A composition for detecting or isolating nucleic acids and a method for detecting or isolating nucleic acids using the same

Abstract

One aspect relates to a composition, a kit, and a method for detecting or isolating nucleic acids using the same. By using the composition, kit, and method for detecting or isolating nucleic acids according to one aspect, high-concentration and high-purity nucleic acids can be detected or isolated rapidly, simply, and accurately at low cost without electricity and special equipment, such as centrifugation, vacuum pumps, and heat controllers, used in conventional column and bead-based methods. Furthermore, by using the composition according to one aspect to detect or isolate one or more nucleic acids selected from the group consisting of miR-141-3p, miR-375-3P, miR-483-5P, and miR-574-3P, prostate cancer (PCa) in patients with benign prostatic hyperplasia (BPH) can be diagnosed rapidly, precisely, and early, and infectious diseases can be diagnosed rapidly, precisely, and early by detecting or isolating nucleic acids derived from microorganisms that cause infectious diseases.

Description

Technology Field

[0001] The present invention relates to a composition for detecting or separating nucleic acids and a method for detecting or separating nucleic acids using the same. Background Technology

[0003] Circulating nucleic acids (NNAs), first described in 1948, are stable circulating DNA and RNA molecules found in bodily fluids such as plasma, urine, and saliva, and have been regarded as promising non-invasive biomarkers for diagnosis, prognosis, and prediction. These molecules originate from necrotic and apoptotic cells and are secreted by various cells to communicate with other cells, even in the early stages of cancer. Changes in the expression of these circulating nucleic acids directly influence physiology and pathology, such as cancer development and progression. Circulating nucleic acids are used as cancer biomarkers in liquid biopsies and do not have the disadvantages of conventional biopsies, such as invasiveness, high risk, high cost, and limitations regarding tumor location and size. Furthermore, because circulating nucleic acids vary heterogeneously depending on the type and stage of cancer, they allow for the monitoring of cancer metastasis and treatment prognosis through molecular characterization. Due to these advantages, circulating nucleic acids are utilized as diagnostic tools for the early or precision diagnosis of cancer and are being proposed as new diagnostic platforms by employing high-sensitivity detection methods such as endpoint, real-time, and droplet-digital PCR. However, the development of diagnostic platforms using circulating nucleic acids was not considered to suffer from reduced sensitivity due to small amounts of circulating nucleic acids in liquid samples. There is a lack of discussion regarding the extraction of high concentrations of circulating nucleic acids using large volumes of samples.

[0004] Prostate cancer (PCa) is one of the most common malignancies in men worldwide and the second leading cause of all cancer-related deaths. Prostate-specific antigen (PSA) testing is widely used for the early diagnosis of prostate cancer, and the mortality rate is significantly decreasing as the number of treatable prostate cancer patients increases. However, due to the low sensitivity and specificity of the PSA test, unnecessary diagnoses and treatments were required, and a PSA level of 4 ngmL -1 to 10 ngmL -1 The PSA gray zone has limitations as it is closely associated with certain non-malignant diseases, such as benign prostatic hyperplasia (BPH) and prostatitis. Therefore, there is a need to discover better biomarkers that can be used for PCa diagnosis. Recently, PCa diagnostic platforms containing circulating miRNAs and circulating mRNAs are being intensively developed due to the various advantages of circulating nucleic acids. miRNAs are non-coding RNA molecules of approximately 22 nucleotides (17-27 nts) that preferentially bind to the 3'UTR of mRNA and act as post-transcriptional regulators influencing important cellular processes such as the cell cycle, proliferation, and apoptosis. In prostate cancer, circulating miRNA-141-3p and miRNA-375-3p are increased compared to healthy controls and are known as promising miRNAs for biomarkers. However, the roles of these miRNAs are still unclear, and there are limitations in identifying prostate cancer-specific miRNA biomarkers among the numerous miRNAs. Furthermore, research to date has focused on circulating miRNAs, and research on circulating mRNAs has not been properly explored.

[0005] One of the critical considerations in circulating RNA analysis is the sample processing method for extracting high concentrations of circulating RNA. Existing separation and purification techniques using phenol-chloroform-based, spin column-based, or bead-based methods have been proposed to capture rare circulating RNA. However, most of these are expensive, time-consuming (> 30 minutes), and labor-intensive, rely on hazardous chemicals such as phenol, and require large or specialized equipment for centrifugation and temperature control. Furthermore, these methods utilize lysis buffers containing chaotropic reagents such as guanidine thiocyanate (GITC), which amplifies the genetic background derived from non-cancerous cells; they are unsuitable for processing large volumes of clinical samples; and they suffer from poor reproducibility due to the use of low concentrations of circulating RNA. Therefore, developing circulating RNA concentration and separation techniques to achieve high reproducibility and establishing a globally common circulating RNA extraction method remains a challenge.

[0006] Accordingly, the inventors developed HAZIS-CirR, a novel platform for the simple and rapid concentration and isolation of circulating RNA from the urine of PCa patients. This platform utilizes adipic acid dihydrazide (ADH), a homofunctional crosslinking agent containing hydrazides as reactive groups, and is based on the covalent and electrostatic interactions between ADH, zeolite, and circulating RNA. Compared to the limited sample usage of existing circulating RNA extraction methods, it allows for the concentration of rare circulating RNA using large-volume samples, enabling the extraction of high-concentration circulating RNA within 20 minutes. Since HAZIS-CirR does not involve a cell lysis process, it does not use chaotropic or harmful reagents, and it does not require large electric centrifuges or temperature controllers. The inventors analyzed various circulating mRNAs and circulating miRNAs using a total of 89 urine samples on HAZIS-CirR. It was demonstrated that circulating RNA can be rapidly, inexpensively, and effectively concentrated and extracted using urine samples from PCa (55 samples), BPH (24 samples), and healthy controls (10 samples), and mRNA and circulating miRNA that can be used as PCa biomarkers in BPH patients were presented. HAZIS-CirR, developed by the inventors, provides a technology for the concentration and isolation of circulating RNA, which is secreted into tumor sites and found in various bodily fluids, as well as for the analysis of high-concentration biomarkers of PCa, including early cancer stages. Prior art literature

[0008] Korean Patent Publication No. 10-2021-0144929 The problem to be solved

[0009] One aspect provides a composition for detecting or separating nucleic acids comprising a compound of Formula 1 or a compound of Formula 2 below:

[0010] [Chemical Formula 1]

[0011] ,

[0012] [Chemical Formula 2]

[0013] .

[0014] The above n is one integer selected from 0 to 10.

[0015] Another aspect is to provide a kit for the detection or isolation of nucleic acids comprising the above composition and a solid support.

[0016] Another aspect is the step of binding nucleic acids in a sample separated from an object to a compound selected from the group consisting of a compound of the following chemical formula 1 and a compound of the following chemical formula 2, the surface of a solid support, and

[0017] The present invention provides a method for detecting or separating nucleic acid, comprising the step of separating the nucleic acid from the surface of the solid support:

[0018] [Chemical Formula 1]

[0019] ,

[0020] [Chemical Formula 2]

[0021] ,

[0022] The above n is one integer selected from 0 to 10.

[0023] Another aspect is a composition for diagnosing prostate cancer (PCa) in a patient with benign prostatic hyperplasia (BPH), comprising the composition for detecting or isolating nucleic acids of claim 1,

[0024] The above nucleic acid provides a composition in which one or more selected from the group consisting of miR-141-3p, miR-375-3P, miR-483-5P and miR-574-3P.

[0025] Another aspect is to provide a kit for diagnosing prostate cancer (PCa) in patients with benign prostatic hyperplasia (BPH), comprising the above composition and a solid support.

[0026] Another aspect is the step of binding nucleic acids in a sample separated from an object to a compound selected from the group consisting of a compound of the following chemical formula 1 and a compound of the following chemical formula 2, the surface of a solid support, and

[0027] A method for providing information for the diagnosis of prostate cancer (PCa) in a patient with benign prostatic hyperplasia (BPH), comprising the step of isolating the nucleic acid from the surface of the solid support,

[0028] The above nucleic acid is one or more selected from the group consisting of miR-141-3p, miR-375-3P, miR-483-5P and miR-574-3P, providing a method:

[0029] [Chemical Formula 1]

[0030] ,

[0031] [Chemical Formula 2]

[0032] ,

[0033] The above n is one integer selected from 0 to 10.

[0034] Another aspect is to provide a composition for diagnosing an infectious disease comprising a composition for detecting or isolating the above nucleic acid.

[0035] Another aspect is to provide a diagnostic kit for infectious diseases comprising a composition for diagnosing infectious diseases and a solid support.

[0036] Another aspect is the step of binding nucleic acids in a sample separated from an object to a compound selected from the group consisting of a compound of the following chemical formula 1 and a compound of the following chemical formula 2, the surface of a solid support, and

[0037] The present invention provides a method for providing information for the diagnosis of an infectious disease, comprising the step of isolating the nucleic acid from the surface of the solid support:

[0038] [Chemical Formula 1]

[0039] ,

[0040] [Chemical Formula 2]

[0041] ,

[0042] The above n is one integer selected from 0 to 10. means of solving the problem

[0044] One aspect provides a composition for detecting or separating nucleic acids comprising a compound of Formula 1 or a compound of Formula 2 below:

[0045] [Chemical Formula 1]

[0046] ,

[0047] [Chemical Formula 2]

[0048] ,

[0049] The above n is one integer selected from 0 to 10.

[0050] In one aspect, the above n may be one integer selected from 0 to 10, 0 to 9, 0 to 8, 0 to 7, 0 to 6, 0 to 5, or 0 to 4.

[0051] When n is 0, the compound of Formula 1 is oxalyl dihydrazide (ODH); when n is 1, the compound of Formula 1 is malonic dihydrazide (MDH); when n is 2, the compound of Formula 1 is succinic dihydrazide (SDH); when n is 3, the compound of Formula 1 is glutaric dihydrazide (GDH); and when n is 4, the compound of Formula 1 is adipic dihydrazide (ADH). The compound of Formula 1 may be derived from nature or synthesized using known organic synthesis methods.

[0052] The compound of Chemical Formula 2 above is carbonic dihydrazide (CDH) and exists with a molecular weight (MW) of 90.08. It may be derived from nature or synthesized using known organic synthesis methods.

[0053] In one aspect, when n is 4, the composition for detecting or separating nucleic acids containing the compound of Formula 1 can separate nucleic acids more simply and quickly than the composition for detecting or separating nucleic acids containing the compound of Formula 1 or the compound of Formula 2 when n is 0 to 3 and 5 to 10.

[0054] The term "nucleic acid" above refers to deoxyribonucleotides or ribonucleotide polymers in linear or cyclic forms and in single-stranded or double-stranded forms.

[0055] In one aspect, the nucleic acid may be one or more nucleic acids selected from the group consisting of DNA, RNA, miRNA, mRNA, siRNA, tRNA, sgRNA, and shRNA, specifically, one or more selected from the group consisting of RNA, miRNA, tRNA, and mRNA, and more specifically, RNA or miRNA.

[0056] The term "detection" above refers to quantifying the concentration of a measured object.

[0057] The above term "separation" refers to physically dividing a clinical sample (e.g., nucleic acid) into at least two separate fractions.

[0059] Another aspect provides a kit for detecting or isolating nucleic acids comprising the above composition and a solid support.

[0060] The above terms, such as "nucleic acid," "detection," and "separation," may be within the aforementioned range.

[0061] In one aspect, it may be one or more selected from the group consisting of zeolite, chromosorb, and diatometic earth, specifically zeolite or diatometic earth, and more specifically zeolite.

[0062] The above solid support is sufficient if its surface has amine groups or can be modified to amine groups, and if the surface of the solid support has hydroxyl groups, it may be modified to amine groups through silanization.

[0063] In addition, in one aspect, the surface of the solid support may be modified with amine groups, and the shape of the solid support may be in the form of a microfluidic platform.

[0064] The term "surface" above is intended to mean an inner part or an outer layer of a solid support. The surface may be in contact with another material, e.g., a gas, liquid, gel, polymer, organic polymer, a second surface of a similar or different material, a metal, or a coat. The surface or an area thereof may be substantially flat. The surface may have surface features, e.g., wells, pits, channels, ridges, raised areas, pegs, pillars, etc.

