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WO2020017632A1 - Méthode de détection d'un acide nucléique cible par chromatographie d'acide nucléique et kit pour la détection d'un acide nucléique cible - Google Patents

Méthode de détection d'un acide nucléique cible par chromatographie d'acide nucléique et kit pour la détection d'un acide nucléique cible Download PDF

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WO2020017632A1
WO2020017632A1 PCT/JP2019/028437 JP2019028437W WO2020017632A1 WO 2020017632 A1 WO2020017632 A1 WO 2020017632A1 JP 2019028437 W JP2019028437 W JP 2019028437W WO 2020017632 A1 WO2020017632 A1 WO 2020017632A1
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nucleic acid
primer
concentration
target
reaction solution
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加藤　真吾
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Keio University
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Keio University
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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
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • 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
    • C12Q1/686—Polymerase chain reaction [PCR]
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • 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 method for detecting a target nucleic acid by nucleic acid chromatography, and a kit for detecting the target nucleic acid.
  • Priority is claimed on Japanese Patent Application No. 2018-137023 filed on July 20, 2018, the content of which is incorporated herein by reference.
  • nucleic acid chromatography which is a method for detecting a target nucleic acid by utilizing hybridization between a target nucleic acid and a detection probe associated with the target nucleic acid, does not require an expensive device and can be easily performed. It is widely used because it can perform genetic tests.
  • a method is known in which a target nucleic acid is amplified by PCR using a primer to which a special tag is added, and the tag portion is hybridized to a single-stranded probe to perform detection (for example, See Non-Patent Document 1, Patent Document 1, and Patent Document 2).
  • An object of the present invention is to provide a method and a kit that can easily detect only a PCR amplification product specific to a target nucleic acid even when non-specific amplification occurs when the target nucleic acid is amplified by PCR. I do.
  • the present inventor has conducted intensive studies on a method capable of simply detecting only a PCR amplification product specific to a target nucleic acid even when non-specific amplification has occurred.As a result, in a method for amplifying a target nucleic acid by PCR, By performing PCR at a concentration of deoxyribonucleotide triphosphate (hereinafter abbreviated as dNTP) in a PCR reaction solution lower than the concentration used in a normal PCR reaction, dNTPs are depleted during the PCR reaction, and target The elongation of the DNA using the nucleic acid as a template is stopped halfway, and a partially single-stranded PCR amplification product can be generated.
  • dNTP deoxyribonucleotide triphosphate
  • the PCR amplification product is immobilized on a solid support. Revealed that the target nucleic acid can be detected by nucleic acid chromatography by hybridizing with a single-stranded probe having a base sequence complementary to a part of the target nucleic acid. , It has led to the completion of the present invention.
  • the present invention includes the following aspects.
  • a method for detecting a target nucleic acid by nucleic acid chromatography A target nucleic acid, a first primer having a base sequence complementary to a part of a minus strand of the target sequence in the target nucleic acid, and a base sequence complementary to a part of a plus strand of the target sequence in the target nucleic acid PCR is performed using a second primer having the following formula: and a dNTP whose concentration in the PCR reaction solution is a concentration calculated by the following equation (1), and elongates from the first primer and the second primer, respectively.
  • dNTPs concentration of C dNTPs are PCR reaction solution, C P1 first primer concentration in the PCR reaction solution, C P2 is a second primer concentration in the PCR reaction solution, L T is the target sequence length (L P1 represents the length of the first primer, L P2 represents the length of the second primer, and n represents a value greater than 1 and 20 or less.)
  • a kit for detecting a target nucleic acid A first detection probe having a base sequence that is complementary to a part of the minus strand of the target sequence in the target nucleic acid so that it can specifically hybridize, and specifically a part of the plus strand of the target sequence.
  • dNTPs concentration of C dNTPs are PCR reaction solution, C P1 first primer concentration in the PCR reaction solution, C P2 is a second primer concentration in the PCR reaction solution, L T is the target sequence length (L P1 represents the length of the first primer, L P2 represents the length of the second primer, and n represents a value greater than 1 and 20 or less.)
