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WO2018048205A1 - Méthode de diagnostic moléculaire basée sur l'amplification par cercle roulant - Google Patents

Méthode de diagnostic moléculaire basée sur l'amplification par cercle roulant Download PDF

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Publication number
WO2018048205A1
WO2018048205A1 PCT/KR2017/009783 KR2017009783W WO2018048205A1 WO 2018048205 A1 WO2018048205 A1 WO 2018048205A1 KR 2017009783 W KR2017009783 W KR 2017009783W WO 2018048205 A1 WO2018048205 A1 WO 2018048205A1
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Prior art keywords
target gene
template
beads
bead
probe
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Korean (ko)
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이혁진
최보람
김지수
정일영
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Ewha Womans University
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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
    • C12Q1/6846—Common amplification features
    • 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
    • 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
    • C12Q2521/00—Reaction characterised by the enzymatic activity
    • C12Q2521/10—Nucleotidyl transfering
    • C12Q2521/101—DNA polymerase
    • 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
    • C12Q2525/00—Reactions involving modified oligonucleotides, nucleic acids, or nucleotides
    • C12Q2525/10—Modifications characterised by
    • C12Q2525/204—Modifications characterised by specific length of the oligonucleotides
    • 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
    • C12Q2531/00—Reactions of nucleic acids characterised by
    • C12Q2531/10—Reactions of nucleic acids characterised by the purpose being amplify/increase the copy number of target nucleic acid
    • C12Q2531/125—Rolling circle
    • 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
    • C12Q2563/00—Nucleic acid detection characterized by the use of physical, structural and functional properties
    • C12Q2563/149—Particles, e.g. beads

Definitions

  • the present invention relates to a rotating circle amplification-based beads for detecting a target gene, a method for preparing the same, a composition for detecting a target gene using the same, a kit, and a method for detecting a target gene using the same.
  • the PCR process goes through three stages: denaturation, annealing, and elongation.
  • denaturation two strands of DNA are heated and separated. Each isolated DNA serves as a template.
  • primers bind to the template DNA.
  • the temperature in the binding step is an important factor in determining the accuracy of the reaction. If the temperature is too high, the primer binds too weakly to the template DNA, resulting in very little product amplified DNA. If the temperature is too low, unwanted primers can be amplified because the primers bind nonspecifically.
  • the kidney stage heat-resistant DNA polymerases make new DNA from template DNA.
  • substances such as DNA or RNA (hereinafter, referred to as 'target substances') that are subject to a PCR process are separated and purified by pretreatment of a sample containing a target substance.
  • pretreatment of a sample is performed by adsorbing a target material to the capture means by flowing the sample through a capture means, such as silica beads, and then rinsing the buffer buffer by the capture means to remove components other than the target material from the capture means, and then removing the elution buffer solution. This is achieved by separating the target substance flowing into the trapping means and adsorbed to the trapping means.
  • PCR-based system is difficult to use in an environment with limited resources, such as developing countries, because it requires a multi-step reaction and set and maintain a variety of temperature conditions as described above. That is, there are many limitations in PCR analysis because of the high cost and experimentation by well-trained analysts. Techniques such as ELISA can also be used to replace this, but have similar drawbacks to PCR for long analysis and limited antigen detection.
  • Rotating ring amplification is a single-stranded circular DNA as a template, and the polymerization is carried out isothermally using primers complementary thereto, so that the template DNA is continuously synthesized.
  • infinite rotation amplification and amplification on a first-order scale have the advantage of overcoming the disadvantages of PCR, which requires cumbersome thermal cycling and expensive equipment.
  • various new analytical methods performed by isothermal processes, including rotation amplification still require much research and development in terms of cost reduction and development into commercial devices that can be used by the public.
