Microparticle probe for single nucleotide polymorphism detection

Abstract

The present invention relates to a microparticle probe for single nucleotide polymorphism. The present invention provides a microparticle probe for single nucleotide polymorphism, comprising: a microparticle; the capture probe is introduced to the surface of the particle and contains a complementary sequence with the target nucleic acid; and a reporter nucleic acid introduced to the surface of the microparticle and generating a signal by an external stimulus.

Description

Technical Field

[0001] The invention relates to a microparticle probe for detecting single nucleotide polymorphism. Background Art

[0002] The genetic codes contained in the genomes of organisms of the same species are not exactly the same, and these differences in nucleotide sequences are called polymorphisms. Deletion or insertion of 1 to 10 nucleotides, duplication of a specific nucleotide sequence, etc. are examples of polymorphisms. If a single nucleotide is replaced by another nucleotide, it is called a single nucleotide (base) polymorphism (SNP).

[0003] Single nucleotide polymorphisms (SNPs) refer to changes in a single base sequence of DNA and are associated with numerous genetic traits. There is approximately one SNP in every 1,000 base sequences, and they are the main reason for differences in the incidence of genetic diseases and drug resistance between individuals. Based on the changes in the four bases (A, G, C, T), SNPs can be divided into a total of four types (C:G->T:A, C:G->G:C, C:A->G:T, T:A->A:T). Among them, the polymorphism between cytosine (C) and thymine (T), namely the C / T type SNP, accounts for about 70% of the total.

[0004] As SNP analysis becomes increasingly important for reasons such as individual disease prediction and personalized medicine, research on effective SNP detection methods has been ongoing, and a variety of methods for identifying SNPs have been developed. Representative examples include single-stranded conformation polymorphism (SSCP) analysis and mini-sequencing as non-allele-specific methods, and RT-PCR using molecular beacons and ligase analysis as allele-specific methods.

[0005] Many SNP screening methods have been developed over the past 20 years, but the ligase-based SNP analysis method has been proposed as a promising method in terms of analytical efficiency. This method has several advantages. First, it enables multiplex analysis and can be combined with microanalysis methods such as flow cytometry, microarray, microfluidics, and capillary to perform SNP analysis on various samples at the same time. In addition, it can be easily combined with methods that use nanoparticles such as gold nanoparticles to enhance signal amplification and specificity, or with optical imaging methods that utilize FRET. In particular, if ligation analysis is combined with PCR, high signals can be obtained even if the sample concentration is very small, and it can be integrated with existing DNA-based automation systems. However, T4 DNA ligase and thermostable DNA ligase have fundamental problems in ensuring the distinction between ligation results of G:C pairing and G:T mismatch at the ligation end part. That is, the method of distinguishing SNPs by whether the ligation is successful or not is fast and simple, and can be used in combination with a variety of methods, but it has the problem of generating false positive results during the ligase reaction, especially in the case of G:T mismatch. That is, DNA ligase will inevitably show positive results in the case of non-complementary G:T mismatches, and ultimately these inaccurate results are the biggest factor that reduces the reliability of the system that uses ligase to determine C / T type SNPs. Deng's research team published the results of ligation-based SNP analysis based on DNA chips using T4 DNA ligase, but their results also showed false positive errors of G:T mismatches being ligated.

[0006] Therefore, a new SNP analysis method is needed that can confirm the ligation reactions of G:C pairs and G:T mismatches without errors. Summary of the invention

[0007]

Technical issues

[0008] In order to solve the above problems, the present invention aims to provide a microparticle probe for single nucleotide polymorphism.

[0009]

Technical solution

[0010] In order to solve the above problems, the present invention provides a microparticle probe for single nucleotide polymorphism, comprising: a microparticle; a capture probe introduced into the surface of the microparticle and containing a complementary sequence to the target nucleic acid; and a reporter nucleic acid introduced into the surface of the microparticle and generating a signal by external stimulation.

[0011] In one embodiment, the capture probe may comprise a guide RNA.

[0012] In one embodiment, the guide RNA comprises a CRISPR RNA (crRNA) having a nucleotide sequence capable of hybridizing to a target site of a gene and a trans-activating crRNA (tracrRNA).

[0013] In one embodiment, the guide RNA binds to the target nucleic acid and, upon encountering a lyase, cleaves the target nucleic acid and the reporter nucleic acid.

[0014] In one embodiment, the reporter nucleic acid may be a nucleic acid labeled with a luminescent substance.

[0015] In one embodiment, the reporter nucleic acid may be a base sequence that is cut when the gene scissors system is used.

[0016] In one embodiment, the microparticles may contain magnetic particles inside.

[0017] In one embodiment, the microparticle may have a core-shell structure having a core including a magnetic substance and a shell layer having a uniform thickness surrounding the core.

[0018] In one embodiment, the microparticles may have a size and specific gravity such that they do not float in water.

[0019] The present invention also provides a kit for analyzing single base polymorphism, comprising a microparticle probe for single nucleotide polymorphism, a lyase and a nucleic acid amplification reagent.

[0020] The present invention also provides a method for analyzing single base polymorphism, comprising the following steps: (a) extracting a target nucleic acid for analyzing single base polymorphism from a sample; (b) amplifying the target nucleic acid; (c) providing a microparticle probe for single nucleotide polymorphism, the microparticle probe comprising a microparticle, a guide RNA introduced into the surface of the microparticle and containing a complementary sequence to the target nucleic acid, and a reporter nucleic acid introduced into the surface of the microparticle and generating a signal by external stimulation; (d) reacting the guide RNA of the microparticle for analyzing single base polymorphism with the target nucleic acid to form a complex of the microparticle probe for single nucleotide polymorphism and the target nucleic acid; (e) combining a lyase with the guide RNA of the microparticle for analyzing single base polymorphism; (f) cutting each base sequence of the target nucleic acid and the reporter nucleic acid through an activation reaction of the lyase; and (g) detecting the target nucleic acid by measuring the opening and closing of the signal emitted by the complex caused by the external stimulation.

[0021] In one embodiment, the detection of the target nucleic acid can be performed by using two or more microparticle probes for analyzing single base polymorphisms and measuring the difference in luminescence of each probe to identify the presence or absence of the target nucleic acid.

[0022] In one embodiment, the microparticle probe for single nucleotide polymorphism may have a microrod shape.

[0023]

Beneficial Effects

[0024] The microparticle probe for single nucleotide polymorphism according to the present invention uses microparticles with higher magnetism than conventional magnetic particles (particularly magnetic beads), so that magnets can be used to efficiently mix or move, and can be manufactured at low cost. Therefore, the present invention can perform the same analysis at a lower cost than existing single base polymorphism analysis equipment.

[0025] Furthermore, the microparticle probe for single nucleotide polymorphism according to the present invention can accurately identify and detect single base polymorphism by introducing gene scissors technology. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1 It is a schematic diagram of an application example of a microparticle probe for detecting nucleic acid according to an example of the present invention.

