Microbiota trapping device, microbiota trapping apparatus, and microbiota trapping method

Abstract

Microbiota trapping device, microbiota trapping apparatus, and microbiota trapping method. Microbiota trapping device comprising: - a first solution reservoir having a first filter designed to trap a residue in a sample; and - a second solution reservoir having a second filter designed to trap bacteria in the sample, the second solution reservoir having a vent above the second filter. Figure for the abstract: Fig. 1

Description

Title of the invention: Microbiota trapping device, microbiota trapping apparatus, and microbiota trapping method Technical field

[0001] The present invention relates to a microbiota trapping device, a microbiota trapping apparatus, and a microbiota trapping method. Prior art

[0002] In recent years, indigenous microbiota, which is a bacterial group residing in the human body, has attracted attention as a novel biomarker or as a target in therapy. Conventional microbiota research inevitably involves the growth of bacteria by culture. Thus, it has been difficult to detect non-culturable bacteria and thus impossible to completely analyze the microbiota. Meanwhile, with a gene analysis technique of microbiota using the next-generation sequencing machine developed in recent years, it has become possible to completely analyze genes from microbiota by amplification without culture. Thus, it has become possible to completely identify the microbiota and make a comparison among a diversity of bacterial species in the microbiota.

[0003] However, as disclosed in Non-Patent Literature 1 (Costea et al., Towards standards for human fecal sample processing in metagenomic studies nature bio-technology, 2017, 1-9), a microbiota DNA extraction technique is still immature. For example, variations in measurement results due to different reagents and / or kits used, and variations in operations due to different engineers involved in the operation, even with the same kit, are major obstacles to the development of microbiota research. It is assumed that the accumulation of microbiota data elements that can be mutually compared with each other will greatly contribute to the promotion of gut microbiota research, and it is desired to automate a DNA extraction technique that is a preprocessing step of a microbiota genetic analysis technique.

[0004] A microbiota DNA extraction technique is broadly divided into a direct extraction method and an indirect extraction method. The direct extraction method is a technique of directly and physically disrupting a sample containing microbiota using agitation with beads, for example, and then extracting DNA from the sample solution and purifying it. The direct extraction method is advantageous for its ease of operation. However, the method is problematic in that DNA from components (e.g., human cells or food residues) other than bacteria contained in the sample would be mixed or the desired DNA would be fragmented into a size of several kb or less due to the physical process carried out.

[0005] The indirect extraction method is a technique of first separating bacterial cells from a sample and then extracting DNA from the separated bacterial cells. Since DNA is extracted from the separated microbiota via a gentle process in the indirect extraction method, it can recover large DNA fragments having a size greater than 20 kb and not having other organisms mixed with them (Non-Patent Literature 2: Kim et al., Robustness of Gut Microbiota of Healthy Adults in Response to Probiotic Intervention Revealed by High-Throughput Pyrosequencing, DNA RESEARCH 2013, 20, 241-253). However, the indirect extraction method is problematic in that it involves a cumbersome operation compared to the direct extraction method.

[0006] The current dominant research on the intestinal microbiota is conducted by comparing the components of the intestinal microbiota. In the future, it is assumed that research will make further progress in analyzing the function of the intestinal microbiota. The length of DNA fragment that is required for the identification of a bacterial species is several hundred bp. Therefore, although many institutions currently use the direct extraction method, the indirect extraction method is more suitable for analyzing the function of the microbiota since it can prepare large DNA fragments with which the analysis of a genetic structure is possible.

[0007] In Non-Patent Literature 2, centrifugation is performed to trap microbiota from a sample after a residue has been removed from the sample through a filter. However, such a step is complex, and in particular, performing a centrifugation process is burdensome for the operator. Statement of the invention

[0008] Accordingly, the present disclosure provides a technique for easily trapping microbiota from a sample. Summary of the invention

[0009] In view of the foregoing, a microbiota trapping device of the present disclosure comprises a first solution reservoir having a first filter configured to trap a residue in a sample, and a second solution reservoir having a second filter configured to trap bacteria in the sample, and the second solution reservoir has a vent above the second filter.

[0010] Other features of the present disclosure will become apparent from the following description of this specification and the accompanying drawings. In addition, embodiments of the present disclosure may be implemented by elements, by a combination of a variety of elements, by the following detailed description, and by the appended claims.

[0011] The description of this specification contains only typical illustrative examples. Therefore, claims or examples of the application of the present disclosure should not be limited in any sense.

[0012] According to the microbiota trapping device of the present disclosure, microbiota can be easily trapped from a sample.

[0013] Problems, configurations, and advantageous effects other than those described above will become apparent from the following description of embodiments.

[0014] In one of its aspects, the invention is related to at least one of the following objects.

[0015] Subject 1: a microbiota trapping device comprising: - a first solution reservoir comprising a first filter designed to trap a residue in a sample; and - a second solution reservoir comprising a second filter designed to trap bacteria in the sample, the second solution tank having a vent above the second filter.

[0016] Object 2: the microbiota trapping device according to object 1, a pore size of the first filter being greater than or equal to 70 pm, and a pore size of the second filter being less than or equal to 1 pm.

[0017] Object 3: The microbiota trapping device according to object 1, further comprising a support which supports the first solution reservoir above the second solution reservoir, the support being configured to support the first solution reservoir such that a gap is formed between the first solution reservoir and the second solution reservoir and an opening of the second solution reservoir serves as a vent.

[0018] Object 4: the microbiota trapping device according to object 1, the second solution reservoir having the vent through a side wall of the second solution reservoir.

