Microorganism detection system and microorganism detection method

Abstract

Provided is a microorganism detection system by which microorganisms contained in a sample can be accurately detected even when the sample is a biological component containing ATP. This pretreatment kit for sorting microorganisms from a sample in order to measure ATP derived from the microorganisms comprises a separation unit which includes separation particles for separating the microorganisms and components other than the microorganisms, which are contained in the sample.

Description

Microbial detection system and microbial detection method

[0001] This invention relates to a system and method for detecting microorganisms contained in a sample.

[0002] For example, in biopharmaceuticals containing biologically derived components such as antibodies and vaccines, microbial contamination can cause serious health problems. Therefore, it is necessary to take a sample of the product at the time of shipment and rigorously inspect it for microbial contamination.

[0003] One method for accurately detecting whether or not microorganisms are present in a sample is to measure the amount of ATP (adenosine triphosphate) contained in the microorganisms, as described in Patent Document 1.

[0004] International Publication No. 2022 / 239867

[0005] However, since bio-derived components such as proteins and peptides that make up biopharmaceuticals such as antibodies and vaccines may themselves contain ATP, there is a risk that product-derived ATP, which is present in the sample at a much higher concentration than microorganisms, may interfere with the measurement, depending on the microbial detection device and microbial detection method described in Patent Document 1.

[0006] Therefore, the present invention was made to solve the above-mentioned problems, and its main objective is to accurately detect microorganisms contained in a sample, even when the product itself, which is the subject of the investigation to determine whether microorganisms are present in the sample, contains biologically derived components that contain ATP.

[0007] In other words, the pretreatment kit (also called a pretreatment unit) according to the present invention is a pretreatment kit for selecting microorganisms from a sample in order to measure ATP derived from microorganisms, and is characterized by comprising a separation unit containing separation particles that separate some or all of the microorganisms contained in the sample from some or all of the non-microorganism components.

[0008] Such a pretreatment kit for microbial detection includes a separation section containing separation particles that separate microorganisms from non-microbial components in the sample, thus allowing for the removal of non-microbial components from the sample and the extraction of microorganisms. As a result, ATP derived from microorganisms can be detected with high accuracy, and even if components other than microorganisms in the sample contain ATP, the microorganisms in the sample can be detected with high accuracy.

[0009] The separation unit, for example, is equipped with separation particles. Specific examples of these separation particles include those that adsorb and separate the target component through one or more actions selected from the group consisting of cation exchange, hydrophobic interaction, and specific interaction.

[0010] When dealing with antibody drugs or biopharmaceuticals, components other than microorganisms contained in these products may be prone to aggregation. Therefore, it is preferable that the pretreatment unit further includes a removal unit for removing the agglutinating components contained in the sample.

[0011] Since the concentration of ATP derived from microorganisms in the sample is very low, it is preferable that the pretreatment unit further includes a concentration unit for concentrating the sample. Furthermore, in order to remove as many non-microorganism components as possible in the concentration process by the concentration unit, it is even more preferable that the concentration unit concentrates the sample by filtering after adding a refolding agent that refolds non-microorganism components such as proteins.

[0012] Specific examples of components other than the aforementioned microorganisms include one or more selected from the group consisting of proteins, peptides, lipids, polysaccharides, nucleic acids, organelles, and metabolites.

[0013] A microorganism detection system comprising a pre-processing unit for selecting microorganisms from a sample, and a measuring unit for measuring the amount of ATP derived from the microorganisms selected by the pre-processing unit, wherein the pre-processing unit includes a separation unit containing separation particles for separating microorganisms from non-microorganism components contained in the sample, is also one embodiment of the present invention.

[0014] A specific embodiment of the microbial detection method according to the present invention is one in which microorganisms are selected using one or more liquid chromatography methods selected from the group consisting of cation exchange chromatography, hydrophobic chromatography, and affinity chromatography.

[0015] It is preferable to further include a step of removing components other than the microorganisms by filtering after adding a diluent to the sample. Alternatively, the process may further include a step of removing components other than the microorganisms by filtering after adding a refolding agent that refolds proteins and other components other than the microorganisms to the sample.

[0016] According to the present invention configured in this way, even if the product to be investigated for the presence of microorganisms contains biological components that include ATP, it is possible to accurately detect microorganisms contained in a sample taken from this product.

