JP2006020513A - Method for detecting nucleic acid - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for clearly and simply detecting a nucleic acid sequence rapidly and precisely in a good reproducibility without requiring complex operations. <P>SOLUTION: This method for detecting the nucleic acid sequence by using a liquid-passing filter, by performing an amplification reaction by using an oligonucleotide for a specific nucleic acid sequence amplification in a specimen solution containing the specific nucleic acid sequence, at the same time with or after the amplification reaction, bringing the above in contact with a bonded material-bonded with a second ligand capable of specifically bonding with the nucleic acid sequence and detecting the complex bonded with the bonded material comprises a process of bonding the first ligand part of the complex with a liquid-passing filter bonded with a first ligand-capturing agent, a process of bonding a labeled physiologically active substance with the second ligand bonded with the liquid-passing filter through the first ligand-capturing agent and the ligand part and a process of measuring the label bonded with the liquid-passing filter. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a method for detecting a nucleic acid sequence using a liquid-permeable filter as a solid phase carrier.

  Technologies for detecting specific nucleic acid molecules present in biological samples include analysis at the nucleic acid molecule level of proteins expressed and functioning in specific organs, research on protein expression control in nerve, brain or immune system information transmission, etc. It is extremely important in genetic diagnosis such as detection of mutant genes for genetic diseases, diagnosis of cancer, detection of virus-related genes.

  However, the conventional technique is complicated and includes many steps, and requires skill, so that a considerable amount of time is often required depending on the operation.

  In addition, since genetic diagnosis is used as a definitive diagnosis, errors are not allowed and rapidity is also required, so it must have high accuracy while maintaining rapidity. It's not enough.

  A typical technique for detecting a specific nucleic acid molecule present in a sample is a hybridization method. Nucleic acid hybridization analysis is a technique for detecting a very small number of target nucleic acids (DNA and RNA) from various types of nucleic acids using probes. In hybridization methods, highly sensitive reporters (enzymes, fluorescent dyes, Even if a radioisotope or the like is used, it is difficult to detect a target nucleic acid molecule having a low copy number (1-1000). Furthermore, since the hybridization method has a big problem of non-specific reaction, operations such as carrier blocking and removal of non-specific signal are indispensable for accurately detecting a specific nucleic acid molecule. Labor is required.

  In the past, attempts have been made to detect biologically specific reactions such as antigen-antibody reactions using a filter composed of glass fibers (see, for example, Patent Document 1), but a method for detecting a specific nucleic acid. There was no specific disclosure regarding.

JP-A-4-318462

  The present invention has been made in view of such circumstances, and is characterized by providing a nucleic acid detection method that is simpler in operation and improved in specificity as compared with conventional nucleic acid detection techniques.

  As a result of diligent research, the present inventors, as a method for simplifying the necessary washing operation in the above-described conventional method, by using a liquid-permeable filter, by simply dropping the reagent sequentially from above, The inventors have found that a specific nucleic acid sequence can be detected, and have completed the present invention.

That is, the present invention has the following configuration.
[1] Using the oligonucleotide for amplifying a specific nucleic acid sequence to which a first ligand is bound to a sample solution containing a specific nucleic acid sequence, the first specifically binds to the nucleic acid sequence simultaneously with and / or after the amplification reaction. In the method of detecting a nucleic acid sequence contained in a sample solution by contacting a conjugate to which two ligands are bound, and detecting the ligand conjugate bound to a specific nucleic acid sequence, the first ligand is captured. Dropping on the surface of the fluid-permeable filter to which the agent is bound to bind the first ligand portion of the complex to the ligand-trapping agent; A solution of a labeled physiologically active substance that binds to the ligand is dropped, and the physiologically active substance having this label is bound to the liquid-permeable filter via the first ligand-trapping agent and the ligand moiety. A step of binding to the second ligand; a step of removing the unbound labeled physiologically active substance by washing the fluid-permeable filter as necessary; and a step of measuring the label bound to the fluid-permeable filter. A method for detecting a nucleic acid sequence using a liquid-permeable filter.

