Methods for extracting nucleic acid from bacteria

Abstract

To provide easier methods and kits for extracting and detecting nucleic acid derived from bacteria in samples without heat processing.SOLUTION: A method for detecting nucleic acid in a sample which may contain bacteria, 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 bacteria, and more particularly to a method and a kit for detecting nucleic acids originating from bacteria 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 acids can be extracted from bacteria 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 bacteria, 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 bacterium is a gram-negative bacterium. [Section 9] Item 9. The method according to item 8, wherein the bacterium is selected from Salmonella, Shigella, and Escherichia coli. [Section 10] Item 10. The method according to any one of Items 1 to 9, 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 11] Item 11. The method according to any one of Items 1 to 10, wherein the concentration of the alkali in the nucleic acid extract is 0.02N to 2N. [Section 12] The method according to any one of items 1 to 11, 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 13] Item 13. The method according to any one of Items 1 to 12, wherein the concentration of the chelating agent in the nucleic acid extract is 0.5 mM to 100 mM. [Section 14] (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 bacteria without heat treatment. [Section 15] A kit for detecting bacteria 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 16] Item 16. The kit according to item 14 or 15, wherein step (1) is carried out at room temperature. [Section 17] Item 17. The kit according to any one of Items 14 to 16, wherein the step (1) is carried out at 30° C. or lower. [Section 18] Item 18. The kit according to any one of Items 15 to 17, wherein the composition for amplifying nucleic acid contains a thermostable DNA polymerase. [Section 19] Item 19. The kit according to any one of Items 15 to 18, further comprising a hybridization probe corresponding to a detection region of the nucleic acid. [Section 20] 20. The kit according to any one of Items 14 to 19, wherein the bacterium is a gram-negative bacterium. [Section 21] Item 21. The kit according to item 20, wherein the bacterium is selected from Salmonella, Shigella, and Escherichia coli. [Section 22] Item 22. The kit according to any one of Items 14 to 21, 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. [Section 23] 23. The kit according to any one of items 15 to 22, wherein the concentration of the alkali in the nucleic acid extract is 0.02N to 2N. [Section 24] 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 14 to 23. [Section 25] 25. The kit according to any one of Items 15 to 24, 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] (Method of detecting bacteria) One aspect of the invention is a method for detecting bacterial nucleic acid that may be present in a sample. The nucleic acid detection method of the present invention includes the step of (1) contacting a sample that may contain bacteria 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 bacteria, and examples thereof include samples derived from specimens discharged from living organisms. The specimens discharged 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 discharged from living organisms that have been suspended in water (sterile water, etc.), physiological saline, buffer solutions, organic solvents, liquid media, or gel media (e.g., Calibrea medium), and those that have been subjected to some kind of treatment, such as the supernatant prepared from the suspension. The buffer solutions are not particularly limited, and examples thereof include Hanks' buffer solution, Tris buffer solution, phosphate buffer solution, glycine buffer solution, HEPES buffer solution, and Tricine buffer solution. The living organisms include humans, pets, livestock, wild animals, insects, and all other animals.

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

[0015] Another embodiment of the sample of the present invention is a sample that may contain cultured cells or culture fluid that may contain bacteria. 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 bacteria. 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 bacteria. 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 bacteria, 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 grasping 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. The treatment may include precipitation, centrifugation, filtration, dilution, and addition of a substance that adsorbs bacteria. 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 bacteria" to be applied to the method of the present invention is not particularly limited, but in the case of a specimen discharged from a living organism that is solid or viscous, such as feces, it is preferable to handle the specimen as a suspension of the specimen discharged from a living organism prepared in advance. The suspension preferably contains a specimen discharged 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, the suspension is 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 specimens of X species (X represents an integer of 2 or more). For example, when the specimen is feces, the specimen of X species may be feces from an individual of X species, or may be feces of X species excreted by the same individual. X is not particularly limited, but for example, in the case of a stool test targeting three bacterial species (Salmonella, Shigella, and Enterohemorrhagic Escherichia coli), it is common in the food hygiene testing industry to mix 50 specimens, which can be used as a reference.

