WO2021141369A1 - Procédé de détection d'arn à base de sonde d'adn simple brin - Google Patents

Procédé de détection d'arn à base de sonde d'adn simple brin Download PDF

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WO2021141369A1
WO2021141369A1 PCT/KR2021/000103 KR2021000103W WO2021141369A1 WO 2021141369 A1 WO2021141369 A1 WO 2021141369A1 KR 2021000103 W KR2021000103 W KR 2021000103W WO 2021141369 A1 WO2021141369 A1 WO 2021141369A1
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dna
rna
stranded dna
present
composition
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PCT/KR2021/000103
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Korean (ko)
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조규봉
임광일
김시원
장윤하
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서강대학교산학협력단
숙명여자대학교산학협력단
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Publication of WO2021141369A1 publication Critical patent/WO2021141369A1/fr

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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
    • C12Q1/6813—Hybridisation assays
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B01L3/50—Containers for the purpose of retaining a material to be analysed, e.g. test tubes
    • B01L3/502—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures
    • B01L3/5027—Containers for the purpose of retaining a material to be analysed, e.g. test tubes with fluid transport, e.g. in multi-compartment structures by integrated microfluidic structures, i.e. dimensions of channels and chambers are such that surface tension forces are important, e.g. lab-on-a-chip
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L2300/00—Additional constructional details
    • B01L2300/08—Geometry, shape and general structure
    • B01L2300/0861—Configuration of multiple channels and/or chambers in a single devices
    • 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
    • C12Q2563/185—Nucleic acid dedicated to use as a hidden marker/bar code, e.g. inclusion of nucleic acids to mark art objects or animals
    • 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
    • C12Q2565/00—Nucleic acid analysis characterised by mode or means of detection
    • C12Q2565/60—Detection means characterised by use of a special device
    • C12Q2565/629—Detection means characterised by use of a special device being a microfluidic device

Definitions

  • the present invention relates to a RNA detection method using a single DNA molecule visualization method.
  • Viruses are contagious pathogens that are smaller than bacteria, and are composed of DNA or RNA, which is genetic material, and proteins surrounding the genetic material. Viruses can be divided into plant viruses, animal viruses, and bacterial viruses (phages) according to the type of host. In most cases, they are divided into DNA virus subfamily and RNA virus subfamily depending on the type of nucleic acid, which are further subdivided into classes, orders, and families. .
  • influenza virus H1N1
  • SARS-CoV respiratory syndrome coronavirus
  • viruses cause infectious diseases.
  • virus detection, early diagnosis of infection, and isolation of infected patients are essential in order to slow down the frequency and spread of infection when a viral infection that causes serious disease occurs.
  • a respiratory infection if it is possible to isolate and treat the patient by quickly determining the presence or absence of the causative virus in the field, certainty of infection can be prevented.
  • Virus detection methods include immunological detection methods such as enzyme-linked immunosorbent assay (ELISA), enzyme immunoassay (EIA) and immunofluorescence assay (IFA), and viral nucleic acid (DNA, RNA) detection method by RT-PCR.
  • ELISA enzyme-linked immunosorbent assay
  • EIA enzyme immunoassay
  • IFA immunofluorescence assay
  • DNA, RNA detection method by RT-PCR.
  • DNA smaller than 1 kb can be analyzed using electrophoresis after amplifying the DNA by generally polymerase chain reaction (PCR).
  • PCR polymerase chain reaction
  • problems such as that it takes a lot of time because a temperature-controlled amplification cycle is required, and that the time consumed for confirming the result through gel electrophoresis is large.
  • the present inventors have tried to develop a method capable of early detection of RNA at the single molecule level.
  • a labeled dsDNA in which the target RNA to be detected was hybridized was prepared, and when visualized using a microchannel including a channel having a slope on one side, it was found that RNA can be detected at the level of a single DNA molecule within a short time.
  • the present invention was completed.
  • Another object of the present invention is to provide a composition for virus detection prepared by a method for preparing a composition for detecting RNA containing labeled DNA.
  • Another object of the present invention is to provide a kit for detecting RNA including labeled DNA.
  • Another object of the present invention is to provide a method for detecting RNA comprising injecting a composition for detecting RNA including labeled DNA into a microchannel.
  • the present inventors prepared labeled dsDNA hybridized with RNA to be detected in order to detect RNA early at the single-molecule level, and when visualized using a microchannel including a channel having a slope on one side, single DNA within a short time It was confirmed that RNA could be detected at the molecular level.
  • the present invention relates to a method for preparing a composition for detecting RNA comprising a marker DNA, a composition for detecting RNA prepared thereby, a kit for detecting RNA comprising the same, and a method for detecting RNA using the same.
