGCATCGCCTAGCGA-3', wherein the nucleotide sequence of the carbaryl quenching probe is as follows: 5 '-CCTAGCGACTGAG-Dabcyl-3'.
The invention also provides application of the carbaryl aptamer fluorescent sensor in carbaryl detection.
Compared with the prior art, the invention has the following beneficial effects:
(1) the carbaryl aptamer is a nucleic acid molecule with a special structure and function, which is obtained by an in-vitro screening technology, has the advantages of high affinity, strong specificity, easiness in storage, easiness in synthesis and modification and the like for target substances carbaryl, and can be used for detecting the carbaryl in a detection sample;
(2) the carbaryl aptamer derivative with the same function as the carbaryl aptamer, which is obtained after nucleotide deletion, addition or replacement is carried out on the basis of the carbaryl aptamer, can be used for preparing products for detecting or assisting in detecting carbaryl, such as carbaryl aptamer fluorescent sensors, and further used for detecting carbaryl in samples, and has the advantages of high detection sensitivity, convenience in instrument preparation and low detection cost.
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FIG. 1 is a secondary structure diagram of a carbaryl aptamer before and after optimization in example 1 of the present invention;
FIG. 2 is a graph showing the concentration optimization of the carbaryl-recognizing probe and the quenching probe in example 2 of the present invention, wherein the abscissa is the mixing ratio of the carbaryl-quenching probe and the recognizing probe, and the ordinate is the fluorescence intensity;
FIG. 3 is a graph showing the fluorescence spectrum of a Carbaryl aptamer fluorescence sensor according to example 2 of the present invention, wherein a is a graph showing the fluorescence spectrum of a FAM-Apta3S group, b is a graph showing the fluorescence spectrum of a FAM-Apta3S + Carbaryl group, c is a graph showing the fluorescence spectrum of a FAM-Apta3S + Quenching group, and d is a graph showing the fluorescence spectrum of a FAM-Apta3S + Quenching + Carbaryl group.
FIG. 4 is a graph of fluorescence spectra and a line graph after incubation with different concentrations of carbaryl in example 3 of the present invention;
FIG. 5 is a diagram showing the specific detection of carbaryl aptamer fluorescence sensor in example 4 of the present invention.
Detailed Description
The present invention will be described in further detail with reference to the accompanying drawings and examples. The following examples are intended to illustrate the invention only and are not intended to limit the scope of the invention.
The experimental procedures used in the examples below are, unless otherwise specified, conventional procedures and the reagents, methods and equipment used are, unless otherwise specified, conventional in the art.
The concentrations mentioned in the following examples (without specific indication) are the final concentrations of the substances contained in the system, and the experimental environments are all under the condition of room temperature (25-30 ℃); the wavelength of the fluorescence test is 500-650nm, the excitation wavelength is 485nm, and the scanning speed is 6000 nm/min.
Example 1
The invention provides a carbaryl aptamer, which has a nucleotide sequence (SEQ ID NO.1) as follows:
5’-ATTGGCACTCCACGCATAGGGTTATGTTAATAAGATGAACCCACGTTCCAGATGGCATCGCCTATGCGTGCTACCGTGAA-3’：
the carbaryl aptamer of the embodiment is obtained by screening through a SELEX technology, and the specific screening comprises the following steps:
1.1, immobilization of the starting library: fixing an initial library by a streptavidin magnetic bead method, taking 1mL of magnetic beads in a 1.5mL of EP tube, and washing for 4 times by using DPBS for later use; 1300pmole of the starting library (100 μ L) was mixed in a molar ratio of 1: 2 to immobilized primer (P1, starting concentration 100. mu.M) in DPBS binding solution (NaCl 136.89 mM; KCl 2.67 mM; Na)2HPO4 8.10mM；KH2PO4 1.47mM；CaCl2 0.90mM；MgCl20.49mM, pH 7.4) followed by PCR slow renaturation, the renaturation procedure was: 10min at 95 ℃; 1min at 60 ℃; 10min at 25 ℃; then adding the mixed solution with good renaturation into the spare magnetic beads, and incubating for 30min by a shaking table; the supernatant was removed by magnetic separation, the initial library-bound magnetic beads were washed four times with 200 μ L DPBS, the EP tube was replaced for each wash, and the washes were collected separately and labeled.
