Sample collection method

Abstract

To efficiently recover a sample by centrifugation.SOLUTION: In a cell recovery method, colored beads B having a predetermined color are added to a cell culture solution S, the cell culture solution S with the colored beads B added thereto is separated into a supernatant L and a precipitate P using a centrifuge 30, the separated supernatant L is removed, and the separated precipitate P is recovered.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present invention relates to a sample recovery method.

Background Art

[0002] Conventionally, there is a technique for recovering cells (appropriately, also referred to as "microorganisms") by centrifuging a cell culture solution. In this technique, after separating the cell culture solution into a medium and a cell pellet by centrifugation, the cells are recovered by removing the medium by decantation.

Prior Art Documents

Patent Documents

[0003]

Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0004] However, in the prior art, it is difficult to efficiently recover a sample by centrifugation. For example, in the prior art, decantation after centrifugation is performed while visually confirming the cell pellet, so it is difficult to perform decantation leaving a small amount of cells that are not visible to the naked eye.

[0005] The present invention has been made in view of the above, and an object thereof is to efficiently recover a sample by centrifugation.

Means for Solving the Problems

[0006] [[ID=​​

[0007] According to the present invention, there is an effect that the sample can be efficiently recovered by centrifugation. [Brief explanation of the drawing]

[0008] [Figure 1] This figure shows an example configuration of a bacterial cell recovery system according to the embodiment. [Figure 2] This figure shows a specific example of colored beads according to the embodiment. [Figure 3] This is a diagram illustrating experimental result 1 according to the embodiment. [Figure 4] This figure illustrates experimental result 2-1 according to the embodiment. [Figure 5] This figure illustrates experimental result 2-2 according to the embodiment. [Figure 6] This figure illustrates experimental result 3 according to the embodiment. [Figure 7] This is a flowchart showing an example of the flow of the bacterial cell recovery process according to the embodiment. [Modes for carrying out the invention]

[0009] A sample recovery method according to one embodiment of the present invention will be described in detail below with reference to the drawings. However, the present invention is not limited to the embodiments described below.

[0010] [Embodiment] The configuration of the bacterial cell recovery system 100 according to this embodiment, the details of each step, and the flow of each step will be described in order below, and finally the effects of the embodiment will be described.

[0011] [1. Configuration of the bacterial cell recovery system 100] Using FIG. 1, the configuration of the cell recovery system 100 according to the embodiment will be described. FIG. 1 is a diagram showing a configuration example of the cell recovery system 100 according to the embodiment. Hereinafter, the configuration example of the entire cell recovery system 100, each step of the cell recovery system 100, and the effects of the cell recovery system 100 will be described in this order.

[0012] (1-1. Configuration example of the entire cell recovery system 100) The cell recovery system 100 includes a cell culture vessel 10, a centrifuge tube 20, a centrifuge 30, and a pipette 40. Hereinafter, the cell culture vessel 10, the centrifuge tube 20, the centrifuge 30, and the pipette 40 will be described in this order.

[0013] (1-1-1. Cell culture vessel 10) The cell culture vessel 10 is a vessel that contains the cell culture solution S. The cell culture solution S is a solution for culturing microorganisms such as cells C. In the example of FIG. 1, the cell culture vessel 10 is an Erlenmeyer flask with a stopper, but the shape, material, capacity, etc. of the cell culture vessel 10 are not limited.

[0014] (1-1-2. Centrifuge tube 20) The centrifuge tube 20 is a vessel that contains the collected cell culture solution S and the added colored beads B. Note that the shape, material, capacity, etc. of the centrifuge tube 20 are not limited.

[0015] (1-1-3. Centrifuge 30) The centrifuge 30 is a device that centrifuges the installed centrifuge tube 20. Note that the type of the centrifuge 30, the centrifugal acceleration, etc. are not limited.

[0016] (1-1-4. Pipette 40) The pipette 40 is a tool for removing the supernatant of the separated cell culture solution S. In the example of FIG. 1, the pipette 40 is a glass Komagome pipette, but the shape, material, capacity, etc. of the pipette 40 are not limited.

[0017] (1-1-5. Others) The cell recovery system 100 shown in FIG. 1 may include a plurality of cell culture vessels 10, a plurality of centrifuge tubes 20, a plurality of centrifuges 30, or a plurality of pipettes 40. Further, the cell culture vessel 10 may be configured to be integrated with the centrifuge tube 20. In the following, as the cell recovery system 100 according to the embodiment, an example of recovering the cells C from the cell culture solution S contained in the cell culture vessel 10 will be described, but it is also applicable to the step of recovering the non-cultured cells C, and the application range of the cell recovery system 100 is not particularly limited.

[0018] (1-2. Steps of the cell recovery system 100) The steps of the above cell recovery system 100 will be described. In the following, the cell culture solution sampling step, the colored bead addition step, the centrifugation step, and the supernatant removal step will be described in this order. Note that each step can also be executed in a different order. Also, some of the steps may be omitted.

[0019] (1-2-1. Cell culture solution sampling step) First, the cell recovery system 100 performs the cell culture solution sampling step shown in FIG. 1(1). For example, in the cell culture solution sampling step, as the cells C, Escherichia coli (E. coli) or Staphylococcus aureus (S. aureus) is used, and a part of the cell culture solution S cultured overnight at 37°C in an SCD (Soybean Casein Digest) liquid medium is sampled into the centrifuge tube 20 using a sterilized whole pipette.