[0027] Figure 2 and Figure 3 The process of detecting target nucleic acid using magnetic particle / gene scissors technology according to one embodiment of the present invention is shown.

[0028] Figure 4 The result of diagnosis performed according to the method by one embodiment of the present invention is virtually shown.

[0029] Figure 5 The preliminary verification results of the possibility of capturing nucleic acids from target samples based on magnetic particles according to one embodiment of the present invention are shown.

[0030] Fig. 6A The figure shows the target nucleic acid capture and amplification process sequentially performed from well 1 to well 4 according to one embodiment of the present invention.

[0031] Figure 6B The result of the capture and amplification process of arbitrary nucleic acid fragments RNA and DNA (see SEQ ID NO: 1) according to one embodiment of the present invention is shown.

[0032] Figure 7 The results show that when free reporter DNA and Cas protein are mixed in the presence of target nucleic acid, the activity of the Cas system is confirmed by coupling magnetic particle probes at different concentrations to observe whether the gRNA changes in a concentration-dependent manner.

[0033] Figure 8 One embodiment of the present invention is shown, showing the results of an experiment based on the presence or absence of a single base polymorphism.

[0034] Fig. 9 The results of measuring the detection limit according to one embodiment of the present invention are shown. DETAILED DESCRIPTION

[0035] The preferred embodiments of the present invention will be described in detail below. When describing the present invention, if it is considered that a detailed description of the relevant known technology may obscure the core content of the present invention, its detailed description will be omitted. Throughout the specification, unless the context clearly indicates otherwise, it should be understood that the singular forms "one", "a kind" and "the" include plural referents; and it should be understood that terms such as "including" or "having" used in this specification are intended to indicate the presence of the stated features, quantities, steps, operations, components, parts or combinations thereof, but do not exclude the possibility of the presence or addition of one or more other features, quantities, steps, operations, components, parts or combinations thereof. In addition, when performing a method or a preparation method, each process constituting the method may occur in an order different from the specified order, unless a specific order is clearly described in the context. That is, each process may occur in a specified order, may be performed substantially simultaneously, or may be performed in reverse.

[0036] The technology disclosed in this specification is not limited to the embodiments described herein, and may be embodied in other forms. However, the embodiments described herein are intended to make the contents disclosed herein complete and detailed so that those skilled in the art can fully understand the spirit of the present technology. In order to clearly express the components of each device, the dimensions of components such as width or thickness are slightly enlarged in the drawings. When the drawings are described as a whole, they are described from the perspective of the observer, and when an element is said to be located on another element, this includes all possible meanings, that is, an element can be directly located on another element, or additional elements can be inserted between them. In addition, those skilled in the art can implement the spirit of the present invention in various other forms without departing from the technical spirit of the present invention. In addition, the same reference numerals in multiple drawings refer to substantially the same elements.

[0037] In this specification, the term "and / or" includes any combination or any one of the multiple listed items. In this specification, "A or B" may include "A", "B" or "both".

[0038] The present invention relates to a microparticle probe for single nucleotide polymorphism, comprising: microparticles; capture probes introduced onto the surface of the microparticles and containing sequences complementary to target nucleic acids; and reporter nucleic acids introduced onto the surface of the microparticles and generating signals under external stimulation.

[0039] Figure 1 is a schematic diagram showing one example of a microparticle probe for single nucleotide polymorphism. Figure 1The microparticle probe 100 for analyzing single base polymorphism includes a microparticle 110 , a capture probe 120 , and a reporter nucleic acid 130 .

[0040] The microparticle 110 provides the surface area required for detecting the target nucleic acid, and for this purpose, the capture probe 120 and the reporter nucleic acid 130 can be introduced on its surface. The microparticle 110 can be made of a polymer such as polymethyl methacrylate (PMMA) or polystyrene (PS), an inorganic material such as silica gel, or a composite material thereof. In addition, in order to facilitate the collection of the detected target nucleic acid or the reaction with the reagent, the microparticle 110 may contain magnetic particles inside.

[0041] The particle 110 may have a single structure or a core-shell structure. Preferably, the particle 110 may have a core-shell structure consisting of a core 112 and a protective shell 114 surrounding the core. The core 112 of the particle 110 may contain a magnetic substance, such as a magnetically responsive metal, which may be a paramagnetic substance. Specifically, the magnetically responsive metal may be an alloy containing iron (Fe), nickel (Ni), cobalt (Co) and manganese (Mn). The magnetically responsive metal may have transition metals such as iron, nickel, cobalt and manganese as main components, and may contain rare earth metals such as gadolinium (Gd), terbium (Tb) and samarium (Sm), as well as elements such as boron (B), silicon (Si), and carbon (C). Representative examples of magnetically responsive metals include iron alloys or cobalt alloys. A specific example of an iron alloy may include Fe 70 B 15 Si 10 C5, and examples of cobalt alloys may include Co 68 Mn7Si 10 B 15 .

[0042] Preferably, the size and specific gravity of the particle 110 are such that it does not float in water so that it can be quickly collected or separated by an external magnet such as a permanent magnet or an electromagnet. In this case, the core 112 can occupy more than 60% of the total volume of the particle 110. For example, the volume of the core 112 can be 60% to 99% of the total volume of the particle 110, and more preferably 75% to 99%. If the core volume is less than 60%, the magnetic properties of the core may be weakened, making it difficult to move using a magnetic rod. If the core volume exceeds 99%, the shell thickness will be reduced, thereby reducing durability.

[0043] In addition, the microparticle 110 can have a size (e.g., diameter or length) of tens to hundreds of microns, and preferably has a specific gravity of 5 or more. If the microparticle 110 is a nanoparticle less than 1 micron, it may float in water even if it is a particle with a high specific gravity (e.g., 7.876 in the case of iron). In this case, in the process of using the microparticle 110 to analyze single base polymorphism, when a magnetic field is applied, the magnetic control of the microparticle cannot be carried out smoothly due to the low magnetic force of the microparticle, which may lead to separation difficulties. When the magnetic bar moves up and down in the hole to promote the immune response, the microparticles will attach and detach. In the process of the magnetic bar moving up and down, due to its light weight, once the microparticles attached to the magnetic bar do not fall back to the bottom of the hole even after the magnetic field is removed, but continue to stay on the magnetic bar through non-specific binding. In this case, when the biological substance in the hole is detected, the repeatability of the quantitative analysis may be reduced.

[0044] In the microparticle probe for single nucleotide polymorphism according to one embodiment of the present invention, its size is much larger than the silica beads used in the traditional analysis of single base dimorphism. Unlike the traditional silica beads, in which the magnetic particles are usually dispersed inside the silica gel, the magnetically responsive metal (magnetic core) here occupies most of the volume of the microparticle and is sensitive to magnetic force, thereby showing excellent repeatability in quantitative analysis.

