WO2017006859A1 - Procédé pour détecter un acide nucléique cible - Google Patents

Procédé pour détecter un acide nucléique cible Download PDF

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WO2017006859A1
WO2017006859A1 PCT/JP2016/069638 JP2016069638W WO2017006859A1 WO 2017006859 A1 WO2017006859 A1 WO 2017006859A1 JP 2016069638 W JP2016069638 W JP 2016069638W WO 2017006859 A1 WO2017006859 A1 WO 2017006859A1
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nucleic acid
tag
acid amplification
primer
sequence
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PCT/JP2016/069638
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Japanese (ja)
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創太郎 佐野
重彦 宮本
高橋　孝治
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株式会社カネカ
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00—Apparatus for enzymology or microbiology
    • C12M1/34—Measuring or testing with condition measuring or sensing means, e.g. colony counters
    • 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
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology

Definitions

  • the present invention relates to a target nucleic acid detection method including amplification of a target nucleic acid using a two-step nucleic acid amplification reaction and detection of a nucleic acid amplification product on a solid phase carrier.
  • the obtained amplification product is diffused and moved in the porous solid phase carrier by a capillary phenomenon, and is captured and bound to the label and the solid phase carrier via the tags attached to both ends of the amplification product. Is detected visually (Patent Document 1).
  • this detection method relies on a tag derived from a primer attached to both ends of the amplification product to perform capture and binding to a label and a solid phase carrier, it is specific to the target nucleic acid.
  • the amplification product and the primer dimer formed by the non-specific reaction cannot be distinguished, and there is a possibility of performing false positive determination based on the detection of the primer dimer.
  • the present invention avoids non-specific detection of a primer dimer in a method for detecting a target nucleic acid including a nucleic acid amplification reaction and detection of a nucleic acid amplification product on a solid phase carrier (for example, a nucleic acid chromatography method).
  • An object of the present invention is to provide a new detection method with high determination accuracy.
  • a method for detecting a target nucleic acid comprising: (A) consisting of a primer containing a sequence homologous to the first base sequence of the target nucleic acid and a primer containing a sequence homologous to the complementary strand sequence of the second base sequence located downstream of the first base sequence Using the first primer set to obtain a first nucleic acid amplification product by performing a nucleic acid amplification reaction using the test DNA as a template, (B) a primer including a sequence homologous to a third base sequence located downstream of the first base sequence and upstream of the second base sequence; a downstream of the third base sequence; and A second primer set comprising primers containing a sequence homologous to the complementary strand sequence of the fourth base sequence located upstream of the second base sequence, wherein any one of the primers can bind to the labeling substance Using the second primer set, which is bound to a first tag and the other primer is bound to a second tag capable of
  • [5] (a) a primer containing a sequence homologous to the first base sequence of the target nucleic acid, and a sequence homologous to the complementary strand sequence of the second base sequence located downstream of the first base sequence A first primer set comprising primers, (B) a primer including a sequence homologous to a third base sequence located downstream of the first base sequence and upstream of the second base sequence; a downstream of the third base sequence; and A second primer set comprising primers containing a sequence homologous to the complementary strand sequence of the fourth base sequence located upstream of the second base sequence, wherein any one of the primers can bind to the labeling substance
  • the second primer set which is bound to a first tag and the other primer is bound to a second tag capable of binding to a solid phase carrier; (C) a labeling substance capable of binding to the first tag, and (D) a solid phase carrier capable of binding to the second tag,
  • a kit for detecting a target nucleic acid comprising: [6] The kit according to [
  • the first tag is a tag made of any low molecular weight compound selected from biotin, digoxigenin, and FITC, and the labeling substance contains a protein that can bind to the tag.
  • the second tag is a tag comprising an oligonucleotide
  • the solid phase carrier comprises an oligonucleotide having a sequence complementary to the oligonucleotide.
  • the Tm value of one or both primers included in the first primer set is higher than the Tm value of one or both primers included in the second primer set. [5 ] To [11].
