Method for paper-based innovative nucleic acid extraction without centrifugation (pine-CX) and method for detecting nucleic acids in samples using same

Abstract

The present invention relates to a method for paper-based innovative nucleic acid extraction without centrifugation (PINE-CX) and a method for detecting nucleic acids in a sample using same. In the method according to an aspect, nucleic acids can be extracted and amplified from a sample with only a simple operation in a short time without centrifugation, and thus nucleic acid extraction and amplification efficiency is remarkably excellent. As a result, nucleic acids in a sample can be detected with high efficiency, and thus the method can be usefully used even in point-of-care testing (mPOCT) with limited resources.

Description

Paper-based nucleic acid extraction method (PINE-CX) without centrifugation and method for detecting nucleic acids in a sample using the same

[0001] [Cross-reference with related applications]

[0002] This application claims the benefit of priority from Republic of Korea Patent Application No. 10-2024-0085553, filed June 28, 2024, the entire contents of which are incorporated herein by reference.

[0003] [Technical Field]

[0004] The present invention relates to a paper-based nucleic acid extraction method (PINE-CX) that does not use centrifugation and a method for detecting nucleic acids in a sample using the same.

[0005] Nucleic acid extraction during sample processing is a crucial step in nucleic acid amplification, as it isolates targets (DNA, RNA, etc.) from complex samples and prevents nucleic acid amplification degradation due to inhibitors within the sample. However, the lysis, washing, and elution processes that are part of typical nucleic acid extraction are complex, time-consuming, and require equipment such as centrifugation, making them unsuitable for resource-constrained point-of-care testing (mPOCT) applications. Therefore, there is a pressing need for an inexpensive, portable, and easy-to-operate nucleic acid extraction technology that can be applied to resource-constrained mPOCT.

[0006] To overcome this challenge, paper-based nucleic acid extraction technologies are being developed to facilitate nucleic acid extraction and processing for mPOCT. Nitrocellulose and filter paper have been applied to nucleic acid extraction, demonstrating potential for nucleic acid extraction and preservation, similar to silica-based membranes in spin columns. Furthermore, paper-based isothermal nucleic acid amplification technology (NAAT) is emerging as a low-cost nucleic acid extraction and detection technology.

[0007] However, in the case of these conventional paper-based nucleic acid extraction technologies, the nucleic acid extraction efficiency is not high yet, so it is difficult to commercialize them, and in particular, Fusion 5 TM Attempts to extract and detect nucleic acids using glass fiber-based paper, such as this, are still lacking.

[0008] Against this backdrop, the inventors of the present invention developed a method for extracting and detecting nucleic acids using glass fiber-based paper, and, using the method, extracted and amplified nucleic acids from a sample in a short period of time with only simple manipulation without centrifugation, resulting in significantly superior nucleic acid extraction and amplification efficiency, thereby enabling high-efficiency detection of nucleic acids in a sample, thereby confirming that it can be usefully utilized even in mPOCT with limited resources, thereby completing the present invention.

[0009] The present invention aims to provide a method for extracting nucleic acid from a sample.

[0010] In addition, the present invention aims to provide a method for detecting nucleic acid in a sample.

[0011] In order to achieve the above object, one aspect of the present invention provides a method for extracting nucleic acid from a sample, comprising: (1) a step of adding a protein-decomposing enzyme to the sample and mixing the sample to dissolve the sample; and (2) a step of mixing the lysate obtained in step (1) and paper made of glass fiber.

[0012] Another aspect of the present invention provides a method for detecting nucleic acid in a sample, comprising the steps of: (1) dissolving the sample by adding a protein-decomposing enzyme to the sample and mixing the sample; (2) mixing the dissolved substance obtained in step (1) with paper made of glass fiber material; (3) separating the paper from the mixture obtained in step (2); and (4) amplifying the nucleic acid in the separated paper itself or in an eluate obtained by eluting the nucleic acid from the separated paper.

[0013] According to a method for extracting nucleic acid from a sample of a certain type and a method for detecting nucleic acid in a sample using the same, nucleic acid can be extracted and amplified from a sample with a simple operation in a short period of time without centrifugation, so that the efficiency of nucleic acid extraction and amplification is significantly superior, and thus nucleic acid in a sample can be detected with high efficiency, so that it can be usefully utilized even in resource-limited mPOCT (point-of-care testing).

[0014] Figure 1 is a diagram of Fusion 5 according to one embodiment. TM This image shows a disk paper (left: diameter 2.00 mm; right: diameter 6.00 mm).

[0015] FIG. 2 is an image schematically illustrating a process of extracting nucleic acid from a sample by performing the PINE-CX method according to one embodiment.

[0016] FIG. 3 is a gel electrophoresis image showing the results of performing LAMP after extracting nucleic acids from an HCV-infected serum sample using the PINE-CX method according to one embodiment.

[0017] FIG. 4 is a gel electrophoresis image showing the results of RT-PCR performed on nucleic acids extracted from HCV-infected serum samples according to the PINE-CX method without centrifugation according to one embodiment (without centrifugation) and on nucleic acids extracted from HCV-infected serum samples according to the PINE-CX method with centrifugation (with centrifugation) (N: negative control; P: positive HCV control).

[0018] Figure 5 is a gel electrophoresis image showing the results of performing LAMP after extracting nucleic acids from HCV-infected serum samples at different elution times using the PINE-CX method according to one embodiment.

[0019] Figure 6 shows the Fusion 5 method for extracting nucleic acids from HCV-infected serum samples using the PINE-CX method according to one embodiment. TM Dissolution of disc paper and Fusion 5 TM This is a gel electrophoresis image showing the results of RT-PCR performed on the disk paper itself.

[0020] FIG. 7 shows the results of extracting nucleic acids from HCV-infected serum samples by varying the amount of PEG added (volume percentage of PEG based on the total volume of lysate and PEG: about 1% (v / v), about 2.5% (v / v), about 5% (v / v), about 10% (v / v), about 30% (v / v)) in the PINE-CX method according to one embodiment, and then using Fusion 5. TM This is a graph showing the Ct values ​​obtained by performing RQ-PCR on the disk paper itself.

[0021] FIG. 8 shows the results of Fusion 5, which was prepared by extracting nucleic acids from HCV-infected serum samples without washing and drying, by varying the amount of PEG added (volume percentage of PEG based on the total volume of lysate and PEG: about 1% (v / v), about 2.5% (v / v), about 5% (v / v)) without adding ethanol when additionally using PEG in the PINE-CX method according to one embodiment. TM This is a graph showing the Ct values ​​obtained by performing RQ-PCR on the disk paper itself.

[0022] FIG. 9 is a graph showing a gel electrophoresis image (top) obtained by performing LAMP after extracting nucleic acids from serially diluted samples containing HCV RNA by performing the PINE-CX method to analyze the detection sensitivity of the PINE-CX method according to one embodiment, and a graph showing Ct values ​​obtained by performing RQ-PCR (bottom) (N: negative control; P: positive HCV control).

[0023] Figure 10 is the same experiment as Figure 9 above, but with a Fusion 5 having a diameter of 6.00 mm. TM This is a gel electrophoresis image obtained by performing LAMP using disk paper (N: negative control; P: positive HCV control).

