Nucleic acid extraction method using porous ionic charged particles

Abstract

The present invention relates to a porous ionic charged particle for nucleic acid separation and a nucleic acid extraction method using the same. More specifically, the porous ionic charged particle of the present invention has anionic properties, so that it can electrically separate negatively charged nucleic acids, adsorb cationic by-products to the particles, and simultaneously absorb water as a solvent. That is, the nucleic acid separation method provides a method for separating and concentrating nucleic acids simply and quickly without using complicated devices such as harmful organic solvents, magnetic beads, and centrifuges, because the separation and concentration of nucleic acids is automatically performed by simply immersing the porous ionic charged particle in a reaction tube containing a biological sample.

Description

[Technical field]

[0001] The present invention relates to porous ionically charged particles and a method for extracting nucleic acid using the same. [Background technology]

[0002] Currently, techniques for extracting nucleic acids from biological samples such as cells, bacteria, or viruses are widely used in conjunction with nucleic acid amplification reaction techniques for basic research in the field of molecular biology, and for the diagnosis, treatment, or prevention of diseases. In addition, techniques for extracting nucleic acids from biological samples are required in various fields such as custom-made drug development, forensic medicine, and environmental hormone detection.

[0003] One example of a conventional nucleic acid extraction technique is a method in which a sample containing cells is treated with sodium dodecyl sulfate (SDS) or proteinase K to solubilize it, then proteins are denatured and decomposed using phenol / chloroform or phenol / chloroform / isoamylalcohol, and by-products are removed by ethanol precipitation to purify the nucleic acids. However, the phenol extraction method has problems such as being time-consuming due to the many processing steps, and the efficiency of nucleic acid extraction is highly dependent on the experience and skill of the researcher, making it extremely unreliable. In addition, in conventional nucleic acid extraction methods, there is a problem that histone proteins and the like remain as by-products during the cell disruption process, which is essential for the cell lysis stage, and act as inhibitors that inhibit the polymerase chain reaction (PCR).

[0004] In recent years, in order to solve such problems, kits using silica or glass fiber that specifically bind to nucleic acids are sometimes used. The silica and glass fiber have a low binding rate with proteins and metabolic products of cells, so that relatively high concentrations of nucleic acids can be obtained. This method has the advantage of being simpler than the phenol method, but it has the disadvantage that it uses chaotropic reagents and ethanol that strongly inhibit enzyme reactions such as polymerase chain reaction (PCR), and therefore these substances must be completely removed, which makes the operation very complicated and time-consuming.

[0005] Recently, a method for directly purifying nucleic acids using a filter has been disclosed in International Patent Publication No. 2000 / 21973. In this method, a sample is passed through a filter, cells are adsorbed onto the filter, the cells adsorbed onto the filter are lysed, and the sample is filtered, after which the nucleic acids adsorbed onto the filter are washed and eluted.

[0006] For example, Korean Patent No. 10-0454869 discloses a method for extracting DNA using a cell lysis buffer, and this conventional nucleic acid extraction method is advantageous in that it can extract nucleic acids reproducibly in a short time with fewer steps, since it is composed of 1) a step of adding a cell lysis buffer to cells to lyse the cells; 2) a step of transferring the lysed cells in step 1 to a filter to fix the nucleic acids; 3) a step of washing the filter in step 2; and 4) a step of recovering the nucleic acids from the filter. However, since the use of a centrifuge is necessary when transferring the lysed cells to the filter, when washing the filter, or when recovering the nucleic acids from the filter, there is a problem that portability and mobility are reduced, and the process of nucleic acid extraction on site is complicated.

