Nucleic acid purification method and kit for nucleic acid purification

Abstract

The present invention addresses the problem of providing a method for purifying a nucleic acid easily, in a short time and with a high level of purity, from a sample containing said nucleic acid, without using a proton-type zeolite, and of providing 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 a zeolite other than a proton-type zeolite.

Description

Nucleic acid purification method and kit for nucleic acid purification

[0001] The present invention relates to a method for purifying nucleic acids and a kit for purifying nucleic acids.

[0002] Currently, nucleic acid amplification techniques, such as PCR and LAMP, have penetrated all fields of biology, including molecular biology and medicine, and are widely used in, for example, genetic diagnosis, DNA testing, food testing, environmental hygiene testing, and animal and plant testing. When amplifying nucleic acids in a sample, it is usually necessary to extract and purify the nucleic acid from the sample. Examples of methods for extracting and purifying nucleic acids from a sample include methods using commercially available nucleic acid extraction kits. On the other hand, when using a typical commercially available nucleic acid extraction kit (e.g., QIAamp Viral RNA Mini Kit (manufactured by Qiagen)), highly pure nucleic acids can be obtained, but multiple devices such as a high-speed centrifuge and a heater are required, making it difficult to use the kit unless the operating equipment is well-equipped. In addition, the operation is complicated, requiring several tens of steps, and it takes approximately one hour to extract and purify nucleic acids (e.g., Non-Patent Documents 1 and 2). In nucleic acid amplification technology, when the number of test samples is large, it is desirable to prepare samples that can be used for nucleic acid amplification in a short time, so extraction and purification methods that require complicated operations are difficult to apply. Furthermore, when using simpler commercially available nucleic acid extraction kits (e.g., Kaneka Simple DNA Extraction Kit (manufactured by Kaneka Corporation), Template Preparer for DNA (manufactured by Nippon Gene Co., Ltd.), ISOSPIN Viral RNA (manufactured by Nippon Gene Co., Ltd.)), nucleic acids can be extracted and purified in a short time, but the purity of the nucleic acids may be low. Furthermore, even when using such kits, heating operations may be required or the operation steps may involve several tens of steps, making it difficult to say that nucleic acids can be extracted and purified easily and with high purity in a short time. For these reasons, there is a demand in the market for a method for extracting and purifying nucleic acids easily and with high purity in a short time without requiring large equipment.

[0003] Also known are methods for extracting and purifying nucleic acids from cell lysates using zeolites. For example, Patent Document 1 discloses a method for preparing a sample for nucleic acid amplification to be used for amplifying nucleic acids contained in a biological sample, comprising the steps of adding a nucleic acid extraction reagent containing an anionic surfactant and / or alkali to a biological sample to obtain a nucleic acid extract, and contacting the nucleic acid extract with zeolite, where the zeolite is a proton-type zeolite and substances adsorbed to the zeolite are removed. Patent Document 2 discloses a method for isolating nucleic acids from a biological solution (e.g., a cell lysate) by pre-treating the clarified lysate with zeolite, which describes that adding zeolite to a cell lysate treated with an alkaline reagent such as sodium dodecyl sulfate (SDS) adsorbs SDS and the like, and then centrifuging the cell lysate to clarify it.

[0004] Patent No. 5290987 Publication Special Publication No. 2005-531329

[0005] 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; Qiagen, Inc., "QIAamp (registered trademark) Viral RNA Mini Protocol and Troubleshooting," April 2010, https: / / www.qiagen.com / jp / resources / download.aspx?id=7d6918b1-77dd-4dac-b694-a9ca8aa58b43&lang=ja-JP

[0006] The present inventors have discovered that there are zeolites to which nucleic acids are adsorbed depending on the components of a sample containing nucleic acids or the purification conditions, and that these zeolites are proton-type zeolites.

[0007] An object of the present invention is to provide a method for purifying nucleic acids from a sample containing nucleic acids simply and quickly with high purity without using proton-type zeolite, and a kit for such purification.

[0008] The present inventors unexpectedly found that when a sample containing nucleic acids is mixed with aprotic zeolite, the aprotic zeolite exhibits extremely low adsorption of the nucleic acids in the sample, while exhibiting high adsorption of substances other than nucleic acids, which led to the completion of the present invention.

[0009] That is, the present invention relates to, for example, the following inventions. [1] A method for purifying nucleic acid, comprising a step of contacting a sample containing nucleic acid with an aprotic zeolite. [2] The purification method according to [1], wherein the sample is derived from at least one specimen selected from the group consisting of blood, cerebrospinal fluid, urine, feces, sputum, saliva, nasal discharge, swab, amniotic fluid, and gargle. [3] The purification method according to [1] or [2], wherein at least a portion of the nucleic acid is not adsorbed to the aprotic zeolite in the contacting step. [4] The purification method according to any of [1] to [3], wherein the aprotic zeolite is at least one selected from the group consisting of ferrierite-type zeolite, ZSM-5-type zeolite, Y-type zeolite, and beta-type zeolite. [5] The aprotic zeolite is ferrierite-NH ４ type zeolite, ferrierite-K type zeolite, ZSM-5-NH ４ type zeolite, Y-Na type zeolite and beta-NH ４[6] The purification method according to any one of [1] to [5], further comprising a step of obtaining a sample containing the nucleic acid by mixing the specimen with a nucleic acid extraction reagent containing a surfactant and / or by heat-treating the specimen before the contacting step. [7] The purification method according to [6], wherein the surfactant contains an anionic surfactant. [8] The purification method according to [7], wherein the anionic surfactant is dodecyl sulfate. [9] The purification method according to any one of [1] to [8], wherein the nucleic acid is RNA and / or DNA.

[10] A kit for purifying nucleic acid, comprising an aprotic zeolite.

