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WO2018168986A1 - Procédé de test de gène et kit de test de gène - Google Patents

Procédé de test de gène et kit de test de gène Download PDF

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Publication number
WO2018168986A1
WO2018168986A1 PCT/JP2018/010136 JP2018010136W WO2018168986A1 WO 2018168986 A1 WO2018168986 A1 WO 2018168986A1 JP 2018010136 W JP2018010136 W JP 2018010136W WO 2018168986 A1 WO2018168986 A1 WO 2018168986A1
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WIPO (PCT)
Prior art keywords
filter
sample
genetic
genetic test
detected
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PCT/JP2018/010136
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English (en)
Japanese (ja)
Inventor
佳子 上倉
道俊 杉本
Original Assignee
東洋紡株式会社
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Priority to CN201880000710.0A priority Critical patent/CN108884488A/zh
Priority to JP2019506251A priority patent/JP7264046B2/ja
Publication of WO2018168986A1 publication Critical patent/WO2018168986A1/fr

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    • 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
    • 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
    • C12M1/12Apparatus for enzymology or microbiology with sterilisation, filtration or dialysis means
    • 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
    • C12M1/26Inoculator or sampler
    • C12M1/28Inoculator or sampler being part of container
    • C12M1/30Sampler being a swab
    • 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
    • C12Q1/68Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6806Preparing nucleic acids for analysis, e.g. for polymerase chain reaction [PCR] assay
    • 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
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • 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
    • G01N33/50Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12NMICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N15/00Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09Recombinant DNA-technology

Definitions

  • the present invention relates to a genetic testing method and a genetic testing kit.
  • Non-Patent Document 1 and Patent Document 1 Various gene testing methods are known (for example, Non-Patent Document 1 and Patent Document 1). Various substances are mixed in samples used for genetic testing. These contaminants affect the inspection method, reduce the sensitivity, cause false positives, and cause troubles in the equipment used for inspection (for example, clogging of the flow path system). There is a possibility. Conventionally, a centrifugal separation method or the like has been used to remove the contaminants, but a dedicated device is required, and the operation is complicated and takes time.
  • Patent Document 2 false positives and clogging are prevented while maintaining sensitivity in an inspection method using a membrane assay method intended for detection with a capture reagent having a specific binding ability (for example, an antibody against a specific antigen, etc.).
  • a capture reagent having a specific binding ability for example, an antibody against a specific antigen, etc.
  • An object of the present invention is to provide a method for preventing false positives and clogging while maintaining sensitivity in a genetic testing method, and enabling highly accurate detection or quantification of an object to be detected, and in such a method It is to provide a kit to be used.
  • the present inventor has found that in the genetic testing method, the above problem can be solved by detecting or quantifying the presence of the analyte in the sample after filtering the sample containing the analyte with a filtration filter. Completed the invention.
  • the outline of the present invention is as follows.
  • [Item 1] A genetic test method characterized by detecting or quantifying the presence of a target to be detected in the sample after filtering the sample containing the target to be detected by a filtration filter.
  • [Item 2] The genetic testing method according to Item 1, wherein the filtration filter is a sintered filter.
  • the sintered filter material is selected from the group consisting of polypropylene, polyethylene, polystyrene, and polymethylmethacrylate.
  • the pore size of the sintered filter is 10 to 100 ⁇ m.
  • the sample is a human or other animal's oral scrapings, pharyngeal wipe, nasal wipe, nasal suction, nasal wash, alveolar wash, stool suspension, rectal wipe, vaginal discharge, uterus Item 8.
  • the genetic test method according to Item 7 which is a sample selected from the group consisting of cervical mucus and urethral scrapings.
  • the genetic test method according to Item 8 wherein the matter that can be examined by detecting or quantifying the presence of the target to be detected is a microorganism causing respiratory infection or a substance derived from the microorganism.
  • the genetic test method according to Item 9 which is a microorganism or a substance derived from the microorganism.
  • the genetic test method according to Item 8 wherein the matter that can be examined by detecting or quantifying the presence of the target to be detected is a causative microorganism of diarrhea or a substance derived from the microorganism.
  • the item that can be examined by detecting or quantifying the presence of the detection target is a pathogenic microorganism selected from the group consisting of norovirus, rotavirus, sapovirus, and diarrhea adenovirus, or a substance derived from the microorganism. 11.
  • ITEM 13 The genetic test
  • the item that can be examined by detecting or quantifying the presence of the detection target is a pathogenic microorganism selected from the group consisting of Neisseria gonorrhoeae, Chlamydia, Mycoplasma, Ureaplasma, HIV and HPV, or a substance derived from the microorganism. 14.
  • Items that can be examined by detecting or quantifying the presence of the detection target include gene polymorphisms of drug metabolizing enzyme genes, gene polymorphisms of alcohol dehydrogenase genes, gene polymorphisms of acetaldehyde dehydrogenase genes, and Item 16.
