State determination method

Abstract

A state determination method includes adding a red dye beet red B to a bacterial culture solution S containing bacteria C, heating the bacterial culture solution S with beet red B, and determining a temperature at which the bacterial culture solution S is heated based on a change in color tone of beet red B.

Description

Technical Field

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

[0002] Techniques for collecting bacterial culture fluids, adding a solubilizing agent, and extracting nucleic acids from the bacterial cells contained in the culture fluid (referred to as "high-temperature and high-pressure methods" depending on the context) are known. Furthermore, in processes requiring heat treatment of a container, techniques are known for determining the temperature at which the container was heated by observing the change in color tone of a temperature-dependent dye, such as Prussian blue or leuco dye (referred to as "heating temperature" or "heating" depending on the context).

[0003] References

[0004] Patent Literature

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

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

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

[0008] Technical issues

[0009] However, the above techniques make it difficult to effectively determine the heating temperature of the bacterial culture solution stored in the container. For example, in the above-mentioned method for confirming the color change of Prussian blue, since the color change occurs at approximately 121°C, which is lower than the approximately 140°C temperature at which nucleic acid can be extracted from bacteria, it is difficult to confirm whether the temperature has actually reached approximately 140°C. Furthermore, in the method for confirming the color change of leuco dye, since the color change occurs at approximately 70°C and the color reversibly returns to the initial color at 20°C, it is difficult to confirm whether the temperature has actually reached approximately 140°C.

[0010] The present invention has been made in view of the above circumstances, and an object thereof is to enable efficient determination of the heating temperature of a sample.

[0011] Solution to the problem

[0012] According to one aspect of the embodiment, a state determination method for determining a temperature at which a sample is heated includes: an adding step of adding a predetermined dye to a sample solution containing the sample; a heating step of heating the sample solution to which the predetermined dye has been added by the adding step; and a determining step of determining the temperature at which the sample solution is heated by the heating step based on a change in hue of the predetermined dye.

[0013] Advantageous Effects of the Invention

[0014] The present invention has the effect of effectively determining the heating temperature of a sample. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] [ Figure 1 ] Figure 1 is a diagram showing a configuration example of a heating determination system according to an embodiment.

[0016] [ Figure 2 ] Figure 2 : is a diagram showing an example of a colored component of beetroot red according to an embodiment.

[0017] [ Figure 3 ] Figure 3 It is a diagram showing an example of Experimental Result 1 of a heating determination experiment according to the embodiment.

[0018] [ Figure 4 ] Figure 4 It is a diagram showing an example of Experimental Result 2 of a heating determination experiment according to the embodiment.

[0019] [ Figure 5 ] Figure 5 It is a diagram showing an example of Experimental Result 3 of a heating determination experiment according to the embodiment.

[0020] [ Figure 6 ] Figure 6 1 is a diagram showing an example of experimental condition 1 of a PCR amplicon measurement experiment according to the embodiment.

[0021] [ Figure 7 ] Figure 7 1 is a diagram showing an example of experimental condition 2 of a PCR amplicon measurement experiment according to the embodiment.

[0022] [ Figure 8 ] Figure 8 3 is a diagram showing an example of experimental condition 3 of a PCR amplicon measurement experiment according to the embodiment.

[0023] [ Figure 9 ] Figure 9 It is a diagram showing an example of Experimental Result 1 of a PCR amplicon measurement experiment according to the embodiment.

[0024] [ Figure 10 ] Figure 10 It is a diagram showing an example of Experimental Result 2 of a PCR amplicon measurement experiment according to the embodiment.

[0025] [ Figure 11 ] Figure 11 : is a flowchart showing an example of the flow of the heating determination step according to the embodiment. DETAILED DESCRIPTION

[0026] The state determination method according to an embodiment of the present invention will be described in detail below with reference to the accompanying drawings. Please note that the present invention is not limited to the embodiment described below.

[0027] First embodiment

[0028] Hereinafter, the configuration of the heating determination system 100 according to the embodiment, the details of each step, the flow of these steps, and finally the effects of the embodiment will be described in sequence.

[0029] 1. Configuration of the Heating Determination System 100

[0030] Will refer to Figure 1 The configuration of the heating determination system 100 according to the embodiment is described. Figure 1 The figure shows a configuration example of the heating determination system 100 according to the embodiment. Hereinafter, the configuration example of the entire heating determination system 100, examples of each step of the heating determination system 100, and effects of the heating determination system 100 will be described in this order.

[0031] (1-1. Configuration Example of Overall Heating Determination System 100)

[0032] The heating determination system 100 includes a bacteria culture container 10, a sealed container 20, and a heating device 30. Hereinafter, the bacteria culture container 10, the sealed container 20, and the heating device 30 will be described in this order.

[0033] (1-1-1. Bacteria Culture Container 10)

[0034] The bacterial culture container 10 is a container for storing a bacterial culture solution S. The bacterial culture solution S is a solution for culturing microorganisms such as bacteria C. Figure 1 In the example, the bacterial culture container 10 is a conical flask with a stopper, but the shape, material, capacity, etc. of the bacterial culture container 10 are not limited.

[0035] (1-1-2. Sealed Container 20)

[0036] The sealed container 20 is a container for sealing the heated solution H. The heated solution H is a solution obtained by adding red dye beetroot red B to the bacterial culture solution S. Figure 1 In the example, the sealed container 20 is a glass tube with a stopper, but the shape, material, capacity, etc. of the sealed container 20 are not limited.