[11] The kit according to

[10] , wherein the aprotic zeolite is at least one selected from the group consisting of ferrierite-type zeolite, ZSM-5-type zeolite, Y-type zeolite, and beta-type zeolite.

[12] The aprotic zeolite is ferrierite-NH ４ type zeolite, ferrierite-K type zeolite, ZSM-5-NH ４ type zeolite, Y-Na type zeolite and beta-NH ４

[13] The kit according to any one of

[10] to

[12] , which is a kit for purifying nucleic acid from at least one specimen selected from the group consisting of blood, cerebrospinal fluid, urine, feces, sputum, saliva, nasal discharge, swabs, amniotic fluid, and gargle, and further comprises a nucleic acid extraction reagent containing a surfactant.

[14] The kit according to

[13] , wherein the surfactant contains an anionic surfactant.

[15] The kit according to

[14] , wherein the anionic surfactant is dodecyl sulfate.

[0010] According to the present invention, it is possible to provide a method for extracting and purifying nucleic acids easily, quickly, and with high purity by efficiently removing substances other than nucleic acids from a sample containing nucleic acids, and a kit for said extraction and purification. In particular, according to the present invention, it is possible to extract and purify nucleic acids easily and with high purity from a sample containing nucleic acids in a short time without using multiple devices such as a high-speed centrifuge and a heater. Furthermore, since aprotic zeolite has extremely low adsorption of nucleic acids in a sample, it is expected that the present invention will extract and purify nucleic acids with high yields. Furthermore, the present invention is highly versatile because it can be applied to the purification of either DNA or RNA, or both.

[0011] Furthermore, as described above, the present invention enables nucleic acids to be purified simply and quickly with high purity, thereby making it possible to obtain an effective sample to be subjected to a nucleic acid amplification reaction, and enabling highly sensitive measurements using nucleic acid amplification techniques.

[0012] Hereinafter, embodiments of the present invention will be described in detail, but the present invention is not limited to the following embodiments.

[0013] [Nucleic Acid Purification Method] The nucleic acid purification method according to this embodiment includes a step of contacting a sample containing nucleic acid with aprotic zeolite (contact step). As shown in the examples below, aprotic zeolite does not adsorb at least a portion of the nucleic acids in the sample containing nucleic acid, while adsorbing substances other than nucleic acids. Therefore, by contacting a sample containing nucleic acid with aprotic zeolite, substances other than nucleic acids can be removed from the sample containing nucleic acid, and nucleic acids can be purified easily, quickly, and with high purity.

[0014] <Sample containing nucleic acid> A sample containing nucleic acid is an aqueous solution or suspension in which nucleic acids and substances other than nucleic acids are dissolved or suspended. The type of nucleic acid contained in the sample is not particularly limited, and may be DNA or RNA, and may be single-stranded or double-stranded. The origin of the nucleic acid is also not limited, and may be, for example, nucleic acid derived from animals, plants, fungi, bacteria, or viruses. The method of the present invention can particularly purify RNA easily, quickly, and with high purity. The pH of the sample containing nucleic acid is not particularly limited.

[0015] Substances other than nucleic acids include proteins (for example, enzymes, glycoproteins, polypeptides, etc.), lipids, sugars (for example, monosaccharides, disaccharides, oligosaccharides, polysaccharides, etc.), salts, and the like.

[0016] Furthermore, the sample containing nucleic acid may be derived from, for example, a specimen obtained from a living organism, or may be derived from a specimen obtained from the environment, such as water, soil, or air. The sample containing nucleic acid is preferably derived from a specimen obtained from a living organism, and more preferably from at least one specimen selected from the group consisting of blood (whole blood, plasma, or serum), cerebrospinal fluid, urine, feces, sputum, saliva, nasal discharge, swab, amniotic fluid, and gargle. The sample containing nucleic acid may be a dilution / suspension of the above specimen or an extract obtained by the extraction step described below. The substance other than nucleic acid may be a component other than nucleic acid derived from these specimens.

[0017] The sample containing nucleic acid may contain a nucleic acid extraction reagent containing a surfactant as described below, and may also contain an inhibitor of a substance that degrades nucleic acid, if necessary. Examples of such inhibitors include nuclease inhibitors.

[0018] <Zeolite> "Zeolite" is a general term for aluminosilicates that have micropores in their crystals, and is a type of SiO ４ and AlO ４ The basic skeleton of this zeolite is a three-dimensional network structure in which regular tetrahedra of the above structure are bonded by sharing oxygen. Zeolites contain cations in their crystals, such as alkali metal ions, alkaline earth metal ions, ammonium ions, and hydrogen ions (protons). A "proton zeolite" refers to a zeolite that contains hydrogen ions as cations in its crystals, and a "non-proton zeolite" refers to a zeolite other than a proton zeolite. The non-proton zeolite in the purification method according to this embodiment does not adsorb at least a portion of the nucleic acids in a sample containing nucleic acids.

[0019] The crystal structure of the aprotic zeolite is not particularly limited, and examples thereof include A-type, ferrierite-type, MCM-22-type, ZSM-5-type, mordenite-type, L-type, Y-type, X-type, beta-type, etc. The aprotic zeolite is preferably at least one selected from the group consisting of ferrierite-type zeolite, ZSM-5-type zeolite, Y-type zeolite, and beta-type zeolite.

[0020] The cations in the crystals of aprotic zeolite are not particularly limited as long as they are other than hydrogen ions. Examples of such cations include alkali metal ions (e.g., sodium ions (Na ＋ ), potassium ions (K ＋ ) and the like), alkaline earth metal ions (e.g., magnesium ions (Mg ２＋ ), calcium ions (Ca ＋ ) etc.), ammonium ion (NH ４ ＋ ) etc.