The immobilization efficiency of the starting library can be calculated by the following formula:
Figure BDA0002764667340000041
(Note: A1 is the ultraviolet absorbance value before the initial library and fixed primer mixture and magnetic beads are incubated; A2 is the initial library and fixed primer mixture and magnetic beads are incubatedUltraviolet absorbance value of supernatant after incubation of initial library and fixed primer mixed liquor and magnetic beads);
starting library (SEQ ID NO. 2):
5 '-ATTGGCACTCCACGCATAGG-NNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNNN-CCTATGCGTGCTACCGTGAA-3' (note: N represents any one of the bases A, T, G, C)
Immobilized primer (P1, SEQ ID NO. 3): 5 '-CCTATGCGTGGAGTGCCAAT-biotin-3'
1.2, primary screening: adding 100 mu L of carbaryl target (100 mu M) into the magnetic beads of the fixed initial library, incubating for 1h by a shaking table, fishing the magnetic beads by a magnet, and recovering supernatant (marked as: resolution 1), namely a primary screening nucleic acid library which contains a compound of DNA and carbaryl;
1.3, purification: reserving 5 mu L of the primary screening nucleic acid library obtained in the step 1.2, and carrying out emulsion PCR amplification on the rest; wherein, the emulsion PCR reaction system (10mL) is as follows: 1mL of PCR-mix, 8. mu. L P2-FAM, 8. mu. L P3-PolyA, 95. mu.L of precipitation 1, 889. mu. LddH2O, 8mL of emulsified oil; sequentially adding the substances into a 50mL centrifuge tube, vertically shaking for 3min on a mixing instrument, standing for 5min to observe that the white emulsion is not layered, and subpackaging into 12 groups of 8-row PCR tubes with each tube of 90-95 μ L until subpackaging is finished; the amplification conditions of the emulsion PCR reaction are as follows: pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 60s, annealing at 60 ℃ for 60s, extension at 72 ℃ for 60s, and amplification for 25 cycles; extending for 5min at 72 ℃;
recovering emulsion PCR products, subpackaging the emulsion PCR products into two tubes of 15mL centrifuge tubes, adding 10mL n-butanol into each tube, shaking and inverting to mix the solution evenly, centrifuging the solution for 10min by using a high-speed centrifuge, recovering green fluorescent dsDNA at the bottom, transferring the dsDNA to a 1.5mL EP tube, if the volume is more than 150 mu L, supplementing about 5 times of n-butanol, inverting and mixing the solution evenly, generating a micro-turbid state, centrifuging the solution for 2min at 12000rpm until the emulsion PCR products are concentrated to about 100uL, and obtaining a dsDNA concentrated solution;
adding 2 XTBE urea sample buffer solution with equal volume to the obtained dsDNA concentrated solution, and carrying out 8% denaturing PAGE electrophoresis; after electrophoresis is finished, cutting off a fluorescent target strip, cutting up the fluorescent target strip, putting the cut fluorescent target strip into a 0.5mL EP tube (the bottom of the EP tube is cut into a 1mm opening), sleeving the cut fluorescent target strip into a 2mL EP tube, centrifuging the EP tube at 12000rpm for 30s, then adding 1mL DPBS into the 2mL EP tube, shaking and shaking back and forth to uniformly mix glue and water, boiling the mixture in a boiling water bath for 10min, and taking out visible water and glue for layering; sucking out the water layer after the first gel boiling, adding the water layer into a filtering device, and filtering the water layer into a new 15mL centrifuge tube; adding 1mL of DPBS into a 2mL EP tube, sealing, and decocting for 10 min; adding a water gel solution into the new filtering device, and pumping into a 15mL tube in the previous step to obtain a target DNA single strand; the ssDNA buffer obtained by gel boiling is about 2mL, a 15mL centrifuge tube is filled with n-butanol for concentration, high-speed centrifugation is carried out for 5min, the lower layer DNA is moved into a 1.5mL EP tube, if the volume is more than 200uL, n-butanol which is 5 times of the volume of the lower layer DNA is added for concentration, and the volume of the ssDNA is about 100-150 uL; then putting the single-stranded.
