WO2023003082A1 - Lab-on-paper for detection of coronavirus and coronavirus diagnosis system using same - Google Patents

Lab-on-paper for detection of coronavirus and coronavirus diagnosis system using same Download PDF

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Publication number
WO2023003082A1
WO2023003082A1 PCT/KR2021/014712 KR2021014712W WO2023003082A1 WO 2023003082 A1 WO2023003082 A1 WO 2023003082A1 KR 2021014712 W KR2021014712 W KR 2021014712W WO 2023003082 A1 WO2023003082 A1 WO 2023003082A1
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Prior art keywords
pad
sample
reaction
detection
isothermal amplification
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PCT/KR2021/014712
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French (fr)
Korean (ko)
Inventor
김종철
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주식회사 에이아이더뉴트리진
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Priority claimed from KR1020210095317A external-priority patent/KR102370553B1/en
Priority claimed from KR1020210095339A external-priority patent/KR102370580B1/en
Priority claimed from KR1020210095332A external-priority patent/KR102370572B1/en
Priority claimed from KR1020210095329A external-priority patent/KR102370566B1/en
Priority claimed from KR1020210095322A external-priority patent/KR102370561B1/en
Application filed by 주식회사 에이아이더뉴트리진 filed Critical 주식회사 에이아이더뉴트리진
Publication of WO2023003082A1 publication Critical patent/WO2023003082A1/en

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    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01LCHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01LCHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L7/00Heating or cooling apparatus; Heat insulating devices
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING 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
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844Nucleic acid amplification reactions
    • C12Q1/686Polymerase chain reaction [PCR]
    • YGENERAL 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
    • Y02TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02ATECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30Against 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 is a lab-on-paper for detecting coronavirus to which a set of primers for detecting novel coronavirus and a composition for detecting the same are applied to a structure based on an integrated system of lateral fluidity capable of simultaneously diagnosing multiple diseases by applying one sample. It is about. Therefore, the detection sensitivity is high even when the nucleic acid is directly applied without purifying the nucleic acid from the sample, and a plurality of target nucleic acids can be simultaneously detected together with the coronavirus, making it possible to quickly and easily diagnose a number of diseases.
  • Coronaviruses refer to viruses belonging to the Coronaviridae family, which have single-stranded RNA as their genome, and are found not only in birds but also in various mammals, including humans. Coronaviruses are known to cause both respiratory and digestive infectious diseases depending on the characteristics of the virus and its host. In humans, it has been problematic as a virus that causes Severe Acute Respiratory Syndrom (SARS), which was a problem worldwide in 2003, and Middle East Respiratory Syndrome Coronavirus (MERS) in 2015, and recently occurred in Wuhan, China. As a result, the coronavirus is attracting attention as SARS-CoV-2, the pathogen of the novel coronavirus disease-19 (COVID-19) that is prevalent worldwide.
  • SARS Severe Acute Respiratory Syndrom
  • MERS Middle East Respiratory Syndrome Coronavirus
  • Coronavirus infection shows a high spread rate and is highly contagious even in an asymptomatic state in the early stages of the outbreak, so it is highly likely to cause mass infection.
  • Treatments and vaccines are currently being developed all over the world, but no cure has been established, so the best countermeasure is to detect infected people early and prevent mass infection. Therefore, it is necessary to develop a technology capable of quickly and accurately diagnosing coronavirus infection for a large number of test subjects.
  • the current universal molecular diagnosis method uses real-time PCR, which is the most common because of its speed, but it is difficult to use easily because it requires large and expensive equipment for on-site diagnosis or primary and secondary medical institutions.
  • three steps are largely required: sample preparation, nucleic acid amplification reaction, and detection.
  • nucleic acid amplification reaction and detection can be reproduced simultaneously by real-time PCR equipment, The preprocessing problem still remains.
  • lab-on-paper technology refers to a technology based on an integrated system that performs sample pretreatment, isothermal amplification, detection, and analysis steps on a single chip. Since all reactions can be automated and quickly performed with small paper and a chip structure embedded in the paper, it has the advantage of being independent of the location such as the detection site.
  • the present invention provides a configuration of a lab-on-paper structure capable of multiple diagnosis and a primer set for detecting coronavirus applied to the lab-on-paper structure.
  • a lab-on-paper for detecting coronavirus includes a sample pad accommodating a biological sample; a first connection pad disposed above the sample pad and connecting the sample pad and the reaction pad; a reaction pad disposed under the first connection pad, containing a primer capable of specifically binding to a target nucleic acid and a reagent for isothermal amplification (LAMP), and wherein the isothermal amplification reaction occurs; a blocking pad disposed above the reaction pad to maintain the reaction temperature and block evaporation of the sample; a second connection pad disposed above the reaction pad and to which gold nanoparticles are fixed; a detection pad disposed under the second connection pad and obtaining target nucleic acid amplified from an isothermal amplification reaction coupled with the gold nanoparticles; and an absorption pad disposed on a side of the detection pad to absorb the remaining sample, and a heating pad disposed below the sample pad, the reaction pad, and the second connection pad,
  • a primer set for detecting coronavirus including
  • a lab-on-paper for detecting coronavirus includes a sample pad accommodating a biological sample; a buffer pad disposed separately from the sample pad and accommodating a rehydration buffer; a first connection pad disposed above the sample pad and connecting the sample pad and the reaction pad; an initiator pad disposed above the buffer pad and connecting the buffer pad and the reaction pad; a reaction pad disposed below the first connection pad and the initiator pad, including a primer capable of specifically binding to a target nucleic acid and a reagent for an isothermal amplification reaction (LAMP), wherein an isothermal amplification reaction occurs; a blocking pad disposed above the reaction pad to maintain the reaction temperature and block evaporation of the sample; a second connection pad disposed above the reaction pad and to which gold nanoparticles are fixed; a detection pad disposed under the second connection pad and obtaining target nucleic acid amplified from an isothermal amplification reaction coupled with the gold nanoparticles; and an absorption pad disposed on a
  • LAMP isothermal
  • the sample pad, the first connection pad, the buffer pad, and the initiator pad; reaction pad; a second connection pad; detection pad; and the absorbent pads may be disposed laterally in contact with at least part of them sequentially.
  • the detection pad may include a plurality of separated detection zones.
  • the gold nanoparticles may contain streptavidin on their surfaces.
  • the sample applied to the sample pad may move laterally to the absorption pad.
  • a coronavirus infection diagnosis system includes a sample pad accommodating a biological sample; a first connection pad disposed above the sample pad and connecting the sample pad and the reaction pad; a reaction pad disposed under the first connection pad, containing a primer capable of specifically binding to a target nucleic acid and a reagent for isothermal amplification (LAMP), and wherein the isothermal amplification reaction occurs; a blocking pad disposed above the reaction pad to maintain the reaction temperature and block evaporation of the sample; a second connection pad disposed above the reaction pad and transporting the isothermal amplification reactant; a detection pad disposed below the second connection pad and obtaining target nucleic acid amplified from the isothermal amplification reaction; an absorption pad disposed on a side of the detection pad and absorbing the remaining sample; a recognition unit disposed above the detection pad, acquiring a fluorescence image from the detection pad, and measuring fluorescence intensity; and an output unit for deriving a significance value using the flu
  • a coronavirus infection diagnosis system includes a sample pad accommodating a biological sample; a buffer pad disposed separately from the sample pad and accommodating a rehydration buffer; a first connection pad disposed above the sample pad and connecting the sample pad and the reaction pad; an initiator pad disposed above the buffer pad and connecting the buffer pad and the reaction pad; a reaction pad disposed below the first connection pad and the initiator pad, including a primer capable of specifically binding to a target nucleic acid and a reagent for an isothermal amplification reaction (LAMP), wherein an isothermal amplification reaction occurs; a second connection pad disposed above the reaction pad and transporting the isothermal amplification reactant; a detection pad disposed below the second connection pad and obtaining target nucleic acid amplified from the isothermal amplification reaction; an absorption pad disposed on a side of the detection pad and absorbing the remaining sample; a recognition unit disposed above the detection pad, acquiring a fluorescence image from the detection pad, and
  • LAMP isothermal
  • the sample pad, the first connection pad, the buffer pad, and the initiator pad; reaction pad; a second connection pad; detection pad; and the absorbent pads may be disposed laterally in contact with at least part of them sequentially.
  • the disease, virus or fungal infection diagnosis system and structure for detecting multiple nucleic acids included therein of the present invention are based on lab-on-paper chip technology. Even without purification, the nucleic acid material can be purified while moving to the reaction pad and immediately applied to the amplification reaction, and multiple target nucleic acids can be simultaneously detected and related diseases can be diagnosed by applying one sample.
  • the system and structure of the present invention include a sample pad 120, a buffer pad 121, a first connection pad 131, an initiator pad 132, a reaction pad 140, and a heating pad 141 , a blocking pad 142, a second connection pad 150, a detection pad 160, an absorption pad 170, and a housing 110 as components.
  • the system includes the structure for detecting multiple nucleic acids according to one embodiment, and may further include a recognition unit 200 and an output unit 120.
  • the sample pad 120 accommodates a sample containing a nucleic acid material.
  • the sample is separated from the human body and includes blood, serum, plasma, saliva, sweat, urine, cell culture fluid, tissue suspension, etc., but is not limited thereto.
  • the sample may be mixed with a cell lysis composition (lysis buffer), and if necessary, after mixing with the cell lysis composition (lysis buffer), it may be additionally purified by a commonly known method such as centrifugation, filtration, and precipitation. .
  • the sample does not include a step of purifying the sample to be applied to the sample pad other than the structure for detecting nucleic acid after being mixed with the composition for cell lysis.
  • the cell lysis composition may be used in the same sense as the cell lysis buffer, and Tris (tris (hydroxymethyl) aminomethane) is 5 mM to 80 mM, 5 mM to 50 mM, or 10 mM to It may be included at 50 mM. Tris may specifically be Tris-HCl, and can reduce rapid pH fluctuations as a buffer in a cell lysis composition.
  • the cell lysis composition may be pH 8.0 to 9.0. When the pH is less than 8.0, the stability of the nucleic acid material may be reduced or the rate of migration may be reduced.
  • the cell lysis composition may include potassium chloride (KCl) at 5 mM to 50 mM, 5 mM to 40 mM, or 10 mM to 20 mM.
  • KCL potassium chloride
  • a high concentration of potassium chloride exceeding 50 mM is helpful for cell lysis, but reduces the aqueous solubility of the eluted nucleic acid material, which may require the addition of a large amount of additional buffer or increase the time taken to move to the reaction pad.
  • potassium chloride is less than 5 mM, cells may not be properly lysed.
  • the cell lysis composition may contain magnesium sulfate (MgSO 4 ) at 1 mM to 30 mM, 1 mM to 20 mM, or 2 mM to 16 mM. It has been confirmed that the stability and movement speed of nucleic acid substances are increased when an appropriate amount of magnesium sulfate is included, and since it does not affect the viscosity, it does not interfere with the flow of the fluid, which is advantageous in pre-processing the sample for paper chip analysis.
  • MgSO 4 magnesium sulfate
  • the cell lysis composition may contain ammonium sulfate ((NH 4 ) 2 SO 4 ) at 5 mM to 50 mM, 5 mM to 40 mM, or 10 mM to 20 mM. High concentrations of ammonium sulfate, greater than 50 mM, may precipitate cell lysates, and pH may become unstable when ammonium sulfate is less than 5 mM.
  • ammonium sulfate (NH 4 ) 2 SO 4 ) at 5 mM to 50 mM, 5 mM to 40 mM, or 10 mM to 20 mM.
  • High concentrations of ammonium sulfate, greater than 50 mM may precipitate cell lysates, and pH may become unstable when ammonium sulfate is less than 5 mM.
  • the cell lysis composition may contain 0.01 mg/ml to 0.1 mg/ml or 0.03 mg/ml to 0.07 mg/ml of a proteolytic enzyme.
  • the proteolytic enzyme degrades the polymer protein so that the polymer protein does not block the pores of the substrate or paper, which is the path for nucleic acid to move, and increases the stability of the nucleic acid material by inhibiting the activities of RNase and DNase.
  • the proteolytic enzyme may be proteinase K.
  • the surfactant may be TritonX-100 or Tween20 (polysorbate 20), and is present in an amount of 0.01 w/w% to 0.2 w/w%, preferably 0.05 w/w% to 0.1 w, based on the weight of the cell lysis composition. Can be included as /w%.
  • the cell lysis composition may be used in a ratio of 1:1 to the volume of the sample.
  • the cell lysis composition may not contain glycerol. Glycerol is sometimes added to prevent protein precipitation, but glycerol increases the viscosity, reduces the fluidity of the cell lysate, and may interfere with the movement of nucleic acid materials.
  • the cell lysis composition may not contain a reducing agent.
  • Reducing agents such as dithiothreitol (DTT) and mercaptoethanol, help to denature proteins and increase the solubility of cell lysates, but interfere with fluorescence or detection reactions when present in the solution flowing into the paper chip. It can be.
  • the heating pad is heated at a temperature of 60 to 80 ° C for 1 to 5 minutes, preferably for 5 minutes, so that the sample pad contains viruses and epithelial cells. Promote dissolution of the sample.
  • the cell lysis composition promotes cell lysis in the sample pad 120 made of a polysulfone membrane (eg, Vivid GF) or a nitrocellulose membrane, and makes it easier to detect nucleic acids.
  • the polysulfone membrane and the nitrocellulose membrane may form a structure in which two or more are laminated.
  • the polysulfone membrane may be asymmetric, and the polysulfone membrane and the nitrocellulose membrane may be porous materials having pores of 0.5 ⁇ m to 1 ⁇ m. It is advantageous that the pores are rather large.
  • Many biological samples have viscosity, and in particular, when the sample is treated with a composition for cell dissolution, the viscosity may increase significantly as nucleic acid materials and proteins are eluted out of the cells. Therefore, it is preferable that the pore size of the sample pad has an appropriate size to rapidly absorb the sample.
  • lateral flow method refers to a method in which a sample is flowed from an application point to a target point by a capillary phenomenon or a diffusion phenomenon in a horizontal direction without using gravity. Since the cell lysate contains a large amount of hydrolytic enzymes capable of degrading nucleic acid material, the yield of nucleic acid material may be reduced if the cell lysate remains in the migration path for a long time. Therefore, in order to be applied to a structure for detecting nucleic acid in a lateral flow type, the flow rate should be excellent and the cell lysate should not precipitate and block the movement path while the sample is moving.
  • composition of the composition for cell lysis of the present invention does not precipitate cell lysate or nucleic acid material even if it does not contain glycerol or a reducing agent, and can transfer nucleic acid material laterally to the reaction pad in high yield.
  • the first connection pad 131 partially contacts the sample pad, is disposed above the sample pad, and connects the sample pad and the reaction pad with a structure having a relatively narrow width compared to the sample pad.
  • the first connection pad may be made of a cellulose membrane and have pores of 0.005 ⁇ m to 0.015 ⁇ m.
  • the buffer pad 121 is a pad to which the rehydration buffer is applied dropwise, and serves to apply water pressure to the structure.
  • the buffer pad may be disposed separately from the sample pad in order to minimize the movement of cell lysis debris such as proteins to the reaction pad and induce the movement of only the isothermal amplification reactants to the detection pad by applying water pressure after the reaction is completed.
  • the rehydration buffer applied to the buffer pad is an additional buffer, for example, 5 mM to 80 mM Tris-HCl, 20 mM to 70 mM potassium chloride, 0.5 mM to 5 mM magnesium sulfate, 1 mM to 30 mM ammonium sulfate, and 0.01 mM It may be an isothermal buffer containing w/w% to 0.2 w/w% Tween®20 to TritonX-100 and having an acidity of pH 8.0 to 9.0 or a phosphate buffer (50 mM Na 2 HPO 4 , pH 7.2).
  • the isothermal buffer may include 20 mM Tris-HCl, 10 mM (NH 4 ) 2 SO 4 , 50 mM KCl, 2 mM MgSO 4 , and 0.1% Tween® 20, and may have a pH of 8.8.
  • the addition buffer may not contain a protease, glycerol, or a reducing agent.
  • the buffer pad is a porous material having pores of 0.5 ⁇ m to 1 ⁇ m to sufficiently receive the rehydration buffer, and is made of cotton, flax, paper, nitrocellulose, cellulose acetate, glass fiber, polysulfone, polyacrylic, polynitrile, polypiperazine, polyamide, polyethersulfone, polyvinylidene fluoride, polyethyleneimine, polydimethylsiloxane or mixtures thereof.
  • the initiator pad 132 partially contacts the buffer pad, is disposed above the buffer pad, and connects the buffer pad and the reaction pad with a structure having a relatively narrow width compared to the buffer pad.
  • the initiator pad may be made of a cellulose membrane and have pores of 0.005 ⁇ m to 0.015 ⁇ m. A portion of the initiator pad in contact with the buffer pad or reaction pad may be coated with low melting point agarose or wax. The coating may block reverse flow to the buffer pad to accelerate lateral fluidity.
  • a heating pad 141 for heating may be disposed below the initiator pad.
  • water pressure is generated to the reaction pad as the low melting point agarose or wax of the initiator pad is melted, so that the isothermally amplified result is easily transferred to the detection pad. let it move
  • the heating may be performed at 60 to 80 °C.
  • the reaction pad is a component corresponding to the paper chip in lab-on-paper, and isothermal amplification reagents including dNTP, DNA polymerase, reverse transcriptase, fluorescent marker, isothermal amplification reaction buffer, etc. for amplification reaction are fixed thereto. Therefore, the solution containing the nucleic acid material penetrates into the reaction pad by the additional buffer applied to the sample pad and is wetted, and the contact material between the isothermal amplification reagent and the sample moves to the reaction pad and is heated by the heating pad at the bottom of the reaction pad. When heated to 60 to 70 ° C., an isothermal amplification reaction or a reverse transcription isothermal amplification reaction occurs.
  • the isothermal amplification reagent is specifically dNTP (1.4mM, dATP, dCTP, dGTP and dTTP), isothermal amplification buffer (1X, 20mM Tris-HCl, 10mM (NH 4 ) 2 SO 4 , 50mM KCl, 2mM MgSO 4 , and 0.1% Tween-20, pH7.5), and Bst 3.0 DNA polymerase (320 U/ml), which are mixed in the reaction pad and then dried or applied to the surface of the reaction pad in powder form, for example, It may be fixed by heating in an oven at about 40 ° C. for about 30 minutes.
  • the reaction pad 140 may partially come into contact with the first connection pad and the initiator pad, and may be disposed under and sideways of the first connection pad and the initiator pad.
  • a heating pad 141 may be disposed below the reaction pad 140 to heat to a temperature at which an isothermal amplification reaction may occur.
  • the heating pad may include a hot wire or a hot plate for heating. The heating may be performed at 60 to 70°C, preferably 60 to 65°C for 20 minutes to 1 hour, preferably 20 minutes to 30 minutes.
  • the reaction pad may have a blocking pad 142 disposed above the reaction pad to serve as a blocking function.
  • the isothermal amplification reaction temperature is maintained by a blocking pad laminated to a series of arranged pads, and evaporation of reagents is blocked to increase reaction efficiency.
  • the blocking pad may be a non-porous membrane or a structure capable of blocking the reaction pad from outside air.
  • ⁇ M) and outer primers (F3 and B3, 0.2 ⁇ M) can be immobilized.
  • the concentration of the primers is based on the volume of the well, and the concentration of the primer set in the well can be changed while maintaining the concentration ratio between the respective primers.
  • the isothermal amplification reaction can occur more intensively by the presence of wells in the reaction pad.
  • the well may have a hydrogel layer formed at the bottom and the primer set fixed to the hydrogel layer.
  • primer sets that specifically bind to different target nucleic acids may be immobilized in each well.
  • the hydrogel layer containing the primer may be formed, for example, by the following method.
  • UV-light crosslinkable poly(ethylene glycol) diacrylate PEGDA, Sigma-Aldrich, MW700
  • PEG poly(ethylene glycol)
  • PEG poly(ethylene glycol)
  • MW600 poly(ethylene glycol)
  • PBS buffer pH7.5
  • the poly(ethylene glycol) is preferably included to increase the porosity of the hydrogel microparticles.
  • the hydrogel solution was applied to the inner surface of each well of the reaction pad and exposed to UV (360 nm wavelength, 35 mJ/cm 2 ) for 1 minute to form a hydrogel coating layer. Since the hydrogel layer has pores, an amplification reaction may occur intensively in the pores by binding to primers in the hydrogel layer.
  • any one of the forward and reverse primers in the primer set is Cy3, Cy5, TAMRA, TEX, TYE, HEX, FAM, TET, JOE, MAX, ROX, VIC, Cy3.5, Texas Red, Cy5.5, TYE, BHQ , Iowa Black RQ, and may be labeled with one or more detectors selected from the group consisting of IRDye.
  • the detector may be labeled differently for each target nucleic acid in order to independently detect the target nucleic acid, and may function as a detector or a fluorescent marker for selective binding depending on the specific structure of the structure.
  • the other one of the forward and reverse primers may be biotin-linked. Since biotin can bind to streptavidin, it exists in a form bound to the amplified target nucleic acid and passes through a pad containing gold particles, for example, the second connection pad according to one embodiment, while gold particles When streptavidin binds to the surface and is captured on the detection pad, the detection result is visualized.
  • streptavidin can be immobilized on the detection pad, and in this case, biotin can bind to streptavidin immobilized on the detection pad. It can be displayed or the fluorescence detection result can be visualized.
  • Biotin may be designed to be present at opposite positions of a detector and a detector in the amplified target nucleic acid. For example, when a detector is bound to the 5' end of a forward primer, biotin may be bound to the 5' end of a reverse primer. there is.
  • the reaction pad is made of a cellulose acetate membrane material, and has a pore size of 0.001 ⁇ m to 0.005 ⁇ m, preferably 0.005 ⁇ m, so that the nucleic acid can remain and sufficiently isothermally amplified while allowing the sample and the additional buffer to flow freely.
  • the reaction pad may include 40 mM to 50 mM sucrose, 0.001 to 0.01% Triton X-100, and 0.1 w/w% to 0.3 w/w% glycerol. This can increase their storage stability when the isothermal amplification reagent and primer set are exposed to moisture or oxygen.
  • the storage stability may mean that it can be stored for 3 weeks or more without degradation products or by-products at 25 ° C to 30 ° C.
  • the second connection pad 150 may partially come into contact with the reaction pad and may be disposed above and sideways of the detection pad. The second connection pad 150 moves the nucleic acid amplified in the reaction pad to the detection pad.
  • the second connection pad 150 includes gold nanoparticles, and the gold nanoparticles combine with nucleic acids amplified in the reaction pad and move to the detection pad.
  • the gold nanoparticles may preferably have streptavidin immobilized on the surface.
  • the second connection pad is cotton, wool, paper, nitrocellulose, glass fiber, polysulfone, polyacrylic, polynitrile, polypiperazine, polyamide, polyethersulfone, polyethersulfone, As a porous material of vinylidene fluoride, polyethyleneimine, polydimethylsiloxane, or a mixture thereof, it may be a porous material having a pore size of 0.01 ⁇ m to 0.05 ⁇ m, preferably, 0.05 ⁇ m.
  • the second connection pad is disposed above the reaction pad and has a relatively narrow width compared to the reaction pad or the detection pad, and connects the reaction pad and the detection pad.
  • the second connection pad may have a cellulose material, and the second connection pad at a portion in contact with the reaction pad may be coated with low melting point agarose or wax.
  • the movement of the isothermal amplification reactant in the reaction pad is blocked, and then the coating melts at the time of heating the second connection pad, and the isothermal amplification reactant moves laterally again, preventing loss of unreacted dielectric material in the sample. .
  • a heating pad 141 for heating may be disposed under the second connection pad.
  • the heating may be performed at 60 to 80° C. for 1 minute to 5 minutes, preferably for 2 minutes.
  • the detection pad 160 may partially come into contact with the second connection pad and may be disposed below and on the side of the second connection pad.
  • a receptor that can bind to the detector is fixed to the detection pad 160 .
  • the receptor may be an antibody, protein, or fragment thereof capable of specifically binding to a detector.
  • the detection pad includes a plurality of detection regions, and the detection regions may be divided into lines or wells.
  • each receptor is independently immobilized.
  • a solution containing a receptor and EDC (1-Ethyl-3-[3-dimethylaminopropyl] carbodiimide) or NHS (N-hydroxysulfosuccinimide) may be applied.
  • EDC 1-Ethyl-3-[3-dimethylaminopropyl] carbodiimide
  • NHS N-hydroxysulfosuccinimide
  • the detection pad is nitrocellulose and may have a pore size of 0.001 ⁇ m to 0.005 ⁇ m, preferably 0.005 ⁇ m, so that the sample can move laterally to the absorption pad.
  • a heating pad 141 may be disposed below the sample pad, the initiator pad, the reaction pad, and the second connection pad.
  • the heating pad is a metal plate having thermal conductivity, and may be made of a material such as iron, stainless steel, aluminum, silver, or copper.
  • the heating pad may be connected with a heating wire, and each heating wire may be independently connected to the sample pad, the reaction pad, the initiator pad, and the heating pad under the second connection pad to control the heating pad.
  • the sample pad, the first connection pad, the initiator pad, the reaction pad, the second connection pad, the detection pad, and the absorption pad may have housings 110 at upper and lower ends so that the sample or buffer is not lost, and the entire structure is a housing. It may be surrounded by a case of a support or non-porous material that can be defined as.
  • the absorbent pad is a porous material, such as cotton, flax, paper, nitrocellulose, cellulose acetate, glass fiber, polysulfone, polyacrylic, polynitrile, polypiperazine, polyamide, polyethersulfone, polyvinylidene fluoride, and polyethyleneimine. , polydimethylsiloxane or mixtures thereof.
  • the absorbent pad is preferably a glass fiber and may have a pore size of 0.1 to 0.5 ⁇ m.
  • the sample pad, the first connection pad, the buffer pad, and the initiator pad; reaction pad; a second connection pad; detection pad; and the absorbent pads are sequentially disposed laterally in partial contact with each other. Specifically, the sample pad, the first connection pad, the buffer pad, and the initiator pad are separated based on the reaction pad and disposed on one side of the reaction pad, followed by the second connection pad, detection pad, and The absorbent pads are sequentially disposed.
  • nucleic acid materials can be purified by filtering cell lysate in addition to nucleic acid materials while the sample reaches the absorption pad due to the arrangement and characteristics of each component of the structure, and the moving direction of the sample is different from the ground. Lateral fluidity, which is a parallel direction, can be realized.
  • nucleic acids exist in the form of proteins such as histones, polymerases, nucleases, and transcription factors.
  • proteins lose their binding to nucleic acids by surfactants such as the cell lysis composition, but by the time the cell lysate reaches the reaction pad by the addition of distilled water or added buffer so that the cell lysate can move to the reaction pad, the surfactant is diluted, allowing the external environment to regain the protein's charge.
  • the protein may bind to the nucleic acid material again to reduce the contact area with the polymerase or interfere with the amplification reaction. Therefore, it is preferable that most cellular components capable of binding to nucleic acids are purified and removed from the reaction pad.
  • the recognition unit 200 acquires a fluorescence image from the detection pad and measures fluorescence intensity from the acquired fluorescence image. Fluorescence intensity can be calculated as a ratio of the actual measured value based on the maximum measurable value, and in order to excite a plurality of fluorescent markers and recognize a fluorescent image, a light source of various wavelength bands, for example, 400 to 700 ⁇ wavelength range, and A filter for selective acquisition of each excitation light may be included.
  • the recognition unit independently recognizes a plurality of detection zones formed on the reaction pad, calculates the fluorescence intensity of each detection zone, and transmits the "difference between the fluorescence intensity value measured in the corresponding detection zone and the fluorescence intensity value of the negative control" to the output unit. .
  • the output unit 210 derives a significance value using the fluorescence intensity value difference transmitted from the recognition unit 200 .
  • the significance value the maximum measurable value in the recognition unit is set to 1, and when the fluorescence intensity difference is greater than or equal to 0.3, the significance value is output as “positive for the target nucleic acid in the corresponding detection region with a probability of 70% or more”, and fluorescence If the difference in intensity value is 0.5 or more, the significance value may be output as "positive with a probability of 90% or more for the target nucleic acid in the corresponding detection region".
  • the output unit uses the derived significance value based on big data including genetic information related to disease, virus, or fungal infection to find a disease, virus, or fungal infection symptom related to a positively output target nucleic acid, and finally It is possible to diagnose whether or not a disease, virus, or fungal infection of an object from which a sample is acquired is detected.
  • the significance value may be output as “negative”.
  • the target nucleic acid may be related to a disease, virus, or fungal infection symptom that is already known when preparing a primer set.
  • the nucleotide sequence to which the primer set specifically binds can be matched with a related disease, virus or fungal infection symptom.
  • Big data can be obtained from open biological databases, and for base sequences, there are Genebank, EMBL (The European Molecular Biology Laboratory), DDBJ (DNA Data Bank of Japan), etc., and genome databases include Entrez Genome, Ensembl, etc. , Metabolic circuit databases include KEGG (Kyoto Encyclopedia of Genes and Genomes) and WikiPathways, but are not limited thereto.
  • a sample for which nucleic acid is to be detected may include mixing with a composition for cell lysis before applying the sample to the sample pad.
  • a composition for cell lysis When mixed with the composition for cell lysis, nucleic acid substances present in cells may be eluted, and thus the amount of detectable nucleic acid substances in the sample may be increased.
  • Tris-HCl Tris-HCl, potassium chloride 5 mM to 50 mM, magnesium sulfate 1 mM to 30 mM, ammonium sulfate 5 mM to 50 mM, protease 0.01 mg/ml to 0.1 mg/ml, TritonX- 100 or Tween20 0.01 w/w% to 0.2 w/w%, by adding dropwise the cell lysate mixed with the cell lysis composition having a pH of 8.0 to 9.0 and operating a heating pad below the sample pad for heating the sample pad Increase the efficiency of sample dissolution. By heating the heating pad below the sample pad at a temperature of 60 to 80° C. for 2 minutes, dissolution of the sample including viruses and epithelial cells in the sample pad is promoted.
  • an additional buffer may be added to the buffer pad.
  • the additional buffer serves to purify the nucleic acid material while allowing the nucleic acid material to move to the reaction pad.
  • the buffer serves to purify the nucleic acid material while allowing the nucleic acid material to move to the detection pad. Since the buffer solution contains an appropriate amount of a buffering component, it is possible to prevent precipitation of proteins or nucleic acids due to rapid changes in salinity or pH that may occur when distilled water is added.
  • Each pad of the structure for detecting nucleic acids of the present invention is made of a porous material having small pores so that nucleic acids can be purified while moving laterally. Therefore, if the protein or nucleic acid material is complexed or aggregated to block the pores, the movement speed of the sample may decrease and the yield of the nucleic acid material may decrease.
  • the addition buffer is, for example, 5 mM to 80 mM Tris-HCl, 20 mM to 70 mM potassium chloride, 0.5 mM to 5 mM magnesium sulfate, 1 mM to 30 mM ammonium sulfate, and 0.01 w/w% to 0.2 w/w % Tween®20 to TritonX-100 and an acidity pH of 8.0 to 9.0 or a phosphate buffer (50 mM Na 2 HPO 4 , pH 7.2).
  • the isothermal buffer may include 20 mM Tris-HCl, 10 mM (NH 4 ) 2 SO 4 , 50 mM KCl, 2 mM MgSO 4 , and 0.1% Tween® 20, and may have a pH of 8.8.
  • the addition buffer may not contain a protease, glycerol, or a reducing agent.
  • the heating pad under the reaction pad may be heated to 60 to 65° C., and allowed to stand for 20 minutes to 1 hour, preferably 20 to 30 minutes, while maintaining the temperature.