[0021] The aprotic zeolite is particularly ferrierite-NH ４ type zeolite, ferrierite-K type zeolite, ZSM-5-NH ４ type zeolite, Y-Na type zeolite and beta-NH ４ It is preferable that the zeolite be at least one selected from the group consisting of ferrierite-NH ４ The term "ferrierite-type zeolite" refers to a zeolite having a ferrierite-type crystal structure and containing ammonium ions as cations.

[0022] <Contacting Step> In the contacting step, a sample containing nucleic acid is contacted with aprotic zeolite to adsorb substances other than nucleic acid. Therefore, in the purification method according to this embodiment, nucleic acid can be purified from a sample containing nucleic acid by the contacting step. In this specification, "purification" means increasing the purity of nucleic acid from a sample containing nucleic acid.

[0023] In the contacting step, "contacting" may mean mixing a sample containing nucleic acid with aprotic zeolite; for example, the sample containing nucleic acid may be added to a container containing aprotic zeolite, or the aprotic zeolite may be added to the sample containing nucleic acid; the order in which the sample containing nucleic acid and the aprotic zeolite are contacted is not particularly limited.

[0024] The amount of zeolite in the contacting 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 per mL of the sample containing nucleic acid. The amount of zeolite in the contacting step may be, for example, 350 mg or less, 340 mg or less, 330 mg or less, 320 mg or less, 310 mg or less, or 300 mg or less per mL of the sample containing nucleic acid.

[0025] The contact time in the contact step is not particularly limited, and may be, for example, 10 seconds to 10 minutes, 20 seconds to 10 minutes, or 30 seconds to 5 minutes.

[0026] The contacting step causes at least a portion of substances other than nucleic acids in a sample containing nucleic acids to be adsorbed to the aprotic zeolite. "Adsorption" refers to a state in which a substance is bound to or encapsulated on the surface or in the pores of the aprotic zeolite, and the adsorbed substances are removed together with the aprotic zeolite when the aprotic zeolite is removed. The contacting step causes at least 30% or more, at least 40% or more, at least 50% or more, at least 60% or more, at least 70% or more, at least 80% or more, or at least 90% or more of the substances other than nucleic acids in a sample containing nucleic acids to be adsorbed to the aprotic zeolite. Furthermore, "at least a portion of the nucleic acid is not adsorbed to the aprotic zeolite" may mean, for example, that when a sample containing only nucleic acid and a sample obtained after contacting the sample containing only nucleic acid with the aprotic zeolite are each subjected to nucleic acid amplification under the same conditions, the difference in the detected copy number of the nucleic acid may be 25% or less, 20% or less, 15% or less, 10% or less, 5% or less, or 1% or less, or preferably 0%, based on the copy number of the former, or may be equal to or greater than the copy number of the nucleic acid detected when nucleic acid amplification is performed using a sample containing only nucleic acid.

[0027] <Extraction Step> The purification method according to this embodiment may include a step (extraction step) of obtaining a sample containing the nucleic acid by mixing the specimen with a nucleic acid extraction reagent containing a surfactant and / or by heat-treating the specimen before the contact step. Cell membranes, viral envelopes, etc. in the specimen are destroyed by the surfactant or heat treatment, allowing the nucleic acid to be dissolved or suspended in the nucleic acid-containing sample. When both mixing the specimen with the nucleic acid extraction reagent containing a surfactant and heat-treating the specimen are performed, the order of these steps is not particularly limited. When the purification method according to this embodiment includes an extraction step, the purification method according to this embodiment can also be considered an extraction method.

[0028] The surfactant is not particularly limited and may be an ionic surfactant (anionic surfactant, cationic surfactant), a nonionic surfactant, or an amphoteric surfactant, but an ionic surfactant is preferred, and an anionic surfactant is more preferred, from the viewpoint of easier destruction of cell membranes, viral envelopes, etc. The nucleic acid extraction reagent may contain one type of surfactant or two or more types of surfactants.

[0029] Examples of anionic surfactants include higher fatty acid salts, linear alkylbenzene sulfonates, α-sulfofatty acid methyl ester salts, α-olefin sulfonates, alkyl sulfate salts, and polyoxyethylene alkyl sulfate salts. Alkyl sulfate salts are preferred, and dodecyl sulfates such as lithium dodecyl sulfate (LDS) and sodium dodecyl sulfate (SDS), or sodium cholate are more preferred.

[0030] Examples of cationic surfactants include alkyltrimethylammonium salts, dialkyldimethylammonium salts, alkyldimethylbenzylammonium salts, and benzyltrimethylammonium salts, and may also be benzalkonium chloride.

[0031] 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, polyoxyalkylphenyl ethers, and the like, and may also include TRITON (registered trademark)-X100, Tween (registered trademark) 40, and the like.

[0032] Examples of amphoteric surfactants include alkylamino fatty acid salts, alkylbetaines, and alkylamine oxides, and may include 3-[(3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate (CHAPSO) and 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS).

[0033] The surfactant content in the nucleic acid extraction reagent may be a normal content sufficient for extracting nucleic acids, and may vary depending on the type of surfactant. For example, it may be 0.01 w / v% or more, 0.1 w / v% or more, 0.5 w / v% or more, or 1 w / v% or more, based on the total amount of the nucleic acid extraction reagent. The surfactant content in the nucleic acid extraction reagent may be, for example, 5 w / v% or less, 4 w / v% or less, or 3 w / v% or less, based on the total amount of the nucleic acid extraction reagent. Note that when multiple types of surfactants are contained in the nucleic acid extraction reagent, the surfactant content in the nucleic acid extraction reagent is the total content of the multiple types of surfactants.

[0034] The nucleic acid extraction reagent may contain an alkali in addition to the surfactant. Examples of alkali include sodium hydroxide, potassium hydroxide, lithium hydroxide, calcium hydroxide, and aqueous ammonia, with sodium hydroxide being preferred. The nucleic acid extraction reagent may contain one type of alkali or two or more types.

