KR101971474B1 - Nucleotide Aptamer for Use in Detection of benzylpenicillin - Google Patents

Abstract

The present invention relates to a nucleic acid plasmid for detecting benzylpenicillin. The nucleic acid aptamer of the present invention is structurally stable, has a long half-life, is easy to synthesize artificially, and can be easily chemically modified at each base sequence position, so that target substances can be detected using various methods. When the nucleic acid plasmid, composition, detection kit or detection method for detecting benzylpenicillin of the present invention is used, benzylpenicillin can be easily detected with high sensitivity.

Description

Nucleotide Aptamer for Use in Detection of Benzylpenicillin < RTI ID = 0.0 >

The present invention relates to a nucleic acid plasmid for detecting benzylpenicillin.

Antibiotics are substances made by living organisms that show antibiotic activity and are classified as antimicrobial substances, antibacterial agents, and antiviral agents. Antibiotics are products that can be commercialized as therapeutic drugs. All organisms, including humans, including animals and plants, fungi, and bacteria, form a specific class of antibiotics. Among them, penicillin was first discovered by Fleming in the summer of 1928 as an antibiotic. The penicillin family has a beta-lactam ring and is known to exhibit antibiotic activity by inhibiting the synthesis of peptidoglycan, the main component of microbial cell walls. Antibiotics that play a different role in addition to penicillin were also found intact, and they were used as a breakthrough device for the cessation and treatment of disease. Therefore, the use of antibiotics has also increased in various livestock industries for the purpose of preventing, treating, and promoting growth of livestock and fish diseases. However, this causes side effects such as human resistance and makes it difficult to treat human diseases. This is becoming more serious as the number of residual offending farms that do not comply with livestock residual antibiotic standards increases.

Among them, benzylpenicillin is a typical penicillin antibiotic, which has a beta-lactam ring and has a benzene ring linked through a peptide bond. Benzylpenicillin can be inhibited by gastric acid, and therefore, it is not orally administered. When it is used in combination with phenoxymethylpenicillin, a high antimicrobial effect can be obtained. Generally, treatment effects can be expected for diseases such as cellulitis and sepsis.

Benzylpenicillin inhibits bacterial cell wall synthesis and leads to cell death. Nonetheless, it is widely used in the shaft and fisheries as well as in the treatment of the human body. It is widely used for the treatment of positive mastitis and for the treatment of infections caused by bacteria. However, when excess amount remains, it causes side effects such as allergic reactions to humans and hinders fermentation in the manufacture of dairy products.

Aptamer refers to a small molecule capable of specifically binding to a specific target substance such as a biomarker or an antibiotic, and is generally a small nucleic acid structure composed of about 20 to 60 nucleotides. Depending on the sequence, it has a variety of stem-ring structures and forms a folding structure to combine with one target substance. Aptamer is selected from a random sequence pool through a series of processes called SELEX (Systematic Evolution of Ligands by Exponential Enrichment). SELEX is a process for finding a good platamer that has high specificity to a target substance and has specificity for a similar structure and can be detected with a small amount of probe. First, it is necessary to synthesize a nucleic acid containing a random sequence through a polymerase chain reaction, and to bind it with a target substance. After the binding, the separation process is performed, and a specific sequence is eluted through a series of amplification steps. This process can be repeated several times to ultimately select a suitable tamtamer for the target material. Aptamers have several advantages when compared to antibodies, since they are smaller in size than antibodies, allowing them to bind to the target material more flexibly. In addition, since it can be synthesized easily in a laboratory, it can be synthesized in a shorter time than an antibody synthesized through a reaction in vivo. Compared with antibodies that are stable in vivo, aptamers are advantageous in that they are stable in environments such as temperature and pH, which are somewhat high, such as common nucleic acids. Therefore, even when developed with a kit or the like, the stability is higher than when antibody is used. In addition, high-pressure liquid chromatography-mass spectrometry, which is currently known to be an accurate method for detecting antibiotics, has a disadvantage in that it takes a long time to detect and can be detected only in a laboratory. However, if a detection system including aptamer is developed, On-site diagnosis will also be possible (Non-Patent Document 1).

