Diagnostic microparticle probe comprising magnetic particles and inactivated gene scissors, and multiple diagnostic system and multiple diagnostic method comprising same

Abstract

The present invention relates to a diagnostic microparticle probe comprising magnetic particles and inactivated gene scissors, and a multiplex diagnosis system and a multiplex diagnosis method comprising the same, the multiplex diagnosis method not only can perform rapid and accurate diagnosis, but also can perform multiple diagnosis by introducing a gene scissors technology into the diagnostic microparticle probe compared to the existing diagnostic methods, and can perform multiple diagnosis. The method can also be used for multiple diagnosis.

Description

Technical Field

[0001] The present invention relates to a diagnostic microparticle probe comprising magnetic particles and inactivated gene scissors, as well as a multiplex diagnostic system and multiplex diagnostic method comprising the same. More specifically, the diagnostic microparticle probe comprising magnetic particles and inactivated gene scissors, as well as a multiplex diagnostic system and multiplex diagnostic method comprising the same, can not only perform rapid and accurate diagnosis compared to existing diagnostic methods by introducing gene scissors technology into the diagnostic microparticle probe, but can also be used for multiplex diagnosis. Background Art

[0002] In vitro diagnostics (IVD) is a technology that diagnoses health conditions by analyzing targets such as blood, urine, and cells collected from the human body, including immunodiagnosis, self-blood glucose monitoring, molecular diagnostic technology, etc. Among these technologies, molecular diagnostic technology is a technology that uses molecular biotechnology to diagnose diseases. Due to the emergence of newly discovered gene mutations and infectious diseases in the fields of genetic diseases and infectious diseases, their scope is gradually expanding.

[0003] Among molecular biotechnologies, the recently developed gene scissors technology is a technology that recognizes specific base sequences of genes in cells and edits them as needed, and has attracted attention because of its ability to treat genetic diseases. The gene scissors technology has been continuously developed (first generation: zinc finger nucleases; second generation: transcription activator-like effector nucleases (TALENs); third generation: CRISPR-Cas), and recent efforts to make the gene scissors technology go beyond the scope of "gene editing" and develop into a faster and more accurate molecular diagnostic technology have attracted attention.

[0004] Meanwhile, due to the current global epidemic situation, as of May 2022, COVID-19 has infected 520 million people and caused 6.3 million deaths worldwide, and even in South Korea, 18 million people have been infected and 24,000 people have died, surpassing the record of the Spanish flu, which is considered to be the worst epidemic in the 20th century. Rapid diagnosis is needed to control this epidemic situation, but because the diagnosis takes about 1 to 2 days in South Korea and usually takes about 4 to 7 days in the United States, it will not be possible to effectively respond to reduce the spread of the disease. In addition, in the process of performing existing diagnostics, the markers partially or repeatedly bind during the reaction process, resulting in nucleic acid amplification, making the sequence used as the probe positive, thereby producing a false positive result. Therefore, there is an urgent need for new instant testing technologies that can provide accurate diagnostic results within 30 to 40 minutes. Summary of the invention

[0005]

Technical issues

[0006] In order to solve the above problems, the purpose of the present invention is to provide a diagnostic microparticle probe comprising magnetic particles and inactivated gene scissors, as well as a multiplex diagnostic system and a multiplex diagnostic method comprising the same. The multiplex diagnostic method introduces gene scissors technology into the diagnostic microparticle probe, which can not only perform rapid and accurate diagnosis compared to existing diagnostic methods, but can also be used for multiplex diagnosis.

[0007]

Technical solution

[0008] In order to solve the above problems, the present invention provides a diagnostic microparticle probe, which comprises: a microparticle; a capture probe introduced into the surface of the microparticle; and an inactivation gene scissors which is bound to the capture probe and contains a gene sequence complementary to a target nucleic acid.

[0009] In one embodiment, the inactivating gene scissors may comprise a guide RNA.

[0010] In one embodiment, the guide RNA may have a nucleic acid sequence that can hybridize to a target site of a target nucleic acid gene.

[0011] In one embodiment, the inactivation gene scissors may have an inactivation target nucleic acid cutting function.

[0012] In one embodiment, the microparticles may contain magnetic particles therein.

[0013] In one embodiment, the microparticle may have a core-shell structure including a core containing a magnetic material and a shell layer surrounding the core and having a uniform thickness.

[0014] In one embodiment, the particles may have a size and specific gravity that prevents them from floating in water.

[0015] The present invention also provides a multiplex diagnostic system comprising the diagnostic microparticle probe; a reporter nucleic acid that complementarily binds to a target nucleic acid and is labeled with biotin; and a reagent for amplifying the nucleic acid.

[0016] In one embodiment, the diagnostic particle probes may be labeled to distinguish one from another by diameter, thickness, length, shape, or an identification code.

[0017] In one embodiment, the diagnostic microparticle probe may be a mixture of two or more diagnostic microparticle probes having different lengths.

[0018] In one embodiment, the reporter nucleic acid may be two or more reporter nucleic acids to which phosphors of different colors are bound.

[0019] The present invention also provides a method for detecting a target nucleic acid, comprising the following steps: (a) extracting the target nucleic acid from a sample; (b) amplifying the target nucleic acid and binding a reporter nucleic acid thereto; (c) providing a diagnostic microparticle probe comprising a microparticle; a capture probe introduced onto the surface of the microparticle; and an inactivated gene scissors bound to the capture probe and containing a gene sequence complementary to the target nucleic acid; (d) reacting the diagnostic microparticle probe with the target nucleic acid to capture the target nucleic acid and the reporter gene; (e) attaching a fluorescent material to the reporter nucleic acid; and (f) detecting the target nucleic acid by measuring a fluorescent signal emitted from the stained reporter nucleic acid.

[0020] In one embodiment, the detection of the target nucleic acid can be a multiplex detection of two or more target nucleic acids using two or more diagnostic microparticle probes.

[0021] In one embodiment, the multiplex detection may include a process of reading the diagnostic particle probes that are labeled to distinguish one from another by length, diameter, thickness, shape, color, or identification code.

[0022] In one embodiment, the method may further comprise washing the diagnostic microparticle probe to which the target nucleic acid and the reporter nucleic acid are attached after step (e).

[0023] In one embodiment, the method for detecting the target nucleic acid can be performed in a microwell.

[0024] In one embodiment, the diagnostic microparticle probes can be moved and immersed between the microwells by an external magnetic force.

[0025]

Beneficial Effects

[0026] According to the diagnostic microparticle probe of the present invention, and the multiplex diagnostic system and multiplex diagnostic method comprising the same, multiplex diagnosis is facilitated by eliminating nonspecific secondary cleavage enzyme activity, and rapid diagnosis can be achieved compared to existing methods. Therefore, the use of the detection method of the present invention is more effective for point-of-care testing (POCT) multiplex diagnosis.

