CN112813071A - Aptamer sequence for specifically recognizing ribavirin and application thereof - Google Patents
Aptamer sequence for specifically recognizing ribavirin and application thereof Download PDFInfo
- Publication number
- CN112813071A CN112813071A CN202110165104.8A CN202110165104A CN112813071A CN 112813071 A CN112813071 A CN 112813071A CN 202110165104 A CN202110165104 A CN 202110165104A CN 112813071 A CN112813071 A CN 112813071A
- Authority
- CN
- China
- Prior art keywords
- ribavirin
- aptamer
- screening
- library
- target
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
Images
Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- 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
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/10—Processes for the isolation, preparation or purification of DNA or RNA
- C12N15/1034—Isolating an individual clone by screening libraries
- C12N15/1048—SELEX
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/94—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving narcotics or drugs or pharmaceuticals, neurotransmitters or associated receptors
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N2310/00—Structure or type of the nucleic acid
- C12N2310/10—Type of nucleic acid
- C12N2310/16—Aptamers
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N21/00—Investigating or analysing materials by the use of optical means, i.e. using sub-millimetre waves, infrared, visible or ultraviolet light
- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/64—Fluorescence; Phosphorescence
- G01N21/6428—Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
- G01N2021/6432—Quenching
Abstract
The invention discloses an aptamer sequence for specifically recognizing ribavirin and application thereof, belonging to the technical field of food safety biology. The invention takes ribavirin as a target, uses the Capture-SELEX technology to enrich the aptamer through multiple screening processes, uses real-time fluorescence quantitative PCR to monitor the screening process, and visually and comprehensively obtains all sequence information of an aptamer library through high-throughput sequencing, thereby obtaining the aptamer sequence with the highest enrichment degree through screening; the affinity and the specificity of the obtained nucleic acid sequence are characterized by a colorimetric method and a fluorescence method, and an aptamer sequence with good affinity and specificity is obtained. Can be used for detecting the content of ribavirin in the bodies of the livestock and poultry breeding animals, foods and medicines used by the livestock and poultry breeding animals and human blood.
Description
Technical Field
The invention relates to an aptamer sequence for specifically recognizing ribavirin and application thereof, belonging to the field of food safety biotechnology.
Background
Ribavirin is a broad-spectrum antiviral drug, can inhibit the replication of RNA viruses, has a strong inhibiting effect on DNA viruses, and is commonly used for treating respiratory diseases and the like. However, the use of the compound as a veterinary drug for treating viral epidemic diseases of animals has a great safety risk, and is prohibited from being sold and used as a veterinary drug.
Abuse of antiviral drugs not only causes the quality and flavor of the cultured animals to be reduced, but also causes drug residues and drug resistance. Abuse of ribavirin in the process of breeding of poultry stocks can cause drug resistance of hepatitis C virus, about 0.4 hundred million HCV carriers exist in China, and the average infection rate is about 3.2%. More than 70% of infected individuals develop hepatitis and liver cirrhosis, and even liver cancer. The standard treatment of chronic HCV infection with long-acting interferon in combination with ribavirin is common, but the failure rate of this approach increases year by year, mainly due to the drug resistance of ribavirin. Additionally, ribavirin inhibits glutathione, damages the erythrocyte membrane to cause anemia, and even worsens existing heart disease. Therefore, the understanding of ribavirin drugs is strengthened, intensive research is carried out on the ribavirin drugs, and the development of a rapid detection method is urgent.
The detection of ribavirin is mainly focused on the detection of ribavirin in blood plasma and the detection of ribavirin in animal food and veterinary medicine, and the detection method is mainly focused on two methods, namely a chromatography method and an immunization method. Among them, chromatography mainly consists of a combination of high performance liquid chromatography and mass spectrometry, and generally requires professional operations and expensive instruments. The other is an immune method based on antigen-antibody interaction, which mainly comprises a radioimmunoassay and an enzyme-linked immunosorbent assay, but the process of preparing the specific antibody is complicated and time-consuming, and the prepared antibody has poor stability.