Small-molecule probes such as platamers can be applied through a variety of techniques, but the development of a ready-to-use property strip kit for livestock slaughter is considered to be the optimal application. In the case of the previously developed kit, the method using the inhibition of the growth of the antibiotic-sensitive microorganisms has a very long incubation time of about 12 hours and can be measured in the laboratory. Some examples include 3M ™ Antibiotics Detection Kit Reader, which was released by 3M, and Sensicheck-Fluoroquinolones, developed by INRGEN. In the latter case, quinolone antibiotics can be detected based on the immunochromatographic method. However, the types of antibiotics that can be detected are limited to the quinolone series .

In the case of detectors and kits using an antigen-antibody reaction, the types of antibiotics that can be detected are all limited, and the detection time is long (Non-Patent Document 2)

Recently, antibiotics have been used at a much higher rate than other veterinary medicines, and a system capable of specifically detecting benzylpenicillin, which occupies the highest proportion, has not been developed. Therefore, the development of aptamer that specifically detects benzylpenicillin will be an important basis for on - site diagnosis of residual antibiotics in livestock farmers.

Currently, the most widely used antibiotic detection kit (MiRA test, MeRA test) requires incubation for at least 4 hours because of the use of bacteria. The platemer specifically binding to benzylpenicillin can be effectively used to develop a system capable of detecting in a short period of time since a separate incubation time is not required. In the case of the SensiCheck method using the immune response, it is selective and can detect the antibiotic in a short time, but it is difficult to improve the sensitivity above a predetermined level. On the other hand, in the case of platemer, there is a strong point that an antibiotic detection system with improved sensitivity can be developed according to the binding force and detection limit of each platemaker.

Yu, X et al., Science of the total environment, 569-570, 23-30 (2016) Erqun Song et al., Biosensors and bioelectronics, 72, 320-325 (2015)

Accordingly, the present inventors have made extensive efforts to develop a nucleic acid plastomer probe capable of detecting benzylpenicillin, which is a typical penicillin antibiotic, which accounts for the largest proportion of residual antibiotics. As a result, the present inventors have completed the present invention by confirming that the nucleic acid abstamer containing a predetermined sequence has an excellent binding ability to benzylpenicillin.

Accordingly, an object of the present invention is to provide a nucleic acid plasmid for detecting benzylpenicillin.

It is still another object of the present invention to provide a composition for detecting benzylpenicillin comprising the above nucleic acid ablator.

It is still another object of the present invention to provide a benzylpenicillin detection kit comprising the nucleic acid platemaker.

It is still another object of the present invention to provide a method for detecting benzylpenicillin using the composition for detecting benzylpenicillin.

According to one aspect of the present invention, there is provided a nucleic acid plasmid for detecting benzylpenicillin comprising the nucleotide sequence of SEQ ID NO: 1.

The benzylpenicillin of the present invention has a beta-lactam ring and has a benzene ring linked through a peptide bond. Benzylpenicillin can be inhibited by gastric acid, and therefore, it is not orally administered. When it is used in combination with phenoxymethylpenicillin, a high antimicrobial effect can be obtained. Generally, treatment effects can be expected for diseases such as cellulitis and sepsis. According to the results of domestic foodstuff residue test in 2013, the distribution of antibiotics in beef, pork, and chicken was the highest, 32.93% in benzylpenicillin and 16.16% in flocassin, All of the antibiotics showed a distribution rate of less than 10%. In the case of benzylpenicillin, the largest proportion of residual antibiotics and 0.004 ppm (about 12 nM) of residual permissible standards are needed to develop a probe for establishing a field detection system. In order to solve this problem in advance, in order to detect benzylpenicillin in advance, the present inventors have developed a nucleic acid platemer probe of a predetermined sequence having a binding force to benzylpenicillin.

The term " nucleic acid plasmid " of the present invention means a nucleic acid molecule capable of binding a specific molecule with high affinity and specificity, and the nucleic acid means DNA, RNA or nucleic acid variant. The nucleic acid aptamer of the present invention is provided in the form of single-stranded DNA, RNA, or the like, and shows a DNA compactor sequence including the sequence represented by SEQ ID NO: 1 or SEQ ID NO: 2, and the thymidine It will be apparent to those skilled in the art that the RNA tyramer sequence substituted with uracil is included in the scope of the present invention.