[0020] (1-2-2. Colored bead addition step) Second, the cell recovery system 100 performs the colored bead addition step shown in FIG. 1(2). For example, in the colored bead addition step, as the colored beads B, red, spherical beads with a particle size of 1.0 μm and made of polystyrene are used, and a suspension of the colored beads B is added to the centrifuge tube 20 in which the cell culture solution S has been sampled.

[0021] (1-2-3. Centrifugation step) Thirdly, the bacterial cell recovery system 100 performs the centrifugation step shown in Figure 1(3). For example, in the centrifugation step, a centrifuge tube 20 to which colored beads B have been added is placed in a centrifuge 30, and the centrifuge tube 20 is rotated at 3000G for 10 minutes to separate the precipitate (sometimes referred to as "precipitate") P containing the colored beads B and bacterial cells C from the supernatant L containing the culture medium components of the bacterial cell culture solution S.

[0022] (1-2-4. Supernatant removal step) Fourth, the bacterial cell recovery system 100 performs the supernatant removal step shown in Figure 1(4). For example, in the supernatant removal step, a pipette 40, which is a sterilized Pasteurized pipette, is used to remove the supernatant L from the centrifuge tube 20 and recover the bacterial cells C contained in the precipitate P.

[0023] (1-2-5. Others) The bacterial cell recovery system 100 can further perform a nucleic acid extraction process to extract nucleic acids from the recovered bacterial cells C. For example, in the nucleic acid extraction process, a solubilizer that promotes the lysis of bacterial cells C is added to the centrifuge tube 20 containing the precipitate P, the resulting suspension is transferred to a heating container, and the sealed heating container is heated at 140°C for 45 seconds to extract nucleic acids from the bacterial cells C.

[0024] (1-3. Effects of the microbial cell recovery system 100) Below, we will first explain the overview of the bacterial cell recovery technology as a reference technology, then the improvements made to the reference technology, and finally, we will explain the effects of the bacterial cell recovery system 100.

[0025] (1-3-1. Overview of Reference Technologies) The bacterial cell recovery technique described in the reference technology involves the following steps: First, the bacterial cell culture is collected in a centrifuge tube. Second, the centrifuge tube containing the bacterial cell culture is placed in a centrifuge and centrifuged. Third, the supernatant containing the culture medium components is removed by decantation of the bacterial cell culture after centrifugation.

[0026] (1-3-2. Improvements to the reference technology) The cell recovery technique in the reference technology has the following shortcomings: Firstly, when a cell culture medium containing a small amount of cells is centrifuged, a precipitate (pellet) cannot be observed, making it impossible to determine whether separation has been achieved. Secondly, decantation after centrifugation is performed while visually checking the cell pellet, making it difficult to recover small amounts of cells that are not visible.

[0027] (1-3-3. Overview of the bacterial cell recovery system 100) In the bacterial cell recovery system 100, colored beads B having a predetermined color are added to the bacterial cell culture medium S containing bacterial cells C. The bacterial cell culture medium S to which the colored beads B have been added is separated into supernatant L and precipitate P by a centrifuge 30. The supernatant L is removed and precipitate P is recovered. In addition, nucleic acids are extracted from the recovered bacterial cells C in the bacterial cell recovery system 100.

[0028] (1-3-4. Effects of the microbial cell recovery system 100) The bacterial cell recovery system 100 is expected to have the following effects. Firstly, by adding colored beads B to the bacterial cell culture medium S, the bacterial cell recovery system 100 makes it possible to recover bacterial cells C contained in the bacterial cell culture medium S even in small amounts where precipitate P cannot be visually confirmed. Secondly, the bacterial cell recovery system 100 makes it possible to efficiently recover bacterial cells C contained in the bacterial cell culture medium S without interfering with the nucleic acid extraction process performed after recovery.

[0029] [2. Details of each step in the bacterial cell recovery system 100] The details of each step of the bacterial cell recovery system 100 shown in Figure 1 will be described below. In the following, each step according to the embodiment will be described in the following order: bacterial cell culture step, bacterial cell culture solution collection step, colored bead addition step, centrifugation step, supernatant removal step, and nucleic acid extraction step.

[0030] (2-1. Bacterial cell culture process) The following describes the bacterial culture process, which is carried out prior to the bacterial culture solution collection process of the bacterial cell recovery system 100, and involves culturing the bacterial cell C sample.

[0031] (2-1-1. Specific examples of bacterial cell culture processes) For example, in the bacterial culture process, bacterial cells C are cultured in a bacterial culture medium S contained in a bacterial culture container 10. An example of the equipment used for culture is that in the bacterial culture process, a sterilized glass Erlenmeyer flask with a stopper is used as the bacterial culture container 10 to culture the bacterial cells C. An example of the bacterial cells C cultured is that in the bacterial culture process, Escherichia coli or Staphylococcus aureus are cultured. An example of the culture conditions is that in the bacterial culture process, bacterial cells C are cultured overnight at 37°C in SCD liquid medium.

