+

WO2018181850A1 - Matériau contenant du papier soluble dans l'eau pour la préparation d'échantillon et procédé de préparation d'échantillon l'utilisant - Google Patents

Matériau contenant du papier soluble dans l'eau pour la préparation d'échantillon et procédé de préparation d'échantillon l'utilisant Download PDF

Info

Publication number
WO2018181850A1
WO2018181850A1 PCT/JP2018/013523 JP2018013523W WO2018181850A1 WO 2018181850 A1 WO2018181850 A1 WO 2018181850A1 JP 2018013523 W JP2018013523 W JP 2018013523W WO 2018181850 A1 WO2018181850 A1 WO 2018181850A1
Authority
WO
WIPO (PCT)
Prior art keywords
water
sample
gene
soluble paper
analysis
Prior art date
Application number
PCT/JP2018/013523
Other languages
English (en)
Japanese (ja)
Inventor
健司 木下
Original Assignee
健司 木下
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by 健司 木下 filed Critical 健司 木下
Priority to JP2019510213A priority Critical patent/JP7093121B2/ja
Publication of WO2018181850A1 publication Critical patent/WO2018181850A1/fr

Links

Images

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/96—Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions

Definitions

  • the present invention relates to the preparation of samples for biological or chemical measurements. More particularly, it relates to sample preparation for genetic or drug analysis in biological samples.
  • the present invention has found that water-soluble paper or its components have an action of inhibiting reaction or suppressing / reducing noise due to substances in a biological sample, and has completed the present invention. Accordingly, the present invention provides an agent or material that suppresses or reduces reaction inhibition or noise caused by substances in biological samples, including water-soluble paper, and suppresses or reduces reaction inhibition or noise caused by substances in biological samples using the same. And a method for detecting, measuring or identifying a specific component (eg, gene or gene product) in a biological sample using the method. Such reaction inhibition or noise suppression / reduction may allow the extraction or purification step in the method to be omitted.
  • a specific component eg, gene or gene product
  • One embodiment of the present invention relates to suppressing the amplification activity inhibition effect of a nucleic acid polymerase by a biological sample or its component using water-soluble paper or its component.
  • a biological sample of a polymerase having 5 ′ ⁇ 3 ′ exonuclease activity containing water-soluble paper or a component thereof, or a suppressor or a suppressor for an amplification activity inhibitory effect by the component, and the suppressor are used.
  • a method for inhibiting amplification activity of a polymerase having 5 ′ ⁇ 3 ′ exonuclease activity by a biological sample or component thereof and a method for detecting, measuring or identifying a gene or gene product in the biological sample or component thereof.
  • the suppressor or suppression material of the amplification activity inhibition effect is useful for gene amplification reaction (for example, PCR) using a nucleic acid polymerase such as a polymerase having 5 ' ⁇ 3' exonuclease activity.
  • Gene analysis accompanied by gene amplification can be performed using a suppressor or suppression material of the amplification activity inhibitory effect.
  • a kit comprising a suppressor or suppressor for an amplification activity inhibitory effect for use in such genetic analysis is provided.
  • the present invention relates to a sampling agent or sampling material for sample preparation used in absorbance measurement, including water-soluble paper.
  • the absorbance measurement is performed, for example, by high performance liquid chromatography (HPLC) -ultraviolet absorption (UV) analysis. It is possible to perform therapeutic drug monitoring in a subject's blood from a dried blood spot (DBS) sample prepared using such a sampling agent or sampling material.
  • HPLC high performance liquid chromatography
  • UV ultraviolet absorption
  • the present invention provides a sampling agent or sampling material for preparing a sample for gene analysis by absorbance measurement and nucleic acid amplification from the same sample.
  • a sampling agent or sampling material for preparing a sample for gene analysis by absorbance measurement and nucleic acid amplification from the same sample.
  • water analysis paper is used to release nucleic acid components from the same blood sample into an aqueous fraction and drug components into an organic solvent fraction for gene analysis and absorbance measurement. At the same time.
  • One aspect of the present invention is a method for preparing a sample for gene analysis by absorbance measurement and nucleic acid amplification from the same sample, comprising dissolving a water-soluble paper spotted with a blood sample in a liquid, Preparing a sample for gene analysis by nucleic acid amplification from a part of the liquid in which the paper is dissolved, and preparing a sample for absorbance measurement from the other part of the liquid in which the aqueous paper is dissolved; Providing a method.
  • the water-soluble paper contains a water-soluble cellulose derivative.
  • the water-soluble paper comprises carboxymethyl cellulose.
  • the present invention is based in part on the unexpected discovery that water-soluble paper or its components have properties that are useful for inhibiting or reducing reaction by substances in biological samples or for reducing or reducing noise.
  • a target drug from a solid sample eg, dry blood spot (DBS) or dry saliva spot (DSS)
  • a biological sample eg, blood sample
  • DW Dermatiled Water
  • Some of them can be used for genetic testing.
  • the present invention provides a sampling agent or sampling material for preparing a sample for absorbance analysis and gene analysis by nucleic acid amplification comprising water-soluble paper or a component thereof from the same sample, wherein the absorbance
  • the measurement is by supercritical fluid chromatography
  • the sample for genetic analysis provides a sampling agent or sampling material that is the extraction residue of the supercritical fluid chromatography.
  • the nucleic acid sample in the water-soluble paper is not decomposed when the drug is extracted, which may be preferable in the present invention.
  • the structure of water-soluble paper is maintained after supercritical carbon dioxide extraction, and can be dissolved in water (distilled water) after that. Useful in the preparation of samples for
  • the present invention provides, for example, the following embodiments.
  • a kit comprising the inhibitor or the inhibitor material according to any one of Items 1 to 4.
  • the kit according to Item 5 for preparing a sample for gene amplification.
  • the kit according to item 7 wherein the gene analysis is a genotype test.
  • the kit according to item 8, wherein the genotype test is a test of a genotype of a drug metabolizing enzyme gene.
  • Sampling agent or sampling material for sample preparation used in absorbance measurement comprising water-soluble paper or its components.
  • the sampling agent or sampling material according to item 10 wherein the absorbance measurement is performed by high performance liquid chromatography (HPLC) -ultraviolet absorption (UV) analysis.
  • HPLC high performance liquid chromatography
  • UV ultraviolet absorption
  • (Item 12) The sampling agent or sampling material according to item 10 or 11, wherein the water-soluble paper contains a water-soluble cellulose derivative.
  • (Item 13) The sampling agent or sampling material according to any one of Items 10 to 12, wherein the water-soluble paper contains carboxymethylcellulose.
  • (Item 14) The sampling agent or the sampling material according to any one of Items 10 to 13, wherein the sample preparation is preparation of a dried blood spot (DBS) sample.
  • BBS dried blood spot
  • a kit comprising the sampling agent or the sampling material according to any one of Items 10 to 14.
  • (Item 16) The kit according to item 15, for use in therapeutic drug monitoring.
  • HPLC high performance liquid chromatography
  • UV ultraviolet absorption
  • (Item 21) The sampling agent or sampling material according to any one of items 17 to 20, wherein the water-soluble paper contains carboxymethylcellulose.
  • (Item 22) The sampling agent or sampling material according to any one of items 17 to 21, wherein the sample for measuring absorbance is a dried blood spot (DBS) sample.
  • (Item 23) A kit comprising the sampling agent or the sampling material according to any one of items 17 to 22.
  • (Item 24) The kit according to item 23, wherein the genotype test of the drug metabolizing enzyme gene and the therapeutic drug monitoring are performed on the same sample.
  • (Item 25) A method for preparing a sample for gene analysis by absorbance measurement and nucleic acid amplification from the same sample, Dissolving a water-soluble paper spotted with a sample (eg, a biological sample such as a blood sample) in a liquid; Preparing a sample for gene analysis by nucleic acid amplification from a part of the liquid in which the water-soluble paper is dissolved; Preparing a sample for absorbance measurement from another part of the liquid in which the water-soluble paper is dissolved. (Item 26) The method according to item 25, wherein the water-soluble paper contains a water-soluble cellulose derivative. (Item 27) The method according to item 25 or 26, wherein the water-soluble paper contains carboxymethyl cellulose. 28.
  • a sample eg, a biological sample such as a blood sample
  • a method for detecting, measuring or identifying a gene or gene product in a sample comprising: (A) providing the sample or component thereof (B) contacting the sample or a component thereof with water-soluble paper or a component thereof; (C) contacting the sample or the component after the contact with the gene or the polymerase using a polymerase having 5 ′ ⁇ 3 ′ exonuclease activity; A method comprising subjecting the gene product to a gene amplification reaction; and (D) detecting, measuring or identifying the gene or gene product based on a result of the amplification reaction.
  • kits for detecting, measuring or identifying a gene or gene product in a sample eg, a biological sample such as a blood sample
  • the kit comprising: (a) water-soluble paper or A kit comprising reagents for a gene amplification reaction, comprising: a component; and (b) a polymerase having 5 ′ ⁇ 3 ′ exonuclease activity.
  • the gene amplification reaction includes a polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • the detection, measurement or identification includes analysis of a genotype.
  • (Item 35) The kit according to any one of items 32 to 34, further comprising the features described in any one or more of items 1 to 9.
  • (Item 36) A method for performing analysis including absorbance measurement and nucleic acid amplification based on the same sample (for example, a biological sample such as a blood sample), wherein the method is brought into contact with water-soluble paper or a component thereof. Contacting the water-soluble paper or its components with an organic solvent to extract an organic fraction; contacting the water-soluble paper or its components with water or a water-containing solvent to extract an aqueous fraction; and the organic A method comprising a step of performing an analysis including nucleic acid amplification using a fraction, and performing an absorbance measurement using the organic fraction.
  • a step of dissolving, a step of preparing a sample for analysis by nucleic acid amplification from a part of the liquid in which the aqueous paper is dissolved, and an absorbance measurement from the other part of the liquid in which the water-soluble paper is dissolved A method comprising: preparing a sample; performing an analysis including nucleic acid amplification using the sample for nucleic acid amplification; and performing the absorbance measurement using the sample for absorbance measurement.
  • the absorbance measurement includes HPLC or LC-MS.
  • a system for performing analysis including absorbance measurement and nucleic acid amplification based on the same sample for example, a biological sample such as a blood sample
  • the system comprising water-soluble paper or a component thereof; Means for contacting the components with an organic solvent to extract an organic fraction; means for contacting the water-soluble paper or its components with water or a water-containing solvent to extract an aqueous fraction; means for performing an analysis including nucleic acid amplification; and A system comprising means for performing absorbance measurements.
  • the means for measuring absorbance includes an instrument that performs HPLC or LC-MS.
