KR100691799B1 - Method for identifying and analyzing certain molecules using by a single strand nucleic acid probe - Google Patents

Abstract

The present invention relates to a method for identifying and analyzing a specific substance using a single-stranded nucleic acid probe, and more specifically, a fixed region (X) -specific substance recognition region (V) -fixed region (Y) or fluorescent substance (F)- After preparing a probe which is a single-stranded nucleic acid consisting of a specific substance recognition site (V), and reacting the produced probe with a specific substance by the treatment of analytical reagent to prepare a specific substance-probe complex, and wash and isolate the complex Then, the present invention relates to a method for identifying and quantifying specific substances using a single-stranded nucleic acid probe consisting of dissociating the probe from the separated complex and searching for the dissociated probe. Specific substance-probe complex of the present invention, the degree of binding is determined according to the specificity and binding strength between them, and the amount of the complex is determined according to the specific material, the specific material is identified by analyzing the probe dissociated from the complex and quantitative changes You can check it. Accordingly, the present invention provides a very useful method that can search for and analyze a specific substance in a sample using a single stranded nucleic acid which is an aptamer.

Single Nucleic Acid Strands, Identification, Quantification, Analytical Methods

Description

Method for identifying and analyzing certain molecules using by a single strand nucleic acid probe             

1 is a schematic diagram for identifying Escherichia coli using a single-stranded nucleic acid probe and PCR instrument analysis of the present invention,

Figure 2 is a schematic diagram identifying the E. coli using the analysis of the single-stranded nucleic acid probe and fluorescence measuring apparatus of the present invention,

Figure 3 is an electrophoresis picture of the quantitative analysis of E. coli using the single-stranded nucleic acid probe and PCR instrument analysis of the present invention,

Figure 4 is a graph illustrating the quantitative analysis of E. coli using the analysis of the single-stranded nucleic acid probe and fluorescence measuring apparatus of the present invention.

The present invention relates to a method for identification and quantitative analysis of a specific substance, and more particularly, single-stranded nucleic acid, which is an aptamer, uses functions having aptamer or nucleic acid properties to form a complex with a specific substance according to a given environment. It relates to a simple and novel way of searching and analyzing specific substances.

The technology to search for and analyze the identification and quantitative change of a specific substance has been developed due to the development of physics and biochemistry, but the problems related to the use and maintenance cost, ease, accuracy, sensitivity, inspection time and process automation of existing methods or devices The need for efficient new methods and devices is very high. Analyzing specific substances is not an ultimate goal in itself, but rather a part of the whole process, which can be useful for microbial and viral identification, cell, protein, and organic material analysis, leading to medical, veterinary, environmental and food engineering. It is widely applied to agriculture and agriculture.

Identification and analysis of specific substances are frequently performed using physical and chemical properties, and analysis using specificity and affinity between materials is usually performed by methods developed based on immunological methods. An immunological method is a technique in which an antibody binds to a specific antigen in a sample to form a complex according to an antigen-antibody reaction, and then the antigen-antibody complex is analyzed using a labeled secondary antibody. For example, ELISA is a method of analyzing a specific antigen by immobilizing a primary antibody in a solid medium and reacting with a sample containing an antigen, followed by processing a labeled secondary antibody that recognizes the complex. When the labeled secondary antibody recognizes and binds to the primary antibody-antigen complex immobilized on the solid medium, the label binds to the solid medium. Probes used in ELISA are labeled with a fluorescent indicator, dioxygenin, horseradish peroxidase, alkaline phosphatase and the like.

Nucleic acids are linear multimers in which nucleotides are covalently linked, and nucleotides are small organic compounds that are phosphoric acid, sugars and purines (adenine or guanine) or pyrimidines (cytosine, thymidine, uracil). Nucleic acids exist in single- or double-stranded strands, which bind together by hydrogen bonds and interactions between nucleotides under specific physical conditions to form unique conformations, which are determined by single-stranded nucleotide sequences. . In general, nucleic acids such as deoxyribonucleic acid (DNA) and ribonucleic acid (RNA) are stores of information for expressing proteins with cell structure and enzyme activity, but in 1982, RNA forms a specific structure. Since the publication of its activity, there have been many reports on the structural properties of a nucleic acid and its specific function. Nucleic acid is composed of four base repeats to maintain a high diversity to form a large number of three-dimensional structure, these three-dimensional structure is stabilized by interacting with a specific material to form a complex.