  • non-specific detection of primer dimers is avoided in a method for detecting a target nucleic acid including a nucleic acid amplification reaction and detection of a nucleic acid amplification product on a solid phase carrier (eg, nucleic acid chromatography). Therefore, it is possible to provide a detection method with high determination accuracy.
  • a solid phase carrier eg, nucleic acid chromatography
  • the schematic diagram of a lateral flow type nucleic acid detection device is shown.
  • 1 solid phase carrier
  • 2 conjugate pad
  • 3 sample pad
  • 4 absorption pad
  • 5 substrate
  • 6 capture means.
  • the region corresponding to the recognition sequence of each primer used in the examples is shown.
  • A It is a photograph figure which shows the result of having detected the amplification product obtained by Nested-PCR method using the outer primer set and the inner primer set in Example 1 using the nucleic acid detection device.
  • (B) It is a photograph figure which shows the result of having detected the amplification product obtained by PCR method using only the inner primer in the comparative example 1 using the nucleic acid detection device. Each figure shows the amount of template DNA added to the reaction system.
  • the target nucleic acid detection method generally includes the following steps: Amplifying the target nucleic acid; A step of labeling the amplified target nucleic acid, and a step of capturing and detecting the labeled target nucleic acid on a solid phase carrier. Hereinafter, each step will be described.
  • the first nucleic acid amplification reaction can be performed using the first primer set, and the copy number of the first nucleic acid amplification product containing the target nucleic acid (if present) can be increased.
  • the copy number of the first nucleic acid amplification product containing the target nucleic acid (if present) can be increased.
  • the directivity for the target nucleic acid can be enhanced, and non-specific nucleic acid amplification different from the target nucleic acid. Can be reduced or avoided.
  • the “target nucleic acid” means any nucleic acid sequence or nucleic acid molecule to be detected, which may be natural or artificially synthesized, and is not particularly limited.
  • the target nucleic acid include a specific trait, a genetic marker indicating the onset or possibility of onset of a specific disease, a gene derived from a pathogen such as a bacterium or virus, a gene derived from an allergen, a single nucleotide polymorphism, Examples include, but are not limited to, base sequences that exhibit congenital or acquired mutations.
  • each primer (so-called forward primer and reverse primer) included in the “first primer set” and the “second primer set” is a common in the nested-PCR method. It can be designed according to a common technique (published by Hiroki Nakayama, “Bio-Experiment Illustrated, 3 Really PCR”, Shujunsha, 1997).
  • the “first primer set” includes a primer having a sequence homologous to a specific base sequence (first base sequence) on one strand of a double-stranded DNA that is a target nucleic acid, or consisting of the sequence, A sequence homologous to the complementary strand sequence of the specific base sequence (second base sequence) located downstream (3 ′ side) from the base sequence of 1 (ie, homologous to the base sequence of the other strand of the double-stranded DNA) Or a primer composed of the sequence.
  • the primer having a “homologous sequence” not only has the same sequence as the first base sequence and the same sequence as the complementary strand sequence of the second base sequence, but also binds to the target nucleic acid and amplifies the nucleic acid.
  • the size of the primer in the first primer set can be 8 bases or more, 12 bases or more, or 15 bases or more in length, and the upper limit is not particularly limited, but 50 bases or less, 40 bases or less, or 30 bases
  • the length can be as follows.
  • the size of each primer may be the same or different.
  • the first primer set has a higher annealing temperature than the annealing temperature in the second nucleic acid amplification reaction using the second primer set by 5 ° C or higher, preferably 10 ° C or higher, more preferably 15 ° C or higher.
  • the target nucleic acid can be amplified under the conditions of one nucleic acid amplification reaction. That is, the Tm value of any one, more preferably both primers included in the first primer set is more than the Tm value of any one, more preferably both primers included in the second primer set. It can be designed to be 5 ° C. or higher, preferably 10 ° C. or higher, more preferably 15 ° C. or higher.
