WO2019150414A1 - Procédé de détection d'un acide nucléique - Google Patents

Procédé de détection d'un acide nucléique Download PDF

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WO2019150414A1
WO2019150414A1 PCT/JP2018/002824 JP2018002824W WO2019150414A1 WO 2019150414 A1 WO2019150414 A1 WO 2019150414A1 JP 2018002824 W JP2018002824 W JP 2018002824W WO 2019150414 A1 WO2019150414 A1 WO 2019150414A1
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sequence
nucleic acid
primer
target
complementary
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PCT/JP2018/002824
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Japanese (ja)
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加藤　真吾
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学校法人 慶應義塾
株式会社ニコン
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Priority to PCT/JP2018/002824 priority Critical patent/WO2019150414A1/fr
Publication of WO2019150414A1 publication Critical patent/WO2019150414A1/fr

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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

Definitions

  • the present invention relates to a method for detecting a nucleic acid.
  • Nucleic acid chromatography in which a probe nucleic acid and a target nucleic acid are hybridized on a piece of test paper is useful as a method for easily detecting the target nucleic acid.
  • a special tag is attached to a primer for amplifying a target, and this tag portion is bound to a single-stranded probe to detect (non-patent document). 1, Patent Documents 1 and 2). In this case, there is a problem of false positives that are detected even when amplification occurs non-specifically.
  • An object of the present invention is to provide a method for detecting a nucleic acid that is unlikely to cause false positives due to nonspecific amplification.
  • the present inventor has primers (jumped) having sequences complementary to the upstream region and the downstream region of a part of the target nucleic acid sequence (target sequence).
  • PCR amplification is performed using a primer (Jumped primer), and the target sequence in the PCR amplification product is detected by hybridizing with a single-stranded probe to detect amplification specific to the target nucleic acid.
  • the present invention has been completed.
  • a PCR reaction is carried out using three types of primers (forward primer, reverse primer, jumped primer).
  • a DNA having a single-stranded DNA (a target sequence portion or a sequence portion complementary to the target sequence) is formed and detected by binding to a single-stranded probe having a sequence complementary to the sequence of this single-stranded portion.
  • Formation of DNA in which a part of the double-stranded DNA becomes a single strand can be enhanced by converting the double-stranded DNA, which is a PCR product, into a single strand (denaturation) and then returning it to a double strand.
  • the gist of the present invention is as follows.
  • the following primers (A) a forward primer for synthesizing the target nucleic acid; (B) a reverse primer for synthesizing the target nucleic acid, and (c) a sequence complementary to the upstream region adjacent to the 5 ′ end of a part of the target nucleic acid sequence (target sequence) and the 3 ′ end of the target sequence PCR amplification is performed using a primer (jumped primer) that has a complementary sequence in the downstream region adjacent to the target sequence but does not include a sequence complementary to the target sequence, and is complementary to the target sequence in the PCR amplification product
  • a method for detecting a nucleic acid comprising detecting a simple sequence by hybridizing with a single-stranded probe.
  • the method further comprises subjecting the PCR amplification product to additional denaturation before detecting the target sequence in the PCR amplification product or a sequence complementary thereto by hybridization with a single-stranded probe.
  • Method. (3) The method according to (2), wherein the denaturation treatment additionally performed on the PCR amplification product is heating, and further includes rapid cooling after the heating.
  • the denaturation treatment additionally performed on the PCR amplification product is an alkali treatment, and further comprises a neutralization treatment after the alkali treatment.
  • primers (A) a forward primer for synthesizing the target nucleic acid; (B) a reverse primer for synthesizing the target nucleic acid, and (c) a sequence complementary to the upstream region adjacent to the 5 ′ end of a part of the target nucleic acid sequence (target sequence) and the 3 ′ end of the target sequence
  • a primer (jumped primer) having a complementary sequence in the downstream region adjacent to but not including a sequence complementary to the target sequence
  • Primer set consisting of (6)
  • a nucleic acid detection kit comprising the primer set of (5).
  • the kit according to (6) further comprising a single-stranded probe that hybridizes with the target sequence in the PCR amplification product or a sequence complementary thereto.
  • the kit according to (7) wherein a single-stranded probe that hybridizes with a target sequence in a PCR amplification product or a sequence complementary thereto is held on a carrier.
  • the carrier is a strip composed of rectangular cellulose fibers, and is complementary to the target sequence in the PCR amplification product in the specimen development direction by dropping the specimen on the carrier or immersing the carrier in the specimen.