[0037] (1-1-3. Heating device 30)

[0038] The heating device 30 is a container for heating the heating solution H. Figure 1In 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.

[0039] (1-1-4. Others)

[0040] Figure 1 The heating determination system 100 shown may include a plurality of bacteria culture containers 10, a plurality of sealed containers 20, or a plurality of heating devices 30. Furthermore, the bacteria culture container 10 and the sealed container 20 may be integrated.

[0041] (1-2. Example of Steps of Heating Determination System 100)

[0042] An example of the steps of heating determination system 100 will be described below. The steps below will be described in the order of bacterial culture fluid collection, dye addition, heating, and dye confirmation. Note that these steps can be performed in a different order. Some of these steps may be omitted.

[0043] (1-2-1. Bacterial culture fluid collection step)

[0044] First, in the heating determination system 100, Figure 1 (1) Bacterial culture liquid collection step. In the bacterial culture liquid collection step, for example, Escherichia coli (E. coli) or Staphylococcus aureus (S. aureus) is used as bacteria C, and a portion of the bacterial culture liquid S cultured overnight at 37° C. in a soybean casein digest (SCD) liquid medium is collected into a sealed container 20 using a sterilized full-volume pipette.

[0045] (1-2-2. Dye Addition Step)

[0046] Second, in the heating determination system 100, Figure 1 (2) Dye addition step. For example, in the dye addition step, beetroot red B is used, and an aqueous solution of beetroot red B is added to the sealed container 20 containing the bacterial culture fluid S, and the container is sealed with a stopper. In this case, a dissolution aid that promotes cell lysis of bacteria C may also be added to the sealed container 20 during the dye addition step.

[0047] (1-2-3. Heating step)

[0048] Third, in the heating determination system 100, Figure 1 (3) Heating step shown. For example, in the heating step, the heating device 30 is preheated to a set temperature of 140°C, the sealed container 20 is placed in the heating device 30, and the sealed container 20 sealed with the heating solution H is heated at 140°C for 45 seconds.

[0049] (1-2-4. Dye confirmation step)

[0050] Fourth, in the heating determination system 100, Figure 1 (4) Dye confirmation step. For example, in the dye confirmation step, visual confirmation is made that the red color of the betalain B sealed in the sealed container 20 has disappeared, thereby confirming that the heated solution H in the sealed container 20 has been heated to 140°C or above. In this case, in the dye confirmation step, the heating temperature can also be determined by measuring the absorbance of the betalain B sealed in the sealed container 20 and based on the decrease in absorbance.

[0051] (1-2-5. Others)

[0052] In the heating determination system 100, a nucleic acid purification step can also be performed to purify the nucleic acid of the bacteria C extracted in the heated solution H. For example, in the nucleic acid purification step, the heated solution H after the heating step can be injected into the column, and the eluent can be injected to purify the extracted nucleic acid of the bacteria C.

[0053] In the heating determination system 100 , a nucleic acid amplification step may be further performed to amplify the nucleic acid of the bacteria C extracted from the heated solution H. For example, in the nucleic acid amplification step, the nucleic acid of the extracted bacteria C may be amplified by adding a polymerase chain reaction (PCR) mixture to the heated solution H after the heating step and performing PCR.

[0054] (1-3. Effects of Heating Determination System 100)

[0055] Hereinafter, an outline of a heating determination technique as a reference technique and improvements required for the reference technique will be described in sequence, and then the effects of the heating determination system 100 will be described.

[0056] (1-3-1. Overview of Reference Technology 1)

[0057] In Reference Technology 1 described in Patent Document 2, a wet heat color-changing indicator composition containing the following substances is formulated into an ink, and the production date, expiration date, and other information are printed on the surface of heat-sealed food packaging: (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, amides such as nicotinamide, and sugars such as starch. In Reference Technology 1, the wet heat color-changing indicator composition exhibits a blue color before heat sterilization but turns black after treatment, thereby confirming heat sterilization.

[0058] (1-3-2. Improvements required for reference technology 1)

[0059] Reference technology 1 requires the following improvements. First, reference technology 1 confirms that the color changes at 121°C, but since the effective processing temperature of the high temperature and high pressure method of nucleic acid extraction technology described in patent document 1 is 140°C, reference technology 1 that changes color at 121°C cannot confirm whether the temperature has actually reached 140°C. Secondly, the change in the color of the dye in reference technology 1 takes about 20 minutes, while the processing time of the high temperature and high pressure method of nucleic acid extraction technology described in patent document 1 is tens of seconds. Therefore, more processing is likely to cause unnecessary fragmentation of genomic DNA and affect the subsequent nucleic acid amplification step by PCR. In summary, it is difficult to apply reference technology 1 as a temperature monitoring method to the high temperature and high pressure method for extracting nucleic acids from bacteria C.

[0060] (1-3-3. Overview of Reference Technology 2)

[0061] Reference Technology 2 described in Patent Document 3 provides a temperature-sensitive, hue-reversible composition comprising three components: a color former composed of a reversible dye that develops color with an acid; a color developer composed of a Lewis acid whose melting point is within the temperature range of the desired hue change; and a sensitizer that develops color upon heating and loses color upon cooling, thereby exhibiting a reversible hue change. In Reference Technology 2, examples of suitable color formers include leuco dyes, examples of suitable color developers include long-chain carboxylic acids, and examples of suitable sensitizers include amides.

