CN113549626A - Aptamer Pr-A08 specifically bound with procymidone and application thereof - Google Patents
Aptamer Pr-A08 specifically bound with procymidone and application thereof Download PDFInfo
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- CN113549626A CN113549626A CN202110852249.5A CN202110852249A CN113549626A CN 113549626 A CN113549626 A CN 113549626A CN 202110852249 A CN202110852249 A CN 202110852249A CN 113549626 A CN113549626 A CN 113549626A
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- 108091023037 Aptamer Proteins 0.000 title claims abstract description 76
- QXJKBPAVAHBARF-BETUJISGSA-N procymidone Chemical compound O=C([C@]1(C)C[C@@]1(C1=O)C)N1C1=CC(Cl)=CC(Cl)=C1 QXJKBPAVAHBARF-BETUJISGSA-N 0.000 title claims abstract description 48
- 239000002773 nucleotide Substances 0.000 claims abstract description 7
- 125000003729 nucleotide group Chemical group 0.000 claims abstract description 7
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- 238000002360 preparation method Methods 0.000 claims description 4
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- TWRXJAOTZQYOKJ-UHFFFAOYSA-L magnesium chloride Substances [Mg+2].[Cl-].[Cl-] TWRXJAOTZQYOKJ-UHFFFAOYSA-L 0.000 description 1
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- LKPLKUMXSAEKID-UHFFFAOYSA-N pentachloronitrobenzene Chemical compound [O-][N+](=O)C1=C(Cl)C(Cl)=C(Cl)C(Cl)=C1Cl LKPLKUMXSAEKID-UHFFFAOYSA-N 0.000 description 1
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- ZLIBICFPKPWGIZ-UHFFFAOYSA-N pyrimethanil Chemical compound CC1=CC(C)=NC(NC=2C=CC=CC=2)=N1 ZLIBICFPKPWGIZ-UHFFFAOYSA-N 0.000 description 1
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- QGHREAKMXXNCOA-UHFFFAOYSA-N thiophanate-methyl Chemical group COC(=O)NC(=S)NC1=CC=CC=C1NC(=S)NC(=O)OC QGHREAKMXXNCOA-UHFFFAOYSA-N 0.000 description 1
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- C12N15/09—Recombinant DNA-technology
- C12N15/11—DNA or RNA fragments; Modified forms thereof; Non-coding nucleic acids having a biological activity
- C12N15/115—Aptamers, i.e. nucleic acids binding a target molecule specifically and with high affinity without hybridising therewith ; Nucleic acids binding to non-nucleic acids, e.g. aptamers
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- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
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Abstract
The invention discloses an aptamer Pr-A08 specifically bound with procymidone, the nucleotide sequence of the aptamer is shown as SEQ ID NO. 1, the secondary structure of the aptamer is provided with a protruding ring and a stem, and the aptamer has a G-quadruplex structure, and Gibbs free energy DG = -12.18; the nucleic acid aptamer specificity and affinity property evaluation is carried out based on a colloidal gold spectrophotometry, and the experimental result shows that the nucleic acid aptamer Pr-A08 can be specifically combined with procymidone and has the characteristics of high specificity and high affinity.
Description
Technical Field
The invention belongs to the technical field of biomedicine, and particularly relates to an aptamer Pr-A08 specifically bound with procymidone and application thereof.