[0027] In addition, the diagnostic microparticle probes according to the present invention, and the multiplex diagnostic systems and multiplex diagnostic methods comprising the same can detect target nucleic acids at a faster rate than existing diagnostic methods, and therefore, they can be effectively used in the diagnosis of infections caused by viruses or bacteria. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The structure of a molecular diagnostic microparticle probe according to an embodiment of the present invention and its binding relationship with target nucleic acid, reporter nucleic acid and fluorescent molecule are shown.

[0029] Figure 2 FIG. 1 is a diagram showing a diagnostic microparticle probe according to an embodiment of the present invention.

[0030] Figure 3 A diagram showing a process of detecting target nucleic acid using magnetic particle / inactivated gene scissors technology according to one embodiment of the present invention.

[0031] Figure 4 A diagram showing a process of detecting a target nucleic acid using magnetic particle / inactivated gene scissors technology according to an embodiment of the present invention, wherein A represents a process with gene amplification and B represents a process without gene amplification.

[0032] Figure 5 To illustrate an embodiment according to the present invention in detail, Figure 3 A diagram of the process of capturing the target gene in the process, where A represents the process with gene amplification and B represents the process without gene amplification.

[0033] Figure 6 The figure is a diagram that virtually shows the diagnosis result of a process according to one embodiment of the present invention.

[0034] Figure 7 The results show the preliminary confirmation of the feasibility of capturing nucleic acids in target samples based on magnetic microparticles.

[0035] Figure 8 A diagram showing a process of capturing and amplifying target nucleic acid step by step from well 1 to well 4 according to an embodiment of the present invention.

[0036] Fig. 9 The figure shows the result of performing a nucleic acid capture and amplification process on influenza B virus RNA according to one embodiment of the present invention.

[0037] Fig.10 The results of introducing amplified DNA according to one embodiment of the present invention into the multiplex diagnostic system of the present invention are shown.

[0038] Fig.11 The results of detecting various viruses using a multiplex diagnostic method according to one embodiment of the present invention are shown.

[0039] Fig.12 The results of simultaneously detecting various viruses using a multiplex diagnostic method according to one embodiment of the present invention are shown. DETAILED DESCRIPTION

[0040] Hereinafter, the preferred embodiments of the present invention will be described in detail. When describing the present invention, if it is judged that a specific description of the relevant known technology may make the gist of the present invention unclear, the detailed description will be omitted. Throughout the specification, it is understood that unless the context clearly indicates otherwise, the singular form includes plural elements; and it is understood that the terms used in this specification, such as "including" or "having", are intended to indicate the existence of the stated features, quantities, steps, operations, components, parts or combinations thereof, rather than excluding the existence or addition of one or more other features, quantities, steps, operations, components, parts or combinations thereof. In addition, when performing the method or preparation method, the order of each process constituting the method may occur in a different order from the specified order unless it is clearly described in the context. That is, each procedural step may occur in a specified order, may be performed substantially simultaneously, or may be performed in the opposite order.

[0041] The technology disclosed in this specification is not limited to the embodiments described herein, and may be implemented in other forms. However, the embodiments introduced herein are provided to make the contents disclosed herein comprehensive and complete, and to make the technical spirit of the present technology fully understood by those skilled in the art. In the accompanying drawings, in order to clearly express the components of each device, the component dimensions such as width or thickness are magnified from certain angles. In general, when describing the accompanying drawings, it is described from the perspective of the observer; when it is pointed out that an element is "above" another element, this includes all the meanings that an element is directly above the other element, or that other elements may intervene between them. In addition, those skilled in the art may implement the spirit of the present invention in a variety of other forms without departing from the scope of the technical spirit of the present invention. In addition, the elements indicated by the same figure mark in multiple drawings represent substantially the same elements.

[0042] In this specification, the term "and / or" includes any one or a combination of multiple items listed. In this specification, "A or B" may include "A", "B" or "both A and B".

[0043] The present invention relates to a diagnostic particle probe, which comprises: a particle; a capture probe introduced onto the surface of the particle; and an inactivation gene scissors which is bound to the capture probe and contains a gene sequence complementary to a target nucleic acid.

[0044] Reference will be made to Figure 1 and 2 A diagnostic microparticle probe according to the present invention is described.

[0045] The particle 110 may be a single structure or a core-shell structure. Preferably, the particle 110 may have a core-shell structure of a core 112 and a protective shell 114 surrounding the core 112. The core 112 of the particle 110 may include a magnetic material, such as a magnetically responsive metal, which may be a paramagnetic material. Specifically, the magnetically responsive metal may be an alloy containing iron (Fe), nickel (Ni), cobalt (Co) or manganese (Mn). The magnetically responsive metal may contain transition metals such as iron, nickel, cobalt and manganese as main components, and rare earth metals such as titanium such as gadolinium (Gd), terbium (Tb), samarium (Sm), and may contain other elements such as boron (B), silicon (Si) and carbon (C). Representative examples of magnetically responsive metals may be iron alloys or cobalt alloys. A specific example of an iron alloy may be Fe. 70 B 15 Si 10 C5, an example of a cobalt alloy may be Co 68 Mn7Si 10 B 15 .

[0046] Preferably, the particle 110 has a size and a specific gravity that prevents it from floating on water, so that it can be quickly collected or separated by an external magnetic force (such as a permanent magnet or an electromagnet). In this case, the core 112 may account for 60% or more of the total volume of the particle 110. For example, the volume of the core 112 may be 60% to 99%, preferably 75% to 99% of the total volume of the particle 110. If the volume of the core is less than 60%, the magnetism of the core may be reduced, making it difficult to move using a magnetic rod; if it exceeds 99%, the thickness of the shell will be reduced, resulting in reduced durability.

[0047] In addition, the microparticle 110 may have a size (e.g., diameter or length) of tens to hundreds of μm, and preferably has a specific gravity of 5 or higher. If the microparticle 130 is a nanoparticle having a size less than 1 micron, it may float in water even if it is a particle with a high specific gravity (e.g., 7.876 for iron). In this case, during the single base polymorphism analysis using the microparticle 110, when a magnetic field is applied, the magnetic control of the microparticle may not be smooth due to the low magnetic force of the microparticle, which may cause difficulty in separation. When the magnetic bar moves up and down in the hole to promote an immune response, the microparticles will detach from and attach to the magnetic bar, and after the magnetic bar moves up and down, even if the magnetic field is removed, the microparticles that have been attached to the magnetic bar will not fall back to the bottom of the hole due to the low weight of the microparticles, and may continue to remain on the magnetic bar through non-specific binding. In this case, when the biological material in the hole is to be detected, the reproducibility of the quantitative analysis may be reduced.

[0048] The diagnostic particle probe according to one embodiment of the present invention is much larger than the size of existing silica beads, and the magnetic responsive metal (magnetic core) occupies most of the volume of the particle. Unlike the existing silica beads, the magnetic particles are usually dispersed inside the silicon. Therefore, it is sensitive to magnetic force and has excellent quantitative analysis reproducibility.