  • Diseases that can be diagnosed using the multi-nucleic acid detection structure are those in which a specific gene can be used as a cause or indicator of a disease, for example, metabolic diseases such as obesity, hypertension, diabetes, genetic diseases, cancer, etc.
  • metabolic diseases such as obesity, hypertension, diabetes, genetic diseases, cancer, etc.
  • viruses or bacteria are related to infectious diseases, and bacteria that can cause fungal infections include bacteria, protozoa, parasites, fungi, etc., but are not limited thereto.
  • the present invention provides a primer set comprising six primers each represented by the nucleotide sequence of SEQ ID NOs: 1 to 6 capable of detecting coronavirus, and applying the primer set to lab-on paper to detect coronavirus can
  • the primer set is for detecting a coronavirus, and the coronavirus may be SARS-CoV-2, a pathogen of novel coronavirus disease-19 (COVID-19).
  • primer refers to a short polynucleotide having a free 3'hydroxyl group, capable of forming a base pair with a complementary template and serving as a starting point for template strand copying.
  • Primers can initiate DNA synthesis in the presence of reagents (DNA polymerase or reverse transcriptase) and four different deoxynucleoside triphospates (dNTPs) for polymerization at an appropriate buffer solution and temperature.
  • Each primer in the primer set is specific to the target gene, and the term "gene specific” or “complementary” refers to 80% or more, 85% or more, 90% or more, 95% or more, or 100% or more of the target gene. It can mean that % base pairing can be achieved.
  • each primer may consist of or include one of the nucleotide sequences of SEQ ID NOs: 1 to 6, and as long as it maintains specific binding to the coronavirus, the bases shown in SEQ ID NOs: 1 to 6 It may have at least 80%, at least 90%, at least 95%, at least 97% or at least 99% homology with the sequence.
  • the primers may also be modified to include additional features within the range of not changing the basic properties to act as starting points for gene synthesis.
  • the primer includes the nucleotide sequence of each defined SEQ ID NO and includes an additional nucleotide sequence of about 1 to 20 bp, 1 to 15 bp, 1 to 10 bp, or 1 to 5 bp, at the 3' end of the primer, It may be further included at the 5' end or inside the nucleotide sequence, and if necessary, a label detectable directly or indirectly by spectroscopic, photochemical, biochemical, immunochemical or chemical means may be further included at each end or a specific base.
  • Examples of the label may be a chromogenic enzyme, a fluorescent substance, a radioactive isotope, or a colloid.
  • the chromogenic enzyme may be peroxidase, alkaline phosphatase or acid phosphatase
  • the fluorescent substance may be thiourea (FTH), 7-acetoxycoumarin-3-yl, fluorescein-5-yl, fluorescein-6 -yl, 2', 7'-dichlorofluorescein-5-yl, 2', 7'-dichlorofluorescein-6-yl, dihydrotetramethylrosamine-4-yl, tetramethylrhodamine-5 -yl, tetramethylrhodamine-6-yl, 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-ethyl or 4,4- Difluoro-5,7-diphenyl-4
  • the primer set of the present invention may be suitable for use for isothermal amplification.
  • isothermal amplification means DNA amplification under one temperature condition.
  • Conventional polymerase chain reaction (PCR) has denaturation, annealing, and extension steps, respectively.
  • PCR polymerase chain reaction
  • the isothermal amplification reaction has the advantage of not requiring a separate special equipment.
  • the isothermal amplification method is a target nucleic acid within an hour within a specific temperature range.
  • the isothermal amplification method has a short execution time and the result can be observed with the naked eye, so amplification can be easily confirmed. .
  • the isothermal amplification may be loop-mediated isothermal amplification.
  • LAMP loop-mediated isothermal amplification
  • the outer primer consists of two types of forward outer (F3) primer and reverse outer (B3) primer, and DNA double strands are formed during the non-cyclic step of the reaction. serves as a release.
  • the inner primer consists of two types, a forward inner primer (FIP) and a backward inner primer (BIP). It is composed of the corresponding nucleotide.
  • These internal primers are not included in the viral RNA, but can be designed to further include specific bases, for example, a series of thymine bases, to increase the efficiency of the reaction.
  • the additional two primers consist of a forward loop (LoopF) primer and two backward loop (LoopB) primers, and are attached to a nucleotide sequence (ring site) to which the inner primer does not bind, thereby intermediating the loop. Accelerates the isothermal amplification reaction.
  • loopF forward loop
  • LoopB backward loop
  • ring site nucleotide sequence
  • the isothermal amplification or loop-mediated isothermal amplification may be a reverse transcription reaction.
  • the isothermal amplification reaction may be performed at 50 to 70 °C, 52 to 70 °C, 52 to 68 °C, 52 to 65 °C, 55 to 65 °C, or 50 to 60 °C.
  • the isothermal amplification method is 10 to 90 minutes, 20 to 90 minutes, 25 to 90 minutes, 10 to 80 minutes, 20 to 80 minutes, 25 to 80 minutes, 10 to 70 minutes, 20 to 70 minutes, 25 to 70 minutes minutes, 10 to 60 minutes, 20 to 60 minutes or 25 to 60 minutes.
  • the primer set contains a very small amount of the target gene of 100 pg or less, for example, 1 pg to 100 pg, 2 pg to 100 pg, 5 pg to 100 pg, or 10 pg to 100 pg.
  • Coronavirus can be detected in samples that have been tested.
  • Another embodiment of the present invention includes a composition or kit for detecting coronavirus including the primer set.
  • the composition or kit for detecting coronavirus may further include appropriate reactants and reagents for performing an isothermal amplification reaction.
  • reactants and reagents may include, for example, additional primers, dNTPs, and enzymes (DNA polymerase or reverse transcriptase), and inorganic salts such as potassium chloride, magnesium sulfate, and ammonium sulfate for reaction efficiency.
  • a surfactant may be further included.
  • the reactants and reagents include 10 to 20 mM Tris-HCl, 1 to 10 mM (NH 4 ) 2 SO 4 , 10 to 50 nM KCl, 0.1 to 2 mM MgSO 4 , and 0.01 to 0.1% Tween-20. It may be a solution of pH 7 to 9.
  • the composition or kit for detecting coronavirus may further include a reagent that can visually check the reaction product.
  • a reagent commonly known or commercially available ones may be used, for example, WarmStart® Colorimetric LAMP Mix, but is not limited thereto.
  • the composition or kit for detecting coronavirus may further include a negative control material or a positive control material in order to accurately read the diagnosis result.
  • a negative control material template RNA or DNA unrelated to coronavirus can be used
  • a positive control material a biological sample obtained from a patient infected with coronavirus or a part of the genome of coronavirus, for example, GenBank accession no.
  • Template RNA or DNA corresponding to at least a part of MN908947.3 Wuhan seafood market pneonia virus isolate Wuhan-Hu-1, complete genome may be used.
  • These control substances can be applied to the gene amplification reaction separately or together with the biological sample in one tube, if necessary.
  • Another embodiment of the present invention mixing the primer set and the biological sample; And it provides a coronavirus detection method comprising the step of amplifying the gene by an isothermal amplification reaction of the mixed solution.
  • the biological sample is obtained from the subject to be tested, obtained through swab or nasal irrigation from the nasopharynx or oropharynx, bronchial lavage or aspirate, intertracheal aspirate and bronchial biopsy, sputum, bronchoalveolar lavage , saliva, blood, urine, feces, etc. can be used.
  • the biological sample may be pretreated to increase the efficiency of the amplification reaction. Pretreatment may be 10-fold, 50-fold, 100-fold, 200-fold, or 400-fold dilution with water, heat treatment after treatment with protenase K, anion exchange chromatography, affinity chromatography, size-specific exclusion chromatography, Genes may be extracted from biological samples or purified to a certain level using commonly known methods such as liquid chromatography, continuous extraction, centrifugation, or gel electrophoresis, or using commercially available kits. When RNA is extracted from a biological sample and used, a step of reverse transcription into cDNA may be further included before amplifying the target gene.
  • the coronavirus detection method includes detecting the amplified gene.
  • nucleic acid extraction and amplification method of the present invention it is possible to perform the sample preparation, nucleic acid amplification reaction, and detection separately, and to detect the sample with one application. can Since each step is not performed separately, the whole process is simplified, and it does not require various samples or devices, and it can be easily performed even if you are not a related technician. can be detected negatively.
  • 1 is an example of the overall structure of the construct for detecting multiple nucleic acids of the present invention.
  • FIG. 2 is another example of the overall structure of the structure for detecting multiple nucleic acids of the present invention.
  • FIG. 3 is a schematic diagram of a principle of obtaining target nucleic acids from a detection pad 160 in a structure including gold particles according to an embodiment.
  • FIG. 4 is a diagram illustrating a principle of obtaining target nucleic acids from the detection pad 160 in the system according to one embodiment.
  • FIG. 5 is a side structural diagram of a part of a structure for detecting multiple nucleic acids including gold particles according to an embodiment.
  • FIG. 6 is a side structural diagram of a structure for detecting multiple nucleic acids included in a system according to one embodiment.
  • FIG. 7 is a perspective view of a portion of a structure for detecting multiple nucleic acids according to one embodiment.
  • FIG. 8 is a perspective view of a portion of a structure for detecting multiple nucleic acids according to another embodiment.
  • FIG 9 is an enlarged structural view of the structure of the detection pad 160 in the structure including gold particles according to one embodiment.
  • FIG. 10 is an enlarged structure diagram of a detection pad 160 in a structure for detecting multiple nucleic acids included in a system according to an embodiment.
  • 11 is an example showing the exterior of the structure for detecting multiple nucleic acids of the present invention surrounded by a case including a buffer pad 121 and an initiator pad 132.
  • FIG. 13 is an example showing the result of detecting SARS-CoV-2 from a blood sample using a structure for detecting multiple nucleic acids according to one embodiment.
  • FIG. 14 is an image showing the result of detecting SARS-CoV-2 by isothermal amplification reaction using the primer set presented in the present invention in order to confirm the effect of the primer set according to an embodiment of the present invention.
  • 16 is an image showing the results of detecting SARS-CoV-2 at 5-minute intervals through an isothermal amplification reaction using the primer set presented in the present invention in order to confirm the effect of the primer set according to an embodiment of the present invention. to be.
  • 17 is an electrophoresis result of detecting SARS-CoV-2 at 5-minute intervals by isothermal amplification using the primer set presented in the present invention to confirm the effect of the primer set according to an embodiment of the present invention. This is an image verified by
  • 19 is an isothermal amplification reaction of SARS-CoV-2 detected in a virtual patient sample under an interfering substance environment using the primer set presented in the present invention to confirm the effect of the primer set according to an embodiment of the present invention. This is an image confirmed by color change and electrophoresis.
  • the cell lysis composition was prepared by mixing as shown in Table 1 below by varying the composition, pH, and ratio of each component.
  • Proteinase K was purchased from Thermofisher (EO0491) as proteinase K, and 0.02 mg/ml of lysozyme (Thermofisher, 90082) was added and used according to the type of bacteria to be detected. pH was adjusted with 0.1M HCl or 0.1M NaOH.
  • Example 1 20 mM Tris HCl 15 mM MgSO 4 15 mM KCl 15 mM (NH 4 ) 2 SO 4 0.1 w/w% Tween20 0.05 mg/ml 8.8 Comparative Example 1 50 mM Tris-HCl 100mM NaCl 1 mM DTT 5% glycerol 0.05 mg/ml 7.0 Comparative Example 2 50 mM Tris-HCl 150mM NaCl 5 mM EDTA 1% NP-40 0.05 mg/ml 8.5 Comparative Example 3 50 mM Tris-HCl 10 mM Na 2 HPO 4 5 mM MgCl2 1% TritonX-100 0.05 mg/ml 8.5 Comparative Example 4 50 mM Tris-HCl 10 mM Na 2 HPO 4 10 mM NaCl 0.5% SDS 1% TritonX-100 - 9.0 Comparative Example 5 50 mM Tris-HCl 10 mM Na 2 H
  • Test Example A-1 Flowability comparison test of cell lysis composition
  • Primers were designed and produced to detect the causative agent of Lyme disease using a loop-mediated isothermal amplification (LAMP) method.
  • LAMP loop-mediated isothermal amplification
  • Primer Set 1 (B.afzelii target) F3 5'-GGTATACTGACAGCAGCTT-3' B3 5′-CTTGCAGCTTAATAATAGCCTT-3′ FIP 5'-GTTGCTCGGTCCTCCATGTTTAAATTTTTAATGTTATCCGTGATATGGTTCCGA-3' BIP 5'-GGATTTCGTATCAATTTTGGAGGCATTTTAAGTTACAAAGGTCCCATTGC-3' FL 5′-ATAAGGCCTTCGGTATTG-3′ BL 5′-ATTCTACGTTCCGATTCTCAGTAT-3′ Primer Set 2 (B.burgdorferi target) F3 5′-AGAGCAGCTGAGGAGCT-3′ B3 5′-CTTCCAGTTGAACACCATCTT-3′ FIP 5'-AAGTCCACGACGGTTGAGACCTTTTTGCAGCCTGCTTAAATTAACA-3' BIP 5′-GAGCAAACGAAGTTGAAGCTATTTTAGCCTGAGCAGTTAGAGC-3′ FL 5′-GA
  • the causative bacteria of Lyme disease, Borrelia afzelii, Borrelia burgdorferi, and Borrelia garinii were mixed in DMEM medium at 2.5 * 10 5 cells/ml in 50 ⁇ l of Example 1 and Comparative Examples 1 to 5. After adding 50 ⁇ l of the cell lysis composition and lightly tapping, 20 ⁇ l was added dropwise to one end of a fiberglass paper (Millipore, MA, USA) cut into 10 cm x 2 cm x 3 mm thickness.
  • the distilled water used here was mixed with RNase, and as a negative control, distilled water was mixed instead of the strain sample and then reacted. Then, a fluorescence image was acquired using a Chemi-Doc XRS + imaging system (Bio-Rad Laboratories, Hercules, CA, USA) with a green light source and a 605/50 filter set, and fluorescence was measured to determine the maximum fluorescence intensity. 1 and the minimum value 0, and after calculating the ratio thereto, the fluorescence intensity value was obtained according to the following formula.
  • Example A-2 Preparation of Lab-on-Paper Structure Containing Gold Particles for Detecting Nucleic Acids
  • the sample pad 120 and buffer pad 121 0.01 ⁇ m cellulose was used as the first connection pad 131 or initiator pad 132
  • the reaction pad 140 was made of 0.005 ⁇ m cellulose acetate
  • the second connection pad 150 was made of 0.05 ⁇ m cellulose
  • the detection pad 160 was made of 0.005 ⁇ m nitrocellulose
  • the absorption pad 170 was made of 0.5 ⁇ m glass fiber.
  • the reaction pad 140 was prepared by making a pad by overlapping cellulose acetate films, immersing the pad in a solution containing 45 mM sucrose, 0.005 w/w% TritonX-100, and 0.2 w/w% glycerol, and drying. Then, a well was formed using a micro-drill, and a hydrogel layer including a primer set was formed on the bottom of the well.
  • hydrogel layer To form the hydrogel layer, first, based on the total volume of the hydrogel solution, UV-light crosslinkable poly (ethylene glycol) diacrylate (PEGDA, Sigma-Aldrich, MW700) 20% v / v, poly (ethylene glycol) ( Mix PEG, Sigma-Aldrich, MW600) 40% v/v and photoinitiator 2-hydroxy-2-methylpropiophenone (Sigma-Aldrich) 5% v/v and buffer (PBS buffer, pH7.5) 35% Then, a hydrogel solution was prepared by mixing each primer set therein. The poly(ethylene glycol) is preferably included to increase the porosity of the hydrogel microparticles. Then, the hydrogel solution was applied to the inner surface of each well of the reaction pad and exposed to UV (360 nm wavelength, 35 mJ/cm 2 ) for 1 minute to form a hydrogel coating layer.
  • PEGDA poly (ethylene glycol) diacrylate
  • PEG Poly (ethylene glycol)
  • dNTP 1.4mM, dATP, dCTP, dGTP and dTTP
  • isothermal amplification buffer (1X, 20mM Tris-HCl, 10mM (NH 4 ) 2 SO 4 , 50mM KCl, 2mM MgSO 4 were added to the surface of the reaction pad 140.
  • 0.1% Tween-20, pH 7.5) and Bst 3.0 DNA polymerase (320 U/ml) were applied, and fixed by heating in an oven at about 38° C. for about 30 minutes.
  • the gold nanoparticles fixed to the second connection pad 150 were prepared as colloidal particles as follows. When the 0.1% HAuCl 4 solution starts to boil while stirring and heating, 0.5% sodium citrate solution is added to reduce the solution to form gold particles, and 1 mg of streptavidin per 100 ml of the gold particle solution is added to condense made it The condensate was precipitated by centrifugation at 10,000 g, dissolved in physiological saline (PBS) containing 0.1% BSA, and stored at an OD450 value of 10.
  • PBS physiological saline
  • the second connection pad 150 was manufactured as follows. Specifically, several layers of cellulose membranes were prepared and cut, 0.4 M Tris (pH 6.5), 0.2% Tween-20, 1% sodium caseinate, 0.1% sodium azide, and 0.05% Proclin. It was immersed in a solution prepared by 300 and kept wet. The prepared gold condensate was prepared by dialysis against a solution having the same composition as the above solution. Then, the cellulose membrane was treated with the dialyzed gold condensate and dried to complete the second connection pad 150.
  • the detection pad 160 is designated by stamping the detection area with polyethylene phthalate ink, and then stamped again with a solution containing an antibody capable of binding to a detector such as FAM, HEX, and Cy5 thereon, and NHS (N- hydroxysulfosuccinimide) solution was applied and reacted to fix the antibody.
  • a detector such as FAM, HEX, and Cy5 thereon
  • NHS N- hydroxysulfosuccinimide
  • a portion of the initiator pad 132 and the second connection pad 150 in contact with the reaction pad was coated with a 5% low melting point agarose (Lonza, NuSieve GTG Agarose) solution.
  • each structure was arranged as shown in FIG. 1 or 2.
  • a structure without the buffer pad 121 and the initiator pad 132 may be disposed as shown in FIG. 1
  • a structure with the buffer pad and the initiator pad may be disposed as shown in FIG. 2 .
  • the perspective view may be the same as that of FIG. 7
  • the perspective view may be the same as that of FIG. 8 .
  • the sample pad 120, the reaction pad 140, and the second connection pad 150 and, optionally, a copper plate connected to a heating pad 141 by a hot wire is disposed at the bottom of the initiator pad 132, and the reaction pad A polyacrylic film was placed on top of the blocking pad 142.
  • Example A-3 Preparation of Fluorescence-Based Lab-on-Paper Structure for Detecting Nucleic Acids
  • the sample pad 120 and buffer pad 121 0.01 ⁇ m cellulose was used as the first connection pad 131 or initiator pad 132
  • the reaction pad 140 was made of 0.005 ⁇ m cellulose acetate
  • the second connection pad 150 was made of 0.05 ⁇ m cellulose
  • the detection pad 160 was made of 0.005 ⁇ m nitrocellulose
  • the absorption pad 170 was made of 0.5 ⁇ m glass fiber.
  • the reaction pad 140 was prepared by making a pad by overlapping cellulose acetate films, immersing the pad in a solution containing 45 mM sucrose, 0.005 w/w% TritonX-100, and 0.2 w/w% glycerol, and drying. Then, a well was formed using a micro-drill, and a hydrogel layer including a primer set was formed on the bottom of the well.
  • hydrogel layer To form the hydrogel layer, first, based on the total volume of the hydrogel solution, UV-light crosslinkable poly (ethylene glycol) diacrylate (PEGDA, Sigma-Aldrich, MW700) 20% v / v, poly (ethylene glycol) ( Mix PEG, Sigma-Aldrich, MW600) 40% v/v and photoinitiator 2-hydroxy-2-methylpropiophenone (Sigma-Aldrich) 5% v/v and buffer (PBS buffer, pH7.5) 35% Then, a hydrogel solution was prepared by mixing each primer set therein. The poly(ethylene glycol) is preferably included to increase the porosity of the hydrogel microparticles. Then, the hydrogel solution was applied to the inner surface of each well of the reaction pad and exposed to UV (360 nm wavelength, 35 mJ/cm 2 ) for 1 minute to form a hydrogel coating layer.
  • PEGDA poly (ethylene glycol) diacrylate
  • PEG Poly (ethylene glycol)
  • dNTP 1.4mM, dATP, dCTP, dGTP and dTTP
  • isothermal amplification buffer (1X, 20mM Tris-HCl, 10mM (NH 4 ) 2 SO 4 , 50mM KCl, 2mM MgSO 4 were added to the surface of the reaction pad 140.
  • 0.1% Tween-20, pH 7.5) and Bst 3.0 DNA polymerase (320 U/ml) were applied, and fixed by heating in an oven at about 38° C. for about 30 minutes.
  • a well having a diameter of 5 mm is formed with a microdrill, polyethylene phthalate ink is applied thereto, a solution containing FAM, HEX, and an antibody capable of binding to Cy5 is added dropwise thereon, and NHS (N-hydroxysulfosuccinimide) solution was applied and reacted to fix the antibody.
  • NHS N-hydroxysulfosuccinimide
  • the initiator pad 132 and the second connection pad 150 were coated with a 5% low-melting point agarose (Lonza, NuSieve GTG Agarose) solution at a portion in contact with the reaction pad.
  • a 5% low-melting point agarose Lisco, NuSieve GTG Agarose
  • each structure was arranged as shown in FIG. 1 or 2.
  • a structure without the buffer pad 121 and the initiator pad 132 may be disposed as shown in FIG. 1
  • a structure with the buffer pad and the initiator pad may be disposed as shown in FIG. 2 .
  • the perspective view may be the same as that of FIG. 7
  • the perspective view may be the same as that of FIG. 8 .
  • the sample pad 120, the reaction pad 140, and the second connection pad 150 and, optionally, a copper plate connected to a heating pad 141 by a hot wire is disposed at the bottom of the initiator pad 132, and the reaction pad A polyacrylic film was placed on top of the blocking pad 142.
  • Test Example A-2 Virus detection from blood samples using structures containing gold particles
  • Virus infection such as SARS-CoV-2 can be easily measured by extracting nucleic acid from a blood sample using the lab-on-paper nucleic acid detection structure of the present invention, amplifying the nucleic acid, and observing the color change of the band.
  • a set of primers capable of selectively binding to the N protein gene characteristic of SARS-CoV-2 or the Rdrp gene can be used.
  • one of the forward or reverse primers of each set is, for example, FAM, HEX, or Cy5 bound, and the other primer is biotin bound.
  • the amplified nucleic acid bound to the gold nanoparticle moves to the detection pad 160 by lateral flow, and the detector bound to the other side of the amplified nucleic acid is specific for FAM, HEX, or Cy5 immobilized on the detection pad 160.
  • the color of the detection area of the detection pad is changed to pink while binding to the receptor that can be bound to it.
  • the principle of binding of the target nucleic acid to the detection pad is illustrated in FIG. 3 .
  • a primer set that selectively binds to a gene characteristic of a virus with a high possibility of cross-detection with SARS-CoV-2 can be introduced and detected together as a negative control.
  • Test Example A-3 Nucleic Acid Extraction and Amplification from Blood Samples Using Structures Containing Gold Particles
  • Nucleic acid was extracted from a blood sample using the lab-on-paper nucleic acid detection construct of the present invention, amplified, and color change was observed to confirm whether it could be used for diagnosis.
  • human blood was purchased and prepared as whole blood from Innovative Research (IWB1K2E10ML, USA), and 18S rRNA primers as a positive control were purchased from Tocris (# 7325, USA). It was confirmed through the data sheet that the whole blood used was not infected with any virus or bacteria.
  • One test sample was prepared by mixing 1 ⁇ l of 0.1 pg/ ⁇ l of SARS-CoV-2 positive control from siTOOLs Biotech with 100 ⁇ l of the human blood.
  • F3 primer bound a detector to the 5' end
  • B3 primer bound biotin to the 5' end.
  • human 18s RNA (A) was introduced with FAM, and N gene (B) with Cy5 as a detector.
  • Lysis buffer (20 mM Tris HCl (pH 8.8), 15 mM MgSO 4 , 15 mM KCl, 15 mM (NH 4 ) 2 SO 4 , 0.1 50 ⁇ l of w/w% Tween20, 0.05 mg/ml proteinase K) was added and lightly tapped, followed by incubation at room temperature for about 5 minutes.
  • the heating pad 141 under the sample pad 120 operated at 60° C., it was slowly added dropwise to the sample pad 120 of the structure for detecting nucleic acids prepared in Example 2 within 5 minutes within 5 minutes, and the addition buffer ( 250 ⁇ l of 20 mM Tris-HCl, 10 mM (NH 4 ) 2 SO 4 , 50 mM KCl, 2 mM MgSO 4 , 0.1% Tween®20, pH 8.8) was slowly added dropwise to the sample pad for about 2 minutes, followed by reaction.
  • the heating pad under the pad was heated to 60 °C and reacted for 30 minutes.
  • the heating pad under the second connection pad and the initiator pad was heated to 65° C., and 250 ⁇ l of the addition buffer was slowly added dropwise to the buffer pad for 2 minutes, and the color change of the detection zone displayed on the detection pad was observed.
  • Test Example A-4 Virus detection from blood samples using fluorescence-based constructs
  • Virus infection such as SARS-CoV-2 can be easily measured by extracting nucleic acid from a blood sample using the lab-on-paper nucleic acid detection structure of the present invention, amplifying the nucleic acid, and measuring fluorescence.
  • a set of primers capable of selectively binding to the N protein gene characteristic of SARS-CoV-2 or the Rdrp gene can be used.
  • either the forward primer or the reverse primer is labeled with a fluorescent marker, and thus can be observed in a sample of a patient infected with SARS-CoV-2.
  • the principle of binding of the target nucleic acid to the detection pad is illustrated in FIG. 4 .
  • a primer set that selectively binds to a gene characteristic of a virus that is highly likely to cross-detect SARS-CoV-2 can be introduced and detected together as a negative control.
  • Test Example A-5 Extraction and amplification of nucleic acids from blood samples using fluorescence-based constructs
  • Nucleic acids were extracted from blood samples using the construct for detecting nucleic acids in lab-on-paper of the present invention, and nucleic acids were amplified to confirm whether they could exhibit fluorescence.
  • human blood was purchased and prepared as whole blood from Innovative Research (IWB1K2E10ML, USA), and 18S rRNA primers as a positive control were purchased from Tocris (# 7325, USA). It was confirmed through the data sheet that the whole blood used was not infected with any virus or bacteria.
  • One test sample was prepared by mixing Borrelia Afzelii with the human blood at 1*10 2 cell/ml, respectively.
  • Primer sets for detection of strains mixed with blood and primer sets used as negative control and Borrelia burgdorferi are shown in Table 4 below.
  • the primers used in this test example were designed to selectively bind to human 18sRNA present in the sample and the mixed strain based on data obtained from Genebank, a nucleotide sequence database.
  • Primer Set 1 (B.afzelii target) F3 5'-GGTATACTGACAGCAGCTT-3' B3 5′-CTTGCAGCTTAATAATAGCCTT-3′ FIP 5'-GTTGCTCGGTCCTCCATGTTTAAATTTTTAATGTTATCCGTGATATGGTTCCGA-3' BIP 5'-GGATTTCGTATCAATTTTGGAGGCATTTTAAGTTACAAAGGTCCCATTGC-3' FL 5′-ATAAGGCCTTCGGTATTG-3′ BL 5′-ATTCTACGTTCCGATTCTCAGTAT-3′ Primer Set 2 (B.burgdorferi target) F3 5′-AGAGCAGCTGAGGAGCT-3′ B3 5′-CTTCCAGTTGAACACCATCTT-3′ FIP 5'-AAGTCCACGACGGTTGAGACCTTTTTGCAGCCTGCTTAAATTAACA-3' BIP 5′-GAGCAAACGAAGTTGAAGCTATTTTAGCCTGAGCAGTTAGAGC-3′ FL 5′-GA
  • F3 primer bound a fluorescent marker to the 5' end
  • the positive control human 18s RNA was FAM
  • Borrelia afzeli B
  • B was HEX
  • Borrelia burgdor Primers targeting Perry C were labeled with Cy5.
  • Lysis buffer (20mM Tris HCl (pH 8.8), 15mM MgSO 4 , 15mM KCl, 15mM (NH 4 ) 2 SO 4 , 0.1 w/w% Tween20, 0.05 mg/ml protease (Protenase K) 50 ⁇ l was added and lightly tapped, followed by incubation at room temperature for about 5 minutes.
  • the heating pad 141 under the sample pad 120 operated at 60° C., it was slowly added dropwise to the sample pad 120 of the structure for detecting nucleic acid prepared in Example 3 within 5 minutes, and the addition buffer ( 250 ⁇ l of 20 mM Tris-HCl, 10 mM (NH 4 ) 2 SO 4 , 50 mM KCl, 2 mM MgSO 4 , 0.1% Tween®20, pH 8.8) was slowly added dropwise to the sample pad for about 2 minutes, followed by reaction.
  • the heating pad under the pad was heated to 60 °C and reacted for 30 minutes.
  • heating pads below the initiator pad and the second connection pad were heated to 65° C., and 250 ⁇ l of the buffer solution was slowly added dropwise to the buffer pad for 2 minutes.
  • the construct as a positive control (A) worked normally, was infected with Borrelia afzeli (B), and was not infected with Borrelia burgdorferi (C). This is the same as intended in the prepared sample, and it was confirmed that a number of diseases, viral or fungal infections can be diagnosed using the system of the present invention.
  • Example B-1 Confirmation of gene amplification
  • the six primer sets were prepared and prepared as a primer mix of the composition and concentration shown in Table 2 below.
  • 1 ⁇ l of prepared SARS-CoV-2 template RNA was mixed with 2 ⁇ l of 10x primer mix, 10 ⁇ l of Colorimetric LAMP 2X Master Mix (WarmStart ® , BioLabs Inc.), and 7 ⁇ l of dH 2 O to prepare a mixture of 20 ⁇ l in total. did No template RNA was added to the control group.
  • the mixed solution has a composition shown in Table 3 below. After the prepared mixture was isothermally amplified at 65° C. for 30 minutes, the reaction was stopped and color change was observed with the naked eye.
  • RNA target detection group control group Colorimetric LAMP 2X Master Mix 10 ⁇ l 10 ⁇ l Primer Mix (10X) 2 ⁇ l 2 ⁇ l target RNA 1 ⁇ l 0 dH2O 7 ⁇ l 8 ⁇ l total volume 20 ⁇ l 20 ⁇ l
  • the analysis ability of the primer set according to the present invention was evaluated using the primer mix of Example B-1.
  • the assay specificity for SARS-CoV-2 of the primer set according to the present invention was evaluated using the primer mix of Example B-1.
  • human coronavirus OC43 HoV OC43
  • human coronavirus 229E HoV 229E
  • MERS virus MERS virus
  • influenza virus H1N1
  • T7 Viral RNA was produced by transcription.
  • the prepared RNA was purified in a conventional manner and used as a template.
  • 10 ⁇ 6 templates were used to prepare a mixed solution for the amplification reaction as in Example B-1, and subjected to an isothermal amplification reaction.
  • the color change of the reaction product was visually confirmed and verified again by electrophoresis. As a result, no color change was observed in the comparison group, and a color change to yellow was observed only for SARS-CoV-2, confirming that the primer set of the present invention has specificity for SARS-CoV-2.
  • the analysis ability of the primer set according to the present invention was evaluated using the primer mix of Example B-1.
  • Example B-1 a mixture for the amplification reaction was prepared as in Example B-1, and 5 minutes, 10 minutes, 15 minutes, 20 minutes, Isothermal amplification reactions were performed for 25 minutes and 30 minutes. Thereafter, the color change was visually confirmed and the result was confirmed through electrophoresis. The color of the reaction product remained pink and was judged as negative, and when it changed to yellow, it was judged as positive.