[2] A method for detecting a nucleic acid sequence, wherein the first ligand bound to the oligonucleotide for amplifying a specific nucleic acid sequence is any one selected from the group consisting of an antigen, an antibody, a fluorescent substance, a luminophore, and an enzyme.

[3] A method for detecting a nucleic acid sequence, wherein the second ligand that specifically binds to the nucleic acid sequence is any one selected from the group consisting of an antigen, an antibody, a fluorescent substance, a luminophore, and an enzyme.

[4] A method for detecting a nucleic acid sequence, wherein the amplification reaction is any method selected from the group consisting of PCR, NASBA, LCR, SDA, RCA, TMA, LAMP and ICAN.

[5] A method for detecting a nucleic acid sequence, wherein the capture agent for the first ligand is any one selected from the group consisting of an antibody, an oligonucleotide, a receptor, a nucleic acid-specific binding substance, and avidin.

[6] A method for detecting a nucleic acid sequence, wherein the physiologically active substance that specifically binds to the second ligand is selected from the group consisting of an antibody, an oligonucleotide, a receptor, a nucleic acid-specific binding substance, and avidin.

[7] A method for detecting a nucleic acid sequence, wherein the label of a physiologically active substance is any one selected from the group consisting of a magnetic substance, an electron carrier, a fluorescent substance, a luminophore, and an enzyme.

[8] A method for detecting a nucleic acid sequence, wherein a fibrous liquid-permeable filter is used as the liquid-permeable filter.

[9] Oligonucleotide to which at least first ligand is bound, conjugate to which second ligand is bound, polymerase, first ligand-capture agent binding liquid-permeable filter, physiological activity to bind to labeled second ligand A specific nucleic acid detection kit containing a substance.

[10] A method for detecting a nucleic acid sequence, comprising using a fibrous liquid-permeable filter to which a ligand-trapping agent is bound via a spacer.

  The present invention provides a method capable of clearly and easily detecting a nucleic acid sequence in a sample. Since the method of the present invention does not require a complicated operation as in the conventional methods, results with good reproducibility can be obtained quickly and easily.

  Hereinafter, the present invention will be described in detail. A chromosome or a fragment thereof containing a specific nucleic acid sequence contained in a sample is not particularly limited as long as it is a target nucleic acid that carries information on a target gene. Examples of the target nucleic acid include viruses, pathogen-derived genes, Alu sequences, exons of genes encoding proteins, introns, promoters, and the like.

  The ligand-binding nucleic acid-specific conjugate in the present invention may be any reagent that specifically reacts with a nucleic acid, and generally an oligonucleotide to which a ligand that hybridizes with a nucleic acid sequence is bound is used.

  In the case of a reaction that replicates a nucleic acid, a primer extension reaction occurs using a target nucleic acid as a template by causing a primer, four types of deoxynucleoside triphosphates (dNTP), and DNA polymerase to act on a target nucleic acid that has been denatured into a single strand. The complementary strand of the sequence is synthesized.

  In the present invention, the ligand-binding nucleic acid-specific conjugate used may be present in the reaction in which the nucleic acid is amplified or may be added after amplification.

  After the reaction between the amplified nucleic acid and the ligand-binding nucleic acid-specific conjugate, it is added onto a fluid-permeable filter to which a ligand capture agent is bound. Further, by adding a labeled target nucleic acid-specific binding physiologically active substance, and preferably, a washing solution on the filter as needed, it is possible to detect the nucleic acid sequence by measuring the labeled substance bound on the filter.

  The amplification method in the present invention can be basically performed by using a conventional method. Usually, four types of chromosomes or fragments thereof containing a specific nucleotide polymorphism site denatured into a single strand are used. By acting with deoxynucleoside triphosphate (dNTP) and DNA polymerase and a set of primers, the target nucleic acid is amplified as a template.