[0024] Figure 11 shows the existing nucleic acid extraction method, Qiagen TM This is a gel electrophoresis image obtained by performing RT-PCR for cases where nucleic acids were extracted by applying a modification of PEG addition to the extraction method, and when nucleic acids were extracted by changing the type of paper used in performing the PINE-CX method according to one embodiment (QIAGEN: Qiagen TM extraction method; Ashless: Application of cellulose filter paper to PINE-CX method; Nylon: Application of nylon filter paper to PINE-CX method) (N: negative control).

[0025] Figure 12 shows Fusion 5 used in performing the PINE-CX method according to one embodiment. TM This diagram shows the results of RT-PCR (gel electrophoresis image at the top) and RQ-PCR (graph and table (Ct value) at the bottom) performed after extracting nucleic acids by varying the number of disk papers (N: negative control; P: positive HCV control).

[0026] Figure 13 is a gel electrophoresis image showing the results of performing RQ-PCR after extracting nucleic acids by varying the sample dissolution time in performing the PINE-CX method according to one embodiment (N: negative control; P: positive HCV control).

[0027] Figure 14 shows Fusion 5 performing the PINE-CX method according to one embodiment. TM If you apply different types of buffer solutions to wash the disc paper, Fusion 5 TM This figure shows the results of RT-PCR (gel electrophoresis image at the top) and RQ-PCR (graph and table (Ct value) at the bottom) performed on the extract from the disk paper (N: negative control; P: positive HCV control).

[0028] Figure 15 shows Fusion 5 performing the PINE-CX method according to one embodiment. TM If you apply different amounts of buffer solution to wash the disc paper, Fusion 5 TM This diagram shows the results of RT-PCR (a), LAMP (b), and RQ-PCR (c) performed on the effluent from the disk (N: negative control; P: positive HCV control).

[0029] Figure 16 shows the extraction of nucleic acids using the PINE-CX method according to one embodiment, using washed Fusion 5TM Fusion 5 obtained by applying different drying times to the disk paper TM This is a gel electrophoresis image showing the results of LAMP performed on the eluate from the disk paper (N: negative control; P: positive HCV control).

[0030] Hereinafter, the present invention will be described in detail.

[0031] 1. Method for extracting nucleic acid from a sample

[0032] One aspect of the present invention provides a method for extracting nucleic acid from a sample.

[0033] Specifically, the method may include (1) a step of adding a protein-decomposing enzyme to a sample and mixing the sample to dissolve the sample; and (2) a step of mixing the lysate obtained in step (1) and paper made of glass fiber.

[0034] The sample may be, for example, a biological sample (or samples), but is not limited thereto. For example, the biological sample may refer to a sample containing biomolecules including nucleic acids. The biological sample may include a solution, cell, tissue, biopsy, powder, or a combination thereof, and may be secretions, body fluids, excretions, etc. derived from living organisms such as animals or plants, environmental samples such as soil, water, air, and in addition, may be collected from compositions or surfaces that are contaminated or may be contaminated with biomolecules, such as food, drugs, investigative evidence, cooking facilities, medical facilities, and facilities in areas where infectious diseases have occurred. The biological sample may include blood, plasma, serum, feces, urine, body fluids, soil, water, air, etc., and such biological samples are complex samples containing various substances, and in particular, may require treatment to stabilize nucleic acids for storage, including decomposing enzymes derived from living organisms.

[0035] The nucleic acid may be DNA, RNA or a combination thereof.

[0036] The term “lysis” can refer to a phenomenon in which the cell membrane of a cell, microorganism, virus, etc. is broken down and the contents of the cell, microorganism, virus, etc. are dispersed or dissolved in a liquid phase.

[0037] In the above step (1), for example, cells, microorganisms (e.g., bacteria, fungi, etc.), viruses, etc. present in the sample may be lysed by the proteolytic enzyme, and nucleic acids present inside the cells, microorganisms, viruses, etc. may be released to the outside. Accordingly, the proteolytic enzyme used in the above step (1) may function as a cell lysing agent without damaging nucleic acids. For example, the proteolytic enzyme may include, but is not limited to, protease K.

[0038] In the above step (1), carrier RNA may be added and mixed, but is not limited thereto. The carrier RNA is RNA used to increase RNA extraction efficiency when the concentration of RNA is low, such as when extracting virus RNA. Poly A may be used as the carrier RNA, but is not limited thereto.

[0039] In the above step (1), the mixing or dissolving may be performed at about 40°C to 70°C or about 50°C to 60°C, for a time of more than about 10 minutes, more than about 10 minutes to 30 minutes, more than about 10 minutes to 15 minutes, more than about 15 minutes, or more than about 15 minutes to 30 minutes.

[0040] According to one specific example, in the above method, when the mixing or dissolution time in step (1) is adjusted within the numerical range, the nucleic acid extraction efficiency is significantly improved, whereas when the mixing or dissolution time in step (1) is outside the numerical range, the nucleic acid extraction efficiency may be reduced.

[0041] The lysate obtained in the above step (1) is, for example, a lysate obtained by dissolving cells, microorganisms, viruses, etc., and may be in the form of a solution, dispersion, suspension, etc., and may include the contents (nucleic acid, etc.) of cells, microorganisms, viruses, etc.

[0042] In the above step (2), the glass fiber paper may be paper containing or made of glass fiber. For example, the glass fiber paper may be FUSION 5. TM However, the present invention is not limited thereto. In addition, for example, the glass fiber may be a silicone-based glass fiber. The glass fiber material paper may have, for example, a membrane or filter structure.

[0043] According to one specific example, in the above method, when the paper made of the glass fiber material is used in step (2), the nucleic acid extraction efficiency is significantly improved, whereas when another type of paper is used, the nucleic acid extraction efficiency may be reduced.

[0044] In addition, according to one specific example, the glass fiber material paper may be a piece of paper having a disk shape (circular shape), and the diameter (diameter) of the disk shape may be about 1 to 50 mm, about 1 to 30 mm, about 1 to 10 mm, about 1 to 6 mm, about 1 to 5 mm, about 1 to 3 mm, about 2 to 10 mm, about 4 to 8 mm, about 6 mm, or about 2 mm, but is not limited thereto.

[0045] The number of the glass fiber material papers mixed in the above step (2) may be about 1 to 20, about 1 to 10, about 1 to 5, about 1 to 4, about 4 or more, about 4 to 20, about 4 to 10, or about 4 to 7, but is not limited thereto.

[0046] According to one specific example, in the above method, when the number of papers made of the glass fiber material mixed in step (2) is about 4 or more, the nucleic acid extraction efficiency can be further improved.

[0047] In the above step (2), ethanol may be added and mixed, but is not limited thereto.

[0048] In addition, in the step (2), polyethylene glycol (PEG) may be further added and mixed. According to one specific example, in the method, when the polyethylene glycol is further added and mixed in the step (2), the nucleic acid extraction efficiency may be improved due to a synergistic effect with the glass fiber material paper. In the case of a conventional general nucleic acid extraction method, when polyethylene glycol is added, there is a problem that the elution efficiency is lowered due to nucleic acid aggregation, so an additional process must be performed to solve this problem, which causes a problem in that the nucleic acid extraction efficiency is lowered. In addition, in the method, when a type of paper other than the glass fiber material paper is used, even if polyethylene glycol is added, the problem of lowering the nucleic acid extraction efficiency may still occur. In contrast, in the above method using glass fiber paper, when the polyethylene glycol is further added and mixed in step (2), the glass fiber paper can better capture the aggregated nucleic acid due to the characteristic of exhibiting an appropriate positive charge, so that the amount of nucleic acid captured on the glass fiber paper increases, thereby increasing the nucleic acid extraction efficiency, and further, when the nucleic acid amplification process is performed on the glass fiber paper itself on which the nucleic acid is captured, the nucleic acid amplification efficiency increases, so that the nucleic acid in the sample can be detected with high efficiency. Therefore, the method can solve the problem of reduced nucleic acid extraction efficiency of the conventional general nucleic acid extraction method using polyethylene glycol or the paper-based nucleic acid extraction method by using the glass fiber paper and polyethylene glycol together.

