KR101448360B1 - DNA aptamer specifically binding to Salmonella Typhymurium and uses thereof - Google Patents

Abstract

The present invention is specifically to sikjungdokgyun ever relates to a single-stranded DNA, Salmonella typhimurium (Salmonella Typhymurium) detecting single-stranded DNA comprising any one or more nucleotide sequences selected from the group consisting of SEQ ID NO: 4 to SEQ ID NO: 8 Provides app tamer. The single-stranded DNA aptamer of the present invention is structurally stable, artificial, easy to synthesize, chemically easily modified at each base sequence position, and is useful as a new next-generation diagnostic probe for Salmonella typhimurium, a food poisoning bacterium .

Description

DNA aptamer specifically binding to Salmonella Typhimurium and uses thereof,

The present invention relates to a single strand DNA aptamer for detecting Salmonella typhimurium which specifically binds to Salmonella typhimurium, a food poisoning bacterium, and a kit for the detection of food poisoning bacteria comprising the same.

Food poisoning is a disease caused by pathogenic bacteria, toxins expressed in pathogenic bacterium, viruses and toxins present in the natural environment, and many pathogens known as causative bacteria of food poisoning are also present. According to the statistics of onset by food poisoning bacteria in Korea, the number of cases and the number of cases caused by pathogenic Escherichia coli and Salmonella spp. Are respectively 1 and 2 (see Table 1).

Onset of food poisoning by causative organism Causative bacteria Number of patients Number of cases Pathogenic Escherichia coli 1,096 10 Salmonella 497 16 Staphylococcus aureus 302 10 Campylobacter Jeju 287 9 Clostridium fur Presence 104 3 Bacillus cereus 70 7 Vibrio parahaemolyticus 12 2 Sum 2,368 57

On the other hand, genetic analysis (PCR) and immunoassay (ELISA) are used in addition to a conventional cell culture method as a method for detecting food poisoning bacteria such as Salmonella and pathogenic Escherichia coli at present. In addition, some companies have developed and marketed diagnostic kits using antibodies. The most common method for diagnosing a diagnostic object is a method of analyzing a small amount of target object by amplifying the inherent gene of the diagnostic object by PCR and amplifying it in vitro . The unique base sequence of the gene is used to target the unique base sequence of the diagnostic target and the application range is expanded by securing a wide range of genetic information database through the recent human genome project. In addition, the technology used for the diagnosis of food poisoning bacteria is based on ELISA analysis in addition to the PCR method described above. ELISA technology uses an antibody that specifically binds to a specific protein as an indicator for recognition. It can treat a variety of specimens by detecting a diagnostic target from a sample such as agricultural or fish food and detecting it using an enzyme-linked antibody. And the like. However, ELISA has a relatively low sensitivity compared to PCR diagnosis, and many studies are in progress to overcome this.

These conventional methods are ones that propose high sensitivity and specificity in themselves, but they have a disadvantage in that detection time is long and separation and purification of target molecules are required, and sometimes expensive detecting equipment is required. Since such conventional diagnostic methods can not detect food poisoning bacteria more conveniently than before, it is essential to develop a detection method having high sensitivity and promptness for ultra-precise quarantine. Therefore, it is necessary to develop a technology to diagnose and detect food poisoning early on the basis of the specimens of salmonella and pathogenic Escherichia coli which occupy the first and second place of pathogen-causing bacteria.

In addition, with the progress of the FTA negotiations with various governments in recent years, agricultural and marine products of the world are expected to be imported and imported on a large scale. Importing and managing agricultural and fishery foods on a large scale increases the likelihood of food poisoning caused by negligent management. When Salmonella typhimurium is present on the surface of food, it enters the body by ingesting food, and bacteria are amplified in large numbers within a few days (72 hours), causing food poisoning. Therefore, early diagnosis of Salmonella typhimurium bacteria present in foods is very important for the prevention of food poisoning, and it is urgent to develop a next-generation diagnosis technology to replace the conventional diagnostic methods described above.

It is an object of the present invention to provide single-stranded DNA aptamers which specifically bind to Salmonella typhimurium and uses thereof.