[0062] (1-3-4. Improvements required for reference technology 2)

[0063] Reference Technology 2 requires the following improvements. First, in Reference Technology 2, the leuco dye changes color at 70°C, and the color reversibly returns to the initial color at 20°C. However, since the effective processing temperature of the high-temperature and high-pressure nucleic acid extraction technology described in Patent Document 1 is 140°C, Reference Technology 2, which changes color at 70°C, cannot confirm whether the temperature has actually reached 140°C. Second, in Reference Technology 2, the leuco dye has the characteristic of developing color when exposed to acid, so its color change can be affected by the properties of the reaction solution, making the leuco dye difficult to use for process control. In summary, it is difficult to apply Reference Technology 2 as a temperature monitoring method to the high-temperature and high-pressure method for extracting nucleic acid from bacteria C.

[0064] (1-3-5. Overview of Heating Determination System 100)

[0065] In the heating determination system 100, a red dye, betalain B, is added to a bacterial culture liquid S containing bacteria C, the bacterial culture liquid S containing betalain B is heated, and the heating temperature of the bacterial culture liquid S is determined based on a change in the color tone of betalain B. Furthermore, in the heating determination system 100, nucleic acid is extracted from the cells of the bacteria C by heating. Furthermore, in the heating determination system 100, the nucleic acid extracted from the cells of the bacteria C is amplified by heating.

[0066] (1-3-6. Effect of Heating Determination System 100)

[0067] First, the heating determination system 100 can confirm the color change at 140° C. That is, since beet red B has a color change even at 140° C., it is expected that the heating determination system 100 can be applied to an autoclave as a temperature monitoring method, which is a typical sterilization method performed at a temperature higher than 120° C.

[0068] Second, heating determination system 100 makes it easy to determine the maximum achievable temperature. While some materials used as indicators may return to their pre-treatment color when the temperature returns to room temperature after treatment, heating determination system 100 allows beetroot red B to retain its color change at the maximum achievable temperature even after returning to room temperature after high-temperature and high-pressure treatment. Therefore, beetroot red B is expected to be applicable to temperature monitoring methods and process control.

[0069] Third, the heating determination system 100 eliminates the need for solvent replacement after nucleic acid extraction. Specifically, when the extracted cell contents are used in subsequent steps, solvent replacement is unnecessary because it has little effect on the extract itself or the PCR enzyme. Consequently, the heating determination system 100 is expected to be useful for process control in high-temperature and high-pressure methods.

[0070] Fourth, the safety of the heating determination system 100 is high because betalain B is a natural dye. That is, in the heating determination system 100, betalain B is a natural dye and is a safe material that is regarded as a food additive and can be used in food factories, etc.

[0071] 2. Details of the Steps of the Heating Determination System 100

[0072] Will describe Figure 1 Detailed steps of heating determination system 100 are shown. Heating determination system 100 is a state determination method for determining the temperature at which a sample is heated. The steps according to this embodiment are described below in the order of a bacterial culture step, a bacterial culture fluid collection step, a dye addition step, a heating step, a dye confirmation step, a nucleic acid purification step, and a nucleic acid amplification step.

[0073] (2-1. Bacterial Culture Step)

[0074] Hereinafter, a bacteria culturing step of culturing bacteria C as a sample, which is performed before the bacteria culture liquid collecting step of the heating determination system 100 , will be described.

[0075] (2-1-1. Specific Example of Bacteria Cultivation Step)

[0076] For example, in the bacterial culturing step, bacteria C are cultured in a bacterial culture liquid S stored in a bacterial culture container 10. An example of a culture apparatus is described below. In the bacterial culturing step, a sterilized Erlenmeyer flask with a glass stopper is used as the bacterial culture container 10 to culture bacteria C. An example of bacteria C to be cultured is described below. In the bacterial culturing step, Escherichia coli and Staphylococcus aureus are cultured. An example of culture conditions is described below. In the bacterial culturing step, bacteria C are cultured overnight at 37° C. in an SCD liquid medium.

[0077] (2-1-2. Cultivation Method of Bacterial Culture Solution S)

[0078] The bacterial culture fluid S for the bacterial culture step is obtained by cultivating a sample comprising nucleic acid. There is no particular limitation on the method for cultivating a sample, and an example thereof includes directly placing a filter having a sample on a solid culture medium and cultivating the sample by a filter (solid phase culture). Other methods for cultivating a sample are, for example, in a liquid culture medium or by dissolving a solid culture medium in water and cultivating the sample in the presence of a solution (liquid phase culture). In addition, the type of liquid culture medium or solid culture medium to be used is selected according to the type of the sample to be cultivated and physiological conditions.

[0079] (2-1-3. Sample of Bacterial Culture Fluid S)

[0080] In the bacterial culture step, the sample to be processed is not particularly limited. For example, the sample to be processed may be a microorganism, an animal cell other than a microorganism (for example, an insect cell, etc.), a plant cell, a mycoplasma, a virus, etc.

[0081] Examples of microorganisms include at least one genus selected from the group consisting of Acinetobacter, Actinomyces, Aerococcus, Aeromonas, Alcaligenes, Bacillus, Bacteriodes, Bordetella, Branhamella, Brevibacterium, Campylobacter, Candida, Capnocytophagia, Chromobacterium, Clostridium, Corynebacterium, Cryptococcus, Deinococcus, Enterococcus, Erysipelothrix, Escherichia, Flavobacterium, Gemella, Haemophilus, Klebsiella ebsiella), Lactobacillus, Lactococcus, Legionella, Leuconostoc, Listeria, Micrococcus, Mycobacterium, Neisseria, Cryptosporidium, Nocardia, Oerskovia, Paracoccus, Pediococcus, Peptostreptococcus Peptostreptococcus, Propionibacterium, Proteus, Pseudomonas, Rahnella, Rhodococcus, Rhodospirillum, Staphylococcus, Streptomyces, Streptococcus, Vibrio, Yersinia, Methylobacterium,Ralstonia and Sphingomonas.