  • the method for detecting a target nucleic acid by nucleic acid chromatography when amplifying a target nucleic acid by PCR, even when non-specific amplification occurs, only a PCR amplification product specific to the target nucleic acid can be conveniently used. Can be detected.
  • the kit for detecting a target nucleic acid according to the present invention can easily carry out the method for detecting a target nucleic acid according to the present invention by nucleic acid chromatography.
  • FIG. 3 is a diagram schematically showing a PCR reaction in a method for detecting a target nucleic acid by nucleic acid chromatography according to the present embodiment.
  • FIG. 3 is a view schematically showing a chromatography step using the nucleic acid chromatography main body of the present embodiment and the nucleic acid chromatography solution.
  • FIG. 3 is a diagram showing the results of HIV-1 detection by the method for detecting a target nucleic acid by nucleic acid chromatography of the present embodiment.
  • FIG. 2 is a diagram showing the results of HIV-2 detection by the method for detecting a target nucleic acid by nucleic acid chromatography of the present embodiment.
  • the method for detecting a target nucleic acid by nucleic acid chromatography comprises: a target nucleic acid; a first primer having a base sequence complementary to a part of a minus strand of the target sequence in the target nucleic acid; PCR using a second primer having a base sequence complementary to a part of the plus strand of the target sequence and a dNTP whose concentration in the PCR reaction solution is a concentration calculated by the following equation (1): Performing the first primer, the step of amplifying a double-stranded nucleic acid having a single-stranded nucleic acid portion not hybridized with the complementary strand extending from the second primer, A double-stranded nucleic acid having the amplified single-stranded nucleic acid portion, and a base sequence immobilized on a solid support, which is complementary to a part of the minus strand of the target sequence so that it can specifically hybridize.
  • a step of hybridizing with a detection probe Detecting the double-stranded nucleic acid hybridized with the detection probe, The above method, wherein at least one of the first primer and the second primer is labeled with a labeling substance.
  • dNTPs concentration of C dNTPs are PCR reaction solution
  • C P2 is a second primer concentration in the PCR reaction solution
  • L T is the target sequence length
  • L P1 represents the length of the first primer
  • L P2 represents the length of the second primer
  • n represents a value greater than 1 and 20 or less.
  • the target nucleic acid used in the detection method of the present invention is a nucleic acid to be detected by the detection method of the present invention.
  • the target nucleic acid may be any of single-stranded or double-stranded DNA (deoxyribonucleic acid), single-stranded or double-stranded RNA (ribonucleic acid), DNA / RNA hybrid, DNA / RNA chimera, and the like. However, double-stranded DNA is preferred.
  • the target nucleic acid is a double-stranded DNA
  • the double-stranded DNA is partially double-stranded and the other part is single-stranded. It may be a single-stranded DNA.
  • the amount of the target nucleic acid is not particularly limited as long as it can be detected by the detection method of the present invention, but is preferably 10 copies or more.
  • the target nucleic acid may be any nucleic acid that can be detected by the detection method of the present invention, and may be a naturally occurring nucleic acid or an artificially synthesized nucleic acid.
  • the naturally occurring target nucleic acid include those present in a sample derived from a living body to be tested.
  • the sample derived from a living body include blood, serum, plasma, urine, sputum, saliva, tissue, cells, and the like, or a sample obtained by extracting a nucleic acid from a sample derived from such a living body.
  • Examples of the naturally-occurring target nucleic acid include, for example, constitution, genetic disease, onset of a specific disease such as cancer, disease diagnosis, treatment prognosis, selection of a drug or treatment, and the like in a human or non-human animal or other organism. May be included.
  • Specific examples of the naturally occurring target nucleic acid include Human Immunodeficiency Virus (hereinafter abbreviated as HIV) -1, HIV-2, and the like.
