WO2013162026A1 - 核酸の増幅方法、および、増幅核酸の検出方法 - Google Patents
核酸の増幅方法、および、増幅核酸の検出方法 Download PDFInfo
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6844—Nucleic acid amplification reactions
- C12Q1/6853—Nucleic acid amplification reactions using modified primers or templates
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- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
- C12Q1/6813—Hybridisation assays
- C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
Definitions
- the present invention relates to a method for amplifying a nucleic acid and a method for detecting a nucleic acid amplified by the method.
- a method for specifically amplifying a target nucleic acid sequence has become a very important technique.
- methods for specifically detecting an amplification product obtained by the nucleic acid amplification method there is a method for detecting by fixing a nucleic acid fragment containing a target sequence to a solid phase.
- this method by capturing the target nucleic acid specifically on the solid phase, nonspecific nucleic acid sequences can be easily removed by washing or the like, and detection specificity can be increased.
- examples of the method for capturing the target nucleic acid on the solid phase include a method using an antigen-antibody combination capable of forming a specific bond or a ligand-receptor combination.
- Non-Patent Document 1 discloses a method for detecting a PCR product amplified using a primer in which the end of one primer is modified with biotin and the other primer is modified with a fluorescent substance. In this method, the PCR product is brought into contact with a solid phase consisting of streptavidin-agarose, bound to the solid phase by forming a streptavidin-biotin complex, and the fluorescence is measured to detect the target amplification product. Is possible.
- a probe composed of an oligonucleotide having a sequence complementary to the target nucleic acid is immobilized on the solid phase, and the target nucleic acid is immobilized via hybridization between the target nucleic acid and the probe.
- a method of indirectly immobilizing on a solid phase In this method, signal intensity is detected by hybridization.
- Such a nucleic acid analysis method can analyze a plurality of target sequences at once by changing the probe sequence.
- Patent Document 1 discloses a method of amplifying a single-stranded nucleic acid by nuclease treatment without performing heat treatment, but this is also complicated and has a problem of single-stranded spheroidization.
- nucleic acid detection methods there is a method based on chromatography described in Patent Document 2 as a method for detecting a target nucleic acid that is excellent in operability and is quick and simple.
- the step of extracting a gene from a cell, a virus or a bacterium, the step of fragmenting an arbitrarily extracted gene, and the step of detecting include an arbitrarily extracted gene or a fragment thereof on a single gene detection device.
- single-stranded nucleic acid is amplified by the NASBA method. The problems in using the single-stranded nucleic acid are as described above.
- Patent Document 3 and Patent Document 4 have a non-natural nucleic acid tag, hairpin structure, or pseudoknot structure that inhibits nucleic acid synthesis by DNA polymerase on the 5 ′ side of the primer region. Even after the PCR reaction, a single-stranded region remains on one side of the double-stranded nucleic acid. It is excellent in that an amplification product having a single-stranded region capable of hybridization at one end of a double-stranded DNA can be prepared only by performing a PCR reaction using a special primer.
- detection requires detection with a fluorescent label or surface plasmon resonance difference imaging, an expensive dedicated device is required, and there is a problem in terms of speed and simplicity.
- the present invention has been made in view of the above problems, and its purpose is to utilize a high degree of specificity of the hybridization method and reduce the time and process required for the PCR product detection process, and a special apparatus. It is an object of the present invention to provide a nucleic acid detection method and a nucleic acid detection device or kit for visual detection that is simple and highly accurate without the need for the above. Furthermore, until now, it was necessary to produce an expensive labeled tag for each target nucleic acid, and there was room for improvement in labor and cost.
- the present inventors use a primer set having a tag region that is not double-stranded in a nucleic acid amplification reaction linked to the 5 ′ end of each primer body.
- the target nucleic acid is amplified as a double-stranded nucleic acid having a single-stranded region at each of both ends, and the amplified fragment is bound to a solid phase having an oligonucleotide probe that can hybridize with one of the single-stranded regions.
- the present invention is to carry out a nucleic acid amplification reaction using a primer in which a tag region that is not double-stranded by a nucleic acid amplification reaction is linked to the 5 ′ end side to obtain a nucleic acid having a single-stranded region at both ends.
- the present invention relates to a method for amplifying nucleic acid.
- the tag region is preferably linked to the primer via a spacer.
- the spacer preferably contains a nucleic acid derivative.
- the nucleic acid derivative is a group consisting of L-type nucleic acid, 3-deoxy-2-hydroxy-dN, modified base nucleic acid, damaged base nucleic acid, phosphate binding site modified nucleic acid, RNA, 2′-OMe-N, and derivatives thereof It is preferable that it is at least 1 or more selected from.
- the L-type nucleic acid is preferably at least one selected from the group consisting of L-type DNA, L-type RNA, and derivatives thereof.
- 3-deoxy-2-hydroxy-dN is preferably linked to the primer by a 2'-5 'bond.
- the modified base nucleic acid comprises a chromophore or biotin.
- the chromophore is preferably at least one selected from the group consisting of pyrene, etheno, pyrrolo, perylene, fluorescein, FITC, Cy3, Cy5, TAMRA, dabsyl, cyanine, and derivatives thereof.
- the damaged base nucleic acid is at least one selected from the group consisting of abasic nucleotides, 5-hydroxymethyl-dN, and derivatives thereof.
- the phosphate binding site modified nucleic acid comprises phosphorothioate or a derivative thereof.
- nucleic acid derivative is linked to the primer by a 5'-5 'bond and linked to the tag region by a 3'-3' bond.
- the spacer comprises a non-nucleic acid derivative.
- the non-nucleic acid derivative preferably has a D-threoninol skeleton.
- At least one selected from the group consisting of azobenzene, biotin, EDTA, and chromophore is introduced into the D-threoninol skeleton.
- the chromophore is preferably at least one selected from the group consisting of pyrene, etheno, pyrrolo, perylene, fluorescein, FITC, Cy3, Cy5, TAMRA, dabsyl, cyanine, and derivatives thereof.
- the non-nucleic acid derivative is at least one selected from the group consisting of a carbon chain (C n ), a peg chain ((CH 2 CH 2 O) n ), a disulfide-containing chain (C n SSC n ), and a dithiol phosphoramidite.
- C n carbon chain
- peg chain ((CH 2 CH 2 O) n )
- disulfide-containing chain C n SSC n
- dithiol phosphoramidite a dithiol phosphoramidite.
- the present invention also relates to a nucleic acid detection method characterized by detecting a nucleic acid having a single-stranded region at both ends obtained by the nucleic acid amplification method.
- the labeling substance is preferably a colored carrier.
- nucleic acid detection device It is preferable to detect a nucleic acid having a single-stranded region at both ends on a nucleic acid detection device.
- the nucleic acid detection device is an array or chromatography.
- the tag region is not double-stranded by the nucleic acid amplification reaction, a nucleic acid having single-stranded regions at both ends and high detection efficiency by hybridization can be obtained.
- the nucleic acid amplification product can be specifically bound to the solid phase using the single-stranded region of the nucleic acid amplification product, and the labeled compound can be combined with the other single-stranded region.
- the nucleic acid amplification product can be easily and quickly visually determined without using a special apparatus. Furthermore, detection sensitivity is improved by detecting structurally stable double-stranded nucleic acids as compared to full-length single-stranded detection.
- the same single-stranded region can be added to any target nucleic acid by using an inexpensive joint primer.
- the same single-stranded region can be used for detection using the same label tag and device. In this case, it is not necessary to produce an expensive label tag for each target nucleic acid, and labor and cost can be greatly improved.
- FIG. 2 is an example of detection results of PCR amplification products by the nucleic acid chromatography-like strip of Example 1.
- FIG. 10 is a detection result of a PCR amplification product by the chromatography-like strip of Example 13.
- FIG. It is a conceptual diagram of the target 1 used in Example 14. It is the conceptual diagram of the target 2 used in Example 14.
- FIG. 10 is a conceptual diagram of the target 1 used in Example 14.
- the present invention is characterized in that a nucleic acid amplification reaction is performed using a primer in which a tag region that is not double-stranded by a nucleic acid amplification reaction is linked to the 5 ′ end side, and a nucleic acid having a single-stranded region at both ends is obtained.
- the present invention relates to a nucleic acid amplification method. Examples of the nucleic acid include DNA and RNA.
- the single-stranded region at both ends of the nucleic acid amplification product preferably contains natural nucleotides.
- the present invention relates to a method for detecting a nucleic acid comprising a step of detecting a nucleic acid having a single-stranded region at both ends produced by the above method.
- a nucleic acid having single-stranded regions at both ends can be obtained by performing a nucleic acid amplification reaction on a sample DNA serving as a template using a specific primer set.
- the nucleic acid having a single-stranded region at both ends is preferably a double-stranded DNA amplification fragment having a single-stranded region at both ends.
- the sample DNA is not particularly limited as long as it can be used as a template for nucleic acid amplification reaction.
- DNA derived from any biological sample such as blood, body fluid, tissue, oral mucosa, hair, nail, cultured cell, animal, plant, microorganism and the like can be used.
- the sample DNA may be genomic DNA, cDNA, mitochondrial DNA, chloroplast DNA, and the like.
- cDNA obtained after reverse transcription using RNA as a template can also be used. These DNAs may be appropriately selected according to the DNA fragment to be amplified. Further, the sample DNA does not need to be purified DNA, and can be directly applied to the nucleic acid amplification reaction without purifying cells or tissues containing the sample DNA.
- a double-stranded DNA amplification fragment having a single-stranded region at both ends must be a product obtained by a nucleic acid amplification method using two or more primers having a tag region that is not double-stranded in a nucleic acid amplification reaction.
- the single-stranded region at both ends of the double-stranded DNA amplified fragment is a region derived from a tag region that is not double-stranded by the nucleic acid amplification reaction among primers used for the nucleic acid amplification reaction.
- FIG. 1 shows nucleic acid amplification primers.
- This primer consists of a primer body region 1 and a tag region 2 that is not double-stranded by a nucleic acid amplification reaction on the 5 'side of the primer body portion. Further, a spacer region may be provided as the polymerase reaction inhibition region 3 between the primer main body region and the tag region.
- the double-stranded DNA amplified fragment includes a first primer set including a sequence that can hybridize to a template of a target nucleic acid, a primer having a common sequence that cannot hybridize to the template, and complementation of the common sequence.
- a product obtained by a nucleic acid amplification method using a second primer set that includes a primer that has a sequence that can hybridize with a sequence and a tag region that is not double-stranded in a nucleic acid amplification reaction is preferable.
- FIG. 2 shows primers constituting the first primer set for PCR.
- the first primer for PCR is composed of a primer body region 4 capable of hybridizing to a target nucleic acid template and a common region 5 having a sequence common to the second primer on the 5 ′ side of the primer body region. It is characterized by.
- FIG. 3 shows primers constituting the second primer set for PCR.
- the second primer includes a primer main body region 6 having a sequence common to the first primer, and a tag region 7 that is not double-stranded by a nucleic acid amplification reaction on the 5 ′ side of the main body region 6.
- a spacer region may be provided as the polymerase reaction inhibition region 8 between the second primer main body region and the tag region.
- the primer main body region means an oligonucleotide region having a base sequence that can function as a primer in a nucleic acid amplification reaction. Specifically, it is a sequence that can hybridize with the 5 ′ end side or 3 ′ end side in the target base sequence of the target nucleic acid, and generally, it is the 5 ′ end side or 3 ′ end side of the target base sequence.
- the base sequence is complementary to the base sequence.
- These primer main body regions may have base deletions, insertions, and mismatch sites as long as they can specifically bind to the target nucleic acid.
- the length of the primer main body region is preferably 8 bases or more, more preferably 12 bases or more, and further preferably 15 bases or more. In addition, there is no particular upper limit to the primer chain length, but from the viewpoint of the synthesis cost and the like, those having 50 bases or less, or 40 bases or less are usually preferred.
- the tag region of the primer preferably contains natural nucleotides.
- Natural nucleotides are nucleotides composed of natural adenine, thymine, guanine, cytosine, uracil base, deoxyribose, ribose sugar moiety, and phosphate group, and each part is artificially modified. A nucleotide that has not been received. Natural nucleotides may be D-type nucleotides or L-type nucleotides. The D-type nucleotide refers to a nucleotide composed of D-type deoxyribose or ribose.
- the L-type nucleotide refers to a nucleotide composed of L-type deoxyribose or ribose.
- a natural nucleotide in the tag region By including a natural nucleotide in the tag region, there is an effect that synthesis is cheap and easy.
- the ratio of the natural nucleotide in the tag region of the primer is preferably 5% or more, more preferably 20% or more, further preferably 50% or more, and preferably 70% or more. Even more preferably, it is most preferably 90% or more.
- the length of the tag region is not particularly limited as long as it has a sufficient length to hybridize with a complementary strand nucleic acid. Usually, it is 5 to 60 bases, preferably 6 to 40 bases.
- the orientation of the nucleic acid in the primer tag region is preferably arranged in the same direction as the primer body region.
- the direction of the nucleic acid in the tag region of the primer in the same direction as the primer main body region, there is an effect that the synthesis is cheap and easy.
- the tag region and the primer body region must be arranged in the same direction even if they are not directly connected. Is preferred.
- the nucleic acid is in the same direction means that adjacent nucleotides are not phosphodiester-bonded between the 5′-position and the 3′-position, not between the 3′-position and 5′-position of the sugar in the nucleotide. It means being.
- the nucleotides are also located at the 5′-position and the 3′-position of the sugar in the main body region. It is formed between.
- the spacer region includes a spacer that inhibits the elongation reaction by the polymerase, and can inhibit the elongation reaction by the polymerase during the nucleic acid amplification reaction and keep the tag region in a single-stranded structure.
- the structure of the spacer is not particularly limited as long as it can inhibit the elongation reaction by polymerase, but preferably contains a nucleic acid derivative or a non-nucleic acid derivative.
- the nucleic acid derivative is not particularly limited as long as it can inhibit the elongation reaction by polymerase and keep the tag region in a single-stranded structure.
- a nucleic acid derivative it has a three-dimensional structure that inhibits the progress of polymerase, such as a nucleic acid that forms an inverted sequence structure such as a 5′-5 ′ bond or a 3′-3 ′ bond, and a strong hairpin structure or pseudoknot structure.
- Nucleic acid, L-type nucleic acid, 3-deoxy-2-hydroxy-dN, modified base nucleic acid, damaged base nucleic acid, phosphate binding site modified nucleic acid, RNA, 2′-OMe-N, BNA (LNA), and derivatives thereof Is mentioned.
- the 5'-5 'bond or the 3'-3' bond is a compound represented by the chemical formula (1)
- the number of inversion structures may be included at least once, and is not particularly limited, but is preferably included even times. If it has an inverted structure of even number of times, the end of the tag region will be 5 'like the normal primer, so non-specific extension reaction from the tag region can be suppressed and effective at the time of detection. is there.
- Halpin structure and “pseudoknot structure” mean a stable loop structure formed by pairing with another single-stranded region in the same molecule.
- L-type nucleic acid has chemical formula (2)
- deoxyribose or ribose which is a sugar constituting a nucleic acid
- L-type DNA is L-type DNA, L-type RNA, and derivatives thereof having a structure of an optical isomer with the natural D-type. Since L-type nucleic acid is not recognized by a commonly used DNA polymerase, it does not function as a template for an extension reaction. Since L-type DNA forms a left-handed double helix structure, it does not form a hybrid with a naturally occurring D-type nucleic acid and can form a hybrid only with L-type nucleic acids.
- 3-deoxy-2-hydroxy-dA deoxyribose does not have a hydroxyl group at the 3 ′ position, and 2′-5 between the 2 ′ position and the adjacent deoxyribose 5 ′ position. 'We are joined by joining. Therefore, since it is not recognized by DNA polymerase, it does not function as an extension reaction template.
- 3-deoxy-2-hydroxy-dN is preferably linked to the primer by a 2'-5 'bond.
- the modified base nucleic acid is a nucleic acid having a modification such as biotin (biotin) or chromophore at the base site of DNA.
- biotin biotin
- chromophores include, but are not limited to, pyrene, etheno, pyrrolo, perylene, fluorescein, FITC, Cy3, Cy5, TAMRA, dabsyl, cyanine and the like.
- modified base nucleic acids include chemical formula (5)
- Biotin-dT represented by the chemical formula (9)
- Damaged base nucleic acids are abasic nucleotides (AP sites: depurine bases, depyrimidine bases), chemical formula (17)
- a nucleic acid having an abasic or modified base such as Abasic or 5-hydroxymethyl-dN. Since they are not recognized by commonly used DNA polymerases, they do not function as templates for extension reactions.
- the phosphate binding site-modified nucleic acid has the chemical formula (19)
- a part of the phosphate group of the nucleic acid is replaced with another atom or molecule, such as phosphorothioate (S oligo), and it is not recognized by DNA polymerase, so it does not function as an extension reaction template.
- S oligo phosphorothioate
- RNA has the chemical formula (20)
- the sugar constituting the nucleic acid is composed of ribose and is not recognized by a commonly used DNA polymerase, it does not function as a template for the extension reaction.
- the sugar moiety constituting the nucleic acid is modified and is not recognized by DNA polymerase, so it does not function as a template for the extension reaction.
- the D-threoninol skeleton has the chemical formula (22)
- nucleic acids are linked with threoninol, and various molecules can be inserted into the amino group of threoninol.
- chromophores such as pyrene, etheno, pyrrolo, perylene, fluorescein, FITC, TET, HEX, JOE, Cy3, Cy5, Dabcyl, cyanine, BHQ , Biotin, EDTA, and other chemical formulas (23)
- the fatty chain as indicated by C n , shows a structure in which carbon chains are linked and its derivative.
- the number of n is not particularly limited, but is preferably 1 to 45, more preferably 2 to 18.
- the peg chain indicates a structure in which polyethylene glycols represented by (CH 2 CH 2 O) n are linked and derivatives thereof.
- the number of n is not particularly limited, but is preferably 1 to 21, and more preferably 1 to 9.
- the disulfide-containing chain is one having a disulfide bond structure represented by C n SSC n .
- the number of n is not particularly limited, but is preferably 1 to 20, and more preferably 1 to 12.
- PNA is a molecule having a peptide structure in the main chain and a structure similar to DNA or RNA, and N- (2-aminoethyl) glycine is bonded to the main chain by an amide bond.
- a purine ring or pyrimidine ring corresponding to the nucleobase is bonded to the main chain via a methylene group and a carbonyl group.
- nucleic acid artificially synthesized by cross-linking the sugar structure of DNA or RNA.
- the tag region consists only of natural nucleotides and the orientation of the nucleic acid in the tag region is the same as that of the primer body region, a spacer that inhibits the polymerase reaction is usually required between the tag region and the primer region.
- the tag region contains L-type nucleic acid, PNA, BNA, etc., and does not become a template for the reaction by DNA polymerase and is not double-stranded after the nucleic acid amplification reaction, the spacer that inhibits the polymerase reaction may be omitted. Is possible.