Background
Procymidone (procymidone) with the chemical name of N- (3, 5-dichlorophenyl) -1, 2-dimethylcyclopropane-1, 2-diformylimide, also known as procymidone and pyrimethanil. It is easily soluble in acetone, chloroform, xylene and dimethylformamide, slightly soluble in ethanol and hardly soluble in water. It is a systemic bactericide, has both protection and treatment effects, and has obvious use effect under low-temperature and high-humidity conditions. The microbial composition is used for crops such as rape, radish, eggplant, cucumber, Chinese cabbage, tomato, sunflower, watermelon, strawberry, onion, peach, cherry, flowers, grape and the like, can prevent and treat gray mold, sclerotinia, gray star disease, flower rot, brown rot, gummy stem blight and the like, and can also be used for resistant original bacteria to thiophanate methyl and carbendazim, but strains are easy to generate resistance to the resistant original bacteria. In recent years, the pesticide residue detection reagent is widely used on vegetables and fruits, and is also one of pesticide varieties with higher pesticide residue detection rate and overproof rate in quality safety monitoring of agricultural products at all levels. The maximum residual limit value in the Chinese chives is 0.2mg/kg, the vegetable oil is 0.5mg/kg, the tomato and the cucumber are 2mg/kg, the eggplant, the pepper, the grape, the mushroom (fresh) and the fresh corn are 5mg/kg, and the strawberry is 10 mg/kg. The procymidone has no influence on human body after being taken in a small amount, but if the procymidone is taken for a long time, the procymidone can affect human health, slightly irritate eyes and skin, and if the procymidone is taken for a long time, the procymidone can be deposited in the human body at a certain amount, thereby causing damage to nerves, blood systems and the like of the human body. The traditional technology for detecting procymidone is generally realized by a gas chromatography-mass spectrometry method and an antibody test strip method, the gas chromatography-mass spectrometry method needs complicated instruments and equipment and high cost, and an antibody has the defects of long and unstable synthesis period and the like.
An Aptamer (Aptamer) is a single-stranded oligonucleotide sequence which has high affinity for a target substance and can be specifically identified and screened from an artificially constructed oligonucleotide library by in vitro ligand index enrichment phylogenetic technology (SELEX). The aptamer can be combined with a target substance in a specific two-dimensional or three-dimensional conformation, and the target substance comprises stem loops, hairpins, G-quadruplexes and other structures, so that the target substance can be detected. The aptamer has the unique advantages that the aptamer has strong specificity and high affinity to a target substance, and the dissociation constant between the aptamer and a target molecule is generally 10-9~10-12mol/L; aptamerThe target range is wide, and the aptamer can be combined with molecules such as enzyme and antibody and can also be combined with small molecules such as metal ions, biotoxins and medicines; the aptamer can be chemically synthesized in vitro in a large amount and rapidly, and is easy to be modified by functional groups; the aptamer has small molecular weight, generally comprises a single-stranded oligonucleotide fragment consisting of 25-100 bases, and has small steric hindrance when being combined with a target substance; the aptamer has good stability and recoverability, and can be repeatedly denatured and renatured. Thus, the advantages possessed by aptamers have led to their widespread use in analytical chemistry, the medical field, as well as in environmental monitoring and food safety control.
Disclosure of Invention
The invention provides an aptamer Pr-A08 specifically bound with procymidone, the nucleotide sequence of the aptamer is shown as SEQ ID NO. 1, the secondary structure of the aptamer is provided with a protruding ring and a stem, and the aptamer has a G-quadruplex structure, and Gibbs free energy DG = -12.18.
The invention also aims to apply the aptamer specifically binding with the procymidone to the preparation of a probe molecule for identifying the procymidone or a kit for detecting the procymidone.