  • nucleic acid chromatography refers to a method in which a nucleic acid chromatography solution containing a target nucleic acid is supplied to a solid phase carrier on which a detection probe capable of capturing the target nucleic acid is immobilized, and the chromatography is performed.
  • the step of performing The nucleic acid chromatography main body for performing the nucleic acid chromatography according to the present invention includes a solid support and one or more detection probes immobilized on the solid support.
  • the solid support is not particularly limited, and a known solid support can be used.
  • the solid support include porous materials mainly composed of polymers such as polyethersulfone, nitrocellulose, nylon, and polyvinylidene fluoride, and cellulosic materials such as filter paper.
  • the form of the nucleic acid chromatography body is not particularly limited, and may be any form, such as a sheet or a fine rod, as long as the liquid for chromatography by capillary phenomenon can be developed and diffused.
  • the detection probe is single-stranded and is at least one selected from the group consisting of a first detection probe and a second detection probe, and the first detection probe and the second detection probe , Or both.
  • the first detection probe includes a detection sequence capable of hybridizing with a part of the minus strand of the target sequence in the target nucleic acid so that the target nucleic acid can be captured.
  • the second detection probe includes a detection sequence capable of hybridizing with a part of the plus strand of the target sequence in the target nucleic acid so that the target nucleic acid can be captured.
  • the detection sequence of the first detection probe has a sequence complementary to a degree capable of specifically hybridizing with a part of the minus strand of the target sequence.
  • the detection sequence of the second detection probe has a base sequence complementary to a degree capable of specifically hybridizing with a part of the plus strand of the target sequence, and It preferably has a base sequence completely complementary to a part of the plus strand.
  • the length of the detection probe is not particularly limited, but is preferably such that the Tm value is preferably 57 to 67 ° C, more preferably 63 to 66 ° C.
  • the length can be 15 to 50 bases.
  • the detection probe is immobilized on a single solid support.
  • the detection probe immobilized on the solid support may be either the first detection probe or the second detection probe, or both.
  • the immobilization mode is not particularly limited, and a known immobilization method is used.
  • the detection probe is immobilized on the solid support by electrostatic interaction between the detection probe and the surface of the solid support, the covalent bond between a functional group in the material of the solid support and a functional group in the detection probe, and the like. can do.
  • the functional group in the material in the solid phase carrier may be a functional group already existing on the solid phase carrier or a functional group previously provided for immobilizing the detection probe.
  • a region on the solid phase carrier on which the detection probe is immobilized (hereinafter abbreviated as a probe region) can be formed in an arbitrary pattern. For example, it may be on a dot or streak having any form. It is preferable that a plurality of probe regions are provided at regular intervals along the developing direction in a strainer leak form perpendicular to the developing direction of the nucleic acid chromatography solution. Further, it is preferable that one probe region corresponds to one type of detection probe.
  • the solid phase carrier can be provided with a marker site for indicating that the nucleic acid chromatography solution has sufficiently developed.
  • a marker site for indicating that the nucleic acid chromatography solution has sufficiently developed.
  • Each of these sites like the probe region, is itself formed of a porous body capable of developing a liquid for chromatography, and at the same time, prevents continuous development of the nucleic acid chromatography solution at each site. Not configured. For example, a plurality of such sites may be provided on one solid support.
  • the nucleic acid chromatography solution is a liquid that diffuses and moves the solid phase carrier of the nucleic acid chromatography main body by a capillary phenomenon, and is a medium for moving the solid phase carrier to the target nucleic acid.
  • the liquid used for the nucleic acid chromatography solution is preferably an aqueous medium.
  • the aqueous medium include water, a buffer, an organic solvent compatible with water, and a mixture of water and one or more of the above-mentioned organic solvents.
  • organic solvent compatible with water examples include lower alcohols having 1 to 4 carbon atoms; dimethyl sulfoxide (DMSO); N, N-dimethylformamide (DMF); esters such as methyl acetate and ethyl acetate; acetone and the like. No.