[0021] The bacteria to be detected in the present invention are not particularly limited, but include gram-negative bacteria and gram-positive bacteria. Examples of gram-positive bacteria include staphylococci, streptococci, pneumococci, mycoplasma, listeria, diphtheria, anthrax, botulinum, tetanus, acid-fast bacteria, tuberculosis, Bacillus cereus, Clostridium perfringens, lactobacillus, streptococci, and enterococci. Examples of gram-negative bacteria include salmonella, shigella, Escherichia coli, gonococci, meningococci, typhoid bacillus, Klebsiella pneumoniae, pertussis bacillus, cholera bacillus, campylobacter, Pseudomonas aeruginosa, bacteroides, spirochetes, leptospira, borrelia, chlamydia, and rickettsia, and are particularly useful for detecting salmonella, shigella, and Escherichia coli, but are not limited thereto. Escherichia coli may be non-pathogenic or pathogenic. Pathogenic E. coli includes diarrheagenic E. coli, such as enteropathogenic E. coli (EPEC), enteroinvasive E. coli (EIEC), enterotoxigenic E. coli (ETEC), enteroaggregative E. coli (EAEC), and enterohemorrhagic E. coli (EHEC), as well as extraintestinal pathogenic E. coli, such as uropathogenic E. coli.

[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 bacteria. Examples of the 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 0.02N 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 extracting agent is not particularly limited as long as it is a concentration that can extract nucleic acid 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 1 mM or more, and even more preferably 5 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 100 mM or less, more preferably 80 mM or less, and even more preferably 50 mM or less.

[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 100 mM, preferably 1 mM to 50 mM, more preferably 2 mM to 30 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 bacteria 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 the bacteria-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 the bacteria-containing sample, the risk of infection to the operator can be reduced. Furthermore, by not performing heat treatment, the decomposition of DNA due to heat treatment 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 bacteria 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 methods 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 may be performed at a temperature suitable for the selected isothermal amplification reaction method. In the case of PCR, one cycle is made up of three steps: DNA denaturation by heat treatment, annealing of a primer to a template single-stranded DNA, and extension using DNA polymerase, and this cycle is repeated to amplify. The cycle conditions are not particularly limited, and amplification is performed in two steps at the same temperature for annealing and extension, 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] (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 bacteria 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; a chelating agent or a solution thereof; and a composition for amplifying a 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.

[0045] 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.

[0046] 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.

[0047] 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.

[0048] 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.

[0049] 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.

[0050] 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.

[0051] 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.

[0052] 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, the solution may contain a surfactant such as Triton X-100, Triton X-114, Tween 20, Nonidet P40, or Briji 58, and may further 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) to reduce reaction inhibition. 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.

[0053] 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.

[0054] 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 bacterial subtypes by labeling each hybridization probe corresponding to a bacterial subtype with a different fluorescent dye. Furthermore, it is also possible to simultaneously identify and determine the presence or absence of bacteria in one reaction solution by labeling each hybridization probe corresponding to nucleic acids derived from multiple bacteria with a different fluorescent dye.

[0055] The double-stranded DNA-binding fluorescent compound is not particularly limited, and includes 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 acid 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. Many intercalators are known in the art. Examples of such dyes include, but are 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, and FUN-1.

[0056] Examples of hybridization probes include TaqMan (registered trademark) 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 (International Publication No. WO 97 / 46707, WO 97 / 46712, WO 97 / 46714), Q probe, and Scorpions (registered trademark) 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

[0057] 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.

[0058] Example 1: Confirmation of nucleic acid extraction effect by extractant The extraction efficiency was compared between extraction using only alkaline solution and extraction using alkaline solution with the addition of a chelating agent. The presence or absence of amplification was confirmed using the centrifugal supernatant of the nucleic acid extract as a template to determine whether DNA was extracted from the bacteria. Since the bacteria precipitate when centrifuged, it is believed that bacterial DNA will be detected in the centrifugal supernatant only if DNA is extracted.