  • a system comprising: a water-soluble paper; a liquid that dissolves the water-soluble paper (eg, water, a water-containing solvent, etc.); a nucleic acid amplification reagent; and a means for measuring absorbance.
  • the means for measuring absorbance includes an instrument performing HPLC or LC-MS.
  • a sampling agent or a sampling material for preparing a sample for gene analysis by absorbance measurement and nucleic acid amplification which contains water-soluble paper or a component thereof, from the same sample, wherein the absorbance measurement is performed using a supercritical fluid.
  • (Item 49) A method for preparing a sample for gene analysis by absorbance measurement and nucleic acid amplification from the same sample, Subjecting a water-soluble paper (eg, DBS or DSS) spotted with a sample (eg, a biological sample such as a blood sample) to supercritical extraction to prepare a sample for absorbance measurement; Preparing a sample for gene analysis by nucleic acid amplification from the residue of the water-critical paper extracted by supercritical extraction (for example, dissolving in distilled water (DW)); Including a method.
  • a sample for gene analysis by nucleic acid amplification from the residue of the water-critical paper extracted by supercritical extraction (for example, dissolving in distilled water (DW)); Including a method.
  • the gene analysis is quantitative analysis.
  • the quantitative analysis is a copy number variation test.
  • (Item 52) The kit according to any of the preceding items, wherein the copy number variation test is performed using real-time PCR.
  • (Item 53) The kit according to any one of the preceding items, wherein the copy number variation test is performed using human RNase P as a standard.
  • (Item 54) The kit according to any one of the above items, wherein the nucleic acid is not extracted or purified in the gene analysis.
  • (Item 55) A method for performing genetic analysis of a blood sample, Contacting a blood sample with water-soluble paper or a component thereof; Collecting a certain area of the carrier, adding the carrier carrying the blood sample to a reaction solution, and performing a gene amplification reaction, and performing no nucleic acid extraction or purification. .
  • (Item 56) The method according to any of the preceding items, further comprising features described in any one or more of the items.
  • (Item 57) The method according to any one of the above items, wherein the gene analysis is a quantitative analysis.
  • (Item 58) The method according to any of the preceding items, wherein the quantitative analysis is a copy number variation test.
  • (Item 59) The method according to any of the preceding items, wherein the copy number variation test is performed using real-time PCR.
  • (Item 60) The method according to any one of the preceding items, wherein the copy number variation test is performed using human RNase P as a standard.
  • an amplification reaction in an assay using a polymerase having 5 ′ ⁇ 3 ′ exonuclease activity, an amplification reaction can be carried out directly using blood or its components, and a blood sample is directly used in Taqman method or the like. Simple detection of gene polymorphism and copy number polymorphism becomes easier.
  • FIG. 1A is a diagram showing the results of gene amplification for a genotype analysis by Taqman probe method from a sample using water-soluble paper (120MDP) and Whatman (registered trademark) 903 Protein save card.
  • the vertical axis represents the gene amplification signal of ADH1B (A).
  • the horizontal axis shows the gene amplification signal of ADH1B (G).
  • ⁇ indicates positive control results.
  • ⁇ shows the result of negative control.
  • X indicates the result of a sample using Whatman (registered trademark) 903 Protein save card.
  • the ⁇ indicates the results for a water-soluble paper sample.
  • FIG. 1A is a diagram showing the results of gene amplification for a genotype analysis by Taqman probe method from a sample using water-soluble paper (120MDP) and Whatman (registered trademark) 903 Protein save card.
  • the vertical axis represents the gene amplification signal of ADH1B (A).
  • the horizontal axis shows the
  • FIG. 1B shows an amplification curve at the time of gene amplification from a sample using water-soluble paper (120MDP) and Whatman (registered trademark) 903 Protein save card for genotype analysis by Taqman probe method.
  • FIG. 2A is a diagram showing the results of gene amplification for genotype analysis by Taqman probe method from samples using various water-soluble papers and filter papers and samples in which blood was dropped as it was into a reaction solution.
  • the vertical axis represents the gene amplification signal of ADH1B (A).
  • the horizontal axis shows the gene amplification signal of ADH1B (G).
  • ⁇ indicates positive control results.
  • ⁇ shows the result of negative control.
  • X indicates the result of a sample in which no gene amplification signal was observed.
  • FIG. 2B is a diagram showing the results of gene amplification for genotype analysis by Taqman probe method from samples using various water-soluble papers, filter papers, FTA cards, and samples obtained by dropping blood into reaction liquids as they are.
  • the vertical axis represents the gene amplification signal of ADH1B (A).
  • the horizontal axis shows the gene amplification signal of ADH1B (G).
  • ⁇ indicates positive control results.
  • ⁇ shows the result of negative control.
  • X indicates the result of a sample in which no gene amplification signal was observed.
  • the triangles indicate the results for samples where gene amplification signals were observed.
  • FIG. 1B The vertical axis represents the gene amplification signal of ADH1B (A).
  • the horizontal axis shows the gene amplification signal of ADH1B (G).
  • ⁇ indicates positive control results.
  • ⁇ shows the result of negative control.
  • X indicates the result of a sample in which no gene amplification signal was observed.
  • FIG. 3 is a diagram showing the results of gene amplification for samples using various water-soluble papers for genotype analysis by the Taqman probe method performed 8 months after sample collection.
  • the vertical axis represents the gene amplification signal of ADH1B (A).
  • the horizontal axis shows the gene amplification signal of ADH1B (G).
  • ⁇ indicates positive control results.
  • ⁇ shows the result of negative control.
  • the triangles indicate the results for samples where gene amplification signals were observed.
  • FIG. 4 shows an exemplary procedure for preparing a sample using water-soluble paper and performing gene analysis. After adding the blood sample to the water-soluble paper, it is dried at room temperature for about 1 hour, cut into a certain size (for example, 2 mm ⁇ ) and dissolved in distilled water.
  • FIG. 5 shows the results of treating a blood sample derived from 8 subjects with water-soluble paper 4 mm ⁇ , water-soluble paper 2 mm ⁇ and FTA, and subjecting it to a gene amplification reaction in the Taqman PCR method to determine the genotype of ADH1B
  • FIG. The vertical axis represents the gene amplification signal of ADH1B (A).
  • the horizontal axis shows the gene amplification signal of ADH1B (G).
  • ⁇ is a plot of the results of a sample determined to be A / A homozygous as a result of gene amplification.
  • ⁇ is a plot of the results of the samples determined to be G / A heterogeneous as a result of gene amplification.
  • FIG. 6 is a diagram showing the results of a blood sample derived from 8 subjects treated with FTA and subjected to a gene amplification reaction in the Taqman PCR method to determine the genotype of ADH1B.
  • the vertical axis represents the gene amplification signal of ADH1B (A).
  • the horizontal axis shows the gene amplification signal of ADH1B (G).
  • ⁇ is a plot of the results of a sample determined to be A / A homozygous as a result of gene amplification.
  • FIG. 7 is a diagram showing the results of treating a blood sample derived from 8 subjects with water-soluble paper 4 mm ⁇ and FTA and subjecting it to a gene amplification reaction in the Taqman PCR method in order to determine the genotype of ADH1B.
  • the vertical axis represents the gene amplification signal of ADH1B (A).
  • the horizontal axis shows the gene amplification signal of ADH1B (G).
  • ⁇ is a plot of the results of a sample determined to be A / A homozygous as a result of gene amplification.
  • ⁇ is a plot of the results of the samples determined to be G / A heterogeneous as a result of gene amplification.
  • ⁇ plots the results of a sample determined to be G / G homozygous as a result of gene amplification.
  • ⁇ is a plot of the negative control results.
  • FIG. 8 is a diagram showing the results of treating a blood sample derived from 8 subjects with 2 mm ⁇ water-soluble paper and FTA and subjecting it to a gene amplification reaction in the Taqman PCR method to determine the genotype of ADH1B.
  • the vertical axis represents the gene amplification signal of ADH1B (A).
  • the horizontal axis shows the gene amplification signal of ADH1B (G).
  • ⁇ is a plot of the results of a sample determined to be A / A homozygous as a result of gene amplification.
  • ⁇ is a plot of the results of the samples determined to be G / A heterogeneous as a result of gene amplification.
  • ⁇ plots the results of a sample determined to be G / G homozygous as a result of gene amplification.
  • ⁇ is a plot of the negative control results.
  • FIG. 9 shows the results of treating blood samples derived from 8 subjects with water-soluble paper 4 mm ⁇ , water-soluble paper 2 mm ⁇ and FTA, and subjecting them to a gene amplification reaction in the Taqman PCR method to determine the ALDH2 genotype.
  • FIG. The vertical axis represents the gene amplification signal of ALDH2 (A).
  • the horizontal axis represents the ALDH2 (G) gene amplification signal.
  • ⁇ is a plot of the results of a sample determined to be A / A homozygous as a result of gene amplification.
  • ⁇ is a plot of the results of the samples determined to be G / A heterogeneous as a result of gene amplification.
  • FIG. 10 is a diagram showing the results of a blood sample derived from 8 subjects treated with FTA and subjected to a gene amplification reaction in the Taqman PCR method in order to determine the ALDH2 genotype.
  • the vertical axis represents the gene amplification signal of ALDH2 (A).
  • the horizontal axis represents the ALDH2 (G) gene amplification signal.
  • ⁇ is a plot of the results of a sample determined to be A / A homozygous as a result of gene amplification.
  • FIG. 11 is a diagram showing the results of treating blood samples derived from 8 subjects with water-soluble paper 4 mm ⁇ and FTA, and subjecting them to a gene amplification reaction in the Taqman PCR method in order to determine the ALDH2 genotype.
  • the vertical axis represents the gene amplification signal of ALDH2 (A).
  • the horizontal axis represents the ALDH2 (G) gene amplification signal.
  • FIG. 12 is a diagram showing the results of treating a blood sample derived from 8 subjects with 2 mm ⁇ water-soluble paper and FTA and subjecting it to a gene amplification reaction in the Taqman PCR method in order to determine the ALDH2 genotype.
  • the vertical axis represents the gene amplification signal of ALDH2 (A).
  • the horizontal axis represents the ALDH2 (G) gene amplification signal.
  • ⁇ is a plot of the results of a sample determined to be A / A homozygous as a result of gene amplification.
  • ⁇ is a plot of the results of the samples determined to be G / A heterogeneous as a result of gene amplification.
  • ⁇ plots the results of a sample determined to be G / G homozygous as a result of gene amplification.
  • ⁇ is a plot of the negative control results.
  • FIG. 13A is a schematic diagram illustrating a sample preparation procedure in Example 3.
  • FIG. 13B shows the results of gene amplification by the Taqman method for samples sampled using saliva and samples prepared from blood using water-soluble paper and FTA cards.
  • the vertical axis represents the gene amplification signal of ADH1B (A).
  • the horizontal axis shows the gene amplification signal of ADH1B (G).
  • ⁇ is a plot of positive control results.
  • ⁇ is a plot of the results of samples sampled using saliva.
  • ⁇ plots the results of a sample using blood.
  • X is the result of the analysis by genomic DNA of the cells remaining in the swab sponge part.
  • ⁇ is a plot of the negative control results.
  • FIG. 14 shows the procedure for preparing a dried blood spot sample in TDM using water-soluble paper and the results of a quantitative test by addition and recovery using the sample.
