Kit for preparing photopolymerization technology-based solution for digital polymerase chain reaction and use thereof

Abstract

An embodiment of the present invention provides: a kit for preparing a solution for digital polymerase chain reaction (PCR), the kit comprising a photopolymerizable monomer, a photoinitiator, and a fluorescent probe; and a use thereof. The kit for preparing a solution for digital polymerase chain reaction (PCR) according to the present invention continuously forms a repeating pattern of liquid portions and gel-like walls in a solution for digital polymerase chain reaction (PCR) in a light-transmitting container having a predetermined space, and thus allows the solution to be partitioned into multiple isolators, even without a microfluidic droplet splitting step. In addition, the photopolymerization-based nucleic acid detection or quantification method according to the present invention allows a solution for digital polymerase chain reaction (PCR) to be three-dimensionally partitioned, and allows fluorescence data to be simply and quickly acquired. Therefore, the photopolymerization-based nucleic acid detection or quantification method according to the present invention can implement digital PCR with improved speed, integration, precision, sensitivity, and specificity.

Description

Kit for preparing a solution for digital polymerase chain reaction based on photopolymerization technology and its use

[0001] The present invention relates to a kit for preparing a solution for digital PCR, and more particularly, to a kit for preparing a solution for digital PCR that can be partitioned into a plurality of isolates by photopolymerization, and a method for detecting or quantifying a nucleic acid and a device for detecting or quantifying a nucleic acid as uses thereof.

[0002] Digital PCR (Digital polymerase chain reaction, dPCR) is a third-generation PCR (polymerase chain reaction) technology that partitions a PCR sample into thousands to hundreds of thousands of pieces, amplifies zero or more nucleic acid molecules in each piece, and then counts the pieces that show a positive reaction to measure the amount of nucleic acid in the sample. Digital PCR (Digital polymerase chain reaction, dPCR) has the advantage of being able to calculate the absolute number of nucleic acids present in a PCR sample. However, in order to obtain accurate results through digital PCR (Digital polymerase chain reaction, dPCR), the PCR sample must be partitioned into thousands to hundreds of thousands of pieces, which leads to long work and detection times, expensive equipment, and a relatively narrow detection concentration range.

[0003] Digital PCR (Digital polymerase chain reaction, dPCR) methods are broadly classified into droplet digital PCR (ddPCR) and chamber digital PCR (cdPCR) depending on the method of fractionating PCR samples. ddPCR is a method of fractionating PCR samples using droplet microfluidics, which sequentially divides PCR samples in flowing oil to generate reaction droplets. ddPCR has several drawbacks, such as long droplet generation time, non-uniformity of the generated droplets, measurement signal errors due to combination of the generated droplets, difficulty in data acquisition due to continuous droplet movement, and risk of sample loss. cdPCR is a method of fractionating PCR samples using microchambers and microchannels manufactured using microfabrication technology, and the PCR sample is transferred and divided into thousands to tens of thousands of microchambers within a microchip using capillary phenomena, etc. The above cdPCR has several disadvantages, such as high loss of PCR samples during the fractionation process, errors in result values ​​that occur when PCR samples are not filled in some microchambers, and the number of fractions of PCR samples being limited to tens of thousands due to the number of microchambers.

[0004] As a prior art of digital PCR (Digital polymerase chain reaction, dPCR), the literature [Eleen Y. Shum et al. Anal. Chem. 2022, 94, 17868-17876] discloses a method of generating droplets using centrifugation and then counting the amplified droplets by irradiating a PCR tube with a light sheet. The prior art uses a light sheet to read the amplified signal, but the droplets are unstable and randomly distributed in three-dimensional space, which may result in errors in the results.

[0005] The present invention has been derived under the conventional technical background, and the purpose of the present invention is to provide a kit for preparing a solution for digital polymerase chain reaction (PCR) that can be partitioned into a plurality of isolates by photopolymerization.

[0006] In addition, an object of the present invention is to provide a method for detecting or quantifying nucleic acids using photopolymerization technology.

[0007] In addition, an object of the present invention is to provide a device for detecting or quantifying nucleic acids using photopolymerization technology.

[0008] To overcome the shortcomings of existing dPCR, the present inventors developed a method for analyzing nucleic acid samples with higher precision without using microfluidics technology. Specifically, the present inventors photopolymerized a digital polymerase chain reaction (PCR) solution containing nucleic acids using photolithography technology to partition the solution into a grid pattern with a repeating liquid portion and gel-like walls, performed a PCR reaction on each isolate generated by the digital polymerase chain reaction (PCR) solution partition, and then counted the isolates that showed an amplification reaction. The present inventors developed a digital PCR method based on photopolymerization technology that surpasses the integration limitations of existing dPCR, reduces errors, and improves dynamic range, accuracy, sensitivity, and specificity.

[0009]

[0010] To solve the above problem, one example of the present invention provides a kit for preparing a solution for digital polymerase chain reaction (PCR), comprising a photopolymerizable monomer, a photoinitiator, and a fluorescent probe.

[0011] The photopolymerizable monomer can form a gel-like polymer by photopolymerization. Furthermore, the digital PCR solution can be partitioned into multiple isolates by photopolymerization. The isolates comprise a gel-like wall formed by photopolymerization and a liquid portion partitioned by the wall and capable of polymerase reaction.

[0012]

[0013] In order to solve the above problem, an example of the present invention provides a method for detecting or quantifying nucleic acids based on photopolymerization, comprising the steps of: (a) preparing a light-transmitting PCR container containing a solution for digital polymerase chain reaction (PCR); (b) irradiating the light-transmitting PCR container containing the solution for digital polymerase chain reaction (PCR) with light to induce a photopolymerization reaction so that the solution for digital polymerase chain reaction (PCR) is compartmentalized into a pattern of a liquid phase portion and a gel phase wall in which the liquid phase portion and the gel phase wall are repeated, thereby generating a plurality of isolates; (c) after compartmentalizing the solution for digital polymerase chain reaction (PCR), applying heat to the light-transmitting PCR container to amplify nucleic acids present in the liquid phase portion for each isolate; and (d) after amplifying the nucleic acids, obtaining fluorescence data from the compartmentalized isolates using a fluorescence imaging technique.

[0014] The above solution for digital polymerase chain reaction (PCR) includes a sample containing a target nucleic acid, a primer, a photopolymerizable monomer, a photoinitiator, a fluorescent probe, a DNA polymerase, a deoxynucleoside triphosphate (dNTP), a metal ion donor compound, and a buffer.

[0015]

[0016] To solve the above problem, one example of the present invention provides a device for detecting or quantifying nucleic acids, including a kit for preparing a solution for digital polymerase chain reaction (PCR).

[0017] The above nucleic acid detection or quantification device may optionally further include a photolithography system or a fluorescence imaging system.

[0018] When using the kit for preparing a solution for digital polymerase chain reaction (PCR) according to the present invention, a pattern in which liquid portions and gel-like walls are repeatedly formed in a light-transmitting vessel having a predetermined space can be continuously partitioned into multiple isolates without going through a microfluidic droplet division step. In addition, when using the photopolymerization-based nucleic acid detection or quantification method according to the present invention, a solution for digital polymerase chain reaction (PCR) can be partitioned three-dimensionally, and fluorescence data can be easily and quickly acquired. Therefore, the photopolymerization-based nucleic acid detection or quantification method according to the present invention can implement digital PCR with improved speed, integration, precision, sensitivity, and specificity.