[0007] Other methods include a nucleic acid extraction method using magnetic beads, a nucleic acid extraction method using a syringe and a filter, and a nucleic acid extraction method using Trizol. However, the nucleic acid extraction method using magnetic beads requires a magnet to fix the nucleic acid to the wall of the tube, and a pump and a valve (automatic device) or multiple tips and a pipette (manual device) must be used to remove the solution. In addition, there is a problem that some of the nucleic acid may be lost during the process of washing and eluting the nucleic acid. Furthermore, in the nucleic acid extraction method using a syringe and a filter, if a force of a certain strength or more is applied during the process of transferring the nucleic acid using the syringe, the filter may be damaged, making it difficult to extract the nucleic acid. In addition, the nucleic acid extraction method using Trizol has a problem that harmful organic solvents such as phenol and chloroform are used.

[0008] Therefore, there is a demand for the development of a nucleic acid extraction method that can solve the problems of conventional nucleic acid extraction methods and easily and efficiently separate high concentrations of nucleic acids. Summary of the Invention [Problem to be solved by the invention]

[0009] The present invention is intended to solve the problems of the conventional technology, and a main object of the present invention is to provide a porous ion-charged particle for nucleic acid extraction, which can improve user convenience by minimizing the use of pipettes and tips, and can rapidly perform reactions on-site, and a nucleic acid extraction method using the same.

[0010] Another object of the present invention is to provide porous ion-charged particles for nucleic acid extraction, which can utilize the electrical properties of nucleic acids and therefore do not require the use of harmful organic solvents, magnetic materials, or complicated devices or equipment, and are therefore highly portable and mobile, making them convenient for use on-site, and a nucleic acid extraction method using the same.

[0011] Yet another object of the present invention is to provide porous ion-charged particles for nucleic acid extraction, which can adsorb cationic by-products onto the particles and absorb water to separate and concentrate only pure nucleic acids, and a nucleic acid extraction method using the same. [Means for solving the problem]

[0012] As a means for achieving the above object of the present invention, the present invention provides porous ionically charged particles for separating nucleic acids.

[0013] The present invention also provides a nucleic acid extraction tube containing the porous ionically charged particles for nucleic acid separation.

[0014] Furthermore, the present invention provides a method for extracting nucleic acids, comprising: introducing a sample containing nucleic acids into a nucleic acid extraction tube together with a direct lysis buffer (DLB) and mixing the sample; introducing porous ion charge particles into the mixture and reacting the mixture for 5 to 30 minutes; and removing the ion charge particles that have completed the reaction to obtain a buffer solution in which nucleic acids are concentrated.

[0015] Furthermore, the present invention provides a method for detecting nucleic acid, comprising: introducing a sample containing nucleic acid into a nucleic acid extraction tube together with a direct lysis buffer and mixing the samples; introducing porous ion charge particles into the mixture and reacting for 5 to 30 minutes; removing the ion charge particles that have completed the reaction to obtain a buffer solution in which nucleic acid is concentrated; introducing a polymerase chain reaction (PCR) mixture into the buffer solution in which nucleic acid is concentrated to perform a polymerase chain reaction (PCR); and detecting the sample in situ during or after the PCR. Effect of the Invention

[0016] According to the porous ion-charged particles for nucleic acid separation and the nucleic acid extraction method using the same of the present invention, nucleic acid separation and concentration are automatically performed simply by immersing the porous ion-charged particles in a reaction tube containing a biological sample, and therefore, it is possible to easily and quickly separate and concentrate nucleic acid without using complicated equipment such as harmful organic solvents, magnetic beads, or a centrifuge.

[0017] In addition, since the present invention can immediately dissolve, purify, and concentrate a trace amount of biological sample without any pretreatment process, it can be effectively applied to samples containing low concentrations of biological materials (pathogens, etc.). Therefore, the present invention can be widely used in various fields in the future, such as various basic research where analysis was previously limited due to the low concentration, diagnostic tests where the amount of sample is extremely limited, clinical diagnosis, environmental monitoring, and pharmacogenetic analysis. [Brief description of the drawings]

