KR20160011511A - SINGLE STRANDED DNA APTAMERS SPECIFICALLY BINDING TO E. coli O157:H7 AND PRODUCTION METHOD THEREOF - Google Patents

Abstract

Disclosed are a single stranded DNA aptamer specifically binding to E. coli O157:H7 and a production method thereof. The method for producing the aptamer comprises systematic evolution of ligands by exponential enrichment (SELEX) and counter SELEX processes. The SELEX process includes the following steps: inducing E. Coli O157:H7-DNA conjugates by mixing a DNA library, including DNAs which have primer areas for PCR on both ends thereof and 40 randomly-arranged nucleotides on the center thereof, and the E. Coli O157:H7 with a conjugation buffer solution (S1); extracting the E. Coli O157:H7-DNA conjugates by centrifuging a mixture solution of the DNA library and the E. Coli O157:H7 (S2); separating DNAs from the Salmonella-DNA conjugate (S3); and amplifying the separated DNAs by polymerase chain reaction (S4). The counter SELEX process comprises the steps of: inducing non-target microorganism-DNA conjugates by mixing the amplified DNAs and a non-target microorganism with a conjugation buffer solution (S5); and extracting DNAs, which are not bound with the non-target microorganism by centrifuging a mixture solution of the concentrated DNAs and the non-target microorganism (S6). After repeating the SELEX process five times, the counter SELEX process is performed once. And then, alternative performance of the SELEX and counter SELEX processes is repeated twice.

Description

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a single-stranded DNA plasmid that specifically binds to E. coli O157: H7,

The present invention relates to a single-stranded nucleic acid aptamer which specifically binds to E. coli O157: H7 (E. coli O157: H7) food poisoning bacteria, and a preparation method thereof.

Food poisoning bacteria can cause food poisoning if they are infected with human body through corrupt vegetable, agricultural, marine products, various foodstuffs and food manufacturing process. Therefore, it is possible to detect these food poisoning bacteria more quickly and accurately in terms of improving food safety and hygiene environment There is a strong interest in developing technology. However, efficient rapid detection methods for food poisoning bacteria have so far suffered from the lack of an ideal ligand.

Rapid detection of foodborne pathogens by bioluminescence and reaction products of microbial metabolism results in relative lack of selectivity and thus can only be used as a general indicator for food poisoning bacteria. In addition, highly selective immunological methods and nucleic acid In addition, in the case of enzyme immunoassay and immunofluorescence measurement, there is a problem in the storage and distribution of the substance due to the instability of the antibody used. There is also a problem and the antibody has disadvantages in selectivity and sensitivity due to the cell wall of food poisoning bacteria.

On the other hand, Aptamer is a small molecule oligonucleotide that specifically and selectively binds to a target molecule. As is well known, nucleic acids are linear polyamides in which the nucleotides are linked by covalent bonds, and the nucleotides are composed of small organic compounds such as phosphoric acid, sugar, and purines (adenine or guanine) or pyrimidines (cytosine, thymidine and uracil). By interaction they form a unique steric structure, which is determined by the single strand base sequence.

Generally, nucleic acids such as DNA and RNA have been reported as a storage material for expressing proteins having activity such as cell structure and enzymes. However, since it has been reported that RNA has activity as an enzyme by forming a specific structure in 1982, There are a lot of reports about the structural characteristics and the specific functions accordingly. The nucleic acid is composed of repeats of four bases and maintains a high diversity to form a large number of three-dimensional structures. These three-dimensional structures interact with specific substances to form a complex and stabilize.

Because of these properties, the nucleic acid can act as a ligand for a specific substance containing a protein, and it is possible to select from a library combining single-stranded nucleic acids arranged in various base sequences, The binding force and the specificity can be used to produce aptamer that binds to a specific substance.

A method for screening a nucleic acid that binds to a specific substance is called SELEX (Systematic Evolution of Ligand by Exponential Enrichment). Through this SELEX, a protein capable of binding to a nucleic acid in a natural state, And molecules capable of binding with very high affinity to various biomolecules including those.

In recent years, various attempts have been made to diagnose and treat diseases using such aptamer. In particular, the use of platemer to detect the presence of various pathogens causing diseases can prevent disease in advance.

However, the aptamer generally binds not only to a single target substance but also to other substances having a similar structure, so that there is a problem that the accuracy of diagnosis of a disease or detection of a pathogen is poor.

It is an object of the present invention to provide a pathogenic bacterium causing food poisoning, in particular, a single stranded DNA plasmid that specifically binds to E. coli O157: H7.

It is still another object of the present invention to provide a method for producing a single stranded DNA plasmid that specifically binds to E. coli O157: H7.