  • the present invention provides a DNA template comprising a binding site that complements a target gene, a binding site that complements a probe for binding to a bead, and a complementary binding site in a template to form a dumbbell form;
  • a probe complementarily hybridizing to the DNA template, wherein the marker bound to the binder of the bead is bound to the 5 ′ end;
  • It provides a bead for rotating circle amplification (Rolling Circle Amplification) based target gene detection comprising a bead comprising a binding agent that binds to the marker.
  • the present invention also provides a method for preparing beads for detecting the rolling circle amplification-based target gene.
  • the present invention also provides a target gene detection composition comprising the target gene detection beads, DNA polymerase, reaction buffer and dNTPs.
  • the present invention also provides a target gene detection kit comprising a rolling circle amplification-based target gene detection beads, a DNA polymerase, a reaction buffer solution and a target gene detection composition comprising dNTPs.
  • the present invention also provides a target gene detection method using the rotating circle amplification based target gene detection beads.
  • the present invention provides a rolling circle amplification-based target gene detection beads, a method for preparing the same, a composition, a kit comprising the same, and a detection method using the same.
  • a rolling circle amplification-based target gene detection beads a method for preparing the same, a composition, a kit comprising the same, and a detection method using the same.
  • the term “template” refers to a binding site (D) that complementarily binds to a target gene at one circular site in a dumbbell form, and a binding site (P) that complementarily binds to a probe for binding to beads at the opposite circular site. It refers to a nucleotide having a dumbbell shape having a binding site (S) in the template for forming a center dumbbell shape.
  • target gene refers to any nucleotide sequence that encodes a known or putative gene product.
  • the target gene may be a gene derived from an animal, a plant, a bacterium, a virus, a fungus, or the like, or a mutated gene accompanying a genetic disease.
  • the target gene may be DNA or RNA, for example, a nucleic acid sequence or molecule may be single or double stranded and may represent a sense or antisense strand.
  • the nucleic acid sequence is dsDNA, A-, B-, or Z-DNA, triple-stranded DNA, RNA, ssRNA made of dsDNA, ssDNA, mixed ssDNA, mixed dsDNA, ssDNA (eg, by fusion, denaturation, helicase, etc.) , dsRNAs, mixed ssRNAs and dsRNAs, dsRNAs made from ssRNAs (eg, via fusion, denaturation, helicase, etc.), messenger RNAs (mRNAs), ribosomal RNAs (rRNAs), transfer RNAs (tRNAs), catalytic RNAs, snRNAs, micros RNA, or protein nucleic acid (PNA).
  • mRNAs messenger RNAs
  • rRNAs ribosomal RNAs
  • tRNAs transfer RNAs
  • catalytic RNAs snRNAs
  • micros RNA micros RNA, or
  • combination site that binds complementarily or “complementary binding site” refers to a site capable of forming complementary base pairs between nucleotide sequences.
  • the term “bead” refers to a material having a spherical substrate and includes nanoparticles, microparticles, nanobeads, or microbeads.
  • the beads may have a diameter of 2 ⁇ m to 7 ⁇ m, specifically the diameter of the particles or beads is 4 ⁇ m to 5 ⁇ m.
  • Beads include resin beads, magnetic beads, metal fine particles and the like.
  • probe refers to a nucleic acid fragment such as RNA or DNA, which corresponds to a few bases to several tens of bases, which can achieve specific binding with a nucleotide sequence. All probes, which may be bound to a marker, and are capable of binding to binding sites that complementarily bind to probes for binding to beads in a template, are all within the scope of the present invention.
  • labeler is not limited to any of the various materials known in the art without limitation as long as it can be used to pair with a label for binding to a gene label, preferably a bead, without disturbing the hybridization of the probe with the gene product. Can be used.
  • binding agent is a substance that recognizes and binds a marker without interfering with the hybridization of the gene product.
  • binding agent is not limited as long as it is easy to bind to beads and has high binding ability with the marker, and various materials known in the art. Can be used.
  • conversion amplification refers to a single-stranded circular DNA as a template, and to perform the polymerization at isothermal temperature using a complementary probe, so that the template DNA is continuously synthesized.