[0049] Meanwhile, the diagnostic microparticle probe 100 of the present invention forms a core-shell structure having a magnetically responsive metal in the center and a shell layer surrounding it, wherein the shell layer 114 may be composed of an organic or inorganic material, and is preferably glass. In addition, capture probes such as antibodies, proteins, nucleic acids, and metabolites may be fixed to the surface of the diagnostic microparticle probe 100. Preferably, for this purpose, functional groups such as acryl, hydroxyl, amino, or carboxyl groups may be introduced into the surface.

[0050] The shell 114 may substantially completely surround the particle 110 , but when necessary or during the manufacturing process, the surface of the particle may not be completely covered by the shell, and some surface areas of the particle may be exposed.

[0051] The shell layer 114 may have a thickness of 1 μm to 100 μm, preferably 1 μm to 50 μm, more preferably 1 μm to 10 μm, and even more preferably 4 μm to 8 μm. If the shell layer 114 has a thickness below the range, the surface of the shell layer 114 is easily broken or cracked; if it has a thickness exceeding the range, problems based on laser characteristics and wavelength may occur during glass cutting.

[0052] The core-shell structure may be formed by applying a liquid shell component to a metal core, or filling a metal core component into a hollow framework.

[0053] In this case, the shell layer 114 may be solidified from a liquid coating solution of an organic or inorganic material. More specifically, the shell layer 114 may be formed by preparing a liquid coating solution in such a way that a shell component in the form of an organic or inorganic material is melted or becomes flowable at a high temperature, or in such a way that it is dissolved in a solvent, and then the liquid coating solution is applied to the metal core. The organic material is mainly a polymer; and the inorganic material may be a metal or a ceramic, particularly glass. For example, in order to form the shell layer 114, a coating solution obtained by dissolving plastic or molten glass in a solvent is applied to the particle 110 by dip coating, spraying, or the like.

[0054] For example, when a glass tube is used as the hollow frame, there are the following methods: a method of stretching the glass tube while injecting metal powder into the glass tube and then melting the metal at a high temperature; a method of injecting molten metal into a glass material while first stretching the glass material; a method of filling a dispersion obtained by dispersing metal powder in an ultraviolet light-curable material into a glass tube and then curing it by irradiating ultraviolet light. In these methods, the hollow frame itself can become a shell layer.

[0055] The microparticles 110 obtained by the above method have excellent surface uniformity. Existing bioanalysis particles use silicon precursors such as TEOS to grow on the surface of core particles to form a shell 114, wherein the shell 114 has a very rough surface. As a result, non-specific binding of the material to be detected may often occur, which may cause unnecessary background noise.

[0056] On the other hand, the glass forming the shell 114, for example, borosilicate glass, can minimize nonspecific binding due to adsorption by chemical reaction with the reaction sample. In particular, the microparticle 130 according to an embodiment of the present invention has a coating obtained from a liquid component as the shell 114, and therefore has a very uniform surface. The surface of the shell 114 has an average roughness (Ra) of 15nm or less, preferably 10nm or less, more preferably 5nm or less, even more preferably 2nm or less, and particularly 1.5nm or less. In addition, Ra can be, for example, 3nm or more, 2nm or more or 1nm or more. If the surface roughness falls within the above range, the nonspecific adsorption of the reaction sample on the surface of the shell 114 can be minimized.

[0057] Considering strength and transparency, the shell 114 of the particle 110 can be made of glass. The glass contains a compound selected from the group consisting of soda lime, borosilicate, aluminosilicate, silicon dioxide, alkali silicate, heat-resistant glass, and quartz as a main component. Preferably, for the purpose of heat resistance, acid resistance, and water resistance experiments, the glass can be borosilicate.

[0058] The microparticles 110 may have various shapes, including regular shapes such as rods, plates, spheres, etc., or irregular shapes. Even if the microparticles 110 have a plate shape, their cross-sections may have a variety of shapes such as stars, polygons, circles, etc., and are not particularly limited. Preferably, for ease of manufacture and easy observation, the microparticles are preferably in the form of microrods, microdisks or microbeads, and are particularly preferably in the form of microrods. If the microparticles 110 have a microrod form, overlapping microparticles can be easily distinguished, and if they are placed in a hole, they will be easy to focus, and the area occupied by individual particles is small, so a large number of microparticles can be observed in a single observation field of view. In addition, if the microparticles have a complex cross-sectional structural shape, such as a star shape, edge breakage may occur due to collisions with each other or with the hole when moving in the hole, so that simple shapes such as microrods are more preferred.

[0059] The microrod may have a length of 10 to 1,000 μm. If the length of the microrod is less than this range, it may be difficult to distinguish particles of different lengths; if the length of the microrod exceeds this range, the particles may overlap, making observation difficult. In addition, the microrod may have a length-to-diameter ratio (aspect ratio) of 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 less than this range, the particles may be approximately spherical, making it difficult to distinguish one from the other; if the aspect ratio is too large, they may be curved.

[0060] In a preferred embodiment, the microparticles 110 can be cut segments of glass-coated metal microwires. Microparticles 110 of various lengths can be obtained by simply cutting the glass-coated metal microwires with a laser.

[0061] The microparticles can be used for diagnosis of specific samples derived from organisms. The samples can be tissue extracts, cell lysates, whole blood, plasma, serum, saliva, eye fluid, cerebrospinal fluid, sweat, urine, milk, ascites, synovial fluid, peritoneal fluid, etc. For rapid diagnosis, the pretreatment of the sample solution can be simplified or omitted in some cases.

[0062] Microparticles can be prepared and used as examples of different sizes, lengths or shapes for POCT diagnostics in specific samples from biologically derived samples.

[0063] Capture probes 120 may be introduced into microparticles 110 to capture biomarkers derived from a sample, particularly nucleic acid-based biomarkers. Biomarkers may be used without limitation as long as they are used in existing scientific or medical fields, such as measuring or evaluating biological therapeutic processes, processes that induce pathogenicity, and pharmacological processes for treatment. Biomarkers may be, for example, polypeptides, peptides, nucleic acids, proteins, or metabolites that can be detected in biological fluids, such as blood, saliva, and urine, and preferably, biomarkers are nucleic acids, wherein the biomarkers are associated with specific diseases and can be detected with high sensitivity and specificity.

[0064] In order to capture the target nucleic acid extracted from the sample, the microparticle according to one embodiment of the present invention may have a capture probe 120 introduced with a shell 114. The capture probe 120 is complementary to the target nucleic acid and thus reacts to specifically bind to the target nucleic acid to immobilize the target nucleic acid on the microparticle 110.

[0065] The capture probe may comprise inactivated genetic scissors comprising a genetic sequence complementary to the target nucleic acid.