  • the kit according to (8), wherein a single-stranded probe that hybridizes with the sequence is immobilized.
  • a single-stranded probe that hybridizes to the target sequence in the PCR amplification product or a sequence complementary thereto is fixed to the strip so as to constitute one or a plurality of test lines perpendicular to the sample development direction.
  • the kit according to (9). (11) The kit according to any one of (6) to (10), further comprising a labeling reagent.
  • the original sequence of the target nucleic acid can be detected, and the possibility of false positives due to nonspecific amplification is reduced.
  • false positives due to non-specific amplification can be reduced in the detection of the target nucleic acid.
  • double-stranded DNA (c and d) having a loop-like single-stranded portion is generated in addition to long double-stranded DNA and short double-stranded DNA (a and b).
  • a, b, and d are synthesized by DNA extension, and a, b, c, and d are generated by once denaturing a double strand synthesized by DNA extension into a single strand and then performing hybridization again.
  • the determination result by the chromatography with the strip 1 of the PCR amplification product which carried out the denaturation by a strong alkali and the neutralization by a weak acid is shown.
  • the strip 1 was coated with the sequences A, B, C, and D selected from the HIV-1 gag and p17 regions in order from the top of the strip.
  • the results of chromatographic determination on strip 1 of PCR amplification products that had been heat-denatured (94 ° C. for 2 minutes) and then ice-cooled are shown.
  • the determination result by the chromatography with the strip 2 of the PCR amplification product which carried out the denaturation by a strong alkali and the neutralization by a weak acid is shown.
  • Strip 2 includes E, which is the sequence of ⁇ phage, sequence F selected from LTR, R3 region of HIV-2, gag of HIV-1, GTR, sequence of HIV-1, LTR, U5 of sequence selected from p17 region H and H ′, which are arrays selected from the region, were applied by changing the position in the order of E, F, G, and H from the top.
  • E is the sequence of ⁇ phage
  • sequence F selected from LTR
  • R3 region of HIV-2 gag of HIV-1, GTR
  • sequence of HIV-1, LTR sequence of HIV-1, LTR
  • U5 of sequence selected from p17 region H and H ′ which are arrays selected from the region
  • the nucleic acid detection method of this embodiment includes the following primers: (A) a forward primer for synthesizing the target nucleic acid; (B) a reverse primer for synthesizing the target nucleic acid, and (c) a sequence complementary to the upstream region adjacent to the 5 ′ end of a part of the target nucleic acid sequence (target sequence) and the 3 ′ end of the target sequence PCR amplification is performed using a primer (jumped primer) that has a complementary sequence in the downstream region adjacent to the target sequence but does not include a sequence complementary to the target sequence, and is complementary to the target sequence in the PCR amplification product Detection of a hybrid sequence with a single-stranded probe.
  • PCR is performed by repeatedly performing a reaction to synthesize a complementary strand of a target nucleic acid with a DNA polymerase from a primer (forward primer and reverse primer) designed in pairs so as to sandwich the region (target nucleic acid) to be amplified. It is a reaction to replicate nucleic acid.
  • PCR consists of heat denaturation (dissociating double-stranded DNA to be templated into single-stranded DNA by heat) and annealing (primer is bound to single-stranded DNA.
  • the forward primer is the template antisense strand
  • the reverse primer is the template.
  • the target nucleic acid is amplified by repeating the three steps of binding to the sense strand and extension reaction (by synthesizing the complementary strand of the original DNA from the primer by DNA polymerase).
  • target nucleic acid may be either a double-stranded DNA or a single-stranded DNA, and the single-stranded DNA may be either a sense strand or an antisense strand.
  • Target nucleic acids include those before PCR amplification and replicates after PCR amplification.
  • the DNA of the target nucleic acid may be genomic DNA, synthetic DNA, mitochondrial DNA, chloroplast DNA, etc., and may be cDNA reverse transcribed from RNA.
  • the length of the target nucleic acid may be any length that can be amplified by PCR, and the length that can be amplified by PCR is said to be several bases to several tens of kilobases, but is preferably 1000 bases or less, 200 to 260 bases are preferred.
  • a template nucleic acid containing the target nucleic acid sequence is used.
  • Samples containing template nucleic acids include whole blood, serum, plasma, urine, feces, cerebrospinal fluid, semen, saliva, tissues, cells, sputum, nasal discharge, biological samples, plant extracts / pulverized products, foods, Examples include, but are not limited to, drinking water, biological preparations, soil, water such as lakes and rivers, drainage, sewage, and the like.