  • the first nucleic acid amplification is performed at a high annealing temperature according to the Tm value of the first primer set.
  • the “Tm value” of each primer in the second primer set means a value calculated based on a region not including the tag region or spacer region described below.
  • the “second primer set” is a specific base sequence located on the downstream side (3 ′ side) of the first base sequence and on the upstream side (5 ′ side) of the second base sequence ( (3rd base sequence) or a primer comprising this sequence, or a primer comprising the sequence, located downstream (3 ′ side) from the third base sequence, and upstream of the second base sequence ( Including a sequence homologous to a complementary strand sequence (that is, a sequence homologous to the base sequence of the other strand of double-stranded DNA) of a specific base sequence (fourth base sequence) located on the 5 ′ side), It can be combined with a primer comprising the sequence.
  • the second primer set can be designed in a region sandwiched between the first primer sets (that is, the first nucleic acid amplification product).
  • the primer having a “homologous sequence” not only has the same sequence as the third base sequence and the same sequence as the complementary strand sequence of the fourth base sequence, but also binds to the target nucleic acid and amplifies the nucleic acid.
  • a base sequence having a deletion, substitution, addition or insertion of one to several bases in the complementary strand sequence of the third base sequence and the fourth base sequence 85% or more, preferably 90% or more, more preferably 95%, when calculated using the base sequence of the above and the complementary strand sequence of the fourth base sequence and BLAST or the like (for example, default or default parameters)
  • a primer having a base sequence having a sequence identity of 99% or more is more preferable.
  • any one of the primers of the second primer set has the same sequence as the entire sequence of any one of the primers of the first primer set or a partial sequence thereof. Also good.
  • the primer size of the second primer set can be 8 bases or more, 12 bases or more, or 15 bases or more in length, and the upper limit is not particularly limited, but 50 bases or less, 40 bases or less, or 30 bases
  • the length can be as follows.
  • the size of each primer may be the same or different.
  • the “primer size” means a size that does not include the tag region and spacer region described below.
  • each primer of the first primer set and the second primer set can be performed using a known primer design software / design site or the like.
  • primer design software / design site for example, OligoEvaluator (Sigma-Aldrich), Primer 3 (National Human Genome Research Institute), Primer-BLAST (NCBI), etc. can be used.
  • each primer of the first primer set and the second primer set can be performed by a known method, and may be performed using a DNA synthesizer or a contracted synthesis service. Can do.
  • the primer included in the first primer set may be referred to as “outer primer” and the primer included in the second primer set may be referred to as “inner primer”.
  • a tag is bound to the 5 'end of each primer in the second primer set.
  • the tag is selected from a first tag that can bind to the labeling substance and a second tag that can bind to the solid phase carrier, and each primer in the second primer set (forward primer and reverse primer) One of the tags is bound to each primer.
  • the “first tag” only needs to be capable of binding to a labeling substance described in detail below, and is a nucleic acid (DNA, RNA, oligonucleotide, etc.), protein, peptide, or low molecular weight compound (eg, biotin, digoxigenin) , FITC, etc.) or a combination thereof can be used, and is not particularly limited.
  • the “second tag” only needs to be capable of binding to the solid phase carrier described in detail below, and is composed of a nucleic acid (DNA, RNA, oligonucleotide, etc.), protein, peptide, compound, or a combination thereof.
  • it contains an oligonucleotide or consists of an oligonucleotide.
  • the primer and the tag can be bound by any means, and can be bound directly or indirectly.
  • a spacer capable of suppressing or stopping the progress of the DNA polymerase reaction is used so that the tag region is not double-stranded together with the primer region by the nucleic acid amplification reaction.
  • the primer and the tag are combined.
  • any spacer can be used as long as it can suppress or stop the progress of the DNA polymerase reaction when contained in the template strand and prevent the tag region from becoming double-stranded.
  • a nucleic acid (or base) serving as a template is required, and the DNA strand does not extend without the template.
  • the spacer has a structure that cannot serve as a template for a DNA extension reaction by DNA polymerase.