[0018] [Figure 1] FIG. 2 is an enlarged view showing the porous structure of the ion-charged particles of the present invention. [Diagram 2] A schematic diagram showing the process of the nucleic acid extraction method of the present invention, in which a biological sample is directly dissolved in a dissolution buffer, nucleic acids and by-products are separated, and ion-charged particles attach cationic by-products while absorbing water to obtain a nucleic acid-enriched buffer solution. [Diagram 3] and [Figure 4] This shows the results of comparing the real-time polymerase chain reaction (Real-time PCR) amplification efficiency of a target gene (glyceraldehyde-3-phosphate dehydrogenase, GAPDH) after applying the nucleic acid extraction method of the present invention (Test 2) and an extraction method using only direct lysis buffer (DLB) (Test 1) to oropharyngeal samples (oropharyngeal cancer samples). [Diagram 5] and [Figure 6]The figure shows the results of comparing the real-time PCR amplification efficiency of a target gene (GAPDH) after applying the nucleic acid extraction method of the present invention (Test 2) and an extraction method using only DLB (Test 1) to a nasopharyngeal sample (nasopharyngeal cancer sample). [Figure 7] and [Figure 8] The figure shows the results of comparing the real-time PCR amplification efficiency of the target gene (GAPDH) after applying an extraction method using cationic charged particles (Test 2) in Comparative Example 1 below and an extraction method using only DLB (Test 1) to an oropharyngeal sample (oropharyngeal cancer sample). [Figure 9] and [Figure 10] The figure shows the results of comparing the real-time PCR amplification efficiency of the target gene (GAPDH) after applying an extraction method using cationic charged particles (Test 2) in Comparative Example 2 below and an extraction method using only DLB (Test 1) to a nasopharyngeal sample (nasopharyngeal cancer sample). [Figure 11] and [Figure 12] 1 is a photograph comparing the size of porous ion-charged particles before and after water absorption. [Figure 13] and [Figure 14] and [Figure 15] A concept image showing what a capsule containing ionic charged particles might look like. [Figure 16] The results show that after separating and concentrating nucleic acids using various forms of polymer absorbents, the nucleic acid separation ability of each method was confirmed by PCR detection tests. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] The present invention will be described and clarified through the following detailed embodiments with the accompanying drawings. The terms and words used in describing the present invention are not to be construed as being limited to their ordinary or dictionary meanings, but are to be construed as being in accordance with the meaning and concept of the technical idea of ​​the present invention in accordance with the principle that the inventor can appropriately define the concept of the term in order to best describe the invention.

[0020] Furthermore, the embodiments described for explaining the present invention and the configurations shown in the drawings are merely the most preferred embodiments of the present invention and do not encompass all of the technical ideas of the present invention, and therefore there may be equivalent modifications that replace these at the time of filing this application.

[0021] Furthermore, in describing the present invention, if a specific description of related publicly known configurations or functions is determined to make the gist of the present invention unclear, the detailed description will be omitted.

[0022] The terms used in this specification are defined as follows:

[0023] The term "porous ionically charged particles" refers to particles that have numerous small pores (micropores) formed on and within the solid surface and that carry an electric charge.

[0024] The "sample" is not limited to a particular type or form, so long as it contains the biomaterial to be detected. Exemplarily, the sample may be a biological sample, such as a biological fluid or biological tissue. Examples of biological fluids include urine, blood, plasma, serum, saliva, semen, feces, sputum, cerebrospinal fluid, tears, mucus, amniotic fluid, and the like. Biological tissues are aggregates of cells, and generally aggregates of intracellular substances that form one of the structural materials of human, animal, plant, bacterial, fungal, or viral structures, and may be connective tissue, epithelial tissue, muscle tissue, and nerve tissue, as specific types of aggregates. In addition, examples of biological tissues may include organs, tumors, lymph nodes, arteries, and individual cells. In addition, the sample may include an environmental sample that contains a low concentration of biomaterial, and may include various forms and types, such as drinking water, food, and the like.