[0019] Subject 5: a microbiota trapping device comprising: - the microbiota trapping device according to object 1; - a pressure applicator configured to apply a first pressure to the first solution tank and apply a second pressure to the second solution tank; and - a control device configured to control the piloting of the pressure applicator.

[0020] Object 6: the microbiota trapping apparatus according to object 5, the device of command being configured for: - causing the pressure applicator to apply the first pressure to the first solution reservoir, so that the first filter traps the residue in the sample, and a filtrate containing the bacteria is introduced into the second solution reservoir, and - make the pressure applicator apply the second pressure to the second solution tank, so that the second filter traps the bacteria in the filtrate.

[0021] Object 7: the microbiota trapping apparatus according to object 5, each of the first pressure and the second pressure being a positive pressure.

[0022] Object 8: the microbiota trapping apparatus according to object 5, the pressure applicator comprising: - a positive pressure generator configured to generate a positive pressure, and - a negative pressure generator configured to generate a negative pressure, the first pressure being the positive pressure generated by the positive pressure generator, and the second pressure being the negative pressure generated by the negative pressure generator.

[0023] Object 9: the microbiota trapping apparatus according to object 6, further comprising: - a dispenser designed to dispense a recovery solution into the second solution tank; and - a collector designed to recover a solution in the second solution tank, the control device being configured to: - causing the second filter to trap the bacteria in the filtrate and then the dispenser to add the recovery solution to the second solution tank to suspend the bacteria in the recovery solution, thereby obtaining a microbiota suspension, and - ensure that the collector recovers the microbiota suspension.

[0024] Subject 10: A method of trapping microbiota using a microbiota trapping device, the device comprising a first solution reservoir having a first filter configured to trap a residue in a sample, and a second solution reservoir having a second filter configured to trap bacteria in the sample, the second solution reservoir also having a vent above the second filter, the method comprising: - introduction of the sample into the first solution tank; - applying a first pressure to the first solution reservoir, so that the first filter traps the residue in the sample and a filtrate containing the bacteria is introduced into the second solution reservoir; and - applying a second pressure to the second solution tank, so that the second filter traps the bacteria in the filtrate.

[0025] Object 11: the method for trapping microbiota according to object 10, a pore size of the first filter being greater than or equal to 70 pm, and a pore size of the second filter being less than or equal to 1 pm.

[0026] Object 12: the method of trapping microbiota according to object 10, each of the first pressure and the second pressure being a positive pressure.

[0027] Object 13: the method of trapping microbiota according to object 10, the first pressure being a positive pressure and the second pressure being a negative pressure.

[0028] Object 14: The microbiota trapping method according to object 10, further comprising adding a recovery solution to the second solution reservoir to suspend the bacteria in the recovery solution, thereby obtaining a microbiota suspension. Brief description of the drawings

[0029] [fig. 1] [fig.l] represents a schematic view of a mi trapping apparatus microbiote according to a first embodiment,

[0030] [fig.2] [fig.2] represents a flow diagram illustrating a trapping process microbiota which uses the microbiota trapping apparatus according to the first embodiment,

[0031] [fig.3] [fig.3] represents a schematic view of a mi trapping device microbiote according to a second embodiment,

[0032] [fig.4A] [fig.4A] represents a schematic view of a part of the configuration of a microbiota trapping device according to a third embodiment,

[0033] [fig.4B] [fig.4B] represents a schematic view of a part of the configuration of a microbiota trapping device according to a third embodiment,

[0034] [fig.5] [fig.5] represents a view illustrating the results of DNA electrophoresis which was extracted from a microbiota in an example extraction test 1,

[0035] [fig.6A] [fig.6A] represents a graph illustrating the results of quantitative PCR carried out on DNA from the microbiota, and,

[0036] [fig.6B] [fig.6B] represents a graph illustrating the results of quantitative PCR carried out on DNA from the microbiota. Detailed description

[0037] In all the drawings for illustrating the following embodiments, elements having the same function are designated by the same reference numeral. Thus, repeated description thereof will be omitted as much as possible. Furthermore, the present disclosure should not be construed as being limited to the description of the following embodiments. Those skilled in the art will readily understand that the The specific configuration of this disclosure may be changed without departing from the spirit and scope thereof.

[0038] There may be instances where the position, size, shape, amplitude, and the like of each structure illustrated in the drawings do not represent the actual position, size, shape, amplitude, and the like of the structure in order to assist in easily understanding the invention. Therefore, the present disclosure is not necessarily limited to the position, size, shape, amplitude, and the like of each structure disclosed in the drawings.

[0039] In addition, the publications cited in this specification partially constitute the description of this specification.

[0040] Each constituent element represented in a singular form in this specification includes a plural form unless explicitly stated otherwise in the context.

[0041] In the present disclosure, "large DNA fragment" refers to DNA having a size greater than or equal to 20 kb which is the upper limit allowing observation by usual agarose gel electrophoresis separation.

[0042] [First embodiment]

[0043] <Configuration de l’appareil de piégeage de microbiote>

[0044] [Fig. 1] is a schematic view of a microbiota trapping apparatus according to a first embodiment. As illustrated in [Fig. 1], the microbiota trapping apparatus comprises a microbiota trapping device 1, a control device 100, a pressure pump 107, and a tube 108. [Fig. 1] illustrates a cross-section of the microbiota trapping device 1. The microbiota trapping device 1 comprises a first solution tank 101, a second solution tank 103, a liquid waste container 105, a pressure vessel 106, and a support 109.

