WO2018043733A1 - Method and kit for detecting pathogenic microorganism - Google Patents

Method and kit for detecting pathogenic microorganism Download PDF

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Publication number
WO2018043733A1
WO2018043733A1 PCT/JP2017/031689 JP2017031689W WO2018043733A1 WO 2018043733 A1 WO2018043733 A1 WO 2018043733A1 JP 2017031689 W JP2017031689 W JP 2017031689W WO 2018043733 A1 WO2018043733 A1 WO 2018043733A1
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Prior art keywords
enzyme
virus
pathogenic microorganism
reaction product
hydrophilic solvent
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PCT/JP2017/031689
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French (fr)
Japanese (ja)
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博行 野地
和仁 田端
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国立研究開発法人科学技術振興機構
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Priority claimed from JP2017099579A external-priority patent/JP7016136B2/en
Application filed by 国立研究開発法人科学技術振興機構 filed Critical 国立研究開発法人科学技術振興機構
Priority to US16/329,829 priority Critical patent/US11008603B2/en
Priority to CN201780054148.5A priority patent/CN109689882A/en
Priority to EP17846736.1A priority patent/EP3508586A4/en
Priority to BR112019004272A priority patent/BR112019004272A2/en
Publication of WO2018043733A1 publication Critical patent/WO2018043733A1/en

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    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING 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
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12MAPPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M1/00Apparatus for enzymology or microbiology
    • C12M1/34Measuring or testing with condition measuring or sensing means, e.g. colony counters
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING 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
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING 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
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/483Physical analysis of biological material
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING 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
    • C12Q2334/00O-linked chromogens for determinations of hydrolase enzymes, e.g. glycosidases, phosphatases, esterases
    • C12Q2334/20Coumarin derivatives
    • C12Q2334/224-Methylumbelliferyl, i.e. beta-methylumbelliferone, 4MU
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/90Enzymes; Proenzymes
    • G01N2333/914Hydrolases (3)
    • G01N2333/924Hydrolases (3) acting on glycosyl compounds (3.2)
    • YGENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02ATECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to a method and kit for detecting pathogenic microorganisms. More specifically, in a minimal volume of hydrophilic solvent coated with a hydrophobic solvent, the pathogenic microorganism is detected by optically detecting the reaction product generated as a result of the reaction by the enzyme on or inside the pathogenic microorganism.
  • the present invention relates to a method for performing detection.
  • Patent Document 1 a simple influenza virus test kit using immunochromatography has been developed (see Patent Document 1). Since the method using immunochromatography can detect influenza virus within minutes to tens of minutes, it is utilized for diagnosis and treatment of infection.
  • Patent Documents 2 and 3 a technique for optically detecting influenza virus based on the reaction between neuraminidase, which is an enzyme of influenza virus, and a chromogenic substrate is known (see Patent Documents 2 and 3).
  • the chromogenic substrate include 4-methylumbelliferyl- ⁇ -D-neuraminic acid (4-Methylumbelliferyl-N-acetyl- ⁇ -D-neuramic acid: 4MU-NANA, see Patent Document 2), 4-alkoxy A derivative of —N-acetylneuraminic acid or 4,7-dialkoxy-N-acetylneuraminic acid (see Patent Document 3) is used.
  • 4-methylumbelliferone which is a fluorescent substance
  • the neuraminidase enzyme activity value can be calculated based on the amount of 4-methylumbelliferone produced, and the number of influenza virus particles can be quantified based on the enzyme activity value.
  • Non-Patent Document 1 discloses a single-molecule enzyme assay using an array of femtoliter-order droplets in which the droplets are covered with oil and directly accessible from the outside. A method is described.
  • the main object of the present invention is to provide a technique that can be used to detect viruses such as influenza viruses with high sensitivity.
  • the present invention provides the following [1] to [25].
  • [1] A method for detecting the pathogenic microorganism in a biological sample isolated from a subject infected with or suspected of being infected with a pathogenic microorganism, A plurality of accommodating portions capable of accommodating the pathogenic microorganisms are opposed to a lower layer portion formed by being separated from each other by a side wall having a hydrophobic upper surface, and a surface of the lower layer portion where the accommodating portion is formed.
  • An introduction procedure for introducing a hydrophilic solvent containing the biological sample and a substance serving as a substrate for a reaction by an enzyme present on the surface or inside of the pathogenic microorganism into a space between the upper layer portion An encapsulation procedure for introducing a hydrophobic solvent into the space, and forming droplets of the hydrophilic solvent that is covered with the hydrophobic solvent and includes the pathogenic microorganism and the substance in the container, A detection procedure for optically detecting a reaction product generated by a reaction between the enzyme and the substance in the droplet, The method, wherein the hydrophilic solvent has a pH value greater than the acid dissociation constant (pKa) of the reaction product.
  • pKa acid dissociation constant
  • the pathogenic microorganism is an influenza virus, the enzyme is neuraminidase, and the substance is 4-methylumbelliferyl- ⁇ -D-neuraminic acid (4-Methylumbelliferyl-N-acetyl- ⁇ -D-neuraminic). acid), and the reaction product is 4-methylumbelliferone [1] or [2].
  • the pathogenic microorganism is coronavirus, severe acute respiratory syndrome (SARS) coronavirus, middle east respiratory syndrome (MERS) virus, mump virus, measles virus, nipavirus, canine distemper virus, human immunodeficiency virus (HIV), From hepatitis B virus, human T cell leukemia virus (HTLV), Ebola virus, hepatitis C virus, Lassa virus, hantavirus, rabies virus, Japanese encephalitis virus, yellow fever virus, dengue virus, rubella virus, rotavirus and norovirus [1] or [2], wherein the enzyme is one or more selected from the group consisting of hemagglutinin esterase, neuraminidase, reverse transcriptase and RNA-dependent RNA polymerase.
  • the substance is selected from the group consisting of a derivative containing 4-methylumbelliferone, a derivative containing fluorescein, a derivative containing resorufin, and a derivative containing rhodamine. [1] or [2].
  • a method for diagnosing the presence or absence of infection with a pathogenic microorganism A procedure for separating a biological sample from a subject suspected of being infected; A plurality of accommodating portions capable of accommodating the pathogenic microorganisms are opposed to a lower layer portion formed by being separated from each other by a side wall having a hydrophobic upper surface, and a surface of the lower layer portion where the accommodating portion is formed.
  • the pathogenic microorganism is an influenza virus, the enzyme is neuraminidase, and the substance is 4-methylumbelliferyl- ⁇ -D-neuraminic acid (4-Methylumbelliferyl-N-acetyl- ⁇ -D-neuraminic). acid) and the reaction product is 4-methylumbelliferone, [6] to [8].
  • the pathogenic microorganism is coronavirus, severe acute respiratory syndrome (SARS) coronavirus, middle east respiratory syndrome (MERS) virus, mump virus, measles virus, nipavirus, canine distemper virus, human immunodeficiency virus (HIV), From hepatitis B virus, human T cell leukemia virus (HTLV), Ebola virus, hepatitis C virus, Lassa virus, hantavirus, rabies virus, Japanese encephalitis virus, yellow fever virus, dengue virus, rubella virus, rotavirus and norovirus Any one of [6] to [8], wherein the enzyme is one or more selected from the group consisting of hemagglutinin esterase, neuraminidase, reverse transcriptase, and RNA-dependent RNA polymerase.
  • the substance is selected from the group consisting of a derivative containing 4-methylumbelliferone, a derivative containing fluorescein, a derivative containing resorufin, and a derivative containing rhodamine. Any one of [6] to [8].
  • a method for detecting drug sensitivity of a pathogenic microorganism in a biological sample isolated from a subject infected with or suspected of being infected with a pathogenic microorganism A plurality of accommodating portions capable of accommodating the pathogenic microorganisms are opposed to a lower layer portion formed by being separated from each other by a side wall having a hydrophobic upper surface, and a surface of the lower layer portion where the accommodating portion is formed.
  • the hydrophilic solvent has a pH value larger than an acid dissociation constant (pKa) of the reaction product.
  • the pathogenic microorganism is an influenza virus, the enzyme is neuraminidase, and the substance is 4-methylumbelliferyl- ⁇ -D-neuraminic acid (4-Methylumbelliferyl-N-acetyl- ⁇ -D-neuraminic). acid), the reaction product is 4-methylumbelliferone, and the inhibitor is a neuraminidase inhibitor [12] or [13].
  • a method of screening for anti-pathogenic microbial agents A plurality of accommodating parts capable of accommodating pathogenic microorganisms are opposed to a lower layer part formed by being separated from each other by a side wall having a hydrophobic upper surface, and a surface on which the accommodating part is formed in the lower layer part Introducing a hydrophilic solvent containing the pathogenic microorganism, a substance serving as a substrate for a reaction by an enzyme present on or inside the pathogenic microorganism, and a candidate compound into a space between the upper layer portion and the upper layer portion When, An enclosing procedure for introducing a hydrophobic solvent into the space and forming droplets of a hydrophilic solvent that is covered with the hydrophobic solvent and includes the pathogenic microorganism, the substance, and the candidate compound in the container.
  • the hydrophilic solvent has a pH value larger than an acid dissociation constant (pKa) of the reaction product.
  • the pathogenic microorganism is an influenza virus, the enzyme is neuraminidase, and the substance is 4-methylumbelliferyl- ⁇ -D-neuraminic acid (4-Methylumbelliferyl-N-acetyl- ⁇ -D-neuraminic). acid), the reaction product is 4-methylumbelliferone, and a neuraminidase inhibitor is screened as the candidate compound [15] or [16].
  • a kit for detecting the pathogenic microorganism in a biological sample isolated from a subject infected with or suspected of being infected with a pathogenic microorganism A space between a lower layer portion in which a plurality of accommodating portions capable of accommodating the pathogenic microorganisms are separated from each other by a side wall having a hydrophobic upper surface, and a surface of the lower layer portion where the accommodating portion is formed
  • An array comprising an upper layer portion facing each other, and A substance that is a substrate for a reaction by an enzyme present on or inside the pathogenic microorganism;
  • a kit comprising a hydrophobic solvent.
  • the pathogenic microorganism is an influenza virus, the enzyme is neuraminidase, and the substance is 4-methylumbelliferyl- ⁇ -D-neuraminic acid (4-Methylumbelliferyl-N-acetyl- ⁇ -D-neuraminic). acid), and the reaction product is 4-methylumbelliferone [18].
  • the pathogenic microorganism is coronavirus, severe acute respiratory syndrome (SARS) coronavirus, Middle East respiratory syndrome (MERS) virus, mump virus, measles virus, nipavirus, canine distemper virus, human immunodeficiency virus (HIV), From hepatitis B virus, human T cell leukemia virus (HTLV), Ebola virus, hepatitis C virus, Lassa virus, hantavirus, rabies virus, Japanese encephalitis virus, yellow fever virus, dengue virus, rubella virus, rotavirus and norovirus [18] The kit according to [18], wherein the enzyme is one or more selected from the group consisting of hemagglutinin esterase, neuraminidase, reverse transcriptase, and RNA-dependent RNA polymerase.
  • the substance is selected from the group consisting of a derivative containing 4-methylumbelliferone, a derivative containing fluorescein, a derivative containing resorufin, and a derivative containing rhodamine.
  • a method of detecting a reaction product by reacting an enzyme with a substance serving as a substrate for a reaction by the enzyme in a hydrophilic solvent in interface with the hydrophobic solvent The method, wherein the hydrophilic solvent has a pH value greater than the acid dissociation constant (pKa) of the reaction product.
  • the hydrophilic solvent contains a pathogenic microorganism,
  • the enzyme is an enzyme having a substrate cleavage activity present on the surface or inside of the pathogenic microorganism,
  • the substance is a chromogenic substrate;
  • the method according to [22] wherein a reaction product generated by cleavage of the chromogenic substrate by the enzyme is optically detected.
  • the pathogenic microorganism is an influenza virus
  • the enzyme is neuraminidase
  • the chromogenic substrate is 4-methylumbelliferyl- ⁇ -D-neuraminic acid (4-Methylumbelliferyl-N-acetyl- ⁇ -D-
  • the pathogenic microorganism is coronavirus, severe acute respiratory syndrome (SARS) coronavirus, middle east respiratory syndrome (MERS) virus, mump virus, measles virus, nipavirus, canine distemper virus, human immunodeficiency virus (HIV), From hepatitis B virus, human T cell leukemia virus (HTLV), Ebola virus, hepatitis C virus, Lassa virus, hantavirus, rabies virus, Japanese encephalitis virus, yellow fever virus, dengue virus, rubella virus, rotavirus and norovirus [23] The method according to [23], wherein the enzyme is one or more selected from the group consisting of hemagglutinin esterase, neuraminidase, reverse transcriptase and RNA-dependent RNA polymerase.
  • the chromogenic substrate is selected from the group consisting of a derivative containing 4-methylumbelliferone, a derivative containing fluorescein, a derivative containing resorufin, and a derivative containing rhodamine.
  • the method of [23] which is one or more of the following.
  • the method of any of [23] to [26], wherein the hydrophilic solvent comprises a biological sample separated from a subject infected with or suspected of being infected with the pathogenic microorganism.
  • pathogenic microorganisms include bacteria and viruses.
  • bacteria include, but are not limited to, coliform bacteria, Vibrio parahaemolyticus, Campylobacter, Enterobacter, and Bacillus bacteria.
  • virus is not particularly limited, but for example, coronavirus, SARS virus, MARS virus, influenza virus, mump virus, measles virus, nipavirus, canine distemper virus, HIV, hepatitis B virus, HTLV, Ebola virus, type C Examples include hepatitis virus, Lassa virus, hantavirus, rabies virus, yellow fever virus, dengue virus, rubella virus, rotavirus and norovirus.
  • the present invention provides a technique that can be used to detect pathogenic microorganisms such as influenza virus with high sensitivity.
  • Pathogenic microorganism detection method The pathogenic microorganism detection method according to the present invention is used to detect pathogenic microorganisms in a biological sample isolated from a subject infected with or suspected of being infected with a pathogenic microorganism. is there.
  • the pathogenic microorganism detection method according to the present invention includes the following procedures.
  • the biological sample is not particularly limited as long as it is a biological material that can contain pathogenic microorganisms to be detected.
  • biological samples include nasal aspirate, nasal swab, throat swab, tracheal swab, saliva, sputum, blood (including whole blood, serum and plasma), urine, cells and tissues, organ extracts, etc. Is mentioned.
  • the pathogenic microorganism is not particularly limited as long as it has an enzyme on the surface or inside thereof and generates a reaction product that can be detected optically as a result of the reaction by the enzyme. More specifically, the pathogenic microorganism has an enzyme having a substrate-cleaving activity for a chromogenic substrate on the surface or inside thereof, and the reaction product as a chromophore is liberated by the cleavage of the chromogenic substrate by the enzyme. .
  • pathogenic microorganisms have an enzyme on the surface or inside that has an activity of synthesizing a polymer by binding a monomer as a substrate, and a reaction generated as a chromophore with the synthesis of the polymer by the enzyme It may be one that generates a product.
  • enzymes on the surface or inside that has an activity of synthesizing a polymer by binding a monomer as a substrate, and a reaction generated as a chromophore with the synthesis of the polymer by the enzyme It may be one that generates a product. Examples of combinations of pathogenic microorganisms and their enzymes include the following.
  • the lower layer portion 10 is formed by a plurality of accommodating portions 13 capable of accommodating virus particles separated from each other by a side wall 12 having a hydrophobic upper surface. Further, the upper layer portion 20 faces the surface of the lower layer portion 10 where the accommodating portion 13 is formed.
  • a hydrophilic solvent 42 is introduced into the space 30 between the lower layer portion 10 and the upper layer portion 20.
  • the hydrophilic solvent 42 may contain virus 2 derived from a biological sample. Further, the hydrophilic solvent 42 includes a substance 3 (hereinafter referred to as “substrate 3”) which is a substrate for a reaction by an enzyme present on the particle surface (or inside) of the virus 2.
  • substrate 3 a substance 3
  • the hydrophilic solvent 42 can be introduced into the space 30 from a through hole (not shown) formed in at least one of the upper layer part 20 and the lower layer part 10, for example. As shown in the drawing, the hydrophilic solvent 42 introduced into the space 30 flows in a direction parallel to the surface where the lower layer portion 10 and the upper layer portion 20 face each other.
  • the hydrophilic solvent 42 has a pH value larger than the acid dissociation constant (pKa) of the reaction product generated from the substrate 3 (details will be described later).
  • the substrate 3 may be any substrate that generates a reaction product having optical characteristics different from those before the reaction after the reaction with the enzyme, and a substance whose absorbance or optical rotation changes before and after the reaction, or exhibits fluorescence after the reaction. The substance that becomes is used.
  • the substrate 3 will be described in detail later.
  • the hydrophilic solvent 42 may contain a buffer substance necessary for optimizing the reaction between the enzyme and the substrate 3. Furthermore, by setting the buffer substance in the hydrophilic solvent 42 to a predetermined concentration or more, the detection of the reaction product can be made more sensitive in the detection procedure (3) (details will be described later).
  • MES 2-Morpholinoethanesulfonic acid
  • ADA N- (2-Acetamido) iminodiacetic acid
  • PIPES Piperazine-1,4-bis (2- ethanesulfonic acid)
  • ACES N- (2-Acetamido) -2-aminoethanesulfonic acid
  • BES N, N-Bis (2-hydroxyethyl) -2-aminoethanesulfonic acid
  • TES N-Tris (hydroxymethyl) methyl- So-called good buffers such as 2-aminoethanesulfonic acid HEPES (4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid), Tris (tris (hydroxymethyl) aminomethane), DEA (diethanolamine) and the like can be used.
  • the hydrophilic solvent 42 may contain a surfactant.
  • a surfactant By including a surfactant, the enzyme present in the virus 2 may be exposed on the surface.
  • the surfactant by including the surfactant, the hydrophilic solvent 42 tends to be easily introduced into the space 30 and the accommodating portion 13.
  • the surfactant is not particularly limited.
  • TWEEN 20 (CAS number: 9005-64-5, polyoxyethylene sorbitan monolaurate) and Triton X-100 (CAS number: 9002-93-1, generic name polyethylene glycol mono) -4-octylphenyl ether (n ⁇ 10)) and the like.
  • the concentration of the surfactant added to the first solvent 20 is not particularly limited, but is preferably 0.01 to 1%.
  • an anionic surfactant an anionic surfactant, a cationic surfactant, a nonionic surfactant, a zwitterionic surfactant, a naturally derived surfactant and the like can be widely used.
  • anionic surfactant examples include a carboxylic acid type, a sulfate ester type, a sulfonic acid type, and a phosphate ester type.
  • specific examples include sodium dodecyl sulfate, sodium laurate, sodium ⁇ -sulfo fatty acid methyl ester, sodium dodecyl benzene sulfonate, sodium dodecyl ethoxylate sulfate, etc.
  • sodium dodecyl benzene sulfonate is used. It is preferable to use it.
  • Examples of the cationic surfactant are classified into a quaternary ammonium salt type, an alkylamine type, and a heterocyclic amine type. Specific examples include stearyl trimethyl ammonium chloride, distearyl dimethyl ammonium chloride, didecyl dimethyl ammonium chloride, cetyl tripyridinium chloride, dodecyl dimethyl benzyl ammonium chloride, and the like.
  • Nonionic surfactants include, for example, polyoxyethylene alkyl ether, polyoxyethylene hydrogenated castor oil, polyoxyethylene mono fatty acid ester, polyoxyethylene sorbitan mono fatty acid ester, sucrose fatty acid ester, polyglycerin fatty acid ester, alkyl polyglycoside , N-methylalkylglucamide and the like.
  • amphoteric surfactants include lauryldimethylaminoacetic acid betaine, dodecylaminomethyldimethylsulfopropylbetaine, and 3- (tetradecyldimethylaminio) propane-1-sulfonate, but 3-[(3-colamide Propyl) dimethylammonio] -1-propanesulfonate (CHAPS), 3-[(3-colamidopropyl) dimethylammonio] -2-hydroxy-1-propanesulfonate (CHAPSO) and the like are preferably used.
  • lauryldimethylaminoacetic acid betaine dodecylaminomethyldimethylsulfopropylbetaine
  • 3- (tetradecyldimethylaminio) propane-1-sulfonate but 3-[(3-colamide Propyl) dimethylammonio] -1-propanesulfonate (CHAPS), 3-[(3-colamidopropyl) dimethylammoni
  • lecithin and saponin are preferable, and among the compounds referred to as lecithin, specifically, phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, phosphatidic acid, phosphatidylglycerol, etc. preferable.
  • a saponin Kiraya saponin is preferable.
  • the virus 2 and the substrate 3 enter the storage unit 13 by this procedure.
  • the number of viruses 2 entering one container 13 can be 0 or 1 at the maximum.
  • the concentration of the virus 2 is higher in the hydrophilic solvent 42, two or more viruses 2 can be introduced into one container 13.
  • the hydrophobic solvent 43 may be any solvent (immiscible solvent) that is difficult to mix with the hydrophilic solvent 42 used in the introduction procedure (1).
  • the hydrophobic solvent 43 include at least one selected from the group consisting of saturated hydrocarbons, unsaturated hydrocarbons, aromatic hydrocarbons, silicone oils, perfluorocarbons, halogenated solvents, and hydrophobic ionic liquids. A mixture or the like can be preferably used.
  • saturated hydrocarbons include alkanes and cycloalkanes. Examples of alkane include decane and hexadecane. Examples of the unsaturated hydrocarbon include squalene.
  • aromatic hydrocarbons include benzene and toluene.
  • the perfluorocarbon examples include Fluorinert (registered trademark) FC40 (manufactured by SIGMA).
  • the halogen solvent examples include chloroform, methylene chloride, chlorobenzene and the like.
