Methods for extracting nucleic acid from virus

Abstract

To provide easier methods and kits for extracting and detecting nucleic acid derived from virus in samples without heat processing.SOLUTION: A method for detecting nucleic acid in a sample which may contain virus, comprises the steps of: (1) contacting the sample with an extraction agent comprising an alkali and a chelating agent to form a nucleic acid extract; (2) mixing the nucleic acid extract obtained in step (1) with a composition for nucleic acid amplification to obtain a reaction solution for nucleic acid amplification; and (3) amplifying nucleic acid using the reaction solution for nucleic acid amplification obtained in step (2).SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to the field of nucleic acid amplification for the detection of viruses, and more particularly to a method and a kit for detecting nucleic acids originating from viruses that may be contained in a sample, without heat treatment of the sample. [Background technology]

[0002] Nucleic acid amplification is a technique for amplifying a few copies of a target nucleic acid to a level where it can be visualized, i.e., hundreds of millions of copies or more, and is widely used not only in the field of life science research, but also in the medical field, such as genetic diagnosis and clinical testing, and in microbial testing of food and the environment. A representative nucleic acid amplification method is PCR (Polymerase Chain Reaction). PCR is a method for amplifying a target nucleic acid in a sample by repeating three steps, which are (1) DNA denaturation by heat treatment (dissociation from double-stranded DNA to single-stranded DNA), (2) annealing of a primer to a template single-stranded DNA, and (3) extension of the primer using DNA polymerase, as one cycle. Annealing and extension may be performed in two steps at the same temperature.

[0003] In addition to PCR, isothermal nucleic acid amplification is sometimes used as a nucleic acid amplification method. Unlike PCR, isothermal nucleic acid amplification is a method in which amplification is performed at a constant temperature without the need to raise and lower the temperature. Representative isothermal nucleic acid amplification methods include LAMP (Loop-mediated isothermal amplification), TMA (Transcription Mediated Amplification), NEAR (Nicking Endonuclease Amplification Reaction), SDA (Strand Displacement Amplification), HDA (Helicase-dependent amplification), NASBA (Nucleic Acid Sequences Based Amplification), and SPIA (Single primer isothermal amplification).

[0004] The samples that are the subject of tests using nucleic acid amplification methods vary depending on the purpose. For the detection of infectious disease-causing bacteria, samples that may contain the causative bacteria include feces, urine, blood, throat swabs, nasal swabs, sputum, saliva, and other biological samples. For food hygiene tests, samples include food, or feces and urine collected from food handlers. For environmental hygiene tests, samples include soil, river water, rainwater, seawater, sewage, and other environmental water, as well as wipes from manufacturing equipment.

[0005] When detecting bacteria from a sample, it is necessary to extract nucleic acid from the bacteria and then perform nucleic acid amplification. There are various methods for extracting nucleic acid from bacteria. For example, Patent Document 1 describes a method for extracting nucleic acids by alkaline heat extraction. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] Patent No. 5884108 Summary of the Invention [Problem to be solved by the invention]

[0007] However, the efficiency of nucleic acid extraction is not sufficient with the alkali treatment described in the method of Patent Document 1 alone, and a separate heat treatment of the sample is required, which takes time. Furthermore, the present inventors have found that if the concentration of alkali is increased to eliminate the heat treatment step and save time, the nucleic acid amplification enzyme is inactivated when the alkali is directly added to the nucleic acid amplification reaction solution, which becomes a cause of inhibiting nucleic acid amplification, and a step of neutralizing the nucleic acid extract is required, so that time saving cannot be achieved either. [Means for solving the problem]

[0008] As a result of intensive research to solve the above problems, the inventors discovered that nucleic acid can be extracted from viruses without prior nucleic acid purification or heat treatment by using an extractant containing an alkali at a concentration that does not inhibit nucleic acid amplification and a chelating agent, and thus arrived at the present invention.