  • sample includes, but is not limited to, samples such as tissues, cells, blood, serum, or plasma.
  • the term “detection”, “detection” or “diagnosis” refers to the identification of the presence or characteristic of the target gene, and thus the pathological state.
  • the present invention provides a DNA template comprising a binding site that complements a target gene, a binding site that complements a probe for binding to a bead, and a complementary binding site in a template to form a dumbbell form;
  • a probe complementarily hybridizing to the DNA template, wherein the marker bound to the binder of the bead is bound to the 5 ′ end;
  • It provides a bead for rotating circle amplification (Rolling Circle Amplification) based target gene detection comprising a bead comprising a binding agent that binds to the marker.
  • the rotating circle amplification-based target gene detection beads according to the present invention have a characteristic of not drying by capturing water molecules through hydrophilic properties by forming a DNA hydrogel while rotating ring amplification proceeds in the presence of a target gene. .
  • the target gene can be quickly identified by visually confirming the volume change, confirming the aggregation of DNA strands, confirming the color change by staining or fluorescence, confirming the viscosity change, confirming the change in the degree of aggregation of the beads, and confirming the change in the sedimentation degree of the beads. Detection is possible.
  • Rolling circle amplification based target gene detection beads are prepared by immobilization on beads after hybridization of the template and probe.
  • the rolling circle amplification-based target gene detection beads form a hydrogel by entanglement while forming long DNA strands while cyclic amplification is performed simultaneously with ligation in the presence of a target gene.
  • the template may have various lengths and lengths in consideration of the length of a target gene such as a gene derived from an animal, a plant, a virus, a bacterium, a pathogen, a length of a mutated gene accompanying a genetic disease, a length of a primer, and the like. Sequences can be designed.
  • the template may have a length of 40 to 160 mer. If the length of the template is too short, the template itself may become unstable and if too long, the RCA efficiency may decrease.
  • the template according to the present invention comprises a binding site (D) that binds complementarily with the target gene.
  • a binding site that complementarily binds to the target gene is formed separately at both ends of the template.
  • the template may comprise a binding site of 5 to 50 mer, preferably 15 to 25 mer in length.
  • the target gene to be detected of the present invention is present in or derived from animals, plants, bacteria, viruses, fungi, etc., preferably present in genetic material including mRNA, dsDNA, and cDNA, mutated genes involved in genetic diseases Or derived therefrom.
  • the target gene to be detected may be, for example, a pathogen gene.
  • the target gene can be targeted to any pathogen that knows the nucleic acid sequence.
  • the pathogen is avian influenza, Dermalogica virus, E. coli and SARS (SARS), hemolytic uremic syndrome (hemolytic uremic syndrome) and hematochezia (bloddy diarrhea) (Escherichia coli O157 : H7), tuberculosis (Mycobacterium tuberculosis), anthrax (Bacillus anthracis ), Pneumonia ( Streptococcus pneumonia ), malaria (Plasmodium), Salmonella, Hepatitis (Hepatitis A, B, C, D and E virus), Yattobacteria ( Francisella) tularensis ), Yersinia pestis ), Yersinia enterocolitica , Ebola virus, MERS-Cov virus, pneumococcal, enterococci, staphylococcus
  • the template according to the invention comprises a binding site (P) which complementarily binds to a probe for binding to the beads.
  • a binding site that complementarily binds to the probe for binding to the gastric beads is formed between complementary binding sites in the template to form a dumbbell shape.
  • the template may comprise a binding site that complementarily binds to a probe for binding to beads of 5 to 50 mer, preferably 10 to 25 mer in length.
  • the binding site that complementarily binds to the probe for binding to the beads may be used as long as the marker that binds to the binding agent of the bead can complementarily bind to the probe bound to the 5 'end without interfering with the detection of the target gene. It is possible.
  • the template according to the invention comprises complementary binding sites (S) in the template for forming a dumbbell shape.