  • a genetic testing method for testing the presence of an object to be detected in a specimen sample comprising: (1) A specimen sample filtration tube provided with a filtration filter constituted by a filter including a sintered filter. [Item 20] The genetic test kit according to Item 19, further comprising a cotton swab or swab as a specimen collection device. [Item 21] The gene test kit according to item 19, further comprising a container filled with a gene test reagent capable of detecting or quantifying the presence of the detection target.
  • the specimen is an intraoral scratch, throat wiping liquid, nasal wiping liquid, nasal suction liquid, alveolar lavage liquid, vaginal secretion, Even for a sample containing a large amount of impurities such as cervical mucus and urethral scrapings, impurities can be removed without reducing the amount of the sample using a sample collection device and / or a filtration filter. As a result, accurate results can be obtained without being affected by impurities.
  • the dedicated equipment and the trouble of operation required for performing the centrifugal separation are unnecessary, and it is easier and more accurate in a short time. The result can be obtained.
  • FIG. It is a figure which shows the gene detection result of Neisseria gonorrhoeae of Example 1. It is a figure which shows the detection result of the internal control in the pertussis detection of Example 2.
  • FIG. It is a figure which shows the gene detection result (contrast in the case of using the sintered filter and membrane filter from which a hole diameter differs) in the mycoplasma * pneumoniae in Example 3.
  • FIG. It is a figure which shows the gene detection result of the internal control in the Mycoplasma pneumoniae detection of Example 3 (comparison when using a sintered filter and a membrane filter with different pore sizes). It is a figure which shows the detection result (comparison with a sintered filter and the centrifugation method) of the gene of Mycoplasma pneumoniae in Example 4.
  • Example 5 it is the figure which showed the real-time RT-PCR analysis result of the influenza A gene.
  • Example 6 it is the figure which showed the detection result of norovirus RNA contained in a stool sample. It is a figure which shows the structure of the genetic test kit of one embodiment of this invention.
  • One embodiment of the present invention is a genetic test method characterized by detecting or quantifying the presence of a target to be detected in the sample after filtering the sample including the target to be detected by a filtration filter. Specifically, after collecting a test object using a sampling tool, put the sampling tool containing the test target in a container, suspend in the liquid in the container, and then remove the sampling tool from the container, A genetic test method characterized in that the filter of the present invention is attached to a container, the liquid inside the container is passed through the filter and eluted out of the container, and the presence of the target to be detected in the eluate is detected or quantified. is there.
  • the gene testing method is not particularly limited, and may be any method that can test a disease or infection based on gene sequence information. Specifically, for example, a partial sequence of a base (eg, DNA, RNA, etc.) peculiar to a specific infection source or disease is detected from a sample, or a polymorphism (eg, single nucleotide polymorphism (SNP)) is detected. The mode to do is mentioned.
  • the detection principle used in the genetic testing method is not particularly limited. For example, PCR methods using allele-specific primers, denaturing gradient gel electrophoresis methods, SSCP methods, RFLP methods, TaqMan PCR methods, invader methods, PCR methods, etc.
  • Various known methods such as a method of performing a melting curve analysis by hybridizing an appropriate probe (for example, Q probe) to the sample amplified in (1) can be used.
  • the detection target is not particularly limited.
  • a specific nucleic acid, a specific oligonucleotide part, a specific polynucleotide part, etc. present in a part of a genome, a structural gene, a regulatory gene, a gene transcript (such as mRNA or a precursor thereof), or a fragment thereof.
  • They may be either DNA or RNA, or both chimeras.
  • the sample including the target to be detected may be a sample collected from a living body (for example, the sample itself including the target to be detected), or DNA or RNA included in the sample may be replicated or amplified by any means. It may be what was done.
  • the method used for replication or amplification is not particularly limited, and various known methods such as PCR, SDA, ICAN, and LAMP can be used.
  • the sample including the detection target may be a sample in which DNA, RNA, or the like included in the specimen is modified or decomposed by some physicochemical means.
  • DNA replication or amplification is performed by the PCR method
  • ⁇ -type DNA polymerase or DNA polymerase belonging to family B
  • KOD DNA polymerase is used.
  • the presence of the target to be detected can be detected or quantified only by a simple filtration operation without extracting or purifying DNA from the sample.
  • RNA is detected by the RT-PCR method
  • the sample including the detection target is filtered using a filtration filter.
  • the filtration filter is not only one type, but also a combination of different materials, different pore sizes or different retained particle sizes However, in the present invention, it is necessary to include at least one type of filtration filter.
  • the filtration filter is not particularly limited, but a sintered filter is preferably used.
  • the material of the sintered filter is not particularly limited, but is preferably selected from the group consisting of polypropylene, polyethylene, polystyrene, and polymethyl methacrylate.
  • the filtration filter is preferably a filter (strong rigidity filter) that is not easily deformed by an external force.