[0009] That is, representative aspects of the present invention are as follows. [Section 1] 1. A method for detecting nucleic acid from a sample that may contain a virus, comprising: (1) contacting the sample with an extractant containing an alkali and a chelating agent to produce a nucleic acid extract; (2) mixing the nucleic acid extract obtained in step (1) with a composition for nucleic acid amplification to obtain a nucleic acid amplification reaction solution; (3) a step of amplifying nucleic acid using the nucleic acid amplification reaction solution obtained in step (2). [Section 2] Item 2. The method according to Item 1, wherein the nucleic acid extract is not heat-treated before being mixed with the composition for amplifying nucleic acid. [Section 3] Item 3. The method according to item 1 or 2, wherein step (1) is carried out at room temperature. [Section 4] The method according to any one of items 1 to 3, wherein the step (1) is carried out at 30° C. or lower. [Section 5] 5. The method according to any one of items 1 to 4, wherein the step (3) is carried out by any one of polymerase chain reaction (PCR) and isothermal nucleic acid amplification. [Section 6] Item 6. The method according to Item 5, wherein step (3) is carried out by polymerase chain reaction (PCR). [Section 7] 7. The method according to any one of Items 1 to 6, wherein the sample is at least one selected from the group consisting of feces, bile, urine, pharyngeal swabs, nasal swabs, sputum, pulmonary aspirates, cerebrospinal fluid, gargle, saliva, tears, blood, cultured cells, culture fluid, food, river water, rainwater, seawater, sewage, bath water, hot spring water, pool water, soil, and samples collected from the environment. [Section 8] Item 8. The method according to any one of Items 1 to 7, wherein the virus is an enveloped or non-enveloped RNA virus. [Section 9] Item 9. The method according to Item 8, wherein the enveloped virus is an influenza virus. [Section 10] Item 9. The method according to item 8, wherein the non-enveloped virus is a norovirus. [Section 11] 11. The method according to any one of items 1 to 10, wherein the alkali is at least one selected from the group consisting of potassium hydroxide, sodium hydroxide, lithium hydroxide, magnesium hydroxide, calcium hydroxide, barium hydroxide, potassium carbonate, sodium carbonate, magnesium carbonate, and calcium carbonate, or a solution of the at least one alkali, or is a Tris buffer solution, a glycine buffer solution, a phosphate buffer solution, a borate buffer solution, or a Good's buffer solution. [Section 12] Item 12. The method according to any one of Items 1 to 11, wherein the concentration of the alkali in the nucleic acid extract is 0.01N to 2N. [Section 13] The method according to any one of items 1 to 12, wherein the chelating agent is at least one selected from the group consisting of ethylenediaminetetraacetic acid (EDTA), cyclohexanediaminetetraacetic acid (CDTA), glycoletherdiaminetetraacetic acid (EGTA), nitrilotriacetic acid (NTA), diethylenetriaminepentaacetic acid (DTPA), hydroxyethylethylenediaminetriacetic acid (HEDTA), triethylenetetraaminehexaacetic acid (TTHA), glutamic acid diacetic acid (GLDA), dihydroxyethylglycine (DHEG), hydroxyethanediphosphonic acid (HEDP), methylenephosphonic acid (NTMP), phosphonobutanetricarboxylic acid (PBTC), ethylenediaminetetramethylenephosphonic acid (EDTMP), citric acid, oxalic acid, phytic acid, and gluconic acid. [Section 14] Item 14. The method according to any one of Items 1 to 13, wherein the concentration of the chelating agent in the nucleic acid extract is 0.5 mM to 100 mM. [Section 15] (a) containing an alkali or a solution thereof and a chelating agent or a solution thereof, or (b) containing alkali and chelating agents or solutions thereof; A nucleic acid extraction kit for extracting nucleic acid from a sample that may contain a virus without heat treatment. [Section 16] A kit for detecting a virus for use in the method according to claim 1, comprising: (a) comprising an alkali or a solution thereof, a chelating agent or a solution thereof, and a composition for amplifying nucleic acid; (b) A kit comprising an alkali and a chelating agent or a solution thereof and a composition for amplifying nucleic acids. [Section 17] Item 17. The kit according to item 15 or 16, wherein step (1) is carried out at room temperature. [Section 18] Item 18. The kit according to any one of Items 15 to 17, wherein the step (1) is carried out at 30° C. or lower. [Section 19] Item 19. The kit according to any one of Items 16 to 18, wherein the composition for nucleic acid amplification contains a thermostable DNA polymerase. [Section 20] 20. The kit according to any one of items 16 to 19, further comprising a hybridization probe corresponding to a detection region of the nucleic acid. [Section 21] 21. The kit according to any one of Items 15 to 20, wherein the virus is an enveloped or non-enveloped RNA virus. [Section 22] Item 22. The kit according to Item 21, wherein the enveloped virus is an influenza virus. [Section 23] Item 22. The kit according to Item 21, wherein the non-enveloped virus is a norovirus. [Section 24] Item 24. The kit according to any one of Items 15 to 23, wherein the alkali is an aqueous potassium hydroxide solution, an aqueous sodium hydroxide solution, an aqueous lithium hydroxide solution, an aqueous magnesium hydroxide solution, an aqueous calcium hydroxide solution, an aqueous barium hydroxide solution, an aqueous potassium carbonate solution, an aqueous sodium carbonate solution, an aqueous magnesium carbonate solution, an aqueous calcium carbonate solution, a Tris buffer solution, a glycine buffer solution, a phosphate buffer solution, a borate buffer solution, or a Good's buffer solution. [Section 25] 25. The kit according to any one of Items 16 to 24, wherein the concentration of the alkali in the nucleic acid extract is 0.01N to 2N. [Section 26] The chelating agent is ethylenediaminetetraacetic acid (EDTA), cyclohexanediaminetetraacetic acid (CDTA), glycol ether diaminetetraacetic acid (EGTA), nitrilotriacetic acid (NTA), diethylenetriaminepentaacetic acid (DTPA), hydroxyethylethylenediaminetriacetic acid (HEDTA), triethylenetetraaminehexaacetic acid (TTHA), glutamic acid diacetic acid (GLDA), dihydroxyethylglycine (DHEG), hydroxyethanediphosphonic acid (HEDP), methylenephosphonic acid (NTMP), phosphonobutanetricarboxylic acid (PBTC), ethylenediaminetetramethylenephosphonic acid (EDTMP), citric acid, oxalic acid, phytic acid, gluconic acid, any one of the kits described in 15 to 25. [Section 27] 26. The kit according to any one of Items 16 to 25, wherein the concentration of the chelating agent in the nucleic acid extract after mixing with a sample is 0.5 mM to 100 mM. Effect of the Invention

[0010] According to the method of the present invention, by using an extractant containing an alkali at a concentration that does not inhibit nucleic acid amplification and a chelating agent, nucleic acids can be detected without heat treatment of the sample. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0011] The present invention will be described in more detail below with reference to the embodiments of the present invention, but the present invention is not limited thereto. It should be understood that the terms used in this specification are used in the sense commonly used in the art unless otherwise specified. In addition, all non-patent literature and patent literature described in this specification are incorporated herein by reference. In this specification, "~" means "more than or equal to, less than or equal to", for example, if "X~Y" is described in the specification, it means "more than or equal to X, less than or equal to Y". In addition, in this specification, "and / or" means either one or both, and in this specification, the singular expression should be understood to include the concept of the plural form, unless otherwise specified.

[0012] (Virus detection methods) One aspect of the invention is a method for detecting viral nucleic acid that may be contained in a sample. The nucleic acid detection method of the present invention includes the step of (1) contacting a sample that may contain a virus with an extractant containing an alkali and a chelating agent to produce a nucleic acid extract.

[0013] The sample to be applied to the present invention is not particularly limited as long as it is a sample that may contain a virus, and examples thereof include samples derived from specimens excreted from living organisms. The specimens excreted from living organisms include feces, bile, urine, pharyngeal swabs, nasal swabs, sputum, lung aspirates, cerebrospinal fluid, gargle, saliva, tears, blood, etc., and further include specimens excreted from living organisms suspended in water (sterile water, etc.), physiological saline, buffer, organic solvent, liquid medium, or gel medium (e.g., Calibrea medium), and those that have been subjected to some treatment, such as the supernatant prepared from the suspension. The buffer is not particularly limited, and examples thereof include Hank's buffer, Tris buffer, phosphate buffer, glycine buffer, HEPES buffer, and Tricine buffer. The living organisms include humans, pets, livestock, wild animals, insects, and all other animals.

[0014] The sample that may contain a virus and is applied to the method of the present invention may be a specimen excreted from a living organism itself, or may be a sample prepared by isolating or concentrating the virus from a specimen excreted from a living organism or by other treatments such as precipitation, centrifugation, filtration, dilution, and addition of a substance that adsorbs the virus.

[0015] Another embodiment of the sample in the present invention is a sample that may contain cultured cells or culture fluid that may contain a virus. The types of cells include HeLa cells, MDCK cells, hCK cells, VeroE6 / TMPRSS2 cells, CHO cells, HEK-293 cells, BHK-21 cells, Sf9 cells, and Sf21 cells, but are not particularly limited thereto, and methods similar thereto are widely included.

[0016] Another embodiment of the sample of the present invention is a food sample that may contain a virus. Food samples include those suspended in water (sterile water, etc.), physiological saline, buffer solution, organic solvent, liquid medium, or gel medium (e.g., Calibrea medium), and those that have been treated in some way, such as the supernatant prepared from the above suspension. Examples of the buffer solution include, but are not limited to, Hank's buffer, Tris buffer, phosphate buffer, glycine buffer, HEPES buffer, and Tricine buffer.

[0017] Another embodiment of the sample in the present invention is river water, rainwater, seawater, sewage, bath water, hot spring water, pool water, soil, etc., which may contain viruses. The sample may be used as is, or may be treated by concentration, separation, etc. The treatment may include precipitation, centrifugation, filtration, dilution, addition of a substance that adsorbs viruses, etc.