[0066] General gene scissors will bind to the target nucleic acid and then cut a part of the target nucleic acid under external stimulation. This behavior is similar to scissors to cut the desired position in the gene, so it is called gene scissors or CRISPR scissors. The present invention focuses on the ability of such gene scissors to bind to the target nucleic acid, and uses gene scissors to bind to the target nucleic acid, but the sequence of the target nucleic acid can be retained by inactivating the nucleic acid cutting function of the gene scissors while binding to the target nucleic acid.

[0067] In this case, the gene scissors can inactivate the target nucleic acid cleavage function by existing techniques. For example, the nucleic acid cleavage function of the gene scissors can be selectively inactivated by heat treatment, acid or alkali treatment, ultraviolet irradiation, insertion of heterologous base sequences, etc.

[0068] The inactivated gene scissors may include guide RNA. Guide RNA refers to RNA that is specific to a target nucleic acid (e.g., RNA that can hybridize to a target site on DNA), and can be used as a common element of a CRISPR system and a cutting enzyme. However, since the cutting function of the CRISPR system (gene scissors) in the present invention has been inactivated, a cutting enzyme is not included. Generally speaking, when the guide RNA selectively binds to the target nucleic acid and then the cutting enzyme is introduced, the target nucleic acid is cut, but in the present invention, since this cutting function has been inactivated, the guide RNA can only be used to selectively bind to the target nucleic acid.

[0069] The capture probe 120 can be fixed to the surface of the microparticle 110 by adsorption, or chemically linked to the surface of the microparticle 110, and preferably linked to a functional group on the surface of the shell 114. For example, the capture probe 114 can be attached to the microparticle 110 by introducing biotin to the surface of the capture probe 114 and introducing avidin, neutravidin or streptavidin that binds to biotin to the microparticle 110. Alternatively, the capture probe 120 can be linked to the microparticle 110 using a hydroxyl group, an amino group or a carboxyl group on the surface of the microparticle 110.

[0070] The present invention also provides a POCT multiplex diagnostic system, which comprises diagnostic microparticle probes; a reporter nucleic acid that complementarily binds to a target nucleic acid and is labeled with biotin; and a reagent for nucleic acid amplification.

[0071] As for the diagnostic microparticle probe, since it is the same as described above, its description is omitted.

[0072] The reporter nucleic acid refers to a core that generates a signal according to the presence or absence of a desired target nucleic acid, and the reporter nucleic acid of the present invention can complementarily bind to the target nucleic acid and can be labeled with biotin.

[0073] As described above, the reporter nucleic acid labeled with biotin can be complementary to a portion or all of the sequence of the target nucleic acid, and in particular, it can be simultaneously amplified and complementary to the target nucleic acid during the amplification process of the target nucleic acid. In this way, most of the target nucleic acid will bind to the reporter nucleic acid labeled with biotin, which means that most of the target nucleic acid bound to the diagnostic microparticle probe will bind to the reporter nucleic acid labeled with biotin.

[0074] In the method of using existing microparticles for diagnosis, the reporter nucleic acid is provided as a part of the probe and bound to the microparticle. That is, the reporter nucleic acid exists as a part of the diagnostic microparticle probe, and a signal is generated by controlling whether the reporter nucleic acid is activated. However, in this case, since the binding and detection of the target nucleic acid and the presence or absence of activation of the reporter nucleic acid are performed at different stages, not only the sensitivity is reduced, but also an additional step must be performed, making the overall diagnostic method complicated.

[0075] However, in the present invention, the target nucleic acid can be selectively attached to the surface of the microparticle by the above-mentioned CRISPR system, and the reporter nucleic acid is complementarily bound to the target nucleic acid, and thus high selectivity is achieved, and sensitivity can also be increased.

[0076] In addition, in the diagnostic method using the existing CRISPR system, the presence or absence of the target nucleic acid is detected by partially or completely cutting the reporter nucleic acid to block the signal when the target nucleic acid is bound as described above, but in this method, if the target nucleic acid concentration is low, only the signal is blocked by cutting the reporter nucleic acid portion on the surface of the microparticle, which may cause the signal intensity to be somewhat reduced, and the signal may still be emitted. As a result, false positives increase significantly and sensitivity may inevitably decrease. However, in the present invention, since the target nucleic acid labeled with the reporter nucleic acid has been captured on the surface of the microparticle by the CRISPR system, even if the concentration of the target nucleic acid is low, some reporter nucleic acids may still generate signals, and therefore, high sensitivity can be achieved.

[0077] The reporter nucleic acid 130 can generate a signal such as a light signal or an electrical signal by external stimulation, including chemical energy, mechanical energy, light energy, and electrical energy. In one embodiment, the reporter nucleic acid 130 can be a nucleic acid labeled with a luminescent material and can generate a luminescent signal such as fluorescence or chemiluminescence by an external photoreaction or chemical reaction.

[0078] Specifically, the reporter nucleic acid 130 can be a DNA complementary to the target nucleic acid. In addition, the reporter nucleic acid can be a 15-mer to 40-mer nucleic acid. In addition, it can be 18-mer to 30-mer, 19-mer to 25-mer or 20-mer to 23-mer.

[0079] After the luminescent material is linked to the reporter nucleic acid, it can generate light through external stimulation such as ultraviolet rays, electron beams, chemical reactions or enzyme-substrate reactions, thereby detecting the presence of the target nucleic acid. Examples of luminescent materials may include fluorescent molecules, quantum dots, metal nanoparticles, magnetic nanoparticles, enzymes, enzyme substrates, etc. Among these, fluorescent molecules can be selected as various luminescent materials in consideration of the convenience of acquisition and application.

[0080] As an example of the fluorescent molecule, the fluorescent molecule is selected from the group consisting of fluorescein isothiocyanate (FITC), fluorescein, fluorescein amide (FAM), phycoerythrin (PE), europium, tetramethylrhodamine isothiocyanate (TRITC), cyanine dye 3 (Cy3), cyanine dye 5 (Cy5), cyanine dye 7 (Cy7), Alexa Fluor dye and rhodamine, which may be commonly used.

[0081] To detect various biomarkers in a sample, various fluorophores such as FITC, PE, and Alexa-647 can be introduced and labeled at the 5′-end of the reporter nucleic acid 130 .

[0082] In addition, in the present invention, the fluorescent material can be supplied after the target nucleic acid is bound to the capture probe and combined with the reporter nucleic acid. In this case, since the fluorescent material that is not bound to the reporter nucleic acid but non-specifically bound can be simply removed by washing, only the specifically bound target nucleic acid and the fluorescent material remain on the surface of the microparticle.

[0083] The multiplex diagnostic system may include a reagent for nucleic acid amplification. The reagent for nucleic acid amplification is used to amplify nucleic acid and is not particularly limited, but may be loop-mediated isothermal amplification (LAMP), single primer isothermal amplification (SPIA), recombinase polymerase amplification (RPA), etc., wherein rapidity and low temperature reaction characteristics are considered, nucleic acid amplification is performed at isothermal, and preferably RPA reagent. For example, RT-RPA reagents may contain specific primers for specifically amplifying the target nucleic acid. In addition, as discussed above, while the target nucleic acid is amplified, the reporter nucleic acid may also be amplified. The reporter nucleic acid of the present invention has a sequence complementary to the target nucleic acid, and therefore, during the period when the target nucleic acid is amplified, the reporter nucleic acid may also be amplified, with the result that most of the target nucleic acid amplified as described above may exist in combination with the reporter nucleic acid.