Upstream primer (P2-FAM, SEQ ID NO. 4): 5 '-FAM-ATTGGCACTCCACGCATAGG-3';
downstream primer (P3-PolyA, SEQ ID NO. 5): 5 '-AAAAAAAAAAAAAAAAAAAAAAAAA- -Spacer 18-TTCACGGTAGCACGCATAGG-3'
1.4, circulation: replacing the initial library with the obtained secondary library and repeating the steps 1.1-1.3 until a nucleic acid library containing the aptamer which is combined with high affinity and high specificity of the carbaryl is obtained.
1.5, monitoring the screening process: adopting Q-PCR monitoring, the concrete steps are as follows: and (3) performing Q-PCR amplification on the cleaning solution obtained by cleaning the magnetic beads combined with the library in the step 1.1 and the solution reserved in the nucleic acid library obtained in the step 1.2, wherein the amplification reaction system is as follows: 10 μ L qPCR-mix, 0.4 μ L P2(10 μ M), 0.4 μ L P3(10 μ M), 2 μ L template, 7.2 μ LddH2And O. The reaction amplification conditions are as follows: pre-denaturation at 95 ℃ for 3 min; denaturation at 95 ℃ for 10s, annealing at 60 ℃ for 30s, extension at 72 ℃ for 30s, and amplification for 35 cycles. The retention calculation formula is as follows:
Figure BDA0002764667340000051
(Note: C1 is the concentration of ssDNA library eluted per selection run for carbaryl; V1 is the volume of carbaryl eluate; C2 is the concentration of ssDNA library dosed per selection run; V1 is the volume dosed per run).
Upstream primer (P2, SEQ ID NO. 6): 5'-ATTGGCACTCCACGCATAGG-3'
Downstream primer (P3, SEQ ID NO. 7): 5'-TTCACGGTAGCACGCATAGG-3'
1.5 Secondary Structure analysis of carbaryl aptamers
The secondary structure of the carbaryl aptamer was predicted by Mfold software, and the predicted structure is shown in fig. 1A.
Example 2
The invention provides a carbaryl aptamer fluorescent sensor, which comprises a carbaryl recognition probe and comprises the following preparation steps:
(1) the method is characterized in that a carbaryl recognition probe and a carbaryl quenching probe are synthesized by the company of Biotechnology engineering (Shanghai) GmbH, wherein the carbaryl recognition probe is obtained by deleting and increasing modification of basic groups of a carbaryl aptamer (SEQ ID NO.1) described in example 1, the secondary structure of the carbaryl recognition probe is shown in figure 1B, and the nucleotide sequence (SEQ ID NO.8) of the carbaryl recognition probe is as follows:
5’-FAM-CTCAGTCGCTAGGGTTATGTTAATAAGATGAACCCACGTTCCAGATGGCATCGCCTAGCGA-3’；
the carbaryl quenching probe is a nucleic acid fragment which is base-complementarily paired with the nucleotide sequence of the carbaryl aptamer (SEQ ID No.1) described in example 1, and the nucleotide sequence (SEQ ID No.9) of the carbaryl quenching probe is:
5’-CCTAGCGACTGAG-Dabcyl-3’；
(2) mixing 100nM carbaryl recognition probe (marked as FAM-Apta3S) and carbaryl Quenching probe (marked as Quenching) with different concentrations respectively, dissolving the mixture in phosphate buffer solution (DPBS), incubating the mixture for 30min at room temperature, then carrying out fluorescence test (carried out in a dark environment) by using Shimadzu RF-6000 fluorescence spectrophotometer, and obtaining the stable carbaryl aptamer fluorescence sensor when the system fluorescence intensity is not reduced along with the increase of the carbaryl Quenching probe concentration.