In order to achieve the above object, one aspect of the invention is SEQ ID NO: 4 to SEQ ID NO: Single-stranded DNA app for Salmonella typhimurium (Salmonella Typhymurium) detection, including any one or more nucleotide sequences selected from a group consisting of 8 Tamer Lt; / RTI >

According to another aspect of the present invention, there is provided a composition for detecting Salmonella typhimurium comprising the single stranded DNA aptamer.

According to another aspect of the present invention, there is provided a kit for detecting food poisoning bacteria comprising the single stranded DNA aptamer.

The single stranded DNA aptamer of the present invention is structurally stable, artificially easy to synthesize, and chemically easily modified at each base sequence position, thus presenting a possibility as a novel next generation diagnostic probe. In addition, the single stranded DNA aptamer of the present invention can greatly improve the stability and sensitivity of the existing diagnostic system, and thus can be effectively utilized for the early diagnosis of Salmonella typhimurium poisoning bacteria.

However, the effects of the present invention are not limited to the above-mentioned effects, and other effects not mentioned can be clearly understood by those skilled in the art from the following description.

1 is a graph showing the dissociation constant (K _D ) of single stranded DNA aptamer of SEQ ID NO: 4 of the present invention.
2 is a graph showing the binding specificity of the single stranded DNA aptamer of SEQ ID NO: 4 of the present invention to Salmonella typhimurium bacteria, wherein (a) is a graph showing the binding specificity of Salmonella typhimurium KCCM 11806, E. coli DH5a and Pseudomonas (B) is the result of comparing the binding specificities of Salmonella typhimurium KCCM 11806 and Salmonella enteritidis KCCM 12021. Fig.

First, terms used in the specification of the present invention will be described.

As used herein, the term "aptamer" refers to a DNA nucleic acid molecule capable of binding to a specific molecule with high affinity and specificity, and refers to a single-stranded DNA nucleic acid molecule.

Hereinafter, the present invention will be described in detail.

One. Salmonella typhimurium ( Salmonella Typhymurium ) Single strand DNA aptamer for detection

One aspect of the invention provides a single stranded DNA aptamer for detection of Salmonella typhimurium.

The single stranded DNA aptamer of the present invention comprises any one or more nucleotide sequences selected from the group consisting of SEQ ID NOS: 4 to 8.

The single-stranded DNA aepta dimmer Salmonella typhimurium to (Salmonella Typhymurium), more preferably specifically and selectively bind to Salmonella typhimurium KCCM 11806. In particular, the single stranded DNA aptamer does not bind to Salmonella typhimurium KCCM 11806 other than Salmonella typhimurium KCCM 11806 because it does not bind to other Salmonella bacteria.

The single-stranded DNA aptamer is preferably, but is not limited to, the nucleotide sequence of SEQ ID NO: 4.

Wherein the single stranded DNA aptamer is a DNA strand having at least one nucleotide sequence selected from the group consisting of SEQ ID NO: 4 to SEQ ID NO: 8 in which at least one of the individual nucleotides constituting the nucleotide sequence is chemically or physically modified . For example, any one or more of the individual nucleotides constituting any one or more of the nucleotide sequences selected from the group consisting of SEQ ID NOS: 4 to 8 may be selected from the group consisting of phosphorothioate DNA, phosphorodithioate DNA, DNA, amide-linked DNA, MMI-linked DNA, 2'-O-methyl RNA, alpha-DNA and methylphosphonate DNA, sugar modified nucleotides such as 2'-O-methyl RNA, 2'-fluoro 2'-O-alkyl DNA, 2'-O-allyl DNA, 2'-O-alkynyl DNA, hexose DNA, pyranosyl RNA and anhydrohexitol DNA, and a base Nucleotides with modifications such as C-5 substituted pyrimidines wherein the substituents are fluoro, bromo, chloro, iodo-, methyl-, ethyl-, vinyl-, formyl-, ethytyl-, propynyl -, alkenyl-, thioglyl-, imidazolyl-, pyridyl-), 7-deazapurines with C-7 substituents (substituents being fluoro-, Bromo, chloro, iodo-, methyl-, ethyl-, vinyl-, formyl-, alkynyl-, alkenyl-, thiazolyl-, imidazolyl-, pyridyl-, inosine or diamino Purine, and the like.