[0084] The diagnostic particle probes may be labeled to distinguish one from another by diameter, thickness, length, shape, or an identification code, etc.

[0085] In the present invention, gene scissors technology such as CRISPR-Cas system is used to capture the target nucleic acid on the surface of the microparticle. In this case, when two or more types of gene scissors, such as CRISPR-Cas system, are used, different target nucleic acids can be captured simultaneously.

[0086] However, since it is difficult to distinguish different types of target nucleic acids by simultaneous capture alone, if the shapes of the particles are different and individual gene scissors, such as the CRISPR-Cas system, are used, different target nucleic acids can be easily distinguished based on the shape differences and can be detected simultaneously.

[0087] For example, if the microparticle probe is a microrod, by using microrods of different lengths and applying gene scissors technology such as CRISPR-Cas, the type and quantity of each nucleic acid can be quickly and independently obtained by analyzing the bright field image and the fluorescent image for the first detection nucleic acid and the second detection nucleic acid, respectively. For example, if a microrod made by cutting a glass-coated microwire into various lengths is used as a microparticle probe, a single base polymorphism can be analyzed with only one fluorescent material. Microparticle probes encoded with their respective length information through image analysis will have the advantage of being able to perform multiple tests at once and maintain high sensitivity and accuracy.

[0088] Different from this, a fluorescent material attached to the reporter nucleic acid can also be used to distinguish various target nucleic acids. When two or more specific bindings to various fluorescent materials are used, the reporter nucleic acid can emit fluorescence of different wavelengths according to the type of each target nucleic acid, and if they are distinguished by polarization analysis or wavelength analysis, the presence or absence of two or more target nucleic acids can be judged at the same time. However, in this method, the types of reporter nucleic acids bound to fluorescent materials are limited, and in some fluorescent materials, the overall efficiency may be lower than the detection method using microparticle shape due to overlapping emission wavelengths or the need for light sources of various wavelengths to emit.

[0089] The present invention also provides a method for detecting a target nucleic acid, comprising the following steps: (a) extracting the target nucleic acid from a sample; (b) amplifying the target nucleic acid and binding a reporter nucleic acid thereto; (c) providing a diagnostic microparticle probe comprising a microparticle; a capture probe introduced onto the surface of the microparticle; and an inactivated gene scissors bound to the capture probe and containing a gene sequence complementary to the target nucleic acid; (d) reacting the diagnostic microparticle probe with the target nucleic acid to capture the target nucleic acid and the reporter gene; (e) attaching a fluorescent material to the reporter nucleic acid; and (f) detecting the target nucleic acid by measuring a fluorescent signal emitted from the stained reporter nucleic acid.

[0090] First, (a) extract the target nucleic acid 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 attached to magnetic silica beads or silica-coated microparticles 110 of the present invention, concentrated, washed with alcohol, and desorbed using an extraction buffer.

[0091] Next, (b) amplify the target nucleic acid. For example, a real-time reverse transcription polymerase chain reaction reagent can be used to specifically amplify the target nucleic acid. In addition, as discussed above, during this process, the reporter nucleic acid can also be amplified at the same time, and after the amplification is completed as described above, the target nucleic acid and the reporter nucleic acid can be complementary.

[0092] Subsequently, (c) a diagnostic microparticle probe is provided. The diagnostic microparticle probe comprises a microparticle; a capture probe introduced onto the surface of the microparticle; and an inactivation gene scissors which is bound to the capture probe and contains a gene sequence complementary to the target nucleic acid. The detailed description of each component of the existing microparticle is as described above.

[0093] (d) by reacting the diagnostic microparticle probe with the target nucleic acid to capture the target nucleic acid and the reporter gene. As discussed above, since the guide RNA included in the gene scissors in the diagnostic microparticle probe can complementarily bind to the target nucleic acid, the target nucleic acid can be selectively captured by the diagnostic microparticle probe. In this case, the reporter nucleic acid complementarily binds to the target nucleic acid, and the reporter nucleic acid can also be attached to the diagnostic microparticle probe. As a result, a diagnostic microparticle probe-nucleic acid complex can be formed.

[0094] In addition, the formation of the complex may include a process of promoting the reaction using an external magnetic force. For example, the well containing the reactants may be heated to an appropriate temperature to promote the reaction, and the particles containing the magnetic material may be controlled by continuously moving a magnetic bar up and down in the well to promote the reaction.

[0095] (e) Attaching a fluorescent material to the reporter nucleic acid A fluorescent material is attached to the reporter nucleic acid and can emit a fluorescent signal by external stimulation in the following step.

[0096] In addition, the fluorescent material should only be attached to the reporter nucleic acid, but it can be non-specifically attached or physically adsorbed to an unwanted position. The abnormal attachment of this fluorescent material is used as a factor indicating a false positive result. In the present invention, this abnormally bound fluorescent material can be easily removed by washing. That is, after step (e), the method may include a process of washing the diagnostic microparticle probe to which the target nucleic acid and the reporter nucleic acid are attached.

[0097] In addition, this washing can remove the reporter nucleic acid that has not bound to the target nucleic acid and residual reagents in the previous process, thereby improving the diagnostic accuracy of the method of the present invention.

[0098] Step (f) may be detecting the target nucleic acid by measuring a fluorescent signal emitted from the stained reporter nucleic acid.

[0099] If the target nucleic acid, the reporter nucleic acid and the fluorescent material are attached to the diagnostic microparticle probe as discussed above, the fluorescent material can generate a signal by external stimulation. In this case, the target nucleic acid can be detected by measuring the switch of the signal emitted by the complex by external stimulation. The signal can be a luminescent signal emitted by the luminescent material in the complex. The external stimulus can be ultraviolet rays, electron beams, chemical reactions, enzyme-substrate reactions, etc. For example, before the target nucleic acid and the reporter nucleic acid are bound to the diagnostic microparticle probe as described above, the diagnostic microparticle probe will not emit light even if an external stimulus such as ultraviolet rays is applied. However, if the target nucleic acid and the reporter nucleic acid are attached to the guide RNA through the above process, and the fluorescent material is bound to the reporter nucleic acid, the diagnostic microparticle probe can emit light by external stimulation as described above. Therefore, by overlapping the fluorescent image with the bright field image and analyzing it, the microparticle probe complexed with the target nucleic acid can be identified so that the target nucleic acid can be distinguished.