[0045] At least a portion of the lower face of the first solution tank 101 is formed with a residue removal filter 102 (i.e., a first filter), and a sample solution (i.e., a sample) is introduced into the first solution tank 101 through an opening thereof. It is also possible to use a filter holder having openings at its upper portion and lower portion as the first solution tank 101 and dispose the residue removal filter 102 at the lower portion of the filter holder.

[0046] The residue removal filter 102 can trap a residue in a sample. The pore size of the residue removal filter 102 can be set to a value greater than or equal to 70 μm, for example. As the material of the residue removal filter 102, either a hydrophobic material or a hydrophilic material can be used. Examples of the hydrophobic material include a polyamide, a fluororesin such as polytetrafluoroethylene, a polyolefin such as polypropylene, a polycarbonate, and a glass. Examples of the hydrophilic material include a polyether sulfone resin, a cellulose acetate, and a nitrocellulose.

[0047] The second solution tank 103 is disposed below the first solution tank 101 and receives a filtrate that has passed through the residue removal filter 102. The second solution tank 103 has a bacteria trapping filter 104 (i.e., a second filter) disposed at its lower portion. The bacteria trapping filter 104 is a membrane filter formed from the aforementioned hydrophilic material, for example. The bacteria trapping filter 104 has a pore size capable of trapping bacteria (typically, about 1 μm in diameter), which may thus be set to a value less than or equal to 1 μm, for example, or less than or equal to 0.45 μm depending on the circumstances.In this way, since the pore size of the bacteria trapping filter 104 located below is smaller than that of the residue removal filter 102, bacteria can be trapped from the filtrate which has been obtained by removing residue from the sample solution.

[0048] The inner diameter of the first solution tank 101 and that of the second solution tank 103 may be set approximately equal, for example. Alternatively, setting the inner diameter of the second solution tank 103 to a value greater than or equal to that of the first solution tank 101 will allow a sample solution filtered through the residue removal filter 102 to be introduced into the second solution tank 103 more reliably.

[0049] The liquid waste container 105 is disposed below the second solution reservoir 103 and receives a filtrate that has passed through the bacteria trapping filter 104. The liquid waste container 105 may have any structure and be disposed at any position that can prevent diffusion of the filtrate from the bacteria trapping filter 104, and may also be coupled to the second solution reservoir 103 via a liquid supply tube, for example.

[0050] The support 109 supports the first solution tank 101 such that a gap 110 is formed between the first solution tank 101 and the second solution tank 103. The size of the gap 110 is not limited to a particular value, but is preferably as small as possible from the point of view of preventing contamination from the outside. In this way, the placement of the gap 110 above the second solution tank 103 allows the opening of the second solution tank 103 to serve as a vent. Thus, even when the first solution tank 101 is pressurized, a pressure inside the second solution tank 103 can be maintained at atmospheric pressure. In this way, pressurization of the first solution tank 101 and pressurization of the second solution tank 103 can be carried out independently.

[0051] The support 109 may be configured to be movable in three axial directions by means of a support actuator (not shown), for example. Accordingly, the positional relationship between the first solution tank 101 and the second solution tank 103 may be changed. Alternatively, the positional relationship between the first solution tank 101 and the second solution tank 103 may be changed by a user. Although [fig.l] illustrates an example in which the support 109 supports the first solution tank 101 by gripping it, the present disclosure is not limited thereto and any structure may be provided that allows the gap 110 to be formed between the first solution tank 101 and the second solution tank 103.For example, bars for supporting the underside of the first solution tank 101 may be placed at the upper edge of the second solution tank 103, and such bars may be used as support 109.

[0052] Although the pressure vessel 106 (i.e., a pressure applicator) in [fig.l] is attached to the first solution tank 101 so as to hermetically seal it by covering its opening, the pressure vessel 106 may be attached to, and detachable from, each of the first solution tank 101 and the second solution tank 103. The pressure vessel 106 may be a cover that covers the opening of the first solution tank 101 or the second solution tank 103. The pressure vessel 106 has an orifice (not shown) for attachment to the tube 108, and is coupled to the pressure pump 107 (i.e., a pressure applicator) via the tube 108. Pressure generated by the pressure pump 107 is supplied to the pressure vessel 106 via the tube 108.It is also possible to couple two or more pressure vessels 106 to the pressure pump 107, and to use the respective pressure vessels 106 for the first solution tank 101 and the second solution tank 103.

[0053] In this manner, pressure generated by the pressure pump 107 can be applied to the solution reservoir 101 or 103 having the pressure vessel 106 attached thereto. This allows the pressure to act on the filter (i.e., the residue removal filter 102 or the bacteria trapping filter 104) placed on the solution reservoir, and thus allows a sample solution to be filtered through the filter.

[0054] Although not illustrated, the microbiota trapping apparatus may also comprise a sample container actuator for moving a sample container containing a sample solution. In such a case, driving the sample container actuator may introduce a sample solution into the first solution reservoir 101. In addition, the microbiota trapping apparatus may also comprise a pressure container actuator for controlling the movement of the pressure container 106 as well as the attachment / detachment of the pressure container 106 to / from the first solution reservoir 101 or to / from the second solution reservoir 103.

[0055] Furthermore, the microbiota trapping apparatus may also comprise a first dispensing nozzle for dispensing a cleaning solution into the first solution reservoir 101 or the second solution reservoir 103, a second dispensing nozzle (i.e., a dispenser or a collector) for dispensing a collection solution for collecting bacteria in the second solution reservoir 103, and a dispensing nozzle actuator for driving the first dispensing nozzle or the second dispensing nozzle. As the cleaning solution and the collection solution, a buffer solution, such as a PBS solution or a TE solution, may be used, for example. With the second dispensing nozzle, it is also possible to obtain a microbiota suspension by pipetting a mixed solution of bacteria and a collection solution up and down.The microbiota suspension can be recovered using the second dispensing nozzle or another dispensing nozzle (i.e., a collector).