  • the pH of the nucleic acid chromatography solution is not particularly limited as long as it allows the detection method of the present invention, and is, for example, pH 6 or more and 8 or less, preferably 7 or more and 8 or less.
  • a buffer corresponding to the set pH can be used.
  • the buffer include an acetate buffer, a citrate buffer, a phosphate buffer, and a phosphate buffered saline (PBS).
  • the target nucleic acid is RNA
  • RT-PCT Reverse Transcription-PCR
  • the target sequence in the target nucleic acid is formed from a plus strand and a minus strand having a sequence complementary to the plus strand (FIG. 1 (a)).
  • the target nucleic acid is denatured by heat, and the first primer and the second primer are respectively bound to the minus strand of the target sequence in the target nucleic acid and the plus strand of the target sequence in the target nucleic acid (FIG. 1 (b)). .
  • a labeling substance is previously bound to at least one of the first primer and the second primer.
  • the labeling substance may be a labeling substance that emits a detectable signal by itself or a labeling substance that emits a signal in combination with other components.
  • Examples of the labeling substance that emits a detectable signal include a fluorescent substance such as FITC and rhodamine.
  • Examples of the labeling substance that emits a signal in combination with other components include enzymes such as peroxidase and alkaline phosphatase.
  • the labeling substance when the labeling substance is biotin, the labeling substance can be detected by reacting colored latex particles to which streptavidin is bound, and then detecting the coloring.
  • the reaction between the labeling substance and another substance that emits a signal in combination with the labeling substance may be performed at the time of detection, or may be performed at the time of starting the development, during the development, or the like.
  • the binding of the labeling substance to the primer can be performed using a known method.
  • the first primer is used to amplify a target nucleic acid by nucleic acid amplification.
  • the first primer specifically hybridizes with a first base sequence constituting a part of a minus strand of a target sequence in a target nucleic acid.
  • the base sequence of the first primer is set to be complementary to the first base sequence of the minus strand of the target sequence with high selectivity and can be hybridized with high selectivity. It is a sequence complementary to the first base sequence.
  • the length of the first primer is not particularly limited and is appropriately determined depending on the length of the target sequence of the target nucleic acid. For example, the length is 15 to 40 bases, and preferably 18 to 30 bases. .
  • the concentration of the first primer in the PCR reaction solution is preferably 0.01 to 10 ⁇ M, more preferably 0.03 to 3 ⁇ M, and particularly preferably 0.1 to 1 ⁇ M.
  • the second primer is used together with the first primer to amplify a target nucleic acid by nucleic acid amplification.
  • the second primer specifically hybridizes with a second base sequence constituting a part of the plus strand of the target sequence in the target nucleic acid.
  • the base sequence of the second primer is set to be complementary to the second base sequence so as to hybridize with high selectivity, and is preferably a sequence completely complementary to the second base sequence.
  • the length of the second primer is not particularly limited and is appropriately determined according to the length of the target sequence of the target nucleic acid. For example, the length is 15 to 40 bases, and preferably 18 to 30 bases. .
  • the concentration of the second primer in the PCR reaction solution is preferably 0.01 to 10 ⁇ M, more preferably 0.03 to 3 ⁇ M, and particularly preferably 0.1 to 1 ⁇ M.
  • ⁇ Amplification step> When PCR is carried out using the first primer and the second primer, as shown in FIGS. 1A and 1B, the first primer and the From the second primer, a template strand containing the primer is formed using dNTP.
  • the minimum concentration C ′ dNTP in the PCR reaction solution of dNTP required when all of the first primer and the second primer are used for the template strand of the target sequence in PCR is calculated by the following equation (2).
  • dNTP means all of deoxyguanosine triphosphate (dGTP), deoxyadenosine triphosphate (dATP), deoxycytidine triphosphate (dCTP), and deoxythymidine triphosphate (dTTP).
  • the concentration of dNTP in the PCR reaction solution refers to the concentration of the dGTP, dATP, dCTP, and dTTP in the PCR reaction solution.
