CN113073100A

CN113073100A - Netilmicin aptamer, screening and application in Netilmicin detection

Info

Publication number: CN113073100A
Application number: CN202110269596.5A
Authority: CN
Inventors: 王鲁梅; 潘超强; 沈国清; 耿雪青
Original assignee: Shanghai Jiaotong University
Current assignee: Zhejiang Shouxin Testing Co ltd
Priority date: 2021-03-12
Filing date: 2021-03-12
Publication date: 2021-07-06
Anticipated expiration: 2041-03-12
Also published as: CN113073100B

Abstract

The invention belongs to the technical field of chemical substance detection, and relates to a netilmicin aptamer, screening and application in netilmicin detection; specifically disclosed are a netilmicin aptamer, a method for screening a netilmicin aptamer, a biosensor for netilmicin detection, and a netilmicin detection method. Compared with the prior art, the detection method has the advantages of low detection limit, high sensitivity, strong specificity, visual rapid qualitative analysis, simple sample pretreatment, low instrument requirement and low cost. The detection method provided by the invention can be used for detecting the content of netilmicin in a water sample, and the detected concentration range is 1.95-200 nmol/L.

Description

Netilmicin aptamer, screening and application in Netilmicin detection

Technical Field

The invention belongs to the technical field of chemical substance detection, and particularly relates to a netilmicin aptamer, screening and application thereof in netilmicin detection.

Background

Netilmicin is a semi-synthetic aminoglycoside antibiotic, and has a chemical composition of 3-N-ethylsisomicin, and is white or off-white powder or loose block. The antibacterial action is basically similar to that of gentamicin, and the antibacterial agent is characterized by being stable to aminoglycoside acetyltransferase AAC (3) and having good antibacterial action on gram-negative bacteria and part of gram-positive bacteria. Because the adverse reaction is less than that of other similar products, the compound preparation has been applied to clinic at abroad and is successfully trial-produced at home, so that the compound preparation is used for preventing, treating and diagnosing diseases, and simultaneously purposefully regulating the physiological function of animals and promoting the growth and development. Is mainly used for treating respiratory tract and digestive tract diseases caused by Escherichia coli, Klebsiella pneumoniae, Proteus vulgaris and other bacteria in clinic and animals, and is also used for preventing secondary feeling caused by Postweaning Multisystemic Wasting Syndrome (PMWS) of various animals such as piglets and the like. The product is not metabolized in vivo, and the abnormal use of the product can cause the problem of excessive netilmicin residue in meat and milk products. Meanwhile, netilmicin has strong affinity with animal tissues, long residual time, severe ototoxicity, nephrotoxicity and neurotoxicity, and allergic reactions such as anaphylaxis, asthma and the like can be caused by inhalation. However, the therapeutic concentration and the toxic concentration of the drugs are very close, and the drugs are limited in clinical application and gather in human bodies and the environment, thereby harming the health of human bodies.

At present, methods used for analyzing netilmicin residue in the environment mainly comprise a microbiological method, a high performance liquid chromatography, a high performance capillary electrophoresis method, a thin layer chromatography, a derivatization ultraviolet spectrophotometry and the like. But the requirements for equipment, professional knowledge and detection time are higher, and the method is not suitable for rapid field detection. Most of the methods have complex sample processing, expensive analytical instruments and low detection sensitivity in some methods. Therefore, the limitations of the conventional analysis method for detecting netilmicin at present are difficult to realize the rapid, real-time, effective, simple and convenient qualitative and quantitative analysis of netilmicin, so that the development of a netilmicin detection technology with low cost, real-time, rapid, simple, convenient, easy, efficient, accurate and stable operation is a problem that needs to be solved urgently by the analysis workers at present.

Disclosure of Invention

Aiming at the defects of a netilmicin detection technology in the prior art, the invention provides a netilmicin aptamer, a screening method and an application in netilmicin detection, and particularly comprises the netilmicin aptamer, a screening method of the netilmicin aptamer, a biosensor for netilmicin detection and a netilmicin detection method.

The biosensor constructed by taking the aptamer as the core provides convenience for qualitative and quantitative analysis of netilmicin in a complex environment medium in the future.

The purpose of the invention is realized by the following technical scheme:

the invention provides a netilmicin aptamer, and the nucleotide sequence of the aptamer is shown in SEQ ID NO. 1.

SEQ ID NO.1：

5’-CTCCTCTGACTGTAACCACGCCCGTCAGTAGTGAGCTGCGTGCCAACATCACGCGGGCCGGCATAGGTAGTCCAGAAGCC-3’

The phenazine aptamer provided by the invention has the following characteristics:

(1) high affinity

The resulting aptamers have high affinity: 194.16 nM;

(2) high specificity

The aptamer exhibits good selectivity for netilmicin in the presence of a netilmicin analog.

The netilmicin aptamer provided by the invention is a nucleic acid aptamer which is obtained by screening based on a magnetic bead SELEX method and is combined with netilmicin with high affinity and high specificity.

The invention also provides a method for screening the netilmicin aptamer, which comprises the following steps:

coupling of S1, netilmicin and carboxyl magnetic beads:

1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride and N-hydroxysuccinimide are used for catalyzing the amidation reaction of the carboxyl magnetic bead and the amino of netilmicin under a 2- (N-morpholinyl) ethanesulfonic acid solution system to form an amido bond, so that the coupling and fixing of metamitron on the carboxyl magnetic bead is realized;

s2, adopting a symmetric PCR amplification technology, then adopting an enzymolysis method to obtain a secondary library, and obtaining the netilmicin aptamer through magnetic bead SELEX screening:

by adopting a symmetric PCR amplification technology, a PCR circulation program and the concentrations of the front primer and the rear primer are set, so that a large amount of target ssDNA products are amplified and enriched, then a secondary library required by a screening process is prepared by a lambda exonuclease through an enzymatic hydrolysis method, and an aptamer sequence combined with netilmicin with high affinity and high specificity is obtained through magnetic bead SELEX screening.

Preferably, in step S1, the coupling of netilmicin and carboxyl magnetic beads specifically comprises the following steps:

s11, dissolving the weighed 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride in 2- (N-morpholinyl) ethanesulfonic acid to obtain a solution a; weighing N-hydroxysuccinimide and dissolving the N-hydroxysuccinimide in 2- (N-morpholinyl) ethanesulfonic acid to obtain a solution b;

s12, adding the solution a and the solution b into the washed carboxyl magnetic beads, and uniformly stirring to obtain a carboxyl magnetic bead solution;

s13, weighing netilmicin sulfate, dissolving the netilmicin sulfate in 2- (N-morpholinyl) ethanesulfonic acid to obtain a netilmicin solution, mixing the netilmicin solution with a carboxyl magnetic bead solution, uniformly mixing, and reacting to obtain carboxyl magnetic beads coupled with the netilmicin.