  • One aspect of the present invention relates to a method for preparing a composition for detecting RNA comprising a marker DNA, comprising the following steps:
  • a circular dsDNA production step of preparing circular double-stranded DNA (dsDNA) from circular single-stranded DNA using a primer complementary to the RNA sequence to be detected;
  • the detection target RNA may include, but is not limited to, viral RNA, miRNA expressed in cells, and various cancer-specific mRNAs.
  • the virus may be an RNA virus, for example, human immunodeficiency virus (HIV) or influenza A virus, but is not limited thereto.
  • HIV human immunodeficiency virus
  • influenza A virus influenza A virus
  • the complementary primer may be a nucleic acid, for example, may be an RNA, but is not limited thereto.
  • the complementary primer may be derived from the RNA virus genome, but is not limited thereto.
  • the step of preparing the probe DNA may include the following steps:
  • An extraction step of extracting circular single-stranded DNA from the culture product is
  • the step of preparing the probe DNA may further include a purification step of purifying the circular single-stranded DNA in order to remove the E. coli genome, the auxiliary phage genome, and other DNA present in the extraction step.
  • probe DNA refers to a DNA to which a target gene sequence to be visualized is hybridized to prepare a marker DNA for single molecule visualization, and an oligonucleotide, cDNA, or circular single-stranded DNA (ssDNA) according to the present invention. may include.
  • visualization target gene refers to DNA or RNA extracted from a target virus to be detected or a target cell to determine whether expression, and a significant portion of the visualization target gene has a sequence complementary to the probe DNA. By their binding, hybridization to a marker DNA is possible through a reverse transcription reaction.
  • phagemid refers to a plasmid having an f1 origin of replication, and the DNA of the phagemid enters the virus particle in a single-stranded circular state and is specific through site-directed mutagenesis. Restriction enzyme sites can be added and foreign genes can be introduced using them.
  • f1 origin of replication means a replication origin derived from f1 phage.
  • auxiliary phage means a phage that does not have infectivity or self-renewal ability alone and helps the proliferation of incomplete phage (phage with a defect), and one of the two phage of the same species is a normal phage, but the other In the case where the phage lacks some of the functions of the phage, the latter can grow using the product of the former if both are mixed and infected in the same cell.
  • the auxiliary phage preferentially amplifies the recombinant phage in the form of single-stranded DNA, so that the progeny phage extracted from E. coli may have the phagemid as a genome. Therefore, circular single-stranded DNA (probe DNA) can be obtained by extracting the genome from these phages in the culture product.
  • the primer in the step of preparing the circularly labeled double-stranded DNA may be generated through fragmentation of the target RNA to be detected, for example, fragmentation of the target virus genome and/or cell-derived RNA.
  • the probe DNA is designed to include a sequence complementary to the primer(s).
  • hybridization from circular single-stranded DNA to circular double-stranded DNA is possible through a reaction using reverse transcriptase using the primer.
  • the present invention is not limited in the detection target RNA, for example, any part on the genome of the virus can be the detection target, and similarly, the sequences for detecting miRNA and mRNA derived from cells are all or part of the RNA sequence. Since it can be determined as a part, the case of probe DNA that can be manufactured is also infinite.
  • the circularly labeled dsDNA may have a G/AATTC sequence
  • the circularly labeled dsDNA can be cut using the restriction enzyme EcoRI to prepare a linear double-stranded labeled DNA.
  • Another aspect of the present invention relates to a composition for detecting RNA comprising a marker DNA.
  • composition for detecting RNA including the labeled DNA may be prepared by the above method.
  • the composition for detecting RNA containing the labeled DNA is a primer, dNTPs and rNTPs, a labeling agent, a reverse transcriptase, a DNA polymerase, a buffer, a chemifluorescent and / or a chemiiluminescent material, etc. may be included, but is not limited thereto.
  • Another aspect of the present invention relates to a kit for detecting a virus comprising the composition for detecting RNA.
  • the kit can detect viral RNA, miRNA expressed in cells, and various cancer-specific mRNAs, but is not limited thereto.
  • the kit may include a probe DNA designed to target a gene specific to a specific virus species so as to reliably detect the virus.
  • the virus may be an RNA virus, for example, human immunodeficiency virus (HIV) or influenza A virus, but is not limited thereto.
  • HIV human immunodeficiency virus
  • influenza A virus influenza A virus
  • the kit may include essential elements necessary for performing analysis at the level of a single molecule, but is not limited thereto.
