Nucleic acid extraction method

Abstract

To efficiently extract a nucleic acid included in a sample.SOLUTION: A nucleic acid extraction method includes adding achromopeptidase to a sample containing cells as an enzyme to promote cell lysis, incubating the sample with the achromopeptidase added thereto, at a predetermined temperature, adding a lysis aid, which aids in cell lysis, to the sample that has been incubated for a predetermined duration, heating the sample with the lysis aid added thereto up to a predetermined temperature, injecting the sample that has been heated for a predetermined duration into a column that contains silica and silicon carbide as adsorption carriers to adsorb nucleic acids, and purifying the nucleic acids.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] The present invention relates to a method for extracting nucleic acids. [Background technology]

[0002] Conventionally, there is a technique for extracting nucleic acids from bacterial cells by collecting a bacterial culture solution, adding a solubilizing agent, and subjecting the solution to high-temperature, high-pressure treatment. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] Patent No. 5624487 Summary of the Invention [Problem to be solved by the invention]

[0004] However, with conventional techniques, it is difficult to efficiently extract nucleic acids contained in samples. For example, with conventional techniques, the amount of nucleic acid recovered from bacteria with a strong cell surface, such as Gram-positive bacteria, is small, and it takes a long time to extract nucleic acids.

[0005] The present invention has been made in view of the above, and has as its object to efficiently extract nucleic acids contained in a sample. [Means for solving the problem]

[0006] The present invention provides a nucleic acid extraction method for extracting nucleic acids from cells, comprising: an incubation step of adding achromopeptidase (registered trademark) to a sample containing the cells as an enzyme that promotes lysis of the cells, and keeping the sample with the achromopeptidase added at a predetermined temperature; and a heating step of adding a solubilizing agent that promotes lysis of the cells to the sample that has been kept warm for a predetermined time in the incubation step, and heating the sample with the solubilizing agent added to it to the predetermined temperature.

[0007] The present invention also provides a nucleic acid extraction method for extracting nucleic acids from cells, comprising: a heating step of adding a solubilizing agent that promotes lysis of the cells to a sample containing the cells, and heating the sample to which the solubilizing agent has been added to a predetermined temperature; and a purification step of injecting the sample that has been heated for a predetermined time in the heating step into a column containing silica and silicon carbide as adsorption carriers that adsorb the nucleic acids, and purifying the nucleic acids. [Effects of the Invention]

[0008] According to the present invention, there is an effect that nucleic acids contained in a sample can be efficiently extracted. [Brief explanation of the drawings]

[0009] [Figure 1] FIG. 1 is a diagram showing a configuration example of a nucleic acid extraction system according to an embodiment. [Figure 2] FIG. 10 is a diagram for explaining Experimental Result 1 according to the embodiment. [Figure 3] FIG. 10 is a diagram for explaining Experimental Result 2 according to the embodiment. [Figure 4] FIG. 10 is a diagram for explaining Experimental Result 3 according to the embodiment. [Figure 5] FIG. 10 is a diagram for explaining Experimental Result 4 according to the embodiment. [Figure 6] FIG. 10 is a diagram for explaining Experimental Result 5 according to the embodiment. [Figure 7] 1 is a flowchart showing an example of the flow of a nucleic acid extraction step according to an embodiment. [Figure 8] 10 is a flowchart showing an example of the flow of a collection process according to the embodiment. [Figure 9] 10 is a flowchart showing an example of the flow of a heat retention process according to the embodiment. [Figure 10] 10 is a flowchart showing an example of the flow of an encapsulation process according to the embodiment. [Figure 11] 10 is a flowchart showing an example of the flow of a heating step according to the embodiment. [Figure 12]1 is a flowchart showing an example of the flow of a purification process according to an embodiment. DETAILED DESCRIPTION OF THE INVENTION

[0010] A nucleic acid extraction method according to one embodiment of the present invention will be described in detail below with reference to the drawings. However, the present invention is not limited to the embodiment described below.

[0011] [Embodiment] The configuration of the nucleic acid extraction system according to the embodiment, the details of each step, and the flow of each step will be explained below in order, and finally, the effects of the embodiment will be explained.

[0012] 1. Configuration of the nucleic acid extraction system 100 The configuration of a nucleic acid extraction system 100 according to an embodiment will be described using Figure 1. Figure 1 is a diagram showing an example of the configuration of a nucleic acid extraction system 100 according to an embodiment. Below, an example of the overall configuration of the nucleic acid extraction system 100, each process of the nucleic acid extraction system 100, and the effects of the nucleic acid extraction system 100 will be described in that order.

[0013] (1-1. Example of the overall configuration of the nucleic acid extraction system 100) The nucleic acid extraction system 100 includes a bacterial cell culture vessel 10, an insulated container 20, an enclosed container 30, a heating device 40, and a purification container 50. Below, the bacterial cell culture vessel 10, the insulated container 20, the enclosed container 30, the heating device 40, and the purification container 50 will be described in this order.

[0014] (1-1-1. Bacteria culture container 10) The bacterial cell culture vessel 10 is a vessel that contains a bacterial cell culture solution S. The bacterial cell culture solution S is a solution for culturing microorganisms having cells C. In the example of FIG. 1, the bacterial cell culture vessel 10 is a stoppered Erlenmeyer flask, but the shape and material of the bacterial cell culture vessel 10 are not limited.

[0015] (1-1-2. Heat insulation container 20) The insulated container 20 is a container for incubating the pretreatment solution I. The pretreatment solution I is a solution in which an enzyme E is added to a bacterial culture solution S. In the example of FIG. 1, the insulated container 20 is a glass tube with a stopper, but the shape and material of the insulated container 20 are not limited.

[0016] (1-1-3. Enclosed container 30) The sealed container 30 is a container that seals the heating solution H. The heating solution H is a solution in which a solubilizing agent D has been added to the pretreatment solution I. In the example of FIG. 1, the sealed container 30 is a glass tube with a stopper, but the shape and material of the sealed container 30 are not limited.

[0017] (1-1-4. Heating device 40) The heating device 40 is a container that heats the heating solution H. In the example of Fig. 1, the heating device 40 is a heat block, but the shape and material of the heating device 40 are not limited.

[0018] (1-1-5. Purification vessel 50) The purification container 50 is a container for purifying a purification solution P. The purification solution P is a solution containing nucleic acids N to be purified. In the example of FIG. 1, the heating device 40 is a column with a cock, but the shape and material of the purification container 50 are not limited.

[0019] (1-1-6. Other) 1 may include a plurality of bacterial cell culture vessels 10, a plurality of heat-retaining vessels 20, a plurality of sealed vessels 30, a plurality of heating devices 40, or a plurality of purification vessels 50. In addition, in the nucleic acid extraction system 100, two or more of the bacterial cell culture vessels 10, the heat-retaining vessels 20, and the sealed vessels 30 may be integrated into one configuration.

[0020] (1-2. Processes of the Nucleic Acid Extraction System 100) The steps of the nucleic acid extraction system 100 will be described below. The steps will be explained in the order of the collection step, the incubation step, the encapsulation step, the heating step, and the purification step. Note that some of the steps may be omitted.