[0017] This is a schematic diagram showing the overall configuration of a microbial detection device according to one embodiment of the present invention. This is a schematic diagram showing the structure of the measuring unit of the microbial detection device of this embodiment. This is a schematic diagram showing the external appearance of the microbial detection device of this embodiment. This is a schematic diagram showing the measurement procedure in the measuring unit of this embodiment. This is a flowchart showing the pre-processing unit in the microbial detection device of this embodiment. This is a schematic diagram showing the concentration unit in the microbial detection device of this embodiment. This is a schematic diagram showing the concentration unit in the microbial detection device of this embodiment. This is a flowchart showing the pre-processing unit in the microbial detection device of this embodiment. This is a flowchart showing the measurement procedure according to another embodiment of the present invention.

[0018] Hereinafter, one embodiment of the microbial detection device according to the present invention will be described with reference to the drawings.

[0019] <Device Configuration> The microorganism detection system (also called a microorganism measuring device) 100 of this embodiment analyzes the amount of ATP (adenosine triphosphate) derived from microorganisms (amol (= 10) by analyzing the light produced by ATP derived from microorganisms contained in the sample. －１８This method detects whether a sample contains microorganisms by measuring the mol (mol) of the sample. The microorganisms to be detected can be one or more species selected from the group consisting of, for example, protozoa, algae, lichens, fungi, bacteria, archaea, molds, yeasts, bacteria, and commensal bacteria. The commensal bacteria can be one or more species selected from the group consisting of, for example, Streptococcus, Micrococcus, Propionibacterium, and Kocuria.

[0020] As shown in Figure 1, for example, this microbial detection device 100 includes a pre-processing unit 1 for pre-treating a sample, a measuring unit 2 for measuring the amount of ATP in the treated liquid processed by the pre-processing unit 1, a control unit 3 for controlling the pre-processing unit 1 and the measuring unit 2, a calculation unit 4 for calculating the amount of ATP based on the output signal from the measuring unit 2, and a display unit 6 for displaying the amount of ATP calculated by the calculation unit 4. In this embodiment, as an example of the control unit 3, it includes a pre-processing control unit 31 for controlling the pre-processing unit and a measurement control unit 32 for controlling the measuring unit, but it is not limited to this.

[0021] These control units 3 and calculation units 4, etc., are performed by a computer COM consisting of, for example, a CPU, memory, input / output interface, AD converter, etc. The display unit 6 may be the one provided in the aforementioned computer COM, or a separately provided display or the like may be used. In Figure 1, the system is shown to have a COM 1 that functions as a pre-processing control unit 31, a COM 2 that functions as a measurement control unit 32 and a calculation unit 4, etc., and only one display unit 6, but it is not limited to this configuration.

[0022] The pre-processing unit 1 is a characteristic configuration of the present invention and will be described in detail later.

[0023] The measuring unit 2 is a so-called ATP meter, and as shown in Figure 2, for example, it includes a holder 23 that holds a plurality of containers 22 for containing a sample, a photodetector 24 fixed in a predetermined position, a holder driving mechanism 25 that moves the holder 23, and a dispensing mechanism 26 that dispenses a luminescent reagent that reacts with ATP to produce light into the containers 22 held by the holder 23.

[0024] As shown in Figure 3, the measuring unit 2 according to this embodiment includes a housing C having a door C2 for inserting and removing the holder 23. The housing C comprises a housing body C1 that houses measuring system equipment necessary for ATP measurement, such as the holder 23, the holder drive mechanism 25, and the dispensing mechanism 26, and an openable and closable door C2 provided on the housing body C1. By opening the door C2, for example, by lifting it upward, the user can access the inside of the housing body C1. By closing the door C2, the inside of the device becomes a darkroom.

[0025] In addition, the housing body C1 is provided with a temperature control mechanism 27 for holding and controlling the temperature of multiple sample tubes FC containing processed liquid processed by the pre-processing unit 1, as shown in Figures 2 and 3; a reagent set section 28 for setting reagent containers RC1 and RC2 containing each reagent; and a pipette tip set section 29 for providing pipette tips PT used in the dispensing mechanism 26.

[0026] The reagent set unit 28 contains a reagent container RC1 containing reaction reagents for extracting ATP from the treated solution pre-treated by the pre-treatment unit 1, and a reagent container RC2 containing a luminescent reagent. The reaction reagents include an ATP elimination solution (for example, a solution containing ATP-degrading enzymes) that eliminates ATP other than that from living microorganisms (live bacteria) in the treated solution, a spore reaction solution that causes bacteria in spore state to germinate, and an ATP extraction solution that extracts ATP from living cells.

[0027] The holder 23 holds, for example, multiple containers 22 in an annular shape, and specifically holds them on the same circle with respect to a predetermined center of rotation. The containers 22 are made of resin and have a bottomed cylindrical shape, and in this embodiment, they are made of resin and have a bottomed circular tubular shape.