Nucleic acid can act as a ligand to a specific substance containing a protein, with high binding capacity and specificity through a constant screening process and sequencing from a library of single stranded nucleic acids arranged in various nucleotide sequences. Nucleic acids that bind to specific substances are being selected. The method of selecting nucleic acid binding to a specific substance is called SELEX (Systematic Evolution of Ligand by Exponential enrichment), and the selected product nucleic acid is called aptamer (Tuerk C. and Gold L .; Science , 249 , pp505-510, 1990), SELEX selects molecules that can bind to nucleic acids in nature as well as to other biomolecules, including proteins that do not bind nucleic acids, with very high affinity. have. Techniques for identifying and analyzing specific substances based on the single-stranded nucleic acids thus selected include molecular beacons (Molecular beacon, Li JJ et al .; Biochem Biophys Res Commun., 292 (1) , pp31-40, 2002), Calorimetric method, Stojanovic MN, Landry DW . ; J. AM. CHEM. SOC., 124 , pp9678-9679, 2002), Electrochemiluminescence and enzymatic method, Bruno JG, Kiel JL .; Biotechniques 32 (1) , pp178-80, pp182-3, 2002), but the techniques for identifying and analyzing specific substances based on the aptamer and nucleic acid properties of single-stranded nucleic acids have been reported. There is no bar.

Accordingly, the present inventors reacted and washed a sample with aptamer, a single-stranded nucleic acid, and then dissociated the single-stranded nucleic acid from a specific substance-aptamer complex, and identified a specific substance and identified a quantitative change by a nucleic acid analysis technique. By this, the present invention was completed.

An object of the present invention is to identify and identify quantitative changes of a specific substance using aptamer or nucleic acid properties that form a complex with a specific substance in accordance with a given environment. It is to provide a system for searching and analyzing various biomolecules including microorganisms, cells, and proteins.

In order to achieve the above object, the present invention is (i) a single strand consisting of a fixed site (X) -specific material recognition site (V) -fixed site (Y) or fluorescent material (F) -specific material recognition site (V) Preparing a probe which is a nucleic acid; (Ii) a second step of preparing a specific substance-probe complex by reacting the specific substance with the probe prepared in the first step by treatment of the assay reagent; (Iii) a third step of washing and separating the specific substance-probe complex prepared in the second step; (Iii) a fourth step of dissociating the probe by treating the separated complex in 60-100 ° C. distilled water for 5-10 minutes; And (iii) provides a method for identifying and quantifying a specific substance using a single-stranded nucleic acid probe consisting of a fifth step of searching for the dissociated probe.

The specific substance includes bacteria, fungi, viruses, cell lines, tissues, proteins, carbohydrates, lipids, polysaccharides, glycoproteins, hormones, receptors, antigens, antibodies, enzymes, and the like.

In addition, the present invention is a probe based on the nucleotide sequence of the single-stranded nucleic acid, which is an aptamer for E. coli, single-stranded nucleic acid or 0.2% BSA assay reagent, E. coli-probe complex for use in the coupling reaction of the probe and E. coli An assay kit is provided for the identification and quantitation of E. coli, including the reagents used, forward primers, and reverse primers.

Hereinafter, the present invention will be described in more detail.

