WO2017078421A1 - Molecular beacons for detecting middle east respiratory syndrome coronavirus and uses thereof - Google Patents

Molecular beacons for detecting middle east respiratory syndrome coronavirus and uses thereof Download PDF

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WO2017078421A1
WO2017078421A1 PCT/KR2016/012574 KR2016012574W WO2017078421A1 WO 2017078421 A1 WO2017078421 A1 WO 2017078421A1 KR 2016012574 W KR2016012574 W KR 2016012574W WO 2017078421 A1 WO2017078421 A1 WO 2017078421A1
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cov
mers
molecular beacon
seq
virus
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Korean (ko)
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한호성
김상태
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서울대학교병원 (분사무소)
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    • 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
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/70—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving virus or bacteriophage
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to a molecular beacon for detecting Middle East Respiratory Syndrome Coronavirus (MERS-CoV), and more particularly, to a molecular beacon for detecting an Msp sequence region of an nsp3 region and a structural protein region of Middle Eastern Respiratory Syndrome coronavirus.
  • the present invention relates to a kit for diagnosing Middle East respiratory syndrome coronavirus infection and a method for detecting Middle East respiratory syndrome coronavirus.
  • MERS-CoV Middle East Respiratory Syndrome Coronavirus
  • ⁇ CoV beta coronavirus
  • nsp3 papain-like protease
  • nsp5 chymotrypsin-like, 3C-like, or main protease
  • nsp12 RNA-dependent RNA polymerase [RdRp]
  • nsp13 helicase
  • EMN Another structural protein, EMN, is encoded by ORF 6, 7, 8 and assembled with virions.
  • M and papain-like proteases and coproteins 4A, 4B and 5 are interferon antagonists known to regulate viral replication and pathogenicity.
  • a molecular beacon is an oligonucleotide hybridization probe that indicates the presence of a specific nucleic acid, and has a hairpin form in which phosphors and quenchers are coupled to both ends. The fluorescence generated in the phosphor remains internally quenched by the quencher, but when hybridization with the target nucleic acid occurs, the fluorescence is generated while the self-binding site of the hairpin structure is opened.
  • Korean Patent No. 10-0607901 discloses a method for preparing a molecular beacon according to a single-stranded nucleic acid sequence, and identifying and quantifying a specific substance using the prepared molecular beacon.
  • Korean Patent Registration No. 10-1449843 discloses a molecular beacon containing graphite nanoparticles having excellent matting properties.
  • Korean Patent No. 10-1249493 discloses a method for detecting a target nucleic acid through a complex of a molecular beacon and a nucleic acid polymerase forming a hairpin structure and a nucleic acid enzyme having a peroxidase activity and a nucleic acid polymerase reaction.
  • no DNA molecular beacon capable of specifically detecting MERS-CoV has yet been disclosed.
  • the present inventors have developed a molecular beacon for detecting MERS-CoV of a specific sequence that can easily and accurately identify whether or not MERS-CoV infection.
  • An object of the present invention is to provide a molecular beacon (molecular beacon) that can specifically detect the Middle East respiratory syndrome coronavirus (MERS-CoV).
  • MERS-CoV Middle East respiratory syndrome coronavirus
  • Another object of the present invention to provide a kit for diagnosing Middle East respiratory syndrome coronavirus (MERS-CoV) infection using the molecular beacon.
  • MERS-CoV Middle East respiratory syndrome coronavirus
  • Still another object of the present invention is to provide a method for detecting Middle East respiratory syndrome coronavirus (MERS-CoV) using the molecular beacon.
  • MERS-CoV Middle East respiratory syndrome coronavirus
  • the present invention comprises a sequence for controlling the signal opening (on / off) of the molecular beacon at both terminal sites, between which detect a specific region of Middle East respiratory syndrome coronavirus (MERS-CoV)
  • MERS-CoV Middle East respiratory syndrome coronavirus
  • an oligonucleotide for detecting Middle East respiratory syndrome coronavirus (MERS-CoV) comprising a complementary nucleotide sequence.
  • the oligonucleotide may further comprise a linker region.
  • the linker region can be used to facilitate the introduction of phosphors or quencher at the ends of the oligonucleotides.
  • the oligonucleotide has a nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2.
  • the 13 nucleotide sequences of the 5'-end and the 24 nucleotide sequences of the 3'-end are sites that control on / off of the molecular beacon signal, and 14 From base to base 35 is the region where the complementary base sequence for detecting the PLpro nsp3 region of MERS-CoV is located (Fig. 2).
  • Bases 14 to 35 are regions where the complementary base sequence for detecting the region of the structural protein M of MERS-CoV is located (FIG. 2).
  • the 5'-terminal portion of the oligonucleotide, a phosphor, a quencher may be coupled to the 3'-terminal portion, respectively, the molecular beacon signal switching regulatory region is a target nucleic acid of MERS-CoV This is an important self-recognition sequence region of the molecular beacon that only distances the quencher from the phosphor when it is recognized.
  • the present invention is oligonucleotide consisting of the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2; A phosphor linked to the 5'-end of the oligonucleotide; And a molecular beacon for detecting Middle East Respiratory Syndrome Coronavirus (MERS-CoV) comprising a quencher linked to the 3′-end of the oligonucleotide.
  • MERS-CoV Middle East Respiratory Syndrome Coronavirus
  • examples of the phosphor include rhodamine and its derivatives, fluorescein and its derivatives, coumarin and its derivatives, acridine and its derivatives, pyrene and its derivatives, erythrocin and its derivatives, eosin And derivatives thereof, but are not limited thereto.
  • the eighth base of the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2 may be a biotin binding site.
  • the present invention also provides a kit for diagnosing Middle East respiratory syndrome coronavirus (MERS-CoV) infection comprising the molecular beacon.
  • MERS-CoV Middle East respiratory syndrome coronavirus
  • the molecular beacon may be immobilized on the surface of the membrane, solution, glass or plastic polymer, other solid support.
  • any method of immobilizing probes known in the art may be used.
  • biotin-streptavidin, crosslinking method with EDC linker of NH 2 -COOH, baking at 80 ° C. or UV crosslinking can be used.
  • a method in which a probe polymer is synthesized by photolithography on an activated solid substrate (WO 92/100092) or a method of covalently bonding an already synthesized probe to the surface of an activated substrate (spotting method) may be used.
  • the molecular beacon of the present invention may be immobilized on the substrate of the microarray.
  • the molecular beacon may be immobilized on the surface of the solid support.
  • the immobilization may be achieved by an interaction between biotin bound to the 8th base of SEQ ID NO: 1 or SEQ ID NO: 2 and streptavidin linked to a solid support.
  • biotin bound to the 8th base of SEQ ID NO: 1 or SEQ ID NO: 2 and streptavidin linked to a solid support.
  • the present invention comprises the steps of obtaining a nucleic acid from a sample; Reacting the nucleic acid with a molecular beacon for detecting the Middle East Respiratory Syndrome coronavirus (MERS-CoV); And it provides a Middle East respiratory syndrome coronavirus (MERS-CoV) detection method comprising the step of detecting the fluorescence generated from the molecular beacon.
  • MERS-CoV Middle East respiratory syndrome coronavirus
  • the step of obtaining a nucleic acid from the sample is any nucleic acid extraction method known in the art, such as phenol-chloroform extraction method, purification method using a solid preparation of nucleic acid or resin packed in a column Adsorption and extraction may be used.
  • the step of obtaining the nucleic acid is the step of separating the RNA from the sample; And obtaining cDNA or RNA from the separated RNA.
  • the method of synthesizing the cDNA may include obtaining a cDNA from the RNA separated by reverse transcriptase. This reverse transcriptase reaction can be linked to a PCR reaction.
  • the molecular beacon system of the present invention may implement the reaction in the form of RNA or cDNA.
  • the reaction is a hybridization reaction between the nucleic acid and the molecular beacon (hybridization), it can be made at room temperature.
  • concentration of the molecular beacon used in the reaction is preferably 10 to 100 pmol, but is not limited thereto.
  • the molecular beacon of the present invention has a target affinity more than an antibody, and is much smaller in size than the antibody and aptamer, and can react with the target molecule with high binding force, is not sensitive to temperature change, is not denatured, and has a small amount of target in a short time. Only nucleic acid molecules can be detected. In addition, since it can be detected by a simple method using fluorescence or UV spectrum, MERS-CoV detection kit using the molecular beacon of the present invention can be widely used for the diagnosis of MERS-CoV infection.
  • 1 is a diagram showing the structure of a molecular beacon (molecular beacon) according to an embodiment of the present invention.
  • Figure 2 is a diagram showing the structure of the oligonucleotide of SEQ ID NO: 1 for detecting the PLpro nsp3 region of MERS-CoV and the oligonucleotide of SEQ ID NO: 2 for detecting the structural protein M region of MERS-CoV.
  • FIG. 3 is a diagram showing a site where biotin binds in the oligonucleotide of SEQ ID NO: 1 or 2.
  • Figure 4 shows the UV spectrum of the nsp3 of MERS-CoV analyzed by the molecular beacon for MERS-CoV detection according to the present invention.
  • Figure 5 shows the fluorescence intensity of the UV spectrum for the nsp3 of MERS-CoV analyzed by the molecular beacon for MERS-CoV detection according to the present invention.
  • Figure 6 shows the fluorescence intensity of the UV spectrum for M of MERS-CoV analyzed by the molecular beacon for MERS-CoV detection according to the present invention.
  • Figure 7 shows the results of observing the nsp3 of the MERS-CoV MERS-CoV with a molecular beacon for detection according to the invention under a confocal microscope (red fluorescence).
  • FIG. 8 is a conceptual diagram of a fluorescent probe reaction of a quantitative molecular beacon amplification system capable of detecting a target nucleic acid factor of MERS-CoV.
  • FIG. 9 is a band separated from 1% agarose gel of a PCR result obtained by detecting concentrations of N regions of MER-CoV analyzed by molecular beacons from a synthetic gene of Middle East respiratory coronavirus.
  • 10 is a graph of nanodrop uv-spectrum detecting the N region of MER-CoV analyzed by molecular beacon from the synthetic gene of Middle East respiratory corona virus.
  • FIG. 11 is a graph of nanodrop uv-spectrum detecting the nsp2 region of MER-CoV analyzed by molecular beacon from synthetic gene of Middle East respiratory coronavirus.
  • MER-CoV 12 is a result of in vitro reaction using a molecular beacon including a complementary nucleotide sequence for detecting nsp3 or M region of MER-CoV according to an embodiment of the present invention.
  • FIG. 13 is a diagram showing the structure of a molecular beacon for detecting MER-CoV M according to an embodiment of the present invention.
  • 57 to 59 in SEQ ID NOs: 1 to 12 to 36 represent important self-recognition functional sequence regions of molecular beacons that only recognize the presence of a target Middle East respiratory viral nucleic acid to allow the distance between the quencher and the phosphor to be greater.
  • Figure 14 shows a picture of the genome similarity between MERS-CoV and SARS coronavirus is an index for selecting a target site of the present invention.
  • Figure 15 shows the primer region and gene map of the target region qRT-PCR of the existing MERS-CoV.
  • 16 is a conceptual diagram of a fluorescent probe reaction of a quantitative molecular beacon amplification system capable of detecting a target nucleic acid factor of MERS-CoV.
  • Figure 17 shows the ultra-molecular beacon system and prior test results of in vitro liquid phase samples for detecting MERS-CoV.
  • FIG. 18 shows a conceptual diagram of UFO-mediated assay for detecting various target sequencing regions and viruses of MERS-CoV and its experimental results (super fluorescence intensity).
  • FIG. 19 shows a conceptual diagram of the FRET assay for detecting MERS-CoV and the results of detecting various viruses using the same.
  • FIG. 20 is a result obtained by attaching a molecular beacon detection system to various target regions of MERS-CoV labeled on a nanoparticle surface.
  • FIG. 21 shows a method using 96 well to in vitro measurements capable of detecting multiple viruses to detect MERS-CoV.
  • FIG. 22 is a conceptual diagram illustrating an ultra-simple analysis technique to which a strip diagnostic kit for detecting MERS-CoV is applied.
  • FIG. 23 shows fluorescence imaging results when a virus present in the air to detect MERS-CoV is coated with a molecular beacon reagent of a Dandelion F.C. multifunctional target coated on a specific paper film or reaction bar.
  • FIG. 24 is a conceptual diagram illustrating a method of coating a molecular beacon detection reagent on a chameleon toy or a model for detecting an acute respiratory virus such as MERS-CoV in the air to determine whether the virus is infected according to color.
  • Figure 25 shows the UV spectrum of PLOpro nsp3 of MERS-CoV analyzed by the molecular beacon for detecting the Middle East respiratory corona virus of the present invention.
  • Figure 26 shows the concentration-specific UV spectrum of MERS-CoV M analyzed by the molecular beacon for detecting the Middle East respiratory corona virus of the present invention.
  • FIG. 27 is an image showing red fluorescence observed by confocal microscopy of a UV spectral sample reacted by concentration of MERS-CoV to nsp3 analyzed by a molecular beacon for detecting a Middle East respiratory corona virus of the present invention.
  • FIG. 28 is a graph showing the amplification degree of the qRT-PCR result detected by the concentration of the N region of MER-CoV analyzed by the molecular beacon for the synthetic gene of the Middle East respiratory corona virus of the present invention.
  • 29 is a diagram showing a probe and a primer for detecting the nsp2 region of MERS-CoV according to an embodiment of the present invention.
  • FIG. 30 is a diagram showing a probe and a primer for detecting the N2 region of MERS-CoV according to an embodiment of the present invention.
  • MERS-CoV The sequence of MERS-CoV was obtained from GenBank and the conserved target regions (nsp3 and M regions) were selected using the dnastar program, and oligonucleotides of SEQ ID NO: 1 and SEQ ID NO: 2 capable of detecting the target region were synthesized ( 2).
  • the synthesized oligonucleotide was treated at 92 ° C. for 2 minutes and at 72 ° C. for 1 minute at 4 ° C. for 10 minutes to form a three-dimensional molecular beacon probe having a three-dimensional structure, and QD565, QD525 and / or QD705 and quencher BHQ1, BHQ2 or Depending on the amine group and morphology of the fluorescent molecular beacon probe attached to both sides of BHQ3, it is connected to a compound such as EDC, which is a near linker, in a solution state, nanoparticle surface, glass surface, membrane or carboxyl compound immobilized on the surface of plastic polymer and solid support.
  • EDC which is a near linker
  • the fluorescence signal of the molecular beacon of the present invention was analyzed for oligonucleotide primers artificially synthesized for specific sites of the nsp2, 3, M and N2 regions of MERS-CoV.
  • biotin-attached probes and primers were mixed on the surface of the streptavidin-coated slide, and then dispensed on a glass plate, followed by confocal microscopy two minutes later, and nsp2, nsp3, M and MERS-CoV were observed. It was found that red fluorescence was observed when the signal of the fluorescent beacon was turned on by binding to the oligonucleotide of N2.
  • nsp2, 3, and M regions of MERS-CoV For specific areas of nsp2, 3, and M regions of MERS-CoV, 1 pmol (10 ⁇ l) of molecular beacon system probes and 1 ⁇ l or more (50 pmol) of sample were mixed in an eppendorf tube and hybridized at room temperature for 2 minutes. Next, 1 ⁇ l was dispensed using nanodrop (Therma, USA) and quantitated by uv-spetrum, and the amount of reaction was the strongest at 100 pmol. It can be seen as a suitable concentration range to confirm the presence of infection.