[0059] (Preparation of Extractant) An extractant having the following composition (twice the final concentration of the nucleic acid extract) was prepared. Condition 1-1 0.5N NaOH Condition 1-2 0.5N NaOH + 20mM EDTA Condition 2-1 0.2N NaOH Condition 2-2 0.2N NaOH + 20mM EDTA Condition 3-1 20mM EDTA

[0060] (Pretreatment of sample) 10 μL of the extractant was mixed with 10 μL of a water suspension of Salmonella at room temperature (25° C.). The nucleic acid extract obtained by mixing was centrifuged at 12,000 rpm for 5 minutes at 4° C., and the supernatant was used as a sample.

[0061] (Preparation of nucleic acid amplification reaction solution) The Enterobacteriaceae Gene Detection Kit - Probe Detection - (Toyobo Co., Ltd. FIK-351) was used as the PCR reaction reagent, and the following volumes were prepared per reaction. PCR Master Mix 14.0μL 10x Primer Mix 2.0μL 10x Probe Mix 2.0μL To the prepared reaction solution (18.0 μL per reaction), 2 μL of the centrifuged supernatant was added. N=3 was performed for each nucleic acid extract.

[0062] (PCR reaction conditions) 20 μL of the reaction solution was reacted using Bio-Rad's CFX96 DEEP WELL under the following temperature cycle. 96℃, 20 seconds (initial denaturation) 96℃, 2 sec - 63℃, 20 sec 10 cycles 96℃, 2 sec - 63℃, 20 sec (fluorescence reading) 35 cycles

[0063] The measurement results were calculated using Bio-Rad's CFX96 analysis software with a threshold value of 30 to calculate the Ct value, and samples from which the Ct value was calculated were determined to be samples from which DNA had been extracted. As a result, no Ct value was calculated with only an alkaline solution or only an EDTA solution, and DNA was not extracted from the Salmonella bacteria. On the other hand, when an extractant containing alkali and EDTA was used, DNA amplification was confirmed, and it was confirmed that DNA could be extracted from the Salmonella bacteria.

[0064] [Table 1]

[0065] Example 2: Comparison with the heat extraction method The extraction method using an extractant was compared with the heat extraction method. The same criteria as in Example 1 were used to determine whether DNA was extracted from bacteria.

[0066] (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

[0067] (Pretreatment of sample) 10 μL of the extractant was mixed with 10 μL of a water suspension of Salmonella at room temperature (25° C.). The nucleic acid extract obtained by mixing was centrifuged at 12,000 rpm for 5 minutes at 4° C., and the supernatant was used as a sample. For the heat-treated sample, 10 μL of sterile water was added to 10 μL of the water suspension of Salmonella, and then heat-treated at 95° C. for 5 minutes, and centrifuged at 12,000 rpm for 5 minutes at 4° C., and the supernatant was used as a sample. Furthermore, as a sample not subjected to heat treatment, a sample in which 10 μL of sterile water was added to 10 μL of the water suspension of Salmonella was centrifuged at 12,000 rpm for 5 minutes at 4° C., and the supernatant was used as a sample.

[0068] (Preparation of nucleic acid amplification reaction solution) The Enterobacteriaceae Gene Detection Kit - Probe Detection - (Toyobo Co., Ltd. FIK-351) was used as the PCR reaction reagent, and the following volumes were prepared per reaction. PCR Master Mix 14.0μL 10x Primer Mix 2.0μL 10x Probe Mix 2.0μL To the prepared reaction solution (18.0 μL per reaction), 2 μL of the centrifuged supernatant was added. N=3 was performed for each nucleic acid extract.