According to one embodiment of the present invention, there is provided a single-stranded DNA stranded DNA having at least one base sequence of SEQ ID NO: 1 to SEQ ID NO: 21, which specifically binds to E. coli O157: H7.

According to an embodiment of the present invention, a SELEX and a counter SELEX process comprises a DNA library comprising DNAs having 40 to 60 bases randomly arranged in the center and having a primer region for PCR at both ends, (S1) of introducing E. coli O157: H7-DNA conjugate by mixing E. coli O157: H7 into a binding buffer solution; (S2) extracting the E. coli O157: H7-DNA conjugate by centrifuging a mixture solution of the DNA library and E. coli O157: H7; Isolating DNA from said Salmonella-DNA complex (S3); And amplifying the isolated DNA through a polymerase chain reaction (S4); (S5) of mixing the amplified DNA with a non-target microorganism into a binding buffer solution to induce a non-target microorganism-DNA conjugate; And a step (S6) of extracting DNA that is not bound to the non-target microorganism by centrifuging a mixture solution of the concentrated DNA and the non-target microorganism, and performing a counter SELEX process And repeating the SELEX process and the counter SELEX process alternately twice. The present invention also provides a method for producing an umbilical cord specifically binding to E. coli O157: H7.

According to the present invention, the binding buffer solution may be a phosphate buffer solution.

In addition, the non-target microorganism according to the present invention may be at least one of E. coli, Staphylococcus aureus, Salmonella enteritidis, and Salmonella typhimurium.

Hereinafter, the present invention will be described in more detail with reference to the accompanying drawings and embodiments. The following specific examples are described for illustrative purposes only, and the scope of the present invention is not limited by the following examples.

According to the present invention, there is provided a single-stranded DNA extramamer which specifically binds to E. coli O157: H7 without binding to E. coli or Staphylococci, which are food-borne bacteria similar to E. coli O157: H7, : H7 can be detected quickly and accurately.

1 is a diagram illustrating a process of manufacturing a single-stranded DNA extruder according to an embodiment of the present invention.
FIG. 2 is a diagram showing electrophoresis results of single-stranded DNA subjected to PCR.
FIG. 3 is a flow cytometric analysis result of a single-stranded DNA extruder according to an embodiment of the present invention.

1, a method of manufacturing a single-stranded DNA extruder according to an embodiment of the present invention will be described in detail.

Formation of DNA pool

For selection of single-stranded nucleic acids that specifically bind to E. coli O157: H7, a DNA library consisting of DNAs having nucleotide sequences randomly arranged at the center with a fixed sequence for primer binding at both ends as shown below 100).

5 '- [Fixed Sequence 1] -N40- [Fixed Sequence 2] -3'

Herein, the fixed sequence 1 and the fixed sequence 2 are fixed regions of the DNAs contained in the DNA library 100, and N40 is a base sequence in which 40 such A, G, T, and C bases are randomly arranged.

The FW primer used in the PCR can bind to the 5 'terminal fixed sequence 1 of the underlined bases of the above base sequence, and the RE primer of the PCR can fix the 3' terminal end of the underlined bases of the above base sequence Base linkage with SEQ ID NO: 2. In addition, the 5 ' -terminal and 3 ' -terminal fixed sequences each contain a specific nucleotide sequence that the restriction enzyme recognizes. According to one embodiment of the present invention, the 5'-terminal fixed sequence includes GAATCC, which is a recognition sequence of restriction enzyme EcoRI, and the 3'-terminal fixed sequence, GGATCC, which is a recognition sequence of restriction enzyme of BamHI have.

Single stranded DNA in the DNA library 100 was denatured by heat treatment at 95 占 폚 for 10 minutes and -20 占 폚 for 10 minutes before binding with the target microorganism 300, E. coli O157: H7. Single stranded DNAs 200 having a three-dimensional structure derived from this thermal denaturation constitute a single stranded DNA pool.

Screening single-stranded DNA with binding specificity (SELEX)

The single stranded DNAs 200 containing the random sequence were put into a high pressure sterilized binding buffer solution (phosphate buffered saline, pH 7.2) for 15 minutes at 121 캜 with the target microorganism 300, E. coli O157: H7 And the mixture is reacted at room temperature to induce binding. This binding buffer solution does not affect the activity of the target microorganism, E. coli O157: H7, so that the target microorganism 300, E. coli O157: H7, can bind to the single stranded DNAs 200 in optimal conditions.

And centrifuged at 10,000 g for 10 minutes to separate the target microorganism-single strand DNA conjugate (310) and unbound single strand DNA (210) in the mixed solution. Centrifugation is the preferred method of isolating the target microorganism-single stranded DNA conjugate (310) in a state that does not affect the living target microorganism as much as possible.