[0032] (2-1-2. Method for culturing bacterial culture medium S) The bacterial culture medium S used in the bacterial culture process described above is obtained by culturing a sample containing nucleic acids. The method of culturing the sample is not particularly limited, and one example is to place a filter on which the sample has been collected directly onto a solid culture medium and culture the sample through the filter (solid-phase culture). Another method of culturing the sample is to culture the sample in the presence of a liquid culture medium or a solution obtained by dissolving a solid culture medium in water (liquid-phase culture). The type of liquid culture medium or solid culture medium used is selected depending on the type of sample to be cultured and the physiological conditions.

[0033] (2-1-3. Sample of bacterial culture medium S) In the above-described bacterial culture process, the sample to be treated is not particularly limited. For example, the sample to be treated may be microorganisms, animal cells other than microorganisms (e.g., insect cells, etc.), plant cells, mycoplasma, viruses, etc.

[0034] Examples of the above microorganisms include Acinetobacter, Actinomyces, Aerococcus, Aeromonas, Alcaligenes, Bacillus, Bacteriodes, Bordetella, Branhamella, Brevibacterium, and Campylobacter. r) species, Candida species, Capnocytophagia species, Chromobacterium species, Clostridium species, Corynebacterium species, Cryptococcus species, Deinococcus species, Enterococcus species, Erysipelothrix species, Escherichia species, Flavobacterium Flavobacterium species, Gemella 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,It is at least one species selected from the group consisting of Rhodospirillum, Staphylococcus, Streptomyces, Streptococcus, Vibrio, and Yersinia, Methylobacterium, Ralstonia, and Sphingomonas.

[0035] Among the microorganisms mentioned above, some take on forms such as spores or spores depending on their growth stage. In the microbial cell recovery system 100, the form of the sample to be processed is not particularly limited. Furthermore, in the microbial cell recovery system 100, the sample to be processed may be of one type or two or more types.

[0036] (2-2. Bacterial culture fluid collection process) The following describes the bacterial culture medium collection process, which is performed after the bacterial culture medium collection process of the bacterial cell recovery system 100, and involves collecting the bacterial culture medium S, which is the sample solution.

[0037] (2-2-1. Specific example of the bacterial culture medium collection process) For example, in the bacterial culture medium collection process, a portion of the bacterial culture medium S contained in the bacterial culture container 10 is collected in a centrifuge tube 20. To give an example of the equipment used for collection, in the bacterial culture medium collection process, a sterilized glass volumetric pipette is used as the collection equipment, and a sterilized polypropylene centrifuge tube 20 is used to collect the bacterial culture medium S.

[0038] (2-2-2. Centrifuge tube 20) In the above bacterial culture solution collection process, the centrifuge tube 20 is not particularly limited. For example, the centrifuge tube 20 may be a centrifuge tube with a capacity of 15 mL to 50 mL, a microtube, or a pressure-resistant glass test tube. Furthermore, the centrifuge tube 20 only needs to be able to withstand a centrifugal acceleration of about 20,000 G when rotated at high speed by the centrifuge 30.

[0039] (2-3. Adding colored beads) The following describes the colored bead addition step, which is performed after the bacterial culture solution collection step of the bacterial cell recovery system 100, and involves adding colored beads B, which are beads having a predetermined color, to the sample solution.

[0040] (2-3-1. Specific example of the colored bead addition process) For example, in the colored bead addition step, a suspension of red, 1.0 μm particle size polystyrene colored beads B is injected into the centrifuge tube 20 from which the bacterial culture medium S has been collected, thereby adding the colored beads B.

[0041] (2-3-2. Color and shape of colored beads B) In the colored bead addition process described above, the color and shape of the colored beads B are not particularly limited. For example, the color of the colored beads B can be any color that is visible to the naked eye, and may be blue, yellow, purple, white, etc., in addition to the red mentioned above. The shape of the colored beads B can be any fine particles within a certain particle size range, and may be spherical, ellipsoidal, polyhedron, have irregularities on the surface of the particles, or have holes penetrating the particles.

[0042] (2-3-3. Material of colored beads B) In the colored bead addition step described above, the material of colored beads B is not particularly limited. For example, the material of colored beads B does not need to have any effect that inhibits extraction or amplification in the nucleic acid extraction step or nucleic acid amplification step (e.g., release of inhibitory substances), and may be latex, polylactic acid, silica, polyethylene, etc., in addition to the polystyrene mentioned above. Similarly, the dye attached to colored beads B does not need to have any effect that inhibits extraction or amplification in the nucleic acid extraction step or nucleic acid amplification step.

[0043] (2-3-4. Specific gravity of colored beads B) In the colored bead addition step described above, the specific gravity of colored beads B is not particularly limited. For example, the specific gravity of colored beads B should be within a predetermined range of approximately 1.1 to 1.4 g / mL relative to the specific gravity of bacterial cells C, which are the sample to be recovered. In other words, the specific gravity of colored beads B should be approximately the same as that of bacterial cells C to be recovered.

[0044] (2-3-5. Particle size of colored beads B) In the colored bead addition step described above, the particle size of the colored beads B is not particularly limited. For example, the particle size of the colored beads B can be within a predetermined range of approximately 1.0 to 32 μm relative to the size of the bacterial cells C, which are the sample to be recovered (e.g., a bacillus 2 μm). In other words, the particle size of the colored beads B can be approximately the same size as the bacterial cells C to be recovered.