  Examples of nucleic acid amplification methods include PCR, NASBA (Nucleic acid sequence-based amplification method; Nature 350, 91 (1991)), LCR (International Publication No. 89/12696, JP-A-2-2934), SDA (Strand Displacement Amplification: Nucleic acid research, Volume 16, 1691 (1992)), RCA (International Publication No. 90/1069), TMA (Transcription mediated amplification method; J. Clin. Microbiol. Volume 31, 3270 (1993)), LAMP (loop-mediated isothermal amplification method: J Clin Microbiol. 42: 1,956 (2004)), ICAN (isothermal and chimeric primer-initiated amplification of nucleic acids: Kekkaku. 78, 533 (2003)).

In particular, the PCR method repeats a cycle consisting of three steps of denaturation, annealing, and extension in the presence of a sample nucleic acid, four types of deoxynucleoside triphosphates, a pair of primers, and a heat-resistant DNA polymerase. In this method, the region of the sample nucleic acid sandwiched between the primers is exponentially amplified. That is, the sample nucleic acid is denatured in the denaturation step, each primer is hybridized with a region on the single-stranded sample nucleic acid complementary to each in the subsequent annealing step, and DNA is started from each primer in the subsequent extension step. A DNA strand complementary to each single-stranded sample nucleic acid serving as a template is extended by the action of the polymerase to form double-stranded DNA. By this one cycle, one double-stranded DNA is amplified into two double-stranded DNAs. Therefore, if this cycle is repeated n times, the region of the sample DNA sandwiched between the pair of primers is theoretically amplified ²ⁿ times. Since the amplified DNA region exists in a large amount, it can be easily detected by a method such as electrophoresis. Therefore, if a gene amplification method is used, it is possible to detect even a very small amount of sample nucleic acid that could not be detected in the past (one molecule is acceptable), which is a very widely used technique recently. .

Labels Labels used in the present invention include magnetic substances, electron carriers, enzymes, biotin, fluorescent substances, haptens, antigens, antibodies, radioactive substances, and luminophores. Examples of the magnetic material include iron oxide, chromium dioxide, cobalt, and ferrite. Examples of electron carriers include ferrocene, PQQ, and redox compounds. Examples of the enzyme include alkaline phosphatase and peroxidase. Examples of the fluorescent material include FITC, 6-FAM, HEX, TET, TAMRA, Texas Red, Cy3, and Cy5. Examples of haptens include biotin and digoxigenin. Examples of radioactive substances include ³² P and ³⁵ S. Examples of luminophores include ruthenium and aequorin. Any label that does not affect the nucleic acid detection reaction may be used. If it does not affect the reaction, it may be bound to any position of the oligonucleotide. The 3 ′ end and 5 ′ end sites are preferred.

Kit In the present invention, the kit includes at least an oligonucleotide to which a first ligand is bound, a conjugate to which a second ligand is bound, a polymerase, a first ligand-capturing agent-bound liquid-permeable filter, a labeled second A specific nucleic acid detection kit containing a physiologically active substance that binds to a ligand is included. The length of the oligonucleotide and primer in the present invention is 9 to 35 bases, preferably 11 to 30 bases.

  Thus, in the method of the present invention, after the reaction between the nucleic acid fragment amplified by the oligonucleotide for amplifying the specific nucleic acid sequence bound to the first ligand and the nucleic acid-specific conjugate bound to the second ligand, Add onto one permeable filter with a ligand capture agent attached. By adding on the liquid-permeable filter, the excess liquid falls down through the filter, and the detection target is bound to the pores on the filter and the upper part of the filter, so that the detection sensitivity is also improved.

  Further, a labeled second ligand-binding physiologically active substance, and if necessary, a washing solution is added onto the filter to remove the substance not captured by the filter, and then the nucleic acid sequence is measured by measuring the labeled substance bound on the filter. Can be detected. Therefore, according to the method of the present invention, a complicated detection operation as in the conventional method is unnecessary, and the target nucleic acid in the sample nucleic acid can be clearly detected.

