WO2023003070A1

WO2023003070A1 - Paper chip capable of one-step diagnosis of multiple nucleic acids

Info

Publication number: WO2023003070A1
Application number: PCT/KR2021/011223
Authority: WO
Inventors: 김종철
Original assignee: 주식회사 에이아이더뉴트리진
Priority date: 2021-07-21
Filing date: 2021-08-23
Publication date: 2023-01-26
Also published as: WO2023003082A1

Abstract

The present invention relates to a structure capable of simultaneously diagnosing multiple diseases by applying a single sample and a system for diagnosing a disease or viral or bacterial infection, comprising same, in which, by applying lab-on-paper technology, the steps of sample preparation, isothermal amplification, detection and analysis can be carried out on a single chip, and by moving the sample in a lateral flow manner, the sample is finally connected to genetic big data related to a disease and strain infection, and whether or not there is a disease or strain infection can be directly determined.

Description

Paper chip capable of one-step multiple nucleic acid diagnosis

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.

As the level of medical service increases and diagnostic equipment develops, technology is being developed that can quickly and easily measure infectious microorganisms that threaten human health, such as new viruses, super bacteria, tuberculosis, and food poisoning bacteria. . Molecular diagnosis that can measure them often requires a diagnostic method with excellent sensitivity and specificity, special equipment or reagents, or involves a complicated process. Immunodiagnostic methods are quick and easy to reproduce with a kit, but have a problem in that the measurement sensitivity is low.

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.

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.

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.

In one aspect, 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.

In one embodiment, in the structure for detecting multiple nucleic acids, 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 pad disposed under the sample pad, initiator pad, reaction pad, and second connection pad. A detection structure is provided.

In one embodiment, in the structure for detecting multiple nucleic acids, 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.

In one embodiment, the detection pad of the structure for detecting multiple nucleic acids may include a plurality of distinct detection zones.

In one embodiment, the gold nanoparticles may contain streptavidin on their surfaces.

In one embodiment, 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

In one embodiment, the sample applied to the sample pad of the structure for detecting multiple nucleic acids may move laterally to an absorption pad.

In one embodiment, 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. mM, 0.01 mg/ml to 0.1 mg/ml of protease, and 0.01 w/w% to 0.2 w/w% of TritonX-100 or Tween20 as a surfactant and mixed with a composition for cell lysis to detect multiple nucleic acids. dragon structure.

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.

In one embodiment, 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 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 system for diagnosing a disease, virus, or fungal infection, including a heating pad disposed below the connection pad.

In one embodiment, in the disease, virus or fungal infection diagnosis system, 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, 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 under the second connection pad.

In one embodiment, in the system, 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.

In one embodiment, the reaction pad of the system may have a blocking pad disposed thereon.

In one embodiment, the detection pad of the system may include a plurality of divided wells.

In one embodiment, 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. .

In one embodiment, the sample applied to the sample pad of the system may move laterally to the absorbent pad.

In one embodiment, 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;

obtaining a fluorescence image of the fluorescent marker bound to the nucleic acid amplification product and measuring fluorescence intensity; determining a significance value using the measured fluorescence intensity value of each target nucleic acid; Provided is an information providing method for diagnosing a virus or fungal infection, outputting whether or not a disease, virus or fungus infection is present from the significance value of each target nucleic acid.

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

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

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.

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.

Hereinafter, each component will be described in detail.

sample pad

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.

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. 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.

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.

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.

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.

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.

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.

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.

1st connection pad

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 μm to 0.015 μm.

buffer pad

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). 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.

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.

initiator pad

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 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.

A heating pad 141 for heating may be disposed under 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.

reaction pad

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.

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 external air.

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.

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 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.

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.

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.

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 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

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.

2nd connection pad

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.

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.

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.

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. 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. .

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.

detection pad

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. 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.

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.

heating 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.

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.

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.

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.

recognition unit

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 group" to the output unit. .

output

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 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. When the fluorescence intensity difference is less than 0.3, 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. 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.

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.

Hereinafter, a method for detecting nucleic acids using the structure for detecting nucleic acid and a system including the structure will be described.

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.

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.

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 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). 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.

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.

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.

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 entire process is simplified, and various samples or devices are not required, and it can be easily performed even without a related technician.

