KR101558474B1 - Nucleic acid aptamer specifically binding to tetracycline compound - Google Patents

Abstract

The present invention relates to a nucleic acid aptamer which specifically binds to a tetracycline compound. The present invention also relates to a method for detecting a tetracycline compound using the nucleic acid aptamer, a composition for detecting a tetracycline compound including the nucleic acid aptamer, and a detection kit. The aptamer according to the present invention specifically binds to a common backbone of a tetracycline compound to show a high affinity to a tetracycline compound. Thus, it is possible to effectively detect residual antibiotic substances from foods, up and down water sources, .

Description

[0001] The present invention relates to a nucleic acid aptamer specifically binding to a tetracycline compound,

The present invention relates to a nucleic acid aptamer which specifically binds to a tetracycline compound.

The present invention also relates to a method for detecting a tetracycline compound using the nucleic acid aptamer, a composition for detecting a tetracycline compound including the nucleic acid aptamer, and a detection kit.

Tetracycline (TC) antibiotics are currently the most widely used antibiotics. These antibiotics are human and veterinary medicines. They are generally used in the treatment of infections caused by bacteria and they are injected directly into the animals in the form of feed or directly administered to prevent infection of fish or marine products. In particular, OTC (oxytetracycline) is the most commonly used antibiotic because it is known to have a higher rate of absorption than TC (tetracycline).

However, residual tetracycline or its analogue present in animal feed residue or excreta is now absorbed in soil or water, and can be affected by various pathways in the environment, which is problematic. In particular, tetracycline residues in honey, milk, and eggs have already been reported in several studies.

In addition, when a concentrated aquatic product in which antibiotics such as a tetracycline compound is remained is ingested for a long period of time, resistance to the living body may occur, which may cause side effects such as impaired immunity. In other words, if antibiotics abuse on animals leads to general food poisoning bacteria becoming "multidrug resistant bacteria" (super bacteria), common antibiotics will not be able to cure food poisoning, and vicious cycle of administering more virulent antibiotics to heal Is repeated. Therefore, it is necessary to develop a method for detecting residual antibiotics to ensure the safety of food as well as to prevent misuse of antibiotics.

Aptamers are single stranded DNA or RNA structures with high specificity and affinity for a particular target. Aptamer has much higher affinity to target and better thermal stability than conventional antibody, which is used in sensor field, and in Since it can be synthesized in vitro , it is possible to omit the troublesome process of producing an antibody by injecting an antigen into an animal. Therefore, it is far superior in terms of cost, and there is no restriction on a target substance, and a biomolecule such as protein, amino acid, It is possible to synthesize aptamers for various targets such as small organic chemicals, bacteria and the like. Thus, aptamers are very suitable for introduction into trace amounts of residual antibiotics, and can be applied to the detection of other specific substances using nano-bio technology. Due to the nature of the aptamer specifically binding to the target substance, a lot of research has been conducted recently to utilize aptamer as a drug development, drug delivery system, and biosensor. However, most of the existing aptamer development goals were mostly targeting target substances of protein related diseases for development of biosensors for medical diagnosis or development of new drugs.

The existing method of detecting residual antibiotics is a method of preparing different test solutions according to the components in which residual antibiotics are dissolved, extracting and purifying residual antibiotics, and quantitatively measuring them by liquid chromatography. The conventional method is a few test methods according to the dissolved substance, and it is troublesome to prepare test solutions separately. In addition, accurate quantification was difficult because the detection substance may be lost during the extraction and purification process. These low molecular weight harmful substance binding specific aptamers such as residual medicines, antibiotics and environmental hormones can be applied not only as a sample in a water source and a river but also as a technology for detecting and diagnosing residual harmful substances in the body such as human body, Antibiotic detection methods have been applied only to food or water.

Accordingly, the present inventors have made intensive researches to overcome the problems of the prior art, and as a result, they have developed a DNA aptamer which is a nucleic acid construct having a specifically high affinity to a tetracycline compound, which is one kind of antibiotic substance. Further, it has been confirmed that residual antibiotic substances can be effectively detected from food, a water supply source, a human body, etc. by producing a composition comprising a DNA aptamer capable of specifically binding to tetracycline and its derivatives, thereby completing the present invention .

One aspect of the present invention is to provide a nucleic acid aptamer which specifically binds to a tetracycline compound.