  • the Middle East respiratory virus MERS-CoV
  • measles virus measles virus
  • enterovirus enterovirus
  • rhinovirus rhinovirus
  • SARS-associated coronavirus SARS-coV
  • varicella virus varicella zoster virus (VSV)
  • adenovirus human parainfluenza virus 1 (HPIV 1), human parainfluenza virus 2 (HPIV 2), human parainfluenza virus 3 (human parainfluenza virus 3: HPIV 3), Influenza virus A (IVA), Influenza virus B (IVB), and respiratory syncytial virus A (RSVA) and respiratory syncytial virus B (RSV)
  • a set of fluorescent probe primers was designed to detect target sequences specific for respiratorysyncytial virus B (RSVB).
  • MERS-CoV specific probe SEQ ID NO: 3
  • MERS-CoV 229e specific probe SEQ ID NO: 4
  • Measles virus specific probes SEQ ID NO: 5
  • Rhinovirus specific probe SEQ ID NO: 7
  • SARS-associated coronavirus (SARS-coV) specific probe SEQ ID NO: 8
  • VSV -Varicella zoster virus
  • Adenovirus specific probes (SEQ ID NO: 10)
  • HPIV 1 specific probe SEQ ID NO: 11
  • HPIV 2 specific probe SEQ ID NO: 12
  • HPIV 3 specific probe SEQ ID NO: 13
  • Influenza virus A (IVA) specific probe SEQ ID NO: 14
  • Influenza virus B (IVB) specific probe SEQ ID NO: 15
  • Respiratory syncytial virus B (RSVB) specific probe SEQ ID NO: 17
  • Probes were selected from the target regions of each viral genome, and the sequences were prepared with nucleic acid fluorescent molecular beacon probes containing 22 oligonucleotide sequences.
  • the synthesized structure of the probe was first treated at 92 ° C. for 2 minutes, and at 72 ° C. for 1 minute at 4 ° C. for 10 minutes.
  • the probe is connected to a solution such as EDC, which is a near linker, to a solution state, a nanoparticle surface, a glass surface, a membrane, or a carboxyl compound immobilized on the surface of a plastic polymer and a solid support.
  • EDC which is a near linker
  • the set was added and immobilized by reacting for 2 minutes at room temperature.
  • the obtained hybrid was added to the sample containing the nucleic acid prepared from each of several viruses as described above in the state in which the oligonucleotide specific for the virus was immobilized, and hybridization reaction was performed at 25 ° C. for 2 minutes.
  • Fig. 8 shows a conceptual diagram for performing real time PCR for quantifying cDNA synthetic virus from fluorescent probes, quencher and higher sensation primers and lower sense primers required to amplify and quantify viruses (Fig. 8).
  • Oligonucleotide primers artificially synthesized for specific regions of the nsp2, 3, M, and N2 regions of MERS-CoV were mixed with several probes and primers with biotin attached to a slide surface coated with streptoavidine, and then dispensed on a glass plate. Then, after 2 minutes of observation by confocal microscopy, it was found that the signal of fluorescent molecular beacon was turned on by binding to the oligonucleotides of nsp2, nsp3, M and N2 of MERS-CoV and red fluorescence was observed (Fig. 9).
  • ONE STEP qPCR should be performed in order to perform suspicious samples in a laboratory under negative pressure with biological defense facilities; GGCACTGAGGACCCACGTT 1ul ,, antisense primer; 1 pL TTGCGACATACCCATAAAAGCA 1 pL, 100 pmol aaactcggcactcggcactgaggacccacgttggccccaaattgctgagcttgctcctacagtgcPCRTRCC11AC (1) in the premix 10 ul for qPCR, 100 pGBAA TFCCC-ACCACHG, 5 minutes, 25 seconds at 95 °C, 30 seconds at 60 °C was carried out in one cycle 45 cycles were shown to be quantified.
  • RNAzol in biopsy or air, and then the total RNA separated from 96well plate or glass plate was analyzed by the molecular beacon technique.
  • an amine group is attached and the carboxyl group of the phosphor is mixed with EDC at a ratio of 1: 1: 78 to induce an amino bond.
  • biotin is attached to the 8mer (oligomer) thibase (T) and 3 ' At the end, a quencher (BHQ) was attached.
  • 10 ⁇ l of fluorescent probe and 1nM of quencher with biotin attached to liquid state are used to obtain fluorescence by detecting a small amount of virus when avidin is attached to the bottom of plate when virus RNA is present in small amount.
  • the synthesized nsp3 of MERS-CoV and a part of the genome of the M region were artificially synthesized (FIG. 12A) and reacted with the probe to 90 ⁇ l at 0, 1, 10, 25, 50, 75, and 100 pmol concentrations. Fluorescence intensities were measured at 565 / 635nm at 25 ° C using a multiplate (BioTek) of Synergy HT.
  • nsp3 areas were 0, 1, 24 ⁇ 6, 56 ⁇ 15, 231 ⁇ 43, 290 ⁇ 11, 450 ⁇ 21 AFI (optional fluorescence) and M was 0, 1, 16 ⁇ 2, 57 ⁇ 9, 189 ⁇ 23, 234 ⁇ 18, 357 ⁇ 43 AFI (optional fluorescence) (FIG. 12B).
  • Targets of ATAGAAGGTGTAAAGGAG SEQ ID NO: 22
  • TGCGCCTCAGTGAGTTGCGTCA SEQ ID NO: 23
  • genomes of specific regions for diagnosing Nsp3 and M regions of the Middle East respiratory virus are located in the 5 'region of the linker gene sequence TTCGCTGTTTACC.
  • SEQ ID NO: 24 is a MERS detection nsp3 (SEQ ID NO: 24) consisting of the base sequence TGTTTGAGGGACAGGTAAACAGCG (nsp3) (SEQ ID NO: 25) and TATTAGACATGATTATACAGCG (SEQ ID NO: 26), which is a 3 'region to recognize a 5' region and some regions of the virus.
  • 62 base) and M (58 base) probes were prepared (FIG. 13).
  • qRT-PCR quantitative reverse transcriptase chain amplification method
  • ORF1a and ORF1b a set of proteins between the start and end points of the protein
  • structural proteins corresponding to the shells of viruses such as E, M, N, and S.
  • ORF a gene part
  • E, M, N, and S use ELISA (immunoenzyme assay) or qRT-PCR method
  • existing CDC or WHO designation method uses hsp3 and N. It exemplifies how to quantify or confirm the presence by analyzing qRT-PCR method, but this takes about two weeks, when the amount of virus to be detected is obtained, and the time and cost of extracting the RNA genome of the virus is high.
  • the method proposed in the present invention has the advantage that the result can be obtained in a short time at a low cost in a small amount at the beginning of the infection for the purpose of detecting it at the beginning of the infection.
  • qRT-PCR is performed on ORF (protein synthesis set), which is a gene part for viral infection, and a primer is required to perform this.
  • Different types of quantum dot nanoparticles connect different sized quantum dot nanoparticles to express various molecular beacons connected with phosphors and quencher to emit fluorescent light like UFO strong light on a specific carrier surface.
  • 10 nm of each probe 1 nM for detecting a virus through a fluorescent probe and a quencher to detect the virus through a molecular beacon technique (Fig. 18a) capable of detecting 500 nm of the UFO molecular beacon method under the same conditions as in Experiment 4 Modified on the surface of iron oxides such as SiO 2 of nanoparticles, and synthesized the bases of artificial SARS-COV N, MERS N, AI H1, RSV A region, and confirmed the reaction degree through 100 pmol. 5, 156 ⁇ 23 and 342 ⁇ 34 are shown (FIG. 18B).
  • Molecular beacons such as SiO 2 of 500 nm nanoparticles can be detected by molecular beacons, which can detect viruses through various phosphors to simultaneously detect target genes to identify a single virus when it is replicated or to distinguish them from their respiratory viruses.
  • each probe 1nM to 10 ⁇ l synthesized in the MB itself, artificial MERS nsp3, S, E, N, nsp5, M base under the same conditions as in Experiment 4, the reaction degree confirmed in 100 pmol Represents 1, 1234 ⁇ 341, 24 ⁇ 5, 56 ⁇ 8, 27 ⁇ 2, 678 ⁇ 78, 23 ⁇ 4, which indicates that the degree of detection of the virus is different depending on the time of replication. 20b).
  • Molecular beacon technique is possible by simply spraying the virus present in the air like dandelion hole seeds in the presence of UV light in the presence of UV light.
  • virus is present in the air
  • RNAzol is sprayed in the atomizer to destroy the particles of the virus.
  • the gene is exposed, at the same time by spraying 10 ⁇ l of the molecular beacon probe 1nM at the same time irradiated with UV to confirm the presence of nsp3 in the air can be confirmed the presence of the virus in the presence of red or green fluorescence (Fig. 23).
  • Molecular Beacon on the surface of artificial chameleon toy with molecular beacon technology that can monitor the virus in real time by changing the color of chameleon like molecular beacon system when virus in air, object surface or water comes into contact with specific reaction object
  • the virus causes the various fluorescence of QD405, 505, 525, 565, 597,635 to change to blue, yellow, green, red, magenta, etc. when exposed to air or liquid at room temperature.
  • was able to detect the presence FIG. 24).
  • MnS-CoV PLpro nsp3 probe 1nM was 10 ⁇ l, and the absorbance of 260nm was rapidly increased depending on the concentration in a specific wavelength range, and 100pmol did not react at all in 229E or SARS-CoV. It is considered that the reaction (Fig. 25).
  • MERS-COV hybridized 1nM probe to 10 ⁇ l for each concentration, and then measured the absorbance of 260nm in the nanodrop reaction zone at 200-800nm with a micropipette.
  • the nanodrop 200 shows M probes having a concentration-dependent increase in absorbance at a specific wavelength band, but are sufficiently detected at concentrations that cannot be detected by the conventional method (FIG. 26).
  • nsp3 region of the respiratory virus MERS-COV, 1 nM of the probe was hybridized to 10 ⁇ l according to the concentration, and then 1 ⁇ l was measured using a micropipette at 200-800nm and 260nm at a nanodrop.
  • the nsp3 probe was measured at 565nm / 635nm when observed with LSM710 confocal microscope (Kalize, Germany) in a concentration-dependent manner at a specific wavelength range, and it was detected in a short time at a concentration that could not be detected by conventional methods. It can be seen (Fig. 27).
  • the results of the nanodrop200 confirm that the N2 probe is absorbed in a concentration-dependent manner in a specific wavelength range and is sufficiently detected at a concentration that cannot be detected by the conventional method (FIG. 10).
  • the N2 probe In the nsp2 region of the respiratory virus, MERS-COV, 1 nM of the probe was hybridized to 10 ⁇ l according to the concentration, and then 1 ⁇ l was measured using a micropipette at 200-800 nm in the nanodrop reaction zone. As shown in the nanodrop200, as shown in FIG. 23, the N2 probe has a concentration-dependent increase in absorbance at a specific wavelength range, indicating that the N2 probe is sufficiently detected at a concentration that cannot be detected by the conventional method (FIG. 11).
  • SEQ ID NO: 1 Oligonucleotide Detecting MERS-CoV nsp3 Region
  • SEQ ID NO 2 Oligonucleotide sensing the MERS-CoV M region
  • SEQ ID NO: 3 MERS-CoV specific probe
  • SEQ ID NO: 4 MERS-CoV 229e specific probe
  • SEQ ID NO 5 Measles virus specific probe
  • SEQ ID NO: 7 rhinovirus specific probe
  • SEQ ID NO: 8 SARS-CoV specific probe
  • SEQ ID NO: 9 Varicella virus (VSV) specific probe
  • SEQ ID NO: 10 adenovirus specific probe
  • SEQ ID NO: 11 Human parainfluenza virus 1 (HPIV 1) specific probe
  • SEQ ID NO: 13 Human parainfluenza virus 3 (HPIV 3) specific probe
  • SEQ ID NO: 16 Respiratory syncytial virus A (RSVA) specific probe
  • SEQ ID NO: 17 Respiratory syncytial virus B (RSVB) specific probe

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Abstract

The present invention relates to a molecular beacon for the detection of Middle East respiratory syndrome coronavirus (MERS-CoV). More specifically, the present invention relates to a molecular beacon for detecting the nsp3 region of the MERS-CoV and the M base sequence region which is a structural protein region, a kit for the diagnosis of a MERS-CoV infection using the same, and a method for detecting MERS-CoV. The molecular beacon of the present invention has a target affinity higher than the affinity of an antibody and is much smaller in size than an antibody and an aptamer so that the beacon can react with a target molecule with a high binding force. In addition, the molecular beacon is not sensitive to temperature change, has no denaturation, and has the advantage of enabling detection with only a small amount of target nucleic acid molecules within a short time. Furthermore, since detection is possible by a simple method using fluorescence or UV spectrum, a kit for detecting MERS-CoV using the molecular beacon of the present invention can be widely used for the diagnosis of a MERS-CoV infection.

Description

중동호흡기증후군 코로나바이러스 검출용 분자비콘 및 이의 용도Molecular Beacon for the Detection of Middle East Respiratory Syndrome Coronavirus and Its Use
본 발명은 중동호흡기증후군 코로나바이러스(MERS-CoV) 검출용 분자비콘(molecular beacon)에 관한 것으로, 보다 구체적으로 중동호흡기증후군 코로나바이러스의 nsp3 영역과 구조단백질 영역인 M 염기서열 부위를 탐지하는 분자비콘, 이를 이용한 중동호흡기증후군 코로나바이러스 감염 진단용 키트 및 중동호흡기증후군 코로나바이러스 검출방법에 관한 것이다.The present invention relates to a molecular beacon for detecting Middle East Respiratory Syndrome Coronavirus (MERS-CoV), and more particularly, to a molecular beacon for detecting an Msp sequence region of an nsp3 region and a structural protein region of Middle Eastern Respiratory Syndrome coronavirus. The present invention relates to a kit for diagnosing Middle East respiratory syndrome coronavirus infection and a method for detecting Middle East respiratory syndrome coronavirus.
최근 우리나라에 전파되었던 중동호흡기증후군 코로나바이러스(MERS-CoV)는 2012년에 새로 발견된 베타코로나바이러스(Betacoronavirus; βCoV)로 약 3만kb의 (+)-센스 단일가닥(single-stranded) RNA 바이러스이며, 5'-복제효소-구조단백질(spike-envelope-membrane-nucleocapsid)-poly(A)-3'[5'-ORF1a/b-S-E-M-N-poly(A)]의 구조를 갖는다. nsp3 (papain-like protease), nsp5 (chymotrypsin-like, 3C-like, 또는 main protease), nsp12 (RNA-dependent RNA polymerase [RdRp]), nsp13 (helicase), 기타 nsp는 바이러스의 복제와 전사에 관여하며, 다른 구조단백질인 E-M-N은 ORF 6,7,8에 의해 암호화하여 비리온과 어셈블리된다. M과 파파인 유사 단백질분해효소와 보조단백질인 4A,4B,5는 인터페론 길항체로 바이러스의 복제와 병원성을 조절하는 것으로 알려져 있다.The Middle East Respiratory Syndrome Coronavirus (MERS-CoV), which has recently spread to Korea, is a newly discovered beta coronavirus (βCoV) in 2012. It is approximately 30,000 kb of positive-sense single-stranded RNA virus. And has a structure of 5'-clonal-membrane-nucleocapsid-poly (A) -3 '[5'-ORF1a / bSEMN-poly (A)]. nsp3 (papain-like protease), nsp5 (chymotrypsin-like, 3C-like, or main protease), nsp12 (RNA-dependent RNA polymerase [RdRp]), nsp13 (helicase), and other nsps are involved in virus replication and transcription Another structural protein, EMN, is encoded by ORF 6, 7, 8 and assembled with virions. M and papain-like proteases and coproteins 4A, 4B and 5 are interferon antagonists known to regulate viral replication and pathogenicity.