[0025] "Nucleic acid" may refer to multiple nucleotides consisting of sugars bound to organic bases (substituted pyrimidines such as cytosine, thymidine, or uracil, or substituted purines such as adenine or guanine). That is, ribonucleic acid (RNA), deoxyribonucleic acid (DNA), methylphosphonate nucleic acid, S-oligo, c-DNA, cRNA, miRNA, siRNA, aptamer, etc. may be mentioned, and the concept may include naturally occurring ones and artificially synthesized ones. However, more typically, it may be DNA, RNA, etc.

[0026] "Direct lysis buffer (DLB)" refers to a buffer used in disrupting cells, and is a composition containing salts and / or surfactants to adjust the acidity and osmotic pressure of the lysate. For example, it may refer to a composition that has a pKa of about 5.8 to about 9 at 25°C and can effectively maintain a pH value of, for example, 6 to 9 (specifically, about 7.5 to 9, more specifically, about 7.8 to 8.5).

[0027] "Cell lysis" refers to the phenomenon in which the cell membrane bursts due to the breakdown of cells, exposing the cellular contents. It is usually used to separate DNA or RNA prior to amplification processes such as PCR.

[0028] "PCR" refers to a reaction in which many identical DNA strands are formed from a single initial template by a cycling process (alternating heating and cooling). In general, a PCR mixture may contain (i) a double-stranded DNA molecule that is a template having a base sequence to be amplified, (ii) a primer (a single-stranded DNA molecule that can bind to a complementary DNA base sequence in the template DNA), (iii) dNTPs, which are a mixture of dATP, dTTP, dGTP, and dCTP (nucleotide subunits that combine to form new DNA molecules during PCR amplification), and (iv) an enzyme that synthesizes new DNA molecules using Taq DNA polymerase (dNTPs).

[0029] The present invention provides porous ionically charged particles for nucleic acid separation.

[0030] According to one embodiment of the present invention, the ionically charged particles may be anionic particles having a functional group or moiety exhibiting a negative charge, and any type of particle having a negative charge may be used.

[0031] Nucleic acids, due to their structure, have negative charge characteristics and are therefore electrically repelled by the anionic charged particles of the present invention and cannot bind to ionic charged particles, whereas dissolution by-products, which have positive charge characteristics, bind to ionic charged particles.

[0032] According to another embodiment of the present invention, the ionically charged particles have a superporous structure and can absorb a solvent, particularly water (H2O).

[0033] In addition, the voids inside the ion-charged particles can have an interconnected structure, allowing water to be absorbed at a very high speed due to capillary action, and the water absorption and swelling speed of the porous ion-charged particles of the present invention can be several hundred times faster than the water absorption and swelling speed of general porous particles.

[0034] According to one embodiment of the present invention, the component of the "porous ion-charged particle" can be any material that is porous and negatively charged, and may be a synthetic resin or a natural resin, without any restrictions on its type.

[0035] The "porous ionically charged particles" of the present invention may be an anionic polymer, specifically, a polyelectrolyte having dissociative groups in the polymer chain and dissociating to form polymer ions upon contact with water.

[0036] According to one embodiment of the present invention, the component of the "porous ion-charged particles" may be one or more selected from the group consisting of polyacrylate, sodium polyacrylate, polyacrylamide, polyvinyl alcohol, polyethylene, polyacrylonitrile, alginate, ethylene maleic anhydride, methyl cellulose, hydroxyethyl cellulose, hydroxypropyl methyl cellulose, cellulose acetate, carboxy methyl cellulose, polymethacrylate (PMA), etc., and may be preferably sodium polyacrylate. Of course, the component is not limited to this.

[0037] More specifically, the porous ionic particle of the present invention has a three-dimensional network structure in which polyelectrolyte materials are cross-linked with each other, and the three-dimensional network structure and osmotic pressure generated by such polyelectrolyte enable the absorption and storage of a solution.

