State determination method

Abstract

A state determination method in which a cell culture solution S containing a cell C and foamed beads B are sealed in a sealing container (20); sealing and sealing the container (20) and heating; an internal state related to at least one of the temperature and the pressure of the sealed container (20) is determined on the basis of a change in the size of the foamed beads (B).

Description

Technical Field

[0001] The present invention relates to a state determination method. Background Art

[0002] A technique is known in which a bacterial culture solution is collected, a solubilization aid is added, and nucleic acids are extracted from the bacterial cells contained in the culture solution under high temperature and high pressure conditions (appropriately, the "high temperature and high pressure method"). Furthermore, a technique is known in which, in a process requiring heat treatment of a container, the temperature of the container during heating is determined by observing the color tone of a pigment such as Prussian blue or leucochrome, which changes color with temperature (appropriately, the "heating temperature" or "heating").

[0003] Prior art literature

[0004] Patent Literature

[0005] Patent Document 1: Japanese Patent No. 5624487

[0006] Patent Document 2: Japanese Patent Application Laid-Open No. 2013-132298

[0007] Patent Document 3: Japanese Patent Application Laid-Open No. 2002-322385 Summary of the Invention

[0008] Problems to be solved by the invention

[0009] However, it is difficult to effectively determine the internal conditions of the bacterial culture solution contained in the container, such as the heating temperature. For example, in the method of confirming the color change of Prussian blue, the temperature of the culture solution is 121°C, which is lower than the 140°C at which nucleic acids can be extracted from bacterial cells.

[0010] Since the color changes around 70°C, it is difficult to confirm whether the temperature has actually reached around 140°C. In addition, in the method for confirming the color change of the leuco pigment, the color changes around 70°C and the hue reversibly changes to the original color at 20°C, so it is difficult to confirm whether the temperature has actually reached around 140°C.

[0011] The present invention has been made in view of the above circumstances, and an object of the present invention is to efficiently identify the state of a sample.

[0012] Means for solving problems

[0013] The present invention provides a state determination method, which is a state determination method for determining the internal state of a container, comprising: a sealing step, wherein a sample solution containing a sample and foamed beads are sealed in the container; a heating step, wherein the container is sealed and heated; and a determination step, wherein the internal state related to at least one of the temperature and pressure of the container in the heating step is determined based on changes in the size of the beads.

[0014] Effects of the Invention

[0015] According to the present invention, there is an effect that the state of a sample can be efficiently determined. BRIEF DESCRIPTION OF THE DRAWINGS

[0016] [ Figure 1 ] is a diagram showing a configuration example of a heating and pressurizing determination system according to an embodiment.

[0017] [ Figure 2 ] is a diagram showing an example of experimental results of a heating and pressurizing confirmation experiment involved in an embodiment.

[0018] [ Figure 3 ] is a diagram showing an example of the relationship between temperature and pressure involved in the embodiment.

[0019] [ Figure 4 ] is a diagram showing an example of experimental condition 1 of a PCR amplicon measurement experiment involved in an embodiment.

[0020] [ Figure 5 ] is a diagram showing an example of experimental condition 2 of a PCR amplicon measurement experiment involved in an embodiment.

[0021] [ Figure 6 ] is a diagram showing an example of experimental condition 3 of a PCR amplicon measurement experiment involved in an embodiment.

[0022] [ Figure 7 ] is a diagram showing an example of the experimental results of a PCR amplicon measurement experiment involved in an embodiment.

[0023] [ Figure 8 ] is a flowchart showing an example of the process of the heating and pressurizing determination step involved in the embodiment. DETAILED DESCRIPTION

[0024] Hereinafter, a state identification method according to an embodiment of the present invention will be described in detail with reference to the accompanying drawings. It should be noted that the present invention is not limited to the embodiment described below.

[0025] [Implementation Method]

[0026] Hereinafter, the configuration of the heating and pressurizing determination system 100 according to the embodiment, the details of each process, and the flow of each process will be described in order, and finally, the effects of the embodiment will be described.

[0027] [1. Configuration of Heating and Pressurizing Determination System 100]

[0028] use Figure 1 The configuration of the heating and pressurizing determination system 100 according to the embodiment will be described. Figure 1 100. The following describes an example of the overall configuration of the heating and pressurizing determination system 100, an example of the process of the heating and pressurizing determination system 100, and the effects of the heating and pressurizing determination system 100.

[0029] (1-1. Overall Configuration Example of the Heating and Pressurizing Determination System 100)

[0030] The heating and pressurization determination system 100 includes a bacterial culture container 10, a sealing container 20, and a heating device 30. The bacterial culture container 10, the sealing container 20, and the heating device 30 will be described below in order.

[0031] (1-1-1. Bacterial Culture Container 10)

[0032] The bacterial culture container 10 is a container for containing bacterial culture solution S. The bacterial culture solution S is a solution for culturing microorganisms such as bacterial cells C. Figure 1 In the example shown, the bacterial cell culture container 10 is a stoppered Erlenmeyer flask, but the shape, material, capacity, etc. of the bacterial cell culture container 10 are not limited.

[0033] (1-1-2. Enclosed in container 20)

[0034] The enclosing container 20 is a container for enclosing the bacterial culture solution S and the foaming beads B. The foaming beads B are beads made of foamed polystyrene. Figure 1 In the example shown, the sealing container 20 is a microtube, but the shape, material, capacity, etc. of the sealing container 20 are not limited.

