Nucleic acid purification method and kit for purifying nucleic acid

Abstract

The purpose of the present invention is to provide: a method for purifying a nucleic acid from a nucleic acid-containing sample in a simple, short-time and high-purity manner without using a protic zeolite; and a kit for the purification. A method for purifying a nucleic acid according to the present invention comprises a step for bringing a sample containing a nucleic acid into contact with an aprotic zeolite.

Description

Technical Field

[0001] This invention relates to methods for purifying nucleic acids and kits for purifying nucleic acids. Background Technology

[0002] Currently, nucleic acid amplification technologies, represented by PCR and LAMP, have permeated all fields of biology, including molecular biology and medicine, and are widely used in applications such as gene diagnosis, DNA identification, food inspection, environmental hygiene inspection, and animal and plant inspection. When amplifying nucleic acids in a sample, it is usually necessary to extract and purify the nucleic acids from the sample. Methods for extracting and purifying nucleic acids from samples include, for example, using commercially available nucleic acid extraction kits. On the other hand, using general commercially available nucleic acid extraction kits (such as the QIAamp Viral RNA Prep Kit (manufactured by Qiagen)) can yield highly pure nucleic acids, but on the other hand, it requires multiple devices such as a high-speed centrifuge and a heater, making it difficult to use such kits in environments without adequate equipment. Furthermore, the operation involves dozens of steps, making it quite complex, and the extraction and purification of nucleic acids takes approximately one hour (e.g., non-patent literature 1-2). In nucleic acid amplification technology, when there are many samples to be tested, it is desirable to prepare samples suitable for nucleic acid amplification in a short time; therefore, complex extraction and purification methods are not suitable. Furthermore, while commercially available nucleic acid extraction kits (such as the Kaneka Simple DNA Extraction Kit, Template Prepper for DNA, and IOSPIN Viral RNA) can extract and purify nucleic acids quickly, the purity of the nucleic acids is sometimes low. Moreover, even with such kits, there are situations where heating is required and the process involves dozens of steps, making it difficult to claim that nucleic acid extraction and purification is simple, quick, and highly pure. Therefore, there is a market demand for methods that can extract and purify nucleic acids easily, quickly, and with high purity without the need for large-scale equipment.

[0003] Furthermore, methods for extracting and purifying nucleic acids from cell lysates and the like using zeolites are also known. For example, Patent Document 1 discloses a method for preparing a nucleic acid amplification sample for amplifying nucleic acids contained in a biological sample, which includes an extraction step of adding a nucleic acid extraction reagent containing an anionic surfactant and / or a base to the biological sample to obtain a nucleic acid extract, and a step of contacting the nucleic acid extract with a proton zeolite, wherein substances adsorbed on the zeolite are removed. Patent Document 2 discloses a pretreatment method for clarifying dissolved substances using zeolites in a method for separating nucleic acids from biological solutions (e.g., cell lysates), and describes a method for clarifying cell lysates by adding zeolites to cell lysates treated with an alkaline reagent such as sodium dodecyl sulfate (SDS) to adsorb SDS, followed by centrifugation.

[0004] Existing technical documents

[0005] Patent documents

[0006] Patent Document 1: Japanese Patent No. 5290987

[0007] Patent Document 2: Japanese Patent Publication No. 2005-531329

[0008] Non-patent literature

[0009] Non-Patent Document 1: National Institute of Infectious Diseases, "Pathogen Detection Manual 2019-nCoV Ver. 2.5", February 15, 2020, https: / / www.niid.go.jp / niid / images / lab-manual / 2019-nCoV20200215.pdf

[0010] Non-Patent Document 2: QIAGEN Co., Ltd., "QIAamp (Registered Trademark) Viral RNA Mini Procedures and Troubleshooting", April 2010, https: / / www.qiagen.com / jp / resources / download.aspx?id=7d6918b1-77dd-4dac-b694-a9ca8aa58b43&lang=ja-JP Summary of the Invention

[0011] The problem that the invention aims to solve

[0012] The inventors have discovered that, depending on the composition of the sample containing nucleic acid or the purification conditions, there exist zeolites that adsorb nucleic acids; these are proton-type zeolites.

[0013] The purpose of this invention is to provide a simple, short-time, and high-purity method for purifying nucleic acids from samples containing nucleic acids without using proton-type zeolites, and a kit for this purification.

[0014] Methods for solving problems

[0015] The inventors discovered that, surprisingly, when a sample containing nucleic acid is mixed with an aprotic zeolite, the adsorption of nucleic acid in the sample is significantly low, while the adsorption of substances other than nucleic acid is high, thus completing the present invention.

[0016] That is, the present invention relates, for example, to the following inventions. [1]

[0018] A method for purifying nucleic acids, comprising the step of contacting a sample containing nucleic acids with an aprotic zeolite. [2]

[0020] According to the purification method described in [1], the above-mentioned sample is derived from at least one sample selected from the group consisting of blood, cerebrospinal fluid, urine, feces, sputum, saliva, nasal mucus, swab specimen, amniotic fluid and mouthwash. [3]

[0022] According to the purification method described in [1] or [2], in the above contact step, at least a portion of the above nucleic acid is not adsorbed by aprotic zeolite. [4]

[0024] According to any one of [1] to [3], the purification method wherein the above-mentioned aprotic zeolite is selected from at least one of the group consisting of magnesium alkali zeolite, ZSM-5 type zeolite, Y type zeolite and β type zeolite. [5]

[0026] According to the purification method described in [4], the above-mentioned aprotic zeolite is selected from at least one of the group consisting of magnesium alkali zeolite-NH4 type zeolite, magnesium alkali zeolite-K type zeolite, ZSM-5-NH4 type zeolite, Y-Na type zeolite and β-NH4 type zeolite. [6]

[0028] The purification method according to any one of [1] to [5] further comprises: obtaining the sample containing nucleic acid by mixing the sample with a nucleic acid extraction reagent containing a surfactant and / or by heating the sample prior to the contact step. [7]

[0030] According to the purification method described in [6], the surfactant mentioned above contains anionic surfactant. [8]

[0032] According to the purification method described in [7], the above-mentioned anionic surfactant is dodecyl sulfate. [9]

[0034] The purification method according to any one of [1] to [8], wherein the above nucleic acid is RNA and / or DNA.