- the primer of the present invention has a stable loop structure such as an inverted structure, a hairpin structure, a pseudoknot structure, an L-type nucleic acid, 3-deoxy-2-hydroxy-dN, a modified base nucleic acid, a damaged base nucleic acid, a phosphate bond Site-modified nucleic acid, RNA, 2′-OMe-N, nucleic acid derivatives such as BNA (LNA), and non-nucleic acid derivatives such as carbon chain, peg chain, disulfide-containing chain, PNA, etc. Alternatively, a plurality of them may be combined.
- a stable loop structure such as an inverted structure, a hairpin structure, a pseudoknot structure, an L-type nucleic acid, 3-deoxy-2-hydroxy-dN, a modified base nucleic acid, a damaged base nucleic acid, a phosphate bond Site-modified nucleic acid, RNA, 2′-OMe-N, nucleic acid derivatives such as BNA
- Primers can also be labeled with various molecules commonly used for labeling oligonucleotides.
- molecules include enzymes, magnetic particles, fluorescent dyes, radioisotopes and the like. These may be used alone or in combination.
- the method for producing the designed primer is not particularly limited, and can be produced by a known method. Specifically, the designed primer can be easily obtained by using a DNA synthesizer or using a commissioned synthesis service.
- the nucleic acid amplification method is not particularly limited as long as it can obtain a nucleic acid having a single-stranded region at both ends by using the above-described primers.
- An example is PCR.
- isothermal amplification methods such as LAMP method and ICAN method can also be used.
- PCR When PCR is used as the nucleic acid amplification method and the tag region and the primer are linked via a spacer, as a combination of reverse primer and forward primer used for PCR, different spacers may be used for both primers. Alternatively, a spacer may be used for the other, without introducing a spacer on the other side, and the 5 ′ end of the primer may be modified with biotin or the like.
- thermostable DNA polymerase examples include, but are not limited to, Ex-Taq (manufactured by Takara Bio Inc.), KOD Plus (manufactured by Toyobo Co., Ltd.), Phusion, PrimeSTAR, KOD FX, Tks Gflex, and the like.
- the PCR reaction conditions such as temperature, time, buffer composition, etc. are not particularly limited, and may be set as appropriate according to the selected DNA polymerase, primer sequence, length of the target sequence, and the like.
- the length of the DNA amplified by the nucleic acid amplification reaction is preferably 20 bases or more, and more preferably 40 bases or more. If it is less than 20 bases, it becomes difficult to design a primer having sufficient specificity, and nonspecific amplification tends to increase.
- FIG. 4 shows a schematic diagram of an amplification reaction when a primer composed of a primer body region and a tag region is used as an example of the amplification reaction.
- the forward primer 10 has a primer main body region 11 having the same sequence as a part of the 5 ′ end side of the target nucleic acid sequence 9 and a tag region 12 on the 5 ′ end side thereof.
- the reverse primer 13 has a primer main body region 14 composed of a sequence complementary to a part of the 3 ′ end side of the target nucleic acid sequence, and a tag region 15 on the 5 ′ end side thereof.
- the sequence of the tag region that binds to both primers preferably has a different sequence.
- the tag region added to both primers does not substantially participate in the PCR reaction, and thus a DNA amplification product 16 having single-stranded regions at both ends is obtained.
- the amplified DNA fragment having a single-stranded region at both ends includes a double-stranded DNA portion identical to the target DNA region, and a single-stranded portion as a tag portion at each 5 ′ end on both sides thereof. It means a DNA amplification product having a region. That is, the DNA amplification fragment will be described in more detail.
- a double-stranded DNA amplification fragment having a single-stranded region composed of a nucleic acid not modified at both ends is shown, and both ends are single-stranded. Each region has a sequence in the same direction as the continuous DNA strand.
- FIG. 5 shows, as an example of the amplification reaction, a schematic diagram of the amplification reaction when a primer composed of a primer body region and a common sequence region and a primer composed of a common sequence and a tag region are used as a joint primer set.
- An amplification product in which single-stranded regions are added to both ends of the target nucleic acid sequence can be obtained by performing PCR by a conventional method using the first and second primer sets.
- the forward first primer 18 has a primer main body region 19 having the same sequence as a part of the 5 ′ end side of the target nucleic acid sequence 17 and a common sequence region 20 on the 5 ′ end side thereof.
- the reverse first primer 21 has a primer main body region 22 composed of a sequence complementary to a part of the 3 ′ end side of the target nucleic acid sequence, and a common sequence region 23 on the 5 ′ end side thereof.
- the common region sequences added to both primers preferably have different sequences.
- the forward second primer 25 in the common sequence region at both ends of the DNA amplification product 24 includes a primer body region 26 having a sequence common to a part of the 5 ′ end side of the double-stranded DNA amplification product 24 having the common region, A tag region 27 is provided on the 5 ′ end side.
- the reverse second primer 28 includes a primer body region 29 having a complementary sequence common to a part of the 3 ′ end side of the double-stranded DNA amplification product 24 having the common region, and a tag region on the 5 ′ end side thereof. 30.
- the sequence of the tag region that binds to both primers preferably has a different sequence.
- the tag region added to both primers does not substantially participate in the PCR reaction, and thus a DNA amplification product 31 having single-stranded regions at both ends is obtained.
- the PCR reaction using the first primer and the PCR reaction using the second primer are carried out continuously as shown in FIG. 5, but the order is to add the first primer and the second primer simultaneously. May be. Alternatively, the second primer may be added later.
- a DNA amplification fragment having a single-stranded region at both ends is a double-stranded DNA portion identical to the target DNA region as shown by 31 in FIG. 5 and a tag portion at each 5 ′ end on both sides thereof. It means a DNA amplification product having a double-stranded region.
- the same primer can be used as the second primer by keeping the common sequence the same, and the same single strand It is possible to create a tag array.
- the DNA amplified fragment will be described in more detail. A double-stranded DNA amplified fragment having a single-stranded region composed of a nucleic acid not modified at both ends is shown, and the single-stranded region at both ends is Each has a sequence in the same direction as the continuous DNA strand.
- a hybridization complex is formed using a single-stranded region of the amplification product obtained using the primer.
- Hybridization means that a molecule containing nucleic acid forms a complex in a complementary manner, and includes DNA / RNA, DNA / RNA, DNA / PNA, L-DNA / L-DNA and the like.
- the nucleic acid detection method of the present invention since the nucleic acid has a single-stranded region, it can be used in a hybridization reaction without performing a single-stranded treatment such as heat treatment after the nucleic acid amplification step.
- One single-stranded region of a double-stranded DNA amplified fragment having a single-stranded region tag containing a natural nucleotide at both ends is hybridized with a first oligonucleotide probe fixed to a capture carrier (solid phase). be able to. Furthermore, it is preferable to include a step of hybridizing the other single-stranded region of the double-stranded DNA amplified fragment with the second oligonucleotide probe to which the labeling substance is bound directly or indirectly.
- sandwich hybridization The formation of a ternary complex consisting of a double-stranded DNA amplified fragment, a first oligonucleotide probe, and a second oligonucleotide probe is called sandwich hybridization.
- the order of the three hybridizations is not particularly limited.
- the length of the first oligonucleotide probe is not particularly limited as long as it can hybridize with the single-stranded region of the double-stranded DNA amplified fragment, but it is preferably 5 to 60 bases long and 10 to 40 bases long. Is more preferable.
- the length of the second oligonucleotide probe is not particularly limited as long as it can hybridize with the single-stranded region of the double-stranded DNA amplified fragment, but it is preferably 5 to 60 bases long and 10 to 40 bases long. Is more preferable.
- the labeling substance that binds to the second oligonucleotide probe is not particularly limited as long as it can detect a double-stranded DNA amplified fragment, but it is a colored carrier and can visually detect a double-stranded DNA amplified fragment. It is preferable.
- colored carriers include colored particles, enzymes, and dye-binding carriers. Among these, it is preferable to use colored particles.
- Colored particles include colloidal particles made of metals such as gold, silver, copper, and platinum, colored latex obtained by coloring latex with pigments and dyes, and silica nano particles in which pigment molecules are immobilized inside silica (silicon dioxide) particles. And particles. Among these, it is preferable to use colloidal gold particles or colored latex particles made of a water-dispersed polymer colored in blue or red. By using such colored particles, visual determination of the amplified DNA fragment can be made easier. In particular, when multiple items are detected at the same time, it is easy to visually determine a large number of items simultaneously by using colored particles of different colors for each item.
- the particle size thereof is not particularly limited, but it has little adverse effect on the formation of a sandwich hybridization complex and the capture of the amplification product containing the target sequence on the solid phase, and detection. In this case, it is preferable that the color is good.
- the particle size of the colored particles is selected from particle sizes smaller than the pore size of the chromatography medium described later. Specifically, a thickness of 500 nm or less is usually used, among which 0.1 nm to 100 nm is preferable, and 1 nm to 50 nm is more preferable.
- An enzyme used as a coloring carrier is a protein that catalyzes a reaction of a substrate that develops color or emits light. Examples thereof include peroxidase, alkaline phosphatase, luciferase and the like, but are not limited thereto as long as they can be detected with the naked eye.
- the hybridization conditions for the single-stranded region at the end of the double-stranded DNA amplified fragment and the first or second oligonucleotide probe are not particularly limited as long as hybridization occurs, but at room temperature, 10 mM phosphate
- the reaction is preferably carried out in a buffer solution. At this time, the efficiency of hybridization is increased by adding a salt such as sodium chloride.
- the presence or absence of the target nucleic acid can be determined.
- the detection is preferably discriminated visually.
- the detection is preferably performed under visible light. Visible light particularly refers to light having a wavelength of 380 to 800 nm.
- the amplification product of the nucleic acid amplification reaction can be used in the hybridization reaction as it is without being subjected to a single strand treatment such as heat denaturation.
- the presence or absence of the target nucleic acid can be easily and quickly discriminated visually without requiring a special device.
- the nucleic acid detection method by forming the sandwich hybridization complex is preferably performed on a nucleic acid detection device.
- the nucleic acid detection device is not particularly limited, but is a device carrying a capture oligonucleotide probe having a sequence complementary to at least a part of a single-stranded tag region at the end of a double-stranded DNA amplified fragment. Is preferred. Examples include, but are not limited to, chromatography, arrays, beads and the like.
- the nucleic acid chromatography device of FIG. 6 has a sample pad 32 (a carrier for adding a DNA amplification product), a conjugate pad 33 (a carrier on which a colored carrier-binding oligonucleotide is arranged) on a member 36 serving as a substrate,
- the carrier 34 chromatography medium holding the capture oligonucleotide and the absorption pad 35 are bonded together using an adhesive or the like.
- a test line 37 coated with a capture oligonucleotide and a control line 38 are provided on the carrier 34.
- the conjugate pad 33 may be omitted.
- the double-stranded DNA amplified fragment is preferably detected by a method comprising a step of hybridizing the first oligonucleotide probe and the DNA amplified fragment.
- step (b) the amplified DNA fragment is diffused in the direction of the arrow.
- step (c) the amplified DNA fragment is captured by hybridization with the immobilized first oligonucleotide probe in the test line 37.
- a step of hybridizing the amplified DNA fragment and the second oligonucleotide probe to which the labeling substance is bound before the step (c).
- the DNA amplified fragment and the second oligonucleotide probe are hybridized on the conjugate pad 33.
- a second oligonucleotide probe in which a DNA amplification fragment and a labeling substance are bound to regions different from the region on the nucleic acid detection device where the first oligonucleotide probe is immobilized.
- E Using a solvent, the amplified DNA fragment is diffused in the direction of the region where the second oligonucleotide probe bound to the labeling substance is disposed, and (f) In the region where the oligonucleotide probe of 2 is arranged, the amplified DNA fragment is hybridized with the second oligonucleotide probe to which the labeling substance is bound, and (g) the complex hybridized in step (f) is obtained.
- the first oligonucleotide probe is diffused on the development medium in the direction in which the one oligonucleotide probe is disposed; In the region where rubber nucleotide probe is fixed, it is preferable to hybridize with said complex with said first oligonucleotide probe.
- step (d) the amplified DNA fragment is arranged on the sample pad 32, and the second oligonucleotide probe is arranged on the conjugate pad 33.
- step (e) the amplified DNA fragment is diffused from the sample pad 32 in the direction of the arrow.
- step (f) the amplified DNA fragment and the second oligonucleotide probe are hybridized in the conjugate pad 33.
- step (g) a complex formed by hybridization of the amplified DNA fragment and the second oligonucleotide probe to which the labeling substance is bound is diffused in the direction of the arrow.
- step (h) the first oligonucleotide probe and the complex are hybridized on the test line 37.
- an oligonucleotide probe having a sequence complementary to one tag region of the amplified DNA fragment is immobilized as a first oligonucleotide probe for capture.
- the first oligonucleotide probe for capture may be bound directly to the membrane, may be bound via a functional group, or may be bound to the membrane via some substance.
- mediators include, but are not limited to, peptides, proteins, and nucleic acids. When the mediator is avidin, biotin modification is required for the capture oligonucleotide.
- An oligonucleotide probe for capturing a colored carrier is immobilized on a control line on the membrane.
- the oligonucleotide probe for the control line has a sequence complementary to the second oligonucleotide probe to which the labeling substance is bound, and the labeling substance is always captured when the solution is developed.
- the oligonucleotide probe for the control line may be directly bonded to the membrane in the same manner as described above, may be bonded via a functional group, or may be bonded to the membrane via any substance.
- the mediator include, but are not limited to, peptides, proteins, and nucleic acids. When the mediator is avidin, biotin modification is required for the capture oligonucleotide.
- the presence of the target nucleic acid in the sample can be visually determined by coloration in the test line. On the other hand, it is possible to visually determine that normal development and color reaction are being performed by coloration in the control line.
- visual observation means observing with the naked eye to judge the color. Further, the determination is preferably performed under visible light. Visible light particularly refers to light having a wavelength of 380 to 800 nm.
- the chromatographic medium examples include qualitative filter paper, quantitative filter paper, liquid separation filter paper, glass fiber filter paper, silica fiber filter paper, and filter paper made of composite fiber filter paper.
- filter paper made of cellulose such as nitrocellulose, a synthetic resin film such as a polyether sulfone membrane, and a porous gel such as silica gel, agarose, dextran, and gelatin can be used.
- a nylon membrane can also be used suitably.
- the form and size of the chromatographic medium are not particularly limited, and may be appropriate in operation and observation of reaction results.
- These carriers can be subjected to various modifications in order to improve hydrophilicity and compound binding.
- the deployment direction in the device is not particularly limited, and may be a horizontal direction as shown in FIG. 6 or a vertical direction. Since the solvent in the nucleic acid amplification reaction can be used as the developing solvent, the reaction solution after the nucleic acid amplification reaction can be dropped directly onto the sample pad 32 in FIG. Alternatively, a developing solution can be added separately to the reaction solution after the amplification reaction and added to the sample pad.
- the developing solvent is not particularly limited as long as it is liquid, but a Good buffer solution such as a phosphate buffer solution or a Tris buffer solution can be used. Further, a salt, a surfactant, a protein, or a nucleic acid may be dissolved in the solvent.
- FIG. 7 as an example of an embodiment of the present invention, the formation of a sandwich hybridization complex on a chromatographic carrier will be described as an example.
- the DNA amplified fragment 16 obtained in the nucleic acid amplification step is used in the next complex formation step without performing a single strand treatment or the like such as heat treatment.
- a first complex 41 is formed by hybridization between an oligonucleotide probe containing a nucleic acid sequence 39 and a colored carrier 40 that can specifically bind to one tag region 12 of the DNA fragment and the DNA amplified fragment 16. Is done.
- the complex 41 may be formed on a development medium before application, like a PCR reaction container, or a DNA amplified fragment is applied on a carrier, and the labeled molecule-binding oligonucleotide is applied during migration by capillary action. It is also possible to form by passing a dried carrier.
- the complex 41 is brought into contact with the capture oligonucleotide probe 43 previously bound to an identifiable position on the chromatography medium 42 made of a porous membrane or the like on the development medium.
- the capture oligonucleotide 43 has a sequence complementary to the other tag sequence 15 of the amplified DNA fragment, and a sandwich hybridization complex is obtained by hybridization of the complex 41 and the capture oligonucleotide. Is formed.
- the order of forming the sandwich hybridization complex is not particularly limited. It is preferable to form a complex 41 with the first oligonucleotide probe for capture after forming the complex 41 of the DNA amplified fragment and the second oligonucleotide probe to which the labeling substance is bound, but the DNA amplified fragment is captured. It is also possible to form a sandwich hybridization complex by concentrating on the development medium with the first oligonucleotide probe for use and then developing the second oligonucleotide to which the labeling substance is bound.
- nucleic acid detection device examples include an array.
- An example of the array is a microarray (DNA chip) as shown in FIG. It is possible to form a ternary complex by sandwich hybridization in a well in which the capture oligonucleotides on the microarray 44 are fixed.
- the nucleic acid detection method and the nucleic acid detection device can be used for techniques in all fields including detecting a nucleic acid (for example, a PCR product) obtained by a nucleic acid amplification method.
- a nucleic acid for example, a PCR product obtained by a nucleic acid amplification method.
- molecular biology research fields pathogen detection, detection of contaminants such as allergens, food quality control (inspection of fake labeled foods, genetically modified foods, etc.), livestock management, genetic mutation (Single nucleotide polymorphism (hereinafter also referred to as “SNP”), insertion, deletion) detection, detection of chromosomal deletion mutation, examination of diseases such as cancer, and the like.
- SNP Single nucleotide polymorphism
- the present invention includes a method for detecting an infectious disease caused by a pathogen, a method for detecting a mixture (for example, allergen) in food, a quality control for food, and a method for managing livestock, which include the nucleic acid detection method according to the present invention as a step. And nucleotide polymorphism detection methods and the like.
- the pathogen detection method should just include the process of detecting the gene which a pathogen specifically has using the nucleic acid detection method concerning this invention.
- the said pathogen is not specifically limited, For example, a pathogenic bacterium, a pathogenic virus, food poisoning bacteria, nosocomial infection bacteria, a virus, etc. can be mentioned, for example. More specifically, for example, viruses such as hepatitis C virus (HCV), cytomegalovirus (CMV), Epstein-Barr virus (EBV), herpes virus, human immunodeficiency virus (HIV), E.
- coli such as O157
- Bacteria such as Mycobacterium tuberculosis, Salmonella typhi, Salmonella or Vibrio parahaemolyticus, or microorganisms such as mycoplasma can be exemplified.
- the nucleic acid detection is performed to determine whether a DNA sample prepared from a sample to be examined for the presence of a pathogen contains a gene specifically possessed by the pathogen. Determine using the method.
- a sample to be examined for the presence of a pathogen can be used as it is as a template for nucleic acid amplification without preparing a DNA sample.
- a bacterium such as E. coli
- a bacterial colony suspension can be used as a template.
- a gene specifically detected by the pathogen is detected, it is determined that the sample contains the pathogen.
- the pathogen detection method according to the present invention can be used for diagnosis of microbial infections.
- the allergen detection method only needs to include a step of detecting a gene encoding the allergen using the nucleic acid detection method according to the present invention.
- the said allergen is not specifically limited,
- the allergen contained in foodstuffs can be mentioned specifically, for example. More specifically, egg white allergen, milk allergen, wheat allergen, buckwheat allergen, peanut allergen and the like can be mentioned.