[0035] (1-1-3. Heating device 30)

[0036] The heating device 30 is a container for heating the heating solution H. Figure 1 In the example, the heating device 30 is a heating block, but the shape, material, heating method, etc. of the heating device 30 are not limited.

[0037] (1-1-4. Others)

[0038] exist Figure 1The heating and pressurizing determination system 100 shown may include a plurality of bacterial culture containers 10, a plurality of sealing containers 20, or a plurality of heating devices 30. In addition, the bacterial culture container 10 may be integrated with the sealing container 20.

[0039] (1-2. Example of Process of Heating and Pressurizing Determining System 100)

[0040] The following describes an example process of the heating and pressurization determination system 100. The following describes the foaming bead insertion process, the bacterial culture fluid collection process, the heating process, and the size confirmation process in sequence. It should be noted that the processes can be performed in a different order. Furthermore, some processes can be omitted.

[0041] (1-2-1. Foam Bead Insertion Process)

[0042] First, in the heating and pressurizing determination system 100, Figure 1 (1) The foaming bead inserting step. For example, in the foaming bead inserting step, foaming beads B made of expanded polystyrene are used and inserted into the sealing container 20. In this case, a plurality of foaming beads B may be inserted into the sealing container 20 in the foaming bead inserting step.

[0043] (1-2-2. Bacterial Culture Fluid Collection Step)

[0044] Second, in the heating and pressurizing determination system 100, the Figure 1 (2) shows a bacterial culture liquid collection step. For example, in the bacterial culture liquid collection step, a portion of the bacterial culture liquid S is collected into the sealing container 20 using a sterilized whole pipette. The bacterial culture liquid S is obtained by culturing Escherichia coli (E. coli) or Staphylococcus aureus (S. aureus) as bacterial cells C in an SCD (Soybean Casein Digest) liquid medium overnight at 37°C. At this time, in the bacterial culture liquid collection step, a lysis aid that promotes cell lysis of the bacterial cells C may be further added to the sealing container 20.

[0045] (1-2-3. Heating process)

[0046] Third, in the heating and pressurizing determination system 100, the Figure 1 (3) The heating step is shown. For example, in the heating step, the heating device 30 is preheated to a set temperature of 140°C, the sealing container 20 is placed in the heating device 30, and the sealing container 20 containing the foaming beads B and the bacterial culture solution S is heated at 140°C for 45 seconds.

[0047] (1-2-4. Size confirmation process)

[0048] Fourth, in the heating and pressurizing determination system 100, the Figure 1 (4) The size confirmation step is shown. For example, in the size confirmation step, the size reduction of the foaming beads B inserted into the sealing container 20 is visually confirmed, thereby confirming that the bacterial culture liquid S in the sealing container 20 is heated to 130°C or above. In this case, in the size confirmation step, the pressure applied to the interior of the sealing container 20 may be determined based on a relationship between temperature and pressure estimated from the capacity or contents of the sealing container 20.

[0049] (1-2-5. Others)

[0050] The heating and pressurization determination system 100 can further perform a nucleic acid purification step to purify the nucleic acids extracted from the bacterial cells C in the heating step. For example, in the nucleic acid purification step, the bacterial culture fluid S after the heating step is injected into a column, and then an eluent is injected to purify the nucleic acids extracted from the bacterial cells C.

[0051] The heating and pressurizing determination system 100 can further perform a nucleic acid amplification step to amplify the nucleic acid of the bacterial cells C extracted in the heating step. For example, in the nucleic acid amplification step, a PCR (Polymerase Chain Reaction) Mix is ​​added to the bacterial culture solution S after the heating step, and a PCR reaction is performed to amplify the nucleic acid extracted from the bacterial cells C.

[0052] (1-3. Effects of Heating and Pressurizing Determination System 100)

[0053] Hereinafter, the effects of the heating and pressurizing determination system 100 will be described after sequentially describing an overview of the heating determination technique as a reference technique and improvements of the reference technique.

[0054] (1-3-1. Overview of Reference Technology 1)

[0055] In Reference Technology 1 described in Patent Document 2, a wet heat color-changing indicator composition containing (A) Prussian blue, (B) a gallic acid ester such as propyl gallate, and (C) at least one compound selected from the group consisting of amino acids such as dicyandiamide and sodium glutamate, aromatic carboxylic acids such as benzoic acid, acid amides such as nicotinamide, and sugars such as starch is formulated into ink. The date of manufacture and expiration date are printed on the surface of retort pouch food packaging. Furthermore, in Reference Technology 1, the wet heat color-changing indicator composition turns blue before heat sterilization and black after treatment, thereby confirming heat sterilization.

[0056] (1-3-2. Improvements based on technique 1)

[0057] Reference technology 1 has the following improvements. First, reference technology 1 confirms color change at 121°C. However, in the nucleic acid extraction technology using the high temperature and high pressure method described in patent document 1, the effective processing temperature is about 140°C, so reference technology 1 that changes color at 121°C cannot confirm whether it actually reaches about 140°C. Second, reference technology 1 requires about 20 minutes to change the color of the pigment. However, the processing time of the nucleic acid extraction technology using high temperature and high pressure treatment described in patent document 1 is tens of seconds. When the processing is performed for more than tens of seconds, the genomic DNA is divided into more parts than necessary, which is likely to affect the subsequent nucleic acid amplification process of the PCR reaction. In summary, reference technology 1 is difficult to apply as a temperature monitoring method in the high temperature and high pressure method for nucleic acid extraction of bacterial cells C.