The aptamer screening technology is to screen a section of single-stranded nucleotide sequence capable of specifically recognizing and binding a target from a random nucleotide sequence synthesized in vitro by using an exponential enrichment phylogenetic evolution technology (SELEX). The aptamer can be folded on the basis of structures such as a hairpin, a stem-loop and a quadruplet to form a complex three-dimensional conformation so as to be specifically combined with a target, and meanwhile, the binding capacity with the target is greatly increased by intermolecular forces such as van der Waals force, hydrogen bond and hydrophobic effect between the aptamer and the target. The Capture-SELEX is to indirectly fix the ssDNA library on a specific carrier through a complementary strand, and when a target exists, an aptamer specifically combined with the target is subjected to three-dimensional structure change, is dissociated from the complementary strand and falls off to form an aptamer-target complex, and then enters a solution. The aptamer specifically combined with the target can be obtained by separating and collecting the solution. Compared with the traditional detection methods (chromatography and immunity method), the aptamer screening technology has the advantages of short screening period, high affinity, good specificity, easy modification, stable performance, easy storage and the like.
Disclosure of Invention
[ problem ] to
The existing chromatography for detecting ribavirin has the problems of expensive equipment and complex operation, and the immunization method has the problems of long period, low affinity, weak specificity and unstable antibody which is difficult to preserve.
[ solution ]
The present invention provides an aptamer for specifically recognizing ribavirin, as shown in a) or b) below:
a) 1-SEQ ID NO: 7, or a nucleotide sequence represented by any one of SEQ ID NOs,
b) the nucleotide sequence which is derived from the a) and has the ability of specifically recognizing ribavirin and is obtained by substituting, deleting or adding one or more bases in the nucleotide sequence defined by the a).
In one embodiment of the present invention, the aptamer for specifically recognizing ribavirin is preferably the nucleotide sequence shown in SEQ ID NO. 1.
The 5 'end or the 3' end of the aptamer can be chemically modified by FITC, amino, biotin or digoxin.
The invention also provides a method for screening the aptamer for specifically recognizing ribavirin, which mainly comprises the following steps:
(1) preparation of aminated Fe3O4Magnetic nanobead, coated with avidin, and aminated Fe3O4Nano magnetic beads;
(2) constructing a random ssDNA library, wherein the length of an initial library is 80bp, and hybridizing the initial ssDNA library with complementary short-chain Bio-P1; the complementary short-chain Bio-P1 refers to biotin-labeled single-stranded DNA which can be complementary with ssDNA in the library;
(3) the library hybridized with the complementary short-chain Bio-P1 is immobilized on the avidin-coated aminated Fe through the action of biotin and avidin3O4The surface of the nanometer magnetic bead;
(4) the method for screening the ribavirin aptamers based on the Capure-SELEX method comprises the following steps: adding ribavirin into the library fixed on the surface of the magnetic beads and incubating, leaving ssDNA which is not combined with the target or is weakly combined on the magnetic beads, and allowing the aptamer combined with the target to enter a supernatant; aptamer is enriched through circular screening, and in the circular screening process, screening pressure is increased through reducing library concentration, shortening incubation time and increasing reverse screening substances;
(5) and (3) carrying out high-throughput sequencing and analysis on the sequences obtained by enrichment to obtain 7 candidate sequences, and characterizing the affinity and specificity of the candidate aptamer by a gold nano colorimetric method and a carbon dot fluorescence quenching method to obtain the aptamer with affinity and specificity to ribavirin.
In one embodiment of the invention, the step (4) is carried out for 15 screening enrichment cycles, wherein 5 screening rounds are included; in the process of back screening, structural analogues (such as acadesine and trifluorothymidine) and coexisting substances (such as adamantane, moroxydine hydrochloride and acyclovir) are added as back screening substances to be incubated with aptamers before target incubation is added, aptamer sequences bound with the back screening substances are removed after entering supernatant, and sequences not bound with the back screening substances are left on magnetic beads to continue incubation and enrichment with target ribavirin.
The invention provides a kit for detecting ribavirin, which contains the aptamer for specifically recognizing ribavirin. Further, the carbon dot solution or a raw material for preparing the carbon dot solution may be contained.
The invention provides a method for detecting ribavirin by utilizing an aptamer technology, which is based on a carbon point fluorescence quenching technology and mainly comprises the following steps: drawing a standard curve by taking the concentration gradient of the target ribavirin as an abscissa and taking the relative fluorescence intensity as an ordinate; the relative fluorescence intensity refers to the fluorescence intensity of an experimental group containing target ribavirin relative to a control group which replaces ribavirin by BB buffer; after a sample to be detected is subjected to proper pretreatment, an aptamer is added, incubation is carried out, a carbon dot solution is added, the fluorescence intensity is measured and calculated, and the fluorescence intensity is substituted into a standard curve to obtain the concentration of ribavirin.