각종 바이러스의 검출용 조직과 배양 세포에서 유전자의 발현을 탐색하기 위한 방법으로 DNA 마이크로어레이나 qPCR 등의 기술들이 소개되어 왔으나, 이들은 복제된 상대적인 바이러스 mRNA에서 측정 가능한 바이러스 게놈을 정량화하는 단계를 필요로 하므로, 많은 시간과 비용이 소요되는 단점이 있다. 한편, 분자비콘(molecular beacon)은 특정 핵산의 존재를 알려 주는 올리고뉴클레오티드 혼성화 프로브로서, 양 말단에 각각 형광체와 소광체가 결합되어 있는 헤어핀(hairpin) 형태를 갖는다. 상기 형광체에서 발생한 형광은 내부적으로 소광체에 의해 소광된 상태로 있게 되나, 표적 핵산과 혼성화 반응이 일어나게 되면 상기 헤어핀 구조의 자가 결합 부위가 열리면서 형광이 발생하게 된다. 분자비콘 관련 기술의 예로서, 대한민국 등록특허 제10-0607901호는 단일가닥 핵산 서열에 따른 분자비콘을 제조하고, 상기 제조된 분자비콘을 이용하여 특정물질을 동정 및 정량하는 방법을 개시하고 있으며, 대한민국 등록특허 제10-1449843호는 우수한 소광 특성을 갖는 흑연 나노입자를 함유하는 분자비콘을 개시한다. 또한, 대한민국 등록특허 제10-1249493호는 과산화효소 활성이 있는 핵산효소와 헤어핀 구조를 형성하는 분자비콘의 복합체와 블로커 핵산 및 핵산중합효소 반응을 통한 표적핵산을 검출하는 방법을 개시하였다. 그러나 아직까지 MERS-CoV를 특이적으로 검출할 수 있는 DNA 분자비콘은 개시된 바 없다. Techniques such as DNA microarray and qPCR have been introduced to detect gene expression in tissues and culture cells for detection of various viruses, but they require quantifying the viral genome measurable from the relative viral mRNAs cloned. Therefore, there is a disadvantage that takes a lot of time and money. Meanwhile, a molecular beacon is an oligonucleotide hybridization probe that indicates the presence of a specific nucleic acid, and has a hairpin form in which phosphors and quenchers are coupled to both ends. The fluorescence generated in the phosphor remains internally quenched by the quencher, but when hybridization with the target nucleic acid occurs, the fluorescence is generated while the self-binding site of the hairpin structure is opened. As an example of a molecular beacon related technology, Korean Patent No. 10-0607901 discloses a method for preparing a molecular beacon according to a single-stranded nucleic acid sequence, and identifying and quantifying a specific substance using the prepared molecular beacon. Korean Patent Registration No. 10-1449843 discloses a molecular beacon containing graphite nanoparticles having excellent matting properties. In addition, Korean Patent No. 10-1249493 discloses a method for detecting a target nucleic acid through a complex of a molecular beacon and a nucleic acid polymerase forming a hairpin structure and a nucleic acid enzyme having a peroxidase activity and a nucleic acid polymerase reaction. However, no DNA molecular beacon capable of specifically detecting MERS-CoV has yet been disclosed.
이에 본 발명자들은 MERS-CoV의 감염 여부를 빠른 시간 내에 간편하면서도 정확하게 확인할 수 있는 특정 서열의 MERS-CoV 검출용 분자비콘을 개발하게 되었다. Therefore, the present inventors have developed a molecular beacon for detecting MERS-CoV of a specific sequence that can easily and accurately identify whether or not MERS-CoV infection.
본 발명의 목적은 중동호흡기증후군 코로나바이러스(MERS-CoV)를 특이적으로 검출할 수 있는 분자비콘(molecular beacon)을 제공하는 것이다. An object of the present invention is to provide a molecular beacon (molecular beacon) that can specifically detect the Middle East respiratory syndrome coronavirus (MERS-CoV).
본 발명의 다른 목적은 상기 분자비콘을 이용한 중동호흡기증후군 코로나바이러스(MERS-CoV) 감염 진단용 키트를 제공하는 것이다.Another object of the present invention to provide a kit for diagnosing Middle East respiratory syndrome coronavirus (MERS-CoV) infection using the molecular beacon.
본 발명의 또 다른 목적은 상기 분자비콘을 이용한 중동호흡기증후군 코로나바이러스(MERS-CoV)의 검출방법을 제공하는 것이다.Still another object of the present invention is to provide a method for detecting Middle East respiratory syndrome coronavirus (MERS-CoV) using the molecular beacon.
상기 목적을 달성하기 위하여, 본 발명은 양 말단 부위에 분자비콘의 신호 개폐(on/off)를 조절하는 서열을 포함하며, 그 사이에 중동호흡기증후군 코로나바이러스(MERS-CoV)의 특정 영역을 감지하는 상보적 염기서열을 포함하는 중동호흡기증후군 코로나바이러스(MERS-CoV) 검출용 올리고뉴클레오티드를 제공한다. In order to achieve the above object, the present invention comprises a sequence for controlling the signal opening (on / off) of the molecular beacon at both terminal sites, between which detect a specific region of Middle East respiratory syndrome coronavirus (MERS-CoV) Provided is an oligonucleotide for detecting Middle East respiratory syndrome coronavirus (MERS-CoV) comprising a complementary nucleotide sequence.
본 발명의 일 실시예에 있어서, 상기 올리고뉴클레오티드는 링커(linker) 영역을 추가로 포함할 수 있다. 상기 링커 영역은 올리고뉴클레오티드의 말단에 형광체 또는 소광체를 도입하기 용이하도록 하기 위해 사용될 수 있다.In one embodiment of the present invention, the oligonucleotide may further comprise a linker region. The linker region can be used to facilitate the introduction of phosphors or quencher at the ends of the oligonucleotides.
본 발명의 일 실시예에 있어서, 상기 올리고뉴클레오티드는 서열번호 1 또는 서열번호 2의 염기서열을 갖는다. 서열번호 1의 올리고뉴클레오티드의 경우, 5'-말단의 13개 염기서열과 3'-말단의 24개 염기서열은 분자비콘 신호의 개폐(on/off)를 조절하는 부위이며, 그 사이에 위치한 14번부터 35번 염기까지가 MERS-CoV의 PLpro nsp3 영역을 감지하는 상보적인 염기서열이 위치하는 영역이다(도 2). 서열번호 2의 올리고뉴클레오티드의 경우에는, 5'-말단의 13개 염기서열과 3'-말단의 22개 염기서열은 분자비콘 신호의 개폐(on/off)를 조절하는 부위이며, 그 사이에 위치한 14번부터 35번 염기까지가 MERS-CoV의 구조단백질 M의 영역을 감지하는 상보적인 염기서열이 위치하는 영역이다(도 2). In one embodiment of the present invention, the oligonucleotide has a nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2. In the case of the oligonucleotide of SEQ ID NO: 1, the 13 nucleotide sequences of the 5'-end and the 24 nucleotide sequences of the 3'-end are sites that control on / off of the molecular beacon signal, and 14 From base to base 35 is the region where the complementary base sequence for detecting the PLpro nsp3 region of MERS-CoV is located (Fig. 2). In the case of the oligonucleotide of SEQ ID NO: 2, 13 nucleotide sequences at the 5'-end and 22 nucleotide sequences at the 3'-end are sites for controlling the on / off of the molecular beacon signal, Bases 14 to 35 are regions where the complementary base sequence for detecting the region of the structural protein M of MERS-CoV is located (FIG. 2).
본 발명의 일 실시예에 있어서, 상기 올리고뉴클레오티드의 5'-말단 부위에는 형광체가, 3'-말단 부위에는 소광체가 각각 결합될 수 있으며, 상기 분자비콘 신호 개폐 조절부위는 MERS-CoV의 표적 핵산이 인식될 경우에만 소광체와 형광체 간의 거리를 멀어지게 하는 분자비콘의 중요 자기인식 기능 염기서열 영역이다.In one embodiment of the present invention, the 5'-terminal portion of the oligonucleotide, a phosphor, a quencher may be coupled to the 3'-terminal portion, respectively, the molecular beacon signal switching regulatory region is a target nucleic acid of MERS-CoV This is an important self-recognition sequence region of the molecular beacon that only distances the quencher from the phosphor when it is recognized.
또한, 본 발명은 상기 서열번호 1 또는 서열번호 2의 염기서열로 이루어진 올리고뉴클레오티드; 상기 올리고뉴클레오티드의 5'-말단에 연결된 형광체; 및 상기 올리고뉴클레오티드의 3'-말단에 연결된 소광체를 포함하는 중동호흡기증후군 코로나바이러스(MERS-CoV) 검출용 분자비콘(molecular beacon)을 제공한다.In addition, the present invention is oligonucleotide consisting of the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2; A phosphor linked to the 5'-end of the oligonucleotide; And a molecular beacon for detecting Middle East Respiratory Syndrome Coronavirus (MERS-CoV) comprising a quencher linked to the 3′-end of the oligonucleotide.
본 발명의 일 실시예에 있어서, 상기 형광체의 예로는, 로다민과 그의 유도체, 플루오레신과 그의 유도체, 쿠마린과 그의 유도체, 아크리딘과 그의 유도체, 피렌과 그의 유도체, 에리트로신과 그의 유도체, 에오신과 그의 유도체 등이 있으나, 이에 제한되는 것은 아니다. In one embodiment of the present invention, examples of the phosphor include rhodamine and its derivatives, fluorescein and its derivatives, coumarin and its derivatives, acridine and its derivatives, pyrene and its derivatives, erythrocin and its derivatives, eosin And derivatives thereof, but are not limited thereto.
본 발명의 일 실시예에 있어서, 상기 서열번호 1 또는 서열번호 2의 염기서열의 8번째 염기는 비오틴(biotin) 결합 부위일 수 있다. In one embodiment of the present invention, the eighth base of the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2 may be a biotin binding site.
또한, 본 발명은 상기 분자비콘을 포함하는 중동호흡기증후군 코로나바이러스(MERS-CoV) 감염 진단용 키트를 제공한다.The present invention also provides a kit for diagnosing Middle East respiratory syndrome coronavirus (MERS-CoV) infection comprising the molecular beacon.
본 발명의 일 실시예에 있어서, 상기 분자비콘은 막, 용액, 유리 또는 플라스틱 중합체, 기타 고체 지지체의 표면에 고정화된 것일 수 있다. 본 발명의 분자비콘을 고체 지지체의 표면에 고정화하는 과정은 당업계에 알려진 임의의 프로브 고정화 방법이 사용될 수 있다. 예를 들면, 비오틴-스트렙타비딘(biotin-streptavidin), NH2-COOH의 EDC링커에 의한 가교결합법, 80 ℃에서의 베이킹 또는 UV 가교화가 사용될 수 있다. 또한, 활성화된 고체 기판 상에서 프로브 중합체가 포토리소그래피에 의하여 합성되는 방법 (WO 92/100092) 또는 이미 합성된 프로브를 활성화된 기판의 표면에 공유 결합시키는 방법 (spotting 법) 등이 사용될 수 있다. 본 발명의 일 실시예에 있어서, 본 발명의 분자비콘은 마이크로어레이의 기판상에 고정화되어 있는 것일 수 있다.In one embodiment of the present invention, the molecular beacon may be immobilized on the surface of the membrane, solution, glass or plastic polymer, other solid support. For immobilizing the molecular beacon of the present invention on the surface of the solid support, any method of immobilizing probes known in the art may be used. For example, biotin-streptavidin, crosslinking method with EDC linker of NH 2 -COOH, baking at 80 ° C. or UV crosslinking can be used. In addition, a method in which a probe polymer is synthesized by photolithography on an activated solid substrate (WO 92/100092) or a method of covalently bonding an already synthesized probe to the surface of an activated substrate (spotting method) may be used. In one embodiment of the present invention, the molecular beacon of the present invention may be immobilized on the substrate of the microarray.
본 발명의 일 실시예에 있어서, 상기 분자비콘은 고체 지지체의 표면에 고정화될 수 있다. 본 발명의 일 실시예에 있어서, 상기 고정화는 상기 서열번호 1 또는 서열번호 2의 8번째 염기에 결합된 비오틴(biotin)과 고체 지지체에 연결된 스트렙타비딘(streptavidin) 사이의 상호작용에 의해 이루어질 수 있으나, 이에 제한되는 것은 아니다. In one embodiment of the present invention, the molecular beacon may be immobilized on the surface of the solid support. In one embodiment of the present invention, the immobilization may be achieved by an interaction between biotin bound to the 8th base of SEQ ID NO: 1 or SEQ ID NO: 2 and streptavidin linked to a solid support. However, it is not limited thereto.
또한, 본 발명은, 검체로부터 핵산을 수득하는 단계; 상기 핵산을 상기 중동호흡기증후군 코로나바이러스(MERS-CoV) 검출용 분자비콘과 반응시키는 단계; 및 상기 분자비콘으로부터 발생하는 형광을 검출하는 단계를 포함하는 중동호흡기증후군 코로나바이러스(MERS-CoV) 검출방법을 제공한다.In addition, the present invention comprises the steps of obtaining a nucleic acid from a sample; Reacting the nucleic acid with a molecular beacon for detecting the Middle East Respiratory Syndrome coronavirus (MERS-CoV); And it provides a Middle East respiratory syndrome coronavirus (MERS-CoV) detection method comprising the step of detecting the fluorescence generated from the molecular beacon.
본 발명의 일 실시예에 있어서, 상기 검체로부터 핵산을 수득하는 단계는 당 업계에 알려진 임의의 핵산 추출법, 예컨대 페놀-클로로포름 추출법, 핵산에 고상제제를 이용한 정제법 또는 칼럼에 충진된 레진을 이용하여 흡착시켜 추출하는 법 등이 사용될 수 있다. 본 발명의 일 실시예에 있어서, 상기 핵산을 수득하는 단계는 검체로부터 RNA를 분리하는 단계; 및 분리된 RNA로부터 cDNA나 RNA를 얻는 단계를 포함하는 것일 수 있다. 상기 cDNA를 합성하는 방법은 역전사효소를 이용하여 분리된 RNA로부터 cDNA를 얻는 단계를 포함하는 것일 수 있다. 이러한 역전사효소 반응은 PCR 반응과 연계될 수 있다. 본 발명의 일 실시예에 있어서, 본 발명의 분자비콘 시스템은 RNA 또는 cDNA형태로 반응을 구현할 수도 있다.In one embodiment of the present invention, the step of obtaining a nucleic acid from the sample is any nucleic acid extraction method known in the art, such as phenol-chloroform extraction method, purification method using a solid preparation of nucleic acid or resin packed in a column Adsorption and extraction may be used. In one embodiment of the present invention, the step of obtaining the nucleic acid is the step of separating the RNA from the sample; And obtaining cDNA or RNA from the separated RNA. The method of synthesizing the cDNA may include obtaining a cDNA from the RNA separated by reverse transcriptase. This reverse transcriptase reaction can be linked to a PCR reaction. In one embodiment of the present invention, the molecular beacon system of the present invention may implement the reaction in the form of RNA or cDNA.