  • the kit of the present invention enables detection of the detection target RNA through a microscope or the like directly from the sample without the need to separately perform PCR analysis or the like.
  • Another aspect of the present invention relates to a method for detecting RNA using the composition for detecting RNA.
  • the RNA detection method may include a contact step of contacting a sample with a composition for detecting RNA including labeled DNA.
  • RNA may include, but is not limited to, viral RNA, miRNA expressed in cells, and various cancer-specific mRNAs.
  • the virus may be an RNA virus, for example, human immunodeficiency virus (HIV) or influenza A virus, but is not limited thereto.
  • HIV human immunodeficiency virus
  • influenza A virus influenza A virus
  • the RNA detection method may include injecting the RNA detection composition into a microchannel.
  • the microchannel includes: a sample attachment unit composed of one or more channels; an inlet connected to one side of the sample attachment part; an injection unit connected to the inlet unit for injecting a sample; and an outlet connected to the other side of the sample attachment, wherein the channel has an inclined surface from an upper connection point on the inlet side toward a lower connection point, a lower surface in the channel is positively charged, and the one or more channels include It may be characterized in that the sample is recovered by communicating with each other at the outlet.
  • FIG. 5 is a schematic plan view of a microchannel including one sample attachment part according to an embodiment of the present invention.
  • the microchannel 1 may be configured to include a sample attachment part 10 , an inlet part 20 , an injection part 30 , and an outlet part 40 .
  • the sample attachment part 10 may be configured to include at least one or more channels 11 .
  • each channel when a channel consists of at least two or more channels, each channel may be configured to be connected in parallel.
  • the channel 11 is a passage for the sample to pass through, and may be configured to have an inclined surface from the upper connection point 12 on the inlet 20 side toward the lower connection point 13 .
  • the height of the upper connection point in the present invention is 1 to 50 um, 1 to 40 um, 1 to 30 um, 10 to 50 um, 10 to 40 um, 10 to 30 um, 20 to 50 um, 20 to 40 um, 20 to 30 um, 25 to 50 um, 25 to 40 um, 25 to 30 um, for example, 27 um.
  • the above range there is an effect that the molecules are more evenly distributed in the sample attachment portion.
  • the lower surface in the channel may be configured to be positively charged.
  • the negatively charged DNA and/or RNA can be detected by attaching to the lower surface of the channel.
  • the material of the channel may include any material used for manufacturing the channel in the art.
  • the inlet part 20 includes an injection part for injecting a sample into the channel, and may be configured to be connected to one side of the sample attachment part 10 .
  • the injection unit 30 is configured to inject a sample, and may be configured to be connected to the inlet unit 20 .
  • the outlet part 40 is an outlet for collecting the sample, and may be configured to be connected to the other side of the sample attachment part.
  • each outlet connected to each channel communicates with each other to collect a sample.
  • the sample may include any sample as long as a single molecule analysis is required in the art.
  • the sample may include the labeled DNA, but is not limited thereto.
  • RNA can be detected by photographing the attached labeled DNA using a microscope.
  • RNA detection method of the present invention overlapping contents related to the composition for detecting RNA are omitted in consideration of the complexity of the present specification.
  • the present invention relates to a virus and cellular RNA detection method using a single DNA molecule visualization method.
  • the method of the present invention has an advantage in that viral and cellular RNA can be detected at the level of a single DNA molecule within a short time by visualizing labeled dsDNA hybridized with a target virus or cellular RNA fragment.
  • ssDNA circular single-stranded DNA
  • dsDNA linear double-stranded
  • 3 is a result of confirming dsDNA synthesized using M13mp18 ssDNA according to an embodiment of the present invention.
  • FIG. 5 is a schematic plan view of a microchannel including one channel according to an embodiment of the present invention.
  • FIG. 6 is a schematic diagram of a microchannel according to an embodiment of the present invention.
  • SEM 7 is a scanning electron microscope (SEM) photograph of an inclination of an inlet portion of a channel according to an embodiment of the present invention.
  • FIG. 9 is a diagram showing the amount of dsDNA synthesized according to an embodiment of the present invention according to the concentration to be visualized.
  • FIG. 10 is a graph showing the number of dsDNA molecules measured using a microchannel according to an embodiment of the present invention with respect to the target DNA concentration.
  • ⁇ phage DNA (48.5 kbp), M13mp18 single-stranded DNA (7.2 kb), and enzymes were purchased from New England Biolabs (Ipswich, MA).
  • DNA primer was purchased from Cosmogenetech (Seoul, Korea), and Top polymerase was purchased from Bioneer (Daejeon, Korea).