In one embodiment of the present invention, in step S12, the magnetic beads are selected from the group consisting of Biotechnology (Shanghai) Inc. (Cat: D110550-0100).

In one embodiment of the present invention, in step S1, netilmicin, carboxyl magnetic beads, 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide, and 2- (N-morpholinyl) ethanesulfonic acid are used in an amount and in an operation such that a solution of 50mg/ml of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride in 2- (N-morpholinyl) ethanesulfonic acid (25mM) system is prepared. Meanwhile, a solution of 50mg/ml N, N-hydroxysuccinimide in a 2- (N-morpholinyl) ethanesulfonic acid (25mM) system is prepared, and the solution is sucked, beaten and mixed evenly. Respectively sucking 50 mu L of a solution of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride in a 2- (N-morpholinyl) ethanesulfonic acid system and 50 mu L N of a solution of N-hydroxysuccinimide in a 2- (N-morpholinyl) ethanesulfonic acid system, adding the mixture into the washed carboxyl magnetic beads, uniformly mixing, and stirring for 1-2h at 25 ℃. Weighing and dissolving netilmicin 1mg in 2- (N-morpholinyl) ethanesulfonic acid (25mM) 100 μ L respectively by an electronic analysis balance, sucking 100 μ L of the netilmicin solution by a pipette gun, adding the netilmicin solution into a carboxyl magnetic bead solution formed by mixing 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide, 2- (N-morpholinyl) ethanesulfonic acid and carboxyl magnetic beads, sucking, beating and uniformly mixing, placing in a shaking table, and setting the conditions of the shaking table as 100 and 200rpm, 25 ℃ and reacting for 12-24 h. After the reaction is finished, the supernatant is discarded, the magnetic beads are washed by 2- (N-morpholinyl) ethanesulfonic acid solution for 3-6 times, then the binding buffer solution is used for washing for 3-6 times, and finally the coupled carboxyl magnetic beads are suspended in 100 mu L of the binding buffer solution and are stored at 4 ℃ for later use by refrigeration.

Preferably, in step S2, the method for obtaining the netilmicin aptamer specifically includes:

s21, weighing the ssDNA library, adding a binding buffer solution, sucking, uniformly mixing, placing at 95 ℃ for denaturation for 10-15min, then placing on an ice block for 10-15min, and finally standing at room temperature for 5-10 min;

s22, mixing the ssDNA library processed in the step S21 with the washed carboxyl magnetic beads coupled with the naftidemicin for incubation reaction;

s23, after the incubation reaction is finished, taking out supernatant, cleaning and eluting the magnetic bead solution to enable the ssDNA to be eluted from the magnetic beads, and collecting eluent;

s24, concentrating and purifying the eluent, collecting ssDNA fragments, measuring the concentration of ssDNA in the recovered solution, and calculating the screening efficiency according to the recovered concentration/input concentration;

s25, taking the ssDNA fragment obtained in the step S24 as a template, carrying out symmetrical PCR amplification, carrying out gel electrophoresis on an amplification product, and continuing to carry out the next step if a correct target band appears; and the concentration of the PCR product is measured, the amount required by the next round of library is calculated, and the PCR product is put into the next round of screening circulation;

s26, converting double strands of the PCR product obtained in the step S25 into single strands by adopting lambda exonuclease, measuring the concentration, calculating the amount required by the library in the next round, and putting the library into the next round of screening circulation;

s27, repeating the steps S21-S26 to carry out multiple rounds of screening, and obtaining the netilmicin aptamer.

Preferably, in step S21, the ss DNA library: a single-stranded DNA synthesized by Biotechnology engineering (Shanghai) GmbH has the sequence: 5 '-CTCCTCTGACTGTAACCACG-N40-GCATAGGTAGTCCAGAAGCC-3'.

Preferably, in step S25, the PCR reaction system for symmetric PCR amplification comprises the following components in a total volume of 50 μ L: ssDNA template 2. mu.L, upstream primer 2. mu.L, downstream primer 2. mu.L, 2 XHiFiTaq PCR StarMix with Dye 25. mu.L and ultrapure water 19. mu.L;

the concentrations of the upstream primer and the downstream primer are respectively 10 mu mol/L;

the sequence of the upstream primer is as follows: 5'-CTCCTCTGACTGTAACCACG-3', the sequence of the downstream primer is as follows: 5'PO 4-GGCTTCTGGACTACCTATGC-3';

during the symmetric PCR amplification, the PCR cycle program is pre-denaturation at 94 ℃ for 2min, and the PCR cycle program comprises 30 cycles, denaturation at 94 ℃ for 30s, annealing at 52 ℃ for 30s, extension at 72 ℃ for 30s, and extension at 72 ℃ for 5min after the cycle is finished.

The invention also provides an application of the netilmicin aptamer, which comprises the following steps: the application of the netilmicin aptamer in preparing a netilmicin detection reagent, a netilmicin detection kit or a netilmicin detection biosensor; or

The application of the netilmicin aptamer in preparing a netilmicin capturing, separating or purifying reagent; or

The application of the netilmicin aptamer in developing a netilmicin detection method.

The invention also provides a biosensor for detecting netilmicin, which takes the netilmicin aptamer as a biological recognition element, and the nucleotide sequence of the netilmicin aptamer is shown as SEQ ID No. 1; the biosensor is used for detecting netilmicin by a fluorescence method based on SYBR Green I.

In one embodiment of the present invention, the biosensor is a fluorescence biosensor.

The invention also provides a netilmicin detection method based on the netilmicin aptamer, which comprises the following steps:

adding SYBR Green I solution into the netilmicin aptamer solution, uniformly mixing, and performing primary incubation to enable the SYBR Green I fluorescent probe to be embedded into a double chain formed by the netilmicin aptamer;

and adding a sample to be detected and a buffer solution into the mixed solution after the first incubation, mixing, incubating for the second time, measuring the fluorescence intensity of the mixed solution after the second incubation, and calculating to obtain the content of the netilmicin in the sample to be detected.

In one embodiment of the present invention, the netilmicin aptamer in step (a) is configured as a 500nM stock solution.

In one embodiment of the present invention, the SYBR Green I solution described in step (B) is formulated into a mother liquor with a concentration of 100 ×;

in one embodiment of the present invention, the 3-propanesulfonic acid buffer described in step (B) may be formulated using routine experimentation.