본 발명의 일 실시예에 있어서, 상기 반응은 핵산과 분자비콘 사이의 혼성화(hybridization) 반응으로, 실온에서 이루어질 수 있다. 또한, 상기 반응에 사용되는 분자비콘의 농도는 10 내지 100 pmol인 것이 바람직하나, 이에 제한되는 것은 아니다.In one embodiment of the invention, the reaction is a hybridization reaction between the nucleic acid and the molecular beacon (hybridization), it can be made at room temperature. In addition, the concentration of the molecular beacon used in the reaction is preferably 10 to 100 pmol, but is not limited thereto.
본 발명의 분자비콘은 항체 이상의 표적 친화력을 가지며, 항체 및 압타머에 비해 크기가 월등히 작아 표적 분자와 높은 결합력으로 반응할 수 있고, 온도 변화에 민감하지 않으며 변성이 없고, 빠른 시간 안에 소량의 표적 핵산 분자만으로도 탐지가 가능한 장점이 있다. 또한, 형광 또는 UV 스펙트럼을 사용하는 간편한 방법으로 검출이 가능하므로, 본 발명의 분자비콘을 이용한 MERS-CoV 검출용 키트는 MERS-CoV 감염 여부의 진단에 널리 사용될 수 있다.The molecular beacon of the present invention has a target affinity more than an antibody, and is much smaller in size than the antibody and aptamer, and can react with the target molecule with high binding force, is not sensitive to temperature change, is not denatured, and has a small amount of target in a short time. Only nucleic acid molecules can be detected. In addition, since it can be detected by a simple method using fluorescence or UV spectrum, MERS-CoV detection kit using the molecular beacon of the present invention can be widely used for the diagnosis of MERS-CoV infection.
도 1은 본 발명의 일 실시예에 따른 분자비콘(molecular beacon)의 구조를 나타낸 그림이다.1 is a diagram showing the structure of a molecular beacon (molecular beacon) according to an embodiment of the present invention.
도 2는 MERS-CoV의 PLpro nsp3 영역을 감지하는 서열번호 1의 올리고뉴클레오티드와, MERS-CoV의 구조단백질 M 영역을 감지하는 서열번호 2의 올리고뉴클레오티드의 구조를 나타낸 그림이다. Figure 2 is a diagram showing the structure of the oligonucleotide of SEQ ID NO: 1 for detecting the PLpro nsp3 region of MERS-CoV and the oligonucleotide of SEQ ID NO: 2 for detecting the structural protein M region of MERS-CoV.
도 3은 서열번호 1 또는 2의 올리고뉴클레오티드에서 비오틴이 결합하는 부위를 나타낸 그림이다. 3 is a diagram showing a site where biotin binds in the oligonucleotide of SEQ ID NO: 1 or 2.
도 4는 본 발명에 따른 MERS-CoV 검출용 분자비콘으로 분석한 MERS-CoV의 nsp3에 대한 UV 스펙트럼을 나타낸 것이다.Figure 4 shows the UV spectrum of the nsp3 of MERS-CoV analyzed by the molecular beacon for MERS-CoV detection according to the present invention.
도 5는 본 발명에 따른 MERS-CoV 검출용 분자비콘으로 분석한 MERS-CoV의 nsp3에 대한 UV 스펙트럼의 형광강도를 나타낸 것이다.Figure 5 shows the fluorescence intensity of the UV spectrum for the nsp3 of MERS-CoV analyzed by the molecular beacon for MERS-CoV detection according to the present invention.
도 6은 본 발명에 따른 MERS-CoV 검출용 분자비콘으로 분석한 MERS-CoV의 M에 대한 UV 스펙트럼의 형광강도를 나타낸 것이다.Figure 6 shows the fluorescence intensity of the UV spectrum for M of MERS-CoV analyzed by the molecular beacon for MERS-CoV detection according to the present invention.
도 7은 본 발명에 따른 MERS-CoV 검출용 분자비콘으로 분석한 MERS-CoV의 nsp3를 공초점 현미경으로 관찰한 결과(적색형광)를 나타낸 것이다. Figure 7 shows the results of observing the nsp3 of the MERS-CoV MERS-CoV with a molecular beacon for detection according to the invention under a confocal microscope (red fluorescence).
도 8은 MERS-CoV의 표적 핵산인자를 탐지할 수 있는 정량적 분자비콘 증폭시스템의 형광프로브 반응 개념도이다. 8 is a conceptual diagram of a fluorescent probe reaction of a quantitative molecular beacon amplification system capable of detecting a target nucleic acid factor of MERS-CoV.
도 9는 중동호흡기 코로나 바이러스의 합성 유전자로부터 분자비콘으로 분석한 MER-CoV의 N영역을 농도별로 탐지한 PCR 결과물을 1% 아가로즈겔에서 분리한 밴드이다. FIG. 9 is a band separated from 1% agarose gel of a PCR result obtained by detecting concentrations of N regions of MER-CoV analyzed by molecular beacons from a synthetic gene of Middle East respiratory coronavirus.
도 10은 중동호흡기 코로나 바이러스의 합성 유전자로부터 분자비콘로 분석한 MER-CoV의 N영역을 농도별로 탐지한 nanodrop uv-spectrum 그래프이다.10 is a graph of nanodrop uv-spectrum detecting the N region of MER-CoV analyzed by molecular beacon from the synthetic gene of Middle East respiratory corona virus.
도 11은 중동호흡기 코로나 바이러스의 합성 유전자로부터 분자비콘로 분석한 MER-CoV의 nsp2영역을 농도별로 탐지한 nanodrop uv-spectrum 그래프이다. FIG. 11 is a graph of nanodrop uv-spectrum detecting the nsp2 region of MER-CoV analyzed by molecular beacon from synthetic gene of Middle East respiratory coronavirus.
도 12는 본 발명의 일 실시예에 따른 MER-CoV의 nsp3 또는 M 영역을 감지하는 상보적인 염기서열을 포함하는 분자비콘을 이용하여 시험관 내에서 반응시킨 결과값이다. 12 is a result of in vitro reaction using a molecular beacon including a complementary nucleotide sequence for detecting nsp3 or M region of MER-CoV according to an embodiment of the present invention.
도 13은 본 발명의 일 실시예에 따른 MER-CoV M을 검출하는 분자비콘의 구조를 나타낸 그림이다. 염기번호 1에서 12 내지 36에서 57/59은 표적 중동호흡기 바이러스 핵산이 존재할 경우만 인식하여 소광체와 형광체 간의 거리가 멀어지게 하는 분자비콘의 중요 자기인식 기능 염기서열 영역을 나타낸다.13 is a diagram showing the structure of a molecular beacon for detecting MER-CoV M according to an embodiment of the present invention. 57 to 59 in SEQ ID NOs: 1 to 12 to 36 represent important self-recognition functional sequence regions of molecular beacons that only recognize the presence of a target Middle East respiratory viral nucleic acid to allow the distance between the quencher and the phosphor to be greater.
도 14는 MERS-CoV와 사스 코로나바이러스의 유전자게놈 유사성에 관한 그림을 나타낸 것으로 본 발명의 표적부위를 선정하는데 지표이다. Figure 14 shows a picture of the genome similarity between MERS-CoV and SARS coronavirus is an index for selecting a target site of the present invention.
도 15는 기존 MERS-CoV의 표적 영역 qRT-PCR의 프라이머 영역과 유전자 지도를 나타낸 것이다.Figure 15 shows the primer region and gene map of the target region qRT-PCR of the existing MERS-CoV.
도 16은 MERS-CoV의 표적 핵산인자를 탐지할 수 있는 정량적 분자비콘 증폭시스템의 형광프로브 반응 개념도이다.16 is a conceptual diagram of a fluorescent probe reaction of a quantitative molecular beacon amplification system capable of detecting a target nucleic acid factor of MERS-CoV.
도 17은 MERS-CoV을 탐지하기 위한 시험관 내 액체상의 시료의 울트라 분자비콘 시스템과 선행 시험 결과값을 나타낸 것이다. Figure 17 shows the ultra-molecular beacon system and prior test results of in vitro liquid phase samples for detecting MERS-CoV.
도 18은 MERS-CoV의 다양한 표적 염기서열 영역과 바이러스를 탐지하기 위한 UFO 매개 분석법의 개념도와 이에 따른 실험 결과값(초강력 형광강도)을 나타낸 것이다.FIG. 18 shows a conceptual diagram of UFO-mediated assay for detecting various target sequencing regions and viruses of MERS-CoV and its experimental results (super fluorescence intensity).
도 19는 MERS-CoV을 탐지하기 위한 FRET 분석법의 개념도와 이를 이용하여 다양한 바이러스를 탐지한 결과값을 나타낸 것이다.19 shows a conceptual diagram of the FRET assay for detecting MERS-CoV and the results of detecting various viruses using the same.
도 20은 나노입자표면에 표지한 MERS-CoV의 다양한 표적영역을 대상으로 분자비콘 탐지시스템을 부착하여 분석한 결과값이다.FIG. 20 is a result obtained by attaching a molecular beacon detection system to various target regions of MERS-CoV labeled on a nanoparticle surface.
도 21은 MERS-CoV을 탐지하기 위해 다중 바이러스를 탐지할 수 있는 96웰(well) 내지 시험관 내 측정을 이용하는 방법을 나타낸 것이다.FIG. 21 shows a method using 96 well to in vitro measurements capable of detecting multiple viruses to detect MERS-CoV.
도 22는 MERS-CoV을 탐지하기 위한 스트립 진단키트를 적용한 초간편 분석 기법을 나타낸 개념도이다.FIG. 22 is a conceptual diagram illustrating an ultra-simple analysis technique to which a strip diagnostic kit for detecting MERS-CoV is applied.
도 23은 MERS-CoV을 탐지하기 위해 공기 속에 존재하는 바이러스를 특정 종이막이나 반응 막대에 코팅된 민들레 홀씨기능 다중 표적인자의 분자비콘시약을 코팅하여 반응시 나타난 형광영상 이미징 결과이다.FIG. 23 shows fluorescence imaging results when a virus present in the air to detect MERS-CoV is coated with a molecular beacon reagent of a Dandelion F.C. multifunctional target coated on a specific paper film or reaction bar.
도 24는 공기중에 MERS-CoV와 같은 급성 호흡기바이러스를 탐지하기 위한 카멜레온 장난감이나 모형에 분자비콘 탐지 시약을 코팅시켜 색상에 따라 바이러스의 감염유무를 확인할 수 있는 기법을 나타낸 개념도이다. FIG. 24 is a conceptual diagram illustrating a method of coating a molecular beacon detection reagent on a chameleon toy or a model for detecting an acute respiratory virus such as MERS-CoV in the air to determine whether the virus is infected according to color.
도 25는 본 발명의 중동호흡기 코로나 바이러스 탐지용 분자비콘으로 분석한 MERS-CoV의 PLOpro nsp3에 대한 UV 스펙트럼을 나타낸 것이다.Figure 25 shows the UV spectrum of PLOpro nsp3 of MERS-CoV analyzed by the molecular beacon for detecting the Middle East respiratory corona virus of the present invention.
도 26은 본 발명의 중동호흡기 코로나 바이러스 탐지용 분자비콘으로 분석한 MERS-CoV의 M에 대한 농도별 UV 스펙트럼을 나타낸 것이다.Figure 26 shows the concentration-specific UV spectrum of MERS-CoV M analyzed by the molecular beacon for detecting the Middle East respiratory corona virus of the present invention.
도 27은 본 발명의 중동호흡기 코로나 바이러스 탐지용 분자비콘으로 분석한 MERS-CoV의 nsp3에 대한 농도별로 반응시킨 UV 스펙트럼 시료를 공초점 현미경으로 관찰한 적색형광을 나타낸 이미지이다. FIG. 27 is an image showing red fluorescence observed by confocal microscopy of a UV spectral sample reacted by concentration of MERS-CoV to nsp3 analyzed by a molecular beacon for detecting a Middle East respiratory corona virus of the present invention.
도 28은 본 발명의 중동호흡기 코로나 바이러스의 합성 유전자에 대해 분자비콘으로 분석한 MER-CoV의 N 영역을 농도별로 탐지한 qRT-PCR 결과물의 증폭정도를 나타낸 그래프이다. 28 is a graph showing the amplification degree of the qRT-PCR result detected by the concentration of the N region of MER-CoV analyzed by the molecular beacon for the synthetic gene of the Middle East respiratory corona virus of the present invention.
도 29는 본 발명의 일 실시예에 따른 MERS-CoV의 nsp2 영역을 감지하는 프로브와 이에 대한 프라이머를 나타낸 그림이다. 29 is a diagram showing a probe and a primer for detecting the nsp2 region of MERS-CoV according to an embodiment of the present invention.
도 30은 본 발명의 일 실시예에 따른 MERS-CoV의 N2 영역을 감지하는 프로브와 이에 대한 프라이머를 나타낸 그림이다. 30 is a diagram showing a probe and a primer for detecting the N2 region of MERS-CoV according to an embodiment of the present invention.
이하 본 발명을 실시예를 통하여 보다 상세하게 설명한다. 그러나 이들 실시예는 본 발명을 예시적으로 설명하기 위한 것으로 본 발명의 범위가 이들 실시예에 한정되는 것은 아니다.Hereinafter, the present invention will be described in more detail with reference to Examples. However, these examples are for illustrative purposes only and the scope of the present invention is not limited to these examples.
<< 실시예Example 1> 1>
본 발명에 따른 According to the invention MERSMERS -- CoVCoV 검출용  For detection 분자비콘의Molecular beacon 제작 making
GenBank로부터 MERS-CoV의 서열을 입수하고 dnastar 프로그램을 이용하여 보존된 표적 영역(nsp3와 M 영역)을 선발하고, 표적 영역을 감지할 수 있는 서열번호 1 및 서열번호 2의 올리고뉴클레오티드를 합성하였다(도 2). The sequence of MERS-CoV was obtained from GenBank and the conserved target regions (nsp3 and M regions) were selected using the dnastar program, and oligonucleotides of SEQ ID NO: 1 and SEQ ID NO: 2 capable of detecting the target region were synthesized ( 2).
상기 합성된 올리고뉴클레오티드를 92℃에서 2분간, 72℃에서 1분간 4℃에서 10분간 처리하여 3차원 구조를 가진 입체 분자비콘 프로브를 만들고, QD565, QD525 및/또는 QD705와 소광체인 BHQ1, BHQ2 또는 BHQ3을 각각 양쪽에 부착시킨 형광 분자비콘 프로브의 아민기와 양식에 따라 용액상태, 나노입자 표면, 유리표면, 막 또는 플라스틱 중합체와 고체 지지체의 표면에 고정화된 카르복실기 화합물에 근거리 링커인 EDC 같은 화합물로 연결시켜 프로브 세트를 첨가하여, 실온에서 2분 동안 반응시킴으로써 고정화하였다. 얻어진 혼성체를 상기한 바와 같은 수종의 각 바이러스로부터 준비한 핵산이 포함되어 있는 시료를 상기 바이러스에 특이적인 올리고뉴클레오티드가 고정화되어 있는 상태에 첨가하고 25℃에서 2분 동안 혼성화 반응을 수행하였다. The synthesized oligonucleotide was treated at 92 ° C. for 2 minutes and at 72 ° C. for 1 minute at 4 ° C. for 10 minutes to form a three-dimensional molecular beacon probe having a three-dimensional structure, and QD565, QD525 and / or QD705 and quencher BHQ1, BHQ2 or Depending on the amine group and morphology of the fluorescent molecular beacon probe attached to both sides of BHQ3, it is connected to a compound such as EDC, which is a near linker, in a solution state, nanoparticle surface, glass surface, membrane or carboxyl compound immobilized on the surface of plastic polymer and solid support. The probe set was added and immobilized by reacting for 2 minutes at room temperature. The obtained hybrid was added to the sample containing the nucleic acid prepared from each of several viruses as described above in the state in which the oligonucleotide specific for the virus was immobilized, and hybridization reaction was performed at 25 ° C. for 2 minutes.