  • the forward primer and the reverse primer are synthetic oligonucleotides (single-stranded DNA) having a base sequence complementary to the template DNA, and are preferably designed in pairs so as to sandwich the region (target nucleic acid) to be amplified.
  • the primer preferably binds to the template DNA in the region (target nucleic acid) to be amplified, but does not bind to the template DNA in other regions.
  • the length of the primer is preferably 15 to 35 mer, and preferably 18 to 27 mer.
  • the T at the 3 'end should be avoided. Moreover, it is good to design so that a secondary structure may not be taken in a primer.
  • the GC content of the primer should be about 40-60%.
  • the bases (G, A, T, C) constituting the primer should be randomly distributed so as not to partially become GC rich or AT rich.
  • the Tm value of the primer should be about 55 to 60 ° C., and the paired primers should be close to each other.
  • PCR amplification is performed using a jumped primer in addition to a forward primer and a reverse primer.
  • the jumped primer has a sequence complementary to the upstream region adjacent to the 5 ′ end of a part of the target nucleic acid sequence (target sequence) and a sequence complementary to the downstream region adjacent to the 3 ′ end of the target sequence. Primer does not contain a sequence complementary to the target sequence. It is desirable that the 5 'end of the jumped primer and the 5' end of the forward primer coincide.
  • FIG. 1 describes the case where the jumped primer has a sequence overlapping with the forward primer, the jumped primer may have a sequence overlapping with the reverse primer.
  • the length of the target sequence is preferably about one third of the length of the target nucleic acid, and preferably 45 to 90 bases.
  • the length of the jumped primer is preferably 30 mer to 70 mer, and preferably 33 mer to 51 mer.
  • the length of the sequence complementary to the upstream region adjacent to the 5 'end of the target sequence in the jumped primer may be 15 mer to 35 mer, and preferably 18 to 27 mer.
  • the length of the sequence complementary to the downstream region adjacent to the 3 'end of the target sequence in the jumped primer may be 15 mer to 35 mer, and preferably 15 to 24 mer.
  • the concentration ratio between the jumped primer and the forward primer (or reverse primer) may be 1/4 to 1/32, and preferably 1/4 to 1/16.
  • DNA polymerase in PCR amplification, DNA polymerase, buffer, Mg ion, dNTP, reverse transcriptase, PCR enhancer, etc. may be used.
  • PCR In PCR, heat denaturation (dissociation of double-stranded DNA used as a template into single-stranded DNA by heat), annealing (binding a primer to single-stranded DNA), extension reaction (by DNA polymerase from the primer to the original DNA) 3) is repeated, and the target nucleic acid is amplified.
  • the temperature and time of each step and the number of cycles may be adjusted as appropriate.
  • a part of the target nucleic acid sequence (target sequence) in the PCR amplification product (target sequence) or a sequence complementary thereto is hybridized with a single-stranded probe and detected.
  • the single-stranded probe may have a sequence complementary to part or all of the target sequence or a sequence complementary thereto.
  • the length of the probe is preferably 14-30 mer.
  • a target nucleic acid (double strand) consisting of a sense strand and an antisense strand is amplified by PCR, and a part of the double-stranded DNA (target sequence or a sequence complementary thereto) is single-stranded. It is considered that this single-stranded portion is hybridized with a single-stranded probe and detected (see “double-stranded c. And loop-shaped single-stranded portion c. d.).
  • the double-stranded DNA of the PCR amplification product is once denatured into a single strand, and then a double strand is rapidly formed. By this step, it is considered that more “double strands having a loop-like single-stranded portion” in FIG. 1 are formed.
  • a PCR amplification product is treated with a strong alkali (for example, NaOH), and then a weak acid (for example, CH 3 And a method of neutralizing with COOH).
  • the PCR amplification product may be heat denatured (for example, heated at 94 ° C.
  • the container containing the PCR amplification product may be rapidly cooled in ice water (for example, 94 ° C. to 10 ° C./sec. It is rapidly cooled to the range of 0 ° C to 4 ° C).
  • ice water for example, 94 ° C. to 10 ° C./sec. It is rapidly cooled to the range of 0 ° C to 4 ° C.
  • the inventor denatures the double-stranded DNA of the PCR amplification product into a single strand by strong alkali or heat, and rapidly changes the single strand again by pH change from alkaline to neutral or rapid cooling. It was found that the detection sensitivity increases when it is returned to double strands.