[0028] As shown in Figure 2, the photodetector 24 detects light emitted from the sample in the container 22 held in the holder 23, and is, for example, a photomultiplier tube (PMT). The photodetector 24 is located below the container 22 held in the holder 23. Above the photodetector 24, an optical system is provided which has a reflector 211 for guiding the light emitted from the sample in the container 22 to the photodetector 24.

[0029] The holder drive mechanism 25 moves the holder 23, and the photodetector 24 detects each container 22 held in the holder 23 at position X ｄｅｔ The components are positioned sequentially. Specifically, the holder drive mechanism 25 rotates the holder 23 around the predetermined rotation center, and as shown in Figure 2, it comprises a mounting base 251 on which the holder 23 is installed, a rotation shaft 252 for rotating the holder 23 installed on the mounting base 251, and an actuator 253 for rotating the rotation shaft 252. In addition, the holder drive mechanism 25 is provided with a rotation position sensor (not shown) for detecting the rotation position of the holder 23. Based on the detection signal from this rotation position sensor, the actuator 253 sets the container 22 to be measured by the control unit 3 to the detection position X ｄｅｔ The rotation is controlled to position the object at a predetermined location. The rotation position sensor detects a landmark position such as the origin, and other positions may be managed by the number of pulses of the motor, which is the actuator 253. This configuration is preferable because it simplifies the configuration of the rotation position sensor.

[0030] As shown in Figures 2 and 3, the dispensing mechanism 26 includes a nozzle 261 for aspirating or dispensing samples and reagents, a pump mechanism 262, such as a syringe, that drives the aspiration or dispensing of the nozzle 261 via a flow path connected to the nozzle 261, and a nozzle moving mechanism 263 that moves the nozzle 261 in a predetermined direction.

[0031] The nozzle 261 is equipped with a tip holder 261H that detachably holds a pipette tip PT for contacting and holding samples and reagents. The tip holder 261H has an internal channel formed therein, with the channel connected to its base end and the pipette tip PT connected to its tip opening.

[0032] Furthermore, the nozzle movement mechanism 263 moves the nozzle 261 linearly in the horizontal direction (X-axis and Y-axis direction) and also moves the nozzle 261 linearly in the vertical direction (Z-axis direction). This nozzle movement mechanism 263 is controlled by the control unit 3 described above.

[0033] The pipette tip PT used for dispensing is removed above the waste box 210 of the holder 23. The waste box 210 is, for example, integrally provided with the holder 23, and in this embodiment, it is provided inside the multiple containers 22 that become dead space in the holder 23. Specifically, the pipette tip PT may be removed by moving the nozzle 261 to a tip removal member (not shown) located above the waste box 210, or a tip removal member may be provided on the movable member 631, and the tip removal may be performed using the tip removal member after moving the movable member 631 above the waste box 210.

[0034] Furthermore, as shown in Figure 2, the measurement unit 2 detects position X ｄｅｔ The system may also be further equipped with a light-shielding mechanism 213 that guides light emitted from the sample in one of the containers 22 to the photodetector 24, while preventing light emitted from the sample in other containers 22 (specifically, the container 22 after measurement has been completed) from being guided to the photodetector 24.

[0035] <Analysis Method> Next, we will explain the operation of the microbial detection device 100 configured as described above, along with the method for detecting microorganisms.

[0036] The sample tube FC containing the processed solution preprocessed by the pretreatment unit is set in the temperature control mechanism 27. With a predetermined number of sample tubes FC set, the door C2 is closed and the measurement is started. In this state, each container 22 held by the holder 23 is empty, but the container 22 for standard solution measurement contains a standard solution with a known ATP amount.

[0037] When the measurement is started, the control unit 3 controls the dispensing mechanism 26 to dispense each reaction reagent, for example, into each sample tube FC held by the temperature control mechanism 27 according to a predetermined sequence. Thereby, a predetermined process (ATP extraction) is performed on the sample in the sample tube FC. Note that the pipette tip PT is replaced for each reaction reagent, and the used pipette tip PT is discarded in the discard box 210.

[0038] Specifically, a mixed solution of an ATP elimination solution and a spore reaction solution is dispensed into the sample in the sample tube FC, and the sample is waited while being kept at a predetermined temperature until the reaction of each reagent is completed. Thereafter, an ATP extraction solution is dispensed into the sample in the sample tube FC, and the sample is waited while being kept at a predetermined temperature until the extraction of ATP is completed. Note that instead of the mixed solution of the ATP elimination solution and the spore reaction solution, the ATP elimination solution and the spore reaction solution may be dispensed separately. For example, as shown in FIG. 4, if an ATP solution and a spore reaction solution are added to the processed solution, and then ATP is extracted with an ATP extraction solution and a luminescent reagent is added for measurement, it is possible to measure the amount of ATP derived from the entire living microorganisms including the microorganisms in the spore state. Also, if the amount of ATP extracted from a control sample to which the spore reaction solution is not added at this time is measured, it is also possible to calculate the amount of ATP derived only from the microorganisms in the spore state.