  • a spacer include artificial nucleic acid bases such as L-type nucleic acids, peptide nucleic acids (PNA), cross-linked nucleic acids (Bridged Nucleic Acid (BNA) or Locked Nucleic Acid (LNA)), and are represented by the following formula I: Spacer with azobenzene structure, fatty chain such as alkylene chain or polyoxyalkylene chain, inverted base (natural nucleobase linked by 5'-5 'or 3'-3' bond), strong hairpin structure, Examples thereof include, but are not limited to, nucleic acid sequences having a pseudoknot structure.
  • 5′-O—C m H 2m —O-3 ′ (In the formula, 5 ′ represents an oxygen atom of a phosphodiester bond on the 5 ′ side, 3 ′ represents a phosphate atom of a phosphodiester bond on the 3 ′ side, and m represents an integer of 2 to 40. To express.) In the formula II, m is preferably 2 or more and 36 or less, more preferably 3 or more and 18 or less, and further preferably 3 or 6.
  • the first nucleic acid amplification reaction and the second nucleic acid amplification reaction can be performed according to a general PCR method. That is, when PCR is performed on the template DNA containing the target nucleic acid using each of the first primer set and the second primer set, the PCR can be performed under PCR conditions that a desired region is amplified. .
  • test DNA is used as template DNA.
  • “Test DNA” means a DNA sample that may contain a target nucleic acid, and is derived from a sample such as an animal, plant, or a part thereof (organ, tissue, cell, etc.), microorganism, virus, food or drink, etc. Examples include (but are not limited to) DNA and cDNA prepared from these samples.
  • the test DNA may be in a form extracted and purified from a sample, or may be in a crudely purified form. Alternatively, a part of cells or tissues can be included in the reaction system as it is as test DNA.
  • the first nucleic acid amplification product produced by the first nucleic acid amplification reaction is used as the template DNA.
  • the number of PCR cycles in the second nucleic acid amplification reaction is preferably smaller than the number of PCR cycles in the first nucleic acid amplification reaction.
  • the DNA polymerase used for PCR is not particularly limited as long as it is a heat-resistant DNA polymerase.
  • commercially available DNA polymerase can be used, and for example, TaKaRa Ex Taq (registered trademark), KOD DNA polymerase, and the like can be suitably used.
  • the temperature, time, buffer composition, etc. can be appropriately selected depending on the DNA polymerase used, the primer sequence, the size of the target nucleic acid amplification region, and the like.
  • the first nucleic acid amplification reaction and the second nucleic acid amplification reaction may be sequentially performed in separate reaction containers, or the first nucleic acid amplification reaction and the second nucleic acid amplification reaction may be performed in the same reaction container. You may carry out continuously.
  • the first nucleic acid amplification reaction and the second nucleic acid amplification reaction are sequentially performed in separate reaction vessels, the obtained first nucleic acid amplification product is taken out after the first nucleic acid amplification reaction (if necessary, This means that after the dilution, a second nucleic acid amplification reaction is performed in addition to a separately prepared second nucleic acid amplification reaction system comprising a DNA polymerase, a second primer set, and the like.
  • performing the first nucleic acid amplification reaction and the second nucleic acid amplification reaction continuously in the same reaction vessel means that the first nucleic acid amplification product is not taken out after the first nucleic acid amplification reaction. It means that two nucleic acid amplification reactions are performed.
  • the second primer set is added to the reaction system in advance before the start of the first nucleic acid amplification reaction. Alternatively, it may be added to the reaction system after the first nucleic acid amplification reaction and before the start of the second nucleic acid amplification reaction.
  • DNA polymerase may be supplemented after the first nucleic acid amplification reaction and before the start of the second nucleic acid amplification reaction.
  • the first nucleic acid amplification reaction and the second nucleic acid amplification reaction are continuously performed in the same reaction vessel for ease of operation.