[0065] The term "amine" above refers to an organic compound and functional group having a non-covalent electron pair on a nitrogen atom as a base, and is a derivative of ammonia in which the position for a hydrogen atom is replaced by a substituent such as an alkyl group or an aryl group.

[0066] The term "modification" refers to the process of reconstructing a form, collectively describing methods of transforming the chemical structure of a material's components and synthesizing or extracting desired materials in the process.

[0067] The term "silanization" above refers to a treatment method in which a silanol radical ≡SiOH in a gas chromatography carrier is substituted with an inert alkyl silicone ≡SiO-R using dimethyl chlorosilane, and in one aspect, the silanization may be a modification of the hydroxyl group to an amine group.

[0068] The above term "solid support" may optionally be inert to the chemical used to attach the nucleic acid. For example, the solid support may be inert to the chemical used to attach the nucleic acid.

[0069] In one aspect, the diameters of the zeolite, the chromosolv, and the diatomite may be 0.1 to 100 μm, 0.1 to 60 μm, 0.1 to 20 μm, 0.5 to 100 μm, 0.5 to 60 μm, 0.2 to 20 μm, 1 to 100 μm, 1 to 60 μm, and 1 to 20 μm.

[0070] When the diameter of the zeolite, the chromosolv, and the diatomite is 0.1 to 100 μm, the solid support can maintain a stable structure even after interaction with the composition for detecting or separating nucleic acids and the nucleic acids.

[0071] In one aspect, one compound selected from the group consisting of the compound of Formula 1 and the compound of Formula 2, the amine group present on the surface of the solid support, and the nucleic acid may interact through one or more attractive forces selected from the group consisting of imine bonds, hydrazone bonds, and electrostatic attraction.

[0072] Specifically, the aldehyde group of one compound selected from the group consisting of the compound of Formula 1 and the compound of Formula 2, or the amine group of a solid support, or the aldehyde group of one compound selected from the group consisting of the compound of Formula 1 and the compound of Formula 2, and the nitrogen base of a nucleic acid may interact through a reactive imine bond, and the aldehyde or ketone group of the nitrogen base of a nucleic acid may interact through a reactive hydrazone bond with one compound selected from the group consisting of the compound of Formula 1 and the compound of Formula 2, and the positive charge of a solid support and the negative charge of a phosphate group of a nucleic acid may interact through a reactive electrostatic attraction.

[0073] The term "reactive bond" above refers to a bond in which, in the presence of a preliminary substrate capable of forming a new bond or a substance capable of breaking the bond, an existing bond is broken to form a bond identical to the preliminary substrate, or an existing bond can be easily broken.

[0074] The term "imine bond" refers to a double bond between carbon and nitrogen, and compounds containing imine bonds are collectively called imine or Schiff bases.

[0075] The term "hydrazone bond" refers to a bond formed by the reaction of a hydrazide and an aldehyde, and "hydrazone" refers to a compound obtained by the reaction of an aldehyde or ketone with a hydrazine, in which a carbonyl group >C=O is changed to >C=N-NH2.

[0076] The term "electrostatic attraction" refers to the attractive force that acts when opposite positive (+) and negative (-) charges meet.

[0077] The term "interaction" above refers to a type of behavior in which two or more objects or targets influence each other, and the influence must manifest in both directions.

[0078] In one aspect, one compound selected from the group consisting of the compound of Formula 1 and the compound of Formula 2 and the solid support may be present in the kit in a weight ratio of 30:1 to 1:10, 30:1 to 1:1, 30:1 to 5:1, 20:1 to 1:10, 20:1 to 1:1, 20:1 to 5:1, 15:1 to 1:10, 15:1 to 1:1, or 15:1 to 5:1.

[0079] When the weight ratio of one compound selected from the group consisting of the compound of Formula 1 and the compound of Formula 2 in the above kit and the solid support is 30:1 to 1:10, the nucleic acid (e.g., RNA) capture efficiency of the one compound selected from the group consisting of the compound of Formula 1 and the compound of Formula 2 and the solid support may be superior.

[0080] The above kit may be performed by one or more techniques selected from the group consisting of reverse transcription polymerase chain reaction (RT-PCR), real-time reverse transcription polymerase chain reaction (Real-time RT-PCR), quantitative polymerase chain reaction (qPCR), competitive reverse transcription polymerase chain reaction (Competitive RT-PCR), real-time quantitative polymerase chain reaction (RT-qPCR), inverse polymerase chain reaction (Inverse PCR), long and accurate polymerase chain reaction (Long and Accurate PCR), RNase protection assay (RPA), Northern blotting, and DNA chips, and specifically, may be performed by one or more techniques selected from the group consisting of reverse transcription polymerase chain reaction (RT-PCR), competitive reverse transcription polymerase chain reaction (Competitive RT-PCR), real-time reverse transcription polymerase chain reaction (Real-time RT-PCR), and real-time quantitative polymerase chain reaction (RT-qPCR). It can be performed, and more specifically, by RT-qPCR or reverse transcription polymerase chain reaction (RT-PCR).

[0081] The above-mentioned kit for the detection or isolation of nucleic acids may additionally include one or more other types of compositions, solutions, or devices suitable for an analysis method, and the kit may be a diagnostic kit characterized by including essential elements necessary to perform the detection or isolation of nucleic acids.

[0082] In one aspect, the kit may be a diagnostic kit characterized by including essential elements necessary for performing DNA chip operations. The DNA chip kit may include a substrate to which cDNA or oligonucleotides corresponding to a gene or a fragment thereof are attached, and reagents, preparations, enzymes, etc., for producing fluorescently labeled probes. Additionally, the substrate may include cDNA or oligonucleotides corresponding to a control gene or a fragment thereof.

[0083] A composition and kit for nucleic acid detection or separation according to one aspect can rapidly and accurately detect a target nucleic acid in a sample at low cost by utilizing a solid support and one compound selected from the group consisting of the compound of Formula 1 and the compound of Formula 2, and interacting with the nucleic acid in the sample. Furthermore, it can separate the target nucleic acid at a high concentration.

[0084] Specifically, in one embodiment, HAZIS-CirR (a compound selected from the group consisting of a compound of Formula 1 or a compound of Formula 2 (adipic acid dihydrazide (ADH), carbonic dihydrazide (CDH), oxalyl dihydrazide (ODH), malonic dihydrazide (MDH), succinic dihydrazide (SDH)) and a solid support (zeolite)) was prepared for nucleic acid (circulating RNA) detection (concentration) and separation. By performing the following steps using the above HAZIS-CirR, nucleic acids were detected and separated within a short time: (1) mixing a sample (a compound of Formula 1 or a compound of Formula 2 according to one aspect, a solid support (zeolite), and urine (a sample containing nucleic acids)) and incubating the mixture; (2) filtering the mixture through a filter to remove uncaptured substances and concentrating the nucleic acids to a high concentration; (3) washing the surface of the solid support to remove contaminants remaining on the solid support; (4) separating the nucleic acids by using a buffer to disrupt the interaction between the compound, the solid support, and the nucleic acids (see Example 1-1(1)).

[0085] In addition, in one embodiment, it was confirmed that in step (1), one compound selected from the group consisting of a compound of Formula 1 or a compound of Formula 2 according to one aspect interacts with a solid support (zeolite) and a nucleic acid through imine bonding, hydrazone bonding, and electrostatic attraction (see Example 1-1(1)).

[0086] In another example, characterization was performed on the HAZIS-CirR to confirm the molecular characteristics and binding mechanisms between adipic acid dihydrazide (ADH), zeolite, and nucleic acid. As a result, it was confirmed that amine groups are formed on the surface of the zeolite, that the zeolite and adipic acid dihydrazide (ADH) bind via imine bonding, and that hydrazide is generated on the surface of the zeolite and can be used as a functional group. In addition, the zeta potential of the zeolite was analyzed, and as a result, it was confirmed that the zeolite and HAZ have high zeta potentials and that HAZ electrically binds to the negative charge of circulating RNA (see Example 1-2(2)).

[0087] In another example, the optimized concentrations of zeolite and adipic acid dihydrazide (ADH) in HAZIS-CirR were measured, and as a result, it was confirmed that RNA capture efficiency was high when 5 mg of zeolite and 50 mg of adipic acid dihydrazide (AHD) were used per 1 mL of sample (see Example 1-2(3)).

[0089] Another aspect is the step of binding nucleic acids in a sample separated from an object to a compound selected from the group consisting of a compound of the following chemical formula 1 and a compound of the following chemical formula 2, the surface of a solid support, and

[0090] A method for detecting or separating nucleic acid is provided, comprising the step of separating the nucleic acid from the surface of the solid support:

[0091] [Chemical Formula 1]

[0092] ,

[0093] [Chemical Formula 2]

[0094] .

[0095] The above n is one integer selected from 0 to 10.

[0096] The above "compound of Chemical Formula 1", "compound of Chemical Formula 2", "nucleic acid", "solid support", "separation", "detection", etc. may be within the aforementioned range.

[0097] In one aspect, the surface of the solid support may be modified with amine groups.

[0098] The term "surface" above is intended to mean an inner part or an outer layer of a solid support. The surface may be in contact with another material, e.g., a gas, liquid, gel, polymer, organic polymer, a second surface of a similar or different material, a metal, or a coat. The surface or an area thereof may be substantially flat. The surface may have surface features, e.g., wells, pits, channels, ridges, raised areas, pegs, pillars, etc.

[0099] The term "amine" above refers to an organic compound and functional group having a non-covalent electron pair on a nitrogen atom as a base, and is a derivative of ammonia in which the position for a hydrogen atom is replaced by a substituent such as an alkyl group or an aryl group.

[0100] The term "modification" refers to the process of reconstructing a form, collectively describing methods of transforming the chemical structure of a material's components and synthesizing or extracting desired materials in the process.

[0101] In one aspect, one compound selected from the group consisting of the compound of Formula 1 and the compound of Formula 2, the amine group present on the surface of the solid support, and the nucleic acid may interact through one or more attractive forces selected from the group consisting of imine bonds, hydrazone bonds, and electrostatic attraction.

[0102] Specifically, the aldehyde group of one compound selected from the group consisting of the compound of Formula 1 and the compound of Formula 2, or the amine group of a solid support, or the aldehyde group of one compound selected from the group consisting of the compound of Formula 1 and the compound of Formula 2, and the nitrogen base of a nucleic acid may interact through a reactive imine bond, and the aldehyde or ketone group of the nitrogen base of a nucleic acid may interact through a reactive hydrazone bond with one compound selected from the group consisting of the compound of Formula 1 and the compound of Formula 2, and the positive charge of a solid support and the negative charge of a phosphate group of a nucleic acid may interact through a reactive electrostatic attraction.

[0103] The term "reactive bond" above refers to a bond in which, in the presence of a preliminary substrate capable of forming a new bond or a substance capable of breaking the bond, an existing bond is broken to form a bond identical to the preliminary substrate, or an existing bond can be easily broken.

[0104] The term "imine bond" refers to a double bond between carbon and nitrogen, and compounds containing imine bonds are collectively called imine or Schiff bases.

[0105] The term "hydrazone bond" refers to a bond formed by the reaction of a hydrazide and an aldehyde, and "hydrazone" refers to a compound obtained by the reaction of an aldehyde or ketone with a hydrazine, in which a carbonyl group >C=O is changed to >C=N-NH2.

[0106] The term "electrostatic attraction" refers to the attractive force that acts when opposite positive (+) and negative (-) charges meet.

[0107] The term "interaction" above refers to a type of behavior in which two or more objects or targets influence each other, and the influence must manifest in both directions.

[0108] The above "individual" refers to all living things, including humans, rats, mice, livestock, etc. As a specific example, it may be mammals, including humans.