<< 실시예Example 2> 2>
MERS-CoV의 nsp2, 3, M 및 N2 영역의 특정 부위에 대해 인공 합성한 올리고뉴클레오티드 프라이머를 대상으로 본 발명의 분자비콘의 형광 신호를 분석하였다.The fluorescence signal of the molecular beacon of the present invention was analyzed for oligonucleotide primers artificially synthesized for specific sites of the nsp2, 3, M and N2 regions of MERS-CoV.
혼성화 후 세척 완충액으로 세척후 200-800nm에서 nanodrop으로 흡광도를 측정하였으며 고유의 피크가 보이는 영역을 토대로 반응유무를 확인하였다.After hybridization, after washing with washing buffer, the absorbance was measured by nanodrop at 200-800nm, and the reaction was confirmed based on the region of inherent peaks.
또한, 스트렙타비딘으로 코팅된 슬라이드 표면에 비오틴이 부착된 수종의 프로브와 프라이머를 혼합하여 이를 유리판에 분주한 다음 2분 후에 공초점 현미경으로 관찰한 결과, MERS-CoV의 nsp2, nsp3, M 그리고 N2의 올리고 뉴클레오티드에 결합에 의해 형광비콘의 신호가 켜져서 적색 형광이 관찰된 것을 알 수 있었다.In addition, several kinds of biotin-attached probes and primers were mixed on the surface of the streptavidin-coated slide, and then dispensed on a glass plate, followed by confocal microscopy two minutes later, and nsp2, nsp3, M and MERS-CoV were observed. It was found that red fluorescence was observed when the signal of the fluorescent beacon was turned on by binding to the oligonucleotide of N2.
<< 실시예Example 3> 3>
MERS-CoV의 nsp2, 3와 M 영역의 특정 영역에 대해 환자검체를 대상으로 분자비콘 시스템 프로브 1pmol (10μl)와 검체 1μl이상 (50pmol)이상을 에펜도로프튜브에 넣고 2분간 실온상태에서 혼성화시킨 다음 이를 nanodrop(Therma, USA)를 이용하여 1μl를 분주하고 uv-spetrum으로 확인하면서 양을 측정한 결과 100pmol에서 가장 강하게 반응하였으며, 이는 해당 바이러스가 존재하는 수 개에서 수천 개가 존재하는 정도이므로 발병 초기에 감염 유무를 확인하기에 적합한 농도범위로 볼 수 있다. For specific areas of nsp2, 3, and M regions of MERS-CoV, 1 pmol (10 μl) of molecular beacon system probes and 1 μl or more (50 pmol) of sample were mixed in an eppendorf tube and hybridized at room temperature for 2 minutes. Next, 1μl was dispensed using nanodrop (Therma, USA) and quantitated by uv-spetrum, and the amount of reaction was the strongest at 100 pmol. It can be seen as a suitable concentration range to confirm the presence of infection.
유리 슬라이드 위에 도말 후 LSM710공초점 현미경하에서 em/ex을 565/635nm파장대에서 확인하여 본 결과 적색 형광의 스팟이 정확히 확인되었으며, 이는 검체양과 형광 이미징과 정확히 일치하는 경향을 보여 분자비콘 시스템으로 최단시간 초스피드로 정량화할 수 있다는 결론을 제시하고 있다. After smearing on a glass slide, em / ex was observed at 565 / 635nm wavelength under the LSM710 confocal microscope. As a result, the spot of red fluorescence was accurately identified, which showed a tendency to exactly match the sample volume and fluorescence imaging. The conclusion is that it can be quantified in ultraspeed.
<실시예 4><Example 4>
중동호흡기 바이러스( MERS - CoV ) 이외 수종의 바이러스에 특이적인 표적서열 을 탐지하기 위한 형광 프라이머의 선정Selection of fluorescent primers to detect target sequences specific to several viruses other than Middle East Respiratory Virus ( MERS - CoV )
본 실시예에서는 중동호흡기 바이러스 (MERS-CoV), 홍역 바이러스 (measles virus), 엔테로바이러스 (enterovirus), 라이노바이러스 (rhinovirus), 사스 연관 코로나바이러스 (SARS-associated coronavirus: SARS-coV), 수두바이러스 (varicella zoster virus : VSV), 아데노바이러스 (adenovirus), 인간 파라인플루엔자바이러스 1 (human parainfluenza virus 1: HPIV 1), 인간 파라인플루엔자바이러스 2 (human parainfluenza virus 2: HPIV 2), 인간 파라인플루엔자바이러스 3 (human parainfluenza virus 3: HPIV 3), 인플루엔자바이러스 A (Influenza virus A : IVA), 인플루엔자바이러스 B (Influenza virus B : IVB) 및 호흡기세포융합바이러스 A (respiratory syncytial virus A : RSVA)와 호흡기세포융합바이러스 B (respiratorysyncytial virus B : RSVB)에 특이적인 표적서열을 탐지할 수 있는 형광 프로브 프라이머 세트를 디자인하였다.In this embodiment, the Middle East respiratory virus (MERS-CoV), measles virus (measles virus), enterovirus (enterovirus), rhinovirus (rhinovirus), SARS-associated coronavirus (SARS-coV), varicella virus ( varicella zoster virus (VSV), adenovirus, human parainfluenza virus 1 (HPIV 1), human parainfluenza virus 2 (HPIV 2), human parainfluenza virus 3 (human parainfluenza virus 3: HPIV 3), Influenza virus A (IVA), Influenza virus B (IVB), and respiratory syncytial virus A (RSVA) and respiratory syncytial virus B (RSV) A set of fluorescent probe primers was designed to detect target sequences specific for respiratorysyncytial virus B (RSVB).
먼저 genebank로부터 호흡기 질환을 야기하는 바이러스의 서열을 입수하고 dnastar 프로그램을 이용하여 이들로부터 각각 보존된 영역을 선발하였다. MERS-CoV의 nsp3와 M영역, N2, 홍역바이러스의 L유전자, 엔테바이러스 UTR(폴리단백질), 라이노바이러스 5UTR (폴리단백질), SARS-CoV(GD69), VZV (ORF54), 아데노바이러스 (HEXON), HPIV1 (HN), HPIV2(HN), HPIV3(HN), IVA (MP), IVB(NP), RSVA(F유전자), RSVB (F 유전자)으로 이들 보존적 영역으로부터 홍역바이러스 ,엔테로바이러스, 라이노바이러스, SARS-Cov, 아데노바이러스, VZV, HPIV1, HPIV2, HPIV3, IVA, IVB, RSVA 및 RSVB에 각각 특이적인 서열의 프라이머쌍을 선정하였다. First, the sequences of viruses causing respiratory diseases were obtained from the genebank, and regions respectively conserved from them were selected using the dnastar program. Nsp3 and M region of MERS-CoV, N2, L gene of measles virus, enterovirus UTR (polyprotein), rhinovirus 5UTR (polyprotein), SARS-CoV (GD69), VZV (ORF54), adenovirus (HEXON) , Measles virus, enterovirus, rhino, from HPV1 (HN), HPIV2 (HN), HPIV3 (HN), IVA (MP), IVB (NP), RSVA (F gene), RSVB (F gene) Primer pairs of sequences specific for virus, SARS-Cov, adenovirus, VZV, HPIV1, HPIV2, HPIV3, IVA, IVB, RSVA and RSVB were selected.
- MERS-CoV 특이적 프로브 (서열번호 3)MERS-CoV specific probe (SEQ ID NO: 3)
ttcgctgtttaccatagaaggtgtaaaggagtttttgtttgagggacaggtaaacagcgttcgctgtttaccatagaaggtgtaaaggagtttttgtttgagggacaggtaaacagcg
- MERS-CoV 229e 특이적 프로브 (서열번호 4)MERS-CoV 229e specific probe (SEQ ID NO: 4)
ttcgctgtcaacttccacactcaatgaatgcggagtggtaaatgtaggaagttgacagcg ttcgctgtcaacttccacactcaatgaatgcggagtggtaaatgtaggaagttgacagcg
- 홍역 바이러스(measles virus) 특이적 프로브 (서열번호 5)Measles virus specific probes (SEQ ID NO: 5)
atggatggatgtrgtgaggaaatggatggatgtrgtgaggaa
- 엔테로바이러스(enterovirus) 특이적 프로브 (서열번호 6)-Enterovirus specific probe (SEQ ID NO: 6)
tcgtaagggcaactctgcagcgtcgtaagggcaactctgcagcg
- 라이노바이러스(rhinovirus) 특이적 프로브 (서열번호 7)Rhinovirus specific probe (SEQ ID NO: 7)
gtcgtaatgagcaattccgggacggtcgtaatgagcaattccgggacg
- 사스 연관 코로나바이러스(SARS-associated coronavirus:SARS-coV) 특이적 프로브 (서열번호 8)SARS-associated coronavirus (SARS-coV) specific probe (SEQ ID NO: 8)
ttcgctgtcatgcaagtcgaagaggtgcaaccatccatgatatcatgacagcg ttcgctgtcatgcaagtcgaagaggtgcaaccatccatgatatcatgacagcg
- 수두바이러스(varicella zoster virus : VSV) 특이적 프로브 (서열번호 9)-Varicella zoster virus (VSV) specific probe (SEQ ID NO: 9)
acccttccatttaaaccactggtatagacccttccatttaaaccactggtatag
- 아데노바이러스(adenovirus) 특이적 프로브 (서열번호 10)Adenovirus specific probes (SEQ ID NO: 10)
gcgctrgayatgacttttgaggtgcgctrgayatgacttttgaggt
- 인간 파라인플루엔자바이러스 1 (human parainfluenza virus 1: HPIV 1) 특이적 프로브 (서열번호 11)Human parainfluenza virus 1: HPIV 1 specific probe (SEQ ID NO: 11)
atgttctgtamtagstgcaggaacaagatgttctgtamtagstgcaggaacaag
- 인간 파라인플루엔자바이러스 2 (human parainfluenza virus 2: HPIV 2) 특이적 프로브 (서열번호 12)Human parainfluenza virus 2: HPIV 2 specific probe (SEQ ID NO: 12)
atctaaccagtatttagcaatgggatctaaccagtatttagcaatggg
- 인간 파라인플루엔자바이러스 3 (human parainfluenza virus 3: HPIV 3) 특이적 프로브 (서열번호 13)Human parainfluenza virus 3: HPIV 3 specific probe (SEQ ID NO: 13)
tggcataaygtgytatcaagaccagtggcataaygtgytatcaagaccag
- 인플루엔자바이러스 A (Influenza virus A :IVA) 특이적 프로브 (서열번호 14)Influenza virus A (IVA) specific probe (SEQ ID NO: 14)
ggctaaagacaagaccratcctgggctaaagacaagaccratcctg
- 인플루엔자바이러스 B (Influenza virus B : IVB) 특이적 프로브 (서열번호 15)Influenza virus B (IVB) specific probe (SEQ ID NO: 15)
aaccagatgatggtcaaagctggrcaaccagatgatggtcaaagctggrc
- 호흡기세포융합바이러스 A (respiratory syncytial virus A : RSVA) 특이적 프로브 (서열번호 16)-Respiratory syncytial virus A (RSVA) specific probe (SEQ ID NO: 16)
ccagcaaagttaytctatcatgtcccagcaaagttaytctatcatgtc
- 호흡기세포융합바이러스 B (respiratory syncytial virus B : RSVB) 특이적 프로브 (서열번호 17)Respiratory syncytial virus B (RSVB) specific probe (SEQ ID NO: 17)
ttyttctgrtcatttgttataggcattyttctgrtcatttgttataggca
1) 프로브의 설계1) Probe Design
각 바이러스 게놈의 표적영역으로부터 프로브를 선정하였으며 그 서열은 염기서열을 22 올리고뉴클레오티드를 포함한 핵산 형광 분자비콘 프로브로 제작되었다.Probes were selected from the target regions of each viral genome, and the sequences were prepared with nucleic acid fluorescent molecular beacon probes containing 22 oligonucleotide sequences.
2) 혼성화 반응2) hybridization reaction
상기 프로브가 합성된 구조를 우선 92℃에서 2분간, 72℃에서 1분간 4℃에서 10분간 처리하고 3차원 구조를 가진 입체 분자비콘 프로브에 QD565, QD525 및 QD705와 소광체인 BHQ1, BHQ2 혹은 BHQ3을 각각 양쪽에 부착시킨 형광 분자비콘 프로브의 아민기와 양식에 따라 용액상태, 나노입자 표면, 유리표면, 막 또는 플라스틱 중합체와 고체 지지체의 표면에 고정화된 카르복실기 화합물에 근거리 링커인 EDC같은 화합물로 연결시켜 프로브 세트를 첨가하여, 실온에서 2분 동안 반응시킴으로써 고정화하였다. 얻어진 혼성체를 상기한 바와 같은 수종의 각 바이러스로부터 준비한 핵산이 포함되어 있는 시료를 상기 바이러스에 특이적인 올리고뉴클레오티드가 고정화되어 있는 상태에 첨가하고 25℃에서 2분 동안 혼성화 반응을 수행하였다.The synthesized structure of the probe was first treated at 92 ° C. for 2 minutes, and at 72 ° C. for 1 minute at 4 ° C. for 10 minutes. Depending on the amine group and the form of the fluorescent molecular beacon probes attached to each side, the probe is connected to a solution such as EDC, which is a near linker, to a solution state, a nanoparticle surface, a glass surface, a membrane, or a carboxyl compound immobilized on the surface of a plastic polymer and a solid support. The set was added and immobilized by reacting for 2 minutes at room temperature. The obtained hybrid was added to the sample containing the nucleic acid prepared from each of several viruses as described above in the state in which the oligonucleotide specific for the virus was immobilized, and hybridization reaction was performed at 25 ° C. for 2 minutes.
<< 실시예5Example 5 > >
수종의 호흡기질환과 관련된 바이러스의 검출Detection of viruses associated with several respiratory diseases
혼성화 후 세척 완충액으로 세척후 200-800nm에서 nanodrop으로 흡광도를 측정하였으며 고유의 피크가 보이는 영역을 토대로 반응유무를 확인하였다.After hybridization, after washing with washing buffer, the absorbance was measured by nanodrop at 200-800nm, and the reaction was confirmed based on the region of inherent peaks.
<< 실시예6Example 6 > >
수종의 Species 호흡기질환탐지하기Respiratory Disease Detection 위한  for 분자비콘Molecular Beacon 기법 반응개념도 Reaction Concept Diagram
바이러스를 증폭하여 정량하는데 요구되는 형광프로브와 소광체 및 상위센서스 프라이머와 하위센스 프라이머로부터 cDNA합성 바이러스를 정량하기 위한 real time PCR을 수행하기 위한 개념도를 나타낸 것이다(도 8).Fig. 8 shows a conceptual diagram for performing real time PCR for quantifying cDNA synthetic virus from fluorescent probes, quencher and higher sensation primers and lower sense primers required to amplify and quantify viruses (Fig. 8).