[0019] Figure 1 schematically illustrates the configuration of a light-sheet photolithography system.

[0020] Figure 2 shows the results obtained by performing the nucleic acid detection and quantification process using 1D dPCR in an embodiment of the present invention. Figure 2 (a) is an image of a microfluidic chip for 1D dPCR made of glass, (b) is a fluorescence image obtained by scanning a microfluidic channel after completing amplification of nucleic acids using a thermal cycler, (c) is an enlarged portion of the fluorescence image of (b), and (d) shows the distribution of the fluorescence signal intensity of each isolate compartmented in the microfluidic channel when amplification of nucleic acids using a thermal cycler is completed.

[0021] FIG. 3 is a schematic diagram of a microfluidic chip used in a nucleic acid detection and quantification process using 2D dPCR in an embodiment of the present invention, and an isolator of a two-dimensional grid pattern formed in a microfluidic chamber of the microfluidic chip.

[0022] Figure 4 is a result obtained by performing a nucleic acid detection and quantification process using 2D dPCR in an embodiment of the present invention.

[0023] Figure 5 is a schematic diagram illustrating a method for detecting and quantifying nucleic acids using photopolymerization-based 3D dPCR proposed in the present invention step by step.

[0024] Figure 6 illustrates a process of creating multiple isolates by three-dimensionally partitioning a polymerase chain reaction (PCR) solution contained in a predetermined container using a light-sheet photolithography system.

[0025] Figure 7 illustrates the process of acquiring fluorescence data from an isolator partitioned into a three-dimensional grid pattern using a light-sheet fluorescence microscopy (LSFM) system.

[0026] Hereinafter, the present invention will be described in detail.

[0027] The term '1D dPCR (1-Dimensional digital polymerase chain reaction)' or 'partitioning in one dimension' or 'partitioning in one-dimensional pattern' used in the present invention includes a process of partitioning a solution for digital polymerase chain reaction (PCR) into a plurality of isolates by forming a repeating pattern of a liquid portion and a gel-like wall surrounding or dividing the liquid portion in one direction (e.g., X-axis direction) in the solution for digital polymerase chain reaction (PCR).

[0028] The term '2D dPCR (2-Dimensional digital polymerase chain reaction)' or '2-dimensional partitioning' or '2-dimensional pattern partitioning' used in the present invention includes a process of partitioning a digital polymerase chain reaction (PCR) solution into a plurality of isolates by forming a repeating pattern of a liquid portion and a gel-like wall surrounding the liquid portion in two directions (e.g., X-axis direction and Y-axis direction).

[0029] The term '3D dPCR (3-Dimensional digital polymerase chain reaction)' or 'partitioning in three dimensions' or 'partitioning in a three-dimensional pattern' used in the present invention includes a process of partitioning a solution for digital polymerase chain reaction (PCR) into a plurality of isolates by forming a repeating pattern of a liquid portion and a gel-like wall surrounding the liquid portion in three directions (e.g., X-axis direction, Y-axis direction, and Z-axis direction).

[0030]

[0031] One aspect of the present invention relates to a kit for preparing a solution for digital polymerase chain reaction (PCR).

[0032] A kit for preparing a solution for digital polymerase chain reaction (PCR) according to an embodiment of the present invention comprises a photopolymerizable monomer, a photoinitiator, and a fluorescent probe. The solution for digital PCR can be partitioned into a plurality of isolates by photopolymerization. The isolates are composed of a gel-like wall formed by photopolymerization and a liquid portion partitioned by the wall and capable of polymerase reaction. In addition, the kit according to an embodiment of the present invention may preferably further include a cross-linking agent. In addition, the kit according to an embodiment of the present invention may preferably further include a polymerase chain reaction accelerator (PCR enhancer). In addition, the kit according to an embodiment of the present invention may preferably further include polymerase chain reaction (PCR) essential elements. In addition, the kit according to an embodiment of the present invention may further include a light-transmitting PCR container for accommodating the solution for digital polymerase chain reaction (PCR).

[0033] The photopolymerizable monomer serves to form a gel-like polymer wall, preferably a hydrogel wall, having a predetermined pattern in a digital polymerase chain reaction (PCR) solution when light is irradiated in a predetermined pattern onto a light-transmitting PCR container containing the digital polymerase chain reaction (PCR) solution. The digital polymerase chain reaction (PCR) solution can be divided into a plurality of liquid portions by the gel-like polymer wall. The type of the photopolymerizable monomer is not particularly limited as long as it forms a gel-like polymer that does not flow and maintains its shape by photopolymerization, and can be selected from various known photopolymerizable monomers. For example, the photopolymerizable monomer may be at least one selected from the group consisting of acrylamide, methacrylamide, gelatin acrylate, gelatin methacrylate (GelMA), ethylene glycol dimethacrylate (EGDMA), ethylene glycol diacrylate (EGDA), acrylated hyaluronic acid, and methacrylated hyaluronic acid.

[0034] The photoinitiator absorbs light energy (e.g., ultraviolet or visible light) to generate free radicals and induce a polymerization reaction of a photopolymerizable monomer. The type of the photoinitiator is not particularly limited as long as it can be easily dispersed or dissolved in water, and may be selected from various known photoinitiators. For example, the photoinitiator may be phenylbis(2,4,6-trimethylbenzoyl)phosphine oxide; 2-Hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone; Riboflavin; Eosin-Y; It may be at least one selected from the group consisting of 2,2'-azobis(2-methyl-N-(2-hydroxyethyl)propionamide); lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP); diphenyl(2,4,6-trimethylbenzoyl)phosphine oxide (TPO); and tetramethylethylenediamine (TEMED). In addition, commercial products of the photoinitiator include Irgacure series products such as Irgacure-2959, Irgacure-819, and Irgacure-500; Sinocure TPO; There is an azo photoinitiator such as VA-086. The photoinitiator is preferably provided in powder form to maintain activity.

[0035] The fluorescent probe above serves to indicate the presence or amount of a target nucleic acid by the size of a fluorescent signal. The fluorescent probe is not particularly limited in type as long as it is used to detect or quantify nucleic acids, and may be selected from a variety of known fluorescent-emitting probes. For example, the fluorescent probe may be selected from a fluorescent dye that binds to double-stranded DNA or a fluorescent substance-labeled oligonucleotide-type hydrolysis probe. The fluorescent substance-labeled oligonucleotide-type hydrolysis probe is configured to include a reporter fluorescent dye at the 5' end and a quencher dye at the 3' end, and emits a fluorescent signal by binding to a specific DNA sequence. Commercial products of the fluorescent probe include SYBR Green I dye, EvaGreen dye, SYTO-82 dye, TaqMan probe, and Amplifluor probe.