[ advantageous effects ]
The invention takes ribavirin as a target, adopts the Capture-SELEX technology to enrich the aptamer through 15 screening processes, adopts real-time fluorescence quantitative PCR to monitor the screening process, obtains all sequence information of an aptamer library through high-throughput sequencing, and is visual and comprehensive, thereby obtaining the aptamer sequence with the highest enrichment degree; the two methods of colorimetry and fluorescence method are used for representing the affinity and specificity of the obtained nucleic acid sequence, the result is reliable, and the aptamer sequence with good affinity and specificity is obtained.
The invention provides a high-specificity detection and identification element which has high stability, good affinity, and is easy to prepare and mark for the detection of ribavirin. Compared with an antibody, the aptamer provided by the invention can be screened in vitro, the screening period is short, the screened sequence has high specificity, the modification is easy, the synthesis is convenient, and the preparation accuracy and the stability are high. Can be used for detecting the content of ribavirin in the bodies of the livestock and poultry breeding animals, foods and medicines used by the livestock and poultry breeding animals and human blood.
The present invention selects the mode of fixing the library and dissociating the target to screen and obtain the aptamer aiming at the small molecular compound target (smaller volume, surface area and binding site).
Drawings
FIG. 1 is a schematic diagram of the Capture-SELEX screening method.
Fig. 2 is an affinity saturation binding curve for aptamers obtained by gold nanopigmentation, (1) APT-1 Kd 34.34 ± 6.038nmol/L, (2) APT-3 Kd 341.7 ± 129.5nmol/L, (3) APT-4 Kd 1780 ± 2267nmol/L, (4) APT-5 Kd 80.76 ± 21.20nmol/L, (5) APT-6 Kd 36.19 ± 15.57nmol/L, (6) APT-7 Kd 88.24 ± 19.87 nmol/L.
Fig. 3 is an affinity saturation binding curve of aptamers obtained by carbon dot fluorescence, (1) APT-1 Kd 69.91 ± 25.33nmol/L, (2) APT-5 Kd 120.3 ± 50.39nmol/L, (3) APT-6 Kd 127.3 ± 85.65nmol/L, and (4) APT-7 Kd 183.0 ± 82.84 nmol/L.
FIG. 4 is a result of the specificity of the aptamer obtained by the carbon dot fluorescence method.
FIG. 5 is a standard curve for identification of aptamer APT-1 for detection of ribavirin, with a linear range of 2.5-50 ng/mL.
Detailed Description
Example 1 screening of aptamers that specifically recognize ribavirin
1.Fe3O4Preparation and characterization of nano magnetic beads
1.1 amination of Fe3O4Preparation and characterization of nano magnetic beads
The preparation method adopts a hot solvent one-step method: weighing 1.0g of ferric chloride hexahydrate and 2.0g of anhydrous sodium acetate, adding the mixture into 30mL of ethylene glycol, slightly shaking and mixing, adding 6.5g of 1, 6-hexanediamine, violently stirring in a water bath kettle at 50 ℃ until a uniform wine red transparent solution is formed, quickly transferring the obtained solution into a 100mL high-pressure reaction kettle with a polytetrafluoroethylene lining, heating at the high temperature of 198 ℃ for 6 hours, taking out the reaction kettle, and cooling at room temperature. After the reaction kettle is cooled, removing supernatant under the action of an external magnetic field, adding 100mL of absolute ethyl alcohol, washing and transferring the obtained lower-layer black solid into a beaker, carrying out magnetic separation after ultrasonic cleaning, adding 100mL of water again by the same method, carrying out magnetic separation after ultrasonic cleaning, and respectively carrying out 3 times of absolute ethyl alcohol and water cleaning according to the method. Finally, the aminated magnetic beads were dried overnight in an oven at 50 ℃ and stored for further use.
1.2 avidin coated aminated magnetic beads
Weighing 5mg of aminated magnetic beads, adding 3mL of PBS buffer (pH 7.4), performing ultrasonic dispersion for 20min, then adding 200 μ L of glutaraldehyde, uniformly mixing, oscillating for 3h at 37 ℃ under the condition of 130rpm in the dark, washing for 4 times by using the PBS buffer after the completion of the ultrasonic dispersion, then adding 200 μ L of 1mg/mL avidin solution, dissolving in 1.8mL of PBS buffer, wherein the final concentration of the avidin solution is 100 μ g/mL. The mixture was placed at 37 ℃ and shaken in the dark at 130rpm for 12 h. Washed with PBS buffer and stored in an environment at 4 ℃ for later use.