In addition, the single-stranded DNA aptamer may comprise a variant of any one or more of the nucleotide sequences selected from the group consisting of SEQ ID NOS: 4 to 8. The mutant is a nucleotide sequence having a sequence similar to that of any one or more nucleotide sequences selected from the group consisting of SEQ ID NO: 4 to SEQ ID NO: 8, but having functional similar to the nucleotide sequences of SEQ ID NO: May be a base sequence having a sequence homology of at least 70%, preferably at least 80%, more preferably at least 90%, and most preferably at least 95% with the nucleotide sequence of SEQ ID NO: 8.

The single stranded DNA aptamer may be labeled with a detectable detector. The detector is for easily identifying, detecting and quantifying whether the single-stranded DNA aptamer is bound to Salmonella typhimurium. It is used for detecting chromosomal enzymes such as chromogenic enzymes (peroxidase, alkaline phosphatase, etc.) (FITC, RITC, rhodamine, Texas Red, fluorescein, phycoerythrin, quantum dots), chromophore and radioactive isotopes ( ¹²⁴ I, ¹²⁵ I, ¹¹¹ In, ^99m Tc, ³² P, ³⁵ S, etc.). When the detector is tagged with the single-stranded DNA aptamer, it is preferable that the detector is attached so that the single-stranded DNA aptamer does not affect the specificity or selectivity for the target. To this end, Link (covalent bonding or crosslinking). The tagging position of the detector can be easily determined by a person skilled in the art through repeated experiments. In particular, the tagging position of the detector can be a 5'-terminal single stranded DNA aptamer.

The length of the single stranded DNA aptamer is not particularly limited and may be 30 to 200 nucleotides, preferably about 100 nucleotides or less, more preferably about 80 nucleotides or less, and most preferably 30 to 40 nucleotides Do. The smaller the total number of nucleotides constituting the single-stranded DNA aptamer, the easier and economical the chemical synthesis and the mass production, the higher the in-vivo stability and the lower the toxicity.

Typically, the single-stranded DNA aptamer can be obtained by in vitro selection for binding of the target Salmonella typhimurium, and a method for selecting an aptamer specifically binding to a target is disclosed in the art . In particular, the screening of aptamers can utilize in vitro or in vivo selection techniques known in the 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). In the SELEX method, by increasing the number of repetitions or using a competitive material, aptamer having a stronger binding property to the target substance can be concentrated and selected. Therefore, the aptamers having different binding strengths, the aptamers having different binding forms, and the aptamers having the same binding but binding forms but different base sequences can be obtained by controlling the number of repetitions or changing the contention state in the SELEX Bingbap. In addition, the SELEX method includes an amplification process by PCR, but mutation can be imparted by using manganese ions in the process, thereby making it possible to perform SELEX with a richer variety.

In particular, the single stranded DNA aptamer can be prepared by 1) amplifying a DNA aptamer using PCR and asymmetric PCR techniques, and selecting a single strand aptamer; And 2) selecting a DNA aptamer that binds to Salmonella typhimurium via the SELEX technique.

In the single-stranded DNA aptamer screening of step 1), asymmetric PCR can be performed to amplify only single-stranded DNA among double-stranded DNA amplified by PCR. Asymmetric PCR allows the single-stranded DNA to be obtained by performing PCR using only one of the forward primer and the reverse primer, for example, a forward primer.

Step 2) comprises a PCR and cloning step for selection of the Salmonella typhimurium-binding DNA aptamer candidate group in the SELEX round with optimal affinity; And DNA sequencing of selected Salmonella typhimurium-bound DNA aptamer candidates.