- the method for detecting allergens will be described in more detail. For example, whether or not a DNA sample prepared from food contains an allergen-encoding gene such as egg, milk, wheat, buckwheat, and peanut Determine using. As a result, when such a gene is detected, it is determined that the food contains an allergen-containing raw material.
- the origin of allergen is not limited to what was illustrated above, For example, if cereal is taken as an example, all of rice, corn, millet, millet, millet, buckwheat, and legumes are included. Since DNA is stable to heat and is detected in trace amounts even in processed foods, the data obtained by the allergen detection method can be used for food display or as food allergy information. In addition, it can be used to detect the presence of trace amounts of food additives such as processing aids and carryovers, or the inclusion of substances not intended by the producer, such as the presence or absence of cross-contamination of production lines.
- the nucleic acid detection method of the present invention is used for parent-child testing of mammals including humans, identification of breeds of livestock, identification of agricultural product varieties, detection of SNPs, detection of diseases (such as cancer) caused by gene mutation, and the like. Can do. Specifically, for example, in the case of livestock, it can be used for purposes such as pedigree registration, individual identification, parent-child determination, and removal of pathogenic gene carrier individuals. It should be noted that the present invention is not limited to the configurations described above, and various modifications are possible within the scope shown in the claims, and can be obtained by appropriately combining technical means disclosed in different embodiments. Embodiments are also included in the technical scope of the present invention.
- tagged primers Ta-S1-F to Ta-S31-F were designed in which a tag sequence (Ta) and any of 31 types of spacers (Sx) were introduced on the 5 'end side of primer F.
- a forward primer not containing a spacer As a forward primer not containing a spacer, a primer Ta-F in which only a tag sequence (Ta) is added to the 5 ′ end side, a primer tin Ta-F modified with Biotin on the 5 ′ end side, and a FITC modification on the 3 ′ end side Primer Ta-Fm was designed.
- the designed forward primer is shown in Table 1.
- the structures of the spacers S1 to S31 in Table 1 correspond to the chemical formulas (1) to (31), respectively.
- the synthesis of these tagged primers was purchased using a custom synthesis system of Tsukuba Oligo Service Co., Ltd. or a custom synthesis system of EUROGENTEC.
- Forward primer F 5′- D d (GGAAACAGCTATGACCATGA) -3 ′ (SEQ ID NO: 1)
- Reverse primer R 5′- D d (CTATGCGGCATCATAGAGAG) -3 ′
- Tag sequence Ta 5′- D d (TGGCAACATTTTTCACTGGGTTTTAG) -3 ′ (SEQ ID NO: 3)
- Primer Ta-Sx-F 5′- D d (TGGCAACATTTTTACTACTGGTTTATAG Sx GGAAAACAGCTATGACCATGA) -3 ′ (SEQ ID NO: 4)
- Primer Ta-F 5′- D d (TGGCAACAATTTTTCACTGGGTTTATAGGGAAACACGCTATGACCATGA) -3 ′ (SEQ ID NO: 5)
- Primer mTa-F 5′-Biotin- D d (TGGCAACAATTTTTCACTGGGTTTATAGGGAAACACGTCATGACCATGA) -3 ′ (SEQ ID
- the amplification product could not be confirmed only by the reaction using the forward primer Ta-Fm which modified the 3 'end and did not contain a spacer.
- Table 1 shows the results of PCR using forward primers having each spacer.
- PCR reaction using various primer sets A PCR reaction is performed in the same manner as in step (1) of the reference example except that Tm-S1-R is used as the reverse primer.
- the target amplification product of 330 bp was amplified.
- Oligonucleotide probe 1 5′-Dd (CTAATAACCCAGTGGAAAAAGTTGCCA) -SH-3 ′ (SEQ ID NO: 10)
- nucleic acid chromatography-like test strip A substrate consisting of a backing sheet, a chromatography medium comprising the nitrocellulose membrane prepared above, a conjugation pad, a versatile sample pad as a sample addition section, and a developed sample As shown in FIG. 6, an absorption pad for absorbing the labeling substance and the labeling substance was attached to prepare a test strip for detecting a PCR amplification product using various tagged primer sets.
- PCR product prepared in step (1) is immediately applied to the sample addition site on the test strip prepared in step (4) without denaturation, and detection by chromatography is performed. went.
- the time required for detection by chromatography was as short as 5 to 15 minutes.
- Table 1 The results are shown in Table 1.
- Table 1 a target nucleic acid-specific colored line is detected on the test strip when pUC19 is added as a specimen in step (1) and a PCR reaction is performed, and a line is added when water is added as a negative control. Primers for which no color detection was observed were indicated as “ ⁇ ”. Chromatography of PCR amplification products using (i) primer Ta-F, (ii) primer Ta-S1-F, (iii) primer Ta-S20-F, and (iv) primer Ta-S23-F as forward primers) The detection result of the graphic-like test strip is shown as a representative example in FIG.
- Array detection using various spacer-added primer sets (1) Immobilization of oligonucleotide probe on solid phase 3 'terminal biotin-modified oligonucleotide probe having a sequence complementary to SEQ ID NO: 8 (SEQ ID NO: 11) was obtained by using streptavidin. Mix with. 1 ⁇ l of the mixed solution was spotted on a nitrocellulose membrane (trade name: Hi-Flow 180, manufactured by Millipore) and air-dried at 40 ° C. for 30 minutes. This probe-immobilized membrane is used as an array for detecting PCR amplification products using various tagged primer sets.
- a nitrocellulose membrane trade name: Hi-Flow 180, manufactured by Millipore
- Table 1 The results are shown in Table 1.
- Table 1 the target nucleic acid-specific colored spots were detected on the array when pUC19 was added as a specimen, and the spots where spot color detection was not observed when water was added as a negative control were designated as “ ⁇ ".
- Tag sequence T1 5′- L d (GACAACGGAGACAGAGCCCAA) -3 ′ (SEQ ID NO: 12)
- Tag sequence T2 5'- Ld (ATGCTACCGTATGCCCCAGTG) -3 '(SEQ ID NO: 13)
- Primer T1-F 5′- L d (GACAACGGAGACAGAGCCCA) -D d (GGAAAACGCTATGACCATGA) -3 ′ (SEQ ID NO: 14)
- Primer T2-R 5′- L d (ATGCTCACCGTATGCCCAGTG) -D d (CTATGCGCATCATAGAGCAG) -3 ′ (SEQ ID NO: 15)
- nucleotide probe 3 5′- L d (TTGGCTCTGTCTCCGTTGTC) -NH 2 -3 ′ (SEQ ID NO: 16)
- nucleic acid chromatography-like test strip A base material comprising a backing sheet, a chromatography medium comprising the nylon membrane prepared above, a conjugation pad, a versatile sample pad as a sample addition section, a developed sample, An absorption pad for absorbing the labeling substance was attached as shown in FIG. 6 to prepare a test strip for detecting a PCR amplification product using a primer set with an L-type DNA tag.
- PCR product detection using test strip The PCR product prepared in step (2) is immediately applied to the sample addition site on the test strip prepared in step (5) without denaturation, and the detection is performed by chromatography. It was. When pUC19 was added as a specimen in step (2), a colored line specific to the target nucleic acid was detected on the test line. On the other hand, when water was added as a negative control, no line was detected. The time required for detection by chromatography was as short as 10 to 15 minutes.
- step (1) of the reference example the forward primer (F) and pUC19 (manufactured by Takara Bio Inc.) are used as a template so that about 330 base pairs are amplified by PCR amplification.
- a reverse primer (R) was designed.
- a polymerase reaction inhibition region (H) having a hairpin structure on its 5 ′ end side, and tagged primers, T3-HF and T4-HR, into which tag sequences T3 and T4 were introduced were synthesized.
- Oligonucleotide probe 5 5′- D d (ATTATGCCGTGGAGAAGCATATCATA) -NH 2 -3 ′ (SEQ ID NO: 23)
- nucleic acid chromatography-like test strip A base material comprising a backing sheet, a chromatography medium comprising the nylon membrane prepared above, a conjugation pad, a versatile sample pad as a sample addition section, a developed sample, An absorption pad for absorbing the labeling substance was attached as shown in FIG. 6 to prepare a test strip for detecting a PCR amplification product using a primer set with a hairpin tag.
- PCR product detection using test strip The PCR product prepared in step (2) is immediately applied to the sample addition site on the test strip prepared in step (5) without denaturation, and the detection is performed by chromatography. It was. When pUC19 was added as a specimen in step (2), a colored line specific to the target nucleic acid was detected on the test line. On the other hand, when water was added as a negative control, no line was detected. The time required for detection by chromatography was as short as 10 to 15 minutes.
- step (1) of the reference example pUC19 (manufactured by Takara Bio Inc.) is used as a template, and a forward primer (approximately 330 base pairs is amplified by PCR amplification) F) and reverse primer (R) were designed.
- a polymerase reaction inhibition region (X) containing azobenzene as an artificial nucleic acid on each 5 ′ end side, and tagged primers T5-XF and T6-XR into which tag sequences T5 and T6 were introduced were synthesized.
- Tag sequence T5 5′- D d (TGGCAACAATTTTTCACTGGGTTTTAG) -3 ′ (SEQ ID NO: 25)
- Tag sequence T6 5′- D d (GGTTAGCTTCCAACCACTGTGTAGCA) -3 ′ (SEQ ID NO: 26)
- Primer T5-XF 5′- D d (TGGCAACATTTTTCACTGGGTTTTAG X GGAAAACAGCTATGACCATGA) -3 ′
- Primer T6-XR 5′- D d (GGTTAGCTTCCAACCACGTGTTAGATCA XTCTATGCGCATCATAGAGCAG) -3 ′ (SEQ ID NO: 28)
- the azobenzene inserted into the primer is represented by the chemical formula (23).
- Primer T5-XF and Primer T6-XR are each 15 pmol and 10 ng pUC19 in a 0.2 ml PCR tube, and 100 ⁇ l PCR is performed according to the instructions of the ExTaq PCR device (Takara Bio).
- a reaction solution was prepared. Then, the tube was set in a thermal cycler (GeneAmp PCR System, manufactured by Applied Biosystems), heat treated at 95 ° C. for 5 minutes, then cycled at 95 ° C. for 30 seconds, 55 ° C. for 30 seconds, and 72 ° C. for 30 seconds.
- the target was amplified by about 330 bp.
- the same reaction was performed without adding pUC19, and was used as a negative control.
- Oligonucleotide probe 7 5′- D d (CTAATAACCCAGTGAAAAATGTTGCCA) -NH 2 -3 ′ (SEQ ID NO: 29)
- nucleic acid chromatography-like test strip A base material comprising a backing sheet, a chromatography medium comprising the nylon membrane prepared above, a conjugation pad, a versatile sample pad as a sample addition section, a developed sample, An absorption pad for absorbing the labeling substance was attached as shown in FIG. 6 to prepare a test strip for detecting a PCR amplification product using an azobenzene insertion primer set.
- PCR product detection using test strip The PCR product prepared in step (2) is immediately applied to the sample addition site on the test strip prepared in step (5) without denaturation, and the detection is performed by chromatography. It was. When pUC19 was added as a specimen in step (2), a colored line specific to the target nucleic acid was detected on the test line. On the other hand, when water was added as a negative control, no line was detected. The time required for detection by chromatography was as short as 10 to 15 minutes.
- Oligonucleotide probe 7 5′- D d (CATAAAACCCATGGAAAAATGTTGCCA) -SH-3 ′ (SEQ ID NO: 31)
- nucleic acid chromatography-like test strip A substrate consisting of a backing sheet, a chromatography medium consisting of the nitrocellulose membrane created above, a conjugation pad, a versatile sample pad as a sample addition section, and a developed sample Then, an absorption pad for absorbing the labeling substance was attached as shown in FIG. 6 to prepare a test strip for detecting a PCR amplification product using an azobenzene insertion primer set.
- PCR product prepared in step (2) of Example 6 was immediately applied to the sample addition site on the test strip prepared in step (3) without denaturation and chromatography. Detection was performed.
- pUC19 was added as a specimen in the step (2) of Example 6, a target nucleic acid-specific colored line was detected on the test line.
- water was added as a negative control, no line was detected.
- the time required for detection by chromatography was as short as 10 to 15 minutes.
- oligonucleotide probe 11 5′- D d (GATCATACACGTGGTTGGAAGCTACC) -3 ′ (SEQ ID NO: 33)
- nucleic acid chromatography-like test strip A substrate consisting of a backing sheet, a chromatography medium comprising the UltraBind affinity membrane prepared above, a conjugation pad, a versatile sample pad as a sample addition section, and a developed sample Then, an absorption pad for absorbing the labeling substance was attached as shown in FIG. 6 to prepare a test strip for detecting a PCR amplification product using an azobenzene insertion primer set.
- PCR product prepared in step (2) of Example 6 was immediately applied to the sample addition site on the test strip prepared in step (2) without denaturation and chromatographed. Detection was performed.
- pUC19 was added as a specimen in the step (2) of Example 6, a target nucleic acid-specific colored line was detected on the test line.
- water was added as a negative control, no line was detected.
- the time required for detection by chromatography was as short as 10 to 15 minutes.
- These 6 kinds of azobenzene insertion primers were purchased by custom synthesis at Tsukuba Oligo Service Co., Ltd.
- Tag sequence T7 5′- D d (TGGCAACAATTTTTCACTGGGTTTTAG) -3 ′ (SEQ ID NO: 34)
- Tag sequence T8 5′- D d (GGTTAGCTTCCAACCACGTGGTAGCA) -3 ′ (SEQ ID NO: 35)
- Primer T7-X-F1 5′- D d (TGGCAACATTTTTCACTGGGTTTTAG X GGAAAACAGCTATGACCATGA) -3 ′ (SEQ ID NO: 36)
- Primer T8-X-R1 5′- D d (GGTTAGCTTCCAACCACGGTGTAGATCA XTCTATGCGGGCATCAGAGCAG) -3 ′ (SEQ ID NO: 37)
- Tag sequence T9 5′- D d (CGCATTGAGCAAGTGTACAGAGCAT) -3 ′ (SEQ ID NO: 38)
- Tag sequence T10 5′- D d (ATTATGCCGTGGAGA
- pUC19 (manufactured by Takara Bio Inc.) is added as a template, (Ii) adding EcoRI methylase gene as a template; (Iii) adding a BamHI methylase gene as a template, (Iv) Add all three types of pUC19 (manufactured by Takara Bio Inc.), EcoRI methylase gene, and BamHI methylase gene as templates, and (V) No template.
- the tube was set in a thermal cycler (GeneAmp PCR System, manufactured by Applied Biosystems), heat-treated at 95 ° C for 5 minutes, then at 95 ° C for 30 seconds, at 55 ° C for 30 seconds, and at 72 ° C for 30 minutes.
- a second cycle was performed 30 times, and DNA fragments each having the target sequence were obtained as follows. (I) about 330 bp, (ii) about 200 bp, (iii) about 100 bp, and (iv) about 330 bp, about 200 bp, about 100 bp, (v) amplification without amplified DNA fragment (as a negative control) did.
- oligonucleotide probe 12-bound latex blue
- oligonucleotide probe 13-bound latex orange
- oligonucleotide probe 14-bound latex green
- Oligonucleotide probe 12 5′- D d (CTAAAACCCAGTGAAAAATGTTGCCA) -NH 2 -3 ′ (SEQ ID NO: 46)
- Oligonucleotide probe 13 5′- D d (TTGCTCTGTACACTTGCTCAATGCG) -NH 2 -3 ′ (SEQ ID NO: 47)
- Oligonucleotide probe 14 5′- D d (TTACACATGACATGCGCAATT) -NH 2 -3 ′ (SEQ ID NO: 48)
- Oligonucleotide probe 15 5′- D d (GATCATACACGTGGTTGGAAGCTACC) -Biotin-3 ′ (SEQ ID NO: 49)
- Oligonucleotide probe 16 5′- D d (TATGATATGCTTCTCCCACGCATAAT) -Biotin-3 ′ (SEQ ID NO: 50)
- Oligonucleotide probe 17 5′- D d (CTCAGCAGGTTTCCTCTAAAGTA) -Biotin-3 ′ (SEQ ID NO: 51)
- PCR products of (i) to (v) prepared in step (2) are immediately denatured to the sample addition site on the test strip prepared in step (5) without denaturation. Each was applied and chromatographic detection was performed. The results are shown below.
- V No coloration was observed in any detection line.
- the time required for detection by chromatography was as short as 10 to 15 minutes.
- KF1 and KR1, common sequences KF2 and KR2, and common sequence-added primers into which common sequences KF3 and KR3 are introduced KF1-Fj1 and KR1-Rj1, KF2-Fj2 and KR2-Rj2, KF3-Fj3 and KR3-Rj3 were synthesized.
- These six kinds of common sequence-added primers (joint primers) were purchased by custom synthesis at Tsukuba Oligo Service Co., Ltd.
- Consensus sequence KF1 5′- D d (TGGGCTGACCTAGAGGCTCTT) -3 ′ (SEQ ID NO: 52)
- Consensus sequence KR1 5′- D d (ATGAAAATGCAGGCCATTCGG) -3 ′ (SEQ ID NO: 53)
- Primer KF1-Fj1 5′- D d (TGGGCTGACCTAGAGGTCCTT GGAAAACAGCTATGACCATGA) -3 ′
- Primer KR1-Rj1 5′- D d (ATGAAAATGCAGGCCATTCGG TCTATGCGCATCATAGAGCAG) -3 ′ (SEQ ID NO: 55)
- Consensus sequence KF2 5′- D d (CCCGGAACAGACACCAGGTTT) -3 ′ (SEQ ID NO: 56)
- Consensus sequence KR2 5′- D d (GAAGCTGTACCGTCA
- each primer is tagged with a polymerase reaction inhibition region (X) containing azobenzene, an artificial nucleic acid, on the 5 ′ end side, and tag sequences T16 and T17, tag sequences T18 and T19, and tag sequences T20 and T21.
- X polymerase reaction inhibition region
- T16-X-KF1 and T17-X-KR1, T18-X-KF2 and T19-X-KR2, and T20-X-KF3 and T21-X-KR3 were synthesized. These 6 kinds of azobenzene insertion primers were purchased by custom synthesis at Tsukuba Oligo Service Co., Ltd.
- Tag sequence T16 5′- D d (TGGCAACAATTTTTCACTGGGTTTTAG) -3 ′ (SEQ ID NO: 64)
- Tag sequence T17 5′- D d (GGTTAGCTTCCAACCACGTGGTAGCA) -3 ′ (SEQ ID NO: 65)
- Primer T16-X-KF1 5′- D d (TGGCAACATTTTTCACTGGGTTTTAG XTGGGCTGACCTAGAGGCTCTT) -3 ′
- Primer T17-X-KR1 5′- D d (GGTTAGCTTCCAACCACGTGTTAGATCA X ATGAAATGCAGGGCCATTCGG) -3 ′ (SEQ ID NO: 67)
- PCR reaction using a joint primer and an azobenzene insertion primer A PCR reaction was performed using the six primer sets prepared in the steps (1) and (2).
- reaction solutions were prepared.
- pUC19 manufactured by Takara Bio Inc.
- EcoRI methylase gene is added as a template
- BamHI methylase gene is added as a template
- Iv Add all three types of pUC19 (manufactured by Takara Bio Inc.), EcoRI methylase gene, and BamHI methylase gene as templates, and (V) No template.