[0021] (1-2-1. Collection process) The collection step shown in FIG. 1(1) will be described. First, in the collection step, a bacterial cell culture step is performed in which bacterial cells are cultured in a bacterial cell culture solution S contained in a bacterial cell culture vessel 10. For example, in the bacterial cell culture step, Escherichia coli (E. coli) or Staphylococcus aureus (S. aureus) are used as bacterial cells and cultured overnight at 37°C in a soybean casein digest (SCD) liquid medium. Second, in the collection step, a bacterial cell culture solution collection step is performed in which a portion of the bacterial cell culture solution S contained in the bacterial cell culture vessel 10 is collected in an insulated container 20. Here, the bacterial cell culture solution collection step can efficiently recover the target, so even if cells C are difficult to culture in the culture step, it can be applied to samples collected directly from the target without culturing them. Furthermore, since the bacterial cell culture solution collection step can be applied even to a small number of cells, it has the effect of shortening the microbial culture time required for detection.

[0022] (1-2-2. Heat retention process) The incubation step shown in FIG. 1(2) will be described. First, in the incubation step, an enzyme addition step is carried out in which an enzyme E that promotes lysis of cells C is added to the bacterial culture solution S collected in the incubation container 20. At this time, achromopeptidase and lysostaphin are added as the enzyme E in the enzyme addition step. Second, in the incubation step, a pretreatment solution preparation step is carried out in which a pretreatment solution I, which is the bacterial culture solution S containing the enzyme E, is prepared. At this time, in the pretreatment solution preparation step, trishydroxymethylaminomethane hydrochloride (Tris-HCl) is added as a buffering agent, and ethylenediaminetetraacetic acid (EDTA) is added as a chelating agent, and the concentration and pH of each component are adjusted. Third, in the incubation step, a pretreatment solution incubation step is carried out in which the incubation container 20 containing the pretreatment solution I is kept warm. For example, in the pretreatment solution incubation process, the insulated container 20 containing the pretreatment solution I is incubated at 37°C for 10 minutes, but the incubation temperature and time may be within a range that can promote the dissolution of cells C and are not particularly limited.

[0023] (1-2-3. Encapsulation process) The encapsulation step shown in FIG. 1(3) will be described. First, in the encapsulation step, a solubilizer addition step is carried out in which a solubilizer D that promotes the dissolution of cells C is added to pretreatment solution I after incubation for a certain period of time. For example, in the solubilizer addition step, sodium dodecyl sulfate (SDS) is added as a surfactant. Second, in the encapsulation step, a heating solution preparation step is carried out in which a heating solution H, which is a bacterial cell culture solution S containing solubilizer D, is prepared. At this time, in the heating solution preparation step, the concentration and pH of each component are adjusted. Third, in the encapsulation step, a heating solution encapsulation step is carried out in which the prepared heating solution H is encapsulated in an encapsulation container 30. For example, in the heating solution encapsulation step, the heating solution H is encapsulated in a glass tube as the encapsulation container 30, and the tube is sealed with a stopper.

[0024] (1-2-4.Heating process) The heating step shown in FIG. 1(4) will be described. First, in the heating step, a heating device preheating step is carried out in which the heating device 40 is preheated to a set temperature. For example, in the heating device preheating step, the heating device is preheated to a set temperature of 140°C. The preheating temperature is not particularly limited as long as it is within a range in which nucleic acid N can be extracted. Second, in the heating step, a sealed container installation step is carried out in which an enclosed container 30 containing a heating solution H is placed in the heating device 40. For example, in the sealed container installation step, the enclosed container 30 is installed by inserting a glass tube, which is the enclosed container 30, into a heat block. Third, in the heating step, a sealed container heating step is carried out in which the installed enclosed container 30 is heated. For example, in the sealed container heating step, the enclosed container 30 containing the heating solution H is heated to 140°C for 45 seconds. The heating temperature and time are not particularly limited as long as they are within a range in which nucleic acid N can be extracted.

[0025] (1-2-5. Purification process) The purification process shown in FIG. 1(5) will be described. First, in the purification process, a purification solution injection step is carried out in which the purification solution P, which is the heating solution H after the heating step and contains the nucleic acid N of the cell C, is injected into a purification vessel 50 filled with an adsorption carrier. In this step, the purification solution P is injected into the purification vessel 50, which has a composite material of silica and silicon carbide as an adsorption carrier. Second, in the purification process, an eluate injection step is carried out in which an eluate is injected into the purification vessel 50 into which the purification solution P has been injected, and the nucleic acid N is extracted. For example, in the eluate injection step, 16S ribosomal ribonucleic acid (rRNA) is extracted. Third, in the purification process, a copy number calculation step is carried out in which the copy number of the purified nucleic acid N is calculated. For example, in the copy number calculation step, the copy number of 16S rRNA is calculated using real-time PCR (Polymerase Chain Reaction) method.

[0026] (1-3. Effects of the Nucleic Acid Extraction System 100) Below, as a reference technology, the background of nucleic acid extraction technology, an overview of the Trizol method and the high-temperature high-pressure method, which are nucleic acid extraction technologies, and improvements of the reference technology will be explained in order, and then the effects of the nucleic acid extraction system 100 will be explained.

[0027] (1-3-1. Background of the reference technology) In recent years, there has been a demand for quick and easy testing methods for microorganisms in food manufacturing. Genetic testing is a faster testing method than conventional culture methods. In particular, genetic testing, which targets 16S rRNA, is useful for determining the level of contamination and identifying bacterial species.

[0028] (1-3-2. Overview of the Trizol method) The Trizol method is a technique for extracting nucleic acids using the following procedure. The Trizol method involves preparing a bacterial culture medium, preparing a lysate, performing Trizol treatment, purifying RNA, and quantifying the total RNA content. The Trizol treatment involves adding Trizol to the lysate-prepared sample, incubating it at 65°C for 20 minutes, then at room temperature for 5 minutes, adding chloroform, incubating it at room temperature for 3 minutes, and centrifuging it at 4°C for 15 minutes. The aqueous layer is then recovered. Isopropyl alcohol is added to the recovered aqueous layer, and the mixture is incubated at room temperature for 10 minutes. The mixture is then centrifuged at 4°C for 10 minutes, and the RNA is collected from the sides and bottom of the tube.

[0029] (1-3-3. Overview of the high-temperature, high-pressure method) The high-temperature, high-pressure method is a nucleic acid extraction technique described in Patent Document 1, and is a simple and rapid nucleic acid extraction technique that extracts nucleic acids by the following procedure. In the high-temperature, high-pressure method, a bacterial cell culture solution is prepared, a heating solution is prepared, a high-temperature, high-pressure treatment is performed, RNA purification is performed, and the total RNA amount is quantified. In this high-temperature, high-pressure treatment, the sample after the preparation of the heating solution is sealed in a glass tube and heated at 140°C for 45 seconds.

[0030] (1-3-4. Improvements to the reference technology) In order to perform the analysis using the above-mentioned genetic testing method, it is necessary to prepare sufficient nucleic acids, but the nucleic acid extraction technique according to the reference technology recovers only a small amount of nucleic acids such as RNA from bacteria with a strong cell surface, such as Gram-positive bacteria. Therefore, the nucleic acid extraction technique according to the reference technology requires the collection of a large number of bacteria for the above-mentioned analysis, and nucleic acids must be extracted from a large number of bacteria, so nucleic acid extraction takes a long time.

[0031] (1-3-5. Overview of the Nucleic Acid Extraction System 100) In the nucleic acid extraction system 100, achromopeptidase is added to a bacterial cell culture solution S containing cells C as an enzyme E that promotes the lysis of cells C, the bacterial cell culture solution S with the added achromopeptidase is kept warm at a predetermined temperature, a solubilizing agent D that promotes the lysis of cells C is added to the bacterial cell culture solution S that has been kept warm for a predetermined time, and nucleic acid N is extracted by heating the bacterial cell culture solution S with the added solubilizing agent D to a predetermined temperature.