  • C 'dNTP the minimum concentration of the PCR reaction solution dNTP required when the first primer and the second primer were used all the template strand
  • C P1 first in PCR reaction solution primer concentration C P2 is a second primer concentration in the PCR reaction solution
  • L T is the length of the target sequence
  • L P1 is the length of the first primer
  • L P2 the length of the second primer
  • n Represents a numerical value larger than 1 and 20 or less.
  • the concentration of dNTP used for elongation of the target sequence in the PCR reaction solution is 1 / n of the concentration of C ' dNTP (n is larger than 1 and represents a numerical value of 20 or less). And That is, in the detection method of the present invention, the concentration of dNTP in the PCR reaction solution is calculated by the following equation (1).
  • n represents a value greater than 1 and 20 or less.
  • n may be any number as long as it is greater than 1 and 20 or less, preferably 2 to 10 and more preferably 2 to 5 inclusive.
  • the dNTP concentration in the PCR reaction solution is adjusted to be not less than 1/20 of the minimum concentration C ′ dNTP in the PCR reaction solution of dNTP required when all of the first primer and the second primer are used for the template strand.
  • concentration less than the minimum concentration C ′ dNTP , as shown in FIG. 1C, dNTP is depleted during the elongation of the template strand and elongation of the template strand becomes complete during the elongation of the template strand. Stop.
  • the double-stranded nucleic acid to be amplified is partially a double-stranded nucleic acid having a single-stranded nucleic acid portion that is not hybridized with a complementary strand extending from the first primer and the second primer.
  • the single-stranded portion of the double-stranded nucleic acid hybridizes with the detection probe immobilized on the solid-phase carrier, whereby the amplified double-stranded nucleic acid can be bound to the solid-phase carrier.
  • the number of PCR cycles is not particularly limited, but is preferably 20 to 60 times, more preferably 30 to 60 times, and particularly preferably 40 to 60 times.
  • the denaturation temperature of PCR is preferably from 90 ° C to 100 ° C, more preferably from 94 ° C to 98 ° C, and particularly preferably about 94 ° C.
  • the annealing temperature of PCR is preferably 55 to 70 ° C, more preferably 55 to 65 ° C, and particularly preferably about 60 ° C.
  • the extension reaction temperature of PCR is preferably from 70 ° C to 78 ° C, more preferably from 72 ° C to 75 ° C.
  • the extension time of the PCR is preferably 10 to 60 seconds, more preferably 15 to 30 seconds.
  • the extension reaction may be performed after the annealing, or the annealing and the extension reaction may be performed simultaneously.
  • the concentration of the polymerase contained in the PCR reaction solution in the PCR reaction solution is preferably 0.0001 U / ⁇ L to 0.2 U / ⁇ L, more preferably 0.001 U / ⁇ L to 0.15 U / ⁇ L, and more preferably 0.004 U / ⁇ L. ⁇ L to 0.1 U / ⁇ L are particularly preferred.
  • a PCR reaction solution containing a double-stranded nucleic acid having a single-stranded nucleic acid portion that has not been hybridized with a complementary strand extending from the first primer and the second primer, respectively, amplified by the PCR is subjected to the nucleic acid chromatography.
  • the labeling substance bound to at least one of the first primer and the second primer is a substance that emits a signal in combination with another substance
  • the other substance hereinafter, referred to as the nucleic acid chromatography solution
  • the labeling substance is biotin
  • colored latex particles bound with streptavidin can be contained in the nucleic acid chromatography solution.
  • the substance for detecting the labeling substance may be added separately from the nucleic acid chromatography solution.
  • the labeling substance bound to the first primer or the second primer is a substance that emits a signal in combination with another substance
  • the substance for detecting the label is two of the substances in the PCR reaction solution. It binds to the labeling substance that is bound to the strand nucleic acid.