Performing gradient optimization on the final concentration of SYBR Green I and the netilmicin aptamer and the time of the SYBR Green I and the netilmicin aptamer in a detection system to obtain the optimal detection condition: the optimal final concentration of SYBR Green I is 0.8X, the optimal final concentration of aptamer is 20nM, and the optimal action time of SYBR Green I and netilmicin aptamer is 8 min.

Preferably, the SYBR Green I solution has a final concentration of 0.8X, and the netilmicin aptamer solution has a final concentration of 20 nM;

the pH value of the buffer solution is 7.0, and MOPS buffer solution is adopted;

the temperature of the first incubation is 25 ℃, and the incubation time is 30 min; the temperature of the second incubation is 25 ℃ and the incubation time is 8 min.

Preferably, the specific method for measuring and calculating the content of the netilmicin in the sample to be detected comprises the following steps: measuring fluorescence intensity F at an excitation wavelength of 485nm and an emission wavelength of 535 nm; and the fluorescence intensity F is measured by using the same operation without adding the aqueous solution of the netilmicin to be measured as a control group₀(ii) a Calculating F as F from fluorescence intensity₀And (4) judging the content of the netilmicin in the solution to be detected according to the size of the F.

The principle of the invention is that when SYBR Green I solution is added into the netilmicin aptamer solution, a SYBR Green I fluorescent probe is embedded into a double chain formed by the netilmicin aptamer, strong fluorescent intensity expression is generated at excitation wavelength 485nm and emission wavelength 535nm, when netilmicin exists in a system, the aptamer is combined with the netilmicin, the structure of the aptamer is changed, the double chain is opened into a single chain, so that the SYBR Green I fluorescent probe cannot be embedded into the aptamer, low fluorescent signal expression is caused, and the content of the netilmicin in the solution to be detected is judged by measuring the attenuation delta F of the fluorescent intensity.

The netilmicin detection method can solve the technical problems of high cost of machines, complex operation, long period, instability and the like in the netilmicin detection method in the prior art.

Compared with the prior art, the invention has the following beneficial effects:

1) among the various methods of signal expression, fluorescence is a simple, rapid, sensitive method of detection. The invention provides the method for detecting the netilmicin, which is simple to operate, high in sensitivity, good in selectivity, low in cost and high in efficiency.

2) The invention provides an aptamer of antibiotic netilmicin and a rapid detection method. The method comprises the steps of establishing a magnetic bead SELEX system, putting an initial aptamer library, performing incubation, separation, elution and PCR (polymerase chain reaction) circulation processes, obtaining a nucleic acid aptamer with high affinity to a target molecule netilmicin through multiple rounds of screening, and constructing a SYBR Green I fluorescence method based on the nucleic acid aptamer to realize the detection of netilmicin. The method comprises the steps of enabling an aptamer of netilmicin obtained through screening to act with an SYBR Green I fluorescent probe, generating strong fluorescent intensity expression at the excitation wavelength of 485nm and the emission wavelength of 535nm, enabling an aptamer to be combined with the netilmicin when the netilmicin exists in a system, enabling the structure of the aptamer to be changed, enabling double chains to be opened into single chains, and accordingly enabling the SYBR Green I fluorescent probe not to be embedded into the aptamer, and enabling the double chains to be low in fluorescent signal expression. The detection method has the advantages of low detection limit, high sensitivity, strong specificity, simple sample pretreatment, low instrument requirement and low cost.

3) Compared with the existing detection technology of netilmicin, the technical progress of the invention is remarkable. The detection method provided by the invention does not need large-scale instruments and equipment, is low in cost, high in detection sensitivity, good in selectivity, simple and efficient to operate, and can be used for detecting the content of netilmicin in a water sample, the detected concentration range is 1.95-200nmol/L, the linear fitting linear equation is Y-23.947X-79.677, and the minimum detection limit is 1.95 nM. In the linear equation, X represents the concentration (nM) of netilmicin in the test sample, and Y represents the decay Δ F of the fluorescence intensity of the sample.

Drawings

Other features, objects and advantages of the invention will become more apparent upon reading of the detailed description of non-limiting embodiments with reference to the following drawings:

FIG. 1: schematic diagram of principle of magnetic bead SELEX method for screening Netilmicin aptamer;

FIG. 2: a schematic diagram of a principle of detecting netilmicin based on an SYBR Green I fluorescence method;

FIG. 3: agarose gel electrophoresis picture of the symmetric PCR product;

FIG. 4: SELEX screening ssDNA recovery efficiency;

FIG. 5: determination of dissociation constant Kd value of netilmicin aptamer;

FIG. 6: a schematic diagram of the optimization result of the buffer solution;

FIG. 7: a schematic diagram of the optimization result of the action time of the SYBR Green I fluorescent dye and the aptamer;

FIG. 8: a schematic diagram of the concentration optimization result of SYBR Green I fluorescent dye;

FIG. 9: a schematic diagram of the concentration optimization results of the netilmicin aptamer;

FIG. 10: a schematic diagram of the sensitivity results of the netilmicin detection system;

FIG. 11: schematic diagram of specific results of netilmicin detection system.

Detailed Description

The present invention will be described in detail with reference to specific examples. The following examples will assist those skilled in the art in further understanding the invention, but are not intended to limit the invention in any way. It should be noted that variations and modifications can be made by persons skilled in the art without departing from the spirit of the invention. All falling within the scope of the present invention.

The invention firstly provides a method for screening metamitron aptamer by a magnetic bead SELEX method, a schematic diagram of the principle is shown in figure 1, and the method comprises the following steps:

(1) coupling of metamitron and carboxyl magnetic beads:

(2) adopting a symmetric PCR amplification technology and an enzymolysis method to obtain a secondary library, and obtaining an aptamer sequence which is combined with netilmicin with high affinity and high specificity through a magnetic bead SELEX screening process:

by adopting a symmetric PCR amplification technology, a PCR circulation program and the concentrations of the front primer and the rear primer are set, so that a large amount of target ssDNA products are amplified and enriched, then a secondary library required by a screening process is prepared by a lambda exonuclease through an enzymatic hydrolysis method, and an aptamer sequence combined with netilmicin with high affinity and high specificity is obtained through a magnetic bead SELEX screening process.