[0056] The control device 100 (i.e., a controller) controls the generation of pressure by the pressure pump 107, the driving of each actuator, and the driving of each dispensing nozzle. The microbiota trapping apparatus of the present embodiment may comprise more than one microbiota trapping device 1, and in such a case, the operation of the more than one microbiota trapping device 1 may be controlled by a single control device 100.

[0057] <Procédé de piégeage de microbiote>

[0058] [Fig. 2] is a flow diagram illustrating a microbiota trapping method using the microbiota trapping apparatus. Hereinafter, it is assumed that each of the aforementioned dispensing nozzles and actuators is present in or on the microbiota trapping apparatus, and the control device 100 controls the driving of each constituent element of the microbiota trapping apparatus so that the microbiota trapping operation is performed automatically.

[0059] In step S1, the control device 100 drives the container actuator sample container so that it moves the sample container, thereby introducing a sample solution into the first solution reservoir 101.

[0060] In step S2, the controller 100 drives the pressure vessel actuator to attach the pressure vessel 106 to the first solution tank 101. Then, the controller 100 drives the pressure pump 107 to generate a positive pressure so that the positive pressure (i.e., a first pressure) is applied to the residue removal filter 102 of the first solution tank 101. This allows the sample solution to be filtered through the residue removal filter 102 and allows the resulting filtrate to be introduced into the second solution tank 103, a residue having been removed. At this time, the target bacteria to be trapped are introduced into the second solution tank 103 together with the filtrate.

[0061] As described above, since the gap 110 is placed between the first solution tank 101 and the second solution tank 103, the second solution tank 103 is maintained under atmospheric pressure conditions even when the first solution tank 101 is pressurized. That is, each of the first solution tank 101 and the second solution tank 103 can be pressurized independently. Thus, while the first solution tank 101 is pressurized to have a residue removed from a sample solution, a solution that has been introduced into the second sample tank 103 is maintained therein without being filtered.

[0062] In step S3, the controller 100 drives the first dispensing nozzle actuator so that the first dispensing nozzle adds a cleaning solution into the first solution tank 101. At this time, the cleaning solution may be added after the pressure vessel 106 is detached, or the pressure vessel 106 may be formed of a material that allows the first dispensing nozzle to penetrate therethrough.

[0063] In step S4, the controller 100 controls the pressure pump 107 to apply pressure to the residue removal filter 102 of the first solution tank 101. This allows bacteria, which have adhered to an inner wall of the first solution tank 101 or the residue removal filter 102, to be removed by cleaning and trapped in the second solution tank 103. Thus, the recovery efficiency of the microbiota is improved. It is noted that each of steps S3 and S4 may be performed more than once or may not be performed at all. The number of times each of steps S3 and S4 is to be performed may be set to any value.

[0064] At the stage where the residue removal step is completed, the first solution tank 101 and the residue removal filter 102 are no longer required. Thus, in step S5, the controller 100 drives the support 109 to discharge the first solution tank 101 from above the second solution tank 103. This can prevent the first solution tank 101 from becoming a physical obstacle in subsequent steps. In addition, the controller 100 detaches the pressure vessel 106 from the first solution tank 101.

[0065] In step S6, the controller 100 drives the pressure vessel actuator to attach the pressure vessel 106 to the second solution tank 103. Then, the controller 100 drives the pressure pump 107 to apply a positive pressure (i.e., a second pressure) to the bacteria trapping filter 104 of the second solution tank 103. This allows the solution containing the bacteria to be filtered through the bacteria trapping filter 104 and thus allows the bacteria to be trapped by the bacteria trapping filter 104, and also allows the resulting filtrate to be introduced into the liquid waste container 105.

[0066] In step S7, the controller 100 drives the first dispensing nozzle actuator so that the first dispensing nozzle adds a cleaning solution into the second solution reservoir 103.

[0067] In step S8, the controller 100 drives the pressure pump 107 to apply pressure to the bacteria trapping filter 104 of the second solution tank 103. This allows substances other than bacteria, which have adhered to the inner wall of the second solution tank 103 or the bacteria trapping filter 104, to be removed by cleaning and discharged as filtrate. Thus, the recovery efficiency of the microbiota is improved. It is noted that each of steps S7 and S8 may be executed more than once or may not be performed at all. The number of times each of steps S7 and S8 is to be executed may be set to any value.

[0068] In step S9, the controller 100 drives the second dispensing nozzle actuator so that the second dispensing nozzle adds a recovery solution into the second solution tank 103.

[0069] In step S10, the controller 100 drives the second dispensing nozzle actuator so that the second dispensing nozzle repeatedly draws and discharges the liquid in the second solution tank 103 inside the second solution tank 103. As a result, a microbiota suspension is obtained in the second solution tank 103. Afterward, the controller 100 drives the second dispensing nozzle actuator so that the second dispensing nozzle dispenses the microbiota suspension into a collection container for recovery.

[0070] Although an example has been described in which the control device 100 drives each constituent element of the microbiota trapping apparatus for automatically trapping microbiota, the present application is not limited to this. For example, a user may manually perform the aforementioned operations. In such a case, the user may attach or detach the pressure vessel 106, operate the pressure pump 107, move the first solution reservoir 101, and operate the first dispensing nozzle and the second dispensing nozzle, for example.