The purpose of the invention is realized by the following technical scheme:
step one, screening
Adopting SELEX technology to screen out a nucleic acid aptamer population capable of being specifically combined with procymidone through a column capable of coupling hydroxyl;
step two, cloning
Designing a primer to perform PCR amplification, ligation transformation and selecting a monoclonal, utilizing a PMD19-T carrier to ligate and transform a PCR product into competent cells, and performing overnight culture on the competent cells connected and transformed on a culture medium plate with ampicillin at 37 ℃ by a streaking method to obtain a single colony;
the aptamer amplification primers are as follows: aptamer Fw: GACATATTCAGTCTGACAGCG, respectively; aptamer Rv: GCTAGACGATATTCGTCCATC, respectively;
step three, identifying positive clone and sequencing
Randomly selecting a single colony on a solid culture medium, culturing the single colony in a liquid culture medium containing ampicillin resistance until the single colony is turbid, carrying out PCR amplification on a bacterial liquid, verifying a PCR product by using agarose gel electrophoresis, selecting a fragment with the same size as a target, and sequencing, wherein a sequencing result shows that a nucleic acid aptamer (Pr-A08) specifically bound with procymidone consists of 82 nucleotides, and the sequence (from a5 'end to a 3' end) of the nucleic acid aptamer is as follows:
gacatattcagtctgacagcgcgtaagaaatcaaggctgctcaccgttgacgggctggatagctagacgatattcgtccatc；
step four, characterization of aptamer (Pr-A08) single-stranded DNA secondary structure
The prediction of the secondary structure of the aptamer Pr-A08 single-stranded DNA molecule by using MFOLD software shows that the secondary structure has outstanding rings and stems, a G-quadruplex structure exists, and Gibbs free energy DG = -12.84, and the secondary structure is as follows:
step five, testing the affinity, specificity and sensitivity to procymidone of aptamer (Pr-A08)
The affinity, specificity and sensitivity of the aptamer are identified by using a colloidal gold spectrophotometry, and the result shows that the affinity constant of Pr-A08 is 27.01 +/-6.519 nmol/L, Pr-A08 has high specificity, and the lowest concentration of procymidone which can be detected is 50 ng/mL.
The invention has the beneficial effects that: compared with the existing procymidone detection technology on the market, the aptamer Pr-A08 screened by the SELEX technology can be identified with high affinity and high specificity and combined with procymidone, and the detection technology based on the aptamer can realize the direct detection of procymidone in food; the specificity and affinity of the aptamer Pr-A08 ensure the accuracy of the detection result of the procymidone remaining in the food.
Drawings
FIG. 1 is a schematic diagram of the secondary structure of the aptamer Pr-A08 of the present invention;
FIG. 2 shows the result of affinity analysis of the aptamer Pr-A08 of the present invention;
FIG. 3 shows the result of the specificity analysis of the aptamer Pr-A08 of the present invention;
FIG. 4 shows the results of analysis of the sensitivity of the aptamer Pr-A08 of the present invention to procymidone.
Detailed Description
The substance of the present invention will be further described with reference to the accompanying drawings and examples, which are provided only for the purpose of better understanding of the present invention and are not intended to limit the scope of the present invention, wherein the methods are conventional unless otherwise specified, and the reagents used are either conventional commercially available reagents or reagents formulated according to conventional methods.
Example 1: screening, cloning, isolation and sequencing of aptamer Pr-A08 and prediction of secondary structure of single-stranded DNA
Coupling of procymidone and immunoaffinity column
1. Diluting procymidone to 50 mu g/mL, wherein the total volume is 1.5 mL;
2. mixing 3mL of immunoaffinity column Sepharose 6B (preserved with acetone) solution with the diluted procymidone solution by using a 15mL centrifuge tube;
3. incubating at 37 deg.C and 20rpm/min for 48 h;
4. adding 5mL of 1mol/L aminoethanol after 48h to seal the sites which are not combined with procymidone, slightly and uniformly mixing, and placing a shaking table (37 ℃, 20 rpm/min) for incubation for 24 h;
5. centrifuging at 4000rpm/min for 2min, and removing the supernatant;
6. respectively and sequentially adding pH8.0 buffer solution, pH4.0 buffer solution and coupling buffer solution to wash the immunoaffinity column, adding 5mL of each washing solution each time, centrifuging at the rotating speed of 4000rpm/min for 2min, and then removing the supernatant;
7. finally, 6mL of coupling buffer solution is added, and the whole mixed solution is transferred to a chromatographic column to form a filter column (for preventing the immunoaffinity column from drying); placing in a refrigerator at 4 deg.C, sealing with sealing film, and standing vertically.
PCR of nucleic acid aptamer libraries (ssDNA)
The ssDNA aptamer library synthesized by TaKaRa company is adopted;
1. preheating a PCR instrument at 94 ℃;
2. mu.L of LssDNA, 28.6. mu.L of deionized water, 5. mu.L of 10 Xbuffer, 3. mu.L of LMgCl 24. mu.L dNTP mixture (2.5. mu. mol/L each), 2. mu.L forward amplification primer (10 mol/L concentration), 2. mu.L reverse amplification primer (10 mol/L concentration) and 0.4. mu.L LTaq enzyme centrifuge tube; the forward amplification primer sequence is 5'-GACATATTCAGTCTGACAGCG-3' and the reverse amplification primer sequence is 5'-GCTAGACGATATTCGTCCATC-3'.