  • Complementary binding sites in the template for forming the stomach dumbbell form the target gene at the opposite end from the binding site that complements the probe for binding to the beads Dumbbell forms through complementary binding in the sequence to include a binding site that binds complementarily with.
  • the template has a total length of 40 to 160 mer
  • the complementary binding site S in the template for forming the dumbbell shape has a total of 10 to 80 mer length, and preferably 15 for forming the dumbbell shape.
  • Complementary binding sites (self-assembled binding sites) of about 40 to 40 mer in length.
  • Table 1 below shows the sequence of the DNA template comprising a binding site that complements the target gene, a binding site that complements the probe for binding to the beads and a complementary binding site in the template to form a dumbbell form Exemplary (bold: binding site that complements the target gene complementarily, light gray background: complementary binding site in the template to form a dumbbell form, dark gray background: complementary to the probe for binding to the beads Binding site).
  • the rolling circle amplification-based target gene detection beads according to the present invention include a probe complementarily hybridizing to the DNA template in which the marker bound to the binder of the beads is bound to the 5 ′ end.
  • the probe sequence may be used as long as the sequence binds to a binding site that complements the probe of the template and does not interfere with the detection of the target gene.
  • the sequence of SEQ ID NO: 25 is used.
  • the marker and the binder of the beads may be a pair of different and complementary molecules, for example biotin, preferably as a marker, and streptavidin, avidin, neutravidin as binders, preferably binding agents to the bead surface.
  • biotin preferably as a marker
  • streptavidin avidin
  • neutravidin as binders
  • binding agents to the bead surface preferably binding agents to the bead surface.
  • Captavidin, polystreptavidin or any other protein capable of binding to biotin can be used.
  • Rolling circle amplification based target gene detection beads includes a bead comprising a binding agent to the marker.
  • the beads may assist in entanglement of the DNA strands as the rotation amplification proceeds depending on the presence of the target gene. Accordingly, by forming the DNA hydrogel, it may be helpful to check the volume change of the naked eye, the aggregation or the fluorescence change according to the RCA reaction.
  • the beads hybridize to the template via a probe in which a binder that binds to the marker is bound to the surface of the bead so that the marker that binds to the binder of the beads complementarily hybridizes to the DNA template bound to the 5 ′ end.
  • the beads are spherical substrates and include nanoparticles, microparticles, nanobeads or microbeads, preferably magnetic beads.
  • the present invention also provides a method for preparing beads for detecting the rolling circle amplification-based target gene.
  • a DNA template comprising a binding site that complements a target gene, a binding site that complements a probe for binding to a bead, and a complementary binding site in a template to form a dumbbell form Hybridizing a marker bound to the binder of the beads with a probe complementarily hybridizing to the DNA template bound to the 5 'end;
  • step (b) providing a method for producing beads for rotating circle amplification-based target gene detection comprising immobilizing a template hybridized with a probe to a bead comprising a binder that binds to a marker in step (a). do.
  • composition for detecting a target gene comprising the rolling circle amplification-based target gene detection beads, DNA polymerase, reaction buffer and deoxynucleotides (dNTPs).
  • the DNA polymerase according to the present invention is selected from the group consisting of E. coli DNA polymerase I, Klenow fragment, phi29 DNA polymerase, vent DNA polymerase, T4, T7 DNA polymerase, Taq polymerase and Splint R polymerase. It may be.
  • the buffer (buffer) is NEB (New England Biolab) buffer series, such as NEB buffer 4, Bst DNA polypolymerase buffer, T4 DNA ligase buffer, T7 DNA ligase buffer Solutions, Splint R ligase buffer, and the like can be used, but is not limited thereto.
  • NEB New England Biolab
  • composition for detecting a target gene may further include a dyeing reagent and a fluorescent reagent to help visually identify the amplified gene product.
  • a dyeing reagent for example, gel-red and trypane blue die may be used.
  • dyes Evans Blue dyes, hematoxylin-eosin stains, crystal violet or methylene blue, but are not limited thereto.