[10]

[0036] A kit for purifying nucleic acids, comprising aprotic zeolite.

[11]

[0038] According to the kit described in

[10] , the aprotic zeolite is selected from at least one of the group consisting of magnesium alkali zeolite, ZSM-5 zeolite, Y-type zeolite and β-type zeolite.

[12]

[0040] According to the kit described in

[11] , the aprotic zeolite is selected from at least one of the group consisting of magnesium alkali zeolite-NH4 type zeolite, magnesium alkali zeolite-K type zeolite, ZSM-5-NH4 type zeolite, Y-Na type zeolite and β-NH4 type zeolite.

[13]

[0042] The kit according to any one of

[10] to

[12] , wherein the kit is for purifying nucleic acids derived from at least one sample selected from the group consisting of blood, cerebrospinal fluid, urine, feces, sputum, saliva, nasal mucus, swab specimens, amniotic fluid and mouthwash, and the kit further comprises a nucleic acid extraction reagent containing a surfactant.

[14]

[0044] According to the kit described in

[13] , the surfactant described above contains anionic surfactant.

[15]

[0046] According to the kit described in

[14] , the anionic surfactant is dodecyl sulfate.

[0047] Invention Effects

[0048] According to the present invention, a method for easily, quickly, and with high purity extracting and purifying nucleic acids by efficiently removing substances other than nucleic acids from a sample containing nucleic acids, as well as a kit for such extraction and purification, can be provided. In particular, according to the present invention, nucleic acids can be easily, quickly, and with high purity extracted and purified from a sample containing nucleic acids without the use of multiple devices such as high-speed centrifuges and heaters. Furthermore, in the case of aprotic zeolites, the adsorption of nucleic acids in the sample is significantly low; therefore, according to the present invention, high yields of nucleic acid extraction and purification are also expected. Moreover, the present invention can also be used for the purification of either or both of DNA and RNA, thus exhibiting high versatility.

[0049] Furthermore, as described above, according to the present invention, nucleic acids can be purified easily, quickly, and with high purity, thus obtaining samples that can be effectively used for nucleic acid amplification reactions, enabling highly sensitive determination using nucleic acid amplification technology. Detailed Implementation

[0050] The following describes in detail the methods for implementing the present invention. However, the present invention is not limited to the following embodiments.

[0051] [Methods for purifying nucleic acids]

[0052] The nucleic acid purification method of this embodiment includes a step of contacting a nucleic acid-containing sample with an aprotic zeolite (contact step). As shown in the examples described later, the aprotic zeolite does not adsorb at least a portion of the nucleic acid in the nucleic acid-containing sample, but adsorbs substances other than nucleic acid. Therefore, by contacting the nucleic acid-containing sample with an aprotic zeolite, substances other than nucleic acid can be removed from the nucleic acid-containing sample, and nucleic acid can be purified easily, quickly, and with high purity.

[0053] <Samples containing nucleic acids>

[0054] The nucleic acid-containing sample is an aqueous solution or aqueous suspension containing dissolved or suspended nucleic acids and other substances. The type of nucleic acid contained in the sample is not particularly limited; it can be DNA or RNA, and can be single-stranded or double-stranded. The source of the nucleic acid is also not limited; for example, it can be nucleic acid derived from animals, plants, fungi, bacteria, or viruses. The method of this invention is particularly effective in purifying RNA simply, quickly, and with high purity. The pH of the nucleic acid-containing sample is not particularly limited.

[0055] Substances other than nucleic acids include proteins (such as enzymes, glycoproteins, polypeptides, etc.), lipids, sugars (such as monosaccharides, disaccharides, oligosaccharides, polysaccharides, etc.), and salts.

[0056] In addition, nucleic acid-containing samples can be derived from samples obtained from living organisms, or from environmental samples such as water, soil, and air. Nucleic acid-containing samples are preferably derived from samples obtained from living organisms, and more preferably from at least one sample selected from the group consisting of blood (whole blood, plasma, or serum), cerebrospinal fluid, urine, feces, sputum, saliva, nasal discharge, swab specimens, amniotic fluid, and mouthwash. Nucleic acid-containing samples can be diluents / suspensions of the above-mentioned samples or extracts obtained through the extraction steps described later. Substances other than nucleic acids can be components derived from these samples that are not nucleic acids.

[0057] Samples containing nucleic acids may contain nucleic acid extraction reagents containing surfactants, as described later. Additionally, they may contain inhibitors that target substances that break down nucleic acids, as needed. Examples of such inhibitors include, for instance, nucleic acid-degrading enzyme inhibitors.

[0058] <Zeolite>

[0059] "Zeolite" is a general term for aluminosilicates with micropores in their crystals, with a three-dimensional network structure formed by the shared oxygen bonds of tetrahedral SiO4 and AlO4 as its basic framework. Zeolites contain cations in their crystals, such as alkali metal ions, alkaline earth metal ions, ammonium ions, and hydrogen ions (protons). "Proton-type zeolites" refer to zeolites that contain hydrogen ions as cations, while "non-proton-type zeolites" refer to zeolites other than proton-type zeolites. In the purification method of this embodiment, the non-proton-type zeolites do not adsorb at least a portion of the nucleic acids in the sample containing nucleic acids.

[0060] The crystal structure of aprotic zeolites is not particularly limited, and examples include type A, magnesium-alkali zeolite, MCM-22, ZSM-5, mordenite, L, Y, X, and β. Preferably, the aprotic zeolite is selected from at least one group consisting of magnesium-alkali zeolite, ZSM-5, Y-type, and β-type zeolites.

[0061] In aprotic zeolite crystals, the cation is not particularly limited as long as it is a cation other than hydrogen ion. Examples of such cations include alkali metal ions (e.g., sodium ions). + ), potassium ions (K) + Alkaline earth metal ions (e.g., magnesium ions (Mg ions)) 2+ ), calcium ions (Ca + (etc.), ammonium ions (NH4+) + )wait.