[0049] In the step (2) above, the polyethylene glycol may be added and mixed so as to have a content (or concentration) of about 0.5% (v / v) to 4% (v / v) or about 1% (v / v) to 2.5% (v / v) based on 100% (v / v) of the volume sum of the melt and the polyethylene glycol. This amount of polyethylene glycol added is significantly lower than that of conventional nucleic acid extraction methods, and the method has an advantage over conventional technologies by increasing nucleic acid extraction efficiency while lowering the amount of polyethylene glycol added.

[0050] According to one specific example, in the above method, when the amount of polyethylene glycol added in step (2) is within the numerical range, the nucleic acid extraction efficiency is further improved, whereas when the amount of polyethylene glycol added in step (2) is outside the numerical range, the nucleic acid extraction efficiency may be relatively reduced.

[0051] In the above step (2), the mixture after mixing may be incubated at room temperature (e.g., about 15°C to 30°C or about 20°C to 25°C) for about 1 to 10 minutes or about 4 to 6 minutes. The incubation may mean placing the mixture in a suitable place corresponding to the temperature and allowing it to stand for the time.

[0052] In the above step (2), nucleic acids may be captured on the glass fiber material paper.

[0053] The above method may further include a step of separating the glass fiber material paper from the mixture obtained in step (2). At this time, the separated glass fiber material paper may have nucleic acid captured therein.

[0054] The method may further include, but is not limited to, a step of washing and / or drying the separated glass fiber material paper.

[0055] In the above washing step, the separated glass fiber material paper may be washed using a washing buffer solution containing guanidinium chloride (e.g., AW1 Buffer), a washing buffer solution containing ethanol (e.g., AW2 Buffer), or a combination thereof, but is not limited thereto. The concentration of ethanol in the washing buffer solution containing ethanol may be about 60 to 100%, about 60 to 80%, or about 70%. For example, in the above method, washing may be performed by placing the separated glass fiber material paper in a tube or container containing one type (each, if two or more types) of the washing buffer solution.

[0056] According to one specific example, in the washing step of the method, when the separated glass fiber material paper is washed using both a washing buffer solution containing the guanidinium chloride (e.g., AW1 Buffer) and a washing buffer solution containing ethanol (e.g., AW2 Buffer), the nucleic acid extraction efficiency can be further improved compared to when washing is performed using each of the washing buffer solutions alone.

[0057] In addition, according to one specific example, in the method, the amount of one type of washing buffer solution used in the washing step (the amount of each type when two or more types are used) may be about 150 to 350 uL, about 150 to 300 uL, about 150 to 250 uL, or about 200 uL, and in this case, the nucleic acid extraction efficiency may be further improved. At this time, the number of the separated glass fiber material papers to be washed may be 1 or more, 2 or more, 3 or more, 4 or more, 1 to 10, 1 to 5, or 1 to 3, but is not limited thereto.

[0058] The drying step may be drying the separated glass fiber material paper (e.g., washed glass fiber material paper) at about 40°C to 70°C or about 50°C to 60°C for a time period of more than about 1 minute, more than about 1 minute to 20 minutes, more than about 1 minute to 15 minutes, more than about 1 minute to 10 minutes, more than about 5 minutes to 15 minutes, more than about 5 minutes to 10 minutes, more than about 10 minutes, or more than about 10 minutes to 20 minutes. Through this process, ethanol present in the separated glass fiber material paper may be evaporated.

[0059] According to one specific example, in the above method, when the drying time is adjusted within the above numerical range, the nucleic acid extraction efficiency can be improved, whereas when the drying time is about 1 minute or less, the nucleic acid extraction efficiency can be significantly reduced.

[0060] In the above method, the addition of ethanol and the washing and drying steps in step (2) as described above are not necessarily required. According to one specific example, the method can extract nucleic acids in a sample with high efficiency even when the addition of ethanol and the washing and drying steps in step (2) are omitted by using polyethylene glycol and glass fiber material paper as described above.

[0061] Accordingly, the method may not perform the addition of ethanol in step (2), the washing step, the drying step, or a combination thereof.

[0062] The method may further include, but is not limited to, a step of eluting nucleic acids from the separated glass fiber material paper. The separated glass fiber material paper may have nucleic acids captured therein.

[0063] The above elution step can be performed by mixing the eluent and the separated glass fiber material paper or by injecting the eluent so that the eluent passes through the separated glass fiber material paper. The eluent can be used without limitation as long as it is a substance that can elute nucleic acid from the separated glass fiber material paper and does not damage the nucleic acid. For example, the eluent can be, but is not limited to, TE buffer (containing 10 mM EDTA and 25 mM Tris-HCl, pH 8.0).

[0064] In the above elution step, the nucleic acid may be eluted from the separated glass fiber material paper using the eluent for a time of less than about 10 minutes, from about 30 seconds to less than 10 minutes, from about 30 seconds to 5 minutes, from about 30 seconds to 3 minutes, from about 30 seconds to 1 minute or less, or from about 1 minute or less.

[0065] According to one specific example, in the above method, even if the elution is performed for a short time of less than 1 minute, the nucleic acid extraction efficiency is high, whereas if the elution time is outside the above numerical range (e.g., about 10 minutes or more), the nucleic acid extraction efficiency may decrease.

[0066] In the above elution step, an eluted solution containing nucleic acids eluted from the separated glass fiber material paper by the eluent can be obtained. The eluted solution contains the nucleic acids but does not contain the separated glass fiber material paper.

[0067] In the above method, the elution step as described above may not be necessarily required. This is because, as described above, a glass fiber material paper in which nucleic acids are captured is obtained by the above method, and a nucleic acid amplification process can be performed on the paper itself. In this case, the nucleic acid extraction and amplification efficiency is excellent, so that the nucleic acids in the sample can be effectively detected.

[0068] Accordingly, the method may or may not include the elution step.

[0069] That is, the nucleic acid extracted from the sample by the above method can exist in a state captured by the glass fiber material paper or in a state contained in the eluate. Therefore, a nucleic acid amplification process can be performed on the glass fiber material paper itself, in which the nucleic acid is captured, obtained by the above method, or on the eluate containing the nucleic acid, thereby enabling the detection of the nucleic acid within the sample.

[0070] The above method may not perform centrifugation throughout the entire process. That is, by using glass fiber paper as described above, the method can extract nucleic acids from a sample with high efficiency without performing centrifugation. Furthermore, when polyethylene glycol is additionally used, the efficiency of extracting nucleic acids from a sample can be further improved without performing centrifugation.

[0071] 2. Method for detecting nucleic acid in a sample

[0072] Another aspect of the present invention provides a method for detecting nucleic acid in a sample.

[0073] The method for detecting nucleic acids in the sample may utilize the method for extracting nucleic acids from the sample as is. Accordingly, the method for detecting nucleic acids in the sample may include all of the processes, steps, components, materials, etc. of the method for extracting nucleic acids from the sample.