[0071] <Avantages techniques>

[0072] As described above, the microbiota trapping device 1 of the present invention can trap bacteria in a sample solution without performing centrifugation, and can thus reduce the burden on the user for operating the device. In addition, automating the operation for microbiota trapping using the microbiota trapping apparatus with the microbiota trapping device 1 can further reduce the burden on the user for operating the device.

[0073] There may be instances where the bacteria trapping filter 104 becomes clogged with microbiota while being trapped by the bacteria trapping filter 104 if the number of bacteria contained in the sample solution is large, which in turn may decrease the filtration rate. In such a case, if the first solution reservoir 101 and the second solution reservoir 103 are structured so that they are tightly coupled together, a decrease in the filtration flow rate through the bacteria trapping filter 104 will cause resistive pressure to act against the pressure applied to the residue removal filter 102, which in turn may disrupt the filtration cleaning performed through the residue removal filter 102.In contrast, the microbiota trapping device 1 of the present embodiment has the spacing 110 between the first solution reservoir 101 and the second solution reservoir 103. Thus, applying pressure to the residue removal filter 102 and applying pressure to the bacteria trapping filter 104 can be performed independently, which can suppress a decrease in the filtration speed through the residue removal filter 102. Therefore, a residue can be effectively removed from the sample solution.

[0074] The spacing 110 is also advantageous for changing the positional relationship between the first solution reservoir 101 and the second solution reservoir 103. If the first solution reservoir 101 and the second solution reservoir 103 are structured so that they are hermetically coupled together without the spacing 110 placed between them, while the first solution reservoir 101 is pressurized, the second solution reservoir 103 would also be pressurized. Therefore, a greater force would be required to evacuate the first solution reservoir 101 from the second solution reservoir 103. Furthermore, if one attempts to separate the first solution reservoir 101 and the second solution reservoir 103 from each other while they are pressurized, a sample would diffuse upon a release of pressure. Then, the valuable sample could be lost due to diffusion or the diffused sample could contaminate the environment. In contrast, in the microbiota trapping device 1 of the present embodiment, the second solution reservoir 103 is maintained under atmospheric pressure conditions due to the presence of the gap 110. Thus, there is no possibility that a sample could diffuse, which might otherwise happen upon a release of pressure when the first solution reservoir 101 is evacuated.

[0075] [Second embodiment]

[0076] The first embodiment mentioned above illustrates an example in which the pressure vessel 106 is attached to the first solution reservoir 101 to pressurize it, and then, the pressure vessel 106 is attached to the second solution reservoir 103 to pressurize it so that a residue is removed from a sample solution by the residue removal filter 102 and then microbiota is trapped by the bacteria trapping filter 104 when the sample solution is filtered therethrough. However, the pressurization method for the second solution reservoir 103 is not necessarily the same as for the first solution reservoir 101. For example, filtration through the bacteria trapping filter 104 may be implemented by applying a positive pressure to the first solution reservoir 101 and applying a negative pressure to the second solution reservoir 103.

[0077] Hereinafter, a second embodiment will describe an example in which the first solution tank 101 and the second solution tank 103 are provided with different pressurization mechanisms.

[0078] <Configuration de l’appareil de piégeage de microbiote>

[0079] [Fig. 3] is a schematic view of a microbiota trapping apparatus according to the second embodiment. As illustrated in [Fig. 3], the microbiota trapping apparatus of the second embodiment differs from the microbiota trapping device 1 of the first embodiment in that a microbiota trapping device 2 comprises a pressure vessel 111 (i.e., a pressure applicator) provided with a discharge port 112, and the discharge port 112 of the pressure vessel 111 and a depressurization pump 113 (i.e., a pressure applicator (or a negative pressure generator)) are coupled together by a tube 114.

[0080] The pressure vessel 111 covers the second solution tank 103 of such that the bacteria trapping filter 104 and the liquid waste container 105 are located in the internal space of the pressure vessel 111. The discharge port 112 and the depressurization pump 113 are coupled together by the tube 114, and the internal space of the pressure vessel 111 is hermetically sealed. A negative pressure generated by the depressurization pump 113 is applied to the pressure vessel 111. Examples of the pressure vessel 111 include a suction filtration vessel.

[0081] Instead of separately providing the pressure pump 107 (i.e., the positive pressure generator) and the depressurization pump 113 (i.e., the negative pressure generator), it is also possible to provide a single pressurization / depressurization pump having both the pressurization and depressurization functions and to couple the pressure vessel 106 and the pressure vessel 111 to the pressurization / depressurization pump.

[0082] <Procédé de piégeage de microbiote>

[0083] The microbiota trapping method of the present embodiment is substantially the same as that of the first embodiment illustrated in [fig.2]. Thus, only the differences will be described below.

[0084] In step S6, instead of switching the attachment target from the pressure vessel 106 to the second solution tank 103 to pressurize it, the depressurization pump 113 is driven to depressurize the pressure vessel 111 in the present embodiment. This allows negative pressure (i.e., a second pressure) to be applied to the bacteria trapping filter 104 and thus allows a solution in the second solution tank 103 to be suction-filtered through the bacteria trapping filter 104, thereby trapping microbiota. In the present embodiment, step S5 shown in [fig.2] may be performed after step S6.

[0085] <Avantages Techniques>

[0086] As described above, the microbiota trapping apparatus of the present embodiment is configured such that a negative pressure is applied to the second solution tank 103 by the pressure vessel 111 and the depressurization pump 113 so that a solution containing bacteria is filtered by suction. This can eliminate the need to change the attachment target of the pressure vessel 106 and can thus perform the microbiota trapping operation more quickly and easily compared with the first embodiment.