2. Screening for aptamers
2.1 construction of libraries and primers
Construction of a random ssDNA library of 80nt length: ATAGGAGTCACGACGACCAG- (40random bases) -TATGTGCGTCTACCTCTTGA, Synthesis of the forward primer P1(SEQ ID NO: 8): 5'-ATAGGAGTCACGACGACCAG-3', 5 ' phosphorylated reverse primer P2(SEQ ID NO: 9): 5 '-P-TCAAGAGGTAGACGCACATA-3', biotin-labeled short complementary to the library: 5 '-bio-TCGTGACTCCTAT-3'. Random library, primers, complementary short chain were all stored in 100. mu. mol/L solution in sterile 1XTE buffer.
2.2 Capture-SELEX screening aptamers
The method for screening the ribavirin aptamers by adopting the Capture-SELEX method based on library immobilization comprises the following steps:
(1) library hybridization: hybridizing an initial ssDNA library and a biotin-labeled complementary strand in a ratio of 1:2, denaturing the library at 95 ℃ for 5min before the beginning of incubation, and performing oscillation incubation at 37 ℃ and 130rpm for 2 h;
(2) fixing magnetic beads: incubating the complemented short chain of the library and the washed magnetic beads at 37 ℃ and 130rpm for 6h, binding biotin with avidin to fix the library on the surfaces of the magnetic beads, and washing the magnetic beads for multiple times by BB buffer solution after the incubation is finished so as to remove the non-specifically bound ssDNA;
(3) and (3) positive screening incubation: adding 10 mu L of prepared 10mmol/L ribavirin solution, oscillating and incubating for 2h at 37 ℃ and 130rpm, competitively binding ssDNA with complementary short chains, leaving ssDNA which is not combined with the target or is weakly combined on magnetic beads, and allowing the aptamer combined with the target to enter into supernatant;
(4) and (3) PCR amplification: and performing PCR amplification by taking ssDNA in the supernatant obtained by magnetic separation after target incubation as a template, wherein the total amplification system is 50 mu L and comprises 40.5 mu L of deionized water, 5 mu L of buffer solution, 2 mu L of template, 0.5 mu L of forward primer, reverse primer, TaqDNA polymerase and 1 mu L of dNTPmix. The PCR amplification conditions were: denaturation at 94 ℃ for 5min, denaturation at 94 ℃ for 30s, annealing at 58 ℃ for 30s, and extension at 72 ℃ for 30s, circulating 18 cycles under the conditions, extension at 72 ℃ for 2min, and cooling at 4 ℃. And finally, separating the PCR product by 8% polyacrylamide gel electrophoresis, verifying the obtained product under a gel imager system, and purifying by using a purification kit.
(5) Preparation and purification of single strands: the concentration of the purified product was determined by a NanoDrop micro ultraviolet-visible spectrophotometer, and 2. mu.L of Lambda exonuclease and 1/10 volumes of Lambda exonuclease buffer were added to excise the 5' -phosphate modified reverse DNA to obtain the secondary library for the next round of screening. And (3) after the enzyme digestion is finished, inactivating the enzyme for 10min at the temperature of 75 ℃ to terminate the reaction, and using 8% modified polyacrylamide gel electrophoresis containing 7mol/L urea to separate enzyme digestion products to verify whether the dsDNA is completely digested into ssDNA. To the cleaved product, 1/10 volumes of NaAC solution and 2 volumes of absolute ethanol were added and mixed, followed by overnight precipitation in a refrigerator at-20 ℃. The precipitated solution was centrifuged at 14000rpm at 4 ℃ and then the supernatant was added to 200. mu.L of 70% ethanol, the mixture was mixed by inversion and centrifuged under the same conditions, the supernatant was dried in a 50 ℃ oven, and then 40. mu.L of 1xTE buffer was added to dissolve and the concentration was measured with a micro UV-visible spectrophotometer.
2.3 circular screening
Rounds 2-15 of the circular screening were performed according to the screening conditions shown in Table 1, in which the screening pressure was increased by decreasing the library concentration, shortening the incubation time, and increasing the back-screening material. Before the target incubation is added in the 6 th, 8 th, 11 th, 13 th and 15 th rounds, the structural analogues of acadesine, trifluorothymidine and coexisting substances of adamantane, moroxydine hydrochloride and acyclovir are added as counter-screening substances to be incubated with the aptamers, the aptamer sequences combined with the counter-screening substances are removed after entering a supernatant, and the sequences not combined with the counter-screening substances are left on magnetic beads to continue to be incubated and enriched with the target ribavirin.