[0045] At the same time, the microparticle probe 100 for analyzing single base polymorphism of the present invention has a magnetic response metal at its center and a shell layer surrounding the center, thereby forming a core-shell structure. Shell layer 114 can be made of organic or inorganic materials, preferably glass. In addition, capture probes such as antibodies, proteins, nucleic acids and metabolites can be fixed on the surface of the microparticle probe 100 for analyzing single base polymorphism. For this purpose, functional groups such as acrylic acid, hydroxyl, amino or carboxyl are preferably introduced into the surface.

[0046] The shell 114 may almost completely surround the particle 110, but if necessary or during the manufacturing process, the surface of the particle may not be completely covered by the shell, and some areas of the particle surface may be exposed.

[0047] The thickness of the shell layer 114 may be between 1 and 100 micrometers, preferably between 1 and 50 micrometers, more preferably between 1 and 10 micrometers, and even more preferably between 4 and 8 micrometers. If the thickness of the shell layer 114 is below the above range, the surface of the shell layer 114 may be easily broken or cracked; if it exceeds the above range, some problems may occur when cutting glass depending on the laser characteristics and wavelength.

[0048] The core-shell structure can be formed by applying a liquid shell component to a metal core, or by filling a metal core component into a hollow mold.

[0049] In this case, the shell layer 114 can be formed by solidifying a liquid coating solution of an organic or inorganic substance. More specifically, the shell layer 114 can be formed by melting or flowing an organic or inorganic type shell component at a high temperature, or dissolving it in a solvent to make a liquid coating solution, and then applying it to the metal core. The organic substance can mainly be a polymer, and the inorganic substance can be a metal or ceramic, especially glass. For example, a coating solution obtained by dissolving plastic in a solvent, or by melting glass to form the shell layer 114, can be applied to the particle 110 by dipping or spraying.

[0050] For example, when a glass tube is used as a hollow mold, there are several methods, including: a method of drawing a glass tube while adding metal powder into the glass tube and melting the metal at a high temperature; a method of first drawing a glass material and then injecting molten metal into the inside thereof; and a method of filling a glass tube with metal powder dispersed in an ultraviolet curing material and hardening it by irradiating ultraviolet light. In these methods, the hollow mold itself can become the shell layer.

[0051] The microparticles 110 obtained by the above method have excellent surface uniformity. For conventional bioanalytical particles, a silica precursor such as TEOS is usually used and the shell 114 is grown on the surface of the core particle, but in this case, the shell 114 has a very rough surface. Therefore, a large amount of non-specific binding may occur to the target substance. This may cause unnecessary background noise.

[0052] On the other hand, the glass constituting the shell 114, such as borosilicate glass, can minimize nonspecific reactions (nonspecific binding) due to adsorption due to chemical reactions with the reaction sample. In particular, the shell 114 of the microparticle 130 according to an embodiment of the present invention is a coating formed by a liquid component and therefore has a very uniform surface. The average roughness (Ra) of the surface of the shell 114 may be 15 nanometers or less, preferably 10 nanometers or less, more preferably 5 nanometers or less, even more preferably 2 nanometers or less, especially 1.5 nanometers or less. In addition, Ra may be, for example, 3 nanometers or more, 2 nanometers or more, or 1 nanometer or more. If the surface roughness is within this range, the nonspecific adsorption of the reaction sample on the surface of the shell 114 can be minimized.

[0053] Considering strength and transparency, the shell 114 of the particle 110 can be made of glass. The glass can be mainly composed of a compound selected from soda lime, borosilicate, aluminosilicate, silicon dioxide, alkali silicate, Pyrex and quartz. Preferably, in order to meet the heat resistance, acid resistance and water resistance required by the experiment, the glass can be borosilicate.

[0054] The microparticle 110 can have various shapes, including regular shapes such as rods, plates, spheres, or irregular shapes. Even if the microparticle 110 has a flat shape, its cross section can also be various shapes such as star-shaped, polygonal, circular, etc., without special restrictions. Preferably, considering the convenience of manufacturing and the convenience of observation, the microparticle is in the shape of a microrod, a microdisk or a microbead, and in particular, the microrod shape is more ideal. If the microparticle 130 is in the shape of a microrod, it is easy to distinguish the microparticles due to overlap, it is easy to focus when placed in the hole, and each particle occupies a small area, so a large number of microparticles can be observed on a single observation screen. On the other hand, if the microparticle has a complex cross-sectional shape, such as a star shape, the edge portion may break due to collisions with each other or collisions with the wall during movement in the hole, so it is more desirable to have a simple shape, such as a microrod.

[0055] The length of the microrod can be 10 to 1000 microns. If the length of the microrod is below the above range, it may not be easy to distinguish particles of different lengths; if the length of the microrod exceeds the above range, the particles may overlap with each other, making observation difficult. In addition, the ratio of the length to the diameter of the microrod (aspect ratio) may be 2 or more, 5 or more, or 10 or more. The upper limit of the aspect ratio may be 20 or less, 10 or less, or 5 or less. If the aspect ratio is lower than the above range, it may be difficult to distinguish from spherical particles because it is similar to this; if the aspect ratio is too large, it may bend.

[0056] As a preferred embodiment, the microparticles 110 may be segments cut from metal microwires coated with glass. Microparticles 110 having various lengths can be obtained by cutting the metal microwires coated with glass with a laser.

[0057] The microparticles are suitable for analyzing single base polymorphisms in specific samples from organisms. The sample can be a tissue extract, a cell lysate, whole blood, plasma, serum, saliva, ocular humor, cerebrospinal fluid, sweat, urine, milk, ascites, synovial fluid, peritoneal fluid, etc. For rapid diagnosis, pretreatment of the sample liquid can be simplified or omitted in some cases.

[0058] Microparticles can be manufactured in different sizes, lengths or shapes for analysis of single base polymorphisms within specific samples from an organism.

[0059] Capture probes 120 can be introduced into microparticles 110 to capture biomarkers derived from the sample, particularly nucleic acid-based biomarkers. These biomarkers can be used without limitation as long as they are used in conventional scientific or medical fields, such as measuring or evaluating biological processes, processes leading to pathogenicity, or pharmacological processes for treatment. Biomarkers can be, for example, polypeptides, peptides, nucleic acids, proteins, or metabolites that can be detected in biological fluids such as blood, saliva, or urine, and preferably, biomarkers are nucleic acids because they can be detected with high sensitivity and specificity as biomarkers associated with specific diseases.

[0060] In order to capture the target nucleic acid extracted from the sample, the microparticle according to one embodiment of the present invention may introduce a capture probe 120 on its shell 114. The capture probe 120 is complementary to the target nucleic acid and specifically binds to the target nucleic acid, thereby playing a role in fixing the target nucleic acid to the microparticle 110.