The present invention provides a method for preparing a probe which is a single-stranded nucleic acid consisting of a fixed site (X) -specific material recognition site (V) -fixed site (Y) or fluorescent material (F) -specific material recognition site (V). Stage 1; (Ii) a second step of preparing a specific substance-probe complex by reacting the specific substance with the probe prepared in the first step by treatment of the assay reagent; (Iii) a third step of washing and separating the specific substance-probe complex prepared in the second step; (Iii) a fourth step of dissociating the probe by treating the separated complex in 60-100 ° C. distilled water for 5-10 minutes; And (iii) provides a method for identifying and quantifying a specific substance using a single-stranded nucleic acid probe consisting of a fifth step of searching for the dissociated probe. As shown in FIG. 1, the probe forms a complex with a specific substance in the Select buffer, and then identifies and quantitatively analyzes a specific substance by analyzing a probe separated from the formed complex by a nucleic acid analysis technique. Samples can also be analyzed by measuring unbound probes.

The first step of the present invention is to prepare a single stranded nucleic acid probe. Specifically, the present invention provides a single-stranded nucleic acid probe consisting of a fixed site (X) -specific material recognition site (V) -fixed site (Y) or a fluorescent material (F) -specific material recognition site (V).

The present invention is a single-stranded nucleic acid probe (probe) is prepared in consideration of the sequencing sequence of the aptamer to recognize a specific substance and the analysis method at the search step, when the analysis by PCR (polymerase chain reaction) technique is fixed A single-stranded nucleic acid consisting of three parts (X) -specific material recognition site (V) -fixing site (Y), and when analyzed by fluorescence analysis method, fluorescent site (F) -specific material recognition site A single-stranded nucleic acid consisting of two parts (V) is prepared.

The specific substance recognition site (V) is based on the nucleotide sequence of the single-stranded nucleic acid selected by SELEX, preferably, is a single-stranded nucleic acid complementary to the target probe containing the site to recognize the specific substance, 16 to 60 bp Single-stranded nucleic acid having a nucleotide sequence of the present invention, among the aptamers (aptamers) are single-stranded nucleic acids selected by SELEX standard method (Bock LC et al .; Nature , 355 (6360) , pp564-6, 1992) in the present invention. It includes the nucleotide sequence of SEQ ID NOs: 3 to 26 analyzed by the inventors to isolate and analyze the single-stranded nucleic acid specifically binding to E. coli (Korean Patent Application No. 2002-0064027).

In case of using the PCR technique in the search step, the fixed region (X or Y) of the probe has a great influence on the degree of amplification of the probe, and its sequencing is very important. The fixed part of the probe consists of characteristic bases and must maintain an appropriate Tm value that binds to the primer during the PCR step. Accordingly, the degree of formation of the PCR product should be able to maintain its own uncontaminated signal value. The base sequence of the fixed region can basically design or jointly use one base sequence for each specific substance to be identified. Considerations in the design of a fixed region include length, base composition, inclusion of non-complementary sequences, and self-complementarity. In a preferred embodiment of the present invention, for example, the X sequence described by SEQ ID NO: 27 (5'-ATA CCA GCT TAT TCA ATT-3 ') and the Y sequence described by SEQ ID NO: 28 (5'-AGA TAG TAA GTG CAA TCT-3 ').

When the fluorescence measurement technique is used in the searching step, the labeling material (F) of the probe may be a radioactive isotope, a fluorescein (Fluorescein), a fluorescent material such as Cy3 or Cy5, or a biotin, and the like. Use The specific substance includes bacteria, fungi, viruses, cell lines, tissues, proteins, carbohydrates, lipids, polysaccharides, glycoproteins, hormones, receptors, antigens, antibodies, enzymes, and the like isolated therefrom. . The cell lines and tissues include those derived from prokaryotes or eukaryotes, and eukaryotes refer to humans, animals and plants. Preferably the specific substance is E. coli. Escherichia coli is a bacterium that resides in the intestines of warm-blooded animals and has a Gram-negative flagella. It is not usually pathogenic in the intestine, but enters the urethra or biliary tract, causing cystitis, pyelitis, and cholecystitis. In children, it causes acute enteritis. It is an indicator of fecal contamination and is often used for water quality tests.