[0069] (PCR reaction conditions) 20 μL of the reaction solution was reacted using Bio-Rad's CFX96 DEEP WELL under the following temperature cycle. 96℃, 20 seconds (initial denaturation) 96℃, 2 sec - 63℃, 20 sec 10 cycles 96℃, 2 sec - 63℃, 20 sec (fluorescence reading) 35 cycles

[0070] The measurement results were calculated using Bio-Rad's CFX96 analysis software with a threshold value of 30 to calculate the Ct value, and samples from which the Ct value was calculated were determined to be samples from which DNA had been extracted. As a result, it was confirmed that DNA can be extracted in the same way as with heat treatment, which requires complicated operations, by using an extractant containing alkali and a chelating agent. Furthermore, it was confirmed that the Ct value was about Ct=3 faster than when extracted by heat treatment, allowing for more sensitive detection.

[0071] [Table 2]

[0072] Example 3: Confirmation of chelate concentration capable of DNA extraction The extraction rate was confirmed by changing the concentration of the chelating agent. Whether DNA was extracted from the bacteria was judged according to the same criteria as in Example 1.

[0073] (Preparation of Extractant) An extractant having the following composition (twice the final concentration of the nucleic acid extract) was prepared. Condition 1 sterile water Condition 2 0.2N NaOH + 0.5mM EDTA Condition 3 0.2N NaOH + 1mM EDTA Condition 4 0.2N NaOH + 2mM EDTA Condition 5 0.2N NaOH + 5mM EDTA Condition 6 0.2N NaOH + 10mM EDTA Condition 7 0.2N NaOH + 40mM EDTA Condition 8 0.2N NaOH + 50mM EDTA Condition 9 0.2N NaOH + 80mM EDTA Condition 10 0.2N NaOH + 100mM EDTA

[0074] (Pretreatment of sample) 10 μL of the extractant was mixed with 10 μL of a water suspension of Salmonella at room temperature (25° C.). The nucleic acid extract obtained by mixing was centrifuged at 12,000 rpm for 5 minutes at 4° C., and the supernatant was used as a sample.

[0075] (Preparation of nucleic acid amplification reaction solution) The Enterobacteriaceae Gene Detection Kit - Probe Detection - (Toyobo Co., Ltd. FIK-351) was used as the PCR reaction reagent, and the following volumes were prepared per reaction. PCR Master Mix 14.0μL 10x Primer Mix 2.0μL 10x Probe Mix 2.0μL To the prepared reaction solution (18.0 μL per reaction), 2 μL of the centrifuged supernatant was added. N=3 was performed for each nucleic acid extract.

[0076] (PCR reaction conditions) 20 μL of the reaction solution was reacted using Bio-Rad's CFX96 DEEP WELL under the following temperature cycle. 96℃, 20 seconds (initial denaturation) 96℃, 2 sec - 63℃, 20 sec 10 cycles 96℃, 2 sec - 63℃, 20 sec (fluorescence reading) 35 cycles

[0077] The measurement results were calculated using Bio-Rad's CFX96 analysis software with a threshold value of 30 to calculate the Ct value, and the samples from which the Ct value was calculated were determined to be samples from which DNA had been extracted. As a result, it was confirmed that DNA was extracted if the chelating agent concentration after sample mixing was 0.5 mM or higher.

[0078] [Table 3]

[0079] Example 4: Confirmation of the alkali concentration that allows DNA extraction The extraction rate was confirmed by changing the alkali concentration. Whether DNA was extracted from the bacteria was judged according to the same criteria as in Example 1.

[0080] (Preparation of Extractant) An extractant having the following composition (twice the final concentration of the nucleic acid extract) was prepared. Condition 1 sterile water Condition 2 20mM EDTA Condition 3 0.001N NaOH + 20mM EDTA Condition 4 0.05N NaOH + 20mM EDTA Condition 5 0.1N NaOH + 20mM EDTA Condition 6 0.5N NaOH + 20mM EDTA Condition 7 1N NaOH + 20mM EDTA

[0081] (Pretreatment of sample) 10 μL of the extractant was mixed with 10 μL of a water suspension of Salmonella at room temperature (25° C.). The nucleic acid extract obtained by mixing was centrifuged at 12,000 rpm for 5 minutes at 4° C., and the supernatant was used as a sample.