[0061] The capture probe 120 may include any base sequence for applying the gene scissors technology. Preferably, the capture probe 120 may be or include a guide RNA (gRNA). Guide RNA refers to a specific RNA targeting DNA (e.g., an RNA capable of hybridizing with a target site of DNA), and can be used as part of a CRISPR system together with a cleavage enzyme. The guide RNA includes two guide RNAs, namely a CRISPR RNA (crRNA) having a nucleotide sequence that can hybridize with a gene target site and an additional trans-activating tracrRNA (trans-activating crRNA), which may be in the form of a partially bound crRNA:tracrRNA complex (dual guide RNA), or in the form of a single guide RNA (sgRNA) in which crRNA (partial or all) and tracrRNA (partial or all) are connected by a linker. The gRNA binds to the target nucleic acid and may cut the target nucleic acid and the reporter nucleic acid when encountering a lyase. Using these characteristics of the guide RNA, the biomarker can be quickly detected by the detection signal change information obtained after the specific biomarker sequence described later is cut. In addition, in this way, it can also be confirmed that the base in the sample has been replaced, which will be described in detail later.

[0062] Specifically, the capture probe 120 can be a nucleic acid with a length of 15 to 60 bases (mer). In addition, it can also be 20 to 45 bases, 25 to 40 bases, or 30 to 41 bases.

[0063] The capture probe 120 can be fixed on the surface of the microparticle 110 by adsorption or chemical connection, and is preferably connected to the functional group on the surface of the shell 114. For example, biotin can be introduced on the surface of the capture probe 120, and avidin, neutravidin or streptavidin that binds to biotin can be introduced on the microparticle 110, so that the capture probe 120 is attached to the microparticle 110. Alternatively, the capture probe 120 can be connected to the microparticle 110 using the hydroxyl group, amino group or carboxyl group on the surface of the microparticle 110.

[0064] If the above-mentioned microparticle probe for analyzing single base polymorphism is used, single base substitution present in nucleic acid in a sample solution can be analyzed rapidly and with high sensitivity.

[0065] The reporter nucleic acid 130 is introduced to the surface of the microparticle 110 together with the capture probe 120, and generates a light signal or an electrical signal through external stimuli including chemical, mechanical, optical and electrical energy. In one embodiment, the reporter nucleic acid 130 may be a nucleic acid labeled with a luminescent substance, which may generate a fluorescent or chemiluminescent signal through external light or chemical reaction.

[0066] Specifically, the reporter nucleic acid 130 can be DNA. In addition, the reporter nucleic acid can be a nucleic acid of 15 to 40 bases in length. In addition, it can be 18 to 30 bases, 19 to 25 bases, or 20 to 23 bases.

[0067] Luminescent substances can detect the presence of target nucleic acids by connecting to detection probes and generating light under external stimulation such as ultraviolet rays, electron beams, chemical reactions, enzyme-substrate reactions, etc. Examples of luminescent substances include fluorescent molecules, quantum dots, metal nanoparticles, magnetic nanoparticles, enzymes, enzyme substrates, etc. Among them, fluorescent molecules can be selected as a variety of luminescent substances from the perspective of acquisition and application.

[0068] Examples of fluorescent molecules include commonly used fluorescent molecules selected from FITC (fluorescein isothiocyanate), fluorescein, FAM (fluorescein amide), PE (phycoerythrin), europium, TRITC (tetramethylrhodamine isothiocyanate), Cy3 (cyanine 3), Cy5 (cyanine 5), Cy7 (cyanine 7), Alexa fluorescent dyes and rhodamine.

[0069] In order to detect multiple biomarkers in a sample, multiple fluorescent groups such as FITC, PE and Alexa-647 can be introduced and labeled at the 5' end of the reporter nucleic acid 130.

[0070] In the case of microparticle probes for target nucleic acid separation and detection according to one embodiment of the present invention, by introducing a gene scissors system, they can be applied to rapid and accurate detection of target nucleic acids, especially single bases that have been replaced.

[0071] When the microparticle probe for detecting nucleic acid is applied to the gene scissors system, the reporter nucleic acid 130 can be the base sequence to be cut in the system. Gene scissors technology is a technology that uses a protein complex with DNA nuclease function to induce DNA insertion, deletion and replacement at a specific position of the genome to cause mutations. Gene scissors technology includes zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs) and CRISPR-Cas systems. In terms of its excellent ability to accurately cut the required part of DNA in a simple and quick way, the preferred gene scissors technology is the CRISPR-Cas system. CRISPR (regularly interspaced short palindromic repeats) as a guide RNA (gRNA) recognizes the target DNA and forms a complex with a lyase such as Cas endonuclease to cut double-stranded DNA. The CRISPR-Cas system in the present invention is not limited to any known one, and can include CRISPR-Cas9, CRISPR-Cas12, CRISPR-Cas13, etc. according to the type of lyase. Among these systems, due to its high sensitivity to mismatches in the guide RNA, the CRISPR-Cas system is preferably CRISPR-Cas12, and more preferably CRISPR-Cas12a using the Cas12a protein as a lyase.

[0072] CRISPR RNA may comprise a base sequence of SEQ ID NO: 1 or 2. In the method for analyzing single base polymorphism of the present invention, two types of probes are input, and then the difference in luminescence of each probe may be measured to determine whether a base substitution has occurred. Therefore, it is desirable that CRISPR RNA have specific sequences, which may consist of two types: one is RNA complementary to DNA that does not exhibit single base dimorphism, and the other is RNA complementary to DNA that exhibits single base dimorphism.

[0073] As an example, the CRISPR RNA may be a base sequence of SEQ ID NO: 1 or 2. In detail, in the polymorphism analysis of CYP1A1, a mutation may occur due to the substitution of an adenine (A) site with a guanine (G). In this case, the unsubstituted one may be CYP1A1-A, and the one that has been substituted and mutated may be CYP1A1-G. As described above, when two microparticle probes containing CRISPR RNA of each base sequence are injected, each probe can emit different light depending on whether a mutation exists, and single base polymorphism analysis can be performed by measuring the difference in light.

[0074] In one embodiment, the reporter nucleic acid 130 can be attached to the surface of the microparticle 110 at a high density so as to react with the subsequently introduced Cas endonuclease (engineered nuclease), which is one of the protein complexes that performs the function of DNA nuclease. For example, the reporter nucleic acid 130 can be attached to the surface of the microparticle 110 at a high density so as to react with the subsequently introduced Cas endonuclease (engineered nuclease), which is one of the protein complexes that performs the function of DNA nuclease. 16 / m 2 , preferably 6 to 7 × 10 16 / m 2 The density of the capture probe 120 and the reporter nucleic acid 130 is introduced onto the surface of the microparticle 110, in which case a strong signal can be generated so that the size and shape of the microparticle 110 can be easily distinguished. The reporter nucleic acid 130 can be composed of any sequence and can be a single-stranded or double-stranded nucleic acid sequence depending on the type of Cas endonuclease. In this case, in one embodiment, the Cas endonuclease works by forming a complex with the assembly of the microparticle probe 100 (i.e., the state in which the capture probe 120 and the reporter nucleic acid 130 are evenly distributed on the surface of the microparticle 110) and the target nucleic acid, and then cutting the reporter nucleic acid 130 distributed near the capture probe 120 assembly and the target nucleic acid.