In one embodiment of the present invention, the method of step (1) comprises the steps of:

(1.1) taking the carboxyl magnetic beads, and carrying out ultrasonic cleaning on the carboxyl magnetic beads for multiple times by using 2- (N-morpholinyl) ethanesulfonic acid;

(1.2) weighing 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride to dissolve in 2- (N-morpholinyl) ethanesulfonic acid, and weighing N, N-hydroxysuccinimide to dissolve in 2- (N-morpholinyl) ethanesulfonic acid;

respectively sucking the solution of N, N-hydroxysuccinimide in a 2- (N-morpholinyl) ethanesulfonic acid system, adding the solution of N-hydroxysuccinimide in the 2- (N-morpholinyl) ethanesulfonic acid system into the washed carboxyl magnetic beads, uniformly mixing, and stirring to obtain a carboxyl magnetic bead solution in which 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide, 2- (N-morpholinyl) ethanesulfonic acid and the carboxyl magnetic beads are mixed;

weighing netilmicin and dissolving in 2- (N-morpholinyl) ethanesulfonic acid to obtain netilmicin solution, sucking the netilmicin solution, adding into 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide, 2- (N-morpholinyl) ethanesulfonic acid and carboxyl magnetic bead solution mixed with carboxyl magnetic beads, sucking, beating and uniformly mixing, reacting to obtain carboxyl magnetic beads coupled with netilmicin after the reaction is finished, removing supernatant, washing the carboxyl magnetic beads coupled with the netilmicin for 3-6 times by using 2- (N-morpholinyl) ethanesulfonic acid solution, then washing for 3-6 times by using binding buffer solution, finally suspending the carboxyl magnetic beads coupled with the netilmicin in the binding buffer solution, and refrigerating and storing for later use.

In one embodiment of the present invention, in step (1.2), the reaction conditions are: 100-200rpm, 25-30 ℃, and reacting for 12-24 h.

In one embodiment of the present invention, the step (2) of obtaining the aptamer sequence with high affinity and high specificity binding to netilmicin by using a symmetric PCR amplification technology and a lambda exonuclease through a magnetic bead SELEX screening process comprises the following steps:

(2.1) weighing the ssDNA library, adding a binding buffer solution, sucking, uniformly mixing, placing at 95 ℃ for denaturation for 10-15min, then quickly transferring on an ice block for 10-15min, and finally standing at room temperature of 25 ℃ for 5-10 min;

(2.2) weighing the carboxyl magnetic beads coupled with the netilmicin, adding a binding buffer solution, and washing the carboxyl magnetic bead solution coupled with the netilmicin for multiple times by using the binding buffer solution;

(2.3) mixing the ssDNA library subjected to the pretreatment in the steps (2.2) and (2.3) with a pretreated carboxyl magnetic bead solution coupled with netilmicin, and incubating;

(2.4) after the incubation is finished, taking out supernatant, adding a binding buffer solution, and washing the obtained magnetic bead solution;

(2.5) adding an elution buffer solution into the washed magnetic bead solution, eluting to enable the ssDNA to be eluted from the magnetic beads, and collecting an eluent;

(2.6) concentrating, purifying and collecting ssDNA in the obtained eluent by adopting a UNIQ-10 column type universal DNA purification kit:

add 4 volumes of absolute ethanol to a Wash Solution bottle. Mu.l of Binding Buffer II was added to 100. mu.l of the DNA solution (i.e., the eluate), mixed well, and left for 2 minutes. The whole was transferred to a UNIQ-10 column, and the column was placed in 2.0ml Collection Tube, left at room temperature for 2 minutes, and centrifuged at 10,000rpm at room temperature for 1 minute by a desk centrifuge. The UNIQ-10 column was removed and the waste liquid in the centrifuge tube was discarded. The column was returned to the same centrifuge tube and 500. mu.l of Wash Solution was added and centrifuged at 10,000rpm for 30 seconds at room temperature. And repeating the steps once. The UNIQ-10 column was removed and all waste liquid in the centrifuge tube was discarded. The column was returned to the same centrifuge tube and centrifuged at 10,000rpm for 30 seconds at room temperature to remove residual Wash Solution. The column is placed into a new clean 1.5ml or 2.0ml centrifuge tube, 100 mul of Elution Buffer is added into the center of the column, the column is placed for 2 minutes at room temperature, and the temperature of the eluent is increased to 55-80 ℃, which is beneficial to improving the Elution efficiency of DNA. Centrifuge at 10,000rpm for 1 minute at room temperature. The liquid in the collection tube is the recovered ssDNA fragments.

(2.7) measuring the ssDNA concentration in the recovered solution by using an ultramicro spectrophotometer, and calculating the screening efficiency, wherein the screening efficiency is calculated by using the recovered concentration/input concentration;

and (2.8) carrying out symmetric PCR amplification by using the purified ssDNA solution as a template, then carrying out 3% agarose gel electrophoresis on the PCR reaction solution, placing the PCR reaction solution under a gel imaging analysis system for observation, and continuing the next step if a correct target band appears. Calculating the amount required as a library of the next round by measuring the concentration of the PCR product, and putting the library into the next round of screening circulation;

(2.9) converting double strands into single strands by using lambda exonuclease, measuring the concentration, calculating the amount required by the library in the next round, and putting the library into the next round of screening circulation;

(2.10) repeating the steps (2.1) to (2.9), and obtaining the netilmicin aptamer through multiple rounds of sample sending and sequencing.

In one embodiment of the present invention, the ssDNA library of step (2.1): a single-stranded DNA synthesized by Biotechnology engineering (Shanghai) GmbH has the sequence:

5’-CTCCTCTGACTGTAACCACG-N40-GCATAGGTA GTCCAGAAGCC-3’

in one embodiment of the present invention, the binding buffer described in steps (2.1) and (2.2) is prepared by: accurately weighing 1.461g of NaCl, 0.788g of Tris-HCl and MgCl₂·6H₂O 0.102g、KCl 0.093g、CaCl₂0.028g in a beaker, dissolve with 100mL of ultrapure water, pipette Tween 2050. mu.L addition, then add ultrapure water to 220mL, adjust pH to 7.6 with NaOH solution to replenish solution to 250 mL; or the solution prepared in equal proportion is adopted.

In one embodiment of the present invention, the elution buffer described in step (2.5) is prepared by: accurately weighing Tris-HCl 0.631g and EDTA.2Na. 2H₂Adding 0.372g of O and 21.02g of Urea into a beaker, dissolving by adding 50mL of ultrapure water, sucking 2020 mu L of Tween by a liquid-transferring gun, adding the ultrapure water to 80mL, and adjusting the pH to 8.0 by using NaOH to supplement the solution to 100 mL; or the solution prepared in equal proportion is adopted.