3. Amplification in PCR apparatus according to the following procedure
(1) Pre-denaturation: 94 ℃ for 5 min; (2) 30 cycles: 94 ℃ for 45 s; at 58 ℃ for 30 s; 72 ℃ for 30 s; (3) post amplification: 72 ℃ for 7 min.
Thirdly, purification, sample loading and elution of ligand library
1. Purification of ligand libraries
(1) Directly mixing the PCR product of the aptamer library with the solution in the gel recovery kit according to the volume ratio of 1:1, and recovering DNA fragments;
(2) after the DNA fragment is recovered, carrying out water bath at 95 ℃ for 10min and carrying out ice bath for 10 min; this denaturation treatment changes the double-stranded DNA into a single-stranded DNA.
2. Affinity chromatography
(1) Adding the single-stranded DNA into a chromatographic column prepared by coupling, and fixing the volume of the whole solution to 5mL by using a binding buffer solution (10 mmol/L KCl); placing the mixture on a shaking table for incubation at 37 ℃ and gently rotating the shaking table for 45min at 40 rpm/min;
(2) discarding a binding buffer solution in the immunochromatographic column, adding ultrapure water for washing, and washing for 3 times, wherein each time is 2 mL;
(3) then, performing linear gradient elution by using elution buffer solution (0.1 mol/L NaCl, 0.1mol/L acetate and pH 4.0) with the volume of 3-4 columns, and collecting elution components;
(4) centrifuging in a centrifuge at 12000rmp/min for 2min, and collecting liquid in the tube by spin-drying;
(5) 50 mu LTE buffer solution is added into the collection tube, the tube is centrifuged for 1min at 12000rmp/min in a centrifuge, and the liquid in the collection tube is transferred into the PCR tube.
Four, PCR optimization and mass amplification of nucleic acid aptamers
Taking the liquid prepared in the third step as a template, and operating according to the following steps:
1. preheating a PCR instrument at 94 ℃;
2. mu.L of template, 28.6. mu.L of deionized water, 5. mu.L of 10 XBuffer, 3. mu.L of MgCl2mu.L of dNTP mix (2.5. mu. mol/L each), 2. mu.L of forward amplification primer, 2. mu.L of reverse amplification primer, and 0.4. mu.L of LTaq enzyme were reacted in a PCR centrifuge tube. The forward amplification primer sequence is 5'-GACATATTCAGTCTGACAGCG-3', and the reverse amplification primer sequence is 5'-GCTAGACGATATTCGTCCATC-3';
3. amplification was performed in a PCR instrument according to the following procedure:
(1) pre-denaturation: 94 ℃ for 5 min; (2) 30 cycles: 94 ℃ for 45 s; at 58 ℃ for 30 s; 72 ℃ for 30 s; (3) post amplification: 72 ℃ for 7 min;
4. after the circulation is finished, the PCR product is extracted and recovered by using a DNA product purification kit of TIANGEN company, and the steps are as follows:
(1) the PCR product and the equal-volume membrane binding buffer are reversed and mixed evenly, then the mixed solution is transferred to an affinity column, the mixed solution is kept stand for 5min at room temperature to ensure that the DNA is fully combined with the silica gel membrane, the mixed solution is centrifuged at 12000rpm/min for 1min, and the waste liquid in the collecting tube is poured off;
(2) adding 700 mu L of rinsing liquid (containing ethanol) into a centrifugal purification column, centrifuging at 12000rpm/min for 1min, and pouring off waste liquid in a collecting pipe;
(3) repeating the step (2);
(4) centrifuging at 12000rpm/min for 3 min;
(5) placing the centrifugal purification column into a new centrifugal tube;
(6) adding 30 muL of ultrapure water, and standing at room temperature for 5 min;
(7) centrifuging at 12000rpm for 1min, and obtaining the bottom solution of the tube as the PCR product of the purified aptamer.