[0082] Some of the above-mentioned microorganisms take the form of spores according to their growth state. In the heating determination system 100, the form of the sample to be processed is not particularly limited. In addition, in the heating determination system 100, the number of species of the sample to be processed may be one or two or more.

[0083] (2-2. Bacterial culture fluid collection step)

[0084] Hereinafter, a bacterial culture liquid collecting step of collecting the bacterial culture liquid S as a sample solution, which is performed after the bacterial culture liquid culturing step of the heating determination system 100 , will be described.

[0085] (2-2-1. Specific Example of Bacterial Culture Fluid Collection Step)

[0086] For example, in the bacterial culture liquid collecting step, a portion of the bacterial culture liquid S stored in the bacterial culture container 10 is collected into the sealed container 20. An example of a collection tool is described below. In the bacterial culture liquid collecting step, a sterilized glass bore pipette is used as a collection tool, and a sterilized stoppered glass tube is used as the sealed container 20 to collect the bacterial culture liquid S.

[0087] (2-2-2. Sealed Container 20)

[0088] In the bacterial culture fluid collection step, sealed container 20 is not particularly limited. For example, sealed container 20 may be a stoppered glass tube, a stoppered plastic tube, a microtube, or the like. Furthermore, any sealed container 20 may be used as long as it has a sealable structure and can withstand temperatures of approximately 140°C during the heating step described below.

[0089] (2-2-3. Others)

[0090] In the bacterial culture liquid collection step, the bacterial culture liquid S may be pretreated before being collected into the sealed container 20. For example, the bacterial culture liquid S may be a suspension containing bacteria C after enzymes are added and cells are cultured for a certain period of time. Alternatively, the bacterial culture liquid S may be a suspension containing bacteria C after centrifugation to remove culture medium components.

[0091] (2-3. Dye Addition Step)

[0092] Hereinafter, a dye adding step of adding a predetermined dye to the bacterial culture liquid S containing the bacteria C as a sample, which is performed after the bacterial culture liquid collecting step of the heating determination system 100 , will be described.

[0093] (2-3-1. Specific Example of Dye Addition Step)

[0094] For example, in the dye addition step, an aqueous solution obtained by dissolving the red dye betaine B in deionized water (DIW) is added. In this dye addition step, betaine B contains at least one of betaine and isobetaine. The dye added in the dye addition step is not particularly limited, as long as it has a visually recognizable color and the color irreversibly disappears when heated within a temperature range of approximately 100°C to 160°C.

[0095] (2-3-2. Beetroot Red B)

[0096] Betaine B will be described. Hereinafter, properties of betaine B, a chemical structure of betaine B, and natural dyes including betaine B will be described in this order.

[0097] (2-3-2-1. Properties of Beetroot Red B)

[0098] First, the properties of beetroot red B will be described. Beetroot red B is obtained by squeezing the red roots of beets (Beta vulgaris LINNE) or by extraction with water, an acidic aqueous solution, or aqueous ethanol at room temperature to low temperatures, and its primary color is betaine-based betaine and isobetaine. Beetroot red B is also a natural dye permitted as a food additive. One property of beetroot red B is that it is a vibrant red dye whose hue varies little with pH (pH 4 to 7) and is readily soluble in water but insoluble in anhydrous ethanol and oils. Furthermore, beetroot red B is unstable, fading in the presence of heat, and also discoloring when exposed to light or metal ions.

[0099] (2-3-2-2. Chemical structure of betaine B)

[0100] Second, refer to Figure 2 Describe the chemical structure of betaine B. Figure 2 : is a diagram showing an example of the dye component of betaine B according to the embodiment. Betaine B is mainly composed of betaine and isobetaine. Figure 2 In the structural formula, structures where the side chain R is glucose are betanin. Isobetaine is a stereoisomer of betanin. Beetroot red B may contain glucan or lactose.

[0101] (2-3-2-3. Natural Dyes)

[0102] Third, beetroot red B is classified as a natural dye considered a food additive. In addition to beetroot red, to which beetroot red B belongs, other natural dyes considered food additives include purple cabbage pigment, carrot pigment, annatto seed extract, sepia pigment, turmeric oleoresin curcumin, cocoa pigment, carotene, gardenia red, gardenia blue, gardenia yellow, chlorophyll, sorghum pigment, cochineal extract, saffron pigment, perilla pigment, sandalwood red, spirulina pigment, onion pigment, tamarind pigment, butterfly pea flower pigment, chili pigment, tomato pigment, roselle pigment, grape skin pigment, hematoxylin pigment, red yeast rice pigment, safflower red, safflower yellow, berry pigment, marigold pigment, purple sweet potato pigment, purple corn pigment, purple yam pigment, and plant carbon black.

[0103] (2-4. Heating step)

[0104] Hereinafter, a heating step performed after the dye adding step of the heating determination system 100 will be described, which heats the sample solution to which a predetermined dye is added and extracts nucleic acid from the cells of the bacteria C as the sample.

[0105] (2-4-1. Specific Example of Heating Step)

[0106] For example, in the heating step, a dissolution aid that promotes cell lysis is added to the bacteria C as a sample in the sealed container 20, which stores the heated solution H to which betalain B was added in the dye adding step, the sealed container 20 is sealed by closing the lid, and the sealed sealed container 20 is heated at 140°C for 45 seconds using a heating device 30 such as a heating block to extract nucleic acid from the cells of the bacteria C.