TABLE 1 aptamer selection conditions
3. Monitoring of screening processes
And (3) monitoring the enrichment condition of the aptamer by using a dissolution curve, adding excessive SYBR fluorescent dye into a PCR reaction system, and only combining SYBR with double-stranded DNA (deoxyribonucleic acid), so that whether the PCR reaction is specific or not can be determined by using the dissolution curve. The experiment used a 10. mu.L system containing 5. mu.L of SYBR Green Master Mix, 1.5. mu.L each of forward primer P1 and reverse primer P2 (10. mu.M), 1.5. mu.L, 3. mu.L enriched library as template, 1. mu.L of ultrapure water. The protocol for PCR was denaturation at 95 ℃ for 5min, followed by 40 cycles of 10s at 95 ℃ and 30s at 60 ℃ and the dissolution curve obtained from the software 7900 v.2.4. Along with the continuous screening, ssDNA specifically bound to the target is continuously enriched, the diversity of the library sequence is reduced, more stable homologous double chains are formed in the PCR process, and the dissolution temperature is continuously increased.
4. Analysis of aptamer sequences
And (4) taking the supernatant obtained in the fifteenth screening as a template to perform PCR, and sending the PCR product to Shanghai Biotechnology Limited for high-throughput sequencing. Sequence information of all sequences is obtained through high-throughput sequencing, the sequence information is analyzed, and high-frequency sequences are found out and are most likely to be aptamers for specifically recognizing ribavirin. Selecting ten aptamer sequences appearing at high frequency, adopting M fold software to analyze the secondary structure of the aptamer sequences, combining the analysis result of the software, removing the sequences and three sequences with highly similar secondary structures, and finally obtaining 7 aptamer sequences enriched most for the subsequent characterization of affinity and specificity.
TABLE 2 candidate aptamer sequences
5. Characterization of aptamer sequences
5.1 aptamer affinity characterization
5.1.1 gold nanopigmentation
The preparation of the gold nanoparticles adopts a citric acid reduction method. Adding 1mL of 1% chloroauric acid aqueous solution into a three-neck round-bottom flask containing 98mL of ultrapure water, stirring vigorously, adding into an oil bath kettle, boiling, quickly adding 1mL of 5% trisodium citrate aqueous solution, reacting for 30min, gradually changing the color of the solution from light yellow to dark wine red, transferring the round-bottom flask to the normal temperature, stirring and cooling to the room temperature, and storing the prepared AuNPs solution at 4 ℃ in a dark place for later use.
Respectively denaturing the candidate aptamers and the target with different concentrations for 10min at 95 ℃, then adding the target into the aptamers, and incubating for 0.5h at 37 ℃ and 200rpm, wherein the final concentrations of the aptamers are respectively 50, 100, 150, 200 and 250nmol, the concentration of the target is 30 μmol, and the total concentration is 300 μ L. Adding into centrifuged gold nanometer, shaking, adding MgCl2Inducing aggregation of the solution, standing for 5min, measuring the change of the light absorption value at 520nm by using an enzyme labeling instrument, using water instead of a target as a blank control group, respectively representing the light absorption values of the control group and an experimental group by using A and A0, and carrying out nonlinear fitting on the change delta A (A-A0) of the relative light absorption values under different aptamer concentrations by using GraphPadprism 5.0 software to draw a saturated binding curve of the aptamer. As shown in FIG. 2, except that APT-2 aptamers do not show binding, the Kd values of APT-1, APT-3, APT-4, APT-5, APT-6 and APT-7 are 34.34 + -6.038 nmol/L, 341.7 + -129.5 nmol/L, 1780 + -2267 nmol/L, 80.76 + -21.20 nmol/L, 36.19 + -15.57 nmol/L and 36.19 + -15.57 nmol/L in this order, and four better affinity aptamers of APT-1, APT-5, APT-6 and APT-7 are selected for further affinity verification.
5.1.2 carbon dot fluorescence method
The required materials were prepared using the candle ash method. Collecting 8mg candle ash from the burning candle, dissolving in 20ml solvent (ethanol: water 1:1), performing ultrasonic treatment for 4.5h at 3000rpm for 1min, removing large particle size, collecting supernatant, performing ultrasonic treatment for 2h, performing ultrasonic treatment at 10000rpm for 5min, collecting precipitate, dissolving in water, and measuring the solution concentration to be 100ng/μ L.