- the tube was set in a thermal cycler (GeneAmp PCR System, manufactured by Applied Biosystems), heat-treated at 95 ° C for 5 minutes, then at 95 ° C for 30 seconds, at 55 ° C for 30 seconds, and at 72 ° C for 30 minutes.
- a second cycle was performed 30 times, and DNA fragments each having the target sequence were obtained as follows. (I) about 360 bp, (ii) about 230 bp, (iii) about 130 bp, and (iv) about 360 bp, about 230 bp, about 130 bp, (v) amplification without amplified DNA fragment (as a negative control) did.
- oligonucleotide probe 18-bound latex blue
- oligonucleotide probe 19-bound latex orange
- oligonucleotide probe 20-bound latex green
- Oligonucleotide probe 18 5′- D d (CTAATAACCCAGTGAAAAATGTTGCCA) -NH 2 -3 ′ (SEQ ID NO: 76)
- Oligonucleotide probes 19 5'- D d (TTGCTCTGTACACTTGCTCAATGCG) -NH 2 -3 '( SEQ ID NO: 77)
- Oligonucleotide probe 20 5′- D d (TTACACATGACATGCCGCAATT) -NH 2 -3 ′ (SEQ ID NO: 78)
- Oligonucleotide probe 21 5′- D d (GATCATACACGTGGTTGGAAGCTACC) -Biotin-3 ′ (SEQ ID NO: 79)
- Oligonucleotide probe 22 5′- D d (TATGATATGTCTCTCCCACGCATAAT) -Biotin-3 ′ (SEQ ID NO: 80)
- Oligonucleotide probe 23 5′- D d (CTCAGCAGCTTTCCTCTAAAGTA) -Biotin-3 ′ (SEQ ID NO: 81)
- nucleic acid chromatography-like test strip A substrate consisting of a backing sheet, a chromatography medium comprising the nitrocellulose membrane prepared above, the conjugation pad produced in step (4), and the versatility of the sample addition part The sample pad, the developed sample and the absorption pad for absorbing the labeling substance were bonded together as shown in FIG. 6 to prepare a test strip for detecting a PCR amplification product using an azobenzene insertion primer set. (7) Detection of PCR products using test strips
- the time required for detection by chromatography was as short as 10 to 15 minutes.
- (1) Synthesis of artificial nucleic acid (azobenzene) insertion primer E. coli DH5 ⁇ into which plasmid pUC19 was introduced was used as a target.
- the forward primer (F) and the reverse primer (R) were designed so that about 330 base pairs were amplified by PCR amplification when pUC19 was used as a template.
- Polymerase reaction inhibition regions (X) containing azobenzene as an artificial nucleic acid on each 5 ′ end side, and tagged primers T25-XF and T26-XR into which tag sequences T25 and T26 were introduced were synthesized.
- These two kinds of azobenzene insertion primers were purchased by custom synthesis at Tsukuba Oligo Service Co., Ltd.
- Tag sequence T25 5′- D d (TGGCAACAATTTTTCACTGGGTTTTAG) -3 ′ (SEQ ID NO: 82)
- Tag sequence T26 5′- D d (GGTTAGCTTCCAACCACGTGGTAGCA) -3 ′ (SEQ ID NO: 83)
- Primer F 5′- D d (GGAAACAGCTATGACCATGA) -3 ′ (SEQ ID NO: 84)
- Primer R 5′- D d (TCTATGCGGGCATCAGACAG) -3 ′ (SEQ ID NO: 85)
- Primer T25-XF 5′- D d (TGGCAACATTTTTCACTGGGTTTTAG X GGAAAACAGCTATGACCATGA) -3 ′ (SEQ ID NO: 86)
- Primer T26-XR 5′- D d (GGTTAGCTTCCAACCACGTGTTAGATCA XTCTATGCGCATCATAGAGCAG) -3 ′
- the target was amplified by about 330 bp. Further, the same reaction was carried out without adding the suspension, and used as a negative control.
- 3′-end FITC-modified oligonucleotide probe 24 (SEQ ID NO: 88, complementary strand of SEQ ID NO: 82) is mixed with the prepared colloidal gold solution, added uniformly to a glass fiber pad, and then vacuum-dried. Dried to make a conjugate pad. Oligonucleotide probe 24: 5′- D d (CTAAAACCCAGTGAAAAATGTTGCCA) -FITC-3 ′ (SEQ ID NO: 88)
- nucleic acid chromatography-like test strip A substrate consisting of a backing sheet, a chromatography medium comprising the nitrocellulose membrane prepared above, a conjugation pad, a versatile sample pad as a sample addition section, and a developed sample Then, an absorption pad for absorbing the labeling substance was attached as shown in FIG. 6 to prepare a test strip for detecting a PCR amplification product using an azobenzene insertion primer set.
- PCR product detection using test strip The PCR product prepared in step (2) is immediately applied to the sample addition site on the test strip prepared in step (5) without denaturation, and the detection is performed by chromatography. It was. When E. coli was added as a specimen in step (2), a target nucleic acid-specific colored line was detected on the test line. On the other hand, when E. coli was not added as a negative control, no line was detected. The time required for detection by chromatography was as short as 10 to 15 minutes.
- SNPs single nucleotide polymorphisms
- WT wild type
- MT mutant type
- Target 1 CACACCCGCATATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAG (SEQ ID NO: 90)
- Target 2 MT: CACACCCGCATATGGTCACTCTCAGCATACA G CTGC T CTGATGCCGCATAG (SEQ ID NO: 91)
- Tag sequence T27 5′- D d (TGGCAACATTTTCACTGGGTTTTAG) -3 ′ (SEQ ID NO: 92)
- Tag sequence T28 5′- D d (GGTTAGCTTCCAACCACGTGGTAGCA) -3 ′ (SEQ ID NO: 93)
- Primer Fwt 5′- D d (CAACCCGCATATGGTGCACT) -3 ′ (SEQ ID NO: 95)
- Primer Rwt 5′- D d (CTATGCGGGCATCAGAGCAG A ) -3 ′ (SEQ ID NO: 96)
- Primer Rmt 5′- D d (CTATGCGGGCATCAGAGCAG C ) -3 ′ (SEQ ID NO: 97)
- Primer T27-X-Fwt 5′- D d
- step (3) PCR reaction using azobenzene insertion primer set PCR reaction using the sample solution containing target 1 or target 2 prepared in step (1) and the three types of primers synthesized in step (2) Went.
- 5 pmol of each of primer T27-X-Fwt, primer T28-X-Rwt, and primer T29-X-Rmt and 1 fmol of each template were placed in a 0.2 ml PCR tube, and an ExTaq PCR kit (manufactured by Takara Bio Inc.) According to the instructions, 100 ⁇ l of a PCR reaction solution was prepared.
- Target 1 (homotype) as a template
- Target 1 and target 2 heterotype
- Target 2 (heterotype) as templates
- Target 2 (homotype) as a template
- No target water addition
- the tube was set in a thermal cycler (GeneAmp PCR System, manufactured by Applied Biosystems), heat-treated at 95 ° C. for 5 minutes, 95 ° C. for 10 seconds, 55 ° C. for 30 seconds, and 72 ° C. for 10 minutes.
- a second cycle was performed 30 times, and DNA fragments each having the target sequence were obtained as follows. (I) About 50 bp, (ii) About 50 bp, (iii) About 50 bp, and (iv) No amplified DNA fragment (as a negative control) was amplified.
- Oligonucleotide probe 26 5′-Dd (CATAAAACCCAGTGAAAAATGTTGCCA) -SH-3 ′ (SEQ ID NO: 101)
- Oligonucleotide probes 27 5'- D d (GATCATACACGTGGTTGGAAGCTAACC) -Biotin-3 '( SEQ ID NO: 102)
- Oligonucleotide probe 28 5′- D d (TTGCTCTGTACACTTGCTCAATGCG) -Biotin-3 ′ (SEQ ID NO: 103)
- nucleic acid chromatography-like test strip On the base material composed of a backing sheet, the chromatography medium composed of the nitrocellulose membrane prepared in step (5), the conjugation pad prepared in step (4), and the sample addition part A general-purpose sample pad and a developed sample and an absorption pad for absorbing a labeled substance are attached as shown in FIG. 6, and a test strip for detecting PCR amplification products using a spacer (azobenzene) insertion primer set is prepared. did.
- PCR products of (i) to (iv) prepared in step (3) are immediately denatured at the sample addition site on the test strip prepared in step (6) without denaturation. Apply and detect by chromatography.
- Polymerase reaction inhibition region (X) containing 5′-5 ′ bond + dA + 3′-3 ′ bond on each 5 ′ terminal side, tag sequences T7 and T8, tag sequences T9 and T10, and tag sequences T11 and T12
- the introduced tagged spacer insertion primers, T7-X-Fwtr and T8-X-Rwtr, T9-X-FFAG and T10-X-RFAG, and T11-X-Fagg and T12-X-Ragg were synthesized. These 6 types of tag spacer insertion primers were purchased by custom synthesis at Tsukuba Oligo Service Co., Ltd.
- Primer T7-X-Fwtr 5′-Dd (TGGCAACATTTTCACTGGGGTTTATAG X CATCACAATCAACTTATGGTGG) -3 ′ (SEQ ID NO: 104)
- Primer T8-X-Rwtr 5′-Dd (GGTTAGCTTCCAACCACGTGTTAGATCA XTTTGGGAGTTGAGAGGGGTA) -3 ′ (SEQ ID NO: 105)
- Primer T9-X-FFAG 5′-Dd (CGCATTGAGCAAGTGTACAGAGCAT X AACGCCATAAACCAGCCCGATT) -3 ′ (SEQ ID NO: 106)
- Primer T10-X-RFAG 5′-Dd (ATTATGCCGTGGAGAAGCATATCATA X CCTCCTGCTCCCCATTCTCTC) -3 ′ (SEQ ID NO: 107)
- Primer T11-X-Fag 5′-Dd (AATTGCCGCATGCCATGCCATTCACTGGGGTT
- Genomic DNA was purified from 2 g of each sample (wheat flour, buckwheat flour, ground peanut). First, a sample was weighed in a centrifuge tube (50 mL volume), 15 mL of CTAB buffer was added, and the mixture was mixed using a homogenizer. Add 30 mL of CTAB buffer, mix by inversion and warm at 55 ° C. for 30 minutes. After the heating treatment, the solution was stirred, and 600 ⁇ L of the homogeneous solution was weighed into a microtube (1.5 mL volume).
- Nucleic acid was extracted from this homogeneous solution by the following method. That is, 500 ⁇ L of a phenol / chloroform mixture (1M Tris / hydrochloric acid (pH 8.0) saturated phenol and chloroform / isoamyl alcohol mixed at a ratio of 1: 1 (v / v)) was added to this homogeneous solution, After mixing by inversion, it was lightly suspended with a vortex mixer. After suspension, the mixture was centrifuged for 15 minutes at a speed of 7,500 ⁇ g at room temperature, and the separated aqueous layer (upper layer) was transferred to a new microtube.
- a phenol / chloroform mixture (1M Tris / hydrochloric acid (pH 8.0) saturated phenol and chloroform / isoamyl alcohol mixed at a ratio of 1: 1 (v / v)
- aqueous layer 500 ⁇ L of a chloroform / isoamyl alcohol mixture (chloroform and isoamyl alcohol mixed at a ratio of 24: 1 (v / v)) was added again, and the mixture was tumbled and lightly suspended with a vortex mixer. The mixture was centrifuged for 15 minutes at a speed of 7,500 ⁇ g at room temperature, and the separated aqueous layer (upper layer) was transferred to a new microtube. An equal volume of isopropyl alcohol was added to the separated aqueous layer, mixed by inversion, centrifuged at room temperature under a speed of 7,500 ⁇ g for 15 minutes, and the supernatant was discarded by decantation.
- chloroform and isoamyl alcohol mixture chloroform and isoamyl alcohol mixed at a ratio of 24: 1 (v / v)
- CTAB buffer was prepared by the following method. Weigh 8 mL of 0.5 mM EDTA (pH 8.0), 20 mL of 1 M Tris / hydrochloric acid (pH 8.0), and 56 mL of 5 M NaCl aqueous solution in a beaker, mix, and then add water to about 150 mL Then, 4 g of cetyltrimethylammonium bromide (CTAB) was added with stirring and completely dissolved. Further, water was added to make a total volume of 200 mL, and sterilized by an autoclave.
- CTAB cetyltrimethylammonium bromide
- DNA sample stock solutions added to the reaction solution were prepared.
- the tube was set in a thermal cycler (GeneAmp PCR System, manufactured by Applied Biosystems), heat-treated at 95 ° C for 5 minutes, then at 95 ° C for 30 seconds, at 55 ° C for 30 seconds, and at 72 ° C for 30 minutes.
- a second cycle was performed 30 times, and DNA fragments each having the target sequence were obtained as follows. (I) about 141 bp, (ii) about 127 bp, (iii) about 95 bp, and (iv) about 141 bp, about 127 bp, about 95 bp, (v) amplification without amplified DNA fragment (as negative control) did.
- Centrifugation was carried out at 6000 rpm for 15 minutes, the supernatant was removed, 5 mM phosphate buffer (pH 7) containing 0.05 M sodium chloride was added and mixed, and then incubated again at 50 ° C. for 40 hours. After incubation, centrifugation (6000 rpm, 15 minutes) was performed, the supernatant was removed, and 5 mM phosphate buffer (pH 7) was added. This buffer replacement was performed again.
- Oligonucleotide probe 29 5′-Dd (CATAAAACCCAGTGAAAAATGTTGCCA) -SH-3 ′ (SEQ ID NO: 110)
- Oligonucleotide probe 30 5′-Dd (TTGCTCTGTACACTTGCTCAATGCG) -SH-3 ′ (SEQ ID NO: 111)
- Oligonucleotide probe 31 5′-Dd (TTACACATGACATGCGCAATT) -SH-3 ′ (SEQ ID NO: 112)
- nucleic acid chromatography-like test strip On the base material composed of a backing sheet, the chromatography medium composed of the nitrocellulose membrane prepared in step (5), the conjugation pad prepared in step (4), and the sample addition part A general-purpose sample pad, a developed sample and an absorption pad for absorbing a labeled substance are attached as shown in FIG. 6, and a spacer (5′-5 ′ bond + 3′-3 ′ bond) insertion primer set is used. A test strip for detection of the PCR amplification product was prepared.
- PCR products (i) to (v) prepared in the step (3) are immediately denatured at the sample addition sites on the test strip prepared in the step (6) without denaturation. Apply and detect by chromatography.
- Target 1 uses plasmid pUC19 DNA (manufactured by Takara Bio Inc.), and target 2 uses a plasmid (pUC19 / gene A) in which gene A (1668 bp) is inserted as an insert at the multicloning site of pUC19.
- Gene A sequence SEQ ID NO: 113
- Target 1 pUC19 sequence
- Target 2 pUC19 sequence / gene A (SEQ ID NO: 115)
- Primer F 5′- D d (GGAAACAGCTATGACCATGA) -3 ′ (SEQ ID NO: 116)
- Primer R 5′- D d (TTTCCCAGTCACGACGTTGT) -3 ′
- Primer R-in 5′- D d (AGTGCGTGCTGGGGCTCTTC) -3 ′
- Primer T27-XF 5′- D d (TGGCAACATTTTTACTACTGGTTTATAG X GGAAAACAGCTATGACCATGA) -3 ′
- Primer T28-XR 5′- D d (GGTTAGCTTCCAACCACGTGTTAGATCA XTTTCCCAGTCACGACGTTGT) -3 ′
- Primer T29-XR-in 5′- D d (CGCATTGAGCAAGTGTACAGAGCAT X AG
- PCR reaction using C6 linker insertion primer set PCR reaction was performed using the target 1 prepared in the above step (1) or the target 2 solution and the three types of primers synthesized in the above step (2). . 5 pmol of each of primer T27-XF, primer T28-XR, and primer T29-XR-in and 1 fmol of each template are placed in a 0.2 ml PCR tube, and ExTaq PCR kit (manufactured by Takara Bio Inc.) ), 100 ⁇ l of a PCR reaction solution was prepared.
- the tube was set on a thermal cycler (GeneAmp PCR System, manufactured by Applied Biosystems), heat-treated at 95 ° C for 5 minutes, then at 95 ° C for 10 seconds, at 55 ° C for 30 seconds, and at 72 ° C for 5 minutes. A cycle of seconds was performed 30 times. The reason why the extension reaction time was shortened to 5 seconds under the PCR reaction conditions is to selectively amplify only those having a short amplification length.
- DNA fragments each having the target sequence were obtained as follows. (I) 118 bp, (ii) 118 bp, 101 bp, (iii) 101 bp, and (iv) no amplified DNA fragment (as a negative control) were amplified.
- test strip for detecting PCR amplification products using a spacer-inserted primer set was prepared in the same manner as in steps (4) to (6) of Example 13.
- Steps (i) to (iv) prepared in step (3) are not immediately denatured, and the sample is immediately added to the sample addition site on the test strip prepared in step (4). Apply and detect by chromatography.
- the presence or absence of gene insertion into the plasmid can be determined, and mutations such as gene insertion and deletion into the genome can be easily detected using the same method.
- Forward second primer 26. Primer region of forward second primer 27.
- Reverse second primer 29. Primer region of reverse second primer 30.
- DNA amplification product having a single-stranded region at both ends 32.
- Sample pad 33. Conjugate pad 34.
- Absorption pad 36 36.
- Base material 37.
- Test line 38. Control line 39.
- Oligonucleotide for labeling molecule binding 40. Labeled molecule 41.
- Carrier holding capture oligonucleotides in each well 45.
- Bead carrier holding capture oligonucleotide Denatured PAGE, stained polyacrylamide gel 47. About 360 mer single strand 48. About 330 mer single strand 49. Chromatographic strip 50.