[0107] (2-4-2. Types of dissolution aids)

[0108] In the heating step of the heating determination system 100, although water alone can achieve the aforementioned effects, for more efficient nucleic acid extraction from the sample, it is preferred to add at least one solubilizing agent selected from the group consisting of surfactants, bases, acids, redox agents, and protein denaturants in addition to water. The solubilizing agent has the ability to dissolve the membrane structure of the sample. When the solubilizing agent acts on the membrane structure of the sample, the sample is easily disrupted, thereby enabling more efficient nucleic acid extraction from the sample. The types of solubilizing agents are described below.

[0109] (2-4-2-1. Surfactants)

[0110] The surfactant used as a dissolution aid may be, for example, ionic or nonionic. Examples of nonionic surfactants include octylphenol ethoxylate (C 14 H 22 O(C2H4O) nIn the nucleic acid extraction step of the heating determination system 100, commercially available octylphenol ethoxylates can be used, and examples thereof include Triton X-100 (C 14 H 22 O(C2H4O) n , n=100).

[0111] Ionic surfactants can be anionic, cationic or zwitterionic. Examples of anionic surfactants include sodium lauryl sulfate (SDS). Examples of cationic surfactants include cetyltrimethylammonium bromide (CTAB). Examples of zwitterionic 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, do not have dissociable hydrogen atoms bound to the positively charged atoms, and the molecule as a whole is uncharged. Typical examples of betaine include trimethylglycine.

[0112] (2-4-2-2. alkali)

[0113] Examples of the base used as the dissolution aid include sodium hydroxide (NaOH) and potassium hydroxide (KOH).

[0114] (2-4-2-3.Acid)

[0115] Examples of acids used as dissolution aids include hydrochloric acid (HCl) and sulfuric acid (H2SO4).

[0116] (2-4-2-4. Redox Agents)

[0117] Examples of the redox agent used as the dissolution aid include aqueous hydrogen peroxide, β-mercaptoethanol, and dithiothreitol.

[0118] (2-4-2-5. Protein denaturant)

[0119] Examples of protein denaturants used as solubility aids include guanidine hydrochloride and urea.

[0120] (2-4-2-6. Others)

[0121] As a component of the dissolution aid, a chelating agent may be used. Examples of the chelating agent used as the dissolution aid include ethylenediaminetetraacetic acid (EDTA).

[0122] Furthermore, among the above-mentioned dissolution aids, the dissolution aid used in the heating determination system 100 preferably contains a surfactant, and more preferably contains one or both of SDS and octylphenol ethoxylate.

[0123] For example, when it is necessary to detect nucleic acid extracted in the nucleic acid extraction step of the heating determination system 100 with high sensitivity, SDS is preferred. On the other hand, when the nucleic acid extracted in the nucleic acid extraction step of the heating determination system 100 is used for an enzyme reaction inhibited by SDS, it is preferred to use octylphenol ethoxylate, which has a milder effect on the membrane structure of the sample than SDS.

[0124] The dissolution aid of the heating determination system 100 may optionally include a buffer. Examples of buffers include Tris-HCl.

[0125] (2-4-3. Types of Nucleic Acids)

[0126] In the heating step, the type of nucleic acid to be extracted is not particularly limited. For example, the nucleic acid to be extracted can be deoxyribonucleic acid (DNA) such as genomic DNA or plasmid DNA, or ribonucleic acid (RNA) such as messenger RNA, transfer RNA or ribosomal RNA.

[0127] (2-5. Dye Confirmation Step)

[0128] Hereinafter, a dye confirmation step will be described, which is a determination step performed after the heating step of the heating determination system 100 for determining the temperature at which the sample solution is heated by the heating step based on a change in color tone of a predetermined dye.

[0129] (2-5-1. Specific Example of Dye Confirmation Step)

[0130] For example, in the dye confirmation step, the maximum temperature at which the sample solution was heated is determined to be 140°C or higher by the disappearance of the red color of betalain B. That is, in the dye confirmation step, when the red color of betalain B disappears and turns yellow as visually observed, it can be confirmed that the heated solution H containing bacteria C has been heated to 140°C or higher.

[0131] In the dye confirmation step, the maximum temperature range of the heated sample solution was determined to be 100° C. to 160° C. by measuring the red absorbance of betaine B. That is, in the dye confirmation step, the absorbance at 535 nm was measured using an absorption photometer, and it was confirmed that when the absorbance was 0.11, the maximum temperature reached 100° C., when the absorbance was 0.10, the maximum temperature reached 110° C., when the absorbance was 0.09, the maximum temperature reached 120° C., when the absorbance was 0.07, the maximum temperature reached 130° C., when the absorbance was 0.05, the maximum temperature reached 140° C., when the absorbance was 0.04, the maximum temperature reached 150° C., and when the absorbance was 0.02, the maximum temperature reached 160° C.

[0132] (2-6. Nucleic Acid Purification Step)

[0133] Hereinafter, a nucleic acid purification step of purifying nucleic acid extracted from cells of bacteria C, which is performed after the heating step of the heating determination system 100 , will be described.

[0134] (2-6-1. Specific Example of Nucleic Acid Purification Step)

[0135] For example, in the nucleic acid purification step, the nucleic acid is purified by injecting the nucleic acid into a column having an adsorption carrier that adsorbs the nucleic acid, and then injecting an eluent for eluting the nucleic acid into the column.