  • Example B-5 Evaluation of analytical performance using virtual specimens
  • samples were taken from the nasopharynx and oropharynx of 5 healthy people who had no history of COVID-19 or contact with patients with COVID-19, did not show any symptoms suggestive of COVID-19, and did not have a history of overseas travel within 2 weeks.
  • After collecting and dividing each sample into two samples only one sample was mixed with 5 pg of SARS-CoV-2 positive control to make a virtual patient sample.
  • An isothermal amplification reaction was performed after preparing a mixed solution for an amplification reaction as in Example B-1 using the ten samples.
  • Example B-6 Analytical performance evaluation using interfering substances
  • Example 6 After randomly dividing 9 samples out of 10 samples collected and prepared in Example 6 into three groups, one group was not treated with interfering substances, and each of the two groups was treated with 0.1 v/v% human blood. and 0.1 v/v% ethanol. Then, each specimen was divided into three samples, and only two samples were prepared as virtual patient specimens as in Example B-5, and a mixed solution for an amplification reaction was prepared and an isothermal amplification reaction was performed.
  • the primer set is not limited to the paper structure of the present invention and can exhibit excellent analytical performance.

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Abstract

The present invention relates to coronavirus diagnosis through single application of a sample, a structure capable of simultaneously diagnosing a plurality of diseases including coronavirus, and a diagnostic system using same and is characterized by the ability to perform sample pretreatment, isothermal amplification, detection, and diagnosis steps on a single chip through the application of lab-on-paper technology and by the structure in which a sample is traveled in a lateral flow-type manner and is finally connected to gene big data relevant to diseases including coronavirus and strain infections, thereby enabling diagnosis of whether a disease and strain infection exists.

Description

코로나바이러스 검출용 랩온 페이퍼 및 이를 이용한 코로나바이러스 진단 시스템Lab-on-paper for detecting coronavirus and coronavirus diagnosis system using the same
본 발명은 1회의 시료를 적용하여 다수의 질환을 동시에 진단할 수 있는 측방 유동성의 일체형 시스템에 기반한 구조물에 대하여 신종 코로나바이러스의 검출용 프라이머 세트 및 이를 검출하기 위한 조성물을 적용한 코로나바이러스 검출용 랩온 페이퍼에 대한 것이다. 따라서 시료로부터 핵산을 정제하지 않고 곧바로 적용함에도 검출 감도가 높고, 코로나바이러스와 함께 복수 개의 표적 핵산을 동시에 검출이 가능하여 다수의 질병을 쉽고 빠르게 진단할 수 있다.The present invention is a lab-on-paper for detecting coronavirus to which a set of primers for detecting novel coronavirus and a composition for detecting the same are applied to a structure based on an integrated system of lateral fluidity capable of simultaneously diagnosing multiple diseases by applying one sample. It is about. Therefore, the detection sensitivity is high even when the nucleic acid is directly applied without purifying the nucleic acid from the sample, and a plurality of target nucleic acids can be simultaneously detected together with the coronavirus, making it possible to quickly and easily diagnose a number of diseases.
코로나 바이러스는 단일가닥 RNA를 게놈으로 가지는 코로나바이러스과에 속하는 바이러스들을 지칭하며 조류뿐만 아니라 사람을 포함한 다양한 포유류에서도 발견된다. 코로나바이러스는 그 종이 다양하고, 바이러스의 특성과 숙주에 따라서 호흡기와 소화기 감염병을 모두 유발하는 것으로 알려져 있다. 사람에서는 2003년 전 세계적으로 문제시되었던 중증급성호흡기증후군(Severe Acute Respiratory Syndrom, SARS)과 2015년 중동호흡기증후군 (Middle East Respiratory Syndrome Coronavirus, MERS)의 원인 바이러스로서 문제된 바 있고, 최근 중국 우한에서 발생하여 전 세계적으로 유행하고 있는 신종 코로나바이러스 감염증-19 (Coronavirus Disease-19, COVID-19)의 병원체인 SARS-CoV-2 로서 코로나바이러스가 주목 받고 있다.Coronaviruses refer to viruses belonging to the Coronaviridae family, which have single-stranded RNA as their genome, and are found not only in birds but also in various mammals, including humans. Coronaviruses are known to cause both respiratory and digestive infectious diseases depending on the characteristics of the virus and its host. In humans, it has been problematic as a virus that causes Severe Acute Respiratory Syndrom (SARS), which was a problem worldwide in 2003, and Middle East Respiratory Syndrome Coronavirus (MERS) in 2015, and recently occurred in Wuhan, China. As a result, the coronavirus is attracting attention as SARS-CoV-2, the pathogen of the novel coronavirus disease-19 (COVID-19) that is prevalent worldwide.
코로나바이러스 감염증은 높은 확산율을 보이고, 발병 초기의 무증상 상태에서도 전염력이 높으므로 집단 감염을 일으킬 가능성이 크다. 현재 전 세계에서 치료제와 백신을 개발하고 있으나 치료책이 확립되지 않았으므로, 최선의 대책은 조기에 감염자를 발견하여 집단 감염을 예방하는 것이다. 따라서 다수의 검사 대상에 대해 신속하고 정확하게 코로나바이러스 감염 여부를 진단할 수 있는 기술의 개발이 필요하다.Coronavirus infection shows a high spread rate and is highly contagious even in an asymptomatic state in the early stages of the outbreak, so it is highly likely to cause mass infection. Treatments and vaccines are currently being developed all over the world, but no cure has been established, so the best countermeasure is to detect infected people early and prevent mass infection. Therefore, it is necessary to develop a technology capable of quickly and accurately diagnosing coronavirus infection for a large number of test subjects.
현재 Real-time PCR을 이용한 진단법이 가장 빠르고 민감한 진단법으로 알려져 있으며, 일반적으로 8시간 이내에 진단이 가능하다. 현재 분자진단 방법은 PCR기술과 마이크로 채널 기술의 발전과 더불어 급속히 발전하고 있으며 Alere사의 AlereTM I 나 Roche의 Cobas Influenza 와 같이 60분 내에 검출이 가능한 제품도 상용화 되어 있다. 하지만 급속분자진단이 가능한 방법론의 경우에는 고가의 분석 장비 혹은 고가의 검사비용이 요구되며 분자진단의 전 과정을 구현하기 위해서는 여러 단계를 요구하고 있으므로 아직 현장 진단에는 한계점을 가지고 있다.Currently, the diagnostic method using real-time PCR is known as the fastest and most sensitive diagnostic method, and it is generally possible to diagnose within 8 hours. Currently, molecular diagnostic methods are developing rapidly with the development of PCR technology and microchannel technology, and products that can be detected within 60 minutes, such as Alere's AlereTM I or Roche's Cobas Influenza, are commercialized. However, in the case of a methodology capable of rapid molecular diagnosis, expensive analysis equipment or expensive test costs are required, and several steps are required to implement the entire process of molecular diagnosis, so on-site diagnosis still has limitations.
현재 보편화된 분자진단 법은 real-time PCR을 이용하는 것으로, 신속성 때문에 가장 보편화되어 있지만 현장진단 또는 1,2차 의료기관에서 사용하기에는 크고 고가의 장비를 요하기 때문에 쉽게 사용하는 것이 어려운 실정이다. 분자 진단을 위해서는 크게 시료의 전처리 (preparation), 핵산 증폭 반응 (reaction)과 검출 (detection)의 3단계가 요구되는데, 핵산 증폭 반응과 검출은 real-time PCR 장비에 의해 동시에 재현하는 것이 가능하지만 시료의 전처리 문제는 여전히 남아 있다.The current universal molecular diagnosis method uses real-time PCR, which is the most common because of its speed, but it is difficult to use easily because it requires large and expensive equipment for on-site diagnosis or primary and secondary medical institutions. For molecular diagnosis, three steps are largely required: sample preparation, nucleic acid amplification reaction, and detection. Although nucleic acid amplification reaction and detection can be reproduced simultaneously by real-time PCR equipment, The preprocessing problem still remains.
한편, 랩온페이퍼(Lab-on-paper) 기술은 시료의 전처리, 등온증폭, 검출, 및 분석 단계를 하나의 칩 위에서 수행하도록 하는 일체형 시스템에 기반한 기술을 의미한다. 작은 페이퍼와 페이퍼에 심은 칩 구조물로 모든 반응을 자동화하여 빠르게 수행할 수 있으므로 검출 현장 등의 장소에 구애받지 않는 장점을 갖는다.On the other hand, lab-on-paper technology refers to a technology based on an integrated system that performs sample pretreatment, isothermal amplification, detection, and analysis steps on a single chip. Since all reactions can be automated and quickly performed with small paper and a chip structure embedded in the paper, it has the advantage of being independent of the location such as the detection site.
본 발명은 다중 진단이 가능한 랩온페이퍼 구조물의 구성과 위 랩온페이퍼 구조물에 적용되는 코로나바이러스 검출용 프라이머 세트를 제공한다.The present invention provides a configuration of a lab-on-paper structure capable of multiple diagnosis and a primer set for detecting coronavirus applied to the lab-on-paper structure.
본 발명의 일 실시예에 따른 코로나바이러스 검출용 랩온 페이퍼는 생물학적 시료를 수용하는 시료패드; 상기 시료패드의 상부에 배치되고, 시료패드와 반응패드를 연결하는 제1연결패드; 상기 제1연결 패드의 하부에 배치되고, 표적 핵산과 특이적으로 결합할 수 있는 프라이머와 등온증폭반응 (LAMP)을 위한 시약을 포함하고, 등온증폭반응이 일어나는 반응패드; 상기 반응패드 상부에 배치되고, 반응온도 유지 및 시료의 증발을 차단하는 차단패드; 상기 반응패드의 상부에 배치되고, 금 나노입자가 고정되어 있는 제2연결패드; 상기 제2연결패드의 하부에 배치되고, 상기 금 나노입자와 결합된 등온증폭반응물로부터 증폭된 표적 핵산을 획득하는 검출패드; 및 상기 검출패드의 측방에 배치되고, 잔류하는 시료를 흡수하는 흡수패드를 포함하고, 상기 시료패드, 반응패드, 및 제2연결패드 하부에 배치된 가열패드를 포함하고, 서열번호 1 내지 6의 염기서열 중 어느 하나로 표시되는 프라이머를 포함하는, 코로나바이러스를 검출하기 위한 프라이머 세트가 적용된 것일 수 있다.A lab-on-paper for detecting coronavirus according to an embodiment of the present invention includes a sample pad accommodating a biological sample; a first connection pad disposed above the sample pad and connecting the sample pad and the reaction pad; a reaction pad disposed under the first connection pad, containing a primer capable of specifically binding to a target nucleic acid and a reagent for isothermal amplification (LAMP), and wherein the isothermal amplification reaction occurs; a blocking pad disposed above the reaction pad to maintain the reaction temperature and block evaporation of the sample; a second connection pad disposed above the reaction pad and to which gold nanoparticles are fixed; a detection pad disposed under the second connection pad and obtaining target nucleic acid amplified from an isothermal amplification reaction coupled with the gold nanoparticles; and an absorption pad disposed on a side of the detection pad to absorb the remaining sample, and a heating pad disposed below the sample pad, the reaction pad, and the second connection pad, A primer set for detecting coronavirus, including a primer represented by any one of the nucleotide sequences, may be applied.
본 발명의 다른 일 실시예에 따른 코로나바이러스 검출용 랩온 페이퍼는 생물학적 시료를 수용하는 시료패드; 상기 시료패드와 분리되어 배치되고, 재수화(rehydration) 완충액을 수용하는 버퍼패드; 상기 시료패드의 상부에 배치되고, 시료패드와 반응패드를 연결하는 제1연결패드; 상기 버퍼패드의 상부에 배치되고, 버퍼패드와 반응패드를 연결하는 개시자 (opener) 패드; 상기 제1연결패드 및 개시자패드의 하부에 배치되고, 표적 핵산과 특이적으로 결합할 수 있는 프라이머와 등온증폭반응 (LAMP)을 위한 시약을 포함하고, 등온증폭반응이 일어나는 반응패드; 상기 반응패드 상부에 배치되고, 반응온도 유지 및 시료의 증발을 차단하는 차단패드; 상기 반응패드의 상부에 배치되고, 금 나노입자가 고정되어 있는 제2연결패드; 상기 제2연결패드의 하부에 배치되고, 상기 금 나노입자와 결합된 등온증폭반응물로부터 증폭된 표적 핵산을 획득하는 검출패드; 및 상기 검출패드의 측방에 배치되고, 잔류하는 시료를 흡수하는 흡수패드를 포함하고, 상기 시료패드, 개시자패드, 반응패드, 및 제2연결패드 하부에 배치된 가열패드를 포함하는 것일 수 있다.A lab-on-paper for detecting coronavirus according to another embodiment of the present invention includes a sample pad accommodating a biological sample; a buffer pad disposed separately from the sample pad and accommodating a rehydration buffer; a first connection pad disposed above the sample pad and connecting the sample pad and the reaction pad; an initiator pad disposed above the buffer pad and connecting the buffer pad and the reaction pad; a reaction pad disposed below the first connection pad and the initiator pad, including a primer capable of specifically binding to a target nucleic acid and a reagent for an isothermal amplification reaction (LAMP), wherein an isothermal amplification reaction occurs; a blocking pad disposed above the reaction pad to maintain the reaction temperature and block evaporation of the sample; a second connection pad disposed above the reaction pad and to which gold nanoparticles are fixed; a detection pad disposed under the second connection pad and obtaining target nucleic acid amplified from an isothermal amplification reaction coupled with the gold nanoparticles; and an absorption pad disposed on a side of the detection pad to absorb the remaining sample, and a heating pad disposed below the sample pad, the initiator pad, the reaction pad, and the second connection pad. .
상기 시료패드 및 제1연결패드패드와 버퍼패드 및 개시자패드; 반응패드; 제2연결패드; 검출패드; 및 흡수패드는 순차적으로 적어도 일부분 접촉되어 측방으로 배치된 것일 수 있다.the sample pad, the first connection pad, the buffer pad, and the initiator pad; reaction pad; a second connection pad; detection pad; and the absorbent pads may be disposed laterally in contact with at least part of them sequentially.
상기 검출패드는 복수 개의 구분된 검출 구역을 포함하는 것일 수 있다.The detection pad may include a plurality of separated detection zones.
상기 금 나노입자는 표면에 스트렙타비딘(streptavidin)을 포함하는 것일 수 있다.The gold nanoparticles may contain streptavidin on their surfaces.
상기 시료패드에 적용된 시료는 흡수 패드까지 측방으로 이동하는 것일 수 있다.The sample applied to the sample pad may move laterally to the absorption pad.
본 발명의 또 다른 일 실시예에 따른 코로나바이러스 감염 진단 시스템은 생물학적 시료를 수용하는 시료패드; 상기 시료패드의 상부에 배치되고, 시료패드와 반응패드를 연결하는 제1연결패드; 상기 제1연결 패드의 하부에 배치되고, 표적 핵산과 특이적으로 결합할 수 있는 프라이머와 등온증폭반응 (LAMP)을 위한 시약을 포함하고, 등온증폭반응이 일어나는 반응패드; 상기 반응패드 상부에 배치되고, 반응온도 유지 및 시료의 증발을 차단하는 차단패드; 상기 반응패드의 상부에 배치되고, 상기 등온증폭반응물을 운반하는 제2연결패드; 상기 제2연결패드의 하부에 배치되고, 상기 등온증폭반응물로부터 증폭된 표적 핵산을 획득하는 검출패드; 상기 검출패드의 측방에 배치되고, 잔류하는 시료를 흡수하는 흡수패드; 상기 검출패드의 상부에 배치되고, 상기 검출패드로부터 형광 이미지를 획득하고, 형광 세기를 측정하는 인식부; 및 상기 인식부로부터 측정된 형광 세기 값을 이용하여 유의성 값을 도출하고, 상기 유의성 값으로 부터, 질병, 바이러스 또는 균 감염 여부를 출력하는 출력부를 포함하고, 상기 시료패드, 반응패드, 및 제2연결패드 하부에 배치된 가열패드를 포함하고, 상기 프라이머는 서열번호 1 내지 6의 염기서열 중 어느 하나로 표시되는 것일 수 있다.A coronavirus infection diagnosis system according to another embodiment of the present invention includes a sample pad accommodating a biological sample; a first connection pad disposed above the sample pad and connecting the sample pad and the reaction pad; a reaction pad disposed under the first connection pad, containing a primer capable of specifically binding to a target nucleic acid and a reagent for isothermal amplification (LAMP), and wherein the isothermal amplification reaction occurs; a blocking pad disposed above the reaction pad to maintain the reaction temperature and block evaporation of the sample; a second connection pad disposed above the reaction pad and transporting the isothermal amplification reactant; a detection pad disposed below the second connection pad and obtaining target nucleic acid amplified from the isothermal amplification reaction; an absorption pad disposed on a side of the detection pad and absorbing the remaining sample; a recognition unit disposed above the detection pad, acquiring a fluorescence image from the detection pad, and measuring fluorescence intensity; and an output unit for deriving a significance value using the fluorescence intensity value measured by the recognition unit and outputting whether or not a disease, virus, or bacterial infection is present from the significance value, wherein the sample pad, the reaction pad, and the second A heating pad disposed below the connection pad, and the primer may be one represented by any one of the nucleotide sequences of SEQ ID NOs: 1 to 6.
본 발명의 또 다른 일 실시예에 따른 코로나바이러스 감염 진단 시스템은 생물학적 시료를 수용하는 시료패드; 상기 시료패드와 분리되어 배치되고, 재수화(rehydration) 완충액을 수용하는 버퍼패드; 상기 시료패드의 상부에 배치되고, 시료패드와 반응패드를 연결하는 제1연결패드; 상기 버퍼패드의 상부에 배치되고, 버퍼패드와 반응패드를 연결하는 개시자 (opener) 패드; 상기 제1연결패드 및 개시자 패드의 하부에 배치되고, 표적 핵산과 특이적으로 결합할 수 있는 프라이머와 등온증폭반응 (LAMP)을 위한 시약을 포함하고, 등온증폭반응이 일어나는 반응패드; 상기 반응패드의 상부에 배치되고, 상기 등온증폭반응물을 운반하는 제2연결패드; 상기 제2연결패드의 하부에 배치되고, 상기 등온증폭반응물로부터 증폭된 표적 핵산을 획득하는 검출패드; 상기 검출패드의 측방에 배치되고, 잔류하는 시료를 흡수하는 흡수패드; 상기 검출패드의 상부에 배치되고, 상기 검출패드로부터 형광 이미지를 획득하고, 형광 세기를 측정하는 인식부; 및 상기 인식부로부터 측정된 형광 세기 값을 이용하여 유의성 값을 도출하고, 상기 유의성 값으로 부터, 질병, 바이러스 또는 균 감염 여부를 출력하는 출력부를 포함하고, 상기 시료패드, 개시자 패드, 반응패드, 및 제2연결패드 하부에 배치된 가열패드를 포함하고, 상기 프라이머는 서열번호 1 내지 6의 염기서열 중 어느 하나로 표시되는 것일 수 있다.A coronavirus infection diagnosis system according to another embodiment of the present invention includes a sample pad accommodating a biological sample; a buffer pad disposed separately from the sample pad and accommodating a rehydration buffer; a first connection pad disposed above the sample pad and connecting the sample pad and the reaction pad; an initiator pad disposed above the buffer pad and connecting the buffer pad and the reaction pad; a reaction pad disposed below the first connection pad and the initiator pad, including a primer capable of specifically binding to a target nucleic acid and a reagent for an isothermal amplification reaction (LAMP), wherein an isothermal amplification reaction occurs; a second connection pad disposed above the reaction pad and transporting the isothermal amplification reactant; a detection pad disposed below the second connection pad and obtaining target nucleic acid amplified from the isothermal amplification reaction; an absorption pad disposed on a side of the detection pad and absorbing the remaining sample; a recognition unit disposed above the detection pad, acquiring a fluorescence image from the detection pad, and measuring fluorescence intensity; and an output unit for deriving a significance value using the fluorescence intensity value measured by the recognition unit and outputting whether or not a disease, virus, or fungus infection is present from the significance value, wherein the sample pad, initiator pad, and reaction pad , and a heating pad disposed below the second connection pad, and the primer may be one represented by any one of the nucleotide sequences of SEQ ID NOs: 1 to 6.
상기 시료패드 및 제1연결패드와 버퍼패드 및 개시자패드; 반응패드; 제2연결패드; 검출패드; 및 흡수패드는 순차적으로 적어도 일부분 접촉되어 측방으로 배치된 것일 수 있다.the sample pad, the first connection pad, the buffer pad, and the initiator pad; reaction pad; a second connection pad; detection pad; and the absorbent pads may be disposed laterally in contact with at least part of them sequentially.
여기서 사용되는 전문용어는 단지 특정 실시예를 언급하기 위한 것이며, 본 발명을 한정하는 것을 의도하지 않는다. 여기서 사용되는 단수 형태들은 문구들이 이와 명백히 반대의 의미를 나타내지 않는 한 복수 형태들도 포함한다. 명세서에서 사용되는 "포함하는"의 의미는 특정 특성, 영역, 정수, 단계, 동작, 요소 및/또는 성분을 구체화하며, 다른 특정 특성, 영역, 정수, 단계, 동작, 요소, 성분 및/또는 군의 존재나 부가를 제외시키는 것은 아니다.The terminology used herein is intended only to refer to specific embodiments and is not intended to limit the present invention. As used herein, the singular forms also include the plural forms unless the phrases clearly indicate the opposite. As used herein, the meaning of "comprising" specifies specific characteristics, regions, integers, steps, operations, elements, and/or components, and other specific characteristics, regions, integers, steps, operations, elements, elements, and/or groups. does not exclude the presence or addition of
다르게 정의하지는 않았지만, 여기에 사용되는 기술용어 및 과학용어를 포함하는 모든 용어들은 본 발명이 속하는 기술분야에서 통상의 지식을 가진 자가 일반적으로 이해하는 의미와 동일한 의미를 가진다. 보통 사용되는 사전에 정의된 용어들은 관련기술문헌과 현재 개시된 내용에 부합하는 의미를 가지는 것으로 추가 해석되고, 정의되지 않는 한 이상적이거나 매우 공식적인 의미로 해석되지 않는다. 여기서 사용되는 전문용어는 단지 특정 실시예를 언급하기 위한 것이며, 본 발명을 한정하는 것을 의도하지 않는다. 여기서 사용되는 단수 형태들은 문구들이 이와 명백히 반대의 의미를 나타내지 않는 한 복수 형태들도 포함한다. 명세서에서 사용되는 "포함하는"의 의미는 특정 특성, 영역, 정수, 단계, 동작, 요소 및/또는 성분을 구체화하며, 다른 특정 특성, 영역, 정수, 단계, 동작, 요소, 성분 및/또는 군의 존재나 부가를 제외시키는 것은 아니다.Although not defined differently, all terms including technical terms and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention belongs. Terms defined in commonly used dictionaries are additionally interpreted as having meanings consistent with related technical literature and currently disclosed content, and are not interpreted in ideal or very formal meanings unless defined. The terminology used herein is intended only to refer to specific embodiments and is not intended to limit the present invention. As used herein, the singular forms also include the plural forms unless the phrases clearly indicate the opposite. As used herein, the meaning of "comprising" specifies specific characteristics, regions, integers, steps, operations, elements, and/or components, and other specific characteristics, regions, integers, steps, operations, elements, elements, and/or groups. does not exclude the presence or addition of
이하, 첨부된 도면을 참조하여 본 발명의 바람직한 실시예에 의한 다중 핵산 검출용 구조물 및 질병, 바이러스 또는 균 감염 진단 시스템에 대하여 설명하기로 한다.Hereinafter, a structure for detecting multiple nucleic acids and a system for diagnosing a disease, virus or fungal infection according to a preferred embodiment of the present invention will be described with reference to the accompanying drawings.
본 발명의 질병, 바이러스 또는 균 감염 진단 시스템 및 이에 포함된 다중 핵산 검출용 구조물은 랩온페이퍼 칩(lab-on-paper chip) 기술에 기반한 것으로서, 본 발명의 시스템 또는 구조물을 이용하면 별도의 핵산을 정제하지 않아도 반응패드까지 이동하면서 핵산 물질이 정제되어 곧바로 증폭 반응에 적용될 수 있고, 1회의 시료를 적용하여 다수의 표적 핵산을 동시에 검출하고 관련 질병을 진단할 수 있다. 이를 실현하기 위해, 본 발명의 시스템 및 구조물은 시료패드(120), 버퍼패드(121), 제1연결패드(131), 개시자패드(132), 반응패드(140), 가열패드(141), 차단패드(142), 제2연결패드(150), 검출패드(160), 흡수패드(170) 및 하우징(110)을 구성요소로 포함한다. 상기 시스템은 일 구체예에 따른 다중 핵산 검출용 구조물을 포함하고, 인식부(200) 및 출력부(120)를 더 포함할 수 있다.The disease, virus or fungal infection diagnosis system and structure for detecting multiple nucleic acids included therein of the present invention are based on lab-on-paper chip technology. Even without purification, the nucleic acid material can be purified while moving to the reaction pad and immediately applied to the amplification reaction, and multiple target nucleic acids can be simultaneously detected and related diseases can be diagnosed by applying one sample. To realize this, the system and structure of the present invention include a sample pad 120, a buffer pad 121, a first connection pad 131, an initiator pad 132, a reaction pad 140, and a heating pad 141 , a blocking pad 142, a second connection pad 150, a detection pad 160, an absorption pad 170, and a housing 110 as components. The system includes the structure for detecting multiple nucleic acids according to one embodiment, and may further include a recognition unit 200 and an output unit 120.
이하 각 구성요소에 대해 상세히 설명한다.Hereinafter, each component will be described in detail.
시료패드sample pad
시료패드(120)는 핵산 물질이 함유된 시료를 수용한다. 상기 시료는 인체로부터 분리된 것으로서 혈액, 혈청, 혈장, 침, 땀, 소변, 세포배양액, 조직현탁액 등이 있으나 이에 제한되지 않는다. 상기 시료는 세포 용해 조성물(lysis buffer)과 혼합된 것일 수 있고, 필요에 따라 상기 세포 용해용 조성물(Lysis buffer)과 혼합 후, 원심분리, 여과, 침전 등 통상적으로 알려진 방법으로 추가 정제될 수 있다. 바람직하게는 상기 시료는 세포 용해용 조성물과 혼합된 후 핵산 검출용 구조물 외에서 상기 시료패드에 적용하기 위해 정제하는 단계를 포함하지 않는다. The sample pad 120 accommodates a sample containing a nucleic acid material. The sample is separated from the human body and includes blood, serum, plasma, saliva, sweat, urine, cell culture fluid, tissue suspension, etc., but is not limited thereto. The sample may be mixed with a cell lysis composition (lysis buffer), and if necessary, after mixing with the cell lysis composition (lysis buffer), it may be additionally purified by a commonly known method such as centrifugation, filtration, and precipitation. . Preferably, the sample does not include a step of purifying the sample to be applied to the sample pad other than the structure for detecting nucleic acid after being mixed with the composition for cell lysis.
상기 세포 용해용 조성물은 세포 용해용 완충액과 같은 의미로 사용될 수 있고, Tris(트리스(히드록시메틸)아미노메탄 (tris(hydroxymethyl)aminomethane))를 5 mM 내지 80 mM, 5mM 내지 50mM, 또는 10mM 내지 50 mM로 포함하는 것일 수 있다. Tris는 구체적으로 Tris-HCl일 수 있고, 세포 용해용 조성물에서 완충제로서 급격한 pH 변동을 줄일 수 있다. The cell lysis composition may be used in the same sense as the cell lysis buffer, and Tris (tris (hydroxymethyl) aminomethane) is 5 mM to 80 mM, 5 mM to 50 mM, or 10 mM to It may be included at 50 mM. Tris may specifically be Tris-HCl, and can reduce rapid pH fluctuations as a buffer in a cell lysis composition.
상기 세포 용해용 조성물은 pH 8.0 내지 9.0일 수 있다. pH가 8.0 미만인 경우, 핵산 물질의 안정성이 감소되거나 이동 속도가 감소될 수 있다.The cell lysis composition may be pH 8.0 to 9.0. When the pH is less than 8.0, the stability of the nucleic acid material may be reduced or the rate of migration may be reduced.
상기 세포 용해용 조성물은 염화칼륨(KCl)을 5 mM 내지 50 mM, 5mM 내지 40mM, 또는 10mM 내지 20 mM로 포함하는 것일 수 있다. 50 mM을 초과하는 고농도의 염화칼륨은 세포 용해에 도움이 되지만, 용출된 핵산 물질의 수용해도를 감소시켜 부가 완충액을 다량 가해야 하거나 반응패드까지 이동하는데 걸리는 시간을 증가시킬 수 있다. 반면 염화칼륨이 5 mM 미만인 경우 세포가 제대로 용해되지 않을 수 있다. The cell lysis composition may include potassium chloride (KCl) at 5 mM to 50 mM, 5 mM to 40 mM, or 10 mM to 20 mM. A high concentration of potassium chloride exceeding 50 mM is helpful for cell lysis, but reduces the aqueous solubility of the eluted nucleic acid material, which may require the addition of a large amount of additional buffer or increase the time taken to move to the reaction pad. On the other hand, if potassium chloride is less than 5 mM, cells may not be properly lysed.
상기 세포 용해용 조성물은 황산마그네슘(MgSO4)을 1 mM 내지 30 mM, 1mM 내지 20mM, 또는 2mM 내지 16 mM 로 포함하는 것일 수 있다. 황산마그네슘을 적정량 포함하는 경우 핵산 물질의 안정성과 이동 속도가 증가하는 것으로 확인되었고, 점도에 영향을 미치지 않기 때문에 유체의 흐름을 방해하지 않아 페이퍼 칩 분석용으로 시료를 전처리하는데 유리하다. The cell lysis composition may contain magnesium sulfate (MgSO 4 ) at 1 mM to 30 mM, 1 mM to 20 mM, or 2 mM to 16 mM. It has been confirmed that the stability and movement speed of nucleic acid substances are increased when an appropriate amount of magnesium sulfate is included, and since it does not affect the viscosity, it does not interfere with the flow of the fluid, which is advantageous in pre-processing the sample for paper chip analysis.
상기 세포 용해용 조성물은 황산암모늄((NH4)2SO4)을 5 mM 내지 50 mM, 5mM 내지 40mM, 또는 10mM 내지 20 mM로 포함하는 것일 수 있다. 50 mM을 초과하는 고농도의 황산암모늄은 세포 용해물을 침전시킬 수 있고, 황산암모늄이 5mM 미만인 경우 pH가 불안정해질 수 있다.The cell lysis composition may contain ammonium sulfate ((NH 4 ) 2 SO 4 ) at 5 mM to 50 mM, 5 mM to 40 mM, or 10 mM to 20 mM. High concentrations of ammonium sulfate, greater than 50 mM, may precipitate cell lysates, and pH may become unstable when ammonium sulfate is less than 5 mM.
상기 세포 용해용 조성물은 단백질분해효소를 0.01 mg/ml 내지 0.1 mg/ml, 또는 0.03 mg/ml 내지 0.07 mg/ml을 포함하는 것일 수 있다. 단백질분해효소는 고분자 단백질을 분해하여 핵산이 이동 경로인 기판이나 페이퍼의 기공을 고분자 단백질이 차단하지 않도록 하고, RNase 및 DNase 활성을 억제하여 핵산 물질의 안정성을 증가시킨다. 상기 단백질분해효소는 프로테이나아제 K(proteinase K)일 수 있다. The cell lysis composition may contain 0.01 mg/ml to 0.1 mg/ml or 0.03 mg/ml to 0.07 mg/ml of a proteolytic enzyme. The proteolytic enzyme degrades the polymer protein so that the polymer protein does not block the pores of the substrate or paper, which is the path for nucleic acid to move, and increases the stability of the nucleic acid material by inhibiting the activities of RNase and DNase. The proteolytic enzyme may be proteinase K.