[0109] In one aspect, the sample may be one or more selected from the group consisting of blood, plasma, serum, tissue, cell, lymph, bone marrow fluid, saliva, ocular fluid, semen, brain extract, cerebrospinal fluid, joint fluid, thymic fluid, ascites fluid, amniotic fluid, urine, cell tissue fluid, and cell culture fluid; specifically, it may be one or more selected from the group consisting of blood, urine, plasma, serum, tissue, cell, lymph, bone marrow fluid, cell tissue fluid, and cell culture fluid; more specifically, it may be one or more selected from the group consisting of tissue, cell, cell tissue fluid, and cell culture fluid.

[0110] By using a nucleic acid detection or separation method according to one aspect, a target nucleic acid in a sample can be detected quickly and accurately at low cost by utilizing the interaction between a compound selected from the group consisting of the compound of Formula 1 and the compound of Formula 2 and the solid support with the nucleic acid in the sample, and furthermore, can be separated at a high concentration.

[0111] In one aspect, the method may further include a step of washing the surface of the solid support prior to the step of isolating the nucleic acid.

[0112] If the above method further includes the washing step prior to the step of separating the nucleic acid, the nucleic acid can be detected or separated more accurately by removing contaminants remaining on the solid support.

[0113] The above washing step can be performed using, for example, PBS buffer, ethanol, distilled water, etc.

[0114] In one aspect, the solid support may be one or more selected from the group consisting of zeolite, chromosorb, and diatometic earth, specifically zeolite or diatometic earth, and more specifically zeolite.

[0115] In addition, in one aspect, the method may further include a step of filtering a solid support and a compound selected from the group consisting of the compound of Formula 1 and the compound of Formula 2, prior to the step of separating the nucleic acid.

[0116] The term "filtration" above refers to a method of separating a mixture of liquid and solid substances using differences in particle size.

[0117] For example, the filtration may be performed using one or more selected from the group consisting of a microfiltration membrane (MF), an ultrafiltration membrane (UF), a nanofiltration membrane (NF), a reverse osmosis membrane (RO), and a polyvinylidene fluoride (PVDF) syringe filter; specifically, it may be performed using one or more selected from the group consisting of a PVDF syringe filter, a microfiltration membrane, and an ultrafiltration membrane; more specifically, it may be performed using a PVDF syringe filter.

[0118] The term "PVDF Syringe Filter" is a disposable filter used to filter suspended matter in a liquid sample using a hydrophilic PVDF membrane, and can filter with a membrane filter (a liquid or solid membrane capable of separating a mixture by separating and passing specific components), and can perform rapid purification and particle removal.

[0119] If the above method further includes the filtration step prior to the step of separating the nucleic acid, according to the method, high concentrations of nucleic acid can be detected or separated more accurately and quickly by removing material not captured on the solid support.

[0120] In one aspect, the filtration step may be performed with a filter having pores of less than 0.1 μm, specifically with a filter having pores of 0.3 μm, and more specifically with a filter having pores of 0.4 μm.

[0121] When the pores of the filter are less than 0.1 μm, fragments and unbound molecules smaller than 0.1 μm pass through the filter and are removed, and one compound selected from the group consisting of the compound of Formula 1 and the compound of Formula 2 and nucleic acids (e.g., circulating RNA) can be concentrated on the filter surface by a solid support.

[0122] In one aspect, the step of isolating the nucleic acid may be performed by using a buffer, specifically an elution buffer, to break the interaction between the compound, the solid support, and the nucleic acid.

[0124] Another aspect is a composition for diagnosing prostate cancer (PCa) in patients with benign prostatic hyperplasia (BPH), comprising a composition for detecting or isolating the above nucleic acid,

[0125] The above nucleic acid provides a composition in which one or more are selected from the group consisting of miR-141-3p, miR-375-3P, miR-483-5P and miR-574-3P.

[0126] The above "nucleic acid," "detection," "separation," etc. may be within the aforementioned range.

[0127] In one aspect, the nucleic acid may be one or more selected from the group consisting of miR-141-3p, miR-375-3P, miR-483-5P and miR-574-3P, and specifically may be miR-141-3p, miR-375-3P, miR-483-5P and miR-574-3P.

[0128] When one or more nucleic acids selected from the group consisting of miR-141-3p, miR-375-3P, miR-483-5P, and miR-574-3P are detected or isolated using the above-mentioned composition for detecting or isolating nucleic acids, it is possible to diagnose prostate cancer (PCa) in patients with benign prostatic hyperplasia (BPH), and when all of miR-141-3p, miR-375-3P, miR-483-5P, and miR-574-3P are detected or isolated, the diagnostic ability of the above-mentioned composition for prostate cancer (PCa) in patients with benign prostatic hyperplasia (BPH) may be superior.

[0129] The term "benign prostatic hyperplasia (BPH)" refers to a condition in which the size of the prostate increases. Symptoms may include frequent urination, difficulty urinating, weak flow, difficulty urinating, or loss of bladder control, and complications may include urinary tract infections, bladder stones, and chronic kidney problems.

[0130] The above term "patient" means human, non-human primates or other animals, particularly mammals such as cattle, horses, pigs, sheep, goats, dogs, cats, mice, rats, etc., and more specifically, the patient is a human.

[0131] The term "prostate cancer (PCa)" refers to a disease in which cells in the prostate gland divide and grow abnormally, eventually becoming a malignant tumor. Malignant tumors are not confined to the prostate but can invade surrounding tissues and metastasize to other organs via blood vessels or lymphatic vessels.

[0132] The term "diagnosis" above includes confirming the presence or absence of disease, the state of the disease, and the prognosis of the disease, and refers to all types of analysis used to derive disease states and decisions.

[0134] Another aspect provides a diagnostic kit for prostate cancer (PCa) in patients with benign prostatic hyperplasia (BPH), comprising the above-mentioned diagnostic composition and a solid support.

[0135] The above terms, such as "diagnosis," "solid support," "benign prostatic hyperplasia (BPH)," "patient," "prostate cancer (PCa)," and "kit," may fall within the aforementioned range.

[0136] When using a composition and kit for diagnosing prostate cancer (PCa) in patients with benign prostatic hyperplasia (BPH) according to a specific pattern, prostate cancer (PCa) in patients with benign prostatic hyperplasia (BPH) can be diagnosed accurately and rapidly.

[0137] Specifically, in one embodiment, the clinical utility of HAZIS-CirR for nucleic acid (circulating miRNA)-based prostate cancer (PCa) diagnosis was confirmed. As a result of nucleic acid analysis, it was confirmed that miR-141-3p, miR-375-3P, miR-483-5P, and miR-574-3P were overexpressed in prostate cancer (PCa) of patients with benign prostatic hyperplasia (BPH), and thus it was confirmed that prostate cancer in patients with benign prostatic hyperplasia can be diagnosed using said nucleic acids (see Example 1-2-(5)).

[0139] Another aspect is the step of binding nucleic acids within a sample separated from an organism to a compound selected from the group consisting of a compound of Chemical Formula 1 and a compound of Chemical Formula 2;

[0140] A method for providing information for the diagnosis of prostate cancer (PCa) in a patient with benign prostatic hyperplasia (BPH), comprising the step of isolating the nucleic acid from the surface of the solid support,

[0141] The above nucleic acid provides a method in which one or more selected from the group consisting of miR-141-3p, miR-375-3P, miR-483-5P and miR-574-3P:

[0142] [Chemical Formula 1]

[0143] ,

[0144] [Chemical Formula 2]

[0145] ,

[0146] The above n is one integer selected from 0 to 10.

[0147] The above "compound of Chemical Formula 1," "compound of Chemical Formula 2," "individual," "nucleic acid," "solid support," "benign prostatic hyperplasia (BPH)," "patient," "prostate cancer (PCa)," "diagnosis," etc. may be within the aforementioned range.

[0148] In one aspect, the sample may be one or more selected from the group consisting of blood, plasma, serum, tissue, cell, lymph, bone marrow fluid, saliva, ocular fluid, semen, brain extract, cerebrospinal fluid, joint fluid, thymic fluid, ascites fluid, amniotic fluid, urine, cell tissue fluid, and cell culture fluid, specifically, it may be one or more selected from the group consisting of blood, urine, plasma, serum, tissue, cell, lymph, bone marrow fluid, cell tissue fluid, and cell culture fluid, and more specifically, it may be tissue, cell, cell tissue fluid, or urine.

[0149] In one aspect, the nucleic acid may be one or more selected from the group consisting of miR-141-3p, miR-375-3P, miR-483-5P and miR-574-3P, and specifically may be miR-141-3p, miR-375-3P, miR-483-5P and miR-574-3P.

[0150] When one or more nucleic acids selected from the group consisting of miR-141-3p, miR-375-3P, miR-483-5P, and miR-574-3P are detected or isolated using the above method for diagnosing prostate cancer (PCa) in patients with benign prostatic hyperplasia (BPH), it is possible to diagnose prostate cancer (PCa) in patients with benign prostatic hyperplasia (BPH), and when all of miR-141-3p, miR-375-3P, miR-483-5P, and miR-574-3P are detected or isolated, the diagnostic ability of the above method for prostate cancer (PCa) in patients with benign prostatic hyperplasia (BPH) may be superior.

[0152] Another aspect provides a composition for diagnosing an infectious disease comprising a composition for detecting or isolating the above nucleic acid.

[0153] The above "nucleic acid," "detection," "separation," etc. may be within the aforementioned range.

[0154] The above term "infectious disease" refers to a disease that develops due to infection by various pathogens such as bacteria, spirochetes, rickettsia, viruses, fungi, and parasites. In one aspect, the infectious disease may be one or more selected from the group consisting of hand, foot, and mouth disease, rubella, epidemic conjunctivitis, influenza, enteritis, measles, impetigo, chickenpox, bacterial meningitis, and tuberculosis; specifically, it may be one or more selected from the group consisting of influenza, enteritis, chickenpox, and tuberculosis; more specifically, it may be tuberculosis.

[0155] The term "tuberculosis" refers to an infectious disease transmitted by a bacterium called Mycobacterium tuberculosis; when an active tuberculosis patient coughs or sneezes, the bacteria released into the air through saliva droplets or droplet nuclei become infectable through respiration.

[0156] In one aspect, the infectious disease is Coxsackie A virus, Rubella virus, Adeno virus, Influenza virus, Norovirus, Measles morbillivirus, Staphylococcus aureus ( Staphylococcus aureus ), Varicella zoster virus, Streptococcus pneumoniae ( Streptococcus pneumoniae ) and Mycobacterium tuberculousis ( Mycobacterium tuberculosis It may be caused by one or more microorganisms selected from the group consisting of ), specifically influenza virus, norovirus, Measles morbillivirus, and Mycobacterium tubulocyrolysis ( Mycobacterium tuberculosis It may be caused by one or more microorganisms selected from the group consisting of ), and more specifically, Mycobacterium tuberculousis ( Mycobacterium tuberculosisIt may be caused by ).

[0157] In one aspect, the nucleic acid may be the IS6110 gene derived from Mycobacterium tuberculosis.

[0159] Another aspect provides a diagnostic kit for infectious diseases comprising a diagnostic composition for infectious diseases and a solid support.

[0160] The above "infectious disease," "solid support," "diagnosis," "kit," etc. may be within the aforementioned scope.

[0161] A kit according to one aspect can rapidly and accurately detect a target nucleic acid in a sample at low cost by utilizing a solid support and one compound selected from the group consisting of the compound of Chemical Formula 1 and the compound of Chemical Formula 2, and the interaction of the solid support with the nucleic acid in the sample, thereby enabling rapid and accurate diagnosis of infectious diseases by detecting or isolating genes derived from microorganisms that cause infectious diseases.

[0162] Specifically, in one embodiment, a microfluidic platform and a zeolite filter were used for pathogen concentration and nucleic acid extraction: (1) mixing a sample (a compound of Formula 1 or a compound of Formula 2 according to one aspect, a solid support (microfluidic platform and zeolite filter) and Brucella Ovis (a sample containing nucleic acid)) and incubating the mixture; (2) filtering the mixture through a filter to remove uncaptured material and concentrating the nucleic acid to a high concentration; (3) washing the surface of the solid support to remove contaminants remaining on the solid support; (4) isolating the nucleic acid by using a buffer to break the interaction between the compound, the solid support and the nucleic acid (see Examples 2-1(2) and 2-1(3)).