[0136] (2-7. Nucleic Acid Amplification Step)

[0137] Hereinafter, the nucleic acid amplification step of amplifying nucleic acid extracted from cells of bacteria C, which is performed after the heating step or after the nucleic acid purification step of the heating determination system 100 , will be described.

[0138] (2-7-1. Specific Example of Nucleic Acid Amplification Step)

[0139] For example, in the nucleic acid amplification step, nucleic acid is amplified by PCR on the solution containing nucleic acid extracted in the heating step. In the nucleic acid amplification step, nucleic acid is amplified by PCR on the solution containing nucleic acid purified in the nucleic acid purification step.

[0140] 3. Results of each experiment

[0141] Will refer to Figures 3 to 10 The experimental results of the heating confirmation system 100 according to the embodiment will be described. Hereinafter, the experimental results related to the heating confirmation experiment and the experimental results related to the PCR amplicon measurement experiment will be described in this order.

[0142] (3-1. Heating confirmation test)

[0143] Will refer to Figures 3 to 5 Experimental results 1 to 3 related to a heating confirmation experiment for confirming a heating temperature by the heating determination system 100 are described. Figures 3 to 5 Graphs showing examples of experimental results of a heating confirmation experiment according to the embodiment. Hereinafter, Experimental Results 1 to 3 of the heating confirmation experiment will be described in sequence while showing the experimental procedure.

[0144] (3-1-1. Experimental procedure for heating confirmation test)

[0145] An example of an experimental procedure for a heating confirmation experiment of the heating determination system 100 will be described. First, a beet red solution with a concentration of 0.05 g / mL was prepared by dissolving "Sun Beet LF", a powder manufactured by San-EiGen FFI, Inc., in deionized water. Second, 2 microliters of the prepared beet red solution was added to 38 microliters of a dissolution aid solution containing 1% SDS and Tris-HCl. Third, 40 microliters of the mixed solution of beet red B and the dissolution aid was sealed in a heat-resistant microtube and heated for 45 seconds. Fourth, the absorbance was measured using an absorption photometer "NanoDrop (UV-VIS)" manufactured by Thermo Fisher Scientific.

[0146] (3-1-2. Experimental results of heating confirmation test 1)

[0147] Will refer to Figure 3 Describe the experimental results of the heating confirmation experiment 1. Figure 3 Various states of the microtubules are shown: the negative control sample "NC" is an unheated mixed solution, "90°C" is a mixed solution heated at 90°C, "100°C" is a mixed solution heated at 100°C, "110°C" is a mixed solution heated at 110°C, "120°C" is a mixed solution heated at 120°C, "130°C" is a mixed solution heated at 130°C, "140°C" is a mixed solution heated at 140°C, "150°C" is a mixed solution heated at 150°C, and "160°C" is a mixed solution heated at 160°C.

[0148] (3-1-3. Examination of Experimental Result 1 of Heating Confirmation Experiment)

[0149] exist Figure 3 In the case of "NC", no disappearance of the red color was observed, but it was visually observed that the degree of disappearance of the red color increased with increasing heating temperature. Figure 3 In the figure, the extent of red disappearance is particularly obvious after 140℃, as shown by the dotted line.

[0150] As can be seen from Experimental Result 1 of the heating confirmation experiment, in the heating confirmation system 100, by visually observing the color change of betalain B when heating at 140°C for 45 seconds under the processing conditions of the above-mentioned high temperature and high pressure method, it can be confirmed that each sample has been heated to above 140°C.

[0151] (3-1-4. Experimental results of heating confirmation test 2)

[0152] Will refer to Figure 4 Describe the experimental results of the heating confirmation experiment 2. Figure 4The absorption spectra of each mixed solution are shown: the negative control sample "NC" is an unheated mixed solution, "90℃" is a mixed solution heated at 90℃, "100℃" is a mixed solution heated at 100℃, "110℃" is a mixed solution heated at 110℃, "120℃" is a mixed solution heated at 120℃, "130℃" is a mixed solution heated at 130℃, "140℃" is a mixed solution heated at 140℃, "150℃" is a mixed solution heated at 150℃, and "160℃" is a mixed solution heated at 160℃.

[0153] (3-1-5. Experimental results of heating confirmation test 3)

[0154] Will refer to Figure 5 Describe the experimental results of the heating confirmation experiment 3. Figure 5 The absorbance of each mixed solution at a wavelength of 535 nm is shown: the negative control sample "NC" is an unheated mixed solution, "90°C" is a mixed solution heated at 90°C, "100°C" is a mixed solution heated at 100°C, "110°C" is a mixed solution heated at 110°C, "120°C" is a mixed solution heated at 120°C, "130°C" is a mixed solution heated at 130°C, "140°C" is a mixed solution heated at 140°C, "150°C" is a mixed solution heated at 150°C, and "160°C" is a mixed solution heated at 160°C.

[0155] (3-1-6. Examination of Experimental Results 2 and 3 of the Heating Confirmation Experiment)

[0156] exist Figure 4 It can be confirmed that the absorbance decreases in the order of "NC", "90℃", "100℃", "110℃", "120℃", "130℃", "140℃", "150℃" and "160℃". Figure 5 In the Figure 4 The absorbance measured at a wavelength of 535 nm, shown by the solid line in the middle, decreases linearly in the order of "NC", "90°C", "100°C", "110°C", "120°C", "130°C", "140°C", "150°C" and "160°C".