실험예Experimental Example 1 One
MERS-CoV의 nsp2, 3, M 및 N2 영역의 특정영역에 대해 인공 합성한 올리고뉴클레오티드 프라이머를 대상으로 스트렙토아비딘으로 코팅된 슬라이드표면에 바이오틴이 부착된 수종의 프로브와 프라이머를 혼합하여 이를 유리판에 분주한 다음 이를 2분 후에 공초점 현미경으로 관찰한 결과 MERS-CoV의 nsp2, nsp3, M 그리고 N2의 올리고 뉴클레오티드에 결합에 의해 형광분자비콘의 신호가 켜져서 적색 형광이 관찰된 것을 알 수 있었다(도 9).Oligonucleotide primers artificially synthesized for specific regions of the nsp2, 3, M, and N2 regions of MERS-CoV were mixed with several probes and primers with biotin attached to a slide surface coated with streptoavidine, and then dispensed on a glass plate. Then, after 2 minutes of observation by confocal microscopy, it was found that the signal of fluorescent molecular beacon was turned on by binding to the oligonucleotides of nsp2, nsp3, M and N2 of MERS-CoV and red fluorescence was observed (Fig. 9).
실험예Experimental Example 2 2
MERS-CoV의 nsp2, 3와 M영역의 특정영역에 대해 환자검체를 대상으로 분자비콘 시스템 프로브 1pmol (10μl)와 검체 1μl이상 (50pmol)이상을 에펜도로프튜브에 넣고 2분간 실온상태에서 혼성화를 시킨 다음 이를 nanodrop(Therma, USA)를 이용하여 1μl를 분주하고 uv-spetrum으로 확인하면서 양을 측정한 결과 도 10, 11처럼 100pmol에서 가장 강하게 반응하였으며 이는 해당 바이러스가 존재하는 수개에서 수천개가 존재하는 정도이므로 발병 초기에 감염 유무를 확인하기에 적합한 농도범위라 사료된다. 유리슬라이드 위에 도말후 LSM710 공초점 현미경하에서 em/ex을 565/635nm파장대에서 확인하여 본 결과 적색형광의 스팟이 정확히 확인되어 이는 검체양과 형광 이미징와의 정확히 일치하는 경향을 보여 분자비콘 시스템으로 최단시간 초스피드로 정량화할 수 있다는 결론을 제시하고 있다.For specific areas of nsp2, 3 and M regions of MERS-CoV, 1 pmol (10 μl) of molecular beacon system probes and 1 μl or more (50 pmol) of sample were placed in eppendorf tubes and hybridized at room temperature for 2 minutes. After dispensing 1μl using nanodrop (Therma, USA) and confirming with uv-spetrum, the amount was measured and the reaction was the strongest at 100 pmol, as shown in Figs. 10 and 11. It is considered to be a suitable concentration range to check for infection at the early stage of the onset. After smearing on a glass slide, the em / ex was observed at 565/635 nm wavelength under the LSM710 confocal microscope. As a result, the spot of red fluorescence was accurately identified, which showed the exact coincidence between the sample volume and fluorescence imaging. It concludes that it can be quantified.
실험예Experimental Example 3 3
MERS-CoV의 N2 특정 영역에 대해 환자검체 시료를 통해 분리한 것을 확인할 목적으로 가상의 N2 영역의 게놈을 인공합성하여 이것을 주형으로 sense primer; GGCACTGAGGACCCACGTT (서열번호 18) 1ul, antisense primer; TTGCGACATACCCATAAAAGCA (서열번호 19) 1ul으로, premix 10ul, 가상의 N2 영역의 주형 유전자 100pmol aaactcggcactgaggacccacgttggccccaaattgctgagcttgctcctacagccagtgcttttatgggtatgtcgcaatttaaa (서열번호 20)을 2ul, dH20 11 ul 해서 PCR(폴리머효소 연쇄반응 장치)에서 94℃에서 5분, 95℃에서 30초, 72℃에서 30초, 58℃에서 30초간을 한 사이클로 하여 30사이클을 수행후 72℃에서 5분간 반응후 4℃에서 유지하여 반응을 종결한 것을 1% 아가로즈 젤을 제작하여 분주한 결과 N2 영역의 밴드를 확인하였다. 이를 토대로 메르스 바이러스의 N2영역 존재는 결론적으로 바이러스가 존재한다는 의미로 시사되어지는데 메르스 바이러스는 취급상 주의를 요하므로 real time PCR를 수행시 RT(역전사과정)을 IN VITRO상에서 수행하지 않고 메르스가 의심되는 시료를 생물학적 방어시설이 구축된 음압상태의 실험실 상태에서 수행하기 위해 ONE STEP qPCR을 수행해야 하므로 본 결과물을 토대로 가상의 N2 염기서열을 대상으로 하이를 정량적으로 측정하기 위한 sense primer; GGCACTGAGGACCCACGTT 1ul,, antisense primer; TTGCGACATACCCATAAAAGCA 1ul으로, qPCR용 premix 10ul, 가상의 N2 영역의 주형 유전자 100pmol aaactcggcactgaggacccacgttggccccaaattgctgagcttgctcctacagccagtgcttttatgggtatgtcgcaatttaaa을 2ul, dH20 11ul 해서 100 pmol FAM-CCCCAAATTGCTGAGCTTGCTCCTACA-BHQ1 VIVAL7 (서열번호 21)의 qPCR(폴리머효소 연쇄반응 장치)에서 94℃에서 5분, 95℃에서 25초, 60℃에서 30초를 한 사이클로 하여 45사이클을 수행한 후 정량화한 것을 나타내었다.For the purpose of confirming that the N2 specific region of MERS-CoV was isolated from the patient sample, artificial genome of the virtual N2 region was artificially synthesized and used as a sense primer; GGCACTGAGGACCCACGTT (SEQ ID NO: 18) 1ul, antisense primer; 1 μl of TTGCGACATACCCATAAAAGCA (SEQ ID NO: 19), 100 μl of premix 10 μl, the template gene of the imaginary N2 region, and 2 μl of the PCR reaction polymerase (95) in a PCR (poly) polymerase (95) in a polymer reaction of the polymerase (DH20 11) in a 95% polymerase chain. 30 seconds, 30 seconds at 72 ℃, 30 seconds at 58 ℃ 30 cycles were carried out after the reaction for 5 minutes at 72 ℃ and then maintained at 4 ℃ to terminate the reaction to produce a 1% agarose gel As a result, the band of the N2 region was confirmed. Based on this, the presence of N2 region of MERS virus suggests that virus exists in conclusion. Since MERS virus requires handling precautions, MRS virus does not perform RT (reverse transcription process) on IN VITRO when real time PCR is performed. ONE STEP qPCR should be performed in order to perform suspicious samples in a laboratory under negative pressure with biological defense facilities; GGCACTGAGGACCCACGTT 1ul ,, antisense primer; 1 pL TTGCGACATACCCATAAAAGCA 1 pL, 100 pmol aaactcggcactcggcactgaggacccacgttggccccaaattgctgagcttgctcctacagccagtgcPCRTRCC11AC (1) in the premix 10 ul for qPCR, 100 pGBAA TFCCC-ACCACHG, 5 minutes, 25 seconds at 95 ℃, 30 seconds at 60 ℃ was carried out in one cycle 45 cycles were shown to be quantified.
실험예Experimental Example 4 4
바이러스를 탐지하기 위하여 생검이나 공기중으로 RNAzol 100μl으로 바이러스외피를 파괴시킨 후 분리된 Total RNA를 96well용 plate나 유리판에서 분자비콘 기법 반응개념으로 MERS-CoV의 nsp3와 M영역를 표적으로 하기 위해 탐지 프로브의 5' 말단에는 아민기를 부착하고 여기에 형광체의 카르복실기와 EDC와 1:1:78 비율로 혼합하여 아미노결합을 유도하고 5' 말단에서 8mer(올리고머) 티염기(T)에 biotin을 부착시키고 3' 말단에는 소광체(BHQ; black quencher hole)를 부착하였다. 액체상태의 바이오틴이 부착된 형광프로브와 소광체의 프로브 1nM를 10μl을 바이러스의 RNA가 소량 존재시 plate 바닥에 아비딘이 부착되어 있는 상태에서 프로브가 결합하고 소량의 바이러스를 탐지하여 형광을 얻기 위한 것으로 이를 반응유무를 얻기 위해 합성된 MERS-CoV의 nsp3와 M영역의 일부 게놈을 인공합성하여 (도 12a) 이를 0, 1, 10, 25, 50, 75, 100pmol농도로 90μl되게 상기 프로브와 반응시켜 시너지 HT의 multiplate(BioTek)를 이용하여 25℃에서 565/635nm파장대에서 형광강도를 측정한 결과 다음과 같이 nsp3영역은 0, 1, 24±6, 56±15, 231±43, 290±11, 450±21AFI(임의 형광도)였고 M은 0, 1, 16±2, 57±9, 189±23, 234±18, 357±43 AFI(임의 형광도)였다(도 12b).In order to detect viruses, the virus was destroyed by 100 μl of RNAzol in biopsy or air, and then the total RNA separated from 96well plate or glass plate was analyzed by the molecular beacon technique. At the 5 'end, an amine group is attached and the carboxyl group of the phosphor is mixed with EDC at a ratio of 1: 1: 78 to induce an amino bond. At the 5' end, biotin is attached to the 8mer (oligomer) thibase (T) and 3 ' At the end, a quencher (BHQ) was attached. 10μl of fluorescent probe and 1nM of quencher with biotin attached to liquid state are used to obtain fluorescence by detecting a small amount of virus when avidin is attached to the bottom of plate when virus RNA is present in small amount. In order to obtain the reaction, the synthesized nsp3 of MERS-CoV and a part of the genome of the M region were artificially synthesized (FIG. 12A) and reacted with the probe to 90 μl at 0, 1, 10, 25, 50, 75, and 100 pmol concentrations. Fluorescence intensities were measured at 565 / 635nm at 25 ° C using a multiplate (BioTek) of Synergy HT. The following nsp3 areas were 0, 1, 24 ± 6, 56 ± 15, 231 ± 43, 290 ± 11, 450 ± 21 AFI (optional fluorescence) and M was 0, 1, 16 ± 2, 57 ± 9, 189 ± 23, 234 ± 18, 357 ± 43 AFI (optional fluorescence) (FIG. 12B).
실험예Experimental Example 5 5
중동호흡기 바이러스의 nsp3와 M영역을 진단하기 위한 특정영역의 게놈인 ATAGAAGGTGTAAAGGAG (서열번호 22), TGCGCCTCAGTGAGTTGCGTCA (서열번호 23)를 대상으로 5‘영역에는 형광체와 자기인식 개폐를 조절하게 링커 유전자 서열인 TTCGCTGTTTACC (서열번호 24)를 3' 영역은 5' 영역과 바이러스의 일부 영역을 인식하게 하는 염기서열인 TGTTTGAGGGACAGGTAAACAGCG(nsp3) (서열번호 25)와 TATTAGACATGATTATACAGCG (서열번호 26) 염기서열로 구성된 메르스 탐지 nsp3(62염기)와 M(58염기)프로브를 제작하였다(도 13).Targets of ATAGAAGGTGTAAAGGAG (SEQ ID NO: 22) and TGCGCCTCAGTGAGTTGCGTCA (SEQ ID NO: 23), genomes of specific regions for diagnosing Nsp3 and M regions of the Middle East respiratory virus, are located in the 5 'region of the linker gene sequence TTCGCTGTTTACC. (SEQ ID NO: 24) is a MERS detection nsp3 (SEQ ID NO: 24) consisting of the base sequence TGTTTGAGGGACAGGTAAACAGCG (nsp3) (SEQ ID NO: 25) and TATTAGACATGATTATACAGCG (SEQ ID NO: 26), which is a 3 'region to recognize a 5' region and some regions of the virus. 62 base) and M (58 base) probes were prepared (FIG. 13).
실험예Experimental Example 6 6
수종의 호흡기질환 바이러스를 대상으로 본 프로브를 활용하는 이유는 이들 바이러스들은 급성기 호흡질환을 야기하는 바이러스인 점, 치명적인 높은 사망률과 전파속도가 아주 높은 이유로 감염 후 호흡기 바이러스의 특정 바이러스를 확인하기 위해서는 어느 정도의 바이러스 titer(기준량)이 존재하는 감염 후 2주 후에 기존방법인 qRT-PCR(정량 역전사유전자연쇄증폭법)으로 확인하는 방법과 달리 감염 후 초동방역과 음압격리를 통한 치료를 하기 위한 시간적이고 임상 안전상 바이러스의 복제초기인 PLOpro의 nsp3와 바이러스의 복제중기단계인 바이러스의 외피 막단백질이 합성과정 유무를 확인하므로 본 메르스의 감염 시간을 예측하기 위하여 표적 염기서열을 선정하기 위함이다(도 14).The reason for using this probe in several respiratory disease viruses is that these viruses cause acute respiratory disease. Two weeks after infection with a degree of viral titer (quantity), unlike the conventional method, qRT-PCR (quantitative reverse transcriptase chain amplification method) is used to confirm the time-based treatment after initial infection and negative pressure isolation. For clinical safety, it is to select a target sequence to predict the infection time of the MERS because nSP3 of PLOpro, which is the initial replication of the virus, and the envelope membrane protein of the virus, which is the intermediate stage of the replication of the virus, are synthesized. ).
실험예Experimental Example 7 7
메르스의 2개의 비구조 단백질영역인 ORF1a와 ORF1b(오알에프:단백질 개시시작점과 종결점 간의 한 세트) 그리고 E, M, N, S 같은 바이러스의 껍질에 해당되는 구조단백질로 구성되어 있는데 일반적으로 바이러스 감염을 위해 유전자부분인 ORF는 qRT-PCR을 수행하고 E, M, N, S는 ELISA(면역효소측정법)으로 하거나 qRT-PCR법을 수행하는데 기존 CDC나 WHO의 지정 방법은 hsp3와 N를 분석하는 qRT-PCR법을 통해 정량하거나 존재유무를 확인하는 방법을 예시하지만 이는 탐지하기 위한 바이러스의 양이 확보되는 시기인 2주 정도 시간이 소요되고 바이러스의 RNA 게놈을 추출하는 시간과 비용이 많이 소요되는 문제점이 있지만 본 발명에서 제시하고자 하는 방법은 감염 초기에 감지하기 위한 목적으로 감염 초기에 소량으로 저비용으로 단시간에 결과를 확보할 수 있다는 장점이 있다(도 15).It consists of two nonstructural protein regions of MERS, ORF1a and ORF1b (a set of proteins between the start and end points of the protein) and structural proteins corresponding to the shells of viruses such as E, M, N, and S. For viral infection, ORF, a gene part, performs qRT-PCR, and E, M, N, and S use ELISA (immunoenzyme assay) or qRT-PCR method, and existing CDC or WHO designation method uses hsp3 and N. It exemplifies how to quantify or confirm the presence by analyzing qRT-PCR method, but this takes about two weeks, when the amount of virus to be detected is obtained, and the time and cost of extracting the RNA genome of the virus is high. Although there is a problem in the present invention, the method proposed in the present invention has the advantage that the result can be obtained in a short time at a low cost in a small amount at the beginning of the infection for the purpose of detecting it at the beginning of the infection. Can (15).