[0018] Another embodiment of the sample in the present invention is a swab sample in the environment. Swab testing is useful for identifying the route of contamination and understanding the contamination status of the facility environment. In the present invention, the swab test is not particularly limited, but for example, a sample obtained by wiping the relevant area or equipment with a cotton swab or the like and dissolving the sample in water or a buffer solution is used. The eluted sample may be used as is, or may be subjected to treatment such as concentration or separation. Examples of the treatment include precipitation, centrifugation, filtration, dilution, and addition of a substance that adsorbs viruses. Examples of the places to be swabbed include cooking utensils such as cutting boards, knives, dishcloths, and tableware, refrigerator handles, toilets, bathroom doorknobs, washrooms, kitchens, faucets, cookers' hands and fingers, washbasins, handrails, and living rooms.

[0019] The form of the "sample that may contain a virus" to be applied to the method of the present invention is not particularly limited, but in the case of a solid or viscous specimen excreted from a living organism, such as feces, it is preferable to handle the specimen as a suspension of the specimen excreted from a living organism prepared in advance. The suspension preferably contains the specimen excreted from a living organism at 20% (w / v) or less. More preferably, the suspension is 1% (w / v) to 20% (w / v), and even more preferably, 1% (w / v) to 10% (w / v).

[0020] In addition to samples consisting of a single specimen or samples prepared from a single specimen, the present invention may also include samples prepared from a mixture of X types of specimens (X represents an integer of 2 or more). For example, when the specimen is saliva or a nasopharyngeal swab, the X type of specimen may be saliva or a nasopharyngeal swab from an individual of X type, or may be saliva or a nasopharyngeal swab of X type excreted by the same individual. X is not particularly limited, but for example, in the case of a test targeting the novel coronavirus (SARS-CoV-2), mixing five specimens leads to more efficient testing and is therefore used to process a large number of specimens, which can be used as a reference.

[0021] In the present invention, the virus to be detected includes, but is not limited to, RNA derived from a pathogenic virus. The target RNA (sometimes referred to as target RNA, etc.) is not particularly limited. Examples of these viruses include non-enveloped RNA viruses that do not have an envelope derived from a lipid bilayer membrane. Examples of such non-enveloped RNA viruses include Astroviridae viruses (e.g., Astrovirus); Caliciviridae viruses (e.g., Sapovirus, Norovirus); Picornaviridae viruses (e.g., Hepatitis A virus, Echovirus, Enterovirus, Coxsackievirus, Poliovirus, Rhinovirus); Hepeviridae viruses (e.g., Hepatitis E virus); Reoviridae viruses (e.g., Rotavirus). Although not limited thereto, the detection method of the present invention is preferably useful for detecting Caliciviridae viruses and Reoviridae viruses, more preferably useful for detecting Norovirus, Sapovirus, and Rotavirus, and particularly useful for detecting Norovirus. Examples of enveloped viruses to be detected in the present invention include influenza viruses, coronaviruses, Japanese encephalitis viruses, Zika viruses, and rubella viruses, with influenza viruses being preferred.

[0022] The extractant includes an alkali and a chelating agent. The extractant is not particularly limited as long as it can be used to extract nucleic acid from a sample, and may be either solid or liquid, but is particularly preferably liquid. When the extractant is liquid, it further includes a solvent. The solvent is not particularly limited, and may be, for example, an aqueous solvent such as water, a lower C1-C4 alcohol (e.g., methanol, ethanol, etc.), or an organic solvent, or a combination thereof. The solvent is preferably an aqueous solvent such as water, a lower C1-C4 alcohol (e.g., methanol, ethanol, etc.), or a combination thereof.

[0023] The alkali is not particularly limited as long as it can be used for nucleic acid extraction from viruses. Examples of alkali include potassium hydroxide, sodium hydroxide, lithium hydroxide, magnesium hydroxide, calcium hydroxide, barium hydroxide, potassium carbonate, sodium carbonate, magnesium carbonate, calcium carbonate, or Tris buffer, glycine buffer, phosphate buffer, borate buffer, Good's buffer (e.g., TAPSO, POPSO, HEPSO, EPPS, Tricine, Bicine, TAPS, CHES, CAPS), etc., and one type can be used alone or two or more types can be used in combination. The alkali is preferably at least one liquid selected from the group consisting of potassium hydroxide, sodium hydroxide, lithium hydroxide, magnesium hydroxide, potassium hydroxide, barium hydroxide, potassium carbonate, sodium carbonate, magnesium carbonate, and calcium carbonate, and more preferably potassium hydroxide, sodium hydroxide, or a combination thereof.

[0024] The alkali may be in the form of a solid or a solution. When the alkali is in the form of a solution, the solvent is not particularly limited, and may be, for example, an aqueous solvent such as water, a lower alcohol of C1 to C4 (e.g., methanol, ethanol, etc.), or an organic solvent, or a combination thereof. The solvent is preferably an aqueous solvent such as water, a lower alcohol of C1 to C4 (e.g., methanol, ethanol, etc.), or a combination thereof.

[0025] The hydrogen ion exponent (pH) (25°C) of the alkali in the extractant is not particularly limited, and may be, for example, pH 8.0 or higher, pH 8.5 or higher, pH 9.0 or higher, pH 9.5 or higher, pH 10.0 or higher, pH 10.5 or higher, pH 11.0 or higher, pH 11.5 or higher, pH 12.0 or higher, pH 12.5 or higher, pH 13.0 or higher, pH 13.5 or higher, or pH 14.0 or higher. The alkali has, for example, a pH of 9 or higher, preferably 11 or higher, and more preferably 13 or higher.

[0026] When the alkali is in the form of a solution, the concentration of the alkali (also referred to as "alkali concentration") is not particularly limited. Here, the alkali concentration is a volume molar concentration (M or mol / L) and can be defined as the molar amount of the alkaline substance (for example, the total amount of the alkaline substance such as potassium hydroxide and / or sodium hydroxide as described above) contained in 1 L of the alkaline solution.

[0027] The hydroxide concentration of the alkali in the extractant may be adjusted appropriately depending on the mixing ratio with the sample, but it is preferably a concentration that does not inhibit nucleic acid amplification when used as a nucleic acid amplification reaction solution, and is preferably a concentration that allows nucleic acid extraction and does not inhibit nucleic acid amplification when used as a nucleic acid amplification reaction solution. For example, the hydroxide concentration of the alkali in the nucleic acid extraction solution is preferably 0.01N to 2N, more preferably 0.05N to 1N, even more preferably 0.05N to 0.5N, and even more preferably 0.05N to 0.2N. When using multiple types of alkali, it is preferable that the total concentration of each hydroxide satisfies the above range. The hydroxide concentration of the alkali in the extractant may be adjusted appropriately depending on the mixing ratio with the sample. For example, when the sample and the extractant are mixed at 1:1, it is desirable that the hydroxide concentration of the alkali in the extractant is twice the hydroxide concentration in the nucleic acid extraction solution.