  • the target nucleic acid can be detected by nucleic acid chromatography, microarray, nucleic acid hybridization, or the like.
  • a labeling substance can be used to detect the PCR amplification product.
  • the primer to be used is preferably bound with a distinguishable labeling substance.
  • PCR amplification is performed using a forward primer or reverse primer to which biotin is bound, and avidin-colored latex beads are bound to biotin bound to the PCR amplification product and colored. If the single-stranded probe is fixed to the carrier, the target sequence in the PCR amplification product or a sequence complementary thereto hybridizes with the single-stranded probe, and coloring is confirmed at that position.
  • the labeling substance that binds to the primer to be used may be a labeling substance that emits a detectable signal, or a labeling substance that emits a signal in combination with other components.
  • the labeling substance that emits a detectable signal per se include fluorescent labeling substances (for example, FITC, rhodamine, etc.).
  • labeling substances that emit signals in combination with other components include haptens such as biotin and digoxigenin, enzymes such as alkaline phosphatase, and peroxidase.
  • a technique for assuming that the primer is provided with a labeling substance can be appropriately selected by those skilled in the art from known means.
  • the primer set of this embodiment includes the following primers: (A) a forward primer for synthesizing the target nucleic acid, (B) a reverse primer for synthesizing the target nucleic acid, and (c) a sequence complementary to the upstream region adjacent to the 5 ′ end of a part of the target nucleic acid sequence (target sequence) and the 3 ′ end of the target sequence A primer (jumped primer) having a complementary sequence in the downstream region adjacent to but not including a sequence complementary to the target sequence Consists of.
  • the forward primer, reverse primer and jumped primer are described above.
  • the primer set of this embodiment is used for the above-mentioned nucleic acid detection method as an example.
  • the primer set may be included in a nucleic acid detection kit.
  • ⁇ Nucleic acid detection kit> The nucleic acid detection kit of this embodiment includes the above primer set.
  • the kit of this embodiment may further include a single-stranded probe that hybridizes with the target sequence in the PCR amplification product or a sequence complementary thereto.
  • the single-stranded probe has been described above.
  • the single-stranded probe that hybridizes with the target sequence in the PCR amplification product or a sequence complementary thereto may be held on a carrier.
  • the chromatographic process may be either a dip (immersion) method or a dropping method
  • the material of the carrier is cellulose, nitrocellulose, nylon, polyethersulfone, silica gel, agarose , Dextrin, gelatin and the like, and the shape of the carrier is preferably flat.
  • the carrier is a strip.
  • the strip is usually an elongated belt-like solid phase carrier composed of cellulose fibers, glass fibers, nylons, ceramic fibers and the like.
  • a single-stranded probe that hybridizes with the target sequence in the PCR amplification product or a sequence complementary thereto may be immobilized in the direction of development of the specimen by dropping the specimen on the carrier or immersing the carrier in the specimen.
  • a single-stranded probe that hybridizes to a target sequence in a PCR amplification product or a sequence complementary thereto, which is perpendicular to the carrier development direction, may be fixed to constitute one or a plurality of test lines.
  • the kit of this embodiment may further contain a labeling reagent.
  • a labeling reagent for example, biotin is bound to the forward primer or the reverse primer, and the labeling reagent may be avidin colored latex beads.
  • the single-stranded probe that hybridizes with the target sequence or a sequence complementary thereto may be fixed to a substrate (carrier) such as glass, metal, or resin to constitute a microarray.
  • a substrate such as glass, metal, or resin
  • mRNA is extracted from a sample, PCR is performed using cDNA synthesized by reverse transcription as a template, using forward primer, reverse primer, and jumped primer, and the target sequence in the PCR amplification product or a sequence complementary thereto Can be detected by hybridizing with a single-stranded probe of a microarray.
  • the PCR amplification product may be labeled by binding the labeling substance to the primer, and the label on the microarray may be detected.
  • the single-stranded probe may be fixed to beads such as glass, metal, and resin.
  • a single-stranded probe may be immobilized on a nitrocellulose membrane (carrier).
  • mRNA is extracted from a sample, and PCR amplification is performed using a cDNA synthesized by reverse transcription as a template, using a forward primer, a reverse primer, and a jumped primer.
  • Complementary sequences can be detected by hybridizing with single stranded probes on the membrane.
  • the PCR amplification product may be labeled by binding the labeling substance to the primer, and the label on the microarray may be detected.