[0062] Aprotic zeolites are particularly preferred to be at least one selected from the group consisting of magnesium-alkali zeolite-NH4 type zeolite, magnesium-alkali zeolite-K type zeolite, ZSM-5-NH4 type zeolite, Y-Na type zeolite, and β-NH4 type zeolite. For example, "magnesium-alkali zeolite-NH4 type zeolite" refers to a zeolite with a magnesium-alkali zeolite-type crystal structure and containing ammonium ions as cations.

[0063] <Contact Steps>

[0064] In the contact step, the sample containing nucleic acid is brought into contact with aprotic zeolite to adsorb substances other than nucleic acid. Therefore, in the purification method of this embodiment, nucleic acid can be purified from a sample containing nucleic acid through the contact step. In this specification, "purification" refers to improving the purity of nucleic acid from a sample containing nucleic acid.

[0065] In the contact step, "contact" can refer to mixing a sample containing nucleic acid with aprotic zeolite. For example, a sample containing nucleic acid can be added to a container containing aprotic zeolite, or aprotic zeolite can be added to a sample containing nucleic acid. There is no particular limitation on the order in which the sample containing nucleic acid comes into contact with the aprotic zeolite.

[0066] The amount of zeolite in the contact step may be, for example, 50 mg or more, 60 mg or more, 70 mg or more, 80 mg or more, 90 mg or more, 100 mg or more, 110 mg or more, or 120 mg or more relative to 1 mL of a nucleic acid-containing sample. The amount of zeolite in the contact step may be, for example, less than 350 mg, less than 340 mg, less than 330 mg, less than 320 mg, less than 310 mg, or less than 300 mg relative to 1 mL of a nucleic acid-containing sample.

[0067] There is no particular limitation on the contact time in the contact step; for example, it can be 10 seconds to 10 minutes, 20 seconds to 10 minutes, or 30 seconds to 5 minutes.

[0068] Through the contact step, at least a portion of the substances other than nucleic acids in the nucleic acid-containing sample is adsorbed by the aprotic zeolite. "Adsorption" refers to the state in which substances are bound to or encapsulated on the surface or in the pores of the aprotic zeolite; the adsorbed substances are removed along with the aprotic zeolite. Through the contact step, at least 30%, at least 40%, at least 50%, at least 60%, at least 70%, at least 80%, and at least 90% of the substances other than nucleic acids in the nucleic acid-containing sample are adsorbed by the aprotic zeolite. Furthermore, "at least a portion of the nucleic acid is not adsorbed by the aprotic zeolite" means, for example, that when a sample containing only nucleic acid and a sample obtained by contacting the sample containing only nucleic acid with the aprotic zeolite are respectively subjected to nucleic acid amplification under the same conditions, the difference in the detected nucleic acid copy number based on the former copy number can be less than 25%, less than 20%, less than 15%, less than 10%, less than 5%, or less than 1%, preferably less than 0%, or greater than the nucleic acid copy number detected when using a sample containing only nucleic acid for nucleic acid amplification.

[0069] <Extraction Steps>

[0070] The purification method of this embodiment may include a step (extraction step) prior to the contact step, in which the sample is mixed with a nucleic acid extraction reagent containing a surfactant and / or the sample is heated to obtain the nucleic acid-containing sample. Through surfactant or heat treatment, cell membranes, viral envelopes, etc., in the sample are disrupted, thus allowing nucleic acids to dissolve or suspend in the nucleic acid-containing sample. The order of the steps of mixing the sample with the nucleic acid extraction reagent containing a surfactant and heating the sample is not particularly limited. Since the purification method of this embodiment includes an extraction step, this purification method can also be understood as an extraction method.

[0071] The surfactant is not particularly limited and can be anionic surfactant (anionic surfactant, cationic surfactant), nonionic surfactant, or amphoteric surfactant. From the viewpoint that it is easier to disrupt cell membranes, viral envelopes, etc., anionic surfactants are preferred, and anionic surfactants are more preferred. The nucleic acid extraction reagent may contain one or more surfactants.

[0072] Examples of anionic surfactants include higher fatty acid salts, linear alkylbenzene sulfonates, α-sulfonyl fatty acid methyl esters, α-olefin sulfonates, alkyl sulfates, and polyoxyethylene alkyl sulfates, with alkyl sulfates being preferred, and more preferably lithium dodecyl sulfate (LDS), sodium dodecyl sulfate (SDS), or sodium cholate.

[0073] Examples of cationic surfactants include alkyltrimethylammonium salts, dialkyldimethylammonium salts, alkyldimethylbenzylammonium salts, benzyltrimethylammonium salts, etc., and benzalkonium chloride, etc.

[0074] Examples of nonionic surfactants include sorbitan fatty acid esters, sucrose fatty acid esters, polyoxyethylene fatty acid esters, polyoxyethylene sorbitan fatty acid esters, polyoxyethylene alkyl ethers, and polyoxyalkyl phenyl ethers, such as TRITON (registered trademark)-X100 and Tween (registered trademark) 40.

[0075] Examples of amphoteric surfactants include alkyl amino fatty acid salts, alkyl betaines, and alkyl amine oxides, such as 3-[(3-cholamidopropyl)dimethylammonium]-2-hydroxy-1-propanesulfonate (CHAPSO) and 3-[(3-cholamidopropyl)dimethylammonium]-1-propanesulfonate (CHAPS).

[0076] The surfactant content in nucleic acid extraction reagents should be at least the amount required for nucleic acid extraction, and can vary depending on the type of surfactant. For example, based on the total amount of the nucleic acid extraction reagent, it can be 0.01 w / v% or higher, 0.1 w / v% or higher, 0.5 w / v% or higher, or 1 w / v% or higher. Alternatively, based on the total amount of the nucleic acid extraction reagent, the surfactant content can be, for example, less than 5 w / v%, less than 4 w / v%, or less than 3 w / v%. It should be noted that if the nucleic acid extraction reagent contains multiple surfactants, the above-mentioned surfactant content refers to the total content of all surfactants.

[0077] Nucleic acid extraction reagents may contain alkalis in addition to surfactants. Examples of alkalis include sodium hydroxide, potassium hydroxide, lithium hydroxide, calcium hydroxide, and ammonia, with sodium hydroxide being preferred. Nucleic acid extraction reagents may contain one or more alkalis.