Another aspect of the present invention is to provide a method for detecting a tetracycline compound using the nucleic acid aptamer, a composition for detecting a tetracycline compound containing the nucleic acid aptamer, and a detection kit.

One aspect of the present invention provides a nucleic acid aptamer that specifically binds to a tetracycline based compound, including a motif consisting of a base sequence selected from the group consisting of GGCGCGG, AGGGA, and AGGA.

The nucleic acid aptamer of the present invention refers to a small single stranded oligonucleotide capable of specifically recognizing a target substance with high affinity. At this time, when the aptamer is RNA, T can be recognized as U in the base sequence.

In the present invention, the tetracycline compound includes tetracycline, chloretetracycline, oxytetracycline, dimethylchloretetracycline, metacycline, doxycycline and minocycline, or derivatives thereof.

The aptamer may include a motif of GGCGCGG, AGGGA or AGGA, preferably the aptamer may comprise a nucleotide sequence of any one of SEQ ID NOS: 10 to 14, more preferably any one of SEQ ID NOS: 4 to 8 It may be composed of a nucleotide sequence.

Also, the aptamer may comprise the nucleotide sequence of SEQ ID NO: 15, and preferably the aptamer comprises SEQ ID NO: 9.

The number of total nucleotides constituting the nucleic acid aptamer may be from 15 to 200 nt, preferably from 15 to 80 nt. When the number of total nucleotides is small, chemical synthesis and mass production are easier, . In addition, the chemical modification is easy, the in-vivo stability is high, and the toxicity is also low. In addition, each nucleotide contained in the nucleic acid aptamer may include one or more chemical modifications that are the same or different. For example, at the 2 'position of the ribose, a nucleotide in which the hydroxyl group is substituted with any atom or group Lt; / RTI > Examples of such an arbitrary atom or group include a hydrogen atom, a fluorine atom or an -O-alkyl group such as -O-CH ₃ , an -O-acyl group such as -O-CHO, ₂ ). ≪ / RTI >

In addition, the aptamer may include a nucleic acid sequence having 90% or more and less than 100% homology with the nucleic acid sequence constituting the aptamer. A nucleic acid sequence having 90% or more and less than 100% homology is a nucleic acid sequence having one or more nucleotides added, deleted, or substituted, and having 90% or more and less than 100% .

Another aspect of the present invention provides a composition for detecting a tetracycline compound, which comprises the nucleic acid aptamer.

Since the nucleic acid aptamer of the present invention specifically binds to the common backbone of the tetracycline compound, the tetracycline compound exhibits high affinity with the tetracycline compound. Thus, the nucleic acid aptamer effectively detects the residual antibiotic substance from foods, up and down water sources, .

Further, another aspect of the present invention provides a method for detecting a tetracycline compound, comprising the step of adding the nucleic acid aptamer to a sample.

In the present invention, a sample is a composition capable of performing analysis, presumably containing or containing a target substance, and is a composition capable of performing analysis, and includes a sample collected from at least one of liquid, soil, air, food, waste, Lt; / RTI > In this case, the liquid may be characterized by water, blood, urine, tears, sweat, saliva, lymph and cerebrospinal fluid. The water may be characterized by precipitation, seawater, lake water, And the waste includes sewage, wastewater, etc., and the animal or plant includes a human body. The plant and animal tissues may include tissues such as mucous membranes, skin, skin, hair, scales, eyeballs, tongue, cheeks, hooves, beaks, snouts, feet, hands, mouths, nipples, ears and nose.

The added nucleic acid aptamer may be 0.1 nM to 100 nM, preferably 0.1 nM to 10 nM, more preferably 1 nM to 4 nM.

Another aspect of the present invention provides a kit for detecting a tetracycline compound, comprising the nucleic acid aptamer.

The kit may take the form of a bottle, a tub, a sachet, an envelope, a tube, an ampoule, etc., which may be partly or wholly made of plastic, glass, paper, foil, / RTI > The container may be fitted with a cap which is initially part of the container or can be fully or partially detachable, which may be attached to the container by mechanical, adhesive, or other means. The container may also be equipped with a stopper, which is accessible to the contents by the injection needle. The kit may include an external package, and the external package may include instructions for use of the components.