In a specific embodiment of the present invention, we have selected single-stranded DNA aptamers that specifically bind to Salmonella typhimurium, a food poisoning bacterium, in order to uncover novel diagnostic probes for food poisoning bacteria. First, the aptamer candidate single-stranded DNA was screened for Salmonella typhimurium KCCM 11806 from a chemically synthesized single-stranded DNA library using the CELL-SELEX method. Since the CELL-SELEX method uses cultured cells as target molecules, the separation and purification steps of specific molecules are omitted, and the structure of the molecules on the cell surface is preserved in a circular form, so that it is advantageous in diagnosis in a subsequent clinical application . After analyzing the nucleotide sequences of the selected aptamer candidate single-stranded DNAs, seven kinds of single-stranded DNA aptamers having specificity for the Salmonella typhimurium and having sequence similarity were selected (see Table 4).

Also, in a specific embodiment of the present invention, in order to confirm the binding ability of the single-stranded DNA aptamer selected as described above, the present inventors used single-stranded DNA aptamers tagged with FAM fluorescent substance to identify the binding ability of Salmonella typhimurium As a result of the measurement of the binding force, it was confirmed that the degree of binding of the single stranded DNA aptamer having the nucleotide sequence of SEQ ID NO: 4 was significantly higher (see FIG. 1). In addition, it was confirmed that the single-stranded DNA aptamer has specificity for binding to Salmonella, especially Salmonella typhimurium, more preferably Salmonella typhimurium KCCM 11806 (see Fig. 2).

Therefore, the single-stranded DNA aptamer consisting of the nucleotide sequences of SEQ ID NOS: 4 to 8 is useful as a new probe against food poisoning bacteria, particularly Salmonella typhimurium KCCM 11806.

2. Composition for detecting Salmonella typhimurium

Another aspect of the present invention provides a composition for detecting Salmonella typhimurium and a method for detecting Salmonella typhimurium using the same.

The composition of the present invention comprises a single-stranded DNA aptamer comprising any one or more base sequences selected from the group consisting of SEQ ID NOS: 4 to 8.

The detection method of the present invention comprises the steps of 1) contacting the composition with a sample suspected of being Salmonella typhimurium; And 2) confirming whether the Salmonella typhimurium and the DNA aptamer are bound to each other.

As for the single-stranded DNA aptamers are the same as described in the above "1. Salmonella typhimurium (Salmonella Typhymurium) detecting single-stranded DNA aptamer for the" item. Thus this regard the "1 Salmonella typhimurium (Salmonella Typhymurium) detecting single-stranded DNA aptamer for" using the original description of the item and the detailed description thereof will be omitted, in the following specific configuration to the sensing composition or a method for detecting .

The sample of step 1) is a target to be analyzed in which the target substance of interest, that is, Salmonella typhimurium, which is a food poisoning bacteria, is presumed to be present or exists and is a liquid, soil, air, food, feed, waste, It may be, but is not limited to, one or more samples. At this time, the liquid may be water, blood, urine, tears, sweat, saliva, lymph and cerebrospinal fluid, and the water may include rain water, seawater, lake water, , The waste includes sewage, wastewater and the like, and the animal or plant includes a human body. In addition, the animal or plant animal is not particularly limited as long as it provides a tissue that can be used for food, feed for plants and animals, and the like.

The binding of step 2) may be detected using a sensor connected to the DNA aptamer and a linker, or may be detected through reaction of a detector attached to the DNA aptamer. At this time, analysis of the detection body can be performed by a generally known detection body analysis method. For example, when a fluorescent substance is used as the detecting substance, Salmonella typhimurium present in the sample is labeled with the fluorescent substance. Therefore, Salmonella typhimurium can be detected by measuring the luminescence or color change of the fluorescent substance have.

3. Kit for detecting food poisoning bacteria

Yet another aspect of the present invention provides a kit for detecting food poisoning bacteria.

The kit for detecting food poisoning bacteria of the present invention comprises a single stranded DNA aptamer comprising any one or more nucleotide sequences selected from the group consisting of SEQ ID NOS: 4 to 8.

As for the single-stranded DNA aptamers are the same as described in the above "1. Salmonella typhimurium (Salmonella Typhymurium) detecting single-stranded DNA aptamer for the" item. Therefore, a detailed description thereof will be omitted because of the description of the item " 1. Single-stranded DNA aptamer for detection of Salmonella typhimurium & quot ;, and a detailed description thereof will be omitted. Hereinafter, Explain.