[0078] Regarding the alkali content in nucleic acid extraction reagents, when the alkali is sodium hydroxide, the base is based on the total amount of nucleic acid extraction reagent, for example, it can be above 10mM, above 25mM, above 50mM, above 75mM, above 100mM, above 125mM, above 150mM, above 175mM, or above 200mM. Regarding the alkali content in reagents containing nucleic acids, when the alkali is sodium hydroxide, the base is based on the total amount of nucleic acid extraction reagent, for example, it can be below 500mM, below 480mM, below 460mM, below 440mM, below 420mM, or below 400mM.

[0079] The pH of nucleic acid extraction reagents is not particularly limited as long as it is a conventional pH that does not decompose nucleic acids. For example, it can be 5-9, 5-7, or 6-7.

[0080] There are no particular limitations on heat treatment as long as it enables the extraction of nucleic acids from the sample or dissolves or suspends the nucleic acids in the nucleic acid-containing sample. For example, it can involve using a heating block to heat the sample. The temperature of the heat treatment can be, for example, 50℃~200℃, 60℃~150℃, or 70℃~100℃. The time of the heat treatment can be, for example, 1 minute~30 minutes, 5 minutes~30 minutes, or 15 minutes~30 minutes.

[0081] <Removal Steps>

[0082] The purification method of this embodiment may include a step of removing aprotic zeolite after the contact step (removal step). In the removal step, aprotic zeolite is removed to obtain a sample containing purified nucleic acid.

[0083] The removal of aprotic zeolites can be achieved, for example, by centrifugation, filtration, or a combination thereof, of the mixture of the nucleic acid-containing sample and the aprotic zeolite after the contact step. Removal by centrifugation can be easily performed, for example, using a commercially available centrifuge column.

[0084] The sample containing purified nucleic acid obtained by the removal step can be used, for example, in nucleic acid amplification analysis.

[0085] [Nucleic Acid Amplification Methods]

[0086] The nucleic acid amplification method of this embodiment includes a step of purifying nucleic acid by implementing the purification method of this embodiment, and a step of performing a nucleic acid amplification reaction on the purified nucleic acid (nucleic acid amplification step).

[0087] <Nucleic Acid Amplification Steps>

[0088] Nucleic acid amplification can be performed using conventional methods. More specifically, it can be performed by incubating a reaction solution containing purified nucleic acid, a primer set containing a base sequence specific to that nucleic acid, nucleic acid synthases such as DNA polymerase, deoxynucleoside triphosphates (dNTPs: dATP, dTTP, dCTP, and dGTP), reverse transcriptase as needed, and labeled probes for detecting the amplification products.

[0089] When the nucleic acid is DNA, nucleic acid amplification reactions can include polymerase chain reaction (PCR), loop-mediated isothermal amplification (LAMP), nicking endonuclease amplification (NEAR), transcription-reverse transcription synergistic (TRC) reaction, whole genome amplification (WGA) reaction, strand substitution amplification (SDA) reaction, helicase-dependent amplification (HDA) reaction, recombinase polymerase amplification (RPA) reaction, and chimeric primer-mediated isothermal amplification (ICAN) reaction. When the nucleic acid is RNA, nucleic acid amplification reactions can include reverse transcription PCR (RT-PCR), reverse transcription LAMP (RT-LAMP) reaction, transcription-mediated amplification (TMA) reaction, or nucleic acid sequence-dependent amplification (NASBA) reaction, etc.

[0090] [Reagent kit for purifying nucleic acids]

[0091] The reagent kit for purifying nucleic acids in this embodiment (hereinafter also referred to as "the purification kit of this embodiment") includes an aprotic zeolite. By using the purification kit of this embodiment, nucleic acids can be purified from a sample containing nucleic acids. The sample containing nucleic acids and the aprotic zeolite are as described above.

[0092] When using a sample derived from a specimen as a nucleic acid-containing sample, the purification kit of this embodiment may include a nucleic acid extraction reagent containing a surfactant. The nucleic acid extraction reagent containing a surfactant is as described above.

[0093] In addition to aprotic zeolite and nucleic acid extraction reagents containing surfactants, the purification kit of this embodiment may also include buffer solution, nucleic acid degrading enzyme inhibitor, and apparatus for purifying nucleic acids (such as centrifuge column).

[0094] [Reagent kit for nucleic acid amplification]

[0095] The kit for amplifying nucleic acids in this embodiment (hereinafter also referred to as "the nucleic acid amplification kit of this embodiment") contains aprotic zeolite, DNA polymerase, and deoxyribonucleoside triphosphate.

[0096] In the nucleic acid amplification kit of this embodiment, in addition to the reagents included in the purification kit of this embodiment, various known reagents for performing nucleic acid amplification reactions can be mixed. When the target nucleic acid is RNA, the nucleic acid amplification kit of this embodiment may also include reverse transcriptase. Other known reagents for performing nucleic acid amplification reactions include, for example, buffers providing conditions suitable for the enzyme reaction, enzymes such as dithiothreitol (DTT), protective agents stabilizing the target nucleic acid sequence, labeled probes for detecting the amplification products, and intercalation agents. Furthermore, the nucleic acid amplification kit of this embodiment may include a device for detecting nucleic acid amplification.

[0097] Example

[0098] The present invention will be described in more detail below based on embodiments. However, the present invention is not limited to these embodiments.

[0099] [Experimental Example 1: Confirmation of RNA adsorption onto various zeolites]

[0100] To replace 1000 μL of a solution containing SARS-CoV-2 transcribed RNA added to a nucleic acid extraction reagent (0.5% sodium dodecyl sulfate (SDS)), 480 mg of zeolite (described later) was added, and the mixture was inverted and mixed. The zeolites used were Tosoh Corporation's models 930NHA (β-NH4 type), 940NHA (β-NH4 type), 840NHA (ZSM-5-NH4 type), 720NHA (magnesium alkali zeolite-NH4 type), 720KOA (magnesium alkali zeolite-K type), 690HOA (mordenite-H type), 320NAA (Y-Na type), or 320HOA (YH type). The inverted and mixed suspension was passed through a 0.45 μm filter to remove the zeolite and adsorbed substances. The resulting filtrate was used as zeolite-treated samples 1-1 to 1-8. 5 μL of zeolite-treated sample and 20 μL of quantitative PCR reagent were used to amplify the target nucleic acid (cDNA of SARS-CoV-2 transcribed RNA) using quantitative PCR. As a positive control, SARS-CoV-2 transcribed RNA was added to nucleic acid extraction reagent (0.5% SDS). Samples 1-9 that were not zeolite-treated were also prepared and amplified using the same quantitative PCR method.