  • the hydrophobic ionic liquid refers to an ionic liquid that does not dissociate at least in water, and examples thereof include 1-Butyl-3-methylimidazolium, Hexafluorophosphate.
  • An ionic liquid refers to a salt that exists as a liquid at room temperature.
  • the hydrophobic solvent 43 may be introduced into the space 30 from a through-hole (not shown) formed in at least one of the upper layer portion 20 and the lower layer portion 10, similarly to the hydrophilic solvent 42. As shown in the drawing, the hydrophobic solvent 43 introduced into the space 30 flows in a direction parallel to the surface where the lower layer portion 10 and the upper layer portion 20 face each other, and the hydrophilic solvent 42 in the space 30 is a hydrophobic solvent. 43 is substituted. As a result, a droplet of the hydrophilic solvent 42 that is covered with the hydrophobic solvent 43 and includes the virus 2 and the substrate 3 is formed in the accommodating portion 13.
  • reaction product 4 is present on or inside the particle of virus 2 (the figure shows the case where enzyme 5 is present on the virus surface).
  • a reaction product 4 is generated.
  • the colored product 4 exhibits optical characteristics different from those of the substrate 3 and exhibits, for example, a shift in absorbance and optical rotation and luminescence (fluorescence).
  • the volume of the container 13 (that is, the volume of the droplet of the hydrophilic solvent 42) is not particularly limited, but is, for example, 10 aL to 100 nL, preferably 1 fL to 1 pL.
  • Virus 2 is an influenza virus (see Table 1), and 4-methylumbelliferyl- ⁇ -D-neuraminic acid (4-Methylumbelliferyl-N-acetyl- ⁇ -D-neuraminic acid: 4MU-NANA) is used as substrate 3 The case where it is used will be described specifically by way of example.
  • Neuraminidase (enzyme 5) is present on the surface of influenza virus particles.
  • 4MU-NANA substrate 3
  • 4-methylumbelliferone reaction product 4
  • 4-Methylumbelliferone is generated as a fluorescent chromophore represented by the following formula, derived from hydrolysis of 4MU-NANA by neuraminidase.
  • the substrate 3 is not limited to 4MU-NANA, and any conventionally known one can be used as long as it releases a chromophore that can be detected optically by neuraminic acid hydrolysis by neuraminidase.
  • 4MU of the reaction product 4 has a hydroxyl group as shown by the following formula.
  • hydrogen is desorbed from the hydroxyl group of 4 MU to bring 4 MU into a charged state, so that the pH value of the hydrophilic solvent 42 is higher than the acid dissociation constant (pKa) of 7.79 of 4 MU. Set larger.
  • the 4MU contained in the droplet of the hydrophilic solvent 42 coated with the hydrophobic solvent 43 cannot move to the hydrophobic solvent 43 due to its charge, and as a result It accumulates in a high concentration in the droplets of the hydrophilic solvent 42.
  • the pH value of the hydrophilic solvent 42 is smaller than the acid dissociation constant (pKa) of 4 MU, 4 MU has a hydroxyl group, and therefore has no charge or when hydrogen is desorbed from the hydroxyl group. In comparison, the charge is reduced.
  • the 4MU contained in the droplet of the hydrophilic solvent 42 has no electric charge or has a small electric charge, the 4MU easily moves to the hydrophobic solvent 43 that is in interface contact with the hydrophilic solvent 42. The organic solvent 42 is lost from the droplet, or the 4MU concentration in the droplet is lowered.
  • the reaction between neuraminidase and 4MU-NANA is performed under pH conditions around 5 which is the optimum pH for the enzyme reaction by neuraminidase, and the detection of released 4MU is maximized with the fluorescence efficiency (quantum efficiency) of 4MU. It was carried out under a pH condition of around 10 which is a pH value.
  • one of the technical features of the present invention is that the reaction of neuraminidase with 4MU-NANA and the detection of 4MU are both carried out under pH conditions higher than 4MU pKa7.79. is there.
  • the reaction between the substrate 3 and the enzyme 5 can proceed if the substrate 3 and the enzyme 5 come into contact with each other even before this procedure.
  • the hydrophilic solvent 42 containing the virus 2 and the substrate 3 is used.
  • the generated reaction product 4 is not accumulated in the minimum volume. For this reason, in the optical detection of the reaction product 4, the influence of the reaction product 4 generated before the encapsulation procedure (2) is small enough to be ignored.
  • reaction product 4 from the droplets of the hydrophilic solvent 42 to the hydrophobic solvent 43 covering the droplets similarly
  • chromogenic substrates that can cause migration problems include:
  • Derivatives containing 4-methylumbelliferone other than 4MU-NANA are derivatives containing 4-methylumbelliferone other than 4MU-NANA.
  • the “derivative” means a compound having 4MU as “chromophore” and “substrate” cleaved by the reaction with the enzyme 5 in the structure.
  • resorufin as a chromophore.
  • the substrate When reacted with the enzyme 5, the substrate is cleaved by the enzyme 5 and resorufin (pKa: 6.0), which is a fluorescent substance, is released as the reaction product 4.
  • resorufin pKa: 6.0
  • the structure of resorufin is shown below.
  • the pH value of the hydrophilic solvent 42 is set larger than the acid dissociation constant (pKa) of the reaction product 4 generated from the derivative. Thereby, each reaction product 4 is brought into a charged state, is prevented from shifting (leaking out) to the hydrophobic solvent 43, and the reaction product 4 is accumulated in a high concentration in the droplets of the hydrophilic solvent 42. (See Example 1).
  • the concentration of the buffer substance is, for example, 50 mM or more, preferably 100 mM or more, more preferably 500 mM or more, and further preferably 1 M or more.
  • the optical detection of the reaction product 4 can be performed using a known means capable of detecting a difference in optical properties between the substrate 3 and the reaction product 4. For example, by detecting a specific absorbance or optical rotation shift using an image sensor, an absorptiometer, or a polarimeter, and by detecting a specific fluorescence wavelength using an image sensor, a fluorescence microscope, or a fluorometer. Product 4 can be detected optically.
  • the number of virus particles and / or subtypes can be determined.
  • influenza virus first, the enzyme activity value of neuraminidase is calculated using the detected fluorescence intensity and a standard curve that defines the relationship between the fluorescence intensity and neuraminidase activity prepared in advance. Next, the number of influenza virus particles is quantified using the calculated enzyme activity value and a standard curve that defines the relationship between the enzyme activity value and the number of virus particles prepared in advance. In addition to determining whether the biological sample separated from the subject contains viruses, the amount of virus can also be determined quantitatively (analog quantification), and whether or not the subject is infected with influenza virus. Can be diagnosed. A standard curve that directly defines the relationship between the fluorescence intensity and the number of virus particles may be used.
  • type A virus has higher neuraminidase activity than type B virus.
  • the inventors of the present invention use the detection method according to the present invention that the type A virus and the type B virus differ by about twice in neuraminidase activity, and that both can be effectively distinguished and detected based on the magnitude of the activity value. Heading.
  • the enzyme activity value of neuraminidase is calculated using the detected fluorescence intensity and a standard curve that defines the relationship between the fluorescence intensity and neuraminidase activity prepared in advance.
  • the subtype of influenza virus can be determined using the calculated enzyme activity value and the relational expression between the enzyme activity value and the subtype prepared in advance.
  • influenza virus it can be determined as type A if the calculated enzyme activity value is greater than or equal to the reference value, and can be determined as type B if it is less than the reference value.
  • the virus subtype in addition to the determination of whether the virus is contained in the biological sample separated from the subject, the virus subtype can also be determined, and the subtype of the infected influenza virus in the subject can be diagnosed.
  • a relational expression that directly defines the relationship between fluorescence intensity and subtype may be used.
  • the number of viruses 2 entering one container 13 can be 0 or 1 at the maximum.
  • the viral load can also be determined quantitatively (digital quantification) based on a defined standard curve.
  • the reaction product 4 can be accumulated in a high concentration in the droplets of the hydrophilic solvent 42, so that even when only one particle of virus 2 is contained in the container 13.
  • the reaction product 4 can be detected with high sensitivity. Therefore, according to the detection method of the present invention, even a very small amount of virus in a biological sample can be detected with high sensitivity, and the amount of pathogenic microorganisms can be determined with high accuracy.
  • reaction product 4 can be accumulated in a high concentration in the droplets of the hydrophilic solvent 42 to obtain a high fluorescence intensity
  • a simple imaging device with relatively low sensitivity can be used for optical detection.
  • optical detection with a camera mounted on a smartphone is expected to be possible.
  • Optical detection by a simple imaging device such as a camera mounted on a smartphone can facilitate the implementation of the pathogenic microorganism detection method according to the present invention in a relatively small hospital, clinic, or individual.
  • the communication means provided in the smartphone is used, the detection information of pathogenic microorganisms is transmitted to the server, and the accumulated information (big data) is analyzed, so that it is possible to grasp the epidemic area, time, subtype, etc. It is expected to be available for prediction.
  • the case where the virus 2 is an influenza virus and 4MU-NANA is used as the substrate 3 has been described as an example.
  • the substrate 3 includes hemagglutinin esterase (on the surface) of these viruses. What is necessary is just to use what hydrolyzes by the enzyme 5) and releases the chromophore (reaction product 4) as mentioned above.
  • the substrate 3 has reverse transcriptase (on the surface or inside thereof) It may be a nucleic acid monomer that is polymerized by the enzyme 5).
  • the fluorescence intensity increased as compared with the nucleic acid monomer is detected in the nucleic acid chain that is a reaction product of polymerization.
  • substrate 3 when the detection target is, for example, Ebola virus, hepatitis C virus, Lassa virus, hantavirus, rabies virus, Japanese encephalitis virus, yellow fever virus, dengue virus, rubella virus, rotavirus or norovirus, substrate 3
  • a fluorescent dye can be prepared by labeling a fluorescent dye to a nucleic acid monomer that is polymerized by the RNA-dependent RNA polymerase (enzyme 5) possessed on the surface or inside of these viruses.
  • the substrate can be appropriately selected depending on the enzyme that is present on the surface or inside the pathogenic microorganism to be detected.
  • the pH value of the hydrophilic solvent used in the introduction procedure (1) may be designed to be larger than the pKa according to the pKa of the selected reaction product.
  • the substrate is a pathogenic microorganism having the same enzyme (neuraminidase) such as influenza virus and mump virus
  • the substrate can be designed differently depending on the substrate specificity of the enzyme of each pathogenic microorganism.
  • 4MU-NANA is used as a chromogenic substrate for detecting influenza virus
  • a chromophore produced by hydrolysis of 4MU-NANA as a chromogenic substrate for detecting mump virus is converted from 4MU to other fluorescent substances such as fluorescein. Use what was changed to.
  • the detection method according to the present invention can also detect both of them separately.
  • the method for detecting drug sensitivity of a pathogenic microorganism in a biological sample isolated from a subject infected with or suspected of being infected with the pathogenic microorganism according to the present invention is as follows. including. (B1) A plurality of accommodating portions capable of accommodating pathogenic microorganisms are separated from each other by a side wall having a hydrophobic upper surface, and a surface where the accommodating portion in the lower layer portion is formed. In the space between the upper layers facing each other, a hydrophilic solvent containing the biological sample, a substance serving as a substrate for a reaction by an enzyme present on or inside the pathogenic microorganism, and an inhibitor of the enzyme Installation procedure to be introduced.
  • a hydrophobic solvent is introduced into the space, and droplets of the hydrophilic solvent that are covered with the hydrophobic solvent and include the pathogenic microorganism, the substance, and the inhibitor are formed in the housing portion.
  • Enclosure procedure to. (B3) a detection procedure for optically detecting a reaction product generated by a reaction between the enzyme and the substance in the droplet, wherein the detection intensity of the reaction product in the presence of the inhibitor is , If it is less than the detected intensity of the reaction product in the absence of the inhibitor, it indicates that the pathogenic microorganism is sensitive to the inhibitor).
  • the introduction procedure (B1) of the method for detecting drug susceptibility of pathogenic microorganisms according to the present invention is based on the above-described method for detecting pathogenic microorganisms only in that an enzyme inhibitor to be evaluated for drug sensitivity is contained in a hydrophilic solvent. Different from the introduction procedure (A1). In the introduction procedure (B1), in addition to the pathogenic microorganism and the substrate, an enzyme inhibitor present on the surface or inside of the pathogenic microorganism enters the container.
  • Encapsulation procedure (B2) The operation of the encapsulation procedure (B2) of the method for detecting drug sensitivity of a pathogenic microorganism according to the present invention is the same as the encapsulation procedure (A2) of the above-mentioned pathogenic microorganism detection method.
  • the encapsulation procedure (B2) droplets of the hydrophilic solvent that are covered with the hydrophobic solvent and include the pathogenic microorganism, the substrate, and the inhibitor are formed in the container.
  • Detection Procedure (B3) In the detection procedure (B3) of the method for detecting drug susceptibility of pathogenic microorganisms according to the present invention, the reaction generated in the droplet of the hydrophilic solvent in the same manner as the detection procedure (A3) of the above-described pathogenic microorganism detection method. Optical detection of the product is performed.
  • the detection intensity of the reaction product in the presence of the inhibitor is compared with the detection intensity of the reaction product in the absence of the inhibitor, It is shown that the formation of reaction products in the droplets is suppressed. That is, the enzyme contained in the pathogenic microorganism is inhibited, which indicates that the pathogenic microorganism is sensitive to the inhibitor.
  • the detection intensity of the reaction product in the absence of the inhibitor is equivalent to the detection intensity of the reaction product in the absence of the inhibitor
  • the reaction in the droplet of the hydrophilic solvent Product formation is shown not to be inhibited by the inhibitor. That is, the enzyme contained in the pathogenic microorganism is not inhibited, indicating that the pathogenic microorganism is resistant to the inhibitor.
  • the pathogenic microorganism is an influenza virus, for example, using 4MU-NANA as a substrate and neuraminidase inhibitors (such as osetamivir, zanamivir) as inhibitors
  • neuraminidase inhibitors such as osetamivir, zanamivir
  • the generated 4MU fluorescence is detected for the two test groups that are identical except under the following conditions.
  • the detection intensity of 4MU in the presence of neuraminidase inhibitor is compared to the detection intensity of 4MU in the absence of neuraminidase inhibitor, if the former is reduced over the latter, the neuraminidase inhibitor will cause the It is shown that the generation of 4MU is suppressed.
  • influenza virus is sensitive to neuraminidase inhibitors.
  • the detection intensity of 4MU in the absence of neuraminidase inhibitor is comparable to the detection intensity of 4MU in the absence of neuraminidase inhibitor, the production of 4MU in the droplet of the hydrophilic solvent is neuraminidase. It is shown that it is not suppressed by the inhibitor. That is, the neuraminidase possessed by the influenza virus is not inhibited, indicating that the influenza virus is resistant to the neuraminidase inhibitor.
  • HE hemagglutinin esterase
  • HIV human immunodeficiency virus
  • hepatitis B virus or human T cell leukemia virus HTLV
  • inhibitors of reverse transcriptase retrovir ( GlaxoSmithKline Inc., Zidovudine / AZT), Videx (Bristol-Myers Squibb Inc., Zidanocin / ddI), Hibid (Hoffmanla Roche) (Zarcitabine / ddC), Zellit (Bristol-Myers Squibb), Stavudine / d4T), Epivir (GlaxoSmithKline, Lamivudine / 3TC) and Combivir (GlaxoSmithKline, Zidovudine / Lamivudine) Vilamune (Boehringer Ingelheim Pharmaceuticals Inc.
  • retrovir GlaxoSmithKline Inc., Zidovudine / AZT
  • Videx Bristol-Myers Squibb Inc., Zidanocin / d
  • RNA-dependent RNA polymerase Fabipyravir, ribavirin, etc.
  • Vibrio parahaemolyticus Vibrio parahaemolyticus, Campylobacter, Enterobacter or Bacillus spp. are targeted for detection, inhibitors of galactosidase (Castanospermine, Conduritol B Epoxide, Bromoconduritol, 2- etc.
  • Deoxy-D-Galactose Deoxy-D-Galactose
  • glucuronidase inhibitor Alsetoguraton, D-glucaro-1,4-lactone , lysophospholipids, etc.
  • chymotrypsin trypsin inhibitor
  • trypsin inhibitor Soybean, egg or the like derived trypsin inhibitor, Arg 4 - Examples include Met 5 -marinostatin, phenylmethylsulfonyl fluoride, aminoethyl benzylsulfonyl fluoride, Aprotinin, Tosyl lysine chloromethyl ketone, tosyl phenylalanine chloromethyl ketone, and inhibitors of xylosidase (Castanospermine, Xyl-amidine, etc.).
  • the inhibitor in the method for detecting drug susceptibility of pathogenic microorganisms according to the present invention, can be appropriately selected depending on the enzyme present on the surface or inside the pathogenic microorganism to be detected.
  • a method for screening an anti-pathogenic microbial drug according to the present invention includes the following procedures.
  • a hydrophilic solvent containing the pathogenic microorganism, a substance serving as a substrate for a reaction by an enzyme existing on or inside the pathogenic microorganism, and a candidate compound is introduced into a space between the upper layers facing each other. Installation procedure to do.
  • (C2) A hydrophobic solvent is introduced into the space, and droplets of the hydrophilic solvent that are covered with the hydrophobic solvent and include the pathogenic microorganism, the substance, and the candidate compound are formed in the container. Enclosure procedure to. (C3) a detection procedure for optically detecting a reaction product generated by the reaction between the enzyme and the substance in the droplet, wherein the detection intensity of the reaction product in the presence of the candidate compound is , If it decreases below the detected intensity of the reaction product in the absence of the candidate compound, it indicates that the candidate compound has anti-pathogenic microbial activity).
  • the introduction procedure (C1) of the screening method for an anti-pathogenic microbial agent according to the present invention is the above-mentioned pathogenic microorganism only in that a candidate compound to be evaluated for anti-pathogenic microbial activity is contained in a hydrophilic solvent. This is different from the detection method introduction procedure (A1). In the introduction procedure (C1), in addition to the pathogenic microorganism and the substrate, the candidate compound enters the housing unit.
  • Encapsulation procedure (C2) The operation of the encapsulation procedure (C2) of the screening method for an anti-pathogenic microorganism according to the present invention is the same as the encapsulation procedure (A2) of the above-mentioned pathogenic microorganism detection method.
  • the encapsulation procedure (C2) droplets of a hydrophilic solvent that are coated with a hydrophobic solvent and include pathogenic microorganisms, substrates, and candidate compounds are formed in the container.
  • Detection Procedure (C3) In the detection procedure (C3) of the screening method for an anti-pathogenic microorganism according to the present invention, the reaction generated in the droplet of the hydrophilic solvent in the same manner as the detection procedure (A3) of the above-mentioned pathogenic microorganism detection method Optical detection of the product is performed.
  • the detection intensity of the reaction product in the presence of the candidate compound is compared with the detection intensity of the reaction product in the absence of the candidate compound. It is shown that the formation of reaction products in the droplets is suppressed. That is, the enzyme possessed by the pathogenic microorganism is inhibited, indicating that the candidate compound has anti-pathogenic microbial activity.
  • the detection intensity of the reaction product in the absence of the candidate compound is equivalent to the detection intensity of the reaction product in the absence of the candidate compound, the reaction in the droplet of the hydrophilic solvent Product formation is shown not to be inhibited by the inhibitor. That is, the enzyme possessed by the pathogenic microorganism is not inhibited, indicating that the candidate compound does not have anti-pathogenic microbial activity.
  • Pathogenic microorganism detection kit is a kit for detecting pathogenic microorganisms in a biological sample isolated from a subject infected with or suspected of being infected with a pathogenic microorganism, A space between a lower layer portion in which a plurality of accommodating portions capable of accommodating the pathogenic microorganisms are separated from each other by a side wall having a hydrophobic upper surface, and a surface of the lower layer portion where the accommodating portion is formed An array comprising an upper layer portion facing each other, and A substance that is a substrate for a reaction by an enzyme present on the surface or inside of the pathogenic microorganism particles; A hydrophilic solvent having a pH value greater than the acid dissociation constant (pKa) of the reaction product produced by the reaction between the enzyme and the substance; A hydrophobic solvent.
  • pKa acid dissociation constant
  • the kit according to the present invention includes the above-described array 1, the substrate 3, the hydrophilic solvent 42, and the hydrophobic solvent 43. Since the substrate 3, the hydrophilic solvent 42, and the hydrophobic solvent 43 are as described above, the configuration of the array 1 will be described in more detail below.
  • the lower layer portion 10 of the array 1 includes a plate-like member 11 and a side wall 12 having a hydrophobic upper surface.
  • a plurality of accommodating portions 13 are formed separated from each other by the side walls 12.
  • the plate member 11 preferably has a hydrophilic surface. “Hydrophilic surface” refers to a surface that has a higher affinity with a hydrophilic solvent than with a hydrophobic solvent.
  • the plate-like member 11 may be a solid material, but for example, glass, silicon, polymer resin, or the like can be used.
  • the side wall 12 is a structure that separates each of the plurality of accommodating portions 13 provided on the surface of the plate-like member 11, preferably on the hydrophilic surface.
  • the side wall 12 has a hydrophobic upper surface. “Hydrophobic” is used herein in the same meaning as “lipophilic” and means that the affinity with a hydrophobic solvent is higher than the affinity with a hydrophilic solvent.
  • the side wall 12 only needs to have a hydrophobic upper surface, that is, a surface facing the upper layer portion 20, and the side surface, that is, the inner wall in the accommodating portion 13, may be hydrophobic or hydrophilic.
  • the side wall 12 may be composed of a hydrophilic structure and a hydrophobic layer formed on the upper surface thereof.
  • a hydrophilic structure for example, glass, silicon, polymer resin, or the like can be used.
  • a hydrophobic layer for example, a water-repellent resin, a fluorine-based polymer resin, or the like can be used.