[0036] The above cross-linking agent forms a network structure by cross-linking the polymer produced by the polymerization reaction of the photopolymerizable monomer, and serves to provide mechanical strength and chemical stability to the gel-like polymer wall. When a monomer having a difunctional acrylic group or methacrylic group, or a monomer in some polymer form, is selected as the photopolymerizable monomer, the gel-like wall formed by the polymerization of the photopolymerizable monomer can have sufficient strength and chemical stability. Meanwhile, when acrylamide or methacrylamide is selected as the photopolymerizable monomer, the kit may preferably include a cross-linking agent in order to form a gel-like wall having appropriate strength and chemical stability. A kit according to an example of the present invention preferably includes a combination of a photopolymerizable monomer selected from acrylamide and methacrylamide and a cross-linking agent selected from N,N'-methylenebisacrylamide.

[0037] A solution for digital polymerase chain reaction (PCR) may contain a photopolymerizable monomer or a photoinitiator, which may reduce the amplification efficiency during the polymerase chain reaction (PCR). The polymerase reaction accelerator (PCR enhancer) serves to alleviate the decrease in amplification efficiency by increasing the activity of DNA polymerase or stabilizing DNA polymerase. The polymerase reaction accelerator (PCR enhancer) may be selected from various known additives added to increase the amplification efficiency or improve the specificity during the polymerase chain reaction (PCR). For example, the polymerase reaction accelerator (PCR enhancer) may be one or more selected from the group consisting of a nonionic surfactant, bovine serum albumin (BSA), skim milk, gelatin, casein, serum, and dimethyl sulfoxide (DMSO). The above nonionic surfactant is not particularly limited in type as long as it increases the activity of DNA polymerase, and may be selected from, for example, polyoxyethylene (20) sorbitan monolaurate, polyoxyethylene (20) sorbitan monooleate, t-octylphenoxypolyethoxyethanol, etc. Commercial products of the above nonionic surfactant include Tween 20, Tween 80, NP-40, Triton X-100, etc.

[0038] The above polymerase chain reaction (PCR) essential elements are a combination of components essential for amplification of target nucleic acids, and are composed of DNA polymerase, deoxynucleoside triphosphate (dNTP), a metal ion donor compound, and a buffer. The metal ion donor compound is not particularly limited in type as long as it provides a metal ion that acts as an essential cofactor of DNA polymerase in an aqueous solution, and for example, magnesium ion (Mg 2+ ) or manganese ion (Mn 2+ ) can be selected from compounds capable of providing a metal ion. The metal ion providing compounds include magnesium salts such as MgCl2, manganese salts such as MnCl2, etc. In addition, the polymerase chain reaction (PCR) essential element may further include a reverse transcriptase to perform reverse transcription PCR when the target nucleic acid is RNA. The polymerase chain reaction (PCR) essential element may preferably be provided in the form of a PCR master mix.

[0039] The above-mentioned light-transmitting PCR container is not particularly limited in type as long as it has a predetermined space for containing a solution for digital polymerase chain reaction (PCR) and is made of a material that allows light such as ultraviolet rays to pass through, and may be selected from, for example, a glass slide combined with an imaging spacer, a microfluidic chip, a cuvette, a small transparent plastic container, etc.

[0040]

[0041] One aspect of the present invention relates to a photopolymerization-based nucleic acid detection or quantification method.

[0042] According to one embodiment of the present invention, a method for detecting or quantifying a nucleic acid comprises the steps of: (a) preparing a light-transmitting PCR vessel containing a digital polymerase chain reaction (PCR) solution; (b) irradiating the light-transmitting PCR vessel containing the digital polymerase chain reaction (PCR) solution with light to induce photopolymerization so that the digital polymerase chain reaction (PCR) solution is compartmentalized into a pattern of a liquid phase portion and a gel phase wall in which the liquid phase portion and the gel phase wall are repeated, thereby generating a plurality of isolates; (c) after compartmentalizing the digital polymerase chain reaction (PCR) solution, applying heat to the light-transmitting PCR vessel to amplify the nucleic acid present in the liquid phase portion for each isolate; and (d) after amplifying the nucleic acid, obtaining fluorescence data from the compartmentalized isolates using a fluorescence imaging technique.

[0043] The above-described solution for digital polymerase chain reaction (PCR) comprises a sample containing a target nucleic acid, a primer, a photopolymerizable monomer, a photoinitiator, a fluorescent probe, a DNA polymerase, a deoxynucleoside triphosphate (dNTP), a metal ion donor compound, and a buffer. In addition, the solution for digital polymerase chain reaction (PCR) may optionally further comprise one or more selected from the group consisting of a cross-linking agent, a PCR enhancer, and a reverse transcriptase. The specific technical characteristics of the components constituting the solution for digital polymerase chain reaction (PCR) refer to the above-described contents.

[0044] The target nucleic acid-containing sample constituting the solution for the above digital polymerase chain reaction (PCR) may be at least one selected from the group consisting of cells, tissues, blood, serum, urine, and saliva isolated from a specimen. In addition, the target nucleic acid-containing sample is preferably a nucleic acid extract extracted from cells, tissues, blood, serum, urine, or saliva isolated from a specimen. The nucleic acid extract can be prepared using various commercial nucleic acid extraction kits. In addition, the target nucleic acid includes all types of nucleic acids to be detected. In addition, the nucleic acid includes not only single-stranded molecules but also double-stranded or triple-stranded nucleic acids, and may include not only DNA and RNA but also nucleotide analogs.

[0045] In the above digital polymerase chain reaction (PCR) solution, the concentration of the photopolymerizable monomer or the combined concentration of the photopolymerizable monomer and the cross-linking agent is not particularly limited. However, considering the mechanical strength and chemical stability of the gel wall, it is preferably 1 to 30% (w / v), and more preferably 5 to 20% (w / v). In general, a polyacrylamide gel having a concentration of 5 to 10% (w / w) is used in DNA electrophoresis to check the quality of PCR results. On the gel wall with a polyacrylamide concentration of 6% (w / v), the migration of target nucleic acids longer than 500 bp may be restricted, and on the gel wall with a polyacrylamide concentration of 10% (w / v) or more, long nucleic acid molecules are separated into their respective liquid spaces, so that crosstalk of target nucleic acids can be prevented during the digital PCR process. However, the concentration of the photopolymerizable monomer or the combined concentration of the photopolymerizable monomer and the cross-linking agent in the solution for digital polymerase chain reaction (PCR) can be selected within a wide range by considering the size of the target nucleic acid, the type of the photopolymerizable monomer, the thickness of the gel wall, etc.

[0046] The inner surface of the above-described light-transmitting PCR vessel is preferably modified by silanization treatment before containing a digital polymerase chain reaction (PCR) solution. Using a light-transmitting PCR vessel whose inner surface is modified by silanization treatment can prevent nucleic acids from adsorbing to the inner surface of the light-transmitting PCR vessel. The silanization treatment is a method of imparting hydrophobicity to the surface by replacing a hydroxyl group present on the surface with an inert alkylsiloxy. The silanization treatment covalently bonds the gel to the inner surface of the vessel, thereby preventing deformation of the gel compartment during the amplification process by thermal cycling, and minimizing diffusion of the amplified amplicon to the surroundings. The above-mentioned light-transmitting PCR container is not particularly limited in type as long as it has a predetermined space for containing a solution for digital polymerase chain reaction (PCR) and is made of a material that allows light such as ultraviolet rays to pass through, and may be selected from, for example, a glass slide combined with an imaging spacer, a chip equipped with a microfluidic channel, a chip equipped with a microfluidic chamber, a cuvette, or a small transparent plastic container.