The liquid-permeable filter may have any form as long as the liquid passes through the filter surface layer when the liquid is dropped on the filter, and examples thereof include woven fabric, non-woven fabric, and porous material. Examples of the filter material include glass, ceramic, metal, and plastic. Examples of the plastic include polyamide, polyamideimide, polyarylate, polyimide, polyetherimide, polyetheretherketone, polyethylene, polyethylene oxide, polyester, polycarbonate, Polystyrene, polysulfone, polyethersulfone, polyparamethylstyrene, polyallylamine, polyvinyl alcohol, polyvinyl ether, polyvinyl butyral, polyvinyl formal, polyphenylene ether, polyphenylene sulfide, polybutylene terephthalate, polypropylene, polymethylpentene, acrylic resin, methacrylic resin , Epoxy resin, xylene resin, guanamine resin, diallyl phthalate tree , Vinyl ester resin, phenol resin, unsaturated polyester resin, furan resin, polyimide, poly -p- hydroxybenzoic acid, polyurethane, maleic acid resins, melamine resins and urea resins resins.
Specific examples include glass fiber woven fabrics, glass fiber nonwoven fabrics, porous glass, porous ceramics (ceramic sintered bodies), metal sintered bodies, closed cell foamed resins, various fiber woven fabrics and nonwoven fabrics. .
Among these, glass fiber filters are preferred, those having an average fiber diameter of 0.3 to 2.0 μm are preferred, and those having an average fiber length of 0.5 to 2 mm are preferred. Specific examples of preferable glass fiber filters are described in JP-A-4-318462, and can be used as they are. Further, details of an example of a plastic filter that can be used are described in JP 2000-65832 A, and this technique can be used as it is.

The filter can be used in a container. The container is made of a liquid-impermeable material and has an opening for limiting the range to which the reagent is applied. The porous carrier is placed below the opening. And the absorption layer which absorbs the reagent which passed the filter is provided in the lower part of the filter. If necessary, a backflow prevention layer may be provided between the porous carrier and the absorption layer to prevent the reagent contained in the absorption layer from flowing back to the porous carrier. The liquid-impermeable material may be any substance that does not allow liquid to permeate. For example, resins such as polyethylene, polystyrene, ABS resin, vinyl chloride resin, polyamide resin, polypropylene, and polycarbonate are liquid-impermeable and easy to mold. Etc. are preferable. The absorbent layer is not particularly limited as long as it can absorb a liquid. Examples of the absorbent layer include cellulose having a high liquid absorbability, and a paper multi-layered body mainly composed of a cellulose derivative. Examples of the backflow prevention layer include a hydrophobic non-woven cloth sheet and a wave material. This technique is specifically described in Japanese Patent Application Laid-Open No. 2004-156985.
In addition, by using such a filter, it is possible to easily develop automatic detection.

  Hereinafter, based on an Example, this invention is demonstrated more concretely. However, the present invention is not limited to the following examples.

Example 1 Detection of Helicobacter pylori23srRNA (1) Synthesis of oligonucleotides for detecting Helicobacter pylori23srRNA Oligonucleotides having the nucleotide sequences shown in SEQ ID NOs: 1 to 3 by the phosphoramidite method using DNA synthesizer type 392 manufactured by PerkinElmer (Hereinafter referred to as oligos 1 to 3) were synthesized. The synthesis was carried out according to the manual, and the deprotection of various oligonucleotides was carried out with ammonia water at 55 ° C. overnight. Oligonucleotide purification was carried out on a Perkin Elmer OPC column. Alternatively, it was commissioned to a DNA synthesis consignment company (Japan Bioservice Co., Ltd., Sawaddy Co., Ltd., Proligo Japan, Sigma Genosis Co., Ltd., etc.).
Oligo 1 is a sense strand, and oligo 2 is an antisense strand, which are used in combination as an oligonucleotide for an amplification reaction. Oligo 3 is used as a probe. Oligo 1 and 2 are used after being labeled if necessary.