[0163] In another example, the operation and efficiency of an adipic acid dihydrazide-based sample pretreatment system for pathogen concentration and nucleic acid extraction were verified. As a result of performing pathogen concentration and nucleic acid extraction using a dimorphic hydrazide, adipic acid dihydrazide showed the highest capture efficiency among the dimorphic hydrazides, and it was confirmed that pathogen concentration and nucleic acid extraction were possible at a similar level in sample preparation using all candidate substances (see Example 2-2(1)).

[0164] In one embodiment, the clinical utility of adipic acid dihydrazide-based sample pretreatment was verified by concentrating Mycobacterium tuberculosis and using three nasopharyngeal swab samples. As a result, it was confirmed that all three samples tested positive for the IS6110 gene in DNA extracted using a column-based kit. Furthermore, it was confirmed that the adipic acid dihydrazide-based sample pretreatment system had a lower Ct value and a higher amount of nucleic acid detected compared to the conventional method. This demonstrates that false negatives caused by low detection sensitivity can be reduced based on high-efficiency pathogen concentration, and confirms that it can be used for infectious diseases requiring rapid diagnosis through a simple and fast process (see Example 2-2(3)).

[0166] Another aspect is the step of binding nucleic acids in a sample separated from an object to a compound selected from the group consisting of a compound of the following chemical formula 1 and a compound of the following chemical formula 2, the surface of a solid support, and

[0167] A method for providing information for the diagnosis of an infectious disease is provided, comprising the step of isolating the nucleic acid from the surface of the solid support:

[0168] [Chemical Formula 1]

[0169] ,

[0170] [Chemical Formula 2]

[0171] ,

[0172] The above n is one integer selected from 0 to 10.

[0173] The above "compound of Chemical Formula 1," "compound of Chemical Formula 2," "organ," "nucleic acid," "solid support," "infectious disease," "diagnosis," etc. may be within the aforementioned range.

[0174] In one aspect, the sample may be one or more selected from the group consisting of blood, plasma, serum, tissue, cell, lymph, bone marrow fluid, saliva, ocular fluid, semen, brain extract, cerebrospinal fluid, joint fluid, thymic fluid, ascites fluid, amniotic fluid, urine, cell tissue fluid, and cell culture fluid; specifically, it may be one or more selected from the group consisting of blood, saliva, urine, plasma, serum, tissue, cell, lymph, bone marrow fluid, cell tissue fluid, and cell culture fluid; more specifically, it may be one or more selected from the group consisting of saliva, tissue, cell, cell tissue fluid, and cell culture fluid. Effects of the invention

[0176] By using a composition, kit, and method for detecting or isolating nucleic acids according to one aspect, high concentrations and high purity nucleic acids can be detected or isolated rapidly, simply, and accurately at low cost without the electricity and special equipment used in conventional column and bead-based methods, such as centrifugation, vacuum pumps, and heat controllers. Furthermore, by using a composition according to one aspect to detect or isolate one or more nucleic acids selected from the group consisting of miR-141-3p, miR-375-3P, miR-483-5P, and miR-574-3P, prostate cancer (PCa) in patients with benign prostatic hyperplasia (BPH) can be diagnosed rapidly, precisely, and early, and infectious diseases can be diagnosed rapidly, precisely, and early by detecting or isolating nucleic acids derived from microorganisms that cause infectious diseases. Brief explanation of the drawing

[0178] Figure 1 is a schematic diagram of HAZIS-CirR. Figure 2 is a figure showing the structure of modified zeolites (amine-modified zeolite (AZ), hydrazide-modified amine-modified zeolite (HAZ), and hydrazide-modified amine-modified zeolite (HAZ) with nucleic acid (NA). Figure 3 is a figure showing a clinical specimen. Figure 4 is a figure showing the results of confirming the shape and size distribution of zeolites using scanning electron microscope (SEM) images and dynamic light scattering (DLS). Figure 5 is a figure showing the optimization of HAZIS-CirR comparing the circulating RNA capture efficiency according to the concentration of amine-modified zeolite (AZ) and the circulating RNA capture efficiency according to the concentration of adipic acid dihydrazide (ADH). Figure 6 shows the copy number and capture efficiency of HAZIS-CirR through standard curves obtained by serial dilution of hsa-mir-21-5p ss mimics. Figure 7 shows the results of the circulating mRNA analysis of PCA3 (prostate cancer antigen 3) and TMPRSS2-ERG gene fusion 3 in HAZIS-CirR. Figure 8 shows the results of the circulating miRNA analysis of PCA3 (prostate cancer antigen 3) and TMPRSS2-ERG gene fusion 3 in HAZIS-CirR. Figure 9 is a figure illustrating a method for concentrating pathogens using isomorphic functional hydrazide. Figure 10 is a figure showing the results of comparing pathogen concentration efficiency using isomorphic functional hydrazide. Figure 11 shows the results of a comparative analysis of the efficiency of DNA and RNA detection using a microfluidic platform, zeolite, and syringe filter. Specific details for implementing the invention

[0179] The present invention will be explained in more detail below through examples. However, these examples are intended to illustrate the invention and the scope of the invention is not limited to these examples.

[0181] Example 1. RNA detection using zeolite

[0182] 1. Experimental Methods and Materials

[0183] (1) Fabrication of a zeolite filter system

[0184] Amine-modified zeolite for pathogen concentration and nucleic acid extraction using zeolite (96096-100G, Sigma-Aldrich, St. Louis, MO, USA) was prepared in two steps as follows: (1) Zeolite washing step; 3 g of zeolite was mixed with 150 mL of ultrapure distilled water at room temperature at 550 rpm for 10 minutes and washed twice. The precipitate was washed two more times with 150 mL of distilled water under the same conditions. After washing, the mixture was separated into a 50 mL conical tube and centrifuged at 1,400 rpm for 1 minute. The supernatant was discarded, and the pure zeolite precipitate was dried and stored. (2) APDMS functionalization step; 3 mL of 3-aminopropyl-methyl-dithoxysilane (APDMS, 371890-50 mL, Sigma-Aldrich) was added to 150 mL of 95% ethanol and stirred at 550 rpm for 3 minutes at room temperature. The pure zeolite was mixed with 95% ethanol containing 3-aminopropyl-methyl-dithoxysilane and stirred at 450 rpm for 4 hours at room temperature. After silylation, the precipitate was washed several times with ultrapure distilled water and 95% ethanol to remove residual 3-aminopropyl-methyl-dithoxysilane. After washing, the mixture was separated into a 50 mL conical tube and centrifuged at 1,400 rpm for 1 minute. The supernatant was discarded, and the amine-modified zeolite precipitate was dried using a vacuum chamber until all remaining ethanol evaporated, and then stored.

[0186] (2) Manufacturing and operation of HAZIS-CirR

[0187] To use zeolite (96096-100G, Sigma-Aldrich, St. Louis, MO, USA) in HAZIS-CirR, amine modification of the zeolite through silanization was performed in several steps. First, to wash the zeolite, 150 mL of ultrapure distilled water (DW) and 95% ethanol-DW (95:5, v / v) were used with 3 g of zeolite, and the mixture was stirred at 550 rpm for 10 minutes at room temperature (RT). After washing, 150 mL of 2% 3-aminopropyl diethoxymethylsilane (APDMS: aminopropyl diethoxymethylsilane, 371890-50 mL, Sigma-Aldrich, St. Louis, MO, USA)-95% ethanol (2:98, v / v) was used for the silanization of the washed zeolite and stirred at 450 rpm for 4 hours at room temperature. After silanization, 150 mL of ultra-high purity DW and 95% ethanol was used for washing the amine-modified zeolite (AZ: amine-modified zeolite) and stirred at 550 rpm for 10 minutes at room temperature. After washing several times, the amine-modified zeolite was completely dried using a vacuum chamber and pump and stored at room temperature until use.

[0188] The process of HAZIS-CirR for circulating RNA concentration and isolation consists of four steps (Fig. 1): (1) sample mixing and incubation, (2) concentration, (3) washing, and (4) isolation. A 1 mL solution containing an hsa-mir-21-5p mimic (Shanghai GenePharma Co., Ltd., Shanghai, China) was used for the optimization of HAZIS-CirR and to confirm clinical utility by analyzing circulating RNA in a 4 mL urine sample using HAZIS-CirR. Specifically, (1) in the sample mixing and incubation step, 2.5 mg, 5 mg, or 10 mg of amine-modified zeolite and 10 mg, 25 mg, or 50 mg of adipic acid dihydrazide (ADH: adipic acid dihydrazide, 8.41689.0050, Merck Millipore) or homobifunctional hydrazides (HHs: homobifunctional hydrazides) were added per 1 mL of sample. Dihydrazide adipic acid was selected by optimization of HAZIS-CirR using isomorphic dihydrazides (Fig. 2): carbonic dihydrazide (CDH: carbonic dihydrazide, C11006-100G, Sigma-Aldrich, St. Louis, MO, USA), oxalyl dihydrazide (ODH: oxalyl dihydrazide, 131296-25G, Sigma-Aldrich, St. Louis, MO, USA), malonic dihydrazide (MDH: malonic dihydrazide, M3206, Tokyo Chemical Industry, Japan), and succinic dihydrazide (SDH: succinic dihydrazide, S5502-25G, Sigma-Aldrich (St. Lurous, MO, USA)). After mixing the samples, incubation for covalent and electrostatic reactions was performed at room temperature for 10 minutes.(2) In the concentration step, 1 mL to 4 mL of incubated samples were passed through a sterile 0.45 μm PVDF (polyvinylidene fluoride) syringe filter (FJ13BSCPV004AL01, GVS Filter Technology, Indianapolis, USA) using a 1 mL to 5 mL syringe (Korea Vaccine Co., Ltd, Yongin, Korea). Circulating RNA captured by amine-modified zeolites by covalent and electrostatic bonding was filtered based on the size of the amine-modified zeolites, and the amine-modified zeolites and uncaptured fragments were passed through the filter and discarded. (3) In the washing step, 3 mL of PBS was used to remove remaining contaminants. (4) In the isolation step, 100 μL of 50 mM sodium bicarbonate (S5761-500G, Sigma-Aldrich, St. Louis, MO, USA) elution buffer (pH 10.6) was used to break the cross-linking of circulating RNA and adipic acid dihydrazide on the amine-modified zeolite. The eluted circulating RNA was collected in a 1.5 mL microcentrifuge tube and stored at sub-zero temperatures (-20°C or -80°C) until further use.

[0190] (3) Evaluation of detection limit and circulating RNA capture efficiency of HAZIS-CirR

[0191] To evaluate the detection limit and circulating RNA capture efficiency of HAZIS-CirR, hsa-mir-21-5p ss mimics were used. The hsa-mir-21-5p ss mimic was 20 μM (1.204 x 10⁻⁶). 13 From copies / reaction) 2 aM(1204 x 10 0It was used by serial dilution into copies / reaction. The hsa-mir-21-5p ss mimic was diluted in a 1.5 mL microcentrifuge tube and stored at sub-zero temperatures (-20°C or -80°C) until use. Material information regarding chemical modification was analyzed by measuring the zeta potential and Fourier Transform-Infrared Spectroscopy (FT-IR) results of nucleic acids with zeolites, amine-modified zeolites, isomorphic hydrazide and amine-modified zeolites (hydrazide-modified amine-modified zeolite, HAZ), and hydrazide-modified amine-modified zeolite (HAZ) using dynamic light scattering (Nano ZS DLS, Malvern Panalytical Ltd, UK) and an FT-IR Fourier Transform-Infrared spectrometer (Vertex 70, Bruker, Germany). Nucleic acids were analyzed using a field emission scanning electron microscope (JSM-7800F Prime, JEOL Ltd, Japan). Scanning electron microscope (SEM) images of zeolite, amine-modified zeolite, hydrazide-modified amine-modified zeolite (HAZ), and hydrazide-modified amine-modified zeolite (HAZ) were obtained.