[0038] According to one embodiment of the present invention, the porous ionic particles may be a polymer made of acrylic acid, and may be manufactured by forming a large mass by gel polymerization and cutting it to the desired particle size. Therefore, the shape of the particles is irregular and atypical, and the particles can be classified and manufactured to the desired particle shape and particle size by subsequent processing. In addition, the absorption capacity of the porous ionic particles changes depending on the ionic properties of the electrolyte with which they come into contact, and ... such as anions (SO4 2- , O.H. - When these substances dissolve in pore water and create an alkaline environment, anionic polymeric substances are produced.

[0039] The acrylic acid-based polymer used in the present invention may contain an anionic functional group such as a carboxyl group (-COO-) in an anionic electrolyte environment.

[0040] The porous ionically charged particles may be of various shapes such as amorphous, spherical, elliptical, polygonal, etc. According to one embodiment of the present invention, the porous ionically charged particles may have an average diameter of about 0.05-1.5 cm, preferably 0.1-1.0 cm, 0.2-0.8 cm, and most preferably 0.3-0.5 cm before solvent absorption.

[0041] According to another embodiment of the present invention, the volume of the porous ionic charged particles may increase by 1,000 to 50,000% (10 to 500 times), preferably 20,000 to 30,000% (200 to 300 times) after absorbing the solution, compared to before absorbing the solvent.

[0042] As an example of the present invention, the pore size (diameter) of the porous ion-charged particles can be, for example, about 0.1 to 10 nm, specifically about 1 to 5 nm, more specifically about 3 to 5 nm. If the pore size is too large, the ability to absorb water (water absorption ability) may decrease, and if the pore size is small, the ability to absorb by-products may decrease, so it may be desirable to use porous ion-charged particles with an appropriate pore size in consideration of this.

[0043] The present invention can also provide a nucleic acid extraction tube containing the porous ionically charged particles for nucleic acid separation.

[0044] The nucleic acid extraction tube is a structure for measuring nucleic acids in a sample containing nucleic acids, and may include a body part for accommodating a sample, a cap for opening and closing an opening of a certain body part, and a connecting part between the body part and the cap, but is not limited thereto, and may also include a form in which the body part does not include a connecting part, and the body part and the cap are not connected by a connecting part and exist separately from each other.

[0045] The material constituting the nucleic acid extraction tube may be at least one of polypropylene (PP), polyethylene (PE), polyvinyl chloride (PVC), polycarbonate (PC), polyester, polymethylmethacrylate (PMMA), polyethylene terephthalate (PET), polyurethane (PU), silicon, glass, ceramic, and Teflon (registered trademark), but is not limited thereto.

[0046] In this case, if moisture absorption by plastics is not an issue, the preferred plastics are acrylonitrile butadiene styrene (ABS), acetal, acrylic fiber, acrylonitrile, cellulose acetate, ethyl cellulose, alkylvinylalcohol, polyaryletherketone, polyetheretherketone, polyetherketone, melamine formaldehyde, phenol formaldehyde, etc. formaldehyde, polyamide (e.g., nylon 6, nylon 66, nylon 12), polyamide-imide, polydicyclopentadiene, polyether-imide, polyethersulfone, polyimide, polyphenyleneoxide, polyphthalamide, methylmethacrylate, polyurethane, polysulfone, and vinyl formal.

[0047] Also, when moisture absorption is a problem, preferred plastics are polystyrene, polypropylene, These may include, but are not limited to, polybutadiene, polybutylene, epoxy, Teflon®, peroxyacetylnitrate (PAN), polyethylene terephthalate (PET), polytetrafluoroethylene (PTFE), chloro-fluoroethylene, polyvinylidene fluoride, liquid crystal polymer, polyester, low-density polyethylene (LDPE), high density polyethylene (HDPE), polymethylpentene, polyphenylene sulfide, polyolefin, polyvinyl chloride (PVC), and chlorinated PVC.