  • the first nucleic acid amplification reaction and the second nucleic acid amplification reaction are continuously performed in the same reaction vessel, and the second primer set is preliminarily set in the reaction system before the start of the first nucleic acid amplification reaction. To be added.
  • the Tm value of any one, more preferably both primers included in the first primer set is more preferably the Tm value of any one, more preferably both primers included in the second primer set. Is preferably high.
  • the annealing temperature in the first nucleic acid amplification reaction using the first primer set can be performed under a temperature condition higher than the annealing temperature in the second nucleic acid amplification reaction using the second primer set.
  • the binding of the second primer set to the target nucleic acid contained in the test DNA and the formation of primer dimers can be suppressed or avoided under the first nucleic acid amplification reaction conditions.
  • the step of labeling the amplified target nucleic acid The second nucleic acid amplification product containing the target nucleic acid (if present) obtained by the nucleic acid amplification includes the first tag and the second tag resulting from the second primer set At each end.
  • the labeling of the second nucleic acid amplification product can be performed by contacting and binding the labeling substance with the first tag that can bind to the labeling substance.
  • the labeling substance is not particularly limited as long as it can detect the second nucleic acid amplification product, but preferably allows visual detection of the second nucleic acid amplification product.
  • Examples of such a labeling substance include colored particles, pigments, enzymes (peroxidase, alkaline phosphatase, luciferase, etc.), and preferably colored particles.
  • Examples of the “colored particles” include, but are not limited to, metal colloid (eg, gold, silver, copper, platinum, etc.) particles, colored latex particles, silica nanoparticles including a pigment, and the like.
  • the size of the labeling substance does not hinder the capture of the second nucleic acid amplification product on the solid phase carrier, and may be any material that develops color at the time of detection. It can select suitably so that it may become a size smaller than the hole diameter of a porous member.
  • the size of the labeling substance can be about 500 nm or less, preferably about 0.1 nm to 250 nm, more preferably about 1 nm to 100 nm.
  • the contact and binding between the labeling substance and the first tag may be direct binding or indirect binding, and the binding means is suitably suitable depending on the labeling substance to be used and the first tag.
  • nucleic acid-nucleic acid interaction, protein-protein interaction, low molecular weight compound-protein interaction, and the like can be used.
  • the first tag includes an oligonucleotide
  • the labeling substance is hybridized by binding the labeling substance to an oligonucleotide containing a sequence complementary to the base sequence of the oligonucleotide, And the first tag can be indirectly coupled.
  • the binding between the labeling substance and the oligonucleotide may be performed via a peptide, protein, nucleic acid, or the like, or may be performed via an appropriate functional group.
  • the hybridization conditions are not particularly limited as long as hybridization occurs.
  • a buffer solution pH 6.5 to 7.5
  • the buffer may further contain a salt such as sodium chloride.
  • the first tag contains biotin, which is a low molecular weight compound, both can be bound by binding the labeling substance to avidin (streptavidin).
  • digoxigenin or FITC which are low molecular compounds, both can be bound by binding the labeling substance to the anti-DIG antibody or anti-FITC antibody.
  • Step of capturing and detecting the labeled target nucleic acid on a solid phase carrier The labeled second nucleic acid amplification product can be captured on a solid phase carrier and detected. Capture (ie, binding) of the second nucleic acid amplification product to the solid phase carrier is performed by bringing the solid phase carrier into contact with a second tag that can bind to the solid phase carrier in the second nucleic acid amplification product. be able to.
  • the solid phase carrier is not particularly limited, but may be made of a resin, metal, polysaccharide, mineral or the like, and may be in the form of a membrane, film, nonwoven fabric, plate, gel or the like.
  • the solid phase carrier has a porous structure so that the second nucleic acid amplification product and the labeling substance in the solution can be developed.
  • the solid phase carrier usable in the present invention include filter paper, nitrocellulose membrane, polyether sulfone membrane, nylon membrane, various dried gels (silica gel, agarose gel, dextran gel, gelatin gel) and the like.
  • the size and form of the solid phase carrier those suitable for various operations and detection can be appropriately selected.