[0193] (4) cDNA synthesis and real-time PCR

[0194] According to the manufacturer's recommended protocol, circulating RNA was reverse transcribed into cDNA using the Sensiscript RT Kit for mRNA (205213, Qiagen, Hilden, Germany) and the Mir-X miRNA First-Strand Synthesis Kit for miRNA (638313, Takara Bio USA, Inc., Kyoto, Japan). The synthesized cDNA was stored at -20°C until use. For the analysis of circulating mRNA, the QuantiTect SYBR green PCR Kit (204145, Qiagen, Hilden, Germany) was used for the detection of prostate cancer antigen 3 (PCA3), transmembrane protease serine 2 (TMPRSS2)-ERG gene fusion 3, and 18S ribosomal RNA (18S rRNA). Forward and reverse primers were synthesized to a standard length of approximately 24 bp (Table 1). The amplification protocol consisted of an initial denaturation step at 95°C for 10 minutes, 40 cycles of 30 seconds at 95°C, 3 seconds at 62°C, and 3 seconds at 72°C, and a cooling step at 40°C for 30 seconds. For cyclic miRNA analysis, the TB Green Advantage qPCR Premix kit (639676, Takara Bio USA, Inc., Kyoto, Japan) was used for the detection of has-miR-21-5p, 141-3p, 375-3p, 148a-3-p, 483-5p, 574-3p, and the non-coding small nuclear RNA component (U6snRNA) of U6 snRNP. Mature sequences were used as forward primers, and the reverse primers for miRNA and U6 snRNA were the primers provided in the kit (Table 1).Amplified products with a TB Green signal were obtained using a QuantStudio 3 Real Time PCR system (Thermo Fisher Scientific Inc., Waltham, MA, USA). Gel electrophoresis was performed to separate PCR products on a 2% agarose gel containing LoadingSTAR (A750, Dyne Bio Inc., Seoul, Korea). The gel was visualized using a ChemiDoc XRS+ system (Bio-Rad, Marnes-la-Coquette, France).

[0195] Target Location Sequence (5'-3') Length (bp) order number mRNA PCA3 Forward GAG AAC AGG GGA GGG AGA G 19 Sequence No. 1 Reverse ACG TTC TGG GAT ACA TGT GC 20 Sequence No. 2 TMPRSS2- ERG fusion Forward CCT GGA GCG CGG CAG GAA GCC TTA TCA GTT G 31 Sequence No. 3 Reverse TCC TGC TGA GGG ACG CGT GGG CTC ATC TTG 30 Sequence No. 4 18S rRNA Forward CCT GGA TAC CGC AGC TAG GA 20 Sequence No. 5 Reverse GCG GCG CAA TAC GAA TGC CCC 21 Sequence number 6 microRNA has-miR-21-5p Forward TAG CTT ATC AGA CTG ATG TTG A 22 Sequence No. 7 has-miR-141-3p Forward TAA CAC TGT CTG GTA AAG ATG G 22 Sequence No. 8 has-miR-375-3p Forward TTT GTT CGT TCG GCT CGC GTG A 22 Sequence No. 9 has-miR-148a-3p Forward TCA GTG CAC TAC AGA AGT TTG T 22 Sequence number 10 has-miR-483-5p Forward AAG ACG GGA GGA AAG AAG GGA G 22 Sequence number 11 has-miR-574-3p Forward CAC GCT CAT GCA CAC ACC CAC A 22 Sequence No. 12 Universal Reverse mRQ 3' Primer (provided in kit) . U6 snRNA Forward U6 Forward Primer (provided in kit) . Reverse U6 Reverse Primer (provided in kit) .

[0197] (5) Analysis of circulating RNA

[0198] Relative quantification (RQ) values ​​of circulating RNA were calculated using the comparative Ct method, in which 18S rRNA for mRNA and U6 snRNA for miRNA were used as reference genes. Relative quantification values ​​of gene expression were analyzed using the following Equations 1 to 3:

[0199] [Mathematical Formula 1]

[0200] △Ct = Ct (Target gene) - Ct (Reference gene),

[0201] [Mathematical Formula 2]

[0202] △△Ct = △Ct (PCa, BPH, or Normal) - △Ct (Average of Normal),

[0203] [Mathematical Formula 3]

[0204] Relative Quantification (RQ) value = 2 -△△Ct .

[0206] All relative quantitative values ​​were automatically calculated by the formula, and P-values ​​and graphs from unpaired t-tests were obtained using GraphPad Prism 8 statistical software (GraphPad Software, San Diego, CA, USA).

[0208] (6) Clinical analysis method

[0209] For clinical analysis, urine samples were collected from 55 PCa patients, 24 BPH patients, and 10 control subjects (Fig. 3). All subjects completed informed consent forms prior to participating in the study. The urine samples used in the experiment were selected from patients of similar age, and the experimental method was conducted as follows: (1) 4 mL of urine sample was prepared, and during the sample mixing and incubation step, 5 mg of amine-modified zeolite and 50 mg of adipic acid dihydrazide were added per 1 mL of sample. After sample mixing, incubation for covalent and electrostatic reactions was performed at room temperature for 10 minutes. (2) In the concentration step, 4 mL of the incubated sample was passed through a sterile 0.45 μm PVDF syringe filter (FJ13BSCPV004AL01, GVS Filter Technology, Indianapolis, USA) using a 5 mL syringe (Korea Vaccine Co., Ltd, Yongin, Korea). Circulating RNA captured by amine-modified zeolites via covalent and electrostatic bonding was filtered based on the size of the amine-modified zeolites, and the amine-modified zeolites and uncaptured fragments were passed through the filter and discarded. (3) In the washing step, 3 mL of PBS was used to remove remaining contaminants. (4) In the isolation step, 100 μL of 50 mM sodium bicarbonate (S5761-500G, Sigma-Aldrich, St. Louis, MO, USA) elution buffer (pH 10.6) was used to break the cross-linking of circulating RNA and adipic acid dihydrazide on the amine-modified zeolites. The eluted circulating RNA was collected in a 1.5 mL microcentrifuge tube and stored at sub-zero temperatures (-20°C or -80°C) until further use.

[0211] 2. Experimental Results

[0212] (1) Circulating RNA Concentration and Separation Using HAZIS-CirR

[0213] HAZIS-CirR is a novel circulating RNA enrichment and isolation platform based on the molecular properties of amine-modified zeolite (AZ) and adipic acid dihydrazide (ADH) and size-based filtration of polyvinylidene fluoride (PVDF) sterile filters. Fig. 1A illustrates HAZIS-CirR designed for the enrichment and isolation of circulating RNA derived from urine samples from individuals with prostate cancer (PCa), benign prostatic hyperplasia (BPH), or healthy controls. The process of HAZIS-CirR for circulating RNA enrichment and isolation consists of four steps (Fig. 1): (1) sample mixing and incubation, (2) enrichment of circulating RNA, (3) washing of circulating RNA, and (4) isolation of circulating RNA. These procedures do not require any additional steps prior to manipulation other than the functionalization of the zeolite, and through direct interaction between the amine-modified zeolite, adipic acid dihydrazide, and circulating RNA, circulating RNA enrichment and isolation are possible within 20 minutes, including a 10-minute incubation time. (1) Covalent and electrostatic coupling between the amine-modified zeolite, adipic acid dihydrazide, and circulating RNA during the sample mixing and incubation steps is shown in FIG. 1B. Adipic acid dihydrazide is water-soluble, odorless, and has low toxicity, and is used as a crosslinking agent in many fields, including the manufacture of mechanical latex films and injectable oxidized hyaluronic acid hydrogels. In this experiment, a simple and rapid method for nucleic acid (NA) enrichment and isolation for molecular diagnostics using adipic acid dihydrazide was proposed for the first time. Adipic acid dihydrazide, a pH-sensitive linker and non-chaotropic reagent, is a symmetric molecule having a C4 backbone and a reactive group of hydrazide (C=ONHNH2).The hydrazide group of adipic acid dihydrazide can react with the aldehyde or ketone group of other molecules to form a reactive hydrazone bond. Additionally, because it has two primary amine groups and one aldehyde group within one hydrazide group, it is nucleophilic and can react with amine-reactive and aldehyde-reactive groups. In HAZIS-CirR, the binding mechanism between the amine-modified zeolite, adipic acid dihydrazide, and circulating RNA can be described as follows: 1) The aldehyde group of adipic acid dihydrazide reacts with the amine group of the amine-modified zeolite and the nitrogenous bases (G, A, and C) of the RNA nucleotide, and the amine group of adipic acid dihydrazide reacts with the aldehyde or ketone group of the nitrogenous bases (G, C, and U) to form a reactive imine bond. 2) The hydrazide group of adipic acid dihydrazide reacts with the aldehyde or ketone group of the nitrogenous base (G, C, and U) of the RNA nucleotide to form a reactive hydrazone bond. 3) The positive charge of the amine-modified zeolite and adipic acid dihydrazide reacts with the negative charge of the phosphate group of the RNA nucleotide through electrostatic attraction. The functional groups of the nitrogenous bases are present in Fig. 1. Circulating RNA captured on the surface of the amine-modified zeolite is injected into a PVDF syringe filter, and debris and unbound molecules smaller than the filter pore size (0.45 μm) pass through the filter and are discarded, while the circulating RNA is concentrated on the filter surface by the amine-modified zeolite. Residual debris is removed by PBS washing, and the circulating RNA is extracted using an elution buffer (pH 10.6) by breaking the reactive bond between the amine-modified zeolite, adipic acid dihydrazide, and circulating RNA. Because HAZIS-CirR does not have a cell lysis step and uses non-chaotropic reagents, there is no contamination by background nucleic acids (NA) that may be released during cell lysis, and there is no degradation of extracted RNA caused by the use of chaotropic reagents.In addition, high concentration and high purity RNA can be extracted quickly and simply without the electricity and special equipment used in conventional column and bead-based methods, such as centrifugation, vacuum pumps, and heat controllers.

[0215] (2) Characterization of HAZIS-CirR

[0216] First, characterization was performed on HAZIS-CirR to confirm the molecular properties and binding mechanisms between the amine-modified zeolite, adipic acid dihydrazide, and nucleic acid. The morphology and size distribution of the zeolite were confirmed using scanning electron microscopy (SEM) images and dynamic light scattering (DLS) (Fig. 4). The zeolites have a size of approximately 2 μm to 18 μm (mostly 6 μm to 14 μm) and maintain a stable structure even after interactions with adipic acid dihydrazide and nucleic acid (HAz-NA) following silanization. Fig. 4C shows the FT-IR results on HAZIS-CirR. Pure zeolite exhibited absorption peaks at 464 (symmetric bending of TO, T: tetrahedral bonded Si or Al), 547 (double six-membered ring of TOT symmetric stretching), 667 (SiOSi symmetric stretching), 777 (symmetric TOT stretching), and 1028 (SiOAl asymmetric stretching relative to TO), as well as 1654 (OH bending) and a broad 3467–3572 (hydroxyl group) cm⁻¹. -1 After silanization, the amine-modified zeolite exhibited widths of 1654 and 3467–3572 cm by NH4 stretching and NH4 bending, respectively. -1 It exhibited an increased absorption peak. These results demonstrate that amine groups are formed on the surface of the amine-modified zeolite. Additionally, the HAZ was 1471 (CN stretching), 1535 (NH bending), 2864 (CH stretching and bending), 2925 (CH stretching), and 3317 (NH stretching) cm⁻¹. -1It showed an additional absorption peak at 1654 (C=O stretching) and a wide 3467–3572 (NH stretching and bending) cm⁻¹ due to adipic acid dihydrazide. -1 The absorption peak was increased at. From these results, it was confirmed that the amine-modified zeolite and adipic acid dihydrazide are bound by the imine bond shown in Fig. 1B, and that hydrazide is generated on the surface of the amine-modified zeolite and can be used as a functional group. In addition, HAZ-NA is a hydrazide (1471, 1535, 2864, 2925, and 3317 cm⁻¹). -1 The absorption peak representing ) is reduced by imine, hydrazine binding and electrostatic attraction, and the absorption peak caused by nucleic acids (broad 3317–3572 cm⁻¹) -1It shows that ) increases. Based on FT-IR analysis, amine-modified zeolite and adipic acid dihydrazide can be used to capture circulating RNA. Figure 4D shows the zeta potentials of pure zeolite, amine-modified zeolite, HAZ, and HAZ-nucleic acid. The zeta potential of the pure zeolite was -44.58 mV, which is attributed to the OH and oxygen-containing groups present on the zeolite surface. The zeta potential of the amine-modified zeolite increased to -11.62 mV compared to the pure zeolite due to immobilized amine groups on the zeolite surface. The zeta potential of the HAZ was -13.82 mV, similar to that of the amine-modified zeolite, and this result is attributed to the interaction between the amine and aldehyde groups within the hydrazide group of the amine-modified zeolite and adipic acid dihydrazide. The zeta potential of HAZ-NA was -33.92 mV, which is attributed to the strong negative charge of the phosphate groups of the nucleic acids binding to HAZ. Based on the analysis of zeta potential, it was confirmed that amine-modified zeolites and HAZ possess high zeta potentials and that HAZ electrically binds to the negative charge of circulating RNA. Therefore, these characterization results indicate that HAZIS-CirR can be used as a novel platform for circulating RNA enrichment and isolation.