In addition, since it does not require a complicated device for performing each step, it is not restricted by location, has the advantage of easy distribution, and is economical because it is possible to detect a large number of nucleic acids by applying one sample.

1 is an example of the overall structure of the construct for detecting multiple nucleic acids of the present invention.

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.

4 is a diagram illustrating a principle of obtaining target nucleic acids from the detection pad 160 in the system according to one embodiment.

5 is a side structural diagram of a part of a structure for detecting multiple nucleic acids including gold particles according to an embodiment.

6 is a side structural diagram of a structure for detecting multiple nucleic acids included in a system according to one embodiment.

7 is a perspective view of a portion of a structure for detecting multiple nucleic acids according to one embodiment.

8 is a perspective view of a portion of a structure for detecting multiple nucleic acids according to another embodiment.

9 is an enlarged structural view of the structure of the detection pad 160 in the structure including gold particles according to one embodiment.

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.

12 is a graph comparing LAMP results according to the type of cell lysis composition.

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.

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.

제조예 1. 세포 용해 조성물(Lysis buffer)의 준비Preparation Example 1. Preparation of cell lysis composition (Lysis buffer)

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·HCl 15mM MgSO₄ 15mM KCl 15mM (NH₄)₂SO₄ 0.1 w/w% Tween2020mM 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	8.8 8.8
비교예 1Comparative Example 1	50mM Tris-HCl 100mM NaCl 1mM DTT 5% 글리세롤50 mM Tris-HCl 100mM NaCl 1 mM DTT 5% glycerol	0.05 mg/ml0.05 mg/ml	7.0 7.0
비교예 2Comparative Example 2	50mM Tris-HCl 150mM NaCl 5mM EDTA 1% NP-4050 mM Tris-HCl 150mM NaCl 5 mM EDTA 1% NP-40	0.05 mg/ml0.05 mg/ml	8.5 8.5
비교예 3Comparative Example 3	50mM Tris-HCl 10mM Na₂HPO₄ 5mM MgCl2 1% TritonX-10050 mM Tris-HCl 10 mM Na ₂ HPO ₄ 5 mM MgCl2 1% TritonX-100	0.05 mg/ml0.05 mg/ml	8.5 8.5
비교예 4Comparative Example 4	50mM Tris-HCl 10mM Na₂HPO₄ 10mM NaCl 0.5% SDS 1% TritonX-10050 mM Tris-HCl 10 mM Na ₂ HPO ₄ 10 mM NaCl 0.5% SDS 1% TritonX-100	--	9.0 9.0
비교예 5Comparative Example 5	50mM Tris-HCl 10mM Na₂HPO₄ 10mM NaCl 0.5% SDS 1% TritonX-10050 mM Tris-HCl 10 mM Na ₂ HPO ₄ 10 mM NaCl 0.5% SDS 1% TritonX-100	--	7.0 7.0

시험예 1. 세포 용해 조성물의 유동성 비교 시험Test 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. 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 유형primer type	서열order
프라이머 세트 1 (B.afzelii 표적)Primer Set 1 (B.afzelii target)	F3F3	5'-GGTATACTGACAGCAGCTT-3' 5'-GGTATACTGACAGCAGCTT-3'
	B3B3	5'-CTTGCAGCTTAATAATAGCCTT-3' 5′-CTTGCAGCTTAATAATAGCCTT-3′
	FIPFIP	5'-GTTGCTCGGTCCTCCATGTTTAAATTTTTAATGTTATCCGTGATATGGTTCCGA-3' 5'-GTTGCTCGGTCCTCCATGTTTAAATTTTTAATGTTATCCGTGATATGGTTCCGA-3'
	BIPBIP	5'-GGATTTCGTATCAATTTTGGAGGCATTTTAAGTTACAAAGGTCCCATTGC-3' 5'-GGATTTCGTATCAATTTTGGAGGCATTTTAAGTTACAAAGGTCCCATTGC-3'
	FLFL	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)	F3F3	5'-AGAGCAGCTGAGGAGCT-3' 5′-AGAGCAGCTGAGGAGCT-3′
	B3B3	5'-CTTCCAGTTGAACACCATCTT-3' 5′-CTTCCAGTTGAACACCATCTT-3′
	FIPFIP	5'-AAGTCCACGACGGTTGAGACCTTTTTGCAGCCTGCTTAAATTAACA-3' 5'-AAGTCCACGACGGTTGAGACCTTTTTGCAGCCTGCTTAAATTAACA-3'
	BIPBIP	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)	F3F3	5'-GCATCGTTGACGTCAGCT-3'5′-GCATCGTTGACGTCAGCT-3′
	B3B3	5'-TGTAGAGGCGATTGTAGC-3'5′-TGTAGAGGCGATTGTAGC-3′
	FIPFIP	5'-CCACGATTACTTGCTGGTAACTTTTGTGACGTGTTCGGTTAAGT-3'5'-CCACGATTACTTGCTGGTAACTTTTGTGACGTGTTCGGTTAAGT-3'
	BIPBIP	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 (2) isothermal amplification reaction