[0087] [Third embodiment]

[0088] The first embodiment and the second embodiment mentioned above each illustrate an example in which the spacer 110 is placed between the underside (i.e., the residue removal filter 102) of the first solution tank 101 and the opening of the second solution tank 103. However, the position of the gap 110 is not limited to this and can be changed as needed as long as each of the first solution tank 101 and the second solution tank 103 can be pressurized independently.

[0089] Hereinafter, a third embodiment will describe a microbiota trapping device in which the position of the spacing 110 is modified.

[0090] <Configuration du dispositif de piégeage de microbiote>

[0091] [Fig.4A] is a schematic view of a part of the configuration of a microbiota trapping device 3 according to the third embodiment. As illustrated in [Fig.4A], in the microbiota trapping device 3, the inner diameter of the second solution tank 103 is larger than the outer diameter of the first solution tank 101. The first solution tank 101 is supported by the holder 109 so that the residue removal filter 102 is located in the second solution tank 103 and the outer wall of the first solution tank 101 is not in contact with the inner wall of the second solution tank 103. Accordingly, the gap 110 is formed between the side of the residue removal filter 102 of the first solution tank 101 and the inner wall of the second solution tank 103.

[0092] [Fig.4B] is a schematic view of a portion of the configuration of a microbiota trapping device 4. As illustrated in [Fig.4B], the gap 110 may be formed as a through-hole in the sidewall of the second solution tank 103. The through-hole (i.e., a vent) is placed above the bacteria trapping filter 104, and may thus maintain the interior of the second solution tank 103 at atmospheric pressure when the second solution tank 103 is not pressurized. In [Fig.4B], the outer diameter of the first solution tank 101 is set substantially equal to the inner diameter of the second solution tank 103 such that one end of the first solution tank 101 may be fitted into the opening of the second solution tank 103.

[0093] With the adoption of the configuration in [fig.4B], it becomes less likely that external material can intrude into the second solution reservoir 103, which can reduce the risk of contamination.

[0094] In the microbiota trapping device 3 of [fig.4A] and the microbiota trapping device 4 of [fig.4B], either the pressurization mechanism of the first embodiment or that of the second embodiment can be adopted for each of the first solution reservoir 101 and the second solution reservoir 103.

[0095] <Procédé de piégeage de microbiote>

[0096] In the present embodiment, the same microbiota trapping method of the first embodiment or the second embodiment can be adopted. So, the description of it is omitted here.

[0097] <Avantages techniques>

[0098] As described above, the gap of the spacing 110 in each of the microbiota trapping devices 3 and 4 of the present embodiment can be reduced compared to those of the first embodiment and the second embodiment. Accordingly, contamination of the second solution reservoir 103 from the outside can be suppressed. Examples

[0099] Hereinafter, Examples of the present disclosure will be described.

[0100] [Example 1]

[0101] <Préparation d’échantillons>

[0102] Four types of pure culture bacterial cells, including Escherichia coli, Pseudomonas aeruginosa, Staphylococcus aureus, and Enterococcus faecalis, were obtained by performing overnight shaking culture using LB culture medium.

[0103] The optical density of each bacterial body solution was measured and was adjusted to achieve OD = 1. The number of bacterial cells, which has a value of 1 x 108 CFU, in 1 mL of a bacterial body solution having an OD = 1 was used as an index, and equal amounts of the four types of bacterial body solutions were mixed to produce a mixed bacterial body solution having a total bacterial number of 1 x 109 CFU or 1 x 1010 CFU as a model microbiota.

[0104] 2.5 g of "Ukigoi" as floating carp (manufactured by MARUKYU CO., LTD.), which is a fishing bait, 1 g of durum wheat flour "Kameliya (registered trademark)" (manufactured by Nisshin Seifun Group Inc.), and 2.5 g of sterile water were mixed into the model microbiota thus produced so as to produce a simulated fecal model sample. Then, the simulated fecal model sample was stored at -20°C for use in the following steps.

[0105] <Préparation du dispositif de piégeage de microbiote>

[0106] In Example 1, a microbiota trapping apparatus was constructed by preparing six microbiota trapping devices 2 of the second embodiment illustrated in [fig.3] and coupling them to their respective control devices 100. As constituent elements of the microbiota trapping apparatus, the following elements were specifically used.

[0107] The first solution reservoir 101 and the residue removal filter 102: a Falcon (registered trademark) cellular sieve (having a mesh with a pore size of 100 pm) manufactured by Corning Incorporated was used.

[0108] The second solution reservoir 103: a support for supporting the bacteria trapping filter 104 was produced with a 3D printer and used as the second solution reservoir 103.

[0109] Bacteria trapping filter 104: Millipore Express (registered trademark) PLUS membrane filter (with a pore size of 0.45 pm) manufactured by Merck KGaA was used.

[0110] Liquid Waste Container 105: A 50 mL Falcon (registered trademark) conical tube manufactured by Corning Incorporated was used.

[0111] Pressure vessel 106: An aluminum pressure vessel was produced and used as pressure vessel 106.

[0112] Pressure vessel 111: A suction-filtration vessel was used.

[0113] Pressure pump 107 and depressurization pump 113: A diaphragm vacuum pump N86KN.18 (i.e., a pressurization / depressurization pump) manufactured by KNF Group was used.

[0114] Tubes 108 and 114: silicone tubes were used.

[0115] <Procédé d’élimination de résidu de l’échantillon>

[0116] A residue removal process of the fecal model sample was carried out with the following procedures. The following steps and tests were conducted with the six microbiota trapping devices 2 (test no. 1 to 6) using the same sample under the same conditions.