[0032] In addition, in the nucleic acid extraction system 100, a solubilizing agent D that promotes dissolution of cells C is added to a bacterial cell culture solution S containing cells C, the bacterial cell culture solution S with added solubilizing agent D is heated to a predetermined temperature, and the bacterial cell culture solution S that has been heated for a predetermined time is injected into a column having silica and silicon carbide as adsorption carriers that adsorb nucleic acid N, thereby purifying nucleic acid N.

[0033] (1-3-6. Effects of the nucleic acid extraction system 100) The nucleic acid extraction system 100 is expected to have the following effects. First, compared to the nucleic acid extraction technology of the reference technology, the nucleic acid extraction system 100 enables the preparation of large amounts of RNA (nucleic acid extraction) from multiple bacterial species. Second, the nucleic acid extraction system 100 makes it possible to increase the amount of RNA recovered from low-concentration bacterial cell samples. Third, the nucleic acid extraction system 100 makes it possible to increase the sensitivity of genetic testing methods and shorten the enrichment process. Fourth, the nucleic acid extraction system 100 makes it possible to complete the preparation of RNA from bacterial cell culture solution S within 30 minutes. Fifth, the nucleic acid extraction system 100 can also be used for nucleic acid extraction in genetic testing methods that require rapidity in particular.

[0034] 2. Details of each step of the nucleic acid extraction system 100 A detailed description will be given of each step of the nucleic acid extraction system 100 shown in Fig. 1. Below, the steps according to the embodiment will be explained in the order of the collection step, the heat retention step, the encapsulation step, the heating step, and the purification step.

[0035] (2-1. Collection process) The details of the collection process of the nucleic acid extraction system 100 will be explained below in the order of the bacterial cell culture process and the bacterial cell culture fluid collection process.

[0036] (2-1-1. Bacterial cell culture process) First, the collection step of the nucleic acid extraction system 100 involves a bacterial cell culture step. For example, in the bacterial cell culture step, bacterial cells are cultured in a bacterial cell culture solution S contained in a bacterial cell culture vessel 10. Regarding an example of the equipment used for culture, in the bacterial cell culture step, a sterilized glass Erlenmeyer flask with a stopper is used as the bacterial cell culture vessel 10, and the bacterial cells are cultured. Regarding an example of the bacterial cells to be cultured, in the bacterial cell culture step, Escherichia coli or Staphylococcus aureus are cultured. Regarding an example of the culture conditions, in the bacterial cell culture step, the cells are cultured overnight at 37°C in an SCD liquid medium.

[0037] (2-1-1-1. Cultivation method of bacterial culture solution S) The bacterial cell culture solution S used in the bacterial cell culture step is obtained by culturing a sample containing nucleic acid N. The method for culturing the sample is not particularly limited, and examples include a method in which a filter on which the sample has been collected is placed directly on a solid medium and the sample is cultured through the filter (solid-phase culture). Another sample culture method includes a method in which the sample is cultured in the presence of a solution in which a liquid medium or solid medium is dissolved in water (liquid-phase culture). The type of liquid or solid medium used is selected depending on the type of sample to be cultured and the physiological conditions.

[0038] (2-1-1-2. Sample of bacterial culture solution S) The above-mentioned bacterial cell culture step is particularly effective for samples to be treated that have a strong cell surface, such as Gram-positive bacteria, but is not limited thereto. For example, the sample to be treated may be a microorganism, an animal cell other than a microorganism (e.g., insect cell), a plant cell, mycoplasma, a virus, etc.

[0039] Examples of the microorganisms include Acinetobacter species, Actinomyces species, Aerococcus species, Aeromonas species, Alcaligenes species, Bacillus species, Bacteriodes species, Bordetella species, Branhamella species, Brevibacterium species, Campylobacter species, and the like. r) species, Candida species, Capnocytophagia species, Chromobacterium species, Clostridium species, Corynebacterium species, Cryptococcus species, Deinococcus species, Enterococcus species, Erysipelothrix species, Escherichia species, Flavobacterium species, Flavobacterium spp., Gemella spp., Haemophilus spp., Klebsiella spp., Lactobacillus spp., Lactococcus spp., Legionella spp., Leuconostoc spp., Listeria spp., Micrococcus spp., Mycobacterium spp., Neisseria spp., Clostridium ... Cryptosporidium species, Nocardia species, Oerskovia species, Paracoccus species, Pediococcus species, Peptostreptococcus species, Propionibacterium species, Proteus species, Pseudomonas species, Rahnella species, Rhodococcus species,The bacterial strain is at least one species selected from the group consisting of Rhodospirillum species, Staphylococcus species, Streptomyces species, Streptococcus species, Vibrio species, and Yersinia species.

[0040] Among the microorganisms described above, there are microorganisms that take the form of spores or spores depending on their growth state. There are no particular limitations on the form of the sample to be processed in the nucleic acid extraction step of the nucleic acid extraction system 100. Furthermore, in the nucleic acid extraction step of the nucleic acid extraction system 100, the sample to be processed may be one type or two or more types.

[0041] (2-1-2. Bacterial culture fluid collection process) Secondly, in the collection step of the nucleic acid extraction system 100, a bacterial cell culture collection step is carried out. For example, in the bacterial cell culture collection step, a portion of the bacterial cell culture S contained in the bacterial cell culture vessel 10 is collected into the insulated container 20. Regarding an example of the tools used for collection, in the bacterial cell culture collection step, a sterilized glass pipette is used as the collection tool, and a sterilized glass tube with a stopper is used as the insulated container 20, and the bacterial cell culture S is collected. Regarding an example of the number of bacterial cells to be collected, in the bacterial cell culture collection step, the number of bacterial cells contained in the insulated container 20 is 1.0 × 10 4 The bacterial culture solution S is collected so that it becomes approximately

[0042] (2-2. Heat retention process) The details of the incubation process of the nucleic acid extraction system 100 will be explained below in the order of the enzyme addition process, the pretreatment solution preparation process, and the pretreatment solution incubation process.

[0043] (2-2-1. Enzyme addition step) First, an enzyme addition step is carried out in the incubation step of the nucleic acid extraction system 100. For example, in the enzyme addition step, an enzyme E that promotes lysis of cells C is added to the bacterial cell culture solution S collected in the incubation container 20. As an example of the tool used for addition, in the enzyme addition step, a sterilized glass pipette is used as the addition tool, and a solution containing enzyme E is added to the bacterial cell culture solution S. As an example of the enzyme to be added, in the enzyme addition step, achromopeptidase is added to the bacterial cell culture solution S, which is a sample containing cells C, as the enzyme E that promotes lysis of cells C. Furthermore, in the enzyme addition step, lysostaphin may be further added in addition to achromopeptidase as the enzyme E.

[0044] (2-2-1-1. Enzymes) In the enzyme addition step, achromopeptidase or both achromopeptidase and lysostaphin are particularly effective as the enzyme E to be added, but are not limited thereto. For example, the enzyme E to be added may be a protease such as proteinase K, or a polysaccharide-degrading enzyme such as chitinase, lysozyme, Zymolyase (registered trademark), or Labiases.