[0100] In this case, the target nucleic acid can be detected by measuring the difference in luminescence of each probe using a microparticle probe for analyzing two or more single base polymorphisms to identify whether the target nucleic acid is present. That is, when an unexpected nucleic acid is present and when a desired target nucleic acid is present, since different types of probes emit light, they can be distinguished by taking a bright field image and a fluorescent image in parallel. Different from this, even in the presence of two or more types of target nucleic acids, they can be distinguished separately and attached to each diagnostic microparticle probe, so that they can be distinguished separately by the shape in the diagnostic microparticle probe on the fluorescent image (see Figure 6 ).

[0101] The method for detecting the target nucleic acid of the present invention can be performed in a microwell. Although the above detection process can be performed in each test tube or container, by performing it in a microwell, the effect of miniaturization and uniformity can be achieved. That is, the diagnostic microparticle probe, buffer, reagent for amplification, washing buffer, etc. can be sequentially loaded into the microwell, and the method for detecting the target nucleic acid can be performed by providing a sample and sequentially moving the diagnostic microparticle probe (see Figures 3 to 5 ).

[0102] Details are as follows.

[0103] 1) Well 1: nucleic acid binds to microparticles

[0104] The sample is placed in well 1 containing magnetic particles, chaotropic salt and lysis solution to destroy the membrane of the sample, release the nucleic acid in the membrane, and make the nucleic acid adhere to the magnetic particles under the action of the chaotropic salt.

[0105] 2) Hole 2: Cleaning and removing impurities

[0106] The magnetic microparticles with attached nucleic acid are transferred to well 2, and unnecessary residual impurities are removed with a solution containing 33% isopropanol and salt.

[0107] 3) Hole 3: Cleaning

[0108] The magnetic particles bound with nucleic acids are washed with 70% ethanol. In this process, salts other than nucleic acids can be removed.

[0109] 4) Well 4: Nucleic acid amplification or hybridization

[0110] The magnetic particles bound with nucleic acid are transferred to well 4 containing reaction buffer, and the nucleic acid is desorbed from the magnetic particles. Then, the cDNA of the target nucleic acid is synthesized and amplified with the RT-RPA reagent contained in well 4, or the target nucleic acid is hybridized with a reporter nucleic acid having a sequence complementary to the target nucleic acid and labeled with biotin. The RT-RPA reagent can contain target-specific primers for specifically amplifying the genes of RSV, influenza virus and coronavirus, and labeling them with biotin.

[0111] At the same time, three different lengths of magnetic microparticle probe / inactivated gene scissors complexes are prepared. Each specific length of magnetic microparticle probe complex is fixed with a guide RNA / dCas9 complex that is specific to a specific virus. When the target gene is amplified by RT-RPA, it is captured by the magnetic microparticle complex that recognizes the double-stranded position of the target gene amplified in the process and forms a capture body.

[0112] On the other hand, when there is no RT-RPA process, the biotin-labeled reporter nucleic acid having a complementary sequence to the target nucleic acid hybridizes with the target nucleic acid and reacts with three magnetic particle complexes having three different lengths to capture the target gene and form a capture body.

[0113] 5) Well 5: Fluorescence staining of magnetic microparticle target nucleic acid capture body

[0114] The magnetic microparticle target nucleic acid capture body that has completed the reaction in well 4 is transferred to well 5 containing streptavidin and R-phycoerythrin complex (SAPE). SAPE will bind to the biotin labeled on the magnetic microparticle target nucleic acid capture body.

[0115] 6) Hole 6: Cleaning

[0116] The magnetic microparticle target nucleic acid capture bodies that have completed the reaction are washed with a washing buffer.

[0117] 7) Hole 7: Cleaning

[0118] The magnetic microparticle target nucleic acid capture bodies that have completed the reaction are washed again with a washing buffer.

[0119] 8) Well 8: Image analysis

[0120] The magnetic microparticle target nucleic acid capture body that has completed the reaction and washing is imaged. In this case, both bright field images and fluorescent images are obtained.

[0121] The above-mentioned diagnostic microparticle probe and the diagnostic method used therein have the following advantages. The present invention overcomes the limitations of POCT analysis using the existing reverse transcription polymerase chain reaction by using CRISPR-Cas gene scissors technology. The existing molecular diagnostic method for POCT analysis consists of a process of on-site sampling, gene extraction, amplification and detection at a trustee institution, and obtaining gene amplification results using expensive equipment. These processes are not suitable for a large number of on-site sample tests and take at least 6 hours to obtain results. However, the present invention introduces CRISPR-Cas gene scissors technology, so that the process of gene extraction, amplification and detection can be realized and automated on-site (the time required for gene extraction and amplification can be significantly shortened by using magnetic microparticles). In addition, the present invention has overcome the non-specific amplification phenomenon seen in existing molecular diagnosis by using inactivated CRISPR-Cas gene scissors technology. Finally, in the existing activated CRISPR-Cas gene scissors technology, the disappearance of the fluorescence signal is judged as positive, but in this case, if the amount of target nucleic acid is small, there is a high probability of false positives. However, in the present invention, since the fluorescence emission is judged as positive by using the inactivated gene scissors technology, it has higher selectivity and sensitivity than the existing method.

[0122] The preferred embodiments of the present invention will be described hereinafter so that those skilled in the art can easily implement them with reference to the accompanying drawings. In addition, when describing the present invention, if it is determined that a specific description of a related known function or known structure may unnecessarily obscure the subject matter of the present invention, its detailed description will be omitted. In addition, for ease of illustration, certain features presented in the drawings have been enlarged, reduced or simplified, and the illustrations and elements are not necessarily drawn to scale. However, those skilled in the art will readily understand these details.

[0123] Example

[0124] Example 1: Preparation of magnetic microparticles for capturing nucleic acids

[0125] In this POCT multiplex diagnosis system, eight wells are used. In order to move nucleic acids between the wells, it is necessary to use magnetic particles that can stably capture nucleic acids extracted from target factors and move them between the wells. In order to stably capture nucleic acids, cylindrical magnetic particles with a size of 10 to 1000 μm or commercial magnetic particles are used as raw materials to prepare magnetic particles with a sufficient surface area for capturing nucleic acids.

[0126] First, the prepared basic magnetic particles (basic core-shell magnetic particles prepared by our company, without capture probes, corresponding to Figure 2 110 and having a cylindrical rod shape with a length of 100 to 500 μm) was washed three times with a 0.1 M sodium hydroxide solution, then washed three times with a 100% ethanol solution, and then ultrasonically cleaned for 2 seconds.

[0127] Next, 200 mg of the magnetic particles were added to 10 ml of ammonia solution (composed of 20 ml of 100% ethanol, 3 ml of deionized water and 1 ml of ammonia), and then 2 ml of TEOS solution was added based on 100 μl of tetraethylorthosilicate (TEOS) for every 10 mg of magnetic particles. The solution was rotated at 20 rpm for 20 minutes as a reaction using a rotating device (FINEPCR, hybridization incubator Combi-H12), and 2 ml of TEOS solution was added for each reaction. The reaction was performed 4 times in total to complete the production of magnetic particles. The magnetic particles obtained after the reaction were washed three times with the previously mentioned ammonia solution and three times with 100% ethanol, and then stored in an ethanol solution.