Cloning, isolation and sequencing of aptamers and prediction of secondary structure of single-stranded DNA
1. Preparation of E.coli DH5 alpha competent cells
(1) Selecting a single DH5 alpha colony, inoculating the colony in 3mL LB culture medium without ampicillin, culturing overnight at 37 ℃, re-inoculating the bacterial liquid in 50mL liquid LB culture medium according to the proportion of 1:100 the next day, and oscillating for 2h at 37 ℃; when the OD600 value reached 0.35, the bacterial culture was harvested;
(2) transferring the bacterial culture to a 50mL pre-cooled sterile polypropylene tube, and allowing the culture to cool on ice for 10 min;
(3) centrifuging at 4000rpm/min at 4 deg.C for 10min, discarding the culture solution, and inverting the tube for 1min to drain the residual culture solution;
(4) adding 150 μ L of ice pre-cooled 0.1mmol/L CaCl2Combining the two tubes of the solution, and carrying out ice bath for 10 min;
(5) centrifuging at 4000rpm/min at 4 deg.C for 10min, discarding the supernatant, and inverting the tube for l min to drain off residual liquid;
(6) firstly, 800 mu L of ice pre-cooled 0.1mol/L CaCl is added2The solution was resuspended in cells, 25. mu.L of pre-cooled 75% glycerol was added, and then stored at-80 ℃ until use.
2. Ligation and conversion of ligation products
(1) Add 1. mu.L Takara pMD19-T simple vector, 4. mu.L aptamer PCR product and 5. mu.L ligase buffer mix to the microfuge tube;
(2) reaction at 16 ℃ overnight incubation;
(3) taking out the competent cells from a refrigerator at-80 deg.C, and immediately placing on ice for 15 min;
(4) adding the system (10 μ L) with the connected carrier into 100 μ L DH5 α competent cells, and placing in ice for 30 min;
(5) heating at 42 deg.C for 90s, and standing in ice for 5 min;
(6) adding 890 μ L of LB liquid medium (without ampicillin) which has been warmed at 37 deg.C, and culturing at 37 deg.C for 60min with slow shaking;
(7) mu.L of the suspension was spread on LB solid medium containing ampicillin, and cultured at 37 ℃ for 16 hours to form single colonies.
3. Cloning, screening and sequencing of aptamers and prediction of secondary structure of single-stranded DNA
Picking the single colony in an LB culture medium containing ampicillin, carrying out shaking culture at 37 ℃ and 150rpm/min until the single colony is turbid, and carrying out PCR amplification on a bacterial liquid; the amplification primers and amplification conditions are the same as those of the aptamer. After plasmid extraction of the positive clones confirmed by PCR, the nucleotide sequence was determined by an automatic nucleotide sequence measuring instrument of Applied Biosystems 3730A; sequencing results showed that the aptamer (Pr-a08) specifically binding to procymidone consisted of 82 nucleotides with the sequence (5 'to 3'):
gacatattcagtctgacagcgcgtaagaaatcaaggctgctcaccgttgacgggctggatagctagacgatattcgtccatc；
sequence length of aptamer Pr-A08 that specifically binds to Pythium: 82 bases, sequence type: nucleic acid, strand number: single strand, topology: linear, sequence type: ssDNA.
The temperature was set to 26 ℃ by MFOLD software, Na+The concentration is 150mmol/L, Mg2+Secondary structure prediction of the nucleic acid aptamer Pr-A08 single-stranded DNA molecule specifically binding to Pythium ultimum was performed at a concentration of 1mmol/L (http:// mfold. rna. albany. edu/q = mfold/DNA-Folding-Form) and QGRS mapping (http:// bioinformatics. ramapo. edu/QGRS/analyze. php); the results show that the aptamer contains prominent loops and stems with gibbs free energy DG = -12.18, and the structure has higher stability (see fig. 1).