[0157] As can be seen from Experimental Results 2 and 3 of the heating confirmation experiment, in the heating confirmation system 100 , the maximum achievable temperature of each sample can be determined by measuring the absorbance of betalain B after the heating treatment.

[0158] (3-2. PCR amplicon determination experiment)

[0159] Will refer to Figures 6 to 10Experimental results 1 and 2 regarding a PCR amplicon measurement experiment for measuring PCR amplicon in the heating determination system 100 are described. Figures 6 to 8 1 is a diagram showing an example of experimental conditions for a PCR amplicon assay experiment according to the embodiment. Figure 9 and Figure 10 1 is a diagram showing an example of experimental results of a PCR amplicon assay experiment according to the embodiment. Hereinafter, Experimental Results 1 and 2 of the PCR amplicon assay experiment will be described in sequence while showing the experimental procedure.

[0160] (3-2-1. Experimental Procedure for PCR Amplicon Assay)

[0161] Will refer to Figures 6 to 8 An example of an experimental procedure for a PCR amplicon determination experiment in the heating determination system 100 is described. First, betaine B is added to a bacterial suspension obtained by culturing Escherichia coli NBRC 3972 strain overnight in an SCD medium, and high-temperature and high-pressure treatment is performed using a high-temperature and high-pressure method (heating at 140°C for 45 seconds). Second, the mixed solution after the high-temperature and high-pressure treatment is diluted to 1 / 100 to prepare a diluted solution. Third, 20 microliters of the diluted solution is mixed with 20 microliters of the PCR mixture shown in Experimental Conditions 1 and 2, and PCR is performed under the temperature cycling conditions shown in Experimental Condition 3. Fourth, the solution after PCR is electrophoresed using the "Agilent 2100 Bioanalyzer Electrophoresis System" manufactured by Agilent Technologies, and the presence or absence of PCR amplification is confirmed.

[0162] (3-2-1-1. Experimental Condition 1)

[0163] First, refer to Figure 6 Describe the experimental conditions for the primers used in the PCR amplicon assay. Figure 6 As shown in the example of , the forward primer is named "16S290f_2" and has a base sequence of "GACACGGCCCAGACTCCTAC." The reverse primer is named "16S 500r+GG" and has a base sequence of "GTATTACCGCGGCTGCTGG." Furthermore, the amplicon base pair size is "211 bp."

[0164] (3-2-1-2. Experimental Condition 2)

[0165] Second, refer to Figure 7 Describe the experimental conditions of the reagents used in the PCR amplicon assay. Figure 7As 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 "deoxynucleoside triphosphate (dNTP) mixture", 4.00 μL / tube of 10-fold concentration of "10× PCR buffer" and 8.60 μL / tube of "Milli-Q water" are mixed, totaling 40.00 μL / tube.

[0166] (3-2-1-3. Experimental Condition 3)

[0167] Third, refer to Figure 8 Experimental Conditions 3 describes the reaction conditions for the PCR amplicon assay experiment. Figure 8 As shown in the example, as the reaction conditions for the PCR amplicon determination experiment, the "activation" process is carried out at 98°C for 120 seconds, and one cycle is performed. The DNA extension reaction consists of three steps:

[0168] "Denaturation," "annealing," and "extension." The "denaturation" process was performed at 98°C for 15 seconds, the "annealing" process was performed at 58°C for 25 seconds, and the "extension" process was performed at 72°C for 15 seconds. These three steps constituted one cycle, and 35 cycles were performed. In addition, an "additional extension" process was performed at 72°C for 120 seconds for one cycle.

[0169] (3-2-2. Experimental Results 1 of PCR Amplicon Measurement Experiment)

[0170] Will refer to Figure 9 Experimental results describing the PCR amplicon assay experiment 1. Figure 9 A is the electrophoresis diagram of the positive control sample "PC", which is a mixed solution without betaine B. Figure 9 B is the electrophoresis diagram of the negative control sample "NC", which is a mixed solution that has not been treated with high temperature and high pressure. Figure 9 C is an electrophoretogram of the "betalain method (1 / 100 dilution, with a solubilizing agent)" of a mixed solution containing betalain B and a solubilizing agent. Figure 9 D is an electrophoretogram of the "betalain method (without solubilizing agent)" of a mixed solution containing betalain B but without a solubilizing agent.

[0171] (3-2-3. Experimental Results 2 of PCR Amplicon Measurement Experiment)

[0172] Will refer to Figure 10Experimental results describing the PCR amplicon assay were presented in 2. Figure 10 In the positive control sample "PC" of the mixed solution without betalain B shown in A, the molar concentration of the PCR amplicon was 307.2 nmol / L. Figure 10 In the negative control sample "NC" of the mixed solution shown in B, which was not subjected to high temperature and high pressure treatment, the molar concentration of the PCR amplicon was 38.8 nmol / L. Figure 10 In the "Beetroot Red Method (1 / 100 dilution, with a solubilizer)" shown in C, which contains a mixed solution of Betalain B and a solubilizer, the molar concentration of the PCR amplicon is 112.9 nmol / L. Figure 10 In the "betalain method (without solubilizing agent)" shown in D, which is a mixed solution containing betalain B but without a solubilizing agent, the molar concentration of the PCR amplicon was 255.0 nmol / L.

[0173] (3-2-4. Examination of Experimental Results 1 and 2 of PCR Amplicon Measurement Experiment)

[0174] Depend on Figure 9 and Figure 10 It can be seen that the PCR results of the positive control sample "PC" ( Figure 9 A and Figure 10 A), PCR results using a mixed solution with betalain B ( Figure 9 D and Figure 10 D) is 83%.