상기 계면활성제는 TritonX-100 또는 Tween20 (폴리소르베이트20)일 수 있고, 세포 용해용 조성물 중량을 기준으로 0.01w/w% 내지 0.2w/w%, 바람직하게는 0.05w/w% 내지 0.1w/w%로 포함될 수 있다.The surfactant may be TritonX-100 or Tween20 (polysorbate 20), and is present in an amount of 0.01 w/w% to 0.2 w/w%, preferably 0.05 w/w% to 0.1 w, based on the weight of the cell lysis composition. Can be included as /w%.
상기 세포 용해용 조성물은 시료의 부피 대비 1:1로 사용되는 것일 수 있다.The cell lysis composition may be used in a ratio of 1:1 to the volume of the sample.
상기 세포 용해용 조성물은 글리세롤을 포함하지 않는 것일 수 있다. 글리세롤은 단백질의 침전을 막기 위해 첨가되기도 하나, 글리세롤은 점도를 증가시켜 세포 용해물의 유동성을 감소시키고, 핵산 물질의 이동에 방해가 될 수 있다.The cell lysis composition may not contain glycerol. Glycerol is sometimes added to prevent protein precipitation, but glycerol increases the viscosity, reduces the fluidity of the cell lysate, and may interfere with the movement of nucleic acid materials.
상기 세포 용해용 조성물은 환원제를 포함하지 않는 것일 수 있다. 환원제는 디티오트레이톨(DTT), 머캅토에탄올 등으로서 단백질을 변성시키고 세포 용해물의 수용해도를 높이는데 도움이 되지만, 페이퍼 칩에 흘러 들어가는 용액 내 존재하는 경우 형광 발광 또는 검출 반응에 방해가 될 수 있다. The cell lysis composition may not contain a reducing agent. Reducing agents, such as dithiothreitol (DTT) and mercaptoethanol, help to denature proteins and increase the solubility of cell lysates, but interfere with fluorescence or detection reactions when present in the solution flowing into the paper chip. It can be.
상기 시료패드의 하부에 배치되고 상기 시료패드를 가열하기 위한 가열패드(141)를 포함한다. 세포 용해용 조성물에 의한 시료의 용해를 효율적으로 하기 위해 가열패드를 60 내지 80℃의 온도에서 1 분 내지 5분 간, 바람직하게는 5 분 간 가열 처리해줌으로써 시료패드에서 바이러스와 상피세포를 포함하는 시료의 용해를 촉진한다.It is disposed under the sample pad and includes a heating pad 141 for heating the sample pad. In order to efficiently dissolve the sample by the cell lysis composition, the heating pad is heated at a temperature of 60 to 80 ° C for 1 to 5 minutes, preferably for 5 minutes, so that the sample pad contains viruses and epithelial cells. Promote dissolution of the sample.
상기 세포 용해용 조성물을 적용하면 폴리설폰 막 (예를 들어, Vivid GF) 또는 니트로셀룰로오스 막으로 이루어진 시료패드(120)에서 세포의 용해를 촉진하고, 핵산을 보다 쉽게 검출할 수 있도록 한다. 상기 폴리설폰 막 및 니트로셀룰로오스 막은 2개 이상이 적층된 구조를 이루는 것일 수 있다. 상기 폴리설폰 막은 비 대칭적인 것일 수 있고, 폴리설폰 막 및 니트로셀룰로오스 막은 0.5 ㎛ 내지 1 ㎛의 기공을 갖는 다공성 재질인 것일 수 있다. 상기 기공은 다소 큰 편인 것이 유리한데, 생물학적 시료는 점도를 갖는 것이 많고, 특히 시료에 세포 용해용 조성물을 처리하게 되면 핵산 물질과 단백질이 세포 밖으로 용출되면서 점도가 현저히 올라갈 수 있다. 따라서 시료패드의 기공 사이즈는 시료를 신속하게 흡수하기에 적절한 크기를 갖는 것이 바람직하다.Application of the cell lysis composition promotes cell lysis in the sample pad 120 made of a polysulfone membrane (eg, Vivid GF) or a nitrocellulose membrane, and makes it easier to detect nucleic acids. The polysulfone membrane and the nitrocellulose membrane may form a structure in which two or more are laminated. The polysulfone membrane may be asymmetric, and the polysulfone membrane and the nitrocellulose membrane may be porous materials having pores of 0.5 μm to 1 μm. It is advantageous that the pores are rather large. Many biological samples have viscosity, and in particular, when the sample is treated with a composition for cell dissolution, the viscosity may increase significantly as nucleic acid materials and proteins are eluted out of the cells. Therefore, it is preferable that the pore size of the sample pad has an appropriate size to rapidly absorb the sample.
상기 세포 용해용 조성물을 이용함으로써 측방 유동식 (lateral flow)으로 핵산을 보다 쉽게 검출할 수 있다. 용어 “측방 유동식”이란 중력을 이용하지 않고, 수평 방향으로 모세관 현상이나 확산 현상에 의해 시료를 적용한 지점으로부터 목표 지점까지 흐르도록 하는 방식을 의미한다. 세포 용해물에는 핵산 물질을 분해할 수 있는 가수분해 효소들이 다량 함유되어 있으므로, 이동 경로에 오래 머무는 경우 핵산 물질의 수득량이 감소할 수 있다. 따라서 측방 유동식 핵산 검출용 구조물에 적용하려면, 흐름도가 우수해야 하고, 시료가 이동하는 동안 세포 용해물이 침전되어 이동 경로를 차단하지 않아야 한다. 또한, 핵산 물질과 염을 형성하여 침전이나 이동속도를 저하시키지 않아야 한다. 본 발명의 세포 용해용 조성물의 조성은 글리세롤 또는 환원제를 포함하지 않아도 세포 용해물이나 핵산 물질이 침전되지 않고, 높은 수율로 핵산 물질을 반응 패드까지 측방으로 이동시킬 수 있다.By using the cell lysis composition, nucleic acids can be more easily detected by lateral flow. The term “lateral flow method” refers to a method in which a sample is flowed from an application point to a target point by a capillary phenomenon or a diffusion phenomenon in a horizontal direction without using gravity. Since the cell lysate contains a large amount of hydrolytic enzymes capable of degrading nucleic acid material, the yield of nucleic acid material may be reduced if the cell lysate remains in the migration path for a long time. Therefore, in order to be applied to a structure for detecting nucleic acid in a lateral flow type, the flow rate should be excellent and the cell lysate should not precipitate and block the movement path while the sample is moving. In addition, it should not form a salt with a nucleic acid material to reduce precipitation or migration speed. The composition of the composition for cell lysis of the present invention does not precipitate cell lysate or nucleic acid material even if it does not contain glycerol or a reducing agent, and can transfer nucleic acid material laterally to the reaction pad in high yield.
제1연결패드1st connection pad
제1연결패드(131)는 시료패드와 일부분 접촉하여 시료패드의 상부에 배치되고, 시료패드에 비해 상대적으로 폭이 좁은 구조물로 시료패드와 반응패드를 연결한다.The first connection pad 131 partially contacts the sample pad, is disposed above the sample pad, and connects the sample pad and the reaction pad with a structure having a relatively narrow width compared to the sample pad.
제1연결패드는 셀룰로오스 막으로 이루어진 재질로서, 0.005 ㎛ 내지 0.015 ㎛의 기공을 갖는 것일 수 있다.The first connection pad may be made of a cellulose membrane and have pores of 0.005 μm to 0.015 μm.
버퍼패드buffer pad
버퍼패드(121)는 재수화 완충액을 적가하는 패드로서, 구조물에 수압을 가하는 역할을 한다. 버퍼패드는 단백질과 같은 세포 용해 잔해물의 반응패드로의 이동을 최소화하고 반응 완료 후 수압을 가하여 등온증폭반응물 만의 검출패드로의 이동을 유도하기 위해 시료패드와 분리되어 배치될 수 있다.The buffer pad 121 is a pad to which the rehydration buffer is applied dropwise, and serves to apply water pressure to the structure. The buffer pad may be disposed separately from the sample pad in order to minimize the movement of cell lysis debris such as proteins to the reaction pad and induce the movement of only the isothermal amplification reactants to the detection pad by applying water pressure after the reaction is completed.
버퍼패드에 적용되는 재수화 완충액은 부가 완충액으로서, 예를 들어 5 mM 내지 80 mM Tris-HCl, 20 mM 내지 70 mM 염화칼륨, 0.5 mM 내지 5 mM 황산마그네슘, 1 mM 내지 30 mM 황산암모늄, 및 0.01w/w% 내지 0.2 w/w% Tween®20 내지 TritonX-100을 포함하고 산도가 pH 8.0 내지 9.0인 등온완충액 또는 인산 완충액 (50mM Na2HPO4, pH 7.2)일 수 있다. 상기 등온완충액은 보다 구체적으로 20 mM Tris-HCl, 10 mM (NH4)2SO4, 50 mM KCl, 2 mM MgSO4, 및 0.1% Tween®20을 포함하고, 산도는 pH 8.8일 수 있다. 상기 부가 완충액은 단백질분해효소, 글리세롤, 또는 환원제를 포함하지 않는 것일 수 있다.The rehydration buffer applied to the buffer pad is an additional buffer, for example, 5 mM to 80 mM Tris-HCl, 20 mM to 70 mM potassium chloride, 0.5 mM to 5 mM magnesium sulfate, 1 mM to 30 mM ammonium sulfate, and 0.01 mM It may be an isothermal buffer containing w/w% to 0.2 w/w% Tween®20 to TritonX-100 and having an acidity of pH 8.0 to 9.0 or a phosphate buffer (50 mM Na 2 HPO 4 , pH 7.2). More specifically, the isothermal buffer may include 20 mM Tris-HCl, 10 mM (NH 4 ) 2 SO 4 , 50 mM KCl, 2 mM MgSO 4 , and 0.1% Tween® 20, and may have a pH of 8.8. The addition buffer may not contain a protease, glycerol, or a reducing agent.
상기 버퍼패드는 재수화 완충액을 충분히 수령할 수 있도록 0.5 ㎛ 내지 1 ㎛의 기공을 갖는 다공성 재질로서, 면, 융, 종이, 니트로셀룰로오스, 셀룰로오스아세테이트, 유리섬유, 폴리설폰, 폴리아크릴, 폴리니트릴, 폴리피페라진, 폴리아미드, 폴리에테르설폰, 폴리비닐리덴플로리드, 폴리에틸렌이민, 폴리디메틸실록산 또는 이들의 혼합물일 수 있다.The buffer pad is a porous material having pores of 0.5 μm to 1 μm to sufficiently receive the rehydration buffer, and is made of cotton, flax, paper, nitrocellulose, cellulose acetate, glass fiber, polysulfone, polyacrylic, polynitrile, polypiperazine, polyamide, polyethersulfone, polyvinylidene fluoride, polyethyleneimine, polydimethylsiloxane or mixtures thereof.
개시자 (opener) 패드initiator pad
개시자 패드(132)는 버퍼패드와 일부분 접촉하여 버퍼패드의 상부에 배치되고, 버퍼패드에 비해 상대적으로 폭이 좁은 구조물로 버퍼패드와 반응패드를 연결한다.The initiator pad 132 partially contacts the buffer pad, is disposed above the buffer pad, and connects the buffer pad and the reaction pad with a structure having a relatively narrow width compared to the buffer pad.
개시자 패드는 셀룰로오스 막으로 이루어진 재질로서, 0.005 ㎛ 내지 0.015 ㎛의 기공을 갖는 것일 수 있다. 개시자 패드에서 버퍼패드 또는 반응패드와 접촉하는 부분은 저융점 아가로오스 또는 왁스로 코팅된 것일 수 있다. 상기 코팅은 버퍼패드로의 역흐름을 차단하여 측방 유동성을 가속화시킬 수 있다.The initiator pad may be made of a cellulose membrane and have pores of 0.005 μm to 0.015 μm. A portion of the initiator pad in contact with the buffer pad or reaction pad may be coated with low melting point agarose or wax. The coating may block reverse flow to the buffer pad to accelerate lateral fluidity.
상기 개시자 패드 하부에는 가열을 위한 가열패드(141)가 배치될 수 있다. 등온증폭이 끝나는 시점에 개시자 패드를 가열하면서 버퍼패드에 부가 완충액을 가하면, 개시자 패드의 저융점 아가로오스 또는 왁스가 녹으면서 반응패드로 수압이 발생하여 등온증폭된 결과물이 검출 패드로 쉽게 이동하도록 한다. 상기 가열은 60 내지 80℃로 수행되는 것일 수 있다.A heating pad 141 for heating may be disposed below the initiator pad. When an additional buffer is added to the buffer pad while heating the initiator pad at the end of isothermal amplification, water pressure is generated to the reaction pad as the low melting point agarose or wax of the initiator pad is melted, so that the isothermally amplified result is easily transferred to the detection pad. let it move The heating may be performed at 60 to 80 °C.
반응패드reaction pad
반응패드는 랩온페이퍼에서 페이퍼 칩에 해당하는 구성요소로서, 증폭반응을 위한 dNTP, DNA 중합효소, 역전사 효소, 형광 표지자, 등온증폭반응 버퍼 등을 포함한 등온증폭반응 시약이 고정되어 있다. 따라서 시료패드에 적용되는 부가 완충액에 의해 반응패드에 핵산 물질을 포함하는 용액이 스며들어 적셔지고, 상기 등온증폭반응 시약과 시료의 접촉물이 반응패드로 이동하여 반응패드 하부에서 가열패드에 의해 온도를 60 내지 70℃로 가열하면 등온증폭반응 또는 역전사 등온증폭반응이 일어난다.The reaction pad is a component corresponding to the paper chip in lab-on-paper, and isothermal amplification reagents including dNTP, DNA polymerase, reverse transcriptase, fluorescent marker, isothermal amplification reaction buffer, etc. for amplification reaction are fixed thereto. Therefore, the solution containing the nucleic acid material penetrates into the reaction pad by the additional buffer applied to the sample pad and is wetted, and the contact material between the isothermal amplification reagent and the sample moves to the reaction pad and is heated by the heating pad at the bottom of the reaction pad. When heated to 60 to 70 ° C., an isothermal amplification reaction or a reverse transcription isothermal amplification reaction occurs.
상기 등온증폭반응 시약은 구체적으로 dNTP(1.4mM, dATP, dCTP, dGTP 및 dTTP), 등온증폭버퍼(1X, 20mM Tris-HCl, 10mM (NH4)2SO4, 50mM KCl, 2mM MgSO4, 및 0.1% Tween-20, pH7.5), 및 Bst 3.0 DNA 중합효소(320U/㎖)를 포함할 수 있고, 이들은 반응패드 내에 혼합 후 건조하거나, 파우더 타입으로 반응패드 표면에 도포하여 예를 들어, 약 40℃오븐에서 약 30분 간 가열하여 고정시킨 것일 수 있다.The isothermal amplification reagent is specifically dNTP (1.4mM, dATP, dCTP, dGTP and dTTP), isothermal amplification buffer (1X, 20mM Tris-HCl, 10mM (NH 4 ) 2 SO 4 , 50mM KCl, 2mM MgSO 4 , and 0.1% Tween-20, pH7.5), and Bst 3.0 DNA polymerase (320 U/ml), which are mixed in the reaction pad and then dried or applied to the surface of the reaction pad in powder form, for example, It may be fixed by heating in an oven at about 40 ° C. for about 30 minutes.
반응패드(140)는 제1연결패드 및 개시자 패드와 일부분 접촉하여, 제1연결패드와 개시자 패드의 하부 및 측방으로 배치될 수 있다. 등온증폭반응이 일어날 수 있는 온도로 가열하기 위해 상기 반응패드(140) 하부에는 가열패드(141)가 배치될 수 있다. 상기 가열패드는 가열을 위한 열선 또는 열판을 포함할 수 있다. 상기 가열은 60 내지 70℃바람직하게는 60 내지 65℃에서 20분 내지 1 시간, 바람직하게는 20분 내지 30분 간 수행되는 것일 수 있다.The reaction pad 140 may partially come into contact with the first connection pad and the initiator pad, and may be disposed under and sideways of the first connection pad and the initiator pad. A heating pad 141 may be disposed below the reaction pad 140 to heat to a temperature at which an isothermal amplification reaction may occur. The heating pad may include a hot wire or a hot plate for heating. The heating may be performed at 60 to 70°C, preferably 60 to 65°C for 20 minutes to 1 hour, preferably 20 minutes to 30 minutes.
상기 반응패드는 등온증폭반응의 효율을 증가시키기 위해 반응패드 상부에 차단패드(142)가 배치되어 차단 역할을 할 수 있다. 일련의 배치된 패드를 라미네이트 처리된 차단패드에 의해 등온증폭반응 온도를 유지하고, 시약의 증발을 차단하여 반응의 효율을 증가시킬 수 있다. 상기 차단 패드는 비공성의 막 또는 외부 공기로부터 반응패드를 차단할 수 있는 구조물일 수 있다.In order to increase the efficiency of the isothermal amplification reaction, the reaction pad may have a blocking pad 142 disposed above the reaction pad to serve as a blocking function. The isothermal amplification reaction temperature is maintained by a blocking pad laminated to a series of arranged pads, and evaporation of reagents is blocked to increase reaction efficiency. The blocking pad may be a non-porous membrane or a structure capable of blocking the reaction pad from outside air.
상기 반응패드에는 복수 개의 웰이 존재하고, 각 웰에는 등온증폭반응을 위한 프라이머 세트로서 정방향 및 역방향내부 프라이머(inner primer: FIP 및 BIP, 1.6μM), 루프 프라이머(loop primer: FL 및 BL, 0.4 μM) 및 외부 프라이머(outer primer: F3 및 B3, 0.2μM)가 고정될 수 있다. 상기 프라이머의 농도는 웰 부피에 대한 농도이고, 웰에서 프라이머 세트의 농도는 각 프라이머 간의 농도 비율을 유지하면서 변경될 수 있다. 반응 패드에 웰이 존재함으로써 등온증폭반응이 보다 집중적으로 일어날 수 있다. 상기 웰은 구체적으로 바닥에 히드로겔 층이 형성되어 있고, 상기 프라이머 세트가 히드로겔 층에 고정된 형태일 수 있다. 특정 표적 핵산의 집중적인 증폭을 위해, 각 웰에는 서로 다른 표적 핵산에 특이적으로 결합하는 프라이머 세트가 고정될 수 있다.A plurality of wells exist in the reaction pad, and in each well, forward and reverse inner primers (inner primer: FIP and BIP, 1.6 μM), loop primers (loop primer: FL and BL, 0.4 μM) are primer sets for isothermal amplification reaction. μM) and outer primers (F3 and B3, 0.2 μM) can be immobilized. The concentration of the primers is based on the volume of the well, and the concentration of the primer set in the well can be changed while maintaining the concentration ratio between the respective primers. The isothermal amplification reaction can occur more intensively by the presence of wells in the reaction pad. Specifically, the well may have a hydrogel layer formed at the bottom and the primer set fixed to the hydrogel layer. For intensive amplification of a specific target nucleic acid, primer sets that specifically bind to different target nucleic acids may be immobilized in each well.
상기 프라이머를 함유하는 히드로겔 층은 예를 들어 하기와 같은 방법으로 형성될 수 있다. 히드로겔 용액 총 부피를 기준으로 UV-광가교성 폴리(에틸렌 글리콜) 디아크릴레이트 (PEGDA, Sigma-Aldrich, MW700) 20% v/v, 폴리(에틸렌 글리콜)(PEG, Sigma-Aldrich, MW600) 40% v/v 및 광개시제 2-하이드록시-2-메틸프로피오페논 (Sigma-Aldrich) 5% v/v 및 버퍼(PBS 완충액, pH7.5) 35%를 혼합하고, 여기에 프라이머 세트를 혼합하여 하이드로겔 용액을 제조한다. 상기 폴리(에틸렌 글리콜)은 히드로겔 미세입자의 공극율을 증가시키기 위하여 포함되는 것이 바람직하다. 그런 다음, 상기 히드로겔 용액을 반응 패드의 각 웰 내부 표면에 도포하고, 1분간 UV 노출(360 nm wavelength, 35 mJ/cm2)하여 히드로겔 코팅층을 형성한다. 히드로겔 층은 공극을 가지므로 히드로겔 층 내 프라이머에 결합하여 공극 내에서 집중적으로 증폭반응이 일어날 수 있다.The hydrogel layer containing the primer may be formed, for example, by the following method. UV-light crosslinkable poly(ethylene glycol) diacrylate (PEGDA, Sigma-Aldrich, MW700) 20% v/v, based on total volume of hydrogel solution, poly(ethylene glycol) (PEG, Sigma-Aldrich, MW600) 40 % v/v and photoinitiator 2-hydroxy-2-methylpropiophenone (Sigma-Aldrich) 5% v/v and buffer (PBS buffer, pH7.5) 35% were mixed, and the primer set was mixed thereto. Prepare a hydrogel solution. The poly(ethylene glycol) is preferably included to increase the porosity of the hydrogel microparticles. Then, the hydrogel solution was applied to the inner surface of each well of the reaction pad and exposed to UV (360 nm wavelength, 35 mJ/cm 2 ) for 1 minute to form a hydrogel coating layer. Since the hydrogel layer has pores, an amplification reaction may occur intensively in the pores by binding to primers in the hydrogel layer.
상기 프라이머 세트에서 정방향 및 역방향 프라이머 중 어느 하나는 Cy3, Cy5, TAMRA, TEX, TYE, HEX, FAM, TET, JOE, MAX, ROX, VIC, Cy3.5, Texas Red, Cy5.5, TYE, BHQ, Iowa Black RQ, 및 IRDye로 이루어지는 군에서 선택되는 하나 이상의 검출자로 표지된 것일 수 있다. 상기 감출자는 표적 핵산을 독립적으로 검출하기 위해, 표적 핵산 마다 다르게 표지 될 수 있고, 구조물의 구체적인 구성에 따라 선택적 결합을 위한 검출자 또는 형광 표지자로 기능할 수 있다. Any one of the forward and reverse primers in the primer set is Cy3, Cy5, TAMRA, TEX, TYE, HEX, FAM, TET, JOE, MAX, ROX, VIC, Cy3.5, Texas Red, Cy5.5, TYE, BHQ , Iowa Black RQ, and may be labeled with one or more detectors selected from the group consisting of IRDye. The detector may be labeled differently for each target nucleic acid in order to independently detect the target nucleic acid, and may function as a detector or a fluorescent marker for selective binding depending on the specific structure of the structure.
상기 프라이머 세트에서 정방향 및 역방향 프라이머 중 나머지 어느 하나는 비오틴이 결합된 것일 수 있다. 비오틴은 스트렙타비딘(streptavidin)에 결합할 수 있으므로, 증폭된 표적 핵산에 결합된 형태로 존재하여 금 입자를 함유하는 패드, 예를 들어 일 구체예에 따른 제2연결패드를 통과하면서, 금 입자의 표면에 스트렙타비딘과 결합하고 검출패드에서 포획되면 검출 결과가 가시화되도록 한다. In the primer set, the other one of the forward and reverse primers may be biotin-linked. Since biotin can bind to streptavidin, it exists in a form bound to the amplified target nucleic acid and passes through a pad containing gold particles, for example, the second connection pad according to one embodiment, while gold particles When streptavidin binds to the surface and is captured on the detection pad, the detection result is visualized.
한편, 스트렙타비딘을 검출패드에 고정시킬 수 있고, 이 경우 비오틴이 검출패드에 고정된 스트렙타비딘에 결합할 수 있으므로, 최종적으로 검출 패드에서 스트렙타비딘과 결합하고 포획되면 색변화로서 직관적으로 표시되거나 형광 검출 결과를 가시화할 수 있다. On the other hand, since streptavidin can be immobilized on the detection pad, and in this case, biotin can bind to streptavidin immobilized on the detection pad. It can be displayed or the fluorescence detection result can be visualized.
비오틴은 증폭된 표적 핵산에서 검출자와 서로 반대 위치에 존재하도록 설계될 수 있고, 예를 들어, 정방향 프라이머의 5' 말단에 검출자를 결합시킨 경우, 역방향 프라이머의 5' 말단에 비오틴을 결합시킬 수 있다.Biotin may be designed to be present at opposite positions of a detector and a detector in the amplified target nucleic acid. For example, when a detector is bound to the 5' end of a forward primer, biotin may be bound to the 5' end of a reverse primer. there is.
상기 반응패드는 셀룰로오스아세테이트 막의 재질로 이루어진 것으로서, 시료 및 부가완충액의 흐름은 자유롭도록 하면서 핵산이 머물면서 충분히 등온증폭반응할 수 있도록, 0.001㎛ 내지 0.005㎛, 바람직하게는, 0.005㎛의 기공 사이즈를 갖는 것일 수 있다.The reaction pad is made of a cellulose acetate membrane material, and has a pore size of 0.001 μm to 0.005 μm, preferably 0.005 μm, so that the nucleic acid can remain and sufficiently isothermally amplified while allowing the sample and the additional buffer to flow freely. may have
상기 반응패드는 40 mM 내지 50 mM 수크로오스, 0.001 내지 0.01% Triton X-100, 및 0.1w/w% 내지 0.3w/w%의 글리세롤을 포함할 수 있다. 이는 수분 또는 산소에 등온증폭반응 시약 및 프라이머세트 등이 노출되었을 때, 이들의 보관 안정성을 증가시킬 수 있다. 상기 보관 안정성이란 25℃내지 30℃에서 분해물이나 부산물 없이 3 주 이상 보관할 수 있는 것을 의미할 수 있다.The reaction pad may include 40 mM to 50 mM sucrose, 0.001 to 0.01% Triton X-100, and 0.1 w/w% to 0.3 w/w% glycerol. This can increase their storage stability when the isothermal amplification reagent and primer set are exposed to moisture or oxygen. The storage stability may mean that it can be stored for 3 weeks or more without degradation products or by-products at 25 ° C to 30 ° C.
제2연결패드2nd connection pad
제2연결패드(150)는 반응패드와 일부분 접촉하여 검출패드의 상부 및 측방으로 배치될 수 있다. 제2연결패드(150)는 반응패드에서 증폭된 핵산을 검출패드로 이동시킨다. The second connection pad 150 may partially come into contact with the reaction pad and may be disposed above and sideways of the detection pad. The second connection pad 150 moves the nucleic acid amplified in the reaction pad to the detection pad.
금 입자 구성을 포함하는 구조물의 경우, 제2연결패드(150)가 금 나노입자를 포함하고, 상기 금 나노입자는 반응패드에서 증폭된 핵산과 결합하여 검출패드로 이동된다. 금 나노입자는 바람직하게는 표면에 스트렙타비딘이 고정된 것일 수 있다. In the case of a structure including gold particles, the second connection pad 150 includes gold nanoparticles, and the gold nanoparticles combine with nucleic acids amplified in the reaction pad and move to the detection pad. The gold nanoparticles may preferably have streptavidin immobilized on the surface.
제2연결패드는 증폭된 핵산이 쉽게 검출패드로 이동할 수 있도록, 면, 융, 종이, 니트로셀룰로오스, 유리섬유, 폴리설폰, 폴리아크릴, 폴리니트릴, 폴리피페라진, 폴리아미드, 폴리에테르설폰, 폴리비닐리덴플로리드, 폴리에틸렌이민, 폴리디메틸실록산 또는 이들의 혼합물인 다공성 재질로서, 0.01㎛ 내지 0.05㎛, 바람직하게는, 0.05㎛의 기공 사이즈를 갖는 다공성 재질일 수 있다.The second connection pad is cotton, wool, paper, nitrocellulose, glass fiber, polysulfone, polyacrylic, polynitrile, polypiperazine, polyamide, polyethersulfone, polyethersulfone, As a porous material of vinylidene fluoride, polyethyleneimine, polydimethylsiloxane, or a mixture thereof, it may be a porous material having a pore size of 0.01 μm to 0.05 μm, preferably, 0.05 μm.
제2연결패드는 반응패드의 상부에 배치되고, 반응패드나 검출패드에 비해 상대적으로 폭이 좁은 구조물로 반응패드와 검출패드를 연결한다.The second connection pad is disposed above the reaction pad and has a relatively narrow width compared to the reaction pad or the detection pad, and connects the reaction pad and the detection pad.
제2연결패드는 셀룰로오스 재질을 갖는 것을 수 있고, 반응패드와 접촉하는 부분의 제2연결패드는 저융점 아가로오스 또는 왁스로 코팅된 것일 수 있다. 패드를 코팅하면 반응패드의 등온증폭반응물의 이동이 차단되었다가, 제2연결패드를 가열하는 시점에서 코팅이 녹아 등온증폭반응물이 측방으로 다시 이동하여 시료 내 미반응 유전물질의 소실을 막을 수 있다.The second connection pad may have a cellulose material, and the second connection pad at a portion in contact with the reaction pad may be coated with low melting point agarose or wax. When the pad is coated, the movement of the isothermal amplification reactant in the reaction pad is blocked, and then the coating melts at the time of heating the second connection pad, and the isothermal amplification reactant moves laterally again, preventing loss of unreacted dielectric material in the sample. .
상기 제2연결패드 하부에는 가열을 위한 가열패드(141)가 배치될 수 있다. 등온증폭이 끝나는 시점에 부가 완충액을 가하면, 반응패드로부터 등온증폭된 결과물이 검출 패드로 쉽게 이동하도록 한다. 상기 가열은 60 내지 80℃로 1 분 내지 5 분간, 바람직하게는 2 분 간 수행되는 것일 수 있다.A heating pad 141 for heating may be disposed under the second connection pad. When an additional buffer is added at the end of the isothermal amplification, the isothermally amplified product is easily transferred from the reaction pad to the detection pad. The heating may be performed at 60 to 80° C. for 1 minute to 5 minutes, preferably for 2 minutes.
검출패드detection pad
검출패드(160)는 제2연결패드와 일부분 접촉하여, 제2연결패드의 하부 및 측방에 배치될 수 있다. 검출패드(160)에는 검출자와 결합할 수 있는 수용체가 고정되어 있다. 상기 수용체는 검출자에 특이적으로 결합할 수 있는 항체, 단백질, 또는 이의 절편일 수 있다.The detection pad 160 may partially come into contact with the second connection pad and may be disposed below and on the side of the second connection pad. A receptor that can bind to the detector is fixed to the detection pad 160 . The receptor may be an antibody, protein, or fragment thereof capable of specifically binding to a detector.
상기 검출패드는 복수 개의 검출 구역을 포함하고, 상기 검출 구역은 선 또는 웰의 형태로 구분될 수 있다. 각 검출 구역 마다 각 수용체가 독립적으로 고정되고, 예를 들어, 폴리에틸렌프탈레이트 성분의 잉크로 스탬핑하여 검출 구역을 만든 후, 여기에 수용체를 포함하는 잉크로 다시 스탬핑 하거나 웰의 경우 수용체를 포함하는 용액을 적용하고, EDC(1-Ethyl-3-[3-dimethylaminopropyl]carbodiimide) 또는 NHS(N-hydroxysulfosuccinimide)를 적용할 수 있다. 하나의 반응패드에는 수십개 내지 수백개 이상의 검출 구역이 형성되어, 1회의 시료를 적용하여 형성된 검출 구역의 수 만큼의 표적 핵산을 검출할 수 있다. 도 9 및 도 10은 반응패드에 형성된 검출 구역의 구조를 상세히 설명하기 위한 것이고, 개수를 한정하는 의미는 아니다.The detection pad includes a plurality of detection regions, and the detection regions may be divided into lines or wells. For each detection zone, each receptor is independently immobilized. For example, after making a detection zone by stamping with polyethylene phthalate-based ink, stamping again with ink containing a receptor here, or in the case of a well, a solution containing a receptor and EDC (1-Ethyl-3-[3-dimethylaminopropyl] carbodiimide) or NHS (N-hydroxysulfosuccinimide) may be applied. Several tens to hundreds of detection zones are formed on one reaction pad, and as many target nucleic acids as the number of detection zones formed by applying a sample once can be detected. 9 and 10 are for explaining the structure of the detection zone formed on the reaction pad in detail, and are not meant to limit the number.