  • a specimen sample suspended in a specimen suspension is placed in a filtration tube, filtered through a filtration filter including a rigid filter attached to the tip, and the filtrate is added with a genetic test reagent (eg, a tube, etc.)
  • a genetic test reagent eg, a tube, etc.
  • the filter receives the pressure of a liquid such as a specimen sample in the filtration tube or air.
  • the tube is made of an elastic material, a person receives pressure by directly pressing the container with his hand.
  • the filtration filter used in the present invention is a container in which a specimen sample suspended in a specimen suspension is placed in a filtration tube, filtered through a filtration filter including a rigid filter attached to the tip, and the filtrate is added with a genetic test reagent. It is preferable that the filter has a rigidity that is difficult to be crushed in the thickness direction of the filter or difficult to be deformed such as a curve when dropped onto (for example, a tube or the like). In other words, it is preferable that the filter has a rigidity that makes it difficult to block the flow path of the filtrate inside the filter even by the pressure of the filtrate.
  • the filtration filter Since the filtration filter is not easily deformed, clogging is unlikely to occur. Further, since it can be laminated or processed to a thickness of about several millimeters to several centimeters, the effective filtration area can be increased thereby, and clogging hardly occurs.
  • the filter is rigid enough not to be crushed in the thickness direction of the filter. For example, even when the pressure applied to the filter by liquid or air when the specimen sample is filtered exceeds 0.2 MPa, The amount of change in thickness does not become 5% or more, preferably 2% or more, and it has rigidity that is difficult to deform such as bending. This means that the radius of curvature of the filter does not become 2 cm or less.
  • filtration filter various metal fibers wound, metal fiber non-woven fabric, plastic foam, metal mesh layered, metal, plastics, ceramic powder, etc. And sintered filters in which metal fibers are bonded with heat or pressure, and any of them can be used in the present invention.
  • the sintered filter can be manufactured, for example, by directly bonding (sintering) powder particles such as metal, ceramic, plastic, and metal fibers by heat and pressure.
  • the sintered filter used in the present invention can be obtained, for example, by sintering a material such as a fiber as a filter raw material by a heat treatment of the melting point of the filter raw material ⁇ 0.3 to 0.7 ° C., Preferably, it can be obtained by sintering by heat treatment of the melting point of the filter material ⁇ 0.5 ° C.
  • sintered filters are 1) inexpensive, 2) pore size can be controlled by particle size, 3) suitable for mass production, and 4) low affinity with biological materials.
  • polypropylene polyethylene (low density polyethylene, high density polyethylene, ultra high molecular weight polyethylene), polystyrene, polymethyl methacrylate resin, alumina, zirconia, silicon, silicon tetrafluoroethylene polymer,
  • resin such as a polypropylene, polyethylene, a polystyrene, a polymethylmethacrylate, is preferable from the surface of workability, and a polypropylene and polyethylene are especially preferable.
  • the filtration filter may be produced by combining two or more types of strong rigidity filters. Moreover, you may combine with filters other than a rigid filter and a rigid filter.
  • the filtration filter can be used for both microfiltration and coarse filtration, but at least one type of filtration filter preferably has a pore size or retained particle size of 200 ⁇ m or less.
  • the filtration filter is configured by stacking a plurality of filters having different pore diameters, the pore diameter of the filter having the smallest pore diameter is 1 ⁇ m or more, and the pore diameter of the filter having the largest pore diameter is What is necessary is just to use the filtration filter which is 200 micrometers or less.
  • the filter having the minimum pore size is 5 ⁇ m or more
  • the filter having the maximum pore size is 150 ⁇ m or less
  • more preferably the filter having the minimum pore size is 10 ⁇ m or more
  • the filter has the maximum pore size.
  • the pore diameter is 100 ⁇ m.
  • the specimen sample containing the detection target may be first passed through a filter having a maximum pore size and then passed through a filter having a smaller pore size.
  • the pore diameter of a polypropylene rigid filter is 5 to 200 ⁇ m
  • the pore diameter of a low density polyethylene rigid filter is 10 to 70 ⁇ m
  • the diameter of a high density polyethylene rigid filter is 10 to 50 ⁇ m
  • an ultrahigh molecular weight polyethylene rigid filter The pore size of the polystyrene rigid filter is 100 to 200 ⁇ m
  • the pore size of the tetrafluoroethylene polymer strong filter is 30 to 100 ⁇ m.
  • a plurality of strong rigidity filters having different materials and / or hole diameters may be used in combination.
  • the pore diameter is not particularly limited, but it can be, for example, 1 ⁇ m to 200 ⁇ m, preferably 5 ⁇ m to 150 ⁇ m, and preferably 10 ⁇ m to 100 ⁇ m. More preferred.
  • a membrane filter can be used as a filter for microfiltration.