[0045] (2-3-6. Specific examples of colored beads B) Using Figure 2, a specific example of the colored beads B used in the colored bead addition process described above will be explained. Figure 2 is a diagram showing a specific example of the colored beads B according to the embodiment.

[0046] (2-3-6-1. Colored beads "A") The colored bead "A" in Figure 2 is colored bead BA, with product name "micromer-blue", manufacturer "micromod", part number "60-02-203", material "latex", color "blue", and particle size "2μm".

[0047] (2-3-6-2. Colored beads "B") The colored bead "B" in Figure 2 is a colored bead BB with the product name "PLA-color particles", manufacturer "micromod", part number "54-00-203", material "colored polylactic acid particles", color "blue", and particle size "2 μm".

[0048] (2-3-6-3. Colored beads "C") The colored bead "C" in Figure 2 is colored bead BC, with product name "sicastar", manufacturer "micromod", part number "74-01-303", material "silica", color "red", and particle size "3μm".

[0049] (2-3-6-4. Colored beads "D") The colored bead "D" in Figure 2 is a colored bead BD with the product name "Polybead Dyed Microsphere", manufactured by "Polysciences", part number "16906-1", material "polystyrene", color "white", and particle size "1.06 μm".

[0050] (2-3-6-5. Colored beads "E") The colored bead "E" in Figure 2 is a colored bead BE with the product name "Polybead Dyed Microsphere", manufacturer "Polysciences", part number "16906-1", material "polystyrene", color "red", and particle size "1.0 μm".

[0051] (2-3-6-6. Colored beads "F") The colored bead "F" in Figure 2 is a colored bead BF with the product name "Polybead Dyed Microsphere", manufactured by "Polysciences", part number "16906-1", material "polystyrene", color "blue", and particle size "1.0 μm".

[0052] (2-3-6-7. Colored beads "G") The colored bead "G" in Figure 2 is colored bead BG, with product name "Polybead Dyed Microsphere", manufacturer "Polysciences", part number "16906-1", material "polystyrene", color "yellow", and particle size "1.0 μm".

[0053] (2-3-6-8. Colored bead "H") The colored bead "H" in Figure 2 is a colored bead BH with the product name "Polybead Dyed Microsphere", manufacturer "Polysciences", part number "16906-1", material "polystyrene", color "purple", and particle size "0.97 μm".

[0054] (2-3-6-9. Colored Beads "I") The colored bead "I" in Figure 2 is a colored bead BI with the product name "Fluorescent Red Polyethylene Microspheres", manufacturer "Cospheric", part number "UVPMS-BR-1.090", material "polyethylene", color "red", and particle size "27-32 μm".

[0055] (2-3-6-10. Colored beads "J") The colored bead "J" in Figure 2 is a colored bead BJ with the product name "Fluorescent Red Polyethylene Microspheres," manufactured by "Cospheric," part number "UVPMS-BB-1.12," material "polyethylene," color "blue," and particle size "27-32 μm."

[0056] (2-3-6-11. Colored beads "K") The colored bead "K" in Figure 2 is a colored bead BK with the product name "Fluorescent Red Polyethylene Microspheres," manufactured by "Cospheric," part number "UVPMS-BV-1.00," material "polyethylene," color "purple," and particle size "27-32 μm."

[0057] (2-3-6-12. Colored beads "L") The colored bead "L" in Figure 2 is a colored bead BL with the product name "Polybead Polystyrene Red Dyed Microsphere 6.00μm", manufactured by "Polysciences", part number "15714-5", material "polystyrene", color "red", and particle size "6μm".

[0058] (2-4. Centrifugal Separation Process) The following describes the centrifugation step performed after the colored bead addition step of the bacterial cell recovery system 100, in which the sample solution to which colored beads B, which are beads having a predetermined color, have been added is separated into supernatant L and precipitate P by centrifuge 30.

[0059] (2-4-1. Specific examples of the centrifugal separation process) For example, in the centrifugation step, a 15 mL centrifuge tube 20 containing a suspension of bacterial culture medium S and red, 1.0 μm particle size polystyrene colored beads B is placed in a centrifuge 30 and centrifuged at 3000 G for 10 minutes.

[0060] (2-4-2. Centrifuge 30) In the centrifugal separation process described above, the type of centrifuge 30 is not particularly limited. For example, the centrifuge tube 20 that the centrifuge 30 is compatible with may be a device capable of accommodating centrifuge tubes with a capacity of 15 mL to 50 mL, or a device capable of accommodating microtubes, pressure-resistant glass test tubes, etc. Furthermore, the centrifugal acceleration of the centrifuge 30 should be approximately 20,000 G as the maximum centrifugal acceleration, but is not particularly limited. Also, the rotor of the centrifuge 30 may be an angle rotor or a swing rotor.

[0061] (2-5. Supernatant removal process) The following describes the supernatant removal process, which is performed after the centrifugation step of the bacterial cell recovery system 100, and involves removing the supernatant L and recovering the precipitate P.