[0075] If the target nucleic acid amplification product obtained after pretreatment of the sample obtained from the patient is reacted with the above-mentioned microparticle probe 100 for separating and detecting the target nucleic acid, the complex can be formed by complementary binding with the gRNA in the capture probe that recognizes the specific target nucleic acid of the single-stranded region to be amplified. If a Cas endonuclease such as the Cas12a protein used in the CRISPR system is applied to this complex, because the target nucleic acid will bind to the spacer sequence of the guide RNA, and then activate the sequence cutting function of the Cas12a protein, thereby cutting the target nucleic acid and the surrounding reporter nucleic acid 130, it can be observed that the fluorescence signal is no longer maintained but disappears.

[0076] Therefore, by simultaneously acquiring and comparing bright field images and fluorescent field images (fluorescent images) before and after the application of Cas endonucleases, it is possible to observe particles whose fluorescence disappears, thereby confirming whether a specific nucleic acid is included or replaced in the sample. By applying the Gene Scissors System in this way, single-base polymorphism analysis can be performed quickly and easily.

[0077] According to another aspect of the present invention, a kit for analyzing single base polymorphism is provided.

[0078] The kit includes the above-mentioned microparticle probe for single nucleotide polymorphism and lyase. The microparticle probe for single nucleotide polymorphism has been described in detail above. As a preferred example of lyase, the Cas endonuclease can form a complex with the guide RNA as part of the CRISPR-Cas system and cut the reporter nucleic acid, for example, it can be Cas12a protein.

[0079] In one embodiment, the kit may further include at least one selected from the following group: magnetic particles for analyzing single base polymorphism, nucleic acid amplification reagents, lysis solutions, and washing solutions.

[0080] The magnetic particles used to analyze single base polymorphisms can be silica beads or silica-coated magnetic particles, which are configured to have the characteristics of silica in the surface area, thereby increasing the surface area and forming a salt bridge in the presence of a lysate containing a high concentration of a high-chaotropic salt (high-chaotropic salt) (such as guanidine salt), thereby producing an effect of easily capturing nucleic acids. Nucleic acid amplification reagents are not limited to specific amplification of target nucleic acids, but nucleic acid amplification can be performed under isothermal conditions, such as loop-mediated isothermal amplification (LAMP), single primer isothermal amplification (SPIA), recombinase polymerase amplification (RPA), etc. Considering the characteristics of rapidity and low temperature reaction, RPA reagents may be preferred. For example, RT-RPA reagents can include target-specific primers for specific amplification of genes for analyzing single base polymorphisms. The lysate can be a sample lysate based on a high-chaotropic salt such as guanidine salt. The washing solution can be an ethanol-based solution for cleaning the sample.

[0081] At the same time, the integrated device for driving the single base polymorphism analysis kit can be combined with the single base polymorphism analysis kit to configure an overall system for single base polymorphism analysis. That is, by providing a magnetic rod to promote the separation and reaction of the sample, the overall system can include an integrated device of the nucleic acid detection kit that can smoothly drive the magnetic particles used to analyze the single base polymorphism in the lysed sample and a particle probe 100 for detecting the signal.

[0082] By using the single-base polymorphism analysis kit, it is possible to start from the coating of the collected samples, regardless of the sample type, through a fully automatic process to complete sample lysis, nucleic acid capture and amplification, capture of amplified target nucleic acid, and activation of Cas12a reaction due to the captured target nucleic acid, etc., and quickly and accurately detect the presence or absence of single-base dimorphism by measuring the reduction in fluorescence signal due to the identified target nucleic acid.

[0083] According to another aspect of the present invention, a method for analyzing single base polymorphism is provided.

[0084] First, (a) the target nucleic acid is extracted from the sample. A lysis solution containing guanidine thiocyanate can be used to destroy the cell membrane of the sample and release the nucleic acid in the cell. The nucleic acid in the solution can be concentrated by attaching to magnetic silica beads or silica-coated microparticles 110 of the present invention, washing with alcohol, and then desorbing using an elution buffer.

[0085] Next, (b) the target nucleic acid is amplified. For example, a reverse transcription real-time polymerase chain reaction (RT-RPA) reagent can be used to specifically amplify the target nucleic acid.

[0086] Then, (c) a microparticle probe for analyzing base polymorphism is provided. The microparticle probe for analyzing single base polymorphism includes a microparticle, a guide RNA introduced to the surface of the microparticle and containing a complementary sequence to the target nucleic acid, and a reporter nucleic acid introduced to the surface of the microparticle and generating a signal by external stimulation. A detailed description of each component of the microparticle is as described above.

[0087] (d) The guide RNA of the microparticle probe for isolating and detecting the target nucleic acid is hybridized with the target nucleic acid to form a microparticle probe-nucleic acid complex for isolating and detecting the target nucleic acid.

[0088] In one embodiment, the microparticle probe for analyzing single base polymorphisms can be fixed with a guide RNA that is only specific to a specific virus and a reporter DNA attached with a 5'-fluorophore. The gRNA of the specific target magnetic particle probe that recognizes the single-stranded region to be amplified can form a complex with the gene showing the single base polymorphism through complementary binding. The formation of the complex may include a process of using an external magnetic force to promote the reaction. For example, in order to promote the reaction, the well containing the reactant can be heated to an appropriate temperature, and the microparticle containing the magnetic substance can be controlled by moving the magnetic rod up and down in the well to promote the reaction.

[0089] (e) Lyase is coupled to the guide RNA of the microparticle probe for isolating and detecting the target nucleic acid. A preferred example of lyase is Cas endonuclease, such as Cas12a.

[0090] The lyase recognizes and binds to the scaffold sequences of the gRNA immobilized on the microparticle probe for isolation and detection of the target nucleic acid.

[0091] (f) Subsequently, the base sequence of each target nucleic acid and the reporter nucleic acid is cut by the activation reaction of the lyase. If the amplified target nucleic acid binds to the gRNA, the Cas protein is activated and can cut each base sequence of the target nucleic acid and the reporter nucleic acid.

[0092] At the same time, the microparticle probe after the reaction is completed can be washed with a washing buffer, and preferably, a process of washing the microparticle probe using an external magnetic force can be included. That is, after the reaction is completed, a step of continuously washing the microparticle probe for analyzing base polymorphism in two or more washing wells using a magnetic strip can be further included. The product is washed. After washing, the presence or absence of single base polymorphism can be accurately confirmed by detecting the luminescent signal from the microparticle probe.