  • the fluoropolymer resin include amorphous fluororesins. Amorphous fluororesin is preferably used because it has high hydrophobicity and low toxicity to biological samples.
  • amorphous fluororesin for example, at least one selected from CYTOP (registered trademark), TEFLON (registered trademark) AF2400, and TEFLON (registered trademark) AF1600 can be suitably used.
  • CYTOP registered trademark
  • TEFLON registered trademark
  • AF1600 TEFLON (registered trademark) AF1600
  • CYTOP registered trademark
  • TEFLON registered trademark
  • the side wall 12 may be made of a hydrophobic material.
  • a fluorine-based polymer resin for example, a paraxylylene-based polymer resin, or the like can be used.
  • the fluoropolymer resin include amorphous fluororesins.
  • the amorphous fluororesin the above-described resins can be suitably used.
  • the side wall 12 should just be comprised so that the some accommodating part 13 may be formed on the plate-shaped member 11, for example, is a plate-shaped structure by which the hole is formed in the position in which the accommodating part 13 is formed. There may be.
  • the housing portion 13 has a part of the surface of the plate-like member 11 as a bottom surface, and the bottom surface is hydrophilic.
  • the shape of the region surrounded by the bottom surface and the side surface of the accommodating portion 13 may be, for example, a cylindrical shape, a prismatic shape, or the like.
  • the bottom surface of the accommodating portion 13 is hydrophilic, and the top surface of the side wall 12 is hydrophobic.
  • the upper layer portion 20 for example, glass, silicon, polymer resin, or the like can be used.
  • the upper layer portion 20 faces the surface of the lower layer portion 10 on which the accommodating portion 13 is formed with a space 30 therebetween. That is, there is a space 30 between the side wall 12 and the hydrophobic layer 22. This space 30 constitutes a flow path. With this configuration, the array 1 has a flow cell structure.
  • the space 30 can be used between the lower layer portion 10 and the upper layer portion 20 as a flow path for flowing a fluid in a direction parallel to the surfaces where the lower layer portion 10 and the upper layer portion 20 face each other.
  • a through hole (not shown) for introducing a fluid into the space 30 may be formed in the lower layer portion 10 or the upper layer portion 20.
  • the lower layer part 10 may have a region where the accommodating part 13 is formed and a region where the accommodating part 13 is not formed.
  • the through-hole may be formed in the area
  • the surface of the upper layer portion 20 constituting the upper surface of the space 30 is hydrophobic
  • the lower surface of the space 30 is the hydrophobic upper surface of the side wall 12 and the accommodating portion 13. Therefore, all portions of the space 30 other than the bottom surface of the accommodating portion 13 are hydrophobic.
  • the hydrophilic solvent 42 can be efficiently introduced into each accommodating portion 13.
  • the hydrophobic solvent 43 is not allowed to enter the respective accommodating portions 13. Therefore, by introducing the hydrophobic solvent 43 into the space 30, it is possible to efficiently form droplets in the respective accommodating portions 13.
  • a positive type photoresist (AZ-4903, AZ Electronic Materials) was spin-coated on the cover glass coated with the amorphous fluororesin and baked at 55 ° C. for 3 minutes, and further baked at 110 ° C. for 5 minutes. Photolithography was performed using a photomask having holes having a diameter of 3 ⁇ m at intervals of 5 ⁇ m. After dry etching with oxygen plasma, a washed cover glass was obtained as DAD. The DAD has a well (accommodating portion) having a diameter of 4 ⁇ m and a depth of 3 ⁇ m (about 1 million pieces / 10 mm 2 ), and the cover glass is exposed on the bottom surface of the well. An array having a flow cell structure as shown in FIG. 1 was prepared using the obtained DAD.
  • 4-MU was dissolved in a buffer solution (33 mM DEA-HCl, 4 mM CaCl 2 ) adjusted to pH 6.5 to 9.0 to 50 ⁇ M.
  • 30 ⁇ L of a buffer solution in which 4-MU was dissolved was introduced into the array, and each well was filled with the buffer solution (see FIG. 1A).
  • 200 ⁇ L of a hydrophobic solvent (FC40) was introduced into the array, and droplets of the hydrophilic solvent coated with the hydrophobic solvent were formed in each well.
  • a fluorescence image of each droplet was taken with a CMOS camera (NeosCMOS, Andor) connected to a fluorescence microscope (IX8, OLYMPUS), and the fluorescence intensity was measured.
  • Photographing was performed by dividing an area of 10 mm 2 for each well into 120 parts. One image includes about 8,600 wells. The fluorescence image was analyzed with image analysis software (Meta-Morph, Molecular Devices), and the fluorescence intensity was calculated.
  • 4-MU was dissolved in a buffer solution (4 mM CaCl 2 , pH 6.5) adjusted to a DEA concentration of 25 mM to 1 M to a concentration of 50 ⁇ M.
  • 30 ⁇ L of a buffer solution in which 4-MU was dissolved was introduced into the array, and each well was filled with the buffer solution (see FIG. 1A).
  • 200 ⁇ L of a hydrophobic solvent (FC40) was introduced into the array, and droplets of the hydrophilic solvent coated with the hydrophobic solvent were formed in each well.
  • Time-lapse photography was performed under a fluorescence microscope, and the fluorescence intensity of each droplet was measured.
  • the fluorescence of 4MU can be detected with higher sensitivity by setting the concentration of the buffer substance in the buffer solution to 50 mM or more. Indicated. It is considered that the migration (leakage) of 4MU FC40 could be suppressed by setting the buffer substance in the buffer solution to a predetermined concentration or more.

Abstract

Provided is a method for detecting pathogenic microorganisms in a biological sample, which is a technique that can be used to perform high-sensitivity detection of pathogenic microorganisms, such as influenza virus, etc., the method including: an introducing step for introducing a hydrophilic solvent that contains the biological sample and substances that serve as substrates for reactions involving enzymes that are present on surfaces of the pathogenic microorganisms or in the interiors thereof into a space between a lower-layer section in which a plurality of accommodating sections that can accommodate the pathogenic microorganisms are formed and an upper-layer section that faces a surface in which the accommodating sections are formed in the lower-layer section; an encapsulating step for introducing a hydrophobic solvent into the space and forming, in the accommodating sections, droplets of the hydrophilic solvent that are coated by the hydrophobic solvent enveloping the pathogenic microorganisms and the substances; and a detecting step for optically detecting reaction products that are generated as a result of the reactions between the enzymes and the substances in the droplets, wherein the hydrophilic solvent has a pH value that is greater than the acid dissociation constant (pKa) of the reaction products.

Description

病原性微生物検出のための方法及びキットMethod and kit for detecting pathogenic microorganisms
 本発明は、病原性微生物検出のための方法及びキットに関する。より詳しくは、疎水性溶媒に被覆された親水性溶媒の極小容積中において、病原性微生物の表面又は内部の酵素による反応の結果生成する反応生成物を光学的に検出することによって病原性微生物の検出を行う方法等に関する。 The present invention relates to a method and kit for detecting pathogenic microorganisms. More specifically, in a minimal volume of hydrophilic solvent coated with a hydrophobic solvent, the pathogenic microorganism is detected by optically detecting the reaction product generated as a result of the reaction by the enzyme on or inside the pathogenic microorganism. The present invention relates to a method for performing detection.
 近年、イムノクロマトグラフィを用いた簡易なインフルエンザウイルス検査キットが開発されている(特許文献1参照)。イムノクロマトグラフィを用いる方法は、数分から数十分の間にインフルエンザウイルスを検出できるので、感染の診断や治療等に活用されている。 Recently, a simple influenza virus test kit using immunochromatography has been developed (see Patent Document 1). Since the method using immunochromatography can detect influenza virus within minutes to tens of minutes, it is utilized for diagnosis and treatment of infection.
 また、従来、インフルエンザウイルスが有する酵素であるノイラミニダーゼと発色基質との反応に基づいて光学的にインフルエンザウイルスを検出する技術が知られている(特許文献2、3参照)。発色基質としては、例えば、4-メチルウンベリフェリル-α-D-ノイラミン酸(4-Methylumbelliferyl-N-acetyl-α-D-neuraminic acid:4MU-NANA、特許文献2参照)や、4-アルコキシ-N-アセチルノイラミン酸又は4,7-ジアルコキシ-N-アセチルノイラミン酸の誘導体(特許文献3参照)などが用いられている。例えば、発色基質として4MU-NANAを用いた方法では、ノイラミニダーゼによる4MU-NANAの分解によって蛍光物質である4-メチルウンベリフェロンが生成する。生成した4-メチルウンベリフェロンの量に基づいてノイラミニダーゼの酵素活性値を算出することができ、さらに酵素活性値に基づいてインフルエンザウイルスの粒子数を定量することができる。 Further, conventionally, a technique for optically detecting influenza virus based on the reaction between neuraminidase, which is an enzyme of influenza virus, and a chromogenic substrate is known (see Patent Documents 2 and 3). Examples of the chromogenic substrate include 4-methylumbelliferyl-α-D-neuraminic acid (4-Methylumbelliferyl-N-acetyl-α-D-neuramic acid: 4MU-NANA, see Patent Document 2), 4-alkoxy A derivative of —N-acetylneuraminic acid or 4,7-dialkoxy-N-acetylneuraminic acid (see Patent Document 3) is used. For example, in the method using 4MU-NANA as a chromogenic substrate, 4-methylumbelliferone, which is a fluorescent substance, is generated by the degradation of 4MU-NANA by neuraminidase. The neuraminidase enzyme activity value can be calculated based on the amount of 4-methylumbelliferone produced, and the number of influenza virus particles can be quantified based on the enzyme activity value.
 本発明に関連して、非特許文献1には、液滴がオイルで覆われており、外部から液滴に直接アクセス可能なフェムトリットルオーダーの液滴のアレイを用いて、一分子酵素アッセイを行う方法が記載されている。 In connection with the present invention, Non-Patent Document 1 discloses a single-molecule enzyme assay using an array of femtoliter-order droplets in which the droplets are covered with oil and directly accessible from the outside. A method is described.
特開2008-275511号JP 2008-275511 A 特開2011-139656号JP 2011-139656 A 特表2002-541858号Special table 2002-541858
 上述のイムノクロマトグラフィに基づくインフルエンザウイルスの検出方法は、簡便ではあるものの、検出のために103~104pfu/ml程度のウイルスが必要であり、検出感度が低いという問題がある。 Although the above-described method for detecting influenza virus based on immunochromatography is simple, it requires a virus of about 10 3 to 10 4 pfu / ml for detection and has a problem of low detection sensitivity.
 そこで、本発明は、インフルエンザウイルス等のウイルスを高感度に検出するために利用可能な技術を提供することを主な目的とする。 Therefore, the main object of the present invention is to provide a technique that can be used to detect viruses such as influenza viruses with high sensitivity.
 上記の課題を解決するために、本発明は、以下の[1]~[25]を提供する。
[1] 病原性微生物に感染した対象又は感染した疑いがある対象から分離された生物試料中の前記病原性微生物を検出する方法であって、
前記病原性微生物を収容可能な複数の収容部が、疎水性の上面を有する側壁によって互いに隔てられて形成されている下層部と、当該下層部における当該収容部が形成されている面に対向している上層部との間の空間に、前記生物試料と、前記病原性微生物の表面又は内部に存在する酵素による反応の基質となる物質と、を含む親水性溶媒を導入する導入手順と、
前記空間に疎水性溶媒を導入して、前記収容部内に、疎水性溶媒で被覆されかつ前記病原性微生物と前記物質を包含する、親水性溶媒の液滴を形成する封入手順と、
前記液滴中における前記酵素と前記物質との反応により生成する反応生成物を光学的に検出する検出手順と、を含み、
前記親水性溶媒が、前記反応生成物の酸解離定数(pKa)よりも大きいpH値を有する、方法。
[2] 前記反応生成物の検出強度に基づいて前記病原性微生物の数及び/又は亜種を決定する手順をさらに含む、[1]の方法。
[3] 前記病原性微生物がインフルエンザウイルスであり、前記酵素がノイラミニダーゼであり、前記物質が4-メチルウンベリフェリル-α-D-ノイラミン酸(4-Methylumbelliferyl-N-acetyl-α-D-neuraminic acid)であり、前記反応生成物が4-メチルウンベリフェロンである、[1]又は[2]の方法。
[4] 前記病原性微生物がコロナウイルス、重症急性呼吸器症候群(SARS)コロナウイルス、中東呼吸器症候群(MERS)ウイルス、ムンプウイルス、麻疹ウイルス、ニパウイルス、イヌジステンパーウイルス、ヒト免疫不全ウイルス(HIV)、B型肝炎ウイルス、ヒトT細胞白血病ウイルス(HTLV)、エボラウイルス、C型肝炎ウイルス、ラッサウイルス、ハンタウイルス、狂犬病ウイルス、日本脳炎ウイルス、黄熱ウイルス、デング熱ウイルス、風疹ウイルス、ロタウイルス及びノロウイルスからなる群より選択される一以上であり、前記酵素がヘマグルチニンエステラーゼ、ノイラミニダーゼ、逆転写酵素及びRNA依存RNAポリメラーゼからなる群より選択される一以上である、[1]又は[2]の方法。
[5] 前記物質が4-メチルウンベリフェロン(4-Methylumbelliferone)を含む誘導体、フルオレセイン(Fluorescein)を含む誘導体、レゾルフィン(Resorufin)を含む誘導体及びローダミン(Rhodamine)を含む誘導体からなる群より選択される一以上である、[1]又は[2]の方法。
In order to solve the above problems, the present invention provides the following [1] to [25].
[1] A method for detecting the pathogenic microorganism in a biological sample isolated from a subject infected with or suspected of being infected with a pathogenic microorganism,
A plurality of accommodating portions capable of accommodating the pathogenic microorganisms are opposed to a lower layer portion formed by being separated from each other by a side wall having a hydrophobic upper surface, and a surface of the lower layer portion where the accommodating portion is formed. An introduction procedure for introducing a hydrophilic solvent containing the biological sample and a substance serving as a substrate for a reaction by an enzyme present on the surface or inside of the pathogenic microorganism into a space between the upper layer portion,
An encapsulation procedure for introducing a hydrophobic solvent into the space, and forming droplets of the hydrophilic solvent that is covered with the hydrophobic solvent and includes the pathogenic microorganism and the substance in the container,
A detection procedure for optically detecting a reaction product generated by a reaction between the enzyme and the substance in the droplet,
The method, wherein the hydrophilic solvent has a pH value greater than the acid dissociation constant (pKa) of the reaction product.
[2] The method according to [1], further comprising a step of determining the number and / or subspecies of the pathogenic microorganisms based on the detected intensity of the reaction product.
[3] The pathogenic microorganism is an influenza virus, the enzyme is neuraminidase, and the substance is 4-methylumbelliferyl-α-D-neuraminic acid (4-Methylumbelliferyl-N-acetyl-α-D-neuraminic). acid), and the reaction product is 4-methylumbelliferone [1] or [2].
[4] The pathogenic microorganism is coronavirus, severe acute respiratory syndrome (SARS) coronavirus, middle east respiratory syndrome (MERS) virus, mump virus, measles virus, nipavirus, canine distemper virus, human immunodeficiency virus (HIV), From hepatitis B virus, human T cell leukemia virus (HTLV), Ebola virus, hepatitis C virus, Lassa virus, hantavirus, rabies virus, Japanese encephalitis virus, yellow fever virus, dengue virus, rubella virus, rotavirus and norovirus [1] or [2], wherein the enzyme is one or more selected from the group consisting of hemagglutinin esterase, neuraminidase, reverse transcriptase and RNA-dependent RNA polymerase.
[5] The substance is selected from the group consisting of a derivative containing 4-methylumbelliferone, a derivative containing fluorescein, a derivative containing resorufin, and a derivative containing rhodamine. [1] or [2].
[6] 病原性微生物の感染の有無を診断する方法であって、
感染した疑いがある対象から生物試料を分離する手順と、
前記病原性微生物を収容可能な複数の収容部が、疎水性の上面を有する側壁によって互いに隔てられて形成されている下層部と、当該下層部における当該収容部が形成されている面に対向している上層部との間の空間に、前記生物試料と、前記病原性微生物の表面又は内部に存在する酵素による反応の基質となる物質と、を含む親水性溶媒を導入する導入手順と、
前記空間に疎水性溶媒を導入して、前記収容部内に、疎水性溶媒で被覆されかつ前記病原性微生物と前記物質とを包含する、親水性溶媒の液滴を形成する封入手順と、
前記液滴中における前記酵素と前記物質との反応により生成する反応生成物を光学的に検出する検出手順、(ここで、前記反応生成物の検出は、前記病原性微生物の感染を示す)と、
を含む、方法。
[7] 前記親水性溶媒が、前記反応生成物の酸解離定数(pKa)よりも大きいpH値を有する、[6]の方法。
[8] 前記反応生成物の検出強度に基づいて病原性微生物の数及び/又は亜種を決定する手順をさらに含む、[6]又は[7]の方法。
[9] 前記病原性微生物がインフルエンザウイルスであり、前記酵素がノイラミニダーゼであり、前記物質が4-メチルウンベリフェリル-α-D-ノイラミン酸(4-Methylumbelliferyl-N-acetyl-α-D-neuraminic acid)であり、前記反応生成物が4-メチルウンベリフェロンである、[6]~[8]のいずれかの方法。
[10] 前記病原性微生物がコロナウイルス、重症急性呼吸器症候群(SARS)コロナウイルス、中東呼吸器症候群(MERS)ウイルス、ムンプウイルス、麻疹ウイルス、ニパウイルス、イヌジステンパーウイルス、ヒト免疫不全ウイルス(HIV)、B型肝炎ウイルス、ヒトT細胞白血病ウイルス(HTLV)、エボラウイルス、C型肝炎ウイルス、ラッサウイルス、ハンタウイルス、狂犬病ウイルス、日本脳炎ウイルス、黄熱ウイルス、デング熱ウイルス、風疹ウイルス、ロタウイルス及びノロウイルスからなる群より選択される一以上であり、前記酵素がヘマグルチニンエステラーゼ、ノイラミニダーゼ、逆転写酵素及びRNA依存RNAポリメラーゼからなる群より選択される一以上である、[6]~[8]のいずれかの方法。
[11] 前記物質が4-メチルウンベリフェロン(4-Methylumbelliferone)を含む誘導体、フルオレセイン(Fluorescein)を含む誘導体、レゾルフィン(Resorufin)を含む誘導体及びローダミン(Rhodamine)を含む誘導体からなる群より選択される一以上である、[6]~[8]のいずれかの方法。
[6] A method for diagnosing the presence or absence of infection with a pathogenic microorganism,
A procedure for separating a biological sample from a subject suspected of being infected;
A plurality of accommodating portions capable of accommodating the pathogenic microorganisms are opposed to a lower layer portion formed by being separated from each other by a side wall having a hydrophobic upper surface, and a surface of the lower layer portion where the accommodating portion is formed. An introduction procedure for introducing a hydrophilic solvent containing the biological sample and a substance serving as a substrate for a reaction by an enzyme present on the surface or inside of the pathogenic microorganism into a space between the upper layer portion,
An encapsulation procedure for introducing a hydrophobic solvent into the space, and forming droplets of the hydrophilic solvent that is covered with the hydrophobic solvent and includes the pathogenic microorganism and the substance in the accommodating portion;
A detection procedure for optically detecting a reaction product produced by a reaction between the enzyme and the substance in the droplet, wherein the detection of the reaction product indicates infection of the pathogenic microorganism; ,
Including a method.
[7] The method according to [6], wherein the hydrophilic solvent has a pH value larger than the acid dissociation constant (pKa) of the reaction product.
[8] The method according to [6] or [7], further comprising a step of determining the number and / or subspecies of pathogenic microorganisms based on the detected intensity of the reaction product.
[9] The pathogenic microorganism is an influenza virus, the enzyme is neuraminidase, and the substance is 4-methylumbelliferyl-α-D-neuraminic acid (4-Methylumbelliferyl-N-acetyl-α-D-neuraminic). acid) and the reaction product is 4-methylumbelliferone, [6] to [8].
[10] The pathogenic microorganism is coronavirus, severe acute respiratory syndrome (SARS) coronavirus, middle east respiratory syndrome (MERS) virus, mump virus, measles virus, nipavirus, canine distemper virus, human immunodeficiency virus (HIV), From hepatitis B virus, human T cell leukemia virus (HTLV), Ebola virus, hepatitis C virus, Lassa virus, hantavirus, rabies virus, Japanese encephalitis virus, yellow fever virus, dengue virus, rubella virus, rotavirus and norovirus Any one of [6] to [8], wherein the enzyme is one or more selected from the group consisting of hemagglutinin esterase, neuraminidase, reverse transcriptase, and RNA-dependent RNA polymerase. Method.
[11] The substance is selected from the group consisting of a derivative containing 4-methylumbelliferone, a derivative containing fluorescein, a derivative containing resorufin, and a derivative containing rhodamine. Any one of [6] to [8].