The aptamer according to the present invention specifically binds to a common backbone of a tetracycline compound to show a high affinity to a tetracycline compound. Thus, it is possible to effectively detect residual antibiotic substances from foods, up and down water sources, .

BRIEF DESCRIPTION OF THE DRAWINGS FIG. 1 is a graph showing binding characteristics of a tetracycline aptamer. FIG. A is the result of the combined assay of the selected aptamers TC29, TC33, TC58, TC60, TC66 and TC94. The ssDNA library was used as a negative control. B is the saturation curve and dissociation constant (Kd) according to ELAA.
Figure 2 is a secondary structure model of the selected aptamers.
3 is a diagram showing the results of indirect competition (ic) -ELAA. A shows the calibration curve for TC using different concentrations of aptamer TC29. B is a diagram showing a competition curve performed with 1 nM TC29 in buffer or milk.
Figure 4 shows the cross-reactivity of 1 nM aptamer TC29 to TC, OTC and CTC.

Hereinafter, the present invention will be described in more detail with reference to examples. However, these examples are for illustrative purposes only, and the scope of the present invention is not limited to these examples.

Example  1: Reagents and devices

(BTC), 1,1'-carbonyldiimidazole, tetracycline (TC), oxytetracycline (OTC), hydrochloride of chlortetracycline (CTC), bovine serum albumin Horseradish peroxidase (HRP), 3,30,5,50-tetramethylbenzidine (TMB) liquid substrate, albumin (OVA) from egg whites and Buffer constituents were purchased from Sigma (USA). The HRP-streptavidin conjugate was purchased from Thermo Scientific (France). The ssDNA random library, primers (aptF, aptR, aptRBioT) and biotinylated aptamers were synthesized by Bioneer (Korea). Taq DNA polymerase and dNTP were purchased from GeneAll (Korea). Milk was purchased at the supermarket.

ELAA (enzyme-linked aptamer assay) was performed on Maxisorp polystyrene microtiter plates (NUNC, Denmark). Enzyme activity was detected at 450 nm using Spectra max plus 384 (Molecular Device, USA). PCR amplification was performed in an automated DNA thermocycler (AxyGen, USA). The concentration of ssDNA or dsDNA was measured using Nanodrop (ASP-2680, ACTgene Inc., USA).

Example  2: TC Lt; RTI ID = 0.0 > Of app tamer  Selection and sequencing

To select an aptamer specifically binding to TC, a random ssDNA containing 40 random nucleotides and flanked with an invariant primer annealing portion: 5'- CGTACGGAATTCGCTAGC-N40-GGATCCGAGCTCCACGTG-3 '(SEQ ID NO: 1) The library was used as an initial pool. Three different primers: aptF 5'-CGTACGGAATTCGCTAGC-3 '(SEQ ID NO: 2), aptR 5'-CACGTGGAGCTCGGATCC-3' (SEQ ID NO: 3) and aptRBioT (same as 5'biotin aptR) were used for PCR amplification. Prior to use, was dissolved in a random DNA library pool of 11.25 ug in binding buffer (20 mM Tris-HCl, 50 mM NaCl, 5 mM KCl, 5 mM MgCl 2, pH 7.2) in 100 uL denaturation at 95 ℃ for 10 minutes denatured, rapidly cooled at 4 [deg.] C for 5 minutes and placed in RT for 5 minutes.

Screening of aptamers for tetracycline detection was carried out by the ELAA method.

)에서 교반하고 침전시킨 후 여과하였다. TC-COOH를 60 ℃에서 건조하고 분광광도계 (spectrophotometer)를 이용하여 확인하였다. 뒤이어, 200 uM TC-COOH를 2 mL N-디메틸포름아미드 (N-dimethylformamide)에 용해시키고, 5 uM BSA를 10 mL의 PBS (100 mM, pH 8.0)에 용해시키며, 100 mM EDC를 0.5 mL 증류수에 용해시켰다. 총 반응 혼합물을 4 ℃에서 4시간동안 교반시킨 후 밤새 4 ℃에서 보관하였다. 투석 (dialysis)은 결합되지 않은 TC를 제거하기 위해, 10,000 MWCO 투석 카세트 (Slide-A-Lyzer, Pierce)를 이용하여 24시간 동안 수행하였다. 결합되지 않은 TC가 분광계를 방해하기 때문에, 복합체의 투석 후에 한하여 신뢰할 수 있는 결과를 얻을 수 있었다. 결과적으로 얻은 TC-BSA 복합체는 분광광도계를 이용하여 확인하고 차후 사용을 위하여 -20 ℃에서 보관하였다. First, the TC-BSA complex was synthesized according to the conventional process modified by the EDC coupling method. Specifically, total 1 mM TC was dissolved in 30 mL of phosphate buffered saline buffer (PBS; 1.47 mM KH ₂ PO ₄ , 10 mM Na ₂ HPO ₄ , 2.7 mM KCl, 137 mM NaCl, pH 7.4) , 1 mM sodium chloroacetate was slowly added to the solution. The reaction mixture was stirred at room temperature (RT, 25 < 0 > C