[0058] (1-3-3. Overview of Reference Technology 2)

[0059] Reference Art 2 described in Patent Document 3 provides a thermosensitive color-reversible composition comprising three components: a color-forming agent comprising a reversible dye that develops color with acid, a color-developing agent comprising a Lewis acid having a melting point in the temperature range of the desired color change, and a sensitizer. This composition develops color when heated and decolorizes when cooled, exhibiting a reversible color change. In Reference Art 2, suitable color-forming agents include leuco-based dyes, suitable color-developing agents include long-chain carboxylic acids, and suitable sensitizers include acid amides.

[0060] (1-3-4. Improvements based on technique 2)

[0061] Reference Technology 2 offers the following improvements. First, in Reference Technology 2, the leuco pigment changes color at 70°C and reversibly returns to its original color at 20°C. However, the effective processing temperature in the high-temperature, high-pressure nucleic acid extraction technique described in Patent Document 1 is approximately 140°C, so Reference Technology 2, which changes color at 70°C, cannot confirm whether the temperature actually reaches approximately 140°C. Second, in Reference Technology 2, since the leuco pigment develops color by reacting with acid, the hue change may be affected by the properties of the reaction solution, making it difficult to use for process control. In summary, Reference Technology 2 is difficult to apply as a temperature monitoring method in the high-temperature, high-pressure method for nucleic acid extraction from bacterial cells C.

[0062] (1-3-5. Overview of Heating and Pressurizing Determination System 100)

[0063] In the heating and pressurization determination system 100, a bacterial culture solution S containing bacterial cells C and foaming beads B are enclosed in a sealed container 20. The sealed container 20 is then sealed and heated. The internal state of the sealed container 20, which is related to at least one of the temperature and pressure of the sealed container 20, is determined based on changes in the size of the foaming beads B. Furthermore, in the heating and pressurization determination system 100, nucleic acids extracted from the cells of the bacterial cells C by heating are purified. Furthermore, in the heating and pressurization determination system 100, nucleic acids extracted from the cells of the bacterial cells C by heating are amplified.

[0064] (1-3-6. Effects of Heating and Pressurizing Determination System 100)

[0065] First, the heating and pressurization determination system 100 can directly confirm the temperature of the solution within a sealed container without leaking. Specifically, since expanded beads B inserted into the sealed container 20 shrink significantly at temperatures above 130°C, the heating and pressurization determination system 100 is expected to be applicable as a temperature monitoring method in autoclaves, a typical sterilization method performed at temperatures exceeding 120°C.

[0066] Second, the heating and pressurization determination system 100 makes it easy to confirm the maximum achieved temperature. Specifically, some materials used as indicators return to their pre-treatment color when returned to room temperature after treatment. However, the heating and pressurization determination system 100 allows the expanded beads B to maintain changes at a maximum achieved temperature of 130°C or higher even when returned to room temperature after high-temperature and high-pressure treatment, and this change can be visually confirmed. Therefore, it is expected to be applicable as a simple temperature monitoring method and process control method.

[0067] Third, the heating and pressurization determination system 100 eliminates the need for solvent replacement after nucleic acid extraction. Specifically, when the extracted cell contents are used in subsequent steps, the system has little effect on the extract itself or the PCR enzyme, eliminating the need for solvent replacement. This makes it promising for process control in the aforementioned high-temperature and high-pressure method.

[0068] [2. Details of Each Step of the Heating and Pressurizing Determination System 100]

[0069] right Figure 1 The following describes in detail each step of the heating and pressurization determination system 100, which serves as a method for determining the internal state of a container. The following describes the steps involved in this embodiment, sequentially including the bacterial cell culture step, the foaming bead insertion step, the bacterial cell culture fluid collection step, the heating step, the nucleic acid purification step, and the nucleic acid amplification step.

[0070] (2-1. Bacterial Cell Cultivation Step)

[0071] Hereinafter, the bacterial cell culture step of culturing bacterial cells C as a sample, which is performed before the bacterial cell culture liquid collection step of the heating and pressurization determination system 100, will be described.

[0072] (2-1-1. Specific example of bacterial cell culture step)

[0073] For example, in the bacterial cell culture step, bacterial cells C are cultured in a bacterial culture solution S contained in a bacterial cell culture container 10. To describe an example of a culture apparatus, in the bacterial cell culture step, a sterilized glass Erlenmeyer flask with a stopper is used as the bacterial cell culture container 10 to culture the bacterial cells C. To describe an example of the cultured bacterial cells C, in the bacterial cell culture step, Escherichia coli or Staphylococcus aureus is cultured. To describe an example of culture conditions, in the bacterial cell culture step, the bacterial cells C are cultured in an SCD liquid medium at 37°C overnight.

[0074] (2-1-2. Cultivation Method of Bacterial Culture Solution S)

[0075] The bacterial culture solution S used in the above-mentioned bacterial culture step is obtained by culturing a sample containing nucleic acid. The method for culturing the sample is not particularly limited. For example, a method in which a filter after capturing the sample is directly placed on a solid culture medium and the sample is cultured through the filter (solid phase culture) can be cited. In addition, as another method for culturing the sample, for example, a method in which the sample is cultured in the presence of a solution obtained by dissolving a liquid culture medium or a solid culture medium in water (liquid phase culture) can be cited. In addition, the type of liquid culture medium or solid culture medium used is selected according to the type of sample to be cultured and the physiological conditions.