As shown in FIG. 2, when the concentration of the carbaryl quenching probe reaches 300nM, the fluorescence intensity of the system reaches the lowest, and then the fluorescence intensity of the system does not decrease with the increase of the concentration of the carbaryl quenching probe, which shows that when the concentration ratio of the carbaryl quenching probe to the recognition probe is 3:1, the fluorescence signal of the recognition probe can be almost completely quenched, and then the carbaryl recognition probe of 100nM and the carbaryl quenching probe of 300nM are mixed and dissolved in the DPBS to obtain the stable carbaryl aptamer fluorescence sensor.
Further, in order to investigate the detectability of carbaryl by a carbaryl aptamer fluorescent sensor, the following tests were performed:
(one) Experimental groups
FAM-Apta3S group: dissolving 100nM carbaryl recognition probe in phosphate buffered saline (DPBS);
FAM-Apta3S + Carbaryl group: mixing 100nM carbaryl recognition probe and 2. mu.M carbaryl standard solution, and dissolving in phosphate buffer solution (DPBS);
FAM-Apta3S + Quenching group: mixing 100nM carbaryl-recognizing probe and 300nM carbaryl-quenching probe, and dissolving them in phosphate buffer solution (DPBS);
FAM-Apta3S + Quenching + Carbaryl group: mixing 100nM carbaryl recognition probe, 300nM carbaryl quenching probe and 2. mu.M carbaryl standard solution, and dissolving in phosphate buffer solution (DPBS);
(II) test treatment and result analysis
The FAM-Apta3S group, the FAM-Apta3S + Carbaryl group, the FAM-Apta3S + Quenching group and the FAM-Apta3S + Quenching + Carbaryl group were incubated at room temperature for 30min, followed by fluorescence testing using Shimadzu RF-6000 fluorescence spectrophotometer (in a dark environment);
as shown in FIG. 3, where a is a fluorescence spectrum characterization chart of FAM-Apta3S set, b is a fluorescence spectrum characterization chart of FAM-Apta3S + Carbaryl set, c is a fluorescence spectrum characterization chart of FAM-Apta3S + Quenching set, and d is a fluorescence spectrum characterization chart of FAM-Apta3S + Quenching + Carbaryl set, the Quenching probe can quench the fluorescence signal of the recognition probe, and when the Carbaryl target exists, the Quenching probe falls off from the recognition probe due to the specific binding of the Carbaryl recognition probe, accompanied by the increase of fluorescence intensity. As shown by the FAM-Apta3S group and the FAM-Apta3S + Carbaryl group, the addition of the 2 muM Carbaryl standard solution can reduce the fluorescent signal of the 100nM Carbaryl recognition probe by 2.52 percent, which indicates that the addition of the target hardly affects the fluorescent signal of the recognition probe; according to the FAM-Apta3S group and the FAM-Apta3S + Quenching group, the 300nM carbaryl Quenching probe can reduce the fluorescent signal of the identification probe by 92.71 percent, and a stable carbaryl aptamer fluorescent sensor is prepared; according to the FAM-Apta3S + Quenching group and the FAM-Apta3S + Quenching + Carbaryl group, the addition of the 2 mu M Carbaryl standard solution can be combined with the Carbaryl recognition probe, so that the recognition probe is stripped from the Quenching probe, the system fluorescence is recovered to 62.6% of the a system, and the constructed fluorescence sensor can be used for detecting Carbaryl.