) And precipitated and filtered. TC-COOH was dried at 60 ° C and confirmed by using a spectrophotometer. Subsequently, 200 μM TC-COOH was dissolved in 2 mL of N-dimethylformamide, 5 μM BSA was dissolved in 10 mL of PBS (100 mM, pH 8.0), and 100 mM EDC was added to 0.5 mL of distilled water ≪ / RTI > The total reaction mixture was stirred at 4 < 0 > C for 4 hours and then kept overnight at 4 < 0 > C. Dialysis was performed for 24 hours using a 10,000 MWCO dialysis cassette (Slide-A-Lyzer, Pierce) to remove unbound TC. Since the unconjugated TC interferes with the spectrometer, reliable results were obtained only after dialysis of the complex. The resulting TC-BSA complex was identified using a spectrophotometer and stored at -20 ° C for further use.

ELAA first adsorbed the TC-BSA complex or BSA to the wells of a microtiter plate in a 50 mM carbonate buffer at pH 9.6 for the coating process. The coating volume was 100 uL / well of 2 ug / mL TC-BSA complex or BSA solution, and plates were incubated overnight at 4 캜. The cultured plates were coated with 200 uL / well of 1% (w / v) OVA solution (blocking buffer) and prepared in binding buffer for 60 min at 37 ° C. The denatured DNA library pool was then added to the BSA-coated wells to remove candidates that could non-specifically bind to BSA. Unbound DNA was collected and added to the TC-BSA coated wells at 37 < 0 > C for 60 minutes. After incubation, the unbound or weakly bound DNA was removed in three washing steps with PBST. Bound DNA was eluted from TC-BSA coated wells with 200 uL elution buffer containing Tris-HCl (40 mM, pH 8.0), EDTA (10 mM), urea (3.5 M), and Tween 20 And ssDNA was recovered. This procedure was repeated three times to elute the bound ssDNA. The eluted oligonucleotides were precipitated using the ethanol precipitation method, and the ssDNA was dissolved in 10 uL of EB buffer (10 mM Tris-HCl, pH 8.5). The eluted ssDNA fractions were subjected to biotinylated reverse transcription in PCR (1 cycle at 95 ° C for 5 minutes, 35 cycles at 95 ° C for 30 seconds, 72 ° C for 30 seconds, and 1 cycle at 72 ° C for 5 minutes) Was amplified using a primer (aptRBioT), which enabled subsequent purification of the aptamer strand by alkaline denaturation in Dynabeads streptavidin M-280. The final amount of ssDNA in each round was determined using Nanodrop. After 12 rounds, ssDNA was amplified using unmodified aptF and aptR primers and cloned in TOPO PCRII (Invitrogen). We sequenced 11 inserted candidate app trams.

As a result, six of the 11 aptamers inserted into the clones were identified and screened for TC binding. The selected six aptamers were designed as TC29, TC33, TC58, TC60, TC66, and TC94. The sequence numbers and sequence information of the six app tamers are shown below.

Among these candidates, we confirmed that three aptamers exhibit good binding affinity to TC. It was confirmed that the aptamers TC29, TC33, and TC58 had a higher binding capacity to the TC than the other aptamers (FIG. 1A).

The sequences for the variable 40-nucleotide (N ₄₀ ) positions of six aptamers using ClustalW were analyzed and the results are shown in Table 2 below.

