WO2022145354A1 - 標的核酸断片の検出方法及びキット - Google Patents

標的核酸断片の検出方法及びキット Download PDF

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WO2022145354A1
WO2022145354A1 PCT/JP2021/048095 JP2021048095W WO2022145354A1 WO 2022145354 A1 WO2022145354 A1 WO 2022145354A1 JP 2021048095 W JP2021048095 W JP 2021048095W WO 2022145354 A1 WO2022145354 A1 WO 2022145354A1
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nucleic acid
well
acid fragment
target nucleic
crispr
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English (en)
French (fr)
Japanese (ja)
Inventor
力也 渡邉
肇 篠田
麻美 牧野
龍也 飯田
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RIKEN
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RIKEN
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Priority to EP21915214.7A priority Critical patent/EP4269581B1/en
Priority to JP2022573049A priority patent/JPWO2022145354A1/ja
Priority to US18/269,264 priority patent/US20240094199A1/en
Publication of WO2022145354A1 publication Critical patent/WO2022145354A1/ja
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C—CHEMISTRY; METALLURGY
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    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/14—Hydrolases (3)
    • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
    • C12N9/22—Ribonucleases [RNase]; Deoxyribonucleases [DNase]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C12Q1/682—Signal amplification
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C12Q1/6825—Nucleic acid detection involving sensors
    • 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
    • C12Q1/6834—Enzymatic or biochemical coupling of nucleic acids to a solid phase
    • C12Q1/6837—Enzymatic or biochemical coupling of nucleic acids to a solid phase using probe arrays or probe chips
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes
    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/536—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase
    • G01N33/542—Immunoassay; Biospecific binding assay; Materials therefor with immune complex formed in liquid phase with steric inhibition or signal modification, e.g. fluorescent quenching
    • 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
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
    • G01N33/54306—Solid-phase reaction mechanisms
    • C—CHEMISTRY; METALLURGY
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    • C12N11/00—Carrier-bound or immobilised enzymes; Carrier-bound or immobilised microbial cells; Preparation thereof
    • C12N11/14—Enzymes or microbial cells immobilised on or in an inorganic carrier
    • C—CHEMISTRY; METALLURGY
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    • C12N2310/00—Structure or type of the nucleic acid
    • C12N2310/10—Type of nucleic acid
    • C12N2310/20—Type of nucleic acid involving clustered regularly interspaced short palindromic repeats [CRISPR]
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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    • C12Q2521/00—Reaction characterised by the enzymatic activity
    • C12Q2521/30—Phosphoric diester hydrolysing, i.e. nuclease

Definitions

  • the present invention relates to a method and a kit for detecting a target nucleic acid fragment.
  • cfDNA cell-free DNA
  • ctDNA blood tumor DNA
  • Exosomes contain various proteins, lipids, microRNAs, DNAs, etc. derived from the cells that secrete them.
  • Non-Patent Documents 1 to 3 report a method for detecting a target nucleic acid fragment with high sensitivity by utilizing such activities of Cas12 and Cas13.
  • Patent Document 1 describes that enzyme activity is detected at the single molecule level using a microchamber having a size on the order of femtolitre.
  • an object of the present invention is to provide a technique capable of detecting a target nucleic acid fragment with high sensitivity without amplifying it.
  • a method for detecting a target nucleic acid fragment in a sample which is a step (a) of contacting the sample with a gRNA complementary to the target nucleic acid fragment, a CRISPR / Cas family protein, and a substrate nucleic acid fragment.
  • the CRISPR / Cas family protein expresses nuclease activity after forming a tripartite complex with the gRNA and the target nucleic acid fragment, and the CRISPR / Cas family protein is immobilized on a solid phase.
  • the substrate nucleic acid fragment is labeled with a fluorescent substance and a dimming substance, and when the fluorescent substance is cleaved by the nuclease activity of the tripartite complex and the fluorescent substance is separated from the extinguishing substance, it emits fluorescence by irradiation with excitation light.
  • the contact is performed in a reaction space having a volume of 10 aL to 100 pL, and as a result, when the target nucleic acid fragment is present in the sample, the tripartite complex is formed and the substrate nucleic acid fragment is cleaved.
  • the step (a) in which the fluorescent substance is separated from the extinguishing substance and the step (b) of irradiating the fluorescent substance with the excitation light to detect the fluorescence are included, and the fluorescence is detected.
  • step (a1) When the fragment is introduced and the CRISPR / Cas family protein does not form a binary complex with the gRNA in advance, the sample, the gRNA and the substrate nucleic acid fragment are introduced into each well of the well array.
  • step (a1) each well of the well array is sealed with a sealing solution, and as a result, when the target nucleic acid fragment is present in the well, the tripartite complex is formed and the substrate nucleic acid fragment is formed.
  • the step (a) is performed in each well of the well array, and each well is arranged in the first well and the bottom of the first well, which is smaller than the first well. It has a second well of volume, the reaction space is the space inside the second well, and the CRISPR / Cas family protein is immobilized on the inner surface of the second well.
  • the CRISPR / Cas family protein has previously formed a two-way complex with the gRNA, the sample and the substrate nucleic acid fragment are introduced into each well of the well array.
  • the step of introducing the sample, the gRNA and the substrate nucleic acid fragment into each well of the well array (a1). ')
  • the step of sealing each well of the well array with a sealing liquid (a2'), and the replacement of the sealing liquid with a water-absorbent organic solvent, and as a result, the contents of the wells are dehydrated.
  • the volume is reduced, the contents are accumulated inside the second well, and when the target nucleic acid fragment is present in the contents, the tripartite complex is formed and the substrate nucleic acid fragment is formed.
  • the method according to [1], comprising a step (a3') of cutting and separating the fluorescent substance from the extinguishing substance.
  • the encapsulant is a fluorinated liquid, a mineral oil, or a linear or branched saturated or unsaturated hydrocarbon having 7 to 17 carbon atoms.
  • the water-absorbent organic solvent is a linear or branched saturated or unsaturated fatty alcohol having 4 to 11 carbon atoms.
  • the CRISPR / Cas family protein is immobilized on the surface of the particles, and the sample, the gRNA and the CRISPR / Cas family protein are mixed in a container before the step (a).
  • [8] The method according to any one of [1] to [7], wherein 0 or 1 of the target nucleic acid fragments is introduced into each reaction space.
  • a substrate having a well formed on the surface having a volume of 10 aL to 100 pL, a gRNA complementary to the target nucleic acid fragment, a CRISPR / Cas family protein immobilized on a solid phase, and a substrate nucleic acid fragment are included.
  • the CRISPR / Cas family protein expresses nuclease activity after forming a tripartite complex with the gRNA and the target nucleic acid fragment, and the substrate nucleic acid fragment is labeled with a fluorescent substance and a dimming substance.
  • a kit for detecting the target nucleic acid fragment which emits fluorescence by irradiation with excitation light when the fluorescent substance is cleaved by the nuclease activity of the tripartite complex and separates from the extinguishing substance. [11] The kit for detecting a target nucleic acid fragment according to [10], wherein the CRISPR / Cas family protein is immobilized on the inner surface of the well.
  • the well has a first well and a second well located at the bottom of the first well and having a smaller volume than the first well, the CRISPR / Cas family.
  • the present invention it is possible to provide a technique capable of detecting a target nucleic acid fragment with high sensitivity without amplifying it.
  • FIG. 1 (a) and 1 (b) are schematic views illustrating a method for detecting a target nucleic acid fragment.
  • FIG. 2A is a top view showing an example of a fluid device.
  • 2 (b) is a cross-sectional view taken along the line b-b'of FIG. 2 (a).
  • 3 (a) to 3 (c) are schematic cross-sectional views illustrating an example of a procedure for carrying out a method for detecting a target nucleic acid fragment.
  • 4 (a) to 4 (d) are schematic views illustrating a method of immobilizing a CRISPR / Cas family protein on the inner surface of a well.
  • FIG. 5A is a top view showing an example of a well array having a first well and a second well.
  • FIG. 5 (b) is a cross-sectional view taken along the line b-b'of FIG. 5 (a).
  • FIG. 6A is a top view showing an example of a well array having a first well and a second well.
  • FIG. 6 (b) is a cross-sectional view taken along the line b-b'of FIG. 6 (a).
  • 7 (a) to 7 (f) are schematic cross-sectional views illustrating each step of forming a well array.
  • 8 (a) and 8 (b) are schematic cross-sectional views illustrating each step of forming a well array.
  • FIG. 9 is a photomicrograph of a well array with a first well and a second well actually manufactured.
  • FIG. 10A is a top view showing an example of a fluid device.
  • 10 (b) is a cross-sectional view taken along the line b-b'of FIG. 10 (a).
  • 11 (a) to 11 (c) are schematic views illustrating a method for detecting a target nucleic acid fragment.
  • 12 (a) to 12 (d) are schematic diagrams illustrating a method for detecting a target nucleic acid fragment.
  • 13 (a) to 13 (d) are typical fluorescence micrographs showing the results of Experimental Example 1.
  • FIG. 14 is a graph showing the results of Experimental Example 1.