[0035] When the alkali is sodium hydroxide, the content of alkali in the nucleic acid extraction reagent may be, for example, 10 mM or more, 25 mM or more, 50 mM or more, 75 mM or more, 100 mM or more, 125 mM or more, 150 mM or more, 175 mM or more, or 200 mM or more, based on the total amount of the nucleic acid extraction reagent. When the alkali is sodium hydroxide, the content of alkali in the nucleic acid-containing reagent may be, for example, 500 mM or less, 480 mM or less, 460 mM or less, 440 mM or less, 420 mM or less, or 400 mM or less, based on the total amount of the nucleic acid extraction reagent.

[0036] The pH of the nucleic acid extraction reagent is not particularly limited as long as it is a normal pH that does not decompose nucleic acids, but may be, for example, 5-9, 5-7, or 6-7.

[0037] The heat treatment is not particularly limited as long as it can extract nucleic acids from a specimen and dissolve or suspend the nucleic acids in a sample containing the nucleic acids, and may be, for example, heat treatment of the specimen using a heat block, etc. The temperature for the heat treatment may be, for example, 50°C to 200°C, 60°C to 150°C, or 70°C to 100°C. The time for the heat treatment may be, for example, 1 minute to 30 minutes, 5 minutes to 30 minutes, or 15 minutes to 30 minutes.

[0038] <Removal Step> The purification method according to this embodiment may include a step of removing the aprotic zeolite (removal step) after the contact step. In the removal step, the aprotic zeolite is removed to obtain a sample containing purified nucleic acids.

[0039] The aprotic zeolite can be removed, for example, by subjecting the mixture of the nucleic acid-containing sample and the aprotic zeolite after the contact step to centrifugation, filtration, or a combination thereof. Removal by centrifugation can be easily carried out, for example, by using a commercially available spin column.

[0040] The sample containing the purified nucleic acid obtained in the removal step can be used, for example, in nucleic acid amplification analysis.

[0041] [Nucleic Acid Amplification Method] The nucleic acid amplification method according to this embodiment comprises a step of purifying nucleic acid by carrying out the purification method according to this embodiment, and a step of performing a nucleic acid amplification reaction on the purified nucleic acid (nucleic acid amplification step).

[0042] <Nucleic Acid Amplification Step> The nucleic acid amplification step can be carried out according to a conventional method. More specifically, the nucleic acid amplification step can be carried out by incubating a reaction solution containing a purified nucleic acid, a primer set containing a base sequence specific to the nucleic acid, a nucleic acid synthesizing enzyme such as DNA polymerase, deoxynucleoside triphosphates (dNTPs: dATP, dTTP, dCTP, and dGTP), if necessary, a reverse transcriptase, a labeled probe for detecting the amplification product, and the like.

[0043] When the nucleic acid is DNA, the nucleic acid amplification reaction may be, for example, a polymerase chain reaction (PCR), a loop-mediated isothermal amplification (LAMP) reaction, a nicking endonuclease amplification reaction (NEAR), a transcription-reverse transcription concerted (TRC) reaction, a whole genome amplification (WGA) reaction, a strand displacement amplification (SDA) reaction, a helicase-dependent amplification (HDA) reaction, a recombinase polymerase amplification (RPA) reaction, or an isothermal chimeric primer-initiated amplification (ICAN) reaction; when the nucleic acid is RNA, the nucleic acid amplification reaction may be, for example, a reverse transcription PCR (RT-PCR), a reverse transcription LAMP (RT-LAMP) reaction, a transcription-mediated amplification (TMA) reaction, or a nucleic acid sequence-based amplification (NASBA) reaction.

[0044] [Kit for purifying nucleic acid] A kit for purifying nucleic acid according to this embodiment (hereinafter also referred to as "purification kit according to this embodiment") includes an aprotic zeolite. By using the purification kit according to this embodiment, nucleic acid can be purified from a sample containing nucleic acid. The sample containing nucleic acid and the aprotic zeolite are as described above.

[0045] When the nucleic acid-containing sample used in the purification kit according to this embodiment is derived from a specimen, the kit may include a nucleic acid extraction reagent containing a surfactant. The nucleic acid extraction reagent containing a surfactant is as described above.

[0046] The purification kit according to this embodiment may include, in addition to the nucleic acid extraction reagent containing aprotic zeolite and a surfactant, a buffer solution, a nuclease inhibitor, a device used for purifying nucleic acids (e.g., a spin column, etc.), and the like.

[0047] [Kit for amplifying nucleic acid] The kit for amplifying nucleic acid according to this embodiment (hereinafter also referred to as "nucleic acid amplification kit according to this embodiment") comprises aprotic zeolite, DNA polymerase, and deoxynucleoside triphosphates.

[0048] The nucleic acid amplification kit according to this embodiment may contain known reagents for carrying out various nucleic acid amplification reactions in addition to those contained in the purification kit according to this embodiment. When the target nucleic acid is RNA, the nucleic acid amplification kit according to this embodiment may further contain a reverse transcriptase. Examples of other known reagents for carrying out various nucleic acid amplification reactions include a buffer solution that provides conditions suitable for the enzymatic reaction, an enzyme such as dithiothreitol (DTT), a protective agent that stabilizes the target nucleic acid sequence, a labeled probe for detecting the amplification product, an intercalator, etc. The nucleic acid amplification kit according to this embodiment may also contain a device used to detect nucleic acid amplification.

[0049] The present invention will be described in more detail below with reference to examples, although the present invention is not limited to these examples.

