WO2023003137A1

WO2023003137A1 - Composition for cell lysis to be applied to structure including lab-on-paper chip

Info

Publication number: WO2023003137A1
Application number: PCT/KR2022/005857
Authority: WO
Inventors: 김종철
Original assignee: 주식회사 에이아이더뉴트리진
Priority date: 2021-07-21
Filing date: 2022-04-25
Publication date: 2023-01-26
Also published as: KR102310223B9; KR102310223B1

Abstract

The present invention relates to a composition for cell lysis which improves lateral fluidity and is to be applied to a structure including a lab-on-paper chip. More specifically, the present invention relates to a composition for cell lysis suitable for application to an integrated system having lateral fluidity, wherein the integrated system can simultaneously purify and detect nucleic acids immediately without purifying nucleic acids from a sample.

Description

Cell lysis composition for application to structures including lab-on-paper chips

The present invention relates to a cell lysis composition for improving lateral fluidity and applying to a structure including a lab-on-paper chip, and more particularly, to a composition for lateral fluidity capable of simultaneously purifying and detecting nucleic acids directly without purifying nucleic acids from a sample. It relates to a composition for cell lysis suitable for application to an all-in-one system.

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.

One aspect provides a cell lysis composition comprising a buffering component, potassium chloride, magnesium sulfate, ammonium sulfate, protease K, and a surfactant.

Another aspect provides a method for detecting nucleic acids by mixing the composition for cell lysis and a sample and then applying the mixture to a nucleic acid detection device.

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

The composition for cell lysis of the present invention enables nucleic acid detection by lateral flow method by applying a sample without including the step of separately purifying the sample, and isothermal amplification reaction required in the process from application of the sample to detection of nucleic acid. This is to improve problems that may occur when mixed with a buffer solution, an additional buffer solution, and the like.

The cell lysis composition (Lysis buffer) may contain Tris (tris (hydroxymethyl) aminomethane) at 5 mM to 80 mM, 5 mM to 50 mM, or 10 mM to 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 migration 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 disturb 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 a proteolytic enzyme at 0.01 mg/ml to 0.1 mg/ml, or 0.03 mg/ml to 0.07 mg/ml. 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, and may be included in an amount of 0.01 w/w% to 0.2 w/w%, preferably 0.05 w/w% to 0.1 w/w%, based on the weight of the cell lysis composition. there is.

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.

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 moved. 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 without including glycerol or a reducing agent, and can transfer nucleic acid material laterally to the reaction pad in high yield.

The composition for cell lysis is sufficient to lyse cells simply by mixing with the sample and reacting for 1 minute to 5 minutes after simply tapping, but in order to increase the efficiency of the cell lysis reaction, the composition is heated at 60 to 80 ° C for 1 minute to 5 minutes. It can be heated for 2 minutes. The heating reaction may be directly performed by a general method such as water bath or incubation after mixing with the sample, or heating may be performed by placing a heating pad 141 below the sample pad 140 .

In one embodiment, the cell lysis composition of the present invention may be suitable for application to a lateral flow type nucleic acid detection device.

In another embodiment, the cell lysis composition of the present invention may be suitable for application to a lab-on-paper chip nucleic acid detection device.

The nucleic acid detection device to which the cell lysis composition of the present invention is applied is based on lab-on-paper chip technology, and the nucleic acid material is purified while moving to the reaction pad without separate nucleic acid purification, and the nucleic acid material is purified immediately for the amplification reaction. It can be applied, and a plurality of target nucleic acids can be simultaneously detected and related diseases can be diagnosed by applying one sample. To realize this, the nucleic acid detection structure of the present invention includes a sample pad 120, a first connection pad 131, a switch pad 132, a reaction pad 140, a heating pad 141, and a blocking pad 142 , a second connection pad 150, a detection pad 160, an absorption pad 170, a detection area 161, and a housing 110 as components.

Hereinafter, each component will be described in detail.

시료패드sample 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 buffer, and, if necessary, after mixing with the cell lysis buffer, it may be further purified by a commonly known method such as centrifugation, filtration, or 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 cell lysis buffer.

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.

제1연결패드1st connection pad

The first connection pad 131 partially contacts the sample pad and is disposed on the top, and connects the sample pad and the reaction pad as a structure having a relatively narrow width compared to the sample pad.

The cell lysate transferred from the sample pad can be rapidly transferred to the reaction pad containing primers and reagents for isothermal amplification within 5 minutes after being dropwise added to the sample.

The first connection pad may be made of a cellulose membrane and have pores of 0.005 μm to 0.015 μm.