In one embodiment of the present invention, the reaction system of the PCR reaction in step (2.8) is prepared as follows:

weighing a PCR reaction system to be 50 mu L; the concentration of the upstream primer and the downstream primer is 10. mu. mol/L. The reaction system of the PCR is ssDNA template 2 uL, upstream primer 2 uL, downstream primer 2 uL, 2 xHiFiTaq PCR StarMix with Dye 25 uL and ultrapure water 19 uL;

in one embodiment of the present invention, in step (2.8), when symmetric PCR amplification is performed, the upstream primer sequence: 5'-CTCCTCTGACTGTAACCACG-3', the sequence is shown in SEQ ID NO.2, the sequence of the downstream primer is as follows: 5'PO4-GGCTTCTGGACTACCTATGC-3' with the sequence shown in SEQ ID No. 3.

In one embodiment of the present invention, in step (2.8), when symmetric PCR amplification is performed, the PCR cycling program is pre-denaturation at 94 ℃ for 2min, 30 cycles of pre-denaturation at 94 ℃ for 30s, annealing at 52 ℃ for 30s, and extension at 72 ℃ for 5min after the end of the cycles.

The invention also provides application of the netilmicin aptamer, which comprises application of the netilmicin aptamer in preparation of a netilmicin detection reagent, a netilmicin detection kit or a netilmicin detection biosensor.

The invention also provides application of the netilmicin aptamer, which comprises application of the netilmicin aptamer in preparation of a netilmicin capture, separation or purification reagent.

The invention also provides application of the netilmicin aptamer, which comprises application of the netilmicin aptamer in development of a netilmicin detection method.

The invention also provides a biosensor for detecting netilmicin, which takes the netilmicin aptamer as a biological recognition element, and the nucleotide sequence of the netilmicin aptamer is shown as SEQ ID No. 1. The method specifically comprises the following steps:

5’-CTCCTCTGACTGTAACCACGCCCGTCAGTAGTGAGCTGCGTGCCAACATCACGCGGGCCGGCATAGGTAGTCCAGAAGCC-3’。

The invention also provides a netilmicin detection method based on the netilmicin aptamer. The netilmicin detection method can solve the technical problems of high cost of machines, complex operation, long period, instability and the like in the netilmicin detection method in the prior art.

The invention discloses a netilmicin detection method, which comprises the following steps:

A. preparing a metamitron aptamer, wherein the nucleotide sequence of the metamitron aptamer is shown in SEQ ID No. 1;

B. adding SYBR Green I solution into a certain amount of netilmicin aptamer solution, uniformly mixing and incubating to ensure that a SYBR Green I fluorescent probe is embedded into a double chain formed by the netilmicin aptamer, adding aqueous solution of netilmicin to be detected and 3-propanesulfonic acid buffer solution, uniformly mixing and incubating, measuring fluorescence intensity F at the excitation wavelength of 485nm and the emission wavelength of 535nm, taking the aqueous solution of netilmicin to be detected which is not added in other operations as a control group, and measuring the fluorescence intensity F₀Calculating F from the fluorescence intensity₀And (4) judging the content of the netilmicin in the solution to be detected according to the size of the F.

A schematic diagram of the principle of detecting netilmicin based on SYBR Green I fluorescence is shown in FIG. 2.

The invention is described in detail below with reference to the figures and specific embodiments.

Example 1

The present embodiment provides a method for screening and obtaining a netilmicin aptamer, comprising the following steps:

A. pipette 100. mu.L of magnetic carboxyl beads into a 1.5mL centrifuge tube, place on a magnetic separation rack, stand for 2min, carefully pipette the supernatant and discard. Then the centrifuge tube was removed, 100. mu.L of ultrapure water was added, the mixture was pipetted and mixed, placed on a magnetic separation rack, allowed to stand for 2min, and the supernatant was carefully aspirated and discarded. 100 μ L of 2- (N-morpholino) ethanesulfonic acid solution was added to the centrifuge tube, the mixture was pipetted and mixed, and the procedure was repeated, and the magnetic beads were washed with 2- (N-morpholino) ethanesulfonic acid for 5 times, and after 5 washes, the supernatant was carefully aspirated and discarded.

B. 10mg of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride was weighed out and dissolved in 1ml of 2- (N-morpholinyl) ethanesulfonic acid (25 mM). At the same time, 50mg of N, N-hydroxysuccinimide was dissolved in 1ml of 2- (N-morpholino) ethanesulfonic acid (25mM), and the solution was pipetted and mixed well. Respectively sucking 50 mu L of the solution of 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride in the 2- (N-morpholinyl) ethanesulfonic acid system and 50 mu L N of the solution of N-hydroxysuccinimide in the 2- (N-morpholinyl) ethanesulfonic acid system, adding the mixture into the washed carboxyl magnetic beads in the step A, uniformly mixing, and stirring for 1-2h at 25 ℃. Weighing 1mg of netilmicin dissolved in 100 mu L of 2- (N-morpholinyl) ethanesulfonic acid (25mM) by an electronic analysis balance, sucking 100 mu L of netilmicin solution in a 2- (N-morpholinyl) ethanesulfonic acid system by a pipette, adding the netilmicin solution into 1- (3-dimethylaminopropyl) -3-ethylcarbodiimide hydrochloride, N-hydroxysuccinimide, 2- (N-morpholinyl) ethanesulfonic acid and carboxyl magnetic bead solution mixed with carboxyl magnetic beads, sucking and uniformly mixing, placing in a shaking table, setting the conditions of the shaking table at 160rpm and 25 ℃, and reacting for 24 hours. After the reaction is finished, the supernatant is discarded, the magnetic beads are washed by 2- (N-morpholinyl) ethanesulfonic acid solution for 3-6 times, then the binding buffer solution is used for washing for 3-6 times, and finally the coupled carboxyl magnetic beads are suspended in 100 mu L of the binding buffer solution and are stored at 4 ℃ for later use by refrigeration.

C. The pipette aspirates 600pmol of ssDNA library into a 1.5mL centrifuge tube, adds 200. mu.L of binding buffer, and aspirates the mixture to mix. Placing at 95 deg.C for denaturation for 10min, rapidly transferring on ice for 10min, and standing at 25 deg.C for 10 min.

D. And (3) sucking 100 mu L of carboxyl magnetic beads coupled with netilmicin (prepared in the step B) into a 1.5mL centrifuge tube, adding 200 mu L of binding buffer solution, sucking, uniformly mixing, placing on a magnetic separation rack, standing for 2min, and carefully sucking the supernatant. The bead solution was washed with binding buffer 5 repeatedly, and the supernatant was discarded in the last step.