[0050] Test Example 1: Confirmation of RNA adsorption to various zeolites To 1,000 μL of a solution prepared by adding SARS-CoV-2 transcript RNA to a nucleic acid extraction reagent (0.5% sodium dodecyl sulfate (hereinafter also referred to as "SDS")), 480 mg of the zeolite described below was added and mixed by inversion. The zeolite used was 930NHA (beta-NH) manufactured by Tosoh Corporation. ４ type), 940NHA (beta-NH ４ type), 840NHA (ZSM-5-NH ４ type), 720NHA (ferrierite-NH ４The suspensions were zeolite-treated samples 1-1 to 1-8. The suspensions were then passed through a 0.45 μm filter to remove zeolite and any substances adsorbed thereto. The resulting filtrates were designated zeolite-treated samples 1-1 to 1-8. Quantitative PCR amplification of the target nucleic acid (cDNA of SARS-CoV-2 transcript RNA) was performed using 5 μL of the zeolite-treated sample and 20 μL of quantitative PCR reaction reagent. Additionally, as a positive control, sample 1-9 was prepared by adding SARS-CoV-2 transcript RNA to a nucleic acid extraction reagent (0.5% SDS) and then not treating it with zeolite. The target nucleic acid was similarly amplified by quantitative PCR.

[0051] Amplification and detection of target nucleic acids by quantitative PCR were carried out using the primer and probe sets shown in Table 1, in a quantitative PCR reaction reagent solution having the composition shown in Table 2, at the temperature and time shown in Table 3. The fluorescence intensity, which increased with the amplification of the target nucleic acid, was measured in real time using a Thermal Cycler Dice® Real Time System III. The probe consisting of the base sequence shown in SEQ ID NO: 3 in Table 1 was labeled with FAM at the 5' end and BHQ1 at the 3' end. The results are shown in Table 4. In Table 4, samples for which no Ct value was detected within the measurement time (40 cycles) were indicated as N.D. In Table 4, the "copy number ratio" indicates the ratio when the copy number for "without zeolite treatment" is set to 100%.

[0052]

[0053]

[0054]

[0055]

[0056] In the samples treated with proton zeolite (samples 1-6 and 1-8), no amplification of the target nucleic acid was detected. This suggests that proton zeolite adsorbs nucleic acids, particularly RNA. On the other hand, in the samples treated with non-proton zeolite (samples 1-1 to 1-5, 1-7), amplification of the target nucleic acid was confirmed, similar to the sample without zeolite treatment (sample 1-9), and the copy number was comparable. This suggests that non-proton zeolite has significantly low adsorption of nucleic acids, particularly RNA.

[0057] Test Example 2: Confirmation of DNA adsorption to various zeolites Zeolite-treated samples 2-1 to 2-9 were obtained in the same manner as in Test Example 1, except that Saccharomyces cerevisiae (hereinafter also referred to as "S. cerevisiae") DNA was added to the nucleic acid extraction reagent (0.5% SDS). Furthermore, for samples 2-1 to 2-9, amplification of the target nucleic acid (S. cerevisiae DNA) by quantitative PCR was performed in the same manner as in Test Example 1, except that 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 were used for the reactions. Furthermore, as a positive control, sample 2-10 was also prepared by adding S. cerevisiae DNA to the nucleic acid extraction reagent, but was not subsequently treated with zeolite. The target nucleic acid was similarly amplified by quantitative PCR. The probe consisting of the base sequence shown in SEQ ID NO: 6 in Table 5 is labeled at the 5' end with FAM and at the 3' end with BHQ1. The results are shown in Table 8. In Table 8, samples for which no Ct value was detected within the measurement time (40 cycles) are indicated as N.D. In Table 8, the "copy number ratio" indicates the ratio when the copy number for "without zeolite treatment" is set to 100%.

[0058]

[0059]

[0060]

[0061]

[0062] Similar results were obtained with DNA as with RNA. In the samples treated with proton-type zeolite (samples 2-6 to 2-7 and 2-9), no amplification of the target nucleic acid was detected, or even if it was detected, the copy number was extremely low. This suggests that proton-type zeolite adsorbs nucleic acids, particularly DNA. On the other hand, in the samples treated with non-proton-type zeolite (samples 2-1 to 2-5, 2-8), amplification of the target nucleic acid was confirmed, similar to the sample without zeolite treatment (sample 2-10), and showed a similar copy number. This suggests that non-proton-type zeolite has significantly low adsorption of nucleic acids, particularly DNA.

[0063] Test Example 3: Confirmation of the Effective pH Range for RNA Adsorption to Various Zeolites Zeolite-treated samples 3-2 to 3-3, 3-5 to 3-6, 3-8 to 3-9, 3-11 to 3-12, 3-14 to 3-15, and 3-17 to 3-18 were obtained in the same manner as in Test Example 1, except that 480 mg of zeolite was added to 1,000 μL of a solution prepared by adding SARS-CoV-2 transcript RNA to a nucleic acid extraction reagent (0.5% SDS) adjusted to a pH of 3 to 12, and the solution was mixed by inversion, and the zeolites used were Tosoh Corporation's model numbers 720NHA and 690HOA. Furthermore, in the same manner as in Test Example 1, the target nucleic acid (cDNA of SARS-CoV-2 transcript RNA) was amplified by quantitative PCR for the zeolite-treated samples. Additionally, as in Test Example 1, samples 3-1, 3-4, 3-7, 3-10, 3-13, and 3-16 were also prepared as positive controls in which SARS-CoV-2 transcript RNA was added to a nucleic acid extraction reagent and then not treated with zeolite, and the target nucleic acid was amplified by quantitative PCR in the same manner as in Test Example 1. The results are shown in Table 9. In Table 9, the "copy number ratio" indicates the ratio when the copy number in the extract "without zeolite treatment" at each pH is set to 100%.