In Table 2, it can be seen that there are a number of G-rich conserved sequences and some conserved sequence motifs. In particular, aptamers TC29, TC33 and TC58 have two conserved sequence motifs. The secondary structure of these app tamers was specified using an m-fold program (Fig. 2). The first conserved consensus sequence (GGCGCGG) and the second common sequence (AGGGA or AGGA) were located at the stem and ring sites of each aptamer. This common sequence motif is a palindromic sequence. Among selected aptamers, TC60 and TC66 had only one common sequence motif (AGGGA) and showed weak affinity for TC compared to the other three aptamers (TC29, TC33 and TC58) with two motifs 1A and Table 1). Moreover, TC94 without these two motifs showed lower affinity to TC (Figure 1A and Table 1).

Example  3: TC For Of app tamer  Identification of binding properties

The ELAA method was performed to compare the binding affinities of selected aptamers to TC. BSA-coated well plates were also used to confirm whether aptamers bind to BSA. A random oligonucleotide (Oligo) library was used as a negative control.

Specifically, fixed TC-BSA complexes at different concentrations (1 ug, 2 ug, 5 ug and 10 ug / mL) were incubated with various concentrations of biotinylated apps And exposed to a treadmill. The coating of the wells was carried out using 2 ug / mL of TC-BSA. The concentration of streptavidin-HRP was 0.5 ug / mL. Binding reactions were performed using a constant quantity of the TC-BSA complex for varying concentrations (1-20 nM) of the selected aptamer.

The aptamer TC29, (dissociation constant) of TC33 and TC58 dissociation constant (K _d) were determined. The binding reaction was carried out as above but using an increased amount of biotinylated aptamer (1-20 nM). The amount of aptamer bound to TC was determined by absorbance measurement and was converted to% binding. Value and equal _{to:% (A / A 100)} = (A / A 100) × 100, where A is 1 nM to an aptamer positive absorbance values of 20 nM, ₁₀₀ is A 10 nM of the absorbance value of the aptamer TC29. K _d was calculated by nonlinear regression analysis.

Figure IB shows a non-linear regression analysis for the combined data. The dissociation constants for the three aptamers were in the nanomolar range. A low K _d value (3.1 nM) was obtained in the aptamer TC29. Other app tamers (TC33 and TC58) showed higher K _d values (6.2 and 4.9 nM, respectively) than TC29.

Example  4: indirect competition ELAA  ( indirect competitive ELAA , ic - ELAA ) TC Detection

We conducted a contention test for TC detection. The indirect competitive assay depends on the concentration of TC-BSA and the concentration of competitor aptamer. In an indirect competition assay, the concentration of the TC-BSA complex coated on the wells of the plate was used at the same concentration as the binding assay of the aptamer for TC and the three concentrations (1, 5, and 10 nM) Were used to examine the sensitivity to TC.

Specifically, competition was performed with addition of unlabeled TC and biotinylated aptamer. The solution was prepared in binding buffer and the competition time was 60 minutes at 37 < 0 > C. After the competition reaction, the staptabidin-HRP solution was added and reacted. Finally, 100 uL of TMB solution was added and the absorbance was measured as described above. The assay was performed five times. The absorbance values were converted to experimental inhibition values (A / A ₀ ) as follows: (A / A ₀ ) = (A / A ₀ ) × 100 where A is the absorbance value of the competition assay and A ₀ is the absorbance value of the non-competitive assay.

As a result, the concentration of free TC causing 50% inhibition (IC ₅₀ ) of binding to immobilized TC at 5 and 10 nM of aptamer was 8 and 22 times higher than IC ₅₀ values obtained from 1 nM of aptamer (Fig. 3A). Thus, a concentration of 1 nM aptamer was selected to provide optimal sensitivity and sufficient absorbance for easy detection signals. These results indicate that sensitivity is increased when using a small amount of aptamer.

Figure 3B shows the dose-response curve obtained under optimal conditions with 2 ug / mL of TC-BSA and 1 nM of aptamer TC29 coated in plate wells in buffer or milk. The calibration curve of this aptamer was calculated as follows: (1) The binding of TC29 in the buffer (%) = - 20.74 × In [TC] - 7.98, (R ² = 0.99); (2) Binding (%) of TC29 in milk = - 16.35 In [TC] + 12.19, (R ² = 0.97).