  • Contact and capture (that is, binding) between the solid phase carrier and the second tag may be direct binding or indirect binding, and the binding means uses the solid phase carrier and the second tag to be used.
  • a suitable one can be selected depending on the case.
  • the second tag contains an oligonucleotide
  • an oligonucleotide containing a sequence complementary to the base sequence of the oligonucleotide is immobilized on a solid phase carrier to serve as a tag capturing means, and both oligonucleotides are hybridized.
  • the solid phase carrier and the second tag can be indirectly bound.
  • Immobilization of the oligonucleotide to the solid phase carrier may be performed via a peptide, protein, nucleic acid, or the like, or may be performed via an appropriate functional group.
  • immobilizing an oligonucleotide on a solid phase carrier the captured second nucleic acid amplification product is detected only in a predetermined region by immobilizing it in a specific region. Negative discrimination can be facilitated.
  • Hybridization conditions can be performed according to the conditions described above.
  • the detection of the second nucleic acid amplification product can be performed by detecting, preferably visually detecting the labeling substance bound to the second nucleic acid amplification product captured on the solid phase carrier.
  • the target nucleic acid is present, the labeling substance of the nucleic acid amplification reaction product captured and bound to the solid phase carrier is detected.
  • the presence or absence of the target nucleic acid in the second nucleic acid amplification reaction product can be determined using the presence or absence of the detection as an index.
  • Nucleic acid detection device The above-mentioned “step of labeling the amplified target nucleic acid” and “step of capturing and detecting the labeled target nucleic acid on a solid phase carrier” can be performed using a nucleic acid detection device utilizing nucleic acid chromatography. By using the nucleic acid detection device, the presence or absence of the target nucleic acid in the second nucleic acid amplification product can be detected and discriminated without requiring a special device, and the result can be obtained easily and quickly. it can.
  • nucleic acid detection device a known nucleic acid detection device (WO2012 / 070618) used for detecting a nucleic acid amplification product labeled by a nucleic acid chromatography method can be used.
  • FIG. 1 shows a schematic diagram of an embodiment of a nucleic acid detection device that can be used in the present invention, but the nucleic acid detection device is not limited to this embodiment.
  • the reference numerals given to the respective members correspond to the reference numerals shown in FIG.
  • the nucleic acid detection device of FIG. 1 includes a sample pad (3) for adding a second nucleic acid amplification product, a conjugate pad (2) having a labeling substance disposed thereon,
  • the solid phase carrier (1) and the absorption pad (4) for capturing the nucleic acid amplification product of 2 are sequentially stacked.
  • means (capturing means) (6) for example, the above-mentioned oligonucleotide etc.
  • the sample pad (3), the conjugate pad (2), the solid phase carrier (1) and the absorption pad (4) are composed of members having a porous structure that can be used as the solid phase carrier.
  • the base material (5) can support various members disposed thereon and can facilitate the operation of the nucleic acid detection device. For example, a material made of resin, metal, mineral, or the like is used. Can do. When the labeling substance is mixed in the developing solution, the conjugate pad (2) can be omitted.
  • the obtained second nucleic acid amplification reaction product is added to the sample pad (3).
  • the reaction solution after the nucleic acid amplification reaction may be dropped as it is, or may be dropped together with an appropriate developing solution (for example, phosphate buffer, Tris buffer, Good buffer, SSC buffer).
  • the developing solution can further contain a surfactant, a salt, a protein, a nucleic acid and the like, if necessary.
  • the second nucleic acid amplification reaction product added to the sample pad (3) develops by a capillary phenomenon from upstream to downstream in the direction indicated by the arrow in FIG.
  • the second nucleic acid amplification reaction product passes through the conjugate pad (2) on which the labeling substance is disposed, the second nucleic acid amplification reaction product comes into contact with the labeling substance and is labeled with the labeling substance via the first tag.