[0064]

[0065] As shown in Table 9, samples treated with protonated zeolite using a nucleic acid extraction reagent adjusted to pH 3-7 showed significantly lower copy numbers of target nucleic acids than samples without zeolite treatment (positive control). This suggests that protonated zeolite adsorbs RNA when an acidic to neutral nucleic acid extraction reagent is used. On the other hand, all samples treated with non-protonated zeolite showed copy numbers similar to those of the positive control sample without zeolite treatment. This suggests that non-protonated zeolite has significantly lower RNA adsorption over the entire pH range.

[0066] [Test Example 4: Confirmation of the Effective pH Range for DNA Adsorption to Various Zeolites] 480 mg of zeolite was added to 1,000 μL of a solution prepared by adding S. cerevisiae DNA to a nucleic acid extraction reagent (0.5% SDS) adjusted to a pH of 3 to 12, and the solution was mixed by inversion. Zeolite-treated samples 4-2 to 4-3, 4-5 to 4-6, 4-8 to 4-9, 4-11 to 4-12, 4-14 to 4-15, and 4-17 to 4-18 were obtained in the same manner as in Test Example 1, except that the zeolite used was Tosoh Corporation's model numbers 720NHA and 690HOA. Furthermore, the target nucleic acid was amplified by quantitative PCR for the above zeolite-treated samples in the same manner as in Test Example 2. Furthermore, as a positive control, S. cerevisiae was added to the nucleic acid extraction reagent in the same manner as in Test Example 2. Samples 4-1, 4-4, 4-7, 4-10, 4-13, and 4-16 were also prepared, to which S. cerevisiae DNA had been added and then not treated with zeolite, and the target nucleic acid was similarly amplified by quantitative PCR. The results are shown in Table 10. In Table 10, the "copy number ratio" indicates the ratio when the copy number in the extract "without zeolite treatment" at each pH is set to 100%.

[0067]

[0068] In the case of DNA, similar results to those in the case of RNA were obtained. Samples treated with protonated zeolite using a nucleic acid extraction reagent adjusted to pH 3-7 showed extremely low copy numbers of target nucleic acids compared to the positive control sample without zeolite treatment. This suggests that protonated zeolite adsorbs DNA when an acidic to neutral nucleic acid extraction reagent is used. On the other hand, all samples treated with aprotonated zeolite showed copy numbers similar to those of the positive control sample without zeolite treatment. This suggests that aprotonated zeolite has significantly lower adsorption of DNA over the entire pH range.

[0069] Test Example 5: Evaluation of DNA Purification Using Various Zeolites. 200 μL of saliva sample was added to 800 μL of nucleic acid extraction reagent (0.5% SDS) and mixed. 240 mg of the zeolite described below was added and further mixed. The mixture was then passed through a 0.45 μm filter to remove the zeolite and any substances adsorbed thereto. The resulting solution was used as a nucleic acid amplification sample (zeolite-treated samples 5-3 to 5-10). 11.5 μL of the zeolite-treated sample was added to 11.5 μL of distilled water (DW) and 2 μL of the target nucleic acid solution described below. The mixture was incubated at 64°C for 50 minutes in a Simprova® Respiratory Infection Panel (manufactured by Eiken Chemical Co., Ltd.) to amplify the target nucleic acid using the LAMP method. The fluorescence intensity, which increased with the amplification of the target nucleic acid, was then measured using a Thermal Cycler Dice® Real Time System III.

[0070] As a positive control, sample 5-1 was prepared by simply adding distilled water to the nucleic acid extraction reagent instead of adding a saliva sample and mixing it, and as a negative control, sample 5-2 (zeolite-untreated sample) was prepared by adding a saliva sample to the nucleic acid extraction reagent but not adding zeolite, and similarly, amplification of the target nucleic acid was performed using the LAMP method.

[0071] The target nucleic acid solution used was Bordetella pertussis (B. pertussis) DNA (2,000 copies / test). The zeolites used were manufactured by Tosoh Corporation and were model numbers 500KOA, 690HOA, 642NAA, 940NHA, 840NHA, 722HOA, 720NHA, or 720KOA. The results are shown in Table 11. In Table 11, samples not detected within the measurement time (50 minutes) are indicated as N.D.

[0072]

[0073] Comparing Sample 5-1 and Sample 5-2, no amplification of the target nucleic acid was detected in Sample 5-2. This suggests that the addition of the saliva sample inhibited amplification of the target nucleic acid, i.e., the presence of substances other than nucleic acids (e.g., inhibitors of nucleic acid amplification) 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), suggesting that zeolite can remove substances other than nucleic acids from the saliva sample.

[0074] Test Example 6: Evaluation of RNA Purification Using Various Zeolites After 170 μL of saliva sample was added to 830 μL of nucleic acid extraction reagent (0.5% SDS) and mixed, 240 mg of zeolite was added and further mixed. The mixture was then passed through a 0.45 μm filter to remove the zeolite and any substances adsorbed thereto. The resulting solution was used as a nucleic acid amplification sample (zeolite-treated samples 6-3 to 6-10). 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 90 minutes using a Loopamp® Novel Coronavirus 2019 (SARS-CoV-2) Detection Reagent Kit (manufactured by Eiken Chemical Co., Ltd.) to amplify the target nucleic acid using the LAMP method. The turbidity value, which increased with the amplification of the target nucleic acid, was then measured in real time using a Loopamp EXIA amplification unit (manufactured by Eiken Chemical Co., Ltd.).

[0075] SARS-CoV-2 transcript RNA (250 copies / test) was used as the target nucleic acid solution. The zeolites used were Tosoh Corporation model numbers 500KOA, 690HOA, 642NAA, 940NHA, 840NHA, 722HOA, 720NHA, or 720KOA. As a positive control, Sample 6-1 was prepared by adding distilled water to the nucleic acid extraction reagent instead of the saliva sample, and similarly subjected to amplification of the target nucleic acid by the LAMP method. Furthermore, as a negative control, Sample 6-2 (zeolite-untreated sample), in which the nucleic acid extraction reagent was mixed with the saliva sample but without the addition of zeolite, was also similarly subjected to amplification of the target nucleic acid by the LAMP method. The results are shown in Table 12.