E. And D, mixing the ssDNA library pretreated in the step C with the solution of the magnetic beads coated with the target substance netilmicin pretreated in the step D, and then placing the mixture on a shaking table to incubate for 1h, wherein the conditions of the shaking table are 160rpm and 25 ℃.

F. After incubation, the supernatant was carefully aspirated. Subsequently, 200. mu.L of binding buffer was added, and the obtained magnetic bead solution was washed 5 times by passing through a magnetic separation rack, and finally, the binding buffer was discarded.

G. And F, adding 200 mu L of elution buffer solution into the magnetic bead solution (namely the screening system) obtained in the step F, eluting under the water bath condition of 80 ℃ to enable the ssDNA to be eluted from the magnetic beads, placing the centrifugal tube on a magnetic separation frame, and collecting the eluent.

H. Repeat step G4 times, collect the eluates, and combine all eluates.

I. And (3) concentrating, purifying and collecting ssDNA in the obtained eluent by adopting a UNIQ-10 column type universal DNA purification kit:

add 4 volumes of absolute ethanol to a Wash Solution bottle. Mu.l of the DNA solution (the eluate obtained in step H) was added to 300. mu.l of Binding Buffer II, mixed well and left for 2 minutes. The whole was transferred to a UNIQ-10 column, and the column was placed in 2.0ml Collection Tube, left at room temperature for 2 minutes, and centrifuged at 10,000rpm at room temperature for 1 minute by a desk centrifuge. The UNIQ-10 column was removed and the waste liquid in the centrifuge tube was discarded. The column was returned to the same centrifuge tube and 500. mu.l of Wash Solution was added and centrifuged at 10,000rpm for 30 seconds at room temperature. And repeating the steps once. The UNIQ-10 column was removed and all waste liquid in the centrifuge tube was discarded. The column was returned to the same centrifuge tube and centrifuged at 10,000rpm for 30 seconds at room temperature to remove residual Wash Solution. The column is placed into a new clean 1.5ml or 2.0ml centrifuge tube, 100 mul of Elution Buffer is added into the center of the column, the column is placed for 2 minutes at room temperature, and the temperature of the eluent is increased to 55-80 ℃, which is beneficial to improving the Elution efficiency of DNA. Centrifuge at 10,000rpm for 1 minute at room temperature. The liquid in the collection tube is the recovered ssDNA fragments.

J. The ssDNA concentration in the recovered solution was measured by a NanoDrop 2000 ultramicro spectrophotometer, and the screening efficiency (recovered concentration/input concentration) was calculated.

K. And (3) carrying out symmetric PCR amplification by taking the purified ssDNA solution as a template, then carrying out 3% agarose gel electrophoresis on 5 mu L of PCR reaction solution, placing the PCR reaction solution under a gel imaging analysis system for observation, and entering subsequent single-strand preparation if a correct target band appears.

L, converting ssDNA double strands into single strands by adopting lambda exonuclease, measuring the concentration, calculating the amount required by the library in the next round, and putting the required amount into the next round of screening circulation; the reaction system and procedure were as follows:

total volume 50 μ L: 10 Xreaction Buffer 5. mu.L,. lambda.exonuclease 10U (1. mu.L), PCR product 44. mu.L, Reaction at 37 ℃ for 30min, Reaction at 80 ℃ for 10min to terminate the Reaction.

Repeating steps C-L until the aptamer binding with high affinity to netilmicin is obtained by screening.

The ssDNA library of step C: for the purpose of synthetic single-stranded DNA, its sequence is: 5 '-CTCCTCTGACTGTAACCACG-N40-GCATAGGTA GTCCAGAAGCC-3'.

The preparation method of the binding buffer solution in the step D comprises the following steps: accurately weighing 1.461g of NaCl, 0.788g of Tris-HCl and MgCl₂·6H2O 0.102g、KCl 0.093g、CaCl₂0.028g in a beaker, dissolve with 100mL of ultrapure water, pipette tips into Tween 2050. mu.L addition, then add ultrapure water to 220mL, adjust pH to 7.6 with NaOH solution to replenish solution to 250 mL.

The preparation method of the elution buffer solution in the step G comprises the following steps: accurately weighing Tris-HCl 0.631g and EDTA.2Na. 2H₂O0.372 g, Urea 21.02g in a beaker, dissolved by adding 50mL of ultrapure water, pipette tips pipette 2020 μ L of Tween addition followed by adding ultrapure water to 80mL, adjusting the pH to 8.0 with NaOH to replenish the solution to 100 mL.

The symmetrical PCR reaction system of the step K is 50 mu L; the concentration of the upstream primer and the downstream primer is 10. mu. mol/L. The reaction system of the PCR is as follows: ssDNA template 2. mu.L, upstream primer 2. mu.L, downstream primer 2. mu.L, 2 XHiFiTaq PCR StarMix with Dye 25. mu.L and ultrapure water 19. mu.L; the sequence of the upstream primer is as follows: 5'-CTCCTCTGACTGTAACCACG-3', downstream primer sequence: 5'PO 4-GGCTTCTGGACTACCTATGC-3';

when asymmetric PCR amplification is further carried out, the PCR cycle program is pre-denaturation at 94 ℃ for 2min, and the PCR cycle program comprises 30 cycles of denaturation at 94 ℃ for 30s, annealing at 52 ℃ for 30s, and extension at 72 ℃ for 5min after the cycle is finished.

In this example, the agarose gel electrophoresis pattern of the symmetric PCR product is shown in FIG. 3. In this example, the efficiency of recovery of screened ssDNA by SELEX is shown in fig. 4, and the determination of the dissociation constant Kd value of netilmicin aptamer is shown in fig. 5.

The aptamer obtained by the magnetic bead SELEX technology has the base sequence of 5'-CTCCTCTGACTGTAACCACGCCCGTCAGTAGTGAGCTGCGTGCCAACATCACGCGGGCCGGCATAGGTAGTCCAGAAGCC-3'.

The aptamer has the following characteristics:

(1) high affinity

The resulting aptamers have high affinity: 194.16 nM;

(2) high specificity

In this embodiment, the netilmicin aptamer is a functional nucleotide sequence synthesized by bio-engineering (shanghai) gmbh, and the synthesized aptamer is centrifuged, added to the ultrapure water in the synthesis report, shaken and mixed to prepare a 500nM mother liquor.