Example 3
The application of the carbaryl aptamer fluorescence sensor prepared in the embodiment 2 in carbaryl detection analysis comprises the following steps:
(1) a carbaryl aptamer fluorescence sensor was prepared according to example 2; mixing a carbaryl recognition probe (100nM, FAM-Apta3S) and a carbaryl Quenching probe (300nM, Quencing) in DPBS to prepare a stable carbaryl aptamer fluorescence sensor;
(2) adding carbaryl aptamer fluorescence sensor into different concentrations of carbaryl target (0.02. mu.M, 0.05. mu.M, 0.1. mu.M, 0.2. mu.M, 0.3. mu.M, 0.5. mu.M, 0.8. mu.M, 1.2. mu.M, 1.5. mu.M, 3.0. mu.M, 5.0. mu.M, 10.0. mu.M, 20.0. mu.M) and incubating for 50 min; measuring the fluorescence intensity of each system (in a dark environment) by using an Shimadzu RF-6000 fluorescence spectrophotometer, drawing a standard curve by taking the fluorescence intensity as a vertical coordinate and the carbaryl concentration as a horizontal coordinate, and performing linear analysis on the standard curve;
(3) taking Nanjing basalt lake water and laboratory tap water, and respectively adding 100nM, 500nM and 1000nM of carbaryl standard solution to prepare a simulation sample; then incubating the constructed carbaryl aptamer fluorescence sensor for 50min at room temperature; and (3) calculating the corresponding target concentration according to the linear equation of the step (2) by measuring the fluorescence intensity of the incubation system.
In this embodiment, the analysis result of the fluorescence data of the carbaryl standard solution test is shown in fig. 4, where fig. 4A is a fluorescence spectrogram under different carbaryl concentrations, it can be seen that the fluorescence intensity of each system corresponds to the carbaryl target concentration, and the lower the carbaryl target concentration is, the weaker the fluorescence intensity of the system is, further verifying that the carbaryl aptamer fluorescence sensor can be used for detecting carbaryl;
FIG. 4B is a plot of fluorescence intensity scattergrams at different carbaryl concentrations, with the inset being a linear correlation plot, from which results it can be seen that carbaryl concentrations are linearly correlated in the range of 100nM to 1500 nM; test on basalt lake water and laboratory tap water simulation samples, the recovery rate can reach 97.7% -107.3%.
Example 4
The specific detection analysis of the carbaryl aptamer sensor comprises the following steps:
4.1, preparing a stable carbaryl aptamer fluorescence sensor according to example 2;
4.2, 20 μ M of various molecules of pesticide residues (Aldicarb, Isoprocarb, Bendiocarb, Methiocarb, 1-napthol, Carbosulfan, Pirimicrab, Carbofuran) and 2 μ M of Carbaryl (Carbaryl) target were incubated with the constructed Carbaryl aptamer fluorescence sensor for 50min at room temperature, and the Blank control (Blank) was ultrapure water, followed by measurement of fluorescence intensity of each system.
The results shown in fig. 5 show that in the incubation containing the carbaryl target, the fluorescence intensity of the system is remarkably enhanced, and the fluorescence intensity of the system containing other pesticide residue molecules with ten times of concentration is almost unchanged, which indicates that the carbaryl aptamer fluorescence sensor can be specifically combined with carbaryl and can be used for carbaryl drug detection.
The above is only a preferred embodiment of the present invention, and the protection scope of the present invention is not limited to the above-mentioned embodiments, and all technical solutions belonging to the idea of the present invention belong to the protection scope of the present invention. It should be noted that modifications and embellishments within the scope of the invention may be made by those skilled in the art without departing from the principle of the invention.