[0093] (g) Detecting the target nucleic acid by measuring the signal switch emitted from the complex caused by external stimulation. The signal may be a luminescent signal emitted from a luminescent substance in the complex. The external stimulus may be ultraviolet light, electron beam, chemical reaction, enzyme-substrate reaction, etc. For example, before being treated with Cas endonuclease, all microparticle probes can be turned on by the fluorescence generated by fluorescent molecules bound to the reporter nucleic acid under external stimulation such as ultraviolet light. Afterwards, when the Cas endonuclease binds to the guide RNA, the Cas endonuclease simultaneously cuts the base sequence of the reporter nucleic acid and the target nucleic acid, thereby turning off the fluorescence of the microparticle probe bound to the target nucleic acid. By superimposing the fluorescent image and the bright field image and analyzing them, the microparticle probe complexed with the target nucleic acid can be identified, thereby distinguishing the target nucleic acid.

[0094] In this case, the detection of the target nucleic acid can be performed by measuring the difference in luminescence of each probe using two or more microparticle probes for analyzing single-base polymorphism, thereby identifying the presence or absence of the target nucleic acid. That is, in the case of a nucleic acid without a single-base substitution and in the case of a nucleic acid with a single-base substitution, since different types of probes emit light respectively, these cases can be distinguished by acquiring a bright field image and a fluorescent field image in parallel.

[0095] In addition, by configuring two or more probes as discussed above, in which a capture probe that specifically binds to each nucleic acid is fixed, analysis of single base polymorphisms can be easily performed. For example, if the microparticle probe is a microrod, then by analyzing the bright field images and fluorescent field images of base-substituted and non-substituted nucleic acids using microrods with different lengths, and using gene scissors technology such as the CRISPR-Cas system, the type and quantity of each nucleic acid can be quickly and independently obtained. For example, if a microrod made of glass-coated microwires cut into various lengths is used as a microparticle probe, analysis of single base polymorphisms can be performed using only one fluorescent substance. By performing image analysis on microparticle probes with information encoded for each length, multiple detections can be achieved at one time, and it has the advantage of maintaining high sensitivity and accuracy.

[0096] That is, if two or more microparticle probes for analyzing single base polymorphisms are used, single base polymorphisms of a plurality of target nucleic acids can be analyzed by a single detection process.

[0097] Figure 2 and Figure 3 Demonstrates the process of detecting target nucleic acids using magnetic particle / gene scissors technology. Figure 2 and Figure 3, shows the target nucleic acid detection process performed in eight wells (wells 1 to 8), wherein a DNA capture step is performed in wells 1 to 3, a DNA amplification and hybridization step is performed in well 4, and an image analysis step is performed in wells 5 to 8. The procedures in each well are described in detail as follows.

[0098] 1) Well 1: Binding of DNA to silica beads

[0099] The sample is placed in well 1 containing magnetic silica beads, chaotropic salt and lysis solution, thereby destroying the cell membrane of the sample and releasing the nucleic acid in the cell, and then the nucleic acid is bound to the magnetic silica beads by the action of the chaotropic salt.

[0100] 2) Well 2: Washing and precipitation in ethanol

[0101] The magnetic silica beads bound with nucleic acid are transferred to well 2, and the reaction of DNA with salt is enhanced by 33% isopropanol, so that the DNA is firmly attached to the magnetic silica beads while removing unwanted residual substances.

[0102] 3) Well 3: Washing

[0103] Wash the magnetic silica beads with nucleic acid bound to them with 70% ethanol. In this process, remove as much residue as possible besides nucleic acid.

[0104] 4) Well 4: Nucleic Acid Amplification and Hybridization

[0105] The magnetic silica beads bound with nucleic acid are transferred to well 4 containing an elution buffer to separate the nucleic acid from the beads. Next, cDNA is synthesized and amplified using the RT-RPA reagent contained in well 4. The RT-RPA reagent may contain a target-specific primer for specifically amplifying a target nucleic acid for single base polymorphism analysis.

[0106] At the same time, guide RNA targeting only a specific base sequence and reporter DNA in the form of single-stranded random DNA with a 5'-fluorophore connection are fixed to each magnetic particle probe of a specific length. As the nucleic acid base to be analyzed is amplified by RT-RPA, the gRNA of the specific target magnetic particle probe that recognizes the single-stranded region amplified in this process complementarily binds to the nucleic acid to be analyzed, thereby forming a complex of the magnetic particle probe and the genetic sequence of the nucleic acid to be analyzed.

[0107] 5) Well 5: Binding of Cas protein and cleavage of reporter DNA sequence

[0108] The circular magnetic particles (RSMP) (i.e., magnetic microparticle probes) that complete the reaction in hole 4 are transferred to hole 5 containing Cas12a endonucleases. Cas12a protein recognition and binding to the scaffold sequence of gRNA fixed on the magnetic particle probe. If gRNA-Cas12a protein complex is formed and target DNA is amplified in hole 4 and combined with the spacer sequence of gRNA, the endonuclease function of Cas12a protein is activated, cutting target DNA and non-specific DNA around it. On the contrary, if target DNA is not amplified in hole 4, gRNA-Cas12a protein complex is formed in hole 5, but target DNA will not be combined with the spacer sequence of guide RNA, so the endonuclease function of Cas12a protein will not be activated.

[0109] 6) Well 6: Washing

[0110] After the reaction is completed, the magnetic microparticle probes are washed with a washing buffer.

[0111] 7) Well 7: Washing

[0112] After the reaction is completed, the magnetic particle probe is washed once more with washing buffer.

[0113] 8) Well 8: Image analysis

[0114] After the reaction and washing are completed, the magnetic microparticle probe is imaged, and both bright field and fluorescent field images are acquired simultaneously.

[0115] The above-mentioned microparticle probes for single nucleotide polymorphisms and methods of using the same may have the following advantages. The present invention overcomes the limitations of the existing reverse transcription-polymerase chain reaction (RT-PCR) method for analyzing single base polymorphisms by using CRISPR-CAS gene scissors technology. Existing molecular diagnostic methods involve the process of collecting samples on site, extracting genes from access mechanisms, amplifying and detecting, and using expensive equipment to obtain the results of gene amplification. Due to this process, existing methods are not suitable for testing a large number of samples on site, and it takes at least 6 hours or more to obtain results. However, the present invention introduces CRISPR-CAS gene scissors technology (the use of magnetic particles can significantly reduce the time required for gene extraction and amplification steps), so that the gene extraction, amplification and detection processes can be carried out on site and may be automated. In addition, the present invention overcomes the non-specific amplification phenomenon that occurs in existing molecular diagnosis by using CRISPR-Cas gene scissors technology.

[0116] The present invention introduces microparticle probe / gene scissors technology for analyzing single base polymorphisms, which can identify and manipulate the exact sequence of genes, and improves sensitivity and specificity compared to existing molecular diagnostic technologies. In addition, the time required for analyzing single base polymorphisms can be reduced to 30 to 40 minutes.

[0117] The preferred embodiments of the present invention will be described below with reference to the accompanying drawings so that those skilled in the relevant art can easily practice them. In addition, when describing the present invention, if it is considered that the detailed description of the relevant known functions or known configurations may obscure the core content of the present invention, the detailed description thereof will be omitted. In addition, for ease of explanation, some features presented in the drawings have been enlarged, reduced or simplified, and the drawings and their components are not necessarily drawn to appropriate scale. However, those skilled in the relevant art will easily understand these details.