[12] 病原性微生物に感染した対象又は感染した疑いがある対象から分離された生物試料中の前記病原性微生物の薬剤感受性を検出する方法であって、
前記病原性微生物を収容可能な複数の収容部が、疎水性の上面を有する側壁によって互いに隔てられて形成されている下層部と、当該下層部における当該収容部が形成されている面に対向している上層部との間の空間に、前記生物試料と、前記病原性微生物の表面又は内部に存在する酵素による反応の基質となる物質と、前記酵素の阻害薬を含む親水性溶媒を導入する導入手順と、
前記空間に疎水性溶媒を導入して、前記収容部内に、疎水性溶媒で被覆されかつ前記病原性微生物と前記物質と前記阻害薬とを包含する、親水性溶媒の液滴を形成する封入手順と、
前記液滴中における前記酵素と前記物質との反応により生成する反応生成物を光学的に検出する検出手順、(ここで、前記阻害薬の存在下における前記反応生成物の検出強度が、前記阻害薬の非存在下における前記反応生成物の検出強度よりも減少する場合、前記病原性微生物が前記阻害薬に感受性を有することを示す)と、
を含む、方法。
[13] 前記親水性溶媒が、前記反応生成物の酸解離定数(pKa)よりも大きいpH値を有する、[12]の方法。
[14] 前記病原性微生物がインフルエンザウイルスであり、前記酵素がノイラミニダーゼであり、前記物質が4-メチルウンベリフェリル-α-D-ノイラミン酸(4-Methylumbelliferyl-N-acetyl-α-D-neuraminic acid)であり、前記反応生成物が4-メチルウンベリフェロンであり、前記阻害薬がノイラミニダーゼ阻害薬である、[12]又は[13]の方法。
[12] A method for detecting drug sensitivity of a pathogenic microorganism in a biological sample isolated from a subject infected with or suspected of being infected with a pathogenic microorganism,
A plurality of accommodating portions capable of accommodating the pathogenic microorganisms are opposed to a lower layer portion formed by being separated from each other by a side wall having a hydrophobic upper surface, and a surface of the lower layer portion where the accommodating portion is formed. A hydrophilic solvent containing the biological sample, a substance serving as a substrate for a reaction by an enzyme present on the surface or inside of the pathogenic microorganism, and an inhibitor of the enzyme; Introduction procedure and
An enclosing procedure for introducing a hydrophobic solvent into the space to form a droplet of a hydrophilic solvent that is coated with the hydrophobic solvent and includes the pathogenic microorganism, the substance, and the inhibitor in the container. When,
A detection procedure for optically detecting a reaction product produced by a reaction between the enzyme and the substance in the droplet, wherein the detection intensity of the reaction product in the presence of the inhibitor is the inhibition Indicating that the pathogenic microorganism is sensitive to the inhibitor if it decreases below the detected intensity of the reaction product in the absence of a drug),
Including a method.
[13] The method according to [12], wherein the hydrophilic solvent has a pH value larger than an acid dissociation constant (pKa) of the reaction product.
[14] The pathogenic microorganism is an influenza virus, the enzyme is neuraminidase, and the substance is 4-methylumbelliferyl-α-D-neuraminic acid (4-Methylumbelliferyl-N-acetyl-α-D-neuraminic). acid), the reaction product is 4-methylumbelliferone, and the inhibitor is a neuraminidase inhibitor [12] or [13].
[15] 抗病原性微生物薬剤をスクリーニングする方法であって、
病原性微生物を収容可能な複数の収容部が、疎水性の上面を有する側壁によって互いに隔てられて形成されている下層部と、当該下層部における当該収容部が形成されている面に対向している上層部との間の空間に、前記病原性微生物と、前記病原性微生物の表面又は内部に存在する酵素による反応の基質となる物質と、候補化合物とを含む親水性溶媒を導入する導入手順と、
前記空間に疎水性溶媒を導入して、前記収容部内に、疎水性溶媒で被覆されかつ前記病原性微生物と前記物質と前記候補化合物とを包含する、親水性溶媒の液滴を形成する封入手順と、
前記液滴中における前記酵素と前記物質との反応により生成する反応生成物を光学的に検出する検出手順、(ここで、前記候補化合物の存在下における前記反応生成物の検出強度が、前記候補化合物の非存在下における前記反応生成物の検出強度よりも減少する場合、前記候補化合物が抗病原性微生物活性を有することを示す)と、
を含む、方法。
[16] 前記親水性溶媒が、前記反応生成物の酸解離定数(pKa)よりも大きいpH値を有する、[15]の方法。
[17] 前記病原性微生物がインフルエンザウイルスであり、前記酵素がノイラミニダーゼであり、前記物質が4-メチルウンベリフェリル-α-D-ノイラミン酸(4-Methylumbelliferyl-N-acetyl-α-D-neuraminic acid)であり、前記反応生成物が4-メチルウンベリフェロンであり、前記候補化合物としてノイラミニダーゼ阻害薬がスクリーニングされる、[15]又は[16]の方法。
[15] A method of screening for anti-pathogenic microbial agents,
A plurality of accommodating parts capable of accommodating pathogenic microorganisms are opposed to a lower layer part formed by being separated from each other by a side wall having a hydrophobic upper surface, and a surface on which the accommodating part is formed in the lower layer part Introducing a hydrophilic solvent containing the pathogenic microorganism, a substance serving as a substrate for a reaction by an enzyme present on or inside the pathogenic microorganism, and a candidate compound into a space between the upper layer portion and the upper layer portion When,
An enclosing procedure for introducing a hydrophobic solvent into the space and forming droplets of a hydrophilic solvent that is covered with the hydrophobic solvent and includes the pathogenic microorganism, the substance, and the candidate compound in the container. When,
A detection procedure for optically detecting a reaction product generated by a reaction between the enzyme and the substance in the droplet, wherein the detection intensity of the reaction product in the presence of the candidate compound is the candidate Indicates that the candidate compound has anti-pathogenic microbial activity if it decreases below the detected intensity of the reaction product in the absence of the compound);
Including a method.
[16] The method according to [15], wherein the hydrophilic solvent has a pH value larger than an acid dissociation constant (pKa) of the reaction product.
[17] The pathogenic microorganism is an influenza virus, the enzyme is neuraminidase, and the substance is 4-methylumbelliferyl-α-D-neuraminic acid (4-Methylumbelliferyl-N-acetyl-α-D-neuraminic). acid), the reaction product is 4-methylumbelliferone, and a neuraminidase inhibitor is screened as the candidate compound [15] or [16].
[18] 病原性微生物に感染した対象又は感染した疑いがある対象から分離された生物試料中の前記病原性微生物を検出するためのキットであって、
前記病原性微生物を収容可能な複数の収容部が疎水性の上面を有する側壁によって互いに隔てられて形成されている下層部と、前記下層部における前記収容部が形成されている面に対して空間を隔てて対向している上層部とを備えるアレイと、
前記病原性微生物の表面又は内部に存在する酵素による反応の基質となる物質と、
前記酵素と前記物質との反応により生成する反応生成物の酸解離定数(pKa)よりも大きいpH値を有する親水性溶媒と、
疎水性溶媒と、を含むキット。
[19] 前記病原性微生物がインフルエンザウイルスであり、前記酵素がノイラミニダーゼであり、前記物質が4-メチルウンベリフェリル-α-D-ノイラミン酸(4-Methylumbelliferyl-N-acetyl-α-D-neuraminic acid)であり、前記反応生成物が4-メチルウンベリフェロンである、[18]のキット。
[20] 前記病原性微生物がコロナウイルス、重症急性呼吸器症候群(SARS)コロナウイルス、中東呼吸器症候群(MERS)ウイルス、ムンプウイルス、麻疹ウイルス、ニパウイルス、イヌジステンパーウイルス、ヒト免疫不全ウイルス(HIV)、B型肝炎ウイルス、ヒトT細胞白血病ウイルス(HTLV)、エボラウイルス、C型肝炎ウイルス、ラッサウイルス、ハンタウイルス、狂犬病ウイルス、日本脳炎ウイルス、黄熱ウイルス、デング熱ウイルス、風疹ウイルス、ロタウイルス及びノロウイルスからなる群より選択される一以上であり、前記酵素がヘマグルチニンエステラーゼ、ノイラミニダーゼ、逆転写酵素及びRNA依存RNAポリメラーゼからなる群より選択される一以上である、[18]のキット。
[21] 前記物質が4-メチルウンベリフェロン(4-Methylumbelliferone)を含む誘導体、フルオレセイン(Fluorescein)を含む誘導体、レゾルフィン(Resorufin)を含む誘導体及びローダミン(Rhodamine)を含む誘導体からなる群より選択される一以上である、[18]のキット。
[18] A kit for detecting the pathogenic microorganism in a biological sample isolated from a subject infected with or suspected of being infected with a pathogenic microorganism,
A space between a lower layer portion in which a plurality of accommodating portions capable of accommodating the pathogenic microorganisms are separated from each other by a side wall having a hydrophobic upper surface, and a surface of the lower layer portion where the accommodating portion is formed An array comprising an upper layer portion facing each other, and
A substance that is a substrate for a reaction by an enzyme present on or inside the pathogenic microorganism;
A hydrophilic solvent having a pH value greater than the acid dissociation constant (pKa) of the reaction product produced by the reaction between the enzyme and the substance;
A kit comprising a hydrophobic solvent.
[19] The pathogenic microorganism is an influenza virus, the enzyme is neuraminidase, and the substance is 4-methylumbelliferyl-α-D-neuraminic acid (4-Methylumbelliferyl-N-acetyl-α-D-neuraminic). acid), and the reaction product is 4-methylumbelliferone [18].
[20] The pathogenic microorganism is coronavirus, severe acute respiratory syndrome (SARS) coronavirus, Middle East respiratory syndrome (MERS) virus, mump virus, measles virus, nipavirus, canine distemper virus, human immunodeficiency virus (HIV), From hepatitis B virus, human T cell leukemia virus (HTLV), Ebola virus, hepatitis C virus, Lassa virus, hantavirus, rabies virus, Japanese encephalitis virus, yellow fever virus, dengue virus, rubella virus, rotavirus and norovirus [18] The kit according to [18], wherein the enzyme is one or more selected from the group consisting of hemagglutinin esterase, neuraminidase, reverse transcriptase, and RNA-dependent RNA polymerase.
[21] The substance is selected from the group consisting of a derivative containing 4-methylumbelliferone, a derivative containing fluorescein, a derivative containing resorufin, and a derivative containing rhodamine. The kit according to [18], which is one or more.
[22] 疎水性溶媒と界面接触する親水性溶媒中において、酵素と、該酵素による反応の基質となる物質とを反応させ、反応生成物を検出する方法であって、
前記親水性溶媒が、前記反応生成物の酸解離定数(pKa)よりも大きいpH値を有する、方法。
[23] 前記親水性溶媒が病原性微生物を含み、
前記酵素が前記病原性微生物の表面又は内部に存在する基質切断活性を有する酵素であり、
前記物質が発色基質であり、
前記酵素による前記発色基質の切断により生成する反応生成物を光学的に検出する、[22]の方法。
[24] 前記病原性微生物がインフルエンザウイルスであり、前記酵素がノイラミニダーゼであり、前記発色基質が4-メチルウンベリフェリル-α-D-ノイラミン酸(4-Methylumbelliferyl-N-acetyl-α-D-neuraminic acid)であり、前記反応生成物が4-メチルウンベリフェロンである、[23]の方法。
[25] 前記病原性微生物がコロナウイルス、重症急性呼吸器症候群(SARS)コロナウイルス、中東呼吸器症候群(MERS)ウイルス、ムンプウイルス、麻疹ウイルス、ニパウイルス、イヌジステンパーウイルス、ヒト免疫不全ウイルス(HIV)、B型肝炎ウイルス、ヒトT細胞白血病ウイルス(HTLV)、エボラウイルス、C型肝炎ウイルス、ラッサウイルス、ハンタウイルス、狂犬病ウイルス、日本脳炎ウイルス、黄熱ウイルス、デング熱ウイルス、風疹ウイルス、ロタウイルス及びノロウイルスからなる群より選択される一以上であり、前記酵素がヘマグルチニンエステラーゼ、ノイラミニダーゼ、逆転写酵素及びRNA依存RNAポリメラーゼからなる群より選択される一以上である、[23]の方法。
[26] 前記発色基質が4-メチルウンベリフェロン(4-Methylumbelliferone)を含む誘導体、フルオレセイン(Fluorescein)を含む誘導体、レゾルフィン(Resorufin)を含む誘導体及びローダミン(Rhodamine)を含む誘導体からなる群より選択される一以上である、[23]の方法。
[27] 前記親水性溶媒が、前記病原性微生物に感染した対象又は感染した疑いがある対象から分離された生物試料を含む、[23]~[26]のいずれかの方法。
[22] A method of detecting a reaction product by reacting an enzyme with a substance serving as a substrate for a reaction by the enzyme in a hydrophilic solvent in interface with the hydrophobic solvent,
The method, wherein the hydrophilic solvent has a pH value greater than the acid dissociation constant (pKa) of the reaction product.
[23] The hydrophilic solvent contains a pathogenic microorganism,
The enzyme is an enzyme having a substrate cleavage activity present on the surface or inside of the pathogenic microorganism,
The substance is a chromogenic substrate;
[22] The method according to [22], wherein a reaction product generated by cleavage of the chromogenic substrate by the enzyme is optically detected.
[24] The pathogenic microorganism is an influenza virus, the enzyme is neuraminidase, and the chromogenic substrate is 4-methylumbelliferyl-α-D-neuraminic acid (4-Methylumbelliferyl-N-acetyl-α-D- The method according to [23], wherein the reaction product is 4-methylumbelliferone.
[25] The pathogenic microorganism is coronavirus, severe acute respiratory syndrome (SARS) coronavirus, middle east respiratory syndrome (MERS) virus, mump virus, measles virus, nipavirus, canine distemper virus, human immunodeficiency virus (HIV), From hepatitis B virus, human T cell leukemia virus (HTLV), Ebola virus, hepatitis C virus, Lassa virus, hantavirus, rabies virus, Japanese encephalitis virus, yellow fever virus, dengue virus, rubella virus, rotavirus and norovirus [23] The method according to [23], wherein the enzyme is one or more selected from the group consisting of hemagglutinin esterase, neuraminidase, reverse transcriptase and RNA-dependent RNA polymerase.
[26] The chromogenic substrate is selected from the group consisting of a derivative containing 4-methylumbelliferone, a derivative containing fluorescein, a derivative containing resorufin, and a derivative containing rhodamine. The method of [23], which is one or more of the following.
[27] The method of any of [23] to [26], wherein the hydrophilic solvent comprises a biological sample separated from a subject infected with or suspected of being infected with the pathogenic microorganism.
 本発明において、「病原性微生物」には、細菌及びウイルスが含まれるものとする。細菌としては、特に限定されないが、例えば大腸菌群や腸炎ビブリオ菌、カンピロバクター、エンテロバクター、バチルス属細菌が挙げられる。また、ウイルスとしては、特に限定されないが、例えば、コロナウイルス、SARSウイルス、MARSウイルス、インフルエンザウイルス、ムンプウイルス、麻疹ウイルス、ニパウイルス、イヌジステンパーウイルス、HIV、B型肝炎ウイルス、HTLV、エボラウイルス、C型肝炎ウイルス、ラッサウイルス、ハンタウイルス、狂犬病ウイルス、黄熱ウイルス、デング熱ウイルス、風疹ウイルス、ロタウイルス及びノロウイルスなどが挙げられる。 In the present invention, “pathogenic microorganisms” include bacteria and viruses. Examples of the bacteria include, but are not limited to, coliform bacteria, Vibrio parahaemolyticus, Campylobacter, Enterobacter, and Bacillus bacteria. In addition, the virus is not particularly limited, but for example, coronavirus, SARS virus, MARS virus, influenza virus, mump virus, measles virus, nipavirus, canine distemper virus, HIV, hepatitis B virus, HTLV, Ebola virus, type C Examples include hepatitis virus, Lassa virus, hantavirus, rabies virus, yellow fever virus, dengue virus, rubella virus, rotavirus and norovirus.
 本発明により、インフルエンザウイルス等の病原性微生物を高感度に検出するために利用可能な技術が提供される。 The present invention provides a technique that can be used to detect pathogenic microorganisms such as influenza virus with high sensitivity.
本発明に係る病原性微生物検出方法の導入手順と封入手順を説明するための図である。It is a figure for demonstrating the introduction procedure and enclosure procedure of the pathogenic microorganism detection method which concern on this invention. ウイルスの粒子表面に存在する酵素と発色基質との反応による反応生成物を説明するための図である。It is a figure for demonstrating the reaction product by reaction of the enzyme which exists on the particle | grain surface of a virus, and a chromogenic substrate. 親水性溶媒のpHが検出感度に及ぼす影響を検討した結果を示すグラフである(実施例1)。It is a graph which shows the result of having examined the influence which pH of a hydrophilic solvent has on detection sensitivity (Example 1). 親水性溶媒の緩衝物質濃度が検出感度に及ぼす影響を検討した結果を示すグラフである(実施例2)。It is a graph which shows the result of having investigated the influence which the buffer substance density | concentration of a hydrophilic solvent has on detection sensitivity (Example 2).
 以下、本発明を実施するための好適な形態について図面を参照しながら説明する。なお、以下に説明する実施形態は、本発明の代表的な実施形態の一例を示したものであり、これにより本発明の範囲が狭く解釈されることはない。 Hereinafter, preferred embodiments for carrying out the present invention will be described with reference to the drawings. In addition, embodiment described below shows an example of typical embodiment of this invention, and, thereby, the range of this invention is not interpreted narrowly.
1.病原性微生物検出方法
 本発明に係る病原性微生物検出方法は、病原性微生物に感染した対象又は感染した疑いがある対象から分離された生物試料中の病原性微生物を検出するために用いられるものである。本発明に係る病原性微生物検出方法は、以下の手順を含む。
(1A)病原性微生物を収容可能な複数の収容部が、疎水性の上面を有する側壁によって互いに隔てられて形成されている下層部と、当該下層部における当該収容部が形成されている面に対向している上層部との間の空間に、前記生物試料と、前記病原性微生物の粒子表面又は内部に存在する酵素による反応の基質となる物質と、を含む親水性溶媒を導入する導入手順。
(2A)前記空間に疎水性溶媒を導入して、前記収容部内に、疎水性溶媒で被覆されかつ前記病原性微生物と前記物質を包含する、親水性溶媒の液滴を形成する封入手順。
(3A)前記液滴中における前記酵素と前記物質との反応により生成する反応生成物を光学的に検出する検出手順。
1. Pathogenic microorganism detection method The pathogenic microorganism detection method according to the present invention is used to detect pathogenic microorganisms in a biological sample isolated from a subject infected with or suspected of being infected with a pathogenic microorganism. is there. The pathogenic microorganism detection method according to the present invention includes the following procedures.
(1A) On the surface where the plurality of accommodating portions capable of accommodating pathogenic microorganisms are separated from each other by a side wall having a hydrophobic upper surface, and the surface where the accommodating portion in the lower layer portion is formed An introduction procedure for introducing a hydrophilic solvent containing the biological sample and a substance serving as a substrate for a reaction by an enzyme present on the surface of or inside the particle of the pathogenic microorganism into a space between the upper layers facing each other. .
(2A) An encapsulation procedure in which a hydrophobic solvent is introduced into the space to form a droplet of a hydrophilic solvent that is covered with the hydrophobic solvent and includes the pathogenic microorganism and the substance in the container.
(3A) A detection procedure for optically detecting a reaction product generated by a reaction between the enzyme and the substance in the droplet.
 本発明において、生物試料とは、検出対象となる病原性微生物が含まれ得る、生体由来の材料であれば特に限定されない。生物試料としては、例えば、鼻腔吸引液、鼻腔ぬぐい液、咽頭ぬぐい液、気管ぬぐい液、唾液、喀痰、血液(全血、血清及び血漿を含む)、尿、細胞や組織、臓器の抽出液等が挙げられる。 In the present invention, the biological sample is not particularly limited as long as it is a biological material that can contain pathogenic microorganisms to be detected. Examples of biological samples include nasal aspirate, nasal swab, throat swab, tracheal swab, saliva, sputum, blood (including whole blood, serum and plasma), urine, cells and tissues, organ extracts, etc. Is mentioned.
 病原性微生物は、表面もしくは内部に酵素を有するものであって、該酵素による反応の結果光学的に検出し得る反応生成物を生成するものであれば特に限定されない。病原性微生物は、より具体的には、発色基質に対する基質切断活性を有する酵素を表面又は内部に有し、該酵素による発色基質の切断によって発色団としての反応生成物を遊離させるものとされる。また、病原性微生物は、基質である単量体を結合させて重合体を合成する活性を有する酵素を表面又は内部に有し、該酵素による重合体の合成に伴って発色団としての反応生成物を生成させるものであってもよい。病原性微生物とその酵素の組み合わせとして、例えば以下が例示される。 The pathogenic microorganism is not particularly limited as long as it has an enzyme on the surface or inside thereof and generates a reaction product that can be detected optically as a result of the reaction by the enzyme. More specifically, the pathogenic microorganism has an enzyme having a substrate-cleaving activity for a chromogenic substrate on the surface or inside thereof, and the reaction product as a chromophore is liberated by the cleavage of the chromogenic substrate by the enzyme. . In addition, pathogenic microorganisms have an enzyme on the surface or inside that has an activity of synthesizing a polymer by binding a monomer as a substrate, and a reaction generated as a chromophore with the synthesis of the polymer by the enzyme It may be one that generates a product. Examples of combinations of pathogenic microorganisms and their enzymes include the following.
Figure JPOXMLDOC01-appb-T000001
Figure JPOXMLDOC01-appb-T000001
[導入手順(A1)]
 図1Aを参照して導入手順(A1)を説明する。本実施形態では、病原性微生物(以下、ウイルスを例に説明する)を収容可能な複数の収容部が疎水性の上面を有する側壁によって互いに隔てられて形成されている下層部と、当該下層部における前記収容部が形成されている面に対して空間を隔てて対向している上層部とを備えるアレイを用いて、本発明に係る病原性微生物検出方法を実施する場合について説明する。
[Introduction procedure (A1)]
The introduction procedure (A1) will be described with reference to FIG. 1A. In the present embodiment, a lower layer part in which a plurality of accommodating parts capable of accommodating pathogenic microorganisms (hereinafter described as an example of a virus) are separated from each other by a side wall having a hydrophobic upper surface, and the lower layer part The case where the pathogenic microorganism detection method according to the present invention is carried out using an array including an upper layer part facing the surface on which the housing part is formed with a space therebetween will be described.