  • the material is cellulose, glass fiber, silica fiber, nitrocellulose, cellulose ester, a mixture of nitrocellulose and cellulose ester, polyethersulfone, polysulfone, tetrafluoride.
  • Suitable materials can be selected from ethylene resin, vinylidene fluoride resin, polycarbonate, polypropylene, polyamide, nylon 6,6, polyester, cotton, stainless steel fiber, etc. Therefore, it is necessary to make the amount of absorption of the sample suspension liquid not large.
  • the minimum pore diameter or the retained particle diameter of the filter having the smallest pore diameter or the retained particle diameter among the filters constituting the filtration filter is about 10 ⁇ m.
  • the filter when two filters are used in combination with another type of filter (hereinafter also referred to as a non-rigid filter) other than the rigid filter and the rigid filter, the filter is used for filtration of a filtration tube described later.
  • the first-stage filter and the second-stage filter are formed from the bottom of the nozzle (in this case, the first-stage filter is the first-stage filter that comes into contact with the specimen sample first, and the specimen sample passes through the first-stage filter.
  • the first stage filter and the second stage filter there are a combination of a strong filter and a strong filter, and a combination of a strong filter and a non-rigid filter.
  • a filter close to the nozzle tip is sometimes referred to as a downstream filter, and a filter that comes into contact with a specimen sample earlier is sometimes referred to as an upstream filter.
  • filters it is preferable to combine filters having different pore sizes. Since all of the strong filters trap contaminants not only on the filter surface but also inside, a certain thickness is required. The required thickness varies naturally depending on the amount of contaminants and the pore size and size of the filter. In general, when 100 to 2000 ⁇ l of a sample sample required for genetic testing is filtered with a sample filtration filter with a diameter of about 5 to 20 mm.
  • the thickness of the rigid filter or non-rigid filter is not limited, but is preferably 0.5 mm to 7 mm, more preferably 1 mm to 5 mm.
  • the total thickness of the sample filtration filter is preferably about 2 mm to 10 mm.
  • a filter having a large pore diameter or retained particle diameter When combining several filtration filters having different pore diameters or retained particle diameters, it is possible to arrange a filter having a large pore diameter or retained particle diameter on the upstream side, a small filter on the downstream side, and so that both filters overlap. It is preferable for avoiding clogging of the specimen sample during filtration. For example, when two filters are combined, it is preferable to use a filter having a large pore diameter or retained particle diameter as the first-stage filter and a filter having a small pore diameter as the second-stage filter.
  • the size of the filter used depends on the size of the sample suspension tube used in the assay, but is, for example, circular and has a diameter of 0.5 to 1.5 cm.
  • the filter described in Patent Document 2 may be used.
  • a sample containing a detection target can be collected using a cotton swab, platinum ear, dropper, spoon tool, or the like as an instrument for collecting the sample and used for the assay.
  • a cotton swab or swab it is preferable to use a cotton swab or swab to collect a specimen and use it for the assay.
  • the swab is mainly used as a sample collection device.
  • the cotton swab is not particularly limited.
  • tip of one side of a axial part is mentioned.
  • the cotton ball portion corresponds to the specimen collection portion.
  • a cotton swab of a type called a brush-like swab in which fibers made of a natural product or an artificial product are arranged in a brush shape and a liquid is collected by using a capillary phenomenon, is commercially available.
  • the brush-like swab can be particularly preferably used in the present invention for the following reasons.
  • the brush-like swab can be produced by, for example, a method called flocking, in which short cut fibers (floc) are attached to the handle of a swab to which an adhesive is applied by static electricity. Also, with this type of swab, it is possible to collect a solid or viscous substance that is a dissolved or mixed material in a liquid. In this case, not only the capillary phenomenon but also a solid or viscous substance is collected in the sample collection site of the instrument in the present invention. It is collected by using the binding by adsorption.
  • the surface area is significantly increased as compared with a conventional cotton swab, the amount of sample collected is large, and it is possible to adsorb more samples including the detection target in the sample. Also, when floating in suspension, unlike conventional cotton swabs, suspension can easily enter between fibers, so the sample is easily released and the sample can be efficiently collected in suspension. Can do. From the above viewpoints, in the genetic testing method of the present invention, it is preferable to use a brush-like swab (also referred to as a brush-like swab), and particularly preferably a flox swab to which short fibers are attached, for collecting a specimen.
  • a brush-like swab also referred to as a brush-like swab
  • flox swab to which short fibers are attached
  • Patent Document 2 As the sample collection device as described above, the one described in Patent Document 2 may be used.
  • matters that can be inspected by detecting or quantifying the presence of the detection target are not particularly limited.
  • examples include microorganisms causing respiratory infections or substances derived from these microorganisms. More specifically, influenza viruses (for example, influenza A virus, influenza B virus, etc.), RS viruses, adenoviruses, group A Examples include pathogenic microorganisms selected from the group consisting of Streptococcus and Mycoplasma pneumoniae, Bordetella pertussis, Chlamydia pneumoniae, or substances derived from the microorganisms.