[0074] Therefore, when the process, steps, configuration, materials, conditions, etc. for improving nucleic acid extraction efficiency in the above-mentioned method for extracting nucleic acid from a sample are applied to the method for detecting nucleic acid from a sample, the efficiency of amplification or detection of nucleic acid from a sample can also be improved by improving the efficiency of nucleic acid extraction.

[0075] Specifically, the method for detecting nucleic acids in a sample may include: (1) a step of dissolving the sample by adding a protein-decomposing enzyme to the sample and mixing the sample; (2) a step of mixing the dissolved substance obtained in step (1) and paper made of glass fiber material; (3) a step of separating the paper from the mixture obtained in step (2); and (4) a step of amplifying nucleic acids in the separated paper itself or in an eluate obtained by eluting nucleic acids from the separated paper.

[0076] In the step (2) above, polyethylene glycol may be further added and mixed. In addition, in the step (2), the polyethylene glycol may be added and mixed so as to have a content (or concentration) of about 0.5% (v / v) to 4% (v / v) or about 1% (v / v) to 2.5% (v / v) based on 100% (v / v) of the volume sum of the melt and the polyethylene glycol.

[0077] In the method for detecting nucleic acid in the sample, matters overlapping with the method for extracting nucleic acid in the sample can be understood in the same way as described in the method for extracting nucleic acid in the sample.

[0078] According to one specific example, the method can omit the step of adding ethanol in step (2) and the step of washing and drying the separated paper by using polyethylene glycol and glass fiber material paper as described above, and can still amplify and detect nucleic acids with high efficiency for both the separated paper itself and the eluate obtained by eluting nucleic acids from the separated paper.

[0079] When amplifying nucleic acids from the separated paper itself in step (4) above, there is no need to go through a step of eluting nucleic acids captured on the separated paper, and nucleic acids can still be detected at an excellent level. Therefore, when amplifying nucleic acids from the separated paper itself in step (4) above, the nucleic acid extraction, amplification, or detection efficiency of the method can be significantly improved.

[0080] When amplifying nucleic acids from an eluate obtained by eluting nucleic acids from the separated paper in step (4), the method may further include a step of eluting nucleic acids from the separated paper in step (3). The elution step is as described in the method for extracting nucleic acids from a sample.

[0081] The above step (4) may be to amplify nucleic acid using primers and / or probes, etc., on the separated paper itself or on the eluate obtained by eluting nucleic acid from the separated paper.

[0082] The above primers and probes bind to the nucleic acid to be detected to amplify a specific region of the nucleic acid, or bind to fluorescent substances, etc. to enable detection of the nucleic acid. Depending on the nucleic acid to be detected, the type, sequence, structure, binding substance, etc. can be designed in various ways. Depending on the nucleic acid to be detected, the above primers and probes can be manufactured by a general method known in the art, or can be purchased and used as commercial products.

[0083] The step of amplifying the nucleic acid may be performed by polymerase chain reaction (PCR), loop-mediated isothermal amplification (LAMP), real-time PCR, reverse transcription polymerase chain reaction (RT-PCR), real-time quantitative polymerase chain reaction (RQ-PCR), multiplex PCR, ligase chain reaction, nucleic acid sequence-based amplification, transcription-based amplification system, strand displacement amplification, amplification via Qβ replicase, or any other suitable method known in the art for amplifying a nucleic acid molecule.

[0084] In one specific example, the PCR may refer to a method of amplifying a target nucleic acid from a primer that specifically binds to the target nucleic acid using a polymerase, and such PCR method is well known in the art, and a commercially available mix composition or kit may be used.

[0085] For example, the nucleic acid amplification can be performed by mixing the separated paper itself or the eluate obtained by eluting the nucleic acid from the separated paper with a reaction solution containing various components known in the art to be necessary for a PCR reaction, and then performing a PCR reaction on the mixture. The reaction solution may contain, for example, a primer and / or probe set, a DNA polymerase, a dNTP mixture, a PCR buffer solution, a DNase inhibitor, sterile water, etc. The DNA polymerase may be, for example, the “Klenow fragment” of E. coli DNA polymerase I, a thermostable DNA polymerase, Taq polymerase, or bacteriophage T7 DNA polymerase, but is not limited thereto. In addition, the reaction solution may further contain a primer and / or probe set of a control marker in addition to the primer and / or probe set. The PCR buffer solution may contain a buffer component such as KCl, Tris-HCl, or MgCl2, and may include, for example, a PCR buffer.

[0086] The method may further include a step of detecting the amplification product. The step of detecting the amplification product may be performed by one or more methods selected from the group consisting of a DNA chip, gel electrophoresis, radioactivity measurement, fluorescence measurement, and phosphorescence measurement. When detecting by gel electrophoresis, agarose gel electrophoresis or acrylamide gel electrophoresis may be used depending on the size of the amplification product. When detecting by fluorescence measurement, PCR may be performed using the primer set labeled with a fluorescent substance, and then fluorescence may be measured using a fluorometer. When detecting by radioactivity measurement, a radioactive substance may be added to the PCR reaction solution to label the amplification product, and then radioactivity may be measured using a radioactivity meter such as a Geiger counter or a liquid scintillation counter. The detection or analysis of the obtained amplification product may be performed by, but is not limited to, restriction enzyme digestion, fluorescence measurement, or phosphorescence measurement.

[0087] In the method for detecting nucleic acid in the sample, if an amplification product is detected in the step of detecting the amplification product, it can be determined that the target nucleic acid is present in the sample, as the target nucleic acid has been detected from the sample.

[0088] The above method for detecting nucleic acids in a sample may not involve centrifugation throughout the entire process. That is, the method for detecting nucleic acids in a sample can efficiently extract nucleic acids in a sample without centrifugation by utilizing glass fiber paper as described above, and further, when polyethylene glycol is additionally utilized, the efficiency of extracting nucleic acids in a sample can be further improved without centrifugation, resulting in highly efficient amplification and detection of nucleic acids in a sample without centrifugation.

[0089] Hereinafter, the present invention will be described in detail by examples.

[0090] However, the following examples specifically illustrate the present invention, and the content of the present invention is not limited by the following examples.

[0091] [Example 1]

[0092] 1.1 Sample collection and HCV RNA extraction

[0093] From May 2021 to October 2023, serum specimens were collected from patients infected with HCV at Seoul Asan Medical Center for HCV viral load tests.

[0094] After routine HCV RNA testing using the Roche Cobas 6800 system (Roche Molecular Diagnostics, CA, USA), the remaining serum samples were separated and stored at -80°C. This study was approved by the Ethics Review Board of Asan Medical Center, Seoul (IRB No. 2017-1742), and RNA was extracted from HCV reference plasma panels of the WHO international standard (OptiQuant AcroMetrix / Life Technology, Benicia, CA, USA and Sera Care, Milford, MA, USA) to evaluate the detection sensitivity of a novel paper-based NAE (nucleic acid extraction) method.

[0095] 1.2 Fusion 5 TM Prepare a paper disc

[0096] Fusion 5, each about 2 mm or 6 mm in diameter TMEach disk (Glass fiber membrane) (Whatman, Florham Park, NJ, USA) was punched using a 2.00 mm Rapid Core Punch from WPI (Sarasota, FL, USA) and a 6.00 mm 510-1 Hole Punch from Peace Korea (Incheon, Korea) (see Fig. 1).

[0097] 1.3 RNA extraction

[0098] For comparison with the novel paper-based NAE, HCV RNA was extracted from approximately 200 μL of archived clinical serum samples remaining from the above HCV RNA test using the QIAamp MinElute Viurs Spin kit (QIAGEN, Hilden, Germany) according to the manufacturer's instructions.