Aptamers (30nmol, 60nmol, 90nmol, 120nmol, 150nmol and 180nmol) with different concentrations were denatured at 95 ℃ for 10min, cooled to room temperature, added with a target (30 μmol), incubated at 37 ℃ and 200rpm for 0.5h to allow sufficient binding, and BB buffer was used as a blank control instead of the target. After incubation was complete, 200. mu.L of carbon dot solution was added to allow fluorescence quenching. Fluorescence values for the experimental and control groups (blank control with water instead of target) were obtained with F and F0, respectively. Relative fluorescence intensity Δ F (F-F) at different aptamer concentrations by GraphPad prism 5.0 software0) Nonlinear fitting is performed to draw a saturated binding curve of the aptamer and calculate the dissociation constant Kd value of the aptamer. As shown in FIG. 3, the Kd values of aptamers APT-1, APT-5, APT-6 and APT-7 were 69.91. + -. 25.33nmol/L, 120.3. + -. 50.39nmol/L, 127.3. + -. 85.65nmol/L and 183.0. + -. 82.84nmol/L, respectively, and APT-1 showed the best affinity in accordance with the results shown in 5.1.1.
5.2 aptamer specificity
Selecting four aptamers (APT-1, APT-5, APT-6 and APT-7) with better binding affinity with ribavirin to carry out specificity analysis, denaturing 50nmol of aptamers at 95 ℃ for 10min, cooling to normal temperature, respectively incubating with a target ribavirin (50 mu mol) and structural analogues of acadesine and trifluorothymidine, coexisting matters of adamantane, moroxydine hydrochloride and acyclovir at 37 ℃ and 200rpm for 0.5h, replacing the target with a BB buffer solution as a blank control group, and adding 200 mu l of carbon dot solution to carry out fluorescence quenching after incubation is finished. F and F0Fluorescence values for the experimental and blank groups, respectively. By Δ F (F-F)0) As an index for determining the degree of binding, APT-1 showed good specificity as shown in FIG. 4.
Combining the above considerations, APT-1 was chosen as a suitable ligand for ribavirin.
Example 2 method for determining ribavirin content in chicken samples using specific aptamers
A fluorescence analysis detection method for ribavirin is constructed based on carbon dot adsorption platform fluorescence quenching.
Diluting a standard ribavirin product with a certain gradient concentration, adding carbon dots to adsorb unbound aptamers, replacing a target with a BB buffer solution as a blank control group, and measuring the fluorescence of the whole system. Taking the target concentration as the abscissa and the relative fluorescence difference as the ordinate, drawing a standard curve, as shown in FIG. 5, in the concentration range of 2.5-50ng/mL, the relative fluorescence intensity and ribavirin concentration have a good linear relationship (R)20.9895), the linear equation is y 24.585x-8.3299ng/mL, and the detection limit is 1.8 ng/mL.
In the chicken sample labeling recovery experiment, the chicken sample is pretreated. After homogenizing 2g of chicken sample, respectively adding 5ng/mg, 10 ng/mg and 25ng/mg of ribavirin into the chicken sample, performing labeling recovery, and taking a blank group with BB buffer solution instead of a target as a negative control to obtain the relative fluorescence difference value of the two (sample group-negative control group). Corresponding concentrations are calculated by comparing with a standard curve, and the recovery rate of ribavirin detection is obtained to be in the range of 86.34-103.87%.
TABLE 3 fluorescence analysis for determining the normalized recovery of ribavirin in chicken samples
Although the present invention has been described with reference to the preferred embodiments, it should be understood that various changes and modifications can be made therein by those skilled in the art without departing from the spirit and scope of the invention as defined in the appended claims.