  • the kit may include DNA polymerase, dNTP, PCR buffer, Mg ion, nucleic acid chromatography development buffer (for nucleic acid chromatography), microarray buffer (for microarray analysis), nucleic acid hybridization buffer, instruction manual, etc. Good.
  • Example 1 Material Platinum Taq (Invitrogen, 10966-034) NaOH (Nacalai Tesque, 31511-05) Acetic acid (Wako Pure Chemical Industries, 015-00257) Custom C-PAS (strip with probe bound), chromatography developer (100 mM NaCl buffer), dye-labeled latex solution (above, TBA)
  • Strips were applied with a region containing the sequence contained in the amplified PCR product as a probe. In HIV-1, LTR and gag sequences are conserved, and the U5 region of LTR, the p17 region in gag is preferred.
  • Strip 1 The strip 1 was coated with the sequences A, B, C, and D selected from the HIV-1 gag and p17 regions in order from the top of the strip (FIGS. 2 and 3). Probes A, B, C, and D all contain the sequence of probe D (SEQ ID NO: 4), and probes A to C are sequences with several bases added at both ends as shown below to function as probes. The required length was confirmed.
  • Lines 10, 12, and 14 are marks used for positioning when A, B, C, and D are applied to the strip 1.
  • A 5′-ATCAATGARGARGCTGCAGAATGGGG-3 ′ (SEQ ID NO: 1)
  • B 5′-CAATGARGARGCTGCAGAATGG-3 ′ (SEQ ID NO: 2)
  • C 5′-ATGARGARGCTGCAGAAT-3 ′ (SEQ ID NO: 3)
  • D 5'-GARGARGCTGCAGA-3 '(SEQ ID NO: 4)
  • Strip 2 the sequence selected from the ⁇ phage is E, the sequence selected from the HIV-2 LTR, R3 region is F, the sequence selected from the HIV-1 gag, p17 region is G, the sequence selected from the HIV-1 LTR, U5 An array, which is an array selected from the region, was applied to the position H (FIGS.
  • HIV-1 is a collection of strains having various base sequences
  • two types of probes H and H ′ different in length by one base were applied at the same position.
  • the sequence of probe G (SEQ ID NO: 7) is a sequence complementary to the sequence of probe A (SEQ ID NO: 1).
  • C Of “double strand having looped single-stranded portion” shown in FIG. And d.
  • the probe G having the complementary strand of the probe A was used.
  • Lines 20 and 22 are marks used for positioning when E, F, G, and H are applied to the strip 2.
  • E 5′-CTGGCCCTTTCCTTTACCAGTTC-3 ′ (SEQ ID NO: 5)
  • F 5'-CACCCAGGCTCTACCTCTC-3-3 (SEQ ID NO: 6)
  • G 5′-CCCATTCTGCAGCCYTCYTCATTG-3 ′ (SEQ ID NO: 7)
  • H 5′-GAGGCTTAAGCAGTGGGTTCC-3 ′
  • SEQ ID NO: 8 H ′, 5′-GAGGCTTTAAGCAGTGGGTTCC-3 ′ (SEQ ID NO: 9)
  • FIG. 6 shows an image of the binding position of the primer to the target nucleic acid sequence and the binding position of the probe.
  • gagM-1FA forward primer
  • gagM-1FA-3FB jumped primer
  • gagM-3RA-Bio biotin-labeled reverse primer
  • 5 ′-[Bio] TTTATAGATTTCTCCYACTGGRAYAGGTGG-3 ′ SEQ ID NO: 12
  • PCR product 10.0 ⁇ l 1N NaOH 1.0 ⁇ l After stirring well, the following solutions were added. 1N CH 3 COOH 1.1 ⁇ l Latex beads (Dye-labeled latex solution) 4.0 ⁇ l Chromatographic developing solution 10.0 ⁇ l Strip 1 or 2 was immersed in a pretreated PCR product solution. Chromatography was performed at room temperature for 10 minutes. After 10 minutes, 10.0 ⁇ l of a new chromatographic developer was added to wash the strip. Depending on the position of the strip band, it was confirmed that the desired product was produced.
  • PCR product 10.0 ⁇ l Latex beads (Dye-labeled latex solution) 4.0 ⁇ l Chromatographic developing solution 10.0 ⁇ l
  • the PCR product is directly reacted with the strip 2 without denaturing the determination.
  • Strip 2 was immersed in the PCR product solution and chromatographed for 10 minutes at room temperature. After 10 minutes, 10.0 ⁇ l of chromatography developing solution was added to wash the strip. The judgment result is shown in 3.2.2.