[0082] (Preparation of nucleic acid amplification reaction solution) The Enterobacteriaceae Gene Detection Kit - Probe Detection - (Toyobo Co., Ltd. FIK-351) was used as the PCR reaction reagent, and the following volumes were prepared per reaction. PCR Master Mix 14.0μL 10x Primer Mix 2.0μL 10x Probe Mix 2.0μL To the prepared reaction solution (18.0 μL per reaction), 2 μL of the centrifuged supernatant was added. N=3 was performed for each nucleic acid extract.

[0083] (PCR reaction conditions) 20 μL of the reaction solution was reacted using Bio-Rad's CFX96 DEEP WELL under the following temperature cycle. 96℃, 20 seconds (initial denaturation) 96℃, 2 sec - 63℃, 20 sec 10 cycles 96℃, 2 sec - 63℃, 20 sec (fluorescence reading) 35 cycles

[0084] The measurement results were calculated using Bio-Rad's CFX96 analysis software with a threshold value of 30 to calculate the Ct value, and samples from which the Ct value was calculated were determined to be samples from which DNA had been extracted. As a result, it was confirmed that DNA could be extracted if the alkali concentration after sample mixing was 0.025N or higher.

[0085] [Table 4]

[0086] Example 5: Extraction confirmation when the type of alkali is changed When NaOH or KOH was selected as the alkali, it was verified whether DNA could be extracted from bacteria. The same criteria as in Example 1 were used to determine whether DNA was extracted from bacteria.

[0087] (Preparation of Extractant) An extractant having the following composition (twice the final concentration of the nucleic acid extract) was prepared. Condition 1 0.3N KOH + 30mM EDTA Condition 2 0.3N NaOH + 30mM EDTA

[0088] (Pretreatment of sample) 10 μL of the extractant was mixed with 10 μL of a water suspension of Salmonella at room temperature (25° C.). The nucleic acid extract obtained by mixing was centrifuged at 12,000 rpm for 5 minutes at 4° C., and the supernatant was used as a sample.

[0089] (Preparation of nucleic acid amplification reaction solution) The Enterobacteriaceae Gene Detection Kit - Probe Detection - (Toyobo Co., Ltd. FIK-351) was used as the PCR reaction reagent, and the following volumes were prepared per reaction. PCR Master Mix 14.0μL 10x Primer Mix 2.0μL 10x Probe Mix 2.0μL To the prepared reaction solution (18.0 μL per reaction), 2 μL of the centrifuged supernatant was added. N=3 was performed for each nucleic acid extract.

[0090] (PCR reaction conditions) 20 μL of the reaction solution was reacted using Bio-Rad's CFX96 DEEP WELL under the following temperature cycle. 96℃, 20 seconds (initial denaturation) 96℃, 2 sec - 63℃, 20 sec 10 cycles 96℃, 2 sec - 63℃, 20 sec (fluorescence reading) 35 cycles

[0091] The measurement results were calculated using Bio-Rad's CFX96 analysis software with a threshold value of 30 to calculate Ct values, and samples from which Ct values ​​were calculated were determined to be samples from which DNA had been extracted. As a result, it was confirmed that DNA could be extracted when KOH was used as the alkali, similar to the case of NaOH.

[0092] [Table 5]

[0093] Example 6: Extraction confirmation when the type of chelating agent is changed A chelating agent was added to 0.2N NaOH to verify whether DNA could be extracted from bacteria. The same criteria as in Example 1 were used to determine whether DNA was extracted from bacteria.