  • a microorganism causing diarrhea or a substance derived from the microorganism can be mentioned, and more specifically, a pathogenic microorganism selected from the group consisting of norovirus, rotavirus, sapovirus and diarrhea adenovirus or a substance derived from the microorganism Is mentioned.
  • a microorganism causing causative sexually transmitted disease or a substance derived from the microorganism can be mentioned, and more specifically, a pathogenic microorganism selected from the group consisting of Neisseria gonorrhoeae, Chlamydia, Mycoplasma, Ureaplasma, HIV and HPV Substances.
  • the gene testing method of the present invention can also test human gene polymorphism, more specifically, drug resistance gene polymorphism (for example, gene polymorphism of drug metabolizing enzyme cytochrome P450 gene (CYP gene)), Used to examine gene polymorphism of alcohol dehydrogenase gene (ADH gene), gene polymorphism of acetaldehyde dehydrogenase gene (ALDH gene), gene polymorphism of methylenetetrahydrofolate reductase gene (MTHFR gene), etc. It is possible.
  • drug resistance gene polymorphism for example, gene polymorphism of drug metabolizing enzyme cytochrome P450 gene (CYP gene)
  • ADH gene alcohol dehydrogenase gene
  • ADH gene polymorphism of acetaldehyde dehydrogenase gene ADH gene
  • gene polymorphism of methylenetetrahydrofolate reductase gene MTHFR gene
  • Specimens to be analyzed by the inspection method of the present invention are not particularly limited.
  • biological samples obtained from humans or other animals can be mentioned.
  • oral rubs, pharyngeal wiping liquid, nasal wiping liquid, nasal aspirate, nasal irrigation liquid, alveolar irrigation liquid, stool suspension and rectal wiping liquid, vaginal discharge, cervical mucus, urethral rub What is necessary is just to select from the group which consists of things.
  • the gene testing method of the present invention does not need to be pretreated by a centrifugation method, which is normally considered essential in the conventional gene testing method.
  • a centrifugation method which is normally considered essential in the conventional gene testing method.
  • the centrifugation method is performed as a pretreatment, in addition to the necessity for a dedicated device, there is a problem that the operation of the centrifugation is complicated and takes time and effort.
  • the genetic testing method of the present invention it is not necessary to prepare a dedicated device for performing such centrifugation, and it is possible to omit complicated operations.
  • the time required for the centrifugation method can be shortened, it is possible to shorten the time from collection of a sample from a subject to obtaining a genetic test result. For example, until the genetic test result is obtained from sample collection.
  • This time can be within 1 day, preferably within half a day, more preferably within 6 hours, even more preferably within 3 hours, and particularly preferably within 2 hours (for example, within 1 hour and a half
  • kits for testing the presence of a target to be detected in a specimen sample comprising a specimen sample filtration tube provided with a filtration filter constituted by a filter including a rigid filter. is there.
  • the kit of the present invention may further include a cotton swab or swab as a specimen collecting device.
  • the kit of the present invention naturally includes a genetic test reagent that can detect or quantify the presence of the detection target. This genetic test reagent is filled in a reagent container such as a tube or bottle, and is included in the kit of the present invention in a form that can be opened at the time of use.
  • FIG. 8 shows an example of a configuration diagram of the genetic test kit of this embodiment.
  • the specimen sample filtration tube 2 includes a sintered filter (21-a, 21-b) inside.
  • the sintered filter (21-a, 21-b) Filtration can be performed.
  • the pore diameters of the sintered filters 21-a and 21-b may be the same or different.
  • the number of sheets of a sintered filter is not limited to this, Three or more sheets may be piled up, or only one sheet may be piled up. Good.
  • tip part containing a sintered filter and the tube main-body part may be comprised separately so that attachment or detachment is possible, and you may make it attach at the time of filtration operation.
  • a tube made of a material having elasticity so that a person can directly press the container with his / her hand to apply pressure can be used.
  • the short fibers are attached to the specimen collecting portion 31 provided at the tip of the swab shaft portion 32.
  • the genetic test reagent 4 is filled in a container body 41 and is sealed with a lid 42 or the like during storage.
  • the genetic test kit of the present invention can be provided by being housed in the packaging box 1, but it may be a kit that is supplied in separate packaging and used as a set at the time of use. Note that the configuration of the genetic test kit of the present invention is not limited to the example shown in FIG. 8, and can include any other than those exemplified above (for example, instruction manuals).
  • Example 1 Detection of Neisseria gonorrhoeae
  • sample A DNA sample extracted from Neisseria gonorrhoeae was prepared with 10 mM Tris-HCl (pH 7.5) to 100 (copy / ⁇ L), and mixed with cervical mucus to prepare a sample. .