[0048] The volume of sample that can be accommodated in a nucleic acid extraction tube may be in the range of 1 to 50,000 μl, but is not limited thereto, and can have a wider range since it can be formed in various sizes according to the user's purpose.

[0049] The body of the nucleic acid extraction tube can be formed into various shapes. The lower part can be formed into various shapes such as a circular arc or a square depending on the purpose of the user.

[0050] Therefore, a nucleic acid extraction tube according to one embodiment of the present invention can include body portions having various forms, volumes and shapes, thereby providing a tube suitable for the user's purpose.

[0051] The present invention also provides a method for extracting nucleic acids, comprising the steps of: introducing a sample containing nucleic acids into a nucleic acid extraction tube together with a direct lysis buffer; and mixing the sample with a direct lysis buffer. adding porous ion charge particles to the mixture and reacting for 5 to 30 minutes; and removing the ion-charged particles that have completed the reaction to obtain a buffer solution in which nucleic acids are concentrated.

[0052] According to one embodiment of the present invention, the porous ionic charged particles are anionic, and the anionic ionic charged particles are capable of adsorbing cationic by-products. The porous ionic charged particles of the present invention are also characterized in that they absorb solvents, particularly water.

[0053] The physical and chemical properties of the materials and particles that can be used as the porous ionically charged particles are similar to those described above.

[0054] According to one example of the present invention, the direct lysis buffer (DLB) can be of any type, as long as it is a buffer that can remove cell membranes and nuclear membranes and elute nucleic acids, and the type, concentration, pH, etc. of the salt and surfactant used can be adjusted as necessary.

[0055] The amount of the porous ionic particles added to the mixture can be appropriately adjusted depending on the type and amount of the sample. According to one example of the present invention, 10 to 500 mg, preferably 10 to 400 mg, 50 to 350 mg, or 100 to 300 mg can be added.

[0056] According to one embodiment of the present invention, the reaction can be carried out for 5 to 20 minutes, for example, 5 to 15 minutes, 5 to 10 minutes, or 10 to 15 minutes, for example, about 10 minutes.

[0057] According to one embodiment of the present invention, the nucleic acid extraction method may be characterized in that the nucleic acid extraction reaction can be carried out at room temperature and does not require an additional washing step.

[0058] In addition, the nucleic acid extraction method of the present invention may further include a step of removing the ionic charged particles to obtain a buffer solution in which nucleic acids are concentrated, and then isolating the nucleic acids from the buffer solution.

[0059] According to another embodiment of the present invention, a method for extracting nucleic acids includes the steps of: introducing a sample containing nucleic acids into a nucleic acid extraction tube together with a direct lysis buffer; and mixing the sample with a direct lysis buffer. adding porous ion charge particles to the mixture and reacting for 5 to 30 minutes; removing the ionic charged particles that have completed the reaction to obtain a nucleic acid-enriched buffer solution; adding a polymerase chain reaction (PCR) mixture to the nucleic acid-concentrated buffer solution and performing a polymerase chain reaction (PCR); detecting the sample in situ during or after the PCR.

[0060] Working Example Production Example 1: Production of porous ionic charged particles In the examples of the present invention, sodium polyacrylate (manufactured by LG Household & Health Care) was used as the anionic polymer, and in the comparative examples, styrene-divinylbenzene copolymers (manufactured by DOW) were used as the cationic polymer.

[0061] Example 1: Verification of nucleic acid detection effect in oral pharyngeal samples (target: GAPDH) A nucleic acid extraction method using ion-charged particles according to one embodiment of the present invention (TEST2; anion) was applied to an oropharyngeal sample (oropharyngeal cancer sample) and then real-time PCR (RT-PCR) was performed. The result (TEST2) was compared with the result (control group, TEST1) of RT-PCR performed after applying only Direct Lysis Buffer (DLB) to the same amount of the same sample, using fluorescent amplification curves. The results are shown in FIGS. 3 and 4.