[0094] (Preparation of Extractant) An extractant having the following composition (twice the final concentration of the nucleic acid extract) was prepared. Condition 1 0.2N NaOH Condition 2 0.2N NaOH + 50mM Cyclohexanediaminetetraacetic acid (CDTA) Condition 3 0.2N NaOH + 50mM diethylenetriaminepentaacetic acid (DTPA) Condition 4 0.2N NaOH + 20mM Triethylenetetraminehexaacetic acid (TTHA)

[0095] (Pretreatment of sample) 10 μL of the extractant was mixed with 10 μL of a water suspension of Salmonella at room temperature (25° C.). The nucleic acid extract obtained by mixing was centrifuged at 12,000 rpm for 5 minutes at 4° C., and the supernatant was used as a sample.

[0096] (Preparation of nucleic acid amplification reaction solution) The Enterobacteriaceae Gene Detection Kit - Probe Detection - (Toyobo Co., Ltd. FIK-351) was used as the PCR reaction reagent, and the following volumes were prepared per reaction. PCR Master Mix 14.0μL 10x Primer Mix 2.0μL 10x Probe Mix 2.0μL To the prepared reaction solution (18.0 μL per reaction), 2 μL of the centrifuged supernatant was added. N=2 was performed for each nucleic acid extract.

[0097] (PCR reaction conditions) 20 μL of the reaction solution was reacted using Bio-Rad's CFX96 DEEP WELL under the following temperature cycle. 96℃, 20 seconds (initial denaturation) 96℃, 2 sec - 63℃, 20 sec 10 cycles 96℃, 2 sec - 63℃, 20 sec (fluorescence reading) 35 cycles

[0098] The measurement results were calculated using Bio-Rad's CFX96 analysis software with a threshold value of 30 to calculate the Ct value, and samples from which the Ct value was calculated were determined to be samples from which DNA had been extracted. As a result, it was confirmed that DNA could not be extracted using NaOH alone, but that DNA could be extracted by adding a chelating agent.

[0099] [Table 6]

[0100] Example 7: Confirmation of DNA extraction from inactivated Salmonella and inactivated Shigella It was confirmed whether DNA could be extracted from Shigella as well as Salmonella. The same criteria as in Example 1 were used to determine whether DNA was extracted from bacteria.

[0101] (Preparation of Extractant) An extractant having the following composition (twice the final concentration of the nucleic acid extract) was prepared. Condition 1 0.2N NaOH + 20mM EDTA Condition 2 sterile water

[0102] (Pretreatment of sample) 10 μL of the extractant was mixed with 10 μL of inactivated Salmonella (NATtrol Salmonella enterica typhimurium (Stool Matrix) / ZeptoMetrix) or inactivated Shigella (NATtrol Shigella sonnei (Stool Matrix) / ZeptoMetrix) at room temperature (25°C). The nucleic acid extract obtained by mixing was centrifuged at 12,000 rpm for 5 minutes at 4°C, and the supernatant was used as a sample.

[0103] (Preparation of nucleic acid amplification reaction solution) The Enterobacteriaceae Gene Detection Kit - Probe Detection - (Toyobo Co., Ltd. FIK-351) was used as the PCR reaction reagent, and the following volumes were prepared per reaction. PCR Master Mix 14.0μL 10x Primer Mix 2.0μL 10x Probe Mix 2.0μL To the prepared reaction solution (18.0 μL per reaction), 2 μL of the centrifuged supernatant was added. N=1 was performed for each nucleic acid extract.

[0104] (PCR reaction conditions) 20 μL of the reaction solution was reacted using Bio-Rad's CFX96 DEEP WELL under the following temperature cycle. 96℃, 20 seconds (initial denaturation) 96℃, 2 sec - 63℃, 20 sec 10 cycles 96℃, 2 sec - 63℃, 20 sec (fluorescence reading) 35 cycles

[0105] The measurement results were calculated using Bio-Rad's CFX96 analysis software with a threshold value of 30 to calculate Ct values, and samples from which Ct values ​​were calculated were determined to be samples from which DNA had been extracted. As a result, it was confirmed that DNA could be extracted from Shigella as well as Salmonella.