Example 2

The present embodiment provides a netilmicin detection method, including the following steps:

A. the Netilmicin aptamer obtained in example 1 was used to prepare a 100nM stock solution.

B. Adding SYBR Green I solution into a certain amount of netilmicin aptamer solution, uniformly mixing and incubating to ensure that a SYBR Green I fluorescent probe is embedded into a double chain formed by the netilmicin aptamer, adding aqueous solution of netilmicin to be detected and 3-propanesulfonic acid buffer solution, uniformly mixing and incubating, and measuring fluorescence intensity F at an excitation wavelength of 485nm and an emission wavelength of 535 nm; and using the aqueous solution without the netilmicin to be detected as the control group to determine the fluorescence intensity F₀(ii) a Calculating F as F from fluorescence intensity₀And (4) judging the content of the netilmicin in the solution to be detected according to the size of the F.

In this embodiment, the SYBR Green I described in step (B) is prepared into a mother liquor with a concentration of 100X;

the preparation method of the 3-propanesulfonic acid buffer solution in the step (B) comprises the following steps: 1.0463g of 3-morpholinopropanesulfonic acid solid was weighed out accurately into a beaker, dissolved by adding 400mL of ultrapure water, the pH of the buffer was adjusted to 7.0 using 1M NaOH solution, and then transferred to a 100mL volumetric flask and made to volume. Placing in a jar with a stopper, and storing at normal temperature.

Further, the preparation scheme of the 1M NaOH solution is as follows: weighing 4.000g of sodium hydroxide solid in a beaker, adding ultrapure water for dissolving, transferring the solution in a 100mL volumetric flask and fixing the volume to obtain 1M sodium hydroxide solution, and storing the solution at normal temperature.

In this embodiment, the method for specifically detecting netilmicin further includes the following steps:

(1) first, a pipette gun sucks 100 μ L of aptamer solution (100nM) into a 1.5mL centrifuge tube, 100 μ L of netilmicin-containing solution to be detected is added to make the final concentration of netilmicin in the system be below 200nM, the solution is uniformly sucked and beaten, and then 295 μ L of 3-propanesulfonic acid buffer solution (pH 7) is added, and the mixture is placed in a constant temperature incubator at 25 ℃ for reaction for 30 min. Then, 5. mu.L of 100 XSYBR Green I solution was added, pipetted and mixed, and incubated at 25 ℃ for 8 min. Measuring fluorescence intensity F at an excitation wavelength of 485nm and an emission wavelength of 535 nm; and using the aqueous solution without the netilmicin to be detected as the control group to determine the fluorescence intensity F₀(ii) a Calculating F as F from fluorescence intensity₀And (4) judging the content of the netilmicin in the solution to be detected according to the size of the F.

(2) And (4) looking up a standard curve according to the calculated delta F value to obtain the content of the netilmicin in the sample.

The fluorescence decay Δ F is directly proportional to the amount of netilmicin at a given concentration.

Example 3

Based on the method of example 2, this example optimizes the buffer type of the system

A test group (F) in which 1. mu.M of netilmicin was present and a blank group (F0) in which netilmicin was not present were set, adding 20nM ofloxacin aptamer solution into a 1.5mL centrifuge tube, respectively selecting 10mM MOPS buffer solution with pH 7.0, 10mM PBS buffer solution with pH 7.4, 50mM Tris-HCl buffer solution with pH 7.4 and ultrapure water ddH2O as buffer systems, making up the volume of the system, incubating for 30 minutes at constant temperature, adding 1x SYBR Green I fluorescent dye, incubating for 10 minutes, sampling in a 96-well micro black enzyme plate, measuring the fluorescence intensity by using an Infinite M200 Pro enzyme labeling instrument to obtain F and F0, calculating to obtain delta F, taking the average value of the fluorescence intensity difference delta F after repeating three experiments as a vertical coordinate, and taking the buffer system buffer solution name as a horizontal coordinate graph, and selecting the corresponding buffer solution (MOPS) at the maximum delta F value as the buffer solution of the reaction system. As shown in fig. 6.

Example 4

Based on the method of example 2, the present example optimizes the reaction time of SYBR Green I fluorescent dye and aptamer of the system

Adding an aptamer solution with the final concentration of 20nM and SYBR Green I fluorescent dye with the concentration level of 1x into a 1.5mL centrifuge tube, supplementing the volume of the system with 3-propanesulfonic acid buffer solution to 500 mu L, incubating at the constant temperature of 25 ℃, setting the incubation Time to be 2,4,6,8,10 and 12 minutes, immediately sampling 200 mu L, measuring the fluorescence intensity F in a 96-hole micro black ELISA plate by using an Infinite M200 Pro ELISA reader, repeating the experiment for three times, drawing by taking the average value of the obtained fluorescence intensity values F as a vertical coordinate and the reaction Time 1 as a horizontal coordinate, and selecting the reaction Time (8min) when the fluorescence intensity value reaches the maximum stability as the optimal reaction Time 1. As shown in fig. 7.

Example 5

Based on the method of example 2, this example optimizes the concentration of SYBR Green I fluorescent dye in the system

For the optimization of the concentration of the fluorescent dye SYBR Green I, firstly setting a test group (F) with 1 mu M netilmicin and a blank group (F0) with ultrapure water replacing netilmicin, respectively adding 20nM netilmicin aptamer solution into a 1.5mL centrifuge tube, supplementing the system volume with an optimal buffer solution (MOPS buffer solution), incubating for 30 minutes at constant temperature, then adding 1,2,3,4,5,10 mu L SYBR Green I fluorescent dye with 100x concentration levels, respectively making the final concentration levels of the SYBR Green I fluorescent dye be 0.2x,0.4x,0.6x,0.8x,1x,2x, and continuing incubating for 8 minutes, sampling in a 96-hole micro black enzyme labeled plate, measuring the fluorescence intensity by using an Infinite M200 Pro enzyme labeled meter, obtaining F and F0 and calculating to obtain delta F, drawing the average value of the difference delta F calculated after three times of experiments as a vertical coordinate, and the SYBR Green I concentration of the fluorescent dye as a horizontal coordinate, the corresponding concentration level (0.8x) at the maximum af value was selected as the optimal concentration of the fluorescent dye SYBR Green I. As shown in fig. 8.