  • This sample was passed through a filtration filter containing a sintered filter (made of polypropylene, pore size 100 ⁇ m) to prepare a sample.
  • water was used as a negative control (NC).
  • Reagents A solution containing the following reagents was prepared. 10 ⁇ M forward primer (SEQ ID NO: 1) 0.4 ⁇ L 100 ⁇ M reverse primer (SEQ ID NO: 2) 0.2 ⁇ L 10 ⁇ M probe (SEQ ID NO: 3, 5 ′ end labeled BODIPY-FL) 0.3 ⁇ L KOD Mix (Gen Cube (R) Test Basic, manufactured by Toyobo) 3 ⁇ L PPD Mix (Gen Cube (R) Test Basic, manufactured by Toyobo) 3 ⁇ L Sample 3 ⁇ L
  • Nucleic acid amplification and melting curve analysis 94 ° C, 2 minutes (1 cycle or more) 97 ° C ⁇ 1 second 58 ° C ⁇ 3 seconds 63 ° C ⁇ 6 seconds (over 60 cycles) 94 ° C, 30 seconds 39 ° C, 30 seconds 39 ° C to 75 ° C (temperature rises at 0.09 ° C / second)
  • FIG. 1 is a graph showing the results of analysis of the change in fluorescence intensity with the subsequent temperature rise, with the temperature on the horizontal axis of the graph and the differential value of the fluorescent signal on the vertical axis.
  • NG DNA indicates the analysis result of the Neisseria gonorrhoeae DNA sample
  • Water indicates the analysis result of water which is NC.
  • Neisseria gonorrhoeae is detected.
  • the same result was obtained even if it used the sample which extract
  • Example 2 Detection of Bordetella pertussis
  • a DNA sample extracted from Bordetella pertussis was prepared with 10 mM Tris-HCl (pH 7.5) to 5 (copy / ⁇ L), and mixed with a pharyngeal wipe suspended in physiological saline. Thus, a pseudo biological sample was obtained. This sample was passed through a filtration filter containing a sintered filter (made of polypropylene, pore size 20 ⁇ m) to prepare a sample. Moreover, the liquid which is not filtered with a sintered filter was also used as a sample.
  • a sintered filter made of polypropylene, pore size 20 ⁇ m
  • Reagents A solution containing the following reagents was prepared. 10 ⁇ M forward primer (SEQ ID NO: 4) 0.4 ⁇ L 100 ⁇ M reverse primer (SEQ ID NO: 5) 0.3 ⁇ L 0.3 ⁇ L of 10 ⁇ M probe (SEQ ID NO: 6, 5 ′ end labeled with BODIPY-FL) KOD Mix (Gen Cube (R) Test Basic, manufactured by Toyobo) 3 ⁇ L PPD Mix (Gen Cube (R) Test Basic, manufactured by Toyobo) 3 ⁇ L IC Mix (Gen Cube Test Basic, manufactured by Toyobo) 1 ⁇ L Sample 3 ⁇ L
  • FIG. 2 shows the results of analysis of the change in the fluorescence intensity of the internal control as the temperature rises after the nucleic acid amplification under the above conditions, with the horizontal axis of the graph being the temperature and the vertical axis being the differential value of the fluorescence signal.
  • FIG. The graph shows the result of the internal control, with the filter, the analysis result obtained by filtering the sample with the sintered filter, and without the filter, the analysis result obtained when the sample was not filtered with the sintered filter.
  • an internal control is detected when there is a filter.
  • internal control is not detected due to PCR inhibition of a substance contained in the sample. From this result, it was clarified that in the genetic test for Bordetella pertussis, a high-sensitivity test result can be obtained only by pretreatment with a sintered filter.
  • Example 3 Detection of pneumonia mycoplasma
  • a DNA sample extracted from Mycoplasma pneumoniae was prepared to 5 (copy / ⁇ L) with 10 mM Tris-HCl (pH 7.5), and mucin was prepared to a final concentration of 0.2%.
  • a pseudo-pharyngeal wipe liquid biological sample was prepared by mixing with the liquid.
  • Sintered filters made of polypropylene with pore sizes of 300 ⁇ m, 100 ⁇ m, and 20 ⁇ m and filters other than sintered filters (commercially used filters for removing dust before nucleic acid extraction: mesh filter (filter for nucleic acid extraction) pore size of 0.45 ⁇ m, membrane A sample was prepared by passing through a filtration filter containing a filter pore size of 0.8 ⁇ m or less. Moreover, the liquid which is not filtered with a sintered filter was also used as a sample.
  • Reagent KOD Mix (Gen Cube (R) Test Basic, Toyobo) 4 ⁇ L 10 ⁇ M forward primer (SEQ ID NO: 7) 0.2 ⁇ L 100 ⁇ M reverse primer (SEQ ID NO: 8) 0.3 ⁇ L 0.4 ⁇ L of 10 ⁇ M probe (SEQ ID NO: 9, 5 ′ end labeled with BODIPY-FL) PPD Mix (Gen Cube (R) Test Basic, manufactured by Toyobo) 3 ⁇ L IC Mix (Gen Cube Test Basic, manufactured by Toyobo) 1.3 ⁇ L Sample 4 ⁇ L
  • Nucleic acid amplification and melting curve analysis 94 ° C, 2 minutes (1 cycle or more) 97 ° C ⁇ 1 second 58 ° C ⁇ 3 seconds 63 ° C ⁇ 6 seconds (over 60 cycles) 94 ° C, 30 seconds 39 ° C, 30 seconds 39 ° C to 75 ° C (temperature rises at 0.09 ° C / second)
  • Mycoplasma pneumonia cutoff value 7.5 Internal control cutoff value: 1.5
  • FIGS. 3 and 4 show results obtained by performing nucleic acid amplification under the above-mentioned conditions, and regarding the change in fluorescence intensity as the temperature rises, the results of various filters are plotted on the horizontal axis and the vertical axis of the graph is the differential value of the fluorescence signal
  • FIG. 3 shows the results of Mycoplasma pneumoniae
  • FIG. 4 shows the results of internal control.
  • Mycoplasma pneumoniae is detected when there is a filter.
  • no filter Mycoplasma pneumoniae has not been detected due to PCR inhibition of the substance contained in the sample.
  • no internal control was detected except for the sintered filters of 20, 100 ⁇ m. From this result, it has been clarified that it is important to use a sintered filter having a specific pore size in order to obtain a highly sensitive test result in the genetic test of Mycoplasma pneumoniae.
  • Example 4 Detection of pneumonia mycoplasma
  • Sample preparation A DNA sample extracted from Mycoplasma pneumoniae was prepared to 10 (copy / ⁇ L) with 10 mM Tris-HCl (pH 7.5) and suspended in physiological saline. A pseudo biological sample was prepared by mixing. This sample was passed through a sintered filter (made of polyethylene, pore size 20 ⁇ m) to prepare a sample. Moreover, the supernatant which was not filtered but centrifuged according to the conventional method (13,000 rpm for 3 minutes) was used as a sample of a comparative example.
  • (2) Nucleic acid amplification and melting curve analysis The sample was added to the following reagents, and Mycoplasma pneumoniae was detected under the following conditions. GENECUBE (registered trademark) manufactured by Toyobo was used for nucleic acid amplification and melting curve analysis.
  • Reagent KOD Mix (Gen Cube (R) Test Basic, Toyobo) 4 ⁇ L 10 ⁇ M forward primer (SEQ ID NO: 7) 0.2 ⁇ L 100 ⁇ M reverse primer (SEQ ID NO: 8) 0.3 ⁇ L 0.4 ⁇ L of 10 ⁇ M probe (SEQ ID NO: 9, 5 ′ end labeled with BODIPY-FL) PPD Mix (Gen Cube (R) Test Basic, manufactured by Toyobo) 3 ⁇ L IC Mix (Gen Cube Test Basic, manufactured by Toyobo) 1.3 ⁇ L Sample 4 ⁇ L
  • Nucleic acid amplification and melting curve analysis 94 ° C, 2 minutes (1 cycle or more) 97 ° C ⁇ 1 second 58 ° C ⁇ 3 seconds 63 ° C ⁇ 6 seconds (over 60 cycles) 94 ° C, 30 seconds 39 ° C, 30 seconds 39 ° C to 75 ° C (temperature rises at 0.09 ° C / second)
  • Mycoplasma pneumonia cutoff value 7.5 Internal control cutoff value: 1.5
  • FIG. 5 is a graph showing the results of analysis of the change in fluorescence intensity with the temperature rise after the nucleic acid amplification under the above conditions, with the horizontal axis of the graph being the temperature and the vertical axis being the differential value of the fluorescence signal. .
  • the graph shows the results of Mycoplasma pneumoniae.
  • Filter filtration is the analysis result obtained by filtering the sample with a sintered filter. Centrifugation is performed by centrifuging the sample at 13,000 rpm for 3 minutes with a centrifuge and using the supernatant as the sample. The analysis results are shown.
  • the fluorescence value of Mycoplasma pneumoniae treated by sintering filter filtration is higher than that of the case treated by centrifugation. Therefore, it has been clarified that the genetic testing method of the present invention can be detected with higher sensitivity while being a simpler method than the conventional centrifugation method.
  • Example 5 Detection of influenza
  • a purified biological sample was prepared by diluting influenza purified RNA 1000-fold and 2000-fold with nasal wiping liquid suspended in sterilized water. This sample was passed through a sintered filter (made of polypropylene, pore size 20 ⁇ m) to prepare a sample. A sample not filtered with a sintered filter was also used as a comparison.