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<120> carbaryl aptamer, aptamer derivative and application thereof
<160> 9
<170> SIPOSequenceListing 1.0
<210> 1
<211> 80
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 1
attggcactc cacgcatagg gttatgttaa taagatgaac ccacgttcca gatggcatcg 60
cctatgcgtg ctaccgtgaa 80
<210> 2
<211> 80
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<220>
<221> misc_feature
<222> (21)..(60)
<223> n = a or g or c or t
<400> 2
attggcactc cacgcatagg nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn 60
cctatgcgtg ctaccgtgaa 80
<210> 3
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 3
cctatgcgtg gagtgccaat 20
<210> 4
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 4
attggcactc cacgcatagg 20
<210> 5
<211> 45
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
aaaaaaaaaa aaaaaaaaaa aaaaattcac ggtagcacgc atagg 45
<210> 6
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
attggcactc cacgcatagg 20
<210> 7
<211> 20
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 7
ttcacggtag cacgcatagg 20
<210> 8
<211> 61
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 8
ctcagtcgct agggttatgt taataagatg aacccacgtt ccagatggca tcgcctagcg 60
a 61
<210> 9
<211> 13
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 9
cctagcgact gag 13

Claims (7)

1. The carbaryl aptamer is characterized in that the nucleotide sequence of the carbaryl aptamer is shown as SEQ ID No. 1.
2. The carbaryl aptamer according to claim 1, wherein: the carbaryl aptamer is coupled with a fluorescent group, a quenching group, biotin, a nano material, sulfydryl, amino or enzyme on a nucleotide sequence.
3. A carbaryl-recognizing probe characterized in that its nucleotide sequence is as set forth in SEQ ID NO. 8.
4. Use of the carbaryl aptamer according to claim 1 or 2 or the carbaryl-recognizing probe according to claim 3 for carbaryl detection.
5. The application of a carbaryl aptamer or carbaryl aptamer derivative in preparing a product for detecting or assisting in detecting carbaryl is characterized in that: the product comprises the carbaryl aptamer of claim 1 or 2 or the carbaryl-recognizing probe of claim 3.
6. The carbaryl aptamer fluorescent sensor is characterized by being prepared by mixing a carbaryl recognition probe and a carbaryl quenching probe in a Duchen phosphate buffer solution, wherein the working concentrations of the carbaryl recognition probe and the carbaryl quenching probe are 100nM and 300nM respectively, and the nucleotide sequence of the carbaryl recognition probe is as follows: 5 '-FAM-CTCAGTCGCTAGGGTTATGTTAATAAGATGAACCCACGTTCCAGATGGCATCGCCTAGCGA-3', wherein the nucleotide sequence of the carbaryl quenching probe is as follows: 5 '-CCTAGCGACTGAG-Dabcyl-3'.
7. Use of the carbaryl aptamer fluorescent sensor of claim 6 in carbaryl detection.
CN202011229380.8A 2020-11-06 2020-11-06 Carbaryl aptamer, aptamer derivative and application thereof Expired - Fee Related CN112375762B (en)

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CN113913431B (en) * 2021-09-09 2023-10-24 湖南工业大学 Phoxim aptamer, aptamer derivative and application thereof

Citations (1)

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Publication number Priority date Publication date Assignee Title
CN104031921A (en) * 2013-08-07 2014-09-10 南京市产品质量监督检验院 Carbaryl aptamer, derivative and application thereof

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Publication number Priority date Publication date Assignee Title
CN104031921A (en) * 2013-08-07 2014-09-10 南京市产品质量监督检验院 Carbaryl aptamer, derivative and application thereof

Non-Patent Citations (4)

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4种有机磷农药DNA适体的筛选及结构分析;王丽等;《南京农业大学学报》;20131130(第06期);121-126 *
Selection of a DNA aptamer for the development of fluorescent aptasensor for carbaryl detection;Yuan Liu等;《Chinese Chemical Letters》;20210113;第32卷(第6期);1957-1962 *
核酸适配体生物传感器在农残检测中的研究进展;李凤球等;《分析试验室》;20180415;第37卷(第4期);488-496 *
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