[0062] As shown in the graph in Figure 3, in the case of TEST1, fluorescence expression began at 26 cycles, and the end point RFU was found to be at a level within a range of approximately 2000. In the case of TEST2 (anion), fluorescence expression began at 23 cycles, and the end point RFU was found to be at a level within a range of approximately 2200. As shown in the graph in Figure 4, in the case of TEST1, fluorescence expression began at 29 cycles, and the end point RFU was found to be at a level within a range of approximately 1300. In the case of TEST2 (anion), fluorescence expression began at 24 cycles, and the end point RFU was found to be at a level within a range of approximately 1900.

[0063] Example 2: Verification of nucleic acid detection effect in nasopharyngeal samples (target: GAPDH) A nucleic acid extraction method using ionic charged particles according to one embodiment of the present invention (TEST2; anion) was applied to a nasopharyngeal sample (nasopharyngeal cancer sample) and then RT-PCR was performed. The result (TEST2) was compared with the result (control group, TEST1) of RT-PCR performed after applying only direct lysis buffer (DLB) to the same amount of the same sample, using the fluorescence amplification curves. The results are shown in Figures 5 and 6.

[0064] As shown in the graph of FIG. 5, in the case of TEST1, it was found that fluorescence expression started at 39 cycles, and the end point RFU was at a level within a range of about 600. On the other hand, in the case of TEST2 (anion), it was found that fluorescence expression started at 27 cycles, and the end point RFU was at a level within a range of about 2300. As shown in the graph of FIG. 6, in the case of TEST1, no fluorescence expression was observed, and in the case of Test2 (anion), it was found that fluorescence expression started at 30 cycles, and the end point RFU was at a level within a range of about 2100.

[0065] From the results of Examples 1 and 2 above, it was found that the nucleic acid extraction method of the present invention using the porous ion-charged particles has superior nucleic acid extraction and detection effects to the nucleic acid extraction method using only DLB.

[0066] Comparative Example 1: Comparison of nucleic acid detection effects in oral pharyngeal samples (target: GAPDH) using cationic charged particles The results of RT-PCR performed after applying a nucleic acid extraction method using DLB and cationic charged particles (TEST2; cationic) to an oropharyngeal sample (oropharyngeal cancer sample) (TEST2) were compared with the results of RT-PCR performed after applying only direct lysis buffer (DLB) to the same amount of the same sample (control group, TEST1). The fluorescence amplification curves are shown in Figures 7 and 8.

[0067] As shown in the graph in Figure 7, in the case of TEST1, fluorescence expression began at the 27th cycle, and the end point RFU was found to be at a level within a range of approximately 2300, while in the case of TEST2 (cation), no fluorescence expression was observed. As shown in the graph in Figure 8, in the case of TEST1, fluorescence expression began at the 29th cycle, and the end point RFU was found to be at a level within a range of approximately 1300, while in the case of TEST2 (cation), no fluorescence expression was observed.

[0068] Comparative Example 2: Comparison of nucleic acid detection effects in nasopharyngeal samples (target: GAPDH) using cationic charged particles A nucleic acid extraction method using DLB and cationic charged particles (TEST2; anionic) was applied to a nasopharyngeal sample (nasopharyngeal cancer sample), and then RT-PCR was performed. The results (TEST2) were compared with the results (control group, TEST1) of RT-PCR performed after applying DLB alone to the same amount of the same sample using fluorescence amplification curves. The results are shown in Figures 9 and 10.

[0069] As shown in the graph of Figure 9, neither TEST1 nor TEST2 (cation) showed any fluorescence expression. Also, as shown in the graph of Figure 10, neither TEST1 nor TEST2 (cation) showed any fluorescence expression.

[0070] From the results of Comparative Examples 1 and 2 above, it was confirmed that, unlike the present invention, when cationic porous ion-charged particles were used, no nucleic acid was extracted or detected at all.