  • the additional signal is that the band signal becomes stronger when neutralized with acid after alkali denaturation or ice-cooled after heat denaturation.
  • the single strand generated by the jumped primer during rapid annealing of the denatured DNA The structure of the double-stranded DNA including the single-stranded portion formed between the DNA and the full-length single-stranded DNA complementary thereto is kinetically fixed, so that the number of DNA molecules that can hybridize with the probe increases. It is thought that.
  • the present invention can be used for genetic testing in fields such as medicine and food.
  • ⁇ SEQ ID NO: 1> The sequence of probe A applied to strip 1 (sequence selected from the gag and p17 regions of HIV-1) is shown.
  • ⁇ SEQ ID NO: 2> The sequence of probe B applied to strip 1 (sequence selected from the gag and p17 regions of HIV-1) is shown.
  • ⁇ SEQ ID NO: 3> The sequence of probe C applied to strip 1 (sequence selected from the gag and p17 regions of HIV-1) is shown.
  • ⁇ SEQ ID NO: 4> The sequence of probe D applied to strip 1 (sequence selected from the gag and p17 regions of HIV-1) is shown.
  • ⁇ SEQ ID NO: 5> The sequence of the probe E applied to the strip 2 ( ⁇ phage sequence) is shown.
  • ⁇ SEQ ID NO: 6> The sequence of probe F applied to strip 2 (sequence selected from LTR and R3 regions of HIV-2) is shown.
  • ⁇ SEQ ID NO: 7> The sequence of the probe G applied to the strip 2 (sequence selected from the gag and p17 regions of HIV-1) is shown.
  • ⁇ SEQ ID NO: 8> The sequence of the probe H applied to the strip 2 (sequence selected from HIV-1 LTR, U5 region) is shown.
  • ⁇ SEQ ID NO: 9> The sequence of the probe H ′ applied to the strip 2 (sequence selected from the LTR, U5 region of HIV-1) is shown.
  • SEQ ID NO: 10 shows the base sequence of the forward primer (gagM-1FA) used for PCR.

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Abstract

L'invention concerne un procédé de détection d'acide nucléique qui supprime l'apparition de faux positifs dus à une amplification non spécifique. Le procédé de détection d'acide nucléique comprend la réalisation d'une amplification par PCR à l'aide des amorces suivantes : (a) une amorce sens pour la synthèse d'un acide nucléique cible ; (b) une amorce antisens pour la synthèse de l'acide nucléique cible ; et (c) une amorce (amorce sautée) qui a une séquence complémentaire d'une région amont adjacente à l'extrémité 5' d'une partie (la séquence cible) de la séquence de l'acide nucléique cible, qui a une séquence complémentaire d'une région aval adjacente à l'extrémité 3' de cette séquence cible, et qui ne contient pas de séquence complémentaire de la séquence cible ; et la détection par hybridation de la séquence cible ou de la séquence complémentaire de celle-ci dans le produit d'amplification PCR avec une sonde simple brin. L'invention concerne également un ensemble d'amorces et un kit de détection d'acide nucléique.
PCT/JP2018/002824 2018-01-30 2018-01-30 Procédé de détection d'un acide nucléique WO2019150414A1 (fr)

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JP2016506748A (ja) * 2013-02-07 2016-03-07 ラトガース，ザ ステート ユニバーシティ オブ ニュー ジャージー 高い選択性の核酸増幅プライマー
JP2016202037A (ja) * 2015-04-17 2016-12-08 国立大学法人東北大学 核酸検出方法

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US20010019825A1 (en) * 1998-09-11 2001-09-06 One Lambda Method for amplification of DNA
JP2007189984A (ja) * 2006-01-20 2007-08-02 Toshiba Corp ハイブリダイゼーションによる核酸の検出方法およびアッセイキット
US20110027786A1 (en) * 2009-07-02 2011-02-03 Satterfield Brent C Primers for nucleic acid extension or amplification reactions
JP2015511818A (ja) * 2012-02-20 2015-04-23 スピーデクス ピーティーワイ リミテッド 核酸の検出
JP2016506748A (ja) * 2013-02-07 2016-03-07 ラトガース，ザ ステート ユニバーシティ オブ ニュー ジャージー 高い選択性の核酸増幅プライマー
JP2015019591A (ja) * 2013-07-16 2015-02-02 国立大学法人東北大学 核酸クロマトグラフィー
JP2016202037A (ja) * 2015-04-17 2016-12-08 国立大学法人東北大学 核酸検出方法

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