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WO2023003070A1 - Puce sur support papier capable de réaliser un diagnostic en une étape de multiples acides nucléiques - Google Patents

Puce sur support papier capable de réaliser un diagnostic en une étape de multiples acides nucléiques Download PDF

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Publication number
WO2023003070A1
WO2023003070A1 PCT/KR2021/011223 KR2021011223W WO2023003070A1 WO 2023003070 A1 WO2023003070 A1 WO 2023003070A1 KR 2021011223 W KR2021011223 W KR 2021011223W WO 2023003070 A1 WO2023003070 A1 WO 2023003070A1
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Prior art keywords
pad
sample
reaction
detection
nucleic acid
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PCT/KR2021/011223
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English (en)
Korean (ko)
Inventor
김종철
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AIthenutrigene Co
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AIthenutrigene Co
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Publication date
Priority claimed from KR1020210095322A external-priority patent/KR102370561B1/ko
Priority claimed from KR1020210095329A external-priority patent/KR102370566B1/ko
Priority claimed from KR1020210095332A external-priority patent/KR102370572B1/ko
Priority claimed from KR1020210095317A external-priority patent/KR102370553B1/ko
Priority claimed from KR1020210095339A external-priority patent/KR102370580B1/ko
Application filed by AIthenutrigene Co filed Critical AIthenutrigene Co
Publication of WO2023003070A1 publication Critical patent/WO2023003070A1/fr
Anticipated expiration legal-status Critical
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    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L3/00—Containers or dishes for laboratory use, e.g. laboratory glassware; Droppers
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B01—PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01L—CHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L7/00—Heating or cooling apparatus; Heat insulating devices
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6844—Nucleic acid amplification reactions
    • C12Q1/686—Polymerase chain reaction [PCR]
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02A—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE
    • Y02A50/00—TECHNOLOGIES FOR ADAPTATION TO CLIMATE CHANGE in human health protection, e.g. against extreme weather
    • Y02A50/30—Against vector-borne diseases, e.g. mosquito-borne, fly-borne, tick-borne or waterborne diseases whose impact is exacerbated by climate change