SEQUENCE LISTING
<110> university of south of the Yangtze river
<120> aptamer sequence for specifically recognizing ribavirin and application thereof
<130> BAA210069A
<160> 10
<170> PatentIn version 3.3
<210> 1
<211> 82
<212> DNA
<213> Artificial sequence
<400> 1
ataggagtca cgacgaccag aaagtaatgc ccggtagtta ttcaaagatg agtaggaaaa 60
gatatgtgcg tctacctctt ga 82
<210> 2
<211> 80
<212> DNA
<213> Artificial sequence
<400> 2
ataggagtca cgacgaccag caagacatgt tgcgtaagac atctcatagt ctacttagag 60
tatgtgcgtc tacctcttga 80
<210> 3
<211> 80
<212> DNA
<213> Artificial sequence
<400> 3
ataggagtca cgacgaccag tcatggctaa ggaactcata ttcatagaag gagtcgtgcg 60
tatgtgcgtc tacctcttga 80
<210> 4
<211> 82
<212> DNA
<213> Artificial sequence
<400> 4
ataggagtca cgacgaccag aaagtaatgc ccggtagtta ttcacagatg agtaggaaaa 60
gatatgtgcg tctacctctt ga 82
<210> 5
<211> 80
<212> DNA
<213> Artificial sequence
<400> 5
ataggagtca cgacgaccag acggtaggga gcatcaaact tcagagtttg aacgttgaag 60
tatgtgcgtc tacctcttga 80
<210> 6
<211> 83
<212> DNA
<213> Artificial sequence
<400> 6
ataggagtca cgacgaccag aaaagtaatg cccggtagtt attcaaagat gagtaggaaa 60
agatatgtgc gtctacctct tga 83
<210> 7
<211> 82
<212> DNA
<213> Artificial sequence
<400> 7
ataggagtca cgacgaccag aaagtaatgc ccggtagtta ttcaacgatg agtaggaaaa 60
gatatgtgcg tctacctctt ga 82
<210> 8
<211> 20
<212> DNA
<213> Artificial sequence
<400> 8
<210> 9
<211> 20
<212> DNA
<213> Artificial sequence
<400> 9
tcaagaggta gacgcacata 20
<210> 10
<211> 13
<212> DNA
<213> Artificial sequence
<400> 10
tcgtgactcc tat 13
Claims (10)
1. An aptamer for specifically recognizing ribavirin, which is characterized by being represented by a) or b) as follows:
a) 1-SEQ ID NO: 7, or a nucleotide sequence represented by any one of SEQ ID NOs,
b) the nucleotide sequence which is derived from the a) and has the ability of specifically recognizing ribavirin and is obtained by substituting, deleting or adding one or more bases in the nucleotide sequence defined by the a).
2. The aptamer for specifically recognizing ribavirin according to claim 1, wherein the 5 'end or the 3' end of the aptamer is chemically modified with FITC, amino, biotin or digoxin.
3. A method for screening an aptamer which specifically recognizes ribavirin, comprising the steps of:
(1) preparation of aminated Fe3O4Magnetic nanobead, coated with avidin, and aminated Fe3O4Nano magnetic beads;
(2) constructing a random ssDNA library, wherein the length of an initial library is 80bp, and hybridizing the initial ssDNA library with complementary short-chain Bio-P1; the complementary short-chain Bio-P1 refers to biotin-labeled single-stranded DNA which can be complementary with ssDNA in the library;
(3) the library hybridized with the complementary short-chain Bio-P1 is immobilized on the avidin-coated aminated Fe through the action of biotin and avidin3O4The surface of the nanometer magnetic bead;
(4) the method for screening the ribavirin aptamers based on the Capure-SELEX method comprises the following steps: adding ribavirin into the library fixed on the surface of the magnetic beads and incubating, leaving ssDNA which is not combined with the target or is weakly combined on the magnetic beads, and allowing the aptamer combined with the target to enter a supernatant;
(5) and (3) carrying out high-throughput sequencing and analysis on the sequences obtained by enrichment to obtain 7 candidate sequences, and characterizing the affinity and specificity of the candidate aptamer by a gold nano colorimetric method and a carbon dot fluorescence quenching method to obtain the aptamer with affinity and specificity to ribavirin.
4. The method for screening aptamers capable of specifically recognizing ribavirin according to claim 3, wherein the aptamers are further enriched in the step (4) by cyclic screening, wherein the screening pressure is increased by reducing the concentration of the library, shortening the incubation time and increasing the back-screening substances in the cyclic screening process; during the reverse screening, before the target incubation is added, the structural analogue and the coexisting substance are added as a reverse screening substance to incubate with the aptamer, the aptamer sequence combined with the reverse screening substance enters into a supernatant and is removed, and the sequence not combined with the reverse screening substance is left on the magnetic beads to continue to incubate and enrich with the target ribavirin.
5. A kit for detecting ribavirin, which comprises the aptamer for specifically recognizing ribavirin according to claim 1 or 2.
6. The kit for detecting ribavirin according to claim 5, which further comprises a carbon dot solution or raw materials for preparing the carbon dot solution.