[0117] Step 1: The fecal model sample was dissolved in 2 mL of PBS solution so that the sample was suspended in the solution and a sample solution was thus obtained.

[0118] Step 2: The membrane filter (i.e., the bacteria trapping filter) was placed on the second solution tank, and the liquid waste container was coupled to the lower part of the second solution tank. In addition, the cell sieve (i.e., the first solution tank and the residue removal filter) was arranged above the second solution tank. The spacing between the second solution tank and the cell sieve was set at about 1 mm. Then, the sample solution was added into the cell sieve.

[0119] Step 3: The pressurization / depressurization pump was set to a pressurization condition so as to pressurize the cell sieve for 1 minute so that the sample solution was filtered and the resulting filtrate was introduced into the second solution reservoir.

[0120] Step 4: 1 mL of PBS solution was added into the cell strainer.

[0121] Step 5: The cell sieve was pressurized for 1 minute as in step 3 to perform the filtration, and the resulting filtrate was introduced into the second solution tank.

[0122] Step 6: The cell sieve was removed from above the second solution reservoir.

[0123] <Piégeage de cellules bactériennes>

[0124] Bacterial cell trapping and membrane filter cleaning were performed with the following procedures.

[0125] Step 7: The second solution tank and the liquid waste container were arranged in the suction-filtration container, and the discharge port of the suction-filtration container and the pressurization / depressurization pump were coupled together with the silicone tube. Afterwards, suction was carried out by the pressurization / depressurization pump so that the vacuum degree in the suction-filtration container was increased (i.e., the suction-filtration container was depressurized) and thus, negative pressure was applied to the membrane filter (i.e., the bacteria trapping filter) and the filtrate was separated.

[0126] Step 8: 3 mL of PBS solution was added to the second solution reservoir.

[0127] Step 9: 3 mL of PBS solution was added to the second solution reservoir.

[0128] Step 10: 2 mL of TE solution was added to the second solution reservoir.

[0129] Step 11: 2 mL of TE solution was added to the second solution reservoir.

[0130] Step 12: 2 mL of TE solution was added to the second solution reservoir.

[0131] Step 13: Depressurization was stopped after the filtrate was separated.

[0132] It should be noted that steps 8 to 12 were carried out while the depressurization by the pressurization / depressurization pump was continued.

[0133] Here, it has been observed that the pressurization time differs depending on the bacteria and suspended matter contained in the sample used. If the sample used is a clean sample close to pure water, it will take ten seconds to filter 3 mL of the solution, for example. Whereas if the sample used contains a large amount of suspended matter, such as feces, it may take about 2 hours to filter 3 mL of the solution. Therefore, the time (i.e., the depressurization time) for carrying out a filtration process can be appropriately adjusted according to the condition and amount of the added sample.In addition, for the purpose of cleaning the membrane filter and the second solution tank, a cleaning solution is added into the second solution tank in the next step after the solution added into the second solution tank has sufficiently passed through it. Spec. fically, cleaning is continued until the amount of solution remaining in the membrane filter becomes less than 200 pL per cleaning. The number of times cleaning should be performed as well as the amount of solution to remain in the membrane filter can be set appropriately by the user.

[0134] <Récupération de la suspension de corps bactérien>

[0135] Bacterial cells were recovered by the following procedures.

[0136] Step 14: 800 pL of a TE solution was added to the second solution reservoir.

[0137] Step 15: A micropipette was used, its volume set at 600 pL, and the bacterial cells and TE solution were pipetted up and down 10 times into the second solution reservoir. As a result, a bacterial body suspension was obtained.

[0138] Step 16: The bacterial body suspension was recovered in the recovery container.

[0139] <Test d’extraction d’ADN>

[0140] Genomic DNA was extracted from the bacterial body suspension thus obtained in accordance with the method described in Non-Patent Literature 2 (i.e., the method using enzymes), and was then subjected to electrophoresis on a 0.8% agarose gel.

[0141] [Fig.5] illustrates the electrophoresis results of DNA extracted from the microbiota. A lane M is a size marker serving as an index of the baseline. Lanes 1 to 6 correspond respectively to tests no. 1 to 6.

[0142] As illustrated in [Fig.5], it was found that long-chain DNA with a size greater than 30 kbp could be extracted in each test.

[0143] <Mesure des proportions d’espèces bactériennes dans la suspension de corps bactérien>

[0144] The DNA from the microbiota extracted in the aforementioned extraction test was subjected to quantitative PCR using a QuantStudio (registered trademark) manufactured by Thermo Fisher Scientific KK so that the proportions of bacterial species contained in the DNA solution were calculated. As a reference example, the steps from residue removal to microbiota recovery were performed on the fecal model sample produced in Example 1 in accordance with the method described in Non-Patent Literature 2 instead of using the microbiota trapping device. Thus, quantitative PCR was performed thereon using a QuantStudio (registered trademark) manufactured by Thermo Fisher Scientific KK

[0145] FIGS. 6A and 6B illustrate the results of quantitative PCR performed on DNA from the microbiota. [Fig.6A] illustrates the results of microbiota recovery using the microbiota trapping device of Example 1 and DNA extraction of the microbiota, and then performing a quantitative PCR on it. [Fig.6B] illustrates the results of recovering the microbiota according to the method described in Non-Patent Literature 2 and extracting DNA from the microbiota, and then performing a quantitative PCR on it. In each of [Fig.6A] and 6B, the results of the six tests (test no. 1 to 6) are illustrated in a graph.