[0118] Examples

[0119] In the example, the experimental steps performed as a single process are arbitrarily divided into a step of cleaving the target factor and capturing and amplifying the nucleic acid, a step of generating a magnetic particle probe for signal detection, and a step of detecting the signal according to the target factor reaction, etc., and each step is described in detail.

[0120] Example 1. Preparation of magnetic microparticles for capturing nucleic acids

[0121] In this single base polymorphism analysis system, eight wells are used, and in order to move nucleic acids between the wells, magnetic particles that can stably capture nucleic acids extracted from target factors and move between the wells must be used. In order to stably capture nucleic acids, cylindrical magnetic particles or commercially available magnetic particles with a size of 10 to 1000 μm are used as raw materials to manufacture magnetic particles with a sufficient surface area to capture nucleic acids.

[0122] First, the prepared basic magnetic particles (produced by our company; cylindrical rod-shaped particles with a length of 100-500 μm) are used as Figure 1 The basic magnetic particles in the core-shell form of the capture probes containing 110 were washed three times with a 0.1 M sodium hydroxide solution, three times with a 100% ethanol solution, and then ultrasonically cleaned for 2 seconds.

[0123] Next, 200 mg of magnetic particles were added to a 10 ml solution containing aqueous ammonia (components included 20 ml of 100% ethanol, 3 ml of deionized water and 1 ml of aqueous ammonia), 100 μl of TEOS was added per 10 mg of magnetic particles, and 2 ml of tetraethoxysilane (TEOS) solution was added. The magnetic particles were produced by adding 2 ml of TEOS solution for each reaction for a total of four reactions, wherein one reaction was set as a process of rotating the solution at 20 rpm for 20 minutes using a rotating device (FINEPCR, Hybridization Incubator Combi-H12). The magnetic particles obtained after the reaction were washed three times with the aforementioned aqueous ammonia solution, washed three times with 100% ethanol, and then stored in an ethanol solution.

[0124] In order to verify the ability of the prepared magnetic microparticles to capture nucleic acids, random samples were prepared to verify the ability to actually capture nucleic acids. Although virtual viruses or harmful bacteria can be assumed as random samples, in this example, random E. coli was selected as the target and whether nucleic acids were captured was confirmed.

[0125] Figure 5 Results are presented that initially confirm the possibility of capturing nucleic acids from target samples based on magnetic microparticles. Figure 5 A) is based on E. coli samples set to any target factor, and compared with the existing commercial E. coli plasmid mini-prep kit (Bioneer, coli introduced with plasmid DNA by using our magnetic microparticles-based nucleic acid capture beads and agarose gel electrophoresis to purify and separate plasmid DNA from E. coli introduced with plasmid DNA.

[0126] Reference Figure 5 A), it can be confirmed that our magnetic particles for nucleic acid purification are capable of purifying and capturing plasmid DNA at 100 and 500 particles.

[0127] Lysis buffer: Based on the buffer composition of well 1 (2M guanidine thiocyanate, 36.7mM Tris-Cl, 16.7mM EDTA, 2% Triton X-100, 33% isopropanol, RNase-free water), 1ml of concentrated cultured E. coli sample was mixed with our magnetic microparticle beads and then rotated for 10 minutes. Bands 1, 2-purification results 1, 2 using commercial plasmid preparation kits; Band 3-purification results based on our 100 (100ea) nucleic acid purification beads, Band 4-purification results based on our 500 (500ea nucleic acid purification beads; Band M: 1kb DNA ladder (Solgent, 1Kb Plus DNA Ladder)).

[0128] Figure 5 B) shows the Figure 5 A) The nucleic acid purification results confirmed by our company's 500 nucleic acid purification beads and plasmid preparation kit under the same conditions. Figure 5 B), it can be confirmed that compared with the plasmid preparation kit, our magnetic particle nucleic acid purification can simultaneously purify and recover E. coli genomic DNA and plasmid DNA.

[0129] Figure 5 C) shows the results confirming the capture ability of our magnetic particles for plasmid DNA at a concentration of 2 μg. Figure 5C), the ability of our magnetic particles for nucleic acid purification to capture nucleic acid samples at a ratio of approximately 7.3% can be used to confirm their ability to capture any nucleic acid.

[0130] Example 2. Target factor cleavage and nucleic acid capture and amplification steps (wells 1 to 4)

[0131] The suitability of our automated device, which performs a single process from target sample lysis to magnetic particle probe imaging, was confirmed. A mobility test was performed to confirm whether the target nucleic acids obtained by lysis and capture can undergo a continuous process through normal mobility between wells.

[0132] FIG. 6 shows the results of nucleic acid capture from a target sample and target site nucleic acid amplification in a multiplex diagnostic method according to one embodiment of the present invention.

[0133] Fig. 6A The target nucleic acid capture and amplification process from well 1 to well 4 is shown. Fig. 6A , lysis / nucleic acid capture was performed in well 1, and nucleic acids were extracted and captured from the virus sample using a solution containing denaturing salts (2 M guanidine thiocyanate, 36.7 mM Tris-Cl, 16.7 mM EDTA, 2% Triton X-100, 33% isopropanol, RNase-free water).

[0134] The first washing process (Wash-1) was performed in well 2, in which the magnetic particles containing captured nucleic acids were washed with 2M guanidine thiocyanate, 36.7mM Tris-Cl, 16.7mM EDTA, 2% Triton X-100, RNase-free water + 66% isopropanol, and the captured nucleic acids were further concentrated in the magnetic particles.

[0135] The second washing process (Wash-2) was performed in well 3 using 70% ethanol to remove the remaining waste attached to the magnetic particles.

[0136] The recovery and amplification of nucleic acids are performed in the well 4. After the nucleic acids attached to the magnetic particles are separated, the separated nucleic acids are amplified. Nucleic acid amplification is performed using a solution based on a recombinase polymerase amplification (RPA) enzyme complex.

[0137] Figure 6BThe results of the nucleic acid capture and amplification process for any nucleic acid such as RNA and DNA fragments (see SEQ ID NO: 1) are shown. The target nucleic acid separated in well 4 was subjected to reverse transcriptase recombinase polymerase amplification (RT-RPA) and RPA reaction at 37°C for 20 minutes when any nucleic acid RNA or DNA fragment was introduced. The RPA reagent composition used at this time was carried out according to the manufacturer's recommended protocol (TwistDx, TwistAmp Liquid Kit). Reference Figure 6B , confirming that each RNA sample and DNA sample was captured and moved between wells, separated in well 4, and then amplified normally by RT-RPA and RPA enzyme complex. In order to more clearly confirm the size of amplified nucleic acids less than 700 bases, TBE PAGE gel electrophoresis was performed.

[0138] Table 1 provides information on the targets used to test for single base polymorphisms.