[0099] 1.4 HCV RNA RT-PCR

[0100] The primers for RT-PCR (reverse transcription polymerase chain reaction) for HCV RNA were a pair of primers specifically designed for the 5'-untranslated region (5'-UTR) of HCV (Integrated DNA Technologies, Coralville, IL, USA) (see Table 1). One-step RT-PCR analysis was performed using AccuPower RT-PCR PreMix (Bioneer, Daejeon, Korea) according to the instructions. Briefly, Fusion 5 TM The disc itself or Fusion 5 TMApproximately 5 μL of the disk eluate was mixed with approximately 20 μL of a reaction solution containing Taq PCR Master Mix, approximately 1.5 μL of forward and reverse primers (approximately 10 μmol / L), distilled water, and Taq PCR Master Mix, and then amplified. Temperature cycling was performed in a C1000 Touch Thermal Cycler instrument (BioRad, Hercules, California) under the following conditions: cDNA synthesis 42°C, 60 min; 95°C, 5 min; and 40 cycles of 95°C for 20 s, 57°C for 30 s, 72°C for 30 s, and a final extension at 72°C for 5 min. PCR amplification products were detected using 2% agarose gel electrophoresis. Electrophoresis was performed according to a general method known in the art.

[0101] PrimerSequence (5' → 3')LocationPCR target sizeForward primerTGCACGGTCTACGAGAC (SEQ ID NO: 1)322-339157 BpReverse primerCGACCGGGTCCTTTCTTGGAT (SEQ ID NO: 2)182-203

[0102] 1.5 HCV RNA RQ-PCR

[0103] Real-Time Quantitative Polymerase Chain Reaction (RQ-PCR) primers for HCV detection are shown in Table 2 below. Approximately 20 μL of the reaction solution for RQ-PCR contained approximately 10 μL of a one-step RQ-PCR probe kit (ABScript II One-Step RT-qPCR Probe Kit, ABclonal, Woburn, MA), approximately 2 μL of a one-step probe enzyme mix (ABclonal), approximately 0.4 μL of forward / reverse primers (approximately 10 μmol / L), a TaqMan probe (IDT) labeled with the fluorophore 6-FAM at the 5' end and the quencher BHQ1 at the 3' end, and Fusion 5 TMThe disk itself or approximately 2 μL of Fusion 5 TM The disc effluent was included. Temperature cycling for RQ-PCR was performed on a BioRad CFX96 Real-Time PCR machine under the following conditions: UDG reaction, 25°C, 5 min; reverse transcription, 50°C, 5 min; amplification, 95°C, 3 min; 5 cycles of 95°C for 20 s, 57°C for 20 s, and 72°C for 20 s; and 35 cycles of 95°C for 20 s and 60°C for 20 s. Fluorescence was measured at 60°C for each cycle. PCR amplification products were detected using 2% agarose gel electrophoresis. Electrophoresis was performed according to general methods known in the art.

[0104] PrimerSequence (5' → 3')HCV-RQ-04-probe5'-6FAM-CCG GGG CAC TCG CAA GCA CCC -3'-BHQ_1(SEQ ID NO: 3)HCV-RQ-F025'-TGC ACG GTC TAC GAG AC-3'(SEQ ID NO: 4)HCV-RQ-R025'-CGA CCG GGT CCT TTC TTG GAT-3'(SEQ ID NO: 5)

[0105] 1.6 HCV RNA LAMP

[0106] The LAMP (Loop-Mediated Isothermal Amplification) primers for HCV detection are shown in Table 3 below. Approximately 25 μL of the reaction solution contained approximately 12.5 μL of WarmStart LAMP 2X Mater Mix (New England Biolab), approximately 2.5 μL of LAMP primer mixture (approximately 10 μM FIP and BIP primers, approximately 4 μM BOP and FOP primers, approximately 6 μM LB and LF primers), approximately 2.5 μL of nuclease-free water, approximately 2.5 μL of guanidine hydrochloride, and Fusion 5 TM The disc itself or Fusion 5 TMApproximately 5 μL of the disc effluent was included. A reverse transcription LAMP reaction was performed on the reaction solution. The reverse transcription LAMP reaction was performed in a BioRad CFX96 Real-Time PCR machine under the following conditions: approximately 25°C for 10 minutes, approximately 55°C for 10 minutes; approximately 72°C for 20 minutes, approximately 68°C for 30 minutes, and approximately 80°C for 5 minutes. The PCR amplification products were detected using 2% agarose gel electrophoresis. The electrophoresis was performed according to a general method known in the art.

[0107] Primer NameSequence (5' → 3')LAMP set 3DN1M-F3CGG GAG AGC CAT AGT GGT(SEQ ID NO: 6)DN1M-R3WGG AWG TGT GCT CAT GAT GCA CG(SEQ ID NO: 7)DN1M-FIPT GAG CGG GTT TDA TCC AAG ATT TTT GCG GAA CCG GTG AGT AC(SEQ ID NO: 8)DN1M-FIP (-)TGA GCG GGT TTD ATC CAA GAT TTT TGC GGA ACC GGT GAG TAC(SEQ ID NO: 9)DN1M-RIPCCG CRA GAC YGC TAG CCG AGT TTT ACC CTA TCA GGC AGT ACC AC(SEQ ID NO: 10)DN1M-LFTCG TCC YGG CRA TTC CGG(SEQ ID NO: 11)DN1M-LRTAG TGT TGG GTC GCG AAA G (SEQ ID NO: 12)

[0108] 1.7 Reproducibility and detection limits

[0109] The precision and limit of detection (LOD) of the developed protocol were ~10 using HCV RNA (AccuSpan HCV RNA Linearity Panel). 1 IU / mL, ~10 2 IU / mL, ~10 3 IU / mL, ~10 4 IU / mL, ~10 5 IU / mL, ~10 6 IU / mL, and ~10 7It was determined by serial dilution to a concentration of IU / mL.

[0110] [Example 2]

[0111] Development of a protocol for paper-based innovative nucleic acid extraction without centrifugation (PINE-CX)

[0112] We developed a paper-based innovative nucleic acid extraction method (PINE-CX) that can extract nucleic acids with high efficiency without centrifugation.

[0113] Specifically, about 100 to 300 μL (specifically, about 200 μL) of HCV-infected serum sample was placed in a 1.5 mL tube, and about 20 to 30 μL (specifically, about 25 μL) of protease K (Sigma Chemicals, St. Louis, MO, USA) and about 1 to 5 μL (specifically, about 3 μL) of carrier RNA (QIAGEN, Hilden, Germany) were added and lysed at about 56°C for about 10 minutes. After that, 2 mm Fusion 5 TM Four disks and about 100 to 200 μL (specifically, about 135 μL) of ethanol were added to the lysate, mixed, and the mixture was incubated at room temperature for about 5 minutes. After incubation, the supernatant was discarded from the culture, and the nucleic acid was captured in the Fusion 5 TM Only the disk was separated, and the separated Fusion 5 TM The disc was washed with approximately 200 μL of washing buffer solution, such as AW1 buffer solution (QIAGEN), and then with approximately 200 μL of AW2 buffer solution (QIAGEN). The washed Fusion 5 TMThe disk was dried at about 56°C for about 10 minutes to evaporate the ethanol, and then eluted with about 30 to 80 μL (specifically, about 50 μL) of TE buffer (10 mM EDTA and 25 mM Tris-HCl, pH 8.0, QIAGEN) at room temperature to obtain the eluate, thereby obtaining Fusion 5 TM Nucleic acids were eluted from the disk. The Fusion 5 TM Fusion 5 with the eluate of the disk and the nucleic acid captured therein TM The disk itself was applied to RT-PCR, RQ-PCR, and LAMP to confirm nucleic acid extraction and amplification. Fusion 5 was used without centrifugation. TM The overall process of extracting and isolating HCV RNA from serum samples using paper discs is shown in Fig. 2.