[0045] (2-2-2. Pretreatment solution preparation process) Second, a pretreatment solution preparation step is carried out in the incubation step of the nucleic acid extraction system 100. For example, in the pretreatment solution preparation step, a pretreatment solution I, which is a bacterial cell culture solution S containing enzyme E, is prepared. An example of the final concentrations of the components of the pretreatment solution I to be prepared is as follows: in the pretreatment solution preparation step, Tris-HCl (pH 8.0) as a buffer is prepared at 10 mM, EDTA as a chelating agent is prepared at 0.1 mM, achromopeptidase as enzyme E is prepared at 400 mg / mL, and lysostaphin as enzyme E is prepared at 20 μg / mL; however, the final concentrations of the components are not particularly limited.

[0046] (2-2-3. Pretreatment solution warming process) Third, the nucleic acid extraction system 100 performs a pretreatment solution warming step. For example, in the pretreatment solution warming step, the insulated container 20 containing the pretreatment solution I is warmed. Regarding an example of the equipment used for the warming, in the pretreatment solution warming step, a thermostatic bath is used to warm the insulated container 20, and the insulated container 20 containing the pretreatment solution I is kept at a constant temperature. Regarding an example of the conditions for the warming, in the pretreatment solution warming step, the insulated container 20 containing the pretreatment solution I is kept at 37°C for 10 minutes. However, the temperature and time for the warming are not particularly limited as long as they are within a range that can promote lysis of the cells C. In this case, in the pretreatment solution warming step, the pretreatment solution I, which is a sample to which achromopeptidase has been added, may be kept warm for 10 minutes or more.

[0047] (2-3. Encapsulation process) The details of the encapsulation process of the nucleic acid extraction system 100 will be explained below in the order of the solubilizing agent addition process, the heating solution preparation process, and the heating solution encapsulation process.

[0048] (2-3-1. Solubilizer Addition Step) First, the encapsulation step of the nucleic acid extraction system 100 involves the addition of a solubilizing agent. For example, in the solubilizing agent addition step, a solubilizing agent D that promotes dissolution of cells C is added to a pretreatment solution I, which is a sample that has been kept warm for a predetermined time in the incubation step. Explaining an example of the equipment used for addition, in the solubilizing agent addition step, a sterilized glass pipette is used as the equipment for addition, and a solution containing solubilizing agent D is added to the pretreatment solution I. Explaining an example of the solubilizing agent D to be added, in the solubilizing agent addition step, a solubilizing agent D containing SDS as a surfactant is added.

[0049] (2-3-2. Heating solution preparation process) Second, a heating solution preparation step is performed in the encapsulation step of the nucleic acid extraction system 100. For example, in the heating solution preparation step, a heating solution H is prepared, which is a bacterial cell culture solution S containing a solubilizing agent D. To explain an example of the final concentration of each component of the heating solution H to be prepared, in the heating solution preparation step, the surfactant SDS is prepared at 1% by mass and the buffer Tris-HCl (pH 8.0) is prepared at 0.05 M by mass, but the final concentration of each component is not particularly limited.

[0050] (2-3-2-1. Types of heating solution H) The following describes the types of heating solution H. The heating solution H of the nucleic acid extraction system 100 can achieve the above-mentioned effect even if it is water alone, but it preferably contains at least one solubilizing agent D selected from the group consisting of surfactants, alkalis, acids, redox agents, and protein denaturants in addition to water, in order to more efficiently extract nucleic acids N from the sample.

[0051] (2-3-2-2. Types of solubilizing agent D) The following describes the types of solubilizing agent D. Solubilizing agent D has the ability to dissolve the membrane structure of a sample. By acting on the membrane structure of a sample, solubilizing agent D makes it easier to destroy the sample, allowing nucleic acid N to be extracted from the sample more efficiently.

[0052] (surfactant) The surfactant used as the solubilizing agent D may be, for example, ionic or nonionic. Examples of nonionic surfactants include octylphenol ethoxylate (CHO(CHO)). In the nucleic acid extraction step of the nucleic acid extraction system 100, commercially available octylphenol ethoxylates can be used, such as Triton X-100 (CHO(CHO), n = 100) manufactured by SIGMA.

[0053] Furthermore, ionic surfactants may be anionic, cationic, or amphoteric. Examples of anionic surfactants include the aforementioned SDS. Examples of cationic surfactants include cetyltrimethylammonium bromide (CTAB). Examples of amphoteric surfactants include betaine. Here, "betaine" refers to a general term for a compound that has a positive charge and a negative charge at non-adjacent positions within the same molecule, with no dissociable hydrogen atoms bonded to the positively charged atom, resulting in the molecule as a whole having no charge. A representative example of betaine is trimethylglycine.

[0054] (alkali) The alkali used as the solubilizing agent D includes, for example, sodium hydroxide (NaOH) or potassium hydroxide (KOH).

[0055] (acid) The acid used as the solubilizing agent D may be, for example, hydrochloric acid (HCl) or sulfuric acid (H2SO4).

[0056] (oxidation-reducing agent) Examples of the oxidation-reducing agent used as the solubilizing agent D include aqueous hydrogen peroxide, β-mercaptoethanol, and dithiothreitol.

[0057] (protein denaturant) Examples of protein denaturants used as solubilizing agent D include guanidine hydrochloride and urea.

[0058] (others) A chelating agent may be used as a component of the solubilizing agent D. Examples of the chelating agent used as the solubilizing agent D include the above-mentioned EDTA.

[0059] Furthermore, among the above-mentioned solubilizing agents D, the solubilizing agent D of the nucleic acid extraction system 100 preferably contains a surfactant, and more preferably contains either or both of SDS and octylphenol ethoxylate.

[0060] For example, SDS is preferably used when it is desired to detect with high sensitivity the nucleic acid extracted in the nucleic acid extraction step of the nucleic acid extraction system 100. On the other hand, when the nucleic acid N extracted in the nucleic acid extraction step of the nucleic acid extraction system 100 is used in an enzymatic reaction that is inhibited by SDS, octylphenol ethoxylate, which acts more mildly than SDS on the membrane structure of the sample, is preferably used.

[0061] The solubilizing agent D of the nucleic acid extraction system 100 may contain a buffer, if necessary. Examples of the buffer include the above-mentioned Tris-HCl.

[0062] (2-3-3. Heating solution sealing process) Third, in the sealing step of the nucleic acid extraction system 100, a heating solution sealing step is carried out. For example, in the heating solution sealing step, the prepared heating solution H is sealed in the sealed container 30. To explain an example of the equipment used for sealing, in the heating solution sealing step, a sterilized glass pipette is used as the equipment for collecting the heating solution H, and a sterilized glass tube is used as the sealed container 30, and the heating solution H is collected. At this time, in the heating solution sealing step, a fixed amount of the heating solution H is collected into each of the multiple sealed containers 30. Then, in the heating solution sealing step, after a fixed amount of the heating solution H has been collected, the glass tube is sealed by plugging it.

[0063] (2-4.Heating process) The heating steps of the nucleic acid extraction system 100 will be described in detail below in the order of the heating device preheating step, the enclosed container installation step, and the enclosed container heating step.

[0064] (2-4-1. Heating device preheating process) First, the heating step of the nucleic acid extraction system 100 includes a heating device preheating step. For example, in the heating device preheating step, the heating device 40 is preheated to a set temperature. As an example of the heating device 40, a heat block, an oil bath, or the like is used as the heating device 40 in the heating device preheating step. As an example of the preheating temperature, the heating device preheating step involves preheating to a set temperature of 140°C, but the preheating temperature is not particularly limited as long as it is within a range in which nucleic acid N can be extracted.