[0062] (2-5-1. Specific example of the supernatant removal process) For example, in the supernatant removal step, a sterile Pasteur pipette is used as pipette 40 to remove the supernatant L containing the culture medium components from centrifuge tube 20.

[0063] (2-5-2. Pipette 40) In the supernatant removal step described above, the type of pipette 40 is not particularly limited. For example, in addition to a Pasteur pipette, pipette 40 may be a dropper, volumetric pipette, measuring pipette, micropipette, capillary, etc.

[0064] (2-6. Nucleic acid extraction process) The following describes the nucleic acid extraction process, which is performed after the supernatant removal process of the bacterial cell recovery system 100, and involves extracting nucleic acids from the recovered bacterial cell C sample.

[0065] (2-6-1. Specific Examples of Nucleic Acid Extraction Processes) For example, in the nucleic acid extraction process, a solubilizing agent is added to the bacterial sample C to prepare a mixture. This mixture is then transferred to a heat-resistant nucleic acid extraction tube, which is sealed by closing its lid. The nucleic acid extraction tube containing the mixture is then heated at 140°C for 45 seconds using a heating device such as a heat block to extract nucleic acids from the cells of bacterial C.

[0066] (2-6-2. Types of Solubilizers) In the nucleic acid extraction step of the bacterial cell recovery system 100, the above effects can be achieved with water alone, but in order to extract nucleic acids from the sample more efficiently, it is preferable to include at least one solubilizer selected from the group consisting of surfactants, alkalis, acids, oxidation-reduction agents, and protein denaturants in addition to water. The solubilizer has the ability to dissolve the membrane structure of the sample. By acting on the membrane structure of the sample, the solubilizer makes the sample easier to break down, allowing for more efficient extraction of nucleic acids from the sample. The types of solubilizers will be described below.

[0067] (2-6-2-1. Surfactants) The surfactant used as a solubilizer may be ionic or nonionic. Examples of nonionic surfactants include octylphenol ethoxylate (C14H22O(C2H4O)n). In the nucleic acid extraction step of the bacterial cell recovery system 100, commercially available octylphenol ethoxylate can be used, such as TritonX-100 (C14H22O(C2H4O)n, n=100) manufactured by SIGMA.

[0068] Furthermore, ionic surfactants may 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" is a general term for compounds in which positive and negative charges are located in non-adjacent positions within the same molecule, and the positively charged atom does not have any dissociable hydrogen atoms bonded to it, resulting in the molecule as a whole having no charge. A typical example of betaine is trimethylglycine.

[0069] (2-6-2-2. Alkaline) Examples of alkalis used as solubilizers include sodium hydroxide (NaOH) or potassium hydroxide (KOH).

[0070] (2-6-2-3. Acids) Examples of acids used as solubilizers include hydrochloric acid (HCl) or sulfuric acid (H2SO4).

[0071] (2-6-2-4. Redox agents) Examples of redox agents used as solubilizers include hydrogen peroxide, β-mercaptoethanol, and dithiothreitol.

[0072] (2-6-2-5. Protein denaturants) Examples of protein denaturants used as solubilizers include guanidine hydrochloride and urea.

[0073] (2-6-2-6. Others) A chelating agent may be used as a component of the solubilizer. Examples of chelating agents used as solubilizers include ethylenediaminetetraacetic acid (EDTA).

[0074] Furthermore, among the solubilizers mentioned above, the solubilizer for the bacterial cell recovery system 100 preferably contains a surfactant, and more preferably contains either SDS or octylphenol ethoxylate, or both.

[0075] For example, if you want to detect nucleic acids extracted in the nucleic acid extraction step of the bacterial cell recovery system 100 with high sensitivity, it is advisable to use SDS. On the other hand, if the nucleic acids extracted in the nucleic acid extraction step of the bacterial cell recovery system 100 are to be used in an enzymatic reaction inhibited by SDS, it is advisable to use octylphenol ethoxylate, which acts more mildly on the membrane structure of the sample than SDS.

[0076] The solubilizer of the bacterial cell recovery system 100 may contain a buffer as needed. Examples of buffers include tris-hydroxymethylaminomethane hydrochloride (Tris-HCl).

[0077] (2-6-3. Types of nucleic acids) In the nucleic acid extraction process described above, the type of nucleic acid extracted is not particularly limited. For example, the extracted nucleic acid may be deoxyribonucleic acid (DNA), such as genomic DNA or plasmid DNA, or ribonucleic acid (RNA), such as messenger RNA, transfer RNA, or ribosomal RNA.

[0078] [3. Results of various experiments] Figures 3 to 6 illustrate the results of various experiments using the bacterial cell recovery system 100 according to this embodiment.

[0079] (3-1. Experimental Results 1) Using Figure 3, we will explain Experimental Result 1, which confirms the behavior of colored beads B using the bacterial cell recovery system 100. Figure 3 is a diagram illustrating Experimental Result 1 according to the embodiment. Below, we will explain the experimental procedure, followed by the state of colored beads B before centrifugation, the state of colored beads B after centrifugation, and a discussion of Experimental Result 1.