[0101] The amplification and detection of the target nucleic acid using quantitative PCR were performed as follows: Using the primer and probe sets shown in Table 1, the reaction was carried out in the quantitative PCR reaction reagent solution with the composition shown in Table 2, at the temperatures and times shown in Table 3. The fluorescence value increasing with the amplification of the target nucleic acid was measured in real time using a Thermal Cycler Dice (registered trademark) real-time system III. It should be noted that the 5' end of the probe composed of the base sequence shown in Serial Number 3 in Table 1 was labeled with FAM, and the 3' end was labeled with BHQ1. The results are shown in Table 4. It should be noted that in Table 4, those without detectable Ct values ​​within the measurement time (40 cycles) are indicated as ND. It should be noted that in Table 4, "copy number ratio" indicates the ratio when the copy number of "no zeolite treatment" is set to 100%.

[0102] [Table 1]

[0103] [Table 2]

[0104] [Table 3]

[0105] [Table 4]

[0106] No amplification of target nucleic acids was detected in samples treated with proton-type zeolites (samples 1-6 and 1-8). This indicates that proton-type zeolites adsorb nucleic acids, especially RNA. On the other hand, amplification of target nucleic acids was also confirmed in samples treated with non-proton-type zeolites (samples 1-1 to 1-5, 1-7) and untreated samples (samples 1-9), showing the same copy number. This indicates that the adsorption of nucleic acids, especially RNA, is significantly lower in the case of non-proton-type zeolites.

[0107] [Experimental Example 2: Confirmation of DNA Adsorption onto Various Zeolites]

[0108] Except for the addition of Saccharomyces cerevisiae (hereinafter also referred to as "S. cerevisiae") DNA to the nucleic acid extraction reagent (0.5% SDS), zeolite-treated samples 2-1 to 2-9 were obtained in the same manner as in Experimental Example 1. Furthermore, except for the primer and probe sets shown in Table 5, the quantitative PCR reaction reagent solutions shown in Table 6, and the temperatures and times shown in Table 7, the target nucleic acid (Saccharomyces cerevisiae DNA) of samples 2-1 to 2-9 was amplified using quantitative PCR in the same manner as in Experimental Example 1. Additionally, as a positive control, Saccharomyces cerevisiae DNA was added to the nucleic acid extraction reagent, and untreated sample 2-10 was also prepared, and the target nucleic acid was amplified using quantitative PCR in the same manner. It should be noted that the 5' end of the probe consisting of the base sequence shown in Serial Number 6 in Table 5 was labeled with FAM, and the 3' end was labeled with BHQ1. The results are shown in Table 8. It should be noted that in Table 8, samples for which no Ct value was detected within the measurement time (40 cycles) are indicated as ND. It should be noted that in Table 8, "Copy Count Ratio" refers to the ratio when the copy count of "Zeolite-Free Treatment" is set to 100%.

[0109] [Table 5]

[0110] [Table 6]

[0111] [Table 7]

[0112] [Table 8]

[0113] Similar results were obtained for DNA as for RNA. In samples treated with proton-type zeolite (samples 2-6–2-7 and 2-9), no amplification of the target nucleic acid was detected, or if detected, the copy number was extremely low. This indicates that proton-type zeolite adsorbs nucleic acids, especially DNA. On the other hand, amplification of the target nucleic acid was also confirmed in samples treated with non-proton-type zeolite (samples 2-1–2-5 and 2-8) and untreated samples (sample 2-10), showing the same level of copy number. This indicates that the adsorption of nucleic acids, especially DNA, is significantly lower in the case of non-proton-type zeolite.

[0114] [Experimental Example 3: Confirmation of the effective pH range for RNA adsorption onto various zeolites]

[0115] 480 mg of zeolite was added to 1000 μL of a solution containing SARS-CoV-2 transcribed RNA in a nucleic acid extraction reagent (0.5% SDS) adjusted to pH 3–12. The mixture was inverted and mixed. The zeolite used was Tosoh Corporation's 720NHA and 690HOA models. Other than this, zeolite-treated samples 3-2–3-3, 3-5–3-6, 3-8–3-9, 3-11–3-12, 3-14–3-15, and 3-17–3-18 were obtained in the same manner as in Experimental Example 1. Furthermore, the target nucleic acid (cDNA of SARS-CoV-2 transcribed RNA) was amplified from the above zeolite-treated samples using quantitative PCR, in the same manner as in Experimental Example 1. In addition, similar to Example 1, as a positive control, SARS-CoV-2 transcribed RNA was added to the nucleic acid extraction reagent, and samples 3-1, 3-4, 3-7, 3-10, 3-13, and 3-16 without zeolite treatment were prepared. The target nucleic acid was amplified using quantitative PCR in the same manner as in Example 1. The results are shown in Table 9. It should be noted that in Table 9, "copy number ratio" indicates the ratio when the copy number of the "zeolite-free" extract at each pH is set to 100%.

[0116] [Table 9]

[0117] As shown in Table 9, samples treated with proton-type zeolite using nucleic acid extraction reagents adjusted to pH 3–7 exhibited extremely low copy numbers of target nucleic acids compared to the untreated samples used as positive controls. This indicates that proton-type zeolite adsorbs RNA when using acidic to neutral nucleic acid extraction reagents. On the other hand, samples treated with non-proton-type zeolite all showed copy numbers comparable to the untreated samples used as positive controls. Therefore, it is speculated that RNA adsorption by non-proton-type zeolite is significantly low across any pH range.