[0047] In the step (b), the compartmentalization of the solution for digital polymerase chain reaction (PCR) can be performed by photolithography. Specifically, the compartmentalization of the solution for digital polymerase chain reaction (PCR) can be performed by a photolithography system or a light-sheet photolithography system consisting of a photomask having a predetermined pattern and an ultraviolet lamp. In the step (b), when light is irradiated in a preset pattern onto a light-transmitting PCR container containing the solution for digital polymerase chain reaction (PCR), a photopolymerization reaction occurs in the portion through which the light is transmitted, and a gel-like polymer wall (e.g., a hydrogel wall) having a width corresponding to the gap of the photomask or the thickness of the light sheet is formed. The wavelength of the light or light sheet used in the above step (b) is preferably 200 to 405 nm, more preferably 350 to 400 nm, considering the induction of a smooth photopolymerization reaction. In addition, the thickness of the light sheet used in the above step (b) is not particularly limited, and is preferably 1 to 1000 μm, more preferably 10 to 200 μm, considering the width of the gel-like wall.

[0048] In the step (b), the solution for digital polymerase chain reaction (PCR) can be partitioned in one dimension, two dimensions, or three dimensions within a light-transmitting PCR vessel. For example, when a microfluidic chip equipped with microfluidic channels is used as the light-transmitting PCR vessel, the solution for digital polymerase chain reaction (PCR) can be partitioned in a one-dimensional lattice pattern in which a liquid portion and a gel-phase wall surrounding or dividing the liquid portion are repeated in one direction (e.g., the X-axis direction). In addition, when a microfluidic chip equipped with microfluidic chambers is used as the light-transmitting PCR vessel, the solution for digital polymerase chain reaction (PCR) can be partitioned in a one-dimensional lattice pattern in which a liquid portion and a gel-phase wall surrounding the liquid portion are repeated in two directions (e.g., the X-axis direction and the Y-axis direction). Additionally, when a cuvette is used as a light-transmitting PCR container, the solution for digital polymerase chain reaction (PCR) can be partitioned into a one-dimensional grid pattern in which the liquid portion and the gel-like wall surrounding it are repeated in three directions (e.g., X-axis direction, Y-axis direction, and Z-axis direction).

[0049] In the step (b) above, when a light-sheet photolithography system is used to compartmentalize a solution for digital polymerase chain reaction (PCR), the compartments can be created one by one, or multiple compartments can be created simultaneously by applying interference lithography technology. When a light-sheet photolithography system is used to compartmentalize a solution for digital polymerase chain reaction (PCR), the size and number of compartments can be easily changed by changing the optical system, such as the slit of the optical system, the cylindrical lens, etc., and by adjusting the moving distance of the stage.

[0050] The step (c) above is preferably performed in a thermal cycler to apply heat to the light-transmitting PCR vessel at a PCR temperature profile. In addition, the process of applying heat to the light-transmitting PCR vessel in the step (c) is preferably performed in the dark with light blocked to prevent the liquid portion of the digital polymerase chain reaction (PCR) solution that has not been photopolymerized from being photopolymerized. In addition, in the step (c), it is preferable to apply heat from all directions, such as the bottom, sides, and top of the light-transmitting PCR vessel, to make the temperature distribution of the plurality of isolates uniform. In addition, the step (c) may further include a process of, after amplifying the nucleic acid, irradiating the entire light-transmitting PCR vessel with light to induce a photopolymerization reaction and fixing the amplified nucleic acid inside the isolates.

[0051] The fluorescence imaging technique used to acquire fluorescence data in the above step (d) can be implemented by a fluorescence imaging system. The type of the fluorescence imaging system is not particularly limited, and considering the ease and speed of fluorescence signal measurement, a confocal laser scanning microscope (CLSM) or a light-sheet fluorescence microscopy (LSFM) is preferable. The light-sheet fluorescence microscopy (LSFM) can capture fluorescence signal images of a three-dimensionally compartmentalized digital polymerase chain reaction (PCR) solution for each isolator layer and reconstruct the results in three dimensions. For example, in the step (b) above, when a solution for digital polymerase chain reaction (PCR) is three-dimensionally partitioned within a light-transmitting PCR vessel by a light-sheet photolithography system, acquisition of fluorescence data in the step (d) may be accomplished by irradiating a light sheet on each isolator layer composed of a plurality of isolators to obtain a fluorescence image in a two-dimensionally partitioned form for each isolator layer, and stacking the fluorescence images for each isolator layer to reconstruct them three-dimensionally.

[0052] A method for detecting or quantifying nucleic acids according to an example of the present invention may preferably further include the step of (e) calculating the concentration of the sample nucleic acid based on Poisson statistics from the acquired fluorescence data.

[0053] Using a nucleic acid detection or quantification method according to one example of the present invention, a solution for digital polymerase chain reaction (PCR) is placed in a light-transmitting PCR vessel at 10 3 ~10 8The nucleic acid detection or quantification method according to one embodiment of the present invention can be compartmentalized into isolates. In the process of compartmentalizing a solution for digital polymerase chain reaction (PCR), sample loss is very small and the homogeneity of the compartmentalized isolates is almost non-existent.

[0054]

[0055] One aspect of the present invention relates to a device for detecting or quantifying nucleic acids based on photopolymerization.

[0056] A nucleic acid detection or quantification device according to one embodiment of the present invention includes a kit for preparing a solution for the digital polymerase chain reaction (PCR) described above. In addition, the nucleic acid detection or quantification device according to one embodiment of the present invention may further optionally include a photolithography system or a fluorescence imaging system. Preferably, the photolithography system is a light-sheet photolithography system. In addition, the fluorescence imaging system may be selected from a fluorescence microscope or a confocal laser scanning microscope (CLSM). In addition, the fluorescence microscope is preferably a light-sheet fluorescence microscope (LSFM).

[0057]

[0058] Hereinafter, the present invention will be described in more detail through examples. However, the following examples are intended only to clearly illustrate the technical features of the present invention and do not limit the scope of protection of the present invention.

[0059]

[0060] 1. Preparation of a solution for polymerase chain reaction (PCR) that can be partitioned by photopolymerization

[0061] A solution for polymerase chain reaction (PCR) was prepared by adding the components listed in Table 1 below to deionized water at preset concentrations in an Eppendorf LoBind tube and mixing.