  • FIG. 15 is a graph showing the results of Experimental Example 2.
  • FIG. 16 is a fluorescence micrograph showing the results of Experimental Example 3.
  • FIG. 17 is a graph showing the results of Experimental Example 4.
  • FIG. 18 is a graph showing the results of Experimental Example 5.
  • FIG. 19 is a graph showing the results of Experimental Example 6.
  • FIG. 20 is a graph showing the results of Experimental Example 7.
  • FIG. 21 is a schematic diagram illustrating a method of immobilizing a CRISPR / Cas family protein on the surface of a particle.
  • 22 (a) and 22 (b) are schematic views illustrating a method of immobilizing a CRISPR / Cas family protein on the surface of a particle.
  • FIG. 23 is a graph showing the results of Experimental Example 8.
  • FIG. 24 is a schematic diagram illustrating a method for detecting a target nucleic acid fragment carried out in Experimental Example 9.
  • FIG. 25 is a fluorescence micrograph showing the results of Experimental Example 9.
  • the invention is a method of detecting a target nucleic acid fragment in a sample, wherein the sample is contacted with a gRNA complementary to the target nucleic acid fragment, a CRISPR / Cas family protein, and a substrate nucleic acid fragment.
  • the target nucleic acid fragment is present in the sample because the step (a) and the step (b) of irradiating the fluorescent substance with the excitation light and detecting the fluorescence are included. Provide a method to show what to do.
  • the CRISPR / Cas family protein expresses nuclease activity after forming a tripartite complex with gRNA and a target nucleic acid fragment. Also, the CRISPR / Cas family proteins are immobilized on the solid phase. Further, the substrate nucleic acid fragment is labeled with a fluorescent substance and a quenching substance, and when the fluorescent substance is cleaved by the nuclease activity of the above-mentioned tripartite complex and the fluorescent substance is separated from the quenching substance, it emits fluorescence by irradiation with excitation light. ..
  • contact of the sample, gRNA, CRISPR / Cas family protein and substrate nucleic acid fragment is performed in a reaction space having a volume of 10aL to 100pL.
  • the target nucleic acid fragment is present in the sample, the above-mentioned tripartite complex is formed, the substrate nucleic acid fragment is cleaved, and the fluorescent substance is separated from the quenching substance.
  • FIGS. 1A and 1B are schematic views illustrating the method of the present embodiment.
  • the case where the CRISPR / Cas family protein is the Cas12a protein will be described as an example.
  • Cas12a protein 110 and gRNA120 are brought into contact with each other in step (a), they bind to form a two-way complex 130.
  • the gRNA 120 partially has a base sequence complementary to the target nucleic acid fragment 140.
  • the substrate nucleic acid fragment 150 is a single-stranded DNA fragment labeled with a fluorescent substance F and a quenching substance Q. No fluorescence is generated even when the substrate nucleic acid fragment 150 is irradiated with excitation light.
  • FIG. 1A is a schematic diagram showing a tripartite complex 100'in which the target site of the target nucleic acid fragment 140 has been cleaved.
  • FIG. 1 (b) the tripartite complex 100'expresses nuclease activity.
  • the substrate nucleic acid fragment 150 existing around the tripartite complex 100' is cleaved.
  • the fluorescent substance F of the substrate nucleic acid fragment 150 is separated from the quenching substance Q.
  • the fluorescent substance F separated from the quenching substance Q emits fluorescence by irradiation with excitation light.
  • the fluorescent substance F is irradiated with excitation light to detect fluorescence.
  • fluorescence it can be determined that the target nucleic acid fragment 140 was present in the sample.
  • the CRISPR / Cas family protein is immobilized on the solid phase.
  • the CRISPR / Cas family protein is immobilized on the solid phase, so that the detection sensitivity of the target nucleic acid fragment is significantly improved.
  • the CRISPR / Cas family protein may be immobilized on the surface of the particles, for example, or may be immobilized on the inner surface of the well, which is the reaction space. Details will be described later.
  • the sample, gRNA120, CRISPR / Cas family protein 110, and substrate nucleic acid fragment 150 may be mixed and contacted in any order.
  • the gRNA 120 and the CRISPR / Cas family protein 110 may be first contacted to form a two-way complex 130 in advance, and then the sample may be contacted.
  • the target nucleic acid fragment 140 if the target nucleic acid fragment 140 is present in the sample, the target nucleic acid fragment 140 binds to the two-way complex 130 to form the three-way complex 100.
  • the substrate nucleic acid fragment 150 may then be contacted.
  • the target nucleic acid fragment 140 and the substrate nucleic acid fragment 150 may be brought into contact at the same time.
  • gRNA120, CRISPR / Cas family protein 110, and a sample may be brought into contact at the same time. Even in this case, if the target nucleic acid fragment 140 is present in the sample, the tripartite complex 100 is finally formed. The substrate nucleic acid fragment 150 may then be contacted.
  • the sample, gRNA 120, CRISPR / Cas family protein 110, and substrate nucleic acid fragment 150 may be contacted at the same time. Even in this case, if the target nucleic acid fragment 140 is present in the sample, the tripartite complex 100 is finally formed, and when the target site of the target nucleic acid fragment 140 is cleaved in the tripartite complex 100, It is converted to a tripartite complex 100', expresses nuclease activity, and cleaves the substrate nucleic acid fragment 150.
  • a biological sample containing impurities can be used as a sample, and the step (a) can be performed without purifying the target nucleic acid fragment from the biological sample.
  • the target nucleic acid fragment can be detected with almost no influence of the contaminants. ..
  • the CRISPR / Cas family protein is immobilized on the solid phase.
  • the CRISPR / Cas family protein may be immobilized on the surface of the particles.
  • the sample, gRNA and CRISPR / Cas family protein are mixed in a container, and the CRISPR / Cas family protein, gRNA and the target nucleic acid fragment are contained on the particles. May further include a step of forming the protein.
  • fixed to the surface of the particle means that the CRISPR / Cas family protein may be fixed in contact with the particle, or the CRISPR / Cas family protein is fixed to the particle via a linker. May be good.
  • the particles are not particularly limited, and examples thereof include resin beads and glass beads.
  • the resin include polyethylene, polypropylene, polystyrene, polycarbonate, cyclic polyolefin, acrylic and the like.
  • the particles may be magnetic beads. If the particles are magnetic beads, the particles can be recovered using a magnetic stand or the like.
  • the size of the particles can be appropriately selected, and for example, particles having a diameter of about 0.1 to 100 ⁇ m can be used.
  • the particles may be colored with a fluorescent dye or the like.
  • a plurality of types of particles colored with different dyes may be used, and a gRNA having a specific base sequence may be bound to a CRISPR / Cas family protein immobilized on the particles colored with a specific dye. This makes it possible to identify the base sequence of the target nucleic acid fragment recognized by the CRISPR / Cas family protein immobilized on the particle by the color of the particle.
  • a functional group existing on the surface of the particle and a functional group existing on the surface of the CRISPR / Cas family protein are covalently bonded using physical adsorption or a chemical linker. Examples thereof include a method using hybridization of a single-stranded nucleic acid fragment, a method using an avidin-biotin bond, and the like. These methods may be used in combination.
  • the functional group when a chemical linker is used examples include a hydroxyl group, an amino group, a thiol group and the like.
  • the CRISPR / Cas family protein may be immobilized on the surface of the particles by utilizing a click reaction using an azide group and an alkyne group.
  • the particles and the CRISPR / Cas family protein are brought into contact with the avidin by contacting the avidin with the biotinylated CRISPR / Cas family protein using a chemical linker. Can be combined.
  • 21 and 22 are schematic views illustrating a method of binding a particle to a CRISPR / Cas family protein using hybridization of a single-stranded nucleic acid fragment.
  • FIG. 21 is a schematic diagram illustrating an embodiment of a method of binding particles to a CRISPR / Cas family protein.
  • the single-stranded DNA fragment 2120 is bound to the particles 2110.
  • the ends of the single-stranded DNA fragment 2120 are biotin-modified.
  • the surface of the particles 2110 is avidin coated. Therefore, when the single-stranded DNA fragment 2120 and the particle 2110 are brought into contact with each other, the single-stranded DNA fragment 2120 binds to the surface of the particle 2110 by avidin-biotin binding.
  • the single-stranded DNA fragment 2120 has a base sequence complementary to a part of the target nucleic acid fragment 140. Therefore, when the particles 2110 to which the single-stranded DNA fragment 2120 is bound and the sample containing the target nucleic acid fragment 140, gRNA, and CRISPR / Cas family protein are mixed in a container, the CRISPR / Cas family protein, gRNA, and target nucleic acid fragment 140 are obtained.
  • the containing tripartite complex 100' is immobilized on the particle 2110 by hybridization of a part of the target nucleic acid fragment 140 with the single-stranded DNA fragment 2120.
  • 22 (a) and 22 (b) are schematic diagrams illustrating another embodiment of binding a particle to a CRISPR / Cas family protein.
  • the particles shown in FIG. 22B are hybridized with a part of the target nucleic acid fragment 140, the single-stranded DNA fragment 2121, and the single-stranded DNA fragment 2120.