The term " nucleic acid ", as used herein, can also be described as a " nucleotide ", and has the same meaning and is defined by a deoxyribonucleotide, a ribonucleotide, a modified nucleotide or base and / or an analogue thereof, or a DNA or RNA polymerase (Scheit, Nucleotide Analogs, John Wiley, New York (1980); Uhlman and Peyman, Chemical Reviews, 90: 543-584 (1990)). If a modification to the nucleotide structure is present, such modification may be added before or after the synthesis of the nucleic acid amplimer. The nucleotide sequence may be terminated by a non-nucleotide component. Nucleic acid aptamers can be further modified after synthesis, e. G. By conjugation with a label.

The nucleic acid aptamers of the present invention are typically obtainable by in vitro selection methods for binding of target molecules. Methods for selecting platemers that specifically bind to target molecules are known in the art. For example, an organic molecule, a nucleotide, an amino acid, a polypeptide, a marker molecule on a cell surface, an ion, a metal, a salt, a polysaccharide may be a suitable target molecule for separating an umbrella that can specifically bind to each ligand . Selection of platemembers may utilize in vivo or in vitro selection techniques known in the art as SELEX (Systematic Evolution of Ligand by Exponential Enrichment) method (Ellington et al., Nature 346, 818-22, 1990; and Tuerk et al. , Science 249, 505-10, 1990). Specific methods for the selection and manufacture of platemers are described in U.S. Patent 5,582,981, WO 00/20040, U.S. Patent 5,270,163, Lorsch and Szostak, Biochemistry, 33: 973 (1994), Mannironi et al., Biochemistry 36: 9726 Blind, Proc. Natl. Acad. Sci. USA 96: 3606-3610 (1999), Huizenga and Szostak, Biochemistry, 34: 656-665 (1995), WO 99/54506, WO 99/27133, WO 97/42317 and U.S. Patent 5,756,291, Are incorporated herein by reference.

SELEX requires a single-stranded oligonucleotide pool or library consisting of randomized sequences. The term " oligonucleotide " in the present invention generally means a nucleic acid polymer comprising less than 100 nucleic acids. Oligonucleotides can be DNA, RNA or DNA / RNA hybrids that are not modified or modified. The oligonucleotide pool is a 100% random or partially random oligonucleotide, preferably the oligonucleotide pool can be composed of random or partially random oligonucleotides in which at least one fixed sequence and / or conserved sequence is included in the random sequence region have. More preferably, the oligonucleotide pool is a random or partially randomized sequence comprising at least one fixed and / or conserved sequence at which the 5 ' and / or 3 ' termini can consist of sequences shared in all molecules of the oligonucleotide pool Lt; / RTI > oligonucleotides. Oligonucleotide pools preferably include fixed sequences as well as random sequence regions, which are essential for efficient replication. The oligonucleotide of the initial pool contains a fixed 5 ' and 3 ' end sequence, into which approximately 20-50 random nucleotides are inserted. In a specific embodiment of the present invention, a library in which 30 random nucleotides are inserted is used, and the nucleotide sequence length in the sequence amplification process can be changed. Random nucleotides can be produced by chemical synthesis and selection from randomly truncated intracellular nucleic acids.

The oligonucleotide may comprise a random sequence portion of a predetermined length and may be composed of a ribonucleotide and / or a deoxyribonucleotide, and may be a native nucleotide or a nucleotide analogue that has not been modified or modified (US Patent No. 5,958,691; No. 5,660,985, US Patent No. 5,958,691, US Patent No. 5,698, 687, US Patent No. 5,817, 635, US Patent No. 5,672,695, and PCT Publication WO 92/07065). Random oligonucleotides can be synthesized from phosphodiester-linked nucleotides using solid state oligonucleotide synthesis techniques well known in the art (Froehler et al., Nucl. Acid Res. 14: 5399-5467 (1986), Froehler et al., Tet. Lett., 27: 5575-5578 (1986)). Random oligonucleotides can also be synthesized using liquid phase methods such as the triester synthesis method (Sood et al., Nucl. Acid Res. 4: 2557 (1977), Hirose et al., Tet. Lett., 28 : 2449 (1978)).