  • the labeling substance of the nucleic acid amplification reaction product captured and bound to the solid phase carrier (1) by the capture means (6) is detected in the region of the capture means (6). If the labeling substance can be visually confirmed, the region of the capturing means (6) is colored due to the labeling substance.
  • the presence or absence of the target nucleic acid in the second nucleic acid amplification reaction product can be determined using the presence or absence of detection (coloration) of the labeling substance as an index.
  • nucleic acid detection kit includes the first primer set, the second primer set, a labeling substance, and a solid phase carrier, and can be used in the nucleic acid detection method of the present invention.
  • the labeling substance and the solid phase carrier can be in the form of the nucleic acid detection device.
  • the nucleic acid detection kit can further contain a PCR buffer solution, dNTPs, DNA polymerase, a nucleic acid chromatography developing solution, and the like.
  • Example 1 Preparation of gold colloid-binding oligonucleotide Gold Colloid (40 nm, 9.0 ⁇ 10 10 (number of particles / ml), manufactured by British Biocell International) and thiol group-containing oligonucleotide represented by SEQ ID NO: 1 are mixed And incubated at 50 ° C. for 16 hours. Centrifugation was performed at 6000 rpm for 15 minutes, the supernatant was removed, 0.05 M sodium chloride, 5 mM phosphate buffer (pH 7) was added and mixed, and then incubated again at 50 ° C. for 40 hours.
  • the prepared gold colloid solution was uniformly added to a glass fiber pad, and then dried with a vacuum dryer to obtain a conjugate pad.
  • the lateral flow type nucleic acid detection device was produced by bonding.
  • an outer primer F (SEQ ID NO: 6) and an outer primer R (SEQ ID NO: 7) were designed so that the recognition sequence of the inner primer was sandwiched between the upstream side (5 ′ end side) of each inner primer.
  • the Tm value of the outer primer was designed to be 10 ° C. higher than the Tm value of the inner primer.
  • Table 1 shows the sequence and Tm value of each primer. Moreover, the area
  • tag sequence 1 SEQ ID NO: 8
  • tag sequence 2 sequence 2
  • spacer Expressed by the above formula (I)
  • azobenzene which is a polymerase reaction inhibition region.
  • Each of No. 9 was ligated to prepare inner primer-Tag1-F (SEQ ID NO: 10) and inner primer-Tag2-R (SEQ ID NO: 11).
  • V Amplification reaction of target nucleic acid TaKaRa Ex Taq (registered trademark) Hot Start Version (Takara Bio) was used as a PCR reagent, 1 ⁇ EX Taq buffer, 0.2 mM dNTPs for each, 0.5 ⁇ M inner primer-Tag1-F, 0.5 ⁇ M inner primer-Tag2-R, 0.11 ⁇ M outer primer F, 0.11 ⁇ M outer primer R, 0.9375 Unit TaKaRa EX Taq (registered trademark), template synthetic DNA (SEQ ID NO: 3) (0 pg / test, 0. Nested-PCR reaction was performed in a reaction solution containing 5 pg / test, 5 pg / test, or 50 pg / test).
  • Reaction conditions were 95 minutes at 95 ° C. for 2 minutes, 55 cycles of 95 ° C. for 10 seconds and 72 ° C. for 20 seconds, then 90 ° C. for 5 seconds, 60 ° C. for 10 seconds, 72 ° C. for 10 seconds. As a set, 15 cycles were performed to obtain an amplification product.
  • the obtained amplification product has a tag sequence consisting of single-stranded DNA resulting from each inner primer.
  • PCR amplification was performed using the synthetic nucleic acid represented by SEQ ID NO: 3 as the template DNA.
  • SEQ ID NO: 3 synthetic nucleic acid represented by SEQ ID NO: 3 as the template DNA.