[0218] (3) Optimization of HAZIS-CirR

[0219] The optimized concentrations of amine-modified zeolite and adipic acid dihydrazide were measured in HAZIS-CirR. Optimizing the concentrations of amine-modified zeolite and adipic acid dihydrazide is essential to avoid low binding affinity at low concentrations and low capture efficiency of circulating RNA caused by intermolecular interference at high concentrations. The standard for optimization was set to 1 mL of sample, and amine-modified zeolite and adipic acid dihydrazide were added in equal proportions according to changes in sample volume. The inventors used different concentrations of amine-modified zeolite and adipic acid dihydrazide and confirmed that RNA capture efficiency was high when 5 mg of amine-modified zeolite and 50 mg of adipic acid dihydrazide were used per 1 mL of sample (Fig. 5). In addition, regarding HAZIS-CirR, RNA was captured using five candidate HH substances (adipic acid dihydrazide (ADH), carbon dihydrazide (CDH), oxalyl dihydrazide (ODH), malonic acid dihydrazide (MDH), and succinic acid dihydrazide (SDH)), and it was confirmed that the capture efficiency was best when using adipic acid dihydrazide (ADH) (Figs. 4B and 4C). Furthermore, the detection limit and capture efficiency of HAZIS-CirR were verified. The copy number and capture efficiency before and after HAZIS-CirR were compared using hsa-mir-21-5p ss mimics at the same concentration. Calculations were performed using the equation values ​​of the standard curve obtained by serial dilution of the hsa-mir-21-5p ss mimics (Fig. 6 and Table 2). The inventors found that the detection limit remains the same as before even after HAZIS-CirR (1.204 Х 10 3 It was confirmed that the concentration and isolation of circulating RNA is possible with high efficiency of 82.03-92.38% (copies / reaction) (Table 3). These optimization data confirmed that HAZIS-CirR can capture circulating RNA with high efficiency without limiting the sample volume.

[0220] Replicate No. 1.204 X 10 N copies reaction -1 (C T ) 8 7 6 5 4 3 #1 14.121 18.533 23.883 27.251 31.902 35.100 #2 14.333 18.933 24.256 27.249 32.396 36.272 #3 14.093 19.323 24.770 28.600 32.791 36.241 Mean 14.182 18.930 24.303 27.700 32.363 35.871 STDEV 0.132 0.395 0.445 0.779 0.446 0.668

[0221] Replicate No. 1.204 X 10 N copies reaction -1 (C T ) 7 6 5 4 3 #1 19.173 24.513 27.513 31.984 35.984 #2 19.057 24.615 27.615 32.849 36.349 #3 19.365 24.487 28.487 32.707 36.407 Mean 19.198 24.539 27.872 32.514 36.247 Capture efficiency (%) 86.81 88.34 91.35 92.38 82.03

[0223] (4) Clinical utility of circulating mRNA in HAZIS-CirR

[0224] The clinical utility of HAZIS-CirR for circulating mRNA-based PCa diagnosis was confirmed by analyzing circulating mRNA from 84 urine samples (Fig. 3). Prostate cancer antigen 3 (PCA3) and transmembrane protease serine 2 (TMPRSS2)-ERG gene fusion 3 were used as circulating mRNA biomarkers, and relative quantification (RQ) values ​​were calculated. The PCA3 gene is a long non-coding RNA overexpressed in prostate cancer cells and has recently been used in clinical applications as a PCa biomarker. The ERG oncogene is known to be overexpressed in more than 50% of PCa cases, and ERG overexpression is induced by fusion with TMPRSS2, prostate-specific, and androgen-regulated genes; the TMPRSS2-ERG gene fusion is a mutation found in 40-70% of PCa cases. Research is underway to validate and apply the potential value of using the two biomarkers together. Figures 7A and 7B show the results of circulating mRNA analysis of PCA3 in 60 samples and TMPRSS2-ERG gene fusion 3 in 33 samples from HAZIS-CirR. In the analysis of circulating mRNA, PCA3 did not differ significantly among PCa (mean RQ = 0.64 ± 0.68), BPH (mean RQ = 0.71 ± 0.46), and healthy controls (mean RQ = 0.93 ± 0.96). These results were contrary to our expectation that there would be a significant difference in PCA3 among PCa, BPH, and healthy controls. TMPRSS2-ERG gene fusion 3 was found to be highly overexpressed in PCa compared to BPH (mean RQ = 1.41 ± 1.81) (P < 0.01) (mean RQ = 6.58 ± 6.23).As a result, in the diagnosis of PCa using circulating mRNA in HAZIS-CirR, TMPRSS2-ERG gene fusion 3 as a biomarker showed a clear difference compared to BPH, whereas PCA3 did not show a significant difference between PCa and BPH and was a healthy control. These results provide some discussion regarding the use of urinary circulating mRNA as a PCa biomarker. First, circulating mRNA is fragmented into short lengths and is easily degraded in urine. Unlike the approximately 160–167 nucleotides (NT) of circulating nucleic acids in plasma, urinary circulating nucleic acids are further fragmented into shorter lengths (less than 100 nt) through glomerular filtration, which leads to the easy degradation of circulating nucleic acids. Oreskovic et al. proposed the development of an extraction platform for small-sized fragmented nucleic acids, and the selection of short-length target sequences is required for the use of urinary circulating nucleic acids in clinical studies. Conventional nucleic acid extraction using silica materials is unsuitable for the extraction of short circulating nucleic acids because it is a method dependent on nucleic acid length, resulting from hydrophobic interactions caused by dehydration of the silica and nucleic acid surfaces and hydrogen bonding between the silica and the nucleic acid backbone. This demonstrates that HAZIS-CirR, which utilizes covalent and electrostatic coupling, may be more suitable as a nucleic acid enrichment and isolation platform capable of extracting small-sized circulating mRNA. However, since primers and target sequences designed under the same conditions used for cell-derived mRNA were selected, it is necessary to select short target sequences to use short circulating mRNA as a diagnostic biomarker for PCa. The second issue is the low circulating mRNA extraction efficiency due to the absence of a lysis step. In existing circulating nucleic acid extraction methods, a lysis step is performed to extract fragmented nucleic acids attached to or contained in biomolecules in order to improve the recovery rate of circulating nucleic acids from small sample volumes.Although the lysis step can extract high concentrations of circulating nucleic acids, contamination by chaotropic reagents of cell-derived genomic nucleic acids and prostate-related nucleic acids extracted from PCa cells and urinary exosomes can reduce sensitivity and specificity in the analysis of pure circulating nucleic acids. In this regard, further analysis and investigation of pure circulating nucleic acids in urine through the development of a platform for circulating nucleic acid concentration and isolation without a lysis process is essential. The inventors anticipate that further analysis and expansion of HAZIS-CirR in various clinical settings will complement the limitations of circulating mRNA analysis and further demonstrate its clinical utility as a cancer biomarker.

[0226] (5) Clinical utility of circulating miRNA in HAZIS-CirR

[0227] To confirm the clinical utility of HAZIS-CirR for circulating miRNA-based PCa diagnosis, circulating miRNAs were analyzed from urine samples (Fig. 8). To confirm clinical relevance, miR-21-5p, miR-141-3p, and miR-375-3p, which are known as existing PCa biomarkers, were used as circulating miRNA biomarkers and relative quantification values. miR-21-5p is one of the oncomiRs and is known to be overexpressed in various cancers, including PCa, as an upregulated oncogenic miRNA. Similarly, miR-141-3p and miR-375-3p have similar expression patterns and are known to be miRNAs overexpressed in PCa. In the analysis of circulating miRNAs, it was confirmed that miR-21-5p (mean RQ = 8.91 ± 12.92) (P < 0.01), miR-141-3p (mean RQ = 12.26 ± 13.37) (p < 0.001), and miR-375-3p (mean RQ = 17.30 ± 18.69) (P < 0.001) are overexpressed in PCa compared to healthy controls (mean RQ = 1.16 ± 0.62, 1.69 ± 1.44, and 1.73 ± 1.64, respectively). These results demonstrate that miR-21-5p, miR-141-3p, and miR-375-3p can be used as biomarkers for PCa. However, even in BPH patients, miR-21-5p (mean RQ = 4.22 ± 4.75) (P < 0.01), miR-141-3p (mean RQ = 5.04 ± 6.68) (P < 0.05), and miR-375-3p (mean RQ = 7.57 ± 8.21) (P ≤ 0.01) were overexpressed compared to healthy controls. This suggests that there must be a significant difference in PCa from BPH patients to use PCa biomarkers. miR-141-3p (P < 0.05) and miR-375-3p (p < 0.05) were overexpressed in PCa compared to BPH patients, and miR-21-5p (P < 0.It was confirmed that there was no statistically significant difference in 075, ns). This result shows that miR-141-3p and miR-375-3p can be used as biomarkers to identify PCa in BPH patients. Although miR-21-5p shows a high expression rate in PCa, it cannot be used as a biomarker to distinguish PCa from BPH because it is not statistically significant compared to BPH. In addition, the inventors analyzed the clinical relevance of miR-148a-3p, miR-483-5p, and miR-574-3p to identify potential biomarkers that can distinguish BPH patients from PCa. It was confirmed that miR-148a-3p (mean RQ 19.45 ± 34.51) (P < 0.01), miR-483-5p (mean RQ = 49.53 ± 60.83) (P ≤ 0.001), and miR-574-3p (mean RQ 17.28 ± 18.82) (P < 0.001) were overexpressed in PCa compared to healthy controls (mean RQ = 2.27 ± 2.73, 1.44 ± 1.58, and 1.35 ± 1.29). However, miR-148a-3p (mean RQ = 9.32 ± 13.51) (P < 0.05), miR-483-5p (mean RQ = 6.98 ± 11.64) (P ≤ 0.05), and miR-574-3p (mean RQ = 4.68 ± 4.34) (P < 0.01) were overexpressed in BPH compared to healthy controls. When comparing PCa and BPH, it was found that miR-483-5p (P < 0.001) and miR-574-3p (P < 0.001) were overexpressed in PCa compared to BPH patients, and there was no significant difference in miR-148a-3p (p < 0.076, ns). These results indicate miR-483-5p and miR-574-3p as biomarkers capable of distinguishing between PCa and BPH.Although miR-148a-3p exhibits a high expression rate in PCa, it cannot be used as a biomarker to distinguish between PCa and BPH because it is not statistically significant compared to BPH. Consequently, it was confirmed that HAZIS-CirR can rapidly concentrate and extract circulating miRNAs from patient urine samples and can be used as a sample preparation platform for PCa diagnosis. Furthermore, it was confirmed that miR-141-3p, miR-375-3P, miR-483-5P, and miR-574-3P can be used as biomarkers to differentiate PCa from BPH patients.

[0228] The inventors expect that HAZIS-CirR will overcome the limitations of low detection sensitivity of existing detection methods by obtaining high concentrations of miRNA through low concentrations and extraction of circulating miRNA present in patient samples, and will be used as a platform for early and precise cancer diagnosis.

[0230] (6) Clinical analysis results

[0231] Clinical analysis confirmed that the PSA levels of PCa patients were relatively higher compared to other groups (Table 4).