[0028] Chelating agents are used to increase the efficiency of nucleic acid extraction. Examples of chelating agents include ethylenediaminetetraacetic acid (EDTA), cyclohexanediaminetetraacetic acid (CDTA), glycoletherdiaminetetraacetic acid (EGTA), nitrilotriacetic acid (NTA), diethylenetriaminepentaacetic acid (DTPA), hydroxyethylethylenediaminetriacetic acid (HEDTA), triethylenetetraaminehexaacetic acid (TTHA), glutamic acid diacetic acid (GLDA), dihydroxyethylglycine (DHEG), hydroxyethanediphosphonic acid (HEDP), methylenephosphonic acid (NTMP), phosphonobutanetricarboxylic acid (PBTC), ethylenediaminetetramethylenephosphonic acid (EDTMP), citric acid, oxalic acid, phytic acid, and gluconic acid, and can be used alone or in combination of two or more.

[0029] The concentration of the chelating agent in the extractant is not particularly limited as long as it is a concentration that allows nucleic acid extraction and does not inhibit nucleic acid amplification when used as a nucleic acid amplification reaction solution. For example, the concentration of the chelating agent in the nucleic acid extract is preferably 0.5 mM or more, more preferably 0.8 mM or more, and even more preferably 1 mM or more. In addition, from the viewpoint of inhibition of nucleic acid amplification, the concentration of the chelating agent in the nucleic acid extract is preferably 10 mM or less, more preferably 8 mM or less, and even more preferably 5 mM or less. Specific values ​​of the concentration of the chelating agent in the nucleic acid extract include, for example, 0.5 mM, 0.8 mM, 1 mM, 1.5 mM, 2 mM, 2.5 mM, 3 mM, 3.5 mM, 4 mM, 4.5 mM, 5 mM, 5.5 mM, 6 mM, 6.5 mM, 7 mM, 7.5 mM, 8 mM, 8.5 mM, 9 mM, 9.5 mM, 10 mM, etc., but are not particularly limited.

[0030] For example, when EDTA is used as a chelating agent, the concentration of EDTA in the nucleic acid extract is 0.5 mM to 10 mM, preferably 0.8 mM to 8 mM, more preferably 1 mM to 5 mM. The concentration of EDTA in the extractant may be appropriately adjusted depending on the mixing ratio with the sample. For example, when mixed with the sample at a 1:1 ratio, the concentration of EDTA in the extractant is desirably twice the concentration of EDTA in the nucleic acid extract.

[0031] In addition to the alkali and chelating agent, the extractant may further include one or more surfactants, reducing agents, metal salts, and the like.

[0032] The extractant can be prepared, for example, by dissolving an alkali and a chelating agent in a solvent. Also, when the alkali is in the form of a solution, the extractant may be prepared by adding the chelating agent to an alkaline solution, or by mixing a solution of the chelating agent with the alkaline solution. Also, when the chelating agent is in the form of a solution, the extractant may be prepared by adding an alkali to a solution of the chelating agent, or by mixing a solution of the alkali with a solution of the chelating agent.

[0033] The mixing ratio of the extractant to the sample is not particularly limited and is, for example, 9:1 to 1:9, preferably 3:1 to 1:3, and more preferably 2:1 to 1:2. The mixing of the sample and the extractant can be carried out at room temperature. Here, room temperature refers to the environmental temperature where an operator works. Here, the upper limit of room temperature is preferably 40° C. or less, more preferably 35° C. or less, and even more preferably 30° C. or less. The lower limit of room temperature is preferably 5° C. or more, more preferably 10° C. or more, and even more preferably 15° C. or more. Furthermore, the conditions for mixing the sample and the extractant are not particularly limited, and the operation may be carried out at low temperatures, for example, on an ice block or crushed ice.

[0034] The sample and the extractant can be mixed by stirring, spinning down, mixing by inversion, pipetting, etc., but the method and the container used are not limited. Furthermore, even if the sample and the extractant are not subjected to heat treatment after mixing, the mixture can be used as a template for nucleic acid amplification to obtain sufficiently high-precision results.

[0035] After mixing the sample and the extractant, it may be used as a template for nucleic acid amplification without being left to stand. The obtained nucleic acid extract can be used as a template for nucleic acid amplification even after a period of time, preferably up to 6 hours, more preferably up to 3 hours, and more preferably up to 2 hours, has elapsed until mixing with the composition for nucleic acid amplification in the next step. Furthermore, the nucleic acid extract may be used as a template for nucleic acid amplification after being stored at room temperature, refrigerated, or frozen. The storage period is preferably within 1 week, more preferably within 3 days, and even more preferably within 1 day at room temperature, and preferably within 2 weeks, more preferably within 1 week, and even more preferably within 3 days in the case of refrigerated storage. In addition, the storage period is preferably within 1 year, more preferably within 3 months, and even more preferably within 1 month in the case of frozen storage.

[0036] According to the method of the present invention, the presence or absence of a virus in a sample can be detected easily and in a short time by using a nucleic acid amplification reaction solution obtained by mixing a nucleic acid extract obtained by contacting a sample with an extractant containing an alkali and a chelating agent with a composition for nucleic acid amplification described later, without requiring prior nucleic acid separation and purification from the sample. As a result, the efficiency of the testing work can be further improved, and the number of tests can be increased, which can contribute to early detection of infectious diseases and prevention of infection. In addition, by omitting the nucleic acid purification step, the opening and closing of the lid of the reaction vessel can also be omitted. As a result, the risk of scattering of a virus-containing sample when opening and closing the lid can be eliminated, and the risk of contamination of other samples can be reduced. This can reduce the risk of false positives and further improve the accuracy of the testing work. In addition, by eliminating the risk of scattering of a virus-containing sample, the risk of infection to the operator can be reduced. Furthermore, by not performing heat treatment, the decomposition of RNA can be suppressed, allowing for more sensitive detection.

[0037] In particular, the effect can be remarkable in tests in which a large number of specimens are processed. For example, according to the present invention, rapid detection can be achieved when a large number of specimens, such as feces, bile, urine, pharyngeal swabs, nasal swabs, sputum, pulmonary aspirates, cerebrospinal fluid, gargle, saliva, tears, blood, cultured cells, culture fluid, food, river water, rainwater, seawater, sewage, bath water, hot spring water, pool water, soil, and samples collected from the environment, are simultaneously processed. The present invention can also be used in life science research, clinical diagnosis, food hygiene tests, environmental tests, and the like.

[0038] The nucleic acid detection method of the present invention includes the step (2) of mixing the nucleic acid extract obtained in the step (1) with a composition for nucleic acid amplification to obtain a nucleic acid amplification reaction solution. The composition for nucleic acid amplification contains components necessary for amplifying nucleic acid. The components contained in the composition for nucleic acid amplification are not particularly limited as long as they can amplify nucleic acid, and can be appropriately selected by those skilled in the art depending on the nucleic acid amplification method selected, and include known components such as DNA polymerase, primers, dNTPs, buffers, and solvents suitable for nucleic acid amplification. Examples of the nucleic acid amplification method selected include polymerase chain reaction (PCR) and isothermal nucleic acid amplification. The components that the composition for amplifying nucleic acid may contain will be described in detail in the explanation of the kit below.