[0065] (2-4-2. Sealed container installation process) Secondly, a sealed container installation step is carried out in the heating step of the nucleic acid extraction system 100. For example, in the sealed container installation step, the sealed container 30 containing the heating solution H is placed in the heating device 40. At this time, in the sealed container installation step, multiple sealed containers 30 are installed by inserting them into a heat block.

[0066] (2-4-3. Sealed container heating process) Third, the heating step of the nucleic acid extraction system 100 involves a sealed container heating step. For example, in the sealed container heating step, the installed sealed container 30 is heated by the heating device 40 to extract the nucleic acid N into the heating solution H. To explain an example of heating conditions, in the sealed container heating step, the sealed container 30 containing the heating solution H is heated at 140°C for 45 seconds, but the heating temperature and time are not particularly limited as long as they are within a range in which the nucleic acid N can be extracted.

[0067] (2-4-3-1. Types of nucleic acid N) In the sealed container heating step, RNA such as 16S rRNA is particularly effective as the nucleic acid N extracted into the heating solution H contained in the sealed container 30, but is not limited thereto. For example, the nucleic acid N to be extracted may be genomic DNA, plasmid DNA, or the like, which is deoxyribonucleic acid (DNA).

[0068] (2-5. Purification process) The purification steps of the nucleic acid extraction system 100 will be described in detail below in the order of the purification solution injection step, the eluate injection step, and the copy number calculation step.

[0069] (2-5-1. Purification solution process) First, the purification step of the nucleic acid extraction system 100 involves a purification solution injection step. For example, in the purification solution injection step, a purification solution P, which is a heating solution H after the heating step and contains nucleic acids N of cells C, is injected into a purification container 50 filled with an adsorption carrier. At this time, in the purification solution injection step, the purification solution P, which is a sample heated for a predetermined time in the heating step, is injected into a column containing silica and silicon carbide as an adsorption carrier that adsorbs nucleic acids N. To explain this by taking a specific example, a composite column of silica and silicon carbide from an RNA purification kit (Single Cell RNA Purification Kit) manufactured by NORGEN is used in the purification solution injection step.

[0070] (2-5-2. Eluate injection process) Second, the purification step of the nucleic acid extraction system 100 involves an elution solution injection step. For example, in the purification solution injection step, an elution solution is injected into the purification container 50 into which the purification solution P has been injected, and nucleic acid N is purified. In this step, the 16S rRNA adsorbed to the column in the purification solution injection step is extracted by dissolving it in the elution solution. To explain this in a specific example, the elution solution injection step uses the elution solution provided with the RNA purification kit described above.

[0071] (2-5-2-1. Types of nucleic acid N) In the eluate injection step, RNA such as 16S rRNA is particularly effective as the nucleic acid N extracted into the eluate, but is not limited thereto. For example, the nucleic acid N to be extracted may be DNA such as genomic DNA or plasmid DNA.

[0072] (2-5-3. Copy number calculation process) Third, a copy number calculation step is carried out in the purification step of the nucleic acid extraction system 100. For example, in the copy number calculation step, the copy number of the purified nucleic acid N is calculated. In this copy number calculation step, the copy number of 16S rRNA is calculated by real-time PCR.

[0073] [3. Experimental Results] 2 to 6, various experimental results using the nucleic acid extraction system 100 according to the embodiment will be described.

[0074] (3-1. Experimental Results 1) Experimental result 1 showing an example of the copy number of 16S rRNA obtained by the nucleic acid extraction system 100 will be described using Figure 2. Figure 2 is a diagram for explaining Experimental result 1 according to the embodiment. Experimental result 1 allows a comparison of the copy number of 16S rRNA for each nucleic acid extraction technique in Escherichia coli (E. coli), a gram-negative bacterium, and Staphylococcus aureus (S. aureus), a gram-positive bacterium.

[0075] (3-1-1. E. coli) The bar graph shown under "E. coli" in Figure 2 shows the copy number of 16S rRNA extracted from E. coli using the Trizol method, which is the reference technology described above (see Figure 2(1-1)), the copy number of 16S rRNA extracted from E. coli using the high-temperature, high-pressure method, which is the reference technology described above (see Figure 2(1-2)), and the copy number of 16S rRNA extracted from E. coli using the nucleic acid extraction system 100 (see Figure 2(1-3)). As shown in Figures 2(1-1) to (1-3), the nucleic acid extraction system 100 can extract about 25 times the copy number of 16S rRNA compared to the nucleic acid extraction technology according to the reference technology.

[0076] (3-1-2. Staphylococcus aureus) The bar graph shown for "S. aureus" in Figure 2 shows the copy number of 16s rRNA extracted from Staphylococcus aureus using the Trizol method, which is the reference technology described above (see Figure 2(2-1)), the copy number of 16s rRNA extracted from Staphylococcus aureus using the high-temperature, high-pressure method, which is the reference technology described above (see Figure 2(2-2)), and the copy number of 16s rRNA extracted from Staphylococcus aureus using the nucleic acid extraction system 100 (see Figure 2(2-3)). As shown in Figures 2(2-1) to (2-3), the nucleic acid extraction system 100 can extract 16S rRNA at a copy number that is approximately 500 to 700 times higher than that obtained using the nucleic acid extraction technology of the reference technology.

[0077] (3-1-3. Discussion of Experimental Result 1) 2, experimental result 1 shows that the nucleic acid extraction system 100 can extract 16S rRNA more effectively from both Escherichia coli, a gram-negative bacterium, and Staphylococcus aureus, a gram-positive bacterium, than the nucleic acid extraction technology of the reference technology. Furthermore, the nucleic acid extraction system 100 can extract 16S rRNA particularly effectively from Staphylococcus aureus, a gram-positive bacterium, than the nucleic acid extraction technology of the reference technology.

[0078] (3-2. Experimental Results 2) Experimental result 2, which shows an example of the copy number of 16S rRNA for each enzyme obtained by the nucleic acid extraction system 100, will be described using FIG. 3. FIG. 3 is a diagram for explaining Experimental result 2 according to the embodiment. Experimental result 2 allows for a comparison of the copy number of 16S rRNA for each enzyme in Staphylococcus aureus, a Gram-positive bacterium. Note that the data labels on the bar graph in FIG. 3 indicate a multiplication factor based on the copy number of 16S rRNA extracted by (3-2-1. No enzyme added), which will be described later. Furthermore, 20 μg / mL of lysostaphin was added as enzyme E in (3-2-1. No enzyme added), (3-2-2. Lysozyme), (3-2-3. Achromopeptidase), and (3-2-4. Labiase), which will be described later.

[0079] (3-2-1. No enzyme added) The bar graph in Figure 3(1) shows the gram-positive bacterium Staphylococcus aureus at a cell count of 1.0 × 10 4 1 shows the copy number of 16S rRNA extracted from pretreatment solution I to which no enzyme E other than lysostaphin was added during the incubation step of the nucleic acid extraction system 100 described above, using bacterial cell culture solution S containing lysostaphin.