[0118] [Experimental Example 4: Confirmation of the effective pH range for DNA adsorption onto various zeolites]

[0119] 480 mg of zeolite was added to 1000 μL of a solution containing Saccharomyces cerevisiae DNA in a nucleic acid extraction reagent (0.5% SDS) adjusted to pH 3–12. The mixture was inverted and mixed. The zeolite used was Tosoh Corporation's 720NHA and 690HOA models. Similar to Example 1, zeolite-treated samples 4-2–4-3, 4-5–4-6, 4-8–4-9, 4-11–4-12, 4-14–4-15, and 4-17–4-18 were obtained. Furthermore, the target nucleic acid was amplified using quantitative PCR on the zeolite-treated samples in the same manner as in Example 2. As a positive control, Saccharomyces cerevisiae DNA was added to the nucleic acid extraction reagent in the same manner as in Example 2, and untreated samples 4-1, 4-4, 4-7, 4-10, 4-13, and 4-16 were also prepared and amplified using quantitative PCR. The results are shown in Table 10. It should be noted that in Table 10, "copy number ratio" refers to the ratio when the copy number of "zeolite-free treatment" in the extract at each pH is set to 100%.

[0120] [Table 10]

[0121] The same results were obtained with DNA as with RNA. Samples treated with proton-type zeolite using nucleic acid extraction reagents adjusted to pH 3–7 showed extremely low copy numbers of the target nucleic acid compared to the untreated samples used as positive controls. This indicates that proton-type zeolite adsorbs DNA when using acidic to neutral nucleic acid extraction reagents. On the other hand, samples treated with non-proton-type zeolite all showed copy numbers comparable to the untreated samples used as positive controls. Therefore, it is speculated that DNA adsorption with non-proton-type zeolite is significantly low across any pH range.

[0122] [Experimental Example 5: Evaluation of DNA Purification Using Various Zeolites]

[0123] Add 200 μL of saliva sample to 800 μL of nucleic acid extraction reagent (0.5% SDS), mix, then add 240 mg of zeolite (described later), mix further, and pass through a 0.45 μm filter to remove the zeolite and adsorbed substances. Use the resulting solution as the sample for nucleic acid amplification (zeolite-treated samples 5-3 to 5-10). Add 11.5 μL of distilled water (DW) and 2 μL of the target nucleic acid solution (described later) to 11.5 μL of the zeolite-treated sample. Incubate using a Simprova (registered trademark) respiratory infection panel (manufactured by Eiken Chemical Co., Ltd.) at 64°C for 50 minutes, and amplify the target nucleic acid using the LAMP method. Then, measure the increase in fluorescence value with target nucleic acid amplification using a ThermalCycler Dice (registered trademark) real-time system III.

[0124] As a positive control, sample 5-1 was prepared by adding only distilled water to the nucleic acid extraction reagent without adding saliva sample and mixing. As a negative control, sample 5-2 (untreated zeolite sample) was prepared by adding saliva sample to the nucleic acid extraction reagent but without adding zeolite and mixing. The target nucleic acid was amplified using the LAMP method in the same way.

[0125] As the target nucleic acid solution, Bordetella pertussis (B. pertussis) DNA (2000 copies / test) was used. The zeolites used were Tosoh Corporation models 500KOA, 690HOA, 642NAA, 940NHA, 840NHA, 722HOA, 720NHA, or 720KOA. The results are shown in Table 11. It should be noted that in Table 11, those not detected within the assay time (50 minutes) are indicated as ND.

[0126] [Table 11]

[0127] When comparing samples 5-1 and 5-2, no amplification of the target nucleic acid was detected in sample 5-2. This indicates that the addition of the saliva sample inhibited the amplification of the target nucleic acid, meaning that substances other than nucleic acids (e.g., inhibitors of nucleic acid amplification) were present in the saliva sample. On the other hand, amplification of the target nucleic acid was confirmed in the samples treated with zeolite (samples 5-3 to 5-10), thus indicating that zeolite can remove substances other than nucleic acids from the saliva sample.

[0128] [Experiment 6: Evaluation of RNA purification using various zeolites]

[0129] Add 170 μL of saliva sample to 830 μL of nucleic acid extraction reagent (0.5% SDS) and mix. Then add 240 mg of zeolite and mix further. Pass the mixture through a 0.45 μm filter to remove the zeolite and any adsorbed substances. Use the resulting solution as the sample for nucleic acid amplification (zeolite-treated samples 6-3 to 6-10). Add 0.5 μL of target nucleic acid solution to 9.5 μL of zeolite-treated sample and react at 62.5 °C for 90 minutes using the Loopamp (registered trademark) Novel Coronavirus 2019 (SARS-CoV-2) Detection Kit (manufactured by Eiken Chemical Co., Ltd.) to amplify the target nucleic acid using the LAMP method. Then, use the Loopamp EXIA amplification unit (manufactured by Eiken Chemical Co., Ltd.) to measure the increase in turbidity value as the target nucleic acid amplifies in real time.

[0130] As the target nucleic acid solution, SARS-CoV-2 transcribed RNA (250 copies / test) was used. Zeolites used were Tosoh Corporation models 500KOA, 690HOA, 642NAA, 940NHA, 840NHA, 722HOA, 720NHA, or 720KOA. As a positive control, sample 6-1 was prepared by mixing the nucleic acid extraction reagent with only distilled water and no saliva sample, and the target nucleic acid was amplified using the same LAMP method. Additionally, sample 6-2 (untreated zeolite sample), which served as a negative control, was mixed with the nucleic acid extraction reagent with saliva sample but no zeolite, and the target nucleic acid was also amplified using the same LAMP method. The results are shown in Table 12.

[0131] [Table 12]

[0132] Similar to Example 5, when comparing samples 6-1 and 6-2, target nucleic acid amplification was detected in sample 6-1, but not in sample 6-2, indicating the presence of substances other than nucleic acids (e.g., substances inhibiting nucleic acid amplification) in the saliva samples. On the other hand, target nucleic acid amplification was confirmed in the samples treated with zeolite (samples 6-3 to 6-10), indicating that zeolite can remove substances other than nucleic acids from saliva samples.