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.

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.

Fluorescence intensity value = II _N

(I _N : Fluorescence intensity of negative control group, I: Fluorescence intensity of sample)

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 2: 금 입자를 포함하는 랩온페이퍼 핵산 검출용 구조물의 제조Example 2: Preparation of Lab-on-Paper Structure Containing Gold Particles for Detecting Nucleic Acids

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.

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.

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.

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.

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 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.

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 3: 형광 기반 랩온페이퍼 핵산 검출용 구조물의 제조Example 3: Preparation of Fluorescence-Based Lab-on-Paper Structure for Detecting Nucleic Acids

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.

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.

시험예 2: 금 입자를 포함하는 구조물을 이용한 혈액 시료로부터 바이러스 검출Test 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.

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.

The principle of binding of the target nucleic acid to the detection pad is illustrated in FIG. 3 .

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 3. 금 입자를 포함하는 구조물을 이용한 혈액 시료로부터 핵산 추출 및 증폭Test Example 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

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.

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.

Primer sets for detecting SARS-CoV-2 mixed in blood are shown in Table 3 below.

유전자gene	프라이머 유형primer type	서열order
SARS-CoV-2의 N 유전자of SARS-CoV-2 N gene	F3F3	5'-ACCGAAGAGCTACCAGACGA-3'5′-ACCGAAGAGCTACCAGACGA-3′
	B3B3	5'-CTGCGTAGAAGCCTTTTGGC-3'5′-CTGCGTAGAAGCCTTTTGGC-3′
	FIPFIP	5'-TCCAGCTTCTGGCCCAGTTCCTTTTTATTCGTGGTGGTGACGGTAA-3'5′-TCCAGCTTCTGGCCCAGTTCCTTTTTATTCGTGGTGGTGACGGTAA-3′
	BIPBIP	5'-TATGGGTTGCAACTGAGGGAGCCTTTTTTCATTGTTAGCAGGATTGCGGG-3'5'-TATGGGTTGCAACTGAGGGAGCCTTTTTTCATTGTTAGCAGGATTGCGGG-3'
	FLFL	5'-ACCATCTTGGACTGAGATCTTTC-3' 5′-ACCATCTTGGACTGAGATCTTTC-3′
	BLBL	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 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)(2) 시료로부터 핵산 검출Nucleic acid detection from samples

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.

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 4. 형광 기반 구조물을 이용한 혈액 시료로부터 바이러스 검출Test 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.

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 .

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 5. 형광 기반 구조물을 이용한 혈액 시료로부터 핵산 추출 및 증폭Test 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.

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.

Primer sets for detecting strains mixed with blood and primer sets used as a 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 the human 18sRNA present in the sample and the mixed strain based on data obtained from Genebank, a nucleotide sequence database.

	프라이머 유형primer type	서열order
프라이머 세트 1 (B.afzelii 표적)Primer Set 1 (B.afzelii target)	F3F3	5'-GGTATACTGACAGCAGCTT-3' 5'-GGTATACTGACAGCAGCTT-3'
	B3B3	5'-CTTGCAGCTTAATAATAGCCTT-3' 5′-CTTGCAGCTTAATAATAGCCTT-3′
	FIPFIP	5'-GTTGCTCGGTCCTCCATGTTTAAATTTTTAATGTTATCCGTGATATGGTTCCGA-3' 5'-GTTGCTCGGTCCTCCATGTTTAAATTTTTAATGTTATCCGTGATATGGTTCCGA-3'
	BIPBIP	5'-GGATTTCGTATCAATTTTGGAGGCATTTTAAGTTACAAAGGTCCCATTGC-3' 5'-GGATTTCGTATCAATTTTGGAGGCATTTTAAGTTACAAAGGTCCCATTGC-3'
	FLFL	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)	F3F3	5'-AGAGCAGCTGAGGAGCT-3' 5′-AGAGCAGCTGAGGAGCT-3′
	B3B3	5'-CTTCCAGTTGAACACCATCTT-3' 5′-CTTCCAGTTGAACACCATCTT-3′
	FIPFIP	5'-AAGTCCACGACGGTTGAGACCTTTTTGCAGCCTGCTTAAATTAACA-3' 5'-AAGTCCACGACGGTTGAGACCTTTTTGCAGCCTGCTTAAATTAACA-3'
	BIPBIP	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 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