  • the present invention also provides a kit for detecting a target gene comprising a composition for detecting a target gene comprising the rolling circle amplification-based target gene detection beads, a DNA polymerase, a reaction buffer, and dNTPs.
  • the target gene detection kit has a characteristic that the beads do not dry by capturing water molecules through hydrophilic properties by forming a DNA hydrogel while the ring amplification proceeds in the presence of the target gene.
  • the target gene can be rapidly identified by visually confirming the volume change, confirming the aggregation of DNA strands, confirming the color change by staining or fluorescence, confirming the viscosity change, confirming the change of the degree of aggregation of the beads, and confirming the change of the sedimentation degree of the beads. Detection is possible.
  • the kit for detecting a target gene according to the present invention may be a kit including essential elements necessary for performing rotation ring amplification.
  • the target gene detection kit according to the present invention may be used in a capillary tube by packing the rotating circle amplification-based target gene detection beads into a capillary tube using a capillary phenomenon. More specifically, the manufactured detection beads may be packed in a capillary tube using a capillary phenomenon, and the detection bead may be performed through a rotation ring amplification reaction in the capillary tube after blocking the lower portion of the capillary tube so that the detection beads do not escape from the capillary tube.
  • kits may further comprise staining reagents or fluorescent reagents to more reliably confirm the presence or absence of a target gene.
  • the present invention also provides a target gene detection method using the rotating circle amplification based target gene detection beads.
  • a DNA template comprising a binding site that complements a target gene, a binding site that complements a probe for binding a bead, and a complementary binding site in a template to form a dumbbell form;
  • a probe complementarily hybridizing to the DNA template, wherein the marker bound to the binder of the bead is bound to the 5 ′ end;
  • target beads including a rotating circle amplification-based target gene detection beads, DNA polymerase, reaction buffer, and deoxynucleotides (dNTPs). Reacting the sample with the composition;
  • dNTPs deoxynucleotides
  • It provides a target gene detection method comprising; (b) confirming the gene amplification by performing a rotation ring amplification in response to the sample.
  • step (a) may be carried out in a capillary tube, and accordingly, in step (b), visually confirm the volume change, confirm the aggregation of DNA strands, confirm the color change by staining or fluorescence, confirm the viscosity change, The presence or absence of the target gene can be confirmed by confirming the change in the degree of aggregation of the beads and the change in the degree of sedimentation of the beads.
  • the target gene detection method can detect a target gene quickly and easily through a reaction at a constant temperature even without a device for temperature change.
  • the rolling circle amplification-based target gene detection beads of the present invention have an advantage of detecting genes such as viruses, bacterial infections, and cancer at a low price by detecting genes quickly and easily.
  • Figure 1 shows a schematic diagram of the process of forming a long DNA strand in the beads in the presence of the target circle for rotating circle amplification (Rolling Circle Amplification) target gene detection.
  • Figure 2 shows a schematic diagram of a dumbbell-shaped template according to the present invention.
  • Figure 3 shows a schematic diagram of target gene detection using a capillary tube containing beads for rotating circle amplification-based target gene detection.
  • Figure 4 shows the overall schematic diagram of the template for the analysis of the target gene cyclic amplification-based molecular diagnostic method according to the present invention, the preparation of beads, the preparation of a solution for the cyclic amplification reaction, packing and diagnostic methods.
  • 5 shows a schematic of hybridization between biotinylated probes and templates.
  • Figure 6 E.Coli having ampicillin resistance according to an embodiment of the present invention The hybridization of the template and biotinylated probe that can bind to the gene is shown through electrophoresis.
  • FIG. 7 shows a schematic diagram of a process for preparing beads for detecting target genes based on rolling circle amplification through immobilization of biotinylated probes and hybridized templates on the bead surface.
  • Figure 8 shows the electrophoresis results showing that the template and biotinylated probes bindable to E.Coli pathogens having ampicillin resistance in accordance with an embodiment of the present invention.