[0076] (2-1-3. Sample of Bacterial Culture Solution S)

[0077] In the above-mentioned bacterial cell culture step, the sample to be processed is not particularly limited. For example, the sample to be processed may be microorganisms, animal cells other than microorganisms (such as insect cells), plant cells, mycoplasma, viruses, etc.

[0078] The microorganisms include, for example, at least one species selected from the group consisting of: Acinetobacter species, Actinomyces species, Aerococcus species, Aeromonas species, Alcaligenes species, Bacillus species, Bacteriodes species, Bordetella species, Branhamella species, Brevibacterium species, Campylobacter species, Candida species, Candida species, Capnocytophagia species, Chromobacterium species, Clostridium species, Corynebacterium species, Cryptococcus species, Deinococcus species, Enterococcus species, Erysipelothrix species, Escherichia species, Flavobacterium species, Gemella species, Haemophilus species Haemophilus species, Klebsiella species, Lactobacillus species, Lactococcus species, Legionella species, Leuconostoc species, Listeria species, Micrococcus species, Mycobacterium species, Neisseria species, Cryptosporidium species, Nocardia species, Oerskovia species , Paracoccus species, Pediococcus species, Peptostreptococcus species, Propionibacterium species, Proteus species, Pseudomonas species, Rahnella species, Rhodococcus species, Rhodospirillum species, Staphylococcus species, Streptomyces species, Streptococcus species,Vibrio species, as well as Yersinia species, Methylobacterium species, Ralstonia species, and Sphingomonas species.

[0079] Some of the aforementioned microorganisms may take on forms such as spores or spores, depending on their growth state. In the heating and pressurization determination system 100, the form of the sample being processed is not particularly limited. Furthermore, the sample being processed in the heating and pressurization determination system 100 may be a single type or two or more types.

[0080] (2-2. Foam Bead Insertion Process)

[0081] Hereinafter, the encapsulation step of enclosing expanded beads in the encapsulation container 20 , that is, the expanded bead inserting step, which is performed before and after the bacterial culture liquid collecting step of the heating and pressurizing determination system 100 , will be described.

[0082] (2-2-1. Specific Example of Foam Bead Insertion Step)

[0083] For example, in the foam bead inserting step, foam beads B, which are expanded polystyrene, are enclosed in the enclosing container 20. In the foam bead inserting step, the foam beads B may be enclosed in the enclosing container 20 from which the bacterial culture solution S has been collected.

[0084] (2-2-2. Foaming beads B)

[0085] There are no particular limitations on the material of the foam beads B. Specifically, the foam beads B may be expandable particles that shrink at a predetermined temperature or higher, and may be made of a foaming material other than the aforementioned expanded polystyrene, such as expanded polyurethane, expanded polyethylene, or expanded polypropylene.

[0086] There are no particular limitations on the color of the expanded beads B. Specifically, the expanded beads B may be any color that can be visually recognized, and may be red, blue, yellow, white, or the like.

[0087] The shape of the expanded beads B is not particularly limited. Specifically, the expanded beads B are preferably spheres of approximately 5 mm, but any size sufficient to be inserted into a sealed container 20 such as a PCR tube may be sufficient. Furthermore, the expanded beads B may be microparticles within a certain particle diameter range and may be ellipsoidal, polyhedral, or spherical in addition to spheres. The particle surface may have irregularities or pores penetrating the particle.

[0088] Furthermore, the expanded beads B preferably do not generate elutions that inhibit biological reactions such as PCR reactions when heated, but this is not particularly limited.

[0089] (2-3. Bacterial Culture Fluid Collection Step)

[0090] Hereinafter, a bacterial culture liquid collecting step of sealing the bacterial culture liquid S as a sample solution, which is performed after the bacterial culture liquid collecting step of the heating and pressurization determining system 100, will be described.

[0091] (2-3-1. Specific Example of Bacterial Culture Fluid Collection Step)

[0092] For example, in the bacterial culture fluid collection step, a portion of the bacterial culture fluid S contained in the bacterial culture container 10 is collected into the sealing container 20. To describe an example of the instrument used for collection, in the bacterial culture fluid collection step, a sterilized glass full-section pipette is used as the collection instrument, and a sterilized glass tube with a stopper is used as the sealing container 20 to collect the bacterial culture fluid S.

[0093] (2-3-2. Enclosed in container 20)

[0094] In the above-described bacterial culture fluid collection step, the sealing container 20 is not particularly limited. For example, the sealing container 20 may be a stoppered glass tube, a stoppered plastic tube, a microtube such as a PCR tube, or the like. Furthermore, the sealing container 20 may be airtight and durable up to temperatures of approximately 180°C during the heating step described below.

[0095] (2-3-3. Others)

[0096] In the above-mentioned bacterial culture liquid collection step, the bacterial culture liquid S may be pretreated before being collected and sealed in the container 20. For example, the bacterial culture liquid S may be a suspension containing bacterial cells C after adding an enzyme and incubating for a certain period of time. Alternatively, the bacterial culture liquid S may be a suspension containing bacterial cells C after centrifugation in a centrifuge to remove medium components.

[0097] (2-4. Heating process)

[0098] The following describes a heating step of sealing and heating the container 20 to extract nucleic acid from cells of the bacterial cells C serving as a sample, which is performed after the foaming bead inserting step and the bacterial culture fluid collecting step of the heating and pressurizing determination system 100 .