[0071] Experimental Example 1. Nucleic acid separation and detection test using various types of polymer absorbents Nucleic acid separation and detection tests were carried out using various types of polymer absorbents, and the results are shown in FIG.

[0072] Specifically, in the nucleic acid extraction method of the present invention, instead of the porous anionic charged particles, DLB alone was used / cationic particles were applied / negative fiber filter was applied / negative ion particle bead type was applied, and then PCR detection tests were performed using solutions concentrated by each separation method (Figure 16, 1. DLB alone was used / 2. Cationic particles (0.1g, 0.2g, 0.3g) / 3. Porous anionic charged particles of the present invention (0.1g, 0.2g, 0.3g) / 4. Negative fiber filter (1, 2, 3) / 5. Anionic particle bead type (3, 6, 9)).

[0073] As shown in FIG. 16, the PCR results confirmed that the use of the porous anionic particles of the present invention provided significantly superior nucleic acid separation and detection effects compared to the use of the anionic fiber filter and anionic particle bead type.

Claims

1. Porous ionic charged particles for nucleic acid separation.

2. The porous ionically charged particles for nucleic acid separation according to claim 1 , wherein the porous ionically charged particles are anionic polyelectrolytes.

3. The porous ion-charged particles may be polyacrylate, sodium polyacrylate, polyacrylamide, polyvinyl alcohol, polyethylene, polyacrylonitrile, alginate, ethylene maleic anhydride, methyl cellulose, hydroxyethyl cellulose, hydroxypropyl methyl cellulose, cellulose acetate, or the like.

2. The porous ionically charged particle for nucleic acid separation according to claim 1, wherein the porous ionically charged particle is at least one selected from the group consisting of carboxymethylcellulose, carboxymethylcellulose, and polymethacrylate (PMA).

4. The particles of claim 3 , wherein the material of the porous ionically charged particles is sodium polyacrylate.

5. A nucleic acid extraction tube comprising the porous ionically charged particles for nucleic acid separation according to any one of claims 1 to 4.

6. A step of mixing a sample containing nucleic acid with a direct lysis buffer in a nucleic acid extraction tube; adding porous ion charge particles to the mixture and reacting for 5 to 30 minutes; removing the ionic charged particles that have completed the reaction to obtain a nucleic acid-enriched buffer solution; A method for extracting nucleic acid comprising the steps of:

7. The method for extracting nucleic acids according to claim 6 , wherein the porous ionically charged particles are anionic polyelectrolytes.

8. The porous ion-charged particles may be polyacrylate, sodium polyacrylate, polyacrylamide, polyvinyl alcohol, polyethylene, polyacrylonitrile, alginate, ethylene maleic anhydride, methyl cellulose, hydroxyethyl cellulose, hydroxypropyl methyl cellulose, cellulose acetate, or the like.

8. The nucleic acid extraction method according to claim 7, wherein the nucleic acid extracting agent is one or more selected from the group consisting of carboxymethyl cellulose, carboxymethyl cellulose, and polymethacrylate (PMA).

9. The nucleic acid extraction method according to claim 8, wherein the material of the porous ionically charged particles is sodium polyacrylate.

10. The method for extracting nucleic acids according to claim 6, wherein the porous ionically charged particles adsorb cationic by-products and absorb the solvent.

11. The nucleic acid extraction method according to claim 6 , wherein the reaction is carried out at room temperature.

12. The method for nucleic acid extraction according to claim 6 , wherein the reaction does not include an additional washing step.

13. A step of mixing a sample containing nucleic acid with a direct lysis buffer in a nucleic acid extraction tube; adding porous ion charge particles to the mixture and reacting for 5 to 30 minutes; removing the ionic charged particles that have completed the reaction to obtain a nucleic acid-enriched buffer solution; adding a polymerase chain reaction (PCR) mixture to the nucleic acid-concentrated buffer solution and performing a polymerase chain reaction (PCR); detecting the sample in situ during or after the PCR; A method for detecting a nucleic acid comprising the steps of:

Images