실험예Experimental Example 8 8
일반적으로 바이러스 감염을 위해 유전자부분인 ORF(단백질합성 세트)를 qRT-PCR을 수행하는데 이를 수행하기 위해 프라이머가 요구되는데 nsp3의 sense primer; TTTTAACGCTCACCCCACG (서열번호 27), antisense primer; CCACAACTTCCACATCAATG (서열번호 28)가 택맨프로브는 FAM-CCCAACCTACATTTACCACTCCGCA-BHQ1 (서열번호 29), M영역은 sense primer;AGGGTGTACCTCTTAATGCCAATTCT (서열번호 30), antisense primer;TCTGTCCTGTCTCCGCCAATA (서열번호 31)가 택맨프로브는 FAM-ACCCCCTGCGCAAAATGCTGGG-BHQ1 (서열번호 32)으로 해서 qRT-PCR을 수행시 기존 택맨 프로브와 사이그로 의 반응성과 정확도가 거의 손색이 없을 정도로 정확하게 우수하였다(도 16).In general, qRT-PCR is performed on ORF (protein synthesis set), which is a gene part for viral infection, and a primer is required to perform this. The sense primer of nsp3; TTTTAACGCTCACCCCACG (SEQ ID NO: 27), antisense primer; CCACAACTTCCACATCAATG (SEQ ID NO: 28) is the TaqMan probe, FAM-CCCAACCTACATTTACCACTCCGCA-BHQ1 (SEQ ID NO: 29), the M area is the sense primer; AGGGTGTACCTCTTAATGCCAATTCT (SEQ ID NO: 30), antisense primer; TCTGTCCTGTCTPROGCCCACGACGGGCCCCCGCTGATGCC When performing qRT-PCR with -BHQ1 (SEQ ID NO: 32), the responsiveness and accuracy of the existing Taqman probe and sieg were almost excellent (Figure 16).
실험예Experimental Example 9 9
메르스 바이러스가 감염초기에 유전자가 소량 존재시 시험관 내에서 바이러스 존재유무를 확인하기란 어려운데 이를 극복하기 위해서는 프로브의 존재하는 형광체의 강도가 기존 프로브의 형광체로는 불가하지만 본 발명에서는 이를 극복하기 위해 기존 형광체인 FAM 대신 QD525나 QD565를 사용하므로 반감기와 강도면에서 우수한 형광체를 해 극복하였고(도 17a) 이를 각각의 프로브 1nM를 10μl으로 소량의 표적 DNA나 RNA를 탐지하는데 유용한 것으로 상기 실험예의 방법과 동일하에서 실제 nsp3는 0, 1, 50, 100pmol에서 0, 5, 156±23, 342±34이고 M은 0, 10±0, 121±15, 290±32으로 나타내었다(도 17b).It is difficult to check the presence of virus in vitro when MERS virus is present in the early stage of infection. To overcome this, the intensity of the fluorescent substance of the probe is not possible with the fluorescent substance of the existing probe, but in the present invention, Since QD525 or QD565 is used instead of FAM, which is a conventional phosphor, it overcomes the excellent phosphor in terms of half-life and intensity (FIG. 17A). This method is useful for detecting a small amount of target DNA or RNA with 10 nL of each probe 1 nM. In the same way, the actual nsp3 is 0, 5, 156 ± 23, 342 ± 34 at 0, 1, 50, and 100 pmol, and M is expressed as 0, 10 ± 0, 121 ± 15, 290 ± 32 (FIG. 17B).
실험예Experimental Example 10 10
특정 담체표면에 UFO강한 빛처럼 형광빛을 내게끔 형광체와 소광체가 연결된 분자비콘을 다양하게 표현하게 사이즈가 다른 양자점 나노입자를 연결하여 급성 호흡기 질환바이러스 다른 종류가 저준위로 있거나 동일 바이러스의 다른 탐지 영역을 감지 가능한 분자비콘기법(도 18a) 을 통해 바이러스를 탐지하기 위한 형광프로브와 소광체를 통해 바이러스를 탐지하기 위한 각각의 프로브 1nM를 10μl으로 실험예 4처럼 동일조건하에서 UFO분자비콘 방법으로 500nm의 나노입자의 SiO2 같은 산화철류의 표면에 개질화하여 인공적인 SARS-COV의 N, 메르스의 N, AI의 H1, RSV의 A 영역의 염기를 합성하여 100 pmol을 통해 확인한 반응정도는 0, 5, 156±23, 342±34을 나타내었다(도 18b).Different types of quantum dot nanoparticles connect different sized quantum dot nanoparticles to express various molecular beacons connected with phosphors and quencher to emit fluorescent light like UFO strong light on a specific carrier surface. 10 nm of each probe 1 nM for detecting a virus through a fluorescent probe and a quencher to detect the virus through a molecular beacon technique (Fig. 18a) capable of detecting 500 nm of the UFO molecular beacon method under the same conditions as in Experiment 4 Modified on the surface of iron oxides such as SiO 2 of nanoparticles, and synthesized the bases of artificial SARS-COV N, MERS N, AI H1, RSV A region, and confirmed the reaction degree through 100 pmol. 5, 156 ± 23 and 342 ± 34 are shown (FIG. 18B).
실험예Experimental Example 11 11
상기 나노입자 표면에 다양한 호흡기질환 바이러스를 동시에 관련된 바이러스와 혼재시 각각의 바이러스에 특이적인 프로브를 합성하여 공여체 형광체를 각각 다르게 부착하여 수용자를 통해 상이한 바이러스와 관계를 동시에 탐지 가능한 분자비콘 기법 반응도를 통해 바이러스를 탐지하기 위한 형광프로브를 통해 반응시 30분전후의 반응결과치는 각각의 프로브 1nM를 10μl으로 실험예 4처럼 동일조건하에서 분당 1회씩 측정한 공여체의 형광수치는 SARS-COV의 N, 메르스의 N, AI의 H1, RSV의 A에서 각각 2, 129±15, 234±34, 145±24, 256±22이고 수용자의 수치는 45±2, 23±6, 67±3, 25±8, 256±22이357±43였으며 30분 후에 반응결과치는 0, 21±2, 33±3, 15±5, 25±2이고 수용자의 수치는 211±25, 321±36, 267±16, 190±18였는데 이는 다양한 바이러스가 존재하더라도 충분히 판정이 가능하고 시간이 경과함에 따라 여기-저기 상태에서 에너지 변화가 충분히 전환하는 것은 바이러스가 구분될 수 있음을 알 수 있다(도 19).When mixed with a virus related to various respiratory diseases simultaneously on the surface of the nanoparticles by synthesizing a probe specific to each virus and attaching donor phosphors differently through the molecular beacon technique reactivity to detect the relationship with different viruses through the recipient at the same time The reaction result of 30 minutes before and after the reaction through the fluorescent probe for detecting the virus was 10 μl of each probe 1 nM, and the fluorescence value of the donor measured once per minute under the same conditions as in Experiment 4 was determined by N, MERS of SARS-COV. N, AI H1, RSV A, 2, 129 ± 15, 234 ± 34, 145 ± 24, 256 ± 22, respectively, and the numbers of the recipients are 45 ± 2, 23 ± 6, 67 ± 3, 25 ± 8, 256 ± 22 was 357 ± 43, and after 30 minutes, the response was 0, 21 ± 2, 33 ± 3, 15 ± 5, 25 ± 2, and the number of recipients was 211 ± 25, 321 ± 36, 267 ± 16, 190 ±. 18, which is enough to determine if a variety of viruses are present. Here over time - it is enough to switch to the energy change in the status can be seen that there can be viruses are separated (Fig. 19).
실험예Experimental Example 12 12
단일 바이러스를 복제시기를 알 수 있도록 표적 유전자를 동시에 감지하거나 관련된 호흡기 바이러스와 혼재시 상호 구분되게 하기 위해 다양한 형광체를 통해 바이러스를 탐지 가능한 분자비콘 기법을 통해 500nm의 나노입자의 SiO2 같은 산화철류의 표면에 개질화하여 각각의 프로브 1nM를 10μl으로 실험예 4처럼 동일조건하에서 MB자체, 인공적인 MERS nsp3, S, E, N, nsp5, M영역의 염기를 합성하여 100 pmol을 대상으로 확인한 반응정도는 1, 1234±341, 24±5, 56±8, 27±2, 678±78, 23±4을 나타내었는데 이는 바이러스가 우선 복제시기에 따라 탐지 정도가 다르게 감지할수 있음을 알 수 있다(도 20b).Molecular beacons such as SiO 2 of 500 nm nanoparticles can be detected by molecular beacons, which can detect viruses through various phosphors to simultaneously detect target genes to identify a single virus when it is replicated or to distinguish them from their respiratory viruses. Modified on the surface, each probe 1nM to 10μl synthesized in the MB itself, artificial MERS nsp3, S, E, N, nsp5, M base under the same conditions as in Experiment 4, the reaction degree confirmed in 100 pmol Represents 1, 1234 ± 341, 24 ± 5, 56 ± 8, 27 ± 2, 678 ± 78, 23 ± 4, which indicates that the degree of detection of the virus is different depending on the time of replication. 20b).
실험예Experimental Example 13 13
바이러스끼리 진화상 동질성을 알기 위한 기존바이러스와 출현 바이러스 간의 관계를 유사성을 규명하기 위해 탐지 가능한 분자비콘 기법 반응개념도를 통해 기존 바이러스와 대조하여 탐지하기 위한 기존 바이러스의 게놈과 MERS-COV의 변이성을 확인하기 위한 형광프로브와 소광체를 통해 바이러스를 탐지법으로 유브이 형광빛이 있는 곳에서 확인한 결과 기존바이러스와 달리 변이성인 경우 분자형광비콘으로 인한 형광빛이 나타냄을 알 수 있다(도 21).Detects the variability of the MERS-COV and the genome of the existing virus for detection against the existing virus by detecting the molecular beacon technique reaction concept to detect the similarity between the existing virus and the emergent virus to know evolutionary homogeneity among viruses As a result of detecting the virus through the fluorescent probe and the quencher to detect the presence of the fluorescent light in the presence of fluorescent light, it can be seen that the fluorescent light due to the molecular fluorescent beacons, unlike the existing virus (Fig. 21).
실험예Experimental Example 14 14
바이러스를 간이적으로 스트립키트로 탐지 가능한 분자비콘 기법 반응 개념도를 통해 이미 알고 있는 바이러스의 S유전자에 대한 항체를 표지하여 대조군으로 사용하고 실험군으로 가상의 복제 초기에 복제하는 nsp3의 영역을 측정하기 위해 가상 유전자 nsp3와 여기에 40nm사이즈의 나노금입자 표면에 분자비콘 시스템인 프로브를 부착형 시료와 반응시켜 스트립에 분리시 대조군은 대조군만이 실험군은 대조군과 동시에 nsp3가 있는 경우 반응하여 밴드가 하나 더 나타내어 2개의 밴드가 나타냄으로 측정하고자 하는 nsp3의 존재를 간이적으로 측정이 가능하였다(도 22).Molecular Beacon Technique for the Simple Detection of Viruses with StripKit Reactions To measure the area of nsp3 that labels antibodies against known S genes of a known virus as a control and replicates them at the beginning of virtual replication in experimental groups When the spontaneous gene nsp3 reacts with the attached sample by attaching a molecular beacon system probe on the surface of the nano-gold particles of 40 nm size to the adherent sample, the control group only reacts with the nsp3 at the same time as the control group. As two bands are shown, the presence of nsp3 to be measured was simplified (FIG. 22).
실험예Experimental Example 15 15
공기속에 존재하는 바이러스를 민들레 홀씨처럼 시약이 분무되면서 간이적으로 공기속에 존재하는 바이러스를 UV 광 존재하에서 가능한 분자비콘 기법으로 공기속에 바이러스가 존재시 분무기속에 RNAzol을 일정하게 분무하면 바이러스의 입자가 파괴되면서 유전자가 노출되는데 이때 분자비콘 프로브 1nM를 10μl를 동시에 분무하면 공기속의 nsp3의 존재를 확인하기 위해 유브이를 조사하면 적색이나 녹색형광이 존재시 바이러스의 존재를 확인할 수 있다(도 23).Molecular beacon technique is possible by simply spraying the virus present in the air like dandelion hole seeds in the presence of UV light in the presence of UV light. When virus is present in the air, RNAzol is sprayed in the atomizer to destroy the particles of the virus. When the gene is exposed, at the same time by spraying 10μl of the molecular beacon probe 1nM at the same time irradiated with UV to confirm the presence of nsp3 in the air can be confirmed the presence of the virus in the presence of red or green fluorescence (Fig. 23).
실험예Experimental Example 16 16
공기, 물체표면이나 물속에 속에 존재하는 바이러스가 특정 반응 물체에 접촉시 분자비콘 시스템처럼 카멜레온의 색상이 변하는 방법을 이용하여 실시간으로 바이러스를 모니터링이 가능한 분자비콘 기법으로 인공 카멜레온 장난감의 표면에 분자비콘 프로브 1nM를 10μl으로 도말하여 실온상태에서 공기나 액체에 노출시 유브이 광존재하에서 QD405, 505, 525, 565, 597,635의 다양한 형광이 청색, 노랑색, 녹색, 적색, 심홍색 등으로 수시로 변하게 하는 것을 통해 바이러스가 존재를 탐지할 수 있었다(도 24).Molecular Beacon on the surface of artificial chameleon toy with molecular beacon technology that can monitor the virus in real time by changing the color of chameleon like molecular beacon system when virus in air, object surface or water comes into contact with specific reaction object By spreading 1 nM of the probe to 10 μl, the virus causes the various fluorescence of QD405, 505, 525, 565, 597,635 to change to blue, yellow, green, red, magenta, etc. when exposed to air or liquid at room temperature. Was able to detect the presence (FIG. 24).
실험예 17 농도별로 바이러스 PLOpro nsp3유전자의 탐지 Experimental Example 17 Detection of Virus PLOpro nsp3 Gene by Concentration
호흡기 바이러스를 대상으로 229E, SARS-COV, MERS-COV PLOpro nsp3를 대상으로 농도별로 프로브 1nM를 10μl으로 혼성화후 200-800nm에서 nanodrop으로 흡광도를 측정하였으며 고유의 피크가 보이는 영역을 토대로 반응유무를 확인하였는데 도 18처럼 nanodrop200에서 확인한 결과 MERS-CoV PLpro nsp3프로브 1nM를 10μl으로 특정 파장대에서 농도의존적으로 260nm흡광도가 급격히 증가되고 229E나 SARS-CoV 경우 100pmol는 전혀 반응을 하지 않았는데 이는 특정 영역에만 정확히 결합해야 반응하기 때문이라 사료되어진다(도 25).The respiratory virus hybridized 1nM to 10μl of 229E, SARS-COV, and MERS-COV PLOpro nsp3 for each concentration, and measured the absorbance with nanodrop at 200-800nm. As shown in nanodrop200, as shown in FIG. 18, MnS-CoV PLpro nsp3 probe 1nM was 10μl, and the absorbance of 260nm was rapidly increased depending on the concentration in a specific wavelength range, and 100pmol did not react at all in 229E or SARS-CoV. It is considered that the reaction (Fig. 25).
실험예 18 농도별로 바이러스 M유전자의 탐지 Experimental Example 18 Detection of Virus M Gene by Concentration
호흡기 바이러스인 MERS-COV의 M영역을 대상으로 농도별로 프로브 1nM를 10μl으로 혼성화 후 마이크로파이펫으로 1μl을 200-800nm에서 nanodrop의 반응구에 260nm 흡광도를 측정하였으며 고유의 피크가 보이는 영역을 토대로 확인하였는데 도 19처럼 nanodrop200에서 확인결과 M프로브가 특정 파장대에서 농도의존적으로 흡광도가 증가되는데 기존 방법으로 탐지할 수 없는 농도에서 충분히 감지됨을 알수 있다(도 26).In the M region of the respiratory virus, MERS-COV, hybridized 1nM probe to 10μl for each concentration, and then measured the absorbance of 260nm in the nanodrop reaction zone at 200-800nm with a micropipette. As shown in FIG. 19, the nanodrop 200 shows M probes having a concentration-dependent increase in absorbance at a specific wavelength band, but are sufficiently detected at concentrations that cannot be detected by the conventional method (FIG. 26).