  • PCR reagents TaKaRa Ex Taq (registered trademark) Hot Start Version (Takara Bio) was used, and 1 ⁇ EX Taq buffer, 0.2 mM dNTPs for each, 0.4 ⁇ M inner primer-Tag1-F, 0.4 ⁇ M inner primer-Tag2- PCR reaction in a reaction solution containing R, 0.9375 Unit TaKaRa EX Taq (registered trademark), template synthetic DNA (SEQ ID NO: 3) (0 pg / test, 0.5 pg / test, 5 pg / test, or 50 pg / test) Went.
  • SEQ ID NO: 3 template synthetic DNA
  • Reaction conditions were in accordance with a conventional method, and after 2 minutes at 95 ° C., 40 cycles were performed with 95 ° C. for 20 seconds, 60 ° C. for 30 seconds, and 72 ° C. for 30 seconds to obtain an amplification product.
  • the obtained amplification product was detected by a lateral flow type nucleic acid detection device in the same manner as in Example 1.
  • Non-specific coloring was also observed in the sample (0 pg / test) to which no template synthetic DNA was added.
  • the amplification product was analyzed by agarose gel electrophoresis for the cause of this coloring, formation of a primer dimer was observed and it was found that this was detected non-specifically.
  • Example 2 Among the PCR conditions described in Example 1, the second amplification step is fixed to 15 cycles that do not cause non-specific amplification by the primer dimer, and the number of cycles of the first amplification step is in the range of 10 to 60 cycles. The sensitivity and specificity when increasing or decreasing were examined.
  • Comparative Example 2 (Comparative Example 2) Among the PCR conditions described in Comparative Example 1, the number of cycles in PCR was changed in the range of 10 to 60 cycles, and the detectability under each PCR condition was evaluated. An amplification product detection test was carried out using a lateral flow type nucleic acid detection device under the same conditions as in Example 1.
  • the present invention can be used for various tests and research including a step of detecting a DNA fragment obtained by a nucleic acid amplification method. More specifically, according to the present invention, molecular biology research fields, pathogen detection, allergen detection in foods and drinks, livestock management, detection of nucleotide polymorphisms, detection of diseases (for example, cancer, etc.) are simplified. And can be done quickly. The present invention is expected to contribute greatly in such fields.

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Abstract

L'objectif de la présente invention concerne un procédé de détection présentant une précision de détermination élevée, ledit procédé de détection étant destiné à détecter un acide nucléique cible et consistant à réaliser une réaction d'amplification d'acide nucléique et à détecter un produit d'amplification d'acide nucléique sur un support solide, la détection non spécifique d'un dimère d'amorce pouvant être évitée. L'invention concerne également un procédé de détection d'un acide nucléique cible qui consiste à réaliser une réaction d'amplification d'acide nucléique par un procédé de PCR nichée et à détecter un produit d'amplification d'acide nucléique sur un support solide.
PCT/JP2016/069638 2015-07-03 2016-07-01 Procédé pour détecter un acide nucléique cible WO2017006859A1 (fr)

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WO2020241785A1 (fr) * 2019-05-29 2020-12-03 藤倉化成株式会社 Composition destinée à être fixée à une phase solide, support en phase solide l'utilisant, procédé de production d'un support en phase solide et procédé d'utilisation d'un support en phase solide

Citations (2)

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JPH04505999A (ja) * 1989-03-22 1992-10-22 セミュ・バイオテクニク・アーベー 医学的健康状態の固相診断法
WO2012070618A1 (fr) * 2010-11-24 2012-05-31 株式会社カネカ Procédé de détection d'acide nucléique amplifié et dispositif de détection

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JPH04505999A (ja) * 1989-03-22 1992-10-22 セミュ・バイオテクニク・アーベー 医学的健康状態の固相診断法
WO2012070618A1 (fr) * 2010-11-24 2012-05-31 株式会社カネカ Procédé de détection d'acide nucléique amplifié et dispositif de détection

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HORNG, YT. ET AL.: "Development of an improved PCR-ICT hybrid assay for direct detection of Legionellae and Legionella pneumophila from cooling tower water specimens", WATER RES., vol. 40, 2006, pages 2221 - 2229, XP027902408, ISSN: 0043-1354 *
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