 アレイ1において、下層部10は、ウイルス粒子を収容可能な複数の収容部13が、疎水性の上面を有する側壁12によって互いに隔てられて形成されている。また、上層部20は、下層部10における収容部13が形成されている面に対向している。 In the array 1, the lower layer portion 10 is formed by a plurality of accommodating portions 13 capable of accommodating virus particles separated from each other by a side wall 12 having a hydrophobic upper surface. Further, the upper layer portion 20 faces the surface of the lower layer portion 10 where the accommodating portion 13 is formed.
 本工程では、下層部10と上層部20との間の空間30に、親水性溶媒42を導入する。親水性溶媒42には、生物試料に由来するウイルス2が含まれ得る。また、親水性溶媒42には、ウイルス2の粒子表面(あるいは内部)に存在する酵素による反応の基質となる物質3(以下「基質3」と称する)が含まれる。親水性溶媒42は、例えば上層部20及び下層部10の少なくとも一方に形成されている貫通孔(図示せず)から空間30内に導入することができる。空間30内に導入された親水性溶媒42は、図に示すように、下層部10と上層部20とが対向する面に平行な方向に流れる。 In this step, a hydrophilic solvent 42 is introduced into the space 30 between the lower layer portion 10 and the upper layer portion 20. The hydrophilic solvent 42 may contain virus 2 derived from a biological sample. Further, the hydrophilic solvent 42 includes a substance 3 (hereinafter referred to as “substrate 3”) which is a substrate for a reaction by an enzyme present on the particle surface (or inside) of the virus 2. The hydrophilic solvent 42 can be introduced into the space 30 from a through hole (not shown) formed in at least one of the upper layer part 20 and the lower layer part 10, for example. As shown in the drawing, the hydrophilic solvent 42 introduced into the space 30 flows in a direction parallel to the surface where the lower layer portion 10 and the upper layer portion 20 face each other.
 親水性溶媒42としては、水が用いられる。親水性溶媒42は、基質3から生じる反応生成物の酸解離定数(pKa)よりも大きいpH値を有するものとされる(詳しくは後述する)。 Water is used as the hydrophilic solvent 42. The hydrophilic solvent 42 has a pH value larger than the acid dissociation constant (pKa) of the reaction product generated from the substrate 3 (details will be described later).
 基質3としては、酵素との反応後に反応前とは異なる光学特性を有する反応生成物を生成するものであればよく、反応前後で吸光度や旋光度が変化する物質や、反応後に蛍光を呈するようになる物質が用いられる。基質3については詳しく後述する。 The substrate 3 may be any substrate that generates a reaction product having optical characteristics different from those before the reaction after the reaction with the enzyme, and a substance whose absorbance or optical rotation changes before and after the reaction, or exhibits fluorescence after the reaction. The substance that becomes is used. The substrate 3 will be described in detail later.
 親水性溶媒42は、酵素と基質3との反応の最適化に必要な緩衝物質を含んでいてもよい。さらに、親水性溶媒42中の緩衝物質を所定の濃度以上に設定することで、検出手順(3)において反応生成物の検出をより高感度化できる(詳しくは後述する)。 The hydrophilic solvent 42 may contain a buffer substance necessary for optimizing the reaction between the enzyme and the substrate 3. Furthermore, by setting the buffer substance in the hydrophilic solvent 42 to a predetermined concentration or more, the detection of the reaction product can be made more sensitive in the detection procedure (3) (details will be described later).
 緩衝物質としては、特に限定されないが、蛍光色素のpKaにあわせてMES(2-Morpholinoethanesulfonic acid)、ADA(N-(2-Acetamido)iminodiacetic acid)、PIPES(Piperazine-1,4-bis(2-ethanesulfonic acid))、ACES(N-(2-Acetamido)-2-aminoethanesulfonic acid)、BES(N,N-Bis(2-hydroxyethyl)-2-aminoethanesulfonic acid)、TES(N-Tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid)HEPES(4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)等のいわゆるグッドバッファー(Good's Buffer)や、Tris(Tris(hydroxymethyl)aminomethane)、DEA(Diethanolamine)等が用いられ得る。 Although it does not specifically limit as a buffer substance, MES (2-Morpholinoethanesulfonic acid), ADA (N- (2-Acetamido) iminodiacetic acid), PIPES (Piperazine-1,4-bis (2- ethanesulfonic acid)), ACES (N- (2-Acetamido) -2-aminoethanesulfonic acid), BES (N, N-Bis (2-hydroxyethyl) -2-aminoethanesulfonic acid), TES (N-Tris (hydroxymethyl) methyl- So-called good buffers such as 2-aminoethanesulfonic acid HEPES (4- (2-hydroxyethyl) -1-piperazineethanesulfonic acid), Tris (tris (hydroxymethyl) aminomethane), DEA (diethanolamine) and the like can be used.
 また、親水性溶媒42は、界面活性剤を含んでいてもよい。界面活性剤を含むことで、ウイルス2の内部に存在する酵素を表面に露出させられる場合がある。また、界面活性剤を含むことで、親水性溶媒42が、空間30及び収容部13内へ導入され易くなる傾向がある。 Further, the hydrophilic solvent 42 may contain a surfactant. By including a surfactant, the enzyme present in the virus 2 may be exposed on the surface. In addition, by including the surfactant, the hydrophilic solvent 42 tends to be easily introduced into the space 30 and the accommodating portion 13.
 界面活性剤としては、特に限定されないが、例えばTWEEN20(CAS番号:9005-64-5、モノラウリン酸ポリオキシエチレンソルビタン)及びTriton X-100(CAS番号:9002-93-1、一般名ポリエチレングリコールモノ-4-オクチルフェニルエーテル(n≒10))などが挙げられる。第一の溶媒20への界面活性剤の添加濃度は、特に限定されないが、好ましくは0.01~1%である。 The surfactant is not particularly limited. For example, TWEEN 20 (CAS number: 9005-64-5, polyoxyethylene sorbitan monolaurate) and Triton X-100 (CAS number: 9002-93-1, generic name polyethylene glycol mono) -4-octylphenyl ether (n≈10)) and the like. The concentration of the surfactant added to the first solvent 20 is not particularly limited, but is preferably 0.01 to 1%.
 さらに、界面活性剤としては、陰イオン性界面活性剤、陽イオン性界面活性剤、非イオン性界面活性剤、両性イオン界面活性剤、天然由来の界面活性剤などを広く用いることができる。 Furthermore, as the surfactant, an anionic surfactant, a cationic surfactant, a nonionic surfactant, a zwitterionic surfactant, a naturally derived surfactant and the like can be widely used.
 陰イオン性界面活性剤としては、例えば、カルボン酸型、硫酸エステル型、スルホン酸型、リン酸エステル型に分類される。このうち、具体的には、例えば、ドデシル硫酸ナトリウム、ラウリン酸ナトリウム、α-スルホ脂肪酸メチルエステルナトリウム、ドデシルベンゼンスルホン酸ナトリウム、ドデシルエトキシレート硫酸ナトリウムなどが挙げられ、中でも、ドデシルベンゼンスルホン酸ナトリウムを用いることが好ましい。 Examples of the anionic surfactant are classified into a carboxylic acid type, a sulfate ester type, a sulfonic acid type, and a phosphate ester type. Of these, specific examples include sodium dodecyl sulfate, sodium laurate, sodium α-sulfo fatty acid methyl ester, sodium dodecyl benzene sulfonate, sodium dodecyl ethoxylate sulfate, etc. Among them, sodium dodecyl benzene sulfonate is used. It is preferable to use it.
 陽イオン性界面活性剤としては、例えば、第四級アンモニウム塩型、アルキルアミン型、複素環アミン型に分類される。具体的には、例えば、ステアリルトリメチルアンモニウムクロライド、ジステアリルジメチルアンモニウムクロライド、ジデシルジメチルアンモニウムクロライド、セチルトリピリジニウムクロライド、ドデシルジメチルベンジルアンモニウムクロライドなどが挙げられる。 Examples of the cationic surfactant are classified into a quaternary ammonium salt type, an alkylamine type, and a heterocyclic amine type. Specific examples include stearyl trimethyl ammonium chloride, distearyl dimethyl ammonium chloride, didecyl dimethyl ammonium chloride, cetyl tripyridinium chloride, dodecyl dimethyl benzyl ammonium chloride, and the like.
 非イオン界面活性剤としては、例えば、ポリオキシエチレンアルキルエーテル、ポリオキシエチレン硬化ひまし油、ポリオキシエチレンモノ脂肪酸エステル、ポリオキシエチレンソルビタンモノ脂肪酸エステル、ショ糖脂肪酸エステル、ポリグリセリン脂肪酸エステル、アルキルポリグリコシド、N-メチルアルキルグルカミドなどが挙げられる。中でも、ドデシルアルコールエトキシレート、ノニルフェノールエトキシレート、ラウロイルジエタノールアマイドの他、Triton X(Triton X-100など)、Pluronic(登録商標)(Pluronic F-123、F-68など)、Tween (Tween 20、40、60、65、80、85など)、Brij(登録商標)(Brij 35、58、98など)、Span (Span 20、40、60、80、83、85)の名前で市販されているものが好ましい。 Nonionic surfactants include, for example, polyoxyethylene alkyl ether, polyoxyethylene hydrogenated castor oil, polyoxyethylene mono fatty acid ester, polyoxyethylene sorbitan mono fatty acid ester, sucrose fatty acid ester, polyglycerin fatty acid ester, alkyl polyglycoside , N-methylalkylglucamide and the like. Among them, in addition to dodecyl alcohol ethoxylate, nonylphenol ethoxylate, lauroyl diethanolamide, Triton X (Triton X-100 etc.), Pluronic (registered trademark) (Pluronic F-123, F-68 etc.), Tween ( Tween 20, 40 , 60, 65, 80, 85, etc.), Brij (registered trademark) (Brij 35, 58, 98, etc.), Span ( Span 20, 40, 60, 80, 83, 85) preferable.
 両性界面活性剤としては、例えば、ラウリルジメチルアミノ酢酸ベタイン、ドデシルアミノメチルジメチルスルホプロピルベタイン、3-(テトラデシルジメチルアミニオ)プロパン-1-スルホナートなどがあるが、3-[(3-コラミドプロピル)ジメチルアンモニオ]-1-プロパンスルホナート(CHAPS)、3-[(3-コラミドプロピル)ジメチルアンモニオ]-2-ヒドロキシ-1-プロパンスルホナート(CHAPSO)などを用いることが好ましい。 Examples of amphoteric surfactants include lauryldimethylaminoacetic acid betaine, dodecylaminomethyldimethylsulfopropylbetaine, and 3- (tetradecyldimethylaminio) propane-1-sulfonate, but 3-[(3-colamide Propyl) dimethylammonio] -1-propanesulfonate (CHAPS), 3-[(3-colamidopropyl) dimethylammonio] -2-hydroxy-1-propanesulfonate (CHAPSO) and the like are preferably used.
 天然由来の界面活性剤としては、例えば、レシチン、サポニンが好ましく、レシチンとして称される化合物のうち、具体的には、ホスファチジルコリン、ホスファチジルエタノールアミン、ホスファチジルイノシトール、ホスファチジルセリン、ホスファチジン酸、ホスファチジルグリセロールなどが好ましい。また、サポニンとしてはキラヤサポニンが好ましい。 As the naturally-occurring surfactant, for example, lecithin and saponin are preferable, and among the compounds referred to as lecithin, specifically, phosphatidylcholine, phosphatidylethanolamine, phosphatidylinositol, phosphatidylserine, phosphatidic acid, phosphatidylglycerol, etc. preferable. Moreover, as a saponin, Kiraya saponin is preferable.
 本手順により、ウイルス2と基質3が収容部13に入る。親水性溶媒42中において、ウイルス2が十分に低い濃度に希釈されている場合、1つの収容部13に入るウイルス2の数は0又は最大で1となり得る。親水性溶媒42中において、ウイルス2の濃度がより高い場合には、1つの収容部13には2以上のウイルス2が導入され得る。 The virus 2 and the substrate 3 enter the storage unit 13 by this procedure. When the virus 2 is diluted to a sufficiently low concentration in the hydrophilic solvent 42, the number of viruses 2 entering one container 13 can be 0 or 1 at the maximum. When the concentration of the virus 2 is higher in the hydrophilic solvent 42, two or more viruses 2 can be introduced into one container 13.
[封入手順(A2)]
 図1Bを参照して封入手順(2)を説明する。本手順では、下層部10と上層部20との間の空間30に疎水性溶媒43を導入する。
[Encapsulation procedure (A2)]
The encapsulation procedure (2) will be described with reference to FIG. 1B. In this procedure, the hydrophobic solvent 43 is introduced into the space 30 between the lower layer portion 10 and the upper layer portion 20.
 疎水性溶媒43は、導入手順(1)で用いた親水性溶媒42と混ざり合いにくい溶媒(非混和性の溶媒)であればよい。疎水性溶媒43として、例えば飽和炭化水素、不飽和炭化水素、芳香族炭化水素、シリコーンオイル、パーフルオロカーボン、ハロゲン系溶媒、及び疎水性イオン液体からなる群より選択される少なくとも1つ又はこれを含む混合物等を好適に用いることができる。飽和炭化水素としては、例えばアルカン、シクロアルカンなどが挙げられる。アルカンとしては、例えばデカン、ヘキサデカン等が挙げられる。不飽和炭化水素としては、例えばスクアレン等が挙げられる。芳香族炭化水素としては、例えばベンゼン、トルエン等が挙げられる。パーフルオロカーボンとしては、例えばフロリナート(登録商標)FC40(SIGMA社製)等が挙げられる。ハロゲン系溶媒としては、例えばクロロホルム、塩化メチレン、クロロベンゼン等が挙げられる。疎水性イオン液体とは少なくとも水中では解離しないイオン液体をさし、例えば1―Butyl―3―methylimidazolium Hexafluorophosphate等が挙げられる。イオン液体とは、室温において液体で存在する塩をさす。 The hydrophobic solvent 43 may be any solvent (immiscible solvent) that is difficult to mix with the hydrophilic solvent 42 used in the introduction procedure (1). Examples of the hydrophobic solvent 43 include at least one selected from the group consisting of saturated hydrocarbons, unsaturated hydrocarbons, aromatic hydrocarbons, silicone oils, perfluorocarbons, halogenated solvents, and hydrophobic ionic liquids. A mixture or the like can be preferably used. Examples of saturated hydrocarbons include alkanes and cycloalkanes. Examples of alkane include decane and hexadecane. Examples of the unsaturated hydrocarbon include squalene. Examples of aromatic hydrocarbons include benzene and toluene. Examples of the perfluorocarbon include Fluorinert (registered trademark) FC40 (manufactured by SIGMA). Examples of the halogen solvent include chloroform, methylene chloride, chlorobenzene and the like. The hydrophobic ionic liquid refers to an ionic liquid that does not dissociate at least in water, and examples thereof include 1-Butyl-3-methylimidazolium, Hexafluorophosphate. An ionic liquid refers to a salt that exists as a liquid at room temperature.
 疎水性溶媒43も、親水性溶媒42と同様に、上層部20及び下層部10の少なくとも一方に形成されている貫通孔(図示せず)から空間30内に導入すればよい。空間30内に導入された疎水性溶媒43は、図に示すように、下層部10と上層部20とが対向する面に平行な方向に流れ、空間30内の親水性溶媒42が疎水性溶媒43によって置換される。これにより、収容部13内に、疎水性溶媒43で被覆されかつウイルス2と基質3を包含する、親水性溶媒42の液滴が形成される。 The hydrophobic solvent 43 may be introduced into the space 30 from a through-hole (not shown) formed in at least one of the upper layer portion 20 and the lower layer portion 10, similarly to the hydrophilic solvent 42. As shown in the drawing, the hydrophobic solvent 43 introduced into the space 30 flows in a direction parallel to the surface where the lower layer portion 10 and the upper layer portion 20 face each other, and the hydrophilic solvent 42 in the space 30 is a hydrophobic solvent. 43 is substituted. As a result, a droplet of the hydrophilic solvent 42 that is covered with the hydrophobic solvent 43 and includes the virus 2 and the substrate 3 is formed in the accommodating portion 13.
 そして、親水性溶媒42の液滴の極小容積中で共存する、ウイルス2の粒子表面又は内部に存在する酵素と基質3との反応が進行し、反応生成物4が生成する。図2を参照して詳しく説明する。ウイルス2の粒子表面又は内部には酵素5が存在している(図には酵素5がウイルス表面に存在する場合を示した)。基質3が、酵素5と接触し反応すると、反応生成物4が生成する。発色生成物4は、基質3と異なる光学特性を示し、例えば吸光度や旋光度のシフトや、発光(蛍光)を示す。 Then, the reaction between the substrate 3 and the enzyme present on the surface of or inside the particle of the virus 2 that coexists in the minimum volume of the droplet of the hydrophilic solvent 42 proceeds to generate a reaction product 4. This will be described in detail with reference to FIG. Enzyme 5 is present on or inside the particle of virus 2 (the figure shows the case where enzyme 5 is present on the virus surface). When the substrate 3 comes into contact with the enzyme 5 and reacts, a reaction product 4 is generated. The colored product 4 exhibits optical characteristics different from those of the substrate 3 and exhibits, for example, a shift in absorbance and optical rotation and luminescence (fluorescence).
 本手順により、ウイルス2と基質3が、極小容積の液滴中に封入されるため、酵素5と基質3との反応によって該液滴中に反応生成物4が極小容積中に生成される。これによって、反応生成物4の光学検出が可能とされる。収容部13の容積(すなわち、親水性溶媒42の液滴の容積)は、特に限定されないが、例えば10aL~100nL、好ましくは1fL~1pLである。 In this procedure, since the virus 2 and the substrate 3 are enclosed in a very small volume droplet, the reaction product 4 is generated in the very small volume by the reaction of the enzyme 5 and the substrate 3. Thereby, the optical detection of the reaction product 4 is enabled. The volume of the container 13 (that is, the volume of the droplet of the hydrophilic solvent 42) is not particularly limited, but is, for example, 10 aL to 100 nL, preferably 1 fL to 1 pL.
 ウイルス2がインフルエンザウイルスであり(表1参照)、基質3に4-メチルウンベリフェリル-α-D-ノイラミン酸(4-Methylumbelliferyl-N-acetyl-α-D-neuraminic acid:4MU-NANA)を用いる場合を例により具体的に説明する。 Virus 2 is an influenza virus (see Table 1), and 4-methylumbelliferyl-α-D-neuraminic acid (4-Methylumbelliferyl-N-acetyl-α-D-neuraminic acid: 4MU-NANA) is used as substrate 3 The case where it is used will be described specifically by way of example.
 インフルエンザウイルスの粒子表面にはノイラミニダーゼ(酵素5)が存在している。4MU-NANA(基質3)が、ノイラミニダーゼと接触し反応すると、蛍光物質である4-メチルウンベリフェロン(反応生成物4)が生成する。 Neuraminidase (enzyme 5) is present on the surface of influenza virus particles. When 4MU-NANA (substrate 3) contacts and reacts with neuraminidase, 4-methylumbelliferone (reaction product 4), which is a fluorescent substance, is produced.
 4MU-NANAのノイラミニダーゼによる加水分解に由来して、下記式で示される、蛍光を呈する発色団として4-メチルウンベリフェロン(4MU)が生成する。基質3は、ノイラミニダーゼによるノイラミン酸の加水分化によって光学的に検出可能な発色団を遊離させるものであれば、4MU-NANAに限られず、従来公知のものを用いることができる。反応生成物4の4MUは、下記式で示されるように水酸基を有する。 4-Methylumbelliferone (4MU) is generated as a fluorescent chromophore represented by the following formula, derived from hydrolysis of 4MU-NANA by neuraminidase. The substrate 3 is not limited to 4MU-NANA, and any conventionally known one can be used as long as it releases a chromophore that can be detected optically by neuraminic acid hydrolysis by neuraminidase. 4MU of the reaction product 4 has a hydroxyl group as shown by the following formula.
Figure JPOXMLDOC01-appb-C000002
Figure JPOXMLDOC01-appb-C000002
 本発明に係る検出方法では、この4MUの水酸基から水素を脱離させ、4MUを電荷を有する状態とするため、親水性溶媒42のpH値を4MUの酸解離定数(pKa)7.79よりも大きく設定する。4MUの水酸基から水素を脱離させることにより、疎水性溶媒43で被覆された、親水性溶媒42の液滴に含まれる4MUが、その電荷のために疎水性溶媒43へ移行できず、結果として親水性溶媒42の液滴中に高濃度に蓄積することとなる。 In the detection method according to the present invention, hydrogen is desorbed from the hydroxyl group of 4 MU to bring 4 MU into a charged state, so that the pH value of the hydrophilic solvent 42 is higher than the acid dissociation constant (pKa) of 7.79 of 4 MU. Set larger. By desorbing hydrogen from the hydroxyl group of 4MU, the 4MU contained in the droplet of the hydrophilic solvent 42 coated with the hydrophobic solvent 43 cannot move to the hydrophobic solvent 43 due to its charge, and as a result It accumulates in a high concentration in the droplets of the hydrophilic solvent 42.
 仮に、親水性溶媒42のpH値が4MUの酸解離定数(pKa)よりも小さい場合、4MUは水酸基を有する状態となるため、電荷を有さないかあるいは水酸基から水素が脱離している場合に比して電荷が小さくなる。親水性溶媒42の液滴に含まれる4MUが電荷を有さない場合あるいは電荷が小さい場合には、親水性溶媒42と界面接触する疎水性溶媒43へ4MUが移行しやすくなるため、4MUが親水性溶媒42の液滴中から失われたり、液滴中の4MU濃度が低下したりする。 If the pH value of the hydrophilic solvent 42 is smaller than the acid dissociation constant (pKa) of 4 MU, 4 MU has a hydroxyl group, and therefore has no charge or when hydrogen is desorbed from the hydroxyl group. In comparison, the charge is reduced. When the 4MU contained in the droplet of the hydrophilic solvent 42 has no electric charge or has a small electric charge, the 4MU easily moves to the hydrophobic solvent 43 that is in interface contact with the hydrophilic solvent 42. The organic solvent 42 is lost from the droplet, or the 4MU concentration in the droplet is lowered.