[0080] (3-1-1. State of colored beads B before centrifugation) Figure 3(1) shows the state after adding 10 μL of a suspension of colored beads BE, which is the colored bead "E" (red) shown in Figure 2, to a 15 mL centrifuge tube 20 containing 10 mL of deionized water (DIW). In Figure 3(1), a red suspension derived from the color of the colored beads BE is observed.

[0081] (3-1-2. State of colored beads B after centrifugation) Figure 3(2) shows the suspension of colored beads BE, which is the colored bead "E" (red) shown in Figure 2, after centrifugation at 3000G for 10 minutes using a centrifuge 30. In Figure 3(2), a dark red precipitate P derived from the color of the colored beads BE is observed.

[0082] (3-1-3. Discussion of Experimental Results 1) From the experimental results 1 in Figures 3(1) and (2), it can be seen that in the bacterial cell recovery system 100, the red suspension before centrifugation is observed as a dark red precipitate P after centrifugation, thus confirming that the colored beads B form precipitate P.

[0083] (3-2. Experimental Results 2) Using Figures 4 and 5, we will explain the experimental results 2 (2-1, 2-2) that confirm the amount of bacterial cells C recovered by the bacterial cell recovery system 100. Below, we will explain the state of the centrifuge tube 20 etc. to which colored beads B have been added, the nucleic acid extraction efficiency using colored beads B, and the discussion of experimental results 2.

[0084] (3-2-1. Condition of centrifuge tube 20, etc.) Figure 4 illustrates the state of the centrifuge tube 20, etc., to which colored beads B have been added. Figure 4 is a diagram illustrating experimental result 2-1 according to the embodiment. Below, the state of the suspension, the state of precipitate P, and the state of the diluent will be described while showing the experimental procedure.

[0085] (3-2-1-1. Suspension state) The suspension is prepared as follows: First, a bacterial suspension (approximately 10%) is obtained from Escherichia coli (E. coli) and Staphylococcus aureus (S. aureus) cultured overnight in SCD medium at 37°C. 6 First, prepare a cell count (cells / mL). Second, add 5 μL each of the suspensions of colored beads BE (colored bead "E": red), BF (colored bead "F": blue), and BL (colored bead "L": red) to 1 mL of the prepared bacterial suspension. Third, prepare a control sample "NC" without the addition of colored bead B. At this point, each of the prepared suspensions (colored bead "E", colored bead "F", colored bead "L", and control sample "NC") will be observed as shown in Figure 4(1).

[0086] In Figure 4(1), the suspension of colored beads BE is observed to be red (see Figure 4(1) "E"), the suspension of colored beads BF is blue (see Figure 4(1) "F"), the suspension of colored beads BL is red (see Figure 4(1) "L"), and the control sample is colorless (see Figure 4(1) "NC").

[0087] (3-2-1-2. State of precipitate P) The precipitated P is recovered as follows. First, each of the prepared suspensions (colored beads "E", colored beads "F", colored beads "L", and control sample "NC") is centrifuged at 13000G for 10 minutes to allow the precipitated P to settle, and 980 μL of the supernatant is removed. At this time, the settled precipitated P (colored beads "E", colored beads "F", colored beads "L", and control sample "NC") is observed as shown in Figure 4(2).

[0088] In Figure 4(2), the precipitate P of colored beads BE is observed to be dark red (see Figure 4(2) "E"), the precipitate P of colored beads BF is dark blue (see Figure 4(2) "F"), the precipitate P of colored beads BL is dark red (see Figure 4(2) "L"), and the precipitate P of the control sample is colorless (see Figure 4(2) "NC").

[0089] (3-2-1-3. State of the diluted solution) The diluents are prepared as follows: First, 20 μL of a solubilizer is added to the recovered precipitate P to prepare a mixture. Second, the prepared mixture is transferred to a nucleic acid extraction tube, sealed, and heated at 140°C for 45 seconds to perform high-temperature and high-pressure treatment. Third, the mixture after high-temperature and high-pressure treatment is diluted 1 / 25 times with deionized water to prepare a diluent. Fourth, the prepared diluents are centrifuged. At this time, each of the prepared diluents (colored bead "E", colored bead "F", colored bead "L", and control sample "NC") is observed as shown in Figure 4(3).

[0090] In Figure 4(3), a dark red precipitate and a light red solution of colored beads BE (see Figure 4(3) "E"), a dark blue precipitate and a light blue solution of colored beads BF (see Figure 4(3) "F"), a dark red precipitate and a light red solution of colored beads BL (see Figure 4(3) "L"), and a colorless solution of the control sample (see Figure 4(3) "NC").

[0091] (3-2-2. Nucleic acid extraction efficiency) Figure 5 illustrates the nucleic acid extraction efficiency using colored beads B. Figure 5 is a diagram illustrating experimental results 2-2 according to the embodiment. Below, the experimental procedure will be shown, followed by an explanation of Escherichia coli and Staphylococcus aureus.

[0092] (3-2-2-1. Nucleic acid extraction efficiency of E. coli) The nucleic acid extraction efficiency of E. coli is calculated as follows: First, for E. coli cells C, the above-described procedures are performed in the order of suspension preparation, precipitate P recovery, and diluent preparation, and the diluent after centrifugation is collected. Second, qPCR (quantitative Polymerase Chain Reaction) is performed on the collected E. coli diluent according to the Takara Bio "Bacteria-tuf gene Quantitative PCR" protocol to quantify the amount of extracted nucleic acid. Third, the amount of extracted nucleic acid from E. coli without the addition of colored beads B is set as 100%, and the nucleic acid extraction efficiency is calculated.