[0146] As illustrated in [Fig.6A], it is confirmed that according to Example 1, the microbiota could be recovered regardless of the bacterial species included therein. In addition, the comparison of [Fig.6A] and 6B can confirm that the result of the quantitative PCR of Example 1 is very similar to that of the reference example. Thus, it is recognized that using the microbiota trapping device of Example 1, the microbiota can be recovered to approximately the same extent as with the conventional microbiota trapping method which involves removing residue through a filter and performing centrifugation.

[0147] [Variation]

[0148] The present disclosure is not limited to the aforementioned embodiments and includes a variety of variations. For example, although the aforementioned embodiments have been described in detail to clearly illustrate the present disclosure, the present disclosure does not include all configurations described in the embodiments. It is possible to replace a portion of a configuration of one embodiment with a configuration of another embodiment. In addition, it is also possible to add, to a configuration of one embodiment, a configuration of another embodiment. Furthermore, it is also possible, for a portion of a configuration of each embodiment, to add, eliminate, or substitute a configuration of another embodiment. List of reference signs

[0149] 1 to 3 Microbiota trapping device

[0150] 100 Control device

[0151] 101 First solution tank

[0152] 102 Residue Removal Filter

[0153] 103 Second solution tank

[0154] 104 Bacteria trapping filter

[0155] 105 Container for liquid waste

[0156] 106 Pressure vessel

[0157] 107 Pressure pump

[0158] 108 Tube

[0159] 109 Support

[0160]

[0161]

[0162]

[0163]

[0164] 110 Spacing 111 Pressure Vessel 112 Discharge Port 113 Depressurization Pump 114 Tube

Claims

Claims

1. A microbiota trapping device (1; 2; 3; 4) comprising: - a first solution reservoir (101) comprising a first filter (102) designed to trap a residue in a sample; and - a second solution reservoir (103) comprising a second filter (104) designed to trap bacteria in the sample, the second solution reservoir (103) having a vent above the second filter (104).

2. A microbiota trapping device (1; 2; 3; 4) according to claim 1, a pore size of the first filter (102) being greater than or equal to 70 pm, and a pore size of the second filter (104) being less than or equal to 1 pm.

3. A microbiota trapping device (1; 2; 3; 4) according to claim 1, further comprising a support (109) which supports the first solution reservoir (101) above the second solution reservoir (103), the support (109) being adapted to support the first solution reservoir (101) such that a gap (110) is formed between the first solution reservoir (101) and the second solution reservoir (103) and an opening of the second solution reservoir (103) serves as a vent.

4. A microbiota trapping device (1; 2; 3; 4) according to claim 1, the second solution reservoir (103) having the vent through a side wall of the second solution reservoir (103).

5. Microbiota trapping apparatus comprising: - the microbiota trapping device (1; 2; 3; 4) according to claim 1; - a pressure applicator (106; 111) configured to apply a first pressure to the first solution reservoir (101) and apply a second pressure to the second solution reservoir (103); and - a control device (100) configured to control the driving of the pressure applicator (106; 111).

6. A microbiota trapping apparatus according to claim 5, the control device (100) being configured to: - cause the pressure applicator (106) to apply the first pressure to the first solution reservoir (101), so that the first filter (102) traps the residue in the sample and a filtrate containing the bacteria is introduced into the second solution reservoir (103) solution, and - causing the pressure applicator (111) to apply the second pressure to the second solution reservoir (103), so that the second filter (104) traps the bacteria in the filtrate.

7. The microbiota trapping apparatus of claim 5, each of the first pressure and the second pressure being a positive pressure.

8. A microbiota trapping apparatus according to claim 5, the pressure applicator (106; 111) comprising: - a positive pressure generator (107) configured to generate a positive pressure, and - a negative pressure generator (113) configured to generate a negative pressure, the first pressure being the positive pressure generated by the positive pressure generator (107), and the second pressure being the negative pressure generated by the negative pressure generator (113).

9. The microbiota trapping apparatus of claim 6, further comprising: - a dispenser configured to dispense a recovery solution into the second solution reservoir (103); and - a collector configured to recover a solution from the second solution reservoir (103), the control device (100) being configured to: - cause the second filter (104) to trap bacteria in the filtrate and then the dispenser to add the recovery solution into the second solution reservoir (103) to suspend the bacteria in the recovery solution, thereby obtaining a microbiota suspension, and - cause the collector to recover the microbiota suspension.

10. A method of trapping microbiota using a microbiota trapping device (1; 2; 3; 4), the device (1; 2; 3; 4) comprising a first solution reservoir (101) having a first filter (102) adapted to trap a residue in a sample, and a second solution reservoir (103) having a second filter (104) adapted to trap bacteria in the sample, the second solution reservoir (103) also having a vent above the second filter (104), the method comprising: - introducing the sample into the first solution reservoir (101) solution; - applying a first pressure to the first solution reservoir (101), so that the first filter (102) traps the residue in the sample and a filtrate containing the bacteria is introduced into the second solution reservoir (103); and - applying a second pressure to the second solution reservoir (103), so that the second filter (104) traps the bacteria in the filtrate.

11. A method of trapping microbiota according to claim 10, a pore size of the first filter (102) being greater than or equal to 70 pm, and a pore size of the second filter (104) being less than or equal to 1 pm.

12. A method of trapping microbiota according to claim 10, each of the first pressure and the second pressure being a positive pressure.

13. A method of trapping microbiota according to claim 10, the first pressure being a positive pressure and the second pressure being a negative pressure.

14. The microbiota trapping method of claim 10, further comprising adding a recovery solution into the second solution reservoir (103) to suspend the bacteria in the recovery solution, thereby obtaining a microbiota suspension.

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