 従来、ノイラミニダーゼと4MU-NANAとの反応は、ノイラミニダーゼによる酵素反応の至適pHである5付近のpH条件下で行い、遊離した4MUの検出を、4MUの蛍光効率(量子効率)が最大化されるpHである10付近のpH条件下で行っていた。これに対して、本発明は、ノイラミニダーゼと4MU-NANAとの反応及び4MUの検出を、いずれも、4MUのpKa7.79よりも大きいpH条件下で行う点を技術的特徴の1つとするものである。 Conventionally, the reaction between neuraminidase and 4MU-NANA is performed under pH conditions around 5 which is the optimum pH for the enzyme reaction by neuraminidase, and the detection of released 4MU is maximized with the fluorescence efficiency (quantum efficiency) of 4MU. It was carried out under a pH condition of around 10 which is a pH value. In contrast, one of the technical features of the present invention is that the reaction of neuraminidase with 4MU-NANA and the detection of 4MU are both carried out under pH conditions higher than 4MU pKa7.79. is there.
 なお、基質3と酵素5の反応は、本手順前においても、基質3と酵素5が接触すれば進行し得るものであるが、本手順においてウイルス2と基質3を包含する親水性溶媒42の液滴が形成される前には、生成した反応生成物4が極小容積中に蓄積されることがない。このため、反応生成物4の光学検出において、封入手順(2)前に生成した反応生成物4の影響は無視できる程度に小さい。 It should be noted that the reaction between the substrate 3 and the enzyme 5 can proceed if the substrate 3 and the enzyme 5 come into contact with each other even before this procedure. However, in this procedure, the hydrophilic solvent 42 containing the virus 2 and the substrate 3 is used. Before the droplet is formed, the generated reaction product 4 is not accumulated in the minimum volume. For this reason, in the optical detection of the reaction product 4, the influence of the reaction product 4 generated before the encapsulation procedure (2) is small enough to be ignored.
 4MUを含む4-メチルウンベリフェリル-α-D-ノイラミン酸の他に、同様に、親水性溶媒42の液滴中から、当該液滴を被覆する疎水性溶媒43への反応生成物4の移行の問題を生じ得る発色基質として以下が挙げられる。 In addition to 4-methylumbelliferyl-α-D-neuraminic acid containing 4MU, the reaction product 4 from the droplets of the hydrophilic solvent 42 to the hydrophobic solvent 43 covering the droplets similarly Examples of chromogenic substrates that can cause migration problems include:
 4-メチルウンベリフェロン(4-Methylumbelliferone)を含む誘導体であって、4MU-NANA以外のもの。ここで、「誘導体」とは、構造中に、「発色団」としての4MUと、酵素5との反応によって切断される「基質」とを有する化合物を意味する。 Derivatives containing 4-methylumbelliferone other than 4MU-NANA. Here, the “derivative” means a compound having 4MU as “chromophore” and “substrate” cleaved by the reaction with the enzyme 5 in the structure.
 フルオレセイン(Fluorescein)を発色団として含む誘導体。酵素5と接触し反応すると、酵素5により基質が切断され、蛍光物質であるフルオレセイン(pKa:6.4)が反応生成物4として遊離する。フルオレセインの構造を以下に示す。 Derivatives containing fluorescein as a chromophore. When the enzyme 5 is brought into contact and reacted, the substrate is cleaved by the enzyme 5 and the fluorescent substance fluorescein (pKa: 6.4) is released as the reaction product 4. The structure of fluorescein is shown below.
Figure JPOXMLDOC01-appb-C000003
Figure JPOXMLDOC01-appb-C000003
 レゾルフィン(Resorufin)を発色団として含む誘導体。酵素5と接触し反応すると、酵素5により基質が切断され、蛍光物質であるレゾルフィン(pKa:6.0)が反応生成物4として遊離する。レゾルフィンの構造を以下に示す。 Derivatives containing resorufin as a chromophore. When reacted with the enzyme 5, the substrate is cleaved by the enzyme 5 and resorufin (pKa: 6.0), which is a fluorescent substance, is released as the reaction product 4. The structure of resorufin is shown below.
Figure JPOXMLDOC01-appb-C000004
Figure JPOXMLDOC01-appb-C000004
 ローダミン(Rhodamine)を発色団として含む誘導体。酵素5と接触し反応すると、酵素5により基質が切断され、蛍光物質であるローダミン(pKa:6.0)が反応生成物4として遊離する。ローダミンの構造を以下に示す。 Derivatives containing rhodamine as a chromophore. When reacted with the enzyme 5, the substrate is cleaved by the enzyme 5, and the fluorescent substance rhodamine (pKa: 6.0) is released as the reaction product 4. The structure of rhodamine is shown below.
Figure JPOXMLDOC01-appb-C000005
Figure JPOXMLDOC01-appb-C000005
 これらの誘導体を基質3として用いる場合には、親水性溶媒42のpH値を、誘導体から生じる反応生成物4の酸解離定数(pKa)よりも大きく設定する。これにより、各反応生成物4を電荷を有する状態とし、疎水性溶媒43への移行(漏れ出し)を防止して、親水性溶媒42の液滴中に反応生成物4を高濃度に蓄積させることができる(実施例1参照)。 When these derivatives are used as the substrate 3, the pH value of the hydrophilic solvent 42 is set larger than the acid dissociation constant (pKa) of the reaction product 4 generated from the derivative. Thereby, each reaction product 4 is brought into a charged state, is prevented from shifting (leaking out) to the hydrophobic solvent 43, and the reaction product 4 is accumulated in a high concentration in the droplets of the hydrophilic solvent 42. (See Example 1).
 さらに、親水性溶媒42の液滴中から疎水性溶媒43への反応生成物4の漏れ出しは、親水性溶媒42中の緩衝物質を所定の濃度以上に設定することで、さらに効果的に防止できる(実施例2参照)。緩衝物質の濃度は、例えば50mM以上、好ましくは100mM以上、より好ましくは500mM以上、さらに好ましくは1M以上とされる。 Further, the leakage of the reaction product 4 from the droplets of the hydrophilic solvent 42 to the hydrophobic solvent 43 can be prevented more effectively by setting the buffer substance in the hydrophilic solvent 42 to a predetermined concentration or more. Yes (see Example 2). The concentration of the buffer substance is, for example, 50 mM or more, preferably 100 mM or more, more preferably 500 mM or more, and further preferably 1 M or more.
[検出手順(A3)]
 本手順では、親水性溶媒42の液滴中に生成した反応生成物4の光学検出を行う。
[Detection Procedure (A3)]
In this procedure, optical detection of the reaction product 4 generated in the droplet of the hydrophilic solvent 42 is performed.
 反応生成物4の光学検出は、基質3と反応生成物4との光学特性の差を検出することが可能な公知の手段を用いて行うことができる。例えば、イメージセンサや吸光度計、旋光度計を用いて特定の吸光度や旋光度のシフトを検出すること、イメージセンサや蛍光顕微鏡、蛍光測定器を用いて特定の蛍光波長を検出することにより、反応生成物4を光学的に検出できる。 The optical detection of the reaction product 4 can be performed using a known means capable of detecting a difference in optical properties between the substrate 3 and the reaction product 4. For example, by detecting a specific absorbance or optical rotation shift using an image sensor, an absorptiometer, or a polarimeter, and by detecting a specific fluorescence wavelength using an image sensor, a fluorescence microscope, or a fluorometer. Product 4 can be detected optically.
 ウイルス2がインフルエンザウイルスであり、基質3に4MU-NANAを用いる場合においては、生成する4MU(反応生成物4)の蛍光検出を行う。 When the virus 2 is an influenza virus and 4MU-NANA is used as the substrate 3, fluorescence detection of 4MU (reaction product 4) to be produced is performed.
 検出された反応生成物4(例えば4MU)の検出強度(例えば蛍光強度)に基づけばウイルスの粒子数及び/又は亜種(サブタイプ)を決定することができる。 Based on the detected intensity (for example, fluorescence intensity) of the detected reaction product 4 (for example, 4MU), the number of virus particles and / or subtypes can be determined.
 具体的には、インフルエンザウイルスの場合、まず、検出された蛍光強度と、予め作成した蛍光強度とノイラミニダーゼ活性との関係を規定した標準曲線とを用いてノイラミニダーゼの酵素活性値を算出する。次に、算出された酵素活性値と、予め作成した酵素活性値とウイルス粒子数との関係を規定した標準曲線を用いてインフルエンザウイルスの粒子数を定量する。これにより、対象から分離された生物試料中にウイルスが含まれているかの判定に加えて、ウイルス量を定量的に決定することもでき(アナログ定量)、対象におけるインフルエンザウイルスの感染の有無及び感染の強度を診断できる。標準曲線には、蛍光強度とウイルス粒子数との関係を直接規定したものを用いてもよい。 Specifically, in the case of influenza virus, first, the enzyme activity value of neuraminidase is calculated using the detected fluorescence intensity and a standard curve that defines the relationship between the fluorescence intensity and neuraminidase activity prepared in advance. Next, the number of influenza virus particles is quantified using the calculated enzyme activity value and a standard curve that defines the relationship between the enzyme activity value and the number of virus particles prepared in advance. In addition to determining whether the biological sample separated from the subject contains viruses, the amount of virus can also be determined quantitatively (analog quantification), and whether or not the subject is infected with influenza virus. Can be diagnosed. A standard curve that directly defines the relationship between the fluorescence intensity and the number of virus particles may be used.
 また、例えばインフルエンザウイルスではB型ウイルスに比してA型ウイルスのほうが高いノイラミニダーゼ活性を有することが知られている。そして、本発明者らは、本発明に係る検出方法を用いてA型ウイルスとB型ウイルスがノイラミニダーゼ活性において2倍程度相違し、両者を活性値の大小に基づいて有効に区別して検出できることを見出している。このような場合、まず、検出された蛍光強度と、予め作成した蛍光強度とノイラミニダーゼ活性との関係を規定した標準曲線とを用いてノイラミニダーゼの酵素活性値を算出する。次に、算出された酵素活性値と、予め作成した酵素活性値とサブタイプとの関係式を用いてインフルエンザウイルスのサブタイプを決定できる。例えばインフルエンザウイルスであれば、算出された酵素活性値が基準値以上であればA型と判定し、基準値未満であればB型と判定できる。これにより、対象から分離された生物試料中にウイルスが含まれているかの判定に加えて、ウイルスサブタイプを決定することもでき、対象における感染インフルエンザウイルスのサブタイプを診断できる。関係式には、蛍光強度とサブタイプとの関係を直接規定したものを用いてもよい。 In addition, for example, in influenza virus, it is known that type A virus has higher neuraminidase activity than type B virus. The inventors of the present invention use the detection method according to the present invention that the type A virus and the type B virus differ by about twice in neuraminidase activity, and that both can be effectively distinguished and detected based on the magnitude of the activity value. Heading. In such a case, first, the enzyme activity value of neuraminidase is calculated using the detected fluorescence intensity and a standard curve that defines the relationship between the fluorescence intensity and neuraminidase activity prepared in advance. Next, the subtype of influenza virus can be determined using the calculated enzyme activity value and the relational expression between the enzyme activity value and the subtype prepared in advance. For example, in the case of influenza virus, it can be determined as type A if the calculated enzyme activity value is greater than or equal to the reference value, and can be determined as type B if it is less than the reference value. Thereby, in addition to the determination of whether the virus is contained in the biological sample separated from the subject, the virus subtype can also be determined, and the subtype of the infected influenza virus in the subject can be diagnosed. A relational expression that directly defines the relationship between fluorescence intensity and subtype may be used.
 さらに、上述の通り、親水性溶媒42中において、ウイルス2が十分に低い濃度に希釈されている場合、1つの収容部13に入るウイルス2の数は0又は最大で1となり得る。この場合、反応生成物4が検出された収容部13の数と、反応生成物4が検出されない収容部13の数との比率を用いて、予め作成した当該比率とウイルス粒子数との関係を規定した標準曲線に基づいて、ウイルス量を定量的に決定することもできる(デジタル定量)。
 封入手順(2)においては、親水性溶媒42の液滴中に反応生成物4を高濃度に蓄積させることができるため、ウイルス2が1粒子のみ収容部13に入っている場合であっても、反応生成物4の検出を高感度に行うことができる。従って、本発明に係る検出方法によれば、生物試料中にごく微量に含まれるウイルスであっても高感度に検出でき、病原性微生物の量を高精度に決定することが可能となる。
Furthermore, as described above, when the virus 2 is diluted to a sufficiently low concentration in the hydrophilic solvent 42, the number of viruses 2 entering one container 13 can be 0 or 1 at the maximum. In this case, using the ratio between the number of storage units 13 in which the reaction product 4 is detected and the number of storage units 13 in which the reaction product 4 is not detected, the relationship between the ratio prepared in advance and the number of virus particles is calculated. The viral load can also be determined quantitatively (digital quantification) based on a defined standard curve.
In the encapsulation procedure (2), the reaction product 4 can be accumulated in a high concentration in the droplets of the hydrophilic solvent 42, so that even when only one particle of virus 2 is contained in the container 13. The reaction product 4 can be detected with high sensitivity. Therefore, according to the detection method of the present invention, even a very small amount of virus in a biological sample can be detected with high sensitivity, and the amount of pathogenic microorganisms can be determined with high accuracy.
 また、親水性溶媒42の液滴中に反応生成物4を高濃度に蓄積させられることにより高い蛍光強度が得られるため、光学検出において比較的感度の低い簡易な撮像装置を用いることができ、例えばスマートフォンに搭載されたカメラなどでの光学検出が可能と期待される。スマートフォンに搭載されたカメラなどの簡易な撮像装置による光学検出は、比較的規模の小さな病院や診療所、個人における本発明に係る病原性微生物の検出方法の実施を容易にし得る。さらに、スマートフォンが備える通信手段を利用すれば、病原性微生物の検出情報をサーバに送信し、蓄積された情報(ビックデータ)を解析することで、流行の地域、時期及びサブタイプなどの把握や予測に供することが可能と期待される。 Further, since the reaction product 4 can be accumulated in a high concentration in the droplets of the hydrophilic solvent 42 to obtain a high fluorescence intensity, a simple imaging device with relatively low sensitivity can be used for optical detection. For example, optical detection with a camera mounted on a smartphone is expected to be possible. Optical detection by a simple imaging device such as a camera mounted on a smartphone can facilitate the implementation of the pathogenic microorganism detection method according to the present invention in a relatively small hospital, clinic, or individual. Furthermore, if the communication means provided in the smartphone is used, the detection information of pathogenic microorganisms is transmitted to the server, and the accumulated information (big data) is analyzed, so that it is possible to grasp the epidemic area, time, subtype, etc. It is expected to be available for prediction.
 上記では、ウイルス2がインフルエンザウイルスであり、基質3に4MU-NANAを用いる場合を例に説明した。本発明において、例えば、コロナウイルス、重症急性呼吸器症候群(SARS)コロナウイルスあるいは中東呼吸器症候群(MERS)ウイルスを検出対象とする場合、基質3には、これらのウイルスが表面に有するヘマグルチニンエステラーゼ(酵素5)によって加水分解を受け、上述のような発色団(反応生成物4)を遊離させるものを用いればよい。 In the above description, the case where the virus 2 is an influenza virus and 4MU-NANA is used as the substrate 3 has been described as an example. In the present invention, for example, when a coronavirus, severe acute respiratory syndrome (SARS) coronavirus or Middle Eastern respiratory syndrome (MERS) virus is a detection target, the substrate 3 includes hemagglutinin esterase (on the surface) of these viruses. What is necessary is just to use what hydrolyzes by the enzyme 5) and releases the chromophore (reaction product 4) as mentioned above.
 また、例えば、ヒト免疫不全ウイルス(HIV)、B型肝炎ウイルスあるいはヒトT細胞白血病ウイルス(HTLV)を検出対象とする場合、基質3には、これらのウイルスが表面又は内部に有する逆転写酵素(酵素5)によって重合化される核酸モノマーであってよい。核酸モノマーに蛍光色素をラベルしておくことで、重合による反応生成物である核酸鎖において、核酸モノマーに比して増加した蛍光強度が検出される。同様に、例えば、エボラウイルス、C型肝炎ウイルス、ラッサウイルス、ハンタウイルス、狂犬病ウイルス、日本脳炎ウイルス、黄熱ウイルス、デング熱ウイルス、風疹ウイルス、ロタウイルスあるいはノロウイルスを検出対象とする場合、基質3には、これらのウイルスが表面又は内部に有するRNA依存RNAポリメラーゼ(酵素5)によって重合化される核酸モノマーに蛍光色素をラベルしたものを用いることができる。なお、核酸モノマーの核酸鎖への重合に伴って、蛍光強度が増加する構成に限らず、反応後に反応前とは異なる光学特性(吸光度、旋光度及び蛍光等)が現れる構成であれば広く採用可能である。 In addition, for example, when human immunodeficiency virus (HIV), hepatitis B virus, or human T cell leukemia virus (HTLV) is to be detected, the substrate 3 has reverse transcriptase (on the surface or inside thereof) It may be a nucleic acid monomer that is polymerized by the enzyme 5). By labeling the nucleic acid monomer with a fluorescent dye, the fluorescence intensity increased as compared with the nucleic acid monomer is detected in the nucleic acid chain that is a reaction product of polymerization. Similarly, when the detection target is, for example, Ebola virus, hepatitis C virus, Lassa virus, hantavirus, rabies virus, Japanese encephalitis virus, yellow fever virus, dengue virus, rubella virus, rotavirus or norovirus, substrate 3 These can be prepared by labeling a fluorescent dye to a nucleic acid monomer that is polymerized by the RNA-dependent RNA polymerase (enzyme 5) possessed on the surface or inside of these viruses. It is not limited to the configuration in which the fluorescence intensity increases with the polymerization of the nucleic acid monomer to the nucleic acid chain, but it is widely adopted if it has a configuration in which optical characteristics (absorbance, optical rotation, fluorescence, etc.) that are different from those before the reaction appear after the reaction. Is possible.
 このように、本発明に係る検出方法において、基質は、検出対象とする病原性微生物が表面又は内部に有する酵素によって適宜選択され得るものである。この際、選択された反応生成物のpKaに応じて、導入手順(1)で用いられる親水性溶媒のpH値を当該pKaよりも大きくなるように設計すればよい。 As described above, in the detection method according to the present invention, the substrate can be appropriately selected depending on the enzyme that is present on the surface or inside the pathogenic microorganism to be detected. At this time, the pH value of the hydrophilic solvent used in the introduction procedure (1) may be designed to be larger than the pKa according to the pKa of the selected reaction product.
 また、基質は、例えば、インフルエンザウイルスとムンプウイルスのように同一酵素(ノイラミニダーゼ)を有する病原性微生物であっても、それぞれの病原性微生物の酵素の基質特異性に応じて異なる設計にされ得る。例えば、インフルエンザウイルスの検出するための発色基質として4MU-NANAを用い、ムンプウイルスを検出するための発色基質としての4MU-NANAにおいてその加水分解により生成する発色団を4MUからフルオレセインなどの他の蛍光物質に変更したものを用いる。このように、基質の一部を変更することで、それぞれの発色基質のノイラミニダーゼに対する親和性が、同一種の酵素であっても、対インフルエンザウイルス酵素と対ムンプウイルスの酵素との間で変化し得る。これによって、本発明に係る検出方法では、両者を区別して検出することも可能である。 In addition, even if the substrate is a pathogenic microorganism having the same enzyme (neuraminidase) such as influenza virus and mump virus, the substrate can be designed differently depending on the substrate specificity of the enzyme of each pathogenic microorganism. For example, 4MU-NANA is used as a chromogenic substrate for detecting influenza virus, and a chromophore produced by hydrolysis of 4MU-NANA as a chromogenic substrate for detecting mump virus is converted from 4MU to other fluorescent substances such as fluorescein. Use what was changed to. In this way, by changing a part of the substrate, the affinity of each chromogenic substrate for neuraminidase can be changed between the enzyme against influenza virus and the enzyme against mump virus even for the same type of enzyme. . Thus, the detection method according to the present invention can also detect both of them separately.
2.病原性微生物の薬剤感受性の検出方法
 本発明に係る病原性微生物に感染した対象又は感染した疑いがある対象から分離された生物試料中の病原性微生物の薬剤感受性を検出する方法は、以下の手順を含む。
(B1)病原性微生物を収容可能な複数の収容部が、疎水性の上面を有する側壁によって互いに隔てられて形成されている下層部と、当該下層部における当該収容部が形成されている面に対向している上層部との間の空間に、前記生物試料と、前記病原性微生物の表面又は内部に存在する酵素による反応の基質となる物質と、前記酵素の阻害薬を含む親水性溶媒を導入する導入手順。
(B2)前記空間に疎水性溶媒を導入して、前記収容部内に、疎水性溶媒で被覆されかつ前記病原性微生物と前記物質と前記阻害薬とを包含する、親水性溶媒の液滴を形成する封入手順。
(B3)前記液滴中における前記酵素と前記物質との反応により生成する反応生成物を光学的に検出する検出手順、(ここで、前記阻害薬の存在下における前記反応生成物の検出強度が、前記阻害薬の非存在下における前記反応生成物の検出強度よりも減少する場合、前記病原性微生物が前記阻害薬に感受性を有することを示す)。
2. Method for Detecting Drug Sensitivity of Pathogenic Microorganism The method for detecting drug sensitivity of a pathogenic microorganism in a biological sample isolated from a subject infected with or suspected of being infected with the pathogenic microorganism according to the present invention is as follows. including.