[0039] Isothermal nucleic acid amplification is a method of amplification at a constant temperature without the need to raise or lower the temperature. Representative isothermal nucleic acid amplification methods include, but are not limited to, LAMP (Loop-mediated isothermal amplification), TMA (Transcription Mediated Amplification), NEAR (Nicking Endonuclease Amplification Reaction), SDA (Strand Displacement Amplification), HDA (Helicase-dependent amplification), NASBA (Nucleic Acid Sequences Based Amplification), and SPIA (Single primer isothermal amplification). Any known method can be used.

[0040] Polymerase chain reaction (PCR) is a method for amplifying target nucleic acid in a sample by repeating three cycles: (1) DNA denaturation by heat treatment (dissociation of double-stranded DNA into single-stranded DNA), (2) annealing of primers to template single-stranded DNA, and (3) extension of primers using DNA polymerase. In some cases, annealing and extension are performed at the same temperature in two steps.

[0041] In order to detect a virus in a sample, it is necessary to detect the amplified nucleic acid during or after nucleic acid amplification, but the detection method is not particularly limited, and any method known to those skilled in the art can be used. Detection methods include agarose gel electrophoresis, detection using an intercalator such as SYBR (registered trademark) Green I or EvaGreen (registered trademark), and detection using a probe such as TaqMan (registered trademark) probe, Q probe, Molecular beacon probe, or Scorpions (registered trademark) probe. In terms of ease of detection, detection using an intercalator or detection using a probe is preferable, and detection using a probe is more preferable.

[0042] The mixing ratio of the nucleic acid extract and the composition for nucleic acid amplification is not particularly limited as long as the nucleic acid can be appropriately amplified. For example, the nucleic acid extract is mixed with the composition for nucleic acid amplification at a ratio of preferably 1 to 50% (v / v), more preferably 1 to 30% (v / v), and even more preferably 1 to 10% (v / v).

[0043] The nucleic acid detection method of the present invention includes the step of (3) amplifying a nucleic acid using the nucleic acid amplification reaction solution obtained in the step (2). Nucleic acid amplification may be performed under reaction conditions according to the selected nucleic acid amplification method, and is not particularly limited. Conditions suitable for the selected nucleic acid amplification method are well known to those skilled in the art. For example, in the case of an isothermal reaction, amplification is performed at a constant temperature, but it may be performed at a temperature suitable for the selected isothermal amplification reaction method. In the case of PCR, amplification is performed by repeating three steps, namely DNA denaturation by heat treatment, annealing of a primer to a template single-stranded DNA, and extension using DNA polymerase, as one cycle, and annealing and extension are performed at the same temperature in two steps. The cycle conditions are not particularly limited, and cycle conditions may be set according to the characteristics of the reaction system and primers. In addition, a reaction for detection may be added after the nucleic acid amplification process. For example, in a detection system using an intercalator such as SYBR (registered trademark) Green I or EvaGreen (registered trademark), a temperature step for melting curve analysis is added.

[0044] When the virus to be detected is an RNA virus, a reverse transcription reaction may be carried out before nucleic acid amplification. The reverse transcription reaction may be carried out by a method known to those skilled in the art, and may be carried out using a known reverse transcriptase such as ReverTra Ace (manufactured by Toyobo). The conditions for the reverse transcription reaction may be appropriately set by those skilled in the art, and may be carried out, for example, at a temperature of 35 to 60°C, preferably 40 to 55°C, more preferably 42 to 50°C, for example, for 30 seconds to 10 minutes, preferably 1 minute to 5 minutes, more preferably 2 minutes to 4 minutes.

[0045] The reverse transcription reaction and the nucleic acid amplification reaction may be carried out by a method such as RT-PCR using a thermostable DNA polymerase that also has reverse transcriptase activity. Here, the DNA polymerase that also has reverse transcriptase activity refers to a DNA polymerase that has both the ability to convert RNA to cDNA and the ability to amplify DNA. Examples of DNA polymerases that also have reverse transcriptase activity include DNA polymerase (Taq) derived from Thermus aquaticus, DNA polymerase (Tth) derived from Thermus thermophilus HB8, DNA polymerase (Z05) derived from Thermus sp Z05, DNA polymerase (Tma) derived from Thermotoga maritima, DNA polymerase (Bca) derived from Bacillus caldotenax, and DNA polymerase (Bst) derived from Bacillus stearothermophilus. Any DNA polymerase with reverse transcription activity known to those skilled in the art or a mutant thereof that does not lose the reverse transcription activity and thermostable DNA polymerase activity can be used. The conditions of the method such as RT-PCR can be appropriately set by those skilled in the art. For example, after performing the reverse transcription reaction under the above conditions, RT-PCR can be performed by performing pre-denaturation at a temperature of, for example, 90 to 99°C, preferably 92 to 97°C, for example, for 5 to 20 seconds, preferably 10 to 15 seconds, and then performing, for example, 1 to 50 cycles, preferably 2 to 45 cycles of heat treatment cycles including heating at a temperature of, for example, 92 to 99°C, preferably 94 to 98°C, for example, for 0.5 to 10 seconds, preferably 1 to 5 seconds, and heating at a temperature of, for example, 50 to 60°C, preferably 52 to 55°C, for example, for 1 to 20 seconds, preferably 5 to 15 seconds.

[0046] (Nucleic acid extraction kit and nucleic acid detection kit) In another aspect of the present invention, there is provided a nucleic acid extraction kit for extracting nucleic acid from a sample that may contain a virus without heat treatment, comprising (a) an alkali or a solution thereof and a chelating agent or a solution thereof, or (b) an alkali and a chelating agent or a solution thereof. In another aspect of the present invention, there is provided a method for detecting a nucleic acid comprising the steps of: (a) detecting an alkali or a solution thereof; (b) detecting a chelating agent or a solution thereof; and (c) detecting a nucleic acid using the composition for amplifying the nucleic acid; (b) A nucleic acid detection kit is provided which comprises an alkali and a chelating agent or a solution thereof, and a composition for amplifying nucleic acid.

[0047] The alkali, chelating agent, and composition for nucleic acid amplification may be the same as those described above for the nucleic acid detection method. The composition for nucleic acid amplification preferably contains a DNA polymerase and a primer pair corresponding to the detection region of the nucleic acid to be detected. It is further preferable that the composition contains a hybridization probe corresponding to the detection region of the nucleic acid to be detected, or an intercalator such as SYBR (registered trademark) Green or EvaGreen (registered trademark) for detection. The kit may further contain a reagent containing a surfactant. In addition, the kit may contain the alkali and the chelating agent in a form in which they coexist in one liquid, or the alkali and the chelating agent in a form in which they exist in separate liquids.