[0093] As shown in the bar graph for "E.Coli" in Figure 5, when colored beads BE (see Figure 5 "E" left) are added, the extraction efficiency is over 120%, when colored beads BF (see Figure 5 "F" left) are added, the extraction efficiency is about 100%, and when colored beads BL (see Figure 5 "L" left) are added, the extraction efficiency is about 100%, allowing for the extraction of a sufficient amount of nucleic acid from the recovered E. coli precipitate P.

[0094] (3-2-2-2. Nucleic acid extraction efficiency of Staphylococcus aureus) The nucleic acid extraction efficiency of Staphylococcus aureus is calculated as follows: First, for Staphylococcus aureus cells C, the above-described procedures are carried out in the order of suspension preparation, precipitate P recovery, and diluent preparation, and the diluent after centrifugation is collected. Second, qPCR is performed on the collected Staphylococcus aureus diluent according to the Takara Bio "Bacteria-tuf gene Quantitative PCR" protocol to quantify the amount of extracted nucleic acid. Third, the nucleic acid extraction efficiency is calculated by setting the amount of extracted nucleic acid from Staphylococcus aureus without the addition of colored beads B as 100%.

[0095] As shown in the bar graph for "S. aureus" in Figure 5, when colored beads BE (see Figure 5 "E" right) are added, the extraction efficiency is slightly less than 80%, when colored beads BF (see Figure 5 "F" right) are added, the extraction efficiency is slightly less than 80%, and when colored beads BL (see Figure 5 "L" right) are added, the extraction efficiency is about 80%, allowing for the extraction of a sufficient amount of nucleic acid from the recovered Staphylococcus aureus precipitate P.

[0096] (3-2-2-3. Discussion of Experimental Results 2) From the experimental results in Figures 4 and 5, it can be confirmed that the bacterial cell recovery system 100 efficiently recovers bacterial cells C from E. coli and Staphylococcus aureus by centrifugation using colored beads B. Furthermore, although some of the dye components of colored beads B elute into the diluted solution from which nucleic acids were extracted, it was confirmed that this does not affect the nucleic acid extraction efficiency.

[0097] (3-3. Experimental Results 3) Using Figure 6, we will explain Experiment 3, which confirms the elution of PCR (Polymerase Chain Reaction) inhibitors during high-temperature and high-pressure treatment of bacterial cells C recovered by the bacterial cell recovery system 100. Figure 6 is a diagram illustrating Experiment 3 according to this embodiment. Below, we will explain the evaluation of PCR inhibitors and the discussion of Experiment 3 in that order.

[0098] (3-3-1. Evaluation of PCR inhibitors) The bar graph in Figure 6 shows the concentration of the PCR inhibitor in the diluted solution after the high-temperature and high-pressure treatment described above for each of the prepared suspensions (colored beads "A", "B", "C", "E", "F", "I", "L", and control sample "NC"), for each solution containing a solubilizer ("L-sol."), deionized water ("DIW"), and a solution containing both a solubilizer and colored bead B ("L-sol. + Beads").

[0099] As shown in the bar graphs in Figure 6, the concentrations for each diluent are approximately 300-330 nmol / L for colored beads BA (see Figure 6 "A"), approximately 330-350 nmol / L for colored beads BB (see Figure 6 "B"), approximately 300-370 nmol / L for colored beads BC (see Figure 6 "C"), and approximately 300-360 nmol / L for colored beads BE (see Figure 6 "E"). In the colored beads BF (see Figure 6 "F"), the elution of PCR inhibitors was confirmed at a concentration of approximately 300 nmol / L for each diluent; in the colored beads BI (see Figure 5 "I"), at a concentration of approximately 300-340 nmol / L for each diluent; in the colored beads BL (see Figure 6 "L"), at a concentration of approximately 300-370 nmol / L for each diluent; and in the control sample (see Figure 6 "NC"), at a concentration of approximately 250-340 nmol / L for each diluent.

[0100] (3-3-2. Discussion of Experimental Result 3) As shown in experimental result 3 of Figure 6, it can be confirmed that in the bacterial cell recovery system 100, when nucleic acids are extracted from bacterial cells C recovered by centrifugation using colored beads B, PCR inhibitors (e.g., dye components, high molecular weight compound components) derived from colored beads B, which affect the amount of nucleic acid extracted, are not eluted.

[0101] [4. Processing flow of the bacterial cell recovery system 100] The process flow of the bacterial cell recovery system 100 according to the embodiment will be explained using Figure 7. Figure 7 is a flowchart showing an example of the bacterial cell recovery process flow according to the embodiment. Note that the steps S101 to S104 below can be performed in a different order. Also, some of the steps S101 to S104 below may be omitted.

[0102] First, the bacterial cell recovery system 100 performs a bacterial cell culture solution collection step (step S101). Second, the bacterial cell recovery system 100 performs a colored bead addition step (step S102). Third, the bacterial cell recovery system 100 performs a centrifugation step (step S103). Fourth, the bacterial cell recovery system 100 performs a supernatant removal step (step S104), and the bacterial cell recovery process is completed.