  • N-trimethoxymethyl silyl propyl-N,N,N-trimethylammonium chloride in 50% methanol was purchased from Gelest (Morrisville, PA), and other reagents were purchased from Sigma-Aldrich (St. Louis, MO).
  • the photosensitive resins SU-8 2015 and SU-8 2005 were purchased from MicroChem.
  • Polydimethyl-siloxane (Sylgard 184, PDMS) was purchased from K1 Solution (Seoul, Korea).
  • Measurements were made with an inverted optical microscope (Olympus IX70, Japan).
  • a 100 X objective lens (Olympus UPlanSApo oil immersion objective) and LED (SOLA SM IIlight engine, Lumencor, Beaverton, OR) were used, and the excitation and emission light was a fluorescent filter set (Semrock, Rochester, NY) passed through Fluorescence images were obtained using a Prime sCMOS camera (Photometrics, Arlington, AZ) and stored in 16-bit TIFF format using Micro-manager software.
  • ImageJ program was used for image processing.
  • a phagemid was used as a backbone to prepare a probe DNA including a nucleotide sequence complementary to a part of the HIV-1 genome.
  • Part of the plasmid DNA containing the HIV-1 genome was restricted (restriction), and the phagemid was ligated in consideration of the direction of the f1 replication origin.
  • 5.3 kb of DNA including 2.3 kb out of 5 kb of the entire HIV-1 genome was prepared in the pBluescript II SK+ (Addgene, Watertown, MA) phagemid.
  • the double-stranded phagemid containing a part of the prepared HIV-1 genome was converted into a circular single-stranded form.
  • the phagemid was introduced into XL1 Blue MRF' (Agilent Technologies, Santa Clara, CA) E. coli through transformation, followed by tetracycline (5 ⁇ g/mL) and ampicillin (ampicillin, 60 ⁇ g/mL). It was applied to LB plate.
  • the tetracycline treatment is to select only E. coli expressing F-pilli that can be infected by a helper phage, and ampicillin was used to select E. coli into which a phagemid containing an ampicillin resistance gene was normally introduced.
  • coli colonies generated after about 15 hours were placed in 30 mL of 2xYT containing the same concentrations of tetracycline and ampicillin as above and cultured in a shaker at 37°C for 2 hours and 30 minutes, and then auxiliary phage (2.05 x 10 8 PFU) was added. After 1 hour and 30 minutes of incubation to infect E. coli by auxiliary phages, uninfected E. coli is removed by treatment with kanamycin (30 ⁇ g/mL), and the amount of progeny phage produced in E. coli is reduced through overnight culture. increased.
  • the phagemid is amplified into a circular single-stranded form, wrapped in a protein produced from the cophage, and released onto the media in the form of phage particles.
  • a solution of 25% PEG and 2.5 M NaCl was mixed with 6 mL (0.2 volume) to concentrate the phages present in the medium. and incubated for 2 hours or more in a refrigerator at 4°C. Then, centrifugation (12,000 g) at 4° C. for 20 minutes, the supernatant was discarded, and only the precipitated phages were obtained.
  • Circular single-stranded DNA was extracted from phage using a DNA clean & concentrator kit (Zymo research, Irvine, US). The extracted DNA is confirmed through gel electrophoresis, and in order to increase the purity, a single-stranded phagemid DNA band is cut and purified using a Zymoclean Gel RNA Recovery kit (Zymo research), and the results are shown in FIG. 1 . As can be seen in FIG. 1 , circular single-stranded DNA (ssDNA) of high purity was obtained.
  • ssDNA circular single-stranded DNA
  • a linear double-stranded DNA was prepared using the 6 kb HIV-1 probe DNA (including the 3 kb complementary strand in the HIV-1 genome) made in the same manner as in Preparation Example 1.
  • HIV-1 RNA which binds to the probe DNA and acts as a primer, was prepared.
  • RNA was extracted from HIV-1 using the Quick-RNA Viral kit (Zymo), and 2 uL out of a total of 35 uL was treated with 0.5 uL of AmbionTM RNaseIII (Thermo Fisher Scientific).
  • a buffer with a NaCl concentration of 10 times lower than that of the existing 10X RNaseIII Reaction buffer provided with the enzyme was made, and 0.5 uL was added to the total volume of 5 uL.
  • 5uL of virus-derived RNA solution fragmented by RNaseIII contains 125ng of probe DNA, 0.3uL of M-MuLV reverse transcriptase, 2uL of 10x reaction buffer, 2uL of 10x DTT, 1uL of dNTP (10mM), RNase Inhibitor, and 0.2uL of Murine (NEB). It was made into a 20 uL solution, and reverse transcription was performed at 42° C. for 1 hour. Since the synthesized DNA has a circular double-stranded form, it was made linear by treatment with 0.5 uL of EcoRI (NEB), a restriction enzyme that cuts only one specific location for single molecule imaging. As can be seen in FIG. 2 , it was confirmed that linear double-stranded DNA (dsDNA) that can be observed under a microscope was synthesized.