[0062] Ingredients Final concentration Acrylamide 9.5% (w / v) N,N'-Methylenebisacrylamide 0.5% (w / v) LAP (photoinitiator) 0.01% (w / v) Tween 20 4% (v / v) Forward primer 10 μM Reverse primer 10 μM SYBR Green I 1X Hisense HotTaq PCR master mix (CellSafe Co., Ltd.) 1X λ gDNA fragment variable

[0063] * LAP (photoinitiator): Lithium phenyl-2,4,6-trimethylbenzoylphosphinate

[0064] * Tween 20 (Tween 20): Commercial product name of polyoxyethylene (20) sorbitan monolaurate

[0065] * Forward primer: Sequence number 1

[0066] * Reverse prime: Sequence number 2

[0067] * HiSense HotTaq PCR master mix: A master mix product consisting of Taq DNA polymerase, Taq antibody, dNTPs (Deoxynucleoside triphosphates), MgCl2, and buffer solution.

[0068] * λ gDNA fragment: A 364 bp partial sequence consisting of the base sequence of sequence number 3 among the gDNA (48,000 bp) of bacteriophage λ. In the nucleic acid detection and quantification example using 1D dPCR described below, the final concentration of the λ gDNA fragment is 20.31 fM, and in the nucleic acid detection and quantification example using 2D PCR, the final concentration of the λ gDNA fragment is designed to be 83 aM and 166 aM.

[0069]

[0070] 2. Fabrication of a light-sheet photolithography system

[0071] The light-sheet photolithography (LSP) system was designed to variably form multiple spaces partitioned into a grid pattern as desired by irradiating a polymerase chain reaction (PCR) solution contained in a predetermined space with a light sheet at a predetermined interval and in a predetermined direction without using a photomask.

[0072] Figure 1 schematically illustrates the configuration of a light-sheet photolithography system. The light-sheet photolithography system is composed of 1) a UV fiber laser having a wavelength of 395 nm suitable for a water-soluble photoinitiator, 2) a collimator for expanding a fiber laser beam, 3) a shutter, 4) a cylindrical lens 1; f= 25 mm, 5) an aspherical lens 8 mm, 6) a cylindrical lens 2; f= 100 mm, 7) a mirror, 8) a slit, and 9) a cylindrical lens 3; f= 150 mm. The light-sheet photolithography system is placed on a motorized stage and can irradiate a light sheet having a wavelength of 395 nm and a width of 50 μm at regular intervals to a specific space while moving in a predetermined direction.

[0073]

[0074] 3. Detection and quantification of nucleic acids using photopolymerization-based 1D dPCR.

[0075] (1) Preparation of a solution for polymerase chain reaction (PCR) that can be partitioned by photopolymerization

[0076] In Table 1 above, a solution for polymerase chain reaction (PCR) was prepared by setting the final concentration of the λ gDNA fragment to 20.31 fM.

[0077]

[0078] (2) Preparation of a microfluidic chip for 1D dPCR and surface modification of the microfluidic channel

[0079] A 1D dPCR microfluidic chip with a linear microfluidic channel measuring 45.6 mm in length, 50 μm in width, and 20 μm in depth was prepared to one-dimensionally partition a polymerase chain reaction (PCR) solution. Furthermore, a surface-modifying solution was prepared by mixing 3-(trimethoxysilyl)propyl methacrylate, acetic acid, methanol, and deionized water in a volume ratio of 2:2:3:3. First, the microfluidic channels of the 1D dPCR microfluidic chip were sequentially washed with deionized water, 1 N sodium hydroxide solution, and deionized water, and dried. Subsequently, the surface-modifying solution was filled into the microfluidic channels of the 1D dPCR microfluidic chip, and the solution was allowed to react for 30 minutes. Afterwards, the surface modification solution was removed from the microfluidic channel, washed with methanol for 10 minutes, and dried to prepare a microfluidic chip for 1D dPCR in which the surface of the microfluidic channel was modified with alkoxysilane.

[0080]

[0081] (3) One-dimensional compartmentalization of solutions for polymerase chain reaction (PCR) using photopolymerization

[0082] The surface-modified microfluidic channels of the 1D dPCR microfluidic chip were filled with a polymerase chain reaction (PCR) solution, and the inlet and outlet wells at both ends of the microfluidic channel were filled with a PDMS (Polydimethylsiloxane) solution, and each well was sealed with PCR tape (PET / acrylic adhesive film; AB-0558, Thermo Fisher Scientific). In addition, a photomask with a grid pattern of alternating 100 μm-wide chrome stripes and 37.5 μm-wide gaps was prepared. The microfluidic chip was then aligned and placed on the photomask and irradiated with UV light from the bottom using a 365 nm wavelength UV lamp (13 mW / cm2, M365LP1-C1, Thorlabs) for approximately 40 min. In the microfluidic channel space corresponding to the gap of the photomask, photopolymerization proceeds by UV irradiation, and the polymerase chain reaction (PCR) solution gels. Upon completion of UV irradiation, the PCR solution filled in the microfluidic channel was compartmentalized into a one-dimensional lattice pattern with a liquid portion with a width of 100 μm and a gel-like wall with a width of 37.5 μm, and approximately 331 isolates were generated in the microfluidic channel.

[0083] Meanwhile, a polymerase chain reaction (PCR) solution filled in a surface-modified microfluidic channel of a microfluidic chip can be partitioned into multiple isolators using a light-sheet photolithography system rather than a photolithography system consisting of a photomask and an ultraviolet lamp. A one-dimensional partitioning method for a polymerase chain reaction (PCR) solution using a light-sheet photolithography system is as follows.

[0084] - After filling the surface-modified microfluidic channel of the microfluidic chip for 1D dPCR with a polymerase chain reaction (PCR) solution, the microfluidic chip is fixed to the XZ plane of the motor stage.

[0085] - A motorized stage is moved in the X-axis direction at a constant interval (e.g., 100 μm) and a light sheet that is perpendicular to the XZ plane and horizontal to the YZ plane is irradiated for about 20 minutes, thereby repeatedly forming a liquid portion with a width of 100 μm and a gel-like wall with a width of 50 μm in the longitudinal direction of the microfluidic channel. Finally, the polymerase chain reaction (PCR) solution filled in the microfluidic channel is partitioned into a one-dimensional lattice pattern in which the liquid portion with a width of 100 μm and the gel-like wall with a width of 50 μm are repeated, and a plurality of isolators are created in the microfluidic channel.

[0086]

[0087] (4) Amplification of nucleic acids using a thermal cycler

[0088] After partitioning the polymerase chain reaction (PCR) solution into a one-dimensional grid pattern using a photolithography system consisting of a photomask and an ultraviolet lamp, the microfluidic chip for 1D dPCR was mounted on a thermal cycler and heated with a temperature profile for PCR as shown in Table 2 below to amplify the nucleic acids present in the liquid portion for each isolate.

[0089] Step Temperature Time Number of Cycles Pre-denaturation 95℃ 5 minutes 1 cycle Denaturation 95℃ 30 seconds 31 cycles Annealing 56.1℃ 30 seconds Extension 72℃ 30 seconds Final extension 72℃ 5 minutes 1 cycle

[0090]

[0091] (5) Extraction of fluorescence signals and quantification of nucleic acids

[0092] After nucleic acid amplification using a thermal cycler, the PCR tape was removed from the microfluidic chip, and the microfluidic chip was inserted into a custom 3D-printed chip insert compatible with the stage of an inverted fluorescence microscope (IX73, Olympus). The entire microfluidic channel of the microfluidic chip was scanned and fluorescence images were acquired using an inverted fluorescence microscope equipped with a motorized XY stage (MS-2000, ASI), an sCMOS camera (Zyla, Andor), and Metamorph software (Molecular Devices). Fluorescence signal values ​​were extracted from the acquired fluorescence images using OriginPro software (OriginLab Corp.), and the nucleic acids were quantified using the Poisson statistical equation. The concentration of the target DNA can be calculated using Equation 1 below.