[0114] As a result, as shown in Fig. 3, the Fusion 5 TM As a result of performing LAMP on the effluent of the disk, when amplified with the control HBV (Hepatitis B virus) detection primer, the nucleic acid was not amplified, but when amplified with the HCV detection primer, the nucleic acid was confirmed to be amplified, so it was confirmed that the Fusion 5 was used without centrifugation. TM It was confirmed that HCV nucleic acid was successfully extracted and amplified from HCV-infected serum samples using paper disks.

[0115] In addition, as shown in Fig. 4, in the PINE-CX method, Fusion 5 obtained by using centrifugation in the washing step, like a general nucleic acid extraction method TM Compared to the case where RT-PCR was performed on the disk effluent, the Fusion 5 obtained by the PINE-CX method without centrifugation at any step including the washing step TMWhen RT-PCR was performed on the disk extract, it was confirmed that there was no significant difference in the level of nucleic acid amplification. This indicates that the PINE-CX method, even without centrifugation, can extract nucleic acids from the sample at a level equivalent to that of nucleic acid extraction methods that utilize centrifugation during washing processes, demonstrating significantly superior nucleic acid extraction efficiency.

[0116] In addition, as shown in Fig. 5, when applying the PINE-CX method, Fusion 5 washed with the buffer solution and then dried TM After immersing the disc in the elution buffer for 1 minute or 10 minutes, Fusion 5 TM The disc itself and Fusion 5 TM LAMP was performed on the effluent from the disk, and it was confirmed that HCV nucleic acid was extracted and amplified at a high level from HCV-infected serum samples despite the short elution time of less than 1 minute. In addition, Fusion 5 TM After soaking the disc in the elution buffer for 10 minutes, Fusion 5 TM As a result of performing LAMP on the effluent of the disk, it was confirmed that the level of nucleic acid amplification was reduced, so when applying the PINE-CX method, Fusion 5 TM It was found that when the disk is eluted with the eluent for a time of less than about 10 minutes (e.g., about 30 seconds to less than 10 minutes, about 30 seconds to 5 minutes, about 30 seconds to 3 minutes, about 30 seconds to 1 minute or less, or about 1 minute or less), the nucleic acid extraction and amplification efficiency can further increase, whereas when the elution time is outside the above numerical range, the nucleic acid extraction and amplification efficiency can further decrease.

[0117] Meanwhile, as confirmed below, the entire process of Fig. 2 was not performed, and the nucleic acid was captured in Fusion 5 TMFusion 5 captures nucleic acids without eluting them from paper discs. TM Even when performing PCR directly on the paper disk itself, nucleic acids can be amplified from the sample, as described in detail below.

[0118] [Example 3]

[0119] Direct use of nucleic acid-capture paper itself and the effect of polyethylene glycol (PEG) on PINE-CX

[0120] RNA was extracted from HCV-infected serum samples using the above PINE-CX method and analyzed using Fusion 5 TM Even when the effluent from the disk was obtained, the separated Fusion 5 TM We confirmed that HCV RNA was still detected on the disk itself (see Figure 6). This was confirmed by Fusion 5 without an elution step. TM Using the disk itself for amplification means faster and easier nucleic acid detection. Meanwhile, nucleic acids captured on paper disks are not easily eluted, making Fusion 5 TM The performance of the improved PINE-CX method without an elution step was verified using the disk itself and polyethylene glycol (PEG).

[0121] Specifically, all other processes except for adding PEG were performed in the same manner as the PINE-CX method performed in Example 2 to obtain Fusion 5 in which nucleic acids were captured. TM Discs were obtained. Specifically, ethanol was added to the lysate of HCV-infected serum samples using Fusion 5 TMIn addition to adding the disk, various amounts of PEG were added (volume percentage of PEG based on the total volume of lysate and PEG: about 1% (v / v), about 2.5% (v / v), about 5% (v / v), about 10% (v / v), about 30% (v / v)), and after incubation, the supernatant was discarded and the nucleic acid was captured in the Fusion 5 TM Only the disk was separated, and the separated Fusion 5 TM After cleaning and drying the disc, Fusion 5 TM After performing RQ-PCR on the disk itself, the Ct value was obtained. As a control, Fusion 5 was obtained by performing the same procedure as above without adding PEG. TM After performing RQ-PCR on the disk itself, the Ct value was obtained.

[0122] As a result, as shown in Fig. 7, it was confirmed that when PEG was added, the nucleic acid extraction efficiency was improved compared to the case without PEG addition, and the average Ct value was much improved. In particular, the Ct value of the control group without PEG addition was 28.0 ± 1.0, the Ct value of the experimental group with about 1% (v / v) PEG addition was 25.4 ± 0.6, and the Ct value of the experimental group with about 2.5% (v / v) PEG addition was 25.6 ± 0.4. That is, it was found that the Ct value of the experimental group with about 1% (v / v) or about 2.5% (v / v) PEG addition was reduced by about 2.5 compared to the Ct value of the control group without PEG addition, which means that the amplification efficiency of the experimental group with about 1% (v / v) or about 2.5% (v / v) PEG addition increased by about 5.6 times compared to the control group, greatly improving the amplification efficiency. Also, Qiagen is mainly used as a general nucleic acid extraction method. TMWhen nucleic acids were extracted and amplified using the extraction method, the average Ct value was about 24.0, which was found to be equivalent to or similar to the amplification efficiency of the experimental group in which PEG was added at about 1% (v / v) or about 2.5% (v / v). In particular, the experimental group in which PEG was added at about 0.5 to 4% (v / v) (e.g., about 1 to 2.5% (v / v)) based on the total volume of the lysate and PEG exhibited a low Ct value as described above, indicating excellent amplification efficiency. On the other hand, in the experimental group in which PEG was added at an amount outside the above numerical range (e.g., the experimental group in which PEG was added at about 5% (v / v), about 10% (v / v), or about 30% (v / v)), the Ct value further increased, confirming that the amplification efficiency further decreased.

[0123] Through the above results, the nucleic acid obtained by the PINE-CX method was captured in Fusion 5 TM It was found that nucleic acid can be amplified by performing PCR directly on the disk itself without going through the elution step, and in this case, by additionally using PEG, the extraction efficiency of nucleic acid from the sample is significantly increased, and thus the nucleic acid amplification efficiency during PCR is also significantly increased. That is, when applying the PINE-CX method, a small amount of PEG is used, and Fusion 5 TM By performing PCR on the disk itself, the extraction and amplification efficiency of nucleic acids from the sample is significantly improved without an elution step, and in this case, when the amount of PEG added is adjusted to about 0.5 to 4% (v / v) (e.g., about 1 to 2.5% (v / v)) based on the total volume of the lysate and PEG, the extraction and amplification efficiency of nucleic acids from the sample is further increased, whereas when the amount of PEG added is outside the above numerical range, the extraction and amplification efficiency of nucleic acids from the sample is relatively decreased.