실험예 19 농도별로 MERS-CoV바이러스 유전자의 형광이미지 Experimental Example 19 Fluorescence Image of MERS-CoV Virus Gene by Concentration
호흡기 바이러스인 MERS-COV의 nsp3영역을 대상으로 농도별로 프로브 1nM를 10μl으로 혼성화 후 마이크로파이펫으로 1μl을 200-800nm에서 nanodrop으로 260nm흡광도를 측정하였으며 고유의 피크가 260nm에서 보이는 영역을 토대로 확인하였는데 도 27처럼 nanodrop200에서 확인결과 nsp3프로브가 특정 파장대에서 농도의존적으로 LSM710 공초점 현미경(칼자이즈,독일)으로 관찰시 565nm/635nm에서 형광을 측정하였는데 기존 방법으로 탐지할 수 없는 농도에서 단시간에 충분히 감지됨을 알수 있다(도 27).In the nsp3 region of the respiratory virus, MERS-COV, 1 nM of the probe was hybridized to 10 μl according to the concentration, and then 1μl was measured using a micropipette at 200-800nm and 260nm at a nanodrop. As shown in nanodrop200, the nsp3 probe was measured at 565nm / 635nm when observed with LSM710 confocal microscope (Kalize, Germany) in a concentration-dependent manner at a specific wavelength range, and it was detected in a short time at a concentration that could not be detected by conventional methods. It can be seen (Fig. 27).
실험예 20 농도별로 바이러스 N2유전자의 탐지 Experimental Example 20 Detection of Virus N2 Gene by Concentration
호흡기 바이러스인 MERS-COV의 N2영역을 대상으로 농도별로 혼성화후 프로브 1nM를 10μl으로 혼성화 후 마이크로파이펫으로 1μl을 200-800nm에서 nanodrop의 반응구에 분주하고 260nm흡광도를 측정하였으며 고유의 피크가 보이는 영역을 토대로 확인하였는데 도 22처럼 nanodrop200에서 확인결과 N2프로브가 특정 파장대에서 농도의존적으로 흡광도가 증가되는데 기존 방법으로 탐지할 수 없는 농도에서 충분히 감지됨을 알 수 있다(도 10).After hybridizing by concentration to the N2 region of the respiratory virus, MERS-COV, hybridize 1nM probe to 10μl, and then disperse 1μl with a micropipette at 200-800nm to the nanodrop reaction zone, and measure the absorbance at 260nm. As shown in FIG. 22, the results of the nanodrop200 confirm that the N2 probe is absorbed in a concentration-dependent manner in a specific wavelength range and is sufficiently detected at a concentration that cannot be detected by the conventional method (FIG. 10).
실험예 21 농도별로 바이러스 nsp2유전자의 탐지 Experimental Example 21 Detection of Virus nsp2 Gene by Concentration
호흡기 바이러스인 MERS-COV의 nsp2영역을 대상으로 농도별로 프로브 1nM를 10μl으로혼성화 후 1μl을 마이크로파이펫으로 나노드랍 반응구에서 200-800nm에서 흡광도를 측정하였으며 고유의 피크가 보이는 영역을 토대로 확인하였는데 도 23처럼 nanodrop200에서 확인결과 N2프로브가 특정 파장대에서 농도의존적으로 흡광도가 증가되는데 기존 방법으로 탐지할 수 없는 농도에서 충분히 감지됨을 알 수 있다(도 11). In the nsp2 region of the respiratory virus, MERS-COV, 1 nM of the probe was hybridized to 10 μl according to the concentration, and then 1 μl was measured using a micropipette at 200-800 nm in the nanodrop reaction zone. As shown in the nanodrop200, as shown in FIG. 23, the N2 probe has a concentration-dependent increase in absorbance at a specific wavelength range, indicating that the N2 probe is sufficiently detected at a concentration that cannot be detected by the conventional method (FIG. 11).
실험예 22 농도별로 바이러스 N2유전자 증폭 Experimental Example 22 Amplification of Virus N2 Gene by Concentration
호흡기 바이러스인 MERS-COV의 N2영역을 인공적으로 합성하여 농도별로 희석후 1,10,100pmol으로 주형으로 정량 유전자연쇄반응을 PCR premix 12μl, 증류수 5μl, N2타켓 주형 합성물 1μl, 프라이머 각 1μl으로 해서 95℃ 2분, 95℃ 30초, 72℃ 30초, 60℃ 30초로 45사이클으로 하여 최종적으로 72℃에서 5분을 유전자연쇄반응을 통해 합성한 것으로 1% 아가로즈에서 반응을 용출한 결과 밴드가 보인 곳에는 농도의존적으로 증가된 밴드를 확인하였다. 따라서 기존 유전자 증폭기법과 동일한 조건하에서도 소량의 표적 유전자만이 있어도 충분히 바이러스의 존재를 확인할 수 있다(도 9).Artificial synthesis of the N2 region of the respiratory virus MERS-COV was diluted by concentration, and then quantitatively determined as a template at 1,10,100 pmol. After 45 cycles of 2 min, 95 ° C 30 sec, 72 ° C 30 sec, and 60 ° C 30 sec, 5 min at 72 ° C was finally synthesized by a gene chain reaction. The reaction was eluted at 1% agarose. Where the concentration-dependently increased bands were identified. Therefore, even if there is only a small amount of the target gene under the same conditions as the conventional gene amplifier method, it is possible to sufficiently confirm the presence of the virus (Fig. 9).
실험예 23 농도별로 바이러스 N2유전자 정량화 Experimental Example 23 Quantification of Virus N2 Genes by Concentration
호흡기 바이러스인 MERS-COV의 N2영역을 인공적으로 합성하여 농도별로 희석후 1,10,100pmol으로 주형으로 실험예 7에서처럼 정량 유전자연쇄반응을 qPCR premix 10μl, 증류수 5μl, N2타켓 택맨프로브2μl, 표적 주형 합성물 1μl, 플라이머 각 1μl으로 해서 95℃ 2분, 95℃ 30초, 60℃ 30초로 45사이클으로 하여 최종적으로 60℃에서 5분을 통해 종결하여 사이클이 증가할수록 유전자 증폭이 증가됨을 확인되었다. 따라서 본 발명에선 MERS-COv를 감지하기 위한 최소의 감염시료에서도 명확히 존재 유무를 확인할 수 있으며 단시간에 정확히 서로 다른 바이러스도 감지할 수 있음을 알 수 있으며 감염 초기에 매우 민감하게 감지할 수 있음을 나타내었다(도 28). Artificially synthesize the N2 region of the respiratory virus MERS-COV and dilute it by concentration to 1,10,100 pmol, and then quantitatively analyze the quantitative gene chain reaction as in Experiment 7. It was confirmed that the gene amplification increased as cycles were increased by ending 45 cycles at 95 ° C for 2 minutes, 95 ° C for 30 seconds, and 60 ° C for 30 seconds with 1 μl and 1 μl for each primer. Therefore, in the present invention, it can be clearly seen that even in the minimum infectious sample for detecting MERS-COv, it can detect exactly different viruses in a short time, and can be detected very sensitively at the early stage of infection. (FIG. 28).
서열번호 1: MERS-CoV nsp3 영역을 감지하는 올리고뉴클레오티드SEQ ID NO: 1: Oligonucleotide Detecting MERS-CoV nsp3 Region
서열번호 2: MERS-CoV M 영역을 감지하는 올리고뉴클레오티드SEQ ID NO 2: Oligonucleotide sensing the MERS-CoV M region
서열번호 3: MERS-CoV 특이적 프로브SEQ ID NO: 3: MERS-CoV specific probe
서열번호 4: MERS-CoV 229e 특이적 프로브SEQ ID NO: 4: MERS-CoV 229e specific probe
서열번호 5: 홍역 바이러스(measles virus) 특이적 프로브SEQ ID NO 5: Measles virus specific probe
서열번호 6: 엔테로바이러스(enterovirus) 특이적 프로브SEQ ID NO 6: Enterovirus specific probe
서열번호 7: 라이노바이러스(rhinovirus) 특이적 프로브SEQ ID NO: 7: rhinovirus specific probe
서열번호 8: SARS-CoV 특이적 프로브SEQ ID NO: 8: SARS-CoV specific probe
서열번호 9: 수두바이러스(VSV) 특이적 프로브SEQ ID NO: 9: Varicella virus (VSV) specific probe
서열번호 10: 아데노바이러스(adenovirus) 특이적 프로브SEQ ID NO: 10 adenovirus specific probe
서열번호 11: 인간 파라인플루엔자 바이러스 1(HPIV 1) 특이적 프로브SEQ ID NO: 11: Human parainfluenza virus 1 (HPIV 1) specific probe
서열번호 12: 인간 파라인플루엔자 바이러스 2(HPIV 2) 특이적 프로브SEQ ID NO: 12 Human parainfluenza virus 2 (HPIV 2) specific probe
서열번호 13: 인간 파라인플루엔자 바이러스 3(HPIV 3) 특이적 프로브SEQ ID NO: 13: Human parainfluenza virus 3 (HPIV 3) specific probe
서열번호 14: 인플루엔자바이러스 A(IVA) 특이적 프로브SEQ ID NO: 14 Influenza virus A (IVA) specific probe
서열번호 15: 인플루엔자바이러스 B(IVB) 특이적 프로브SEQ ID NO: 15 Influenza virus B (IVB) specific probe
서열번호 16: 호흡기세포융합바이러스 A(RSVA) 특이적 프로브SEQ ID NO: 16: Respiratory syncytial virus A (RSVA) specific probe
서열번호 17: 호흡기세포융합바이러스 B(RSVB) 특이적 프로브SEQ ID NO: 17 Respiratory syncytial virus B (RSVB) specific probe
서열번호 18: MERS-CoV N2 센스 프라이머(sense primer)SEQ ID NO: 18 MERS-CoV N2 sense primer
서열번호 19: MERS-CoV N2 안티센스 프라이머(antisense primer)SEQ ID NO: 19 MERS-CoV N2 antisense primer
서열번호 20: MERS-CoV N2 템플레이트(template)SEQ ID NO: 20 MERS-CoV N2 Template
서열번호 21: MERS-CoV N2 프로브SEQ ID NO: 21 MERS-CoV N2 probe
서열번호 22: MERS-CoV N2 nsp3 검출 시퀀스(detection sequence)SEQ ID NO: 22 MERS-CoV N2 nsp3 detection sequence
서열번호 23: MERS-CoV M 검출 시퀀스(detection sequence)SEQ ID NO: 23 MERS-CoV M detection sequence
서열번호 24: 링커 유전자(linker gene)SEQ ID NO: 24 linker gene
서열번호 25: nsp3 검출(detection)SEQ ID NO: 25 nsp3 detection
서열번호 26: M 검출(detection)SEQ ID NO: 26 M detection
서열번호 27: nsp3 센스 프라이머(sense primer)SEQ ID NO: 27 nsp3 sense primer
서열번호 28: nsp3 안티센스 프라이머(antisense primer)SEQ ID NO: 28 nsp3 antisense primer
서열번호 29: nsp3 택맨프로브(TaqMan probe)SEQ ID NO: 29 nsp3 TaqMan probe
서열번호 30: M 센스 프라이머(sense primer)SEQ ID NO: 30 M sense primer
서열번호 31: M 안티센스 프라이머(antisense primer)SEQ ID NO: 31 M antisense primer
서열번호 32: M 택맨프로브(TaqMan probe)SEQ ID NO: 32 M TaqMan probe

Claims (10)

  1. 서열번호 1의 염기서열로 이루어진 중동호흡기증후군 코로나바이러스(MERS-CoV) 검출용 올리고뉴클레오티드.Oligonucleotide for detecting Middle East respiratory syndrome coronavirus (MERS-CoV) consisting of the nucleotide sequence of SEQ ID NO: 1.
  2. 서열번호 2의 염기서열로 이루어진 중동호흡기증후군 코로나바이러스(MERS-CoV) 검출용 올리고뉴클레오티드.Oligonucleotide for detecting Middle East respiratory syndrome coronavirus (MERS-CoV) consisting of the nucleotide sequence of SEQ ID NO: 2.
  3. 제1항에 있어서,The method of claim 1,
    상기 올리고뉴클레오티드는 중동호흡기증후군 코로나바이러스의 nsp3 (non-structural protein 3) 유전자 부위를 감지하는 영역을 포함하는 것을 특징으로 하는 올리고뉴클레오티드.The oligonucleotide is oligonucleotide, characterized in that it comprises a region for detecting the nsp3 (non-structural protein 3) gene region of the Middle East respiratory syndrome coronavirus.
  4. 제2항에 있어서,The method of claim 2,
    상기 올리고뉴클레오티드는 중동호흡기증후군 코로나바이러스의 구조단백질 M 유전자 부위를 감지하는 영역을 포함하는 것을 특징으로 하는 올리고뉴클레오티드.The oligonucleotide is oligonucleotide, characterized in that it comprises a region for detecting the structural protein M gene region of Middle East respiratory syndrome coronavirus.
  5. 서열번호 1 또는 서열번호 2의 염기서열로 이루어진 올리고뉴클레오티드; 상기 올리고뉴클레오티드의 5'-말단에 연결된 형광체; 및 Oligonucleotides consisting of the nucleotide sequences of SEQ ID NO: 1 or SEQ ID NO: 2; A phosphor linked to the 5'-end of the oligonucleotide; And
    상기 올리고뉴클레오티드의 3'-말단에 연결된 소광체를 포함하는 중동호흡기증후군 코로나바이러스(MERS-CoV) 검출용 분자비콘(molecular beacon).Middle east respiratory syndrome coronavirus (MERS-CoV) detection including a quencher linked to the 3 '-end of the oligonucleotide molecular beacon (molecular beacon).
  6. 제5항에 있어서,The method of claim 5,
    서열번호 1 또는 서열번호 2의 염기서열의 8번째 염기는 비오틴(biotin) 결합 부위인 것을 특징으로 하는 분자비콘(molecular beacon).Molecular beacon, characterized in that the eighth base of the nucleotide sequence of SEQ ID NO: 1 or SEQ ID NO: 2 is a biotin binding site.
  7. 제5항 또는 제6항의 분자비콘을 포함하는 중동호흡기증후군 코로나바이러스(MERS-CoV) 감염 진단용 키트.A kit for diagnosing Middle East Respiratory Syndrome Coronavirus (MERS-CoV) infection comprising the molecular beacon of claim 5 or 6.
  8. 제7항에 있어서,The method of claim 7, wherein
    상기 분자비콘은 고체 지지체의 표면에 고정화되어 있는 것을 특징으로 하는 키트.The molecular beacon is a kit, characterized in that the immobilized on the surface of the solid support.
  9. 검체로부터 핵산을 수득하는 단계;Obtaining nucleic acid from a sample;
    상기 핵산을 제5항 또는 제6항의 분자비콘과 반응시키는 단계; 및Reacting the nucleic acid with the molecular beacon of claim 5 or 6; And
    상기 분자비콘으로부터 발생하는 형광을 검출하는 단계를 포함하는 중동호흡기증후군 코로나바이러스(MERS-CoV) 검출방법.Middle East respiratory syndrome coronavirus (MERS-CoV) detection method comprising the step of detecting the fluorescence generated from the molecular beacon.
  10. 제9항에 있어서,The method of claim 9,
    상기 반응은 핵산을 10 내지 100 pmol의 분자비콘과 실온에서 혼성화시키는 것을 특징으로 하는 방법.The reaction is characterized in that the nucleic acid hybridizes with 10 to 100 pmol molecular beacon at room temperature.
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