Abstract
Description
(1)各種プライマーの合成
本参考例では、テンプレートとしてpUC19(タカラバイオ社製)を用い、PCR増幅により約330塩基対が増幅するようにフォワードプライマーFおよびリバースプライマーRを設計した。
フォワードプライマーF:5’-Dd(GGAAACAGCTATGACCATGA)-3’(配列番号1)
リバースプライマーR:5’-Dd(CTATGCGGCATCAGAGCAG)-3’(配列番号2)
タグ配列Ta:5’-Dd(TGGCAACATTTTTCACTGGGTTTATAG)-3’(配列番号3)
プライマーTa-Sx-F:5’-Dd(TGGCAACATTTTTCACTGGGTTTATAG Sx GGAAACAGCTATGACCATGA)-3’(配列番号4)
プライマーTa-F:5’-Dd(TGGCAACATTTTTCACTGGGTTTATAGGGAAACAGCTATGACCATGA)-3’(配列番号5)
プライマーmTa-F:5’-Biotin-Dd(TGGCAACATTTTTCACTGGGTTTATAGGGAAACAGCTATGACCATGA)-3’(配列番号6)
プライマーTa-Fm:5’-Dd(TGGCAACATTTTTCACTGGGTTTATAGGGAAACAGCTATGACCATGA)-FITC-3’(配列番号7)
前記工程(1)で作製したプライマーを用いたPCR反応を行った。20pmolのフォワードプライマーTa-S1-F~Ta-S31-F、Ta-F、mTa-F、Ta-Fmのいずれか、20pmolのリバースプライマーR、および10pgのpUC19を0.2mlのPCR用チューブに入れ、ExTaq PCRキット(タカラバイオ社製)の説明書に従い、100μlのPCR反応液を調製した。その後、チューブをサーマルサイクラー(GeneAmp PCR System、アプライドバイオシステム社製)にセットし、95℃で5分間熱処理後、95℃で30秒、55℃で30秒、72℃で30秒のサイクルを35回行い、目的の約330bpの核酸を増幅した。
1×TAEバッファー(ニッポンジーン社製)とアガロース(和光純薬社製)を用いて、2%アガロースゲルを作製した。ゲルを電気泳動槽に入れ、1×TAEバッファーで満たした後、前記工程(2)で得られたPCR反応液を5μlとローディングバッファー1μlを混合し、ゲルのウェルにアプライした。100Vの電圧をかけて30分間泳動を行った後、臭化エチジウムブロマイド溶液(ナカライテスク社製)にゲルを浸して、15分間染色を行った。染色後UV照射撮影装置を用いて、254nmのUVを照射し、二本鎖DNAの増幅産物の確認を行った。
前記工程(2)で得られたPCR反応液を7μlずつPCRチューブに取り、TBE-Urea sample buffer(invitrogen社製)7μlと混合した。その混合液を70℃、3分間熱処理を行った。電気泳動槽に6% TBE-Urea Gel(invitrogen社製)をセットし、TBEバッファー(1.08%(w/v)トリス、0.55%(w/v)ホウ酸、0.037%(w/v)EDTA・2Na(2H2O))を泳動槽に入れた。熱処理を行った増幅産物を各ウェルにアプライした。
(1)各種プライマーセットを用いたPCR反応
リバースプライマーにTm-S1-Rを用いること以外は、参考例の工程(1)と同様にPCR反応を行い、目的の330bpの増幅産物を増幅した。
タグ配列Tm:5’-Dd(GGTTAGCTTCCAACCACGTGTAGATCA)-3’(配列番号8)
プライマーTm-S1-R:5’-Dd(GGTTAGCTTCCAACCACGTGTAGATCA S1 CTATGCGGCATCAGAGCAG)-3’(配列番号9)
Gold Colloid(40nm、9.0×1010(粒子数/ml)、British BioCell International社製)とチオール基含有オリゴヌクレオチドプローブ(配列番号5、タグTa配列番号3の相補鎖)を混合し、50℃で16時間インキュベートした。6000rpmで15分間遠心分離し、上清を除去、0.05M塩化ナトリウムを含む5mMリン酸(pH7)バッファーを添加し混和後、再度50℃で40時間インキュベートした。
オリゴヌクレオチドプローブ1:5’-Dd(CTATAAACCCAGTGAAAAATGTTGCCA)-SH-3’(配列番号10)
配列番号8に相補的な配列(配列番号11)を有する3’末端ビオチン修飾オリゴヌクレオチドプローブを、ストレプトアビジンと混合する。その混合液をニトロセルロースメンブレン(商品名:Hi-Flow 180、ミリポア社製)にディスペンサーを用いてライン上に塗布し、40℃で30分間風乾した。
オリゴヌクレオチドプローブ2:5’-Dd(GATCATACACGTGGTTGGAAGCTAACC)-Biotin-3’(配列番号11)
バッキングシートから成る基材に、上記で作成したニトロセルロースメンブレンからなるクロマトグラフィー媒体、コンジュゲーションパッド、試料添加部である汎用性のサンプルパッド、展開した試料や標識物質を吸収するための吸収パッドを図6に示す様に貼り合わせ、各種タグ付きプライマーセットを用いたPCR増幅産物の検出用テストストリップを作製した。
工程(1)で作製したPCR産物を変性することなく、それぞれ直ちに工程(4)で作製したテストストリップ上の試料添加部位にアプライし、クロマトグラフィーによる検出を行った。クロマトグラフィーによる検出に要した時間は、5~15分と短時間であった。
(1)オリゴヌクレオチドプローブの固相への固定化
配列番号8に相補的な配列(配列番号11)を有する3’末端ビオチン修飾オリゴヌクレオチドプローブを、ストレプトアビジンと混合する。その混合液をニトロセルロースメンブレン(商品名:Hi-Flow 180、ミリポア社製)に1μlスポットし、40℃で30分間風乾した。このプローブ固定化メンブレンを、各種タグ付きプライマーセットを用いたPCR増幅産物の検出用アレイとして用いる。
実施例1の工程(1)で作製したPCR産物を変性することなく、それぞれ直ちに工程(1)で作製したアレイ上にアプライし、更に実施例1の工程(2)で作製した金コロイド溶液を滴下して15~20分静置し、アレイによる検出を行った。アレイを用いたドットブロット法による検出に要した時間は、15~20分と短時間であった。
EX Taq以外のPCRキットとして、KOD plus、Phusion、PrimeSTAR、KOD FX、Tks Gflexを用いて参考例、および実施例1~2と同様の実験を行ったところ、EX Taq PCRを使用した場合と同様の結果が確認された。
本実施例では、テンプレートとしてpUC19(タカラバイオ社製)を用い、PCR増幅により約330塩基対が増幅するようにフォワードプライマー(F)およびリバースプライマー(R)を参考例と同様に設計した。更にそれぞれの5’末端側に非天然型(L型)DNA鎖からなるタグ配列T1およびT2を導入したL-DNAタグ付きプライマー、T1-FおよびT2-Rを設計した。L-DNAタグ付きプライマーの合成は、0.2μMスケールのカラムを用いて、一般的なホスホロアミダイト法に基づいて、DNA自動合成機(H-8-SE:Gene World)で合成した。
タグ配列T1:5’-Ld(GACAACGGAGACAGAGCCAA)-3’(配列番号12)
タグ配列T2:5’-Ld(ATGCTACCGTATGCCCAGTG)-3’(配列番号13)
プライマーT1-F:5’-Ld(GACAACGGAGACAGAGCCAA)-Dd(GGAAACAGCTATGACCATGA)-3’(配列番号14)
プライマーT2-R:5’-Ld(ATGCTACCGTATGCCCAGTG)-Dd(CTATGCGGCATCAGAGCAG)-3’(配列番号15)
前記工程(1)で実施し作製したプライマーセットを用いたPCR反応を行った。プライマーT1-FとプライマーT2-Rを各15pmolと、10ngのpUC19とを0.2mlのPCR用チューブに入れ、ExTaq PCRデバイス(タカラバイオ社製)の説明書に従い、100μlのPCR反応液を調製した。その後、チューブをサーマルサイクラー(GeneAmp PCR System、アプライドバイオシステム社製)にセットし、95℃で5分間熱処理後、95℃で30秒、55℃で30秒、72℃で30秒のサイクルを35回行い、目的の約330bpを増幅した。また、pUC19を添加しないで同様の反応を行い、ネガティブコントロールとした。
カルボキシル基含有ポリスチレンラテックス(固形分10%(w/w)、Bangs社製)とアミノ基含有L型オリゴヌクレオチドプローブ(配列番号16、配列番号12の相補鎖)を、水溶性カルボジイミドを必要量添加したMES緩衝液中で混合し、結合後、モノエタノールアミンでブロッキングを行った。前記反応液を遠心分離後、上清を除去し、得られた沈殿を水洗した。洗浄後、界面活性剤を含むHEPES緩衝液に再懸濁し、グラスファイバー製パッドに均一になるように添加した後、真空乾燥機にて乾燥させ、コンジュゲーションパッドとした。
ヌクレオチドプローブ3:5’-Ld(TTGGCTCTGTCTCCGTTGTC)-NH2-3’(配列番号16)
カルボキシル基修飾ナイロンメンブレン(日本ポール社製、6mmx60mm)を水溶性カルボジイミドにより処理し、脱イオン水で洗浄した。活性化したメンブレンの一端から30mmの箇所に、配列番号13に対して相補的な配列(配列番号17)を有するアミノ基含有L型オリゴヌクレオチドプローブを、ディスペンサーを用いてライン上に塗布し、15分間風乾した。その後、メンブレンをTris緩衝液で処理し、ブロッキング後、メンブレンを水洗、乾燥した。
ヌクレオチドプローブ4:5’-Ld(CACTGGGCATACGGTAGCAT)-NH2-3’(配列番号17)
バッキングシートから成る基材に、上記で作成したナイロンメンブレンからなるクロマトグラフィー媒体、コンジュゲーションパッド、試料添加部である汎用性のサンプルパッド、展開した試料や標識物質を吸収するための吸収パッドを図6に示す様に貼り合わせ、L型DNAタグ付きプライマーセットを用いたPCR増幅産物の検出用テストストリップを作製した。
工程(2)で作製したPCR産物を変性することなく、直ちに工程(5)で作製したテストストリップ上の試料添加部位にアプライし、クロマトグラフィーによる検出を行った。工程(2)で検体としてpUC19を添加した場合、テストライン上に標的核酸特異的な着色ラインが検出された。一方、ネガティブコントロールとして水を添加した場合、ラインの検出は認められなかった。また、クロマトグラフィーによる検出に要した時間は、10~15分と短時間であった。
参考例の工程(1)と同様に、テンプレートとしてpUC19(タカラバイオ社製)を用い、PCR増幅により約330塩基対が増幅するようにフォワードプライマー(F)およびリバースプライマー(R)を設計した。その5’末端側にヘアピン構造を有するポリメラーゼ反応阻害領域(H)、およびタグ配列T3およびT4を導入したタグ付きプライマー、T3-H-FおよびT4-H-Rを合成した。
ポリメラーゼ反応阻害配列H:5’-Dd(AGGCGAGGTCGCGAGCGCACATGTGCGCTCGCGACCTC GCCT)-3’(配列番号18)
タグ配列T3:5’-Dd(TATGATATGCTTCTCCACGCATAAT)-3’(配列番号19)
タグ配列T4:5’-Dd(TGCTCTGTACACTTGCTCAAT)-3’(配列番号20)
プライマーT3-H-F:5’- Dd(TATGATATGCTTCTCCACGCATAATAGGCGAGGTCGCGAGCGCACATG TGCGCTCGCGACCTCGCCTGGAAACAGCTATGACCATGA)-3’(配列番号21)
プライマーT4-H-R:5’- Dd(TGCTCTGTACACTTGCTCAATAGGCGAGGTCGCGAGCGCACATGTGC GCTCGCGACCTCGCCTCTATGCGGCATCAGAGCAG)-3’(配列番号22)
前記工程(1)で実施し作製したプライマーセットを用いたPCR反応を行った。プライマーFとプライマーRを各15pmolと、10ngのpUC19とを0.2mlのPCR用チューブに入れ、ExTaq PCRデバイス(タカラバイオ社製)の説明書に従い、100μlのPCR反応液を調製した。その後、チューブをサーマルサイクラー(GeneAmp PCR System、アプライドバイオシステム社製)にセットし、95℃で5分間熱処理後、95℃で30秒、55℃で30秒、72℃で30秒のサイクルを35回行い、目的の約330bpを増幅した。また、pUC19を添加しないで同様の反応を行い、ネガティブコントロールとした。
カルボキシル基含有ポリスチレンラテックス(固形分10%(w/w)、Bangs社製)とアミノ基含有オリゴヌクレオチドプローブ(配列番号23、配列番号19の相補鎖)を、水溶性カルボジイミドを必要量添加したMES緩衝液中で混合し、結合後、モノエタノールアミンでブロッキングを行った。前記反応液を遠心分離後、上清を除去し、得られた沈殿を水洗した。洗浄後、界面活性剤を含むHEPES緩衝液に再懸濁し、グラスファイバー製パッドに均一になるように添加した後、真空乾燥機にて乾燥させ、コンジュゲーションパッドとした。
オリゴヌクレオチドプローブ5:5’-Dd(ATTATGCGTGGAGAAGCATATCATA)-NH2-3’(配列番号23)
カルボキシル基修飾ナイロンメンブレン(日本ポール社製、6mmx60mm)を水溶性カルボジイミドにより処理し、脱イオン水で洗浄した。活性化したメンブレンの一端から30mmの箇所に、配列番号20の相補的な配列(配列番号24)を有するアミノ基含有L型オリゴヌクレオチドプローブを、ディスペンサーを用いてライン上に塗布し、15分間風乾した。その後、メンブレンをTris緩衝液で処理し、ブロッキング後、メンブレンを水洗、乾燥した。
オリゴヌクレオチドプローブ6: 5’-Dd(ATTGAGCAAGTGTACAGAGCA)-NH2-3’(配列番号24)
バッキングシートから成る基材に、上記で作成したナイロンメンブレンからなるクロマトグラフィー媒体、コンジュゲーションパッド、試料添加部である汎用性のサンプルパッド、展開した試料や標識物質を吸収するための吸収パッドを図6に示す様に貼り合わせ、ヘアピンタグ付きプライマーセットを用いたPCR増幅産物の検出用テストストリップを作製した。
工程(2)で作製したPCR産物を変性することなく、直ちに工程(5)で作製したテストストリップ上の試料添加部位にアプライし、クロマトグラフィーによる検出を行った。工程(2)で検体としてpUC19を添加した場合、テストライン上に標的核酸特異的な着色ラインが検出された。一方、ネガティブコントロールとして水を添加した場合、ラインの検出は認められなかった。また、クロマトグラフィーによる検出に要した時間は、10~15分と短時間であった。
参考例の工程(1)と同様に、テンプレートとしてpUC19(タカラバイオ社製)を用い、PCR増幅により約330塩基対が増幅するようにフォワードプライマー(F)およびリバースプライマー(R)を設計した。それぞれの5’末端側に人工核酸であるアゾベンゼンを含むポリメラーゼ反応阻害領域(X)、およびタグ配列T5およびT6を導入したタグ付きプライマー、T5-X-FおよびT6-X-Rを合成した。この2種のアゾベンゼン挿入プライマーはつくばオリゴサービス株式会社にて受託合成を行い購入した。本検討で作製したプライマーセットを示す。
タグ配列T5:5’-Dd(TGGCAACATTTTTCACTGGGTTTATAG)-3’(配列番号25)
タグ配列T6:5’-Dd(GGTTAGCTTCCAACCACGTGTAGATCA)-3’(配列番号26)
プライマーT5-X-F:5’-Dd(TGGCAACATTTTTCACTGGGTTTATAG X GGAAACAGCTATGACCATGA)-3’(配列番号27)
プライマーT6-X-R:5’-Dd(GGTTAGCTTCCAACCACGTGTAGATCA X TCTATGCGGCATCAGAGCAG)-3’(配列番号28)
なお、プライマーに挿入されたアゾベンゼンは前記化学式(23)で表される。
前記工程(1)で実施し作製したプライマーセットを用いたPCR反応を行った。プライマーT5-X-FとプライマーT6-X-Rを各15pmolと、10ngのpUC19とを0.2mlのPCR用チューブに入れ、ExTaq PCRデバイス(タカラバイオ社製)の説明書に従い、100μlのPCR反応液を調製した。その後、チューブをサーマルサイクラー(GeneAmp PCR System、アプライドバイオシステム社製)にセットし、95℃で5分間熱処理後、95℃で30秒、55℃で30秒、72℃で30秒のサイクルを35回行い、目的の約330bpを増幅した。また、pUC19を添加しないで同様の反応を行い、ネガティブコントロールとした。
カルボキシル基含有ポリスチレンラテックス(固形分10%(w/w)、Bangs社製)とアミノ基含有オリゴヌクレオチドプローブ(配列番号29、配列番号25の相補鎖)を、水溶性カルボジイミドを必要量添加したMES緩衝液中で混合し、結合後、モノエタノールアミンでブロッキングを行った。前記反応液を遠心分離後、上清を除去し、得られた沈殿を水洗した。洗浄後、界面活性剤を含むHEPES緩衝液に再懸濁し、グラスファイバー製パッドに均一になるように添加した後、真空乾燥機にて乾燥させ、コンジュゲーションパッドとした。
オリゴヌクレオチドプローブ7:5’-Dd(CTATAAACCCAGTGAAAAATGTTGCCA)-NH2-3’(配列番号29)
カルボキシル基修飾ナイロンメンブレン(日本ポール社製、6mmx60mm)を水溶性カルボジイミドにより処理し、脱イオン水で洗浄した。活性化したメンブレンの一端から30mmの箇所に、配列番号26の相補的な配列(配列番号30)を有するアミノ基含有D型オリゴヌクレオチドプローブを、ディスペンサーを用いてライン上に塗布し、15分間風乾した。その後、メンブレンをTris緩衝液で処理し、ブロッキング後、メンブレンを水洗、乾燥した。
オリゴヌクレオチドプローブ8:5’-Dd(GATCATACACGTGGTTGGAAGCTAACC)-NH2-3’(配列番号30)
バッキングシートから成る基材に、上記で作成したナイロンメンブレンからなるクロマトグラフィー媒体、コンジュゲーションパッド、試料添加部である汎用性のサンプルパッド、展開した試料や標識物質を吸収するための吸収パッドを図6に示す様に貼り合わせ、アゾベンゼン挿入プライマーセットを用いたPCR増幅産物の検出用テストストリップを作製した。
工程(2)で作製したPCR産物を変性することなく、直ちに工程(5)で作製したテストストリップ上の試料添加部位にアプライし、クロマトグラフィーによる検出を行った。工程(2)で検体としてpUC19を添加した場合、テストライン上に標的核酸特異的な着色ラインが検出された。一方、ネガティブコントロールとして水を添加した場合、ラインの検出は認められなかった。また、クロマトグラフィーによる検出に要した時間は、10~15分と短時間であった。
Gold Colloid(40nm、9.0×1010(粒子数/ml)、British BioCell International社製)とチオール基含有オリゴヌクレオチドプローブ(配列番号31、配列番号25の相補鎖)を混合し、50℃で16時間インキュベートした。6000rpmで15分間遠心分離し、上清を除去、0.05M 塩化ナトリウム、5mMリン酸バッファー、pH7を添加し混和後、再度50℃で40時間インキュベートした。
調製した金コロイド溶液をグラスファイバー製パッドに均一になるように添加した後、真空乾燥機にて乾燥させ、コンジュゲーションパッドとした。
オリゴヌクレオチドプローブ7:5’-Dd(CTATAAACCCAGTGAAAAATGTTGCCA)-SH-3’(配列番号31)
配列番号26に相補的な配列(配列番号31)を有する3’末端ビオチン修飾オリゴヌクレオチドプローブを、ストレプトアビジンと混合する。その混合液をニトロセルロースメンブレン(商品名:Hi-Flow 180、 ミリポア社製)にディスペンサーを用いてライン上に塗布し、40℃で30分間風乾した。
オリゴヌクレオチドプローブ10:5’-Dd(GATCATACACGTGGTTGGAAGCTAACC)-Biotin-3’(配列番号32)
バッキングシートから成る基材に、上記で作成したニトロセルロースメンブレンからなるクロマトグラフィー媒体、コンジュゲーションパッド、試料添加部である汎用性のサンプルパッド、展開した試料や標識物質を吸収するための吸収パッドを図6に示す様に貼り合わせ、アゾベンゼン挿入プライマーセットを用いたPCR増幅産物の検出用テストストリップを作製した。
実施例6の工程(2)で作製したPCR産物を変性することなく、直ちに工程(3)で作製したテストストリップ上の試料添加部位にアプライし、クロマトグラフィーによる検出を行った。実施例6の工程(2)で検体としてpUC19を添加した場合、テストライン上に標的核酸特異的な着色ラインが検出された。一方、ネガティブコントロールとして水を添加した場合、ラインの検出は認められなかった。また、クロマトグラフィーによる検出に要した時間は、10~15分と短時間であった。
配列番号26に相補的な配列(配列番号33)を有するオリゴヌクレオチドプローブをUltraBindアフィニティメンブレン(日本ポール社製)にディスペンサーを用いてライン上に塗布し、80℃で1時間風乾した。