[0139] Table 1:

[0140] +293 cells (human embryonic kidney cells) genomic DNA (CYP1A1-A) +Mutation (plasmid, CYP1A1-G)

[0141] Table 2 below shows the primers used to amplify the target sequence (115 bp).

[0142] Table 2:

[0143]

[0144] Table 3 below shows the information of guide RNA.

[0145] Table 3

[0146]

[0147] Table 4 below shows the reporter DNA used for fluorescence signal detection.

[0148] Table 4

[0149]

[0150] Based on the above results, it was indirectly confirmed that the target signal can be detected based on the amplified nucleic acid and the single base polymorphism can be analyzed.

[0151] Example 3. Preparation of magnetic particle probes for signal detection and functional verification (wells 4 to 5)

[0152] In the magnetic particle probe for signal detection, the guide RNA (gRNAs) that recognizes the target factor target site sequence and the gRNAs / target sequence / Cas12a complex play the role of the cutting sequence. In this case, the magnetic particle probe is produced in a state mixed with single-stranded reporter DNA, which plays a role in signal detection. Therefore, when the gRNAs that recognize the target site sequence are mixed with the reporter DNA and coupled to the magnetic particles, it is necessary to confirm whether they can play the role of gRNAs.

[0153] An experiment was designed to confirm the results of gRNAs or fluorescently labeled reporter DNA coupled to magnetic particles separately. The sequence information of gRNAs and reporter DNA can be found in SEQ ID NO:3 to SEQ ID NO:6 in the sequence listing.

[0154] Figure 7 The results of the Cas system activity were demonstrated by mixing free reporter DNA and Cas protein in the presence of target nucleic acid and coupling magnetic particle probes at different concentrations to see whether there is concentration-dependent gRNA variation. The purpose of this experiment was to verify the concept of the Cas system. To construct the free reporter DNA, a fluorescent factor was bound to one end and a quencher that absorbs the fluorescent signal was bound to the other end, and then tested. Figure 7 , if the gRNA sequence is coupled to the magnetic particle surface according to the concentration, it can be confirmed that the free reporter DNA (whose function of the fluorescent factor is masked by the quencher) will be cleaved at the quencher position according to the gRNA concentration, and the fluorescent signal is detected.

[0155] Figure 8 The experimental results based on the presence or absence of single base polymorphisms are shown. If a base that does not show single base polymorphisms is used (gRNA_A), a decrease in the probe designated as Target A can be observed, and this can be distinguished when taking bright field images and fluorescent images. However, since these polymorphisms do not appear in most bases, the change in light amount is very small compared to NC, which can minimize false positives.

[0156] In addition, using a base that exhibits single base polymorphism (gRNA_G), it was confirmed that the total amount of fluorescence was very low because fluorescence was observed only in certain bases. In particular, the probe designated as Target G was observed very weakly, so it was easy to distinguish them.

[0157] These results show that the amount of light from the probe changes significantly only when a polymorphism is present, confirming that single-base polymorphisms can be accurately analyzed while minimizing false positives.

[0158] Example 4. Verification of the detection limit of magnetic particle probes for signal detection

[0159] for Fig. 9 In the case of Example 3, the detection limit was confirmed by reducing the amount of gRNA used to 1 / 10 of that in Example 3. In Example 3, the amount of gRNA injected was 0.0125 pmol / μL, while in Example 4, 0.00125 pmol / μL was used to measure the detection limit. The results are shown in Table 9, and it was found that when there was a polymorphic base similar to Example 3, the change in light amount was greater.

[0160] That is, for the present invention, it was found to have a detection limit of 0.00125 pmol / μL, which means that even with an extremely small amount of sample, it is easy to analyze whether a single base polymorphism exists.

[0161] As mentioned above, although the specific parts of the present invention are described in detail, it is obvious to those skilled in the art that such specific description is only a preferred embodiment and is not intended to limit the scope of the present invention. Therefore, the actual scope of the present invention will be defined by the appended claims and their equivalents.

Claims

1. A microparticle probe for single nucleotide polymorphism, comprising: particle; a capture probe introduced onto the surface of the microparticle and containing a complementary sequence to the target nucleic acid; as well as A reporter nucleic acid is introduced onto the surface of the microparticle and generates a signal upon external stimulation.

2. The microparticle probe of claim 1, wherein the capture probe comprises a guide RNA. 3 . The microparticle probe according to claim 2 , wherein the guide RNA comprises a CRISPR RNA (crRNA) having a nucleotide sequence capable of hybridizing to a target site of a gene and a trans-activating crRNA (tracrRNA). 4 . The microparticle probe according to claim 2 , wherein the guide RNA binds to the target nucleic acid and, when encountering a lyase, cleaves the target nucleic acid and the reporter nucleic acid. The microparticle probe according to claim 1 , wherein the reporter nucleic acid is a nucleic acid labeled with a luminescent substance. 6 . The microparticle probe according to claim 1 , wherein the reporter nucleic acid becomes a base sequence to be cleaved when used in a gene scissors system. The microparticle probe according to claim 1 , wherein the interior of the microparticle contains magnetic particles. 8 . The microparticle probe according to claim 1 , wherein the microparticle has a core-shell structure having a core including a magnetic substance and a shell layer surrounding the core with a uniform thickness.

9. The particle probe according to claim 1, wherein the particles have a size and a specific gravity that are not buoyant in water.

10. A kit for analyzing single base polymorphism, comprising the microparticle probe for single nucleotide polymorphism according to any one of claims 1 to 9, a lyase and a nucleic acid amplification reagent.

11. A method for analyzing single base polymorphism, comprising the following steps: (a) extracting a target nucleic acid for analyzing single base polymorphism from a sample; (b) amplifying the target nucleic acid; (c) providing a microparticle probe for single nucleotide polymorphism, the microparticle probe comprising a microparticle, a guide RNA introduced onto the surface of the microparticle and containing a complementary sequence to a target nucleic acid, and a reporter nucleic acid introduced onto the surface of the microparticle and generating a signal upon external stimulation; (d) reacting the guide RNA of the microparticle for analyzing single nucleotide polymorphism with the target nucleic acid to form a complex of the microparticle probe for single nucleotide polymorphism and the target nucleic acid; (e) combining a lyase with a guide RNA of a microparticle for analyzing a single base polymorphism; (f) cleaving each base sequence of the target nucleic acid and the reporter nucleic acid through an activation reaction of the lyase; and (g) detecting the target nucleic acid by measuring the on / off of a signal emitted by the complex caused by the external stimulus.

12. The method for analyzing single base polymorphism according to claim 11, wherein the detection of the target nucleic acid is performed by using microparticle probes for analyzing two or more single base polymorphisms and measuring the difference in luminescence of each probe to identify the presence or absence of the target nucleic acid. 13 . The method for analyzing single base polymorphism according to claim 11 , wherein the microparticle probe for analyzing single base polymorphism has a microrod shape.

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