  • Step (c) The solid phase carrier is immersed in the nucleic acid chromatography solution, and the nucleic acid chromatography solution is developed on the solid phase carrier.
  • Step (d) The double-stranded nucleic acid developed on the solid-phase carrier is bound to the detection probe immobilized on the solid-phase carrier at its single-stranded portion, and is captured.
  • the captured double-stranded nucleic acid is detected by a detection method according to the labeling substance.
  • the conditions for performing the nucleic acid chromatography are not particularly limited as long as the development of the nucleic acid chromatography solution, complementary hybridization, and the like are not inhibited.
  • the temperature can be, for example, 5 to 40 ° C, preferably 15 to 37 ° C.
  • the time required for the nucleic acid chromatography can be appropriately set according to the shape of the nucleic acid chromatography main body, the type of the substrate, and the composition of the liquid for chromatography. For example, it can be set for 2 to 50 minutes.
  • the kit of the present invention is a kit for detecting a target nucleic acid, A first detection probe having a base sequence that is complementary to a part of the minus strand of the target sequence in the target nucleic acid so that it can specifically hybridize, and specifically a part of the plus strand of the target sequence.
  • n represents a value greater than 1 and 20 or less.
  • n may be any number as long as it is greater than 1 and 20 or less, preferably 2 to 10 and more preferably 2 to 5 inclusive.
  • the kit for detecting a target nucleic acid of the present invention can be used for the method for detecting a target nucleic acid by nucleic acid chromatography.
  • a target nucleic acid, a first detection probe having a sequence complementary to a portion capable of specifically hybridizing with a part of the minus strand of the target sequence, and specifically hybridizable with a part of the plus strand of the target sequence A solid support on which at least one detection probe selected from the group consisting of second detection probes having a base sequence complementary to a certain extent is immobilized, and complementary to a part of the minus strand of the target sequence.
  • the first primer having a base sequence, the second primer having a base sequence complementary to a part of the plus strand of the target sequence, and the labeling substance include the target nucleic acid, the solid phase carrier, and the first A primer, a second primer, and a labeling substance are used, respectively.
  • the dNTP concentration in the kit of the present invention is a concentration calculated by the following formula (1).
  • dNTPs concentration of C dNTPs are PCR reaction solution
  • C P2 is a second primer concentration in the PCR reaction solution
  • L T is the target sequence length
  • L P1 represents the length of the first primer
  • L P2 represents the length of the second primer
  • n represents a value greater than 1 and 20 or less.
  • the concentration of the first primer and the concentration of the second primer are preferably 0.01 to 10 ⁇ M in the PCR reaction solution, more preferably 0.03 to 3 ⁇ M, Particularly preferred is a concentration of 0.1-1 ⁇ M.
  • the kit of the present invention can contain other components as necessary.
  • Other components include a substance for detecting the labeling substance, a reagent for amplifying the target nucleic acid, a ribonuclease inhibitor, a negative control for the target nucleic acid, and the like.
  • Example 1 [Detection of HIV-1] (1) Production of nucleic acid chromatography main body For the first detection consisting of the base sequence represented by SEQ ID NO: 1 on a Hi-Flow Plus membrane sheet (3.5 mm wide, 60 mm long; manufactured by Merck Millipore) A solution containing the probe and a second detection probe consisting of the base sequence represented by SEQ ID NO: 2 was spotted using a spotter (GENESHOT; manufactured by NGK) to immobilize the detection probe.
  • GENESHOT manufactured by NGK
  • gagC-1F manufactured by Eurofin
  • gagC-1RB-bio Efin
  • SEQ ID NO: 5 RT-PCR targeting the base sequence represented by SEQ ID NO: 5 RT-PCR was performed as follows as a base sequence of the plus strand of the sequence.
  • the concentration of dNTP in the PCR reaction solution was determined as follows. A value obtained by subtracting 25 bases, which is the length of gagC-1F as the first primer, from 191 bases, which is the length of the target sequence represented by SEQ ID NO: 5, is 166 bases, and the target represented by SEQ ID NO: 5 A value obtained by subtracting 20 nucleotides, which is the length of gagC-1RB-bio as the second primer, from 191 nucleotides, which is the sequence length, is 171 nucleotides.