オリゴヌクレオチドプローブ11:5’-Dd(GATCATACACGTGGTTGGAAGCTAACC)-3’(配列番号33)
バッキングシートから成る基材に、上記で作成したUltraBindアフィニティメンブレンからなるクロマトグラフィー媒体、コンジュゲーションパッド、試料添加部である汎用性のサンプルパッド、展開した試料や標識物質を吸収するための吸収パッドを図6に示す様に貼り合わせ、アゾベンゼン挿入プライマーセットを用いたPCR増幅産物の検出用テストストリップを作製した。
実施例6の工程(2)で作製したPCR産物を変性することなく、直ちに工程(2)で作製したテストストリップ上の試料添加部位にアプライし、クロマトグラフィーによる検出を行った。実施例6の工程(2)で検体としてpUC19を添加した場合、テストライン上に標的核酸特異的な着色ラインが検出された。一方、ネガティブコントロールとして水を添加した場合、ラインの検出は認められなかった。また、クロマトグラフィーによる検出に要した時間は、10~15分と短時間であった。
標的核酸のテンプレートとしてpUC19(タカラバイオ社製)、EcoRIメチラーゼ遺伝子、および、BamHIメチラーゼ遺伝子の3種類を用い、PCR増幅により約330塩基対、約200塩基対、および、約100塩基対が増幅するようにフォワードプライマー(F1)とリバースプライマー(R1)、フォワードプライマー(F2)とリバースプライマー(R2)、および、フォワードプライマー(F3)とリバースプライマー(R3)の3組のプライマーをそれぞれ設計した。それぞれの5’末端側に人工核酸であるアゾベンゼンを含むポリメラーゼ反応阻害領域(X)、およびタグ配列T7とT8、タグ配列T9とT10、および、タグ配列T11とT12を導入したタグ付きプライマー、T7-X-F1とT8-X-R1、T9-X-F2とT10-X-R2、および、T11-X-F3とT12-X-R3を合成した。この6種のアゾベンゼン挿入プライマーはつくばオリゴサービス株式会社にて受託合成を行い購入した。
タグ配列T7:5’-Dd(TGGCAACATTTTTCACTGGGTTTATAG)-3’(配列番号34)
タグ配列T8:5’-Dd(GGTTAGCTTCCAACCACGTGTAGATCA)-3’(配列番号35)
プライマーT7-X-F1:5’-Dd(TGGCAACATTTTTCACTGGGTTTATAG X GGAAACAGCTATGACCATGA)-3’(配列番号36)
プライマーT8-X-R1:5’-Dd(GGTTAGCTTCCAACCACGTGTAGATCA X TCTATGCGGCATCAGAGCAG)-3’(配列番号37)
タグ配列T9:5’-Dd(CGCATTGAGCAAGTGTACAGAGCAT)-3’(配列番号38)
タグ配列T10:5’-Dd(ATTATGCGTGGAGAAGCATATCATA)-3’(配列番号39)
プライマーT9-X-F2:5’-Dd(CGCATTGAGCAAGTGTACAGAGCAT X AGCATTATGAATTATATGGT)-3’(配列番号40)
プライマーT10-X-R2:5’-Dd(ATTATGCGTGGAGAAGCATATCATA X TTGTTTACATTTATAGCATC)-3’(配列番号41)
タグ配列T11:5’-Dd(AATTGCGCATGTCCATGTGTAA)-3’(配列番号42)
タグ配列T12:5’-Dd(TACTTTAGAGGAAACTGCTGAG)-3’(配列番号43)
プライマーT11-X-F3:5’-Dd(AATTGCGCATGTCCATGTGTAA X TGGTTTTAAAACTCTGATAC)-3’(配列番号44)
プライマーT12-X-R3:5’-Dd(TACTTTAGAGGAAACTGCTGAG X AGTATGATGAGGGTGTAACA)-3’(配列番号45)
前記工程(1)で実施し作製した3組のプライマーセットを用いたPCR反応を行った。プライマーT7-X-F1、プライマーT8-X-R1、プライマーT9-X-F2、プライマーT10-X-R2、プライマーT11-X-F3、および、プライマーT12-X-R3を各15pmolと、各テンプレート10ngを0.2mlのPCR用チューブに入れ、ExTaq PCRデバイス(タカラバイオ社製)の説明書に従い、100μlのPCR反応液を調製した。反応液は次の5種類を用意した。
(i)テンプレートとしてpUC19(タカラバイオ社製)を添加、
(ii)テンプレートとしてEcoRIメチラーゼ遺伝子を添加、
(iii)テンプレートとしてBamHIメチラーゼ遺伝子を添加、
(iv)テンプレートとしてpUC19(タカラバイオ社製)、EcoRIメチラーゼ遺伝子、および、BamHIメチラーゼ遺伝子の3種類全て添加、そして、
(v)テンプレートなし。
カルボキシル基含有ポリスチレンラテックス(青色)(固形分10%(w/w)、Bangs社製)とアミノ基含有オリゴヌクレオチドプローブ12(配列番号46、配列番号34の相補鎖)、カルボキシル基含有ポリスチレンラテックス(オレンジ色)(固形分10%(w/w)、Bangs社製)とアミノ基含有オリゴヌクレオチドプローブ13(配列番号47、配列番号38の相補鎖)、および、カルボキシル基含有ポリスチレンラテックス(緑色)(固形分10%(w/w)、Bangs社製)とアミノ基含有オリゴヌクレオチドプローブ14(配列番号48、配列番号42の相補鎖)を、それぞれ水溶性カルボジイミドを必要量添加したMES緩衝液中で混合し、結合後、モノエタノールアミンでブロッキングを行った。前記反応液を遠心分離後、上清を除去し、得られた沈殿を水洗した。洗浄後、界面活性剤を含むHEPES緩衝液に再懸濁し、オリゴヌクレオチドプローブ12結合ラテックス(青色)、オリゴヌクレオチドプローブ13結合ラテックス(オレンジ色)、オリゴヌクレオチドプローブ14結合ラテックス(緑色)を作製した。
オリゴヌクレオチドプローブ12:5’-Dd(CTATAAACCCAGTGAAAAATGTTGCCA)-NH2-3’(配列番号46)
オリゴヌクレオチドプローブ13:5’-Dd(TTGCTCTGTACACTTGCTCAATGCG)-NH2-3’(配列番号47)
オリゴヌクレオチドプローブ14:5’-Dd(TTACACATGGACATGCGCAATT)-NH2-3’(配列番号48)
配列番号35に相補的な配列(配列番号49)を有する3’末端ビオチン修飾オリゴヌクレオチドプローブ15、配列番号39に相補的な配列(配列番号50)を有する3’末端ビオチン修飾オリゴヌクレオチドプローブ16、および、配列番号43に相補的な配列(配列番号51)を有する3’末端ビオチン修飾オリゴヌクレオチドプローブ17を、それぞれストレプトアビジンと混合する。それらの混合液をニトロセルロースメンブレン(商品名:Hi-Flow 135、ミリポア社製)上の3箇所にディスペンサーを用いて、上流側から順に互いに離れた位置でライン上に塗布し、40℃で30分間風乾した。三本の検出ラインを作製した。
オリゴヌクレオチドプローブ15:5’-Dd(GATCATACACGTGGTTGGAAGCTAACC)-Biotin-3’(配列番号49)
オリゴヌクレオチドプローブ16:5’-Dd(TATGATATGCTTCTCCACGCATAAT)-Biotin-3’(配列番号50)
オリゴヌクレオチドプローブ17:5’-Dd(CTCAGCAGTTTCCTCTAAAGTA)-Biotin-3’(配列番号51)
バッキングシートから成る基材に、上記で作成したニトロセルロースメンブレンからなるクロマトグラフィー媒体、工程(3)で作製したコンジュゲーションパッド、試料添加部である汎用性のサンプルパッド、展開した試料や標識物質を吸収するための吸収パッドを図6に示す様に貼り合わせ、アゾベンゼン挿入プライマーセットを用いたPCR増幅産物の検出用テストストリップを作製した。
工程(2)で作製した(i)~(v)のPCR産物をそれぞれ変性することなく、直ちに工程(5)で作製したテストストリップ上の試料添加部位にそれぞれアプライし、クロマトグラフィーによる検出を行った。その結果は以下に示す。
(i):一本目の検出ラインのみ青色に着色。
(ii):二本目の検出ラインのみオレンジ色に着色。
(iii):三本目の検出ラインのみ緑色に着色。
(iv):一本目の検出ラインが青色に、二本目の検出ラインがオレンジ色に、三本目の検出ラインが緑色にそれぞれ着色。
(v):どの検出ラインも着色は認められなかった。
また、クロマトグラフィーによる検出に要した時間は、10~15分と短時間であった。
標的核酸のテンプレートとしてpUC19(タカラバイオ社製)、EcoRIメチラーゼ遺伝子、および、BamHIメチラーゼ遺伝子の3種類を用い、PCR増幅により約330塩基対、約200塩基対、および、約100塩基対が増幅するようにフォワードプライマー(Fj1)とリバースプライマー(Rj1)、フォワードプライマー(Fj2)とリバースプライマー(Rj2)、および、フォワードプライマー(Fj3)とリバースプライマー(Rj3)の3組のプライマーをそれぞれ設計した。それぞれの5’末端側に共通配列KF1とKR1、共通配列KF2とKR2、および、共通配列KF3とKR3を導入した共通配列付加プライマー、KF1-Fj1とKR1-Rj1、KF2-Fj2とKR2-Rj2、および、KF3-Fj3とKR3-Rj3を合成した。この6種の共通配列付加プライマー(ジョイントプライマー)はつくばオリゴサービス株式会社にて受託合成を行い購入した。
共通配列KF1:5’-Dd(TGGGCTGACCTAGAGGTCTT)-3’(配列番号52)
共通配列KR1:5’-Dd(ATGAAATGCAGGCCATTCGG)-3’(配列番号53)
プライマーKF1-Fj1:5’-Dd(TGGGCTGACCTAGAGGTCTT GGAAACAGCTATGACCATGA)-3’(配列番号54)
プライマーKR1-Rj1:5’-Dd(ATGAAATGCAGGCCATTCGG TCTATGCGGCATCAGAGCAG)-3’(配列番号55)
共通配列KF2:5’-Dd(CCGGAACAGACACCAGGTTT)-3’(配列番号56)
共通配列KR2:5’-Dd(GAAGCTGTACCGTCACATGA)-3’(配列番号57)
プライマーKF2-Fj2:5’-Dd(CCGGAACAGACACCAGGTTT AGCATTATGAATTATATGGT)-3’(配列番号58)
プライマーKR2-Rj2:5’-Dd(GAAGCTGTACCGTCACATGA TTGTTTACATTTATAGCATC)-3’(配列番号59)
共通配列KF3:5’-Dd(ATACCGATGAGTGTGCTACC)-3’(配列番号60)
共通配列KR3:5’-Dd(TGGCCTGTGTGACACTATGC)-3’(配列番号61)
プライマーKF3-Fj3:5’-Dd(ATACCGATGAGTGTGCTACC TGGTTTTAAAACTCTGATAC)-3’(配列番号62)
プライマーKR3-Rj3:5’-Dd(TGGCCTGTGTGACACTATGC AGTATGATGAGGGTGTAACA)-3’(配列番号63)
工程(1)で作成したジョイントプライマーセットがそれぞれ増幅する3種類のPCR増幅断片に結合できるように、それぞれのジョイントプライマーと同一の共通配列を有するプライマーを3組設計した。それぞれのプライマーは、5’末端側に人工核酸であるアゾベンゼンを含むポリメラーゼ反応阻害領域(X)、およびタグ配列T16とT17、タグ配列T18とT19、および、タグ配列T20とT21を導入したタグ付きプライマー、T16-X-KF1とT17-X-KR1、T18-X-KF2とT19-X-KR2、および、T20-X-KF3とT21-X-KR3を合成した。この6種のアゾベンゼン挿入プライマーはつくばオリゴサービス株式会社にて受託合成を行い購入した。
タグ配列T16:5’-Dd(TGGCAACATTTTTCACTGGGTTTATAG)-3’(配列番号64)
タグ配列T17:5’-Dd(GGTTAGCTTCCAACCACGTGTAGATCA)-3’(配列番号65)
プライマーT16-X-KF1:5’-Dd(TGGCAACATTTTTCACTGGGTTTATAG X TGGGCTGACCTAGAGGTCTT )-3’(配列番号66)
プライマーT17-X-KR1:5’-Dd(GGTTAGCTTCCAACCACGTGTAGATCA X ATGAAATGCAGGCCATTCGG )-3’(配列番号67)
タグ配列T18:5’-Dd(CGCATTGAGCAAGTGTACAGAGCAT)-3’(配列番号68)
タグ配列T19:5’-Dd(ATTATGCGTGGAGAAGCATATCATA)-3’(配列番号69)
プライマーT18-X-KF2:5’-Dd(CGCATTGAGCAAGTGTACAGAGCAT X CCGGAACAGACACCAGGTTT)-3’(配列番号70)
プライマーT19-X-KR2:5’-Dd(ATTATGCGTGGAGAAGCATATCATA X GAAGCTGTACCGTCACATGA)-3’(配列番号71)
タグ配列T20:5’-Dd(AATTGCGCATGTCCATGTGTAA)-3’(配列番号72)
タグ配列T21:5’-Dd(TACTTTAGAGGAAACTGCTGAG)-3’(配列番号73)
プライマーT20-X-KF3:5’-Dd(AATTGCGCATGTCCATGTGTAA X ATACCGATGAGTGTGCTACC)-3’(配列番号74)
プライマーT21-X-KR3:5’-Dd(TACTTTAGAGGAAACTGCTGAG X TGGCCTGTGTGACACTATGC)-3’(配列番号75)
前記工程(1)および(2)で実施し作製した6組のプライマーセットを用いたPCR反応を行った。プライマーKF1-Fj1、プライマーKR1-Rj1、プライマーKF2-Fj2、プライマーKR2-Rj2、プライマーKF3-Fj3、プライマーKR3-Rj3、プライマーT16-X-KF1、プライマーT17-X-KR1、プライマーT18-X-KF2、プライマーT19-X-KR2、プライマーT20-X-KF3、プライマーT21-X-KR3を各8pmolと、各テンプレート10ngを0.2mlのPCR用チューブに入れ、ExTaq PCRデバイス(タカラバイオ社製)の説明書に従い、100μlのPCR反応液を調製した。
(i)テンプレートとしてpUC19(タカラバイオ社製)を添加、
(ii)テンプレートとしてEcoRIメチラーゼ遺伝子を添加、
(iii)テンプレートとしてBamHIメチラーゼ遺伝子を添加、
(iv)テンプレートとしてpUC19(タカラバイオ社製)、EcoRIメチラーゼ遺伝子、および、BamHIメチラーゼ遺伝子の3種類全て添加、そして、
(v)テンプレートなし。
カルボキシル基含有ポリスチレンラテックス(青色)(固形分10%(w/w)、Bangs社製)とアミノ基含有オリゴヌクレオチドプローブ18(配列番号76、配列番号64の相補鎖)、カルボキシル基含有ポリスチレンラテックス(オレンジ色)(固形分10%(w/w)、Bangs社製)とアミノ基含有オリゴヌクレオチドプローブ19(配列番号77、配列番号68の相補鎖)、および、カルボキシル基含有ポリスチレンラテックス(緑色)(固形分10%(w/w)、Bangs社製)とアミノ基含有オリゴヌクレオチドプローブ20(配列番号78、配列番号72の相補鎖)を、それぞれ水溶性カルボジイミドを必要量添加したMES緩衝液中で混合し、結合後、モノエタノールアミンでブロッキングを行った。前記反応液を遠心分離後、上清を除去し、得られた沈殿を水洗した。洗浄後、界面活性剤を含むHEPES緩衝液に再懸濁し、オリゴヌクレオチドプローブ18結合ラテックス(青色)、オリゴヌクレオチドプローブ19結合ラテックス(オレンジ色)、オリゴヌクレオチドプローブ20結合ラテックス(緑色)を作製した。
オリゴヌクレオチドプローブ18:5’-Dd(CTATAAACCCAGTGAAAAATGTTGCCA)-NH2-3’(配列番号76)
オリゴヌクレオチドプローブ19:5’-Dd(TTGCTCTGTACACTTGCTCAATGCG)-NH2-3’(配列番号77)
オリゴヌクレオチドプローブ20:5’-Dd(TTACACATGGACATGCGCAATT)-NH2-3’(配列番号78)
配列番号65に相補的な配列(配列番号79)を有する3’末端ビオチン修飾オリゴヌクレオチドプローブ21、配列番号69に相補的な配列(配列番号80)を有する3’末端ビオチン修飾オリゴヌクレオチドプローブ22、および、配列番号73に相補的な配列(配列番号81)を有する3’末端ビオチン修飾オリゴヌクレオチドプローブ23を、それぞれストレプトアビジンと混合する。それらの混合液をニトロセルロースメンブレン(商品名:Hi-Flow 135、ミリポア社製)上の3箇所にディスペンサーを用いて、上流側から順に互いに離れた位置でライン上に塗布し、40℃で30分間風乾した。三本の検出ラインを作製した。
オリゴヌクレオチドプローブ21:5’-Dd(GATCATACACGTGGTTGGAAGCTAACC)-Biotin-3’(配列番号79)
オリゴヌクレオチドプローブ22:5’-Dd(TATGATATGCTTCTCCACGCATAAT)-Biotin-3’(配列番号80)
オリゴヌクレオチドプローブ23:5’-Dd(CTCAGCAGTTTCCTCTAAAGTA)-Biotin-3’(配列番号81)
バッキングシートから成る基材に、上記で作成したニトロセルロースメンブレンからなるクロマトグラフィー媒体、工程(4)で作製したコンジュゲーションパッド、試料添加部である汎用性のサンプルパッド、展開した試料や標識物質を吸収するための吸収パッドを図6に示す様に貼り合わせ、アゾベンゼン挿入プライマーセットを用いたPCR増幅産物の検出用テストストリップを作製した。
(7)テストストリップによるPCR産物の検出
(i):一本目の検出ラインのみ青色に着色。
(ii):二本目の検出ラインのみオレンジ色に着色。
(iii):三本目の検出ラインのみ緑色に着色。
(iv):一本目の検出ラインが青色に、二本目の検出ラインがオレンジ色に、三本目の検出ラインが緑色に着色。
(v):どの検出ラインも着色は認められなかった。
本実施例では、標的としてプラスミドpUC19を導入した大腸菌(E.coli DH5α)を用いる。pUC19がテンプレートとなるときに、PCR増幅により約330塩基対が増幅するようにフォワードプライマー(F)およびリバースプライマー(R)を設計した。それぞれの5’末端側に人工核酸であるアゾベンゼンを含むポリメラーゼ反応阻害領域(X)、およびタグ配列T25およびT26を導入したタグ付きプライマー、T25-X-FおよびT26-X-Rを合成した。この2種のアゾベンゼン挿入プライマーはつくばオリゴサービス株式会社にて受託合成を行い購入した。
タグ配列T25:5’-Dd(TGGCAACATTTTTCACTGGGTTTATAG)-3’(配列番号82)
タグ配列T26:5’-Dd(GGTTAGCTTCCAACCACGTGTAGATCA)-3’(配列番号83)
プライマーF:5’-Dd(GGAAACAGCTATGACCATGA)-3’(配列番号84)
プライマーR:5’-Dd(TCTATGCGGCATCAGAGCAG)-3’(配列番号85)
プライマーT25-X-F:5’-Dd(TGGCAACATTTTTCACTGGGTTTATAG X GGAAACAGCTATGACCATGA)-3’(配列番号86)
プライマーT26-X-R:5’-Dd(GGTTAGCTTCCAACCACGTGTAGATCA X TCTATGCGGCATCAGAGCAG)-3’(配列番号87)
プラスミドpUC19を導入した大腸菌(E.coli DH5α)のコロニーを取り、1ml水中に混和した。前記工程(1)で作製したプライマーセットを用いたPCR反応を行った。プライマーFとプライマーRを各5pmolと、前記大腸菌のけん濁液1μlとを0.2mlのPCR用チューブに入れ、ExTaqPCRデバイス(タカラバイオ社製)の説明書に従い、25μlのPCR反応液を調製した。その後、チューブをサーマルサイクラー(GeneAmp PCR System、アプライドバイオシステム社製)にセットし、95℃で5分間熱処理後、95℃で30秒、55℃で30秒、72℃で30秒のサイクルを30回行い、目的の約330bpを増幅した。また、けん濁液を添加しないで同様の反応を行い、ネガティブコントロールとした。
Gold Colloid(10nm、5.7×1012(粒子数/ml)、British BioCell International社製)と抗FITC抗体溶液(5mMリン酸バッファー、pH7)を混合し、20分、室温で静置した。1%BSAおよび0.1%PEGを含む溶液を1/2量添加し、10000rpmで25分間遠心分離し、上清を除去、1%BSAおよび0.1%PEGを含む溶液を添加し混和後、10000rpmで25分間遠心分離した。遠心後に上清を除去し、5mMリン酸バッファー(pH7)を添加した。このバッファー置換を再度行った。
オリゴヌクレオチドプローブ24:5’-Dd(CTATAAACCCAGTGAAAAATGTTGCCA)-FITC-3’(配列番号88)
配列番号83に相補的な配列(配列番号89)を有する3’末端ビオチン修飾オリゴヌクレオチドプローブ25を、ストレプトアビジンと混合する。その混合液をニトロセルロースメンブレン(商品名:Hi-Flow 180、 ミリポア社製)にディスペンサーを用いてライン上に塗布し、40℃で30分間風乾した。
オリゴヌクレオチドプローブ25:5’-Dd(GATCATACACGTGGTTGGAAGCTAACC)-Biotin-3’(配列番号89)
バッキングシートから成る基材に、上記で作成したニトロセルロースメンブレンからなるクロマトグラフィー媒体、コンジュゲーションパッド、試料添加部である汎用性のサンプルパッド、展開した試料や標識物質を吸収するための吸収パッドを図6に示す様に貼り合わせ、アゾベンゼン挿入プライマーセットを用いたPCR増幅産物の検出用テストストリップを作製した。
工程(2)で作製したPCR産物を変性することなく、直ちに工程(5)で作製したテストストリップ上の試料添加部位にアプライし、クロマトグラフィーによる検出を行った。工程(2)で検体として大腸菌を添加した場合、テストライン上に標的核酸特異的な着色ラインが検出された。一方、ネガティブコントロールとして大腸菌を添加しない場合、ラインの検出は認められなかった。また、クロマトグラフィーによる検出に要した時間は、10~15分と短時間であった。
本実施例では、標的として2種類の50塩基対の合成DNAを準備した。この2つのDNAは、末端から20番目の一塩基だけ異なる一塩基多型(SNP)である。標的1を野生型(WT)、標的2を変異型(MT)と仮定してSNP検出試験を行う。
標的1(WT):CACACCGCATATGGTGCACTCTCAGTACAATCTGCTCTGATGCCGCATAG(配列番号90)
標的2(MT):CACACCGCATATGGTGCACTCTCAGTACAAGCTGCTCTGATGCCGCATAG(配列番号91)
前記工程(1)で合成した標的1もしくは標的2をテンプレートとしたときに、PCR増幅により約50塩基対が増幅するようにフォワードプライマー(Fwt)、リバースプライマー(Rwt)、およびリバースプライマー(Rmt)を設計した。それぞれの5’末端側に人工核酸であるアゾベンゼンを含むポリメラーゼ反応阻害領域(X)、およびタグ配列T27、T28、T29を導入したタグ付きプライマー、T27-X-Fwt、T28-X-Rwt、T29-X-Rmtを合成した。この3種のアゾベンゼン挿入プライマーはつくばオリゴサービス株式会社にて受託合成を行い購入した。
タグ配列T27:5’-Dd(TGGCAACATTTTTCACTGGGTTTATAG)-3’(配列番号92)
タグ配列T28:5’-Dd(GGTTAGCTTCCAACCACGTGTAGATCA)-3’(配列番号93)
タグ配列T29:5’-Dd(CGCATTGAGCAAGTGTACAGAGCAT)-3’(配列番号94)
プライマーFwt:5’-Dd(CACACCGCATATGGTGCACT)-3’(配列番号95)
プライマーRwt:5’-Dd(CTATGCGGCATCAGAGCAGA)-3’(配列番号96)
プライマーRmt:5’-Dd(CTATGCGGCATCAGAGCAGC)-3’(配列番号97)
プライマーT27-X-Fwt:5’-Dd(TGGCAACATTTTTCACTGGGTTTATAG X CACACCGCATATGGTGCACT)-3’(配列番号98)
プライマーT28-X-Rwt:5’-Dd(GGTTAGCTTCCAACCACGTGTAGATCA X TCTATGCGGCATCAGAGCAGA)-3’(配列番号99)
プライマーT29-X-Rmt:5’-Dd(CGCATTGAGCAAGTGTACAGAGCAT X TCTATGCGGCATCAGAGCAGC)-3’(配列番号100)
前記工程(1)で作製した標的1、もしくは、標的2を含む試料溶液と、前記工程(2)で合成した3種のプライマーを用いてPCR反応を行った。
プライマーT27-X-Fwt、プライマーT28-X-Rwt、および、プライマーT29-X-Rmtを各5pmolと、各テンプレート1fmolを0.2mlのPCRチューブに入れ、ExTaq PCRキット(タカラバイオ社製)の説明書に従い、100μlのPCR反応液を調整した。
(i)テンプレートとして標的1(ホモ型)、
(ii)テンプレートとして標的1、および、標的2(ヘテロ型)、
(iii)テンプレートとして標的2(ホモ型)、
(iv)標的なし(水添加)
Gold Colloid(40nm、9.0×1010(粒子数/ml)、British BioCell International社製)と、チオール基含有オリゴヌクレオチドプローブ26(配列番号101、配列番号92の相補鎖)を混合し、50℃で16時間インキュベートした。6000rpmで15分間遠心分離し、上清を除去、0.05M塩化ナトリウムを含む5mMリン酸バッファー(pH7)を添加し混和後、再度50℃で40時間インキュベートした。インキュベート後、遠心(6000rpm、15分間)を行い、上清を除去し、5mMリン酸バッファー(pH7)を添加した。このバッファー置換を再度行った。
オリゴヌクレオチドプローブ26:5’-Dd(CTATAAACCCAGTGAAAAATGTTGCCA)-SH-3’(配列番号101)
配列番号93に相補的な配列(配列番号102)を有する3’末端ビオチン修飾オリゴヌクレオチドプローブ27、および、配列番号94に相補的な配列(配列番号103)を有する3’末端ビオチン修飾オリゴヌクレオチドプローブ28、それぞれストレプトアビジンと混合する。それらの混合液をニトロセルロースメンブレン(商品名:Hi-Flow 135、ミリポア社製)上の、上流側から順に互いに離れた2箇所の位置でディスペンサーを用いて塗布し、40℃で30分間風乾し、二本の検出ラインを作製した。
オリゴヌクレオチドプローブ27:5’-Dd(GATCATACACGTGGTTGGAAGCTAACC)-Biotin-3’(配列番号102)
オリゴヌクレオチドプローブ28:5’-Dd(TTGCTCTGTACACTTGCTCAATGCG)-Biotin-3’(配列番号103)
バッキングシートから成る基材に、工程(5)で作成したニトロセルロースメンブレンからなるクロマトグラフィー媒体、工程(4)で作製したコンジュゲーションパッド、試料添加部である汎用性のサンプルパッド、展開した試料や標識物質を吸収するための吸収パッドを図6に示す様に貼り合わせ、スペーサー(アゾベンゼン)挿入プライマーセットを用いたPCR増幅産物の検出用テストストリップを作製した。
工程(3)で作製した(i)~(iv)のPCR産物を変性することなく、直ちに工程(6)で作製したテストストリップ上の試料添加部位にそれぞれアプライし、クロマトグラフィーによる検出を行った。
(i):一本目の検出ラインのみ着色。
(ii):一本目のライン、二本目の検出ライン両方とも着色。
(iii):二本目の検出ラインのみ着色。
(iv):どの検出ラインも着色は認められなかった。
本実施例では、試料として薄力小麦粉(日清製粉社製)、そば粉(おびなた社製)、および、落花生(ル・モンド・アリコ社製)の3種類を用いた。各試料から、PCR増幅により、それぞれ約141塩基対、約127塩基対、および、約95塩基対のDNAが増幅するようにフォワードプライマー(Fwtr)とリバースプライマー(Rwtr)、フォワードプライマー(FFAG)とリバースプライマー(RFAG)、および、フォワードプライマー(Fagg)とリバースプライマー(Ragg)の3組のプライマーをそれぞれ設計した。それぞれの5’末端側に5’-5’結合+dA+3’-3’結合を含むポリメラーゼ反応阻害領域(X)、およびタグ配列T7とT8、タグ配列T9とT10、および、タグ配列T11とT12を導入したタグ付きスペーサー挿入プライマー、T7-X-FwtrとT8-X-Rwtr、T9-X-FFAGとT10-X-RFAG、および、T11-X-FaggとT12-X-Raggを合成した。この6種のタグスペーサー挿入プライマーはつくばオリゴサービス株式会社にて受託合成を行い購入した。
プライマーT7-X-Fwtr:5’-Dd(TGGCAACATTTTTCACTGGGTTTATAG X CATCACAATCAACTTATGGTGG)-3’(配列番号104)
プライマーT8-X-Rwtr:5’-Dd(GGTTAGCTTCCAACCACGTGTAGATCA X TTTGGGAGTTGAGACGGGTTA)-3’(配列番号105)
プライマーT9-X-FFAG:5’-Dd(CGCATTGAGCAAGTGTACAGAGCAT X AACGCCATAACCAGCCCGATT)-3’(配列番号106)
プライマーT10-X-RFAG:5’-Dd(ATTATGCGTGGAGAAGCATATCATA X CCTCCTGCCTCCCATTCTTC)-3’(配列番号107)
プライマーT11-X-Fagg:5’-Dd(AATTGCGCATGTCCATGTGTAA X CGAAGGAAACCCCGCAATAAAT)-3’(配列番号108)
プライマーT12-X-Ragg:5’-Dd(TACTTTAGAGGAAACTGCTGAG X CGACGCTATTTACCTTGTTGAG)-3’(配列番号109)
各試料(小麦粉、そば粉、粉砕した落花生)2gからそれぞれゲノムDNAを精製した。まず、遠沈管(50mL容)に試料を量り採り、CTAB緩衝液15mLを加え、ホモジナイザーを用いて混合した。CTAB緩衝液30mLを加え、転倒混和後55℃で30分間加温する。加温処理後、溶液を撹拌し、均質となった溶液600μLをマイクロチューブ(1.5mL容)に量り採った。
前記工程(1)で実施し作製した3組のプライマーセットを用いたPCR反応を行った。プライマーT7-X-Fwtr、プライマーT8-X-Rwtr、プライマーT9-X-FFAG、プライマーT10-X-RFAG、プライマーT11-X-Fagg、および、プライマーT12-X-Raggを各15pmolと、各DNA試料原液を0.2mlのPCR用チューブに入れ、ExTaq PCRデバイス(タカラバイオ社製)の説明書に従い、100μlのPCR反応液を調製した。
(i)テンプレートとして小麦粉のDNA試料原液を添加、
(ii)テンプレートとしてそば粉のDNA試料原液を添加、
(iii)テンプレートとして落花生のDNA試料原液を添加、
(iv)テンプレートとして小麦粉、そば粉、落花生3種類全てのDNA試料原液を添加、そして、
(v)テンプレートなし。
Gold Colloid(40nm、9.0×1010(粒子数/ml)、British BioCell International社製)と、チオール基含有オリゴヌクレオチドプローブ29(配列番号110、配列番号34の相補鎖)、チオール基含有オリゴヌクレオチドプローブ30(配列番号111、配列番号38の相補鎖)、および、チオール基含有オリゴヌクレオチドプローブ31(配列番号112、配列番号42の相補鎖)を、それぞれ混合し、50℃で16時間インキュベートした。6000rpmで15分間遠心分離し、上清を除去、0.05M塩化ナトリウムを含む5mMリン酸バッファー(pH7)を添加し混和後、再度50℃で40時間インキュベートした。インキュベート後、遠心(6000rpm、15分間)を行い、上清を除去し、5mMリン酸バッファー(pH7)を添加した。このバッファー置換を再度行った。
オリゴヌクレオチドプローブ29:5’-Dd(CTATAAACCCAGTGAAAAATGTTGCCA)-SH-3’(配列番号110)
オリゴヌクレオチドプローブ30:5’-Dd(TTGCTCTGTACACTTGCTCAATGCG)-SH-3’(配列番号111)
オリゴヌクレオチドプローブ31:5’-Dd(TTACACATGGACATGCGCAATT)-SH-3’(配列番号112)
実施例9の工程(4)と同様の方法を用いて、オリゴヌクレオチドプローブ15、16、17をそれぞれライン上に塗布し、ニトロセルロースメンブレン上に乾燥固定した。
バッキングシートから成る基材に、工程(5)で作成したニトロセルロースメンブレンからなるクロマトグラフィー媒体、工程(4)で作製したコンジュゲーションパッド、試料添加部である汎用性のサンプルパッド、展開した試料や標識物質を吸収するための吸収パッドを図6に示す様に貼り合わせ、スペーサー(5’-5’結合+3’-3’結合)挿入プライマーセットを用いたPCR増幅産物の検出用テストストリップを作製した。
工程(3)で作製した(i)~(v)のPCR産物を変性することなく、直ちに工程(6)で作製したテストストリップ上の試料添加部位にそれぞれアプライし、クロマトグラフィーによる検出を行った。
(i):一本目の検出ラインのみ着色。
(ii):二本目の検出ラインのみ着色。
(iii):三本目の検出ラインのみ着色。
(iv):一本目の検出ライン、二本目の検出ライン、および、三本目の検出ラインが全て着色。
(v):どの検出ラインも着色は認められなかった。
(1)ターゲットDNA(pUC19、および、インサートを含むpUC19)の準備
本実施例では、標的として2種類のプラスミドDNAを準備した。標的の概要を図13、図14に示す。標的1はプラスミドpUC19 DNA(タカラバイオ社製)、標的2はpUC19のマルチクローニングサイトにインサートとして遺伝子A(1668bp)を挿入したプラスミド(pUC19/遺伝子A)を用いる。
遺伝子A配列(配列番号113)
標的1:pUC19配列(配列番号114)
標的2:pUC19配列/遺伝子A(配列番号115)
図13、図14で示すように、pUC19配列上のマルチクローニングサイトを挟んだ位置にフォワードプライマー(F)、および、リバースプライマー(R)を、インサート遺伝子A配列上にリバースプライマー(R‐in)を設計した。フォワードプライマーFとリバースプライマーRを用いてPCR反応を行うと、標的1のpUC19では118塩基対の増幅産物が得られ、インサートを含む標的2のpUC19/遺伝子Aでは1768塩基対の増幅産物が得られる。また、フォワードプライマーFとリバースプライマーR-inを用いてPCR反応を行うと、標的2(インサート挿入)で101塩基長の増幅産物が生ずる。それぞれの5’末端側にC6リンカーを含むポリメラーゼ反応阻害領域(X)、および実施例12に記載したタグと同様のタグ配列T27、T28、T29を導入したタグ付きプライマー、T27-X-F、T28-X-R、T29-X-R-inを合成した。この3種のC6リンカー挿入プライマーはつくばオリゴサービス株式会社にて受託合成を行い購入した。
プライマーF:5’-Dd(GGAAACAGCTATGACCATGA)-3’(配列番号116)
プライマーR:5’-Dd(TTTCCCAGTCACGACGTTGT)-3’(配列番号117)
プライマーR-in:5’-Dd(AGTGCGTGCTGGGCTCTTC)-3’(配列番号118)
プライマーT27-X-F:5’-Dd(TGGCAACATTTTTCACTGGGTTTATAG X GGAAACAGCTATGACCATGA)-3’(配列番号119)
プライマーT28-X-R:5’-Dd(GGTTAGCTTCCAACCACGTGTAGATCA X TTTCCCAGTCACGACGTTGT)-3’(配列番号120)
プライマーT29-X-R-in:5’-Dd(CGCATTGAGCAAGTGTACAGAGCAT X AGTGCGTGCTGGGCTCTTC)-3’(配列番号121)
前記工程(1)で準備した標的1、もしくは、標的2溶液と前記工程(2)で合成した3種のプライマーを用いてPCR反応を行った。プライマーT27-X-F、プライマーT28-X-R、および、プライマーT29-X-R-inを各5pmolと、各テンプレート1fmolを0.2mlのPCRチューブに入れ、ExTaq PCRキット(タカラバイオ社製)の説明書に従い、100μlのPCR反応液を調整した。
(i)テンプレートとして標的1、
(ii)テンプレートとして標的1、および、標的2、
(iii)テンプレートとして標的2、
(iv)標的なし(水添加)
実施例13の工程(4)~(6)と同様の方法で、スペーサー挿入プライマーセットを用いたPCR増幅産物の検出用テストストリップを作製した。
工程(3)で作製した(i)~(iv)のPCR産物を変性することなく、直ちに工程(4)で作製したテストストリップ上の試料添加部位にそれぞれアプライし、クロマトグラフィーによる検出を行った。
(i):一本目の検出ラインのみ着色。
(ii):一本目のライン、二本目の検出ライン両方とも着色。
(iii):二本目の検出ラインのみ着色。
(iv):どの検出ラインも着色は認められなかった。
2.タグ領域
3.ポリメラーゼ反応阻害領域(スペーサー領域)
4.第1プライマー(ジョイントプライマー)のプライマー領域
5.第1プライマー(ジョイントプライマー)の共通領域
6.第2プライマーの共通領域
7.第2プライマーのタグ領域
8.第2プライマーのポリメラーゼ反応阻害領域(スペーサー領域)
9.標的核酸配列
10.フォワードプライマー
11.フォワードプライマーのプライマー領域
12.フォワードプライマーのタグ領域
13.リバースプライマー
14.リバースプライマーのプライマー領域
15.リバースプライマーのタグ領域
16.両末端に一本鎖領域を有するDNA増幅産物
17.標的核酸配列
18.フォワード第1プライマー
19.フォワード第1プライマーのプライマー領域
20.フォワード第1プライマーのタグ領域
21.リバース第1プライマー
22.リバース第1プライマーのプライマー領域
23.リバース第1プライマーのタグ領域
24.第1プライマーによる二本鎖PCR産物
25.フォワード第2プライマー
26.フォワード第2プライマーのプライマー領域
27.フォワード第2プライマーのタグ領域
28.リバース第2プライマー
29.リバース第2プライマーのプライマー領域
30.リバース第2プライマーのタグ領域
31.両末端に一本鎖領域を有するDNA増幅産物
32.サンプルパッド
33.コンジュゲートパッド
34.捕捉用オリゴヌクレオチドを保持した担体
35.吸収パッド
36.基材
37.テストライン
38.コントロールライン
39.標識分子結合用オリゴヌクレオチド
40.標識分子
41.PCR産物-標識分子複合体
42.多孔質メンブレン
43.捕捉用オリゴヌクレオチド
44.捕捉用オリゴヌクレオチドを各ウェルに保持した担体(マイクロアレイ)
45.捕捉用オリゴヌクレオチドを保持したビーズ担体
46.変性PAGE、染色後のポリアクリルアミドゲル
47.約360merの一本鎖
48.約330merの一本鎖
49.クロマトグラフィー様ストリップ
50.テストライン
51.テストライン1
52.テストライン2
53.テストライン3
Claims (24)
- 核酸増幅反応で二本鎖化されないタグ領域が5’末端側に連結されたプライマーを用いて核酸増幅反応を行い、両末端に一本鎖領域を有する核酸を得ることを特徴とする、核酸の増幅方法。
- タグ領域が、スペーサーを介してプライマーと連結されていることを特徴とする、請求項1に記載の核酸の増幅方法。
- スペーサーが、核酸誘導体を含むことを特徴とする、請求項2に記載の標的核酸の増幅方法。
- 核酸誘導体が、L型核酸、3-deoxy-2-hydroxy-dN、修飾塩基核酸、損傷塩基核酸、リン酸結合部位修飾核酸、RNA、2’-OMe-N、および、それらの誘導体からなる群から選択される少なくとも1以上であることを特徴とする、請求項3に記載の核酸の増幅方法。
- L型核酸が、L型DNA、L型RNA、および、それらの誘導体からなる群から選択される少なくとも1以上であることを特徴とする、請求項4に記載の核酸の増幅方法。
- 3-deoxy-2-hydroxy-dNが、2’-5’結合によりプライマーと連結されていることを特徴とする、請求項4に記載の核酸の増幅方法。
- 修飾塩基核酸が、発色団またはビオチンを含むことを特徴とする、請求項4に記載の核酸の増幅方法。
- 発色団が、ピレン、エテノ、ピロロ、ぺリレン、フルオレセイン、FITC、Cy3、Cy5、TAMRA、ダブシル、シアニン、および、それらの誘導体からなる群から選択される少なくとも1以上であることを特徴とする、請求項7に記載の核酸の増幅方法。
- 損傷塩基核酸が、脱塩基ヌクレオチド、5-ヒドロキシメチル-dN、および、それらの誘導体からなる群から選択される少なくとも1以上であることを特徴とする、請求項4に記載の核酸の増幅方法。
- リン酸結合部位修飾核酸が、ホスホロチオエート又はその誘導体を含むことを特徴とする、請求項4に記載の核酸の増幅方法。
- 核酸誘導体が、5’-5’結合でプライマーと連結され、かつ、3’-3’結合でタグ領域と連結されていることを特徴とする、請求項3~10のいずれかに記載の核酸の増幅方法。
- スペーサーが、非核酸誘導体を含むことを特徴とする、請求項2に記載の標的核酸の増幅方法。
- 非核酸誘導体が、D-threoninol骨格を有することを特徴とする、請求項12に記載の核酸の増幅方法。
- D-threoninol骨格に、アゾベンゼン、ビオチン、EDTA、および、発色団からなる群から選択される少なくとも1以上が導入されていることを特徴とする、請求項13に記載の核酸の増幅方法。
- 発色団が、ピレン、エテノ、ピロロ、ぺリレン、フルオレセイン、FITC、Cy3、Cy5、TAMRA、ダブシル、シアニン、および、それらの誘導体からなる群から選択される少なくとも1以上であることを特徴とする、請求項14に記載の核酸の増幅方法。
- 非核酸誘導体が、炭素鎖(Cn)、ペグ鎖((CH2CH2O)n)、ジスルフィド含有鎖(CnSSCn)、および、ジチオールフォスフォロアミダイトからなる群から選択される少なくとも1以上であることを特徴とする、請求項12に記載の核酸の増幅方法。
- スペーサーが複数種類および/又は複数個であることを特徴とする、請求項2に記載の核酸の増幅方法。