  • FIG. 10 is a diagram showing E. ampicillin resistance using a rotating circle amplification-based target gene detection beads comprising a template capable of binding to the E.Coli gene having ampicillin resistance according to an embodiment of the present invention. The result of detecting Coli is shown.
  • FIG. 11 shows a result of detecting Zika virus using a rotating circle amplification-based target gene detection beads including a template capable of binding to Zika virus according to an embodiment of the present invention.
  • FIG. 12 shows a schematic diagram of packing a rotating circle amplification-based target gene detection bead into a capillary tube through a capillary phenomenon and visually observing it.
  • FIG. 13 E.Coli having ampicillin resistance according to an embodiment of the present invention The result of visually confirming the presence of E.Coli having ampicillin resistance by using a capillary packed with a rolling circle amplification-based target gene detection bead containing a template capable of binding to a gene is shown.
  • FIG. 14 shows a long tandem RCA product formed by rotating ring amplification in capillaries packed with target gene detection beads, and the resulting DNA products are entangled with each other by physical entanglement. Accordingly, when the capillary is inverted, the degree of dropping of the amplification mixture including the beads is different due to gravity, and when the amplification occurs, the amplification mixture does not flow due to entanglement.
  • FIG. 15 illustrates a gene using a capillary packed with a bead-packed bead-based target gene detection capillary including a template capable of binding to an E. Coli gene having ampicillin resistance according to an embodiment of the present invention. Indicates the presence or absence of the result by fluorescence.
  • Template_Zika virus is a template for forming a binding site that complements a target gene (the pathogen gene of Zika virus), that is, a complementary site of a pathogen (Pathogen complementary site (SEQ ID NOs: 9, 20 mer, bold), and a dumbbell shape).
  • Template_Ampicillin resistant E.coli ie, template that specifically binds to E.Coli with ampicillin resistance, based on the E.Coli gene sequence with Ampicillin resistance (Template_Ampicillin resistant E. coli, SEQ ID NO: 2) was prepared.
  • Template_Ampicillin resistant E. coli is a binding site that complementarily binds to a target gene (the pathogen gene of E.
  • coli ie, the pathogen complementary site (SEQ ID NO: 13, 23 mer, bold), Complementary binding sites in the template to form a dumbbell shape, ie, complementary regions in the template (SEQ ID NO: 14 and SEQ ID NO: 15, total 47 mer, light gray background), which complementarily bind to the probe for binding to the beads Consisting of binding sites (SEQ ID NOs: 16, 17 mer, dark gray background).
  • FIG. 5 A schematic diagram of the preparation of a template hybridized with a marker bound (eg biotin) probe is shown in FIG. 5.
  • a marker bound probe eg biotin
  • Example 2 Bead On the surface Biotinylated With probe Hybridized Template Through immobilization A rotary ring Preparation of Beads for Detection of Targeting Circle Amplification
  • the template hybridized with the biotinylated probe prepared in Example 1 was fixed to the magnetic beads, thereby immobilizing the template on the magnetic beads.
  • a schematic diagram of the above immobilization step is shown in FIG. 7.
  • the binder (eg streptavidin) beads were discarded by centrifugation after Vortexing, and the supernatant was discarded, and the mixed template with the probe prepared in Example 1 was kept at room temperature for about 15 minutes. Immobilize the template on the beads.
  • the target gene was detected using the rotating ring amplification-based target gene detection beads prepared in Example 2.
  • a schematic diagram of the above detection step is shown in FIG. 9. Complementary binding of the target gene detection beads to the spin ring amplification of the target gene results in the formation of a closed form dumbbell-shaped template by the enzyme T4 ligase. Long strands of DNA strands are formed.
  • ligation of 10 uM Zika virus RNA pathogen (SEQ ID NO: 5, Bioneer) was carried out simultaneously using ligation and rolling circle amplification (RCA) reaction using a beta-based target gene detection beads.