  • (2) Nucleic acid amplification and melting curve analysis The above samples were added to the following reagents, and influenza A was detected under the following conditions. As nucleic acid amplification, Rotor-Gene Q (registered trademark) was used for real-time RT-PCR using TaqMan probe.
  • a solution containing the following reagents was prepared. 1 ⁇ L of 10 ⁇ M forward primer (SEQ ID NO: 10) 10 ⁇ M reverse primer (SEQ ID NO: 11) 1 ⁇ L 0.4 ⁇ L of 10 ⁇ M probe (SEQ ID NO: 12, 5 ′ end is FAM, 3 ′ end is BHQ1), QRZ-101 (THUNDERBIRD (R) Probe One-step qRT-PCR Kit, manufactured by Toyobo) Sample 4 ⁇ L
  • RT-PCR conditions 50 ° C, 10 minutes, 95 ° C, 1 minute (more than one cycle) 95 ° C / 15 seconds 60 ° C / 45 seconds (over 50 cycles)
  • FIG. 6 is a table showing the analysis results of real-time RT-PCR under the above conditions. With the filter, the analysis result when the sample is filtered with the sintered filter is shown, and without the filter, the analysis result when the sample is not filtered with the sintered filter is shown. As is clear from FIG. 6, the cut-off value (Ct value) of the influenza sample when treated by sintered filter filtration rises faster than when the filter is not filtered. From this result, it became clear that a high-sensitivity test result can be obtained only by pretreatment with a sintered filter.
  • Example 6 Detection of Norovirus RNA
  • a sample prepared by suspending a stool sample containing Norovirus G2 type collected by Phloxwav in purified water was passed through a filtration filter containing a sintered filter (made of polyethylene, pore size 100 ⁇ m) to prepare a sample. .
  • a sample not filtered with a sintered filter was also used as a comparison.
  • Nucleic acid amplification and melting curve analysis The above sample was added to the following reagents, and Norovirus G2 type was detected under the following conditions. GENECUBE (registered trademark) manufactured by Toyobo was used for nucleic acid amplification and melting curve analysis.
  • a reaction solution containing the components shown below was prepared using GeneCube (registered trademark) Test Basic (Toyobo Co., Ltd.). The reaction solution was prepared in accordance with the instruction manual for GeneCube (registered trademark) Test Basic.
  • 0.5 ⁇ M COG2F primer (SEQ ID NO: 13) 1.5 ⁇ M COG2R primer (SEQ ID NO: 14) 0.3 ⁇ M hybridization probe (SEQ ID NO: 15, 3 ′ end labeled with BODIPY-FL) 0.05 unit / ⁇ L Reverse Ace (Toyobo) Sample 3 ⁇ L
  • Reverse transcription reaction, nucleic acid amplification and melting curve analysis 42 ° C. 180 seconds (reverse transcription reaction) 94 ° C, 30 seconds (more than one cycle) 98 ° C ⁇ 1 second 60 ° C ⁇ 10 seconds 63 ° C ⁇ 10 seconds (over 60 cycles) 94 ° C, 30 seconds 39 ° C, 30 seconds 39 ° C to 75 ° C (temperature rises at 0.09 ° C / second)
  • G2 cutoff value 7 Internal control cutoff value: 1.5
  • FIG. 7 is a graph showing the change in fluorescence intensity as the temperature rises after the RT-PCR under the above conditions, with the horizontal axis of the graph representing the temperature and the vertical axis representing the differential value of the fluorescence signal. is there.
  • norovirus G2 is detected when there is a filter.
  • the fluorescence value is low and below the cut-off value, which is weakly positive. From this result, it was revealed that it is important to use a sintered filter in order to obtain a highly sensitive test result in the Norovirus G2 gene test.
  • the method and kit of the present invention can be used for disease detection and diagnosis.
  • Genetic test kit body Packaging box
  • Specimen filter tube 21-a, 21-b: Sintered filter 3: Swab (Flox wab with short fibers attached)
  • Specimen collection part with short fibers attached 32: Swab shaft part 4: Genetic test reagent 41: Container body of genetic test reagent 42: Cover part of genetic test reagent

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Abstract

Le but de la présente invention est de fournir : un procédé de test de gène permettant de détecter ou de quantifier une substance à détecter à une précision élevée tout en empêchant la génération de faux positifs ou le bouchage et en conservant une sensibilité élevée ; et un kit à utiliser dans le procédé. Par conséquent, la présente invention concerne un procédé de test de gène caractérisé en ce qu'il comprend le filtrage d'un échantillon contenant une substance à détecter à travers un filtre, puis la détection ou la quantification de la substance présente dans l'échantillon. Dans un mode de réalisation spécifique, un filtre fritté est utilisé en tant que filtre dans le procédé et le kit de test de gène selon la présente invention.
PCT/JP2018/010136 2017-03-15 2018-03-15 Procédé de test de gène et kit de test de gène WO2018168986A1 (fr)

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