7. Use of an aptamer recognizing ribavirin as claimed in claim 1 or 2 for detecting ribavirin.
8. Use of the kit of claim 5 or 6 for the detection of ribavirin.
9. Use according to claim 7 or 8, characterized in that it is based on the carbon spot fluorescence quenching technique.
10. The use according to claim 9, for detecting the content of ribavirin in the bodies of, food and drugs for, and human blood of livestock animals.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110165104.8A CN112813071B (en) | 2021-02-06 | 2021-02-06 | Aptamer sequence for specifically recognizing ribavirin and application thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202110165104.8A CN112813071B (en) | 2021-02-06 | 2021-02-06 | Aptamer sequence for specifically recognizing ribavirin and application thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN112813071A true CN112813071A (en) | 2021-05-18 |
| CN112813071B CN112813071B (en) | 2022-09-27 |
Family
ID=75862008
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN202110165104.8A Active CN112813071B (en) | 2021-02-06 | 2021-02-06 | Aptamer sequence for specifically recognizing ribavirin and application thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN112813071B (en) |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114540359A (en) * | 2022-01-17 | 2022-05-27 | 江南大学 | Aptamer capable of specifically recognizing trimethoprim and screening and application thereof |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103031305A (en) * | 2011-09-30 | 2013-04-10 | 江南大学 | Screening and application of oligonucleotide aptamer for specific recognition of Salmonella Typhimurium |
| CN106834295A (en) * | 2017-03-21 | 2017-06-13 | 江南大学 | The wide spectrum aptamer and its directed screening method of a kind of specific recognition bacteria lipopolysaccharide |
-
2021
- 2021-02-06 CN CN202110165104.8A patent/CN112813071B/en active Active
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103031305A (en) * | 2011-09-30 | 2013-04-10 | 江南大学 | Screening and application of oligonucleotide aptamer for specific recognition of Salmonella Typhimurium |
| CN106834295A (en) * | 2017-03-21 | 2017-06-13 | 江南大学 | The wide spectrum aptamer and its directed screening method of a kind of specific recognition bacteria lipopolysaccharide |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN114540359A (en) * | 2022-01-17 | 2022-05-27 | 江南大学 | Aptamer capable of specifically recognizing trimethoprim and screening and application thereof |
Also Published As
| Publication number | Publication date |
|---|---|
| CN112813071B (en) | 2022-09-27 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| US20230045556A1 (en) | Nucleic acid complexes for screening barcoded compounds | |
| CN107541516B (en) | Nucleic acid aptamer group for specifically recognizing three marine toxins | |
| CN105886512B (en) | Oligonucleotide aptamer group for high-specificity recognition of clenbuterol hydrochloride, salbutamol and ractopamine | |
| CN107090457A (en) | Detect the multivariate analysis of sample | |
| CA2873099A1 (en) | Aptamer-based multiplexed assays | |
| CN106645050B (en) | It is a kind of for detecting the aptamers and its application of lactoferrin content | |
| CN108138244A (en) | Virus group capture microarray dataset, design and construction method and application method | |
| CN111662900B (en) | Sulfadimidine aptamer screening method, kit and application | |
| CN113215167A (en) | Aptamer and application thereof in detection of cells infected by iridovirus of micropterus salmoides | |
| CN112813071B (en) | Aptamer sequence for specifically recognizing ribavirin and application thereof | |
| CN110643611A (en) | Aptamer, construction method thereof and application thereof in detection of grouper iridovirus | |
| Pianowski et al. | Nucleic acid encoding to program self-assembly in chemical biology | |
| CN109628458B (en) | Oligonucleotide aptamer capable of specifically recognizing histamine and application thereof | |
| CN110129413A (en) | A kind of nano magnetic particle and its preparation method and application of selectivity capture and purifying microRNA | |
| TW201321520A (en) | Method and system for virus detection | |
| CN114457083A (en) | Single-stranded DNA (deoxyribonucleic acid) aptamer group for specifically recognizing malachite green and application thereof | |
| CN110819632B (en) | Aptamer for binding to trastuzumab | |
| CN109810980B (en) | Nucleic acid aptamer for specifically recognizing tyramine and application thereof | |
| CN112961859B (en) | Aptamer for specifically recognizing amantadine and application thereof | |
| WO2015151349A1 (en) | Soba-allergen-binding nucleic acid molecule and use therefor | |
| WO2015151350A1 (en) | Egg-allergen-binding nucleic acid molecule and use therefor | |
| CN111876423B (en) | Aptamer of nitrobenzene, aptamer derivative and application of aptamer derivative | |
| CN111363749A (en) | Aptamer for detecting Chinese softshell turtle iridovirus and construction method and application thereof | |
| WO2019200580A1 (en) | Parallel liquid-phase hybrid capture method for simultaneously capturing positive-sense and antisense strands of genomic target region | |
| CN108396029B (en) | A group of oligonucleotide aptamers capable of specifically recognizing Escherichia coli O157H 7 in different growth stages |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| PB01 | Publication | ||
| PB01 | Publication | ||
| SE01 | Entry into force of request for substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |