WO2017009719A2 - 나노입자를 이용한 핵산 검출용 키트 및 핵산 검출 방법 - Google Patents

나노입자를 이용한 핵산 검출용 키트 및 핵산 검출 방법 Download PDF

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WO2017009719A2
WO2017009719A2 PCT/IB2016/001261 IB2016001261W WO2017009719A2 WO 2017009719 A2 WO2017009719 A2 WO 2017009719A2 IB 2016001261 W IB2016001261 W IB 2016001261W WO 2017009719 A2 WO2017009719 A2 WO 2017009719A2
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nucleic acid
nanoparticles
primer
amplification
detecting
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French (fr)
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WO2017009719A3 (ko
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황상현
임지현
강수진
이도훈
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National Cancer Center Japan
National Cancer Center Korea
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National Cancer Center Japan
National Cancer Center Korea
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Priority claimed from KR1020150121109A external-priority patent/KR101817155B1/ko
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Publication of WO2017009719A3 publication Critical patent/WO2017009719A3/ko
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/6848—Nucleic acid amplification reactions characterised by the means for preventing contamination or increasing the specificity or sensitivity of an amplification reaction
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813—Hybridisation assays
    • C12Q1/6816—Hybridisation assays characterised by the detection means
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]
    • 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
    • C12Q1/6876—Nucleic acid products used in the analysis of nucleic acids, e.g. primers or probes

Definitions

  • the present application contains Korean Patent Application No. 10-2015-0099378, filed on July 13, 2015, and Korean Patent Application No. 10-2015-0121109, filed on August 27, 2015.
  • the present invention relates to a kit for detecting nucleic acid and a method for detecting nucleic acid using nanoparticles, and more particularly, a method for detecting nucleic acid, including amplifying and labeling nucleic acid, capturing and centrifuging the nanoparticle, and a method for detecting the nucleic acid. It is about a kit for detecting nucleic acids using the method.
  • Nucleic acid assays include clinical validity, such as sensitivity, specificity, precision, and accuracy, and clinical validity and usefulness, such as clinical sensitivity, specificity, and negative predictive values.
  • nucleic acid testing There are two major targets for nucleic acid testing: nucleic acids derived from human microorganisms and those derived directly from the human body. The former accounts for more than 80% of the market for nucleic acid testing. The latter is a genetic test that analyzes nucleic acids related to disease induction and treatment, with market share of less than 20%, but the potential value and marketability are expected to be very high.
  • Nucleic acid detection, using nanoparticles is one of the methods of conventional charged polyanion and silicic acid detection.
  • the next generation technology that can significantly improve the low speed and high cost method, which is the disadvantage of the method using the silica membrane technology, manufactures nanoparticles and performs coating treatment to capture nucleic acid to selectively bind the nucleic acid molecules to target nucleic acid molecules.
  • a detection system has been developed competitively.
  • Prior art has disclosed that US patent US20140100131 detects a target gene. Magnetic particles are used as a method, and US Patent US20100009383 relates to a method for detecting a target biomolecule, and after collecting the target material with the magnetic particles, centrifugation can be carried out to detect the collected target material.
  • Conventional methods are inconvenient to add additional DNA probes or antibodies, and additional separation and washing processes are required, requiring the invention of more acidic nucleic acid detection methods.
  • the present inventors then used gels and cards to amplify the target nucleic acid and nanoparticles.
  • nucleic acid detection method has been invented a nucleic acid detection method and nucleic acid detection kit that can be more quickly, simpler, more sensitive and reliable discrimination of negative and positive judgments for specific diseases.
  • the present invention provides a kit for detecting nucleic acids quickly and accurately.
  • Another object of the present invention is to provide a nanoparticle complex for detecting nucleic acids.
  • Another object of the present invention is to provide a gel card for the detection of nanoparticle complexes.
  • Another object of the present invention is a method of detecting nucleic acids quickly and accurately.
  • the present invention provides a gel card in which a primer set specifically binding to a nucleic acid to be detected; capture nanoparticles; It provides a nucleic acid detection kit containing.
  • the present invention also provides a detectable nanoparticle complex comprising nanoparticles to which the target nucleic acid is trapped or bound for use in a target nucleic acid detection assay.
  • the present invention also provides a gel card in which a glass bead component and a gel component are sequentially laminated for detecting a nanoparticle composite.
  • the present invention also relates to the use of a detectable nanoparticle complex comprising nanoparticles in which the target nucleic acid is trapped or bound for use in a target nucleic acid detection assay.
  • the present invention also relates to the use of a gel card in which a glass bead component and a gel component are sequentially laminated to detect the nanoparticle composite.
  • the present invention also provides a method for amplifying a target nucleic acid with a primer set that specifically binds a target nucleic acid; (L) capturing or binding the nucleic acid to the nanoparticles by adding the nanoparticles to the nucleic acid amplified in the step ( ⁇ ⁇ ); (c) the step (L) of the step of placing the complex in which the nucleic acid is trapped or bonded to the nanoparticles in the gel card container; ( ⁇ ) provides a method for detecting nucleic acid using nanoparticles, including centrifugally separating the complex prepared in step (c); and comparing the position of the precipitate in the gel gel container with a control.
  • the primer set of the step ( ⁇ ) is a biotin-labeled forward primer and a fluorescence-labeled reverse primer.
  • the step (VII) is polymerase.
  • PCR chain reaction
  • isothermal amplification reaction it may be amplification of nucleic acid by chain reaction (PCR) or isothermal amplification reaction.
  • the isothermal amplification reaction is characterized by HDA (Helicase-Dependent Amplification),
  • RPA Recombinase Polymerase Amplification
  • RCA Rolling Circle Amplification
  • LAMP Loop mediated isothermal amplification
  • TMA Acid-Sequence-Based Amplification
  • TMA Transcription Mediated Amplification
  • SMART Signal Mediated Amplification of RNA Technology
  • Amplification, Single Primer Isothermal Amplification (SPIA), and Circular Helicase-Dependent Amplification (cHDA) may be performed by any one method, preferably Helical-Dependent Amplification (HDA), Recombinase Polymerase Amplification, rolling circle amplification (RCA), loop mediated isothermal amplification (LAMP), and more preferably HDA (Helicase-Dependent).
  • HDA Helical-Dependent Amplification
  • RCA rolling circle amplification
  • LAMP loop mediated isothermal amplification
  • HDA Helicase-Dependent
  • RPA Recombinase Polymerase Amplification
  • the nanoparticles of the present invention are provided. [22] According to another preferred embodiment of the present invention, the nanoparticles of the present invention
  • the nanoparticles of the present invention can be bound via antigen-antibody reactions to antigens bound to avidin, amine, streptavidin, or primers that can capture nucleic acids on the surface.
  • antigen-antibody reactions to antigens bound to avidin, amine, streptavidin, or primers that can capture nucleic acids on the surface.
  • primers that can capture nucleic acids on the surface.
  • equivalents such as digoxigenin / anti-digoxigenin antibody, Cy3 / anti-Cy3 antibody
  • aptamers is characterized by being coated with any one selected from the group consisting of oligonucleotides.
  • the gel component is selected from the group consisting of IgG-agarose, agarose, agar, agar, cellulose acetate and polyacrylamide gel. It can be either.
  • the nucleic acid detection method of the present invention may additionally include a step of processing an enhancer, and the separation and washing processes included in the existing method are unnecessary.
  • the present invention relates to a method for detecting nucleic acid and detecting kit nucleic acid using nanoparticles.
  • the present invention relates to a nucleic acid detection method comprising amplifying and labeling nucleic acid, capturing with nanoparticles, and centrifuging the nucleic acid, and a kit for detecting nucleic acid using the same.
  • the present invention does not include a separation step.
  • Nucleic acid detection method is effective because it can quickly and easily determine the negative and positive determination of a certain disease, and it is effective because it is sensitive and reliable.
  • FIG. 1 is a schematic diagram showing a nucleic acid detection method of the present invention.
  • FIG. 2 is a schematic diagram and NHS specifically showing the nucleic acid detection method of the present invention
  • FIG. 3 is a photograph showing a positive / negative result according to a nucleic acid detection method using a reverse primer labeled with phosphoramide method.
  • 5 is a photograph showing a result of nucleic acid detection with or without an enhancer.
  • the picture shows that it can be identified.
  • FIG. 8 is a photograph of a nucleic acid detection method of the present invention using an HPV clinical specimen (tube 1-3)
  • HPV DNA negative sample HPV DNA negative sample
  • No. 4-6 tube HPV DNA positive sample
  • the inventors then amplified the target nucleic acid and combined it with nanoparticles and gels. After injection into the gel-card, a simple method of centrifugation is designed to quickly and cleanly detect specific nucleic acids related to disease.
  • nucleic acid refers to any length of nucleotides.
  • polymer By polymer, it includes DNA and RNA.
  • the term “capture” or “capture” means that the present invention captures, adsorbs, electrostatic bonds, ions of at least a portion (or linker portion of the agent) to the nanoparticles or surfaces thereof. Integral, covalent, complementary oligonucleotides, antigen-antibody reactions, or immobilization.
  • the term "primer” means oligonucleotide.
  • the primer can act as a starting point for the synthesis under conditions in which the synthesis of the primer extension product complementary to the nucleic acid chain (template) is present, i.e. the presence of polymerizers such as nucleotides and DNA polymerase, and at suitable temperature and pH conditions.
  • the primer is a deoxyribonucleotide and is a single chain.
  • the primer used in the present invention may include naturally occurring dNMP (ie, dAMP, dGMP, dCMP and dTMP), modified nucleotides or non-natural nucleotides. Primers may also contain ribonucleotides. Primers should be long enough to prime the synthesis of extension products in the presence of a polymer.
  • the suitable length of the primer is typically 15-30 nucleotides, depending on a number of factors, such as silver, the field of application, and the source of the primer. Short primer molecules are generally common to form a highly stable hybrid complex with the template.
  • annealing or “priming” means that oligodioxynucleotides or nucleic acids are apposition to the template nucleic acid, where the polymerase polymerizes the nucleotides to form the template nucleic acid or its To form nucleic acid molecules that are complementary to one part.
  • inhancer refers to a substance that helps determine the sample's positive and negative qualities more accurately and clearly. For example, fluorescence is bound to nucleic acids or primers. It may be a substance that stands out or develops color.
  • gel card can be defined with reference to the gel column described in the patent WO 1999/050673.
  • a gel card is a sequential order of components and gel components that cause density differences. It is a laminated gel card, more preferably
  • the present invention provides a nucleic acid detection method comprising a primer set specifically binding to a nucleic acid to be detected; a capture nanoparticle; and a gel card in which a component and a gel component sequentially causing density differences are stacked; Provide the kit.
  • the primer set may be a biotin-labeled forward primer and a fluorescence-labeled reverse primer.
  • the primers included in the nucleic acid detection kit of the present invention are labeled with biotin.
  • primers included in the kit of the present invention can be labeled with fluorescence, and primers labeled with fluorescence can be identified with fluorescence to determine the presence or absence of nucleic acid through the location of the band. can do.
  • the labeling of the primer can be used to visually discriminate between the negative and the positive, and the fluorescent or negative can be distinguished (see FIG. 6).
  • the fluorescent label of the primer is not limited thereto, but Cy3, Cy5, TAMRA, TEX, TYE, HEX, FAM, TET, JOE, MAX, ROX, VIC, Cy3.5, Texas Red, Cy5.5, TYE Can be one or more selected from the group consisting of BHQ, Iowa Black RQ, and IRDye.
  • Cy dyes are catalog of amersham biosicences for sale.
  • the positive sample forms a band below the negative sample
  • the positive sample is characterized by forming a band above the negative sample. This difference is judged to be different depending on the position of the fluorescent label of the primer.
  • a positive sample when a nucleic acid is detected using a primer labeled with NHS modification, a positive sample forms a bend below (see FIG. 2), and a fluorescent label is used by phosphoramide method.
  • a positive sample formed a band on the stomach (see FIG. 3).
  • the nanoparticles of the present invention are magnetic particles, gold (Au) nanoparticles, silver (Ag) nanoparticles, platinum (R) nanoparticles, quantum dots, upward conversion.
  • Nanoparticles (upconversion nanoparticle, UCNP) graphene (graphene)-nanoparticle complex, color dyed particles (late dyed) and latex (latex) is characterized in that any one selected from the group consisting of nanoparticles.
  • Dynabead (MyOne Streptavidin CI) is used as a nanoparticle, and in addition, magnetic particles, metal particles, quantum dots, upward conversion nanoparticles, and graphene capable of capturing nucleic acid on the surface of nanoparticles are used.
  • -Nanoparticle composites, color-dyed nanoparticles or latex nanoparticles can be used, more preferably magnetic particles or metal particles.
  • the nanoparticles of the present invention are antibodies that can be bound by antigen-antibody reactions against antigens that are bound to avidin, amine, streptavidin, or primers that can capture nucleic acids on the surface. (E.g,
  • It is characterized by being coated with any one selected from the group consisting of digoxigenin / anti-digoxigenin antibodies, Cy3 / anti-Cy3 antibodies and their equivalents), aptamers and oligonucleotides.
  • a primer containing an antigen When amplifying the target nucleic acid, a primer containing an antigen is used, and the antibody binds to the antigen on the surface of the nanoparticle, thereby allowing the nanoparticle to capture the target nucleic acid through the antigen-antibody reaction.
  • the component that causes the difference in density serves to support the gel component from below, and sinks when the density of the nucleic acid-nanoparticle composite is greater than the component that causes the density difference.
  • the low density of the nucleic acid-nanoparticle complex serves to separate the negative and positive ones using density differences to prevent them from sinking, which eliminates the need for separate washing or separation.
  • the above-mentioned components that cause the density difference of the present invention are not limited as long as they can cause the difference in density.
  • glass beads, quartz based matrix, percoll, and colloidal silica solution are used.
  • it may be a glass bead.
  • the gel component of the present invention is IgG-agarose (agarose), agarose (agarose),
  • the ingredient is IgG-agarose or
  • It can be agarose.
  • IgG-agarose was used.
  • the minimum time and high resolution for positive and negative discrimination in IgG-agarose were shown, preferably in the range of 15 to 25 ⁇ and more preferably in the range of 20 ⁇ .
  • the nucleic acid detection kit of the present invention further provides an enhancer, an interchelating agent.
  • interchelating agents include SYBR green, ethidium bromide, biotium gelred, and biotium. gelgreen, JOJO, POPO, SYTO, BOBO,
  • TOTO series actinomycin, adriamycin, anthracene, benzopyrene, propidium diiodide-intertwining, distamycin, netroxin and acridine, psoralen, berberine, It can be proflavine, daunomycin, doxorubicin, thalidomine, cyanine dye or LDS 751.
  • the interchelating dyes such as SYBR green, bromide ether, biotium gelred, biotium gelgreen, JOJO, POPO, SYTO, BOBO and
  • It can be any one selected from a group of TOTOs.
  • the nucleic acid detection kit of the present invention is preferably a biotin-labeled direction.
  • a primer set consisting of a primer and a fluorescent surface local directional primer, the primer set specifically binding to a nucleic acid to be detected; capture nanoparticles;
  • An in situ nucleic acid detection kit comprising an interchelating agent; and a gel card in which a gel component and a component that cause density differences are sequentially stacked.
  • the present invention provides a detectable nanoparticle complex comprising nanoparticles in which a target nucleic acid is trapped or bound for use in a target nucleic acid detection assay.
  • the present invention provides a gel card in which a glass bead component and a gel component are sequentially laminated for detecting a nanoparticle composite.
  • the gel card in which the glass beads and the gel component are laminated is contained in a test container, and the test container can be used as any solid container, and can be processed in various types, for example, a test tube and a microplate. Any container such as a well may be used.
  • a method for detecting nucleic acid using nanoparticles comprising the steps of: separating and comparing the position of the precipitate in the gel card vessel with a control.
  • the primer set may be a biotin-labeled forward primer and a fluorescence-labeled reverse primer.
  • the primers included in the nucleic acid detection kit of the present invention may be labeled with biotin. Primers labeled with biotin are better at nanoparticles
  • the primer set is biotin-labeled.
  • It can be a biotin-labeled forward primer and a Cy3-labeled reverse primer.
  • Biotin binds to nanoparticles coated with a substance capable of specifically binding to biotin (for example, avidin) through a labeled forward primer, and the precipitate is either visible or fluorescent by a reverse primer labeled with Cy3. You can check the location. Cy3 also acts as an enhancer to more clearly differentiate the amplification.
  • HPV nucleic acid was detected by a primer set represented by SEQ ID NOs: 1 and 2 or a primer set represented by SEQ ID NOs: 3 and 4.
  • the nucleic acid of the present invention includes DNA and RNA, and the amplification of DNA can be used by any known amplification method. Preferably, it may be a PCR or an isothermal amplification method. Any known amplification method may be used, preferably a method of synthesizing after cDNA synthesis, and more preferably a reverse transcription PCR (rt-PCR) method.
  • the step ( ⁇ ) of the present invention is characterized by amplifying a nucleic acid by polymerase chain reaction (PCR) or isothermal amplification reaction.
  • PCR polymerase chain reaction
  • Nucleic acid of the present invention includes DNA and RNA, and the amplification of DNA can be any conventionally known amplification method. Preferably, it may be PCR or isothermal amplification.
  • the isothermal amplification reaction of the present invention is performed by HDA (Helicase-Dependent Amplification),
  • RPA Recombinase Polymerase Amplification
  • RCA Rolling Circle Amplification
  • LAMP Loop mediated isothermal amplification
  • TMA Acid-Sequence-Based Amplification
  • TMA Transcription Mediated Amplification
  • SMART Signal Mediated Amplification of RNA Technology
  • Amplification, Single Primer Isothermal Amplification (SPIA) and circular Helicase-Dependent Amplification (cHDA) are performed by any one method selected from the group consisting of. '
  • PCR Polymerase Chain Reaction
  • isothermal amplification technology A technique capable of amplifying DNA / RNA at isothermal temperature without changing the temperature. Since the technology does not need time to change the temperature, a large amount of DNA can be amplified in a short time, and thus it is highly utilized as a rapid detection technology of nucleic acid.
  • Loop-mediated isothermal amplification is a technique that amplifies a specific target sequence using 4-6 primers.
  • RNA is amplifies by repeating the reaction that synthesizes RNA by self-sustained again.
  • SDA 4 primers are required and Hinc's cognitive base sequence (GTTGAC) is used.
  • GTTGAC Hinc's cognitive base sequence
  • RCA is a method in which ⁇ 29 DNA polymerase cyclically extends a primer to amplify a polymer's circular nucleic acid into a long strand. RCA is tightly coupled and used as an important foundation for DNA diagnostics among other isothermal amplification techniques. Recently, RCA is used for genetic analysis, immunoassay, sequencing, SNP scoring and genetic expression analysis.
  • IMDA The primer is attached to both sides of the double-stranded nucleic acid and stretched as it is. HDA can be separated into single strands using helicase
  • cHDA is a technology that uses DNA polymerase and helicase together to perform all reactions at a temperature.
  • the nucleic acid is amplified by HDA and RPA reaction.
  • the isothermal amplification reactions of the present invention preferably include HDA (Helicase-Dependent Amplification), Recombinase Polymerase Amplification (RPA), Rolling Circle Amplification (RCA) and LAMP (Loop mediated isothermal amplification), more preferably HDA (Helicase-Dependent Amplification), RPA (Recombinase Polymerase Amplification) 0 ].
  • any known amplification method can be used for amplifying RNA, and may be preferably a rt-PCR response that amplifies after synthesizing cDNA by using a NASBA reaction with an RNA template.
  • the nanoparticles are not limited as long as they are nanoparticles capable of capturing or binding acid. Any nanoparticles capable of coating or binding a substance capable of capturing nucleic acid can be applied. There is a difference between the density and the density of the medium, since it determines the positive or negative of a particular nucleic acid.
  • a glass bead is used as the medium, and since the medium density thereof is 1.04 g /, all particles having a higher density are possible. However, it can be applied by using a medium of different density or by using particles smaller than the density of the medium.
  • the nanoparticles of the present invention are magnetic particles, gold (Au) nanoparticles, silver (Ag) nanoparticles, platinum (Pt) nanoparticles, quantum dots, upward conversion.
  • the nanoparticles may be upconversion nanoparticles (UCNP), graphene-nanoparticle complexes, color dyed particles or latex nanoparticles.
  • UCNP upconversion nanoparticles
  • graphene-nanoparticle complexes color dyed particles or latex nanoparticles.
  • Dynabead (MyOne Streptavidin CI) is used as a nanoparticle, and in addition, magnetic particles, metal particles, quantum dots, upward conversion nanoparticles, and graphene capable of capturing nucleic acid on the surface of nanoparticles are used.
  • -Nanoparticle composites, colored dyed nanoparticles or latex nanoparticles can be used. More preferably, they can be magnetic or metal particles.
  • the nanoparticles of the present invention are antibodies that can be bound by antigen-antibody reactions against antigens bound to avidin, amine, streptavidin, and primers, which can capture nucleic acids on the surface.
  • the coating may be one selected from the group consisting of digoxigenin / anti-digoxigenin antibodies, Cy3 / anti-Cy3 antibodies and their equivalents, aptamers and oligonucleotides.
  • a primer containing an antigen is used, and the antibody to the antigen is bound to the surface of the nanoparticle, thereby allowing the nanoparticle to capture the target nucleic acid through the antigen-antibody reaction.
  • the gel component of the present invention is IgG-agarose (agarose), agarose (agarose),
  • It may be any one selected from the group consisting of polyacrylamide gels.
  • IgG-agarose was used, and 10-30.
  • Minimal time and high discrimination in positive and negative discrimination in IgG-agarose of may preferably be between 15 and 25 ⁇ , more preferably between 20 ⁇ .
  • an enhancer of an intercalating agent may be further processed.
  • the enhancer can be an interchelating agent
  • Interchelating agents include SYBR green, ethidium bromide, biotium gelred, biotium gelgreen, JOJO, POPO, SYTO, BOBO,
  • TOTO family actinomycin, adriamycin, anthracene, benzopyrene, propidium diiodide-intertwining, distamycin, netroxin and acridine, psoralen, berberine, With proflavine, daunomycin, doxorubicin, thalidomine, cyanine dyes and LDS 751
  • It may be any one selected from the group consisting of SYBR green, ethidium bromide, biotium gelred, biotium gelgreen, JOJO, POPO, SYTO, BOBO and TOTO
  • Biotium Biotium
  • nanoparticles were mixed and treated on a gel-card.
  • FIG. 5 it was confirmed that the positive and negative distinction was more pronounced when the enhancer was treated.
  • fluorescent dyes can be combined with primers and used as an enhancer. Fluorescent dyes can be combined with primers. Examples of fluorescent dyes include Cy3, Cy5, TAMRA, TEX, TYE, HEX, FAM, TET, JOE, MAX, ROX, VIC, Cy3.5, Texas Red, Cy5.5, TYE, BHQ, Iowa Black RQ, IRDye series and equivalents.
  • Fig. 2 is a schematic representation of the present invention. If it contains nucleic acid, it will be captured by the target nucleic acid or the nanoparticles, and then centrifuged using a density difference to place the nucleic acid on the lower side. Because of the lowness, a precipitate is placed in the center of the tube.
  • the present invention is characterized in that, before step (d), the separation step and the washing step are not included.
  • step (a) at least two or more labeled with different colors of fluorescence are used.
  • the primer is treated to check for the presence of nucleic acid in multiples.
  • two or more primers labeled with different fluorescence can be treated to determine the presence or absence of nucleic acid due to multiple symptoms. This can be an effective method for detecting nucleic acids, since it can save time and money.
  • Nucleic acid can be prepared according to the manufacturer's instructions using a DNA Micro kit, QIAGEN, Valencia, CA, USA, or ChargeSwitch gDNA ⁇ ⁇ Serum Kit, Life Technologies, NY, USA.
  • Human Papillomavirus (HPV) DNA standards were obtained from the Ministry of Food and Drug Safety and
  • Nucleic acid was prepared by applying to the National Institute for Biological Standards and Control (NIBSC).
  • the PCR primer set of Table 1 was used to obtain the amplified product of each sample.
  • the primer set of the present invention is not only PCR amplified but also isothermal amplified (preferably helicase dependent amplification (HDA), recombinase polymerase amplificatkm (RPA)). ) was also made possible and modified with reference to known literature (Virol J. 2010 Aug 19; 7: 194).
  • HDA helicase dependent amplification
  • RPA recombinase polymerase amplificatkm
  • PCR reaction mixture is shown in Table 2, and the amplification condition is 95 to 10 minutes, [95 to 30 seconds,
  • the RPA semi-formulation solution is shown in Table 3 above, and isothermally amplified at 37 to 40 minutes using the primer set and twist amplifier basic kit (TwistDx, Cambridge, UK) described in Table 1 above.
  • the HDA reaction composition is shown in Table 4 above, and isothermally amplified at 65 to 60 minutes using the primer set and isoamp III universal tHDA kit (Biohelix) described in Table 1 above.
  • PCR products were analyzed on a Mupid-a (Advance, Japan) electrophoresis apparatus using 1.5% (w / v) agarose gel.
  • 1.5 g of agarose was added to a triangular flask (250 id) ⁇ A].
  • 0.5 X TBE tris boric acid EDTA
  • the solution was melted in a microwave for 2 to 3 minutes and poured into the gel container for 30 minutes. After confirming that the gel was completely solid, the gel was collected.
  • the gel was placed in an electrophoresis apparatus and filled with a 0.5 ⁇ complete solution, loaded with 4 ⁇ PCR product 6 ⁇ and 0.8 6 bromo phenol blue (BPB) dye, each loaded and electrophoresed at 100 V for 25 minutes. Removed and stained with EtBr (ethidium bromide) for 10 minutes and washed EtBr that failed to bind DNA with distilled water for 10 minutes.
  • EtBr ethidium bromide
  • the agarose gel was placed on a transilluminator to confirm PCR amplification.
  • the nanoparticles are capable of capturing nucleic acid and can be used theoretically as long as the particles are larger than the density of the medium.
  • Example 3 in order to capture nucleic acid molecules,
  • Dynabead (MyOne Streptavidin CI) was used as a nanoparticle, and the surface of the Dynabead was treated with straptavidin to bind biotin-labeled primers. Clinical Diagnostics, NJ, USA) was purchased and used.
  • the primer set consisting of SEQ ID NOs: 1 and 2 described in Table 1 was used to amplify HPV nucleic acid from the HPV sample through isothermal amplification.
  • the primer represented by SEQ ID NO: 2 is labeled with a fluorescent substance of Cy3 at the 5 'end.
  • the labeling method was labeled with NHS (N-hydroxysuccinimide) modification method.
  • Primers labeled by NHS modification can be represented by the following formula (1).
  • FIG. 2 shows a schematic diagram of the above process.
  • PCR products were banded on the lower side.
  • HPV nucleic acid was amplified from the HPV sample through round amplification.
  • the primer represented by SEQ ID NO: 4 was provided with a fluorescent substance of Cy3 at the 5 'end. Labeled, the labeling method was carried out by the phosphoramide method
  • the primers labeled by the phosphoramide method may be represented by the following formula (2).
  • the method for detecting nucleic acids using nanoparticles of the present invention can be used as clinical specimens.
  • Nucleic acid can be detected quickly and sensitively, and the fluorescence labeling method can easily change the voice and positive judgment.
  • the gel-card was treated with nucleic acid and nanoparticles according to the method of Example 3-2. Thereafter, centrifugation was performed at 100 rpm, 200 rpm, 400 rpm, 600 rpm, 800 rpm, 1200 rpm and 1600 rpm for 2 minutes, respectively.
  • the detection of HPV16 DNA 136 bp was confirmed that the optimal centrifugation rate for detecting nucleic acid was 600rpm to 1200rpm, and more preferably 800rpm x 2min (about 55g) was the most desirable. (Not shown).
  • the centrifugation rate can be adjusted according to the length of the nucleic acid to be detected or the size / density of the nanoparticles used to control the g-force.
  • the nucleic acid and the nanoparticles were treated in the gel-card according to the method of Example 3-2.
  • the centrifugation speed was set at 800 rpm (about 55 g) and observed for 8 minutes at 1 minute intervals.
  • the centrifugation time of the present invention can be controlled by the size of nanoparticles used, density, semi-culture, concentration of medium, centrifugal force, etc.
  • the nucleic acid was treated by treating the gel-card prepared in Example 3 with 2 to 6 ⁇ (preferably, 4 / ⁇ amplified product in the produced master mix. If too much nucleic acid is treated, there is less nucleic acid binding to the nanoparticles, so the band for determining the positive and negative is not clear and difficult to distinguish.
  • the amount of nucleic acid treated on the gel-card can be adjusted according to the type and amount of nanoparticles reacted, and the entire master mix is also available. .
  • agarose from 10 to 30 ⁇ agarose. More preferably, it may be a gel-card containing agarose (result not shown).
  • enhancers can be added for more distinctiveness.
  • Nanoparticles and amplified products were mixed and treated on gel-cards, in which case not only positive and negative judgments were possible with the naked eye, but also fluorescence, and the detection results were clearer (see Fig. 6).
  • HPV 16 DNA standard WHO International Standard 1 st WHO International
  • Standard for Human Papillomavirus (HPV) Type 16 DNA, 10 7 copies /) was diluted to make 10 2 , 10 3 , 10 and 10 5 copies / ⁇ and amplified by PCR.
  • Fig. 7A Whether the amplified product was properly diluted was observed through the method of Example 2, and the result is shown in Fig. 7A.
  • These amplified products 4 ⁇ and Dynabead 4 ⁇ were mixed and anti-mouse IgG agarose 1 / 50 20 ⁇ was added to the gel-card, and the sponsorship was separated at 37 minutes for 5 minutes. At this time, 2, 4, and 6 minutes were observed to descend (Fig. 7B).
  • the HPV nucleic acid was amplified from the sample.
  • the primer represented by SEQ ID NO: 2 was labeled with a fluorescent substance of Cy3 at the 5 'end.
  • amplified product 4 ⁇ and Dynabead 4 are mixed and anti-mouse IgG agarose 1/5020
  • the present invention is more industrially feasible because it can quickly and simply determine the negative and positive determination of a specific disease by using a nucleic acid detection method that does not include a separation step.

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Abstract

본 발명은 나노입자를 이용한 핵산 검출용 키트 및 핵산 검출 방법에 관한 것으로, 보다 상세하게는 핵산을 증폭 및 표지시킨 후 나노입자로 포획하여 원심분리하는 단계를 포함하는 핵산 검출 방법 및 이의 방법을 이용한 핵산 검출용 키트에 관한 것이다. 본 발명은 분리 단계를 포함하지 않는 핵산 검출법을 통해 특정 질병에 대한음성, 양성의 판단을 보다 신속하고 간단하며, 민감하고 신뢰도 높게 판별할 수 있어 효과적이다.

Description

명세서
발명의명칭:나노입자를이용한핵산검출용키트및핵산검출 방법
기술분야
[1] 본출원은 2015년 07월 13일출원된대한민국특허출원제 10-2015-0099378호 및 2015년 08월 27일출원된대한민국특허출원제 10-2015-0121109호를
우선권으로주장하고，상기명세서전체는본출원의참고문헌이다.
[2] 본발명은나노입자를이용한핵산검출용키트및핵산검출방법에관한 것으로，보다상세하게는핵산을증폭및표지시킨후나노입자로포획하여 원심분리하는단계를포함하는핵산검출방법및이의방법을이용한핵산 검출용키트에관한것이다.
배경기술
[3] 초고속핵산검사의필요성은많은분야에서점점증가하고있다.핵산검사는 궁극적으로병의조기발견을통해전체의료비용을낮추는목적으로사용되고 있으며，향후병원균검사，제노타이핑，암진단등을위한여러종류의핵산에 대한동시분석 ·수요ᅵ증가와현장검사용으로사용할수있는제품에대한 수요가급증할것으로기대된다.이를해결하기위해서는제노타이핑및암 진단에대한특이성과민감도가높은 DNA/RNA표지의개발이많이이루어져야 할것이다.핵산검사가널리이용되기위해서해결해야할문제들이많이 남아있지만,시기만문제일뿐핵산검사가진단시장을지배하는시대가곧올 것으로기대되며，그시기를앞당기기위해저비용의핵산검사를위한장치와 효율적인 DNA표지를개발하는것이연구의증심으로자리매김하고있다.
[4] 핵산검사는민감도,특이성,정밀성，정확성등의분석적타당성 (analytical validity)과임상적민감도와특이성，음성예측률 (negative predictive value)등의 임상적타당성 (clinical validity)및유용성 (clinical utility)를만족하여야한다.핵산 검사의대상을크게두가지로나누면인체에감염된미생물에서유래된핵산과 인체에서직접유래된핵산이다.전자는감염여부를진단하는검사로핵산검사 시장의 80%이상올차지하고있다.후자는질환유발과치료에관련된핵산을 분석하는유전자검사 (genetic test)이며시장점유율은 20%미만이지만잠재적 가치와시장성은매우높올것으로전망하고있다.
[5] 여러핵산검출방법중나노입자를아용한핵산검출은기존의음전하형태의 수지 (negative charged polyanion)및실리? 막 (silica membrane)기술을이용한 방법의단점인저속,고비용방법을획기적으로개선할수있는차세대기술로， 나노입자를제조하고,핵산을포획할수있는코팅처리를실시하여대상핵산 분자와선택적으로결합하여핵산을검출하는시스템이경쟁적으로개발되고 있다.선행문헌으로미국특허 US20140100131에서는목적유전자를검출하는 방법으로자성입자를사용하며，미국특허 US20100009383는목적하는 생체분자를검출하기위한방법에관한것으로,자성입자로타겟물질을회수한 후,원심분리를수행하여웅집된타겟물질을검출할수있다.하지만,기존의 방법은추가적인 DNA프로브나항체를첨가해주어야하는불편함이있고,분리 및세척과정이추가적으로필요하여，보다산속한핵산검출방법에대한 발명이필요하였다.
[6] 이에，본발명자들은증폭된목적핵산과나노입자를사용하여겔-카드를
제조하였고,이를원심분리후육안 /형광으로확일할수있음을입증하였으며, 이러한핵산검출법이특정질병에대한특정핵산을검출하는데현저한효과가 있음을확인하였다.따라서，본발명은분리단계를포함하지않는핵산 검출법을통해특정질병에대한음성，양성의판단을보다신속하고간단하며 , 민감하고신뢰도높게관별할수있는핵산검출방법및핵산검출용키트를 발명하였다.
발명의상세한설명
기술적과제
[7] 본발명은빠르고정확하게핵산을검출하는키트를제공한다.
[8] 본발명의또다른목적은핵산을검출하기위한，나노입자복합체를제공하는 것이다.
[9] 본발명의또다른목적은나노입자복합체를검출하기위한겔카드를
제공하는것이다.
[10] 본발명의또다른목적은핵산을빠르고정확하게검출하는방법을
제공하는데있다.
과제해결수단
[11] 상술한본발명의과제를해결하기위하여본발명은검출하고자하는핵산에 특이적으로결합하는프라이머세트;포획나노입자;및밀도차이를발생시키는 성분과겔성분이순차적으로적층된겔카드；를포함하는핵산검출용키트를 제공한다.
[12] 본발명은또한，목적핵산검출용어세이 (assay)에사용하기위한，목적핵산이 포획되거나또는결합된나노입자를포함하는검출가능한나노입자복합체를 제공한다.
[13] 본발명은또한，나노입자복합체를검출하기위한,유리구슬 (glass bead) 성분과겔성분이순차적으로적층된겔카드를제공한다.
[14] 본발명은또한,목적핵산검출용어세이 (assay)에사용하기위한，목적핵산이 포획되거나또는결합된나노입자를포함하는검출가능한나노입자복합체의 용도에관한것이다.
[15] 본발명은또한，상기나노입자복합체를검출하기위한,유리구슬 (glass bead) 성분과겔성분이순차적으로적층된겔카드의용도에관한것이다. [16] 본발명은또한 목적핵산에특이적으로결합하는프라이머세트로목적 핵산을증폭시키는단계; (L)상기 (~ι )단계에서증폭된핵산에나노입자를 첨가하여핵산을나노입자에포획또는결합시키는단계; (c )상기 (L)단계에서 나노입자에핵산이포획또는결합된복합체를겔카드용기에넣는단계; (Ξ ) 상기 (c )단계에서제조된흔합물을원심분리하는단계;및 (口)겔카드용기내 침전물의위치를대조군과비교하는단계를포함하는나노입자를이용한핵산 검출방법을제공한다.
[17]
[18] 본발명의바람직한다른일실시예에따르면，상기상기 (π )단계의프라이머 세트는바이오틴 -표지정방향프라이머 (biotin-labeled forward primer)와형광 표지역방향프라이머 (fluorescence-labeled reverse primer)인것일수있다.
[19]
[20] 본발명의바람직한다른일실시예에따르면，상기 (ᅳ I )단계는중합효소
연쇄반웅 (PCR)또는등온증폭반웅으로핵산을증폭하는것일수있다.
[21] 상기등온증폭반웅은 HDA (Helicase-Dependent Amplification),
RPA(Recombinase Polymerase Amplification), RCA(Rolling Circle Amplification), LAMP(Loop mediated isothermal amplification), NASBA(Nucleic
Acid-Sequence-Based Amplification), TMA(Transcription Mediated Amplification), SMART(Signal Mediated Amplification of RNA Technology), SDA(Strand
Displacement Amplification), IMDA(Isothermal Multiple Displacement
Amplification), SPIA(Single Primer Isothermal Amplification)및 cHDA(circular Helicase-Dependent Amplification)로이루어지는군에서선택되는어느하나의 방법으로수행되는것일수있으며，바람직하게는 HDA (Helicase-Dependent Amplification), RPA(Recombinase Polymerase Amplification), RCA(Rolling Circle Amplification), LAMP(Loop mediated isothermal amplification)의방법으로 수행되는것일수있고,더욱바람직하게는 HDA (Helicase-Dependent
Amplification)또는 RPA(Recombinase Polymerase Amplification)의방법으로 수행되는것일수있다.
[22] 본발명의바람직한또다른일실시예에따르면，본발명의나노입자는
자성입자 (magnetic bead),금 (Au)나노입자,은 (Ag)나노입자，백금 (Pt)나노입자， 양자점 (Quantum dot),상방전환나노입자 (upconversion nanoparticle, UCNP), 그래핀 (graphene)-나노입자복합체，색염색나노입자 (color dyed particles)및 라텍스 (latex)나노입자로이루어지는군에서선택되는어느하나인것일수 있다.
[23] 또한，본발명의나노입자는표면에핵산을포획할수있는아비딘 (avidin), 아민 (amine),스트랩타비딘 (streptavidin)，프라이머에결합된항원에항원 -항체 반웅을통해결합될수있는항체 (예를들어， digoxigenin/anti-digoxigenin antibody, Cy3/anti-Cy3 antibody둥과같은등가물)，앱타머 (aptamer)및 올리고뉴클레오타이드로이루어지는군에서선택되는어느하나로코팅처리가 된것을특징으로한다.
[24] 본발명에서겔성분은 IgG-아가로스 (agarose),아가로스 (agarose),한천 (寒天), 셀를로오스아세테이트 (cellulose acetate)및폴리아크릴아마이드 (polyacrylamide gel)로이루어지는군에서선택되는어느하나인것일수있다.
[25] 본발명의핵산검출방법은인핸서를처리하는단계를추가적으로포함할수 있으며,기존의방법에는포함되었던분리과정및세척과정이불필요하다. 발명의효과
[26] 본발명은나노입자를이용한핵산검출용및키트핵산검출방법에관한
것으로,보다상세하게는핵산을증폭및표지시킨후나노입자로포획하여 원심분리하는단계를포함하는핵산검출방법및이의방법을이용한핵산 검출용키트에관한것이다.본발명은분리단계를포함하지않는핵산 검출법을통해특정질병에대한음성，양성의판단을보다신속하고간단하며, 민감하고신뢰도높게판별할수있어효과적이다.
도면의간단한설명
[27] 도 1은본발명의핵산검출방법을나타낸모식도이다.
[28] 도 2는본발명의핵산검출방법을구체적으로나타낸모식도와 NHS
변형법으로표지된역방향프라이머를사용한핵산검출방법에따른양성 /음성 결과를나타낸사진이다.
[29] 도 3은포스포라미티드법으로표지된역방향프라이머를사용한핵산검출 방법에따른양성 /음성결과를나타낸사진이다ᅳ
[30] 도 4는원심분리시간에따른핵산검출결과를나타낸사진이다.
[31] 도 5는인핸서사용유무에따른핵산검출결과를나타낸사진이다.
[32] 도 6은 HPV DNA핵산검출을실시한결과에대해서육안또는형광으로
식별이가능함을보여주는사진이다.
[33] 도 7은 DNA농도에따른검출민감도를측정한결과를나타낸사진이다.
[34] 도 8은 HPV임상검체로본발명의핵산검출법을실시한사진이다 (1-3번튜브
： HPV DNA음성샘플， 4-6번튜브： HPV DNA양성샘플).
발명의실시를위한최선의형태
[35] 이하,본발명을더욱상세히설명한다.
[36] 상술한바와같이,기존의질병관련특정핵산의음성및양성진단의경우, 통상적으로 4내지 6시간이라는오랜시간이걸리고,정확도가떨어지는 문제점이초래되어왔다.따라서，양성인환자에게빠른처방이어려웠다.
[37] 또한,미국공개특허 2014-0100131의경우，목적유전자를검출하는방법에 관해서는항체를사용하며 ,나노입자로반드시자성입자만을사용하여야하며， 분리단계가추가적으로포함되어빠른검출이어려웠다.
[38] 이에본발명자들은목적핵산을증폭하고이를나노입자및겔성분과함께 겔-카드에주입한후，원심분리하는간단한방법을통해질병관련특정핵산을 신속하고정화하게검출하는방법을고안하였다.
[39] 본발명에서사용되는용어는다음과같이정의된다.
[40] 본발명에서 "핵산 (nucleic acid)"이란임의의길이의뉴클레오타이드의
중합체를의미하고, DNA및 RNA를포함한다.
[41] 본발명에서 "포획또는결합 (capture)"이란본발명에서하나이상의제제의 적어도일부 (또는이제제와결합된링커부)를나노입자또는그의표면에포집， 흡착，정전기적결합，이온적결합，공유결합,상보적인올리고뉴클레오타이드 결합，항원 -항체반웅을통한결합또는고정시키는것일수있다.
[42] 본발명에서 "프라이머"란올리고뉴클레오타이드를의미하는것으로，
핵산쇄 (주형)에상보적인프라이머연장산물의합성이유도되는조건，즉， 뉴클레오타이드와 DNA증합효소와같은중합제의존재,그리고적합한온도와 pH의조건에서합성의개시점으로작용할수있다.바람직하게는，프라이머는 디옥시리보뉴클레오타이드이며단일쇄이다.본발명에서이용되는프라이머는 자연 (naturally occurring) dNMP (즉， dAMP, dGMP, dCMP및 dTMP),변형 뉴클레오타이드또는비 -자연뉴클레오타이드를포함할수있다.또한， 프라이머는리보뉴클레오타이드도포함할수있다.프라이머는，중합제의존재 하에서연장산물의합성을프라이밍시킬수있을정도로충분히길어야한다. 프라이머의적합한길이는다수의요소，예컨대,은도，웅용분야및프라이머의 소스 (source)에따라결정되지만전형적으로 15-30뉴클레오타이드이다.짧은 프라이머분자는주형과층분히안정된혼성복합체를형성하기위하여 일반적으로보다낮은온도를요구한다.용어 "어닐링"또는 "프라이밍 "은주형 핵산에올리고디옥시뉴클레오타이드또는핵산이병치 (apposition)되는것올 의미하며,상기병치는증합효소가뉴클레오타이드를중합시켜주형핵산또는 그의일부분에상보적인핵산분자를형성하게한다.
[43] 본발명에서 "인핸서 (inhancer)"란검체의양성및음성을판단할때,이를더욱 정확하고명확하게판단할수있도록돕는물질을의미한다.예를들어 ,핵산 또는프라이머에결합하여형광을띄거나또는발색하는물질일수있다.
[44] 본발명에서 "겔카드 (gel card)"란 WO 1999/050673특허에기재된 gel column을 참고하여정의될수있다.본발명에서겔-카드란，밀도차이를발생시키는 성분과겔성분이순차적으로적층된겔카드이며,더욱바람직하게는
유리구슬 (glass bead)과아가로스겔을순차적으로용기에담은것올의미한다.
[45] 본발명은검출하고자하는핵산에특이적으로결합하는프라이머세트;포획 나노입자 (capture nanoparticle);및밀도차이를발생시키는성분과겔성분이 순차적으로적층된겔카드；를포함하는핵산검출용키트를제공한다.
[46] 상기프라이머세트는바이오틴 -표지정방향프라이머 (biotin-labeled forward primer)와형광표지역방향프라이머 (fluorescence-labeled reverse primer)일수 있다.본발명의핵산검출용키트에포함되는프라이머는바이오틴으로표지된 、
것일수있으며，바이오틴으로표지된프라이머는나노입자에더욱잘
포획되거나또는결합될수있어핵산을검출하는데용이하다.또한,본발명의 키트에포함되는프라이머는형광으로표지될수있으며,형광으로표지된 프라이머는형광으로확인하여밴드의위치를통해핵산의유무를판별할수 있다.
[47] 본발명의일실시예에서는프라이머의표지방법에따라육안으로음성또는 양성을구분할수있으며，형광으로도음성또는양성을구분할수있다 (도 6 참조).
[48] 상기프라이머의형광표지는이에한정하지는않으나， Cy3, Cy5, TAMRA, TEX, TYE, HEX, FAM, TET, JOE, MAX, ROX, VIC, Cy3.5, Texas Red, Cy5.5, TYE, BHQ, Iowa Black RQ및 IRDye로이루어지는군에서선택되는어느하나 이상일수있다.
[49] 또한，상기프라이머의형광표지는크게두가지방법， NHS
변형법 (N-hydroxysuccinimide modification)또는
포스포라미티드법 (phosphoramidite synthesis)으로이루어질수있다.
[50] Cy염색약의표지방법은판매제조사 amersham biosicences의카달로그
"labelling of oligonucleotides with CyDye fluors for fluorescent applications using the
LEAD seeker homogeneous imaging system, amersham biosiecnces, V0I.L6, 2000"에 나온방법으로실시될수있다.
[51] 본원발명에서 NHS변형법으로표지된프라이머를사용하여핵산을검출하면, 양성검체가음성검체보다아래에밴드를형성하는것올특징으로하며;
포스포라미티드법으로형광표지된프라이머를사용하여핵산을검출하면， 양성검체가음성검체보다위에밴드를형성하는것을특징으로한다.이러한 차이는프라이머의형광표지위치에따라상이해지는것으로판단된다.
[52] 본발명의일실시예에서는 NHS변형법으로형광표지된프라이머를사용하여 핵산을검출할때，양성검체가아래에벤드를형성하였으며 (도 2참조), 포스포라미티드법으로형광표지된프라이머를사용하여핵산을검출할때, 양성검체가위에밴드를형성하였다 (도 3참조).
[53] 본발명의상기나노입자는자성입자 (magnetic bead),금 (Au)나노입자,은 (Ag) 나노입자,백금 (R)나노입자，양자점 (Quaantum dot),상방전환
나노입자 (upconversion nanoparticle, UCNP)그래핀 (graphene)-나노입자복합체， 색염색나노입자 (color dyed particles)및라텍스 (latex)나노입자로이루어지는 군에서선택되는어느하나인것을특징으로한다.
[54] 본발명의일실시예에서는나노입자로 Dynabead (MyOne Streptavidin CI)를 사용하였으며，이외에도나노입자표면에핵산을포획할수있는처리가가능한 자성입자，금속입자，양자점,상방전환나노입자,그래핀-나노입자복합체,색 염색나노입자또는라텍스나노입자가사용될수있다.보다바람직하게는 자성입자，금속입자일수있다. [55] 본발명의상기나노입자는표면에핵산을포획할수있는아비딘 (avidin), 아민 (amine),스트렙타비딘 (streptavidin),프라이머에결합된항원에대하여 항원 -항체반응을통해결합될수있는항체 (예를들어,
digoxigenin/anti-digoxigenin antibody, Cy3/anti-Cy3 antibody및이의등가물 ), 앱타머 (aptamer)및올리고뉴클레오타이드로이루어지는군에서선택되는어느 하나로코팅처리가된것을특징으로한다.
[56] 목적핵산을증폭할때，항원을포함하는프라이머를사용하고，나노입자의 표면에상기항원에대한항체를결합시키면，항원 -항체반웅을통하여 나노입자가목적핵산을포획할수있다.
[57] 상기밀도차이를발생시키는성분은겔성분을아래에서지지해주는역할을 하며，밀도차이를발생시키는성분보다핵산 -나노입자복합체의밀도가클경우 가라앉으며，밀도차이를발생시키는성분보다핵산-나노입자복합체의밀도가 낮으면가라앉지않도록음성및양성을밀도차이를이용하여판별할수있도록 분리해주는역할을한다.이로인해,별도의세척과정이나분리과정이필요하지 않게된다.
[58] 본발명의상기밀도차이를발생시키는성분은밀도차이를발생시킬수있는 물질이면한정되지않으나，바람직하게는유리구슬 (glass bead), quartz based matrix,퍼콜 (percoll),콜로이드실리카용액 (colloidal silica media),피콜 (ficoll)로 이루어지는군에서선택되는어느하나인것을특징으로하며，더욱
바람직하게는유리구슬 (glass bead)일수있다.
[59] 본발명의상기겔성분은 IgG-아가로스 (agarose),아가로스 (agarose),
한천 (寒天)，셀를로오스아세테이트 (cellulose acetate)및폴리아크릴아마이드 겔 (polyacrylamide gel)로이루어지는군에서선택되는어느하나인것을
특징으로하며 ,바람직하게는성분은 IgG-아가로스 (agarose)또는
아가로스 (agarose)일수있다.
[60] 본발명의일실시예에서는 IgG-아가로스를사용하였으며, 10내지 30 ^의
IgG-아가로스에서양성및음성을판별하는데최소의시간및높은구분력을 나타냈으며，바람직하게는 15내지 25 ^의양일수있으며，보다바람직하게는 20 ^의양일수있다.
[61] 본발명의핵산검출용키트는인터킬레이팅제제인인핸서를추가적으로
포함할수있다.인핸서를추가적으로포함하여，양성과음성의구분을더욱 뚜렷하게할수있는효과가있으며，이에한정되지는않으나，바람직한 인터킬레이팅제제로는 SYBR그린,브롬화에티듐 (ethidium bromide), biotium gelred, biotium gelgreen, JOJO계열， POPO계열， SYTO계열， BOBO계열,
TOTO계열，악티노마이신,아드리아마이신，안트라센,벤조피렌,프로피디움 디이오다이드 -인터트위닝 (propidium diiodide-intertwining),디스타마이신, 네트롭신과아크리딘，프소랄렌,베르베린 (berberine),프로플라빈 (proflavine), 다우노마이신,독소루비신，탈리도마이신,시아닌염료또는 LDS 751일수있다. 더욱바람직하게는인터킬레이팅염색약인 SYBR그린,브름화에티듬， biotium gelred, biotium gelgreen, JOJO계열, POPO계열, SYTO계열， BOBO계열및
TOTO계열로이루어진군에서선텍된어느하나일수있다.
[62] 본발명의핵산검출용키트는바람직하게는바이오틴 -표지정방향
프라이머와형광표지역방향프라이머로구성되고，검출하고자하는핵산에 특이적으로결합하는프라이머세트;포획나노입자 (capture nanoparticle);
인터킬레이팅제제;및밀도차이를발생시키는성분과겔성분이순차적으로 적층된겔카드;를포함하는인시츄 (in situ)핵산검출용키트이다.
[63] 본발명은목적핵산검출용어세이 (assay)에사용하기위한，목적핵산이포획 또는결합된나노입자를포함하는검출가능한나노입자복합체를제공한다.
[64] 본발명은나노입자복합체를검출하기위한，유리구슬 (glass bead)성분과겔 성분이순차적으로적층된겔카드를제공한다.
[65] 상기유리구슬성분과겔성분이적층된겔카드는테스트용기내에담겨있는 형태이며，테스트용기는어떠한고체용기로도사용할수있으며，다른여러 유형으로가공할수있다ᅳ예를들면시험관,마이크로플레이트웰등의어떠한 용기도사용가능하다.
[66] 본발명자들은 ("! )목적핵산에특이적으로결합하는프라이머세트로목적 핵산을증폭시키는단계; 一)상기 ( π )단계에서증폭된핵산에나노입자를 첨가하여핵산을나노입자에포획또는결합시키는단계 ; ( C )상기 ( 1一)단계에서 나노입자에핵산이포획또는결합된복합체를겔카드용기에넣는단계; ( Ξ ) 상기 (1= )단계에서제조된흔합물을원심분리하는단계；및 (口)겔카드용기내 침전물의위치를대조군과비교하는단계를포함하는나노입자를이용한핵산 검출방법을제공한다.
[67] 상기프라이머세트는바이오틴 -표지정방향프라이머 (biotin-labeled forward primer)와형광표지역방향프라이머 (fluorescence-labeled reverse primer)일수 있다.본발명의핵산검출용키트에포함되는프라이머는바이오틴으로표지된 것일수있으며,바이오틴으로표지된프라이머는나노입자에더욱잘
포획되거나또는결합될수있어핵산을검출하는데용이하다.
[68] 더욱바람직하게상기프라이머세트는바이오틴 -표지정방향
프라이머 (biotin-labeled forward primer)와 Cy3-표지역방향프라이머 (Cy3-labeled reverse primer)일수있다 .
[69] 바이오틴이표지된정방향프라이머를통해바이오틴과특이적으로결합할수 있는물질 (예를들어 avidin등)로코팅된나노입자에결합하고， Cy3가표지된 역방향프라이머에의하여육안또는형광에서침전물의위치를확인할수있다. 또한， Cy3는증폭물을더욱명확하게구분할수있는인핸서의역할을
수행하기도한다.
[70] 본발명의일실시예에서는서열번호 1및 2로표시되는프라이머세트또는 서열번호 3및 4로표시되는프라이머세트로 HPV핵산올검출하였다. [71] 본발명의핵산은 DNA와 RNA를포함하며 , DNA의증폭은기존에알려진증폭 방법이면무엇이든사용가능하다.바람직하게는 PCR또는등온증폭법일수 있다.또한, RNA의증폭에있어서도기존에알려진증폭방법이면무엇이든 사용가능하며 ,바람직하게는 cDNA를합성한후증폭하는방법일수있으며 , 더욱바람직하게는 rt-PCR(reverse transcription PCR)방법일수있다.
[72] 본발명의상기 (π )단계는중합효소연쇄반웅 (PCR)또는등온증폭반웅으로 핵산을증폭하는것을특징으로한다.
[73] 본발명의핵산은 DNA와 RNA를포함하며， DNA의증폭은기존에알려진증폭 방법이면무엇이든사용가능하다.바람직하게는 PCR또는등온증폭법일수 있다.
[74] 본발명의상기등온증폭반웅은 HDA (Helicase-Dependent Amplification),
RPA(Recombinase Polymerase Amplification), RCA(Rolling Circle Amplification), LAMP(Loop mediated isothermal amplification), NASBA(Nucleic
Acid-Sequence-Based Amplification), TMA(Transcription Mediated Amplification), SMART(Signal Mediated Amplification of RNA Technology), SDA(Strand
Displacement Amplification), IMDA(Isothermal Multiple Displacement
Amplification), SPIA(Single Primer Isothermal Amplification)및 cHDA(circular Helicase-Dependent Amplification)로이루어지는군에서선택되는어느하나의 방법으로수행되는것을특징으로한다. '
[75] PCR(Polymerase Chain Reaction)은선택성이우수하고신속한분석방법이지만 반웅동안에주기적으로온도를변화시켜주어야한다.온도변화없이도 ' 등온에서 DNA/RNA증폭이가능한기술을등온증폭기술이라고한다.둥온증폭 기술은온도를변화할시간이필요없기때문에단시간내에대량의 DNA증폭이 가능하여핵산의신속검출기술로서활용도가높다.대표적인것이
loop-mediated isothermal amplification (LAMP)인데， LAMP는 4-6개의 primer를 이용하여특정한 target sequence를증폭하는기술로서 magnesium
pyrophosphate이나 SYBR green올첨가하면증폭후눈으로도핵산증폭판정이 가능하기때문에향후현장용신속검출기술에웅용가능성이매우높다.
[76] NASBA와 TMA의경우 RNA template을사용하여 cDNA합성후
self-sustained에의해다시 RNA를합성해내는반웅을반복하여증폭하는 방법이다ᅳ SMART의경우 target에의존하는방법으로온도변화없이 target DNA나 RNA를증폭시켜검출할수있는방법이다. SDA의경우 4개의 primer를 필요로하며제한효소를사용, Hinc의인지염기서열 (GTTGAC)을웅용한 방법이다. RCA는 Φ29 DNA polymerase가순환적으로 primer를연장하여 고분자의원형핵산을결과적으로긴가닥으로증폭시키는방법이다. RCA는 단단하게결합하여다른등온증폭기술중 DNA진단에서중요한기반기술로 사용된다.최근주목받고있는방법으로현재유전검사부터면역분석법, sequencing, SNP scoring,유전발현분석등에사용되고있다. IMDA는 double-stranded nucleic acid에양쪽으로 primer가달라붙어그대로신장하는 방법이다. HDA는 helicase를사용하여단일가닥으로분리시킴으로써
denaturation time이따로필요치않고모든반웅을한온도에서시킬수있는 방법이다. SPIA는 RNA에서 cDNA가합성되고 RNaseH에의해 RNA가 제거되면서 SPIA용 primer가붙으며 DNA polymerase에의해증폭이일어난다. SPIA primer와 polymerase의지속적인증폭반웅을통해한가닥의 cDNA에서 여러가닥이생성되는방법이다. cHDA는 DNA polymerase와 helicase를함께 사용하는기술로한온도에서모든반응을시키는방법이다.
[77] 본원발명의일실시예에서는 HDA및 RPA반웅으로증폭하여핵산을
검출하였으며, LAMP및 RCA반웅도현장분자진단에적합한증폭반웅증 하나이므로，바람직하게본발명의등온증폭반웅은 HDA (Helicase-Dependent Amplification), RPA(Recombinase Polymerase Amplification), RCA(Rolling Circle Amplification)및 LAMP(Loop mediated isothermal amplification)일수있으며 ,보다 바람직하게는 HDA (Helicase-Dependent Amplification), RPA(Recombinase Polymerase Amplification)0]다.
[78] 또한， RNA의증폭에있어서도기존에알려진증폭방법이면무엇이든사용 가능하며,바람직하게는 RNA를 template로하는 NASBA반웅을이용하거나, cDNA를합성한후증폭하는 rt-PCR반웅일수있다.
[79] 상기나노입자는첵산을포획또는결합시킬수있는나노입자라면제한되지 않는다 핵산을포획할수있는물질을코팅또는결합시킬수있는나노입자면 무엇이든적용할수있다.다만，본원발명이밀도차이를이용하여특정핵산의 양성또는음성을판단하는것이기때문에,매질의밀도와차이가있어야한다. 본원발명의일실시예에서는매질로유리구슬 (glass bead)을사용하며，이의 매질밀도가 1.04 g/ 이기때문에그보다큰밀도를가진입자면모두가능하다. 하지만，다른밀도의매질을이용하거나,매질의밀도보다작은입자를이용하여 적용할수도있다.
[80] 바람직하게본발명의나노입자는자성입자 (magnetic bead),금 (Au)나노입자， 은 (Ag)나노입자，백금 (Pt)나노입자,양자점 (Quantum dot),상방전환
나노입자 (upconversion nanoparticle, UCNP),그래핀 (graphene)-나노입자복합체， 색염색나노입자 (color dyed particles)또는라텍스 (latex)나노입자일수있다.
[81] 본발명의일실시예에서는나노입자로 Dynabead (MyOne Streptavidin CI)를 사용하였으며,이외에도나노입자표면에핵산을포획할수있는처리가가능한 자성입자，금속입자，양자점,상방전환나노입자,그래핀-나노입자복합체，색 염색나노입자또는라텍스나노입자가사용될수있다.보다더욱바람직하게는 자성입자,금속입자일수있다.
[82] 본발명의상기나노입자는표면에핵산을포획할수있는아비딘 (avidin), 아민 (amine)，스트랩타비딘 (streptavidin),프라이머에결합된항원에대하여 항원 -항체반응을통해결합될수있는항체 (예를들어, digoxigenin/anti-digoxigenin antibody, Cy3/anti-Cy3 antibody및이의등가물)， 앱타머 (aptamer)및올리고뉴클레오타이드로이루어지는군에서선택되는어느 하나로코팅처리가된것일수있다.
[83] 목적핵산을증폭할때，항원을포함하는프라이머를사용하고,나노입자의 표면에상기항원에대한항체를결합시키면，항원 -항체반웅을통하여 나노입자가목적핵산올포획할수있다.
[84] 본발명의상기겔성분은 IgG-아가로스 (agarose),아가로스 (agarose),
한천 (寒天),샐를로오스아세테이트 (cellulose acetate)및
폴리아크릴아마이드 (polyacrylamide gel)로이루어지는군에서선택되는어느 하나일수있다.
[85] 본발명의일실시예에서는 IgG-아가로스 (agarose)를사용하였으며， 10내지 30
의 IgG-아가로스에서양성및음성을판별하는데최소의시간및높은 구분력을나타냈으며,바람직하게는 15내지 25 ^의양일수있으며，보다 바람직하게는 20 ^의양일수있다.
[86] 본발명의상기 = )단계에서인터킬레이팅제제 (intercalating agent)의인핸서를 추가적으로처리할수있다.
[87] 양성및음성의구분을명확하게하기위하여본원발명에서는인핸서를
추가로처리할수있으며，인핸서는인터킬레이팅제제일수있으며 ,
인터킬레이팅제제는 SYBR그린，브름화에티듐 (ethidium bromide), biotium gelred, biotium gelgreen, JOJO계열， POPO계열, SYTO계열， BOBO계열,
TOTO계열，악티노마이신，아드리아마이신,안트라센,벤조피렌,프로피디움 디이오다이드 -인터트위닝 (propidium diiodide-intertwining),디스타마이신, 네트롭신과아크리딘，프소랄렌，베르베린 (berberine),프로플라빈 (proflavine), 다우노마이신，독소루비신，탈리도마이신，시아닌염료및 LDS 751으로
이루어지는군에서선택되는어느하나일수있으며 ,더욱바람직하게는 인터킬레이팅염색약인 SYBR그린，브롬화에티듐， biotium gelred, biotium gelgreen, JOJO계열, POPO계열， SYTO계열, BOBO계열및 TOTO계열로
이루어진군에서선택된어느하나일수있다.
[88] 본발명의일실시예에서는증폭된핵산에인핸서로 GelRed (10,000X,
Biotium)와나노입자를섞고겔-카드에처리하였다.그결과,도 5에보이는바와 같이,인핸서를처리했을경우,양성과음성이구분이더욱뚜렷해지는효과를 확인할수있었다.
[89] 한편,인터킬레이팅제제뿐만아니라，프라이머에형광염료를결합하여이를 인핸서로사용할수있다.형광염료는프라이머에결합할수있는것은모두 가능하며 ,형광염료의예로는 Cy3, Cy5, TAMRA, TEX, TYE, HEX, FAM, TET, JOE, MAX, ROX, VIC, Cy3.5, Texas Red, Cy5.5, TYE, BHQ, Iowa Black RQ, IRDye 계열및이와등가물이있다.
[90] 상기용기내침전물의위치를대조군과비교하는단계는 NHS변형법으로형광 표지한프라이머를사용하였을경우,용기내침전물이음성대조군보다 아래쪽에위치해있으면양성，음성대조군과유사한위치에침전물이위치해 있으면음성으로판단할수있다.도 2는본원발명올나타낸모식도로,검체 내에목적핵산이포함되어있으면，목적핵산이나노입자에포획된후 밀도차이를이용한원심분리를통하여핵산이아래쪽에위치하게된다.반면， 목적핵산이포함되어있지않으면，나노입자와결합하지않아상대적으로 밀도가낮으므로튜브의중앙부에침전물이위치하게된다.
[91] 반면，포스포라미티드법으로형광표지한프라이머를사용하였을경우，용기내 침전물이음성대조군보다아래쪽에위치해있으면음성,음성대조군과유사한 위치에침전물이위치해있으면양성으로판단할수있다.이와관련된실험 결과는도 3에나타나있다.
[92] 본발명은상기 (d)단계전에，따로분리과정및세척과정을포함하지않는것을 특징으로한다.
[93] 선행문헌인미국공개특허 2014-0100131의경우，목적유전자를검출하기위한 방법에관한별명으로써 ,자성입자와 capture항체를사용하며，분리단계를거쳐 목적유전자를검출한다.또한,선행문헌인미국공개특허 2010-0009383에서도 자성입자와 capture항체를사용하고,분리단계를거쳐야만목적생물분자를 검출할수있다.반면,본원발명은입자의제한이없으며，항체를사용하지않을 수있고,분리및세척단계가필요하지않아더욱쉽고간단하며빠르게목적 핵산을검출할수있다.
[94] 본발명은상기 (a)단계에서，각각다른색의형광으로표지된둘이상의
프라이머를처리하여，다중으로핵산의존재유무를확인하는것을특징으로 한다.
[95] 핵산을증폭하는단계에서，각각다른색을띄는형광으로표지된둘이상의 프라이머를처리하여，다증으로핵산의존재유무를확인할수있다.따라서， 한번의실험올통해다중핵산의존재유무를판별할수있으며,이를통해 시간과비용을절감할수있으므로효과적인핵산검출방법이다.
발명의실시를위한형태
[96] 이하,실시예를통하여본발명을더욱상세히설명하고자한다.이들실시예는 오로지본발명을예시하기위한것으로서，본발명의범위가이들실시예에 의해제한되는것으로해석되지않는것은당업계에서통상의지식을가진자에 있어서자명할것이다ᅳ
[97] [실시예 1]
[98] 시료로부터 genomic DNA추출및증폭 ' [99] 자궁경부검체로부터 genomic DNA를추출하는경우,핵산추출키트 (QIAamp
DNA Micro kit, QIAGEN, Valencia, CA, USA또는 ChargeSwitch gDNA Ι βί Serum Kit, Life Technologies, NY, USA)를이용하여제조사의지시대로핵산을 추출하였고，인유두종바이러스 (HPV) DNA표준품은식품의약품안전처및
NIBSC(National Institute for Biological Standards and Control)에분양신청을하여 핵산을준비하였다
[100] [표 1]
프라이머정보
Figure imgf000015_0001
[101] 상기표 1의 PCR프라이머세트를이용하여각시료의증폭산물을얻었다.본 발명의프라이머세트는 PCR증폭뿐만아니라등온증폭 (바람직하게는 helicase dependent amplification(HDA), recombinase polymerase amplificatkm(RPA))도 가능하도록제작되었고，기존에알려진문헌을참고하여변형하였다 (Virol J. 2010 Aug 19;7: 194).
[102] [표 2]
PCR반웅조성액
Components of PCR Volume
Forward/reverse primer (10 pmole/^£) 1 id
HotStarTaq plus Master Mix Q id
Template DNA (1 5≠
Deionized water 4 id
Total 20 (d [표 3]
RPA반웅조성액
Figure imgf000016_0001
[105] 1) PCR
[106] PCR반웅조성액은상기표 2와같으며 ,증폭조건은 95에서 10분， [95에서 30초,
55에서 30초， 72에서 30초]를한사이클로하여 40회반복하여증폭하였다.
[107] 2) RPA
[108] RPA반웅조성액은상기표 3과같으며，상기표 1에기재된프라이머세트및 트위스트엠프기본키트 (TwistDx, Cambridge, UK)을이용하여, 37에서 40분간 등온증폭하였다.
[109] 3) HDA
[110] HDA반웅조성액은상기표 4와같으며，상기표 1에기재된프라이머세트및 아이소엠프 III유니버셜 tHDA키트 (Biohelix)를이용하여 65에서 60분간등온 증폭하였다.
[111] [실시예 2] [112] 전기영동에의한증폭산물의확인
[113] 1.5%(w/v)아가로스겔을이용하여 Mupid-a(Advance, Japan)전기영동장치로 PCR산물을분석하였다.아가로스 1.5 g을삼각플라스크 (250 id)<A]넣고, 0.5 X TBE(tris boric acid EDTA)완층용액 100 μί을채운후에전자렌지에서 2내지 3분 동안녹인후용액을겔용기에부어서 30분정도굳혔다.겔이완전히굳은것을 확인하고겔을수거하였다.수거한겔을전기영동장치에넣고 0.5 Χ ΤΒΕ 완층용액으로채운다.그리고 PCR산물 4 ^와 6Χ BPB(bromo phenol blue) dye 0.8 ^를섞어서 4 씩로딩하고 100 V에서 25분동안전기영동하였다.그후에 겔을떼어내어 EtBr(ethidium bromide)로 10분간염색하고다시 10분간증류수로 DNA와결합하지못한 EtBr을씻어내었다.마지막으로 UV
트랜스일루미네이터 (transilluminator)위에아가로스겔을올려놓고 PCR증폭 여부를확인하였다.
[114] PCR증폭산물에대하여전기영동을실시한결과， 136bp의밴드가
확인되었으며，상기실시예 1에기재된방법으로부터정상적으로목적 DNA가 증폭됨을확인할수있었다 (결과미도시).
[115] [실시예 3]
[116] 3-1.나노입자및겔 -카드준비
[117] 나노입자는핵산을포획가능하면서,매질의밀도보다큰입자이면이론적으로 모두사용가능하다.본실시예 3에서는핵산분자를포획하기위해서
나노입자로 Dynabead (MyOne Streptavidin CI)를사용하였다.이 Dynabead의 표면은스트랩타비딘이처리되어있어바이오틴 (biotin)이표지된프라이머와 결합하게된다.또한，겔-카드는 polyspecific Ortho BioVue System (Ortho Clinical Diagnostics, NJ, USA)의제품을구입하여사용하였다.
[118] 3-2. NHS변형법으로표지된프라이머를이용한핵산검출
[119] 상기표 1에기재된서열번호 1및 2로이루어지는프라이머세트를이용하여 등온증폭을통해 HPV검체로부터 HPV핵산을증폭하였다.서열번호 2로 표시되는프라이머에는 5'말단에 Cy3의형광물질이표지되었으며,표지방법은 NHS(N-hydroxysuccinimide)변형법으로표지하였다. NHS변형법으로표지된 프라이머는하기화학식 1과같이나타날수있다.
[120] [화학식 1] [121] Cy3 Amino MpdifierC6 pep^guanosine
Figure imgf000018_0001
[122] 증폭된핵산 4 ^와 Dynabead 를섞은후겔카드에넣고 37에서 5분간
반웅시킨후 2분내지 4분간원심분리하였다.도 2는상기과정을모식도로 나타낸것이며,그결과，도 2에보이는바와같이，핵산이검출된 (양성) 겔 -카드에서는 PCR산물이아랫쪽에밴드를이루는것을볼수있으며,핵산이 검출되지않은 (음성)겔 -카드에서는윗쪽에밴드를이루는것을확인할수 있었다.
[123] 3-3.포스포라미티드법으로표지된프라이머를이용한핵산검출
[124] 상기표 1에기재된서열번호 3및 4로이루어지는프라이머세트를이용하여 둥은증폭을통해 HPV검체로부터 HPV핵산을증폭하였다.서열번호 4로 표시되는프라이머에는 5'말단에 Cy3의형광물질을표지되었으며,표지방법은 포스포라미티드법으로실시되었다ᅳ포스포라미티드법으로표지된프라이머는 하기화학식 2와같이나타날수있다.
[125] [화학식 2]
Cy3 Deox guanosine
Figure imgf000018_0002
[127] 증폭된산물 4 ^와 Dynabead 4 를섞은후겔카드에넣고， 37에서 5분
반웅시킨후원심분리하였다.이때, 2분， 4분및 6분째각각내려가는정도를 관찰하였다.그결과，도 3에나타난바와같이，음성에해당하는검체인 1번 튜브에서는밴드가내려가고，양성에해당하는검체인 2번튜브에서는밴드가 위에형성되는것을확인할수있었다.
[128] 이로서，본원발명의나노입자를이용한핵산검출방법은임상검체로도
빠르고민감하게핵산을검출할수있으며，형광표지방법에따라음성과양성 판단방식을용이하게변경할수있다.
[129] [실시예 4]
[130] 핵산검출방법의최적화
[131] 1)원심분리속도
[132] 핵산을검출하기에가장최적의원심분리속도를측정하기위하여,상기
실시예 3-2의방법에따라겔-카드에핵산과나노입자를처리하였다.그후， 2분동안각각 lOOrpm, 200rpm, 400rpm, 600rpm, 800rmp, 1200rpm및 1600rmp으로 원심분리를실시하였다.
[133] 그결과, HPV16 DNA 136 bp를검출하는데 600rpm내지 1200rpm이핵산을 검출하기에최적의원심분리속도임을확인하였으며，더욱바람직하게는 800 rpm x 2분 (약 55 g)이가장바람직한것으로확인되었다 (결과미도시).
[134] 원심분리속도는검출하고자하는핵산의길이또는사용하는나노입자의 크기 /밀도에따라최적화하여 g-force를조절가능하다.
[135] 2)원심분리시간
[136] 핵산을검출하기에가장최적의원심분리시간을측정하기위하여，상기
실시예 3-2의방법에따라겔-카드에핵산과나노입자를처리하였다.원심분리 속도는 800 rpm (약 55g)로설정하고， 1분간격으로각각 8분까지관찰하였다.
[137] 그결과，도 3에나타난바와같이 (2분, 4분， 6분만도시함)，최초 2분의
원심분리로도양성및음성의구분이가능하였으며， 2분내지 6분간원심분리를 하였을때구분이가능하였으며， 6분이상원심분리를실시할경우，양성및 음성구분이명확하지않은것으로확인되었다.
[138] 본발명의원심분리시간은사용나노입자의크기，밀도,반웅양，매질의농도, 원심력등에의해조절가능하다.
[139] 3)핵산의양
[140] 상기실시예 1을통해핵산을증폭한후，생성된 master mix에서증폭된산물 2 내지 6 ^ (가장바람직하게는， 4 /^를실시예 3에서준비한겔-카드에처리하여 핵산을검출하였다.너무적은핵산을처리하면,나노입자와결합하는핵산이 적어양성및음성을판단하기위한밴드가선명하지않아구분이어렵다.
[141] 본발명의겔-카드를이용한핵산검출에서겔-카드에처리하는핵산의양은 반응하는나노입자의종류와양에따라증폭된산물의양을조절가능하며， master mix전체또한이용가능하다.
[142] 4)아가로스의양
[143] 핵산을검출하기에가장최적의겔 -카드내 agarose의양을측정하기위하여 각각 5ιΛ, \0βί, 20μί, 30βί, 40μί및 의 agarose (anti-mouse IgG agarose)가 적층되어있는겔 -카드내에상기실사예 1의방법으로수득한 PCR증폭산물과 나노입자를처리하였다.
[144] 그결과, 10내지 30 ^의아가로스에서양성및음성을판별하는데최소의시간 및높은구분력을나타냈다.더욱바람직하게는아가로스 가포함된 겔-카드일수있다 (결과미도시 ).
[145] 5)인핸서첨가
[146] 양성및음성판정에있어서，보다확연한구분을위하여인핸서를첨가할수 있다.
[147] 첫번째로，실시예 1의방법으로증폭된핵산에인핸서로 GelRed (ΙΟ,ΟΟΟΧ, Biotium) 1/100 1 ^와나노입자를섞고겔-카드에처리하였다.그결과，도 4에 보이는바와같이,인핸서를처리했을경우，양성과음성이구분이더욱 뚜렷해지는효과를확인할수있었다 (도 5참조).
[148] 두번째로,인핸서로프라이머에형광인 Cy3를붙여핵산을증폭한후，
나노입자와증폭산물을흔합하여겔-카드에처리하였다.이경우에는육안으로 양성및음성을판단할수있을뿐만아니라,형광에서도관측가능하였으며 , 검출결과가더욱분명하게나타났다 (도 6참조).
[149] [실시예 5]
[150] 핵산검출민감도측정
[151] HPV 16 DNA standard (WHO International Standard 1 st WHO International
Standard for Human Papillomavirus (HPV) Type 16 DNA, 107 copies/ )를희석하여 102, 103, 10및 105 copies/^를만든후 PCR로증폭하였다.
[152] 증폭된산물이제대로희석되었는지는，실시예 2의방법을통해관찰하였으며， 도 7의 A에결과를도시하였다.이들증폭된산물 4 ^와 Dynabead 4 ^를섞고 anti-mouse IgG agarose 1/50 20 ^를추가한겔-카드에넣고， 37에서 5분반웅시킨 후원심분리하였다.이때， 2분, 4분， 6분째각각내려가는정도를관찰하였다 (도 7 B).
[153] 그결과도 7에서보이는바와같이, 102내지 105 copies//^에서모두핵산의 양성및음성판정이가능함을확인하였다.즉， 102 copies/^의핵산만으로도 핵산검출이가능하였다.
[154] [실시예 6]
[155] HPV임상검체에서의핵산검출
[156] HPV임상검체에서도민감하고특이적으로핵산을검출할수있는지확인하기 위하여，자궁암선별검사를위해 HPV DNA검사가의뢰된검체로검사를 시행하고남은잔여검체를이용하여핵산검출을실시하였다. HPV DNA양성 및음성검체는로슈 Cobas 4800 HPV test를시행한검체에서각각 3개씩 선정하였다.
[157] 상기실시예에기재한방법과같이총 6개의검체를상기표 1에기재된
서열번호 1및 2로이루어지는프라이머세트를이용하여등온증폭을통해 HPV 검체로부터 HPV핵산을증폭하였다.서열번호 2로표시되는프라이머에는 5' 말단에 Cy3의형광물질이표지되었으며,표지방법은
NHS(N-hydroxysuccinimide)변형법으로표지하였다.
[158] 그후증폭된산물 4 ^와 Dynabead 4 를섞고 anti-mouse IgG agarose 1/5020
^를추가한겔-카드에넣고， 37에서 5분반웅시킨후원심분리하였다.이때,
2분, 4분및 6분째각각내려가는정도를관찰하였다.
[159] 그결과，도 8에나타난바와같이,음성에해당하는검체인 1내지 3번
류브에서는밴드가윗쪽에형성됨을확인하였으며，양성에해당하는검체인 4 내지 6번류브에서는밴드가아랫쪽에형성됨을확인할수있었다.이로서，임상 검체로도빠르고민감하게핵산을검출할수있음이확인되었다.
산업상이용가능성
[160] 본발명은분리단계를포함하지않는핵산검출법을통해특정질병에대한 음성，양성의판단을보다신속하고간단하며，민감하고신뢰도높게판별할수 있어산업상이용가능성이높다.

Claims

청구범위
[청구항 1] -검출하고자하는핵산에특이적으로결합하는프라이머세트;
포획나노입자 (capture nanoparticle);및
밀도차이를발생시키는성분과겔성분이순차적으로적층된겔카드; 를포함하는핵산검출용키트.
[청구항 2] 제 1항에있어서，상기프라이머세트는바이오틴 -표지정방향
프라이머 (biotin-labeled forward primer)와형광표지역방향 프라이머 (fluorescence-labeled reverse primer)인것을'특징으로하는핵산 검출용키트.
[청구항 3] 제 2항에있어서，상기형광표지프라이머는 NHS
변형법 (N-hydroxysuccinimide modification)또는
포스포라미티드법 (phosphoramidite syntheis)을통해형광표지된것을 특징으로하는핵산검출용키트.
[청구항 4] 제 2항에있어서，상기형광표지는 Cy3, Cy5, TAMRA, TEX, TYE, HEX,
FAM, TET, JOE, MAX, ROX, VIC, Cy3.5, Texas Red, Cy5.5, TYE, BHQ,
Iowa Black RQ및 IRDye로이루어지는군에서선택되는어느하나로 표지된것을특징으로하는핵산검출용키트.
[청구항 5] 제 3항에있어서, NHS변형법으로형광표지된프라이머를사용하여
핵산을검출하면,양성검체가음성검체보다아래에밴드를형성하는 것을특징으로하며;
포스포라미티드법으로형광표지된프라이머를사용하여핵산을 검출하면，양성검체가음성검체보다위에밴드를형성하는것을 특징으로하는핵산검출용키트.
[청구항 6] 제 1항에있어서，상기나노입자는자성입자 (magnetic bead),금 (Au)
나노입자，은 (Ag)나노입자，백금 (Pt)나노입자,양자점 (Quaantum dot), 상방전환나노입자 (upcon version nanoparticle, UCNP)
그래핀 (graphene)-나노입자복합체,색염색나노입자 (color dyed particles) 및라텍스 (latex)나노입자로이루어지는군에서선택되는어느하나인 것올특징으로하는핵산검출용키트.
[청구항 7] 제 1항에있어서,상기밀도차이를발생시키는성분은유리구슬 (glass bead), quartz based matrix,퍼콜 (percoll),콜로이드실리카용액 (colloidal silica media),피콜 (ficoll)로이루어지는군에서선택되는어느하나인것을 특징으로하는핵산검출용키트.
[청구항 8] 제 1항에있어서 ,상기겔성분은 IgG-아가로스 (agarose),
아가로스 (agarose),한천 (寒天)，샐를로오스아세테이트 (cellulose acetate) 및폴리아크릴아마이드 (polyacrylamide gel)로이루어지는군에서 선텍되는어느하나인것올특징으로하는핵산검출용키트. [청구항 9] 제 1항에있어서,인터킬레이팅제제의인핸서를추가적으로포함하는 핵산검출용키트.
[청구항 1이 제 9항에있어서,상기인터킬레이팅제제는 SYBR그린,브롬화
에티듐 (ethidium bromide), biotium gelred, biotium gelgreen, JOJO계열, POPO계열， SYTO계열， BOBO계열, TOTO계열,악티노마이신, 아드리아마이신,안트라센,벤조피렌，프로피디움
디이오다이드 -인터트위닝 (propidium diiodide-intertwining),디스타마이신, 네트롭신과아크리딘,프소랄렌,베르베린 (berberine),
프로풀라빈 (proflavine),다우노마이신，독소루비신,탈리도마이신,시아닌 염료및 LDS 751로이루어지는군에서선택되는어느하나이상인것을 특징으로하는핵산검출용키트.
[청구항 11] 바이오틴 -표지정방향프라이머와형광표지역방향프라이머로
구성되고，검출하고자하는핵산에특이적으로결합하는프라이머세트; 포획나노입자 (capture nanoparticle);
인터킬레이팅제제;및
밀도차이를발생시키는성분과겔성분이순차적으로적층된겔카드; 를포함하는인시츄 (in situ)핵산검출용키트.
[청구항 12] 핵산검출용어세이 (assay)에사용하기위한，목적핵산이포획되거나
또는결합된나노입자를포함하는검출가능한나노입자복합체.
[청구항 13] 제 12항의나노입자복합체를검출하기위한,유리구슬 (glass bead)성분과
겔성분이순차적으로적층된겔카드.
[청구항 14] Π)목적핵산에특이적으로결합하는프라이머세트로목적핵산을
증폭시키는단계;
( Iᅳ)상기 (π)단계에서증폭된핵산에나노입자를첨가하여핵산을 나노입자에포획또는결합시키는단계;
(ττ )상기 (L)단계에서나노입자에핵산이포획또는결합된복합체를 겔카드용기에넣는단계;
(Ξ)상기 ([)단계에서제조된혼합물을원심분리하는단계；및
(口)겔카드용기내침전물의위치를대조군과비교하는단계를포함하는 나노입자를이용한핵산검출방법.
[청구항 15] 제 W항에있어서，상기 (ᅳ1 )단계의프라이머세트는바이오틴 -표지
정방향프라이머 (biotin-labeled forward primer)와형광표지역방향
프라이머 (fluorescence-labeled reverse primer)인것을특징으로하는
나노입자를이용한핵산검출방법.
[청구항 16] 제 14항에있어서，상기 Π)단계는중합효소연쇄반웅 (PCR)또는
둥온증폭반웅으로핵산을증폭하는것을특징으로하는나노입자를 이용한핵산검출방법.
[청구항 17] 제 16항에있어서，상기등온증폭반웅은 HDA (Helicase-Dependent Amplification), RPA(Recombinase Polymerase Amplification), RCA(Rolling Circle Amplification), LAMP(Loop mediated isothermal amplification), NASBA(Nucleic Acid-Sequence-Based Amplification), TMA(Transcription Mediated Amplification), SMART(Signal Mediated Amplification of RNA Technology), SDA(Strand Displacement Amplification), IMDA(Isothermal Multiple Displacement Amplification), SPIA(Single Primer Isothermal Amplification)및 cHDA(circular Helicase-Dependent Amplification)로 이루어지는군에서선택되는어느하나의방법으로수행되는것을 특징으로하는핵산검출방법 .
[청구항 18] 제 14항에있어서，상기나노입자는자성입자 (magnetic bead),금 (Au)
나노입자，은 (Ag)나노입자，백금 (Pt)나노입자,양자점 (Quantum dot), 상방전환나노입자 (upconversion nanoparticle, UCNP),
그래핀 (graphene)-나노입자복합체，색염색나노입자 (color dyed particles) 및라텍스 (latex)나노입자로이루어지는군에서선택되는어느하나인 것을특징으로하는핵산검출방법.
[청구항 19] 제 18항에있어서,상기나노입자는표면에핵산을포획할수있는
아비딘 (avidin),아민 (amine),스트렙타비딘 (streptavidin),프라이머에 결합된항원에항원 -항체반웅을통해결합될수있는항체, 앱타머 (aptamer)및올리고뉴클레오타이드로이루어지는군에서 선택되는어느하나로코팅처리가된것올특징으로하는핵산검출방법 .
[청구항 20] 제 14항에있어서,상기겔성분은 IgG-아가로스 (agarose),
아가로스 (agarose),한천 (寒天),셀를로오스아세테이트 (cellulose acetate) 및폴리아크릴아마이드 (polyacrylamide gel)로이루어지는군에서 선택되는어느하나인것을특징으로하는핵산검출방법.
[청구항 21] 제 14항에있어서，상기 0= )단계에서인터킬레이팅제제 (intercalating
agent)의인핸서를추가적으로처리하는것을특징으로하는핵산검출 방법.
[청구항 22] 제 14항에있어서，상기 (e)단계전에별도의분리과정또는세척과정을 포함하지않는것을특징으로하는핵산검출방법ᅳ
[청구항 23] 제 14항내지제 22항중어느한항에있어서，상기 Π )단계에서,각기
다른색의형광으로표지된둘이상의프라이머세트를처리하여 , 다중으로핵산의존재유무를확인하는것을특징으로하는핵산검출 방법.
PCT/IB2016/001261 2015-07-13 2016-09-06 나노입자를 이용한 핵산 검출용 키트 및 핵산 검출 방법 Ceased WO2017009719A2 (ko)

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