  • the main difference is that the human complex 100'is fixed to the particles 2110.
  • FIG. 22A shows a state in which the number of molecules of the tripartite complex formed on one target nucleic acid fragment is increased by using a plurality of types of gRNA for one type of target nucleic acid fragment. Shows. As described above, this makes it possible to increase the number of molecules of the tripartite complex per target molecule of nucleic acid fragment, so that the detection sensitivity of the target nucleic acid fragment can be further increased.
  • a tripartite complex 100' that recognizes the target nucleic acid fragments (target sequences) 140, 140', and 140', respectively, is bound to one long nucleic acid fragment.
  • single-stranded nucleic acid fragments 2121, 2122, and 2123 having a base sequence complementary thereto are hybridized in the vicinity of the target nucleic acid fragments (target sequences) 140, 140 ′′, and 140 ′′, respectively.
  • the region of the single-stranded nucleic acid fragments 2121, 2122, and 2123 that does not hybridize with the long nucleic acid fragment has a base sequence complementary to the single-stranded nucleic acid fragment 2120 bound to the particle 2110. is doing.
  • the region where the single-stranded nucleic acid fragments 2121, 2122, and 2123 hybridize with the long nucleic acid fragment is protected from the nuclease activity of the tripartite complex 100'and is not cleaved. ..
  • the long nucleic acid fragment is cleaved by the nuclease activity of the tripartite complex 100'(trans cleavage).
  • the complex of the excised tripartite complex 100'and the single-stranded nucleic acid fragment 2121 hybridizes with the single-stranded DNA fragment 2120 bound to the particle 2110. Is fixed to the particles 2110.
  • the complex of the tripartite complex 100'and the single-stranded nucleic acid fragment 2122, and the complex of the tripartite complex 100'and the single-stranded nucleic acid fragment 2123 are similarly single-stranded DNA fragments. By hybridization with 2120, it is fixed to the particles 2110.
  • the CRISPR / Cas family protein can be immobilized on the particles 2110.
  • the order of mixing the particles 2110, the single-stranded nucleic acid fragment 2120, the sample containing the target nucleic acid fragment 140, the gRNA, the CRISPR / Cas family protein, and the single-stranded nucleic acid fragments 2121, 2122, and 2123 is not limited.
  • the single-stranded nucleic acid fragment 2120 is mixed with the sample containing the target nucleic acid fragment 140, these are hybridized, then the gRNA and the CRISPR / Cas family protein are mixed, and finally the particles 2110 are added. It may be mixed. Further, the gRNA and the CRISPR / Cas family protein may form a two-way complex in advance.
  • the sample containing the target nucleic acid fragment 140, gRNA and the CRISPR / Cas family protein may be mixed to form a tripartite complex, and then the particles 2110 to which the single-stranded DNA fragment 2120 is bound may be mixed.
  • single-stranded nucleic acid fragments 2121, 2122, and 2123 are mixed with a sample containing a long target nucleic acid, and after these are hybridized, gRNA and CRISPR / Cas family are used.
  • the proteins may be mixed, followed by the particles 2110 to which the single-stranded DNA fragment 2120 is bound.
  • single-stranded nucleic acid fragments 2120, 2121, 2122, 2123 are mixed with a sample containing a long target nucleic acid, these are hybridized, gRNA and CRISPR / Cas family proteins are mixed, and finally particles 2110 are mixed. You may. Further, the gRNA and the CRISPR / Cas family protein may form a two-way complex in advance.
  • the particles 2110 are magnetic beads, the particles can be recovered using a magnetic stand or the like. It is also easy to clean the particles.
  • the container for mixing the sample, gRNA, CRISPR / Cas family protein, etc. is not particularly limited, and for example, a plastic tube, a well array, or the like can be used.
  • the target nucleic acid fragment present at a low concentration in the sample is efficiently bound to the CRISPR / Cas family protein to form a tripartite complex. Can be formed. Subsequently, the particles may be dispensed into a tiny reaction space and contacted with the substrate nucleic acid fragment.
  • the detection sensitivity of the target nucleic acid fragment is significantly improved. That is, the CRISPR / Cas family protein is immobilized on the solid phase, so that the target nucleic acid fragment in the sample can be concentrated.
  • sample The sample is not particularly limited and may be appropriately selected depending on the intended purpose.
  • a biological sample such as saliva, blood, urine, amniotic fluid, malignant ascites, pharyngeal swab, nasal swab, or on cultured cells. Qing and the like can be mentioned.
  • Target nucleic acid fragment examples include viral genome, cfDNA, ctDNA, microRNA, exosome-derived DNA and the like.
  • target nucleic acid fragment examples include viral genome, cfDNA, ctDNA, microRNA, exosome-derived DNA and the like.
  • a nucleic acid fragment containing a hotspot region of an oncogene as a target nucleic acid fragment, it is possible to detect a mutation in the oncogene contained in the sample.
  • the CRISPR / Cas family protein is Cas12 protein
  • a double-stranded DNA fragment can be detected as a target nucleic acid fragment.
  • the CRISPR / Cas family protein is Cas13 protein
  • a single-stranded RNA fragment or a single-stranded DNA fragment can be detected as a target nucleic acid fragment.
  • the guide RNA is not particularly limited as long as it can be used for the CRISPR / Cas family protein to be used, and is limited to CRISPR RNA (crRNA) and trans-activated CRISPR RNA (tracrRNA). It may be a complex with, a single gRNA (sgRNA) which is a combination of tracrRNA and crRNA, or may be only crRNA.
  • the crRNA can be, for example, the following base sequence.
  • the base sequence obtained by removing the protospacer adjacent motif (PAM) sequence from the target base sequence is used as the spacer base sequence.
  • PAM protospacer adjacent motif
  • a base sequence in which a scaffold sequence is linked is designed at the 3'end of the spacer base sequence, and the complementary strand thereof is used as the base sequence of crRNA.
  • the base sequence obtained by removing the PAM sequence from the target base sequence is "5'-GCCAAGCGCACCTAATTTCC-3'" (SEQ ID NO: 1)
  • the base sequence of crRNA for Cas12a protein is "5'-AAUUUCUACUAAGUGUAGAUGGAAAUUAGGUGCGCUUGGC-3'”. (SEQ ID NO: 2) can be used.
  • the crRNA can be, for example, the following base sequence.
  • a base sequence in which a scaffold sequence is linked is designed at the 3'end of a base sequence complementary to the target base sequence, and the complementary strand is used as the base sequence of crRNA.
  • the target base sequence is "5'-AUGGAUUACUUGGUAGAACAGCAAUCUA-3'" (SEQ ID NO: 3)
  • the base sequence of crRNA for Cas13a protein is "5'-GAUUUAGACUACCCCAAAAACGAAGGGGACUAAAACUAGAUUGCUGUUCUACCAAGUAAUCCAU-3'" (SEQ ID NO: 4). Can be done.
  • the target base sequence is a partial base sequence of the base sequence of the target nucleic acid fragment. At least one target base sequence is set in one type of target nucleic acid fragment. By setting a plurality of types of target base sequences in one type of target nucleic acid fragment, a tripartite complex of a plurality of molecules can be formed on one molecule of the target nucleic acid fragment.
  • the number of molecules of the tripartite complex formed on one target nucleic acid fragment can be increased.
  • the present invention is a method for increasing the detection sensitivity in the above-mentioned method for detecting a target nucleic acid fragment, and by using a plurality of types of gRNA, a plurality of molecules are placed on one target nucleic acid fragment.
  • a method including a step of forming a tripartite complex of.
  • the CRISPR / Cas family protein can be used as long as it expresses nuclease activity after forming a tripartite complex with gRNA and a target nucleic acid fragment. As mentioned above, more precisely, it forms a tripartite complex and expresses nuclease activity after the CRISPR / Cas family protein cleaves the target nucleic acid fragment.
  • CRISPR / Cas family proteins examples include Cas12 protein and Cas13 protein.
  • the Cas12 protein and Cas13 protein may be Cas12 protein, Cas13 protein, orthologs of these proteins, variants of these proteins, and the like.
  • CRISPR / Cas family proteins that can be used in the method of the present embodiment, for example, Lachnospiraceae bacterium ND2006-derived Cas12a protein (LbCas12a, UniProtKB accession number: A0A182DWE3), Acidaminococcus. Derived Cas12a protein (AsCas12a, UniProtKB accession number: U2UMQ6), Francisella tularensis subsp.