[0093] [Calculation Formula 1]

[0094]

[0095] In the above calculation formula 1, C DNA is the measured concentration of target DNA, P is the fraction of isolates showing a positive fluorescence signal relative to the total number of isolates, and v d is the volume of each isolator. The volume occupied by the gel-like walls in the isolator volume is excluded from the calculation.

[0096] As a result of quantification using Poisson statistics, the experimental λ-gDNA template concentration was calculated to be 2018 copies / ㎕. Fig. 2 shows the results obtained by performing the nucleic acid detection and quantification process using 1D dPCR in an embodiment of the present invention. Fig. 2 (a) is an image of a microfluidic chip for 1D dPCR made of glass, (b) is a fluorescence image obtained by scanning a microfluidic channel after completing nucleic acid amplification using a thermal cycler, (c) is an enlarged portion of the fluorescence image of (b), and (d) shows the distribution of fluorescence signal intensity of each isolate compartmented in the microfluidic channel when nucleic acid amplification using a thermal cycler is completed.

[0097]

[0098] 4. Detection and quantification of nucleic acids using photopolymerization-based 2D dPCR.

[0099] (1) Preparation of a solution for polymerase chain reaction (PCR) that can be partitioned by photopolymerization

[0100] In Table 1 above, the final concentration of the λ gDNA fragment was set to 83 aM and 166 aM, and two polymerase chain reaction (PCR) solutions with different concentrations were prepared.

[0101]

[0102] (2) Preparation of a microfluidic chip for 2D dPCR and surface modification of the microfluidic chamber

[0103] A 2D dPCR microfluidic chip with a microfluidic chamber measuring 7,500 μm in width, 7,500 μm in height, and 100 μm in depth was prepared to two-dimensionally partition a polymerase chain reaction (PCR) solution. Furthermore, a 2D dPCR microfluidic chip with an alkoxysilane-modified inner surface of the microfluidic chamber was prepared using the same method as for modifying the microfluidic channel surface of the 1D dPCR microfluidic chip.

[0104]

[0105] (3) Two-dimensional compartmentalization of polymerase chain reaction (PCR) solutions using photopolymerization

[0106] The surface-modified microfluidic chamber of the 2D dPCR microfluidic chip was filled with a polymerase chain reaction (PCR) solution, and the inlet and outlet wells at both ends of the microfluidic chamber were filled with a PDMS (Polydimethylsiloxane) solution, and each well was sealed with PCR tape (PET / acrylic adhesive film; AB-0558, Thermo Fisher Scientific). In addition, a photomask with a grid pattern consisting of alternating 100 μm × 100 μm chrome squares surrounding 37.5 μm wide gaps was prepared. The microfluidic chip was then aligned and placed on the photomask and irradiated with UV light from the bottom using a 365 nm wavelength UV lamp (13 mW / cm2, M365LP1-C1, Thorlabs) for approximately 40 min. In the space corresponding to the gap of the photomask in the microfluidic chamber, photopolymerization proceeds by UV irradiation, and the polymerase chain reaction (PCR) solution gels. When the UV irradiation is complete, the PCR solution filled in the microfluidic chamber is compartmentalized into a two-dimensional grid pattern with repeating 100 μm × 100 μm square liquid portions and 37.5 μm wide gel walls surrounding them, and approximately 2,916 isolates are generated in the microfluidic channel.

[0107] FIG. 3 is a schematic diagram of a microfluidic chip used in a nucleic acid detection and quantification process using 2D dPCR in an embodiment of the present invention, and an isolator having a two-dimensional grid pattern formed in a microfluidic chamber of the microfluidic chip. FIG. 3 (a) is an image of a glass microfluidic chip for 2D dPCR used in a nucleic acid detection and quantification process using 2D dPCR in an embodiment of the present invention, and FIG. 3 (b) is a schematic diagram of an isolator having a two-dimensional grid pattern formed when a polymerase chain reaction (PCR) solution filled in a microfluidic chamber of a microfluidic chip for 2D dPCR is compartmentalized using a photolithography system consisting of a photomask and an ultraviolet lamp in an embodiment of the present invention.

[0108] Meanwhile, a polymerase chain reaction (PCR) solution filled in a surface-modified microfluidic chamber of a microfluidic chip can be partitioned into multiple isolators using a light-sheet photolithography system rather than a photolithography system consisting of a photomask and an ultraviolet lamp. A two-dimensional partitioning method for a polymerase chain reaction (PCR) solution using a light-sheet photolithography system is as follows.

[0109] - After filling the surface-modified microfluidic chamber of the microfluidic chip for 2D dPCR with a polymerase chain reaction (PCR) solution, the microfluidic chip is fixed to the XZ plane of the motor stage.

[0110] - A motorized stage is moved in the X-axis direction at a constant interval (e.g., 100 μm) and a light sheet that is perpendicular to the XZ plane and horizontal to the YZ plane is irradiated for about 20 minutes to repeatedly form a 100 μm wide liquid portion and a 50 μm wide gel wall in the horizontal direction of the microfluidic chamber.

[0111] - After rotating the microfluidic chip by 90°, it is fixed again on the XZ plane of the motorized stage. While moving the motorized stage in the X-axis direction by a constant interval (e.g., 100 μm), a light sheet that is perpendicular to the XZ plane and horizontal to the YZ plane is irradiated for about 20 minutes to repeatedly form a 100 μm wide liquid portion and a 50 μm wide gel wall in the vertical direction of the microfluidic chamber. Finally, the polymerase chain reaction (PCR) solution filled in the microfluidic chamber is partitioned into a two-dimensional lattice pattern in which a 100 μm × 100 μm square liquid portion and a 50 μm wide gel wall surrounding it are repeated, and a plurality of isolators are created in the microfluidic chamber.

[0112]

[0113] (4) Amplification of nucleic acids using a thermal cycler

[0114] After partitioning the polymerase chain reaction (PCR) solution into a two-dimensional grid pattern using a photolithography system consisting of a photomask and an ultraviolet lamp, the 2D dPCR microfluidic chip was mounted on a thermal cycler and heated with a PCR temperature profile as shown in Table 2 above to amplify the nucleic acids present in the liquid portion for each isolate.

[0115]

[0116] (5) Extraction of fluorescence signals and quantification of nucleic acids

[0117] The entire microfluidic chamber of the 2D dPCR microfluidic chip was scanned and fluorescence images were acquired using the same method used for nucleic acid detection and quantification using 1D dPCR. Fluorescence signal values ​​were then extracted from the fluorescence images, and nucleic acids were quantified using Equation 1, which applies the Poisson statistical equation.