[0048] The origin of the DNA polymerase that can be included in the composition for amplifying nucleic acid is not particularly limited as long as it is capable of amplifying nucleic acid, and specific examples include Taq DNA polymerase, Tth DNA polymerase, Bst DNA polymerase, KOD DNA polymerase, Pfu DNA polymerase, Pwo DNA polymerase, Tbr DNA polymerase, Tfi DNA polymerase, Tfl DNA polymerase, Tma DNA polymerase, Tne DNA polymerase, Vent DNA polymerase, DEEPVENT DNA polymerase, PrimeSTAR DNA polymerase, Phi29 DNA polymerase, Klenow Fragment, Bsu DNA polymerase, and mutants thereof. It is preferable to use a thermostable DNA polymerase, and more preferable examples are Taq DNA polymerase, Tth DNA polymerase, KOD DNA polymerase, Pfu DNA polymerase, PrimeSTAR DNA polymerase or a mutant thereof, and particularly preferable examples are Taq DNA polymerase and Tth DNA polymerase and a mutant thereof. Furthermore, in order to enhance the effect of suppressing non-specific reactions, it is preferable to suppress the enzyme activity of DNA polymerase by using it in combination with an anti-DNA polymerase antibody, or by introducing a thermolabile block group into the DNA polymerase by chemical modification, or by using it in combination with an aptamer, so that it can be applied to hot start and warm start nucleic acid amplification. In addition, the DNA polymerase contained in the nucleic acid detection kit is not limited to one type, and multiple DNA polymerases may be mixed.

[0049] In the present specification, the term "DNA polymerase mutant" refers to a mutant having, for example, 85% or more, preferably 90% or more, more preferably 95% or more, even more preferably 98% or more, and especially preferably 99% or more sequence identity with respect to the amino acid sequence of the wild-type DNA polymerase from which it is derived, and having the activity of amplifying DNA similar to that of the wild-type DNA polymerase. Here, the method of calculating the identity of the amino acid sequence can be performed by any means known in the art. For example, it can be calculated using an analysis tool that is commercially available or available via an electric communication line (Internet). For example, the identity of the amino acid sequence can be calculated by using the default (initial setting) parameters in the homology algorithm BLAST (Basic local alignment search tool) of the National Center for Biotechnology Information (NCBI) http: / / www.ncbi.nlm.nih.gov / BLAST / . Furthermore, the mutant that can be used in the present invention may be a polypeptide consisting of an amino acid sequence in which one or several amino acids have been substituted, deleted, inserted and / or added (hereinafter, these are also collectively referred to as "mutation") in the amino acid sequence of the wild-type DNA polymerase from which it is derived, and may have DNA amplifying activity similar to that of the wild-type DNA polymerase. Here, "one or several" may be, for example, 1 to 80, preferably 1 to 40, more preferably 1 to 10, even more preferably 1 to 5, and even more preferably 1 to 3, but is not particularly limited thereto.

[0050] The components contained in the composition for amplifying nucleic acids are not particularly limited, and include conventionally known components, and the proportions thereof are not limited either. It is preferable that the composition contains at least one selected from the group consisting of one or more types of deoxynucleotide triphosphates or derivatives of deoxynucleotide triphosphates, buffers, and salts.

[0051] The buffer that can be included in the composition for amplifying nucleic acid is not particularly limited, and examples thereof include TRIS, TRICINE, BIS-TRICINE, BICINE, HEPES, MOPS, TES, TAPS, PIPES, and CAPS. The pH is adjusted to 6 to 10, more preferably 7 to 9, with sulfuric acid, hydrochloric acid, acetic acid, phosphoric acid, etc. The concentration of the buffer to be added is 10 to 200 mM, more preferably 20 to 150 mM. At this time, a salt solution is added to obtain ionic conditions suitable for the reaction. Examples of the salt solution include potassium chloride, potassium acetate, potassium sulfate, ammonium sulfate, ammonium chloride, and ammonium acetate.

[0052] Examples of deoxynucleotide triphosphates or derivatives of deoxynucleotide triphosphates that can be included in the composition for nucleic acid amplification include dATP, dCTP, dGTP, and dTTP, each of which is added at 0.1 to 0.5 mM, most commonly about 0.2 mM. dUTP may be used instead of and / or as a part of dTTP. Since dUTP is incorporated into the nucleic acid amplification product reacted with dUTP, the nucleic acid amplification product containing dUTP can be decomposed by applying Uracil-N-glycosyrase (UNG) before the nucleic acid amplification reaction. Therefore, it is possible to prevent carryover contamination that occurs when the amplification product obtained in the previous reaction is mixed with the subsequent reaction solution. In this case, Uracil-N-glycosyrase (UNG) may be contained in the reaction solution.

[0053] It is preferable to add metal ions necessary for DNA polymerase activity to the composition for nucleic acid amplification. Examples of magnesium salts include magnesium chloride, magnesium sulfate, and magnesium acetate, and examples of manganese salts include manganese chloride, manganese sulfate, and manganese acetate, and it is preferable to add about 1 to 10 mM. Since a chelating agent is contained in the nucleic acid extract, metal ions consumed by the chelating agent in the nucleic acid amplification reaction solution may be replenished. For example, in the case of EDTA, which binds to divalent metal ions at a molar ratio of 1:1, Mg is added in consideration of the amount of EDTA brought into the PCR reaction solution. 2+ The amount of ions can be adjusted. Furthermore, other components that can coexist in PCR may be added.

[0054] The components contained in the composition for amplifying nucleic acid may contain known substances added for the purpose of improving reactivity. The substances to be added include proteins such as bovine serum albumin (BSA). The amount of bovine serum albumin added is not limited as long as the effect of the present invention is exhibited, but is preferably at least 0.5 mg / mL or more, more preferably at least 1 mg / mL or more. In a sample containing a large amount of impurities, a concentration of bovine serum albumin of preferably 2 mg / mL or more, more preferably 3 mg / mL or more, enables good detection. Furthermore, it may contain a surfactant such as Triton X-100, Triton X-114, Tween 20, Nonidet P40, or Briji 58. Furthermore, to reduce reaction inhibition, it may contain a chelating agent such as ethylene glycol-bis(2-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) or 1,2-bis(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid (BAPTA). Other examples of additives include glycerol, polyols, protease inhibitors, single-stranded binding protein (SSB), T4 gene 32 protein, tRNA, sulfur- or acetic acid-containing compounds, glycerol, ethylene glycol, propylene glycol, trimethylene glycol, formamide, acetamide, betaine (trimethylglycine), L-carnitine, ectoine, trehalose, dextran, polyvinylpyrrolidone (PVP), tetramethylammonium chloride (TMAC), tetramethylammonium hydroxide (TMAH), tetramethylammonium acetate (TMAA), polyethylene glycol, gelatin, and the like.