Example 6

Based on the method of example 2, this example optimizes the concentration of the netilmicin aptamer in the system

For the optimization of the concentration of the netilmicin aptamer solution, a test group (F) with 1 μ M netilmicin and a blank group (F0) with ultrapure water instead of netilmicin are also set, 500nM

ofloxacin aptamer solution

0,5,10,15,20,25,30,35,50 μ L is added into a 1.5mL centrifuge tube respectively to make the

final concentration reach

0,5,10,15,20,25,30,35,50nM respectively, the optimal buffer solution (MOPS buffer solution) is used to complement the system volume and incubate for 30 minutes at constant temperature, then the optimal concentration SYBR Green I fluorescent dye is added and incubate for 8 minutes continuously, the fluorescence intensity is measured by an Infinite M200 Pro enzyme linked immunosorbent assay instrument in a 96-hole micro black enzyme linked plate, F and F0 are obtained and Δ F is calculated, the average value of the fluorescence intensity difference Δ F calculated after three times is taken as a vertical coordinate, the concentration of aptamer solutions was plotted on the abscissa and the corresponding concentration value (20nM) at the maximum af value was selected as the optimal concentration for the ofloxacin aptamer solution. As shown in fig. 9.

Example 7

The embodiment provides a method for detecting netilmicin by using an SYBR Green I fluorescence method, which comprises the following steps:

(1) first, 20nM aptamer solution and netilmicin at a concentration of 0,5,10,20,40,80,120,160,200,400,600,800,1000,2000nM were mixed with a certain amount of MOPS buffer solution with pH 7 and shaken in a 1.5mL centrifuge tube, and the reaction was incubated at 25 ℃ for 30 minutes. Then, 0.8X SYBR Green I fluorescent dye was added and incubation continued for 8 minutes, and samples were taken in 96-well microtiter black plate and fluorescence intensity was measured with Infinite M200 Pro microplate reader to obtain F and F0 and calculate Δ F.

(2) Standard curves were plotted with different concentrations of netilmicin plotted against the corresponding enhanced Δ F. As shown in fig. 10.

(3) Preparing a sample detection system: and (3) adding 100 mu L of sample to be detected into the detection system centrifugal tube prepared by the method in the step (1) to replace the netilmicin solution in the step (1). After treatment as described above,. DELTA.F was measured.

(4) And (4) looking up a standard curve according to the calculated delta F value to obtain the content of the netilmicin in the sample.

(5) And (3) verification: the method of the invention is used for measuring each part of water samples (tap water and lake water) containing netilmicin with the concentration of 5nmol/L, 120nmol/L and 200nmol/L respectively, and the obtained average recovery rate ranges from 97% to 111%, thereby proving the reliability of the method. As shown in tables 1 and 2.

The concentration range of the metamitron in the water sample measured by the method is 1.95-200nmol/L, the linear fitting linear equation is Y-23.947X-79.677, and the lowest detection limit is 1.95 nM. In the linear equation, X represents the concentration (nM) of netilmicin in the sample to be tested, Y represents the ratio of Δ F in the sample, and the lowest detection limit is 1.95 nmol/L.

TABLE 1 spiking recovery in ultrapure water samples

TABLE 2 recovery of spiked tap water samples

Example 8

This example provides a specificity assay for netilmicin

In the study of system specificity, the test uses a mixture of various different classes of interferents and a compound containing netilmicin instead of netilmicin; a blank set was set up and ultrapure water was used in place of various materials. In an optimized detection system, 1 mu M netilmicin or other different interferents is added into the system, firstly, the netilmicin or is evenly vibrated and mixed with 20nM ofloxacin aptamer and a proper amount of MOPS buffer solution, the incubation is carried out for 30 minutes, SYBR Green I fluorescent dye with an optimized concentration level is added, the reaction is carried out for 8 minutes, the fluorescence intensity is. The average value of fluorescence intensity difference delta F obtained after repeating the steps for three times is taken as a vertical coordinate, the names of various substances are plotted as horizontal coordinates, and the specificity of the detection system is judged by comparing and observing the delta F. As shown in fig. 11.

The embodiments described above are described to facilitate an understanding and use of the invention by those skilled in the art. It will be readily apparent to those skilled in the art that various modifications to these embodiments may be made, and the generic principles described herein may be applied to other embodiments without the use of the inventive faculty. Therefore, the present invention is not limited to the above embodiments, and those skilled in the art should make improvements and modifications within the scope of the present invention based on the disclosure of the present invention.

Sequence listing

<110> Shanghai university of transportation

<120> Netilmicin aptamer, screening and application in Netilmicin detection

<130> KAG45940

<160> 3

<170> SIPOSequenceListing 1.0

<210> 1

<211> 80

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

ctcctctgac tgtaaccacg cccgtcagta gtgagctgcg tgccaacatc acgcgggccg 60

gcataggtag tccagaagcc 80

<210> 2

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 2

ctcctctgac tgtaaccacg 20

<210> 3

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

ggcttctgga ctacctatgc 20

Claims

1. The netilmicin aptamer is characterized in that the nucleotide sequence of the aptamer is shown in SEQ ID NO. 1.

2. A method for screening a netilmicin aptamer, comprising the following steps:

coupling of S1, netilmicin and carboxyl magnetic beads:

3. The method for screening the netilmicin aptamer according to claim 2, wherein in step S1, the coupling of netilmicin to the carboxyl magnetic beads comprises the following steps:

4. The method for screening the netilmicin aptamer according to claim 2, wherein in step S2, the netilmicin aptamer is obtained by a method comprising:

5. The method for screening Netilmicin aptamers according to claim 4, wherein in step S21, the ss DNA library: is single-stranded DNA with the sequence: 5 '-CTCCTCTGACTGTAACCACG-N40-GCATAGGTA GTCCAGAAGCC-3'.

6. The method for screening Netilmicin aptamer according to claim 4, wherein in step S25, the PCR reaction system used for the symmetric PCR amplification comprises the following components in a total volume of 50 μ L: ssDNA template 2. mu.L, upstream primer 2. mu.L, downstream primer 2. mu.L, 2 XHiFiTaq PCR StarMix with Dye 25. mu.L and ultrapure water 19. mu.L;

7. Use of the netilmicin aptamer according to claim 1, comprising: the application of the netilmicin aptamer in preparing a netilmicin detection reagent, a netilmicin detection kit or a netilmicin detection biosensor; or

8. The biosensor for detecting the netilmicin is characterized in that the biosensor takes a netilmicin aptamer as a biological recognition element, and the nucleotide sequence of the netilmicin aptamer is shown as SEQ ID No. 1; the biosensor is used for detecting metamitron by a SYBR Green I-based fluorescence method.

9. A netilmicin assay based on the netilmicin aptamer of claim 1, comprising the steps of:

10. The netilmicin assay as claimed in claim 9, wherein the SYBR Green I solution has a final concentration of 0.8X, the netilmicin aptamer solution has a final concentration of 20 nM;