[0118] Figure 4 is a result obtained by performing a nucleic acid detection and quantification process using 2D dPCR in an embodiment of the present invention. (a) of Figure 4 is a fluorescence image obtained by scanning a microfluidic chamber after partitioning a polymerase chain reaction (PCR) solution without λ-gDNA into a two-dimensional lattice shape using photopolymerization and completing amplification of nucleic acids using a thermal cycler, (b) is a fluorescence image obtained by scanning a microfluidic chamber after partitioning a polymerase chain reaction (PCR) solution with a λ-gDNA concentration of 83 aM into a two-dimensional lattice shape using photopolymerization and completing amplification of nucleic acids using a thermal cycler, (c) is a fluorescence image obtained by scanning a microfluidic chamber after partitioning a polymerase chain reaction (PCR) solution with a λ-gDNA concentration of 166 aM into a two-dimensional lattice shape using photopolymerization and completing amplification of nucleic acids using a thermal cycler, and (d) is a fluorescence image obtained by scanning a microfluidic chamber after partitioning a polymerase chain reaction (PCR) solution with a λ-gDNA concentration of 166 aM into a two-dimensional lattice shape using photopolymerization and completing amplification of nucleic acids using a thermal cycler, This graph shows the linear relationship between the λ-gDNA concentration and the λ-gDNA concentration measured in the nucleic acid detection and quantification process using 2D dPCR. As shown in (d) of Fig. 4, when nucleic acid detection and quantification were performed using 2D dPCR, R 2 The value was 0.979, which was very close to 1, and this result means that the accuracy of the photopolymerization technology-based nucleic acid detection or quantification method proposed in the present invention is very high.

[0119]

[0120] 5. Detection and quantification of nucleic acids using photopolymerization-based 3D dPCR.

[0121] When using the nucleic acid detection or quantification method based on photopolymerization technology proposed in the present invention, a polymerase chain reaction (PCR) solution can be compartmentalized into a three-dimensional lattice pattern to generate thousands to millions of isolates. Fig. 5 is a step-by-step schematic diagram of the nucleic acid detection and quantification method using photopolymerization-based 3D dPCR proposed in the present invention. As shown in Fig. 5, the nucleic acid detection and quantification method using photopolymerization-based 3D dPCR largely consists of the steps of preparing a polymerase chain reaction (PCR) solution that can be compartmentalized by photopolymerization and selecting a container to accommodate the same; three-dimensional compartmentalization of the PCR solution using a light-sheet photolithography system; amplification of nucleic acids through PCR thermal cycling; and measurement of fluorescence signals and quantification of nucleic acids using light-sheet fluorescence microscopy (LSFM).

[0122]

[0123] (1) Preparation of a polymerase chain reaction (PCR) solution that can be partitioned by photopolymerization and selection of a container to accommodate it.

[0124] A solution for polymerase chain reaction (PCR) that can be partitioned by photopolymerization essentially includes, in addition to a typical PCR master mix consisting of DNA polymerase (Taq DNA Polymerase), dNTPs (Deoxynucleoside triphosphates), MgCl2, and a buffer solution, a sample containing a target nucleic acid, a primer, a fluorescent probe, a photopolymerizable monomer capable of forming a polymer on a gel (particularly, a hydrogel) by photopolymerization, a photoinitiator, and preferably, a cross-linking agent, a polymerase reaction accelerator (PCR enhancer), etc. In a method for detecting and quantifying nucleic acids using photopolymerization-based 3D dPCR, a container for accommodating the solution for polymerase chain reaction (PCR) must have a high light transmittance and an internal space that can be partitioned three-dimensionally, and examples thereof include a cuvette used in spectroscopic measurements.

[0125]

[0126] (2) Three-dimensional compartmentalization of polymerase chain reaction (PCR) solutions using a light-sheet photolithography system.

[0127] Figure 6 illustrates a process for generating multiple isolates by three-dimensionally partitioning a polymerase chain reaction (PCR) solution contained in a predetermined container using a light-sheet photolithography system. As shown in Figure 6, three light-sheet photolithography units are used to three-dimensionally partition a polymerase chain reaction (PCR) solution contained in a cuvette. When a cuvette containing a polymerase chain reaction (PCR) solution is fixed on the XZ plane, and then a light sheet parallel to the YZ plane and a light sheet parallel to the XZ plane are irradiated at preset intervals to first partition into a two-dimensional grid pattern in which a square liquid portion and a gel-like wall surrounding it are repeated, and then a light sheet parallel to the XY plane is irradiated at preset intervals to partition into a three-dimensional grid pattern in which a liquid portion in the shape of a cube (for example, a cube whose width, length, and height are all 100 ㎛) and a gel-like wall surrounding it (for example, a gel-like wall having a width of 50 ㎛) are repeated, a large number of isolates are generated in the cuvette. In the case of partitioning a polymerase chain reaction (PCR) solution three-dimensionally using a light-sheet photolithography system, 10 3 10 inland 6 It can produce high density isolates of dogs.

[0128]

[0129] (3) Amplification of nucleic acid through PCR thermal cycling

[0130] After dividing the solution for polymerase chain reaction (PCR) into a three-dimensional grid pattern, the 3D dPCR container (e.g., cuvette) is placed in a thermal cycler and heated with a temperature profile for PCR, thereby amplifying the nucleic acids present in the liquid portion for each isolate.

[0131]

[0132] (4) Measurement of fluorescence signals and quantification of nucleic acids using light-sheet fluorescence microscopy (LSFM)

[0133] When detecting and quantifying nucleic acids using photopolymerization-based 3D dPCR, isolates have a structure in which spaces partitioned by a two-dimensional lattice pattern in a three-dimensional space are vertically stacked, requiring a method for effectively counting positive and negative fluorescent signals within the three-dimensional lattice pattern. Detection of target DNA for each isolate in a one-dimensional or two-dimensional lattice patterned PCR solution can be performed by acquiring fluorescence images using a standard fluorescence microscope and using commercial image analysis software. Acquisition of fluorescence data for each isolate in a three-dimensional lattice patterned PCR solution can be performed using a confocal laser scanning microscope (CLSM). However, CLSM is large, slow, and has a typical vertical resolution of less than about 1 μm, making it excessively expensive and expensive for imaging isolates composed of relatively thick layers (tens to hundreds of μm) in 3D dPCR. Therefore, a customized light-sheet fluorescence microscopy (LSFM) system is needed to effectively acquire two-dimensional image stacks of each of hundreds to thousands of partitioned isolator layers in a three-dimensional grid pattern and to reconstruct the results in three dimensions.