  • Cas12a protein (FnCas12a, UniProtKB accession number: A0Q7Q2) derived from novicida
  • Cas12b protein (AaCas12b, UniProtKB accession number: T0D7A2) derived from Acidobacterristris (AaCas12b, UniProtKB accession number: T0D7A2), Le ),
  • Cas13a protein derived from Lachnospiraceae bacterium NK4A179 (LbaCas13a, NCBI accession number: WP_022785443.1), Leptotricia buccalis C-1013-b derived Cas13a protein (LbaCya Cas13b protein (BzoCas13b, NCBI accession number: WP_002664492), Cas13b protein from Prevotella intermedia (PinCas13b, NCBI accession number: WP_036860899), Prevotella bacteria Cas
  • Cas13b protein derived from MA2016 PsmCas13b, NCBI accession number: WP_036929175)
  • Cas13b protein derived from Riemera anatipestifer RanCas13b, NCBI accession number: WP_004919755
  • Prevotella Prevotella saccharolytica ⁇ Cas13b ⁇ (PsaCas13b ⁇ NCBI ⁇ :WP_051522484) ⁇ Prevotella intermedia ⁇ Cas13b ⁇ (Pin2Cas13b ⁇ NCBI ⁇ :WP_061868553) ⁇ Capnocytophaga canimorsus ⁇ Cas13b ⁇ (CcaCas13b ⁇ NCBI ⁇ :WP_013997271) , Porphyromonas gulae-derived Cas13b protein (PguCas13b, NCBI accession number: WP_039434803), Prevotella sp.
  • Cas13b protein derived from P5-125 (PspCas13b, NCBI accession number: WP_044065294), Cas13b protein derived from Porphyromonas gingivalis (PigCas13b, NCBI accession number: WP_0534444117), Prevotella ), Csm6 protein derived from Enteroccus italicus (EiCsm6, NCBI accession number: WP_007208953.1), Csm6 protein derived from Lactobacillus salivalius (LsCsm6, NCBI accession number: WP_081509) Accession number: WP_11229148.1) and the like can be mentioned.
  • the CRISPR / Cas family protein may be a variant of the Cas family protein described above.
  • the mutant for example, a mutant having increased nuclease activity after forming a tripartite complex can be used.
  • the substrate nucleic acid fragment is labeled with a fluorescent substance and a quenching substance, and when the fluorescent substance is cleaved by the nuclease activity of the tripartite complex and the fluorescent substance separates from the quenching substance, it emits fluorescence by irradiation with excitation light. ..
  • the substrate nucleic acid fragment may be appropriately selected according to the substrate specificity of the CRISPR / Cas family protein to be used.
  • the Cas12 protein is cleaved using single-stranded DNA as a substrate. Therefore, when Cas12 protein is used, single-stranded DNA may be used as the substrate nucleic acid fragment.
  • Cas13 protein is cleaved using single-strand RNA as a substrate. Therefore, when Cas13 protein is used, it is preferable to use single-strand RNA as a substrate nucleic acid fragment.
  • the combination of the fluorescent substance and the quenching substance a combination that can quench the fluorescence of the fluorescent substance when they are brought close to each other is used.
  • a combination that can quench the fluorescence of the fluorescent substance when they are brought close to each other is used.
  • FAM, HEX, or the like is used as the fluorescent substance
  • Iowa Black FQ (IDT), TAMRA, or the like can be used as the quenching substance.
  • the base sequence of the single-stranded RNA to be cleaved has specificity depending on the type of Cas13 protein. Specifically, for example, it has been reported that the LwaCas13a protein, CcaCas13b protein, LbaCas13a protein, and PsmCas13b protein recognize and cleave the base sequences of AU, UC, AC, and GA in the substrate nucleic acid fragment, respectively.
  • LwaCas13a protein, CcaCas13b protein, LbaCas13a protein, and PsmCas13b protein are used as CRISPR / Cas family proteins, and different gRNAs are bound to each CRISPR / Cas family protein as gRNA to form a substrate.
  • a single-stranded RNA containing the base sequences of AU, UC, AC, and GA as the nucleic acid fragment
  • four types are used in one reaction space.
  • Target nucleic acid fragments can be detected. That is, multicolor detection can be performed.
  • reaction space As a method for accurately detecting a target substance such as a target nucleic acid fragment, a technique for performing an enzymatic reaction in a large number of minute reaction spaces is being studied. These methods are called digital measurements. In digital measurement, a sample is divided into a large number of minute reaction spaces to detect signals.
  • the signal from each reaction space is binarized, only the presence or absence of the target substance is determined, and the number of molecules of the target substance is measured. According to the digital measurement, the detection sensitivity and the quantitativeness can be remarkably improved as compared with the conventional ELISA, the real-time PCR method and the like.
  • the method of this embodiment is preferably performed by digital measurement. More specifically, the contact of the sample, the CRISPR / Cas family protein, the gRNA, and the substrate nucleic acid fragment is divided into minute reaction spaces.
  • the volume per reaction space is 10aL to 100pL, for example, 10aL to 10pL, for example, 10aL to 1pL, for example, 10aL to 100fL, and for example, 10aL to 10fL. You may.
  • the reaction space is within the above range, it is possible to detect the presence of the target nucleic acid fragment with high sensitivity without amplifying it.
  • Digital measurement can be performed by performing the method of the present embodiment under the condition that 0 or 1 target nucleic acid fragment is introduced into one reaction space. That is, the number of reaction spaces in which the signal is detected can be made to correspond to the number of molecules of the target nucleic acid fragment in the sample.
  • the reaction space may be, for example, a droplet.
  • the reaction space may be a well formed on the substrate.
  • FIG. 2A is a top view showing an example of a fluid device having a substrate having a well having a volume of 10 aL to 100 pL per well formed on the surface thereof.
  • 2 (b) is a cross-sectional view taken along the line b-b'of FIG. 2 (a).
  • the fluid device 200 has a substrate 210 having a well 211 having a volume of 10aL to 100pL formed on its surface, a spacer 220, and a lid having a liquid inlet 231 formed therein. It has a member 230. There are a plurality of wells 211, forming a well array 212. The space between the substrate 210 and the lid member 230 functions as a flow path for the sample, gRNA, CRISPR / Cas family protein, substrate nucleic acid fragment, and the like.
  • the shape of the well is not particularly limited as long as the volume is within the above-mentioned range, and the well may be, for example, a cylinder, a polyhedron composed of a plurality of faces (for example, a rectangular parallelepiped, a hexagonal column, an octagonal column, etc.). You may.
  • a plurality of wells 211 having the same shape and size form the well array 212.
  • the same shape and the same size may be as long as they have the same shape and the same capacity as required for digital measurement, and variations of the degree of manufacturing error are acceptable.
  • Detection method 3 (a) to 3 (c) are schematic cross-sectional views illustrating an example of a procedure for carrying out the method of the present embodiment using the fluid device 200.
  • the sample, gRNA, and CRISPR / Cas family protein are mixed to form a tripartite complex.
  • the assay solution 310 is prepared by mixing the tripartite complex and the substrate nucleic acid fragment, and immediately introduced from the liquid inlet 231 of the fluid device 200.
  • the space inside the well 211 and between the substrate 210 and the lid member 230 is filled with the assay solution 310.
  • the sealant 320 is introduced from the liquid introduction port 231.
  • an organic solvent containing a lipid 321 is used as the encapsulant 320.
  • the lipid 321 natural lipids derived from soybeans, Escherichia coli and the like, artificial lipids such as dioleoylphosphatidylethanolamine (DOPE) and dioleoylphosphatidylglycerol (DOPG) can be used. Hexadecane or chloroform can be used as the organic solvent in this case.
  • DOPE dioleoylphosphatidylethanolamine
  • DOPG dioleoylphosphatidylglycerol
  • Hexadecane or chloroform can be used as the organic solvent in this case.
  • each well 211 becomes an independent reaction space.
  • the well array 212 may be irradiated with excitation light to measure the fluorescence.
  • a lipid membrane can be further laminated on the first lipid membrane 322 to form a lipid bilayer membrane.
  • a membrane-forming aqueous solution 330 for forming the lipid bilayer membrane 324 is introduced from the liquid inlet 231.
  • a 10 mM pH buffer solution (pH 5 to 9) for example, a 10 mM sodium chloride aqueous solution, or the like can be used.
  • the membrane-forming aqueous solution 330 is introduced, the second lipid membrane 323 is laminated on the first lipid membrane 322 to form the lipid bilayer membrane 324.
  • each well 211 becomes an independent reaction space.
  • the well array 212 may be irradiated with excitation light to measure the fluorescence.
  • the lipid vesicles can be fused to the lipid bilayer membrane 324.
  • the contents of the exosome can be released into the well 211.
  • gRNA, CRISPR / Cas family protein, and substrate nucleic acid fragment are first sealed in well 211, and the opening of well 211 is sealed with a lipid bilayer membrane 324, and then exosomes are used as a sample. It may be brought into contact with the bilayer membrane 324.
  • the exosome is fused to the lipid bilayer membrane 324, and the contents of the exosome are released into the well 211.
  • the target nucleic acid fragment is present in the contents of the exosome, a tripartite complex is formed inside the well 211, the substrate nucleic acid fragment is cleaved, and fluorescence is detected by irradiation with excitation light.
  • the CRISPR / Cas family protein is immobilized on the solid phase.
  • the CRISPR / Cas family protein may be immobilized on the inner surface of the well.
  • the CRISPR / Cas family protein 110 may be pre-immobilized on the inner surface of the well 211 in the method of the present embodiment.
  • the CRISPR / Cas family protein 110 may bind to the gRNA 120 to form a bipartite complex 130.
  • the possibility that the tripartite complex 100 exists inside the well 211 when the samples are brought into contact with each other can be improved, and the detection sensitivity can be dramatically improved.