[0099] (2-4-1. Specific example of heating step)

[0100] For example, in the heating process, a dissolution aid that promotes cell lysis is added to the bacteria C in the sealing container 20 enclosing the bacterial culture solution S and the foaming beads B. The sealed container 20 is sealed by closing the lid, and the sealed container 20 is heated at 140°C for 45 seconds using a heating device 30 such as a heating block, thereby extracting nucleic acids from the cells of the bacteria C.

[0101] (2-4-2. Types of dissolution aids)

[0102] During the heating process of the heating and pressurizing determination system 100, water alone can achieve the aforementioned effects. However, for more efficient nucleic acid extraction from the sample, it is preferable to include, in addition to water, at least one dissolution aid selected from the group consisting of surfactants, bases, acids, redox agents, and protein denaturants. Dissolution aids have the ability to dissolve the membrane structure of the sample. By acting on the membrane structure of the sample, dissolution aids facilitate the disruption of the sample, enabling more efficient nucleic acid extraction from the sample. The following describes the types of dissolution aids.

[0103] (2-4-2-1. Surfactants)

[0104] The surfactant used as the dissolution aid may be, for example, ionic or nonionic. Examples of nonionic surfactants include octylphenol ethoxylate (C 14 H 22 O(C2H4O) n In the nucleic acid extraction step of the heating and pressurizing determination system 100, commercially available octylphenol ethoxylates can be used, for example, Triton X-100 (C 14 H 22 O(C2H4O) n , n=100) etc.

[0105] In addition, ionic surfactants can be anionic, cationic, or amphoteric. Examples of anionic surfactants include sodium dodecyl sulfate (SDS). Examples of cationic surfactants include cetyltrimethylammonium bromide (CTAB). Examples of amphoteric surfactants include betaine. Here, "betaine" refers to a general term for compounds that have positive and negative charges at non-adjacent positions in the same molecule, have no dissociable hydrogen atoms bonded to the atoms with the positive charge, and have no charge as a whole. A representative example of betaine is trimethylglycine.

[0106] (2-4-2-2. alkali)

[0107] Examples of the base used as the dissolution aid include sodium hydroxide (NaOH) and potassium hydroxide (KOH).

[0108] (2-4-2-3.Acid)

[0109] Examples of the acid used as the dissolution aid include hydrochloric acid (HCl) and sulfuric acid (H 2 SO 4 ).

[0110] (2-4-2-4. Redox Agents)

[0111] Examples of the redox agent used as the dissolution aid include aqueous hydrogen peroxide, β-mercaptoethanol, and dithiothreitol.

[0112] (2-4-2-5. Protein denaturant)

[0113] Examples of protein denaturants used as solubilization aids include guanidine hydrochloride and urea.

[0114] (2-4-2-6. Others)

[0115] A chelating agent may be used as a component of the dissolution aid. Examples of the chelating agent used as the dissolution aid include ethylenediaminetetraacetic acid (EDTA).

[0116] Furthermore, among the above-mentioned dissolution aids, the dissolution aid of the heating and pressurization determination system 100 preferably includes a surfactant, and more preferably includes one or both of SDS and octylphenol ethoxylate.

[0117] For example, SDS can be used to detect nucleic acids extracted in the nucleic acid extraction step of the heating and pressurization determination system 100 with high sensitivity. In contrast, when nucleic acids extracted in the nucleic acid extraction step of the heating and pressurization determination system 100 are used in an enzyme reaction inhibited by SDS, octylphenol ethoxylate can be used because it acts more gently on the membrane structure of the sample than SDS.

[0118] The dissolution aid of the heating and pressurizing determination system 100 may contain a buffer as needed. Examples of the buffer include tris-HCl.

[0119] (2-4-3. Types of Nucleic Acids)

[0120] The type of nucleic acid extracted in the heating step is not particularly limited. For example, the extracted nucleic acid may include deoxyribonucleic acid (DNA) such as genomic DNA or plasmid DNA, or ribonucleic acid (RNA) such as messenger RNA, transfer RNA, or ribosomal RNA.

[0121] (2-5. Dimension confirmation process)

[0122] The following describes the size confirmation process, which is implemented after the heating process of the heating determination system 100 and is a determination process for determining the internal state related to at least one of the temperature and pressure of the sealing container 20 in the heating process based on the change in the size of the foaming beads B.

[0123] (2-5-1. Specific example of the size confirmation process)

[0124] For example, during the size confirmation step, the shrinkage of the foaming beads B can be used to confirm that the temperature at which the sealing container 20 is heated is 130°C or higher. Specifically, during the size confirmation step, if the foaming beads B significantly shrink in size, decreasing to less than half of their original size, it can be confirmed that the bacterial culture solution S containing the bacterial cells C has been heated to 130°C or higher. In this case, since the size changes in response to temperatures of 130°C or higher during the size confirmation step, the temperature at which the bacterial culture solution S containing the bacterial cells C has been heated can be estimated based on the size of the foaming beads B after shrinkage.

[0125] Furthermore, during the size confirmation step, the pressure of the sealed container 20 can be determined based on the relationship between temperature and pressure. Specifically, during the size confirmation step, it can be determined that the maximum pressure reaches 2757 hPa at a maximum temperature of 130°C, 3706 hPa at a maximum temperature of 140°C, 4906 hPa at a maximum temperature of 150°C, 6403 hPa at a maximum temperature of 160°C, 8249 hPa at a maximum temperature of 170°C, and 10498 hPa at a maximum temperature of 180°C.