  • FIG. 10 shows the detection results for ampicillin resistant E. coli. As shown in FIG. 10, it was confirmed that the target gene was detected by the reaction of the gel well by the long DNA strand formation and entanglement through amplification (SP), and the product produced over time was confirmed to be large. . In addition, it was confirmed that the amplification was not performed even if the target gene exists in the bead (NC) to which the non-target template was fixed, and the target gene was detected using the rotating ring amplification-based target gene detection beads of the present invention. It became.
  • Example 3 a mixture of the mixed solution in the tube and the rotating ring amplification-based target gene detection beads (for detecting ampicillin-resistant E. coli) was packed onto a capillary tube using capillary action and lysed at 25 ° C for 15 minutes. The gate and the RCA (rolling circle amplification) reaction were performed at the same time, and then the capillary tube was turned upside down. Then, the detection of the target gene was visually confirmed by aggregation of the magnetic beads in the control group and the sample group. For reference, a schematic diagram of the above process is shown in FIG. 12.
  • Figure 13 shows the detection results for ampicillin resistant E. coli.
  • hydrophilic long-stranded DNA strands formed by spin ring amplification do not trap water molecules and are dried and maintained in volume, while long-stranded DNA strands are not formed if spin ring amplification does not proceed. It was found that it did not form and was easily dried to reduce volume (NC). That is, the target gene was detected by visually confirming the volume change.
  • the target gene was detected by confirming aggregation and fluorescence changes through entanglement of DNA strands as in FIGS. 14 and 15.
  • FIG. 14 shows a long tandem RCA product formed by rotating ring amplification in capillaries packed with target gene detection beads, and the resulting DNA products are entangled with each other by physical entanglement. Accordingly, when the inverted capillary is inverted, the degree of dropping of the amplification mixture including the beads is changed by gravity, and when the amplification occurs, the amplification mixture does not flow due to entanglement.
  • FIG. 15 shows the presence of the gene in the capillary packed with beads for target gene detection based on rolling circle amplification comprising a template capable of binding to the E.Coli gene having ampicillin resistance according to an embodiment of the present invention. It is the result of confirming the presence or absence by fluorescence. As a result of the rotational amplification in the presence of the pathogen gene, a long strand of DNA was produced, and its presence was confirmed by fluorescence using SYBR dye (eg, SYBR gold) (SP-Sample, NC-Negative control). )).
  • SYBR dye eg, SYBR gold

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  • Biochemistry (AREA)
  • Physics & Mathematics (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Analytical Chemistry (AREA)
  • Immunology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Chemical Kinetics & Catalysis (AREA)
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Abstract

La présente invention concerne une bille pour la détection de gène cible sur la base d'une amplification par cercle roulant, son procédé de production, une composition et un kit de détection de gène cible l'utilisant, et un procédé de détection de gène cible l'utilisant. La présente invention permet une détection de gène rapide et simple, permettant ainsi un diagnostic d'une maladie, rapide et à coût abordable, telle qu'une infection virale ou bactérienne ou un cancer.
PCT/KR2017/009783 2016-09-06 2017-09-06 Méthode de diagnostic moléculaire basée sur l'amplification par cercle roulant Ceased WO2018048205A1 (fr)

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KR1020160114621A KR102366854B1 (ko) 2016-09-06 2016-09-06 회전환 증폭 기반 분자 진단 방법
KR10-2016-0114621 2016-09-06

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KR102572521B1 (ko) * 2021-02-18 2023-08-30 한국과학기술원 등온증폭 및 커피링 현상에 기반한 표적 핵산의 검출용 조성물 및 검출방법
KR20230082063A (ko) 2021-12-01 2023-06-08 김채원 손가락에 끼워 잡는 강아지 자동 리드줄

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* Cited by examiner, † Cited by third party
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CN111139289A (zh) * 2019-12-25 2020-05-12 中国海洋大学 基于哑铃环模板的恒温滚环扩增方法

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