[0039] Regarding the calibration solution, during the waiting time after the reagent dispensing to the above sample, each reaction reagent is dispensed into a standard solution with a known ATP amount and a zero solution with an ATP amount of zero according to a predetermined sequence. Specifically, after dispensing an ATP elimination solution and a spore reaction solution into the container for standard solution measurement and the container for blank measurement, respectively, an ATP extraction solution is dispensed, and then a standard solution or a zero solution is dispensed. Note that the dispensing order of each reaction reagent to the zero solution is not limited to the above.

[0040] At this time, for example, the mixing ratios of the sample, the ATP elimination solution, the spore reaction solution, and the ATP extraction solution, the mixing ratios of the standard solution, the ATP elimination solution, the spore reaction solution, and the ATP extraction solution, and the mixing ratios of the zero solution, the ATP elimination solution, the spore reaction solution, and the ATP extraction solution are made the same. Specifically, their mixing ratios are made to be a predetermined value. By making the liquid volumes of each reagent the same among the sample, the standard solution, and the zero solution in this way, the pH in the liquid can be made the same. As a result, the preconditions of the liquid before dispensing the luminescent reagent can be made the same, and accurate light intensity can be detected.

[0041] Then, the dispensing mechanism 26 separately collects the pretreated samples in each specimen tube FC into each container 22 held by the holder 23.

[0042] And the control unit 3 controls the holder drive mechanism 25 to move the container 22 to be measured to the detection position X ｄｅｔ After moving the container 22 to be measured to the detection position X ｄｅｔ After moving the container 22 to be measured to the detection position X, the control unit 3 controls the dispensing mechanism 26 to introduce the luminescent reagent into the container 22 at the detection position X ｄｅｔ Thereby, luminescence measurement is performed by detecting the light emitted from the sample in the container 22 at the detection position X by the photodetector 24. Before the luminescence measurement of each container 22, blank measurement and standard solution measurement are performed, and zero point calibration and span calibration are performed. ｄｅｔ The light intensity signal obtained by the photodetector 24 is subjected to arithmetic processing by the calculation unit 4 to calculate the amount of ATP (amol).

[0043] Specifically, this calculation unit 4 subtracts the "light intensity signal obtained before adding the luminescent reagent" from the "light intensity signal obtained after adding the luminescent reagent" to the sample to remove the afterglow of the container 22 and calculate a value related to the amount of ATP.

[0044] Specifically, this calculation unit 4 subtracts the "light intensity signal obtained before adding the luminescent reagent" from the "light intensity signal obtained after adding the luminescent reagent" to the sample to remove the afterglow of the container 22 and calculate a value related to the amount of ATP.

[0045] "The light intensity signal obtained after adding the luminescent reagent to the sample" is an integrated average signal that is the average value of the integrated signal from the time when the luminescent reagent is introduced to a predetermined time (for example, several seconds to several tens of seconds). "The light intensity signal obtained before adding the luminescent reagent" is an integrated average signal that is the average value of the integrated signal up to a predetermined time (for example, several seconds to several tens of seconds) before introducing the luminescent reagent. Here, "the light intensity signal obtained before adding the luminescent reagent" is based on the light stored in the container 22. For example, if the container 22 is placed outside the microorganism detection device 100 before the measurement starts, light such as ultraviolet light or fluorescent light may be stored in the container 22. Therefore, the calculation unit 4 subtracts "the second light intensity signal which is only the light intensity signal derived from the container 22" from "the first light intensity signal including the light intensity signal derived from the container 22 and the light intensity signal derived from ATP". Thereby, the microorganism detection device 100 can accurately calculate only the light intensity signal derived from the microorganism. Note that the signal processing in the blank measurement and the standard solution measurement is the same. Also, the light intensity signal is not limited to the integrated average signal, and may be simply an integrated signal from the time when the luminescent reagent is introduced to a predetermined time (for example, several seconds to several tens of seconds), or may be a signal subjected to other arithmetic processing.

[0046] In this way, the calculation unit 4 calculates the amount of ATP (amol) for each container 22 by the following formula (Equation 1).