  • the concentration of gagC-1F as the first primer in the PCR reaction solution was set to 0.2 ⁇ M
  • the concentration of the second primer in the PCR reaction solution was set to 0.2 ⁇ M
  • n was set to 4.21.
  • the dNTP concentration in the PCR reaction solution was set to 16 ⁇ M (4 ⁇ M for each dNTP concentration).
  • the solution A was placed in a thermal cycler (GeneAmp PCR system 9700, manufactured by Thermo Fischer) maintained at 56 ° C., and the following solution B was placed on the thermal cycler. added.
  • the concentration of each dNTP (dATP, dCTP, dGTP, dTTP) in the solution A was adjusted to 0.5 mM so that the concentration of dNTP in the PCR reaction solution was 16 ⁇ M (4 ⁇ M for each dNTP).
  • dNTP dATP, dCTP, dGTP, dTTP
  • Liquid A 20 ⁇ M gagC-1F 0.25 ⁇ l 20 ⁇ M gagC-1RB-bio 0.25 ⁇ l 0.5 mM dNTP 0.2 ⁇ l Ribonuclease inhibitor (RNasin; manufactured by Promega) 0.1 ⁇ l 8.2 ⁇ l of water
  • Liquid B 2.5 ⁇ l of 10 ⁇ PCR Buffer (Invitrogen) 2.0 mM 50 mM MgCl 2 Water 10.35 ⁇ l Platinum Taq DNA polymerase (Invitrogen) 0.1 ⁇ l SuperScript III (Invitrogen) 0.05 ⁇ l
  • PCR was performed as follows. After performing the reverse transcription reaction at 56 ° C. for 10 minutes, heat denaturation at 97 ° C. was performed for 2 minutes, and the PCR reaction was performed 5 times with a cycle of heat denaturation at 97 ° C. for 5 seconds and annealing extension reaction at 56 ° C. for 10 seconds. . Thereafter, the PCR reaction was performed 30 times with a cycle of heat denaturation at 94 ° C. for 5 seconds and annealing extension reaction at 56 ° C. for 10 seconds. After the completion of the PCR reaction, the PCR reaction solution was kept at 4 ° C. As a control, PCR was similarly performed on a sample containing no HIV-1 RNA.
  • the lower end of the chromatography main body prepared in (1) was immersed in each of the nucleic acid chromatography solutions obtained above, and the nucleic acid chromatography solution was developed. After the development, the solution was dried for about 10 minutes. The result is shown in FIG. As shown in FIG. 3, when 0.5 mM dNTP was used, bands could be confirmed in all of 1000 copies, 100 copies, and 10 copies of HIV-1.
  • Example 2 [Detection of HIV-2]
  • a probe consisting of the base sequence represented by SEQ ID NO: 6 was used as the first detection probe, and a probe consisting of the base sequence represented by SEQ ID NO: 7 was used as the second detection probe.
  • HIV-2-1FH manufactured by Eurofin
  • gagC-1RB-bio was used as the second primer.
  • HIV-2-1RZ-b-bio manufactured by Eurofin
  • HIV-2 was used instead of HIV-1 as the target nucleic acid.
  • Nucleic acid chromatography was performed in the same manner as in Example 1, except that the base sequence represented by 10 was used as the base sequence of the plus strand of the target sequence.
  • the concentration of dNTP in the PCR reaction solution was determined as follows. The value obtained by subtracting 21 bases, which is the length of the first primer HIV-2-1FH, from 112 bases, which is the length of the target sequence represented by SEQ ID NO: 10, is 91 bases, and is represented by SEQ ID NO: 10. The value obtained by subtracting the length of 21 bases, which is the length of the second primer HIV-2-1RZ-b-bio, from the length of 112 bases of the target sequence is 91 bases.