[0076]

[0077] Similar to Test Example 5, when Samples 6-1 and 6-2 were compared, amplification of the target nucleic acid was detected in Sample 6-1, but not in Sample 6-2, suggesting the presence of substances other than nucleic acids in the saliva specimen (e.g., inhibitors of nucleic acid amplification).On the other hand, amplification of the target nucleic acid was confirmed in the samples treated with zeolite (Samples 6-3 to 6-10), suggesting that zeolite can remove substances other than nucleic acids from the saliva specimen.

[0078] Test Example 7: Application of DNA purified with aprotic zeolite to PCR. 200 μL of a solution obtained by adding Streptococcus pneumoniae (S. pneumoniae) to a saliva sample was mixed with 800 μL of a nucleic acid extraction reagent (0.5% SDS), and 240 mg of zeolite (Model No. 720NHA) from Tosoh Corporation was added and mixed by inversion. The suspension mixed by inversion was passed through a 0.45 μm filter to remove the zeolite and any substances adsorbed thereto, yielding Sample 7-4. Target nucleic acid amplification was performed by quantitative PCR using 5 μL of the obtained Sample 7-4 and 20 μL of quantitative PCR reaction reagent. At the same time, a solution in which S. pneumoniae had been added to saliva was mixed with the nucleic acid extraction reagent, and the purified DNA was used to amplify Sample 7-3, which was obtained without zeolite treatment, and S. A solution of S. pneumoniae added to physiological saline was mixed with a nucleic acid extraction reagent, and sample 7-1 obtained without zeolite treatment and sample 7-2 obtained with zeolite treatment were also prepared, and target nucleic acids were similarly amplified by quantitative PCR.

[0079] Amplification and detection of target nucleic acids by quantitative PCR were carried out using the primer and probe sets shown in Table 13, in a quantitative PCR reaction reagent solution having the composition shown in Table 14, at the temperature and time shown in Table 15. The fluorescence intensity, which increased with the amplification of the target nucleic acid, was measured in real time using a LightCycler® 480 System. The probe consisting of the base sequence shown in SEQ ID NO: 9 in Table 13 was labeled with FAM at the 5' end and TAMRA at the 3' end. The results are shown in Table 16. In Table 16, the "copy number ratio" indicates the ratio when the copy number of sample 7-1 is set to 100%.

[0080]

[0081]

[0082]

[0083]

[0084] The copy numbers of target nucleic acids in Samples 7-1 and 7-2 were similar. These results suggest that S. pneumoniae was extracted using the nucleic acid extraction reagent, and that the nucleic acid (DNA) of S. pneumoniae was not adsorbed to 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 copy number of target nucleic acid than Samples 7-1 to 7-2, which did not contain a saliva sample. On the other hand, Sample 7-4, which was treated with aprotic zeolite, had a copy number of target nucleic acid similar to that of a sample not containing a saliva sample. This indicates that treatment with aprotic zeolite can remove substances other than nucleic acids (e.g., nucleic acid amplification inhibitors) contained in saliva samples. In other words, it suggests that nucleic acids can be extracted and purified with high purity.

[0085] Test Example 8: Application of RNA Purified with Non-Protonated Zeolite to PCR. 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 (Model No. 720NHA) from Tosoh Corporation was added and mixed by inversion. The suspension mixed by inversion was passed through a 0.45 μm filter to remove the zeolite and any substances adsorbed thereto, yielding samples 8-4 and 8-6. Target nucleic acids were amplified by quantitative PCR using 5 μL of the resulting samples 8-4 and 8-6 and 20 μL of quantitative PCR reaction reagent. Samples 8-3 and 8-5, obtained without zeolite treatment, and a solution obtained by adding influenza A virus to physiological saline were mixed with the nucleic acid extraction reagent. The target nucleic acids were similarly amplified by quantitative PCR using sample 8-1, obtained without zeolite treatment, and sample 8-2, obtained with zeolite treatment.

[0086] Amplification of the target nucleic acid by quantitative PCR was carried out using the primer set shown in Table 17 in a quantitative PCR reaction reagent solution having the composition shown in Table 2 above, at the temperature and time shown in Table 3 above. The fluorescence value, which increased with the amplification of the target nucleic acid, was measured in real time using a LightCycler (registered trademark) 480 System. The probe consisting of the base sequence shown in SEQ ID NO: 12 in Table 17 was labeled with FAM at the 5' end and MGB at the 3' end. The results are shown in Table 18. In Table 18, the "copy number ratio" indicates the ratio when the copy number of sample 8-1 is set to 100%.

[0087]

[0088]

[0089] The results showed that the copy numbers of target nucleic acids in Samples 8-1 and 8-2 were similar, suggesting that influenza A virus was extracted using the nucleic acid extraction reagent and that the nucleic acid (RNA) of influenza A virus was not adsorbed to zeolite and could be detected. Compared to Samples 8-1 to 8-2, which did not contain saliva samples, the copy numbers of detected target nucleic acids in Samples 8-3 and 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 copy numbers of target nucleic acids comparable to those in 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 saliva samples. In other words, it suggests that nucleic acids can be purified to a high purity.