  • a functional group present on the inner surface of the well 211 and a CRISPR / Cas family protein present on the surface of the well 211 using physical adsorption or a chemical linker examples thereof include a method of covalently bonding with a functional group to be used, a method of utilizing an avidin-biotin bond, and the like.
  • the functional group when a chemical linker is used include a hydroxyl group, an amino group, a thiol group and the like.
  • the CRISPR / Cas family protein may be immobilized on the inner surface of the well 211 by utilizing a click reaction using an azide group and an alkyne group.
  • the inner surface of the well 211 is biotinylated in advance, and the avidin is brought into contact with the biotinylated CRISPR / Cas family protein using a chemical linker to bring the inner surface of the well 211 into contact.
  • CRISPR / Cas family proteins can be bound to.
  • the gRNA 120 may be fixed to the inner surface of the well 211.
  • an additional sequence that functions as a linker may be added to the gRNA 120.
  • wells with immobilized gRNA are easier to store than wells with immobilized protein.
  • the step (a) may be performed in each well of the well array.
  • the reaction space for contacting the sample, gRNA, CRISPR / Cas family protein and substrate nucleic acid fragment may be the space inside each of the wells.
  • the CRISPR / Cas family protein may be immobilized on the inner surface of each of the above wells.
  • the step (a) may have the following steps (a1) and (a2).
  • step (a1) when the CRISPR / Cas family protein has previously formed a two-way complex with the gRNA, a sample and a substrate nucleic acid fragment are introduced into each well of the well array. If the CRISPR / Cas family protein has not previously formed a two-way complex with gRNA, a sample, gRNA and substrate nucleic acid fragment are introduced into each well of the well array.
  • each well of the well array is sealed with a sealing liquid.
  • the sealing liquid will be described later.
  • the fluorescent substance is irradiated with the excitation light.
  • fluorescence when fluorescence is detected, it can be determined that the target nucleic acid fragment was present in the sample.
  • the step (a) may be performed in each well of the well array.
  • Each well may then have a first well and a second well located at the bottom of the first well and having a smaller volume than the first well.
  • the reaction space for contacting the sample, gRNA, CRISPR / Cas family protein and substrate nucleic acid fragment may be the space inside the second well.
  • the CRISPR / Cas family protein may be immobilized on the inner surface of the second well.
  • the step (a) may have the following steps (a1'), (a2'), (a3').
  • step (a1') when the CRISPR / Cas family protein has previously formed a two-way complex with gRNA, a sample and a substrate nucleic acid fragment are introduced into each well of the well array. If the CRISPR / Cas family protein has not previously formed a two-way complex with gRNA, a sample, gRNA and substrate nucleic acid fragment are introduced into each well of the well array.
  • each well of the well array is sealed with a sealing liquid.
  • the sealing liquid will be described later.
  • the sealing liquid is replaced with a water-absorbent organic solvent.
  • the water-absorbent organic solvent will be described later.
  • the fluorescent substance is irradiated with the excitation light.
  • fluorescence when fluorescence is detected, it can be determined that the target nucleic acid fragment was present in the sample.
  • each well in the well array has a first well and a second well located at the bottom of the first well and having a smaller volume than the first well. As the volume of the contents of the well becomes smaller, the contents may be accumulated in the second well.
  • FIG. 5 (a) and 5 (b) are schematic views illustrating an example of a well array having a first well and a second well.
  • 5 (a) is a top view
  • FIG. 5 (b) is a cross-sectional view taken along the line b-b'of FIG. 5 (a).
  • the well array 500 is formed on one surface of the substrate 510.
  • Each well of the well array 500 has a first well 520 and a second well 530 located at the bottom of the well 520 and having a smaller capacity than the well 520. As will be described later, as the volume of the contents of the first well 520 becomes smaller, the contents accumulate in the second well 530.
  • the target substance can be detected with high sensitivity, and the time required for detection is shortened.
  • the volume ratio of the first well 520 and the second well 530 (volume of well 520: volume of well 530) may be 10: 1 to 1,000,000: 1.
  • the volume of the first well 520 may be 1 to 1,000 pL, and the volume of the second well 530 may be 0.1 to 1,000 fL.
  • FIG. 6 (a) and 6 (b) are schematic views illustrating another example of a well array having a first well and a second well.
  • 6 (a) is a top view
  • FIG. 6 (b) is a cross-sectional view taken along the line bb'of FIG. 6 (a).
  • the well array 600 is formed on one surface of the substrate 510.
  • a plurality of second wells 530 may be arranged per first well 520. Also in this case, when the volume of the contents of the first well 520 becomes smaller, the contents are accumulated in the second well 530.
  • the shapes of the first well 520 and the second well 530 are not particularly limited, and may be, for example, a cylinder, a polyhedron composed of a plurality of faces (for example, a rectangular parallelepiped, a hexagonal column, an octagonal column, etc.). good.
  • the plurality of first wells 520 have the same shape and size
  • the plurality of second wells 530 have the same shape and size.
  • the same shape and the same size may be used as long as they have the same shape and the same capacity to the extent required for digital measurement, and variations of the degree of manufacturing error are allowed.
  • FIGS. 8 (a) and 8 (b) are schematic cross-sectional views illustrating each step of manufacturing the well array 600.
  • the film 700 is laminated on the surface of the substrate 510.
  • Examples of the material of the substrate 510 include glass, resin, and the like.
  • Examples of the resin include polyethylene, polypropylene, polystyrene, polycarbonate, cyclic polyolefin, acrylic and the like.
  • polycarbonate is also used as a material for CDs and DVDs that can be mass-produced at low cost, and is suitable from the viewpoint of manufacturing well arrays at low cost.
  • the inventors have clarified that the use of polycarbonate as the material of the substrate 510 is preferable for detecting fluorescence with a microscope because the refractive index of light is close to that of glass.
  • Examples of the material of the film 700 include fluororesin, cyclic polyolefin, silicone resin and the like.
  • the resist film 710 is laminated on the surface of the film 700. Subsequently, the resist film 710 is exposed by irradiating the resist film 710 with active energy rays using the mask of the well array pattern. Subsequently, it is developed with a developing solution, and as shown in FIG. 4D, the resist film 710 at the portion forming the well is removed.
  • the film 700 masked with the resist film 710 is etched to form a second well 530 in the film 700.
  • the resist film 710 is removed by washing the substrate to obtain an array of wells 530.
  • a well array of second wells is obtained. That is, the array of minute wells produced in the steps up to this point can also be used to detect the target nucleic acid fragment.
  • the well array of the first well is laminated on the well array of the second well by the following steps.
  • As the resist film 710 a sheet-type resist can be preferably used.
  • FIG. 8 (b) the resist film 710 is exposed by irradiating the resist film 710 with active energy rays using a mask with a well array pattern. Subsequently, it is developed with a developing solution to remove the resist film 710 of the portion forming the first well 520. The result is a well array 600 with a first well 520 and a second well 530.
  • FIG. 9 is a photomicrograph of a well array having a first well and a second well actually manufactured by the inventors.
  • Digital measurement can be performed by introducing 0 or 1 target nucleic acid fragment per reaction space. That is, the number of wells in which fluorescence is detected can be made to correspond to the number of molecules of the target nucleic acid fragment in the sample.
  • the encapsulant is preferably immiscible with water.
  • Immiscible with water means that water and the encapsulant are sufficiently mixed and then allowed to stand to separate into an aqueous phase and an organic phase.
  • the sealing liquid preferably has low water absorption. Low water absorption means that the volume change of the organic layer is 1% or less when it is mixed with water of equal volume and allowed to stand at 20 ° C. and separated into an aqueous phase and an organic phase.
  • the sealing liquid a substance having a boiling point of about 100 ° C. or higher and liquid at room temperature can be used.
  • Specific encapsulants include fluorine such as FC-40, FC-43, FC-770, FC-72, FC-3283 (all manufactured by 3M), and von Bryn (registered trademark) oil (Solvey). Examples thereof include system liquids, mineral oils (Sigmar Aldrich Co., Ltd.), straight-chain or branched-chain saturated or unsaturated hydrocarbons having 7 to 17 carbon atoms. These may be used alone or in combination of two or more.
  • Linear or branched saturated or unsaturated hydrocarbons having 7 to 17 carbon atoms include heptane (C 7 H 16 ), octane (C 8 H 18 ), nonan (C 9 H 20 ), and decan (C 9 H 20).
  • examples of the isomer of octane include 1-octane, 2-methylheptane, 3-methylheptane, 2,2-dimethylhexane, 2,3-dimethylhexane, 2,3,3-trimethylpentane and the like.
  • examples of the isomers of octene include 1-octene, 2-methyl-1-heptene, 2,3-dimethyl-1-hexene, 2-ethyl-1-hexene, and 2,3,3-trimethyl-1. -Butene etc. can be mentioned.
  • Water-absorbent organic solvent As the water-absorbent organic solvent, a water-absorbent organic solvent having a boiling point of about 100 ° C. or higher, liquid at room temperature, and immiscible with water can be used, and is linear or branched with 4 to 11 carbon atoms. Examples include chain saturated or unsaturated aliphatic alcohols. Immiscible with water means that water and an organic solvent are sufficiently mixed and then allowed to stand to separate into an aqueous phase and an organic phase. Further, water absorption means that water is dissolved.