In one embodiment of the present invention, the nucleic acid overtamer of the present invention comprises the nucleotide sequence shown in SEQ ID NO: 2. The sequence of SEQ ID NO: 2 represents a DNA plasmid sequence, and an RNA abstamer sequence in which thymine is replaced with uracil in the nucleic acid sequence of the DNA plasmid can be used. In addition, the nucleic acid aptamer of the present invention is also interpreted to include a sequence that exhibits substantial identity with the sequence described in the sequence of SEQ ID NO: 2. The above-mentioned substantial identity is determined by aligning the above-described sequence of the present invention with any other sequence as much as possible, and analyzing the aligned sequence using an algorithm commonly used in the art, at least 80% Homology, more preferably at least 90% homology, and most preferably at least 95% homology. Alignment methods for sequence comparison are well known in the art. Various methods and algorithms for alignment are described by Smith and Waterman, Adv. Appl. Math. 2: 482 (1981), Needleman and Wunsch, J. Mol. Bio. 48: 443 (1970); Pearson and Lipman, Methods in Mol. Biol. 24: 307-31 (1988); Higgins and Sharp, Gene 73: 237-44 (1988); Higgins and Sharp, CABIOS 5: 151-3 (1989); Corpet et al., Nuc. Acids Res. 16: 10881-90 (1988); Huang et al., Comp. Appl. BioSci. 8: 155-65 (1992) and Pearson et al., Meth. Mol. Biol. 24: 307-31 (1994). The NCBI Basic Local Alignment Search Tool (BLAST) (Altschul et al., J. Mol. Biol. 215: 403-10 (1990)) is accessible from National Center for Biological Information (NBCI) It can be used in conjunction with sequence analysis programs such as blastx, tblastn and tblastx. BLSAT is available at http://www.ncbi.nlm.nih.gov/BLAST/. A method for comparing sequence homology using this program can be found at http://www.ncbi.nlm.nih.gov/BLAST/blast_help.html.

The aptamer according to the present invention can detect benzylpenicillin more specifically and sensitively than conventional antibodies, and can be applied to a sensor system by various methods using an effective signal conversion system such as gold nanoparticles or photon points It will be possible. These applications will enable us to detect benzylpenicillin more rapidly and sensitively than conventional diagnostic systems.

According to another aspect of the present invention, there is provided a composition for detecting benzylpenicillin comprising the above-described nucleic acid platemater. The composition for detecting benzylpenicillin of the present invention may contain carriers and / or preservatives, stabilizers and the like well known in the art for stable storage and preservation of the nucleic acid platemer in addition to the nucleic acid platemer described above.

In one embodiment of the invention, the nucleic acid aptamer of the present invention is attached with a detectable label. By attaching a detectable label to the nucleic acid platemer, it is possible to easily observe and measure the binding and the degree of binding between the target and the nucleic acid platemer. The detectable label may be a moiety that can be detected by a detection method known in the art, and is not particularly limited.

In one embodiment of the invention, the detectable label of the present invention is an optical label, an electrochemical label, a radioactive isotope, or a combination thereof. The label may be attached to the specific base or 3 ' end or 5 ' end of the plummeter. The optical label may be, for example, a fluorescent substance. The fluorescent material may be selected from the group consisting of fluorescein, 6-FAM, rhodamine, Texas red, tetramethylrhodamine, carboxydodamine, carboxyrotamine 6G, carboxyrodone, carboxydodamine 110, cascade blue Cascade Blue), Cascade Yellow, Comarine, Cy2 (cyanine 2), Cy3, Cy3.5, Cy5, Cy5.5, Cy-chrome, Picoeritrin, PerCP (Peridinin chlorophyll- , PerCP-Cy5.5, JOE (6-carboxy-4 ', 5'-dichloro-2', 7'-dimethoxyfluorescein), NED, ROX (5- Hex, Lucifer Yellow, Marina Blue, Oregon Green 488, Oregon Green 500, Oregon Green 514, Alexa Floor, 7-amino-4-methylcomarine- -Acetic acid, BODIPY FL, BODIPY FL-Br 2, BODIPY 530/550, conjugated cargoes thereof, and combinations thereof. For example, the fluorescent material may be fluorescein, Cy3 or Cy5. In addition, the optical label may include a fluorescent donor dye and a fluorescent acceptor dye separated by an appropriate distance, and the fluorescence emitted by the donor is fluorescence resonance energy transfer (FRET) pair . The donor pigments may include FAM, TAMRA, VIC, JOE, Cy3, Cy5 and Texas Red. The acceptor dye can be selected such that its excitation spectrum overlaps the donor emission spectrum. The receiver can also be a non-fluorescent receiver that quenches a wide range of donors. Other examples of donor-acceptor FRET pairs are known in the art. The electrochemical label includes an electrochemical label known in the art. For example, the electrochemical label may be methylene blue.