[0126] (2-6. Nucleic Acid Purification Step)

[0127] The following describes the nucleic acid purification step of purifying nucleic acids extracted from cells of bacterial cells C, which is performed after the heating step by the heating determination system 100 .

[0128] (2-6-1. Specific example of nucleic acid purification step)

[0129] For example, in the nucleic acid purification step, after injection into a column having an adsorption carrier for adsorbing nucleic acid, the nucleic acid is purified by injecting an elution solution for eluting the nucleic acid into the column.

[0130] (2-7. Nucleic Acid Amplification Step)

[0131] The following describes the nucleic acid amplification step of amplifying nucleic acids extracted from cells of bacterial cells C, which is performed after the heating step or the nucleic acid purification step in the heating determination system 100 .

[0132] (2-7-1. Specific Example of Nucleic Acid Amplification Step)

[0133] For example, in the nucleic acid amplification process, nucleic acid is amplified by performing a PCR reaction on a solution containing nucleic acid extracted in a heating process. Alternatively, in the nucleic acid amplification process, nucleic acid is amplified by performing a PCR reaction on a solution containing nucleic acid purified in a nucleic acid purification process.

[0134] [3. Various experimental results]

[0135] use Figures 2 to 7 Various experimental results of the heating and pressurizing determination system 100 according to the embodiment will be described. Hereinafter, the experimental results related to the heating and pressurizing confirmation experiment and the experimental results related to the PCR amplicon measurement experiment will be described in sequence.

[0136] (3-1. Heating and Pressurizing Confirmation Test)

[0137] use Figure 2 and Figure 3 , the experimental results related to the heating and pressurizing confirmation experiment for confirming the heating temperature and the like using the heating and pressurizing determination system 100 will be described. Figure 2 This is a diagram showing an example of experimental results of a heating and pressurizing confirmation experiment according to the embodiment. Figure 3 : is a graph showing an example of the relationship between temperature and pressure according to the embodiment. The following describes the experimental procedure and the experimental results of the heating and pressurizing confirmation experiment in sequence.

[0138] (3-1-1. Experimental procedures for the heating and pressurization confirmation test)

[0139] An example of the experimental steps involved in a heating confirmation test using heating and pressurization determination system 100 will be described. First, insert 6.5 mm foam beads B made of expanded polystyrene into a PCR tube serving as sealing container 20. Second, inject 40 μL of a dissolution aid solution containing 1% SDS and Tris-HCl into the PCR tube containing the foam beads B. Third, seal the PCR tube containing the foam beads B and dissolution aid and heat for 60 seconds. Fourth, visually inspect the condition of the foam beads B after the heat treatment.

[0140] (3-1-2. Experimental results of the heating and pressurizing confirmation test)

[0141] use Figure 2 , the experimental results of the heating and pressurizing confirmation experiment are explained. Figure 2 The diagram shows the states of the PCR tubes after heating, including a mixed solution that was not heated, i.e., the negative control sample "NC", a mixed solution heated at 90°C, i.e., "90°C", a mixed solution heated at 100°C, i.e., "100°C", a mixed solution heated at 110°C, i.e., "110°C", a mixed solution heated at 120°C, i.e., "120°C", a mixed solution heated at 130°C, i.e., "130°C", a mixed solution heated at 140°C, i.e., "140°C", a mixed solution heated at 150°C, i.e., "150°C", and a mixed solution heated at 160°C, i.e., "160°C".

[0142] (3-1-3. Examination of Experimental Results of Heating and Pressurizing Confirmation Test)

[0143] exist Figure 2 In the experiment, no size reduction of the expanded beads B was observed at "NC", "90°C", "100°C", "110°C" and "120°C", but a significant size reduction of the expanded beads B was observed at "130°C", "140°C", "150°C" and "160°C". Figure 2 In the figures, the sizes of the expanded beads B can be confirmed as "130°C" > "140°C" > "150°C" > "160°C".

[0144] According to the experimental results of the heating and pressurizing confirmation experiment, in the heating and pressurizing confirmation system 100, by visually observing the change in the size of the expanded beads B, it was possible to confirm that each sample was heated to 130° C. or higher.

[0145] (3-1-4. Relationship between temperature and pressure)

[0146] use Figure 3 , the relationship between the temperature and pressure inside the sealed container 20 is described. Figure 3As shown, the theoretical relationship between the internal pressure [hPa] and the temperature [°C] of the reaction tube can be derived based on information such as the volume of the sealing container 20, the volume of the foam beads B, and the composition or amount of the contents.

[0147] exist Figure 3 In the example, a temperature of 20°C indicates an internal pressure of 23 hPa in the reaction tube, a temperature of 40°C indicates an internal pressure of 73 hPa in the reaction tube, a temperature of 60°C indicates an internal pressure of 199 hPa in the reaction tube, a temperature of 80°C indicates an internal pressure of 475 hPa in the reaction tube, a temperature of 100°C indicates an internal pressure of 1022 hPa in the reaction tube, a temperature of 110°C indicates an internal pressure of 1450 hPa in the reaction tube, a temperature of 120°C indicates an internal pressure of 2018 hPa in the reaction tube, and a temperature of 130°C indicates an internal pressure of The temperature “140℃” indicates the pressure in the reaction tube is “2757hPa”, the temperature “140℃” indicates the pressure in the reaction tube is “3706hPa”, the temperature “150℃” indicates the pressure in the reaction tube is “4906hPa”, the temperature “160℃” indicates the pressure in the reaction tube is “6403hPa”, the temperature “170℃” indicates the pressure in the reaction tube is “8249hPa”, the temperature “180℃” indicates the pressure in the reaction tube is “10498hPa”, the temperature “190℃” indicates the pressure in the reaction tube is “13210hPa”, and the temperature “200℃” indicates the pressure in the reaction tube is “16450hPa”.