Example 2: affinity, specificity and sensitivity detection of aptamer Pr-A08
1. Aptamer Pr-A08 affinity assay
The colloidal gold spectrophotometry takes colloidal gold as a marker, and utilizes the characteristic that colloidal gold is aggregated in a high-salt environment to cause the red shift of the wavelength of the colloidal gold and the rightward shift of a characteristic peak to measure the light absorption value of the colloidal gold solution under a specific wavelength to identify.
(1) Preparation of 25nm colloidal gold
1) Preparing 1% sodium citrate solution, weighing 0.1g of sodium citrate, and adding 10mL of ultrapure water;
2) diluting 25 μ L of chloroauric acid solution (containing 49% Au) 45 times to 1125 μ L (the step is performed under the condition of keeping out light);
3) adding 99mL of purified water into a clean conical flask, wherein the purified water, the sodium citrate solution and the chloroauric acid solution are filtered through 45-micrometer filter membranes;
4) adding 1mL of chloroauric acid solution into a conical flask, uniformly mixing, adjusting the temperature to 80 ℃ by using an electrothermal sleeve, and heating to boil (in the process, magnetic stirring is carried out by using a rotor);
5) when the solution is boiled quickly, the temperature is adjusted to 100 ℃, the solution is heated to be completely boiled, and 1.5mL of 1% sodium citrate solution is added;
6) adjusting the temperature to 0 deg.C, adjusting the temperature to 70 deg.C after the solution is colorless-black-purple-red and the color is stable, and heating for 10 min;
7) after cooling to room temperature, the mixture was transferred to 4 ℃ and stored away from light.
(2) Diluting procymidone to 1 mu g/mL by using ultrapure water, wherein each tube contains 50 mu L of procymidone;
(3) sending the aptamer Pr-A08 to Beijing Optimalaceae biology company for synthesis; when the aptamer-containing test tube is used, the short-time centrifugation is firstly carried out, so that the aptamer is totally gathered at the bottom of the test tube. According to the instructions, the nucleic acid aptamer was dissolved sufficiently with sterile water to have a concentration of 10-4The storage solution of mol/L can be divided into small parts to avoid repeated freeze thawing; then diluting the aptamer to 50nmol/L, 100nmol/L, 200 nmol/L, 400nmol/L, 800 nmol/L and 1600 nmol/L by 1 XPBS, adding the aptamer to the Pythium solution in the step (2), adding 50 mu L of aptamer to each tube, and incubating for 30min at room temperature; meanwhile, blank controls without aptamer and with 50 μ L of 1 × PBS are set, and each sample is subjected to 3 parallel experiments;
(4) concentrating the colloidal gold prepared in the step (1) by 4 times, centrifuging 4mL of colloidal gold at 4 ℃ and 8000rpm/min for 12min, discarding 3mL of supernatant, and uniformly blowing the rest 1mL of colloidal gold by using a pipette for later use;
(5) adding 50 mu L of concentrated colloidal gold into each tube in the step (3), and incubating for 30min at room temperature in a dark place;
(6) adding NaCl with the final concentration of 40mmol/L into each tube in the step (5), uniformly mixing, measuring the absorbance value at 520nm by using a microplate reader, and calculating (A' -A)0)/A0Wherein A' is the absorbance value corresponding to the aptamer with different concentrations, A0Is the absorbance value without addition of an aptamer; with (A' -A)0)/A0As a result of analysis and calculation of affinity constants using GraphPad Prism8 software in ordinate and aptamer concentration in abscissa, it was revealed that the aptamers were presentOf Pr-A08Kd= 27.01 ± 6.519nmol/L (see fig. 2), the aptamer having an affinity, wherein a smaller affinity constant indicates a higher affinity of the aptamer having an affinity.