According to another aspect of the present invention, there is provided a benzylpenicillin detection kit comprising the nucleic acid abstamator described above.

The kit of the present invention can be prepared by using the above nucleic acid aptamer as a polymer compound of silica, semiconductor, plastic, gold, silver, magnetic molecule, nylon, poly (dimethylsiloxane), PDMS, cellulose, nitro Cellulose or a glass slide or the like. There is no particular restriction on the shape of the support, but may be in the form of a thin plate that can be held by hand, such as, for example, a glass slide, or in the form of a bead that is less than 0.1 mm in size, . The surface of the support may be functionalized with functional groups such as an aldehyde group, a carboxyl group, an epoxy group, an isothiocyanate group, an N-hydroxysuccinimidyl group, and an activated ester group, particularly an epoxy group. However, after the probe is fixed, it can be stabilized through a process of blocking the residual functional group to reduce the background signal.

The kits of the present invention may include buffer solutions and containers for performing and analyzing detection as needed, such as bottles, tubs, sachets, envelopes, tubes or ampoules, May take the same form and may be formed partly or wholly of plastic, glass, paper, foil or wax, and the like. The container may initially be a part of the container or may be fitted with a fully or partially detachable cap which may be attached to the container by mechanical, adhesive or other means. The container may also be equipped with a stopper 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.

According to another aspect of the present invention, the present invention provides a method for detecting benzylpenicillin comprising the steps of:

(a) contacting the composition for detecting benzylpenicillin with a sample to be tested; And

(b) measuring the combined signal of the composition for detecting benzylpenicillin and the target sample after step (a).

The " subject sample " of the present invention means an object to be judged as to whether benzylpenicillin is detected or not, and is not particularly limited. Specific examples thereof include those obtained from polycarbonate plastic, epoxy resin, phenoplast resin, phenol resin, unsaturated polyester resin, thermal paper, polyol, modified polyamide, automobile tire or flame retardants and the like But are not limited to, benzylpenicillin.

The binding signal measurement in step (b) of the present invention can be measured using known methods according to the characteristics of the detection signal used, and it is determined whether or not an increased signal is shown compared to the control group to confirm the presence of benzylpenicillin .

The present invention improves the existing long-term, low-sensitivity detection system to develop a small nucleic acid molecule probe that binds to benzylpenicillin in a shorter period of time, thereby becoming the basis of an effective benzylpenicillin detection system. Currently, benzylpenicillin accounts for the largest proportion of antibiotics remaining in livestock, and diagnostic probes have not been developed. The aptamer according to the present invention is the first platemper specifically binding to benzylpenicillin and has a detection limit of 13 nM, which is the same level as the residual tolerance standard. Therefore, it will be an effective invention in establishing a system for diagnosing residual antibiotics in the slaughterhouse.