In the second step of the present invention, a specific material-probe complex is prepared by reacting a specific material with a probe by treatment with an analyte. At this time, the reaction solution is composed of a salt composition, a component which prevents nonspecific binding, etc., in which the probe may bind well with a specific substance. The assay reagent comprises (i) 20-100 mM, preferably 50 mM Tris, hydrochloric acid (pH 7.4), (ii) for secondary structure stabilization of single-stranded nucleic acids for buffering against pH. The material contains potassium chloride, sodium chloride, and magnesium chloride at 0 to 200 mM, preferably 5 mM potassium chloride, 100 mM sodium chloride, 1 mM magnesium chloride, (iii) 0.05 to 0.2% sodium azide, and preferably A nucleic acid containing 0.1% sodium azide and (iii) inhibiting 0.1% to 0.3%, preferably 0.2% of bovine serum albumin or nonspecific binding for nonspecific binding inhibition, which is a nucleic acid determined in consideration of the specificity of the probe. . The assay reagent is a reagent comprising three or more of the four basic components mentioned above. Preferably, an assay reagent comprising a solution containing 50 mM Tris hydrochloric acid (pH 7.4), 5 mM potassium chloride, 100 mM sodium chloride, 1 mM magnesium chloride, 0.1% sodium azide and 0.2% bovine serum albumin is used. The reaction temperature is ideally carried out at a temperature lower than the SELEX operating temperature, preferably the reaction is carried out at a temperature of 20 to 37 ℃. In general, the protein and single-stranded nucleic acid is excessively treated to prevent nonspecific binding of the target probe, and preferably the yeast tRNA, salmon sperm DNA or human placental DNA of the target probe. DNA) can be used.

The third step of the present invention is the step of washing and separating the specific substance-probe complex prepared in the second step. This can be separated by various methods such as membrane filtration, adsorption or centrifugation. In the second step, when the sample as a specific substance is a microorganism and a cell, the probe solution is mixed with a selection buffer containing the sample and reacted at room temperature or 37 ° C. for 30 minutes, and then directly centrifuged or filtered using a 0.42 μm membrane. Thus, the complex may be washed with 0 to 1 × Celex buffer, distilled water or 0 to 500 mM EDTA solution to remove the probe according to the unbound probe and binding force, and then used for the dissociation reaction in the fourth step. In the case of protein, the probe, protein complex, and non-binding probe are separated by 0.45 μm nitrocellulose membrane or filtration method using PVDF (polyvinylidiene fluoride) filter, labeling protein or adsorption method performed by adsorbing protein sample onto ELISA plate. You may.

The fourth step of the present invention is the step of dissociating the probe from the specific substance-probe complex which is washed and separated. The probe is dissociated from the complex by treating the specific substance-probe complex obtained in the third step in distilled water having a temperature of 60 to 100 ° C. for 5 to 10 minutes.

Specific substance-probe complexes are determined by the specificity and binding strength between them, from which specific substances can be identified and quantified. The specific substance recognition site of the probe and the specificity and binding force of the specific substance determine the binding degree of the specific substance-probe complex, and the amount of the specific substance determines the amount of the specific substance-probe complex. Probes dissociated from the complex are determined by the amount of the specific substance-probe complex and probe analysis is determined by nucleic acid analysis techniques. Therefore, by analyzing the result of nucleic acid analysis, it is possible to confirm the kind and quantitative change of a specific substance.

The fifth step of the present invention is to search for dissociated probes, and various methods such as FIGS. 1 and 2 may be selected according to the type of probe.

Figure 1 is a measurement method based on the PCR technique, the E. coli and the probe is reacted, the E. coli-probe complex is separated by filtration to dissociate the probe, the dissociated probe is reverse with the forward primer corresponding to the fixed part of the probe By amplifying the primers, the amplification products are measured by various techniques including electrophoresis.

Figure 2 after the E. coli and the labeled probe is reacted, the E. coli-probe complex is separated by filtration to dissociate the probe, and the dissociated labeled probe is placed in a cubic (fluorescent tube). Mounted on the fluorometer, the light emitted from the lamp is irradiated to the cubic through an excitation filter (excitation filter). The fluorescence emitted from the cubic can be analyzed for a specific material by using a fluorescence value measured through an emission filter.