  • the water-absorbent organic solvent may be a monohydric alcohol or a divalent or higher alcohol.
  • Specific water-absorbing organic solvents include butanol (C 4 H 10 O), pentanol (C 5 H 12 O), hexanol (C 6 H 14 O), heptanol (C 7 H 16 O), and octanol. (C 8 H 18 O), nonanol (C 9 H 20 O), decanol (C 10 H 22 O), undecanol (C 11 H 24 O), pentanediol (C 5 H 12 O 2 ) and the like can be mentioned. These may be any isomer. In addition, these may be used alone or in combination of two or more.
  • examples of the isomer of octanol include 1-octanol, isooctyl alcohol, 2-ethylhexanol and the like.
  • examples of the isomer of pentanediol include 1,5-pentanediol, 1,2-pentanediol, 2,3-pentanediol and the like.
  • FIG. 10 (a) shows a well array having a first well and a plurality of wells arranged at the bottom of the first well and having a second well having a smaller capacity than the first well on the surface. It has a substrate, a lid member arranged to face the well array, and a spacer for separating the substrate and the lid member, and the space between the well array and the lid member is a flow through which a fluid flows. It is a top view which shows an example of the fluid device forming a path.
  • 10 (b) is a cross-sectional view taken along the line bb'of FIG. 10 (a).
  • the fluid device 1000 is located at the bottom of the first well 520 and the first well 520 and has a smaller capacity than the first well 520. It has a substrate 510 on which a well array 500 having a plurality of wells having wells 530 is arranged on the surface, a spacer 1010, and a lid member 1020 forming a liquid inlet 1021.
  • the space 1030 between the substrate 510 and the lid member 1020 functions as a flow path for flowing a sample, a detection reagent, a sealing liquid, a water-absorbent organic solvent, and the like.
  • FIGS. 11 (a) to 11 (c) are schematic cross-sectional views illustrating an example of a procedure for carrying out a method for detecting a target nucleic acid fragment using the fluid device 200 shown in FIG.
  • RNA fragment a single-strand RNA fragment (tgRNA) is detected as a target nucleic acid fragment.
  • Cas13a protein is used as the CRISPR / Cas family protein.
  • crRNA is used as gRNA.
  • the two-way complex 130 of Cas13a protein and crRNA is immobilized on the inner surface of each well 211 of the well array 212. As shown in FIG. 11A, the two-way complex 130 is introduced from the liquid inlet 231 of the fluid device 200.
  • the space inside the well 211 and between the substrate 210 and the lid member 230 is filled with the two-way complex 130 (Cas13a-crRNA).
  • the Cas13a protein is biotinylated.
  • the inner surface of the well 211 (here, only the bottom surface) is biotinylated, and avidin is further bound. Therefore, as shown in FIG. 11B, the two-way complex introduced inside the well 211 is fixed to the bottom surface of the well 211.
  • the sample and the substrate nucleic acid fragment 150 are introduced from the liquid inlet 231 of the fluid device 200.
  • the target nucleic acid fragment 140 (tgRNA) in the sample is introduced into the well 211.
  • the target nucleic acid fragment 140 in the sample binds to the two-way complex to form the three-way complex 100'(Cas13a-crRNA-tgRNA).
  • each well of the well array is sealed with a sealing liquid.
  • the sealant 320 is introduced from the liquid introduction port 231.
  • the opening of the well 211 is sealed with the sealant 320 with the inside of the well 211 filled with the tripartite complex 100'and the substrate nucleic acid fragment 150.
  • each well forms an independent reaction space.
  • the substrate nucleic acid fragment 150 is cleaved by the nuclease activity of the tripartite complex 100', and the fluorescent substance F separates from the quenching substance Q.
  • the fluorescent substance F separates from the quenching substance Q.
  • the efficiency of forming the tripartite complex is significantly improved as compared with the case where Cas13a is not immobilized on the solid phase. That is, the detection sensitivity of the target nucleic acid fragment is significantly improved.
  • each well of the well array has a first well 520 and a second well 530.
  • the main difference is that the assay solution is concentrated using a water-absorbent organic solvent.
  • FIGS. 12 (a) to 12 (d) are schematic cross-sectional views illustrating an example of a procedure for carrying out a method for detecting a target nucleic acid fragment using the fluid device 1000 shown in FIG.
  • a single-strand RNA fragment tgRNA
  • Cas13a protein is used as the CRISPR / Cas family protein.
  • crRNA is used as gRNA.
  • the two-way complex 130 of Cas13a protein and crRNA is immobilized on the inner surface (here, only the bottom surface) of the second well 530.
  • the assay solution 310 containing the sample and the substrate nucleic acid fragment 150 is prepared. It is introduced from the liquid introduction port 1021 of the fluid device 1000. As a result, as shown in FIG. 12 (a), the inside of the first well 520, the inside of the second well 530, and the space 1030 between the substrate 510 and the lid member 1020 are filled with the assay solution 310.
  • the assay solution 310'containing the sample, crRNA and the substrate nucleic acid fragment 150 may be introduced from the liquid inlet 1021 of the fluid device 1000.
  • each well of the well array is sealed with a sealant 320 (not shown).
  • the sealant 320 is introduced from the liquid introduction port 1021.
  • the opening of the well 520 is sealed with the sealant 320 with the assay solution 310 filled inside the first well 520.
  • each well forms an independent reaction space.
  • the encapsulant 320 is replaced with the water-absorbent organic solvent 1230.
  • the contents of the well are dehydrated, the volume is reduced (concentrated), and a tripartite complex 100'of Cas13a-crRNA-tgRNA is formed in the assay solution 310 to form a substrate nucleic acid. Cut the fragment 150.
  • the fluorescent substance F bound to the substrate nucleic acid fragment 150 separates from the quenching substance Q and becomes fluorescent when irradiated with the excitation light. Fluorescence detected in a well indicates the presence of a target substance in that well.
  • the substrate nucleic acid fragment 150 is not cleaved and fluorescence does not occur because the tripartite complex 100'is not formed.
  • the water-absorbent organic solvent 1230 may be replaced with the sealant 320 again. This allows the dehydration of the well contents (assay solution 310) to be stopped. That is, the detection method of the present embodiment may further include a step of replacing the water-absorbent organic solvent 1230 with the encapsulant 320.
  • the probability of capturing the target nucleic acid fragment 140 is improved, and the detection is performed in the small volume second well 530.
  • the target nucleic acid fragment 140 can be detected with high sensitivity, and the time required for detection is shortened.
  • the present invention comprises a substrate with wells having a volume of 10aL to 100pL formed on the surface, a gRNA complementary to a target nucleic acid fragment, a CRISPR / Cas family protein immobilized on a solid phase, and a substrate.
  • a kit for detecting a target nucleic acid fragment including a nucleic acid fragment is provided.
  • the CRISPR / Cas family protein expresses nuclease activity after forming a tripartite complex with the gRNA and the target nucleic acid fragment, and the substrate nucleic acid fragment is a fluorescent substance and quenching. It is labeled with a substance, and when it is cleaved by the nuclease activity of the tripartite complex and the fluorescent substance separates from the quenching substance, it emits fluorescence by irradiation with excitation light.
  • the above-mentioned method for detecting a target nucleic acid fragment can be suitably carried out.
  • the substrate on which the well is formed on the surface, the target nucleic acid fragment, the gRNA, the CRISPR / Cas family protein, and the substrate nucleic acid fragment are the same as those described above.
  • the CRISPR / Cas family protein may be immobilized on the inner surface of the well.
  • the well has a first well and a second well located at the bottom of the first well and having a smaller volume than the first well, and the CRISPR / Cas family protein is the first. It may be fixed to the inner surface of the two wells.
  • the method of binding the CRISPR / Cas family protein to the inner surface of the well is the same as that described above. Further, the same applies to the well having the first well and the second well.
  • the CRISPR / Cas family protein may be immobilized on the surface of the particles.
  • the method of binding the CRISPR / Cas family protein to the particles and the surface of the particles is the same as described above.
  • the CRISPR / Cas family protein may be a Cas12 protein or a Cas13 protein.
  • the specific Cas12 protein or Cas13 protein is the same as described above.
  • the target nucleic acid fragment is a single-stranded RNA fragment chemically synthesized by outsourcing (IDT) (SEQ ID NO: 5) or a full-length RNA of the N gene of SARS-CoV2 synthesized by in vitro transcription (IVT) (SEQ ID NO: 9). It was used.
  • gRNA DNA fragment encoding gRNA (crRNA) was prepared by PCR amplification using an overlapping primer containing a T7 promoter sequence, a 20-base target sequence, and a scaffold sequence as a template. Subsequently, the obtained DNA fragment was subjected to an in vitro transcription reaction to prepare crRNA.
  • the nucleotide sequences of the crRNA used are shown in SEQ ID NO: 4 and SEQ ID NO: 8.
  • Substrate nucleic acid fragments (single-stranded RNA fragments) were chemically synthesized by outsourcing (IDT).