- 請求項1~17のいずれかに記載の核酸の増幅方法で得られた、両末端に一本鎖領域を有する核酸を検出することを特徴とする核酸の検出方法。
- 片方の一本鎖領域と相補的な配列を含む第1のオリゴヌクレオチドプローブを固相に固定する工程、および、前記第1のオリゴヌクレオチドプローブを、両末端に一本鎖領域を有する核酸とハイブリダイズさせる工程を含むことを特徴とする、請求項18に記載の核酸の検出方法。
- 更に、他方の一本鎖領域と相補的な配列を含む第2のオリゴヌクレオチドプローブを標識物質と結合する工程、および、前記第2のオリゴヌクレオチドプローブを、両末端に一本鎖領域を有する核酸とハイブリダイズさせる工程を含むことを特徴とする、請求項19に記載の核酸の検出方法。
- 更に、目視で核酸を判別する工程を含むことを特徴とする、
請求項20に記載の核酸の検出方法。 - 標識物質が着色担体であることを特徴とする、
請求項20または21に記載の核酸の検出方法。 - 両末端に一本鎖領域を有する核酸を、核酸検出デバイス上で検出することを特徴とする、請求項18~22のいずれかに記載の核酸の検出方法。
- 核酸検出デバイスが、アレイ又はクロマトグラフィーである、請求項23に記載の核酸の検出方法。
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Citations (12)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPH05252998A (ja) | 1992-03-13 | 1993-10-05 | Internatl Reagents Corp | Pcr増幅dnaの測定法 |
JP2002534434A (ja) * | 1998-12-30 | 2002-10-15 | オリゴス・イーティーシー・インコーポレイテッド | 酸安定性骨格で修飾された末端がブロックされた核酸及びその治療的使用 |
JP2003504018A (ja) * | 1999-07-02 | 2003-02-04 | インビトロゲン・コーポレーション | 核酸合成の感度および特異性の増大のための組成物および方法 |
JP2006201062A (ja) | 2005-01-21 | 2006-08-03 | Kainosu:Kk | 核酸の検出あるいは定量方法 |
WO2006095550A1 (ja) | 2005-03-04 | 2006-09-14 | Kyoto University | Pcrプライマー、それを利用したpcr法及びpcr増幅産物、並びにpcr増幅産物を利用するデバイス及びdna-タンパク複合体 |
JP2007111048A (ja) * | 2005-10-05 | 2007-05-10 | Qiagen Gmbh | プルーフリーディング特性を有するdnaポリメラーゼを用いるポリメラーゼ連鎖反応のための方法 |
JP2008525037A (ja) * | 2004-12-23 | 2008-07-17 | アイ−スタツト・コーポレイシヨン | 分子診断システム及び方法 |
JP2009296948A (ja) | 2008-06-13 | 2009-12-24 | Olympus Corp | Pcr用プライマー、標的核酸の検出方法及び標的生体分子の検出方法 |
WO2010106997A1 (ja) * | 2009-03-19 | 2010-09-23 | 株式会社カネカ | 核酸の検出方法及びキット、デバイス |
WO2012070618A1 (ja) * | 2010-11-24 | 2012-05-31 | 株式会社カネカ | 増幅核酸検出方法及び検出デバイス |
WO2013039228A1 (ja) * | 2011-09-14 | 2013-03-21 | 日本碍子株式会社 | 標的核酸の検出方法 |
WO2013038534A1 (ja) * | 2011-09-14 | 2013-03-21 | 日本碍子株式会社 | 標的核酸の検出方法 |
Family Cites Families (36)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US5310650A (en) | 1986-09-29 | 1994-05-10 | Abbott Laboratoires | Method and device for improved reaction kinetics in nucleic acid hybridizations |
ATE157404T1 (de) | 1989-03-17 | 1997-09-15 | Abbott Lab | Verfahren und vorrichtung zur hybridisierung von nukleinsäure mit verbesserter reaktionskinetik |
US5629158A (en) | 1989-03-22 | 1997-05-13 | Cemu Bitecknik Ab | Solid phase diagnosis of medical conditions |
GB8920097D0 (en) * | 1989-09-06 | 1989-10-18 | Ici Plc | Amplification processes |
US5475098A (en) | 1994-06-14 | 1995-12-12 | The United States Of America As Represented By The Department Of Health And Human Services | Distinctive DNA sequence of E. coli 0157:H7 and its use for the rapid, sensitive and specific detection of 0157:H7 and other enterohemorrhagic E. coli |
US5925518A (en) | 1995-05-19 | 1999-07-20 | Akzo Nobel N.V. | Nucleic acid primers for amplification of a mycobacteria RNA template |
US5874216A (en) | 1996-02-23 | 1999-02-23 | Ensys Environmental Products, Inc. | Indirect label assay device for detecting small molecules and method of use thereof |
CZ293215B6 (cs) * | 1996-08-06 | 2004-03-17 | F. Hoffmann-La Roche Ag | Rekombinantní tepelně stálá DNA polymeráza, způsob její přípravy a prostředek, který ji obsahuje |
US6124092A (en) | 1996-10-04 | 2000-09-26 | The Perkin-Elmer Corporation | Multiplex polynucleotide capture methods and compositions |
US6136610A (en) | 1998-11-23 | 2000-10-24 | Praxsys Biosystems, Inc. | Method and apparatus for performing a lateral flow assay |
WO2001021637A1 (fr) | 1999-09-20 | 2001-03-29 | Makoto Komiyama | Oligonucleotide sensible a la lumiere |
JP2001157598A (ja) | 1999-12-01 | 2001-06-12 | Matsushita Electric Ind Co Ltd | 遺伝子検出方法およびその方法を利用した装置 |
EP1130113A1 (en) | 2000-02-15 | 2001-09-05 | Johannes Petrus Schouten | Multiplex ligation dependent amplification assay |
GB0016814D0 (en) | 2000-07-07 | 2000-08-30 | Lee Helen | Improved dipstick assays (3) |
DE10046184A1 (de) | 2000-09-18 | 2002-04-04 | November Ag Molekulare Medizin | Verfahren zum Nachweis mindestens einer Nukleinsäuresequenz |
US20030198980A1 (en) | 2001-12-21 | 2003-10-23 | Applera Corporation | Heteroconfigurational polynucleotides and methods of use |
US7452699B2 (en) * | 2003-01-15 | 2008-11-18 | Dana-Farber Cancer Institute, Inc. | Amplification of DNA in a hairpin structure, and applications |
JP3923917B2 (ja) | 2003-03-26 | 2007-06-06 | 株式会社東芝 | ターゲットの製造方法、標的配列検出方法、ターゲットおよび標的配列検出用アッセイキット |
FR2853077B1 (fr) | 2003-03-28 | 2005-12-30 | Vedalab | Procedes immunochromatographiques en phase solide |
CA2522753C (en) | 2003-04-18 | 2014-06-10 | Becton, Dickinson And Company | Immuno-amplification |
ATE504660T1 (de) * | 2003-05-07 | 2011-04-15 | Coris Bioconcept Sprl | Einstufige oligochromatographische vorrichtung und verfahren zu ihrer verwendung |
CN1459506A (zh) | 2003-05-30 | 2003-12-03 | 山东大学 | 中国对虾抗菌肽基因的重组表达与应用 |
CA2528172C (en) | 2003-06-03 | 2012-10-30 | Bayer Healthcare Llc | Native analyte as reference in lateral flow assays |
AU2006215038B2 (en) | 2005-02-18 | 2011-07-07 | Kyushu University, National University Corporation | Gene detection method |
CN100513577C (zh) | 2005-07-04 | 2009-07-15 | 北京大学 | 一种pcr方法 |
US8703445B2 (en) | 2005-12-29 | 2014-04-22 | Abbott Point Of Care Inc. | Molecular diagnostics amplification system and methods |
RU2477137C2 (ru) * | 2006-03-08 | 2013-03-10 | АРКЕМИКС Эл Эл Си | Связывающие комплемент аптамеры и средства против с5, пригодные для лечения глазных нарушений |
US8377379B2 (en) | 2006-12-15 | 2013-02-19 | Kimberly-Clark Worldwide, Inc. | Lateral flow assay device |
JPWO2009034842A1 (ja) | 2007-09-11 | 2010-12-24 | 株式会社カネカ | 核酸検出方法、および核酸検出キット |
US8008019B2 (en) | 2007-11-28 | 2011-08-30 | Luminex Molecular Diagnostics | Use of dual-tags for the evaluation of genomic variable repeat regions |
JP2009162535A (ja) * | 2007-12-28 | 2009-07-23 | Sekisui Medical Co Ltd | 固相作製用試薬及び該試薬を使用して作製した固相 |
CN102686728B (zh) | 2009-06-29 | 2018-09-18 | 卢米耐克斯公司 | 具有发夹构象的嵌合引物及其使用方法 |
SG184946A1 (en) * | 2010-05-07 | 2012-11-29 | Quantibact As | Method for generating a double stranded nucleic acid with a single stranded overhang |
CN101845511A (zh) | 2010-06-12 | 2010-09-29 | 中国人民解放军军事医学科学院微生物流行病研究所 | 一种核酸检测方法及其专用试剂盒 |
US9029103B2 (en) | 2010-08-27 | 2015-05-12 | Illumina Cambridge Limited | Methods for sequencing polynucleotides |
WO2013040491A2 (en) * | 2011-09-15 | 2013-03-21 | Shafer David A | Probe: antiprobe compositions for high specificity dna or rna detection |
-
2013
- 2013-04-26 EP EP13782032.0A patent/EP2843058B1/en active Active
- 2013-04-26 JP JP2014512726A patent/JP6219816B2/ja active Active
- 2013-04-26 US US14/396,466 patent/US9783844B2/en active Active
- 2013-04-26 CN CN201380022347.XA patent/CN104254620B/zh active Active
- 2013-04-26 WO PCT/JP2013/062488 patent/WO2013162026A1/ja active Application Filing
-
2017
- 2017-09-29 JP JP2017190135A patent/JP2018038406A/ja active Pending
Patent Citations (12)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JPH05252998A (ja) | 1992-03-13 | 1993-10-05 | Internatl Reagents Corp | Pcr増幅dnaの測定法 |
JP2002534434A (ja) * | 1998-12-30 | 2002-10-15 | オリゴス・イーティーシー・インコーポレイテッド | 酸安定性骨格で修飾された末端がブロックされた核酸及びその治療的使用 |
JP2003504018A (ja) * | 1999-07-02 | 2003-02-04 | インビトロゲン・コーポレーション | 核酸合成の感度および特異性の増大のための組成物および方法 |
JP2008525037A (ja) * | 2004-12-23 | 2008-07-17 | アイ−スタツト・コーポレイシヨン | 分子診断システム及び方法 |
JP2006201062A (ja) | 2005-01-21 | 2006-08-03 | Kainosu:Kk | 核酸の検出あるいは定量方法 |
WO2006095550A1 (ja) | 2005-03-04 | 2006-09-14 | Kyoto University | Pcrプライマー、それを利用したpcr法及びpcr増幅産物、並びにpcr増幅産物を利用するデバイス及びdna-タンパク複合体 |
JP2007111048A (ja) * | 2005-10-05 | 2007-05-10 | Qiagen Gmbh | プルーフリーディング特性を有するdnaポリメラーゼを用いるポリメラーゼ連鎖反応のための方法 |
JP2009296948A (ja) | 2008-06-13 | 2009-12-24 | Olympus Corp | Pcr用プライマー、標的核酸の検出方法及び標的生体分子の検出方法 |
WO2010106997A1 (ja) * | 2009-03-19 | 2010-09-23 | 株式会社カネカ | 核酸の検出方法及びキット、デバイス |
WO2012070618A1 (ja) * | 2010-11-24 | 2012-05-31 | 株式会社カネカ | 増幅核酸検出方法及び検出デバイス |
WO2013039228A1 (ja) * | 2011-09-14 | 2013-03-21 | 日本碍子株式会社 | 標的核酸の検出方法 |
WO2013038534A1 (ja) * | 2011-09-14 | 2013-03-21 | 日本碍子株式会社 | 標的核酸の検出方法 |
Non-Patent Citations (3)
Title |
---|
ANALYTICAL BIOCHEMISTRY, vol. 193, 1991, pages 231 - 235 |
LIANG, X. ET AL.: "Nick Sealing by T4 DNA Ligase on a Modified DNA Template: Tethering a Functional Molecule on D-Threoninol", CHEM. EUR. J., vol. 17, 2011, pages 10388 - 10396, XP055176515 * |
See also references of EP2843058A4 |
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WO2015076356A1 (ja) * | 2013-11-22 | 2015-05-28 | 株式会社カネカ | 短鎖rnaの検出方法 |
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US10392652B2 (en) | 2013-11-22 | 2019-08-27 | Kaneka Corporation | Micro RNA detection method using two primers to produce an amplified double stranded DNA fragment having a single stranded region at one end |
WO2015175789A1 (en) * | 2014-05-14 | 2015-11-19 | Mcruer Robert N | Translocation control for sensing by a nanopore |
US10457979B2 (en) | 2014-05-14 | 2019-10-29 | Stratos Genomics, Inc. | Translocation control for sensing by a nanopore |
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JP2017063728A (ja) * | 2015-09-30 | 2017-04-06 | 富士フイルム株式会社 | プライマーの設計方法、プライマー、プライマーセット、dna増幅方法および解析方法 |
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EP2843058A4 (en) | 2015-10-28 |
US9783844B2 (en) | 2017-10-10 |
US20150203905A1 (en) | 2015-07-23 |
JPWO2013162026A1 (ja) | 2015-12-24 |
EP2843058B1 (en) | 2019-12-18 |
CN104254620B (zh) | 2022-11-08 |
JP6219816B2 (ja) | 2017-10-25 |
CN104254620A (zh) | 2014-12-31 |
EP2843058A1 (en) | 2015-03-04 |
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