The results are shown in Table 3 below.

matrix LOD
( ug / L) LOQ
( ug / L) SD
(Average, %) Linearity  (Linearity), R ² Buffer 9.1 27.3 3.4 0.9991 milk 11.1 74.2 4.6 0.9677

As shown in Table 3, the LOD and LOQ of the aptamer TC29 with the highest sensitivity to TC were 9.1 and 27.3 ug / L in the buffer, respectively. LOD and LOQ in milk were 11.1 and 74.2 ug / L, respectively.

Example  5: TC For Of app tamer  Identification of specificity

In the aptamer assay, the specificity was determined as to whether the aptamer could selectively respond to the target substance. Specificity of selected aptamers to TC was assessed using cross-reactivity. Cross - reactivity was achieved by indirect competition of oxytetracycline and chlortetracycline, structurally similar to TC, using ELAA.

The 50% B / B 0 value and cross-reactivity of each material are shown in Figure 4 and Table 4 below.

Reactant 50% B / B ₀  ( ug / ml ) Cross-reactivity (%) Tetracycline 0.067 100 Oxytetracycline 0.118 56.7 Chlortetracycline 0.100 67.0 Cross-reactivity (%) = A ₁ / A ₂ × 100.
A ₁ is TC concentration that represents 50% B / B ₀ . A ₂ is the cross-reactant concentration representing 50% B / B ₀ . B / B ₀ is the ratio of the B value when there is free reactant to the maximum B ₀ value when no free reactant is present.

As shown in FIG. 4 and Table 4, aptamer TC29 showed cross-reactivity of more than 50% for both OTC and CTC. This result indicates that the aptamer TC29 can bind to the common TC backbone contained in TC, OTC and CTC.

Example  6: ic - ELAA  And HPLC In milk TC Detection

The TC recovery in the sample was carried out using two different methods, ic-ELAA and HPLC-UV. Milk supplemented with 0.5, 2 and 4 times the free TC of maximum residue levels (MRLs) (100 ug / L in milk) was prepared as a sample. Recovery rates of TC in milk samples were analyzed simultaneously with ic-ELAA and HPLC-UV, and the TC concentrations obtained from the two methods were compared.

Specifically, milk samples were purchased at local supermarkets and stored at 4 ° C prior to use. Milk contains a cation, such as a protein capable of forming a chelating complex with TC, fat, carbohydrate and Ca ^{2 +} and Mg ^2. To remove such material, 5 mL of milk sample was mixed with 5 mL of McIlvaine buffer containing 0.02M EDTA (pH 5.0) and 0.5% (v / v) tree Trifluoroacetic acid was added. The milk samples were then centrifuged at 8,000 rpm at 4 ° C for 20 minutes to remove fat and protein. 1 M NaOH was added to the supernatant to titrate to pH 7.0. The supernatant was filtered through a 0.45 um filter (Corning, Germany) and evaporated at 40 < 0 > C using gentle nitrogen blow-down. After extraction, the precipitate was resuspended in binding buffer. Detection of TC in milk samples was verified by ic-ELAA using aptamer TC29 as described above, and HPLC-UV system was performed by a conventionally known method for its analysis.

The recovery averages of TC added to milk measured by ic-ELAA and HPLC-UV are shown in Table 5 below.

Way Added TC Concentration
 ( ug / L) Measured TC Concentration
( ug / L, mean ± SD). Recovery rate
(%, Mean ± SD) ic-ELAA 50 46.7 ± 1.5 93.3 ± 2.9 100 99.5 ± 1.3 99.5 ± 1.3 200 195.2 ± 5.6 97.6 ± 2.8 400 405.2 ± 7.2 101.3 ± 1.8 HPLC-UV 50 44.8 ± 0.4 89.0 ± 1.2 100 90.4 ± 1.3 90.4 ± 1.3 200 181.2 + - 4.4 90.6 ± 2.2 400 369.4 ± 4.6 92.4 ± 1.2

As shown in Table 5, the recovery averages of TC added to milk were 92.1% and 90.6% in ic-ELAA and HPLC-UV, respectively. In both methods, the recovery rate of TC in milk was 90% or more.