[0106] [Table 7]

[0107] Example 8: Confirmation of stability of extracted DNA We confirmed whether the extracted DNA was stable at room temperature, 4°C, and -20°C. We compared the Ct values ​​of the extracted DNA immediately after mixing the extractant with the sample and the DNA stored at room temperature, 4°C, and -20°C for one day.

[0108] (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

[0109] (Pretreatment of sample) 50 μL of extraction agent was mixed with 50 μL of a water suspension of Salmonella at room temperature (25°C). 20 μL was dispensed into four tubes, and one tube was immediately centrifuged at 12,000 rpm for 5 minutes at 4°C to obtain the supernatant as a sample. The remaining three tubes were stored at room temperature, 4°C, and -20°C for one day, and then centrifuged at 12,000 rpm for 5 minutes at 4°C to obtain the supernatant as a sample.

[0110] (Preparation of nucleic acid amplification reaction solution) The Enterobacteriaceae Gene Detection Kit - Probe Detection - (Toyobo Co., Ltd. FIK-351) was used as the PCR reaction reagent, and the following volumes were prepared per reaction. PCR Master Mix 14.0μL 10x Primer Mix 2.0μL 10x Probe Mix 2.0μL To the prepared reaction solution (18.0 μL per reaction), 2 μL of the centrifuged supernatant was added. N=3 was performed for each nucleic acid extract.

[0111] (PCR reaction conditions) 20 μL of the reaction solution was reacted using Bio-Rad's CFX96 DEEP WELL under the following temperature cycle. 96℃, 20 seconds (initial denaturation) 96℃, 2 sec - 63℃, 20 sec 10 cycles 96℃, 2 sec - 63℃, 20 sec (fluorescence reading) 35 cycles

[0112] The measurement results were calculated using Bio-Rad's CFX96 analysis software with a threshold value of 30 as the Ct value. As a result, no decrease in the Ct value was confirmed when DNA-extracted samples stored at room temperature, 4°C, or -20°C were used, and detection was possible.

[0113] [Table 8]

[0114] Example 9: Confirmation of DNA extraction from fecal positive samples DNA was extracted from bacteria contained in fecal samples, and detection by nucleic acid amplification reaction was compared with an extraction method using heat treatment.

[0115] (Preparation of Extractant) An extractant having the following composition (twice the final concentration of the nucleic acid extract) was prepared. 0.3N NaOH + 30mM EDTA

[0116] (Pretreatment of sample) Three Salmonella-positive feces samples and three Enterohemorrhagic E. coli-positive feces samples were suspended in 50 μL of sterile water and then dispensed into 25 μL portions. 25 μL of sterile water was added to one portion and heat-treated at 95°C for 5 minutes. One portion was used as a sample without centrifugation, and the remaining portion was centrifuged at 12,000 rpm for 5 minutes at 4°C, and the supernatant was used as a sample. 25 μL of extractant was added to the other portion, and one portion was used as a sample without centrifugation, and the remaining portion was centrifuged at 12,000 rpm for 5 minutes at 4°C, and the supernatant was used as a sample.

[0117] (Preparation of nucleic acid amplification reaction solution) The Enterobacteriaceae Gene Detection Kit - Probe Detection - (Toyobo Co., Ltd. FIK-351) was used as the PCR reaction reagent, and the following volumes were prepared per reaction. PCR Master Mix 14.0μL 10x Primer Mix 2.0μL 10x Probe Mix 2.0μL To the prepared reaction solution (18.0 μL per reaction), 2 μL of the extracted DNA that had been treated as described above was added.