Immediately after centrifugation, the supernatant is removed to remove unbound single stranded DNA (210) that is not bound to the target microorganism. The target microbe-single stranded DNA conjugate 310 is present in the remaining mixed solution. Single stranded DNA of the target microorganism-single strand DNA conjugate (310) becomes the plasmid candidate DNA according to the present invention.

The single-stranded DNA was eluted from the target microorganism by adding the same volume of sterilized distilled water to the mixed solution containing only the microorganism-single stranded DNA conjugate 310, heated at 95 ° C for 10 minutes and heat-treated. The solution was centrifuged at 10,000g for 10 minutes Separate. The eluted plastomer candidate single-stranded DNA (220) floats on the upper layer of the solution after centrifugation, so only the supernatant is extracted to perform PCR amplification.

Since the amount of single-stranded DNA (220) bound to food poisoning bacteria was very small, the concentration of the template was varied in the range of 0.1% to 30% during the PCR amplification, and the gel elution Gel elution was performed to collect pure DNA bands, and SELEX was performed.

2 is a graph showing the results of DNA electrophoresis analysis before and after centrifugal separation.

C1 and C2 are the results of DNA analysis in the DNA library before the start of SELEX, S1-1 is the single-stranded DNA extracted from the first SELEX, S1-2 is the single-stranded DNA of S1-1 is concentrated 20 times using the centrifugal filter It is the result of one time analysis. S5-1 and S5-2 are the analysis results when the single-stranded DNA extracted from the 5th-order SELEX and the single-stranded DNA of S5-1 were 20-fold concentrated using a centrifugal filter.

As can be seen in FIG. 2, DNA bands become more apparent as the number of SELEXs increases, but become remarkably clear when subjected to a concentration process by a centrifugal filter. The DNA band (S1-2) enriched in the first SELEX is more apparent than the DNA (S5-1) not enriched in the fifth SELEX and the DNA band (S5-2) enriched in the fifth SELEX The band is clearly observed.

Thus, by concentrating DNA using a centrifugal filter in SELEX, the success rate of SELEX is significantly increased.

Single-stranded DNA was obtained by selectively amplifying only the single-stranded DNA bound in the primary SELEX and then selectively binding the target microorganism with the same procedure five times.

Excitation of competitor candidate single-stranded DNA binding to non-target microorganisms (counter-SELEX)

After the end of the fifth SELEX, a counter-SELEX was performed to exclude single-stranded DNA binding to non-target microorganisms.

In one embodiment of the present invention, the cells obtained by mixing Escherichia coli, Salmonella, Staphylococcus aureus and Campylobacter jejuni were subjected to counter-SELEX under the same conditions as those of SELEX.

Namely, the plasmid candidate single-stranded DNAs 220 are mixed with the non-target microorganism 400 and the binding buffer solution to induce the binding, and the non-target microorganism-single strand DNA conjugate 410 and the non- When the single-stranded DNA 230 is separated and PCR-amplified only by unbonded single-stranded DNA 230, it is possible to specifically bind to E. coli O157: H7 and to bind to non-target microorganisms such as E. coli and Staphylococcus aureus Single stranded DNA can be obtained.

After the first counter-SELEX, perform two SELEX and two counter-SELEX alternately. That is, after the first counter-SELEX, a sixth-order SELEX, a second-order counter-SELEX, a seventh-order SELEX, and a third-order counter-SELEX were sequentially performed.

Verification of binding specificity between platemaker and E. coli O157: H7

In order to confirm the binding specificity between the SELTEX and the SELTEX-developed SELTEX and E. coli O157: H7, the binding strength and selectivity were firstly determined using a fluorescence detector. After SELEC-counter SELEX was performed, cloning and sequencing were performed, and 28 kinds of nucleotide sequences were ligated to the 5'-terminal of FAM labeled with fluorescein. Then, 1.0 nmol plamma candidate and 10 ⁸ cfu of food poisoning bacteria were added to SELEX And the binding specificity was determined by analyzing the fluorescence intensity. Thus, only the final eight plambers among the 28 candidate groups were selected.

To confirm the binding specificity of the finally obtained single-stranded DNA plasmid and E. coli O157: H7, a flow cytometer was used. Fluorescently labeled tympanum was used to confirm the binding strength between E. coli O157: H7 and tympanic membrane during flow cytometry.

The nucleotide sequences of single-stranded DNA in which specific binding was confirmed by flow cytometry are shown in Table 1 below.