상기 검출패드는 니트로셀룰로오스로서, 시료가 흡수패드까지 측방으로 이동할 수 있도록, 0.001㎛ 내지 0.005㎛, 바람직하게는, 0.005㎛의 기공 사이즈를 갖는 것일 수 있다. The detection pad is nitrocellulose and may have a pore size of 0.001 μm to 0.005 μm, preferably 0.005 μm, so that the sample can move laterally to the absorption pad.
가열패드heating pad
상기 시료패드, 개시자패드, 반응패드, 및 제2연결패드 하부에는 가열패드(141)가 배치될 수 있다. 상기 가열패드는 열전도성을 갖는 금속판으로서, 철, 스테인리스, 알루미늄, 은, 구리 등의 재질로 이루어진 것일 수 있다. 상기 가열패드는 열선으로 연결될 수 있고, 각 열선은 상기 시료패드, 반응패드, 개시자패드 및 제2연결패드 하부의 가열패드에 독립적으로 연결되어 가열이 되는 패드를 제어할 수 있다.A heating pad 141 may be disposed below the sample pad, the initiator pad, the reaction pad, and the second connection pad. The heating pad is a metal plate having thermal conductivity, and may be made of a material such as iron, stainless steel, aluminum, silver, or copper. The heating pad may be connected with a heating wire, and each heating wire may be independently connected to the sample pad, the reaction pad, the initiator pad, and the heating pad under the second connection pad to control the heating pad.
상기 시료패드, 제1연결패드, 개시자패드, 반응패드, 제2연결패드, 검출패드, 및 흡수패드는 시료나 완충액이 소실되지 않도록 상하단에 하우징(110)이 존재할 수 있고, 전체 구조물은 하우징으로 정의될 수 있는 지지체 또는 비공성 재질의 케이스로 둘러싸인 것일 수 있다.The sample pad, the first connection pad, the initiator pad, the reaction pad, the second connection pad, the detection pad, and the absorption pad may have housings 110 at upper and lower ends so that the sample or buffer is not lost, and the entire structure is a housing. It may be surrounded by a case of a support or non-porous material that can be defined as.
상기 시료와 완충액 등을 흡수하여 역흐름을 차단하고 측방 유동성이 유발될 수 있도록 기여한다. 흡수패드는 다공성 재질로서, 면, 융, 종이, 니트로셀룰로오스, 셀룰로오스아세테이트, 유리섬유, 폴리설폰, 폴리아크릴, 폴리니트릴, 폴리피페라진, 폴리아미드, 폴리에테르설폰, 폴리비닐리덴플로리드, 폴리에틸렌이민, 폴리디메틸실록산 또는 이들의 혼합물일 수 있다. 상기 흡수패드는 바람직하게는 유리섬유로서 0.1 내지 0.5㎛의 기공 사이즈를 갖는 것일 수 있다.It absorbs the sample and the buffer solution to block reverse flow and contribute to inducing lateral fluidity. The absorbent pad is a porous material, such as cotton, flax, paper, nitrocellulose, cellulose acetate, glass fiber, polysulfone, polyacrylic, polynitrile, polypiperazine, polyamide, polyethersulfone, polyvinylidene fluoride, and polyethyleneimine. , polydimethylsiloxane or mixtures thereof. The absorbent pad is preferably a glass fiber and may have a pore size of 0.1 to 0.5 μm.
상기 시료패드 및 제1연결패드와 버퍼패드 및 개시자패드; 반응패드; 제2연결패드; 검출패드; 및 흡수패드는 순차적으로 서로 일부분 접촉되어 측방으로 배치된다. 구체적으로, 시료패드 및 제1연결패드와 버퍼패드 및 개시자패드는 반응패드를 기준으로 분리되어 반응패드의 일 측면에 배치되고, 반응패드의 다른 측면에 이어서 제2연결패드, 검출패드, 및 흡수패드가 순차적으로 배치된다. the sample pad, the first connection pad, the buffer pad, and the initiator pad; reaction pad; a second connection pad; detection pad; and the absorbent pads are sequentially disposed laterally in partial contact with each other. Specifically, the sample pad, the first connection pad, the buffer pad, and the initiator pad are separated based on the reaction pad and disposed on one side of the reaction pad, followed by the second connection pad, detection pad, and The absorbent pads are sequentially disposed.
본 발명에 따른 구조물은, 구조물의 각 구성요소의 배치와 특성에 의해 시료가 흡수패드까지 도달하는 동안 핵산 물질 외에 세포 용해물을 여과하여 핵산 물질을 정제할 수 있고, 시료의 이동 방향이 지면과 평행한 방향인 측방 유동성을 실현할 수 있다. 세포 내에서 핵산은 히스톤, 중합효소, 핵산분해효소, 전사인자 등의 단백질이 결합된 형태로 존재한다. 세포 용해 조성물과 같은 계면활성제에 의해 많은 단백질이 핵산과의 결합을 소실하게 되나, 세포 용해물이 반응패드까지 이동할 수 있도록 추가로 가해지는 증류수 또는 부가 완충액에 의해 반응패드에 도달할 쯤에는 계면활성제가 희석되어 외부 환경이 단백질의 전하를 다시 회복할 있게 된다. 이 경우 단백질은 다시 핵산 물질과 결합하여 중합효소와의 접촉 면적을 축소시키거나 증폭 반응을 방해할 수 있다. 따라서 반응패드에는 핵산과 결합할 수 있는 대부분의 세포 구성 요소가 정제되어 제거되는 것이 바람직하다.According to the structure according to the present invention, nucleic acid materials can be purified by filtering cell lysate in addition to nucleic acid materials while the sample reaches the absorption pad due to the arrangement and characteristics of each component of the structure, and the moving direction of the sample is different from the ground. Lateral fluidity, which is a parallel direction, can be realized. In cells, nucleic acids exist in the form of proteins such as histones, polymerases, nucleases, and transcription factors. Many proteins lose their binding to nucleic acids by surfactants such as the cell lysis composition, but by the time the cell lysate reaches the reaction pad by the addition of distilled water or added buffer so that the cell lysate can move to the reaction pad, the surfactant is diluted, allowing the external environment to regain the protein's charge. In this case, the protein may bind to the nucleic acid material again to reduce the contact area with the polymerase or interfere with the amplification reaction. Therefore, it is preferable that most cellular components capable of binding to nucleic acids are purified and removed from the reaction pad.
인식부recognition unit
인식부(200)는 검출패드로부터 형광 이미지를 획득하고, 획득한 형광 이미지로부터 형광 세기를 측정한다. 형광 세기는 측정 가능한 최대 값을 기준으로 실제 측정된 값의 비율로 산출할 수 있고, 복수 개의 형광 표지자를 여기하고 형광 이미지를 인식하기 위해, 다양한 파장대, 예를 들어 400 내지 700 λ파장대의 광원과 각 여기 광의 선택적인 획득을 위한 필터를 포함할 수 있다. 인식부는 반응패드에 형성된 복수 개의 검출 구역을 독립적으로 인식하여 각 검출 구역의 형광 세기를 산출하고, "해당 검출 구역에서 측정된 형광 세기 값과 음성대조군의 형광 세기 값의 차이"를 출력부로 전송한다.The recognition unit 200 acquires a fluorescence image from the detection pad and measures fluorescence intensity from the acquired fluorescence image. Fluorescence intensity can be calculated as a ratio of the actual measured value based on the maximum measurable value, and in order to excite a plurality of fluorescent markers and recognize a fluorescent image, a light source of various wavelength bands, for example, 400 to 700 λ wavelength range, and A filter for selective acquisition of each excitation light may be included. The recognition unit independently recognizes a plurality of detection zones formed on the reaction pad, calculates the fluorescence intensity of each detection zone, and transmits the "difference between the fluorescence intensity value measured in the corresponding detection zone and the fluorescence intensity value of the negative control" to the output unit. .
출력부output
출력부(210)는 인식부(200)로부터 전송된 형광 세기 값 차이를 이용하여 유의성 값을 도출한다. 유의성 값은 상기 인식부에서 측정 가능한 최대 값을 1로 설정하고, 형광 세기 값 차이가 0.3 이상인 경우 상기 유의성 값은 "70% 이상의 확률의 해당 검출 구역의 표적 핵산에 대해 양성"으로 출력하고, 형광 세기 값 차이가 0.5 이상인 경우 상기 유의성 값은 "해당 검출 구역의 표적 핵산에 대해 90% 이상의 확률의 양성"으로 출력할 수 있다. 상기 출력부는 질병, 바이러스 또는 균 감염과 관련된 유전자 정보를 포함하는 빅 데이터에 기반하여, 상기 도출된 유의성 값을 이용해 양성으로 출력된 표적 핵산과 관련된 질병, 바이러스 또는 균 감염 증상을 찾아내고, 최종적으로 시료가 획득된 개체의 질병, 바이러스 또는 균 감염 여부를 진단할 수 있도록 한다. 형광 세기 값 차이가 0.3 미만인 경우 상기 유의성 값은 "음성"으로 출력할 수 있다.The output unit 210 derives a significance value using the fluorescence intensity value difference transmitted from the recognition unit 200 . As for the significance value, the maximum measurable value in the recognition unit is set to 1, and when the fluorescence intensity difference is greater than or equal to 0.3, the significance value is output as “positive for the target nucleic acid in the corresponding detection region with a probability of 70% or more”, and fluorescence If the difference in intensity value is 0.5 or more, the significance value may be output as "positive with a probability of 90% or more for the target nucleic acid in the corresponding detection region". The output unit uses the derived significance value based on big data including genetic information related to disease, virus, or fungal infection to find a disease, virus, or fungal infection symptom related to a positively output target nucleic acid, and finally It is possible to diagnose whether or not a disease, virus, or fungal infection of an object from which a sample is acquired is detected. When the fluorescence intensity difference is less than 0.3, the significance value may be output as “negative”.
상기 표적 핵산은 프라이머 세트를 제작할 때에 이미 알고 있는 질병, 바이러스 또는 균 감염 증상과 관련된 것일 수 있고, 다른 예로는, 상업적으로 판매되거나 획득할 수 있는 프라이머 세트를 무작위로 적용하여 반응패드를 제작한 후, 시료로 부터 획득된 유의성 값을 빅 데이터에 기반하여 도출한 후 해당 프라이머 세트가 특이적으로 결합하는 염기서열과 관련 질병, 바이러스 또는 균 감염 증상을 매칭시킬 수 있다.The target nucleic acid may be related to a disease, virus, or fungal infection symptom that is already known when preparing a primer set. In another example, after preparing a reaction pad by randomly applying a commercially available or obtainable primer set, , After the significance value obtained from the sample is derived based on big data, the nucleotide sequence to which the primer set specifically binds can be matched with a related disease, virus or fungal infection symptom.
빅 데이터는 공개된 생물학적 데이터베이스로부터 획득될 수 있고, 염기서열의 경우 Genebank, EMBL(The European Molecular Biology Laboratory), DDBJ(DNA Data Bank of Japan) 등이 있고, 유전체데이터베이스로 Entrez Genome, Ensembl 등이 있고, 대사회로 데이터베이스로 KEGG(Kyoto Encyclopedia of Genes and Genomes), WikiPathways 등이 있으나 이에 제한되지 않는다. Big data can be obtained from open biological databases, and for base sequences, there are Genebank, EMBL (The European Molecular Biology Laboratory), DDBJ (DNA Data Bank of Japan), etc., and genome databases include Entrez Genome, Ensembl, etc. , Metabolic circuit databases include KEGG (Kyoto Encyclopedia of Genes and Genomes) and WikiPathways, but are not limited thereto.
이하, 상기 핵산 검출용 구조물 상기 구조물을 포함하는 시스템을 이용하여 핵산을 검출하는 방법을 서술한다.Hereinafter, a method for detecting nucleic acids using the structure for detecting nucleic acid and a system including the structure will be described.
핵산을 검출하고자 하는 시료는 시료패드에 적용하기 전에 세포 용해용 조성물과 혼합하는 단계를 포함할 수 있다. 세포 용해용 조성물과 혼합되면 세포 내에 존재하는 핵산 물질이 용출될 수 있으므로 시료 내에 검출 가능한 핵산 물질의 양을 증가시킬 수 있다.A sample for which nucleic acid is to be detected may include mixing with a composition for cell lysis before applying the sample to the sample pad. When mixed with the composition for cell lysis, nucleic acid substances present in cells may be eluted, and thus the amount of detectable nucleic acid substances in the sample may be increased.
시료패드에 5 mM 내지 80 mM Tris-HCl, 염화칼륨 5 mM 내지 50 mM, 황산마그네슘 1 mM 내지 30 mM, 황산암모늄 5 mM 내지 50 mM, 단백질분해효소 0.01 mg/ml 내지 0.1 mg/ml, TritonX-100 또는 Tween20 0.01w/w% 내지 0.2w/w%, pH 8.0 내지 9.0인 세포 용해용 조성물과 혼합된 세포 용해물을 적가하고, 상기 시료패드를 가열하기 위한 시료패드 하방의 가열패드를 작동시킴으로서 시료의 용해를 효율성을 높인다. 상기 시료패드 하방의 가열패드를 60 내지 80℃의 온도에서 2분간 가열 처리해줌으로써 시료패드에서 바이러스와 상피세포를 포함하는 시료의 용해를 촉진한다.5 mM to 80 mM Tris-HCl, potassium chloride 5 mM to 50 mM, magnesium sulfate 1 mM to 30 mM, ammonium sulfate 5 mM to 50 mM, protease 0.01 mg/ml to 0.1 mg/ml, TritonX- 100 or Tween20 0.01 w/w% to 0.2 w/w%, by adding dropwise the cell lysate mixed with the cell lysis composition having a pH of 8.0 to 9.0 and operating a heating pad below the sample pad for heating the sample pad Increase the efficiency of sample dissolution. By heating the heating pad below the sample pad at a temperature of 60 to 80° C. for 2 minutes, dissolution of the sample including viruses and epithelial cells in the sample pad is promoted.
또한 버퍼패드에 부가완충액을 가할 수 있다. 상기 부가완충액은 핵산 물질이 반응패드까지 이동할 수 있도록 하면서, 핵산 물질을 정제하는 역할을 한다. 상기 완충액은 핵산 물질이 검출패드까지 이동할 수 있도록 하면서, 핵산 물질을 정제하는 역할을 한다. 상기 완충액은 적절한 양의 완충 성분을 함유하므로, 증류수를 첨가하는 경우 발생할 수 있는 급격한 염도나 pH 변화로 인한 단백질이나 핵산 물질의 침전 현상을 예방할 수 있다. 본 발명의 핵산 검출용 구조물의 각 패드는 핵산이 측방으로 이동하면서 정제될 수 있도록 작은 기공을 갖는 다공성 재질로 이루어진다. 따라서 단백질이나 핵산 물질이 착염이나 응집되어 기공을 막게 되면 시료의 이동속도가 감소하고 핵산 물질의 수득률이 저하될 수 있다.In addition, an additional buffer may be added to the buffer pad. The additional buffer serves to purify the nucleic acid material while allowing the nucleic acid material to move to the reaction pad. The buffer serves to purify the nucleic acid material while allowing the nucleic acid material to move to the detection pad. Since the buffer solution contains an appropriate amount of a buffering component, it is possible to prevent precipitation of proteins or nucleic acids due to rapid changes in salinity or pH that may occur when distilled water is added. Each pad of the structure for detecting nucleic acids of the present invention is made of a porous material having small pores so that nucleic acids can be purified while moving laterally. Therefore, if the protein or nucleic acid material is complexed or aggregated to block the pores, the movement speed of the sample may decrease and the yield of the nucleic acid material may decrease.
상기 부가 완충액은 예를 들어 5 mM 내지 80 mM Tris-HCl, 20 mM 내지 70 mM 염화칼륨, 0.5 mM 내지 5 mM 황산마그네슘, 1 mM 내지 30 mM 황산암모늄, 및 0.01w/w% 내지 0.2 w/w% Tween®20 내지 TritonX-100을 포함하고 산도가 pH 8.0 내지 9.0인 등온완충액 또는 인산 완충액 (50mM Na2HPO4, pH 7.2)일 수 있다. 상기 등온완충액은 보다 구체적으로 20 mM Tris-HCl, 10 mM (NH4)2SO4, 50 mM KCl, 2 mM MgSO4, 및 0.1% Tween®20을 포함하고, 산도는 pH 8.8일 수 있다. 상기 부가 완충액은 단백질분해효소, 글리세롤, 또는 환원제를 포함하지 않는 것일 수 있다.The addition buffer is, for example, 5 mM to 80 mM Tris-HCl, 20 mM to 70 mM potassium chloride, 0.5 mM to 5 mM magnesium sulfate, 1 mM to 30 mM ammonium sulfate, and 0.01 w/w% to 0.2 w/w % Tween®20 to TritonX-100 and an acidity pH of 8.0 to 9.0 or a phosphate buffer (50 mM Na 2 HPO 4 , pH 7.2). More specifically, the isothermal buffer may include 20 mM Tris-HCl, 10 mM (NH 4 ) 2 SO 4 , 50 mM KCl, 2 mM MgSO 4 , and 0.1% Tween® 20, and may have a pH of 8.8. The addition buffer may not contain a protease, glycerol, or a reducing agent.
상기 핵산 증폭반응이 원활히 일어날 수 있도록, 반응패드 하부의 가열패드를 60 내지 65 ℃로 가열하고, 온도를 유지하면서 20 분 내지 1 시간 바람직하게는 20 분 내지 30 분 동안 방치할 수 있다.To facilitate the nucleic acid amplification reaction, the heating pad under the reaction pad may be heated to 60 to 65° C., and allowed to stand for 20 minutes to 1 hour, preferably 20 to 30 minutes, while maintaining the temperature.
상기 다중 핵산 검출용 구조물을 이용해서 진단할 수 있는 질병은 특정 유전자가 원인이나 질병의 지표로서 이용될 수 있는 것으로, 예를 들어, 비만, 고혈압, 당뇨병 등과 같은 대사성 질환, 유전질환, 암 등이 있고, 바이러스 또는 균은 감염성 질환과 관련 있는 것으로, 균 감염증을 유발할 수 있는 균으로는 세균, 원충, 기생충, 진균 등을 포함하나 이에 제한되지 않는다.Diseases that can be diagnosed using the multi-nucleic acid detection structure are those in which a specific gene can be used as a cause or indicator of a disease, for example, metabolic diseases such as obesity, hypertension, diabetes, genetic diseases, cancer, etc. In addition, viruses or bacteria are related to infectious diseases, and bacteria that can cause fungal infections include bacteria, protozoa, parasites, fungi, etc., but are not limited thereto.
본 발명은 코로나바이러스를 검출할 수 있는 각각 서열번호 1 내지 6의 염기서열로 표시되는 6 개의 프라이머를 포함하는, 프라이머 세트를 제공하며, 상기 프라이머 세트를 랩온 페이퍼에 적용하여 코로나바이러스가 검출되는 것일 수 있다.The present invention provides a primer set comprising six primers each represented by the nucleotide sequence of SEQ ID NOs: 1 to 6 capable of detecting coronavirus, and applying the primer set to lab-on paper to detect coronavirus can
상기 프라이머 세트는 코로나바이러스를 검출하기 위한 것으로서, 상기 코로나바이러스는 신종 코로나바이러스 감염증-19 (Coronavirus Disease-19, COVID-19)의 병원체인 SARS-CoV-2일 수 있다.The primer set is for detecting a coronavirus, and the coronavirus may be SARS-CoV-2, a pathogen of novel coronavirus disease-19 (COVID-19).
용어 "프라이머"는 자유 3말단 수산화기(free 3'hydroxyl group)를 가지는 짧은 폴리뉴클레오티드로서, 상보적인 주형(template)과 염기쌍(base pair)을 형성할 수 있고 주형 가닥 복사를 위한 시작 지점으로 기능할 수 있다. 프라이머는 적절한 완충용액 및 온도에서 중합반응을 위한 시약(DNA 중합효소 또는 역방향 전사효소) 및 상이한 4가지 dNTP(deoxynucleoside triphospate)의 존재 하에서 DNA 합성을 개시할 수 있다. The term "primer" refers to a short polynucleotide having a free 3'hydroxyl group, capable of forming a base pair with a complementary template and serving as a starting point for template strand copying. can Primers can initiate DNA synthesis in the presence of reagents (DNA polymerase or reverse transcriptase) and four different deoxynucleoside triphospates (dNTPs) for polymerization at an appropriate buffer solution and temperature.
상기 프라이머 세트에서 각각의 프라이머는 표적하는 유전자에 특이적으로서, 용어 "유전자에 특이적" 또는 "상보적인"은 표적 유전자에 80% 이상, 85% 이상, 90% 이상, 95% 이상, 또는 100% 염기쌍을 이룰 수 있는 것을 의미할 수 있다. Each primer in the primer set is specific to the target gene, and the term "gene specific" or "complementary" refers to 80% or more, 85% or more, 90% or more, 95% or more, or 100% or more of the target gene. It can mean that % base pairing can be achieved.
상기 프라이머 세트에서 각 프라이머는 서열번호 1 내지 6의 염기서열 중 하나의 염기서열로 이루어지거나 이를 포함하는 것일 수 있고, 코로나바이러스에 특이적으로 결합을 유지하는 한, 서열번호 1 내지 6으로 기재된 염기서열과 80% 이상, 90% 이상, 95% 이상, 97% 이상 또는 99% 이상의 상동성을 가질 수 있다. 상기 프라이머는 또한, 유전자 합성의 개시점으로서 작동하는데 기본적인 성질을 변화시키지 않는 범위에서 추가의 특징을 더 포함하도록 개조될 수 있다. 따라서, 상기 프라이머는 각 정의된 서열번호의 염기서열을 포함하면서 약 1 내지 20 bp, 1 내지 15 bp, 1 내지 10 bp, 또는 1 내지 5 bp의 추가적인 염기 서열을, 상기 프라이머의 3' 말단, 5' 말단 또는 염기서열 내부에 더 포함할 수 있고, 필요한 경우 각 말단 또는 특정 염기에 분광학적, 광화학적, 생화학적, 면역화학적 또는 화학적 수단에 의해 직접적으로 또는 간접적으로 검출 가능한 표지를 더 포함할 수 있다. In the primer set, each primer may consist of or include one of the nucleotide sequences of SEQ ID NOs: 1 to 6, and as long as it maintains specific binding to the coronavirus, the bases shown in SEQ ID NOs: 1 to 6 It may have at least 80%, at least 90%, at least 95%, at least 97% or at least 99% homology with the sequence. The primers may also be modified to include additional features within the range of not changing the basic properties to act as starting points for gene synthesis. Accordingly, the primer includes the nucleotide sequence of each defined SEQ ID NO and includes an additional nucleotide sequence of about 1 to 20 bp, 1 to 15 bp, 1 to 10 bp, or 1 to 5 bp, at the 3' end of the primer, It may be further included at the 5' end or inside the nucleotide sequence, and if necessary, a label detectable directly or indirectly by spectroscopic, photochemical, biochemical, immunochemical or chemical means may be further included at each end or a specific base. can
상기 표지의 예로는, 발색효소, 형광물질, 방사성 동위원소 또는 콜로이드일 수 있다. 상기 발색효소는 퍼록시다제, 알칼라인 포스파타제 또는 산성 포스파타제일 수 있고, 상기 형광물질은 티오우레아(FTH), 7-아세톡시쿠마린-3-일, 플루오레신-5-일, 플루오레신-6-일, 2',7'-디클로로플루오레신-5-일, 2',7'-디클로로플루오레신-6-일, 디하이드로 테트라메틸로사민-4-일, 테트라메틸로다민-5-일, 테트라메틸로다민-6-일, 4,4-디플루오로-5,7-디메틸-4-보라-3a,4a-디아자-s-인다센-3-에틸 또는 4,4-디플루오로-5,7-디페닐-4-보라-3a,4a-디아자-s-인다센-3-에틸, Cy3, Cy5, 폴리 L-라이신-플루오레세인 이소티오시아네이트(FITC), 로다민-B-이소티오시아네이트(RITC), PE(Phycoerythrin) 또는 로다민일수 있고, 상기 방사성 동위원소는 32P일 수 있으나, 이에 제한되지 않는다.Examples of the label may be a chromogenic enzyme, a fluorescent substance, a radioactive isotope, or a colloid. The chromogenic enzyme may be peroxidase, alkaline phosphatase or acid phosphatase, and the fluorescent substance may be thiourea (FTH), 7-acetoxycoumarin-3-yl, fluorescein-5-yl, fluorescein-6 -yl, 2', 7'-dichlorofluorescein-5-yl, 2', 7'-dichlorofluorescein-6-yl, dihydrotetramethylrosamine-4-yl, tetramethylrhodamine-5 -yl, tetramethylrhodamine-6-yl, 4,4-difluoro-5,7-dimethyl-4-bora-3a,4a-diaza-s-indacene-3-ethyl or 4,4- Difluoro-5,7-diphenyl-4-bora-3a,4a-diaza-s-indacene-3-ethyl, Cy3, Cy5, poly L-lysine-fluorescein isothiocyanate (FITC) , Rhodamine-B-isothiocyanate (RITC), PE (Phycoerythrin), or rhodamine, and the radioactive isotope may be 32 P, but is not limited thereto.
본 발명의 프라이머 세트는 등온증폭 (isothermal amplification)용으로 사용하기에 적합할 수 있다. 용어 “등온증폭(isothermal amplification)"은 하나의 온도 조건하에서 DNA를 증폭시키는 것을 의미한다. 기존의 중합효소 연쇄반응(PCR)이 변성 (Denaturation), 어닐링 (Annealing) 및 신장 (Extension) 단계가 각각 다른 온도와 조건을 요구함에 따라 특수한 PCR 장치로 수행해야 하는 것과 달리, 등온증폭 반응은 별도의 특수한 기기가 필요하지 않은 장점을 갖는다. 일반적으로 등온 증폭 방법은 특정 온도범위 내에서 1시간 내에 목표 핵산을 10^9개까지 증폭시킬 수 있으며, 상기 온도를 유지시킬 수 있는 항온 장치만 있으면 수행할 수 있다. 또한 등온 증폭 방법은 수행 시간이 짧고 결과를 육안으로 관찰할 수 있으므로 간편하게 증폭을 확인할 수 있다.The primer set of the present invention may be suitable for use for isothermal amplification. The term “isothermal amplification” means DNA amplification under one temperature condition. Conventional polymerase chain reaction (PCR) has denaturation, annealing, and extension steps, respectively. Unlike the special PCR equipment that requires different temperatures and conditions, the isothermal amplification reaction has the advantage of not requiring a separate special equipment.In general, the isothermal amplification method is a target nucleic acid within an hour within a specific temperature range. Can amplify up to 10 ^ 9, and can be performed with a constant temperature device capable of maintaining the temperature.In addition, the isothermal amplification method has a short execution time and the result can be observed with the naked eye, so amplification can be easily confirmed. .
일 구체예에서, 상기 등온증폭은 고리매개 등온증폭(Loop-mediated isothermal amplification)일 수 있다. 용어 "고리매개 등온증폭방법(loop-mediated isothermal amplification, LAMP)"은 외부 프라이머, 내부 프라이머, 및 반응 속도를 향상시키기 위한 추가의 루프 프라이머를 사용하여 등온에서 수행되는 증폭 방법이다. 상기 LAMP 반응에서 사용되는 프라이머 중 외부 프라이머는 정방향 외부(forward outer, F3) 프라이머와 역방향 외부(backward outer, B3) 프라이머 2종으로 구성되고 반응의 비순환기(non-cyclic step) 동안 DNA 이중 가닥을 풀어주는 역할을 한다. 내부 프라이머는 정방향 내부 프라이머(forward inner primer, FIP)와 역방향 내부 프라이머(backward inner primer, BIP) 2종으로 구성되고 고리매개 등온증폭반응에 필수적인 고리(loop)를 만들 수 있도록 정방향 및 역방향 염기서열에 해당하는 뉴클레오티드로 구성된다. 이들 내부 프라이머는 바이러스 RNA 에는 포함되지 않으나 반응의 효율성을 높이기 위하여 특정 염기, 예를 들어, 일련의 티민 염기를 더 포함하도록 설계될 수 있다. 추가 2종의 프라이머는 정방향 고리(forward loop, LoopF) 프라이머와 역방향 고리(backward loop, LoopB) 프라이머 2종으로 구성되며 내부(inner) 프라이머가 결합하지 않는 염기서열(고리 부위)에 부착하여 고리매개등온증폭 반응을 가속화시킨다. 상기 고리매개 등온 증폭 방법은 내부 프라이머가 주형에 먼저 결합하여 신장된 후, 각 DNA 나선의 바깥쪽으로 외부 프라이머가 결합되어 신장된다. 이때, 외부 프라이머의 신장으로 인해 가닥(strand) 변위가 발생하고, 그에 따라 먼저 합성된 가닥은 주형에서 떨어지는데, 떨어진 합성 가닥이 5' 말단에서부터 고리(loop) 구조를 형성하고, 이어서 3' 말단에도 동일한 과정으로 고리 구조가 형성되며, 이를 매개로 증폭 반응이 일어난다. 상기 등온증폭 또는 고리매개 등온증폭 반응은 역전사 반응일 수 있다.In one embodiment, the isothermal amplification may be loop-mediated isothermal amplification. The term “loop-mediated isothermal amplification (LAMP)” refers to an amplification method performed isothermally using an external primer, an internal primer, and an additional loop primer to enhance the reaction rate. Among the primers used in the LAMP reaction, the outer primer consists of two types of forward outer (F3) primer and reverse outer (B3) primer, and DNA double strands are formed during the non-cyclic step of the reaction. serves as a release. The inner primer consists of two types, a forward inner primer (FIP) and a backward inner primer (BIP). It is composed of the corresponding nucleotide. These internal primers are not included in the viral RNA, but can be designed to further include specific bases, for example, a series of thymine bases, to increase the efficiency of the reaction. The additional two primers consist of a forward loop (LoopF) primer and two backward loop (LoopB) primers, and are attached to a nucleotide sequence (ring site) to which the inner primer does not bind, thereby intermediating the loop. Accelerates the isothermal amplification reaction. In the loop-mediated isothermal amplification method, internal primers are first bound to the template and extended, and then external primers are coupled to the outside of each DNA helix and then extended. At this time, strand displacement occurs due to extension of the external primer, and accordingly, the first synthesized strand is separated from the template. The separated synthetic strand forms a loop structure from the 5' end, and then also from the 3' end In the same process, a ring structure is formed, through which an amplification reaction takes place. The isothermal amplification or loop-mediated isothermal amplification may be a reverse transcription reaction.
상기 등온증폭 반응은 50 내지 70℃, 52 내지 70℃, 52 내지 68℃, 52 내지 65℃, 55 내지 65℃, 또는 50 내지 60℃에서 수행될 수 있다. 또한, 상기 등온증폭방법은 10 내지 90분, 20 내지 90분, 25 내지 90분, 10 내지 80분, 20 내지 80분, 25 내지 80분, 10 내지 70분, 20 내지 70분, 25 내지 70분, 10 내지 60분, 20 내지 60분 또는 25 내지 60분 동안 수행될 수 있다.The isothermal amplification reaction may be performed at 50 to 70 °C, 52 to 70 °C, 52 to 68 °C, 52 to 65 °C, 55 to 65 °C, or 50 to 60 °C. In addition, the isothermal amplification method is 10 to 90 minutes, 20 to 90 minutes, 25 to 90 minutes, 10 to 80 minutes, 20 to 80 minutes, 25 to 80 minutes, 10 to 70 minutes, 20 to 70 minutes, 25 to 70 minutes minutes, 10 to 60 minutes, 20 to 60 minutes or 25 to 60 minutes.