  • dsDNA linear double-stranded DNA
  • a linear dsDNA was synthesized based on the circular M13mp18 ssDNA in the same manner and applied to the performance analysis of the single molecule detection system in the following experiment.
  • M13mp18 ssDNA and the above-prepared linear M13mp18 dsDNA were diluted 20-fold, mixed with 50 nM YOYO-1 (0.5% BME) 1:1, applied on a positively charged surface and observed under a microscope, and the results are shown in FIG. 4 It was.
  • SU-8 2015 which is a negative photoresist
  • the well-coated wafer was soft baked at 95° C. for 5 minutes on a hot plate.
  • the first pattern was fixed on a mask aligner.
  • the mask and wafer were properly positioned and exposed to UV (350-450 nm) for 40 seconds.
  • exposure bake was performed at 95° C. for 6 minutes on a hot plate, and immersed in SU-8 developer for 7 minutes.
  • the first pattern was engraved by washing thoroughly with isopropyl alcohol and then with distilled water.
  • SYLGARD ® 184 silicone elastomer base and 3 g of SYLGARD ® 184 silicone elastomer curing agent were mixed, poured on the wafer overlaid with the two types of patterns prepared above, and placed in an oven at 65° C. overnight to prepare a channel did.
  • the prepared channel was treated in an air plasma generator (Femto Science Cute Basic, Korea) at 100 W for 30 seconds to make the surface hydrophilic.
  • the hydrophilicized channels were stored in water and air-dried before use to remove moisture. A 2 mm hole was drilled in the channel from which the water was removed and used as a sample injection part.
  • a 22 x 22 mm cover glass was mounted on a Teflon rack and fixed with Teflon tape.
  • a microchannel including a channel inlet without a slope (slope) was prepared by using the same method as in Preparation Example 3, except for the step of Preparation Example 3-1.
  • ⁇ DNA (NEB) was diluted to 10 pg/ul, mixed with 50 nM YOYO-1 (5% BME) 1:1 and 2 ul was flowed into the microchannel injection part. It was observed under the microscope, and the results are shown in FIG. 8 .
  • the comparative example (upper) was difficult to distinguish because the DNA was agglomerated at the entrance, but in the example (lower), the DNA was not agglomerated at the inlet and spread widely, so to distinguish/measure a single DNA molecule Ease of use was confirmed.
  • DNA solutions of 39 fmol/uL, 19.5 fmol/uL, 9.75 fmol/uL, and 4.875 fmol/uL were prepared by diluting the solution concentration of the DNA to be visualized from 78 fmol/uL to 2 times (M13mp18 ssDNA concentration was 78 fmol/uL) fixed in uL). Then, mixed with 50 nM YOYO-1 (5% BME) at a 1:1 ratio, 2 ul was flowed into the microchannel injection part. It was observed under the microscope. All narrow channels were measured under a microscope and analyzed in Image J. DNA length information was obtained and analyzed using 'Mol Length' among JAVA Plugins linked in Image J, and the results are shown in FIG. 9 .
  • the ⁇ DNA solution was diluted to 16 fg/uL, 8 fg/uL, 4 fg/uL, 2 fg/uL, 1 fg/uL, and 0.5 fg/uL concentrations. Then, 50 nM YOYO-1 (5% BME) was mixed with 1:1 and 2 ul was flowed into the microchannel injection part. It was observed under the microscope. All narrow channels were measured under a microscope and analyzed in Image J. DNA length information was obtained and analyzed using 'Mol Length' among JAVA Plugins linked in Image J, and the results are shown in FIG. 10 .
  • microchannel 10 sample attachment part
  • the present invention relates to a RNA detection method using a single DNA molecule visualization method.

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Abstract

La présente invention concerne un procédé de détection d'ARN viral et cellulaire au moyen d'un procédé de visualisation de molécule d'ADN unique. Le procédé de la présente invention peut avantageusement détecter un ARN viral et cellulaire à un niveau moléculaire d'ADN unique en un court laps de temps en visualisant un ADN double brin marqueur dans lequel un fragment d'ARN viral ou cellulaire à détecter est hybridé.
PCT/KR2021/000103 2020-01-07 2021-01-06 Procédé de détection d'arn à base de sonde d'adn simple brin WO2021141369A1 (fr)

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