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.

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.

Fluorescence intensity value = II _N

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

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.

[Description of code]

110: housing structure

120: sample pad

121: buffer pad

131: first connection pad

132: initiator pad

140: reaction pad

141: heating pad

142: blocking pad

150: second connection pad

160: detection pad

170: absorbent pad

161: detection area

200: recognition unit

210: output unit

Claims

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

A structure for detecting multiple nucleic acids, comprising an absorption pad disposed on a side of the detection pad and absorbing remaining samples, and including a heating pad disposed below the sample pad, the reaction pad, and the second connection pad.
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 and absorbing the remaining sample;

A structure for detecting multiple nucleic acids, comprising a heating pad disposed below the sample pad, the initiator pad, the reaction pad, and the second connection pad.
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 sequentially arranged at least partially in contact with each other laterally.
The structure for detecting multiple nucleic acids according to claim 2, wherein the detection pad includes a plurality of distinct detection zones.
The structure for detecting multiple nucleic acids according to claim 2, wherein the gold nanoparticles contain streptavidin on the surface.
The method according to claim 2, wherein the reaction pad includes forward and reverse primer sets, one of the forward and reverse primers is biotin-coupled, 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 multiple nucleic acids labeled with one or more fluorescent markers selected from the group consisting of IRDye detection structure.
The structure for detecting multiple nucleic acids according to claim 2, wherein the sample applied to the sample pad moves laterally to the absorption pad.
The method according to claim 2, wherein the sample 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, protease 0.01 A structure for detecting multiple nucleic acids, which is mixed with a composition for cell lysis containing 0.01 w/w% to 0.2 w/w% of TritonX-100 or Tween20 as a surfactant.
A diagnostic kit for disease, virus or fungal infection, comprising the structure for detecting multiple nucleic acids of claim 2.
Applying a biological sample to the sample pad of the structure for detecting multiple nucleic acids of claim 2 and amplifying a target nucleic acid; and

A method for providing information for diagnosing a disease, virus or fungal infection, comprising detecting the nucleic acid amplification product on a detection pad.
The method according to claim 10, further comprising the step of adding an additional buffer dropwise to the buffer pad after applying the biological sample.
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 fungus infection is present from the significance value, and the sample pad, reaction pad, and second connection A system for diagnosing a disease, virus or fungal infection comprising a heating pad disposed under the pad.
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 bacterial infection is present from the significance value,

Including a heating pad disposed below the sample pad, the initiator pad, the reaction pad, and the second connection pad,

Disease, viral or fungal infection diagnosis system.
The method according to claim 13, 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.
The system for diagnosing a disease, virus or fungus infection according to claim 13, wherein the detection pad includes a plurality of divided wells.
The method according to claim 13, wherein the reaction pad includes forward and reverse primer sets, one of the forward and reverse primers is biotin-coupled, 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 labeled with one or more fluorescent markers selected from the group consisting of IRDye, a disease, Virus or fungal infection diagnosis system.
The system for diagnosing a disease, virus or fungus infection according to claim 13, wherein the sample applied to the sample pad moves laterally to the absorbent pad.
Applying a biological sample to the sample pad of the disease, virus or fungal infection diagnosis system of claim 13 and amplifying a target nucleic acid;

obtaining a fluorescence image of the fluorescent marker bound to the nucleic acid amplification product and measuring fluorescence intensity;

determining a significance value using the measured fluorescence intensity value of each target nucleic acid;

A method for providing information for diagnosing a virus or fungal infection, outputting whether or not a disease, virus or fungus infection is present from the significance value of each target nucleic acid.
The method according to claim 18, further comprising the step of adding an additional buffer to the buffer pad dropwise after applying the biological sample.