The food poisoning bacteria to be detected by the kit for detecting food poisoning bacteria may be Salmonella, and the food poisoning bacteria are more preferably Salmonella typhimurium, and the food poisoning bacteria are most preferably Salmonella typhimurium KCCM 11806, but are not limited thereto. The single-stranded DNA aptamer contained in the kit for detecting food poisoning bacteria does not bind to other non-Salmonella bacteria other than food poisoning bacteria such as Salmonella typhimurium, and thus it is possible to selectively detect food poisoning bacteria contaminated in foods, environmental samples or biological samples.

The kit for detecting food poisoning bacteria may further include reagents necessary for the hybridization reaction. The reagents necessary for the hybridization reaction may be those routinely used in the art, and are not particularly limited. In addition, the kit may include a user's manual describing optimal reaction performance conditions.

The food-borne disease detection kit may take the form of a bottle, a tub, a sachet, an envelope, a tube, an ampoule, and the like, which may be partially or wholly made of plastic, , Wax, and the like. The container may be part of it 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.

Hereinafter, the present invention will be described in detail by the following examples.

However, the following examples are illustrative of the present invention, and the contents of the present invention are not limited by the following examples.

Salmonella typhimurium ( Salmonella Typhymurium ) Selection of apt tamer specific to KCCM 11806

<1-1> Synthesis of Single Stranded DNA Library by PCR

First, a library of 60-mer long single-stranded DNAs represented by the nucleotide sequence of SEQ ID No. 1 below was prepared. The single-stranded DNA library was custom-made on Bioneer (Korea).

[SEQ ID NO: 1]

5'- GCGCGGATCCCGCGC - (N) ₃₀ - CGCGCGAAGCTTGCG -3 '

The underlined portion in SEQ ID NO: 1 is a sequence that is fixed as a portion to which a primer binds when PCR is performed, and the (N) ₃₀ portion is a portion where A, G, C and T bases are arbitrarily arranged. The sequence at which the bases are arbitrarily arranged determines the sequence of the aptamer molecules to be separated.

Asymmetric PCR was performed using the single-stranded DNA library prepared as described above as a template to amplify the DNA library.

The sequences of the primers used in the PCR are shown in Table 2 and Table 2 below. Herein, the underlined portions of the forward primer and the reverse primer sequence were used by inserting restriction enzyme sites ( BamH1 and HindIII ) for further gene recombination.

primer Base sequence SEQ ID NO: Forward primer 5'-GCGC GGATCC CGCGC-3 ' (Bam HI G / GATCC) 2 Reverse primer 5'-GCGC AAGCTT CGCGC-3 ' (Hind Ⅲ A / AGCTT) 3

The asymmetric PCR was carried out by performing PCR with different concentrations of the used primers to induce the production of single strand DNA. The asymmetric PCR was performed by adding a forward primer 10 times more than the reverse primer.

The amplified PCR product was electrophoresed on 2.5% agarose gel and visually confirmed.

<1-2> Extraction of single-stranded DNA library

The single stranded DNA library amplified in Example <1-1> was isolated by the Crush and Soak method. The PCR product obtained in Example <1-1> was electrophoresed on a 12% acrylamide gel to separate double strand and single strand DNA. After electrophoresis, the single strand DNA band was cut after staining the nucleic acid with EtBr. The digested gel was pulverized into Crush and Soak buffer (500 mM NH ₄ OAc, 0.1% SDS, 0.1 mM EDTA) to extract single-stranded DNA. The extract was centrifuged to separate the solid gel, and single strand DNA was purified through ethanol precipitation or the like to extract a single strand DNA library.

The single stranded DNA library thus extracted was quantified using a UV / Vis spectrophotometer and used in Cell-SELEX.