[0080] (3-2-2. Lysozyme) The bar graph in Figure 3(2) shows the gram-positive bacterium Staphylococcus aureus at a cell count of 1.0 × 10 4 The figures show the copy numbers of 16S rRNA extracted from pretreatment solution I, which was prepared by adding 10 mg / mL, 20 mg / mL, and 80 mg / mL of lysozyme as the enzyme E other than lysostaphin during the incubation step of the nucleic acid extraction system 100 described above, using a bacterial culture solution S containing lysostaphin. As shown in Figure 3(2), the nucleic acid extraction system 100, which added lysozyme as the enzyme E, extracted 5.3 times as many copies of 16S rRNA at 10 mg / mL, 2.7 times as many copies at 20 mg / mL, and 0.0 times as many copies at 80 mg / mL compared to the step shown in Figure 3(1) in which no enzyme E other than lysostaphin was added. Here, the decrease in copy number as the concentration of enzyme E increased is due to RNA degradation caused by the nuclease activity contained in the enzyme E product.

[0081] (3-2-3. Achromopeptidase) The bar graph in Figure 3(3) shows the gram-positive bacterium Staphylococcus aureus at a cell count of 1.0 × 10 4The figures show the copy numbers of 16S rRNA extracted from pretreatment solution I, which was prepared by adding achromopeptidase at 10 μg / mL, 20 μg / mL, 100 μg / mL, and 400 μg / mL as the enzyme E other than lysostaphin during the incubation process of the nucleic acid extraction system 100 described above. As shown in Figure 3(3), the nucleic acid extraction system 100, which added achromopeptidase as the enzyme E, extracted 8.3-fold more 16S rRNA copies at 10 μg / mL, 8.5-fold more 16S rRNA copies at 20 μg / mL, 9.1-fold more 16S rRNA copies at 100 μg / mL, and 0.9-fold more 16S rRNA copies at 400 μg / mL than the process shown in Figure 3(1) in which no enzyme E other than lysostaphin was added. The decrease in copy number as the concentration of enzyme E increased is due to RNA degradation caused by the nuclease activity contained in the enzyme E product.

[0082] (3-2-4. Rabiase) The bar graph in Figure 3(4) shows the gram-positive bacterium Staphylococcus aureus at a cell count of 1.0 × 10 4 The figures show the copy number of 16S rRNA extracted from pretreatment solution I to which 10 mg / mL of labiase was added as an enzyme E other than lysostaphin during the incubation step of the nucleic acid extraction system 100 described above, using a sample corresponding to bacterial culture solution S containing lysostaphin. As shown in Figure 3(4), the nucleic acid extraction system 100 to which labiase was added as enzyme E was able to extract 1.3 times more copies of 16S rRNA at 10 mg / mL compared to the step shown in Figure 3(1) in which no enzyme E other than lysostaphin was added.

[0083] (3-2-5. Discussion of Experimental Result 2) 3, experimental result 2 shows that, compared to a process in which enzyme E other than lysostaphin is not added, nucleic acid extraction system 100 can effectively extract 16S rRNA from Staphylococcus aureus, a Gram-positive bacterium, when an excessive amount of enzyme E is not added. In particular, nucleic acid extraction system 100 can more effectively extract 16S rRNA from Staphylococcus aureus, a Gram-positive bacterium, when an appropriate amount of achromopeptidase is added as enzyme E.

[0084] (3-3. Experimental Results 3) Experimental result 3, which shows an example of the 16S rRNA preservation rate for each enzyme obtained by the nucleic acid extraction system 100, will be described using FIG. 4. FIG. 4 is a diagram for explaining Experimental result 3 according to the embodiment. Experimental result 3 compares the 16S rRNA preservation rate for each enzyme in Staphylococcus aureus, a Gram-positive bacterium, and makes it possible to confirm the trade-off in RNA degradation due to the nuclease activity contained in Enzyme E products. Note that 20 μg / mL of lysostaphin was added as Enzyme E in (3-3-1. No enzyme added), (3-3-2. Lysozyme), (3-3-3. Achromopeptidase), and (3-3-4. Labiase), which will be described later.

[0085] (3-3-1. No enzyme added) The bar graph in Figure 4(1) shows the number of cells extracted from the gram-positive bacterium Staphylococcus aureus (1.0 × 10 5 The figure shows the RNA preservation rate when purified RNA corresponding to the above was used and no enzyme E other than lysostaphin was added to the RNA. As shown in Figure 4(1), when no enzyme E other than lysostaphin was added, the 16S rRNA preservation rate was 100%.

[0086] (3-3-2. Lysozyme) The bar graph in Figure 4(2) shows the number of cells extracted from the gram-positive bacterium Staphylococcus aureus (1.0 × 10 5 The figure shows the RNA preservation rate when purified RNA corresponding to the above was used and lysozyme was added to the RNA as an enzyme E other than lysostaphin at 10 mg / mL, 20 mg / mL, and 80 mg / mL. As shown in Figure 4(2), in the nucleic acid extraction system 100 to which lysozyme was added as enzyme E, the preservation rate of 16S rRNA was slightly reduced at 10 mg / mL, and was nearly 0% at 20 mg / mL and 80 mg / mL.

[0087] (3-3-3. Achromopeptidase) The bar graph in Figure 4(3) shows the number of cells extracted from the gram-positive bacterium Staphylococcus aureus (1.0 × 10 5The figure shows the RNA preservation rate when purified RNA corresponding to the above was used and achromopeptidase was added to the RNA as an enzyme E other than lysostaphin at 10 μg / mL, 20 μg / mL, 100 μg / mL, and 400 μg / mL. As shown in Figure 4(3), in the nucleic acid extraction system 100 to which achromopeptidase was added as enzyme E, the 16S rRNA preservation rate was nearly 100% at 10 μg / mL and 20 μg / mL, but was slightly reduced at 100 μg / mL, and was nearly 0% at 400 μg / mL.

[0088] (3-3-4. Rabiase) The bar graph in Figure 4(4) shows the number of cells extracted from the gram-positive bacterium Staphylococcus aureus (1.0 × 10 5 The figure shows the RNA preservation rate when purified RNA corresponding to the above was used and 10 mg / mL of labiase was added to the RNA as an enzyme E other than lysostaphin. As shown in Figure 4 (4), in the nucleic acid extraction system 100 to which labiase was added as enzyme E, the preservation rate of 16S rRNA was 10% or less at 10 mg / mL.

[0089] (3-3-5. Discussion of Experimental Result 3) Experimental result 3 in Figure 4 confirms conditions under which the impact of RNA degradation due to the nuclease activity of enzyme E is minimal in the nucleic acid extraction system 100. That is, in the nucleic acid extraction system 100, when 10 mg / mL to 20 mg / mL of achromopeptidase is added as enzyme E other than lysostaphin to Gram-positive bacteria Staphylococcus aureus, 16S rRNA can be extracted particularly effectively.

[0090] (3-4. Experimental Results 4) Experimental result 4, which shows an example of the amount of residual RNA obtained by the nucleic acid extraction system 100, will be described using FIG. 5. FIG. 5 is a diagram for explaining Experimental result 4 according to the embodiment. In Experimental result 4, achromopeptidase was used as enzyme E for Staphylococcus aureus, a gram-positive bacterium, and the amount of 16S rRNA preserved at each concentration of achromopeptidase was compared with and without the addition of EDTA, allowing the relationship between RNA degradation due to the nuclease activity contained in the enzyme E product and EDTA to be confirmed. Note that the "remaining amount of RNA (%)" in the graph of FIG. 5 is set to 100% when the amount of residual RNA is not added with achromopeptidase.

[0091] (3-4-1. No EDTA added) The white bar graph in Figure 5 shows the amount of RNA preserved when purified RNA extracted from the Gram-positive bacterium Staphylococcus aureus was used, and achromopeptidase was added to the RNA as an enzyme E other than lysostaphin, without the addition of the chelating agent EDTA. As shown in Figure 5, when EDTA was not added, the amount of 16S rRNA preserved increased when the achromopeptidase concentration was 10 μg / mL to 40 μg / mL, decreased at 100 μg / mL or higher, and was almost 0% at 400 μg / mL or higher.