This method is useful as a system for searching and analyzing biomolecules including microorganisms, cells and proteins.

In addition, the present invention is a single-stranded nucleic acid produced by a probe containing a specific binding sequence for E. coli, single-stranded nucleic acid or 0.2% BSA assay reagent, E. coli-probe complex used in the coupling reaction of the probe and E. coli It provides an analysis kit for identifying and quantitating E. coli, including a membrane for separating the E. coli-probe complex, a solution used in dissociation of the E. coli-probe complex, a forward primer, and a reverse primer.

The present invention provides a system for searching and analyzing biomolecules including microorganisms, cells, and proteins in a sample using a single stranded nucleic acid probe.

The system for searching and analyzing the biomolecules is composed of a probe, an assay reagent, and a method for searching for a probe that is a single stranded nucleic acid of the present invention.

The biomolecules include microorganisms such as bacteria and viruses, cell lines, proteins or enzymes isolated therefrom, and the like.

The single-stranded nucleic acid probe refers to a single-stranded nucleic acid consisting of a fixed site-specific material recognition site-fixing site or a fluorescent material-specific material recognition site, and the related reagent is the selection buffer, 0.2% bovine serum albumin (BSA). It contains an analysis reagent containing a selection buffer or a single nucleic acid strand, the method of searching for a signal material can use a nucleic acid analysis technique and the obtained data can be analyzed and used in various bioinformatics methods, Preferred examples include fluorometry and PCR analysis, and biomolecules can be analyzed using the system for identification and analysis.

Hereinafter, the present invention will be described in more detail with reference to Examples. The following examples are merely illustrative of the present invention and are not intended to limit the scope of the present invention.

Example 1. Selection of single stranded nucleic acid

SELEX libraries, primers and single stranded nucleic acids were prepared as follows. The HPLC purified SELEX library contains a nucleotide sequence of 60 nucleotides randomly arranged between a primer binding sequence of 18 nucleotides (5'-ATA CCA GCT TAT TCA ATT (N) 60 AGA TAG TAA GTG CAA TCT-3 ': MWG). -Biotech AG). Double stranded or single-stranded forward primer (5'-ATA CCA GCT TAT TCA ATT-3 ') as depicted in SEQ ID NO: 1 and reverse primer (5'-AGA TTG CAC TTA CTA TCT-3') as depicted in SEQ ID NO: 2 When performing PCR (polymerase chain reaction) for the synthesis of strand DNA, it was used as a reaction primer. On the other hand, the fixed region of the single-stranded nucleic acid probe of the present invention is the X sequence described in SEQ ID NO: 27 (5'-ATA CCA GCT TAT TCA ATT-3 ') and the Y sequence described in SEQ ID NO: 28 (5'-AGA TAG TAA GTG CAA TCT-3 ').

The selection of aptamers, single-stranded nucleic acids for the target material, was performed by SELEX standard method (Bock LC et al., Nature , 355 (6360), pp564-6, 1992). Aptamers are oligonucleotides that show specific binding ability to a specific substance, and thus, a highly specific sequence can be obtained by repeating a selection process.

The single-stranded nucleic acid synthesized in the above was selected at a concentration of 10 ¹⁵ nucleotide sequences / 1 ml, and at a concentration of 200 pmol / 200 μl from the next time (50 mM TrisCl (pH 7.4), 5 mM KCl, 100). mM NaCl, 1 mM MgCl ₂ , 0.1% NaN ₃ ) was heated at 80 ° C. for 10 minutes, and then left on ice for 10 minutes. The reaction solution was prepared by adding 5 times yeast tRNA (yeast tRNA, Life Technologies) and 0.2% BSA (bovine serum albumin, Merck) of the used single-stranded nucleic acid. Single stranded nucleic acid was added to a selection buffer containing 10 ⁶ Escherichia coli and allowed to react at 37 ° C for 30 minutes. Single-stranded nucleic acid binding to E. coli was centrifuged and separated, and washed with 1 ml selection buffer (containing 0.2% BSA). Solution I (selection buffer, 10 mM EDTA) was treated with the reaction to separate the single stranded nucleic acid, followed by complete separation of the single stranded nucleic acid by phenol extraction and ethanol precipitation. In order to perform the second process, asymmetric PCR was performed on the single-stranded nucleic acid obtained by ethanol precipitation using a forward primer and a reverse primer to prepare a single-stranded nucleic acid, and then the above-described method was repeatedly performed. .