(B1) A plurality of accommodating portions capable of accommodating pathogenic microorganisms are separated from each other by a side wall having a hydrophobic upper surface, and a surface where the accommodating portion in the lower layer portion is formed. In the space between the upper layers facing each other, a hydrophilic solvent containing the biological sample, a substance serving as a substrate for a reaction by an enzyme present on or inside the pathogenic microorganism, and an inhibitor of the enzyme Installation procedure to be introduced.
(B2) A hydrophobic solvent is introduced into the space, and droplets of the hydrophilic solvent that are covered with the hydrophobic solvent and include the pathogenic microorganism, the substance, and the inhibitor are formed in the housing portion. Enclosure procedure to.
(B3) a detection procedure for optically detecting a reaction product generated by a reaction between the enzyme and the substance in the droplet, wherein the detection intensity of the reaction product in the presence of the inhibitor is , If it is less than the detected intensity of the reaction product in the absence of the inhibitor, it indicates that the pathogenic microorganism is sensitive to the inhibitor).
[導入手順(B1)]
 本発明に係る病原性微生物の薬剤感受性の検出方法の導入手順(B1)は、薬剤感受性の評価対象となる酵素阻害薬が親水性溶媒に含まれる点でのみ、上述の病原性微生物検出方法の導入手順(A1)と異なる。導入手順(B1)では、病原性微生物及び基質に加えて、病原性微生物の表面又は内部に存在する酵素の阻害薬が収容部に入ることとなる。
[Introduction procedure (B1)]
The introduction procedure (B1) of the method for detecting drug susceptibility of pathogenic microorganisms according to the present invention is based on the above-described method for detecting pathogenic microorganisms only in that an enzyme inhibitor to be evaluated for drug sensitivity is contained in a hydrophilic solvent. Different from the introduction procedure (A1). In the introduction procedure (B1), in addition to the pathogenic microorganism and the substrate, an enzyme inhibitor present on the surface or inside of the pathogenic microorganism enters the container.
[封入手順(B2)]
 本発明に係る病原性微生物の薬剤感受性の検出方法の封入手順(B2)の操作は、上述の病原性微生物検出方法の封入手順(A2)に同じである。封入手順(B2)では、収容部内に、疎水性溶媒で被覆されかつ病原性微生物、基質及び阻害薬を包含する、親水性溶媒の液滴が形成されることとなる。
[Encapsulation procedure (B2)]
The operation of the encapsulation procedure (B2) of the method for detecting drug sensitivity of a pathogenic microorganism according to the present invention is the same as the encapsulation procedure (A2) of the above-mentioned pathogenic microorganism detection method. In the encapsulation procedure (B2), droplets of the hydrophilic solvent that are covered with the hydrophobic solvent and include the pathogenic microorganism, the substrate, and the inhibitor are formed in the container.
[検出手順(B3)]
 本発明に係る病原性微生物の薬剤感受性の検出方法の検出手順(B3)では、上述の病原性微生物検出方法の検出手順(A3)と同様にして、親水性溶媒の液滴中に生成した反応生成物の光学検出を行う。
[Detection Procedure (B3)]
In the detection procedure (B3) of the method for detecting drug susceptibility of pathogenic microorganisms according to the present invention, the reaction generated in the droplet of the hydrophilic solvent in the same manner as the detection procedure (A3) of the above-described pathogenic microorganism detection method. Optical detection of the product is performed.
 阻害薬の存在下における前記反応生成物の検出強度を、阻害薬の非存在下における前記反応生成物の検出強度と比較し、前者が後者よりも減少する場合、阻害薬によって親水性溶媒の液滴中における反応生成物の生成が抑制されていることが示される。すなわち、病原性微生物が有する酵素が阻害されていることから、病原性微生物が阻害薬に感受性を有することが示される。
 一方、阻害薬の非存在下における前記反応生成物の検出強度が、阻害薬の非存在下における前記反応生成物の検出強度と比較して同等である場合、親水性溶媒の液滴中における反応生成物の生成は阻害剤によって抑制されていないことが示される。すなわち、病原性微生物が有する酵素は阻害されていないことから、病原性微生物が阻害薬に耐性を有することが示される。
When the detection intensity of the reaction product in the presence of the inhibitor is compared with the detection intensity of the reaction product in the absence of the inhibitor, It is shown that the formation of reaction products in the droplets is suppressed. That is, the enzyme contained in the pathogenic microorganism is inhibited, which indicates that the pathogenic microorganism is sensitive to the inhibitor.
On the other hand, when the detection intensity of the reaction product in the absence of the inhibitor is equivalent to the detection intensity of the reaction product in the absence of the inhibitor, the reaction in the droplet of the hydrophilic solvent Product formation is shown not to be inhibited by the inhibitor. That is, the enzyme contained in the pathogenic microorganism is not inhibited, indicating that the pathogenic microorganism is resistant to the inhibitor.
 具体的には、病原性微生物がインフルエンザウイルスである場合には、例えば、基質として4MU-NANA、阻害薬としてノイラミニダーゼ阻害剤(オセタミビル、ザナミビル等)を用いて、ノイラミニダーゼ阻害剤の存在下と非存在下の条件以外を同一とした2試験群について、生成する4MUの蛍光検出を行う。ノイラミニダーゼ阻害剤の存在下における4MUの検出強度を、ノイラミニダーゼ阻害剤の非存在下における4MUの検出強度と比較し、前者が後者よりも減少する場合、ノイラミニダーゼ阻害剤によって親水性溶媒の液滴中における4MUの生成が抑制されていることが示される。すなわち、インフルエンザウイルスが有するノイラミニダーゼが阻害されていることから、インフルエンザウイルスがノイラミニダーゼ阻害剤に感受性を有することが示される。
 一方、ノイラミニダーゼ阻害剤の非存在下における4MUの検出強度が、ノイラミニダーゼ阻害剤の非存在下における4MUの検出強度と比較して同等である場合、親水性溶媒の液滴中における4MUの生成はノイラミニダーゼ阻害剤によって抑制されていないことが示される。すなわち、インフルエンザウイルスが有するノイラミニダーゼは阻害されていないことから、インフルエンザウイルスがノイラミニダーゼ阻害剤に耐性を有することが示される。
Specifically, when the pathogenic microorganism is an influenza virus, for example, using 4MU-NANA as a substrate and neuraminidase inhibitors (such as osetamivir, zanamivir) as inhibitors, the presence or absence of neuraminidase inhibitors The generated 4MU fluorescence is detected for the two test groups that are identical except under the following conditions. When the detection intensity of 4MU in the presence of neuraminidase inhibitor is compared to the detection intensity of 4MU in the absence of neuraminidase inhibitor, if the former is reduced over the latter, the neuraminidase inhibitor will cause the It is shown that the generation of 4MU is suppressed. That is, since neuraminidase possessed by influenza virus is inhibited, it is shown that influenza virus is sensitive to neuraminidase inhibitors.
On the other hand, when the detection intensity of 4MU in the absence of neuraminidase inhibitor is comparable to the detection intensity of 4MU in the absence of neuraminidase inhibitor, the production of 4MU in the droplet of the hydrophilic solvent is neuraminidase. It is shown that it is not suppressed by the inhibitor. That is, the neuraminidase possessed by the influenza virus is not inhibited, indicating that the influenza virus is resistant to the neuraminidase inhibitor.
 このような薬剤感受性の検出を行い得る病原微生物と酵素阻害薬の組合わせとしては、例えば、コロナウイルス、重症急性呼吸器症候群(SARS)コロナウイルスあるいは中東呼吸器症候群(MERS)ウイルスを検出対象とする場合、これらのウイルスが表面に有するヘマグルチニンエステラーゼ(HE)の阻害薬(HE抗体, 3,4-dichloroisocoumarin, 9-O-Acetylated Polysialoside等)が挙げられる。 Examples of combinations of pathogenic microorganisms and enzyme inhibitors that can detect such drug susceptibility include coronavirus, severe acute respiratory syndrome (SARS) coronavirus, or Middle East respiratory syndrome (MERS) virus as detection targets. In this case, hemagglutinin esterase (HE) inhibitors (HE antibody, 3,4-dichloroisocoumarin, 9-O-Acetylated Polysialoside, etc.) on the surface of these viruses can be mentioned.
 また、例えば、ヒト免疫不全ウイルス(HIV)、B型肝炎ウイルスあるいはヒトT細胞白血病ウイルス(HTLV)を検出対象とする場合、これらのウイルスが表面又は内部に有する逆転写酵素の阻害薬(レトロビル(グラクソ・スミスクライン・Inc.、ジドブジン/AZT)、ヴァイデックス(ブリストル・マイヤーズスクイブ・Inc.、ジダノシン/ddI)、ハイビッド(ホフマンーラ・ロシュ社)(ザルシタビン/ddC)、ゼリット(ブリストル・マイヤーズスクイブ社、スタブジン/d4T)、エピビル(グラクソ・スミスクライン社、ラミブジン/3TC)およびコンビビル(グラクソ・スミスクライン社、ジドブジン/ラミブジン)ビラミューン(ベーリンガーインゲルハイム・ファーマシューティカルズ・Inc.、ネビラピン)、レスクリプター(ファルマシア/アップジョン社、デラビルジン)およびサスティバ(デュポン・ファーマ・Co.、エファビレンズ)等)が挙げられる。
 同様に、例えば、エボラウイルス、C型肝炎ウイルス、ラッサウイルス、ハンタウイルス、狂犬病ウイルス、日本脳炎ウイルス、黄熱ウイルス、デング熱ウイルス、風疹ウイルス、ロタウイルスあるいはノロウイルスを検出対象とする場合、これらのウイルスが表面又は内部に有するRNA依存RNAポリメラーゼの阻害薬(ファビピラビル、リバビリン等)が挙げられる。
In addition, for example, when human immunodeficiency virus (HIV), hepatitis B virus or human T cell leukemia virus (HTLV) is to be detected, inhibitors of reverse transcriptase (retrovir ( GlaxoSmithKline Inc., Zidovudine / AZT), Videx (Bristol-Myers Squibb Inc., Zidanocin / ddI), Hibid (Hoffmanla Roche) (Zarcitabine / ddC), Zellit (Bristol-Myers Squibb), Stavudine / d4T), Epivir (GlaxoSmithKline, Lamivudine / 3TC) and Combivir (GlaxoSmithKline, Zidovudine / Lamivudine) Vilamune (Boehringer Ingelheim Pharmaceuticals Inc. , Nevirapine), Rescriptor (Pharmacia / Upjohn, delavirdine), and SUSTIVA (DuPont Pharma · Co., efavirenz), and the like).
Similarly, for example, when detecting Ebola virus, hepatitis C virus, Lassa virus, hantavirus, rabies virus, Japanese encephalitis virus, yellow fever virus, dengue virus, rubella virus, rotavirus or norovirus, these viruses Inhibitors of RNA-dependent RNA polymerase (Fabipyravir, ribavirin, etc.) possessed on the surface or inside.
 さらに、例えば、大腸菌群、腸炎ビブリオ菌、カンピロバクター、エンテロバクターあるいはバチルス属菌を検出対象とする場合、これらの細菌が表面又は内部に有するガラクトシダーゼの阻害薬(Castanospermine、Conduritol B Epoxide、Bromoconduritol、2-Deoxy-D-Galactose等)、グルクロニダーゼの阻害薬(アセトグラトン、D-glucaro-1,4-lactone、リゾリン脂質類等)、キモトリプシン、トリプシンの阻害薬(大豆、鶏卵等由来トリプシンインヒビター、Arg4-Met5-マリノスタチン、Phenylmethylsulfonyl fluoride、Aminoethyl benzylsulfonyl fluoride、Aprotinin、Tosyl lysine chloromethyl ketone、tosyl phenylalanine chloromethyl ketone等)、キシロシダーゼの阻害薬(Castanospermine、Xyl-amidine等)が挙げられる。 Further, for example, when the coliform group, Vibrio parahaemolyticus, Campylobacter, Enterobacter or Bacillus spp. Are targeted for detection, inhibitors of galactosidase (Castanospermine, Conduritol B Epoxide, Bromoconduritol, 2- etc. Deoxy-D-Galactose), glucuronidase inhibitor (Asetoguraton, D-glucaro-1,4-lactone , lysophospholipids, etc.), chymotrypsin, trypsin inhibitor (soybean, egg or the like derived trypsin inhibitor, Arg 4 - Examples include Met 5 -marinostatin, phenylmethylsulfonyl fluoride, aminoethyl benzylsulfonyl fluoride, Aprotinin, Tosyl lysine chloromethyl ketone, tosyl phenylalanine chloromethyl ketone, and inhibitors of xylosidase (Castanospermine, Xyl-amidine, etc.).
 このように、本発明に係る病原性微生物の薬剤感受性の検出方法において、阻害薬は、検出対象とする病原性微生物が表面又は内部に有する酵素によって適宜選択され得るものである。 Thus, in the method for detecting drug susceptibility of pathogenic microorganisms according to the present invention, the inhibitor can be appropriately selected depending on the enzyme present on the surface or inside the pathogenic microorganism to be detected.
3.抗病原性微生物薬剤のスクリーニング方法
 本発明に係る抗病原性微生物薬剤をスクリーニングする方法は、以下の手順を含む。
(C1)病原性微生物を収容可能な複数の収容部が、疎水性の上面を有する側壁によって互いに隔てられて形成されている下層部と、当該下層部における当該収容部が形成されている面に対向している上層部との間の空間に、前記病原性微生物と、前記病原性微生物の表面又は内部に存在する酵素による反応の基質となる物質と、候補化合物とを含む親水性溶媒を導入する導入手順。
(C2)前記空間に疎水性溶媒を導入して、前記収容部内に、疎水性溶媒で被覆されかつ前記病原性微生物と前記物質と前記候補化合物とを包含する、親水性溶媒の液滴を形成する封入手順。
(C3)前記液滴中における前記酵素と前記物質との反応により生成する反応生成物を光学的に検出する検出手順、(ここで、前記候補化合物の存在下における前記反応生成物の検出強度が、前記候補化合物の非存在下における前記反応生成物の検出強度よりも減少する場合、前記候補化合物が抗病原性微生物活性を有することを示す)。
3. Screening Method for Anti-Pathogenic Microbial Drug A method for screening an anti-pathogenic microbial drug according to the present invention includes the following procedures.
(C1) On the surface where the plurality of accommodating portions capable of accommodating pathogenic microorganisms are separated from each other by a side wall having a hydrophobic upper surface, and on the surface where the accommodating portion in the lower layer portion is formed A hydrophilic solvent containing the pathogenic microorganism, a substance serving as a substrate for a reaction by an enzyme existing on or inside the pathogenic microorganism, and a candidate compound is introduced into a space between the upper layers facing each other. Installation procedure to do.
(C2) A hydrophobic solvent is introduced into the space, and droplets of the hydrophilic solvent that are covered with the hydrophobic solvent and include the pathogenic microorganism, the substance, and the candidate compound are formed in the container. Enclosure procedure to.
(C3) a detection procedure for optically detecting a reaction product generated by the reaction between the enzyme and the substance in the droplet, wherein the detection intensity of the reaction product in the presence of the candidate compound is , If it decreases below the detected intensity of the reaction product in the absence of the candidate compound, it indicates that the candidate compound has anti-pathogenic microbial activity).
[導入手順(C1)]
 本発明に係る抗病原性微生物薬剤のスクリーニング方法の導入手順(C1)は、抗病原性微生物活性の評価対象となる候補化合物が親水性溶媒に含まれる点でのみ、上述の病原性微生物検出方法の導入手順(A1)と異なる。導入手順(C1)では、病原性微生物及び基質に加えて、候補化合物が収容部に入ることとなる。
[Introduction procedure (C1)]
The introduction procedure (C1) of the screening method for an anti-pathogenic microbial agent according to the present invention is the above-mentioned pathogenic microorganism only in that a candidate compound to be evaluated for anti-pathogenic microbial activity is contained in a hydrophilic solvent. This is different from the detection method introduction procedure (A1). In the introduction procedure (C1), in addition to the pathogenic microorganism and the substrate, the candidate compound enters the housing unit.
[封入手順(C2)]
 本発明に係る抗病原性微生物薬剤のスクリーニング方法の封入手順(C2)の操作は、上述の病原性微生物検出方法の封入手順(A2)に同じである。封入手順(C2)では、収容部内に、疎水性溶媒で被覆されかつ病原性微生物、基質及び候補化合物を包含する、親水性溶媒の液滴が形成される。
[Encapsulation procedure (C2)]
The operation of the encapsulation procedure (C2) of the screening method for an anti-pathogenic microorganism according to the present invention is the same as the encapsulation procedure (A2) of the above-mentioned pathogenic microorganism detection method. In the encapsulation procedure (C2), droplets of a hydrophilic solvent that are coated with a hydrophobic solvent and include pathogenic microorganisms, substrates, and candidate compounds are formed in the container.
[検出手順(C3)]
 本発明に係る抗病原性微生物薬剤のスクリーニング方法の検出手順(C3)では、上述の病原性微生物検出方法の検出手順(A3)と同様にして、親水性溶媒の液滴中に生成した反応生成物の光学検出を行う。
[Detection Procedure (C3)]
In the detection procedure (C3) of the screening method for an anti-pathogenic microorganism according to the present invention, the reaction generated in the droplet of the hydrophilic solvent in the same manner as the detection procedure (A3) of the above-mentioned pathogenic microorganism detection method Optical detection of the product is performed.
 候補化合物の存在下における前記反応生成物の検出強度を、候補化合物の非存在下における前記反応生成物の検出強度と比較し、前者が後者よりも減少する場合、候補化合物によって親水性溶媒の液滴中における反応生成物の生成が抑制されていることが示される。すなわち、病原性微生物が有する酵素が阻害されていることから、候補化合物が抗病原性微生物活性を有することが示される。
 一方、候補化合物の非存在下における前記反応生成物の検出強度が、候補化合物の非存在下における前記反応生成物の検出強度と比較して同等である場合、親水性溶媒の液滴中における反応生成物の生成は阻害剤によって抑制されていないことが示される。すなわち、病原性微生物が有する酵素は阻害されていないことから、候補化合物が抗病原性微生物活性を有しないことが示される。
The detection intensity of the reaction product in the presence of the candidate compound is compared with the detection intensity of the reaction product in the absence of the candidate compound. It is shown that the formation of reaction products in the droplets is suppressed. That is, the enzyme possessed by the pathogenic microorganism is inhibited, indicating that the candidate compound has anti-pathogenic microbial activity.
On the other hand, when the detection intensity of the reaction product in the absence of the candidate compound is equivalent to the detection intensity of the reaction product in the absence of the candidate compound, the reaction in the droplet of the hydrophilic solvent Product formation is shown not to be inhibited by the inhibitor. That is, the enzyme possessed by the pathogenic microorganism is not inhibited, indicating that the candidate compound does not have anti-pathogenic microbial activity.
4.病原性微生物検出キット
 本発明に係るキットは、病原性微生物に感染した対象又は感染した疑いがある対象から分離された生物試料中の病原性微生物を検出するためのキットであって、
前記病原性微生物を収容可能な複数の収容部が疎水性の上面を有する側壁によって互いに隔てられて形成されている下層部と、前記下層部における前記収容部が形成されている面に対して空間を隔てて対向している上層部とを備えるアレイと、
前記病原性微生物の粒子表面又は内部に存在する酵素による反応の基質となる物質と、
前記酵素と前記物質との反応により生成する反応生成物の酸解離定数(pKa)よりも大きいpH値を有する親水性溶媒と、
疎水性溶媒と、を含む。
4). Pathogenic microorganism detection kit The kit according to the present invention is a kit for detecting pathogenic microorganisms in a biological sample isolated from a subject infected with or suspected of being infected with a pathogenic microorganism,
A space between a lower layer portion in which a plurality of accommodating portions capable of accommodating the pathogenic microorganisms are separated from each other by a side wall having a hydrophobic upper surface, and a surface of the lower layer portion where the accommodating portion is formed An array comprising an upper layer portion facing each other, and
A substance that is a substrate for a reaction by an enzyme present on the surface or inside of the pathogenic microorganism particles;
A hydrophilic solvent having a pH value greater than the acid dissociation constant (pKa) of the reaction product produced by the reaction between the enzyme and the substance;
A hydrophobic solvent.
 本発明に係るキットは、上述したアレイ1と、基質3、親水性溶媒42及び疎水性溶媒43を含むものである。基質3、親水性溶媒42及び疎水性溶媒43については既に説明した通りであるので、以下にはアレイ1の構成をさらに詳しく説明する。 The kit according to the present invention includes the above-described array 1, the substrate 3, the hydrophilic solvent 42, and the hydrophobic solvent 43. Since the substrate 3, the hydrophilic solvent 42, and the hydrophobic solvent 43 are as described above, the configuration of the array 1 will be described in more detail below.
 アレイ1の下層部10は、板状部材11と疎水性の上面を有する側壁12とを備えている。下層部10には、複数の収容部13が側壁12によって互いに隔てられて形成されている。 The lower layer portion 10 of the array 1 includes a plate-like member 11 and a side wall 12 having a hydrophobic upper surface. In the lower layer portion 10, a plurality of accommodating portions 13 are formed separated from each other by the side walls 12.
 板状部材11は親水性表面を有していることが好ましい。「親水性表面」とは、親水性溶媒との親和性が疎水性溶媒との親和性よりも高い表面を指す。板状部材11としては、固体材料であればよいが、例えばガラス、シリコン、高分子樹脂等を用いることができる。 The plate member 11 preferably has a hydrophilic surface. “Hydrophilic surface” refers to a surface that has a higher affinity with a hydrophilic solvent than with a hydrophobic solvent. The plate-like member 11 may be a solid material, but for example, glass, silicon, polymer resin, or the like can be used.