[0055] Components that may be included in the composition for nucleic acid amplification may include a primer according to the principle of the selected nucleic acid amplification reaction. The primer is selected to have a primer sequence corresponding to the detection region of the nucleic acid to be detected. The type of nucleic acid amplification reaction method is not limited, but an example of a primer when PCR is used is a primer pair corresponding to the detection region of the nucleic acid to be detected, and two pairs of primers in which one primer is complementary to the nucleic acid extension product of the other primer can be mentioned. In addition, as another embodiment, so-called multiplex PCR in which two or more pairs of the above primers are included can be mentioned. Furthermore, when the target nucleic acid is a subtype, it may include a degenerate primer, or may include multiple primers corresponding to the mutation of the subtype, and the number of primers in one primer and the other primer in the primer pair may be different. As for the concentration of the primers used, the concentration of each primer is preferably 0.01 μM or more and 3 μM or less with respect to the entire nucleic acid amplification reaction solution, and more preferably, the concentration of each primer is 0.05 μM or more and 2 μM or less.

[0056] In another embodiment, the present invention further preferably includes at least one type of labeled hybridization probe or double-stranded DNA binding fluorescent compound. This allows the analysis of the amplification product to be monitored by monitoring a fluorescent signal instead of normal electrophoresis, reducing the analysis labor. Furthermore, there is no need to open the reaction vessel, making it possible to reduce the risk of contamination. It is also possible to identify virus subtypes by labeling each hybridization probe corresponding to a virus subtype with a different fluorescent dye. Furthermore, it is also possible to simultaneously identify and determine the presence or absence of viruses in one reaction solution by labeling each hybridization probe corresponding to nucleic acids derived from multiple viruses with different fluorescent dyes.

[0057] The double-stranded DNA-binding fluorescent compound is not particularly limited, and examples include dyes that bind to phosphate groups on the surface of DNA and dyes that intercalate between bases (intercalators). An intercalator refers to any molecule that binds to nucleic acids in a reversible, non-covalent manner by intercalating into double-stranded DNA, thereby indicating the presence and amount of nucleic acid. In general, an intercalator is a dye that intercalates into double-stranded DNA and emits fluorescence. Numerous intercalators are known in the art, including, but not limited to, Ethidium bromide, cyanine dyes (e.g., TOTO®, YOYO®, BOBO, and POPO), SYBR® Green I, SYBR® Green ER, SYBR® Green Gold, SYBR® DX, PicoGreen®, LCGeen®, EvaGreen®, SYTOX® Green, ResoLight, propidium iodide, Acridine orange, 7-amino-actinomycin D, CyQUANT® GR, SYTO® 9, SYTO® 10, SYTO® 13, SYTO® 14, SYTO® 82, FUN-1, and the like.

[0058] Examples of hybridization probes include TaqMan Probe (U.S. Pat. No. 5,210,015, U.S. Pat. No. 5,538,848, U.S. Pat. No. 5,487,972, U.S. Pat. No. 5,804,375), Molecular beacon probe (U.S. Pat. No. 5,118,801), FRET hybridization probe (WO 97 / 46707, WO 97 / 46712, WO 97 / 46714), Q probe, and Scorpions probe. The concentration of the fluorescently labeled probe is preferably 0.01 μM or more and 1.0 μM or less. More preferably, it is 0.013 μM or more and 0.75 μM or less, and even more preferably, it is 0.02 μM or more and 0.5 μM or less. EXAMPLES

[0059] The present invention will be described in more detail below with reference to examples, although the present invention is not limited to the following examples.

[0060] Example 1: RNA extraction from norovirus We confirmed whether it is possible to extract RNA from norovirus using an extraction agent, and whether detection was possible using nucleic acid amplification reactions when using an extraction solution or sterilized water.

[0061] (Preparation of Extractant) An extractant having the following composition (twice the final concentration of the nucleic acid extract) was prepared. 0.2N NaOH + 20mM EDTA

[0062] (Pretreatment of sample) Norovirus Group II (Recombinant) (Stool Matrix) (ZeptoMetrix) diluted 25-fold with sterile water was mixed with equal amounts of extraction agent or sterile water at room temperature (25°C) to prepare samples.

[0063] (Preparation of nucleic acid amplification reaction solution) The PCR reaction reagent used was the Norovirus Detection Kit G1 / G2 - Rapid Probe Detection Quick Step - (Toyobo Co., Ltd. FIK-273), and the following volumes were prepared per reaction. Pretreatment solution 3μL Reaction solution 30μL Enzyme solution 5μL Primer solution (G1 / G2) 5 μL Probe solution (G1 / G2) 5μL To the prepared reaction solution (48.0 μL per reaction), 2 μL of the extracted RNA that had been treated as described above was added.

[0064] (PCR reaction conditions) 50 μL of the reaction solution was reacted using Bio-Rad's CFX96 DEEP WELL under the following temperature cycle. 42℃, 4 minutes (reverse transcription reaction) 95℃, 10 seconds (pre-denaturation) 98℃, 1 sec - 52℃, 10 sec 2 cycles 95℃, 1 sec - 52℃, 10 sec 8 cycles 95℃, 1 sec - 52℃, 10 sec (fluorescence reading) 30 cycles

[0065] The measurement results were calculated using Bio-Rad's CFX96 analysis software, with the threshold value set at 30, to calculate the Ct value. RNA was not extracted from norovirus using sterilized water, and was not detectable after the nucleic acid amplification reaction. However, by using an extractant, RNA was extracted, making it possible to detect the virus after the nucleic acid amplification reaction.

[0066] [Table 1]

[0067] Example 2: RNA extraction from influenza virus We confirmed whether RNA can be extracted from influenza virus using an extractant, and whether detection was possible by nucleic acid amplification reaction when using an extract solution or sterilized water.

[0068] (Preparation of Extractant) An extractant having the following composition (twice the final concentration of the nucleic acid extract) was prepared. 0.2N NaOH + 20mM EDTA

[0069] (Pretreatment of sample) Influenza A H1N1, External Run Control, Medium (ZeptoMetrix) diluted 2.5-fold with sterile water was mixed with equal amounts of extractant or sterile water at room temperature (25°C) to prepare samples.

[0070] (Preparation of nucleic acid amplification reaction solution) The PCR reaction reagent was prepared using the THUNDERBIRD Probe One-step qRT-PCR Kit (QRZ-101, Toyobo Co., Ltd.) at the following volumes per reaction: Primers and probes were used with sequences listed in the WHO information for molecular diagnosis of influenza virus. 2×Reaction Buffer 10μL DNA Polymerase 0.5μL 0.5μL RT Enzyme Mix 10μM GRAM / 7Fw(Forward Primer) 1μL 10μM GRAM / 161Rv(Reverse Primer) 1μL 10μM GRAM probe / 52 / +(Probe) 0.4μL RNase-free water 4.6μL To the prepared reaction solution (19.0 μL per reaction), 1 μL of the extracted RNA that had been treated as described above was added.