  • the concentration of the first primer, HIV-2-1FH, in the PCR reaction solution was 0.2 ⁇ M
  • the concentration of the second primer, HIV-2-1RZ-b-bio, in the PCR reaction solution was 0.2 ⁇ M.
  • N was set to 2.275
  • the dNTP concentration in the PCR reaction solution was set to 16 ⁇ M according to the above equation (1). Therefore, the concentration of each dNTP (dATP, dCTP, dGTP, dTTP) used in the solution A is 0.5 mM so that the concentration of dNTP in the PCR reaction solution becomes 16 ⁇ M (each dNTP concentration is 4 ⁇ M).
  • FIG. 4 shows the results. As shown in FIG. 4, when 0.5 mM dNTP was used, bands could be confirmed in any of 10, 100, and 1000 copies.
  • Example 1 Nucleic acid chromatography was performed in the same manner as in Example 1 except that 25 mM dNTP was used instead of 0.5 mM dNTP in Example 2.
  • FIG. 4 shows the results.
  • ddATP dideoxyadenosine triphosphate
  • the present invention it is possible to provide a method and a kit that can easily detect only a PCR amplification product specific to a target nucleic acid even when non-specific amplification occurs when the target nucleic acid is amplified by PCR. it can.

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Abstract

Cette méthode de détection d'un acide nucléique cible par chromatographie d'acide nucléique comprend : une étape consistant à effectuer une PCR à l'aide d'un acide nucléique cible, d'une amorce ayant une séquence de base complémentaire d'une séquence cible dans l'acide nucléique, et du triphosphate de désoxyribonucléotide pour lequel la concentration de celui-ci dans une solution de réaction de PCR est inférieure à la concentration utilisée en PCR normale, et à amplifier un acide nucléique double brin ayant un brin complémentaire qui s'étend à partir de l'amorce, et une partie d'acide nucléique simple brin qui n'est pas hybridée ; une étape consistant à hybrider l'acide nucléique double brin ayant la partie d'acide nucléique simple brin amplifiée, et une sonde de détection immobilisée sur un support en phase solide et ayant une séquence de base complémentaire à une partie de la séquence cible ; et une étape consistant à détecter la sonde de détection et l'acide nucléique double brin hybridé.
PCT/JP2019/028437 2018-07-20 2019-07-19 Méthode de détection d'un acide nucléique cible par chromatographie d'acide nucléique et kit pour la détection d'un acide nucléique cible Ceased WO2020017632A1 (fr)

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WO2014156513A1 (fr) * 2013-03-29 2014-10-02 日本碍子株式会社 Procédé de détection d'une mutation

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Publication number Priority date Publication date Assignee Title
WO2014156513A1 (fr) * 2013-03-29 2014-10-02 日本碍子株式会社 Procédé de détection d'une mutation

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
HARIDAS, T. K. ET AL.: "Development of a PCR based nucleic acid lateral flow assay device for detection of Mycobacterium Tuberculosis complex", INTERNATIONAL JOURNAL OF PHARMTECH RESEARCH, vol. 6, no. 5, 2014, pages 1695 - 1702 *
XU, Y. ET AL.: "Fluorescent probe-based lateral flow assay for multiplex nucleic acid detection", ANALYTICAL CHEMISTRY, vol. 86, no. 12, 17 June 2014 (2014-06-17), pages 5611 - 5614, XP055520587, ISSN: 0003-2700, DOI: 10.1021/ac5010458 *
ZHANG, H. ET AL.: "Rapid detection of methicillin-resistant Staphylococcus aureus in pork using a nucleic acid-based lateral flow immunoassay", INTERNATIONAL JOURNAL OF FOOD MICROBIOLOGY, vol. 243, 11 December 2016 (2016-12-11), pages 64 - 69, XP029892293, ISSN: 0168-1605, DOI: 10.1016/j.ijfoodmicro.2016.12.003 *

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