[0133] [Experimental Example 7: Application of DNA purified using aprotic zeolite in PCR]

[0134] 200 μL of a solution obtained by adding Streptococcus pneumoniae (S. pneumoniae) to a saliva sample was mixed with 800 μL of nucleic acid extraction reagent (0.5% SDS), and 240 mg of zeolite (Tosoh Corporation, Model 720NHA) was added. The mixture was then inverted and mixed. The resulting suspension was passed through a 0.45 μm filter to remove the zeolite and any adsorbed substances, yielding sample 7-4. 5 μL of sample 7-4 and 20 μL of quantitative PCR reagent were used for amplification of the target nucleic acid using quantitative PCR. Additionally, samples 7-3 (prepared by mixing a solution obtained by adding Streptococcus pneumoniae to saliva with the nucleic acid extraction reagent without zeolite treatment), 7-1 (prepared by mixing a solution obtained by adding Streptococcus pneumoniae to physiological saline with the nucleic acid extraction reagent without zeolite treatment), and 7-2 (prepared by zeolite treatment) were also prepared and amplified using quantitative PCR in the same manner.

[0135] The amplification and detection of the target nucleic acid using quantitative PCR were performed as follows: Using the primer and probe sets shown in Table 13, the reaction was carried out in the quantitative PCR reaction reagent solution with the composition shown in Table 14, at the temperatures and times shown in Table 15. The fluorescence value increasing with the amplification of the target nucleic acid was measured in real time using a LightCycler 480 system. It should be noted that the probes with the base sequence shown in Serial Number 9 in Table 13 have FAM-labeled 5' ends and TAMRA-labeled 3' ends. The results are shown in Table 16. It should be noted that in Table 16, "Copy Number Ratio" indicates the ratio when the copy number of sample 7-1 is set to 100%.

[0136] [Table 13]

[0137] [Table 14]

[0138] [Table 15]

[0139] [Table 16]

[0140] The copy numbers of the target nucleic acid in samples 7-1 and 7-2 were equivalent. This result indicates that *Streptococcus pneumoniae* was extracted using the nucleic acid extraction reagent, and the *Streptococcus pneumoniae* nucleic acid (DNA) was not adsorbed onto the zeolite and could be detected. Comparing samples 7-3 and 7-4, sample 7-3, which was not treated with aprotic zeolite, had a lower detected copy number of the target nucleic acid compared to samples 7-1 to 7-2 without added saliva. On the other hand, sample 7-4, which was treated with aprotic zeolite, showed a target nucleic acid copy number equivalent to that of the sample without added saliva. This indicates that by using aprotic zeolite, substances other than nucleic acids (e.g., nucleic acid amplification inhibitors) contained in the saliva sample can be removed. In other words, it demonstrates that nucleic acids can be extracted and purified with high purity.

[0141] [Experimental Example 8: Application of RNA purified using aprotic zeolite in PCR]

[0142] 200 μL of a solution obtained by adding influenza A virus to different saliva samples 1 or 2 was mixed with 800 μL of nucleic acid extraction reagent (0.5% SDS), and 240 mg of zeolite (Tosoh Corporation, model 720NHA) was added. The mixture was then inverted and mixed. The inverted suspension was passed through a 0.45 μm filter to remove the zeolite and adsorbed substances, yielding samples 8-4 and 8-6. 5 μL of samples 8-4 and 8-6, along with 20 μL of quantitative PCR reagent, were used for amplification of the target nucleic acid using quantitative PCR. Simultaneously, samples 8-3 and 8-5 (without zeolite treatment), sample 8-1 (a solution obtained by mixing influenza A virus with nucleic acid extraction reagent in physiological saline without zeolite treatment), and sample 8-2 (with zeolite treatment) were also amplified using quantitative PCR in the same manner.

[0143] The amplification of the target nucleic acid using quantitative PCR was performed as follows: using the primer set shown in Table 17, the reaction was carried out in the quantitative PCR reaction reagent solution composed of the components shown in Table 2, at the temperature and time specified in Table 3. The fluorescence value as the target nucleic acid was amplified was measured in real time using a LightCycler 480 system. It should be noted that the probe composed of the base sequence shown in Serial No. 12 in Table 17 has a 5' end labeled with FAM and a 3' end labeled with MGB. The results are shown in Table 18. It should be noted that in Table 18, "Copy Number Ratio" indicates the ratio when the copy number of sample 8-1 is set to 100%.

[0144] [Table 17]

[0145] [Table 18]

[0146] The copy numbers of the target nucleic acid in samples 8-1 and 8-2 were identical, indicating that the influenza A virus was extracted using the nucleic acid extraction reagent and that the influenza A virus nucleic acid (RNA) was not adsorbed onto the zeolite and could be detected. Compared to samples 8-1 to 8-2 without saliva, the copy numbers of the target nucleic acid in samples 8-3 or 8-5, which were not treated with aprotic zeolite, were halved. On the other hand, samples 8-4 and 8-6, which were treated with aprotic zeolite, showed the same copy numbers of the target nucleic acid as the samples without saliva. This indicates that aprotic zeolite treatment can remove substances other than nucleic acids (e.g., nucleic acid amplification inhibitors) contained in the saliva sample. In other words, it demonstrates that nucleic acids can be purified to a high purity.

[0147] [Experiment 9: Purification of RNA using aprotic zeolite with other samples]

[0148] Nucleic acid extraction reagent (0.5% SDS) and various samples (urine, cerebrospinal fluid, nasal swab specimens, nasopharyngeal swab specimens, serum, whole blood, feces, sputum) were mixed separately. Then, 240 mg of zeolite (described later) was added and mixed further. The mixture was then passed through a 0.45 μm filter to remove the zeolite and any adsorbed substances. The resulting solution was used as the sample for nucleic acid amplification (zeolite-treated sample). 0.5 μL of target nucleic acid solution was added to 9.5 μL of the zeolite-treated sample. The mixture was reacted at 62.5 °C for 35 minutes using the Loopamp (registered trademark) Novel Coronavirus 2019 (SARS-CoV-2) Detection Kit (manufactured by Eiken Chemical Co., Ltd.). The increase in turbidity as the target nucleic acid was amplified was measured in real time using the Loopamp EXIA amplification unit (manufactured by Eiken Chemical Co., Ltd.).