[0071] (PCR reaction conditions) 20 μL of the reaction solution was reacted using Bio-Rad's CFX96 DEEP WELL under the following temperature cycle. 50℃, 10 minutes (reverse transcription reaction) 95℃, 1 min (pre-denaturation) 95℃, 15 sec - 60℃, 45 sec (fluorescence reading) 45 cycles

[0072] The measurement results were calculated using Bio-Rad's CFX96 analysis software, with the threshold value set at 30, to calculate the Ct value. RNA was not extracted from influenza viruses using sterilized water, and was not detectable after the nucleic acid amplification reaction. However, by using an extractant, RNA was extracted and could be detected after the nucleic acid amplification reaction.

[0073] [Table 2] [Industrial Applicability]

[0074] According to the present invention, it is possible to detect viral nucleic acid contained in a sample simply and with high sensitivity. Use of the method and kit of the present invention can greatly contribute to the fields of life science research, clinical diagnosis, food hygiene testing, environmental testing, etc.

Claims

1. 1. A method for detecting nucleic acid from a sample that may contain a virus, comprising: (1) contacting the sample with an extractant containing an alkali and a chelating agent to produce a nucleic acid extract; (2) mixing the nucleic acid extract obtained in step (1) with a composition for nucleic acid amplification to obtain a nucleic acid amplification reaction solution; (3) a step of amplifying nucleic acid using the nucleic acid amplification reaction solution obtained in step (2).

2. The method according to claim 1 , wherein the nucleic acid extract is not heat-treated prior to mixing with the composition for amplifying nucleic acid.

3. The method according to claim 1 or 2, wherein step (1) is carried out at room temperature.

4. The method according to claim 3, wherein step (1) is carried out at 30° C. or lower.

5. 2. The method according to claim 1, wherein the step (3) is carried out by any one of the following methods: polymerase chain reaction (PCR) and isothermal nucleic acid amplification.

6. 6. The method of claim 5, wherein step (3) is carried out by polymerase chain reaction (PCR).

7. The method according to claim 1, wherein the sample is at least one selected from the group consisting of feces, bile, urine, throat swabs, nasal swabs, sputum, pulmonary aspirates, cerebrospinal fluid, gargle, saliva, tears, blood, cultured cells, culture fluid, food, river water, rainwater, seawater, sewage, bath water, hot spring water, pool water, soil, and samples collected from the environment.

8. The method of claim 1, wherein the virus is an enveloped or non-enveloped RNA virus.

9. The method of claim 8, wherein the enveloped virus is an influenza virus.

10. The method of claim 8, wherein the non-enveloped virus is a Norovirus.

11. 2. The method of claim 1, wherein the alkali is at least one selected from the group consisting of potassium hydroxide, sodium hydroxide, lithium hydroxide, magnesium hydroxide, calcium hydroxide, barium hydroxide, potassium carbonate, sodium carbonate, magnesium carbonate and calcium carbonate, or a solution of said at least one alkali, or is a Tris buffer, a glycine buffer, a phosphate buffer, a borate buffer or a Good's buffer.

12. The method according to claim 1, wherein the concentration of the alkali in the nucleic acid extract is 0.01N to 2N.

13. 2. The method of claim 1, wherein the chelating agent is at least one selected from the group consisting of ethylenediaminetetraacetic acid (EDTA), cyclohexanediaminetetraacetic acid (CDTA), glycoletherdiaminetetraacetic acid (EGTA), nitrilotriacetic acid (NTA), diethylenetriaminepentaacetic acid (DTPA), hydroxyethylethylenediaminetriacetic acid (HEDTA), triethylenetetraaminehexaacetic acid (TTHA), glutamic acid diacetic acid (GLDA), dihydroxyethylglycine (DHEG), hydroxyethanediphosphonic acid (HEDP), methylenephosphonic acid (NTMP), phosphonobutanetricarboxylic acid (PBTC), ethylenediaminetetramethylenephosphonic acid (EDTMP), citric acid, oxalic acid, phytic acid, and gluconic acid.

14. The method according to claim 1, wherein the concentration of the chelating agent in the nucleic acid extract is 0.5 mM to 100 mM.

15. (a) containing an alkali or a solution thereof and a chelating agent or a solution thereof, or (b) containing alkali and chelating agents or their solutions; A nucleic acid extraction kit for extracting nucleic acid from a sample that may contain a virus without heat treatment.

16. A kit for detecting a virus for use in the method according to claim 1, comprising: (a) comprising an alkali or a solution thereof, a chelating agent or a solution thereof, and a composition for amplifying nucleic acid; (b) A kit comprising an alkali and a chelating agent or a solution thereof and a composition for amplifying nucleic acids.

17. The kit according to claim 15 or 16, wherein step (1) is carried out at room temperature.

18. The kit according to claim 17, wherein step (1) is carried out at 30°C or lower.

19. The kit according to claim 16, wherein the composition for amplifying nucleic acids comprises a thermostable DNA polymerase.

20. 17. The kit of claim 16, further comprising a hybridization probe corresponding to a detection region of the nucleic acid.

21. The kit according to claim 15 or 16, wherein the virus is an enveloped or non-enveloped RNA virus.

22. The kit of claim 21 , wherein the enveloped virus is an influenza virus.

23. The kit of claim 21 , wherein the non-enveloped virus is a Norovirus.

24. The kit according to claim 15 or 16, wherein the alkali is an aqueous solution of potassium hydroxide, an aqueous solution of sodium hydroxide, an aqueous solution of lithium hydroxide, an aqueous solution of magnesium hydroxide, an aqueous solution of calcium hydroxide, an aqueous solution of barium hydroxide, an aqueous solution of potassium carbonate, an aqueous solution of sodium carbonate, an aqueous solution of magnesium carbonate, an aqueous solution of calcium carbonate, a Tris buffer, a glycine buffer, a phosphate buffer, a borate buffer, or a Good's buffer.

25. The kit according to claim 16, wherein the concentration of the alkali in the nucleic acid extract is 0.01N to 2N.

26. 17. The kit of claim 15 or 16, wherein the chelating agent is ethylenediaminetetraacetic acid (EDTA), cyclohexanediaminetetraacetic acid (CDTA), glycoletherdiaminetetraacetic acid (EGTA), nitrilotriacetic acid (NTA), diethylenetriaminepentaacetic acid (DTPA), hydroxyethylethylenediaminetriacetic acid (HEDTA), triethylenetetraaminehexaacetic acid (TTHA), glutamic acid diacetic acid (GLDA), dihydroxyethylglycine (DHEG), hydroxyethanediphosphonic acid (HEDP), methylenephosphonic acid (NTMP), phosphonobutanetricarboxylic acid (PBTC), ethylenediaminetetramethylenephosphonic acid (EDTMP), citric acid, oxalic acid, phytic acid, gluconic acid.

27. The kit according to claim 16, wherein the concentration of the chelating agent in the nucleic acid extract after mixing with the sample is 0.5 mM to 100 mM.

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