[0118] (PCR reaction conditions) 20 μL of the reaction solution was reacted using Bio-Rad's CFX96 DEEP WELL under the following temperature cycle. 96℃, 20 seconds (initial denaturation) 96℃, 2 sec - 63℃, 20 sec 10 cycles 96℃, 2 sec - 63℃, 20 sec (fluorescence reading) 35 cycles

[0119] The measurement results were compared with the heat treatment method by calculating the Ct value using Bio-Rad's CFX96 analysis software with a threshold value of 30. As a result, it was confirmed that DNA was extracted in the same way as with heat treatment when the extractant was used, and that it could be amplified and detected by the nucleic acid amplification reaction. Furthermore, when comparing the detected Ct values, the result showed that the rise (Ct value) was faster when the extractant was used compared to the heat treatment method. By eliminating the heat treatment process, there was no concern about DNA degradation due to heat treatment, and it is thought that the detected Ct value was faster when the extractant was used. In addition, it was confirmed that amplification and detection was possible not only with the supernatant of the nucleic acid extract, but also under conditions without centrifugation, in which the feces itself was brought into the nucleic acid amplification reaction system as a sample.

[0120] [Table 9]

[0121] Example 10: Stability confirmation of nucleic acid extract of DNA containing feces To confirm whether DNA extracted from bacteria contained in fecal samples was stable, we checked samples stored for one day after DNA extraction.

[0122] (Sample preparation) The nucleic acid extract prepared in Example 9 was stored at room temperature for one day and used as a sample.

[0123] (Preparation of nucleic acid amplification reaction solution) The Enterobacteriaceae Gene Detection Kit - Probe Detection - (Toyobo Co., Ltd. FIK-351) was used as the PCR reaction reagent, and the following volumes were prepared per reaction. PCR Master Mix 14.0μL 10x Primer Mix 2.0μL 10x Probe Mix 2.0μL To the prepared reaction solution (18.0 μL per reaction), 2 μL of the nucleic acid extract stored for one day was added.

[0124] (PCR reaction conditions) 20 μL of the reaction solution was reacted using Bio-Rad's CFX96 DEEP WELL under the following temperature cycle. 96℃, 20 seconds (initial denaturation) 96℃, 2 sec - 63℃, 20 sec 10 cycles 96℃, 2 sec - 63℃, 20 sec (fluorescence reading) 35 cycles

[0125] The measurement results were calculated using Bio-Rad's CFX96 analysis software with a threshold value of 30 as the Ct value, and compared with the Ct value of Example 9. As a result, amplification was confirmed in all samples even after one day at room temperature, and no decrease in Ct value was confirmed. This result shows that the extracted DNA, including fecal samples, can be used even after one day of storage at room temperature.

[0126] [Table 10] [Industrial Applicability]

[0127] According to the present invention, it is possible to easily and sensitively detect nucleic acids derived from bacteria contained in a sample. 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 bacteria, 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 bacterium is a gram-negative bacterium.

9. 9. The method of claim 8, wherein the bacteria is selected from Salmonella, Shigella, and Escherichia coli.

10. 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.

11. The method according to claim 1, wherein the concentration of the alkali in the nucleic acid extract is 0.02N to 2N.

12. 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.

13. 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.

14. (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 bacteria without heat treatment.

15. A kit for detecting bacteria 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.

16. The kit according to claim 14 or 15, wherein step (1) is carried out at room temperature.

17. The kit according to claim 16, wherein step (1) is carried out at 30°C or lower.

18. The kit according to claim 15, wherein the composition for amplifying nucleic acids comprises a thermostable DNA polymerase.

19. 16. The kit of claim 15, further comprising a hybridization probe corresponding to a detection region of the nucleic acid.

20. The kit according to claim 14 or 15, wherein the bacterium is a gram-negative bacterium.

21. 21. The kit of claim 20, wherein the bacteria is selected from Salmonella, Shigella, and Escherichia coli.

22. The kit according to claim 14 or 15, 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.

23. The kit according to claim 15, wherein the concentration of the alkali in the nucleic acid extract is 0.02N to 2N.

24. 16. The kit of claim 14 or 15, 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.

25. The kit according to claim 15, 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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