  • the 5'end of the substrate nucleic acid fragment was labeled with FAM, a fluorescent material, and the 3'end was labeled with Iowa Black FQ (IDT), a quencher.
  • the base sequence of the chemically synthesized substrate nucleic acid fragment (single-stranded RNA fragment) was "5'-(FAM) UUUUU (IABkFQ) -3'" (where "IABkFQ" indicates Iowa Black FQ). ..
  • the well array A was produced by the same procedure as in FIGS. 7 (a) to 7 (f) described above. First, as shown in FIG. 7A, the glass substrate 510 was immersed in an 8M potassium hydroxide solution for about 24 hours to form a hydroxyl group on the surface.
  • a fluororesin (CYTOP, manufactured by AGC Co., Ltd.) was spin-coated on the surface of the glass substrate 510 to form a film 700.
  • the spin coating conditions were set to 1,000 rpm (revolutions per minute) for 30 seconds. Under this condition, the film thickness of the film 700 is about 1.8 ⁇ m.
  • the silanol group of the film 700 (CYTOP) and the hydroxyl group on the glass surface were dehydrated and condensed by baking on a hot plate at 180 ° C. for 1 hour, so that the film 700 was brought into close contact with the surface of the glass substrate 510.
  • a resist product name "AZ-P4903", manufactured by AZ Electrical Materials
  • AZ-P4903 manufactured by AZ Electrical Materials
  • the glass substrate 710 was baked on a hot plate at 110 ° C. for 1 hour to evaporate the organic solvent in the resist film 710, whereby the resist film 710 was brought into close contact with the surface of the film 700.
  • the resist film 710 was exposed to the resist film 710 by irradiating the resist film 710 with ultraviolet rays at 250 W for 14 seconds with an exposure machine (manufactured by Union Optical Co., Ltd.) using a mask with a well array pattern. Subsequently, it was immersed in a developing solution (AZ developer, manufactured by AZ Electrical Materials) for 1.5 minutes for development. As a result, the resist film 710 at the portion forming the well was removed.
  • AZ developer manufactured by AZ Electrical Materials
  • the film 700 masked with the resist film 710 is subjected to 30 under the conditions of O 2 200 sccm, pressure 5 Pa, and output 50 W using a Reactive ion etching apparatus (manufactured by YAC).
  • Wells 530 were formed on the film 700 by dry etching for a minute.
  • the resist film 710 was removed by immersing the glass substrate 510 in acetone, washing with isopropanol, and then washing with pure water to remove the resist film 710, and the array of wells 530 (well array A).
  • the well array A had a shape in which 1,500,000 columnar wells 530 having a diameter of 3.5 ⁇ m and a depth of 1.8 ⁇ m were arranged in 1 cm 2 .
  • Well 530 The volume per well was 17 fL.
  • a well array B having a first well and a second well was prepared by the same procedure as in FIGS. 7 (a) to 7 (f), 8 (a) and 8 (b).
  • the well array obtained in the same manner as in FIGS. 7 (a) to 7 (f) is dry-etched (O 2 13 sccm, pressure 14 Pa, output 125 W) for 5 seconds using a reactive ion etching apparatus (manufactured by Samco). Therefore, the surface of the well array was hydrolyzed.
  • a sheet-type resist product name "SU-8 3020CF DFR Type-S", KAYAKU Advanced Materials, Inc.
  • a laminating roller As shown in FIG. 8A, a resist film 710 was formed.
  • the resist film 710 was exposed by irradiating ultraviolet rays for 20 seconds with an exposure machine (manufactured by Union Optical Co., Ltd.) using a mask with a well array pattern. Subsequently, it was immersed in a developing solution (product name "SU8 developer", KAYAKU Advanced Materials, Inc.) for 8 minutes for development. As a result, the resist film 710 at the portion forming the well was removed, and a well array B having a first well 520 and a second well 530 was obtained.
  • a developing solution product name "SU8 developer", KAYAKU Advanced Materials, Inc.
  • the well array B consists of a well array in which 1,500,000 columnar wells 530 having a diameter of 3.5 ⁇ m and a depth of 1.8 ⁇ m are arranged in 1 cm 2 and a columnar well 520 having a diameter of 40 ⁇ m and a depth of 20 ⁇ m. It had a shape in which 40,000 well arrays were stacked in 1 cm 2 .
  • the well array B had a shape in which 12 to 18 wells 530 were arranged at the bottom of each well 520.
  • FIG. 9 is a photomicrograph of the prepared well array B.
  • an assay solution in which the above-mentioned tripartite complex solution and a solution of the substrate nucleic acid fragment were mixed was prepared, and immediately introduced from the liquid inlet of each fluid device A. As a result, the assay solution was introduced into each well of the well array.
  • FIG. 13 (a) is a representative fluorescence micrograph showing the result of the assay solution in which the final concentration of the target nucleic acid fragment is 0 pM.
  • FIG. 13 (b) is a representative fluorescence micrograph showing the results of an assay solution in which the final concentration of the target nucleic acid fragment is 0.3 pM.
  • FIG. 13 (c) is a representative fluorescence micrograph showing the results of an assay solution in which the final concentration of the target nucleic acid fragment is 3 pM.
  • FIG. 13 (d) is a representative fluorescence micrograph showing the result of the assay solution in which the final concentration of the target nucleic acid fragment is 30 pM.
  • the scale bar is 50 ⁇ m.
  • FIG. 14 is a graph showing the relationship between the number of wells in which fluorescence was detected and the final concentration of the target nucleic acid fragment.
  • the vertical axis shows the number of wells in which fluorescence was detected, and the horizontal axis shows the final concentration of the target nucleic acid fragment.
  • a sealant (hexadecane, Sigma-Aldrich) was introduced from the liquid inlet of the fluid device B.
  • a sealant hexadecane, Sigma-Aldrich
  • FIG. 15 is a representative graph showing the percentage of wells showing a predetermined fluorescence intensity (relative value) based on a photograph of a well array in which fluorescence of sTG was detected.
  • the horizontal axis of the graph shows the concentration of alkaline phosphatase (ALP).
  • Biotinogenesis was performed as follows. First, a thiol group was modified on the glass surface of the bottom surface of the well 530 of the fluid device A by a silane coupling treatment using (3-mercaptopropyl) -trimethoxysilane. Subsequently, biotin was bound to the above thiol group by a maleimide reaction using biotin-dPEG 11 -maleimide. By the above operation, the inner surface of the well 530 of the fluid device A was biotinylated.
  • avidin was suspended in buffer A having the composition shown in Table 1 above so that the final concentration was 1 mg / mL, and introduced from the liquid inlet of the fluid device A. As a result, avidin was introduced into each well of the well array and bound to biotin bound to the inner surface of well 530.
  • the lysine residue of Cas13a protein or the N-terminal of Cas13a protein was modified with NHS- PEG4 -biotin to biotinylated.
  • the cysteine residue of the biotinylated Cas13a protein was labeled with Alexa488-maleimide.
  • the biotinylated Cas13a protein was introduced from the liquid inlet of the fluid device A.
  • the biotinylated Cas13a protein was introduced into each well of the well array, bound to avidin bound to the inner surface of well 530, and immobilized.
  • Alexa647 was dissolved in water, introduced from the liquid inlet of the fluid device A, and observed with a fluorescence microscope.
  • FIG. 16 is a fluorescence micrograph of the fluid device A.
  • the upper part of FIG. 16 is a photograph of the fluid device A taken from the upper surface
  • the lower part of FIG. 16 is a cross-sectional photograph of the fluid device A in the dotted line portion on FIG.
  • the lower photograph of FIG. 16 is upside down, and the upper side of the photograph is the bottom surface of the fluid device A.
  • the fluorescence of Alexa488 indicates the location of the Cas13a protein.
  • the fluorescence of Alexa647 indicates the location of the water introduced into the well. As a result, it was clarified that the Cas13a protein can be immobilized only on the inner surface of the well 530.
  • the final concentration of the substrate nucleic acid fragment is 10 ⁇ M
  • the final concentration of the target nucleic acid fragment (SEQ ID NO: 5) is 3 pM, 0.3 pM, 30 fM, and 3 fM, respectively.
  • a solution dissolved in buffer A having the composition shown in the above was prepared and used as an assay solution.
  • each assay solution was introduced from the liquid inlet of each fluid device A.
  • the assay solution was introduced into each well of the well array.
  • FIG. 17 is a graph showing the relationship between the number of wells in which fluorescence was detected and the final concentration of the target nucleic acid fragment (tgRNA).
  • the vertical axis shows the number of wells in which fluorescence was detected, and the horizontal axis shows the final concentration of the target nucleic acid fragment.
  • a substrate nucleic acid fragment was added to the buffer A so that the final concentration was 10 ⁇ M
  • a target nucleic acid fragment was added so that the final concentration was 30 pM
  • an assay solution was further added with impurities.
  • Contaminants include 10v / v% phosphate buffered saline (PBS), 70v / v% virus transport solution (VTM, catalog number "SGVTM-3R", Sugiyamagen Co., Ltd.), final concentration based on the assay solution.