[0124] In addition, as shown in Fig. 8, when applying the PINE-CX method, a small amount of PEG is added (e.g., PEG addition amount: about 1% (v / v), about 2.5% (v / v), about 5% (v / v) based on the total volume of the dissolved substance and PEG), and Fusion 5 TM In the way PCR is performed on the disk itself, the nucleic acid extraction process, i.e. the Fusion 5 in which the nucleic acids are captured TM In the process of obtaining the disk, the lysate of the HCV infected serum sample was dissolved in Fusion 5 without adding ethanol. TM Add only the disk and mix, and separate Fusion 5 from the mixture. TM When performing PCR directly on the disk without going through the washing and drying process, the nucleic acid obtained by the PINE-CX method of Example 2, which performs the ethanol addition, washing and drying process as described above without using PEG, is captured on Fusion 5 TM We confirmed that there was no statistical difference in the average Ct values ​​compared to performing PCR on the disk itself.

[0125] Through the above results, it was found that when PEG is used to extract nucleic acids from a sample using the PINE-CX method, the ethanol addition, washing, and drying processes can be omitted, thereby significantly improving the nucleic acid extraction efficiency.

[0126] [Example 4]

[0127] 4.1 Reproducibility and detection limit analysis

[0128] The reproducibility of the PINE-CX method was analyzed by running the method twice a day for 5 days (10 repetitions at each concentration) using serially diluted samples containing HCV RNA. Specifically, nucleic acids were extracted from serially diluted HCV-infected serum samples using the PINE-CX method performed in Example 2 and analyzed using Fusion 5.TM The disc effluent was obtained. Thereafter, the agarose gel electrophoresis results of the nucleic acid amplification product of the effluent and the amplification product of the sample HCV RNA were compared to determine the limit of detection (LOD) of the PINE-CX method.

[0129] As a result, as shown in Fig. 9, when using the PINE-CX method, the concentration of nucleic acid in the sample is ~10 1 Even at significantly low levels of IU / mL (n=10), clear visibility was observed in all replicate experiments, demonstrating excellent reproducibility and nucleic acid detection sensitivity. In addition, the sensitivity of the PINE-CX method was confirmed to be similar to that of the existing QIAamp DNA Micro kit in testing most samples. Specifically, it was found to be effective in detecting very low concentrations of HCV RNA (approximately 10 1 IU / mL, approximately 10 2 The Ct values ​​of (IU / mL) were approximately 29.25 ± 0.96 and approximately 27.76 ± 1.52, which were strongly detected in all repeated experiments and were similar to the Ct values ​​of the existing QIAamp DNA Micro kit extraction (approximately 28.32 and approximately 26.62). In addition, the Ct values ​​of 6 mm Fusion 5 TM We confirmed that similar results were obtained when the PINE-CX method was applied using a disk (see Fig. 10).

[0130] 4.2 Evaluation of PINE-CX using clinical HCV infection samples

[0131] The PINE-CX method and the Roche Cobas 6800 HCV test (Roche HCV, Roche Molecular Diagnostics, Pleasanton, CA, USA) were performed on clinical HCV-infected serum samples and the results were compared. Specifically, nucleic acids were extracted from HCV-infected serum samples using the PINE-CX method performed in Example 2 and tested on Fusion 5TM After obtaining the effluent from the disk, the nucleic acid was amplified for the effluent to determine whether it was HCV positive or not, and this was compared with the results of the Roche Cobas 6800 HCV test.

[0132] Among 60 clinical HCV-infected serum samples, 30 (approximately 1.5 x 10) were HCV-positive as a result of the PINE-CX test. 1 IU / mL ~ 3.0 x 10 7 IU / mL), 30 negative results were obtained, which was completely consistent with the research results of the Roche Cobas 6800 HCV test, and it was confirmed that it accurately determined the presence or absence of HCV infection in all samples.

[0133] 4.3 Analytical specificity

[0134] To evaluate the analytical specificity of the above PINE-CX method, Fusion 5 was obtained by extracting nucleic acids from HCV-infected serum samples using the PINE-CX method performed in Example 2. TM For the disc extract, PCR was performed using detection primers for various viruses, including HCV, to analyze whether amplification occurred.

[0135] As a result, when nucleic acids were extracted from HCV-infected serum samples using the PINE-CX method, and then PCR was performed using detection primers for human rhinovirus (HRV) (n=2), influenza A (n=1), influenza B (n=1), human bocavirus (n=2), adenovirus (n=2), hepatitis B virus (HBV) (n=2), and human BK virus (n=2), no amplification products were found. However, when PCR was performed using HCV detection primers, an amplification product was found. Through this, it was confirmed that target nucleic acids can be specifically detected using the PINE-CX method.

[0136] [Example 5]

[0137] 5.1 Performance Evaluation of Common Nucleic Acid Extraction Methods with PEG Addition

[0138] Qiagen, a conventional nucleic acid extraction method with a modification that adds PEG to the lysate of HCV-infected serum samples TM HCV RNA nucleic acid was extracted from HCV-infected serum samples using the extraction method, and then RT-PCR and RQ-PCR were performed (repeated 6 times).

[0139] As a result, as shown in Table 4 and Figure 11, the existing nucleic acid extraction method, Qiagen TM When applying a modification of the PEG addition method to the extraction method, we confirmed that nucleic acid amplification was not achieved in real-time PCR. This indicates that PEG addition does not unconditionally increase nucleic acid extraction efficiency. Therefore, it was confirmed that the combination of the PINE-CX method and PEG addition improved nucleic acid extraction efficiency.

[0140] Repeated Assessment QIAGEN TM Extraction method (Ct value)QIAGEN TM (+1% PEG)replicate_129.3829.27replicate_228.08fail (no amplification)replicate_326.42fail (no amplification)replicate_425.71fail (no amplification)replicate_526.00fail (no amplification)replicate_625.81fail (no amplification)Total Mean26.90Total SD1.5

[0141] 5.2 Performance Evaluation of the PINE-CX Nucleic Acid Extraction Method According to Paper Type

[0142] Fusion 5 of glass fiber material in the PINE-CX method of Example 3 aboveTM After extracting nucleic acids from HCV-infected serum samples using a type of paper other than disk paper, i.e., cellulose filter paper (Whatman quantitative filter paper, Grade 41, Whatman, Maidstone, Kent, UK) or nylon filter paper (Roche, Basel, Switzerland), RT-PCR and RQ-PCR were performed on the paper itself on which the obtained nucleic acids were captured.

[0143] As a result, as shown in Table 5 and Fig. 11, when a type of paper other than glass fiber paper was used in the PINE-CX method, it was confirmed that the nucleic acid amplification efficiency in PCR was low regardless of the use of PEG, indicating that nucleic acid extraction was not performed well. Through this, it was found that the nucleic acid extraction efficiency was significantly improved by using glass fiber paper in the PINE-CX method.

[0144] replicate_1replicate_2replicate_3replicate_4replicate_5replicate_6AshlessN / A29.81N / A29.33N / A30.33Ashless (+1% PEG)29.20N / A30.3230.34N / AN / ANylonN / A29.0629.51N / A29.6N / ANylon (+1% PEG)31.09N / AN / A29.13N / AN / A

[0145] (N / A: fail (no amplification))

[0146] [Example 6]

[0147] 6.1 Optimizing the number of paper disks in the PINE-CX method

[0148] In the PINE-CX method performed in Example 2 above, Fusion 5 TMFusion 5, which was obtained after extracting nucleic acids from HCV-infected serum samples by changing the number of disk papers (2 mm), and capturing the nucleic acids TM RT-PCR and RQ-PCR were performed on the disk itself.