2. Aptamer Pr-A08 specific detection
(1) Procymidone was diluted to 1 μ g/mL with ultrapure water, 50 μ L was added per tube while setting the nonspecific target control sample at the same concentration as procymidone: dimethomorph, quintozene and glyphosate, setting a blank control without adding procymidone and adding 50 mu L of ultrapure water, and repeating 3 parallel experiments on each target;
(2) the aptamers were diluted to 400nmol/L with 1 XPBS, 50. mu.L per tube was added in step (1), and incubated at room temperature for 30 min;
(3) concentrating the colloidal gold prepared in the step 1 by 4 times, centrifuging 4mL of colloidal gold at 4 ℃ and 8000rpm/min for 12min, discarding 3mL of supernatant, and uniformly blowing the rest 1mL of colloidal gold by using a pipette for later use;
(4) adding 50 mu L of concentrated colloidal gold into each tube in the step (2), and incubating for 30min at room temperature in a dark place;
(5) in step (4), NaCl was added to each tube to a final concentration of 40mmol/L, absorbance values were measured at 520nm and 620nm using a microplate reader to calculate A620nm/A520nm, and (A '-A'0)/A’0Wherein A 'is the ratio of the absorbance value at 620nm to the absorbance value at 520nm corresponding to the aptamers with different concentrations, wherein A'0Is the ratio of the absorbance value at 620nm to the absorbance value at 520nm without the addition of a target; the results are shown in FIG. 3, which shows that (A ' -A ' was added to the tube of procymidone '0)/A’0The value of (a) is much greater than that of the addition of other non-specific targets, and the aptamer Pr-A08 is capable of binding specifically to Pythium.
3. Aptamer Pr-A08 sensitivity detection
(1) Diluting procymidone to different concentration gradients of 50ng/mL, 100ng/mL, 200ng/mL, 400ng/mL, 600ng/mL and 800ng/mL by using ultrapure water, and adding 50 mu L of procymidone into each tube; setting that no procymidone is added and ultrapure water is added as a blank control, and repeating 3 parallel experiments for each concentration;
(2) diluting the aptamer to 400nmol/L with 1 XPBS, adding 50 μ L of aptamer per tube to the step (1), and incubating at room temperature for 30 min;
(3) concentrating the colloidal gold prepared in the step 1 by 4 times, centrifuging 4mL of colloidal gold at 4 ℃ and 8000rpm/min for 12min, discarding 3mL of supernatant, and uniformly blowing the rest 1mL of colloidal gold by using a pipette for later use;
(4) adding 50 mu L of concentrated colloidal gold into each tube in the step (2), and incubating for 30min at room temperature in a dark place;
(5) adding NaCl with a final concentration of 40mmol/L into each tube in the step (4), measuring absorbance values at the wavelengths of 520nm and 620nm by using a microplate reader, and calculating A = A620nm/A520 nm; the result shows that when the concentration of the procymidone reaches 50ng/mL, the value of A620nm/A520nm is obviously higher than that of a blank control, the value of A620nm/A520nm gradually increases with the increase of the concentration of the procymidone, and the sensitivity of the aptamer Pr-A08 is higher (see figure 4).
The aptamer Pr-A08 of the invention has good affinity, specificity and sensitivity, and can be used for detecting procymidone in subsequent actual samples.
Sequence listing
<110> university of Kunming science
<120> aptamer Pr-A08 specifically binding to procymidone and application thereof
<160> 3
<170> SIPOSequenceListing 1.0
<210> 1
<211> 82
<212> DNA
<213> Artificial sequence (Artificial)
<400> 1
gacatattca gtctgacagc gcgtaagaaa tcaaggctgc tcaccgttga cgggctggat 60
agctagacga tattcgtcca tc 82
<210> 2
<211> 21
<212> DNA
<213> Artificial sequence (Artificial)
<400> 2
gacatattca gtctgacagc g 21
<210> 3
<211> 21
<212> DNA
<213> Artificial sequence (Artificial)
<400> 3
gctagacgat attcgtccat c 21
Claims (3)
1. An aptamer Pr-A08 specifically combined with procymidone has a nucleotide sequence shown in SEQ ID NO. 1.
2. The aptamer Pr-A08 that specifically binds to Pythium according to claim 1, wherein: the secondary structure has prominent rings and stems and has a G-quadruplex structure with gibbs free energy DG = -12.18.
3. The use of the aptamer Pr-A08 of claim 1 in the preparation of a reagent for identifying procymidone or a kit for detecting procymidone.
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