[0232] Demographics in HAZIS-CirR Age (median, IQR) Initial PSA value Circulating mRNA (PCA3) PCa (35) 70 (63-72) 6.60 (4.00-12.69) BPH (20) 68 (63.3-74) 4.62 (3.35-7.09) Normal (5) 64 (60-68.8) 2.31 (1.50-6.29) Circulating mRNA(TMPRSS2-ERG gene fusion) PCa (22) 70 (68-72.8) 5.06 (3.70-8.93) BPH (11) 70 (63.5-75) 3.13 (1.64-4.70) Circulating miRNAs PCa (30) 70 (62.5-73) 6.56 (4.30-12.09) BPH (22) 68 (62.5-74) 4.45 (3.13-6.61) Normal (10) 68.0 (66-71) 2.31 (0.86-6.98)

[0234] Example 2. Concentration of pathogens and nucleic acid extraction using a microfluidic platform or zeolite

[0235] 1. Experimental Methods and Materials

[0236] (1) Fabrication of a microfluidic platform

[0237] For sample preparation, a microfluidic platform composed of spiral-shaped microchannels was used as previously reported. The microfluidic chip was credit card size (70.16 mm x 85 mm x 0.5 mm), designed using AutoCAD (Autodesk, Inc., San Rafael, CA), and fabricated using a CO2 laser cutter (VLS3. 50 Universal Laser Systems, Scottsdale, AZ). A hydrophilic thin film outer layer (Kemafoil hydrophilic lm, HNW-100, COVEME, Italy) and a double-sided tape inner layer (Adhesive 300LSE-9495LE, 3M, St. Paul, MN) were assembled after O2 plasma treatment (10 min, 100 W, 60 sccm). Acrylic adapters for sample injection and discharge were attached to the inlet and outlet of the microfluidic chip, and Tygon tubing (AAD04103, Saint-Gobain PPL Corp, USA) was placed in the adapter holes using epoxy. For internal surface silanization, 2% 3-aminopropyl diethoxymethylsilane (APDMS: aminopropyl diethoxymethylsilane, 371890-50 mL, Sigma-Aldrich, St. Louis, MO, USA) was injected into ultrapure distilled water (DW) using a syringe, incubated at 65°C for 60 minutes, and then washed three times with 1 mL of ultrapure distilled water. The fabricated microfluidic chip was stored at room temperature (21°C to 22°C) until use.

[0239] (2) Use of microfluidic platforms in pathogen enrichment and nucleic acid extraction

[0240] Adipic acid dihydrazide, one of the homodifunctional hydrazides, was used as a core material for pathogen concentration and nucleic acid extraction within a microfluidic platform. Since homodifunctional hydrazides have two hydrazide groups on both sides, they can directly bind to pathogens and nucleic acids through electrostatic and covalent bonding. Homodifunctional hydrazides act as cross-linkers through interaction with the silanized inner surface of the microfluidic chip. For the homodifunctional hydrazide-based one-step pathogen concentration and DNA extraction platform, Brucella Ovis (ATCC 25840) diluted in 1 mL of PBS was mixed with 100 mg of adipic acid dihydrazide and then injected into a 100 μL microfluidic platform using a syringe pump (KD Scient, MaIUCon, Hollciston). To facilitate the interaction between pathogens, the microfluidic internal surface, and adipic acid dihydrazide, the chip was incubated at room temperature for 20 minutes. Afterward, foreign matter was washed by injecting 1 mL of PBS and air, and 1 mL of lysis buffer containing 50 mg of adipic acid dihydrazide (20 μL Protease K, 100 mM pH 8.0 Tris-HCl, 10 mM ethylenediaminetetraacetic acid, 1% sodium dodecyl sulfate, and 10% Triton X-100) was injected into a 750 μL chip. The chip was then incubated at 56°C for cell lysis and DNA extraction. After washing away foreign matter with 1 mL of PBS and air, the captured DNA was leached to 50 μL min of pH 10.6 elution buffer (10 mM sodium bicarbonate). -1It was collected from. For the adipic acid dihydrazide-based RNA extraction platform, heat-inactivated SARS-Related Corona 2 (SARS-CoV-2) culture medium (ZeptoMetrix, Buffalo, NY, USA) diluted in 200 μL of PBS was mixed with 50 mg of adipic acid dihydrazide, 200 μL of lysis buffer, 10 μL of DNase (only RNA), and approximately 350 μL of PBS, after which 100 μL min was injected using a syringe pump. -1 It was injected into a microfluidic platform. After incubation at room temperature (20 minutes) for cell lysis and RNA concentration, foreign matter was washed by injecting 1 mL of PBS and air. The captured RNA was dissolved in 50 μL min of pH 10.6 elution buffer (10 mM sodium bicarbonate) -1 It was eluted. The eluted nucleic acid was stored at sub-zero temperatures (-20°C or -80°C) until use.

[0242] (3) Use of zeolite filters in pathogen concentration and nucleic acid extraction

[0243] Pathogen concentration and nucleic acid extraction using amine-modified zeolite, adipic acid dihydrazide, and a 0.45 μm PVDF syringe filter were performed as follows: Specifically, a 1 mL solution containing Brucella Ovis (ATCC 25840) or a clinical sample was used for the optimization and evaluation of clinical efficacy, and 5 mg of amine-modified zeolite and 50 mg of adipic acid dihydrazide were added to 1 mL of the solution containing Brucella Ovis or the clinical sample. Incubation for electrostatic interaction was performed at room temperature for 20 minutes to capture the pathogen. The pathogens captured on the surface of the amine-modified zeolite by electrostatic coupling were filtered through a 0.45 μm syringe filter using a 1 mL syringe (Korea Vaccine Co., Ltd., Yongin, Korea). Residues not captured by the amine-modified zeolite were filtered out, and the remaining residues were washed with 1 ml of PBS. For the first step nucleic acid extraction, 50 mg adipic acid dihydrazide, 20 μL protease K, 10 μL DNase (only RNA), 100 mM Tris-HCiam (pH 8.0), 10 mM ethylenediaminetetraacetic acid, 1% sodium dodecyl sulfate, and 10% Triton X-100 were placed in a syringe filter, and the syringe filter was incubated at 56°C (for DNA) or room temperature (for RNA) for 20 minutes. Nucleic acids released by the cell lysis buffer were captured on the surface of the amine-modified zeolite by electrostatic and covalent bonds, and uncaptured residues were removed through the filter. The remaining residue was washed with 3 mL of PBS. The concentrated pathogens were washed in 50 mM sodium bicarbonate (S5761-500G, Sigma-Aldrich) at a high pH (pH 10) prior to the nucleic acid extraction step.6) The pathogen was eluted by breaking the cross-links between the pathogen, amine-modified zeolite, and adipic acid dihydrazide using 100 μL of elution buffer. The eluted pathogen and nucleic acid were collected in a 1.5 mL microcentrifuge tube and stored at -20°C or -80°C until use.

[0245] (4) Evaluation of detection limit and capture efficiency of Brucella Ovis and SARS-CoV-2 cultures

[0246] Brucella Ovis, a isomorphic, difunctional hydrazide-based sample preparation system (1 x 10 5 mL -1 Up to 1 x 10 0 CFUs mL -1 To evaluate the detection limit and capture efficiency of ) and heat-inactivated SARS-Related Coronavirus 2 (SARS-CoV-2) cultures, serially diluted PBS (0.96 x 10⁶ 5 PFUs mL -1 Up to 0.96 x 10 0 PFUs mL -1 Brucella Ovis was used for DNA and RNA analysis in the method using zeolite and syringe filters, and Brucella Ovis was used for DNA analysis and heat-inactivated SARS-Related Coronavirus 2 (SARS-CoV-2) culture medium was used for RNA analysis in the method using a microfluidic platform. The prepared samples were used immediately or stored at sub-zero temperatures (-20°C or -80°C) until use.

[0247] The ability of isomorphic dihydrazide candidate substances (adipic acid dihydrazide, carbonate dihydrazide, oxalyl dihydrazide, malonic acid dihydrazide, and succinic acid dihydrazide) to directly bind to pathogens and nucleic acids was compared, and each substance was tested under the same conditions as adipic acid dihydrazide.

[0249] (5) Existing method for nucleic acid extraction and PCR detection

[0250] Conventional methods such as column-based nucleic acid extraction kits and PCR were used to compare and validate the preparation of homozygous dihydrazide-based samples. For DNA extraction, DNA was extracted from Brucella Ovis using the QIAamp DNA Mini and Blood Mini kits, and all procedures were performed according to the manufacturer's instructions. Samples were used with starting volumes of 100 μL to 200 μL, and 100 μL was eluted using Qiagen AE elution buffer. The eluted DNA was stored at -20°C until use.

[0251] For endpoint and quantitative PCR (qPCR), the sequences and detailed information of the synthesized forward primers, reverse primers, and probes are shown in Table 5. DNA and RNA were obtained from Brucella Ovis and heat-inactivated SARS-associated coronavirus 2 (SARS-CoV-2) cultures. Endpoint PCR for DNA was performed using the Taq PCR Core Kit (201225, Qiagen, Hilden, Germany) according to the modified manufacturer's instructions. Endpoint PCR amplification was performed at 95°C for 15 minutes, followed by 40 cycles of 30 seconds at 95°C, 30 seconds at 60°C, and 30 seconds at 72°C, and then terminated with a final extension of 10 minutes at 72°C using a T100 Thermal Cycler (Bio-Rad, Hercules, CA, USA). All endpoint PCR reactions were performed using 5 μL of DNA in 25 μL of PCR reaction buffer, and gel electrophoresis was performed to separate PCR products on a 2% agarose gel containing LoadingSTAR (A750, Dyne Bio Inc., Seoul, Korea). The agarose gels were visualized using a ChemiDoc XRS+ system (Bio-Rad, Marnes-la-Coquette, France), and qPCR for DNA was performed using Brilliant III SYBR Green QPCR Master Mix (600882, Agilent Technologies, Wilmington, DE, USA) with modified manufacturer's instructions.qPCR amplification consisted of 40 cycles of 10 minutes at 95°C, followed by 10 seconds at 95°C, 20 seconds at 60°C, and 20 seconds at 72°C; termination consisted of 15 seconds at 95°C, 1 minute at 60°C, and 1 minute at 95°C in a melting curve phase with a 1% ramp rate, followed by 15 seconds at 60°C for 15 seconds in a CFX96 Touch Deep Well Real-Time PCR Detection System (Bio-Rad, Hercules, CA, USA). All qPCR reactions used 5 μL of DNA in 20 μL of PCR reaction buffer, and TaqMan RT-qPCR for viral RNA was performed using a LightCycler® Multiplex RNA Virus Master (07083173001, Roche Diagnostics, Mannheim, Germany) with modified manufacturer's instructions. TaqMan RT-qPCR amplification was performed on a CFX96 Touch Deep Well Real-Time PCR detection system in 40 cycles of 5 minutes at 95°C, 5 seconds at 95°C, and 20 seconds at 60°C, and all TaqMan RT-qPCR reactions used 5 μL of DNA in 20 μL of PCR reaction buffer.

[0252] Target Location Sequence (5'-3') Length (bp) Sequence number DNA Brucella Ovis ( pgk ) Forward GCT TTC GAC CGA CCT CAT TG 20 Sequence No. 13 Reverse CCG CCA GCG GTT GAG ATA TA 20 Sequence No. 14 MTB ( IS6110 ) Forward CTA ACC GGC TGT GGG TAG 18 Sequence number 15 Reverse GTC TTT CAG GTC GAG TAC 18 Sequence number 16 RNA Brucella Ovis ( IS711 ) Forward GCT TGA AGC TTG CGG ACA GT 20 Sequence number 17 Reverse GGC CTA CCG CTG CGA AT 17 Sequence No. 18 SARS-CoV-2 ( N ) Forward TGG CAG TAA CCA GAA TGG AGA AC 23 Sequence No. 19 Reverse AGT GAG AGC GGT GAA CCA AGA 21 Sequence number 20 Probe CGC GAT CAA AAC AAC GTC GGC C 22 Sequence number 21

[0254] (6) Clinical analysis method

[0255] For clinical analysis, oral swabs were performed using OMNIgene, and the ORAL OMR-110 kit (DNA Genotek, Ottawa, Canada) was used according to the manufacturer's instructions. The swab was wiped back and forth along the subject's palate, upper gums, and back of the tongue for about 10 seconds (5 to 6 times per area, a total of 20 times), and then wiped again to touch the mouth. After inserting the swab into a tube containing a stabilizing solution, the sample was immediately sent to the laboratory and stored at -80°C until analysis. The oral swab sample (1 mL) was liquefied in a 1:1 ratio liquid solution (4% NALC, 1·45% sodium citrate and 2·67% NaOH) according to Ganoza et al (9) and conventional methods, and used to prepare an adipic acid dihydrazide-based sample.