[0134] Figure 7 illustrates a process for acquiring fluorescence data from an isolator partitioned into a three-dimensional grid pattern using a light-sheet fluorescence microscopy (LSFM) system. As shown in Figure 7, the LSFM system has a structure similar to that of a light-sheet photolithography (LSP) system and can be built into an inverted microscope with a motorized stage. The optical system of the LSFM system is similar to that of the LSP system, but differs in that it uses a laser with a wavelength of 488 nm, rather than ultraviolet light with a wavelength of 395 nm, to excite the fluorescent probe. The beam width of the laser irradiated in the LSFM system is preferably about 100 μm, considering the thickness of the isolator in each layer. The process for acquiring fluorescence data from an isolator partitioned into a three-dimensional grid pattern using the LSFM system is as follows. First, when the light sheet is irradiated on the first layer (z=1) in the LSFM system, the DNA-bound fluorescent probe is excited and fluoresces only within the positive isolations where the target DNA is present. The fluorescence images of each compartment are captured by an objective lens positioned perpendicular to the light sheet and a high-sensitivity digital camera. Next, the motorized stage is lowered and the light sheet is irradiated, capturing images of the second isolate layer. This process is repeated until the last isolate layer (z=n) is captured, and this continuous image sequence is used to reconstruct a three-dimensional image of the isolate. The optical system, motorized stage, and image acquisition are computer-controlled, and the dPCR results are counted based on the intensity of the fluorescence signal.Afterwards, isolates having a fluorescence signal higher than a threshold value can be determined as positive results, and isolates having a fluorescence signal lower than the threshold value can be determined as negative results. Finally, Poisson statistics is applied to determine the concentration of target DNA, C. DNA is calculated using the aforementioned calculation formula 1.

[0135]

[0136] While the present invention has been described through the above-described embodiments, it should be understood that the invention is not necessarily limited to these embodiments. Various modifications and variations are possible without departing from the scope and spirit of the invention. Therefore, the scope of protection of the present invention should be interpreted to encompass all embodiments falling within the scope of the appended claims.

Claims

1. A kit for preparing a solution for digital polymerase chain reaction (PCR), Containing a photopolymerizable monomer, a photoinitiator and a fluorescent probe, The above photopolymerizable monomer can form a gel-like polymer by photopolymerization, The above solution for digital PCR can be partitioned into multiple isolates by photopolymerization, A kit characterized in that the above-mentioned isolator is composed of a gel-like wall formed by photopolymerization and a liquid portion partitioned by the wall and capable of polymerase reaction.

2. In the first paragraph, the photopolymerizable monomer is at least one selected from the group consisting of acrylamide, methacrylamide, gelatin acrylate, gelatin methacrylate (GelMA), ethylene glycol dimethacrylate (EGDMA), ethylene glycol diacrylate (EGDA), acrylated hyaluronic acid, and methacrylated hyaluronic acid. The photoinitiator is Phenylbis(2,4,6-trimethylbenzoyl)phosphine oxide; 2-Hydroxy-4'-(2-hydroxyethoxy)-2-methylpropiophenone; Riboflavin; Eosin-Y; 2,2'-Azobis(2-methyl-N-(2-hydroxyethyl)propionamide; Lithium phenyl-2,4,6-trimethylbenzoylphosphinate (LAP); A kit characterized by comprising at least one selected from the group consisting of diphenyl(2,4,6-trimethylbenzoyl)phosphine oxide (TPO); and tetramethylethylenediamine (TEMED).

3. A kit according to claim 1, characterized in that the fluorescent probe is selected from a fluorescent dye that binds to double-stranded DNA or a fluorescent substance-labeled oligonucleotide type hydrolysis probe.

4. A kit according to claim 1, characterized in that it further comprises a cross-linking agent or a polymerase reaction promoter (PCR enhancer).

5. A kit according to claim 4, characterized in that the polymerase reaction promoter (PCR enhancer) is at least one selected from the group consisting of a nonionic surfactant, bovine serum albumin (BSA), skim milk, gelatin, casein, serum, and dimethyl sulfoxide (DMSO).

6. A kit according to claim 1, characterized in that it further comprises polymerase chain reaction (PCR) essential elements comprising a DNA polymerase, a deoxynucleoside triphosphate (dNTP), a metal ion providing compound, and a buffer.

7. (a) a step of preparing a light-transparent PCR vessel containing a solution for digital polymerase chain reaction (PCR); (b) a step of irradiating the light-transparent PCR vessel containing the solution for digital polymerase chain reaction (PCR) with light to induce a photopolymerization reaction so that the solution for digital polymerase chain reaction (PCR) is partitioned into a liquid phase portion and a gel phase wall in a repeating pattern, thereby generating a plurality of isolates; (c) a step of applying heat to the light-transparent PCR vessel after partitioning the solution for digital polymerase chain reaction (PCR) to amplify nucleic acids present in the liquid phase portion for each isolate; and (d) a step of acquiring fluorescence data from the partitioned isolates using a fluorescence imaging technique after amplifying the nucleic acids. The above digital polymerase chain reaction (PCR) solution is characterized by including a target nucleic acid-containing sample, a primer, a photopolymerizable monomer, a photoinitiator, a fluorescent probe, a DNA polymerase, a deoxynucleoside triphosphate (dNTP), a metal ion-providing compound, and a buffer. A photopolymerization-based nucleic acid detection or quantification method using the kit of claim 1.

8. A method for detecting or quantifying nucleic acids based on photopolymerization, characterized in that in paragraph 7, the solution for digital polymerase chain reaction (PCR) further comprises at least one selected from the group consisting of a cross-linking agent, a polymerase reaction promoter (PCR enhancer), and a reverse transcriptase.

9. A method for detecting or quantifying nucleic acids based on photopolymerization, characterized in that the inner surface of the light-transmitting PCR container in paragraph 7 is modified by silanization treatment before the digital polymerase chain reaction (PCR) solution is received.

10. A method for detecting or quantifying nucleic acids based on photopolymerization, characterized in that in the 7th paragraph, the light-transmitting PCR container is selected from a glass slide combined with an imaging spacer, a chip equipped with a microfluidic channel, a chip equipped with a microfluidic chamber, a cuvette, or a transparent plastic container.

11. A photopolymerization-based nucleic acid detection or quantification method, characterized in that in the step (b), the compartmentalization of the solution for digital polymerase chain reaction (PCR) is performed by a photolithography system or a light-sheet photolithography system comprising a photomask and an ultraviolet lamp.

12. A method for detecting or quantifying nucleic acids based on photopolymerization, characterized in that in the 7th paragraph, step (c) further includes a process of inducing a photopolymerization reaction by irradiating light to the entire light-transmitting PCR container after amplifying the nucleic acid and fixing the amplified nucleic acid inside the isolator.

13. A method for detecting or quantifying nucleic acids based on photopolymerization, characterized in that the fluorescence imaging system for implementing the fluorescence imaging technique in claim 7 is a confocal laser scanning microscope (CLSM) or a light-sheet fluorescence microscope (LSFM).

14. A method for detecting or quantifying nucleic acids based on photopolymerization, characterized in that in the step (b), when a solution for digital polymerase chain reaction (PCR) is three-dimensionally partitioned within a light-transmitting PCR container by a light-sheet photolithography system, the acquisition of fluorescence data in the step (d) is performed by irradiating a light sheet to each isolator layer composed of a plurality of isolators to obtain a fluorescence image in a two-dimensionally partitioned form for each isolator layer, and stacking the fluorescence images for each isolator layer to reconstruct them three-dimensionally.

15. A device for detecting or quantifying nucleic acids, comprising a kit for preparing a solution for digital polymerase chain reaction (PCR) selected from any one of claims 1 to 9.

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