[0092] (3-4-2. EDTA addition) The hatched bars in Figure 5 show the amount of RNA preserved when purified RNA extracted from the Gram-positive bacterium Staphylococcus aureus was used, and achromopeptidase was added to the RNA as an enzyme E other than lysostaphin, and 5 mM of EDTA was also added as a chelating agent. As shown in Figure 5, when EDTA was added, the amount of 16S rRNA preserved was 100% or more, even when the achromopeptidase concentration was 400 μg / mL or higher.

[0093] (3-4-3. Discussion of Experimental Result 4) From Experimental Result 4 in Figure 5, even when achromopeptidase is added as enzyme E in the nucleic acid extraction system 100, it is possible to confirm that the addition of EDTA, a chelating agent, prevents RNA degradation due to nuclease activity, and that this is an effective condition for the process using enzyme E in the nucleic acid extraction system 100.

[0094] (3-5. Experimental Results 5) Experimental result 5, which shows an example of the recovery rate of RNA by the nucleic acid extraction system 100, will be described using Figure 6. Figure 6 is a diagram for explaining Experimental result 5 according to the embodiment. Experimental result 5 uses purified RNA extracted from Staphylococcus aureus, a gram-positive bacterium, and shows the recovery rate of RNA for each column material for that RNA. As shown in Figure 6, the "RNA recovery rate (%)" is the number of copies of RNA recovered from the column, i.e., extracted, relative to the number of copies of RNA tested, i.e., injected into the column.

[0095] (3-5-1. Silica column) The broken line graph in Figure 6 shows the recovery rate of RNA when purified RNA extracted from Staphylococcus aureus, a gram-positive bacterium, is used, a solution containing the RNA is injected into a silica column packed with silica to adsorb it, and the RNA is extracted by injecting the eluate into the column. As shown in Figure 6, when a silica column is used, the copy number of the test RNA is low at 1.0 x 10 6 ~1.0×10 9 The recovery rate is approximately 5% to 20%.

[0096] (3-5-2. Composite column) The solid line graph in Figure 6 shows the recovery rate of RNA when purified RNA extracted from Staphylococcus aureus, a gram-positive bacterium, is used, a solution containing the RNA is injected into a composite column packed with a composite material of silica and silicon carbide to adsorb the RNA, and the RNA is extracted by injecting the eluate into the column. As shown in Figure 6, when a composite column is used, the copy number of the test RNA is low at 1.0 x 105 ~1.0×10 10 The recovery rate is approximately 50% to 70%.

[0097] (3-5-3. Discussion of Experimental Result 5) Experimental result 5 in Figure 6 shows that when a composite column packed with a composite material of silica and silicon carbide is used in the nucleic acid extraction system 100, approximately six times the copy number of 16S rRNA can be extracted, even at low concentrations, compared to a silica column.

[0098] 4. Processing flow of the nucleic acid extraction system 100 7 to 12, the process flow of the nucleic acid extraction system 100 according to the embodiment will be described. Below, the flow of the nucleic acid extraction process, which is the entire process of the nucleic acid extraction system 100, will be explained, followed by the flow of the collection process, the flow of the heat retention process, the flow of the encapsulation process, the flow of the heating process, and the flow of the purification process, in that order.

[0099] (4-1. Nucleic acid extraction process flow) The flow of the nucleic acid extraction process, which is the entire process of the nucleic acid extraction system 100, will be described with reference to Figure 7. Figure 7 is a flowchart showing an example of the flow of the nucleic acid extraction process according to the embodiment. Note that the following steps S101 to S105 can also be performed in a different order. Furthermore, some steps of the following steps S101 to S105 may be omitted.

[0100] First, the nucleic acid extraction system 100 performs a collection step (step S101). Second, the nucleic acid extraction system 100 performs an incubation step (step S102). Third, the nucleic acid extraction system 100 performs an encapsulation step (step S103). Fourth, the nucleic acid extraction system 100 performs a heating step (step S104). Fifth, the nucleic acid extraction system 100 performs a purification step (step S105), thereby completing the nucleic acid extraction step.

[0101] (4-2. Collection process flow) The flow of the collection step of the nucleic acid extraction system 100 will be described with reference to Fig. 8. Fig. 8 is a flowchart showing an example of the flow of the collection step according to the embodiment. Note that the following steps S201 to S202 can also be performed in a different order. Furthermore, some of the following steps S201 to S202 may be omitted.

[0102] First, a bacterial cell culture step is carried out (step S201) in the collection step of the nucleic acid extraction system 100. Second, a bacterial cell culture solution collection step is carried out (step S202) in the collection step of the nucleic acid extraction system 100, and the collection step is then completed.

[0103] (4-3. Flow of the heat retention process) The flow of the heat retention step of the nucleic acid extraction system 100 will be described with reference to Fig. 9. Fig. 9 is a flowchart showing an example of the flow of the heat retention step according to the embodiment. Note that the following steps S301 to S303 can also be performed in a different order. Furthermore, some of the following steps S301 to S303 may be omitted.

[0104] First, an enzyme addition step is carried out (step S301) in the heat retention step of the nucleic acid extraction system 100. Second, a pretreatment solution preparation step is carried out (step S302) in the heat retention step of the nucleic acid extraction system 100. Third, a pretreatment solution heat retention step is carried out (step S303) in the heat retention step of the nucleic acid extraction system 100, and the heat retention step is completed.

[0105] (4-4. Encapsulation process flow) The flow of the encapsulation step of the nucleic acid extraction system 100 will be described with reference to Fig. 10. Fig. 10 is a flowchart showing an example of the flow of the encapsulation step according to the embodiment. Note that the following steps S401 to S403 can also be performed in a different order. Furthermore, some of the following steps S401 to S403 may be omitted.

[0106] First, a solubilizing agent adding step is carried out (step S401) in the encapsulating step of the nucleic acid extraction system 100. Second, a heating solution preparing step is carried out (step S402) in the encapsulating step of the nucleic acid extraction system 100. Third, a heating solution encapsulating step is carried out (step S403) in the encapsulating step of the nucleic acid extraction system 100, and the encapsulating step is completed.

[0107] (4-5. Heating process flow) The flow of the heating step of the nucleic acid extraction system 100 will be described with reference to Fig. 11. Fig. 11 is a flowchart showing an example of the flow of the heating step according to this embodiment. Note that the following steps S501 to S503 can also be performed in a different order. Furthermore, some of the following steps S501 to S503 may be omitted.

[0108] First, a heating device preheating step is carried out (step S501) in the heating step of the nucleic acid extraction system 100. Second, a sealed container installation step is carried out (step S502) in the heating step of the nucleic acid extraction system 100. Third, a sealed container heating step is carried out (step S503) in the heating step of the nucleic acid extraction system 100, and the heating step is then completed.

[0109] (4-6. Refining process flow) The flow of the purification process of the nucleic acid extraction system 100 will be described with reference to Fig. 12. Fig. 12 is a flowchart showing an example of the flow of the purification process according to the embodiment. Note that the following steps S601 to S603 can also be performed in a different order. Furthermore, some of the following steps S601 to S603 may be omitted.