  • FIG. 15 shows the detailed preparation procedure of a dried blood spot sample in TDM using water-soluble paper and the result of quantitative test by addition recovery using the dried blood spot sample.
  • FIG. 16 shows a procedure for preparing a dried blood spot sample in TDM using a filter paper paper disk for antibiotic testing, and the results of a quantitative test by addition recovery using the sample.
  • FIG. 17 shows a procedure for preparing a dried blood spot sample in TDM using water-soluble paper and FTA card.
  • FIG. 18 shows the result of detecting the caffeine concentration in peripheral blood by HPLC-UV from a DBS sample prepared using water-soluble paper and FTA card.
  • FIG. 19 is a diagram showing various analysis procedures from a blood sample.
  • FIG. 20 illustrates a procedure in the case where genetic polymorphism testing and therapeutic drug monitoring (TDM) are simultaneously performed using water-soluble paper.
  • FIG. 21 is a schematic diagram of a sampling kit used in Example 6.
  • FIG. 22 is a schematic diagram illustrating a procedure for sampling the peripheral blood sample (1) in the sixth embodiment.
  • FIG. 23 is a schematic diagram illustrating a procedure for sampling the peripheral blood sample (2) in the sixth embodiment.
  • FIG. 24 is a schematic diagram showing a procedure for collecting an oral mucosal cell sample in Example 6.
  • FIG. 25 is a schematic diagram showing the sampling procedure for the oral mucosal cell sample (3) in Example 6.
  • FIG. 26 is a schematic diagram showing the sampling procedure for the oral mucosal cell sample (4) in Example 6.
  • FIG. 27 is a schematic diagram summarizing the processing procedure of each sample in Example 6.
  • FIG. 28 shows the results of gene amplification of RNase P by TaqMan (registered trademark) PCR in the peripheral blood sample (1) in Example 6.
  • FIG. 29 shows the results of gene amplification of RNase P by TaqMan (registered trademark) PCR in the peripheral blood sample (2) in Example 6.
  • FIG. 22 shows the results of gene amplification of RNase P by TaqMan (registered trademark) PCR in the oral mucosa cell sample (3) in Example 6.
  • FIG. 22 shows the results of gene amplification of RNase P by TaqMan (registered trademark) PCR in the oral mucosa cell sample (4) in Example 6.
  • water-soluble paper refers to paper having the property of being decomposed by contact with water and dissolved or dispersed in water (water decomposability). Whether or not it is water-degradable is determined by, for example, contacting 0.05 to 0.1 mg of water-soluble paper with 10 ⁇ l of water (reaction solution), and then setting the turbidity of the water (reaction solution) at 660 nm to Bio -It shall have water-decomposability when it is 0.4 or less when measured using a Smart Spec (trademark) 3000 manufactured by RAD.
  • water-soluble paper examples include 60 MDP (manufactured by Nippon Paper Industries, Inc.), 30 MDP (manufactured by Nippon Paper Industries, Inc.), 120 MDP (manufactured by Nippon Paper Industries, Inc.), 30 MDP-S (manufactured by Nippon Paper Industries, Inc.), MDP series products such as 60MDP-S (Nippon Paper Papillia), 30CD-2 (Nippon Paper Papillia), 60CD-2 (Nippon Paper Papillia), 120CD-2 (Nippon Paper Papillia), etc.
  • Water-degradable paper such as CD-2 series, water-degradable paper (available from TT Trading Co., Ltd.
  • Water-soluble paper is preferable because it is uniformly dispersed during the reaction in the reaction solution used for gene amplification or specific gene detection, and further settles, so that it does not interfere with physical operation or optical detection. In another preferred embodiment, one that allows optical detection is preferred.
  • water-soluble paper may also be referred to as “hydrolytic paper”, and both are used interchangeably.
  • Ordinary paper is mainly made of vegetable fibers (for example, cellulose) and entangled with fibers. Between these fibers, hydrogen bonds are generated by the OH groups of cellulose, but the bonds are released in water, so paper generally loses physical strength due to water, and the fibers disperse in water over time. To do.
  • Water-soluble paper is paper that has been manufactured to further enhance its water-soluble properties.
  • water-soluble paper forms paper by forming paper using a derivative obtained by introducing a hydrophilic functional group into cellulose, for example, a water-soluble cellulose derivative such as hydroxyethyl cellulose or carboxymethyl cellulose (CMC). It can be produced by making cellulose itself easily soluble in water.
  • the CMC may be Sunrose (registered trademark) (Nippon Paper Industries) or the like.
  • CMC is obtained by substituting a carboxymethyl group for an OH group, and is solubilized by the polarity of the carboxyl group.
  • Glucose which is a unit of cellulose, has three OH groups. Among these, when an average of 0.3 or more per glucose unit is substituted with a carboxymethyl group, water solubility begins to occur.
  • Such a water-soluble cellulose derivative may be obtained by substituting OH groups in cellulose fibers at various ratios. Moreover, such a water-soluble cellulose derivative can be included in paper fiber in an arbitrary ratio.
  • the paper fibers can be bound by using a water-soluble binder instead of physical entanglement, whereby the water-soluble paper can be produced by eliminating the binding between the fibers in water.
  • a water-soluble binder instead of physical entanglement
  • Such water-related properties of paper are described in Textile and Industrial Vol. 1 1968 No. 10 P604-608, Toshiharu Emae, Papa Gikyo Magazine 58 (8), Papyrus, 105-109 (2004).
  • a special component contained in the water-soluble paper is a blood of a polymerase having the effect of the present invention, eg, 5 ′ ⁇ 3 ′ exonuclease activity. Or it is understood that it has contributed partially to suppression of the amplification activity inhibitory effect by the component.
  • Such water-soluble paper can be used as a preferred carrier in the present invention.
  • Water-soluble papers described in literatures such as those described above, or water-soluble papers produced using the described methods can also be used in the present invention.
  • Commonly commercially available tissues (water soluble) and paper used for goldfish scooping poi can also be used.
  • the water-soluble paper includes a water-soluble cellulose derivative. In a further embodiment, the water-soluble paper comprises carboxymethyl cellulose.
  • a sample for biological or chemical analysis of a biological sample can be prepared by dropping a biological sample (for example, blood) onto water-soluble paper and drying it.
  • a biological sample for example, blood
  • the biological analysis is genetic analysis.
  • the chemical analysis is a pharmacokinetic analysis.
  • Gene analysis refers to examining the state of nucleic acids (DNA, RNA, etc.) in a biological sample.
  • genetic analysis can include those that utilize nucleic acid amplification reactions. Examples of gene analysis including these include sequencing, genotyping / polymorphism analysis (SNP analysis, copy number polymorphism, restriction fragment length polymorphism, repeat number polymorphism), expression analysis, fluorescence quenching probe ( Quenching Probe (Q-Probe), SYBR green method, melting curve analysis, real-time PCR, quantitative RT-PCR, and the like.
  • SNP analysis genotyping / polymorphism analysis
  • Q-Probe Quenching Probe
  • SYBR green method melting curve analysis
  • real-time PCR quantitative RT-PCR
  • Such gene analysis generally involves a nucleic acid amplification reaction.
  • a biological sample eg, blood
  • a reaction solution for a nucleic acid amplification reaction eg, PCR
  • an amplification reaction eg, 5 ′ ⁇ 3 ′ exonuclease such as Taq DNA polymerase
  • active polymerase may be inhibited.
  • a sample prepared by dropping a biological sample (for example, blood) on water-soluble paper inhibition of such amplification reaction is suppressed.
  • water-soluble paper has the ability to remove substances that inhibit PCR amplification (heme, polysaccharides, polyphenols, fulvic acids, dyes, ions, etc.). It is done.
  • a nucleic acid polymerase In the nucleic acid amplification reaction that can be used in one embodiment of the present invention, a nucleic acid polymerase is used.
  • the DNA polymerase used for gene analysis can be used without particular limitation as long as it is a polymerase excellent in heat resistance for synthesizing nucleic acid by primer addition, represented by Taq DNA polymerase.
  • the nucleic acid amplification reaction particularly used in the present invention uses a polymerase having 5 ′ ⁇ 3 ′ exonuclease activity.
  • Examples of such a DNA polymerase include Taq DNA polymerase derived from Thermus aquaticus, Tth DNA polymerase, or a mixture of at least one of the above-mentioned DNA polymerases.
  • C derived from Tth DNA polymerase and Carboxyothermus hydrogenoforms. Since the therm DNA polymerase also has RT activity, when RT-PCR is performed in One tube-One step, it has the characteristic that it can be covered with one kind of enzyme. In one embodiment, methods using other polymerases may be used, but it is recommended as a simple method that the sample be used directly with a polymerase having 5 ' ⁇ 3' exonuclease activity, such as Taq DNA polymerase.
  • amplification inhibition contained in a biological sample occurs.
  • a biological sample such as a blood sample
  • water-soluble paper or its components as in the present invention, the problem of amplification inhibition contained in such a biological sample (for example, a blood sample) has been solved unexpectedly.
  • a biological sample such as a blood sample can be directly applied to an analysis method using a polymerase having 5 ' ⁇ 3' exonuclease activity (for example, a so-called Taqman method or a technique equivalent thereto). This is something that could not be achieved with the prior art.
  • Examples of the reaction using a polymerase having 5 ′ ⁇ 3 ′ exonuclease activity such as Taq DNA polymerase include a PCR (Polymerase Chain Reaction) method, a LAMP (Loop-Mediated Isothermal Amplification), and loop-mediated isothermal amplification.
  • the buffer is not particularly limited, but EzWay TM (KOMA Biotechnology), Ampdirect (registered trademark) (manufactured by Shimadzu Corporation), Phusion (registered trademark) Blood Direct PCR kit buffer (New ENGLAND Bio-Labs), MasterAmp Among the registered trademark PCR kit (manufactured by Epicentre), one that does not diminish the effect of removing amplification inhibition can be used.
  • the primers used for the reaction in the present invention can be appropriately designed by a known method when a gene to be amplified or a specific gene to be detected is determined.
  • the primer used for the reaction in the present invention is not particularly limited as long as it can specifically amplify a gene to be amplified or a specific gene to be detected.
  • the amplification of the gene contained in the test sample or the detection of the specific gene contained in the test sample is preferably performed on a plate-like or tube-like insoluble carrier.
  • insoluble carriers include tubes made of plastic, glass and the like that are insoluble in the reaction solution, as well as 96-wells.
  • the tube shape means a hollow state, and may be a shape such as a PCR tube with a bottom or an Eppendorf tube.
  • the reaction solution is put into a plate-like or tube-like insoluble carrier.
  • a reaction solution containing a reagent such as a buffer, a polymerase and a primer is put therein, and in the case of a plate-shaped insoluble carrier, the reaction solution is placed on the surface thereof.
  • carrier may contact directly, and amplification methods, such as PCR method mentioned above, are performed.
  • a water-degradable carrier carrying a test sample may be placed on an insoluble carrier, and then the reaction solution may be placed on the insoluble carrier.
  • the nucleic acid-containing biomaterial is transferred from the base material to the carrier, and a certain area (in this case, a 4 mm diameter can be used, but may be an appropriate size, for example, 2 to 6 mm diameter, 3 to 5 mm diameter, or about 4 mm It may be a diameter.)
  • the carrier is dissolved in a tube-shaped insoluble container and heated as necessary to elute the nucleic acid from the carrier or accelerate the disappearance of the carrier, and then the buffer, polymerase, primer, etc. Add amplification reagents to amplify genes or detect specific genes.
  • any known technique such as a known real-time PCR can be used.
  • These methods are methods for detecting the amplification amount of DNA in real time using a label such as a fluorescent reagent, and typically require an apparatus in which a thermal cycler and a spectrofluorometer are integrated. Such devices are commercially available.
  • a label such as a fluorescent reagent
  • thermal cycler and a spectrofluorometer are integrated.
  • Such devices are commercially available.
  • There are several methods depending on the fluorescent reagent to be used and examples include the intercalator method, the TaqMan TM probe method, the Molecular Beacon method, and the like.
  • a fluorescent reagent or fluorescent probe called intercalator, TaqMan TM probe or Molecular Beacon probe is added to a PCR reaction system containing a primer pair for amplifying a template genomic DNA and a genomic region containing a target SNP site.
  • the TaqMan TM probe method also referred to as TaqMan TM method
  • the TaqMan TM probe is an oligonucleotide that can be hybridized to the amplification region of the target nucleic acid with both ends modified with a fluorescent substance and a quencher.
  • the amount of amplification product produced can be monitored by measuring the fluorescence intensity, thereby estimating the original template DNA amount.
  • Taqman assay Copy number analysis by Taqman assay is a technique known in the art, for example.
  • the Taqman assay is based on a 5'-nuclease assay, also referred to as a fluorogenic 5'-nuclease assay; Holland et al., Proc. Natl. Acad. Sci. USA 88: 7276-7280 (1991); and Heid You can also refer to et al., Genome Research 6: 986-994 (1996).
  • oligonucleotide primers are used to create an amplicon specific for the PCR reaction.
  • a third oligonucleotide (TaqMan probe) is designed to hybridize with the nucleotide sequence in the amplicon located between the two PCR primers.
  • the probe may have a structure that cannot be extended by the DNA polymerase used in the PCR reaction, and is usually (although not essential) co-labeled with a fluorescent reporter dye and a quencher moiety in close proximity to each other. Light emission from the reporter dye is quenched by the quenching moiety when the phosphor and quencher are in close proximity, as on the probe.
  • the probe may be labeled only with a fluorescent reporter dye or another detectable moiety.
  • thermostable DNA-dependent DNA polymerase having 5'-3 'nuclease activity is used.
  • the 5'-3 'nuclease activity of the DNA polymerase cleaves the labeled probe that hybridizes with the amplicon in a template-specific manner.
  • the resulting probe fragment dissociates from the primer / template complex and the reporter dye is released from the quenching effect of the quencher moiety.
  • Approximately one molecule of reporter dye is released for each newly synthesized amplicon molecule, and the amount of fluorescent reporter dye released is directly proportional to the amount of amplicon template by detecting the unquenched reporter dye.
  • a basis for quantitative interpretation of the data in form is provided.
  • CT threshold cycle
  • the fluorescence level is recorded during each PCR cycle and is proportional to the amount of product amplified to that point in the amplification reaction.
  • the PCR cycle when the fluorescence signal was first recorded as statistically significant or when the fluorescence signal exceeds some other arbitrary level (eg, arbitrary fluorescence level; AFL) is the threshold Cycle (CT).
  • Protocols and reagents for 5'-nuclease assays are known to those of skill in the art and are described in various references. For example, the 5′-nuclease reaction and probe are described in US Pat. No. 6,214,979 (Gelfand et al.); US Pat. No.
  • TaqMan TM PCR can be performed using commercially available kits and equipment such as ABI PRISM® 7700 Sequence Detection System (Applied Biosystems, Foster City, Calif., USA), LightCycler® (Roche Applied). Sciences, Mannheim, Germany).
  • the 5′-nuclease assay procedure is performed on a real-time quantitative PCR instrument such as, but not limited to, an ABI PRISM® 7700 Sequence Detection System.
  • This system typically consists of a thermocycler, laser, charge-coupled device (CCD), camera, and computer.
  • This system amplifies samples in a microtiter plate format such as 96 wells on a thermocycler. During amplification, for all wells, the laser-induced fluorescence signal is collected in real time through a fiber optic cable and detected with a CCD camera.
  • the system includes software for instrument operation and data analysis.
  • the Taqman assay reported for CYP2D6 can be used (Bodin et al., J Biomed Biotechnol. 3: 248-53 2005).
  • a new reverse primer can be designed by Primer Express or the like, and copy number analysis can be performed again.
  • a Taqman probe having a 5 'end labeled with FAM and a No Fluorescence Quencher and MGB linked to the 3' end can be used.
  • RNase P assay (ThermoFisher) labeled with an appropriate label (for example, VIC) can be used.
  • All Taqman assays can be performed according to the reported protocol, available from the manufacturer, and copy number calculations can be performed by the ⁇ Ct method (Bodin et al., 2005). As one example, it is assumed that the sample having the median value of ⁇ Ct is 2 copies and can be used as a calibrator, but is not limited thereto. All samples can be tested in duplicate and the average copy number can be used in a scatter plot analysis, but the number of samples can be increased or decreased as needed.
  • TaqPath TM ProAmp TM Master Mix can be used as a real-time PCR reagent in the present invention.
  • TaqPath TM ProAmp TM Master Mix features exceptional data quality (even in the presence of PCR inhibitors, can provide high specificity, dynamic range, and reproducibility in genotyping and copy number variation (CNV) analysis ) And resistance to PCR inhibitors (capable of handling samples prepared from humans and animals (buccal swabs, blood, and card punches)).
  • CNV genotyping and copy number variation
  • the present invention provides a suppressor or suppression material for the inhibitory effect on the amplification activity of blood of a polymerase having 5 ′ ⁇ 3 ′ exonuclease activity or its component, including water-soluble paper or its component.
  • a polymerase having 5 ′ ⁇ 3 ′ exonuclease activity is useful because it is used for quantitative analysis using this exonuclease activity represented by the Taqman method.
  • Typical examples of the polymerase to be used include Taq DNA polymerase and Tth DNA polymerase, and Taq DNA polymerase is preferably used.
  • the water-soluble paper or its components that can be used in the present invention contains a water-soluble cellulose derivative (for example, carboxymethyl cellulose).
  • a water-soluble cellulose derivative for example, carboxymethyl cellulose.
  • the present invention provides a kit containing the inhibitor or inhibitor material of the present invention.
  • a kit is for preparing a sample for gene amplification or for use in gene analysis (eg genotyping such as genotyping of drug metabolizing enzyme genes).
  • reagents polymerase, buffer, etc.
  • necessary for gene amplification and / or appropriate primers are optionally included.
  • the present invention provides a method for detecting, measuring or identifying a gene or gene product in a sample or component thereof.
  • the method includes: (A) providing a sample (eg, a biological sample such as a blood sample) or component thereof; (B) contacting the biological sample or component thereof with aqueous paper or component thereof; (C) contacting.
  • the present invention provides a kit for detecting, measuring or identifying a gene or gene product in a sample or a component thereof.
  • the kit includes reagents for a gene amplification reaction comprising: (a) water-soluble paper or components thereof; and (b) a polymerase having 5 ' ⁇ 3' exonuclease activity.
  • the sample may be a biological sample such as a blood sample, for example.
  • the gene amplification reaction includes, but is not limited to, polymerase chain reaction (PCR). 5 ′ ⁇ 3 ′ exonuclease
  • PCR polymerase chain reaction
  • Any assay using an active polymerase can include those exemplified herein and any other assay.
  • the water-soluble paper that can be utilized in this aspect of the present invention may be any as long as it can achieve its purpose, such paper can include any of those described herein (polymerase (Suppression of the amplification activity inhibitory effect) can also be used.
  • detection, measurement or identification includes genotype analysis.
  • gene amplification / specific gene detection is performed, or using the method of the present invention (for example, amplification method), human 3 billion base pairs
  • SNPs single nucleotide polymorphisms
  • future risk factors can also be diagnosed.
  • genetic factors such as strength against alcohol can be examined by SNP typing the alcohol dehydrogenase gene (ADH1B) and the aldehyde dehydrogenase gene (ALDH2).
  • ADH1B gene polymorphism 47th arginine (C G C) is converted to histidine (C A C) (G of the second nucleic acid in the codon is converted to A), and guanine (G) is converted to SNP site.
  • C G C histidine
  • G guanine
  • SNP site Those having an ADH1B * 1 allele and those mutated and having an adenine (A) are referred to as an ADH1B * 2 allele.
  • the rate of acetaldehyde production increases due to an increase in the activity of ADH, which increases alcohol sensitivity.
  • ALDH2 is (converted to the first G nucleic acids in codon A) 487 th glutamic acid (G AG) lysine (A AG) converted and to have a guanine (G) in the SNP site Those with ALDH2 * 1 allele and those with mutation and adenine (A) are called ALDH2 * 2 allele. Also in ALDH2, mutant ALDH2 * 2 alleles are frequently seen in Asia, but this shows that ALDH2 activity decreases in mutant carriers. As a result, the metabolism of acetaldehyde is delayed and alcohol sensitivity is increased.
  • the sampling technique of the present invention or the amplification method using the same can be used in liquid samples such as saliva, sweat, blood and feces. It can also be applied to microbiological tests such as bacteria and viruses.
  • blood samples include whole blood samples (eg, dried whole blood spot samples) as well as samples that have undergone some treatment or lack any components in whole blood. Examples include plasma, serum, blood clot, blood cell fraction, red blood cell fraction, platelet fraction and the like. Even in the case of a sample of some blood components, if it contains a nucleic acid amplification inhibitor, the effect of inhibiting inhibition of amplification of the present invention can be expected.
  • a method for performing gene analysis of a blood sample wherein nucleic acid is not extracted or purified.
  • the method comprises a step of bringing a blood sample into contact with water-soluble paper or a component thereof, and a step of collecting a certain area of the carrier, adding the carrier carrying the blood sample to a reaction solution, and performing a gene amplification reaction.
  • the gene analysis may be a quantitative analysis, and accurate quantification may be possible by the action of suppressing the amplification inhibitory effect of water-soluble paper or its components.
  • the quantitative analysis may be a copy number polymorphism test, which may be performed using real-time PCR. The copy number variation test can be performed using human RNase P as a standard.
  • the present invention provides a sampling agent or sampling material for use in absorbance measurements comprising water-soluble paper or components thereof.
  • a sampling agent or sampling material for use in absorbance measurements comprising water-soluble paper or components thereof.
  • noise in absorbance measurements is reduced.
  • FTA card eg FTA card, available from GE Healthcare.
  • Any form can be used as long as it is planned, such as dry blood spot (DBS) sample preparation.
  • DBS dry blood spot
  • the target absorbance measurement is high-performance liquid chromatography (HPLC) -ultraviolet absorption (UV) analysis, analysis using mass spectrometry such as liquid chromatography-mass spectrometry (LC-MS), and supercritical fluid extraction.
  • HPLC high-performance liquid chromatography
  • UV ultraviolet absorption
  • LC-MS liquid chromatography-mass spectrometry
  • supercritical fluid extraction -Supercritical fluid chromatography
  • SFE-SFC Ultraviolet absorption
  • SFE Supercritical fluid extraction
  • SFC Supercritical fluid extraction
  • MS Mass spectrometry
  • the water-soluble paper that can be utilized in this aspect of the present invention may be any as long as it can achieve its purpose, such paper can include any of those described herein (polymerase (Suppression of inhibitory effect on amplification activity of nucleic acid) and (Nucleic acid amplification and analysis using suppression of inhibitory effect on amplification activity of polymerase) can also be used.
  • kits In another aspect, the present invention provides a kit comprising the sampling agent or sampling material of the present invention.
  • This kit can be used for pharmacological monitoring, therapeutic drug monitoring and the like.
  • the kit of the present invention may comprise a means for preparing a sample for monitoring.
  • the kit of the present invention includes a carrier comprising water-soluble paper or components thereof affixed on a mount.
  • a carrier comprising water-soluble paper or components thereof affixed on a mount.
  • the mount of the kit is configured such that it can be folded to prevent exposure of the water-soluble paper or a carrier containing its components.
  • the mount on a substantially rectangular shape is divided into at least three sections, and the carrier is arranged in the end section, so that the second section covers one surface of the carrier, and the third section is the carrier. It is the structure which prevents exposure of both surfaces of a support
  • a plastic member can be provided in the second compartment to promote drying of the carrier.
  • the present invention provides a sampling agent or sampling material for preparing a sample for gene analysis by absorbance measurement and nucleic acid amplification comprising water-soluble paper or a component thereof from the same sample.
  • the sample can be, for example, a dried blood spot (DBS) sample. Sampling for such dual or multiplex assays is one of the features when using water-soluble paper or its components.
  • DBS dried blood spot
  • the gene analysis performed in the assay of the present invention can be any assay with nucleic acid amplification, such as Taqman (TM) analysis, and the absorbance measurement is high performance liquid chromatography (HPLC) -ultraviolet absorption (UV) analysis, supercritical Examples thereof include a method involving chromatography such as fluid chromatography (SFC) -ultraviolet absorption (UV) analysis or a method of directly measuring absorbance.
  • HPLC high performance liquid chromatography
  • SFC fluid chromatography
  • UV ultraviolet absorption
  • the water-soluble paper that can be used in this aspect of the present invention is not limited as long as the object can be achieved, and examples of such paper include any of those described in the present specification. Those described in (Suppression of inhibitory effect of polymerase amplification activity) and (Sampling for absorbance measurement) can also be used.
  • the present invention provides a kit containing the sampling agent or sampling material of the present invention.
  • This kit can prepare samples for performing two or more assays including absorbance analysis and gene analysis by nucleic acid amplification.
  • this kit is a kit for performing two or more assays including absorbance analysis and gene analysis by nucleic acid amplification.
  • means for absorbance measurement, reagents for nucleic acid amplification, means for monitoring, etc. can be included.
  • the two or more assays comprise genotyping of drug metabolizing enzyme genes and therapeutic drug monitoring, characterized in that they are performed on the same sample.
  • the present invention is a method of preparing a sample for genetic analysis by absorbance measurement and nucleic acid amplification from the same sample, wherein the sample (eg, a biological sample such as a blood sample) is spotted A step of dissolving water-soluble paper in a liquid, a step of preparing a sample for gene analysis by nucleic acid amplification from a portion of the liquid in which the water-soluble paper is dissolved, and another portion of the liquid in which the water-soluble paper is dissolved Preparing a sample for absorbance measurement from.
  • the sample eg, a biological sample such as a blood sample
  • the water-soluble paper that can be utilized in this aspect of the present invention may be any as long as it can achieve its purpose, such paper can include any of those described herein (polymerase Those described in (Suppression of inhibitory effect on amplification activity) and (Sampling for absorbance measurement) can also be used.
  • the present invention provides a method of performing an analysis that includes absorbance measurement and nucleic acid amplification based on the same sample (eg, a biological sample such as a blood sample).
  • This method comprises contacting the sample with water-soluble paper or a component thereof; contacting the water-soluble paper or a component thereof with an organic solvent; and extracting an organic fraction; water or a water-containing solvent with the water-soluble paper or a component thereof.
  • Absorbance measurements utilized herein can include HPLC or LC-MS.
  • the water-soluble paper that can be utilized in this aspect of the present invention may be any as long as it can achieve its purpose, such paper can include any of those described herein (polymerase (Suppression of inhibitory effect on amplification activity), (Nucleic acid amplification and analysis using suppression of inhibitory effect on polymerase amplification activity), and (Sampling for absorbance measurement) can also be used.
  • the present invention provides a method for performing an analysis including absorbance measurement and nucleic acid amplification using the same sample (for example, a biological sample such as a blood sample).
  • the method comprises dissolving a water-soluble paper spotted with the sample in a liquid, preparing a sample for analysis by nucleic acid amplification from a part of the liquid in which the water-soluble paper is dissolved, and Preparing a sample for absorbance measurement from the other part of the dissolved liquid, performing an analysis including nucleic acid amplification using the sample for nucleic acid amplification, and preparing a sample for the absorbance measurement. And measuring the absorbance using the method.
  • Absorbance measurements utilized herein can include HPLC or LC-MS.
  • the water-soluble paper that can be utilized in this aspect of the present invention may be any as long as it can achieve its purpose, such paper can include any of those described herein (polymerase (Suppression of the inhibitory effect on amplification activity) and (sampling for absorbance measurement) can also be used.
  • the present invention provides a system for performing analysis including absorbance measurement and nucleic acid amplification based on the same sample (for example, a biological sample such as a blood sample).
  • the system comprises a water-soluble paper or a component thereof; means for contacting the water-soluble paper or a component thereof with an organic solvent; and extracting an organic fraction; contacting the water-soluble paper or a component thereof with water or a water-containing solvent.
  • the present invention provides a system for performing analysis including absorbance measurement and nucleic acid amplification using the same sample (for example, a biological sample such as a blood sample).
  • This system includes a water-soluble paper on which the sample has been spotted as necessary; a liquid that dissolves the water-soluble paper (eg, water, a water-containing solvent, etc.); a nucleic acid amplification reagent; and a means for measuring absorbance.
  • the water-soluble paper or its component can include any of those described in the present specification, and (for suppressing the amplification activity inhibition effect of polymerase) and (for absorbance measurement) Can also be used.
  • any organic solvent for example, alcohol such as methanol, nonpolar solvent, etc.
  • water or any water-containing solvent can be used.
  • the analysis including nucleic acid amplification may include a reagent for performing a nucleic acid amplification reaction (an arbitrary polymerase, a buffer as necessary, a primer as necessary).
  • the means for measuring the absorbance can include an instrument for performing HPLC or LC-MS, if necessary, in addition to the absorbance meter.
  • the water-soluble paper that can be utilized in this aspect of the present invention may be any as long as it can achieve its purpose, such paper can include any of those described herein (polymerase Also, those described in (Suppression of the inhibitory effect on the amplification activity), (Sampling for absorbance measurement) and (Sampling for performing two or more assays from the same sample) can be used.
  • the present invention provides a sampling agent or sampling material for sample preparation for supercritical extraction or supercritical fluid chromatography.
  • Supercritical extraction or supercritical fluid chromatography can be performed for pharmacokinetic analysis as described below.
  • supercritical extraction can be performed from a sampling agent or sampling material comprising water-soluble paper and the supercritical fluid phase can be subjected to chromatography. Further, the residue of the water-soluble paper after the supercritical extraction can be further dissolved in water and used as a sample for analysis of the remaining component (for example, nucleic acid).
  • Supercritical extraction is a method of extracting a substance from a sample using a supercritical fluid (supercritical fluid) as a solvent.
  • a supercritical fluid is a state of a substance placed at a temperature and pressure higher than a critical point (critical point). In such a supercritical state, the kinetic energy is sufficient to shake off the intermolecular force, but the intermolecular distance is close because of the high pressure, that is, the influence of intermolecular force must be considered. , It becomes a peculiar state. For this reason, supercritical fluids have unique properties that are neither liquid nor gas, and have advantages different from using organic solvents in operations such as extraction and chromatography. In particular, the supercritical fluid can change the solubility of the fluid by adjusting the temperature and pressure, and the target substance can be selectively extracted.
  • supercritical carbon dioxide As the supercritical fluid, supercritical carbon dioxide (scCO 2 ) is most commonly used. Carbon dioxide, a supercritical fluid, dissolves various substances well, and if the supercritical carbon dioxide that dissolves the target product is made below the critical point, carbon dioxide is vaporized. possible. The vaporized carbon dioxide can be recovered and reused. Since carbon dioxide has a low critical temperature of 31 ° C., it can be extracted while preventing the destruction of molecules (in particular, biomolecules such as nucleic acids and proteins) and / or denaturation.
  • molecules in particular, biomolecules such as nucleic acids and proteins
  • Supercritical fluid chromatography is a kind of column chromatography and is characterized by using a supercritical fluid as a mobile phase. SFC is basically performed in a system similar to high performance liquid chromatography, except that a supercritical fluid is used as a main mobile phase. Carbon dioxide is a common substance used as a supercritical fluid. In SFC, separation (clearly separated from the peaks of other substances) and the supercritical fluid are characterized by the low viscosity and high diffusivity. There is a tendency for the ability of detection (high sensitivity to be obtained with sharp peaks) to be improved over HPLC. The measurement time varies greatly depending on the measurement parameter, but is generally about several minutes to several tens of minutes / times.
  • the detector includes an ultraviolet / visible absorbance detector (Ultra-Violet / Visible Light Absorbance Detector), diode array detector (Diode Array Detector), mass spectrometer (MS), circular dichroism detector (CD).
  • a detector used in HPLC such as a flame ionization detector (FID) used in gas chromatography (GC) can be used.
  • the mobile phase is a liquid mixed with carbon dioxide
  • various liquids can be used as long as they do not adversely affect the column and apparatus, and they are used in HPLC such as alcohols (especially methanol), acetonitrile, and dichloromethane. Can be used.
  • HPLC high-density liquids
  • alcohols especially methanol
  • acetonitrile especially acetonitrile
  • dichloromethane can be used.
  • the polarity of supercritical carbon dioxide changes depending on the degree of pressurization, it is possible to use only carbon dioxide as the mobile phase and perform analysis by changing the pressure.
  • SFC system a system provided by Shimadzu Corporation, Waters, Thar Instruments, INC, JASCO, Novasep, Gilson, etc. can be used.
  • a dedicated column such as Shim-pack UC (Shimadzu Corporation) can be used, but an HPLC column can also be used.
  • a method in which a sample containing water-soluble paper (eg, DBS) is subjected to supercritical fluid chromatography, the remaining sample is dissolved in distilled water, and used for genetic testing.
  • a sample containing water-soluble paper for example, DBS
  • a sample containing water-soluble paper for example, DBS
  • a part is used as a sample for genetic testing, and supercritical fluid extraction is performed from the other part.
  • a sample method for pharmacokinetic analysis is provided.
  • distilled water dissolution (partially genetic testing) after DBS, solvent extraction (eg water-ethyl acetate) followed by organic phase (HPLC).
  • solvent extraction immersion in methanol or the like ⁇ HPLC
  • solvent extraction immersion in methanol or the like ⁇ HPLC
  • SFC supercritical fluid extraction ⁇ supercritical fluid chromatography
  • the present invention provides a sampling agent or sampling material for sample preparation for pharmacokinetic analysis.
  • Pharmacokinetic analysis can include absorbance measurements.
  • the pharmacokinetic analysis can be therapeutic drug monitoring (TDM).
  • TDM therapeutic drug monitoring
  • any feature described in the present specification can be adopted as long as the object can be achieved, (suppression of polymerase amplification activity inhibition effect), (polymerase amplification activity) As described in Nucleic Acid Amplification and Analysis with Suppression of Inhibitory Effects, (Sampling for Absorbance Measurement), (Sampling to Perform Two or More Assays from the Same Sample), and (Multiple Assay Using the Same Sample) Can also be used.
  • pharmacokinetic analysis methods include separation analysis methods (high-performance liquid chromatography (HPLC), gas chromatography (GC) and their separation methods and detection methods (for example, combination with UV measurement), immunological methods, and the like) (For example, fluorescence polarization immunoassay (FPIA), enzyme immunoassay (EIA), radioimmunoassay (RIA)), atomic absorption photometry, etc., supercritical fluid extraction (SFE) ⁇ supercritical fluid chromatography (SFC) ).
  • separation analysis methods high-performance liquid chromatography (HPLC), gas chromatography (GC) and their separation methods and detection methods (for example, combination with UV measurement), immunological methods, and the like)
  • FPIA fluorescence polarization immunoassay
  • EIA enzyme immunoassay
  • RIA radioimmunoassay
  • SFE supercritical fluid extraction
  • SFC supercritical fluid chromatography
  • a sample for a method including absorbance analysis for example, high performance liquid chromatography (HPLC) -ultraviolet absorption (UV) analysis
  • HPLC high performance liquid chromatography
  • UV ultraviolet absorption
  • DBS dry blood spot
  • drugs that should be TDM include caffeine, cardiac glycosides and antiepileptic drugs, immunosuppressants, theophylline, antiarrhythmic drugs, antibacterial drugs (aminoglycosides, glycopeptides), lithium drugs, breast cancer drugs (anti Estrogen tamoxifen, aromatase inhibitor letrozole) and the like.
  • a sampling agent or sampling material is provided for preparing samples for pharmacokinetic analysis and gene analysis by nucleic acid amplification from the same sample.
  • water analysis paper is used to release nucleic acid components from the same blood sample into an aqueous fraction and drug components into an organic solvent fraction for gene analysis and absorbance measurement. At the same time.
  • a dried blood spot sample (DBS) is dissolved in distilled water, some as samples for genetic testing, and some others as solvent extraction (eg water-ethyl acetate) Then, the organic phase is analyzed by HPLC or the like to prepare a sample for pharmacokinetic analysis.
  • DBS dried blood spot sample
  • solvent extraction immersion in methanol or the like is performed from a dried blood spot sample (DBS), and the sample is used for pharmacokinetic analysis by HPLC, and the remaining DBS is dissolved in distilled water. A part of the sample is used for genetic testing.
  • DBS dried blood spot sample
  • a sample for analyzing the kinetics and the polymorphism of the drug metabolizing enzyme gene is prepared from the same biological sample (for example, blood).
  • Water-soluble paper dissolves in distilled water but does not dissolve in organic solvents such as methanol. Therefore, after extraction of the drug fraction with organic solvent, it can be dissolved in distilled water to recover nucleic acids. .
  • reagents described in the examples were used specifically for the reagents, but equivalent products from other manufacturers (Sigma-Aldrich, Wako Pure Chemicals, Nakarai, R & D Systems, USCN Life Science INC, etc.) can be substituted.
  • Example 1 Suppression of nucleic acid amplification inhibitory effect by water-soluble paper
  • sample preparation A blood sample was taken from the subject.
  • the blood sample was diluted as follows. Thereafter, the diluted blood was directly subjected to a PCR reaction, or the diluted blood was dropped on a filter paper or water-soluble paper and dried.
  • X10 10 ⁇ L of whole blood (100,000 copies) was diluted 10-fold with 90 ⁇ L of DW.
  • X100 X10 diluted blood 10 ⁇ L (10000 copies) was diluted 10 times with DW 90 ⁇ L.
  • 10 ⁇ L (1000 copies) of X1000: X100 diluted blood was diluted 10-fold with 90 ⁇ L of DW.
  • the water-soluble paper used in this example was 60 MDP and 120 MDP (Nippon Paper Papillia).
  • the filter paper was ADVANTEC qualitative filter paper No. 2 (Advantech Toyo Co., Ltd.).
  • Whatman (registered trademark) 903 Protein save card was used as a blood sampling material.
  • TaqMan PCR method In the following procedure, detection of the gene polymorphism of the alcohol dehydrogenase gene was performed by the TaqMan (registered trademark) probe method using a real-time PCR apparatus ABI7300 (manufactured by Applied Biosystems).
  • an oligonucleotide called a Taqman probe is used in addition to a normal PCR amplification primer set.
  • One end of the Taqman probe is modified with a fluorescent substance, and the other is modified with a quencher substance that suppresses fluorescence.
  • the Taqman probe specifically hybridized to the template DNA is degraded by the 5'-3 'exonuclease activity of the replication enzyme with the replication by the DNA polymerase in the PCR reaction.
  • fluorescence is emitted from the fluorescent molecules released from the quencher substance.
  • fluorescence detection fluorescence is generated by exciting fluorescent molecules with light from a halogen tungsten lamp, and the fluorescence is detected by an optical detection means such as a CCD camera through a filter that passes only the wavelength of the fluorescence.
  • the fluorescence intensity indicates the amount of the probe hybridized to the template DNA
  • the change in the amount of the template DNA for each cycle can be traced.
  • the amount of PCR amplification product reaches an amount that allows fluorescence detection, the amplification curve begins to rise, the signal rises exponentially, and then reaches a steady state.
  • the amount of amplified product reaches an amount that can be detected earlier, so that the amplification curve rises in an earlier cycle. For example, the number of cycles until reaching half of the steady-state signal can be used as an index of the initial amount of template DNA.
  • the reaction can be performed using a plurality of Taqman probes specific to different sequences.
  • the fluorescent substances that modify the respective Taqman probes can generate fluorescence of different wavelengths and can be detected separately.
  • alleles for example, alleles X and Y
  • X and Y present in the sample in one reaction.
  • the amount of template DNA can be determined, and the genotype (XX, YY, or XY) can be determined.
  • the fluorescence intensity in the amplification of the sample is obtained.
  • the fluorescence intensity in the amplification of the sample is obtained.
  • Genomic DNA was amplified in TaqMan PCR as follows for the alcohol dehydrogenase gene ADH1B.
  • composition of the TaqMan PCR reaction solution was as shown in Table 2 below.
  • Thunderbird qPCR Mix contains Taq DNA polymerase as a polymerase for DNA synthesis.
  • the KOD FX Neo buffer was not used. From the development process of the experimental method so far, it is considered possible to carry out the PCR reaction without using KOD FX Neo buffer (10%) when the dry water-soluble paper pieces of whole blood are not directly used as the PCR reaction solution. It was because of it.
  • the PCR reaction cycle was as shown in Table 3 below.
  • PCR reaction was carried out using an aqueous solution in which recombinant (recombinant) DNA in which the gene region of ADH1B (A / G) was incorporated into plasmid DNA was dissolved ( ⁇ in FIGS. 1 to 3). Distilled water was used as a negative control ( ⁇ in FIGS. 1 to 3).
  • FTA Elute whole blood & 10-fold diluted and saliva (oral mucosal cells)
  • dried filter paper blood were treated according to the FTA Elute protocol and subjected to the same PCR reaction as above. Specifically, it is as follows.
  • a sample obtained by dripping and drying blood collected using a disposable lancet for blood collection (completely disposable skin puncture device) on FTA Elute is placed in a 1.5-mL Eppendorf tube using a 4 mm ⁇ biopsy trepan (Ky Industries Co., Ltd.). Then, after washing three times with 0.5 mL of distilled water, 100 ⁇ L of distilled water was added to the punch piece and heated at 95 ° C. for 15 minutes to prepare a sample solution. 4 ⁇ L was subjected to TaqMan PCR.
  • G / G homo appears to have a difference between FTA and water-soluble paper (60, 120 MDP), but reflects a slight variation in the total blood volume depending on the sampling position. From the Ct of the amplification curve, it is considered that the difference is several times. A / A homozygous is observed with almost the same sensitivity as G / A homozygous. From this result, it is considered that there is no difference between 4 mm ⁇ and 2 mm ⁇ punches of water-soluble paper. Although no difference in sensitivity between FTA and water-soluble paper is observed, the recovery rate of genomic DNA is estimated to be 1/10 of that of water-soluble paper from the dilution factor. Compared to water-soluble paper, such as the three water washing steps and the expansion of a 4 mm ⁇ punch disk, there are significant disadvantages.
  • Dried water-soluble paper blood 4 mm ⁇ punch piece (mm ⁇ indicates the diameter (mm) of the punch piece, the same shall apply hereinafter) was added to 0.5 and 1.0 mL of distilled water (DW), treated at 95 ° C. for 5 minutes, 4 ⁇ L was used for PCR reaction.
  • DW distilled water
  • the blood was dropped on water-soluble paper, dried, and then subjected to PCR reaction after 8 months.
  • a blood sample (derived from 7 subjects) was dropped and dried on dry water-soluble paper (60 MDP)
  • 4 mm ⁇ punch piece of water-soluble paper was added to 100 ⁇ L of DW, treated at 95 ° C. for 5 minutes, diluted 10 times The subsequent 4 ⁇ L was subjected to TaqMan PCR.
  • FIG. 1A shows an amplification curve in the amplification reaction.
  • a blood sample (x10, 100, 1000) was diluted with 200 ⁇ L of DW, treated at 95 ° C. for 5 minutes, and 4 ⁇ L was subjected to PCR reaction. The result is shown in FIG. 2A. No gene amplification signal was obtained for samples using all these blood directly (FIG. 2A, x). It is thought that there is an inhibitor of the PCR amplification reaction in the blood.
  • FIG. 2A shows the result of adding 6 mm ⁇ punch pieces of filter paper, which was dried by dropping blood (x10, 100, 1000), to DW200 ⁇ L, treating at 95 ° C. for 5 minutes, and subjecting 4 ⁇ L of supernatant to PCR reaction. No gene amplification signal was obtained for all these dry filter paper blood samples (FIG. 2A, x). It is considered that the PCR amplification inhibitor in the blood elutes in the heat-treated supernatant.
  • a water-soluble paper (60, 120 MDP) 6 mm ⁇ punch piece that has been dried by dropping blood (x10, 100, 1000) was added to 200 ⁇ L of DW, treated at 95 ° C. for 5 minutes, and 4 ⁇ L of the supernatant was subjected to PCR. Shown in Gene amplification signals were not detected with x1000 (20 copies), but gene amplification signals were detected with x10 and x100 (FIG. 2A, ⁇ ). With x10 and x100, it was possible to detect with the theoretical copy number.
  • a solution was prepared by diluting whole blood 10 times, 100 times, 1000 times with distilled water. A fixed amount (10 ⁇ L) of each diluted solution was dropped onto Advantech qualitative filter paper No. 2, water-soluble paper 120 MDP and 60 MDP. 4 ⁇ L of a solution obtained by heating 10 ⁇ L of each diluted whole blood (10 times, 100 times, 1000 times) at 95 ° C. for 5 minutes was used for the PCR reaction solution.
  • Qualitative Filter Paper No. 2 and water-soluble paper 120MDP and 60MD each diluted whole blood was dropped and dried, 200 ⁇ L of distilled water was added to each 6 mm ⁇ punch piece, and 4 ⁇ L of a solution heated at 95 ° C. for 5 minutes was used for the PCR reaction solution.
  • FTA Elute whole blood & 10-fold dilution and saliva (oral mucosa cells) was subjected to the same PCR reaction as above.
  • a sample obtained by dripping and drying blood collected using a disposable lancet for blood collection (completely disposable skin puncture device) on FTA Elute is punched with a 4 mm ⁇ biopsy trepan (Kay Industry Co., Ltd.)
  • the punch piece was put in a 1.5 mL Eppendorf tube, washed with 0.5 mL of distilled water three times, 100 ⁇ L of distilled water was added to the punch piece, and heated at 95 ° C. for 15 minutes to prepare a sample solution. 4 ⁇ L of the sample solution was subjected to TaqMan PCR. Other parts followed manufacturer's protocol.
  • PCR reaction inhibitors cannot be removed by preparing a dried blood sample (DBS) using qualitative filter paper.
  • DBS dried blood sample
  • Gene detection was possible with sufficient sensitivity in dry blood samples obtained by dripping blood diluted 10-fold and 100-fold using water-soluble paper 120MDP and 60MDP.
  • the reason why it was not possible to detect the dried blood samples of water-soluble paper 120MDP and 60MDP using blood diluted 1000 times was considered to be that a sufficient amount of genomic DNA could not be subjected to the PCR reaction.
  • the water-soluble paper 120MDP and 60MDP trap capture, adsorb the PCR reaction inhibiting substance, dissolve in distilled water, and do not elute by heat treatment at 95 ° C. for 5 minutes.
  • FIG. 3 The results of the PCR reaction after 8 months are shown in FIG. In all seven samples, gene amplification of 10 3 to 10 4 copies was detected (FIG. 3). Because the detection level was almost constant, blood samples dropped on water-soluble paper and dried could not only be stored for a long time in an amplifiable state, but also amplified with quantitative properties. It has been shown to remain possible for a long time.
  • Example 2 Comparative experiment of FTA and water-soluble paper using whole blood
  • FTA card GE Healthcare
  • the amplification reaction was performed in the same manner as in Example 1 above, and it was examined whether or not a gene amplification signal was detected for each sample prepared by each method. However, when the genotype was detected for the ALDH2 gene, the probe for ALDH2 was used.
  • Blood samples obtained from 8 subjects were sampled with water-soluble paper 4 mm ⁇ , 2 mm ⁇ and FTA, respectively, as described above, and subjected to amplification reaction of each gene for genotyping of ADH1B or ALDH2.
  • FIG. 5 shows a sample sampled by FTA
  • FIG. 7 shows a sample sampled by FTA and water-soluble paper 4 mm ⁇ punch
  • FIG. 8 shows a sample sampled by FTA and water-soluble paper 2 mm ⁇ punch. The result about gene amplification of is shown.
  • G / G homo appears to have a difference between FTA and water-soluble paper (4 mm ⁇ and 2 mm ⁇ ), which is considered to reflect a slight variation in the total blood volume depending on the sampling position. From the Ct of the amplification curve (data not shown), it is considered that the difference is several times. From this result, it is considered that there is no difference between 4 mm ⁇ and 2 mm ⁇ punches of water-soluble paper. Although no difference in sensitivity between FTA and water-soluble paper is observed, the recovery rate of genomic DNA is estimated to be 1/10 of that of water-soluble paper from the dilution factor. When FTA is used, the demerits are large compared to water-soluble paper such as three washing steps and expansion of a 4 mm ⁇ punch disk.
  • FIG. 9 shows the results for all samples sampled with water-soluble paper 4 mm ⁇ , 2 mm ⁇ and FTA.
  • FIG. 10 shows a sample sampled by FTA
  • FIG. 11 shows a sample sampled by FTA and water-soluble paper 4 mm ⁇ punch
  • FIG. 12 shows a sample sampled by FTA and water-soluble paper 2 mm ⁇ punch. The result about gene amplification of is shown.
  • the genotype of the target gene ALDH2 is determined in the same manner as the result of the comparison experiment between ADH1B FTA and water-soluble paper (punch diameter difference) shown in FIGS. It was shown that it was possible.
  • Example 3 Comparison with gene analysis using saliva
  • a saliva sample was collected from the oral mucosa of the subject by a swab, transferred to a carrier as shown in FIG. 13A, and a sample for gene analysis was prepared.
  • a comparative experiment was conducted using four types of water-soluble paper (120MDP, 60MDP, 120CD2, 60CD2) with different materials (thickness, manufacturing method, etc.) as the carrier. After transferring from the swab to the water-soluble paper, the polyurethane sponge part of the swab was removed, 100 mL of DW was added, and the heat-treated supernatant was added dropwise to the PCR reaction solution to estimate the number of residual oral mucosa cells.
  • the blood sample was prepared in the same manner as in Example 2 above, and FTA Elute and water-soluble paper (60MDP) were used.
  • Example 4 Noise reduction effect in HPLC-UV by water-soluble paper
  • the purpose of this example is to demonstrate the noise reduction effect in HPLC-UV by water-soluble paper.
  • HPLC high performance liquid chromatography
  • Agilent 1200 HPLC System manufactured by Agilent Technologies
  • HPLC high performance liquid chromatography
  • the analytical columns used were TSKgel ODS-100V (5 mm, 4.6 ⁇ 150 mm, TOSOH) and TSKgel guardgel ODS-100V (5 mm, 3.2 ⁇ 15 mm, TOSOH).
  • the column oven was set to 30 ° C., the mobile phase was flowed at a flow rate of 1.5 mL / min, and detection was performed at a wavelength of 280 nm.
  • the above analysis conditions were referred to the following documents.
  • FIG. 18 shows the results of a DBS sample prepared using water-soluble paper and FTA card. Whether water-soluble paper or FTA Elute was used, caffeine in peripheral blood could be measured at a concentration close to the theoretical drug concentration with very high quantitativeness. Here, it is understood that when water-soluble paper is used, the signal of the target compound is detected equally, but there are few other signals and noise is reduced.
  • Example 5 Use of water-soluble paper in simultaneous sample preparation for multiple analyzes A biological sample can be analyzed by a plurality of analysis techniques (FIG. 19).
  • peripheral blood is collected, dropped onto water-soluble paper (120 MDP), and dried at room temperature for 1 hour.
  • the remainder of the 4 mm ⁇ punch piece dissolved in 40 ⁇ L of distilled water is subjected to 10 minutes of ultrasonic treatment, dissolved in 960 ⁇ L of methanol and subjected to 10 minutes of Voltex. Thereafter, the mixture is centrifuged for 5 minutes, and the blood drug concentration is measured by HPLC-UV using the supernatant.
  • Water-soluble paper dissolves in distilled water, but does not dissolve in organic solvents such as methanol. Therefore, it is possible to recover nucleic acid by dissolving in distilled water after extraction of the drug fraction with organic solvent. .
  • Example 6 Inspection of copy number polymorphism reference RNase P by real-time PCR
  • CNV copy number reference assay
  • sample Collection of peripheral blood and saliva (including human oral mucosal cells) from the test subjects (3 subjects) was performed according to the following manual.
  • a dried peripheral blood sample (Dried Blood Spot) was collected by holding a BD Microtina safety lancet in one hand and pressing the tip against the other hand, for example, the tip of the middle finger, for puncture.
  • the blood of the fingertip was brought into contact with a sampling kit with 120 MDP fixed, and the sampling sheet (self-made kit with water-soluble paper 120 MDP attached: see FIG. 21) was returned to three folds and dried for 30 minutes or more. It was dried taking care not to allow other people's splashing saliva to adhere to the sample (see FIGS. 22 and 23).
  • Control dry saliva sample (3) (Dried Saliva Spot) had a swab stick and rubbed the outside / inside of the gums and the inside of the cheek about 10 times with one side of the sponge. I rubbed it about 10 times on the other side as well. Both sides of the swab were moistened with saliva. Next, holding the base of the stick, one side of the sponge was pressed against the water-soluble paper part of the sampling sheet, and the cover part was covered and sandwiched for about 5 seconds (see FIG. 24). The used swab was returned to the original bag and discarded. The sampling sheet was returned to three folds and dried for more than 30 minutes.
  • composition of the PCR reaction solution of the dried blood spot (1) was a 1.2 mm diameter punch piece of dried blood spot, TaqPath (registered trademark) ProAmp (registered trademark) Master Mix 2x (Thermo Fisher Scientific Inc.): 5 ⁇ L, 20 ⁇ TaqMan (registered trademark) Copy Number Reference Assay, human, RNase P (Thermo Fisher Scientific Inc.): 0.5 ⁇ L, sterile purified water: 4.5 ⁇ L was added to make a total volume of 10 ⁇ L.
  • composition of the PCR reaction solution of the dried blood spot (2) was prepared as follows: Test sample preparation solution obtained by treating a 1.2 mm diameter punch piece of the dried blood spot with 100 ⁇ L of distilled water: 4 ⁇ L, TaqPath (registered trademark) ProAmp (registered trademark) Master Mix 2x (Thermo Fisher Scientific Inc.): 5 ⁇ L, 20 ⁇ TaqMan® Copy Number Reference Assay, human, RNase P (Thermo Fisher Scientific Inc.): 0.5 ⁇ L, sterilized purified water: 0.5 ⁇ L, total volume 10 ⁇ L It was.
  • the composition of the PCR reaction solution of the dried saliva spot (4) is: 4 ⁇ L of test sample preparation solution obtained by treating a 2 mm diameter punch piece of the dried saliva spot with distilled water 100 ⁇ L, TaqPath (registered trademark) ProAmp (registered trademark) Master Mix 2x (Thermo Fisher Scientific Inc.): 5 ⁇ L, 20 ⁇ TaqMan® Copy Number Reference Assay, human, RNase P (Thermo Fisher Scientific Inc.): 0.5 ⁇ L, sterile purified water: 0.5 ⁇ L did.
  • the difference between (1) and (2) is about 6 cycles, and the difference in the amount of template DNA is theoretically (1) is 64 times that of (2).
  • the greatest merit of performing a genetic test by directly putting a small piece of dried blood spot (1) into a TaqMan (registered trademark) PCR reaction solution is that it simplifies the operation and reduces the risk of contamination. Furthermore, reaction inhibition is not observed even when water-soluble paper is mixed with blood in the PCR reaction solution.
  • the average Ct value of the amplification curve was 31.3 ⁇ 1.6 cycles, and the dried saliva spot (4) was 28.0 ⁇ 1.4 cycles.
  • the difference in the amount of template DNA is theoretically (3) 9 times that in (4), and the difference in Ct value converted from the theoretical amount of genomic DNA introduced into the PCR reaction system is about 3 cycles. It agrees with the Ct difference of values (3 cycles) Therefore, inhibition by adding water-soluble paper directly to the PCR reaction solution is not observed (see FIGS. 30 and 31).
  • Saliva can be collected non-invasively, and no big difference between blood and template DNA is observed. However, if detection of viruses / bacteria in the blood, cancer cell-derived gene fragments (cell-free DNA, cfDNA) or microRNA floating in the blood can be detected, cancer may be detected early.
  • Homocystinuria maple syrup urine disease, citrullinemia type 1, argininosuccinic aciduria, propionic acidemia, methylmalonic acidemia, glutaric aciduria type 1, isovaleric acidemia, 3-hydroxy- 3-methylglutaric aciduria (HMG-CoA lyase deficiency), multiple carboxylase deficiency, 3-methylcrotonylglycineuria, CPT-1 deficiency, VLCAD deficiency, MCAD deficiency, TFP (MTP) deficiency It can be applied to genetic diagnosis of urea cycle disorders, congenital copper metabolism disorders, etc.), and will bring about great development.
  • the present invention can be used in sample preparation for research or clinical use, for example, sample preparation for genotype determination and sample preparation for pharmacokinetic monitoring.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Zoology (AREA)
  • Engineering & Computer Science (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • Genetics & Genomics (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Molecular Biology (AREA)
  • Biochemistry (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Medicinal Chemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Physics & Mathematics (AREA)
  • Analytical Chemistry (AREA)
  • Biophysics (AREA)
  • Biomedical Technology (AREA)
  • Immunology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Enzymes And Modification Thereof (AREA)

Abstract

La présente invention concerne : un agent ou un matériau d'inhibition d'une réaction par une substance dans un échantillon biologique ou de suppression ou de réduction du bruit provoqué par cette dernière ; un procédé d'inhibition d'une réaction par une substance dans un échantillon biologique ou de suppression ou de réduction du bruit provoqué ainsi par l'utilisation de l'agent ou du matériau ci-dessus mentionné ; et un procédé de détection, de mesure ou d'identification d'un constituant spécifique (par exemple, d'un gène ou d'un produit génique) dans un échantillon biologique avec son utilisation. La présente invention concerne la suppression d'un effet inhibiteur d'un échantillon biologique ou de son constituant sur l'activité d'amplification d'un acide nucléique polymérase avec l'utilisation d'un papier soluble dans l'eau ou son constituant. Dans un autre aspect, la présente invention concerne un agent d'échantillonnage ou un matériau d'échantillonnage, ledit agent d'échantillonnage ou matériau d'échantillonnage contenant un papier soluble dans l'eau, pour la préparation d'un échantillon à utiliser dans la mesure d'absorbance. Dans encore un autre aspect, la présente invention concerne un agent d'échantillonnage ou un matériau d'échantillonnage pour la préparation d'échantillons, lesdits échantillons devant être utilisés dans la mesure d'absorbance et dans l'analyse de gène par amplification d'acide nucléique, à partir d'un échantillon unique.
PCT/JP2018/013523 2017-03-31 2018-03-30 Matériau contenant du papier soluble dans l'eau pour la préparation d'échantillon et procédé de préparation d'échantillon l'utilisant WO2018181850A1 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
JP2019510213A JP7093121B2 (ja) 2017-03-31 2018-03-30 水溶紙を含むサンプル調製用材料およびそれを用いるサンプル調製方法

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
JP2017-070962 2017-03-31
JP2017070962 2017-03-31

Publications (1)

Publication Number Publication Date
WO2018181850A1 true WO2018181850A1 (fr) 2018-10-04

Family

ID=63676470

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/JP2018/013523 WO2018181850A1 (fr) 2017-03-31 2018-03-30 Matériau contenant du papier soluble dans l'eau pour la préparation d'échantillon et procédé de préparation d'échantillon l'utilisant

Country Status (2)

Country Link
JP (1) JP7093121B2 (fr)
WO (1) WO2018181850A1 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021049601A1 (fr) * 2019-09-12 2021-03-18 積水メディカル株式会社 Méthode de détection d'acide nucléique par pcr en temps réel
WO2021167058A1 (fr) * 2020-02-21 2021-08-26 積水メディカル株式会社 Procédé de détection d'acide nucléique cible à l'aide d'un morceau de papier filtre contenant du sang séché

Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH0813385A (ja) * 1994-06-24 1996-01-16 Lintec Corp 水分散性基材の製造方法
JP2001258556A (ja) * 2000-03-21 2001-09-25 Shimadzu Corp 核酸合成法
JP2006262777A (ja) * 2005-03-24 2006-10-05 Japan Science & Technology Agency 夾雑物を含む試料を用いた核酸合成方法及び添加剤
JP2012157295A (ja) * 2011-02-01 2012-08-23 Mukogawa Gakuin 遺伝子の増幅方法、特定遺伝子の検出方法
WO2018025856A1 (fr) * 2016-08-02 2018-02-08 学校法人武庫川学院 Procédé de dépistage génétique simple, procédé de mesure du nombre de copies, et technologie de support associée

Family Cites Families (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008093533A1 (fr) * 2007-01-30 2008-08-07 Olympus Corporation Procédé de détermination du changement dans les nombres de copies de la séquence d'acide nucléique
WO2013157215A1 (fr) * 2012-04-17 2013-10-24 国立大学法人山口大学 Procédé pour estimer la susceptibilité au cancer de l'endomètre
US20180073084A1 (en) * 2015-03-16 2018-03-15 Daiichi Sankyo Company, Limited Method for predicting responsiveness to phosphatidylserine synthase 1 inhibitor

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JPH0813385A (ja) * 1994-06-24 1996-01-16 Lintec Corp 水分散性基材の製造方法
JP2001258556A (ja) * 2000-03-21 2001-09-25 Shimadzu Corp 核酸合成法
JP2006262777A (ja) * 2005-03-24 2006-10-05 Japan Science & Technology Agency 夾雑物を含む試料を用いた核酸合成方法及び添加剤
JP2012157295A (ja) * 2011-02-01 2012-08-23 Mukogawa Gakuin 遺伝子の増幅方法、特定遺伝子の検出方法
WO2018025856A1 (fr) * 2016-08-02 2018-02-08 学校法人武庫川学院 Procédé de dépistage génétique simple, procédé de mesure du nombre de copies, et technologie de support associée

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
HAYASHIDA, MARIKO ET AL.: "Genotyping of Polymorphisms in Alcohol and Aldehyde Dehydrogenase Genes by Direct Application of PCR-RFLP on Dried Blood without DNA Extraction", ANALYTICAL SCIENCES, vol. 26, 2010, pages 503 - 505, XP055461972 *
IMAI MIHO ET AL.: "Establishment of copy number variation analysis method of CYP2D6 using quantitative real-time PCR", DVD ABSTRACTS OF THE PHARMACEUTICAL SOCIETY OF JAPAN, vol. 138, 5 March 2018 (2018-03-05) *
OTA TOMOKO ET AL.: "High Performance and straight forward genotyping of the cytochrone P450 2C9 and vitamin K Epoxide reductase complex subunit 1 VKORC1 polymorphisms", JAPANESE JOURNAL OF PHARMACEUTICAL HEALTH CARE AND SCIENCES, vol. 40, 2014, pages 402 - 408 *
SAKAGUCHI YUI ET AL.: "Development of high throughput SNP typing method", DVD ABSTRACT OF THE PHARMACEUTICAL SOCIETY OF JAPAN, vol. 138, 5 March 2018 (2018-03-05) *

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2021049601A1 (fr) * 2019-09-12 2021-03-18 積水メディカル株式会社 Méthode de détection d'acide nucléique par pcr en temps réel
JP2021040576A (ja) * 2019-09-12 2021-03-18 積水メディカル株式会社 リアルタイムｐｃｒによる核酸検出方法
EP4029938A4 (fr) * 2019-09-12 2023-10-11 Sekisui Medical Co., Ltd. Méthode de détection d'acide nucléique par pcr en temps réel
WO2021167058A1 (fr) * 2020-02-21 2021-08-26 積水メディカル株式会社 Procédé de détection d'acide nucléique cible à l'aide d'un morceau de papier filtre contenant du sang séché

Also Published As

Publication number Publication date
JPWO2018181850A1 (ja) 2020-02-13
JP7093121B2 (ja) 2022-06-29

Similar Documents

Publication Publication Date Title
RU2753883C2 (ru) Набор зондов для анализа образцов днк и способы их использования
KR20190004768A (ko) 메틸화된 dna 분석에 의한 폐 종양의 검출
EP2652147B1 (fr) Procédé en une étape d'élution d'adn à partir d'échantillons de sang
JP4617403B2 (ja) 配列の相違を検出する方法
JPH10332701A (ja) 核酸融解温度測定法
US10053686B2 (en) Methods for one step nucleic acid amplification of non-eluted samples
US20210317439A1 (en) Automated isolation and chemical reaction(s) of nucleic acids
JP2012520086A (ja) アブスクリプションに基づく分子検出
JP7336091B2 (ja) 簡便な遺伝子検査法およびコピー数計測法ならびにその支援技術
JP2022145606A (ja) 核酸の正確な並行定量のための高感度な方法
US20160108459A1 (en) Automated isolation and chemical reaction(s) of nucleic acids
WO2018181850A1 (fr) Matériau contenant du papier soluble dans l'eau pour la préparation d'échantillon et procédé de préparation d'échantillon l'utilisant
JP7560112B2 (ja) Ｐｖａスポンジを用いるリキッドバイオプシーの調製方法
CN103124797B (zh) 用于在相同反应容器内的细胞裂解和pcr的方法
US20150191800A1 (en) Detection of cytomegalovirus dna using amplification from blood samples
CA3225580A1 (fr) Procedes et compositions pour l'analyse d'acide nucleique
CN103124796B (zh) 用于pcr反应缓冲液中的细胞裂解的方法
RU2796350C1 (ru) Молекулярно-генетическая система детекции делеции экзона 7 гена SMN1, пригодная для проведения неонатального скрининга
WO2023040997A1 (fr) Procédé de test monogénique et son application
Oh et al. Efficiency of EGFR mutation analysis for small microdissected cytological specimens using multitech DNA extraction solution
JP3705499B2 (ja) ターゲット核酸断片の分析方法及びターゲット核酸断片の分析キット
JP5731167B2 (ja) 一本鎖ｃＤＮＡの合成方法、マイクロアレイ用試料の調製方法および核酸の検出方法
WO2024195263A1 (fr) Procédé de mesure du niveau de méthylation de cpg du gène
RU2402612C2 (ru) Способ скрининга новорожденных на моногенные заболевания и биочип для осуществления этого способа
WO2018151709A1 (fr) Isolation automatisée et réaction(s) chimique(s) d'acides nucléiques

Legal Events

Date Code Title Description
121 Ep: the epo has been informed by wipo that ep was designated in this application

Ref document number: 18775643

Country of ref document: EP

Kind code of ref document: A1

ENP Entry into the national phase

Ref document number: 2019510213

Country of ref document: JP

Kind code of ref document: A

NENP Non-entry into the national phase

Ref country code: DE

122 Ep: pct application non-entry in european phase

Ref document number: 18775643

Country of ref document: EP

Kind code of ref document: A1

点击 这是indexloc提供的php浏览器服务，不要输入任何密码和下载