Figure 1 shows single-stranded DNA synthesis for the progression of SELEX [(A) using asymmetric polymerase chain reaction with different primer concentrations using a template containing random sequences followed by 12% SDS-PAGE electrophoresis (M: Marker), (B) After DNA extraction, size is checked through 2.5% agarose gel.
FIG. 2 shows a SELEX reaction scheme using reduced graphene oxide and single-stranded tympanic membrane screening conditions for each round. [As the circuit is repeated, the concentration of the reaction buffer solution is increased and the reaction time is reduced, Want to screen].
Figure 3 shows a single stranded plasmid sequence extracted through four screening steps. [A total of 30 sequences were analyzed to finally select six plasmother candidates].
Fig. 4 shows the secondary structure of the No. 1 platemer (SEQ ID NO: 2) showing the highest frequency among the sequence candidates. (Gibbs free energy (G) = -15.06 kJ / mol)
Figure 5 shows the adsorption saturation concentration and reaction time using FAM-labeled plumbers. [(A) Reduced graphene oxide was treated at different concentrations to obtain adsorbed 1 μM aptamers at a concentration of 4 μg / , (B) the fluorescence intensity was extinguished during the reaction for at least 15 minutes as measured by different reaction times for treating 4 ug / ul of graphene oxide.
Fig. 6 shows the trend of signal recovery according to the concentration of platamer. [From the signal recovered from the graphene of the FAM-labeled plummeter, the dissociation constant (Kd), which is an indicator of the binding force, is derived. (Kd = 383.4 nM).
FIG. 7 shows the fluorescence signal transition according to the concentration of benzylpenicillin. [Recovered fluorescence intensity is saturated with increasing concentration, and a linear graph can be obtained in a concentration range up to 50 nM. The detection limit of the corresponding platemer was confirmed to be 13 nM].
Fig. 8 shows the analysis of the specificity of the platemer. [Comparison of fluorescence recovery signals of amoxicillin, cephalothin and other antibiotics tetracycline, oxytetracycline, and kanamycin, which are beta-lactam antibiotics such as benzylpenicillin Showed 90% or more difference in binding, confirming that the sequence has specificity for similar structures or other antibiotics].
FIG. 9 shows the results of analysis of the secondary structure of the squamous cell (60mer) of SEQ ID NO: 2 and the squamous cell (25mer) of SEQ ID NO: 1 using the M-fold program.
10 is a graph showing the type and intensity of the platemer of SEQ ID NO: 1 according to the concentration of benzylpenicillin.
FIG. 11 is a graph showing a relative value of the binding efficiency of the squamous cell (60mer) of SEQ ID NO: 2 and the squamous cell (25mer) of SEQ ID NO: 1 to benzylpenicillin.

Hereinafter, embodiments of the present invention will be described in detail. The following examples illustrate the invention and are not intended to limit the scope of the invention. It is to be understood that both the foregoing general description and the following detailed description of the present invention are exemplary and explanatory and are intended to provide further explanation of the invention as claimed. Only.

Example  1: random for screening Single strand  Securing DNA library

A SELEX procedure is required to screen for an abdominal tyramine that binds specifically to benzylpenicillin. Prior to this, a single-stranded DNA library must be obtained. Polymerase chain reaction was carried out with 15 nucleotides (primer) at each end and 30 template sequences with 30 random sequences in the middle. In the case of a general symmetric polymerase chain reaction, double-stranded DNA is synthesized exponentially by a forward primer and a reverse primer. In the asymmetric polymerase chain reaction, however, two primers are treated at different concentrations to produce single-stranded DNA. In this invention, asymmetric polymerase chain reaction (PCR) was performed by adding 100 uM and 10 uM of a forward primer (5'-ATGCGGATCCCGCGC-3 ', Bam H Site) and a reverse primer (5'-GCGCAAGCTTCGCGC-3' . All oligonucleotides were synthesized and purified on PAGE by Bioneer (Korea). Crush and soak method was used to obtain single strand DNA only after amplification. The amplified products were loaded with 12% DNA native gel, developed with constant voltage, and only single strand DNA was generated by staining gel with EtBr after electrophoresis. The resulting mixture was pulverized and dissolved in Crush and soak buffer (500 mM NH4OAc, 0.1% SDS, 0.1 mM EDTA). The solid gel was separated by centrifugation, and a single stranded DNA library was obtained by ethanol precipitation. A UV / Vis spectrophotometer (Fig. 1).

Example  2: Reduction Graphene oxide  Used Single strand  DNA Abtamer  Screening ( SELEX )

In this screening process, a total of 4 rounds of SELEX were performed under the condition of binding buffer (20 mM Tris, pH 8.0).