<110> Hoseo University Academic Cooperation Foundation <120> Nucleic acid aptamer specifically binding to tetracycline          compound <130> 1-1 <160> 15 <170> Kopatentin 2.0 <210> 1 <211> 76 <212> DNA <213> Artificial Sequence <220> <223> invarible pimer annealing part containing 40 random nucleotide <400> 1 cgtacggaat tcgctagcnn nnnnnnnnnn nnnnnnnnnn nnnnnnnnnn nnnnnnnngg 60 atccgagctc cacgtg 76 <210> 2 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> aptF primer <400> 2 cgtacggaat tcgctagc 18 <210> 3 <211> 18 <212> DNA <213> Artificial Sequence <220> <223> aptR primer <400> 3 cacgtggagc tcggatcc 18 <210> 4 <211> 76 <212> DNA <213> Artificial Sequence <220> <223> aptamer TC 29 full sequence <400> 4 cgtacggaat tcgctagcgg cgcggcaagg tgtggactcc aggcggtagg gatgtgctgg 60 atccgagctc cacgtg 76 <210> 5 <211> 76 <212> DNA <213> Artificial Sequence <220> <223> aptamer TC 33 full sequence <400> 5 cgtacggaat tcgctagcgg cgcggctagg tcgactcctg cgtatacgag gatggcgtgg 60 atccgagctc cacgtg 76 <210> 6 <211> 76 <212> DNA <213> Artificial Sequence <220> <223> aptamer TC 58 full sequence <400> 6 cgtacggaat tcgctagcgg cgcggcatcg atgggactcc aggcggtagg gatgtcgtgg 60 atccgagctc cacgtg 76 <210> 7 <211> 76 <212> DNA <213> Artificial Sequence <220> <223> aptamer TC 60 full sequence <400> 7 cgtacggaat tcgctagcga cagggacggc cagtacgacc cagaattctg cccactgggg 60 atccgagctc cacgtg 76 <210> 8 <211> 76 <212> DNA <213> Artificial Sequence <220> <223> aptamer TC 66 full sequence <400> 8 cgtacggaat tcgctagcga gagggacgtt ccatgttcgt atccgcagtc ctgagcgtgg 60 atccgagctc cacgtg 76 <210> 9 <211> 76 <212> DNA <213> Artificial Sequence <220> <223> aptamer TC 94 full sequence <400> 9 cgtacggaat tcgctagcca cacggccggt gtcgctcggc tcaataccgc cccggtgtgg 60 atccgagctc cacgtg 76 <210> 10 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> aptamer TC 29 N40 sequence <400> 10 ggcgcggcaa ggtgtggact ccaggcggta gggatgtgct 40 <210> 11 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> aptamer TC 33 N40 sequence <400> 11 ggcgcggcta ggtcgactcc tgcgtatacg aggatggcgt 40 <210> 12 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> aptamer TC 58 N40 sequence <400> 12 ggcgcggcat cgatgggact ccaggcggta gggatgtcgt 40 <210> 13 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> aptamer TC 60 N40 sequence <400> 13 gacagggacg gccagtacga cccagaattc tgcccactgg 40 <210> 14 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> aptamer TC 66 N40 sequence <400> 14 gagagggacg ttccatgttc gtatccgcag tcctgagcgt 40 <210> 15 <211> 40 <212> DNA <213> Artificial Sequence <220> <223> aptamer TC 94 N40 sequence <400> 15 cacacggccg gtgtcgctcg gctcaatacc gccccggtgt 40

Claims (10)

A nucleic acid aptamer which specifically binds to a tetracycline compound, characterized by comprising a nucleotide sequence of any one of SEQ ID NOS: 10, 12, 13 and 14. The nucleic acid aptamer according to claim 1, wherein the tetracycline compound is selected from the group consisting of tetracycline, chloretetracycline, oxytetracycline, dimethylchloretetracycline, methacycline, doxycycline and minocycline. delete 2. The nucleic acid aptamer of claim 1, wherein the aptamer comprises a nucleotide sequence selected from the group consisting of SEQ ID NOS: 4, 6, 7 and 8. delete delete A composition for detecting a tetracycline compound, comprising a nucleic acid aptamer according to any one of claims 1, 2 and 4. A method for detecting a tetracycline compound, comprising the step of adding a nucleic acid aptamer of any one of claims 1, 2 and 4 to a sample. [Claim 9] The method according to claim 8, wherein the added nucleic acid aptamer is 0.1 nM to 100 nM. A kit for detecting tetracyclic compounds, comprising a nucleic acid aptamer according to any one of claims 1, 2 and 4.

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