반응패드reaction pad

The reaction pad 140 is a component corresponding to a paper chip in lab-on-paper, and isothermal amplification reagents including dNTPs, DNA polymerases, reverse transcriptases, fluorescent markers, and isothermal amplification buffers for amplification reactions are fixed thereto. Therefore, the reaction pad is permeated with a solution containing a nucleic acid material by the additional buffer applied to the sample pad, and the contact material between the isothermal amplification reaction reagent and the sample moves to the reaction pad and is heated by the heating pad below 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 may be mixed in a reaction pad and then dried or applied to the surface of a reaction pad in a powder type, for example, It may be fixed by heating in an oven at about 35 to 40 ° C. for about 30 minutes.

The reaction pad 140 may partially come into contact with the first connection pad and may be disposed below and to the side of the first connection 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 65° C. for 20 minutes to 1 hour, preferably for 20 minutes to 30 minutes. The heating pad 141 may be disposed only on the reaction pad.

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. An 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 fluorescent markers selected from the group consisting of IRDye. The fluorescent marker may be labeled differently for each target nucleic acid in order to independently detect the target nucleic acid.

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 the second connection pad, binds to streptavidin on the surface of the gold particle and is detected when captured on the detection pad Make the results visible. 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.

제2연결패드2nd 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 reaction pad. The second connection pad 150 includes gold nanoparticles, and the gold nanoparticles combine with the nucleic acid 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, flax, paper, nitrocellulose, glass fiber, polyacrylic, polynitrile, polypiperazine, polyamide, polyethersulfone, polyvinylidene fluoride so that the amplified nucleic acid can easily move to the detection pad. A porous material made of lead, polyethyleneimine, polydimethylsiloxane, or a mixture thereof, may have a pore size of 0.01 μm to 0.05 μm, preferably 0.05 μm.

The second connection pad may have a cellulose material and may be coated with low melting point agarose or wax.

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 or wells are formed on one reaction pad, and target nucleic acids as many as the number of detection zones formed by applying a sample once can be detected. 6 is for explaining in detail the structure of the detection zone formed on the reaction pad, and is 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.

The sample pad, the first connection 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 solution is not lost, and the entire structure may be defined as a housing. It may be surrounded by a support or a case made of a non-porous material.

The absorbent pad absorbs a sample and a buffer solution to block reverse flow and contribute to induce lateral fluidity. Absorbent pads are porous materials, such as cotton, flax, paper, nitrocellulose, cellulose acetate, glass fiber, polyacrylic, polynitrile, polypiperazine, polyamide, polyethersulfone, polyvinylidene fluoride, polyethyleneimine, and polydimethyl It may be a siloxane or a mixture thereof. The absorbent pad is preferably a glass fiber and may have a pore size of 0.1 to 0.5 μm.

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.

Hereinafter, a method of detecting a nucleic acid using the structure for detecting a nucleic acid will be described.

A sample for which nucleic acid is to be detected may include mixing with a cell lysis buffer prior to application to the sample pad. When mixed with the cell lysis buffer, the nucleic acid material present in the cells can be eluted, and thus the amount of the nucleic acid material detectable in the sample can 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%, the cell lysate mixed with the cell lysis composition having a pH of 8.0 to 9.0 may be added dropwise, and an additional buffer may be added. The addition buffer serves to purify the nucleic acid material while allowing the nucleic acid material to move to the reaction pad. The addition buffer serves to purify the nucleic acid material while allowing the nucleic acid material to move to the conversion pad. Since the addition buffer 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 more specifically 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 below 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.

By using the cell lysis composition of the present invention, the lateral fluidity of the sample can be improved and the detection reaction can occur more efficiently in a structure including a lab-on-paper chip.

If the nucleic acid detection device to which the cell lysis composition of the present invention is applied is used, sample preparation, nucleic acid amplification reaction, and detection are not separately performed, and the sample is applied once to detect. can be done at once. 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.

Figure 2 is a graph showing the comparison of LAMP results according to the type of cell lysis composition.

FIG. 3 is a diagram illustrating the principle of obtaining target nucleic acids from the detection pad 160.

4 is a side structural diagram of a part of the structure for detecting multiple nucleic acids of the present invention.

5 is a perspective view of a part of the structure for detecting multiple nucleic acids of the present invention.

6 is an enlarged structural diagram of the structure of the detection pad 160 .

7 is an example showing the appearance of a structure for detecting multiple nucleic acids of the present invention surrounded by a case.

7 is an example showing the result of detecting SARS-CoV-2 from a blood sample.

The present invention contains 5 mM to 80 mM Tris-HCl, 5 mM to 50 mM potassium chloride, 1 mM to 30 mM magnesium sulfate, 5 mM to 50 mM ammonium sulfate, 0.01 mg/ml to 0.1 mg/ml protease, and an interface. The acidity of the composition for cell lysis containing 0.01w/w% to 0.2w/w% of TritonX-100 or Tween20 as an activator is pH 8.0 to 9.0, and relates to a composition for cell lysis.