  • PBS phosphate buffered saline
  • VTM virus transport solution
  • a 3 ng / ⁇ L non-target nucleic acid fragment, 10 v / v% saliva was used. Since saliva contains RNase, 1 mM of the surfactant Triton X-100 was added in advance, and then the mixture was heated at 90 ° C. for 5 minutes to inactivate RNase. Also, for comparison, an assay solution containing no impurities was prepared.
  • each assay solution was introduced from the liquid inlet of each fluid device A.
  • the assay solution was introduced into each well of the well array.
  • FIG. 18 is a graph showing the number of wells in which fluorescence was detected when each assay solution was used. The vertical axis shows the number of wells in which fluorescence was detected.
  • "w / o” is the result of the assay solution without impurities
  • "w / PBS” is the result of the assay solution with PBS added
  • "w / VTM” is the virus transport solution.
  • "W / ntgRNAs” is the result of the assay solution to which the non-target nucleic acid fragment was added
  • w / saliva is the result of the assay solution to which saliva was added.
  • an assay in which a substrate nucleic acid fragment is added to the buffer A so that the final concentration is 10 ⁇ M, and a target nucleic acid fragment (tgRNA) is added so that the final concentration is 30 pM, 3 pM, 300 fM, 80 fM, 30 fM, and 8 fM.
  • tgRNA target nucleic acid fragment
  • a substrate nucleic acid fragment was added to saliva so that the final concentration was 5 ⁇ M, and a target nucleic acid fragment (tgRNA) was added so that the final concentration was 30 pM, 3 pM, 300 fM, and 30 fM to prepare an assay solution. Since saliva contains RNase, 1 mM of the surfactant Triton X-100 was added in advance, and then the mixture was heated at 90 ° C. for 5 minutes to inactivate RNase.
  • tgRNA target nucleic acid fragment
  • each assay solution was introduced from the liquid inlet of each fluid device A.
  • the assay solution was introduced into each well of the well array.
  • FIG. 19 is a graph showing the relationship between the number of wells in which fluorescence was detected and the final concentration of the target nucleic acid fragment (tgRNA).
  • the vertical axis shows the number of wells in which fluorescence was detected, and the horizontal axis shows the final concentration of the target nucleic acid fragment.
  • Cas13a protein and gRNA are mixed with buffer A having the composition shown in Table 1 above so that the final concentration of Cas13a protein is 40 nM and the final concentration of gRNA is 25 nM. Formed.
  • a substrate nucleic acid fragment was added to the buffer A so that the final concentration was 10 ⁇ M, and a new coronavirus was added so that the final concentration was 3 pM, 0.3 pM, 0.03 pM, 0.003 pM, and 0 pM.
  • the assay solution was prepared.
  • each assay solution was introduced from the liquid inlet of each fluid device A.
  • the assay solution was introduced into each well of the well array.
  • FIG. 20 is a graph showing the relationship between the number of wells in which fluorescence was detected and the final concentration of the new coronavirus.
  • the vertical axis shows the number of wells in which fluorescence was detected, and the horizontal axis shows the final concentration of the new coronavirus.
  • a single-stranded DNA fragment having a biotin-modified 3'end (base sequence shown in SEQ ID NO: 7) and a target nucleic acid fragment (SEQ ID NO: 5) were mixed and incubated at 70 ° C. for hybridization.
  • the single-stranded DNA fragment (SEQ ID NO: 7) had a base sequence complementary to a part of the target nucleic acid fragment (SEQ ID NO: 5).
  • streptavidin-coated magnetic beads product name "Dynabeads M280", Veritas
  • a solution of the hybridized single-stranded DNA fragment (SEQ ID NO: 7) and the target nucleic acid fragment (SEQ ID NO: 5). And a mixed solution was obtained.
  • buffer B (20 mM HEPES (pH 7.5), 20 mM KCl, 2 mM so that the final concentration of the single-stranded DNA fragment (SEQ ID NO: 7) is 12 nM and the final concentration of the magnetic beads is 1 mg / mL. It was mixed in MgCl 2 , 50 ⁇ M Triton X-100). Further, four kinds of mixed solutions were prepared so that the final concentrations of the target nucleic acid fragment (SEQ ID NO: 5) were 0.3 pM, 30 fM, 3 fM, and 0.8 fM, respectively.
  • buffer B is dispensed into four new plastic tubes, and Cas13a protein, gRNA (SEQ ID NO: 4), substrate nucleic acid fragment and each of the above mixtures are mixed therein, and four kinds of assay solutions are prepared. Obtained. The final concentration of the substrate nucleic acid fragment was adjusted to 5 ⁇ M.
  • a well array C in which 2,800,000 columnar wells having a diameter of 4.0 ⁇ m and a depth of 3.0 ⁇ m were arranged per 1 cm 2 was prepared.
  • the volume of the well array C per well was 50 fL.
  • the fluid device C was produced in the same manner as the above-mentioned fluid device A except that the well array C was used instead of the well array A.
  • each of the above assay solutions was introduced from the liquid inlet of each fluid device C and placed on a magnet sheet.
  • each assay solution was introduced into each well of the well array.
  • magnetic beads bound to the tripartite complex containing the target nucleic acid fragment were concentrated and captured in each well.
  • each fluid device C was sealed with a sealant, and each well became an independent reaction space. After a few minutes, a well array of each fluid device C was observed under a fluorescence microscope.
  • FIG. 23 is a graph showing the relationship between the number of wells in which fluorescence was detected and the final concentration of the target nucleic acid fragment (tgRNA).
  • the vertical axis shows the number of wells in which fluorescence was detected, and the horizontal axis shows the final concentration of the target nucleic acid fragment.
  • the detection sensitivity was about 0.16 fM.
  • FIG. 23 for comparison, the results when the two-way complex measured in Experimental Example 1 was not immobilized, and the two-way complex measured on the inner surface of the well measured in Experimental Example 4 were fixed. The result when it is converted is also shown.
  • the detection sensitivity was further significantly improved from about 3.3 fM when immobilized on the inner surface of the well to about 0.16 fM. ..
  • FIG. 24 is a schematic diagram illustrating a detection method of this experimental example.
  • streptavidin-coated magnetic beads product name "Dynabeads MyOne Streptavidin T1", Veritas
  • the lysine residue of Cas13a protein or the N-terminal of Cas13a protein was modified with NHS-PEG4-biotin and biotinylated.
  • the biotinylated Cas13a protein and gRNA were buffered with buffer F (20 mM HEPES-KOH (pH 6.8) so that the final concentration of the biotinylated Cas13a protein was 3 ⁇ M and the final concentration of gRNA was 0.75 ⁇ M. ), 60 mM NaCl, 6 mM MgCl 2 , 50 ⁇ M Triton X-100) to form a two-way complex.
  • the above-mentioned two-way complex was diluted with buffer F containing a substrate nucleic acid fragment and Alexa647-maleimide to obtain a mixed solution.
  • concentration of the biotinylated Cas13a protein in the mixed solution was 60 nM
  • concentration of gRNA was 12 nM
  • concentration of the substrate nucleic acid fragment was 12 ⁇ M
  • concentration of Alexa647-maleimide was 60 ⁇ M.
  • buffer E (20 mM HEPES-KOH (pH 7.5), 100 mM KCl, 10 mM MgCl 2 ) containing the target nucleic acid fragment (SEQ ID NO: 9) so as to have a final concentration of 300 fM. , 50 ⁇ M Triton X-100) 100 ⁇ L.
  • 20 ⁇ L of 0.5 mg / mL magnetic beads was further added, and the mixture was mixed for 10 seconds by pipetting. Subsequently, it was incubated for 3 minutes to prepare an assay solution.
  • an assay solution to which magnetic beads were not added was also prepared.
  • a well array D in which 2,000,000 columnar wells having a diameter of 3.5 ⁇ m and a depth of 3.5 ⁇ m were arranged per 1 cm 2 was prepared.
  • the volume of the well array D per well was 30 fL.
  • the assay solution was dropped directly onto the well array D, and a sealing agent was further dropped to seal each well before imaging.
  • Two well arrays D were prepared, 105 ⁇ L of each of the above assay solutions was dropped onto each well array D, and a magnet was placed at the bottom of the well array D. As a result, each assay solution was introduced into each well of the well array D. In addition, when the assay solution contained magnetic beads, the magnetic beads bound to the tripartite complex containing the target nucleic acid fragment were concentrated and captured in each well.
  • each well array D was observed under a fluorescence microscope.
  • FIG. 25 (a) is a typical fluorescence micrograph showing the results of an assay solution containing 300 fM of a target nucleic acid fragment and no magnetic beads.
  • FIG. 25 (b) is a representative fluorescence micrograph showing the results of an assay solution containing 300 fM of target nucleic acid fragments and magnetic beads.
  • the present invention it is possible to provide a technique capable of detecting a target nucleic acid fragment with high sensitivity without amplifying it.
  • Film forming aqueous solution 520 ... First well, 530 ... Second well, 700, 710 ... film, 1030 ... space, 1230 ... water-absorbent organic solvent, 2110 ... particles, 2120, 2121,122, 2123 ... single-stranded DNA fragment, F ... fluorescent substance, Q ... dimming substance ..

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