일 구체예에서, 상기 프라이머 세트를 이용하면 100 pg 이하, 예를 들어, 1 pg 내지 100 pg, 2 pg 내지 100 pg, 5 pg 내지 100 pg 또는 10 pg 내지 100 pg의 매우 소량의 표적 유전자가 포함된 검체에서 코로나바이러스 검출이 가능하다. 또한, 일반적인 등온증폭반응 조건에서 빠른 시간 내에 코로나바이러스 검출이 가능하다. 예를 들어, 상기 프라이머 세트의 각 프라이머 0.2 내지 1.6 μM을 이용하여, 60 내지 65 ℃에서 약 30 분, 예를 들어, 30 분 내지 60 분, 30 분 내지 50 분, 또는 30 분 내지 40 분 동안 등온증폭 반응하여 검출 결과를 확인할 수 있다.In one embodiment, the primer set contains a very small amount of the target gene of 100 pg or less, for example, 1 pg to 100 pg, 2 pg to 100 pg, 5 pg to 100 pg, or 10 pg to 100 pg. Coronavirus can be detected in samples that have been tested. In addition, it is possible to detect coronavirus in a short time under general isothermal amplification conditions. For example, using 0.2 to 1.6 μM of each primer of the primer set, 60 to 65 ° C. for about 30 minutes, for example, 30 to 60 minutes, 30 to 50 minutes, or 30 to 40 minutes The detection result can be confirmed by the isothermal amplification reaction.
본 발명의 다른 구체예는, 상기 프라이머 세트를 포함하는 코로나바이러스 검출용 조성물 또는 키트를 포함한다.Another embodiment of the present invention includes a composition or kit for detecting coronavirus including the primer set.
상기 코로나바이러스 검출용 조성물 또는 키트는 등온증폭 반응을 수행하기 위한 적절한 반응물 및 시약을 더 포함할 수 있다. 이러한 반응물 및 시약은, 예를 들어 추가의 프라이머, dNTP, 및 효소 (DNA 중합효소 또는 역방향 전사효소)등을 포함할 수 있고, 반응의 효율을 위해 염화칼륨, 황산마그네슘, 황산암모늄과 같은 무기염류나 계면활성제를 더 포함할 수 있다. 일 구체예에서, 상기 반응물 및 시약은 10 내지 20mM Tris-HCl, 1 내지 10mM (NH4)2SO4, 10 내지 50nM KCl, 0.1 내지 2mM MgSO4, 및 0.01 내지 0.1% Tween-20을 포함하는 pH 7 내지 9의 용액일 수 있다.The composition or kit for detecting coronavirus may further include appropriate reactants and reagents for performing an isothermal amplification reaction. These reactants and reagents may include, for example, additional primers, dNTPs, and enzymes (DNA polymerase or reverse transcriptase), and inorganic salts such as potassium chloride, magnesium sulfate, and ammonium sulfate for reaction efficiency. A surfactant may be further included. In one embodiment, the reactants and reagents include 10 to 20 mM Tris-HCl, 1 to 10 mM (NH 4 ) 2 SO 4 , 10 to 50 nM KCl, 0.1 to 2 mM MgSO 4 , and 0.01 to 0.1% Tween-20. It may be a solution of pH 7 to 9.
일 구체예에서, 상기 코로나바이러스 검출용 조성물 또는 키트는 반응 결과물을 육안으로 확인할 수 있는 시약을 더 포함할 수 있다. 그러한 시약은 통상적으로 알려지거나 상업적으로 구매 가능한 것을 사용할 수 있고, 예를 들어, WarmStart® Colorimetric LAMP Mix가 있으나 이에 제한되지 않는다.In one embodiment, the composition or kit for detecting coronavirus may further include a reagent that can visually check the reaction product. As such a reagent, commonly known or commercially available ones may be used, for example, WarmStart® Colorimetric LAMP Mix, but is not limited thereto.
상기 코로나바이러스 검출용 조성물 또는 키트는 진단 결과를 정확하게 판독하기 위해서 음성대조물질 또는 양성대조물질을 더 포함할 수 있다. 음성대조물질로는 코로나바이러스와 무관한 주형 RNA 또는 DNA를 이용할 수 있고, 양성대조물질로는 코로나바이러스 감염 환자로부터 수득된 생물학적 시료 또는 코로나바이러스의 유전체 일부, 예를 들어, GenBank accession no. MN908947.3인 Wuhan seafood market pneonia virus isolate Wuhan-Hu-1, complete genome의 적어도 일부에 해당하는 주형 RNA 또는 DNA를 이용할 수 있다. 이들 대조 물질은 필요에 따라 하나의 튜브에서 생물학적 시료와 함께 또는 개별적으로 유전자 증폭 반응에 적용될 수 있다. The composition or kit for detecting coronavirus may further include a negative control material or a positive control material in order to accurately read the diagnosis result. As a negative control material, template RNA or DNA unrelated to coronavirus can be used, and as a positive control material, a biological sample obtained from a patient infected with coronavirus or a part of the genome of coronavirus, for example, GenBank accession no. Template RNA or DNA corresponding to at least a part of MN908947.3 Wuhan seafood market pneonia virus isolate Wuhan-Hu-1, complete genome may be used. These control substances can be applied to the gene amplification reaction separately or together with the biological sample in one tube, if necessary.
본 발명의 또 다른 구체예는, 상기 프라이머 세트 및 생물학적 시료를 혼합하는 단계; 및 상기 혼합액을 등온증폭 반응으로 유전자를 증폭하는 단계를 포함하는, 코로나바이러스 검출 방법을 제공한다.Another embodiment of the present invention, mixing the primer set and the biological sample; And it provides a coronavirus detection method comprising the step of amplifying the gene by an isothermal amplification reaction of the mixed solution.
상기 생물학적 시료는 검사 대상인 개체로부터 수득된 것으로서, 비인두 또는 구인두로부터 스왑 또는 비 세척을 통해 수득된 것, 기관지 세척 또는 흡인물, 기관 간의 흡인물 및 기관지 생검, 객담, 기관지 폐포세척액 (bronchoalveolar lavage), 침, 혈액, 소변, 대변 등을 이용할 수 있다. The biological sample is obtained from the subject to be tested, obtained through swab or nasal irrigation from the nasopharynx or oropharynx, bronchial lavage or aspirate, intertracheal aspirate and bronchial biopsy, sputum, bronchoalveolar lavage , saliva, blood, urine, feces, etc. can be used.
상기 생물학적 시료는 증폭 반응의 효율을 높이기 위해 전처리를 가할 수 있다. 전처리는 물로 10배, 50배, 100배, 200배, 또는 400배 희석하는 것일 수 있고, protenase K를 처리 후 열처리하는 것일 수 있고, 음이온 교환 크로마토그래피, 친화도 크로마토그래피, 크기별 배제 크로마토그래피, 액체 크로마토그래피, 연속추출, 원심분리 또는 젤 전기영동 등의 방법과 같은 통상적으로 알려진 것을 이용하거나 상업적으로 판매하는 키트를 이용해 생물학적 시료로부터 유전자를 추출하거나 일정 수준으로 정제하는 것일 수 있다. 생물학적 시료로부터 RNA를 추출하여 사용하는 경우, 표적 유전자를 증폭하기 전에 cDNA로 역전사하는 단계를 더 포함할 수 있다. 상기 코로나바이러스 검출 방법은 증폭된 유전자를 검출하는 단계를 포함한다.The biological sample may be pretreated to increase the efficiency of the amplification reaction. Pretreatment may be 10-fold, 50-fold, 100-fold, 200-fold, or 400-fold dilution with water, heat treatment after treatment with protenase K, anion exchange chromatography, affinity chromatography, size-specific exclusion chromatography, Genes may be extracted from biological samples or purified to a certain level using commonly known methods such as liquid chromatography, continuous extraction, centrifugation, or gel electrophoresis, or using commercially available kits. When RNA is extracted from a biological sample and used, a step of reverse transcription into cDNA may be further included before amplifying the target gene. The coronavirus detection method includes detecting the amplified gene.
본 발명의 핵산 추출 및 증폭 방법을 이용하면, 시료의 전처리 (preparation), 핵산 증폭 반응 (reaction)과 검출 (detection)을 각각 별도로 수행하지 않고, 시료의 1회 적용으로 검출까지 한 번에 수행할 수 있다. 각 단계를 별도로 수행하지 않으므로, 전 과정이 단순화되어 다양한 시료나 장치를 요하지 않고, 관련 기술자가 아니어도 쉽게 수행할 수 있으며, 특히 본 발명에서 제시하는 프라이머 세트를 적용하여 SARS-CoV-2를 특이적으로 검출할 수 있다.Using the nucleic acid extraction and amplification method of the present invention, it is possible to perform the sample preparation, nucleic acid amplification reaction, and detection separately, and to detect the sample with one application. can Since each step is not performed separately, the whole process is simplified, and it does not require various samples or devices, and it can be easily performed even if you are not a related technician. can be detected negatively.
또한, 각 단계를 수행하기 위한 복잡한 장치를 요구하지 않으므로, 장소에 제약을 받지 않고, 배포가 용이한 장점을 갖고, 1회의 시료를 적용하여 코로나바이러스를 포함한 다수의 핵산 검출이 가능하므로 경제적이다.In addition, since it does not require a complicated device to perform each step, it is not restricted by location, has the advantage of easy distribution, and is economical because it is possible to detect multiple nucleic acids, including coronavirus, by applying one sample.
도 1은 본 발명의 다중 핵산 검출용 구조물의 전체 구조의 예시이다.1 is an example of the overall structure of the construct for detecting multiple nucleic acids of the present invention.
도 2는 본 발명의 다중 핵산 검출용 구조물의 전체 구조의 다른 예시이다.2 is another example of the overall structure of the structure for detecting multiple nucleic acids of the present invention.
도 3은 일 구체예에 따른 금 입자를 포함하는 구조물에서 검출패드(160)에 표적 핵산이 획득되는 원리를 도식화한 것이다.FIG. 3 is a schematic diagram of a principle of obtaining target nucleic acids from a detection pad 160 in a structure including gold particles according to an embodiment.
도 4는 일 구체예에 따른 시스템에서 검출패드(160)에 표적 핵산이 획득되는 원리를 도식화한 것이다.4 is a diagram illustrating a principle of obtaining target nucleic acids from the detection pad 160 in the system according to one embodiment.
도 5는 일 구체예에 따른 금 입자를 포함하는 다중 핵산 검출용 구조물 일부분의 측면 구조도이다.5 is a side structural diagram of a part of a structure for detecting multiple nucleic acids including gold particles according to an embodiment.
도 6은 일 구체예에 따른 시스템이 포함하는 다중 핵산 검출용 구조물의 측면 구조도이다.6 is a side structural diagram of a structure for detecting multiple nucleic acids included in a system according to one embodiment.
도 7은 일 구체예에 따른 다중 핵산 검출용 구조물 일부분의 사시도이다.7 is a perspective view of a portion of a structure for detecting multiple nucleic acids according to one embodiment.
도 8은 다른 구체예에 따른 다중 핵산 검출용 구조물 일부분의 사시도이다.8 is a perspective view of a portion of a structure for detecting multiple nucleic acids according to another embodiment.
도 9는 일 구체예에 따른 금 입자를 포함하는 구조물에서 검출패드(160)의 구조를 확대한 구조도이다.9 is an enlarged structural view of the structure of the detection pad 160 in the structure including gold particles according to one embodiment.
도 10은 일 구체예에 따른 시스템이 포함하는 다중 핵산 검출용 구조물에서 검출패드(160)의 구조를 확대한 구조도이다.10 is an enlarged structure diagram of a detection pad 160 in a structure for detecting multiple nucleic acids included in a system according to an embodiment.
도 11은 버퍼패드(121)와 개시자패드(132)를 포함하는 경우 케이스로 둘러싸인 본 발명의 다중 핵산 검출용 구조물의 외관을 보여주는 예시이다.11 is an example showing the exterior of the structure for detecting multiple nucleic acids of the present invention surrounded by a case including a buffer pad 121 and an initiator pad 132.
도 12는 세포 용해용 조성물의 종류에 따른 LAMP 결과를 비교하여 그래프로 나타낸 것이다.12 is a graph comparing LAMP results according to the type of cell lysis composition.
도 13은 일 구체예에 따른 다중 핵산 검출용 구조물을 이용하여 혈액 시료로부터 SARS-CoV-2를 검출한 결과를 보여주는 예시이다.13 is an example showing the result of detecting SARS-CoV-2 from a blood sample using a structure for detecting multiple nucleic acids according to one embodiment.
도 14은 본 발명의 일 실시예에 따른 프라이머 세트에 대한 효과를 확인하기 위하여 본 발명에서 제시하는 프라이머 세트를 이용하여 SARS-CoV-2를 등온증폭반응으로 검출한 결과를 나타낸 이미지이다.14 is an image showing the result of detecting SARS-CoV-2 by isothermal amplification reaction using the primer set presented in the present invention in order to confirm the effect of the primer set according to an embodiment of the present invention.
도 15는 본 발명의 일 실시예에 따른 프라이머 세트에 대한 효과를 확인하기 위하여 본 발명에서 제시하는 프라이머 세트를 이용하여 SARS-CoV-2를 등온증폭반응으로 검출한 결과를 전기영동으로 확인한 이미지이다.15 is an image confirming the result of detecting SARS-CoV-2 by isothermal amplification reaction by electrophoresis using the primer set presented in the present invention in order to confirm the effect of the primer set according to an embodiment of the present invention. .
도 16은 본 발명의 일 실시예에 따른 프라이머 세트에 대한 효과를 확인하기 위하여 본 발명에서 제시하는 프라이머 세트를 이용하여 SARS-CoV-2를 등온증폭반응으로 5분 간격으로 검출한 결과를 나타낸 이미지이다.16 is an image showing the results of detecting SARS-CoV-2 at 5-minute intervals through an isothermal amplification reaction using the primer set presented in the present invention in order to confirm the effect of the primer set according to an embodiment of the present invention. to be.
도 17는 본 발명의 일 실시예에 따른 프라이머 세트에 대한 효과를 확인하기 위하여 본 발명에서 제시하는 프라이머 세트를 이용하여 SARS-CoV-2를 등온증폭반응으로 5분 간격으로 검출한 결과를 전기영동으로 확인한 이미지이다.17 is an electrophoresis result of detecting SARS-CoV-2 at 5-minute intervals by isothermal amplification using the primer set presented in the present invention to confirm the effect of the primer set according to an embodiment of the present invention. This is an image verified by
도 18는 본 발명의 일 실시예에 따른 프라이머 세트에 대한 효과를 확인하기 위하여 본 발명에서 제시하는 프라이머 세트를 이용하여 가상의 환자 검체에서 SARS-CoV-2를 등온증폭반응으로 검출한 결과를 색 변화와 전기영동으로 확인한 이미지이다.18 shows the results of detecting SARS-CoV-2 by isothermal amplification reaction in a virtual patient sample using the primer set presented in the present invention in order to confirm the effect of the primer set according to an embodiment of the present invention. It is an image confirmed by change and electrophoresis.
도 19은 본 발명의 일 실시예에 따른 프라이머 세트에 대한 효과를 확인하기 위하여 본 발명에서 제시하는 프라이머 세트를 이용하여 방해 물질 환경 하에 가상의 환자 검체에서 SARS-CoV-2를 등온증폭반응으로 검출한 결과를 색 변화와 전기영동으로 확인한 이미지이다.19 is an isothermal amplification reaction of SARS-CoV-2 detected in a virtual patient sample under an interfering substance environment using the primer set presented in the present invention to confirm the effect of the primer set according to an embodiment of the present invention. This is an image confirmed by color change and electrophoresis.
이하, 본 발명을 하기 실시예에 의거하여 보다 자세하게 설명하나, 이들은 본 발명을 설명하기 위한 것일 뿐 이들에 의하여 본 발명의 범위가 어떤 식으로든 제한되는 것은 아니다.Hereinafter, the present invention will be described in more detail based on the following examples, but these are only for explaining the present invention, and the scope of the present invention is not limited in any way by these.
[A. 랩온 페이퍼 구조물에 대한 실험][A. Experiments on Lab-on-Paper Structures]
제조예 A-1. 세포 용해 조성물(Lysis buffer)의 준비Preparation Example A-1. Preparation of cell lysis composition (lysis buffer)
세포 용해 조성물은, 구성 성분, pH 및 각 성분 비 등을 달리하여 하기의 표 1과 같이 혼합하여 준비하였다. 단백질분해효소는 프로테이나아제 K로서 Thermofisher (EO0491)에서 구매하였고, 검출하려는 균의 종류 따라 lysozyme (Thermofisher, 90082) 0.02 mg/ml을 첨가하여 사용하였다. pH는 0.1M HCl 또는 0.1M NaOH로 조절하였다.The cell lysis composition was prepared by mixing as shown in Table 1 below by varying the composition, pH, and ratio of each component. Proteinase K was purchased from Thermofisher (EO0491) as proteinase K, and 0.02 mg/ml of lysozyme (Thermofisher, 90082) was added and used according to the type of bacteria to be detected. pH was adjusted with 0.1M HCl or 0.1M NaOH.
조성Furtherance 단백질 분해효소proteolytic enzyme pHpH
실시예 1Example 1 20mM Tris·HCl15mM MgSO4
15mM KCl
15mM (NH4)2SO4
0.1 w/w% Tween20
20 mM Tris HCl 15 mM MgSO 4
15 mM KCl
15 mM (NH 4 ) 2 SO 4
0.1 w/w% Tween20
0.05 mg/ml0.05 mg/ml 8.88.8
비교예 1Comparative Example 1 50mM Tris-HCl
100mM NaCl
1mM DTT
5% 글리세롤
50 mM Tris-HCl
100mM NaCl
1 mM DTT
5% glycerol
0.05 mg/ml0.05 mg/ml 7.07.0
비교예 2Comparative Example 2 50mM Tris-HCl
150mM NaCl
5mM EDTA
1% NP-40
50 mM Tris-HCl
150mM NaCl
5 mM EDTA
1% NP-40
0.05 mg/ml0.05 mg/ml 8.58.5
비교예 3Comparative Example 3 50mM Tris-HCl
10mM Na2HPO4
5mM MgCl2
1% TritonX-100
50 mM Tris-HCl
10 mM Na 2 HPO 4
5 mM MgCl2
1% TritonX-100
0.05 mg/ml0.05 mg/ml 8.58.5
비교예 4Comparative Example 4 50mM Tris-HCl
10mM Na2HPO4
10mM NaCl
0.5% SDS
1% TritonX-100
50 mM Tris-HCl
10 mM Na 2 HPO 4
10 mM NaCl
0.5% SDS
1% TritonX-100
-- 9.09.0
비교예 5Comparative Example 5 50mM Tris-HCl
10mM Na2HPO4
10mM NaCl
0.5% SDS
1% TritonX-100
50 mM Tris-HCl
10 mM Na 2 HPO 4
10 mM NaCl
0.5% SDS
1% TritonX-100
-- 7.07.0
시험예 A-1. 세포 용해 조성물의 유동성 비교 시험Test Example A-1. Flowability comparison test of cell lysis composition
고리매개 등온증폭방법(loop-mediated isothermal amplification, LAMP)을 사용하여 라임병 원인균을 검출하기 위해 프라이머를 설계 및 제작하였다. 라임병 원인균으로는 보렐리아 아프젤리(B. Afzelii), 보렐리아 부르그도르페리(B. Burgdorferi), 및 보렐리아 가리니이(B.garinii)를 이용하였고, 사용된 프라이머는 하기의 표 2에 표시하였다. Primers were designed and produced to detect the causative agent of Lyme disease using a loop-mediated isothermal amplification (LAMP) method. As the causative bacteria of Lyme disease, Borrelia Afzelii, Borrelia Burgdorferi, and Borrelia garinii were used, and the primers used are shown in Table 2 below. did
프라이머
유형
primer
type
서열order
프라이머 세트 1
(B.afzelii 표적)
Primer Set 1
(B.afzelii target)
F3F3 5'-GGTATACTGACAGCAGCTT-3' 5'-GGTATACTGACAGCAGCTT-3'
B3 B3 5'-CTTGCAGCTTAATAATAGCCTT-3' 5′-CTTGCAGCTTAATAATAGCCTT-3′
FIPFIP 5'-GTTGCTCGGTCCTCCATGTTTAAATTTTTAATGTTATCCGTGATATGGTTCCGA-3' 5'-GTTGCTCGGTCCTCCATGTTTAAATTTTTAATGTTATCCGTGATATGGTTCCGA-3'
BIPBIP 5'-GGATTTCGTATCAATTTTGGAGGCATTTTAAGTTACAAAGGTCCCATTGC-3' 5'-GGATTTCGTATCAATTTTGGAGGCATTTTAAGTTACAAAGGTCCCATTGC-3'
FL FL 5'-ATAAGGCCTTCGGTATTG-3' 5′-ATAAGGCCTTCGGTATTG-3′
BLBL 5'-ATTCTACGTTCCGATTCTCAGTAT-3' 5′-ATTCTACGTTCCGATTCTCAGTAT-3′
프라이머 세트 2
(B.burgdorferi 표적)
Primer Set 2
(B.burgdorferi target)
F3 F3 5'-AGAGCAGCTGAGGAGCT-3' 5′-AGAGCAGCTGAGGAGCT-3
B3B3 5'-CTTCCAGTTGAACACCATCTT-3' 5′-CTTCCAGTTGAACACCATCTT-3′
FIPFIP 5'-AAGTCCACGACGGTTGAGACCTTTTTGCAGCCTGCTTAAATTAACA-3' 5'-AAGTCCACGACGGTTGAGACCTTTTTGCAGCCTGCTTAAATTAACA-3'
BIP BIP 5'-GAGCAAACGAAGTTGAAGCTATTTTAGCCTGAGCAGTTAGAGC-3' 5′-GAGCAAACGAAGTTGAAGCTATTTTAGCCTGAGCAGTTAGAGC-3
FLFL 5'-TGAATGCGATCCTGGT-3' 5′-TGAATGCGATCCTGGT-3′
BLBL 5'-TATGGAGCTAATGTTGCTAAT-3'5′-TATGGAGCTAATGTTGCTAAT-3′
프라이머 세트 3
(B.garinii 표적)
Primer Set 3
(B. garinii target)
F3 F3 5'-GCATCGTTGACGTCAGCT-3'5′-GCATCGTTGACGTCAGCT-3
B3B3 5'-TGTAGAGGCGATTGTAGC-3'5′-TGTAGAGGCGATTGTAGC-3′
FIPFIP 5'-CCACGATTACTTGCTGGTAACTTTTGTGACGTGTTCGGTTAAGT-3'5'-CCACGATTACTTGCTGGTAACTTTTGTGACGTGTTCGGTTAAGT-3'
BIP BIP 5'-GATAAGAGTGCCGCTGATAAGTTTTTAGCCCAGCAGATAAGGG-3'5′-GATAAGAGTGCCGCTGATAAGTTTTTAGCCCAGCAGATAAGGG-3
FLFL 5'-TAACAACGGTTGCGGTCG-3' 5′-TAACAACGGTTGCGGTCG-3′
BLBL 5'-TGACGATGAGGTCAATTCAT-3'5′-TGACGATGAGGTCAATTCAT-3′
※ F3, forward primer; B3, backward primer; FIP, forward inner primer; BIP, backward inner primer; FL, forward loop primer; BL, backward loop primer※ F3, forward primer; B3, backward primer; FIP, forward inner primer; BIP, backward inner primer; FL, forward loop primer; BL, backward loop primer
(2) 등온증폭반응(2) Isothermal amplification reaction
라임병 원인균인 보렐리아 아프젤리, 보렐리아 부르그도르페리, 및 보렐리아 가리니이 각 균주를 DMEM 배지에 2.5 * 105 cell/ml로 혼합한 것 50㎕에 실시예 1 및 비교예 1 내지 5의 세포 용해 조성물을 50㎕첨가하고 가볍게 탭핑(tapping)한 후, 가로 10cm*세로 2cm*두께 3mm로 자른 섬유유리 재질의 페이퍼(Millipore, MA, USA)의 한 쪽 끝에 20㎕를 덜어 적가하였다. 그런 다음, 페이퍼를 약 30도의 경사로 기울인 상태로 부가 완충액(20 mM Tris-HCl, 10 mM (NH4)2SO4, 50 mM KCl, 2 mM MgSO4, 0.1% Tween® 20, pH 8.8) 500㎕를 약 1분간 천천히 적가하여 페이퍼의 반대쪽 끝으로 용액이 이동하게 하였다. 실시예 1을 적용하되 부가 용액 대신 증류수를 이용한 것을 비교예 6으로 하였다.The causative bacteria of Lyme disease, Borrelia afzelii, Borrelia burgdorferi, and Borrelia garinii, were mixed in DMEM medium at 2.5 * 10 5 cells/ml in 50 μl of Example 1 and Comparative Examples 1 to 5. After adding 50 μl of the cell lysis composition and lightly tapping, 20 μl was added dropwise to one end of a fiberglass paper (Millipore, MA, USA) cut into 10 cm x 2 cm x 3 mm thickness. Then, with the paper tilted at an angle of about 30 degrees, add buffer (20 mM Tris-HCl, 10 mM (NH 4 ) 2 SO 4 , 50 mM KCl, 2 mM MgSO 4 , 0.1% Tween® 20, pH 8.8) 500 [mu]l was slowly added dropwise over about 1 minute to allow the solution to migrate to the opposite end of the paper. Example 1 was applied, but using distilled water instead of the addition solution was used as Comparative Example 6.
페이퍼가 반대쪽 끝까지 적셔지면 1cm를 잘라서 부가 완충액 100㎕에 담궈 볼텍싱하고 원심분리 한 후 상등액 2.0㎕를 취해 25.0㎕ PCR 튜브에 정방향(forward) 및 역방향(backward) 내부 프라이머(inner primer: FIP 및 BIP, 1.6μM) 각각 1.0㎕, 외부 프라이머(outer primer: F3 및 B3, 0.2μM) 각각 1.0㎕, deoxynucleotide solution mixture(1.4mM, dATP, dCTP, dGTP 및 dTTP) 3.5㎕, 10X 등온증폭버퍼 Ⅱ2.5㎕(1X), MgSO4(8μM) 1.5㎕, Bst 3.0 DNA 중합효소(320U/㎖), 0.5㎕, EvaGreen(1X) 0.5㎕, 및 증류수 9.5㎕를 혼합하고, 60℃의 온도에서 30분 동안 역전사 등온증폭반응을 수행하였다. 여기서 사용된 증류수는 RNase가 혼합된 것을 사용하였고, 음성대조군으로는 균주 시료 대신 증류수를 혼합한 후 반응시킨 것으로 하였다. 그런 다음, 녹색 광원 및 605/50 필터가 세팅된 Chemi-Doc XRS + 이미징 시스템 (Bio-Rad Laboratories, Hercules, CA, USA)을 이용하여 형광 이미지를 획득하고, 형광을 측정하여 형광 세기를 최대 값 1 및 최소값 0으로 하고 그에 대한 비율로 산출한 후 하기의 식에 따라 형광세기 값을 구하였다. When the paper is wetted to the opposite end, cut 1 cm, immerse in 100 μl of addition buffer, vortex, centrifuge, take 2.0 μl of the supernatant, and put forward and reverse inner primers (FIP and BIP) in a 25.0 μl PCR tube. , 1.6 μM) 1.0 μl each, outer primers (F3 and B3, 0.2 μM) 1.0 μl each, deoxynucleotide solution mixture (1.4 mM, dATP, dCTP, dGTP and dTTP) 3.5 μl, 10X isothermal amplification buffer Ⅱ2.5 [mu]l (1X), MgSO 4 (8 [mu]M) 1.5 [mu]l, Bst 3.0 DNA polymerase (320 U/ml), 0.5 [mu]l, EvaGreen (1X) 0.5 [mu]l, and distilled water 9.5 [mu]l were mixed and incubated at 60°C for 30 minutes. Reverse transcription isothermal amplification was performed. The distilled water used here was mixed with RNase, and as a negative control, distilled water was mixed instead of the strain sample and then reacted. Then, a fluorescence image was acquired using a Chemi-Doc XRS + imaging system (Bio-Rad Laboratories, Hercules, CA, USA) with a green light source and a 605/50 filter set, and fluorescence was measured to determine the maximum fluorescence intensity. 1 and the minimum value 0, and after calculating the ratio thereto, the fluorescence intensity value was obtained according to the following formula.
형광 세기 값 = I-IN Fluorescence intensity value = II N
(IN: 음성대조군의 형광 세기, I : 시료의 형광 세기)(I N : Fluorescence intensity of negative control group, I: Fluorescence intensity of sample)
그 결과, 실시예 1의 lysis buffer를 사용한 경우 1 분내 가장 많은 유전 물질이 이동한 것으로 확인되었다 (도 12).As a result, when the lysis buffer of Example 1 was used, it was confirmed that the most genetic material moved within 1 minute (FIG. 12).
실시예 A-2: 금 입자를 포함하는 랩온페이퍼 핵산 검출용 구조물의 제조Example A-2: Preparation of Lab-on-Paper Structure Containing Gold Particles for Detecting Nucleic Acids
본 발명의 랩온페이퍼 핵산 검출용 구조물을 제조하기 위해, 시료패드(120) 및 버퍼패드(121)로는 0.5 ㎛ 폴리설폰, 제1연결패드(131) 또는 개시자패드(132)로는 0.01㎛ 셀룰로오스, 반응패드(140)로는 0.005㎛ 셀룰로스아세테이트, 제2연결패드(150)로는 0.05㎛ 셀룰로오스, 검출패드(160)로는 0.005㎛ 니트로셀룰로오스, 및 흡수패드(170)로는 0.5 ㎛ 유리섬유 재질로 준비하였다.To prepare the lab-on-paper nucleic acid detection structure of the present invention, 0.5 μm polysulfone was used as the sample pad 120 and buffer pad 121, 0.01 μm cellulose was used as the first connection pad 131 or initiator pad 132, The reaction pad 140 was made of 0.005 μm cellulose acetate, the second connection pad 150 was made of 0.05 μm cellulose, the detection pad 160 was made of 0.005 μm nitrocellulose, and the absorption pad 170 was made of 0.5 μm glass fiber.
반응패드(140)는 셀룰로오스아세테이트 막을 겹쳐서 패드로 만들고 이를 45 mM 수크로오스, 0.005 w/w% TritonX-100, 및 0.2 w/w% 글리세롤을 포함하는 용액에 담근 후 건조시켜 준비하였다. 그리고 여기에 미세 드릴로 웰을 형성하고, 상기 웰의 바닥에 프라이머 세트를 포함하는 히드로겔 층을 형성하였다. 상기 히드로겔 층을 형성하기 위해 우선 히드로겔 용액 총 부피를 기준으로 UV-광가교성 폴리(에틸렌 글리콜) 디아크릴레이트 (PEGDA, Sigma-Aldrich, MW700) 20% v/v, 폴리(에틸렌 글리콜)(PEG, Sigma-Aldrich, MW600) 40% v/v 및 광개시제 2-하이드록시-2-메틸프로피오페논 (Sigma-Aldrich) 5% v/v 및 버퍼(PBS 완충액, pH7.5) 35%를 혼합하고, 여기에 각 프라이머 세트를 혼합하여 하이드로겔 용액을 제조하였다. 상기 폴리(에틸렌 글리콜)은 히드로겔 미세입자의 공극율을 증가시키기 위하여 포함되는 것이 바람직하다. 그런 다음, 상기 히드로겔 용액을 반응패드의반응 패드의 각 웰 내부 표면에 도포하고, 1분간 UV 노출(360 nm wavelength, 35 mJ/cm2)하여 히드로겔 코팅층을 형성하였다.The reaction pad 140 was prepared by making a pad by overlapping cellulose acetate films, immersing the pad in a solution containing 45 mM sucrose, 0.005 w/w% TritonX-100, and 0.2 w/w% glycerol, and drying. Then, a well was formed using a micro-drill, and a hydrogel layer including a primer set was formed on the bottom of the well. To form the hydrogel layer, first, based on the total volume of the hydrogel solution, UV-light crosslinkable poly (ethylene glycol) diacrylate (PEGDA, Sigma-Aldrich, MW700) 20% v / v, poly (ethylene glycol) ( Mix PEG, Sigma-Aldrich, MW600) 40% v/v and photoinitiator 2-hydroxy-2-methylpropiophenone (Sigma-Aldrich) 5% v/v and buffer (PBS buffer, pH7.5) 35% Then, a hydrogel solution was prepared by mixing each primer set therein. The poly(ethylene glycol) is preferably included to increase the porosity of the hydrogel microparticles. Then, the hydrogel solution was applied to the inner surface of each well of the reaction pad and exposed to UV (360 nm wavelength, 35 mJ/cm 2 ) for 1 minute to form a hydrogel coating layer.