(2) Amplification of Helicobacter pylori23srRNA by PCR
Amplification reaction by PCR method Using a DNA solution extracted from a stomach biopsy material by the phenol-chloroform method as a sample, the following reagents were added, and Helicobacter pylori23srRNA was detected under the following conditions.

The 25μl solution containing the following reagents reagents were prepared.
rTaq DNA polymerase reaction solution Oligo 1 (5 ′ end labeled with biotin) 5 pmol,
5 pmol of oligo 2,
Oligo 3 (3 ′ end labeled with FITC) 15 pmol,
X10 buffer 2.5 μl,
2.5 μl of 2 mM dNTP,
₂ μl of 25 mM MgCl ₂
rTaq DNA polymerase 1.3U (manufactured by Toyobo)
Extracted DNA solution 100ng

Amplification conditions 95 ° C for 5 minutes,
95 ° C for 30 seconds,
67 ° C for 30 seconds,
72 ° C, 30 seconds (40 cycles)
72 ℃, 2 minutes,
95 ℃, 3 minutes,
15 ° C for 15 minutes.

(3) Detection using liquid-permeable filter 20 μl of the amplification reaction solution was added to 30 μl of a solution of POD-labeled anti-FITC antibody (manufactured by DAKO Cytomation) and reacted at room temperature for 5 minutes. As a result, the FITC-labeled oligonucleotide bound to the amplified Helicobacter pylori23s rRNA fragment and the POD-labeled anti-FITC antibody bind. When this reaction solution is added to a glass fiber nonwoven fabric filter to which avidin is bonded, Helicobacter pylori23srRNA amplified on the filter is captured. Next, after sequentially adding a cleaning solution and a POD substrate solution from the top, color development on the filter surface was visually confirmed. In addition, the glass fiber nonwoven fabric filter which the avidin couple | bonded was performed according to Example 1 of Unexamined-Japanese-Patent No. 2004-156985.

Example 2
Detection of Cytochrome P450 2D6 gene (1) Synthesis of oligonucleotide for detecting Cytochrome P450 2D6 gene The DNA synthesizer type 392 manufactured by PerkinElmer is used to have the nucleotide sequences shown in SEQ ID NOs: 4 to 6 by the phosphoramidite method. Oligonucleotides (hereinafter referred to as oligos 4 to 6) were synthesized. The synthesis was carried out according to the manual, and the deprotection of various oligonucleotides was carried out with ammonia water at 55 ° C. overnight. Oligonucleotide purification was carried out on a Perkin Elmer OPC column. Alternatively, it was commissioned to a DNA synthesis consignment company (Japan Bioservice Co., Ltd., Sawaddy Co., Ltd., Proligo Japan, Sigma Genosis Co., Ltd., etc.). Oligo 4 is a sense strand, and oligo 5 is an antisense strand, which are used in combination as an oligonucleotide for an amplification reaction. Oligo 6 is used as a probe. Oligos 4 and 5 are used by labeling as necessary.

(2) Amplification of Cytochrome P450 2D6 by PCR
Amplification reaction by PCR method Using a DNA solution extracted from a stomach biopsy material by the phenol-chloroform method as a sample, the following reagents were added, and Cytochrome P450 2D6 was detected under the following conditions.

The 25μl solution containing the following reagents reagents were prepared.
rTaq DNA polymerase reaction solution Oligo 4 (5 ′ end labeled with biotin) 5 pmol,
Oligo 5 5 pmol,
Oligo 6 (3 ′ end labeled with FITC) 15 pmol,
X10 buffer 2.5 μl,
2.5 μl of 2 mM dNTP,
₂ μl of 25 mM MgCl ₂
rTaq DNA polymerase 1.3U (manufactured by Toyobo)
Extracted DNA solution 100ng

Amplification conditions 95 ° C for 5 minutes,
95 ° C for 30 seconds,
60 ° C for 30 seconds,
72 ° C, 30 seconds (40 cycles)
72 ℃, 2 minutes,
95 ℃, 3 minutes,
15 ° C for 15 minutes.