<1-3> Selection of aptamers binding to Salmonella typhimurium KCCM 11806

The single-stranded DNA library extracted in Example <1-2> was subjected to Cell-SELEX 10 times to react with Salmonella typhimurium KCCM 11806 under the conditions shown in Table 3 below. The cell-SELEX was centrifuged at the last step of each step and washed three times with wash buffer (50 mM Tris-HCl (pH 7.4), 5 mM KCl, 1 mM MgCl ₂ ) to obtain Salmonella typhimurium KCCM 11806 Unbound DNA aptamer was removed and only the DNA aptamer bound to Salmonella typhimurium KCCM 11806 was isolated.

One 2 3 4 5 6 7 8 9 10 Salt (NaCl) concentration (mM) 50 100 150 200 250 300 350 400 450 500 Number of cells () 10 ⁸ 10 ⁸ 10 ⁸ 10 ⁸ 10 ⁴ 10 ⁴ 10 ⁴ 10 ⁴ 10 ⁴ 10 ⁴ Library concentration (㎍) 2 2 2 2 One One One One 0.5 0.5 Library coupling time (minutes) 60 60 60 60 60 30 30 30 30 30

Selected single-stranded DNA aptamer sequencing and specific binding assays

<2-1> Sequence analysis of aptamers

And the nucleic acid sequences of single stranded DNA aptamers selected in Example 1 were analyzed by performing genetic recombination. First, the nucleic acid aptamer selected in the 10-times screening experiment as described in the above Example <1-3> was treated with a restriction enzyme designated in advance and inserted into a T-vector as a plasmid vector for E. coli to induce the junction. The ligated plasmid vector was transformed into E. coli and amplified. After the amplified plasmid vector was separated and purified, the sequence of inserted DNA aptamers was analyzed.

As a result, five new aptamer sequences specifically binding to Salmonella typhimurium KCCM 11806 were identified as shown in Table 4 below. In the five aptamer sequences, (A / T) GGG (C / A ) Sequences were commonly identified.

SEQ ID NO: order Poverty SEQ ID NO: 4 CCGATGTCCGTT AGGGC TCCTCCATAGATC 12 SEQ ID NO: 5 CGCACTGAAG TGGGA TACCGCCTAAACGGG 3 SEQ ID NO: 6 CAGACCGTA AGGGA TGCCGCCTATTCACCG 3 SEQ ID NO: 7 CGCACTGAAG TGGGA TACCGCCGGAACGG One SEQ ID NO: 8 CAGACCGTA AGGGA TGCCGCGACAACACC 2

<2-2> Measuring the bond strength of an aptamer

Of the five new aptamer sequences identified in Example <2-1>, FAM (carboxyfluorescein), which is a FITC-based fluorescent labeling substance, is typically attached to the 5'-terminal of the aptamer of SEQ ID NO: (0 μM, 1 μM, 3 μM, 5 μM, 8 μM, 11 μM and 14 μM) in a buffer solution (50 nM HEPES, 50 mM NaCl) 1.0x10 ⁸ Salmonella typhimurium KCCM 11806 was suspended in the binding buffer to which different concentrations of aptamers were added and cultured for 1 hour. Then, the fluorescence emission signal of the aptamer of SEQ ID NO: 4 at each concentration And the dissociation constant (K _D ) was analyzed to confirm the binding ability of the aptamer of SEQ ID NO: 4 to Salmonella typhimurium KCCM 11806.

As a result, it was confirmed that the aptamer of SEQ ID NO: 4 had a K _D value of 0.53 ± 0.11 μM, and that the aptamer of SEQ ID NO: 4 had a binding force of micromolar concentration (μM) (FIG.

<2-3> Identify the specific cohesion of the aptamer

In order to confirm the specificity of Salmonella typhimurium KCCM 11806 of the aptamer of SEQ ID NO: 4, Escherichia coli DH5α, Pseudomonas aeruginosa , and Salmonella enteric bacteria, which are the same gram negative bacterium, Salmonella Enteritidis KCCM 12021 was cross-reacted to confirm the binding of aptamers.