200 pmol of the single-stranded DNA library constructed by the method of Example 1 was treated with amoxicillin, cephalothin, kanamycin (1 mM each) and reduced graphene oxide, which are beta-lactam antibiotics, at the same time, Min to adsorb single-stranded DNA that does not bind to the negative target on the reduced graphene oxide surface. And centrifuged to remove DNA bound to the negative targets present in the supernatant. The remaining reduced graphene oxide was centrifuged again using 1X Binding buffer to remove nonspecific DNA, followed by treatment with benzylpenicillin and incubation for 30 minutes to elute single-stranded DNA. In order to increase the concentration of the binding buffer and reduce the reaction time, SELEX was performed in four stages (Step 2). In the final round, the DNA eluted in the final round was subjected to symmetric polymerase chain reaction to double-stranded DNA And BamH and Hind in the primer were cut with restriction enzymes and cloned DNA was obtained by gel elution. The pET-28a vector was also treated with a restriction enzyme to insert a cleaved fragment. After a ligation reaction for 16 hours, the colonies obtained by the transformation were inoculated and the DNA present in the cells was extracted. When the sequence derived here was analyzed, the sequence was found to be the most frequent (FIG. 3).

(5'-ATGCGGATCCCGCGCGGGTCTGAGGAGTGCGCGGTGCCAGTGAGTGCGCGAAGCTTGCGC-3 ': SEQ ID NO: 2, hereinafter referred to as "60mer abtamer")

Example  3: Extracted Abtamer  Sequencing

The plasmid sequences extracted by the above method consist of 60 nucleotides having a forward primer (5'-ATGCGGATCCCGCGC-3 ') and a reverse primer (5'-GCGCAAGCTTCGCGC-3') at both ends. When the structure is predicted using the M-FOLD program, the aptamer has a large ring structure with a Gibbs free energy (G) of -15.06 kJ / mol and one side open at the center. On either side of it were linear structures connected by triple bonds of guanine and cytosine, and several small ring structures were present (FIG. 4).

Example  4: Reduction Grapina  Reaction conditions using oxide

For the characterization of the 60-mer platamer, an alphamer sequence linked with FAM (6-Carboxyfluorescein) was ordered and synthesized (5'-FAM-ATGCGGATCCCGCGCGGGTCTGAGGAGTGCGCGGTGCCAGTGAGTGCGCGAA GCTTGCGC-3 '). The binding density was measured by reducing graphene oxide by concentration of fibrin glutamate (FAM) -conjugated glutamate oxide on the basis of the characteristics of graphene oxide which extinguished the fluorescence by FRET phenomenon. As a result of measuring the minimum reaction time after measuring the binding saturation concentration, it was confirmed that all the fluorescence signals were not extinguished when the reaction time was 15 minutes (FIG. 5).

Example  5: Synthesized Abtamer  Analysis of binding force between sequence and benzylpenicillin

The fluorescence intensity of the restored fluorescence was measured by treating the abdomen with a concentration range of 0 to 1 uM by adding 4 ug / ul of reduced graphene oxide and treating with benzylpenicillin (1 mM) The binding force of penicillin was analyzed. Fluorescence intensity was increased with benzylpenicillin throughput and it was confirmed that the fluorescence intensity was saturated at the concentration level of 500 nM. Finally, fitting with Graphpad Prism 6 showed a dissociation constant of about 390 nM (FIG. 6).

Example  6: Synthesized Abtamer  Detection limit of sequence

After the reduction graphene oxide and platemer were saturat- ed at the optimized concentration, the fluorescence intensity of the recovered platamer was measured by treating the concentration of benzylpenicillin in each tube to analyze the detection limit of benzylpenicillin in the 60-mer plamer sequence Respectively. Fluorescence intensity was saturated as the concentration of benzylpenicillin increased and fluorescence intensities recovered in a small concentration range were measured. As a result, a linear graph was obtained in the concentration range of 0 ~ 50 nM. This graph confirms that the 60mer aptamer has a detection limit of 13 nM, which is the same level as the residual tolerance standard for benzyl penicillin (Fig. 7).