그런 다음 반응패드(140)의 표면에 dNTP(1.4mM, dATP, dCTP, dGTP 및 dTTP), 등온증폭버퍼(1X, 20mM Tris-HCl, 10mM (NH4)2SO4, 50mM KCl, 2mM MgSO4, 및 0.1% Tween-20, pH7.5)를 함유하는 파우더와 Bst 3.0 DNA 중합효소(320U/㎖)를 도포하고, 약 38℃오븐에서 약 30분 간 가열하여 고정하였다. Then, dNTP (1.4mM, dATP, dCTP, dGTP and dTTP), isothermal amplification buffer (1X, 20mM Tris-HCl, 10mM (NH 4 ) 2 SO 4 , 50mM KCl, 2mM MgSO 4 were added to the surface of the reaction pad 140. , and 0.1% Tween-20, pH 7.5) and Bst 3.0 DNA polymerase (320 U/ml) were applied, and fixed by heating in an oven at about 38° C. for about 30 minutes.
제2연결패드(150)에 고정된 금 나노입자는 콜로이드 입자로서 하기와 같이 제조하였다. 0.1% HAuCl4용액을 교반 가열하면서 끓기 시작하면 0.5% 소듐시트르산 용액을 첨가하여 용액을 환원시켜 금 입자를 만들고, 여기에 스트렙타비딘(streptavidin)을 금 입자용액 100㎖당 각각 1mg을 첨가하여 축합시켰다. 축합체를 10,000g에서 원심분리하여 침전시키고 0.1% BSA를 함유하는 생리식염수(PBS)로 용해하여 OD450값이 10이 되도록 만들어 보관하였다. The gold nanoparticles fixed to the second connection pad 150 were prepared as colloidal particles as follows. When the 0.1% HAuCl 4 solution starts to boil while stirring and heating, 0.5% sodium citrate solution is added to reduce the solution to form gold particles, and 1 mg of streptavidin per 100 ml of the gold particle solution is added to condense made it The condensate was precipitated by centrifugation at 10,000 g, dissolved in physiological saline (PBS) containing 0.1% BSA, and stored at an OD450 value of 10.
제2연결패드(150)는 하기와 같이 제조하였다. 구체적으로, 셀룰로오스 막을 여러 겹쳐 준비하여 절단하고, 0.4 M의 Tris (pH 6.5), 0.2%의 Tween-20, 1%의 카제인나트륨, 0.1%의 소듐아지드(sodium azide), 및 0.05%의 Proclin 300으로 제조된 용액에 담궈 적셔 두었다. 상기 제조된 금 축합체는 상기 용액과 동일한 조성의 용액에 투석하여 준비하였다. 그런 다음, 상기 셀룰로오스 막에 상기 투석한 금 축합체를 처리하고 건조하여 제2연결패드(150)를 완성하였다.The second connection pad 150 was manufactured as follows. Specifically, several layers of cellulose membranes were prepared and cut, 0.4 M Tris (pH 6.5), 0.2% Tween-20, 1% sodium caseinate, 0.1% sodium azide, and 0.05% Proclin. It was immersed in a solution prepared by 300 and kept wet. The prepared gold condensate was prepared by dialysis against a solution having the same composition as the above solution. Then, the cellulose membrane was treated with the dialyzed gold condensate and dried to complete the second connection pad 150.
검출패드(160)는 폴리에틸렌프탈레이트 잉크로 검출 구역을 스탬핑하여 지정하고, 그 위에 FAM, HEX, 및 Cy5 등의 검출자에 결합할 수 있는 항체를 함유하는 용액으로 다시 스탬핑 한 후, NHS(N-hydroxysulfosuccinimide) 용액을 적용하고 반응시켜 항체를 고정하였다.The detection pad 160 is designated by stamping the detection area with polyethylene phthalate ink, and then stamped again with a solution containing an antibody capable of binding to a detector such as FAM, HEX, and Cy5 thereon, and NHS (N- hydroxysulfosuccinimide) solution was applied and reacted to fix the antibody.
상기 개시자패드(132) 및 제2연결패드(150)의 반응패드와 접촉하는 부분에는 5%의 저융점 아가로오스(Lonza, NuSieve GTG Agarose) 용액으로 코팅하였다.A portion of the initiator pad 132 and the second connection pad 150 in contact with the reaction pad was coated with a 5% low melting point agarose (Lonza, NuSieve GTG Agarose) solution.
그런 다음, 각 구조물의 구성요소를 도 1 또는 2와 같이 배치하였다. 버퍼패드(121) 및 개시자패드(132)가 없는 구조는 도 1과 같이 배치할 수 있고, 버퍼패드 및 개시자패드가 있는 구조는 도 2와 같이 배치할 수 있다. 도 1의 구조물의 경우 사시도는 도 7과 같을 수 있고, 도 2의 구조물의 경우 사시도는 도 8과 같을 수 있다. 상기 시료패드(120), 반응패드(140), 및 제2연결패드(150)와 선택적으로 개시자패드(132)의 하단에는 가열패드로(141)로 열선이 연결된 구리판을 배치하고, 반응패드의 상부에는 차단패드(142)로 폴리아크릴 막을 배치하였다.Then, the components of each structure were arranged as shown in FIG. 1 or 2. A structure without the buffer pad 121 and the initiator pad 132 may be disposed as shown in FIG. 1 , and a structure with the buffer pad and the initiator pad may be disposed as shown in FIG. 2 . In the case of the structure of FIG. 1 , the perspective view may be the same as that of FIG. 7 , and in the case of the structure of FIG. 2 , the perspective view may be the same as that of FIG. 8 . The sample pad 120, the reaction pad 140, and the second connection pad 150 and, optionally, a copper plate connected to a heating pad 141 by a hot wire is disposed at the bottom of the initiator pad 132, and the reaction pad A polyacrylic film was placed on top of the blocking pad 142.
실시예 A-3: 형광 기반 랩온페이퍼 핵산 검출용 구조물의 제조Example A-3: Preparation of Fluorescence-Based Lab-on-Paper Structure for Detecting Nucleic Acids
본 발명의 랩온페이퍼 핵산 검출용 구조물을 제조하기 위해, 시료패드(120) 및 버퍼패드(121)로는 0.5 ㎛ 폴리설폰, 제1연결패드(131) 또는 개시자패드(132)로는 0.01㎛ 셀룰로오스, 반응패드(140)로는 0.005㎛ 셀룰로스아세테이트, 제2연결패드(150)로는 0.05㎛ 셀룰로오스, 검출패드(160)로는 0.005㎛ 니트로셀룰로오스, 및 흡수패드(170)로는 0.5 ㎛ 유리섬유 재질로 준비하였다.To prepare the lab-on-paper nucleic acid detection structure of the present invention, 0.5 μm polysulfone was used as the sample pad 120 and buffer pad 121, 0.01 μm cellulose was used as the first connection pad 131 or initiator pad 132, The reaction pad 140 was made of 0.005 μm cellulose acetate, the second connection pad 150 was made of 0.05 μm cellulose, the detection pad 160 was made of 0.005 μm nitrocellulose, and the absorption pad 170 was made of 0.5 μm glass fiber.
반응패드(140)는 셀룰로오스아세테이트 막을 겹쳐서 패드로 만들고 이를 45 mM 수크로오스, 0.005 w/w% TritonX-100, 및 0.2 w/w% 글리세롤을 포함하는 용액에 담근 후 건조시켜 준비하였다. 그리고 여기에 미세 드릴로 웰을 형성하고, 상기 웰의 바닥에 프라이머 세트를 포함하는 히드로겔 층을 형성하였다. 상기 히드로겔 층을 형성하기 위해 우선 히드로겔 용액 총 부피를 기준으로 UV-광가교성 폴리(에틸렌 글리콜) 디아크릴레이트 (PEGDA, Sigma-Aldrich, MW700) 20% v/v, 폴리(에틸렌 글리콜)(PEG, Sigma-Aldrich, MW600) 40% v/v 및 광개시제 2-하이드록시-2-메틸프로피오페논 (Sigma-Aldrich) 5% v/v 및 버퍼(PBS 완충액, pH7.5) 35%를 혼합하고, 여기에 각 프라이머 세트를 혼합하여 하이드로겔 용액을 제조하였다. 상기 폴리(에틸렌 글리콜)은 히드로겔 미세입자의 공극율을 증가시키기 위하여 포함되는 것이 바람직하다. 그런 다음, 상기 히드로겔 용액을 반응패드의반응 패드의 각 웰 내부 표면에 도포하고, 1분간 UV 노출(360 nm wavelength, 35 mJ/cm2)하여 히드로겔 코팅층을 형성하였다.The reaction pad 140 was prepared by making a pad by overlapping cellulose acetate films, immersing the pad in a solution containing 45 mM sucrose, 0.005 w/w% TritonX-100, and 0.2 w/w% glycerol, and drying. Then, a well was formed using a micro-drill, and a hydrogel layer including a primer set was formed on the bottom of the well. To form the hydrogel layer, first, based on the total volume of the hydrogel solution, UV-light crosslinkable poly (ethylene glycol) diacrylate (PEGDA, Sigma-Aldrich, MW700) 20% v / v, poly (ethylene glycol) ( Mix PEG, Sigma-Aldrich, MW600) 40% v/v and photoinitiator 2-hydroxy-2-methylpropiophenone (Sigma-Aldrich) 5% v/v and buffer (PBS buffer, pH7.5) 35% Then, a hydrogel solution was prepared by mixing each primer set therein. The poly(ethylene glycol) is preferably included to increase the porosity of the hydrogel microparticles. Then, the hydrogel solution was applied to the inner surface of each well of the reaction pad and exposed to UV (360 nm wavelength, 35 mJ/cm 2 ) for 1 minute to form a hydrogel coating layer.
그런 다음 반응패드(140)의 표면에 dNTP(1.4mM, dATP, dCTP, dGTP 및 dTTP), 등온증폭버퍼(1X, 20mM Tris-HCl, 10mM (NH4)2SO4, 50mM KCl, 2mM MgSO4, 및 0.1% Tween-20, pH7.5)를 함유하는 파우더와 Bst 3.0 DNA 중합효소(320U/㎖)를 도포하고, 약 38℃오븐에서 약 30분 간 가열하여 고정하였다.Then, dNTP (1.4mM, dATP, dCTP, dGTP and dTTP), isothermal amplification buffer (1X, 20mM Tris-HCl, 10mM (NH 4 ) 2 SO 4 , 50mM KCl, 2mM MgSO 4 were added to the surface of the reaction pad 140. , and 0.1% Tween-20, pH 7.5) and Bst 3.0 DNA polymerase (320 U/ml) were applied, and fixed by heating in an oven at about 38° C. for about 30 minutes.
검출패드(160)는 미세 드릴로 지름 5mm의 웰을 형성하고, 여기에 폴리에틸렌프탈레이트 잉크를 적용하고, 그 위에 FAM, HEX, 및 Cy5에 결합할 수 있는 항체를 함유하는 용액을 적가한 후, NHS(N-hydroxysulfosuccinimide) 용액을 적용하고 반응시켜 항체를 고정하였다.In the detection pad 160, a well having a diameter of 5 mm is formed with a microdrill, polyethylene phthalate ink is applied thereto, a solution containing FAM, HEX, and an antibody capable of binding to Cy5 is added dropwise thereon, and NHS (N-hydroxysulfosuccinimide) solution was applied and reacted to fix the antibody.
상기 개시자 패드(132) 및 제2연결패드(150)는 반응패드와 접촉하는 부분에 5%의 저융점 아가로오스(Lonza, NuSieve GTG Agarose) 용액으로 코팅하였다.The initiator pad 132 and the second connection pad 150 were coated with a 5% low-melting point agarose (Lonza, NuSieve GTG Agarose) solution at a portion in contact with the reaction pad.
그런 다음, 각 구조물의 구성요소를 도 1 또는 2와 같이 배치하였다. 버퍼패드(121) 및 개시자패드(132)가 없는 구조는 도 1과 같이 배치할 수 있고, 버퍼패드 및 개시자패드가 있는 구조는 도 2와 같이 배치할 수 있다. 도 1의 구조물의 경우 사시도는 도 7과 같을 수 있고, 도 2의 구조물의 경우 사시도는 도 8과 같을 수 있다. 상기 시료패드(120), 반응패드(140), 및 제2연결패드(150)와 선택적으로 개시자패드(132)의 하단에는 가열패드로(141)로 열선이 연결된 구리판을 배치하고, 반응패드의 상부에는 차단패드(142)로 폴리아크릴 막을 배치하였다.Then, the components of each structure were arranged as shown in FIG. 1 or 2. A structure without the buffer pad 121 and the initiator pad 132 may be disposed as shown in FIG. 1 , and a structure with the buffer pad and the initiator pad may be disposed as shown in FIG. 2 . In the case of the structure of FIG. 1 , the perspective view may be the same as that of FIG. 7 , and in the case of the structure of FIG. 2 , the perspective view may be the same as that of FIG. 8 . The sample pad 120, the reaction pad 140, and the second connection pad 150 and, optionally, a copper plate connected to a heating pad 141 by a hot wire is disposed at the bottom of the initiator pad 132, and the reaction pad A polyacrylic film was placed on top of the blocking pad 142.
시험예 A-2: 금 입자를 포함하는 구조물을 이용한 혈액 시료로부터 바이러스 검출Test Example A-2: Virus detection from blood samples using structures containing gold particles
본 발명의 랩온페이퍼 핵산 검출용 구조물을 이용하여 혈액 시료로부터 핵산을 추출하고 이를 증폭하여 밴드의 색변화를 관찰함으로써 SARS-CoV-2와 같은 바이러스 감염 여부를 쉽게 측정할 수 있다.Virus infection such as SARS-CoV-2 can be easily measured by extracting nucleic acid from a blood sample using the lab-on-paper nucleic acid detection structure of the present invention, amplifying the nucleic acid, and observing the color change of the band.
SARS-CoV-2를 검출하기 위해 SARS-CoV-2에 특징적인 N 단백질 유전자, 또는 Rdrp 유전자에 선택적으로 결합할 수 있는 프라이머 세트를 이용할 수 있다. 이러한 프라이머 세트를 사용하는 경우, 각 세트의 정방향 프라이머 또는 역방향 프라이머 중 어느 하나는 예를 들어, FAM, HEX, 또는 Cy5가 결합한 형태이고, 다른 프라이머는 비오틴으로 결합된 것이다. 시료 내에 상기 프라이머 세트가 특이적으로 결합할 수 있는 표적 핵산이 존재하는 경우, 반응패드 (140)에서 증폭되고, 제2연결패드(150)를 통과하면서 증폭된 핵산에 결합된 비오틴이 금 나노입자의 스트렙타비딘과 특이적으로 결합한다. 측방 흐름에 의해 금 나노입자와 결합한 증폭된 핵산은 검출패드(160)로 이동하고, 증폭된 핵산의 다른 측에 결합된 검출자가 상기 검출패드(160)에 고정된 FAM, HEX, 또는 Cy5에 특이적으로 결합할 수 있는 수용체에 결합하면서 검출 패드의 검출 구역을 분홍색으로 색변화 시킨다.To detect SARS-CoV-2, a set of primers capable of selectively binding to the N protein gene characteristic of SARS-CoV-2 or the Rdrp gene can be used. In the case of using such a primer set, one of the forward or reverse primers of each set is, for example, FAM, HEX, or Cy5 bound, and the other primer is biotin bound. When a target nucleic acid to which the primer set can specifically bind exists in the sample, it is amplified in the reaction pad 140, and biotin bound to the amplified nucleic acid while passing through the second connection pad 150 is gold nanoparticles. It binds specifically to streptavidin. The amplified nucleic acid bound to the gold nanoparticle moves to the detection pad 160 by lateral flow, and the detector bound to the other side of the amplified nucleic acid is specific for FAM, HEX, or Cy5 immobilized on the detection pad 160. The color of the detection area of the detection pad is changed to pink while binding to the receptor that can be bound to it.
검출패드에 표적 핵산이 결합하는 원리를 도식화하면 도 3과 같다.The principle of binding of the target nucleic acid to the detection pad is illustrated in FIG. 3 .
신뢰도를 높이기 위해 검출패드에 추가의 검출 구역을 형성한 후, SARS-CoV-2와 교차검출 가능성이 높은 바이러스에 특징적인 유전자에 선택적으로 결합하는 프라이머 세트를 도입하여 이를 음성대조군으로서 함께 검출할 수 있다.In order to increase reliability, after forming an additional detection zone on the detection pad, a primer set that selectively binds to a gene characteristic of a virus with a high possibility of cross-detection with SARS-CoV-2 can be introduced and detected together as a negative control. there is.
시험예 A-3. 금 입자를 포함하는 구조물을 이용한 혈액 시료로부터 핵산 추출 및 증폭Test Example A-3. Nucleic Acid Extraction and Amplification from Blood Samples Using Structures Containing Gold Particles
(1)(One) 시험 시료 및 페이퍼 칩의 준비Preparation of test samples and paper chips
본 발명의 랩온페이퍼 핵산 검출용 구조물을 이용하여 혈액 시료로부터 핵산을 추출하고 이를 증폭하여 색변화를 관찰함으로써 진단용으로 활용할 수 있는지 확인하였다.Nucleic acid was extracted from a blood sample using the lab-on-paper nucleic acid detection construct of the present invention, amplified, and color change was observed to confirm whether it could be used for diagnosis.
먼저 인간 혈액을 전혈로서 Innovative research (IWB1K2E10ML, USA)에서 구입하여 준비하고, 양성 대조군인 18S rRNA의 프라이머는 Tocris(# 7325, USA)에서 구입하였다. 사용된 전혈은 일체의 바이러스나 박테리아에 감염된 것이 아님을 data sheet를 통해 확인하였다. 상기 인간 혈액 100㎕에 siTOOLs Biotech 사의 SARS-CoV-2 positive control 0.1pg/㎕를 1㎕를 혼합하여 1 개의 시험 시료를 준비하였다. First, human blood was purchased and prepared as whole blood from Innovative Research (IWB1K2E10ML, USA), and 18S rRNA primers as a positive control were purchased from Tocris (# 7325, USA). It was confirmed through the data sheet that the whole blood used was not infected with any virus or bacteria. One test sample was prepared by mixing 1 μl of 0.1 pg/μl of SARS-CoV-2 positive control from siTOOLs Biotech with 100 μl of the human blood.
혈액에 혼합된 SARS-CoV-2의 검출을 위한 프라이머 세트는 하기의 표 3과 같다. Primer sets for detecting SARS-CoV-2 mixed in blood are shown in Table 3 below.
유전자gene 서열번호sequence number 프라이머 유형Primer type 서열order
SARS-CoV-2의
N 유전자
of SARS-CoV-2
N gene
1One F3 F3 5'-ACCGAAGAGCTACCAGACGA-3'5′-ACCGAAGAGCTACCAGACGA-3′
22 B3 B3 5'-CTGCGTAGAAGCCTTTTGGC-3'5′-CTGCGTAGAAGCCTTTTGGC-3′
33 FIP FIP 5'-TCCAGCTTCTGGCCCAGTTCCTTTTTATTCGTGGTGGTGACGGTAA-3'5′-TCCAGCTTCTGGCCCAGTTCCTTTTTATTCGTGGTGGTGACGGTAA-3′
44 BIPBIP 5'-TATGGGTTGCAACTGAGGGAGCCTTTTTTCATTGTTAGCAGGATTGCGGG-3'5'-TATGGGTTGCAACTGAGGGAGCCTTTTTTCATTGTTAGCAGGATTGCGGG-3'
55 FL FL 5'-ACCATCTTGGACTGAGATCTTTC-3' 5′-ACCATCTTGGACTGAGATCTTTC-3′
66 BL BL 5'-TACACCAAAAGATCACATTGGCA-3'5′-TACACCAAAAGATCACATTGGCA-3′
※ F3, forward primer; B3, backward primer; FIP, forward inner primer; BIP, backward inner primer; FL, forward loop primer; BL, backward loop primer상기 각 프라이머 세트에서 F3 프라이머는 5' 말단에 검출자를 결합하였고, B3 프라이머 5' 말단에는 비오틴을 결합하였다. 양성대조군인 인간 18s RNA (A)는 FAM, 및 N 유전자(B)는 Cy5를 검출자로서 도입하였다.※ F3, forward primer; B3, backward primer; FIP, forward inner primer; BIP, backward inner primer; FL, forward loop primer; BL, backward loop primer In each of the above primer sets, the F3 primer bound a detector to the 5' end, and the B3 primer bound biotin to the 5' end. As a positive control, human 18s RNA (A) was introduced with FAM, and N gene (B) with Cy5 as a detector.
(2) 시료로부터 핵산 검출(2) Detecting nucleic acids from samples
상기 시험 시료와 SARS-CoV-2를 혼합하지 않은 음성대조군 시료를 각각 50㎕ 취하여 Lysis buffer (20mM Tris·HCl (pH 8.8), 15mM MgSO4, 15mM KCl, 15mM (NH4)2SO4, 0.1 w/w% Tween20, 0.05 mg/ml 단백질분해효소 (Protenase K)) 50㎕를 첨가하고 가볍게 탭핑(tapping)한 후, 5분 정도 상온에서 인큐베이션 하였다. 그런 다음, 시료패드(120) 하부의 가열패드(141)를 60℃로 작동시킨 상태에서 실시예 2에서 제조된 핵산 검출용 구조물의 시료패드(120)에 5 분 내로 천천히 적가하고, 부가 완충액 (20 mM Tris-HCl, 10 mM (NH4)2SO4, 50 mM KCl, 2 mM MgSO4, 0.1% Tween®20, pH 8.8) 250㎕를 시료패드에 약 2 분 동안 천천히 적가한 후, 반응패드 하부의 가열패드를 60℃로 가열하여 30분 간 반응시켰다. 그런 다음, 제2연결패드와 개시자패드 하부의 가열패드를 65℃로 가열하고 상기 부가 완충액 250㎕을 버퍼패드에 2 분 동안 천천히 적가하고 검출 패드에 표시되는 검출 구역의 색변화를 관찰하였다. 50 μl of each of the test sample and the negative control sample in which SARS-CoV-2 was not mixed was added to Lysis buffer (20 mM Tris HCl (pH 8.8), 15 mM MgSO 4 , 15 mM KCl, 15 mM (NH 4 ) 2 SO 4 , 0.1 50 μl of w/w% Tween20, 0.05 mg/ml proteinase K) was added and lightly tapped, followed by incubation at room temperature for about 5 minutes. Then, with the heating pad 141 under the sample pad 120 operated at 60° C., it was slowly added dropwise to the sample pad 120 of the structure for detecting nucleic acids prepared in Example 2 within 5 minutes within 5 minutes, and the addition buffer ( 250 μl of 20 mM Tris-HCl, 10 mM (NH 4 ) 2 SO 4 , 50 mM KCl, 2 mM MgSO 4 , 0.1% Tween®20, pH 8.8) was slowly added dropwise to the sample pad for about 2 minutes, followed by reaction. The heating pad under the pad was heated to 60 °C and reacted for 30 minutes. Then, the heating pad under the second connection pad and the initiator pad was heated to 65° C., and 250 μl of the addition buffer was slowly added dropwise to the buffer pad for 2 minutes, and the color change of the detection zone displayed on the detection pad was observed.
그 결과, 도 13과 같이 시험 시료 및 음성대조군에서 모두 양성대조군(A) 밴드를 확인함으로써 구조물이 정상적으로 작동함을 확인하였고, 시험 시료에서는 SARS-CoV-2 (B)가 검출된 반면, 음성대조군 시료에서는 양성대조군(A) 만 관찰되어 본 발명의 시스템을 이용하여 다수의 질병, 바이러스 또는 균 감염 여부를 진단할 수 있음을 확인하였다.As a result, as shown in FIG. 13, it was confirmed that the structure worked normally by confirming the band of the positive control group (A) in both the test sample and the negative control group, and SARS-CoV-2 (B) was detected in the test sample, while the negative control group In the sample, only the positive control group (A) was observed, confirming that multiple diseases, viral or fungal infections can be diagnosed using the system of the present invention.
시험예 A-4. 형광 기반 구조물을 이용한 혈액 시료로부터 바이러스 검출Test Example A-4. Virus detection from blood samples using fluorescence-based constructs
본 발명의 랩온페이퍼 핵산 검출용 구조물을 이용하여 혈액 시료로부터 핵산을 추출하고 이를 증폭하여 형광을 측정함으로써 SARS-CoV-2와 같은 바이러스 감염 여부를 쉽게 측정할 수 있다.Virus infection such as SARS-CoV-2 can be easily measured by extracting nucleic acid from a blood sample using the lab-on-paper nucleic acid detection structure of the present invention, amplifying the nucleic acid, and measuring fluorescence.
SARS-CoV-2를 검출하기 위해 SARS-CoV-2에 특징적인 N 단백질 유전자, 또는 Rdrp 유전자에 선택적으로 결합할 수 있는 프라이머 세트를 이용할 수 있다. 이러한 프라이머 세트를 사용하는 경우, 정방향 프라이머 또는 역방향 프라이머 중 어느 하나는 형광 표지자로 표지되고, 그에 따른 SARS-CoV-2에 감염된 환자의 시료에서 관찰할 수 있다. 검출 패드에 표적 핵산이 결합하는 원리를 도식화하면 도 4와같다.To detect SARS-CoV-2, a set of primers capable of selectively binding to the N protein gene characteristic of SARS-CoV-2 or the Rdrp gene can be used. When using such a primer set, either the forward primer or the reverse primer is labeled with a fluorescent marker, and thus can be observed in a sample of a patient infected with SARS-CoV-2. The principle of binding of the target nucleic acid to the detection pad is illustrated in FIG. 4 .
신뢰도를 높이기 위해 검출패드에 추가의 웰을 형성한 후, SARS-CoV-2와 교차검출 가능성이 높은 바이러스에 특징적인 유전자에 선택적으로 결합하는 프라이머 세트를 도입하여 이를 음성대조군으로서 함께 검출할 수 있다.After forming additional wells on the detection pad to increase reliability, a primer set that selectively binds to a gene characteristic of a virus that is highly likely to cross-detect SARS-CoV-2 can be introduced and detected together as a negative control. .
시험예 A-5. 형광 기반 구조물을 이용한 혈액 시료로부터 핵산 추출 및 증폭Test Example A-5. Extraction and amplification of nucleic acids from blood samples using fluorescence-based constructs
(1)(One) 시험 시료 및 페이퍼 칩의 준비Preparation of test samples and paper chips
본 발명의 랩온페이퍼 핵산 검출용 구조물을 이용하여 혈액 시료로부터 핵산을 추출하고 이를 증폭하여 형광을 나타낼 수 있는지 확인하였다.Nucleic acids were extracted from blood samples using the construct for detecting nucleic acids in lab-on-paper of the present invention, and nucleic acids were amplified to confirm whether they could exhibit fluorescence.
먼저 인간 혈액을 전혈로서 Innovative research (IWB1K2E10ML, USA)에서 구입하여 준비하고, 양성 대조군인 18S rRNA의 프라이머는 Tocris(# 7325, USA)에서 구입하였다. 사용된 전혈은 일체의 바이러스나 박테리아에 감염된 것이 아님을 data sheet를 통해 확인하였다. 상기 인간 혈액에 보렐리아 아프젤리(B. Afzelii) 를 각각 1*102 cell/ml로 혼합하여 1 개의 시험 시료를 준비하였다. First, human blood was purchased and prepared as whole blood from Innovative Research (IWB1K2E10ML, USA), and 18S rRNA primers as a positive control were purchased from Tocris (# 7325, USA). It was confirmed through the data sheet that the whole blood used was not infected with any virus or bacteria. One test sample was prepared by mixing Borrelia Afzelii with the human blood at 1*10 2 cell/ml, respectively.
혈액에 혼합된 균주의 검출을 위한 프라이머 세트와 음성 대조군으로 사용된 및 보렐리아 부르그도르페리(B. Burgdorferi)의 프라이머 세트는 하기의 표 4와 같다. 본 시험예에서 사용된 프라이머는 염기서열 데이터베이스인 Genebank로부터 획득된 데이터에 기반하여 시료 내 존재하는 인간의 18sRNA 및 상기 혼합된 균주에 선택적으로 결합하는 것으로 설계된 것이다.Primer sets for detection of strains mixed with blood and primer sets used as negative control and Borrelia burgdorferi (B. Burgdorferi) are shown in Table 4 below. The primers used in this test example were designed to selectively bind to human 18sRNA present in the sample and the mixed strain based on data obtained from Genebank, a nucleotide sequence database.
프라이머
유형
primer
type
서열order
프라이머 세트 1
(B.afzelii 표적)
Primer Set 1
(B.afzelii target)
F3F3 5'-GGTATACTGACAGCAGCTT-3' 5'-GGTATACTGACAGCAGCTT-3'
B3 B3 5'-CTTGCAGCTTAATAATAGCCTT-3' 5′-CTTGCAGCTTAATAATAGCCTT-3′
FIPFIP 5'-GTTGCTCGGTCCTCCATGTTTAAATTTTTAATGTTATCCGTGATATGGTTCCGA-3' 5'-GTTGCTCGGTCCTCCATGTTTAAATTTTTAATGTTATCCGTGATATGGTTCCGA-3'
BIPBIP 5'-GGATTTCGTATCAATTTTGGAGGCATTTTAAGTTACAAAGGTCCCATTGC-3' 5'-GGATTTCGTATCAATTTTGGAGGCATTTTAAGTTACAAAGGTCCCATTGC-3'
FL FL 5'-ATAAGGCCTTCGGTATTG-3' 5′-ATAAGGCCTTCGGTATTG-3′
BLBL 5'-ATTCTACGTTCCGATTCTCAGTAT-3' 5′-ATTCTACGTTCCGATTCTCAGTAT-3′
프라이머 세트 2
(B.burgdorferi 표적)
Primer Set 2
(B.burgdorferi target)
F3 F3 5'-AGAGCAGCTGAGGAGCT-3' 5′-AGAGCAGCTGAGGAGCT-3
B3B3 5'-CTTCCAGTTGAACACCATCTT-3' 5′-CTTCCAGTTGAACACCATCTT-3′
FIPFIP 5'-AAGTCCACGACGGTTGAGACCTTTTTGCAGCCTGCTTAAATTAACA-3' 5'-AAGTCCACGACGGTTGAGACCTTTTTGCAGCCTGCTTAAATTAACA-3'
BIP BIP 5'-GAGCAAACGAAGTTGAAGCTATTTTAGCCTGAGCAGTTAGAGC-3' 5′-GAGCAAACGAAGTTGAAGCTATTTTAGCCTGAGCAGTTAGAGC-3
FLFL 5'-TGAATGCGATCCTGGT-3' 5′-TGAATGCGATCCTGGT-3′
BLBL 5'-TATGGAGCTAATGTTGCTAAT-3'5′-TATGGAGCTAATGTTGCTAAT-3′
※ F3, forward primer; B3, backward primer; FIP, forward inner primer; BIP, backward inner primer; FL, forward loop primer; BL, backward loop primer상기 각 프라이머 세트에서 F3 프라이머는 5' 말단에 형광 표지자를 결합하였고, 양성대조군인 인간 18s RNA (A)는 FAM, 보렐리아 아프젤리(B)는 HEX, 및 보렐리아 부르그도르페리(C)를 표적하는 프라이머는 Cy5로 표지하였다.※ F3, forward primer; B3, backward primer; FIP, forward inner primer; BIP, backward inner primer; FL, forward loop primer; BL, backward loop primer In each primer set, the F3 primer bound a fluorescent marker to the 5' end, and the positive control human 18s RNA (A) was FAM, Borrelia afzeli (B) was HEX, and Borrelia burgdor Primers targeting Perry (C) were labeled with Cy5.