(3) Detection using liquid-permeable filter 20 μl of the amplification reaction solution was added to 30 μl of a solution of POD-labeled anti-FITC antibody (manufactured by DAKO Cytomation) and reacted at room temperature for 5 minutes. As a result, the FITC-labeled oligonucleotide bound to the amplified Cytochrome P450 2D6 fragment binds to the POD-labeled anti-FITC antibody. When this reaction solution is added to a liquid-permeable filter to which avidin is bound, Cytochrome P450 2D6 amplified on the filter is captured. Next, after sequentially adding a cleaning solution and a POD substrate solution from the top, color development on the filter surface was visually confirmed.

  As described above, after the reaction between the amplified nucleic acid and the ligand-binding nucleic acid-specific conjugate, it is added onto a liquid-permeable filter to which a ligand capture agent is bound, and is further labeled with a target nucleic acid-specific binding physiologically active substance, The genotype could be clearly and easily determined easily and quickly by measuring the label bound to the filter by adding a washing solution on the filter as needed.

  As described above, the present invention provides a method that can clearly and easily detect a nucleic acid sequence in a sample. Since the method of the present invention does not require a complicated operation as in the conventional methods, results with good reproducibility were obtained quickly and easily.

Claims (10)

  A second ligand that specifically binds to the nucleic acid sequence simultaneously with and / or after the amplification reaction using the oligonucleotide for amplifying the specific nucleic acid sequence in which the first ligand is bound to a sample solution containing the specific nucleic acid sequence In a method of detecting a nucleic acid sequence contained in a sample solution by contacting a conjugate to which is bound, and detecting a complex with a conjugate to which a second ligand bound to a specific nucleic acid sequence is bound, Dropping the first ligand capturing agent onto the surface of the liquid-permeable filter to which the first ligand-capturing agent is bound to bind the first ligand portion of the complex to the ligand-trapping agent; the liquid-permeable filter; A solution of a labeled physiologically active substance that binds to the second ligand is dropped on the surface of the substrate, and the physiologically active substance having this label is passed through the first ligand-trapping agent and the ligand moiety. liquid Step of binding to a second ligand bound to Iruta; characterized by comprising the step of measuring the bound labeled liquid permeability filter, detection methods of nucleic acid sequences using liquid permeability filter.   The method according to claim 1, wherein the first ligand bound to the oligonucleotide for amplifying the specific nucleic acid sequence is any one selected from the group consisting of an antigen, an antibody, a fluorescent substance, a luminophore and an enzyme.   The method according to claim 1, wherein the second ligand that specifically binds to the nucleic acid sequence is any one selected from the group consisting of an antigen, an antibody, a fluorescent substance, a luminophore, and an enzyme.   2. The method according to claim 1, wherein the amplification reaction is any method selected from the group consisting of PCR, NASBA, LCR, SDA, RCA, TMA, LAMP and ICAN.   The method according to claim 1, wherein the capture agent for the first ligand is any one selected from the group consisting of an antibody, an oligonucleotide, a receptor, a nucleic acid-specific binding substance, and avidin.   The method according to claim 1, wherein the physiologically active substance that specifically binds to the second ligand is selected from the group consisting of an antibody, an oligonucleotide, a receptor, a nucleic acid-specific binding substance, and avidin.   The method according to claim 1, wherein the label of the physiologically active substance is selected from the group consisting of a magnetic substance, an electron carrier, a fluorescent substance, a luminophore, and an enzyme.   The method according to claim 1, wherein a fibrous liquid-permeable filter is used as the liquid-permeable filter.   Includes at least an oligonucleotide bound to the first ligand, a conjugate bound to the second ligand, a polymerase, a first ligand-capture agent binding liquid-permeable filter, and a physiologically active substance that binds to the labeled second ligand Specific nucleic acid detection kit.   The method according to any one of claims 1 to 9, wherein a fibrous liquid-permeable filter to which a ligand-trapping agent is bound via a spacer is used.