[0110] First, a purification solution preparation step is carried out (step S601) in the purification process of the nucleic acid extraction system 100. Second, an elution solution injection step is carried out (step S602) in the purification process of the nucleic acid extraction system 100. Third, a copy number calculation step is carried out (step S603) in the purification process of the nucleic acid extraction system 100, and the purification process is completed.

[0111] 5. Effects of the embodiment Finally, the effects of the embodiment will be described below: Effects 1 to 8 corresponding to the steps according to the embodiment will be described below.

[0112] (5-1. Effect 1) First, in the process according to the embodiment described above, achromopeptidase is added to a sample containing cells C as enzyme E that promotes lysis of cells C, the sample with added achromopeptidase is kept warm at a predetermined temperature, a solubilizing agent D that promotes lysis of cells C is added to the sample that has been kept warm for a predetermined time, and the sample with added solubilizing agent D is heated to a predetermined temperature, thereby extracting nucleic acid N from cells C. Therefore, in the process according to the embodiment, by effectively promoting lysis of cells C, nucleic acid N contained in the sample can be efficiently extracted.

[0113] (5-2. Effect 2) Secondly, in the process according to the embodiment described above, a solubilizing agent D that promotes dissolution of cells C is added to a sample containing cells C, the sample to which solubilizing agent D has been added is heated to a predetermined temperature, and the sample that has been heated for a predetermined time is injected into a column having silica and silicon carbide as adsorption carriers that adsorb nucleic acid N, and nucleic acid N is purified, thereby extracting nucleic acid N from cells C. Therefore, in the process according to the embodiment, nucleic acid N can be efficiently extracted from a sample by effectively adsorbing nucleic acid N to the column.

[0114] (5-3. Effect 3) Thirdly, in the steps according to the above-described embodiment, the nucleic acid N is RNA. Therefore, in the steps according to the embodiment, RNA can be efficiently extracted as the nucleic acid N contained in the sample.

[0115] (5-4. Effect 4) Fourth, in the steps according to the above-described embodiments, the cells C are cells C possessed by Gram-positive bacteria. Therefore, in the steps according to the embodiments, the lysis of the cells C of Gram-positive bacteria is effectively promoted, thereby enabling efficient extraction of nucleic acids N contained in the sample.

[0116] (5-5. Effect 5) Fifth, in the process according to the embodiment described above, the solubilizing agent D contains SDS as a surfactant. Therefore, in the process according to the embodiment, the nucleic acid N contained in the sample can be efficiently extracted by effectively promoting the lysis of the cells C during high-temperature and high-pressure treatment.

[0117] (5-6. Effect 6) Sixth, in the process according to the embodiment described above, EDTA is further added as a chelating agent to the sample containing the cells C. Therefore, in the process according to the embodiment, even if the enzyme E that promotes the lysis of the cells C is at a high concentration, the nucleic acid N contained in the sample can be efficiently extracted.

[0118] (5-7. Effect 7) Seventh, in the steps according to the above-described embodiment, lysostaphin is further added to the sample containing the cells C as the enzyme E. Therefore, in the steps according to the embodiment, the nucleic acid N contained in the sample can be efficiently extracted by using a more effective enzyme E to promote lysis of the cells C.

[0119] (5-8. Effect 8) Eighth, in the process according to the embodiment described above, the sample to which achromopeptidase has been added is kept warm for 10 minutes or more. Therefore, in the process according to the embodiment, by keeping the sample warm for a more effective time, the lysis of the cells C is promoted, and the nucleic acid N contained in the sample can be efficiently extracted.

[0120] 〔system〕 The information including the processing procedures, control procedures, specific names, various data and parameters shown in the above documents and drawings can be changed arbitrarily unless otherwise specified.

[0121] Furthermore, the components of each device shown in the figure are functional concepts and do not necessarily have to be physically configured as shown. In other words, the specific form of distribution and integration of each device is not limited to that shown. In other words, all or part of them can be functionally or physically distributed and integrated in any unit depending on various loads, usage conditions, etc.

[0122] 〔others〕 Some examples of combinations of the disclosed technical features are set out below.

[0123] (1) A nucleic acid extraction method for extracting nucleic acids from cells, comprising: an incubation step of adding achromopeptidase to a sample containing the cells as an enzyme that promotes lysis of the cells, and keeping the sample with the achromopeptidase added at a predetermined temperature; and a heating step of adding a solubilizing agent that promotes lysis of the cells to the sample that has been kept warm for a predetermined time in the incubation step, and heating the sample with the solubilizing agent added to it to the predetermined temperature.

[0124] (2) A nucleic acid extraction method for extracting nucleic acids from cells, comprising: a heating step of adding a solubilizing agent that promotes the dissolution of the cells to a sample containing the cells, and heating the sample to which the solubilizing agent has been added to a predetermined temperature; and a purification step of injecting the sample heated for a predetermined time in the heating step into a column containing silica and silicon carbide as adsorption carriers that adsorb the nucleic acids, thereby purifying the nucleic acids.

[0125] (3) The nucleic acid extraction method according to (1) or (2), wherein the nucleic acid is a ribonucleic acid.

[0126] (4) The nucleic acid extraction method according to any one of (1) to (3), wherein the cells are cells of a gram-positive bacterium.

[0127] (5) The nucleic acid extraction method according to any one of (1) to (4), wherein the solubilizing agent contains sodium dodecyl sulfate as a surfactant.

[0128] (6) The nucleic acid extraction method according to (1), wherein the incubation step further comprises adding ethylenediaminetetraacetic acid as a chelating agent to the cell-containing sample.

[0129] (7) The nucleic acid extraction method according to (1), wherein the incubation step further comprises adding lysostaphin as the enzyme to the cell-containing sample.

[0130] (8) The nucleic acid extraction method according to (1), wherein the incubation step involves incubating the sample containing the achromopeptidase for 10 minutes or more. [Explanation of symbols]

[0131] 10 Bacterial culture container 20 Thermal container 30 Enclosure 40 Heating device 50 Refining vessel 100 Nucleic Acid Extraction System

Claims

1. A nucleic acid extraction method for extracting nucleic acid from cells of Gram-positive bacteria, comprising: an incubation step of adding achromopeptidase to a sample containing the cells as an enzyme that promotes lysis of the cells, and incubating the sample to which the achromopeptidase has been added at a predetermined temperature; a sealing step of adding a solubilizing agent that promotes lysis of the cells to the sample that has been kept warm for a predetermined time in the warming step, and sealing a solution containing the sample and the solubilizing agent in a container; a heating step of heating the container containing the sample to which the solubilizing agent has been added to a predetermined temperature and for a predetermined time by high-temperature, high-pressure treatment; a purification step of injecting the sample heated for a predetermined time in the heating step into a column containing silica and silicon carbide as adsorption carriers that adsorb the nucleic acid, thereby purifying the nucleic acid; A nucleic acid extraction method comprising:

2. The nucleic acid is a ribonucleic acid. The nucleic acid extraction method according to claim 1.

3. The solubilizing agent contains sodium dodecyl sulfate as a surfactant. The nucleic acid extraction method according to claim 1.

4. The heat-retaining step includes: further adding ethylenediaminetetraacetic acid as a chelating agent to the cell-containing sample; The nucleic acid extraction method according to claim 1.

5. The heat-retaining step includes: lysostaphin is further added as the enzyme to the sample containing the cells; The nucleic acid extraction method according to claim 1.

6. The heat-retaining step includes: Incubating the sample to which the achromopeptidase has been added for 10 minutes or more. The nucleic acid extraction method according to claim 1.

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