[0148] That is, the pressure applied to the interior of the sealing container 20 can be determined based on the heating temperature estimated from the size of the heated foamed beads B or the set temperature of the heating device 30. On the other hand, when applying the pressure applied to the foamed beads B used as the comparison object, the heating temperature can also be determined based on the size of the heated foamed beads B.

[0149] (3-2. PCR amplicon determination experiment)

[0150] use Figures 4 to 7 , the experimental results related to the PCR amplicon measurement experiment in which the PCR amplicon is measured using the heating and pressurizing determination system 100 are described. Figures 4 to 6 This is a diagram showing an example of experimental conditions for a PCR amplicon measurement experiment according to an embodiment. Figure 7 1 is a diagram showing an example of experimental results of a PCR amplicon measurement experiment according to an embodiment. The experimental results of the PCR amplicon measurement experiment will be described below in order while showing the experimental procedure.

[0151] (3-2-1. Experimental steps for PCR amplicon determination experiment)

[0152] use Figures 4 to 6, an example of the experimental steps related to the PCR amplicon determination experiment of the heating and pressurizing determination system 100 is described. First, the foaming beads B and the bacterial culture solution S are sealed in the sealing container 20, and a high-temperature and high-pressure treatment (140°C, heating for 45 seconds) using the high-temperature and high-pressure method is performed. Second, the bacterial culture solution S after the high-temperature and high-pressure treatment is diluted to 1 / 100 to prepare a dilution solution. Third, 20 μL of the dilution solution and 20 μL of the PCR Mix shown in experimental conditions 1 and 2 are mixed, and a PCR reaction is performed under the temperature cycle conditions shown in experimental condition 3. Fourth, the solution after the PCR reaction is electrophoresed using the "Agilent2100 Bioanalyzer Electrophoresis System" manufactured by Agilent Technologies to confirm whether PCR amplification occurs.

[0153] (3-2-1-1. Experimental Condition 1)

[0154] First, use Figure 4 , the experimental condition 1 related to the primers for the PCR amplicon measurement experiment is described. Figure 4 In the example shown in FIG, the forward primer is named "16S290f_2" and has a base sequence of "GACACGGCCCAGACTCCTAC." Furthermore, the reverse primer is named "16S500r+GG" and has a base sequence of "GTATTACCGCGGCTGCTGG." Furthermore, the number of base pairs in the amplicon is "211 bp."

[0155] (3-2-1-2. Experimental Condition 2)

[0156] Second, use Figure 5 , the experimental condition 2 related to the reagents for the PCR amplicon measurement experiment is described. Figure 5 As shown in the example, as reagents for the PCR amplicon measurement experiment, 1.0 U / μL, 0.20 μL / tube of "Platinum Taq DNA Polymerase", 20 μL / tube of "High Temperature and High Pressure Treatment Solution", 10.0 μM, 0.8 μL / tube of "Forward Primer", 10.0 μM, 0.8 μL / tube of "Reverse Primer", 50 mM, 1.60 μL / tube of "Magnesium Sulfate MgSO4", 2.00 mM, 4.00 μL / tube of "dNTP (deoxynucleoside triphosphate) Mix", 10-fold concentration, 4.00 μL / tube of "10× PCR Buffer", and 8.60 μL / tube of "Milli-Q Water" are mixed, totaling 40.00 μL / tube.

[0157] (3-2-1-3. Experimental Condition 3)

[0158] 3. Use Figure 6 , Experimental Condition 3 related to the reaction conditions of the PCR amplicon determination experiment is described. Figure 6 As shown in the example of , as the reaction conditions of the PCR amplicon measurement experiment, the "activation" process is implemented for one cycle at 98°C for 120 seconds. The DNA chain extension reaction includes three processes: "denaturation", "annealing" and "extension". The "denaturation" process is implemented at 98°C for 15 seconds, the "annealing" process is implemented at 58°C for 25 seconds, and the "extension" process is implemented at 72°C for 15 seconds. These processes constitute one cycle, and 35 cycles are performed. In addition, the "add. extension" process is implemented for one cycle at 72°C for 120 seconds.

[0159] (3-2-2. Experimental Results of PCR Amplicon Measurement Experiment)

[0160] use Figure 7 , Experimental result 1 of the PCR amplicon determination experiment is described. Figure 7 A is the electrophoresis diagram of the bacterial culture solution S without the foaming beads B, that is, the positive control sample "PC". Figure 7 B is the electrophoresis diagram of the bacterial culture solution S that was not subjected to high temperature and high pressure treatment, that is, the negative control sample "NC". Figure 7 C is an electrophoretic diagram of the "foaming beads method (with a solubilizing agent, 1 / 100 dilution)" in which the foaming beads B were inserted and a solubilizing agent was added to the bacterial culture solution S. Figure 7 D is the electrophoresis diagram of the "foaming beads method (with a solubilizing agent)" in which the foaming beads B were inserted and the bacterial culture solution S was not added with a solubilizing agent.