 側壁12は、板状部材11の表面上、好ましくは親水性表面上に設けられている、複数の収容部13の各々を隔てる構造物である。側壁12は、疎水性の上面を有している。「疎水性」とは、ここでは「親油性」と同じ意味で用いられ、疎水性溶媒との親和性が親水性溶媒との親和性よりも高いことをいう。 The side wall 12 is a structure that separates each of the plurality of accommodating portions 13 provided on the surface of the plate-like member 11, preferably on the hydrophilic surface. The side wall 12 has a hydrophobic upper surface. “Hydrophobic” is used herein in the same meaning as “lipophilic” and means that the affinity with a hydrophobic solvent is higher than the affinity with a hydrophilic solvent.
 なお、側壁12は、その上面、すなわち上層部20と対向する面が疎水性であればよく、側面、すなわち収容部13内の内壁は、疎水性であっても親水性であってもよい。 The side wall 12 only needs to have a hydrophobic upper surface, that is, a surface facing the upper layer portion 20, and the side surface, that is, the inner wall in the accommodating portion 13, may be hydrophobic or hydrophilic.
 例えば、側壁12は、親水性の構造物と、その上面に形成されている疎水性層とにより構成されていてもよい。親水性の構造物には、例えばガラス、シリコン、高分子樹脂等を用いることができる。疎水性層には、例えば撥水性の樹脂、フッ素系高分子樹脂等を用いることができる。フッ素系高分子樹脂としては、例えばアモルファスフッ素樹脂等が挙げられる。アモルファスフッ素樹脂は、高い疎水性を有し、かつ、生体試料に対する毒性が低いという理由で、好ましく用いられる。 For example, the side wall 12 may be composed of a hydrophilic structure and a hydrophobic layer formed on the upper surface thereof. For the hydrophilic structure, for example, glass, silicon, polymer resin, or the like can be used. For the hydrophobic layer, for example, a water-repellent resin, a fluorine-based polymer resin, or the like can be used. Examples of the fluoropolymer resin include amorphous fluororesins. Amorphous fluororesin is preferably used because it has high hydrophobicity and low toxicity to biological samples.
 上記アモルファスフッ素樹脂としては、例えば、CYTOP(登録商標)、TEFLON(登録商標)AF2400、およびTEFLON(登録商標)AF1600から選択した少なくとも1つを好適に用いることができる。中でも、微細加工が容易であるという理由で、CYTOP(登録商標)が最も好ましい。 As the amorphous fluororesin, for example, at least one selected from CYTOP (registered trademark), TEFLON (registered trademark) AF2400, and TEFLON (registered trademark) AF1600 can be suitably used. Among them, CYTOP (registered trademark) is most preferable because it is easy to perform microfabrication.
 また例えば、側壁12は、疎水性の材料により構成されていてもよい。側壁12として、例えばフッ素系高分子樹脂、パラキシリレン系高分子樹脂等を用いることができる。フッ素系高分子樹脂としては、例えばアモルファスフッ素樹脂等が挙げられる。アモルファスフッ素樹脂としては上述の樹脂を好適に用いることができる。 For example, the side wall 12 may be made of a hydrophobic material. As the side wall 12, for example, a fluorine-based polymer resin, a paraxylylene-based polymer resin, or the like can be used. Examples of the fluoropolymer resin include amorphous fluororesins. As the amorphous fluororesin, the above-described resins can be suitably used.
 側壁12は、板状部材11上に複数の収容部13が形成されるように構成されていればよく、例えば収容部13が形成される位置に孔が形成されている板形状の構造物であってもよい。 The side wall 12 should just be comprised so that the some accommodating part 13 may be formed on the plate-shaped member 11, for example, is a plate-shaped structure by which the hole is formed in the position in which the accommodating part 13 is formed. There may be.
 収容部13は、板状部材11の表面の一部を底面としており、底面が親水性である。収容部13の底面及び側面によって囲まれた領域の形状は、例えば円柱形状、角柱形状等であってもよい。 The housing portion 13 has a part of the surface of the plate-like member 11 as a bottom surface, and the bottom surface is hydrophilic. The shape of the region surrounded by the bottom surface and the side surface of the accommodating portion 13 may be, for example, a cylindrical shape, a prismatic shape, or the like.
 本実施形態では、収容部13の底面が親水性であり、かつ側壁12の上面が疎水性である。これにより、導入手順(1)において、親水性溶媒42を効率よく収容部13の中に導入することができるとともに、封入手順(2)において疎水性溶媒43が収容部13に入り込むことを防止することができる。 In this embodiment, the bottom surface of the accommodating portion 13 is hydrophilic, and the top surface of the side wall 12 is hydrophobic. Thereby, in the introduction procedure (1), the hydrophilic solvent 42 can be efficiently introduced into the housing part 13 and the hydrophobic solvent 43 is prevented from entering the housing part 13 in the sealing procedure (2). be able to.
 上層部20は、例えばガラス、シリコン、高分子樹脂等を用いることができる。上層部20は、下層部10における収容部13が形成されている面に対して空間30を隔てて対向している。すなわち、側壁12と疎水性層22との間に空間30があいている。この空間30は、流路を構成する。この構成により、アレイ1はフローセル構造となっている。 For the upper layer portion 20, for example, glass, silicon, polymer resin, or the like can be used. The upper layer portion 20 faces the surface of the lower layer portion 10 on which the accommodating portion 13 is formed with a space 30 therebetween. That is, there is a space 30 between the side wall 12 and the hydrophobic layer 22. This space 30 constitutes a flow path. With this configuration, the array 1 has a flow cell structure.
 空間30は、下層部10と上層部20との間に、下層部10と上層部20とが互いに対向する面と平行な方向に流体を流すための流路として用いられうる。 The space 30 can be used between the lower layer portion 10 and the upper layer portion 20 as a flow path for flowing a fluid in a direction parallel to the surfaces where the lower layer portion 10 and the upper layer portion 20 face each other.
 下層部10又は上層部20には、空間30に流体を導入するための貫通孔(図示せず)が形成されていてもよい。例えば、下層部10は、収容部13が形成されている領域と、収容部13が形成されていない領域とを有していてもよい。そして、下層部10における収容部13が形成されていない領域、又は上層部20におけるこの領域と対向する部分に、貫通孔が形成されていてもよい。 A through hole (not shown) for introducing a fluid into the space 30 may be formed in the lower layer portion 10 or the upper layer portion 20. For example, the lower layer part 10 may have a region where the accommodating part 13 is formed and a region where the accommodating part 13 is not formed. And the through-hole may be formed in the area | region in which the accommodating part 13 in the lower layer part 10 is not formed, or the part in the upper layer part 20 facing this area | region.
 本実施形態においては、空間30の上面を構成する上層部20の表面が疎水性であり、空間30の下面が側壁12の疎水性上面及び収容部13である。したがって、空間30のうち、収容部13の底面以外の部分は全て疎水性となっている。これにより、導入手順(1)において、親水性溶媒42を効率よく各収容部13内に導入することができる。また、封入手順(2)において、各収容部13内に疎水性溶媒43を入り込ませることがない。したがって、疎水性溶媒43を空間30内に導入することにより、各収容部13に液滴を効率よく形成させることができる。 In the present embodiment, the surface of the upper layer portion 20 constituting the upper surface of the space 30 is hydrophobic, and the lower surface of the space 30 is the hydrophobic upper surface of the side wall 12 and the accommodating portion 13. Therefore, all portions of the space 30 other than the bottom surface of the accommodating portion 13 are hydrophobic. Thereby, in the introduction procedure (1), the hydrophilic solvent 42 can be efficiently introduced into each accommodating portion 13. Further, in the enclosing procedure (2), the hydrophobic solvent 43 is not allowed to enter the respective accommodating portions 13. Therefore, by introducing the hydrophobic solvent 43 into the space 30, it is possible to efficiently form droplets in the respective accommodating portions 13.
[試験例1:親水性溶媒のpH値の検討]
 以下の手順によって、親水性溶媒のpH値が検出感度に及ぼす影響を検討した。
 まず、金らの報告(" Quantifying genetically inserted fluorescent protein in single iPS cells to monitor Nanog expression using electroactive microchamber arrays", Lab on Chip, 2014, Issue 4, Vol.14, p.730-736)に従って、Droplet array device(DAD)を作成した。カバーガラス(24mm×32mm)を洗浄、乾燥した後、アモルファスフッ素樹脂(CYTOP 816AP、旭硝子)をスピンコートし、180℃で1時間焼成した。アモルファスフッ素樹脂がコートされたカバーガラスに、ポジ型フォトレジスト(AZ-4903、AZ Electronic Materials)をスピンコートして、55℃で3分間焼成した後、さらに110℃で5分間焼成した。直径3μmの穴を5μm間隔で有するフォトマスクを用いてフォトリソグラフィーを行った。酸素プラズマでドライエッチング後、洗浄したカバーガラスをDADとして得た。DADは、直径4μm、深さ3μmのウェル(収容部)を有し(約100万個/10mm2)、ウェルの底面はカバーガラスが露出している。得られたDADを用いて図1に示すようなフローセル構造のアレイを作成した。
[Test Example 1: Examination of pH value of hydrophilic solvent]
The influence of the pH value of the hydrophilic solvent on the detection sensitivity was examined by the following procedure.
First, according to Kim et al.'S report ("Quantifying genetically inserted fluorescent protein in single iPS cells to monitor Nanog expression using electroactive microchamber arrays", Lab on Chip, 2014, Issue 4, Vol. 14, p.730-736). A device (DAD) was created. After the cover glass (24 mm × 32 mm) was washed and dried, an amorphous fluororesin (CYTOP 816AP, Asahi Glass) was spin-coated and baked at 180 ° C. for 1 hour. A positive type photoresist (AZ-4903, AZ Electronic Materials) was spin-coated on the cover glass coated with the amorphous fluororesin and baked at 55 ° C. for 3 minutes, and further baked at 110 ° C. for 5 minutes. Photolithography was performed using a photomask having holes having a diameter of 3 μm at intervals of 5 μm. After dry etching with oxygen plasma, a washed cover glass was obtained as DAD. The DAD has a well (accommodating portion) having a diameter of 4 μm and a depth of 3 μm (about 1 million pieces / 10 mm 2 ), and the cover glass is exposed on the bottom surface of the well. An array having a flow cell structure as shown in FIG. 1 was prepared using the obtained DAD.
 pH6.5~9.0に調整したバッファー溶液(33mM DEA-HCl, 4mM CaCl2)に4-MUを50μMになるように溶解した。
 4-MUを溶解したバッファー溶液30μLをアレイに導入し各ウェルにバッファー溶液を充填した(図1A参照)。続いて、アレイに疎水性溶媒(FC40)を200μL導入し、各ウェルに疎水性溶媒で被覆された親水性溶媒の液滴を形成させた。
 蛍光顕微鏡(IX8, OLYMPUS)に接続されたCMOSカメラ(Neo sCMOS, Andor)で、各液滴の蛍光画像を撮影し、蛍光強度を測定した。各ウェルにつき10mm2の領域を120分割して撮影を行った。1枚の画像には約8,600個のウェルが含まれる。蛍光画像を画像解析ソフト(Meta-Morph, Molecular Devices)で解析し、蛍光強度を算出した。
4-MU was dissolved in a buffer solution (33 mM DEA-HCl, 4 mM CaCl 2 ) adjusted to pH 6.5 to 9.0 to 50 μM.
30 μL of a buffer solution in which 4-MU was dissolved was introduced into the array, and each well was filled with the buffer solution (see FIG. 1A). Subsequently, 200 μL of a hydrophobic solvent (FC40) was introduced into the array, and droplets of the hydrophilic solvent coated with the hydrophobic solvent were formed in each well.
A fluorescence image of each droplet was taken with a CMOS camera (NeosCMOS, Andor) connected to a fluorescence microscope (IX8, OLYMPUS), and the fluorescence intensity was measured. Photographing was performed by dividing an area of 10 mm 2 for each well into 120 parts. One image includes about 8,600 wells. The fluorescence image was analyzed with image analysis software (Meta-Morph, Molecular Devices), and the fluorescence intensity was calculated.
 結果を図3に示す。バッファー溶液のpH値を、4MUのpKa(7.79)よりも大きく設定することで、4MUの蛍光をより高感度に検出できた。pH値8以上では、4MUが電荷を有する状態となり、4MUのFC40への移行(漏れ出し)が抑制されたためと考えられる。 The results are shown in FIG. By setting the pH value of the buffer solution to be larger than 4MU pKa (7.79), 4MU fluorescence could be detected with higher sensitivity. When the pH value is 8 or more, it is considered that 4MU has a charge and the transition (leakage) of 4MU to FC40 is suppressed.
[試験例2:親水性溶媒の緩衝物質濃度の検討]
 以下の手順によって、親水性溶媒の緩衝物質濃度が検出感度に及ぼす影響を検討した。
[Test Example 2: Examination of buffer substance concentration of hydrophilic solvent]
The influence of the buffer substance concentration of the hydrophilic solvent on the detection sensitivity was examined by the following procedure.
 DEAの濃度を25mM~1Mに調整したバッファー溶液(4mM CaCl2、pH6.5)に4-MUを50μMになるように溶解した。
 4-MUを溶解したバッファー溶液30μLをアレイに導入し各ウェルにバッファー溶液を充填した(図1A参照)。続いて、アレイに疎水性溶媒(FC40)を200μL導入し、各ウェルに疎水性溶媒で被覆された親水性溶媒の液滴を形成させた。
 蛍光顕微鏡下でタイムラプス撮影を行い、各液滴の蛍光強度を測定した。
4-MU was dissolved in a buffer solution (4 mM CaCl 2 , pH 6.5) adjusted to a DEA concentration of 25 mM to 1 M to a concentration of 50 μM.
30 μL of a buffer solution in which 4-MU was dissolved was introduced into the array, and each well was filled with the buffer solution (see FIG. 1A). Subsequently, 200 μL of a hydrophobic solvent (FC40) was introduced into the array, and droplets of the hydrophilic solvent coated with the hydrophobic solvent were formed in each well.
Time-lapse photography was performed under a fluorescence microscope, and the fluorescence intensity of each droplet was measured.
 結果を図4に示す。DEA濃度1Mでは、露光による退色の影響を差し引くと、蛍光強度の経時的な減少はみられなかった。DEA濃度500mM,100mMでも、蛍光強度の減少は、観察後30分まで顕著に抑制された。DEA濃度50mMでは観察後10分で蛍光強度が維持されていたが、25mMでは蛍光強度の減少がみられた。 The results are shown in FIG. At a DEA concentration of 1M, when the influence of fading due to exposure was subtracted, no decrease in fluorescence intensity with time was observed. Even at DEA concentrations of 500 mM and 100 mM, the decrease in fluorescence intensity was remarkably suppressed until 30 minutes after observation. At a DEA concentration of 50 mM, the fluorescence intensity was maintained 10 minutes after observation, but at 25 mM, a decrease in fluorescence intensity was observed.
 本発明に係る検出方法において検出に要する時間は数分程度であることを考慮すると、バッファー溶液中の緩衝物質の濃度を50mM以上に設定することで、4MUの蛍光をより高感度に検出できることが示された。バッファー溶液中の緩衝物質を所定の濃度以上に設定することで、4MUのFC40の移行(漏れ出し)を抑制できたと考えられる。 Considering that the time required for detection in the detection method according to the present invention is about several minutes, the fluorescence of 4MU can be detected with higher sensitivity by setting the concentration of the buffer substance in the buffer solution to 50 mM or more. Indicated. It is considered that the migration (leakage) of 4MU FC40 could be suppressed by setting the buffer substance in the buffer solution to a predetermined concentration or more.
1:アレイ、2:ウイルス、3:基質、4:反応生成物、5:酵素、10:下層部、11:板状部材、12:側壁、13:収容部、20:上層部、30:空間、42:親水性溶媒、43:疎水性溶媒 1: Array, 2: Virus, 3: Substrate, 4: Reaction product, 5: Enzyme, 10: Lower layer part, 11: Plate member, 12: Side wall, 13: Storage part, 20: Upper layer part, 30: Space 42: hydrophilic solvent, 43: hydrophobic solvent

Claims (7)

  1.  病原性微生物に感染した対象又は感染した疑いがある対象から分離された生物試料中の前記病原性微生物を検出する方法であって、
    前記病原性微生物を収容可能な複数の収容部が、疎水性の上面を有する側壁によって互いに隔てられて形成されている下層部と、当該下層部における当該収容部が形成されている面に対向している上層部との間の空間に、前記生物試料と、前記病原性微生物の表面又は内部に存在する酵素による反応の基質となる物質と、を含む親水性溶媒を導入する導入手順と、
    前記空間に疎水性溶媒を導入して、前記収容部内に、疎水性溶媒で被覆されかつ前記病原性微生物と前記物質を包含する、親水性溶媒の液滴を形成する封入手順と、
    前記液滴中における前記酵素と前記物質との反応により生成する反応生成物を光学的に検出する検出手順と、を含み、
    前記親水性溶媒が、前記反応生成物の酸解離定数(pKa)よりも大きいpH値を有する、方法。
    A method for detecting the pathogenic microorganism in a biological sample isolated from a subject infected with or suspected of being infected with a pathogenic microorganism,
    A plurality of accommodating portions capable of accommodating the pathogenic microorganisms are opposed to a lower layer portion formed by being separated from each other by a side wall having a hydrophobic upper surface, and a surface of the lower layer portion where the accommodating portion is formed. An introduction procedure for introducing a hydrophilic solvent containing the biological sample and a substance serving as a substrate for a reaction by an enzyme present on the surface or inside of the pathogenic microorganism into a space between the upper layer portion,
    An encapsulation procedure for introducing a hydrophobic solvent into the space, and forming droplets of the hydrophilic solvent that is covered with the hydrophobic solvent and includes the pathogenic microorganism and the substance in the container,
    A detection procedure for optically detecting a reaction product generated by a reaction between the enzyme and the substance in the droplet,
    The method, wherein the hydrophilic solvent has a pH value greater than the acid dissociation constant (pKa) of the reaction product.
  2.  前記病原性微生物がインフルエンザウイルスであり、前記酵素がノイラミニダーゼであり、前記物質が4-メチルウンベリフェリル-α-D-ノイラミン酸(4-Methylumbelliferyl-N-acetyl-α-D-neuraminic acid)であり、前記反応生成物が4-メチルウンベリフェロンである、請求項1記載の方法。 The pathogenic microorganism is influenza virus, the enzyme is neuraminidase, and the substance is 4-methylumbelliferyl-α-D-neuraminic acid The method of claim 1, wherein the reaction product is 4-methylumbelliferone.
  3.  病原性微生物に感染した対象又は感染した疑いがある対象から分離された生物試料中の前記病原性微生物を検出するためのキットであって、
    前記病原性微生物を収容可能な複数の収容部が疎水性の上面を有する側壁によって互いに隔てられて形成されている下層部と、前記下層部における前記収容部が形成されている面に対して空間を隔てて対向している上層部とを備えるアレイと、
    前記病原性微生物の表面又は内部に存在する酵素による反応の基質となる物質と、
    前記酵素と前記物質との反応により生成する反応生成物の酸解離定数(pKa)よりも大きいpH値を有する親水性溶媒と、
    疎水性溶媒と、を含むキット。
    A kit for detecting the pathogenic microorganism in a biological sample isolated from a subject infected with or suspected of having the pathogenic microorganism,
    A space between a lower layer portion in which a plurality of accommodating portions capable of accommodating the pathogenic microorganisms are separated from each other by a side wall having a hydrophobic upper surface, and a surface of the lower layer portion where the accommodating portion is formed An array comprising an upper layer portion facing each other, and
    A substance that is a substrate for a reaction by an enzyme present on or inside the pathogenic microorganism;
    A hydrophilic solvent having a pH value greater than the acid dissociation constant (pKa) of the reaction product produced by the reaction between the enzyme and the substance;
    A kit comprising a hydrophobic solvent.
  4.  疎水性溶媒と界面接触する親水性溶媒中において、酵素と、該酵素による反応の基質となる物質とを反応させ、反応生成物を検出する方法であって、
    前記親水性溶媒が、前記反応生成物の酸解離定数(pKa)よりも大きいpH値を有する、方法。
    A method of detecting a reaction product by reacting an enzyme with a substance that is a substrate for a reaction by the enzyme in a hydrophilic solvent that is in interface contact with the hydrophobic solvent,
    The method, wherein the hydrophilic solvent has a pH value greater than the acid dissociation constant (pKa) of the reaction product.
  5.  前記親水性溶媒が病原性微生物を含み、
    前記酵素が前記病原性微生物の表面又は内部に存在する基質切断活性を有する酵素であり、
    前記物質が発色基質であり、
    前記酵素による前記発色基質の切断により生成する反応生成物を光学的に検出する、請求項4記載の方法。
    The hydrophilic solvent comprises pathogenic microorganisms;
    The enzyme is an enzyme having a substrate cleavage activity present on the surface or inside of the pathogenic microorganism,
    The substance is a chromogenic substrate;
    The method according to claim 4, wherein a reaction product produced by cleavage of the chromogenic substrate by the enzyme is optically detected.
  6.  前記病原性微生物がインフルエンザウイルスであり、前記酵素がノイラミニダーゼであり、前記発色基質が4-メチルウンベリフェリル-α-D-ノイラミン酸(4-Methylumbelliferyl-N-acetyl-α-D-neuraminic acid)であり、前記反応生成物が4-メチルウンベリフェロンである、請求項5記載の方法。 The pathogenic microorganism is an influenza virus, the enzyme is neuraminidase, and the chromogenic substrate is 4-methylumbelliferyl-α-D-neuraminic acid (4-Methylumbelliferyl-N-acetyl-α-D-neuraminic acid) 6. The method of claim 5, wherein the reaction product is 4-methylumbelliferone.
  7.  前記親水性溶媒が、前記病原性微生物に感染した対象又は感染した疑いがある対象から分離された生物試料を含む、請求項5又は6記載の方法。

     
    7. The method of claim 5 or 6, wherein the hydrophilic solvent comprises a biological sample isolated from a subject infected with or suspected of being infected with the pathogenic microorganism.

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