[0128] In order to verify the nucleic acid capture ability of magnetic microparticles prepared for nucleic acid capture, an arbitrary sample was prepared and the actual nucleic acid capture ability was verified. Assuming a hypothetical virus or harmful strain as an arbitrary sample, any E. coli was selected as a target to confirm whether nucleic acid capture existed.

[0129] Figure 7 Results showing preliminary confirmation of the feasibility of capturing nucleic acids from target samples based on magnetic microparticles. Figure 7 A comparison is based on existing commercial E. coli plasmid miniprep kits (Bioneer, coli into which plasmid DNA was introduced), and the results of purification and separation of plasmid DNA from Escherichia coli into which plasmid DNA was introduced by magnetic microparticle beads for nucleic acid capture prepared based on our company's magnetic microparticles based on agarose gel electrophoresis.

[0130] refer to Figure 7 A. Under the conditions of 100 and 500 of our magnetic particles for nucleic acid purification, it can be seen that plasmid DNA can be purified and captured.

[0131] Lysis buffer: 1 ml of concentrated E. coli sample cultured in buffer composition (2 M guanidine thiocyanate, 36.7 mM Tris-Cl, 16.7 mM EDTA, 2% Triton X-100, 33% isopropanol, RNase-free water) constituting well 1 was mixed with our company's magnetic particles, and then rotated for 10 minutes; lanes 1 and 2 are purification results 1 and 2 based on commercial plasmid preparation kits, lane 3 is the result of purification based on 100 beads used for nucleic acid purification by our company, lane 4 is the result of purification based on 500 beads used for nucleic acid purification by our company, and lane M is a 1 kb DNA standard (Solgent, DNA standard above 1 Kb).

[0132] Figure 7 B shows that Figure 7 A Results of nucleic acid purification using a plasmid preparation kit and 500 of our company's nucleic acid purification beads under the same conditions. Figure 7 B. It was confirmed that genomic DNA and plasmid DNA of Escherichia coli can be purified and recovered by nucleic acid purification based on our magnetic particles, compared with a plasmid preparation kit.

[0133] Figure 7 C shows the results of the ability of our magnetic particles to capture 2 μg of plasmid DNA. Figure 7 C. Based on the fact that the nucleic acid sample was captured at a ratio of 7.3% by our magnetic particles for nucleic acid purification, the ability to capture any nucleic acid was confirmed.

[0134] Example 2: Target factor cleavage and nucleic acid capture amplification steps (wells 1 to 4)

[0135] We confirmed the feasibility of applying our automated equipment to perform the process from target sample lysis to magnetic microparticle probe imaging as a single process. We conducted a mobility test to confirm whether the target nucleic acid obtained by lysis and capture can be processed sequentially through the normal movement sequence between wells.

[0136] Fig. 9 The figure shows the results of confirming the nucleic acid capture of the target sample and the nucleic acid amplification of the target site in the multiplex diagnosis method according to one embodiment of the present invention.

[0137] Figure 8 The process of capturing and amplifying the target nucleic acid is shown in sequence from well 1 to well 4. Figure 8 Lysis / nucleic acid capture was performed in well 1, and nucleic acids were extracted and captured from the virus sample using a solution containing chaotropic salts (2 M guanidine thiocyanate, 36.7 mM Tris-Cl, 16.7 mM EDTA, 2% Triton X-100, 33% isopropanol, RNase-free water).

[0138] The first wash (wash-1) is performed in hole 2, wherein the magnetic particles containing the captured nucleic acids are washed with a solution of 2 M guanidine thiocyanate, 36.7 mM Tris-Cl, 16.7 mM EDTA, 2% Triton X-100, and RNase-free water + 66% isopropanol, and the captured nucleic acids are further concentrated on the magnetic particles.

[0139] The second wash (Wash-2) was performed in Well 3, in which 70% ethanol was used to remove residual waste attached to the magnetic particles.

[0140] Nucleic acid recovery and amplification are performed in well 4. The nucleic acid attached to the magnetic particles is desorbed, and then the desorbed nucleic acid is amplified. A solution based on a recombinase polymerase amplification (RPA) enzyme complex is used for nucleic acid amplification.

[0141] Fig. 9 The results of the capture and amplification process for nucleic acid of IBV RNA (a type of influenza virus) are shown. In well 4, the desorbed target nucleic acid was subjected to reverse transcription recombinase polymerase amplification (RT-RPA) reaction for 20 minutes at 37°C with the addition of IBV RNA. The RPA kit composition used in this case was performed according to the experimental manual recommended by the manufacturer (TwistDx, TwistAmp liquid kit). Reference Fig. 9 , it can be confirmed that after the RNA sample is moved and captured between the wells, the nucleic acid is detached in well 4 and then the nucleic acid amplification is performed normally by the RT-RPA enzyme complex.

[0142] Table 1 below shows the primer sequences used for target sequence amplification.

[0143] [Table 1]

[0144]

[0145] Based on the results, it was able to indirectly confirm that target signal detection and POCT diagnosis based on amplified nucleic acid were possible.

[0146] Example 3: Construction and functional verification of magnetic particle probes for signal detection (wells 4-5)

[0147] In this magnetic particle probe for signal detection, dCas9 is coupled to the surface of the magnetic particle, and the magnetic particle probe is constructed in the form of a complex formed by guide RNA (gRNA) and dCas9, which plays a role in identifying the target site sequence of the target factor.

[0148] The inactivating gene scissors (dCas9) used in this case were Alt-R SpdCas9 protein V3 purchased from IDT.

[0149] When the target nucleic acid is amplified by RT-RPA in well 4, the magnetic particle probe for signal detection located in well 7 is transferred to well 4, where the amplified nucleic acid can be captured by the gRNA / dCas9 complex.

[0150] The magnetic microparticle target nucleic acid capture body that has completed the reaction in well 4 is transferred to well 5 containing a streptavidin-R-phycoerythrin complex (SAPE). SAPE binds to biotin labeled on the magnetic microparticle target nucleic acid capture body.

[0151] Fig.10 The results show that when the target nucleic acid is present, the activity of the Cas system is confirmed by a magnetic particle probe to check whether the fluorescence intensity changes depending on the presence or absence of the target nucleic acid. In this experiment conducted to confirm the concept of the Cas system, it can be confirmed that the target nucleic acid labeled with biotin has been captured by the gRNA / dCas9 complex of the magnetic particle probe, and a fluorescent signal is detected.

[0152] Table 2 below shows relevant information of guide RNA.

[0153] [Table 2]

[0154]

[0155]

[0156] Example 4: Confirmation of virus detection by signal detection using magnetic particle probes

[0157] Based on the previous examples, confirm the possibility of detecting the target factor and confirm whether a diagnosis can be made for the single target factor.