[0161] (3-2-3. Examination of Experimental Results of PCR Amplicon Measurement Experiment)

[0162] according to Figure 7 , compared with the results of PCR using bacterial culture fluid S without the insertion of foaming beads B ( Figure 7 A) compared to the positive control sample "PC", the results of PCR using the bacterial culture solution S inserted with the foaming beads B ( Figure 7 D) No significant reduction in PCR amplicons was found.

[0163] Based on the experimental results of the PCR amplicon measurement experiment, it was confirmed that the heating and pressurization determination system 100 had no influence on the nucleic acid extraction process and the nucleic acid amplification process.

[0164] [4. Processing Flow of Heating and Pressurizing Determination System 100]

[0165] use Figure 8 , the process flow of the heating and pressurizing determination system 100 involved in the embodiment is described. Figure 8 This is a flowchart showing an example of the process of the heating and pressurizing determination step according to the embodiment. It should be noted that the following steps S101 to S104 can be performed in a different order. In addition, some steps in the following steps S101 to S104 can be omitted.

[0166] First, in the heating and pressurizing determination system 100, a foaming bead insertion process is performed (step S101). Second, in the heating and pressurizing determination system 100, a bacterial culture fluid collection process is performed (step S102). Third, in the heating and pressurizing determination system 100, a heating process is performed (step S103). Fourth, in the heating and pressurizing determination system 100, a size confirmation process is performed (step S104), and the heating and pressurizing determination process is terminated. It should be noted that in the heating and pressurizing determination system 100, a nucleic acid purification process or a nucleic acid amplification process may also be performed after the size confirmation process.

[0167] [5. Effects of Implementation]

[0168] Finally, the effects of the embodiment will be described. Hereinafter, effects 1 to 7 corresponding to the steps according to the embodiment will be described.

[0169] (5-1. Effect 1)

[0170] First, in the process according to the above embodiment, a sample solution containing a sample and foaming beads B are sealed in a sealing container 20, the sealing container 20 is sealed and then heated, and the internal state of the sealing container 20 related to at least one of the temperature and pressure is determined based on changes in the size of the foaming beads B. Therefore, in the process according to the embodiment, the state of the sample can be effectively determined.

[0171] (5-2. Effect 2)

[0172] Second, in the step according to the embodiment, the shrinkage of the expanded beads B confirms that the temperature of the sealing container 20 is heated to 130° C. or higher. Therefore, in the step according to the embodiment, the state of the sample can be effectively confirmed during the heating treatment at 140° C. or higher.

[0173] (5-3. Effect 3)

[0174] Third, in the process according to the embodiment, the expanded beads B are made of expanded polystyrene. Therefore, in the process according to the embodiment, the state of the sample can be determined efficiently by using the easily available expanded beads B.

[0175] (5-4. Effect 4)

[0176] Fourthly, in the process according to the above embodiment, the sealing container 20 is a PCR tube. Therefore, in the process according to the embodiment, the state of the sample can be determined efficiently during the process of handling a small amount of sample.

[0177] (5-5. Effect 5)

[0178] Fifth, in the steps according to the above embodiment, the sample solution is a bacterial culture solution S containing bacterial cells C. Therefore, in the steps according to the embodiment, the state of the sample can be efficiently determined during the high temperature and high pressure method for extracting nucleic acid from cells of bacterial cells C.

[0179] (5-6. Effect 6)

[0180] Sixth, in the steps of the above embodiment, nucleic acids extracted from cells of bacterial cell C are purified. Therefore, in the steps of the embodiment, the state of the sample can be determined efficiently without affecting the purification of nucleic acids extracted from cells of bacterial cell C.

[0181] (5-7. Effect 7)

[0182] Seventh, in the steps of the above embodiment, nucleic acids extracted from cells of bacterial cell C are amplified. Therefore, in the steps of the embodiment, the state of the sample can be determined efficiently without affecting the amplification of nucleic acids extracted from cells of bacterial cell C.

[0183] [system]

[0184] The processing steps, control steps, specific names, and information including various data or parameters shown in the above documents or drawings may be changed arbitrarily unless otherwise specified.

[0185] Furthermore, the components of the devices shown in the diagrams are conceptual and functional, and are not necessarily physically configured as shown. That is, the specific configuration of the distribution or integration of the devices is not limited to that shown. In other words, all or part of them may be functionally or physically distributed / integrated in arbitrary units, depending on various loads or usage conditions.

[0186] Explanation of symbols

[0187] 10 Bacteria culture container

[0188] 20 sealed in container

[0189] 30 Heating device

[0190] 100 Heating and pressurizing determination system

Claims

1. A state determination method, which is a state determination method for determining the internal state of a container, comprising: a sealing step of sealing a sample solution containing a sample and foamed beads in the container; a heating step, wherein the container is sealed and heated; and A determining step of determining the internal state related to at least one of the temperature and the pressure of the container in the heating step based on the change in the size of the beads.

2. The state determination method according to claim 1, wherein: The determining step determines that the temperature at which the container is heated is 130° C. or higher based on the shrinkage of the beads.

3. The state determination method according to claim 1, wherein: The beads are expanded polystyrene.

4. The state determination method according to claim 1, wherein: The container is a PCR (Polymerase Chain Reaction) tube.

5. The state determination method according to any one of claims 1 to 4, wherein: The sample solution is a bacterial culture solution containing bacterial cells.

6. The state determination method according to claim 5, further comprising A purification step of purifying the nucleic acid extracted from the cells of the bacterial body by the heating step.

7. The state determination method according to claim 5, further comprising an amplification step of amplifying the nucleic acid extracted from the cells of the bacterial body by the heating step.

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