After performing the last 8 times of the Selex process, PCR was performed on the final isolated single-stranded nucleic acid using a forward primer and a reverse primer to synthesize double-stranded nucleic acid. After the PCR product was cloned into the T-vector to determine the nucleotide sequence, single-stranded nucleic acid E18 having high binding ability with E. coli was selected (SEQ ID NO: 20; Korean Patent Application No. 2002-0064027).

Example 2. Fabrication of Probes

Probes were prepared in consideration of the nucleic acid analysis method, and in the case of the PCR technique, a standard asymmetric PCR was performed with a forward primer and a reverse primer using a selected plasmid cloned with a single stranded nucleic acid as a template. Isolated 1 pg plasmid, PCR reaction solution [10 pM forward primer, 1 pM reverse primer, dNTP mixture (5mM dATP, 5mM CTP, 5mM dGTP, 5 mM dTTP)] by standard asymmetric PCR method 94 ℃ 30 seconds, 52 ℃ A probe was prepared by repeating 30 cycles under conditions of 30 seconds, 72 ° C. and 20 seconds. Probes were finally prepared by removing unbound primers according to the protocol of the PCR product purification kit (QIAquick PCR Purification Kit, Qiagen). In the case of the fluorescence measurement method, Cy-3 was labeled at the 5 'end of the selected aptamer by HPLC purification.

Example 3. Reaction

The reaction was carried out in three steps: first reaction, second reaction and third reaction. Samples were prepared by diluting E. coli (Promega) and standard methods (Sambrook J., Fritsh EF, and Maniatis T. 1989. Molecular cloning (2nd edition) .Cold spring Harbor Laboratory Press. 3: A.1-A.6 Colony forming unit (cfu) was measured.

In the first reaction, a 150 μl selection buffer (containing 0.2% BSA) containing the purified probe solution and the sample were mixed and reacted at 37 ° C. for 30 minutes. In the second reaction, the E. coli-probe complex was washed three times with a selective buffer by centrifugation and the reaction product was harvested by centrifugation for 15,000 × g and 5 minutes. Alternatively, the reaction solution was filtered using a 0.45 μm nitrocellulose membrane or a PVDF (polyvinylidiene fluoride) filter as a filtration method, and washed three times with a selective buffer. In the third reaction, in the case of centrifugation, the pellet was placed in a pellet containing a specific substance-probe complex in 1,000 µl of distilled water. In the case of filtration, the membrane was placed in 1,000 µl of distilled water and treated for 5 to 10 minutes at 60 to 100 ° C. It was.

Example 4 Exploration and Analysis

4-1. Search and Analysis by PCR Method

When the dissociated probe in Example 3 was analyzed by a PCR method, double stranded nucleic acid was synthesized by performing PCR using a forward primer and a reverse primer. Dissociated Probe, PCR Reaction Solution, 100 pM Forward Primer, 100 pM Reverse Primer, dNTP Mixture (5 mM dATP, 5 mM CTP, 5 mM dGTP, 5 mM dTTP) by 94 ° C 30 sec, 52 ° C 30 sec, 72 by standard PCR method Eight cycles were repeated at 20 ° C. for 20 seconds to synthesize double-stranded nucleic acids, followed by electrophoresis on 2% agarose gel.

As a result of electrophoresis, the band degree was formed in proportion to the amount of E. coli sample. The degree of binding of the E. coli-probe complex is determined according to the specificity and binding strength between the E. coli and the E. coli recognition site of the probe, and the amount of binding is determined according to the amount of E. coli. The amount of probe dissociated in the E. coli-probe complex determines the amount of PCR product. Therefore, by analyzing the degree of the band formed can be confirmed E. coli and quantitative changes.