(2)(2) 시료로부터 핵산 검출Nucleic acid detection from samples
상기 시료 50㎕를 취하여 Lysis buffer (20mM Tris·HCl (pH 8.8), 15mM MgSO4, 15mM KCl, 15mM (NH4)2SO4, 0.1 w/w% Tween20, 0.05 mg/ml 단백질분해효소 (Protenase K)) 50㎕를 첨가하고 가볍게 탭핑(tapping)한 후, 5분 정도 상온에서 인큐베이션 하였다. 그런 다음, 시료패드(120) 하부의 가열패드(141)를 60℃로 작동시킨 상태에서 실시예 3에서 제조된 핵산 검출용 구조물의 시료패드(120)에 5 분 내로 천천히 적가하고, 부가 완충액 (20 mM Tris-HCl, 10 mM (NH4)2SO4, 50 mM KCl, 2 mM MgSO4, 0.1% Tween®20, pH 8.8) 250㎕를 시료패드에 약 2 분 동안 천천히 적가한 후, 반응패드 하부의 가열패드를 60℃로 가열하여 30분 간 반응시켰다. 그런 다음, 개시자패드 및 제2연결패드 하부의 가열패드를 65℃로 가열하고 상기 부가 완충액 250㎕을 버퍼패드에 2 분 동안 천천히 적가하였다. Lysis buffer (20mM Tris HCl (pH 8.8), 15mM MgSO 4 , 15mM KCl, 15mM (NH 4 ) 2 SO 4 , 0.1 w/w% Tween20, 0.05 mg/ml protease (Protenase K)) 50 μl was added and lightly tapped, followed by incubation at room temperature for about 5 minutes. Then, with the heating pad 141 under the sample pad 120 operated at 60° C., it was slowly added dropwise to the sample pad 120 of the structure for detecting nucleic acid prepared in Example 3 within 5 minutes, and the addition buffer ( 250 μl of 20 mM Tris-HCl, 10 mM (NH 4 ) 2 SO 4 , 50 mM KCl, 2 mM MgSO 4 , 0.1% Tween®20, pH 8.8) was slowly added dropwise to the sample pad for about 2 minutes, followed by reaction. The heating pad under the pad was heated to 60 °C and reacted for 30 minutes. Then, heating pads below the initiator pad and the second connection pad were heated to 65° C., and 250 μl of the buffer solution was slowly added dropwise to the buffer pad for 2 minutes.
그런 다음, 인식부(200)인 400 내지 700 λ의 파장대 광원 필터가 세팅된 Chemi-Doc XRS + 이미징 시스템 (Bio-Rad Laboratories, Hercules, CA, USA)을 이용하여 각 형광 표지자에 해당하는 파장대에서 형광 이미지를 획득하고, 상기 형광 이미지로부터 형광을 측정하여 형광 세기를 최대 값 1 및 최소값 0으로 하고 그에 대한 비율로 산출한 후 하기의 식에 따라 형광세기 값을 구하였다. Then, using a Chemi-Doc XRS + imaging system (Bio-Rad Laboratories, Hercules, CA, USA) with a light source filter for the wavelength band of 400 to 700 λ, which is the recognition unit 200, is used in the wavelength band corresponding to each fluorescent marker. A fluorescence image was acquired, fluorescence was measured from the fluorescence image, the fluorescence intensity was set to a maximum value of 1 and a minimum value of 0, and the fluorescence intensity value was obtained according to the following formula after calculating the ratio thereto.
형광 세기 값 = I-IN Fluorescence intensity value = II N
(IN: 음성대조군의 형광 세기, I : 시료의 형광 세기)(I N : Fluorescence intensity of negative control group, I: Fluorescence intensity of sample)
검출된 형광세기 값에 기반하여 도출된 유의성 값을 산출하면 하기의 표 5와 같다.Calculating the significance value derived based on the detected fluorescence intensity value is shown in Table 5 below.
형광 세기 값fluorescence intensity value 유의성 값significance value
AA 0.780.78 90% 이상의 확률의 양성 (정상작동)More than 90% probability positive (normal operation)
BB 0.730.73 90% 이상의 확률의 양성More than 90% probability positive
CC 0.050.05 음성voice
상기 유의성 값에 기반하여 판단하면, 양성대조군(A)로서 구조물은 정상적으로 작동하였고, 보렐리아 아프젤리(B)에 감염되었고, 및 보렐리아 부르그도르페리(C)에는 감염되지 않는다는 결과가 출력된다. 이는 제조된 시료에서 의도한 바와 동일하여, 본 발명의 시스템을 이용하여 다수의 질병, 바이러스 또는 균 감염 여부를 진단할 수 있음을 확인하였다.Judging based on the significance value, the construct as a positive control (A) worked normally, was infected with Borrelia afzeli (B), and was not infected with Borrelia burgdorferi (C). This is the same as intended in the prepared sample, and it was confirmed that a number of diseases, viral or fungal infections can be diagnosed using the system of the present invention.
[B: 코로나바이러스 검출용 프라이머에 대한 평가][B: Evaluation of primers for detecting coronavirus]
상기 [표 3]에 개시한 프라이머 6개의 프라이머 세트에 대한 효과를 확인하기 위하여 페이퍼 구조물 외에 별도로 하기의 같은 실험을 진행하였다.In order to confirm the effect of the primer set of 6 primers disclosed in [Table 3], the following experiment was conducted separately in addition to the paper structure.
실시예 B-1: 유전자 증폭 확인Example B-1: Confirmation of gene amplification
상기 6 개의 프라이머 세트는 하기의 표 2와 같은 조성과 농도의 primer mix 로서 제조하여 준비하였다. 준비된 SARS-CoV-2 주형 RNA 1㎕에 10x primer mix 2㎕, Colorimetric LAMP 2X Master Mix 10㎕ (WarmStart®, BioLabs Inc.), 및 dH2O 7㎕을 혼합하여 총 20㎕가 되도록 혼합액을 제조하였다. 대조군에는 주형 RNA를 넣지 않았다. 상기 혼합액은 하기의 표 3과 같은 조성을 갖는다. 제조된 혼합액을 65 ℃에서 30 분 동안 등온증폭반응한 후, 반응을 멈추고 육안으로 색 변화를 확인하였다.The six primer sets were prepared and prepared as a primer mix of the composition and concentration shown in Table 2 below. 1μl of prepared SARS-CoV-2 template RNA was mixed with 2μl of 10x primer mix, 10μl of Colorimetric LAMP 2X Master Mix (WarmStart ® , BioLabs Inc.), and 7μl of dH 2 O to prepare a mixture of 20μl in total. did No template RNA was added to the control group. The mixed solution has a composition shown in Table 3 below. After the prepared mixture was isothermally amplified at 65° C. for 30 minutes, the reaction was stopped and color change was observed with the naked eye.
프라이머primer 10X 농도(STOCK) (μM)10X concentration (STOCK) (μM) 1X 농도(FINAL) (μM)1X concentration (FINAL) (μM)
FIPFIP 16 16 1.6 1.6
BIPBIP 16 16 1.61.6
F3F3 2 2 0.20.2
B3B3 2 2 0.20.2
FLFL 4 4 0.40.4
BLBL 4 4 0.40.4
RNA 표적 검출군RNA target detection group 대조군control group
Colorimetric LAMP 2X Master MixColorimetric LAMP 2X Master Mix 10㎕10 μl 10㎕10 μl
Primer Mix(10X)Primer Mix (10X) 2㎕2 μl 2㎕2 μl
표적 RNAtarget RNA 1㎕1 μl 00
dH2OdH2O 7㎕7 μl 8㎕8 μl
총 부피total volume 20㎕20 μl 20㎕20 μl
실시예 B-2: 분석능 평가Example B-2: Analytical Performance Evaluation
상기 실시예 B-1의 primer mix를 이용하여 본 발명에 따른 프라이머 세트의 분석능을 평가하였다.The analysis ability of the primer set according to the present invention was evaluated using the primer mix of Example B-1.
구체적으로, siTOOLs Biotech 사의 SARS-CoV-2 positive control을 사용하여 5pg, 10pg, 20pg, 및 40pg으로 연속 희석하여 각 농도마다 1 ㎕를 준비하였다. 여기에 상기 실시예 2와 같이 등온증폭반응을 실시하였다. 이후 색 변화를 육안으로 확인하고 전기영동을 통해 그 결과를 확인하였다. 반응산물의 색이 분홍색으로 남아있으면 음성으로, 노란색으로 변하였으면 양성으로 판단하였다. 또한 전기영동으로 증폭 결과물을 직접 확인하였다.Specifically, using SARS-CoV-2 positive control from siTOOLs Biotech, 5pg, 10pg, 20pg, and 40pg were serially diluted to prepare 1 μl for each concentration. Here, an isothermal amplification reaction was performed as in Example 2 above. Thereafter, the color change was visually confirmed and the result was confirmed through electrophoresis. When the color of the reaction product remained pink, it was judged negative, and when it turned yellow, it was judged positive. In addition, the amplification result was directly confirmed by electrophoresis.
그 결과, 도 14에 나타낸 바와 같이 시험된 모든 농도에서 반응산물의 색이 노란색으로 변하였고, 도 15에 나타낸 바와 같이 전기영동으로 증폭 결과물을 존재함을 확인할 수 있었다.As a result, as shown in FIG. 14, the color of the reaction product turned yellow at all concentrations tested, and as shown in FIG. 15, it was confirmed that the amplification product was present by electrophoresis.
실시예 B-3: 분석 특이도 평가Example B-3: Evaluation of assay specificity
상기 실시예 B-1의 primer mix를 이용하여 본 발명에 따른 프라이머 세트의 SARS-CoV-2에 대한 분석 특이도를 평가하였다. 본 평가를 위한 비교군으로 인간 코로나 바이러스 OC43(HCoV OC43), 인간 코로나 바이러스 229E(HCoV 229E), 메르스 바이러스 (MERS-CoV), 및 인플루엔자 바이러스 (H1N1) 유전자를 통상적인 방법으로 합성하고, T7 전사를 통해 바이러스 RNA를 제작하였다. 제작된 RNA를 통상적인 방법으로 순수분리하고, 이를 주형으로 사용하였다. SARS-CoV-2 및 상기 비교군 각각에 대해 10^6개 수준의 주형을 사용하여 상기 실시예 B-1과 같이 증폭 반응을 위한 혼합액을 준비하고, 등온증폭반응 하였다. 반응 산물의 색 변화를 육안으로 확인하고, 전기영동으로 다시 검증하였다. 그 결과 비교군에서 색 변화가 관찰되지 않고, SARS-CoV-2에 대해서만 노란색으로의 색 변화가 관찰되어, 본 발명의 프라이머 세트는 SARS-CoV-2에 대한 특이성이 있음을 확인하였다.The assay specificity for SARS-CoV-2 of the primer set according to the present invention was evaluated using the primer mix of Example B-1. As a comparative group for this evaluation, human coronavirus OC43 (HCoV OC43), human coronavirus 229E (HCoV 229E), MERS virus (MERS-CoV), and influenza virus (H1N1) genes were synthesized by a conventional method, and T7 Viral RNA was produced by transcription. The prepared RNA was purified in a conventional manner and used as a template. For each of SARS-CoV-2 and the control group, 10^6 templates were used to prepare a mixed solution for the amplification reaction as in Example B-1, and subjected to an isothermal amplification reaction. The color change of the reaction product was visually confirmed and verified again by electrophoresis. As a result, no color change was observed in the comparison group, and a color change to yellow was observed only for SARS-CoV-2, confirming that the primer set of the present invention has specificity for SARS-CoV-2.
실시예 B-4: 분석능 평가Example B-4: Analytical Performance Evaluation
상기 실시예 B-1의 primer mix를 이용하여 본 발명에 따른 프라이머 세트의 분석능을 평가하였다.The analysis ability of the primer set according to the present invention was evaluated using the primer mix of Example B-1.
구체적으로 siTOOLs Biotech 사의 SARS-CoV-2 positive control 5pg을 주형으로 사용하여 실시예 B-1과 같이 증폭 반응을 위한 혼합액을 준비하고, 5분 간격으로 5분, 10분, 15분, 20분, 25분, 및 30분 동안 등온증폭반응을 실시하였다. 이후 색 변화를 육안으로 확인하고 전기영동을 통해 그 결과를 확인하였다. 반응산물의 색이 분홍색으로 남아있으며 음성으로, 노란색으로 변하였으면 양성으로 판단하였다. Specifically, using 5 pg of SARS-CoV-2 positive control from siTOOLs Biotech as a template, a mixture for the amplification reaction was prepared as in Example B-1, and 5 minutes, 10 minutes, 15 minutes, 20 minutes, Isothermal amplification reactions were performed for 25 minutes and 30 minutes. Thereafter, the color change was visually confirmed and the result was confirmed through electrophoresis. The color of the reaction product remained pink and was judged as negative, and when it changed to yellow, it was judged as positive.
그 결과, 도 16에 나타낸 바와 같이 30 분 후 반응산물의 색이 노란색으로 변하였고, 도 17에 나타낸 바와 같이 전기영동으로 증폭 결과물을 존재함을 확인할 수 있었다.As a result, as shown in FIG. 16, the color of the reaction product turned yellow after 30 minutes, and as shown in FIG. 17, it was confirmed that the amplification product was present by electrophoresis.
실시예 B-5: 가상 검체를 이용한 분석능 평가Example B-5: Evaluation of analytical performance using virtual specimens
실제 임상 검체에서 분석이 가능할지 평가하기 위해, 가상의 검체를 제조하여 본 발명에 따른 프라이머 세트의 분석능을 평가하였다.In order to evaluate whether analysis can be performed on actual clinical specimens, virtual specimens were prepared to evaluate the analytical ability of the primer set according to the present invention.
구체적으로, COVID-19 병력이나 COVID-19 환자와 접촉한 적이 없고, COVID-19를 의심할 만한 증상을 나타내지 않았으며, 2 주 내에 해외 방문 이력이 없는 건강한 사람 5명의 비인두와 구인두에서 검체를 채취하고 각 검체를 두 개의 시료로 나눈 후, 하나의 시료에만 SARS-CoV-2 positive control 5 pg을 혼합하여 가상의 환자 검체로 하였다. 상기 열 개의 시료를 이용하여 실시예 B-1과 같이 증폭 반응을 위한 혼합액을 준비하고 등온증폭반응을 실시하였다.Specifically, samples were taken from the nasopharynx and oropharynx of 5 healthy people who had no history of COVID-19 or contact with patients with COVID-19, did not show any symptoms suggestive of COVID-19, and did not have a history of overseas travel within 2 weeks. After collecting and dividing each sample into two samples, only one sample was mixed with 5 pg of SARS-CoV-2 positive control to make a virtual patient sample. An isothermal amplification reaction was performed after preparing a mixed solution for an amplification reaction as in Example B-1 using the ten samples.
그 결과, 도 18에 나타낸 바와 같이 가상의 환자 검체에서만 반응산물의 색이 노란색으로 변하고, 나머지 시료에서는 색이 변하지 않았다. 따라서, 실제 임상 검체에서도 우수한 분석능을 발휘할 수 있음을 확인하였다.As a result, as shown in FIG. 18, the color of the reaction product changed to yellow only in the virtual patient sample, and the color did not change in the other samples. Therefore, it was confirmed that excellent analytical performance can be exhibited even in actual clinical specimens.
실시예 B-6: 방해물질을 이용한 분석능 평가Example B-6: Analytical performance evaluation using interfering substances
실제 임상 검체에 혼합될 수 있는 다양한 생체 성분과, 검체의 정제에 유입될 수 있는 용매 등에 의해 분석능이 영향을 받을 수 있는지 확인하였다.It was confirmed whether the analytical performance could be affected by various biocomponents that could be mixed with actual clinical specimens and solvents that could flow into the purification of specimens.
상기 실시예 6에서 채취하여 제조한 10 개의 검체 중 임의의 9 개의 검체를 세 개의 군으로 나눈 후, 하나의 군에는 방해물질을 처리하지 않고, 두 개의 군에 각각 0.1 v/v%의 인간 혈액 및 0.1 v/v%의 에탄올을 혼합하였다. 그런 다음, 각 검체를 세 개의 시료로 나누어 두 개의 시료만 실시예 B-5와 같이 가상의 환자 검체로 제조하고, 증폭 반응을 위한 혼합액을 준비하고 등온증폭반응을 실시하였다.After randomly dividing 9 samples out of 10 samples collected and prepared in Example 6 into three groups, one group was not treated with interfering substances, and each of the two groups was treated with 0.1 v/v% human blood. and 0.1 v/v% ethanol. Then, each specimen was divided into three samples, and only two samples were prepared as virtual patient specimens as in Example B-5, and a mixed solution for an amplification reaction was prepared and an isothermal amplification reaction was performed.
그 결과, 도 19에 나타낸 바와 같이 세 개의 군 모두 가상의 환자 검체에서만 반응산물의 색이 노란색으로 변하고, 나머지 시료에서는 색이 변하지 않았다. 따라서, 분석능에 영향을 미칠 가능성이 있는 환경에서도 우수한 분석능을 발휘할 수 있음을 확인하였다.As a result, as shown in FIG. 19, in all three groups, the color of the reaction product changed to yellow only in the virtual patient sample, and the color did not change in the other samples. Therefore, it was confirmed that excellent analytical performance can be exhibited even in an environment that may affect analytical performance.
따라서 상기 실험을 통하여 상기 프라이머 세트는 본 발명의 페이퍼 구조물에 한정되는 것이 아니며, 우수한 분석능을 나타낼 수 있다는 점을 알 수 있다.Therefore, through the above experiments, it can be seen that the primer set is not limited to the paper structure of the present invention and can exhibit excellent analytical performance.
[부호의 설명][Description of code]
110: 하우징 구조물110: housing structure
120: 시료패드; 121: 버퍼패드120: sample pad; 121: buffer pad
131: 제1연결패드; 132: 개시자패드131: first connection pad; 132: initiator pad
140: 반응패드; 141: 가열패드; 142: 차단패드140: reaction pad; 141: heating pad; 142: blocking pad
150: 제2연결패드150: second connection pad
160: 검출패드; 161: 검출구역160: detection pad; 161: detection area
170: 흡수패드170: absorbent pad
200: 인식부200: recognition unit
210: 출력부210: output unit
SEQUENCE LISTING SEQUENCE LISTING
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Claims (9)

  1. 생물학적 시료를 수용하는 시료패드; a sample pad accommodating a biological sample;
    상기 시료패드의 상부에 배치되고, 시료패드와 반응패드를 연결하는 제1연결패드; a first connection pad disposed above the sample pad and connecting the sample pad and the reaction pad;
    상기 제1연결 패드의 하부에 배치되고, 표적 핵산과 특이적으로 결합할 수 있는 프라이머와 등온증폭반응 (LAMP)을 위한 시약을 포함하고, 등온증폭반응이 일어나는 반응패드; a reaction pad disposed under the first connection pad, containing a primer capable of specifically binding to a target nucleic acid and a reagent for isothermal amplification (LAMP), and wherein the isothermal amplification reaction occurs;
    상기 반응패드 상부에 배치되고, 반응온도 유지 및 시료의 증발을 차단하는 차단패드; a blocking pad disposed above the reaction pad to maintain the reaction temperature and block evaporation of the sample;
    상기 반응패드의 상부에 배치되고, 금 나노입자가 고정되어 있는 제2연결패드; a second connection pad disposed above the reaction pad and to which gold nanoparticles are fixed;
    상기 제2연결패드의 하부에 배치되고, 상기 금 나노입자와 결합된 등온증폭반응물로부터 증폭된 표적 핵산을 획득하는 검출패드; 및 a detection pad disposed under the second connection pad and obtaining target nucleic acid amplified from an isothermal amplification reaction coupled with the gold nanoparticles; and
    상기 검출패드의 측방에 배치되고, 잔류하는 시료를 흡수하는 흡수패드를 포함하고, 상기 시료패드, 반응패드, 및 제2연결패드 하부에 배치된 가열패드를 포함하고,An absorption pad disposed on the side of the detection pad and absorbing the remaining sample, and a heating pad disposed under the sample pad, the reaction pad, and the second connection pad,
    서열번호 1 내지 6의 염기서열 중 어느 하나로 표시되는 프라이머를 포함하는, 코로나바이러스를 검출하기 위한 프라이머 세트가 적용된A primer set for detecting coronavirus, including a primer represented by any of the nucleotide sequences of SEQ ID NOs: 1 to 6, is applied.
    코로나바이러스 검출용 랩온 페이퍼Lab-on-paper for detecting coronavirus
  2. 생물학적 시료를 수용하는 시료패드;a sample pad accommodating a biological sample;
    상기 시료패드와 분리되어 배치되고, 재수화(rehydration) 완충액을 수용하는 버퍼패드;a buffer pad disposed separately from the sample pad and accommodating a rehydration buffer;
    상기 시료패드의 상부에 배치되고, 시료패드와 반응패드를 연결하는 제1연결패드;a first connection pad disposed above the sample pad and connecting the sample pad and the reaction pad;
    상기 버퍼패드의 상부에 배치되고, 버퍼패드와 반응패드를 연결하는 개시자 (opener) 패드;an initiator pad disposed above the buffer pad and connecting the buffer pad and the reaction pad;
    상기 제1연결패드 및 개시자패드의 하부에 배치되고, 표적 핵산과 특이적으로 결합할 수 있는 프라이머와 등온증폭반응 (LAMP)을 위한 시약을 포함하고, 등온증폭반응이 일어나는 반응패드; a reaction pad disposed below the first connection pad and the initiator pad, including a primer capable of specifically binding to a target nucleic acid and a reagent for an isothermal amplification reaction (LAMP), wherein an isothermal amplification reaction occurs;
    상기 반응패드 상부에 배치되고, 반응온도 유지 및 시료의 증발을 차단하는 차단패드;a blocking pad disposed above the reaction pad to maintain the reaction temperature and block evaporation of the sample;
    상기 반응패드의 상부에 배치되고, 금 나노입자가 고정되어 있는 제2연결패드; a second connection pad disposed above the reaction pad and to which gold nanoparticles are fixed;
    상기 제2연결패드의 하부에 배치되고, 상기 금 나노입자와 결합된 등온증폭반응물로부터 증폭된 표적 핵산을 획득하는 검출패드; 및a detection pad disposed under the second connection pad and obtaining target nucleic acid amplified from an isothermal amplification reaction coupled with the gold nanoparticles; and
    상기 검출패드의 측방에 배치되고, 잔류하는 시료를 흡수하는 흡수패드를 포함하고,An absorption pad disposed on a side of the detection pad and absorbing the remaining sample;
    상기 시료패드, 개시자패드, 반응패드, 및 제2연결패드 하부에 배치된 가열패드를 포함하는, 코로나바이러스 검출용 랩온 페이퍼Lab-on-paper for detecting coronavirus, including a heating pad disposed below the sample pad, the initiator pad, the reaction pad, and the second connection pad
  3. 청구항 2에 있어서, 상기 시료패드 및 제1연결패드패드와 버퍼패드 및 개시자패드; 반응패드; 제2연결패드; 검출패드; 및 흡수패드는 순차적으로 적어도 일부분 접촉되어 측방으로 배치된 것인, 코로나바이러스 검출용 랩온 페이퍼.The method according to claim 2, wherein the sample pad, the first connection pad pad, the buffer pad and the initiator pad; reaction pad; a second connection pad; detection pad; and the absorbent pads are sequentially disposed at least partially in contact with each other, and the lab-on-paper for detecting coronavirus.
  4. 청구항 2에 있어서, 상기 검출패드는 복수 개의 구분된 검출 구역을 포함하는 것인, 코로나바이러스 검출용 랩온 페이퍼.The lab-on-paper for detecting coronavirus according to claim 2, wherein the detection pad includes a plurality of separated detection zones.
  5. 청구항 2에 있어서, 상기 금 나노입자는 표면에 스트렙타비딘(streptavidin)을 포함하는 것인, 코로나바이러스 검출용 랩온 페이퍼.The lab-on-paper for detecting coronavirus according to claim 2, wherein the gold nanoparticles contain streptavidin on the surface.
  6. 청구항 2에 있어서, 상기 시료패드에 적용된 시료는 흡수 패드까지 측방으로 이동하는 것인, 코로나바이러스 검출용 랩온 페이퍼.. The lab-on-paper for detecting coronavirus according to claim 2, wherein the sample applied to the sample pad moves laterally to the absorbent pad.
  7. 생물학적 시료를 수용하는 시료패드; a sample pad accommodating a biological sample;
    상기 시료패드의 상부에 배치되고, 시료패드와 반응패드를 연결하는 제1연결패드; a first connection pad disposed above the sample pad and connecting the sample pad and the reaction pad;
    상기 제1연결 패드의 하부에 배치되고, 표적 핵산과 특이적으로 결합할 수 있는 프라이머와 등온증폭반응 (LAMP)을 위한 시약을 포함하고, 등온증폭반응이 일어나는 반응패드; a reaction pad disposed under the first connection pad, containing a primer capable of specifically binding to a target nucleic acid and a reagent for isothermal amplification (LAMP), and wherein the isothermal amplification reaction occurs;
    상기 반응패드 상부에 배치되고, 반응온도 유지 및 시료의 증발을 차단하는 차단패드; a blocking pad disposed above the reaction pad to maintain the reaction temperature and block evaporation of the sample;
    상기 반응패드의 상부에 배치되고, 상기 등온증폭반응물을 운반하는 제2연결패드; a second connection pad disposed above the reaction pad and transporting the isothermal amplification reactant;
    상기 제2연결패드의 하부에 배치되고, 상기 등온증폭반응물로부터 증폭된 표적 핵산을 획득하는 검출패드;a detection pad disposed below the second connection pad and obtaining target nucleic acid amplified from the isothermal amplification reaction;
    상기 검출패드의 측방에 배치되고, 잔류하는 시료를 흡수하는 흡수패드;an absorption pad disposed on a side of the detection pad and absorbing the remaining sample;
    상기 검출패드의 상부에 배치되고, 상기 검출패드로부터 형광 이미지를 획득하고, 형광 세기를 측정하는 인식부; 및a recognition unit disposed above the detection pad, acquiring a fluorescence image from the detection pad, and measuring fluorescence intensity; and
    상기 인식부로부터 측정된 형광 세기 값을 이용하여 유의성 값을 도출하고, 상기 유의성 값으로 부터, 질병, 바이러스 또는 균 감염 여부를 출력하는 출력부를 포함하고, 상기 시료패드, 반응패드, 및 제2연결패드 하부에 배치된 가열패드를 포함하고, An output unit for deriving a significance value using the fluorescence intensity value measured by the recognition unit and outputting whether or not a disease, virus or fungus infection is present from the significance value, and the sample pad, reaction pad, and second connection Including a heating pad disposed under the pad,
    상기 프라이머는 서열번호 1 내지 6의 염기서열 중 어느 하나로 표시되는The primer is represented by any one of the nucleotide sequences of SEQ ID NOs: 1 to 6
    코로나바이러스 감염 진단 시스템.Coronavirus infection diagnostic system.
  8. 생물학적 시료를 수용하는 시료패드;a sample pad accommodating a biological sample;
    상기 시료패드와 분리되어 배치되고, 재수화(rehydration) 완충액을 수용하는 버퍼패드;a buffer pad disposed separately from the sample pad and accommodating a rehydration buffer;
    상기 시료패드의 상부에 배치되고, 시료패드와 반응패드를 연결하는 제1연결패드;a first connection pad disposed above the sample pad and connecting the sample pad and the reaction pad;
    상기 버퍼패드의 상부에 배치되고, 버퍼패드와 반응패드를 연결하는 개시자 (opener) 패드;an initiator pad disposed above the buffer pad and connecting the buffer pad and the reaction pad;
    상기 제1연결패드 및 개시자 패드의 하부에 배치되고, 표적 핵산과 특이적으로 결합할 수 있는 프라이머와 등온증폭반응 (LAMP)을 위한 시약을 포함하고, 등온증폭반응이 일어나는 반응패드;a reaction pad disposed below the first connection pad and the initiator pad, including a primer capable of specifically binding to a target nucleic acid and a reagent for an isothermal amplification reaction (LAMP), wherein an isothermal amplification reaction occurs;
    상기 반응패드의 상부에 배치되고, 상기 등온증폭반응물을 운반하는 제2연결패드; a second connection pad disposed above the reaction pad and transporting the isothermal amplification reactant;
    상기 제2연결패드의 하부에 배치되고, 상기 등온증폭반응물로부터 증폭된 표적 핵산을 획득하는 검출패드;a detection pad disposed below the second connection pad and obtaining target nucleic acid amplified from the isothermal amplification reaction;
    상기 검출패드의 측방에 배치되고, 잔류하는 시료를 흡수하는 흡수패드;an absorption pad disposed on a side of the detection pad and absorbing the remaining sample;
    상기 검출패드의 상부에 배치되고, 상기 검출패드로부터 형광 이미지를 획득하고, 형광 세기를 측정하는 인식부; 및a recognition unit disposed above the detection pad, acquiring a fluorescence image from the detection pad, and measuring fluorescence intensity; and
    상기 인식부로부터 측정된 형광 세기 값을 이용하여 유의성 값을 도출하고, 상기 유의성 값으로 부터, 질병, 바이러스 또는 균 감염 여부를 출력하는 출력부를 포함하고,An output unit for deriving a significance value using the fluorescence intensity value measured by the recognition unit and outputting whether or not a disease, virus or bacterial infection is present from the significance value,
    상기 시료패드, 개시자 패드, 반응패드, 및 제2연결패드 하부에 배치된 가열패드를 포함하고,A heating pad disposed below the sample pad, the initiator pad, the reaction pad, and the second connection pad,
    상기 프라이머는 서열번호 1 내지 6의 염기서열 중 어느 하나로 표시되는The primer is represented by any one of the nucleotide sequences of SEQ ID NOs: 1 to 6
    코로나바이러스 감염 진단 시스템.Coronavirus infection diagnostic system.
  9. 청구항 8에 있어서, 상기 시료패드 및 제1연결패드와 버퍼패드 및 개시자패드; 반응패드; 제2연결패드; 검출패드; 및 흡수패드는 순차적으로 적어도 일부분 접촉되어 측방으로 배치된 것인, 질병, 바이러스 또는 균 감염 진단 시스템.The method according to claim 8, wherein the sample pad, the first connection pad, the buffer pad and the initiator pad; reaction pad; a second connection pad; detection pad; and the absorbent pads are sequentially disposed at least partially in contact with each other, the system for diagnosing a disease, virus or fungus infection.
PCT/KR2021/014712 2021-07-21 2021-10-20 Lab-on-paper for detection of coronavirus and coronavirus diagnosis system using same WO2023003082A1 (en)

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KR1020210095332A KR102370572B1 (en) 2021-07-21 2021-07-21 Colorimetric diagnostic method using strutcture with opener-based paper chip for controlling lateral flow that can diagnose multiple nucleic acids in one step
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