Concretely, FAM (carboxyfluorescein) of the FITC series was attached to the 5'-terminal of the aptamer of SEQ ID NO: 4, and the binding buffer (50 nM HEPES, 50 mM NaCl) 4 [mu] M and 8 [mu] M). 1.0 × ^{10 8} Salmonella typhimurium KCCM 11806, E. coli DH5a, Pseudomonas aeruginosa and Salmonella enteritidis were suspended in the binding buffer to which different concentrations of aptamers were added as described above, followed by incubation for 1 hour , The fluorescence emission signal of the aptamer of SEQ ID NO: 4 at each concentration was collected and the dissociation constant (K _D ) was analyzed to confirm the binding specificity of the aptamer of SEQ ID NO: 4 to Salmonella typhimurium KCCM 11806 .

As a result, the highest fluorescence emission signal of Salmonella typhimurium KCCM 11806 of the aptamer of SEQ ID NO: 4 was collected in the experiment group treated with the same concentration (Fig. 2).

From the above results, it can be seen that the DNA aptamers of SEQ ID NOS: 4 to 8 of the present invention selected by Cell-SELEX specifically bind to the surface of Salmonella typhimurium KCCM 11806.

While the present invention has been particularly shown and described with reference to exemplary embodiments thereof, it is to be understood that the scope of the invention is not limited to the disclosed exemplary embodiments. It will be possible to change it appropriately.

<110> Industry-University Cooperation Foundation Hanyang University <120> DNA aptamer specifically binding to Salmonella Typhimurium and          uses thereof <130> HY120089N <160> 8 <170> Kopatentin 1.71 <210> 1 <211> 60 <212> DNA <213> Artificial Sequence <220> <223> ssDNA library template sequence <400> 1 gcgcggatcc cgcgcnnnnn nnnnnnnnnnnnnnnnnnnnnnnnncgcgc gaagcttgcg 60                                                                           60 <210> 2 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> Forward primer for the library amplification <400> 2 gcgcggatcc cgcgc 15 <210> 3 <211> 15 <212> DNA <213> Artificial Sequence <220> <223> Reverse primer for the library amplification <400> 3 gcgcaagctt cgcgc 15 <210> 4 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Aptamer 1 <400> 4 ccgatgtccg ttagggctcc tccatagatc 30 <210> 5 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Aptamer 2 <400> 5 cgcactgaag tgggataccg cctaaacggg 30 <210> 6 <211> 30 <212> DNA <213> Artificial Sequence <220> <223> Aptamer 3 <400> 6 cagaccgtaa gggatgccgc ctattcaccg 30 <210> 7 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Aptamer 4 <400> 7 cgcactgaag tgggataccg ccggaacgg 29 <210> 8 <211> 29 <212> DNA <213> Artificial Sequence <220> <223> Aptamer 5 <400> 8 cagaccgtaa gggatgccgc gacaacacc 29

Claims (9)

SEQ ID NO: 4 to SEQ ID NO: including any one or more nucleotide sequences selected from a group consisting of 8, and Salmonella typhimurium (Salmonella Typhymurium) KCCM 11806 single stranded Salmonella typhimurium (Salmonella Typhymurium) for detection of specifically binding to DNA aptamer. The single stranded DNA aptamer for detecting Salmonella typhimurium according to claim 1, wherein the aptamer has the nucleotide sequence of SEQ ID NO: 4. The method according to claim 1, wherein the aptamer is tagged with at least one detection element selected from the group consisting of a chromogenic enzyme, a fluorescent substance and a radioisotope. The single stranded DNA app for detection of Salmonella typhimurium Tamer. delete A composition for detecting Salmonella typhimurium comprising the single stranded DNA aptamer of claim 1. 6. The composition for detecting Salmonella typhimurium according to claim 5, wherein the composition comprises DNA aptamer having the nucleotide sequence of SEQ ID NO: 4. A kit for detecting food poisoning bacteria comprising the single stranded DNA aptamer of claim 1. The kit for detecting food poisoning bacteria according to claim 7, wherein the kit comprises DNA aptamer having the nucleotide sequence of SEQ ID NO: 4. Contacting a sample suspected of having Salmonella typhimurium KCCM 11806 with the single stranded DNA aptamer of claim 1; And
And confirming whether the Salmonella typhimurium KCCM 11806 binds to the single stranded DNA aptamer.

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