SEQ ID NO: Base sequence One TCGTG CAGCA GGGGC TGTGT CGCGG TCGGT AGTGC TGTGG TGCG 2 GCGTG CGGAG CCGGT ATGGG AGGTC TGGAT ATCTG CGGGG CGTG 3 TCGTG TCGGT GTCGT CCGGT GGCTG TGGGG CTGGG TGTTG CGCG 4 ACGTG CAGCA GGGGC TGTGT CGCGG TCGGT AGTGC TGTGG TGCG 5 GCGTG TCGGT GTCGT CAGGT GGCTG TGGGG CTGGG TGTTG CGCG 6 GCGTG TCGGT GTCGT CTGGT GGCTG TGGGG CTGGG TGTTG CGCG 7 GCGTG CAGCA GGGGC TGTGT CGCTG TGGGT ATTGG TGTGG TGCG 8 GCGTG CAGCA GGGGC TGTGT CGCGG TCGGT AGGGC TGTGG TGCG

As shown in FIG. 2 and FIG. 3, the result of flow cytometry analysis of E. coli O157: H7 bound to a fluorescently labeled tympanic membrane (FIG. 2) In comparison, in the case of the combination of platemer and other food poisoning bacteria, only a small amount of the fluorescence signal detection area in green appears in a small part, and in the case of the platemaker-E. coli O157: H7 complex, . Based on these results, specific binding of E. typhimurium with E. coli O157: H7 produced by the method of producing the platemer of the present invention can be confirmed.

100: DNA library 200: modified single stranded DNA
300: Target microorganism 400: Non-target microorganism

<110> Republic of Korea (Management: Rural Development Administraion) <120> SINGLE STRANDED DNA APTAMERS SPECIFICALLY BINDING TO E. coli          O157: H7 AND PRODUCTION METHOD THEREOF <130> RDK-14P001 <160> 8 <170> Kopatentin 2.0 <210> 1 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> aptamer specifically binding to E. coli O157: H7 <400> 1 tcgtgcagca ggggctgtgt cgcggtcggt agtgctgtgg tgcg 44 <210> 2 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> aptamer specifically binding to E. coli O157: H7 <400> 2 gcgtgcggag ccggtatggg aggtctggat atctgcgggg cgtg 44 <210> 3 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> aptamer specifically binding to E. coli O157: H7 <400> 3 tcgtgtcggt gtcgtccggt ggctgtgggg ctgggtgttg cgcg 44 <210> 4 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> aptamer specifically binding to E. coli O157: H7 <400> 4 acgtgcagca ggggctgtgt cgcggtcggt agtgctgtgg tgcg 44 <210> 5 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> aptamer specifically binding to E. coli O157: H7 <400> 5 gcgtgtcggt gtcgtcaggt ggctgtgggg ctgggtgttg cgcg 44 <210> 6 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> aptamer specifically binding to E. coli O157: H7 <400> 6 gcgtgtcggt gtcgtctggt ggctgtgggg ctgggtgttg cgcg 44 <210> 7 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> aptamer specifically binding to E. coli O157: H7 <400> 7 gcgtgcagca ggggctgtgt cgctgtgggt attggtgtgg tgcg 44 <210> 8 <211> 44 <212> DNA <213> Artificial Sequence <220> <223> aptamer specifically binding to E. coli O157: H7 <400> 8 gcgtgcagca ggggctgtgt cgcggtcggt agggctgtgg tgcg 44

Claims (4)

A single-stranded DNA plasmid having at least one base sequence selected from the group consisting of SEQ ID NO: 1 to SEQ ID NO: 8, which specifically binds to E. coli O157: H7. SELEX and Counter SELEX processes,
In the SELEX process,
A step of introducing E. coli O157: H7-DNA conjugate by mixing a DNA library containing DNAs having 40 to 60 bases randomly arranged at the center with PCR primer regions and E. coli O157: H7 in a binding buffer solution (S1);
(S2) of extracting the E. coli O157: H7-DNA conjugate by centrifuging a mixed solution of the DNA library and E. coli O157: H7,
Isolating DNA from said Salmonella-DNA complex (S3); And
Amplifying the isolated DNA through a polymerase chain reaction (S4); Lt; / RTI &gt;
The counter SELEX process includes:
Mixing the amplified DNA with a non-target microorganism in a binding buffer solution to induce a non-target microorganism-DNA complex (S5); And
Centrifuging the mixed solution of the concentrated DNA and the non-target microorganism to extract DNA not bound to the non-target microorganism (S6)
Wherein the SELEX process is repeated five times and then the counter SELEX process is repeated once, and the SELEX process and the counter SELEX process are alternately repeated. Gt; 3. The method of claim 2,
Wherein the binding buffer solution is a phosphate buffer solution. 3. The method of claim 2,
Wherein said non-target microorganism is at least one of Campylobacter jejuni, Staphylococcus aureus, and Salmonella.

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