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.

Preparation 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·HCl15mM MgSO₄ 15mM KCl 15mM (NH₄)₂SO₄ 0.1 w/w% Tween2020 mM Tris HCl 15 mM MgSO ₄ 15 mM KCl 15 mM (NH ₄ ) ₂ SO ₄ 0.1 w/w% Tween20	0.05 mg/ml0.05 mg/ml	8.8 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

Preparation Example 2. Preparation of Lab-on-Paper Structure for Detecting Nucleic Acids

In order to prepare the lab-on-paper structure for detecting nucleic acids of the present invention, a 0.5 μm polysulfone film was used as the sample pad 120, a 0.01 μm cellulose film was used as the first connection pad 131, and a 0.005 μm cellulose acetate film was used as the reaction pad 140. , A 0.05 μm cellulose film was prepared as the second connection pad 150, a 0.005 μm nitrocellulose film as the detection pad 160, and a 0.5 μm glass fiber film as the absorption pad 170.

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 fluorescent marker such as FAM, HEX, and Cy5 thereon, and NHS (N- hydroxysulfosuccinimide) solution was applied and reacted to fix the antibody.

Then, the components of each structure are arranged as shown in FIG. 1, a copper plate connected to a heating pad 141 is placed at the bottom of the reaction pad 140, and a blocking pad 142 is placed on the top of the reaction pad. A polyacrylic membrane was placed on the

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. 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 (see Figure 1). 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. 2).

Test Example 2. Virus detection using blood samples

Virus infection such as SARS-CoV-2 can be easily measured by extracting nucleic acid from a blood sample using the lab-on-paper nucleic acid detection structure of the present invention, amplifying the nucleic acid, and measuring fluorescence.

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.

Test Example 3. Nucleic acid extraction and amplification using blood samples

(1) Preparation of test samples and paper chips

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 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) Detecting nucleic acids 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, while the heating pad 141 was operated at 60° C., it was slowly added dropwise to the sample pad 120 of the structure for detecting nucleic acid prepared in Preparation Example 2, and added buffer (20 mM Tris-HCl, 10 mM ( 500 μl of NH ₄ ) ₂ SO ₄ , 50 mM KCl, 2 mM MgSO ₄ , 0.1% Tween® 20, pH 8.8) was slowly added dropwise over 1 to 3 minutes, followed by reaction for 30 minutes. Then, the color change of the detection area displayed on the detection pad was observed.

As a result, as shown in FIG. 8, it was confirmed that the structure worked normally by confirming the positive control (A) band 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.

[Description of code]

110: housing structure

120: sample pad

131: first connection pad

140: reaction pad

141: heating pad

142: blocking pad

150: second connection pad

160: detection pad

170: absorbent pad

161: detection area

Claims

5 mM to 80 mM Tris-HCl, 5 mM to 50 mM potassium chloride, 1 mM to 30 mM magnesium sulfate, 5 mM to 50 mM ammonium sulfate, 0.01 mg/ml to 0.1 mg/ml protease, and TritonX as a surfactant -100 or Tween20 0.01 w / w% to 0.2 w / w%,

The acidity of the composition for cell lysis is pH 8.0 to 9.0, the composition for cell lysis.
The method according to claim 1, 10 mM to 50 mM Tris-HCl, potassium chloride 10 mM to 20 mM, magnesium sulfate 2 mM to 16 mM, ammonium sulfate 10 mM to 20 mM, protease 0.03 mg / ml to 0.07 mg / ml, and as a surfactant 0.05w/w% to 0.1w/w% of TritonX-100 or Tween20, a composition for cell lysis.
The method according to claim 1, wherein the composition for cell lysis is a composition for cell lysis suitable for application to a lateral flow type nucleic acid detection device.
The method according to claim 1, wherein the composition for cell lysis is a cell lysis composition suitable for application to a lab-on-paper chip (lab-on-paper chip) nucleic acid detection device.
The method according to claim 1, wherein the cell lysis composition for cell lysis composition characterized in that it does not contain glycerol.
The method according to claim 1, wherein the cell lysis composition is cell lysis composition characterized in that it does not contain a reducing agent.
The method according to claim 1, wherein the composition for cell lysis and the sample and the composition for cell lysis to be used in a volume ratio of 1: 1.
Mixing the cell lysis composition of claim 1 and the sample;

applying the mixture to a sample pad;

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® to the sample pad. Adding dropwise an addition buffer solution containing 20 to TritonX-100 and having an acidity of pH 8.0 to 9.0; and

A method for detecting nucleic acids comprising heating a reaction pad to 60 to 65° C. and incubating for 20 minutes to 1 hour.