[0090] Test Example 9: RNA Purification Using Non-Protonated Zeolite with Other Samples Nucleic Acid Extraction Reagent (0.5% SDS) was mixed with various samples (urine, cerebrospinal fluid, nasal swab, nasopharyngeal swab, serum, whole blood, feces, and sputum), followed by the addition of 240 mg of the zeolite described below. The mixture was then passed through a 0.45 μm filter to remove the zeolite and any substances adsorbed thereto. The resulting solution was used as a nucleic acid amplification sample (zeolite-treated sample). 0.5 μL of the target nucleic acid solution was added to 9.5 μL of the zeolite-treated sample, and the mixture was incubated at 62.5°C for 35 minutes using a Loopamp® Novel Coronavirus 2019 (SARS-CoV-2) Detection Reagent Kit (manufactured by Eiken Chemical Co., Ltd.). The turbidity value, which increased with amplification of the target nucleic acid, was measured in real time using a Loopamp EXIA Amplification Unit (manufactured by Eiken Chemical Co., Ltd.).

[0091] SARS-CoV-2 transcript RNA (250 copies / test) was used as the target nucleic acid solution. The zeolites used were Tosoh Corporation model numbers 690HOA and 720NHA. As a positive control, a sample was prepared by adding distilled water to the nucleic acid extraction reagent instead of the saliva sample and mixing, and similarly subjected to amplification of the target nucleic acid by the LAMP method. Furthermore, as a negative control, a sample was prepared by adding a saliva sample to the nucleic acid extraction reagent but mixing without adding zeolite (zeolite-untreated sample), and similarly subjected to amplification of the target nucleic acid by the LAMP method. The results are shown in Tables 19 to 26.

[0092]

[0093] In samples untreated with zeolite, target nucleic acids were not detected or the detection time was significantly delayed compared to samples without added specimens. This is thought to be because substances other than nucleic acids contained in various specimens were not removed. On the other hand, target nucleic acids were detectable in zeolite-treated samples. This is thought to be because zeolite was able to remove substances other than nucleic acids contained in various specimens. Among various specimens, non-protonated zeolite was found to be more effective than protonated zeolite for urine, nasal swabs, nasopharyngeal swabs, serum, and whole blood.

[0094] Test Example 10: Evaluation of nucleic acid purification depending on the type of surfactant in the nucleic acid extraction reagent After mixing 890 μL of a nucleic acid extraction reagent containing various surfactants shown in Table 27 with 110 μL of a saliva sample, 240 mg of zeolite (model number 720NHA) manufactured by Tosoh Corporation was added and further mixed, and the mixture was passed through a 0.45 μm filter to remove the zeolite and any substances adsorbed thereto, yielding samples 10-20 to 10-28. The resulting samples 10-20 to 10-28 were used as samples for nucleic acid amplification (non-proton zeolite-treated samples). 0.5 μL of the 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 a Loopamp (registered trademark) Novel Coronavirus 2019 (SARS-CoV-2) Detection Reagent Kit (manufactured by Eiken Chemical Co., Ltd.). The turbidity value, which increased with the amplification of the target nucleic acid, was measured in real time using a Loopamp EXIA amplification unit (manufactured by Eiken Chemical Co., Ltd.).

[0095] SARS-CoV-2 transcript RNA (250 copies / test) was used as the target nucleic acid solution. As positive controls, samples 10-1 to 10-10 were prepared by adding distilled water to various nucleic acid extraction reagents instead of saliva samples and mixing them. Target nucleic acid amplification was similarly performed using the LAMP method. Furthermore, as negative controls, samples 10-11 to 10-19 (zeolite-untreated samples), which were prepared by adding saliva samples to the nucleic acid extraction reagents but without adding zeolite, were also similarly subjected to amplification of target nucleic acids using the LAMP method. The results are shown in Table 27.

[0096]

[0097] In samples 10-11 to 10-19 (zeolite-untreated samples), in which saliva samples were simply mixed with a nucleic acid extraction reagent, target nucleic acids could not be detected. On the other hand, amplification of target nucleic acids could be detected in aprotic zeolite-treated samples 10-20 to 10-28, regardless of whether a nucleic acid extraction reagent containing any surfactant was used. This demonstrates that aprotic zeolites can remove substances other than nucleic acids (e.g., nucleic acid amplification inhibitors) contained in saliva samples, regardless of whether a nucleic acid extraction reagent containing any surfactant is used.

Claims

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

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

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

4. The purification method according to claim 1 or 2, wherein the aprotic zeolite is at least one selected from the group consisting of ferrierite-type zeolite, ZSM-5-type zeolite, Y-type zeolite, and beta-type zeolite.

5. The aprotic zeolite is ferrierite-NH ４ type zeolite, ferrierite-K type zeolite, ZSM-5-NH ４ type zeolite, Y-Na type zeolite and beta-NH ４ 5. The purification method according to claim 4, wherein the zeolite is at least one selected from the group consisting of zeolites of the above type.

6. The purification method according to claim 2, further comprising a step of obtaining a sample containing the nucleic acid by mixing the specimen with a nucleic acid extraction reagent containing a surfactant and / or by heat-treating the specimen prior to the contact step.

7. The purification method according to claim 6, wherein the surfactant comprises an anionic surfactant.

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 an aprotic zeolite.

11. The kit according to claim 10, wherein the aprotic zeolite is at least one selected from the group consisting of ferrierite-type zeolite, ZSM-5-type zeolite, Y-type zeolite, and beta-type zeolite.

12. The aprotic zeolite is ferrierite-NH ４ type zeolite, ferrierite-K type zeolite, ZSM-5-NH ４ type zeolite, Y-Na type zeolite and beta-NH ４ 12. The kit according to claim 11, wherein the zeolite is at least one selected from the group consisting of zeolites of the above type.

13. The kit according to claim 10, which is for purifying nucleic acid from at least one specimen selected from the group consisting of blood, cerebrospinal fluid, urine, feces, sputum, saliva, nasal discharge, swabs, amniotic fluid, and gargle, and further comprises a nucleic acid extraction reagent containing a surfactant.

14. The kit of claim 13, wherein the surfactant comprises an anionic surfactant.

15. The kit of claim 14, wherein the anionic surfactant is dodecyl sulfate.

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