[0149] SARS-CoV-2 transcribed RNA (250 copies / test) was used as the target nucleic acid solution. The zeolite used was Tosoh Corporation's 690HOA and 720NHA. As a positive control, samples were prepared by adding only distilled water to the nucleic acid extraction reagent without adding saliva samples, and the target nucleic acid was amplified using the LAMP method in the same way. Additionally, samples used as negative controls, obtained by adding saliva samples to the nucleic acid extraction reagent but without adding zeolite (zeolite-untreated samples), were also amplified using the LAMP method in the same way. The results are shown in Tables 19–26.

[0150] [Table 19]

[0151] [Table 20]

[0152] [Table 21]

[0153] [Table 22]

[0154] [Table 23]

[0155] [Table 24]

[0156] [Table 25]

[0157] [Table 26]

[0158] In the absence of zeolite-treated samples, target nucleic acids were not detected or the detection time was significantly delayed compared to samples without zeolite treatment. This is believed to be due to the failure to remove substances other than nucleic acids contained in various samples. On the other hand, in the presence of zeolite-treated samples, target nucleic acids could be detected. This is believed to be due to the ability of zeolite to remove substances other than nucleic acids contained in various samples. Regarding urine, nasal swab specimens, nasopharyngeal swab specimens, serum, and whole blood samples, it was confirmed that non-proton zeolite was more effective than proton zeolite.

[0159] [Experimental Example 10: Evaluation of Nucleic Acid Purification Based on the Type of Surfactant in Nucleic Acid Extraction Reagents]

[0160] 890 μL of nucleic acid extraction reagent containing the various surfactants shown in Table 27 was mixed with 110 μL of saliva sample. Then, 240 mg of Tosoh Corporation's 720NHA zeolite was added, and the mixture was further mixed. The mixture was then passed through a 0.45 μm filter to remove the zeolite and the substances adsorbed on it, yielding samples 10⁻²⁰ to 10⁻²⁸. These samples 10⁻²⁰ to 10⁻²⁸ were used as samples for nucleic acid amplification (non-proton zeolite-treated samples). 0.5 μL of target nucleic acid solution was added to 9.5 μL of the zeolite-treated sample, and the mixture was reacted at 62.5 °C for 35 minutes using the Loopamp (registered trademark) Novel Coronavirus 2019 (SARS-CoV-2) Detection Kit (manufactured by Eiken Chemical Co., Ltd.). The turbidity value, increasing with the amplification of the target nucleic acid, was measured in real time using the Loopamp EXIA amplification unit (manufactured by Eiken Chemical Co., Ltd.).

[0161] As the target nucleic acid solution, SARS-CoV-2 transcribed RNA (250 copies / test) was used. As positive controls, samples 10⁻¹ to 10⁻¹⁰, obtained by mixing various nucleic acid extraction reagents with distilled water but without saliva samples, were also prepared and amplified using the LAMP method. Additionally, samples 10⁻¹¹ to 10⁻¹⁹ (untreated zeolite samples), obtained as negative controls by mixing nucleic acid extraction reagents with saliva samples but without zeolite, were also amplified using the LAMP method. The results are shown in Table 27.

[0162] [Table 27]

[0163] Samples 10⁻¹¹ to 10⁻¹⁹ (untreated samples) containing only saliva in the nucleic acid extraction reagent failed to detect the target nucleic acid. On the other hand, samples 10⁻²⁰ to 10⁻²⁸ treated with aprotic zeolite using a nucleic acid extraction reagent containing any surfactant showed detectable amplification of the target nucleic acid. This indicates that using a nucleic acid extraction reagent containing any surfactant, aprotic zeolite can effectively remove substances other than nucleic acids (e.g., nucleic acid amplification inhibitors) from saliva samples.

Claims

1. A method for purifying nucleic acids, comprising the step of contacting a sample containing nucleic acids with an aprotic zeolite.

2. The purification method according to claim 1, wherein, The sample is derived from at least one sample selected from the group consisting of blood, cerebrospinal fluid, urine, feces, sputum, saliva, nasal mucus, swab specimens, amniotic fluid, and mouthwash.

3. The purification method according to claim 1 or 2, wherein, During the contact step, at least a portion of the nucleic acid is not adsorbed by aprotic zeolite.

4. The purification method according to claim 1 or 2, wherein, The aprotic zeolite is selected from at least one of the following groups: magnesium-alkali zeolite, ZSM-5 zeolite, Y-type zeolite, and β-type zeolite.

5. The purification method according to claim 4, wherein, The aprotic zeolite is selected from at least one group consisting of magnesium-alkali zeolite-NH4 type zeolite, magnesium-alkali zeolite-K type zeolite, ZSM-5-NH4 type zeolite, Y-Na type zeolite and β-NH4 type zeolite.

6. The purification method according to claim 2, wherein, It also includes the step of obtaining the nucleic acid-containing sample by mixing the sample with a nucleic acid extraction reagent containing a surfactant and / or by heating the sample prior to the contact step.

7. The purification method according to claim 6, wherein, The surfactant contains anionic surfactants.

8. The purification method according to claim 7, wherein, The anionic surfactant is dodecyl sulfate.

9. The purification method according to claim 1 or 2, wherein, The nucleic acid is RNA and / or DNA.

10. A kit for purifying nucleic acids, comprising aprotic zeolite.

11. The kit according to claim 10, wherein, The aprotic zeolite is selected from at least one of the following groups: magnesium-alkali zeolite, ZSM-5 zeolite, Y-type zeolite, and β-type zeolite.

12. The kit according to claim 11, wherein, The aprotic zeolite is selected from at least one group consisting of magnesium-alkali zeolite-NH4 type zeolite, magnesium-alkali zeolite-K type zeolite, ZSM-5-NH4 type zeolite, Y-Na type zeolite and β-NH4 type zeolite.

13. The kit according to claim 10, wherein, The kit is for purifying nucleic acids from at least one sample selected from the group consisting of blood, cerebrospinal fluid, urine, feces, sputum, saliva, nasal mucus, swab specimens, amniotic fluid, and mouthwash. The kit also includes a nucleic acid extraction reagent containing a surfactant.

14. The kit according to claim 13, wherein, The surfactant contains anionic surfactants.

15. The kit according to claim 14, wherein, The anionic surfactant is dodecyl sulfate.

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