[0175] From Experimental Results 1 and 2 of the PCR amplicon measurement experiment, it was confirmed that the heating determination system 100 did not affect the nucleic acid extraction step and the nucleic acid amplification step.

[0176] 4. Processing Flow of Heating Determination System 100

[0177] Will refer to Figure 11 A step flow of the heating determination system 100 according to the embodiment is described. Figure 11 1 is a flowchart showing an example of the process of the heating determination step according to the embodiment. Note that the following steps S101 to S104 may be performed in a different order. In addition, some of the following steps S101 to S104 may be omitted.

[0178] First, in the heating determination system 100, a bacterial culture fluid collection step (step S101) is performed. Second, in the heating determination system 100, a dye addition step (step S102) is performed. Third, in the heating determination system 100, a heating step (step S103) is performed. Fourth, in the heating determination system 100, a dye confirmation step (step S104) is performed, and the heating determination step is completed. Please note that in the heating determination system 100, after the dye confirmation step is performed, the nucleic acid purification step and the nucleic acid amplification step can be performed.

[0179] 5. Effect of the implementation plan

[0180] Finally, the effects of the embodiment will be described. Hereinafter, effects 1 to 8 corresponding to the steps of the embodiment will be described.

[0181] (5-1. Effect 1)

[0182] First, in the steps according to the above embodiment, a predetermined dye is added to a sample solution containing a sample, the sample solution containing the predetermined dye is heated, and the heating temperature of the sample solution is determined based on the change in color tone of the predetermined dye. Therefore, in the steps according to the embodiment, the heating temperature of the sample can be effectively determined.

[0183] (5-2. Effect 2)

[0184] Second, in the step according to the above embodiment, the predetermined dye is the red dye beetroot red B. Therefore, in the step according to the embodiment, by using a dye having high visibility and high safety, the heating temperature of the sample can be efficiently determined.

[0185] (5-3. Effect 3)

[0186] Third, in the steps according to the above embodiment, betaine B contains at least one of betaine and isobetaine. Therefore, in the steps according to the embodiment, by using a dye whose hue reversibly changes according to the heating temperature, the heating temperature of the sample can be efficiently determined.

[0187] (5-4. Effect 4)

[0188] Fourth, in the steps according to the above embodiment, the maximum temperature at which the sample solution is heated is determined to be 140°C or higher by the disappearance of the red color of betalain B. Therefore, in the steps according to the embodiment, the heating temperature of the sample can be effectively determined during the heating treatment at 140°C or higher.

[0189] (5-5. Effect 5)

[0190] Fifth, in the steps according to the above embodiment, the maximum temperature at which the sample solution is heated within the range of 100°C to 160°C is determined by measuring the absorbance of the red color of betalain B. Therefore, in the steps according to the embodiment, the heating temperature of the sample can be effectively determined in the heating treatment within the range of 100°C to 160°C.

[0191] (5-6. Effect 6)

[0192] Sixth, in the steps according to the above embodiment, the sample solution is a bacterial culture solution S containing bacteria C. Therefore, in the steps according to the embodiment, when nucleic acid is extracted from cells of bacteria C by the high temperature and high pressure method, the heating temperature of the sample can be effectively determined.

[0193] (5-7. Effect 7)

[0194] Seventh, in the step according to the above embodiment, the nucleic acid extracted from the cells of the bacterium C is purified. Therefore, in the step according to the embodiment, the heating temperature of the sample can be effectively determined without affecting the purification of the nucleic acid extracted from the cells of the bacterium C.

[0195] (5-8. Effect 8)

[0196] Eighth, in the step according to the above embodiment, nucleic acid extracted from the cells of bacteria C is amplified. Therefore, in the step according to the embodiment, the heating temperature of the sample can be effectively determined without affecting the amplification of nucleic acid extracted from the cells of bacteria C.

[0197] system

[0198] Unless otherwise specified, the processing methods, control methods, specific names, and information including various data and parameters shown in the above text and drawings may be changed as needed.

[0199] Furthermore, the components of the various devices shown in the drawings are functional concepts and are not necessarily arranged as shown. That is, the specific distribution and integration of the devices are not limited to those shown in the drawings. In other words, depending on the load, usage conditions, and other factors, they can be functionally or physically distributed and integrated into any unit, in whole or in part.

[0200] Reference Signs List

[0201] 10 Bacterial culture container

[0202] 20 airtight containers

[0203] 30 Heating device

[0204] 100 Heating Determination System

Claims

1. A state determination method for determining a temperature at which a sample is heated, comprising: an adding step of adding a predetermined dye to the sample solution containing the sample; a heating step of heating the sample solution to which the predetermined dye is added in the adding step; as well as A determining step of determining a temperature at which the sample solution is heated by the heating step based on a change in the color tone of the predetermined dye.

2. The state determination method according to claim 1, wherein The predetermined dye is a red dye beetroot red.

3. The state determination method according to claim 2, wherein The betaine includes at least one of betanin and isobetaine.

4. The state determination method according to claim 2, wherein In the determining step, it is determined that the maximum temperature to which the sample solution is heated is 140° C. or higher by the disappearance of the red color of betalain.

5. The state determination method according to claim 2, wherein In the determining step, the maximum temperature at which the sample solution is heated is determined to be in the range of 100° C. to 160° C. by measuring the red absorbance of betalain.

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

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

8. The state determination method according to claim 6, further comprising an amplification step of amplifying the nucleic acid extracted from the bacterial cells by the heating step.

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