As shown in FIG. 3, a band was formed in the probe and the E. coli reaction group as shown in FIG. 3, and the bend could not be confirmed as a result of testing the same method using Listeria monocytogenes ( ATCC # 19118) as a sample. . Also, as a result of comparing 100 cfu of E. coli and 10,000 cfu of E. coli, it was confirmed that the band degree was relatively higher than that of 100 cfu of E. coli 10,000 cfu. there was. In other words, the E. coli-probe complex is formed, the bandage density varies depending on the number of E. coli, which correlates with the number of microorganisms. Limits of detection of microbial testing using aptamers and PCR techniques are theoretically possible to detect even 1 cfu microorganisms, but in this example 100 cfu.

4-2. Exploration and analysis by fluorometry

When the dissociated probe was analyzed by a fluorimeter, fluorescence was measured by a fluorimeter (Turner Biosystem, Picofluor ^™ , Handheld Dual Channel Fluorometer). The instrument was set to zero experimental group containing only E. coli samples, and the experimental group in which the same amount of the probe was placed in the sample was set to 999, and then the comparison group in which the sample reacted with the probe was measured.

As a result of fluorescence analysis, the degree of fluorescence varied in proportion to the amount of specific substances. The degree of binding of the E. coli-probe complex is determined according to the specificity and binding strength between the E. coli and the E. coli recognition site of the probe, and the amount of binding is determined according to the amount of E. coli. The amount of probe dissociated from the E. coli-probe complex affects the degree of fluorescence. Thus, by analyzing the degree of fluorescence it is possible to confirm the E. coli and quantitative changes.

As shown in FIG. 4, the fluorescence was formed in the probe and the E. coli reaction group as shown in FIG. 4, and the fluorescence degree was 100 100 for the E. coli and 10,000 cfu. Higher fluorescence than cfu was found to correlate with cfu of E. coli. That is, the E. coli-probe complex was formed and the degree of fluorescence differs depending on the number of E. coli, which is correlated with the number of microorganisms and the number of microorganisms could be determined using this. The limit of detection of microbial testing using aptamers and fluorescence measurement technology is theoretically possible to detect even 1 cfu microorganism, but in this example is 100 cfu.

By using the above method, probes having recognition sites for various substances can be used to search for and analyze identification and quantitative changes of various substances in a sample.

In the present invention described above, the single-stranded nucleic acid, which is an aptamer, is capable of identifying identification and quantitative change of a specific substance by using aptamer properties or nucleic acid properties, which form a complex with a specific material according to a given environment. The method and system can be used to search and analyze microorganisms, cells, biomolecules including proteins, and the like, and ultimately find wide application in medicine, veterinary medicine, environmental engineering, food engineering, agriculture, and the like.

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Claims (8)

(Iii) a first step of preparing a probe that is a single stranded nucleic acid consisting of a fixed site (X) -specific material recognition site (V) -fixed site (Y) or a fluorescent material (F) -specific material recognition site (V); (Ii) specific substances and the above by treatment of analytical reagents consisting of 50 mM tris hydrochloric acid (pH 7.4), 5 mM potassium chloride, 100 mM sodium chloride or 1 mM magnesium chloride, 0.1% sodium azide, 0.2% bovine serum albumin or single stranded nucleic acid. A second step of preparing a specific substance-probe complex by reacting the probe prepared in the first step; (Iii) a third step of washing and separating the specific substance-probe complex prepared in the second step with 0-1 x Serex buffer or 0-500 mM EDTA solution; (Iii) a fourth step of dissociating the probe by treating the separated complex in 60-100 ° C. distilled water for 5-10 minutes; And (iii) a fifth step of searching for the dissociated probe by a polymerase chain reaction (PCR) technique or a fluorescence measurement technique. delete delete The method of claim 1, wherein the specific substance of the second step is at least one selected from the group consisting of bacteria, viruses, cell lines, tissues, proteins and glycoproteins isolated therefrom. The method of claim 4, wherein the specific substance is E. coli. delete delete delete

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