[0257] (7) Working principle of isomorphic dihydrazide-based sample preparation

[0258] Concentrating cells and genetic material from liquid samples is an important technique that must be employed to enhance the detection sensitivity of infectious diseases. Non-chaotropic and pH-sensitive isomorphic dihydrazides are applied to solid supports to concentrate and extract biomolecules quickly, simply, and inexpensively. Figure 9 illustrates the principle of an isomorphic dihydrazide-based sample preparation system for pathogen concentration and nucleic acid extraction, utilizing a microfluidic chip or zeolite filter as the solid support. Isomorphic dihydrazides include adipic acid dihydrazide, carbon dihydrazide, oxalyl dihydrazide, malonic acid dihydrazide, and succinic acid dihydrazide; they possess two reactive hydrazide groups and act as crosslinkers between biomolecules and solid supports. As carbonyl-reactive crosslinkers, isomorphic dihydrazides have been used in protein-related research, such as the preparation of antibodies and glycoprotein conjugates through hydrazone binding. This study utilizes the hydrazone binding of the isodifunctional hydrazide and additional covalent and electrostatic bonding between the two nucleophilic amine groups and the aldehyde group of the hydrazide. First, liquid samples such as plasma, urine, and nasopharyngeal swab stabilization solution are mixed with the isodifunctional hydrazide at room temperature to prepare the sample. The prepared sample, injected into a microfluidic chip or zeolite, was added for pathogen enrichment. The solid support surface can form an imine bond with one side of the isodifunctional hydrazide. The two nucleophilic amine groups of the immobilized or suspended isodifunctional dihydrazide, as well as the amine groups on the internal surface of the microfluidic chip and the zeolite surface, carry a positive charge, thereby capturing and enriching negatively charged pathogens. The concentrated pathogen is a nucleic acid extracted using a lysis buffer containing a homozygous hydrazide, and the binding mechanism is as follows: (1) The positively charged solid support and the homozygous hydrazide electrostatically bind to the negatively charged nucleic acid.(2) The carbonyl group of the homodifunctional hydrazide forms an imine bond with the amine group of the solid support and the nucleic acid. (3) The hydrazide group of the homodifunctional hydrazide forms a hydrazone bond with the carbonyl group of the nucleic acid. The captured nucleic acid was extracted using an elution buffer to separate the nucleic acid from the solid support. The adipic acid dihydrazide-based pathogen concentration and nucleic acid extraction system using two solid supports requires only a single temperature (56°C) for pathogen lysis and does not require special equipment such as centrifuges and temperature controllers. In addition, it can rapidly concentrate pathogens into liquid clinical specimens, and the first step of sample preparation, including nucleic acid extraction, can be completed within one hour.

[0260] 2. Experimental Results

[0261] (1) Efficiency for pathogen concentration and nucleic acid extraction

[0262] To verify the operation and efficiency of the adipic acid dihydrazide-based sample pretreatment system, pathogen concentration and nucleic acid extraction using isodifunctional hydrazides were performed using a Brucella Ovis. First, the efficiency of sample preparation using the isodifunctional hydrazide candidates adipic acid dihydrazide, carbonate dihydrazide, oxalyl dihydrazide, malonic acid dihydrazide, and succinic acid dihydrazide was evaluated. As a result, among the isodifunctional hydrazides, adipic acid dihydrazide showed the highest capture efficiency, and it was confirmed that pathogen concentration and nucleic acid extraction were possible at a similar level in sample preparation using all candidate substances (Fig. 10).

[0264] (2) Detection limit using conventional PCR

[0265] To verify the sensitivity of the adipic acid dihydrazide-based sample pretreatment system, the detection limit was determined using serially diluted Brucella Ovis and heat-inactivated SARS-CoV-2 (SARS-CoV-2) culture medium (Fig. 11). Adipic acid dihydrazide-based sample preparation for DNA extraction was performed using a microfluidic chip and a zeolite filter, and it was confirmed that the sample gradually amplified according to the concentration gradient. In addition, the adipic acid dihydrazide-based sample pretreatment system compared with a conventional column-based DNA extraction kit (1 × 10⁻⁶ 3 CFUs ML -1 Compared to ), DNA (1 × 10 2 CFUs mL -1 It was confirmed that the detection sensitivity was 10 times higher. This implies that the low detection sensitivity resulting from the use of a limited volume is improved by increasing pathogen concentration and extracting nucleic acids to detect infectious diseases. Furthermore, the detection limit of the extracted RNA was determined using a one-step adipic acid dihydrazide-based sample pretreatment system; as a result, the detection limits of the microfluidic platform and the zeolite filter were 1 × 10, respectively. 2 CFUs mL -1 and 0.96 × 10 2 PFUs mL -1 It was confirmed that... These results indicate that an adipic acid dihydrazide-based sample pretreatment system can be used for high-concentration and high-efficiency DNA and RNA detection.

[0267] (3) Clinical utility of adipic acid dihydrazide-based sample pretreatment

[0268] We confirmed the clinical utility of an adipic acid dihydrazide-based sample preparation system for the diagnosis of infectious diseases by concentrating Mycobacterium tuberculosis (MTB) and extracting DNA from three nasopharyngeal swab samples (Table 6). Specifically, nasopharyngeal swab samples were obtained from clinically diagnosed tuberculosis patients, and a stabilization solution was used as the liquid sample. All three samples tested positive for the IS6110 gene (Genbank No. NC_000962.3) in DNA extracted using a column-based kit. However, compared to conventional methods, the adipic acid dihydrazide-based sample pretreatment system was found to have lower Ct values ​​and higher nucleic acid detection amounts. These results demonstrate that false negatives caused by low detection sensitivity can be reduced based on high-efficiency pathogen concentration, and the system can be used for infectious diseases requiring rapid diagnosis through a simple and fast process.

[0269] Mycobacterium tuberculosis (MTB) (Nasopharyngeal swab) Column-based Sample preparation (100 μL) HHs-basedSample preparation (1 mL) Sample #1( Ct value) 29.25 26.35 Sample #2( Ct value) 34.01 31.81 Sample #3( Ct value) 34.19 31.68

Claims

Claim 1 A composition for detecting or separating nucleic acids comprising a compound of the following Chemical Formula 1 or a compound of the following Chemical Formula 2; and a solid support, wherein the solid support is an amine-modified zeolite, Composition: Water: [Chemical Formula 1] ,[Chemical Formula 2] , above, n is one integer selected from 0, 1, 2 and 4. Claim 2 delete Claim 3 A composition for detecting or separating nucleic acids according to claim 1, wherein the nucleic acid is one or more nucleic acids selected from the group consisting of DNA, RNA, miRNA, mRNA, siRNA, tRNA, sgRNA and shRNA. Claim 4 A kit for detecting or isolating nucleic acids comprising the composition of claim 1. Claim 5 delete Claim 6 A kit for detecting or separating nucleic acids according to claim 4, wherein one compound selected from the group consisting of the compound of Formula 1 and the compound of Formula 2, the amine group and the nucleic acid interact through one or more attractive forces selected from the group consisting of imine bonds, hydrazone bonds, and electrostatic attraction. Claim 7 delete Claim 8 delete Claim 9 A kit for detecting or separating nucleic acids according to claim 4, wherein the diameter of the zeolite is 0.1 to 100 μm. Claim 10 A kit for detecting or separating nucleic acids according to claim 4, wherein one compound selected from the group consisting of the compound of Formula 1 and the compound of Formula 2 and the solid support are present in a weight ratio of 30:1 to 1:

10. Claim 11 A method for detecting or separating nucleic acids comprising: a step of binding nucleic acids in a sample separated from an organism to a compound selected from the group consisting of a compound of Formula 1 and a compound of Formula 2 below; and a step of separating said nucleic acids from the surface of said solid support, wherein the solid support is an amine-modified zeolite. [Formula 1] ,[Chemical Formula 2] , above, n is one integer selected from 0, 1, 2 and 4. Claim 12 delete Claim 13 A method for detecting or separating nucleic acids according to claim 11, wherein the sample is one or more samples selected from the group consisting of blood, plasma, serum, tissue, cell, lymph fluid, bone marrow fluid, saliva, ocular fluid, semen, brain extract, cerebrospinal fluid, joint fluid, thymic fluid, ascites fluid, amniotic fluid, urine, cell tissue fluid, and cell culture fluid. Claim 14 A method for detecting or separating nucleic acids according to claim 11, wherein the bond is one compound selected from the group consisting of the compound of Formula 1 and the compound of Formula 2, and one or more attractive forces selected from the group consisting of an imine bond, a hydrazone bond, and an electrostatic attraction between the amine group and the nucleic acid. Claim 15 A method for detecting or separating nucleic acids according to claim 11, further comprising the step of washing the surface of the solid support prior to the step of separating the nucleic acid. Claim 16 A method for detecting or separating nucleic acids according to claim 11, further comprising, prior to the step of separating the nucleic acid, a step of filtering a solid support and a compound selected from the group consisting of the compound of Formula 1 and the compound of Formula 2 combined. Claim 17 A method for detecting or separating nucleic acids according to claim 16, wherein the filtration step is performed with a filter having pores of less than 0.1 μm. Claim 18 A composition for diagnosing prostate cancer (PCa) in a patient with benign prostatic hyperplasia (BPH), comprising a composition for detecting or isolating a nucleic acid according to claim 1, wherein the nucleic acid is one or more selected from the group consisting of miR-141-3p, miR-375-3P, miR-483-5P and miR-574-3P. Claim 19 A kit for diagnosing prostate cancer (PCa) in a patient with benign prostatic hyperplasia (BPH), comprising the composition for diagnosing prostate cancer of claim 18. Claim 20 A method for providing information for the diagnosis of prostate cancer (PCa) in a patient with benign prostatic hyperplasia (BPH), comprising: a step of combining a nucleic acid in a sample separated from an individual and a compound selected from the group consisting of a compound of Chemical Formula 1 and a compound of Chemical Formula 2 below with the surface of a solid support; and a step of separating said nucleic acid from the surface of said solid support, wherein the nucleic acid is one or more selected from the group consisting of miR-141-3p, miR-375-3P, miR-483-5P and miR-574-3P, and the solid support is an amine-modified zeolite. [Chemical Formula 1] ,[Chemical Formula 2] , above, n is one integer selected from 0, 1, 2 and 4. Claim 21 A composition for diagnosing an infectious disease comprising the composition for detecting or isolating nucleic acid of claim 1. Claim 22 A composition according to claim 21, wherein the infectious disease is one or more selected from the group consisting of hand, foot, and mouth disease, rubella, epidemic conjunctivitis, influenza, enteritis, measles, impetigo, chickenpox, bacterial meningitis, and tuberculosis. Claim 23 A composition according to claim 21, wherein the nucleic acid is an IS6110 gene derived from Mycobacterium tuberculosis. Claim 24 A diagnostic kit for infectious diseases comprising the composition for diagnosing infectious diseases of claim 21. Claim 25 A method for providing information for the diagnosis of an infectious disease, comprising: a step of binding a nucleic acid in a sample separated from an organism to a compound selected from the group consisting of a compound of Chemical Formula 1 and a compound of Chemical Formula 2; and a step of separating the nucleic acid from the surface of the solid support, wherein the solid support is an amine-modified zeolite. ,[Chemical Formula 2] , above, n is one integer selected from 0, 1, 2 and 4.

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