[0158] In order to confirm the signal detection of a single target factor, three types of magnetic particle probes targeting respiratory viruses were constructed, and the signal detection results of influenza A, influenza B and COVID-19 virus targets were confirmed.

[0159] Fig.11 Results showing that influenza A, influenza B, and COVID-19 viruses can be detected using corresponding magnetic particle probes for signal detection.

[0160] It can be confirmed that the fluorescence signal intensity value (3412.74) of the corresponding magnetic particle probe group for signal detection in which the influenza A target nucleic acid exists is significantly higher than the fluorescence signal intensity value (808.79) of the negative control group in which the influenza A target nucleic acid does not exist.

[0161] In addition, in the experiment using the magnetic particle probe for influenza B detection, it can be confirmed that the fluorescence signal intensity value for influenza B detection is 7384.3 compared to the fluorescence signal intensity value of the negative control (505.836); in the experiment using the magnetic particle probe for COVID-19 detection, it can be confirmed that the fluorescence signal intensity value for COVID-19 detection is 9215.27 compared to the fluorescence signal intensity value of the negative control (446.189). Therefore, it can be confirmed that when each respiratory virus is present, the RNA of the corresponding virus will be extracted and amplified, and captured by a magnetic particle probe with a corresponding length for signal detection, and the fluorescence signal is detected.

[0162] Example 5: Multiplex detection validation of magnetic particle probes for signal detection

[0163] Based on previous examples where it was confirmed that each respiratory virus could be detected, the possibility of multiplex detection of three respiratory viruses was confirmed.

[0164] The magnetic particle probes with a length of 200 μm were used to detect influenza A, the magnetic particle probes with a length of 300 μm were used to detect influenza B, and the magnetic particle probes with a length of 400 μm were used to detect COVID-19. When all three magnetic particle probes with different lengths were present, experiments were performed to verify the possibility of multiplex diagnosis.

[0165] against Fig.12 The influenza A positive group shown in the experiment was conducted to specifically detect the influenza A target in the presence of three different lengths of magnetic particle probes. It can be confirmed that the fluorescence signal intensity value of the magnetic particle probe for influenza B (2136.23) and the fluorescence signal intensity value of the magnetic particle probe for COVID-19 (2046.03) are lower than the fluorescence signal intensity value of the magnetic particle probe for influenza A (2507.45).

[0166] against Fig.12 Influenza B positive, experiments were conducted to specifically detect influenza B targets in the presence of three different lengths of magnetic particle probes. It can be confirmed that compared with the fluorescence signal intensity value of the magnetic particle probe for influenza B (5194.37), the fluorescence signal intensity value of the magnetic particle probe for influenza A (4231.45) and the fluorescence signal intensity value of the magnetic particle probe for COVID-19 (3657.74) are lower.

[0167] against Fig.12The COVID-19 positive samples were tested in an experiment to specifically detect the COVID-19 target in the presence of three different lengths of magnetic particle probes. It can be confirmed that the fluorescence signal intensity value of the magnetic particle probe for influenza A (9215.27) and the fluorescence signal intensity value of the magnetic particle probe for influenza B (8388.55) are lower than the fluorescence signal intensity value of the magnetic particle probe for COVID-19 (12443.7). Fig.12 The graphical analysis results can confirm that when each target is present or all targets are present, they can identify each target and use it as a magnetic particle probe for each target.

[0168] As described in detail above, specific parts of the present invention have been described. It will be apparent to those skilled in the art that these specific descriptions are only preferred embodiments, and the scope of the present invention is not limited thereto. Accordingly, the essential scope of the present invention is defined by the appended claims and their equivalents.

Claims

1. A diagnostic microparticle probe comprising: particle; a capture probe introduced onto the surface of the microparticle; and Inactivated gene scissors, which are bound to the capture probe and contain a gene sequence complementary to the target nucleic acid.

2. The diagnostic microparticle probe of claim 1, wherein the inactivating gene scissors comprise a guide RNA.

3. The diagnostic microparticle probe according to claim 2, wherein the guide RNA has a nucleic acid sequence capable of hybridizing to a target site of a target nucleic acid gene. The diagnostic microparticle probe according to claim 1 , wherein the inactivation gene scissors have an inactivation target nucleic acid cleavage function.

5. The diagnostic microparticle probe of claim 1, wherein the microparticle contains magnetic particles therein. 6 . The diagnostic microparticle probe according to claim 1 , wherein the microparticle has a core-shell structure comprising a core containing a magnetic material and a shell layer surrounding the core and having a uniform thickness.

7. The diagnostic particle probe of claim 1, wherein the particles have a size and specific gravity that prevents them from floating in water.

8. A POCT multiple diagnosis system, comprising The diagnostic microparticle probe according to any one of claims 1 to 7; a reporter nucleic acid that complementarily binds to the target nucleic acid and is labeled with biotin; and Reagents for amplifying nucleic acids.

9. The POCT multiplex diagnosis system according to claim 8, wherein the diagnostic microparticle probes are labeled to be distinguished from each other by diameter, thickness, length, shape or identification code.

10. The POCT multiplex diagnosis system according to claim 9, wherein the diagnostic microparticle probes are a mixture of two or more diagnostic microparticle probes having different lengths.

11. The POCT multiplex diagnosis system according to claim 8, wherein the reporter nucleic acid is two or more reporter nucleic acids to which phosphors of different colors are bound.

12. A method for detecting a target nucleic acid, comprising the following steps: (a) extracting target nucleic acid from a sample; (b) amplifying the target nucleic acid and binding a reporter nucleic acid thereto; (c) providing a diagnostic microparticle probe comprising a microparticle; a capture probe introduced onto the surface of the microparticle; inactivating gene scissors, which are bound to the capture probe and contain a gene sequence complementary to the target nucleic acid; (d) reacting the diagnostic microparticle probe with the target nucleic acid to capture the target nucleic acid and the reporter gene; (e) attaching a fluorescent material to the reporter nucleic acid; (f) detecting the target nucleic acid by measuring a fluorescent signal emitted from the stained reporter nucleic acid.

13. The method for detecting a target nucleic acid according to claim 12, wherein the detection of the target nucleic acid is a multiplex detection of two or more of the target nucleic acids using two or more of the diagnostic microparticle probes.

14. The method for detecting a target nucleic acid according to claim 13, wherein the multiplex detection comprises a process of reading the diagnostic microparticle probes that are labeled to be distinguished from each other by length, diameter, thickness, shape, color or identification code. 15 . The method for detecting a target nucleic acid according to claim 12 , further comprising washing the diagnostic microparticle probe to which the target nucleic acid and the reporter nucleic acid are attached after step (e).

16. The method for detecting a target nucleic acid according to claim 12, wherein the method for detecting the target nucleic acid is performed in a microwell. 17 . The method for detecting a target nucleic acid according to claim 16 , wherein the diagnostic microparticle probe is moved and immersed between the microwells by an external magnetic force.

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