[0103] [5. Effects of the Embodiment] Finally, the effects of the embodiment will be described. Below, effects 1 to 6 corresponding to the steps of the embodiment will be described.

[0104] (5-1. Effect 1) Firstly, in the process according to the embodiment described above, colored beads B having a predetermined color are added to the bacterial culture medium S, and the bacterial culture medium S to which the colored beads B have been added is separated into supernatant L and precipitate P using a centrifuge 30, the supernatant L is removed and the precipitate P is recovered. Therefore, in the process according to the embodiment, bacterial cells C can be efficiently recovered by centrifugation.

[0105] (5-2. Effect 2) Secondly, in the process according to the embodiment described above, colored beads B having a specific gravity within a predetermined range relative to the specific gravity of the bacterial cells C to be recovered are added. Therefore, in the process according to the embodiment, by using colored beads B with a specific gravity similar to that of the bacterial cells C, the bacterial cells C can be efficiently recovered by centrifugation.

[0106] (5-3. Effect 3) Thirdly, in the steps of the embodiment described above, colored beads B having a particle size within a predetermined range relative to the size of the bacterial cells C to be recovered are added. Therefore, in the steps of the embodiment, by using colored beads B of a size similar to that of the bacterial cells C, the bacterial cells C can be efficiently recovered by centrifugation.

[0107] (5-4. Effect 4) Fourth, in the process according to the embodiment described above, the colored beads B are latex, polylactic acid, silica, polystyrene, or polyethylene. Therefore, in the process according to the embodiment, by using widely available colored beads B, the bacterial cells C can be efficiently recovered by centrifugation.

[0108] (5-5. Effect 5) Fifth, in the steps of the embodiment described above, the sample solution is a bacterial culture medium S. Therefore, in the steps of the embodiment, bacterial cells C in particular can be efficiently recovered from among the various samples to be analyzed by centrifugation.

[0109] (5-6. Effect 6) Sixth, in the steps according to the embodiment described above, nucleic acids are extracted from the recovered bacterial cells C. Therefore, in the steps according to the embodiment, bacterial cells C can be efficiently recovered by centrifugation, and nucleic acids can be extracted even more efficiently.

[0110] 〔system〕 Unless otherwise specified, the processing procedures, control procedures, specific names, and various data and parameters shown in the above documents and drawings may be changed at will.

[0111] Furthermore, the components of each illustrated device are functionally conceptual and do not necessarily need to be physically configured as shown. In other words, the specific forms of distribution and integration of each device are not limited to those shown. That is, all or part of them can be functionally or physically distributed and integrated in any unit according to various loads and usage conditions.

[0112] 〔others〕 Some examples of the combinations of technical features that will be disclosed are listed below.

[0113] (1) A sample recovery method for recovering a sample from a sample solution containing a sample, comprising: an addition step of adding beads having a predetermined color to the sample solution; a separation step of separating the sample solution to which the beads have been added into a supernatant and a precipitate using a centrifuge; and a removal step of removing the supernatant and recovering the precipitate.

[0114] (2) The sample recovery method according to (1), wherein the addition step involves adding beads having a specific gravity within a predetermined range relative to the specific gravity of the sample to be recovered.

[0115] (3) The sample recovery method according to (1) or (2), wherein the addition step involves adding beads having a particle size within a predetermined range relative to the size of the sample to be recovered.

[0116] (4) The sample recovery method according to any one of (1) to (3), wherein the beads are latex, polylactic acid, silica, polystyrene, or polyethylene.

[0117] (5) The sample recovery method according to any one of (1) to (4), wherein the sample solution is a bacterial culture medium.

[0118] (6) A sample recovery method according to any one of (1) to (5), further comprising an extraction step of extracting nucleic acids from the bacterial cells recovered by the removal step. [Explanation of Symbols]

[0119] 10 Bacterial culture container 20 Centrifuge tubes 30 Centrifugal Separators 40 pipettes 100 bacterial cell recovery system

Claims

1. A sample recovery method for recovering bacterial cells from a bacterial culture medium containing bacterial cells, An addition step of adding beads having a predetermined color that can be visually identified to the bacterial culture medium, A separation step is to use a centrifuge to separate the bacterial culture solution to which the beads have been added into a supernatant and a precipitate, A removal step of removing the supernatant and recovering the precipitate, Includes, The aforementioned beads are The specific gravity is 1.1 to 1.4 g / mL, and is selected to be approximately the same as that of the bacterial cells to be recovered. The particle size is 1.0 to 32 μm, and is selected to be approximately the same size as the bacterial cells to be recovered. Sample collection method.

2. The aforementioned beads are latex, polylactic acid, silica, polystyrene, or polyethylene. The sample recovery method according to claim 1.

3. An extraction step in which nucleic acids are extracted from the bacterial cells recovered by the removal step, It further includes, The extraction step is, As a solubilizing agent, sodium dodecyl sulfate is added when high-sensitivity detection of nucleic acids is desired, and octylphenol ethoxylate is added when nucleic acids are used in enzymatic reactions. The sample recovery method according to claim 1.

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