[0149] As a result, as shown in Fig. 12, in the PINE-CX method, Fusion 5 TM When nucleic acid was extracted using four disk papers (2 mm), the PCR result Ct value was the lowest, confirming the best nucleic acid amplification efficiency. Through this, it was confirmed that Fusion 5 in the PINE-CX method TM It was found that the nucleic acid extraction efficiency could be significantly improved by using four or more disk papers (2 mm).

[0150] 6.2 Optimization of sample lysis time in the PINE-CX method

[0151] In the PINE-CX method performed in Example 2 above, the lysis time of the HCV-infected serum sample with a protein-degrading enzyme (e.g., proteinase K) was changed (5 minutes, 10 minutes, 15 minutes) to extract nucleic acids from the sample, and Fusion 5 was obtained. TM RQ-PCR was performed on the effluent from the disk.

[0152] As a result, as shown in Fig. 13, it was confirmed that nucleic acid amplification did not occur well in the PCR results when the sample was dissolved for 10 minutes or less in the PINE-CX method to extract nucleic acids. Through this, it was found that the nucleic acid extraction efficiency can be significantly improved by adjusting the sample dissolution time in the PINE-CX method to more than 10 minutes (e.g., more than 10 minutes to 30 minutes or less, more than 10 minutes to 15 minutes or less, more than 15 minutes, or more than 15 minutes to 30 minutes or less).

[0153] 6.3 Optimization of washing buffer in PINE-CX method

[0154] In the PINE-CX method performed in Example 2 above, the washing buffer was changed and applied to extract nucleic acid from an HCV-infected serum sample, resulting in Fusion 5 TM RT-PCR and RQ-PCR were performed on the effluent from the disk.

[0155] As a result, as shown in Fig. 14, in the PINE-CX method, AW1 was used alone as a washing buffer, and Fusion 5 TM When the disk paper was washed, it was confirmed that the Ct value of the PCR results increased relatively, and the nucleic acid amplification efficiency was relatively reduced, and both AW1 and AW2 were used as washing buffers for Fusion 5 TM When the disk paper was washed, it was confirmed that the Ct value of the PCR results was relatively reduced, and the nucleic acid amplification efficiency was relatively increased. Through this, it was confirmed that both AW1 and AW2 were used as washing buffers in the PINE-CX method and Fusion 5 TM It was found that the nucleic acid extraction efficiency could be further improved by washing the disk paper.

[0156] 6.4 Optimization of the amount of washing buffer in the PINE-CX method

[0157] In the PINE-CX method performed in Example 2 above, the amount of washing buffer was changed (about 100 uL, about 200 uL, about 400 uL), and Fusion 5 obtained by extracting nucleic acid from an HCV-infected serum sample TM RT-PCR, LAMP, and RQ-PCR were performed on the effluent from the disk.

[0158] As a result, as shown in Fig. 15, in the PINE-CX method, Fusion 5 TMWhen the amount of buffer used to wash the disk paper was adjusted to approximately 200 uL, it was confirmed that the nucleic acid amplification efficiency of PCR or LAMP results was further increased. Through this, in the PINE-CX method, Fusion 5 TM It was found that the nucleic acid extraction efficiency could be further improved by adjusting the amount of buffer used to wash the disc paper to about 150 to 350 uL, about 150 to 300 uL, about 150 to 250 uL, or about 200 uL.

[0159] 6.5 Optimization of drying time in the PINE-CX method

[0160] In the PINE-CX method performed in Example 2 above, the drying time was changed (10 minutes or 1 minute at about 56°C), and Fusion 5 was obtained by extracting nucleic acids from HCV-infected serum samples. TM LAMP was performed on the disc itself and the extract from the disc.

[0161] As a result, as shown in Fig. 16, the washed Fusion 5 in the PINE-CX method TM After drying the disc paper for 1 minute, Fusion 5 TM LAMP was performed on the extract from the disk paper, and it was confirmed that the nucleic acid amplification efficiency was significantly reduced compared to when dried for 10 minutes. These results are based on Fusion 5 TM The same thing happened when I did a LAMP on the disk itself.

[0162] Through this, the washed Fusion 5 in the PINE-CX method TM It was found that the nucleic acid extraction efficiency can be further improved by drying the disk paper for a time of more than 1 minute but less than 20 minutes, more than 1 minute but less than 15 minutes, more than 1 minute but less than 10 minutes, more than 5 minutes but less than 15 minutes, more than 5 minutes but less than 10 minutes, more than 10 minutes, or more than 10 minutes but less than 20 minutes.

Claims

1. (1) A step of adding a protein-decomposing enzyme to the sample and mixing to dissolve the sample; and (2) A method for extracting nucleic acid from a sample, comprising a step of mixing the lysate obtained in step (1) and paper made of glass fiber material.

2. In claim 1, A method for extracting nucleic acid from a sample, wherein in step (2) above, polyethylene glycol (PEG) is further added and mixed.

3. In claim 2, A method for extracting nucleic acid from a sample, wherein in the step (2), the polyethylene glycol is added in an amount of 0.5% (v / v) to 4% (v / v) based on 100% (v / v) of the volume sum of the melt and the polyethylene glycol.

4. In claim 1, A method for extracting nucleic acid from a sample, wherein the method does not involve centrifugation.

5. In claim 1, A method for extracting nucleic acid from a sample, wherein the dissolution in step (1) is performed for a time exceeding 10 minutes.

6. In claim 1, A method for extracting nucleic acid from a sample, wherein in step (2), the mixture is incubated at 15°C to 30°C for 1 to 10 minutes after mixing.

7. In claim 1, A method for extracting nucleic acid from a sample, wherein the nucleic acid is captured on the paper in the step (2).

8. In claim 1, A method for extracting nucleic acid from a sample, wherein the method further comprises a step of separating the paper from the mixture obtained in step (2).

9. In claim 8, A method for extracting nucleic acid from a sample, wherein the method further comprises a step of washing or drying the separated paper.

10. In claim 8, A method for extracting nucleic acid from a sample, wherein the method further comprises a step of eluting nucleic acid from the separated paper. 11.(1) A step of adding a protein-decomposing enzyme to a sample and mixing to dissolve the sample; (2) A step of mixing the melt obtained in step (1) and the paper made of glass fiber material; (3) a step of separating the paper from the mixture obtained in step (2); and (4) A method for detecting nucleic acid in a sample, comprising a step of amplifying nucleic acid from the separated paper itself or an eluate obtained by eluting nucleic acid from the separated paper.

12. In claim 11, A method for detecting nucleic acid in a sample, wherein in step (2) above, polyethylene glycol is further added and mixed.

13. In claim 12, A method for detecting nucleic acid in a sample, wherein in the step (2), the polyethylene glycol is added in an amount of 0.5% (v / v) to 4% (v / v) based on 100% (v / v) of the volume sum of the melt and the polyethylene glycol.

14. In claim 11, A method for detecting nucleic acid in a sample, wherein the method does not perform centrifugation.

15. In claim 11, A method for detecting nucleic acid in a sample, wherein the step of amplifying the nucleic acid uses polymerase chain reaction (PCR) or loop-mediated isothermal amplification (LAMP).

16. In claim 11, A method for detecting nucleic acid in a sample, wherein the method further comprises a step of detecting an amplification product.

Images