Definitions

  • the present invention relates to a structure based on a lateral fluidity integrated system capable of simultaneously diagnosing multiple diseases by applying a single sample, and has high detection sensitivity even when directly applied without purifying nucleic acids from a sample, and detects a plurality of target nucleic acids. Simultaneous detection is possible, so multiple diseases can be diagnosed easily and quickly.
  • 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.
  • 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 absorbent pad disposed at 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. to provide.
  • LAMP isothermal amplification
  • the sample pad and the first connection 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.
  • Another aspect is a sample pad for receiving 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 and absorbing the remaining sample, comprising a heating
  • 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 of the structure for detecting multiple nucleic acids may include a plurality of distinct detection zones.
  • the gold nanoparticles may contain streptavidin on their surfaces.
  • the reaction pad of the structure for detecting multiple nucleic acids includes forward and reverse primer sets, one of the forward and reverse primers is biotin-conjugated, and the other primer is Cy3, Cy5, TAMRA, or TEX. , TYE, HEX, FAM, TET, JOE, MAX, ROX, VIC, Cy3.5, Texas Red, Cy5.5, TYE, BHQ, Iowa Black RQ, and labeled with one or more fluorescent markers selected from the group consisting of IRDye may have been
  • the sample applied to the sample pad of the structure for detecting multiple nucleic acids may move laterally to an absorption pad.
  • the sample of the structure for detecting multiple nucleic acids includes 5 mM to 80 mM Tris-HCl (pH 8.0 to 9.0), 5 mM to 50 mM potassium chloride, 1 mM to 30 mM magnesium sulfate, and 5 mM to 50 mM ammonium sulfate.
  • Another aspect provides a kit for diagnosing a disease, virus or fungus infection, including the construct for detecting multiple nucleic acids.
  • Another aspect includes applying a biological sample to the sample pad of the structure for detecting multiple nucleic acids and amplifying a target nucleic acid; and detecting the nucleic acid amplification product on a detection pad, providing an information providing method for diagnosing a disease, virus or fungal infection.
  • the information providing method may further include adding a buffer solution dropwise to the buffer pad after applying the biological sample.
  • Another aspect is a sample pad for receiving a biological sample; A first connection pad disposed on top of the singi 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; 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
  • the sample pad and the first connection pad; reaction pad; a second connection pad; detection pad; and the absorbent pad may be disposed laterally in contact with at least a portion in sequence.
  • Another aspect is a sample pad for receiving 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 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,
  • the sample pad, the first connection pad, the buffer pad, the initiator pad, the reaction pad, the second connection pad, the detection pad, and the absorption pad may be surrounded by a housing structure.
  • the sample pad, the first connection pad, the buffer pad, the initiator pad, the reaction pad, the second connection pad, the detection pad, and the absorption pad may be surrounded by a housing structure.
  • reaction pad of the system may have a blocking pad disposed thereon.
  • the detection pad of the system may include a plurality of divided wells.
  • the reaction pad of the system includes forward and reverse primer sets, one of the forward and reverse primers is biotin-conjugated, and the other primer 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 fluorescent markers selected from the group consisting of IRDye. .
  • the sample applied to the sample pad of the system may move laterally to the absorbent pad.
  • the sample of the system is 5 mM to 80 mM Tris-HCl (pH 8.0 to 9.0), potassium chloride 5 mM to 50 mM, magnesium sulfate 1 mM to 30 mM, ammonium sulfate 5 mM to 50 mM, proteolysis 0.01 mg/ml to 0.1 mg/ml of enzyme, and 0.01 w/w% to 0.2 w/w% of TritonX-100 or Tween20 as a surfactant may be mixed with a composition for cell lysis.
  • Another aspect may include applying a biological sample to a sample pad of the disease, virus, or fungal infection diagnosis system and amplifying a target nucleic acid;
  • the information providing method may further include adding a buffer solution dropwise to the buffer pad after applying the biological sample.
  • 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 under 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 external 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 is 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 group" 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 associated with 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 buffer components, 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.
  • 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 entire process is simplified, and various samples or devices are not required, and it can be easily performed even without a related technician.
  • 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.
  • 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 20mM 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 HPO
  • Test Example 1 Fluidity 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.
  • 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.
  • 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 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.
  • 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 4 Virus detection from blood samples using fluorescence-based constructs
  • Virus infection such as SARS-CoV-2 can be easily measured by extracting nucleic acids from a blood sample using the lab-on-paper nucleic acid detection structure of the present invention, amplifying the nucleic acids, 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 5 Extraction and amplification of nucleic acids from blood samples using fluorescence-based structures
  • 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 detecting strains mixed with blood and primer sets used as a negative control and Borrelia burgdorferi are shown in Table 4 below.
  • the primers used in this test example were designed to selectively bind to the 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.

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Abstract

La présente invention concerne une structure capable de diagnostiquer simultanément de multiples maladies par application d'un échantillon unique et un système pour diagnostiquer une maladie ou une infection virale ou bactérienne, comprenant celle-ci, dans lequel, par application d'une technologie de laboratoire sur support papier, les étapes de préparation d'échantillon, d'amplification isotherme, de détection et d'analyse peuvent être effectuées sur une puce unique, et par déplacement de l'échantillon de manière à flux latéral, l'échantillon est finalement connecté à des mégadonnées génétiques associées à une maladie et à une infection par souche, et il est possible de déterminer directement s'il existe ou s'il n'existe pas de maladie ou d'infection par souche.
PCT/KR2021/011223 2021-07-21 2021-08-23 Puce sur support papier capable de réaliser un diagnostic en une étape de multiples acides nucléiques Ceased WO2023003070A1 (fr)

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KR10-2021-0095339 2021-07-21
KR1020210095322A KR102370561B1 (ko) 2021-07-21 2021-07-21 원스텝 다중 핵산 비색 검출이 가능한 페이퍼 칩 검출방법
KR1020210095329A KR102370566B1 (ko) 2021-07-21 2021-07-21 원스텝 형광 다중 핵산진단 방법
KR1020210095332A KR102370572B1 (ko) 2021-07-21 2021-07-21 원스텝 다중 핵산 진단이 가능한 유체흐름조절 오프너 기반 페이퍼 칩 비색진단법
KR10-2021-0095332 2021-07-21
KR10-2021-0095322 2021-07-21
KR1020210095317A KR102370553B1 (ko) 2021-07-21 2021-07-21 원스텝 다중 핵산 비색 검출이 가능한 측면유동 페이퍼 칩 검출방법
KR1020210095339A KR102370580B1 (ko) 2021-07-21 2021-07-21 원스텝 다중 핵산 진단이 가능한 유체흐름조절 오프너 기반 페이퍼 칩 구조물
KR10-2021-0095329 2021-07-21
KR10-2021-0095317 2021-07-21

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