Example 7: 60mer Abtamer  Sequence specificity analysis

In order to analyze the specificity of the 60-mer platemer, the binding force between the negative targets and the platemer was compared based on 200 nM antibiotics, 40 times the detection limit of benzylpenicillin.

The experiment was carried out by the same method of adsorbing the reduced graphene oxide and the plastomer through the agitation at room temperature and measuring the fluorescence signal of the recovered platamer by treating various kinds of antibiotics. Specificity analysis of amoxicillin, cephalothin and kanamycin which were screened at the SELEX stage showed 13.3%, 8% and 1.8% low binding strength of benzylphenicin 100% . In addition, it was confirmed that tetracycline and oxytetracycline, which are other antibiotics that had not undergone the screening process, had a low binding strength of 9% and 3.4%, respectively. The 60-mer aptamer of the present invention exhibited more than 87.7% difference when compared to other antibiotics of similar structure, and showed a specific binding only to benzylpenicillin (FIG. 8).

Example  8: Nucleic acid for detection of benzylpenicillin Abtamer  Confirm the bonding strength of the part where the inner ring structure is formed

(5'-ATGCGGATCCCGGCGGGGTCTGAGGAGTGCGCGGTGCCAGTGAGGGGGGGAAGCTTGCGC-3 ') of SEQ ID NO: 2, a portion in which a ring structure is formed except for the primer portion at each end of 5' and 3 ' CCGCGCGGGTCTGAGGAGTGCGCGG-3 ').

After the secondary structure analysis, it was confirmed that the 25mer aptamer maintained the same structure as the 60mer squidramer, and the entire sequence of 60mer had a four-dimensional steric structure of a general oligonucleotide sequence such as a G-quadruplex structure, and the structure was composed of benzylpenicillin Lt; RTI ID = 0.0 > of < / RTI > Comparing fluorescence recovery values using extinction and recovery methods using FAM (6-carboxyfluorescein) labeled platamer, 25-mercaptoterum showed a binding strength of 51.13% compared to 60-mercaptamer.

The secondary structure of the 60-mer plamer sequence and the 25-mer plamer sequence was analyzed using the M-fold program to confirm that the structure of the corresponding portion was maintained (FIG. 9).

Also, as the concentration of benzylpenicillin increased, the fluorescence intensity of the 25-mer plammer increased, but the ratio was lower than that of the 60-mercaptoma (FIG. 10).

As a result of comparing the binding efficiency between the 60-mer plastomer sequence and the 25-mer plamer sequence with relative values, it was confirmed that the 25-mer plumer sequence had a binding force of 51.13% based on the 60-mer plamer sequence 100% (FIG. 11).

<110> Industry-University Cooperation Foundation Hanyang University <120> Nucleotide Aptamer for Use in Detection of benzylpenicillin <130> P16U10C1714 <160> 2 <170> Kopatentin 2.0 <210> 1 <211> 25 <212> DNA <213> Artificial Sequence <220> The modified aptamer sequence (25-mer) <400> 1 ccgcgcgggt ctgaggagtg cgcgg 25 <210> 2 <211> 60 <212> DNA <213> Artificial Sequence <220> Benzylpenicillin-specific aptamer sequense (60-mer) <400> 2 atgcggatcc cgcgcgggtc tgaggagtgc gcggtgccag tgagtgcgcg aagcttgcgc 60                                                                           60

Claims (9)

delete delete A nucleic acid plasmid for detecting benzylpenicillin comprising the nucleotide sequence of SEQ ID NO: 2.
A composition for detecting benzylpenicillin comprising the nucleic acid abstamator of claim 3.
5. The method of claim 4,
Wherein the nucleic acid aptamer is attached with a detectable label.
6. The method of claim 5,
Wherein the detectable label is an optical label, an electrochemical label, a radioisotope, or a combination thereof.
5. The method of claim 4,
A composition for detecting 12 nM or more of benzylpenicillin.
A benzylpenicillin detection kit comprising the nucleic acid abstamator of claim 3.
A method for detecting benzylpenicillin comprising the steps of:
(a) contacting the composition of claim 4 with a sample of interest; And
(b) measuring the combined signal of the composition and the target sample after step (a).

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