[0047]

[0048] Sample [[ID=?]] signal is the signal obtained in the sample measurement, and is the signal obtained by subtracting "the light intensity signal obtained before adding the luminescent reagent to the sample" from "the light intensity signal obtained after adding the luminescent reagent to the sample". STD signal is the signal obtained in the standard solution measurement, and is the signal obtained by subtracting "the light intensity signal obtained before adding the luminescent reagent to the standard solution" from "the light intensity signal obtained after adding the luminescent reagent to the standard solution". Zero signal It seems there is a formatting issue with the "?". Please check and correct it if needed. Also, I've done my best to translate accurately while following the rules. If there are any specific terms or concepts that need further clarification, feel free to let me know.This signal is obtained from a blank measurement and is the signal obtained by subtracting the "light intensity signal obtained before adding the luminescent reagent to the zero solution" from the "light intensity signal obtained after adding the luminescent reagent to the zero solution." Note that while an emission peak is usually observed when the luminescent reagent is added during a blank measurement, it may not always be observed.

[0049] The above calculations eliminate variations in the amount of light detected in each of the containers 22 for measuring the standard solution, the container for measuring the blank, and the container for measuring the sample, allowing for an accurate calculation of the amount of ATP.

[0050] The user determines whether or not microorganisms are present in the sample based on the amount of ATP calculated as described above. Specifically, for example, a predetermined threshold may be set for the ATP concentration in the sample, which is determined from the concentration ratio of the sample in the pretreatment process and the amount of ATP calculated. If the ATP concentration in the sample exceeds this threshold, it may be determined that microorganisms are present.

[0051] After the light emission measurement of one container 22 is completed, the control unit 3 controls the holder drive mechanism 25 to detect the next container 22 to be measured at the detection position X ｄｅｔ The samples are then moved to the next container. In this manner, the luminescence measurement of the samples in each container 22 is performed sequentially. Here, the pipette tip PT is replaced after each luminescence measurement of the sample in each container 22, and the used pipette tip PT is discarded in the waste box 210. Note that when the risk of contamination is small, such as when dispensing the same reagent continuously, the pipette tip PT may be used without being replaced each time.

[0052] After all samples have been measured, the door C2 is opened and the sample tube FC held in the temperature control mechanism 27 is replaced, and the container 22 held in the holder 23 is replaced. When replacing the container 22 held in the holder 23, the holder 23 is removed from the main body of the device by holding the holding hole 3h of the holder 23. Since the used and discarded pipette tips PT are placed in the waste box 210 of the holder 23, the discarded pipette tips PT can also be removed from the main body of the device at the same time as the holder 23 is removed from the main body of the device. Alternatively, the measurement unit may automatically perform the replacement of the sample tube FC and container 22 according to a pre-programmed schedule.

[0053] <Features> The pre-processing unit 1, as shown in Figure 5, for example, selects microorganisms to be detected from a sample obtained by sampling a part of the product, and obtains a concentrated liquid (processed liquid) by concentrating the selected microorganisms. The pre-processing unit 1 includes, for example, a separation unit 11 that separates the microorganisms contained in the sample from at least some of the non-microorganism components that hinder the detection of microorganisms, and the microorganisms are selected by this separation unit 11. In this embodiment, microorganisms refer to, for example, live bacteria, dead bacteria, and ATP derived from microorganisms, and non-microorganism components refer to one or more types of components other than these. Examples of non-microorganism components include, for example, proteins, peptides, lipids, polysaccharides, nucleic acids, etc. that constitute products such as antibody drugs, biopharmaceuticals, supplements, and foods, or components in the manufacturing process of these products, but are not limited to these, and may also include, for example, extracellular organelles such as exosomes and liposomes, and metabolites such as polyphenols.

[0054] The separation unit 11 is a liquid chromatograph equipped with a separation column containing separation particles formed from one or more materials selected from the group consisting of, for example, resin, magnetic material, glass, metal, and ceramic. The liquid chromatograph is equipped with, for example, the aforementioned separation column and a liquid flow mechanism for supplying a sample or other solvent to the separation column.

[0055] The separation column is preferably an adsorption column that adsorbs components other than microorganisms by bringing the sample directly into contact with the adsorbed particles, and does not adsorb microorganisms. Examples of such adsorption columns include those that adsorb and remove components other than microorganisms in the sample using various chromatography methods such as cation exchange chromatography, hydrophobic chromatography (reverse-phase chromatography), and affinity chromatography.

[0056] The adsorption particles packed into this adsorption column can be those that adsorb the target component through cation exchange, hydrophobic interaction, specific interaction, etc. Examples of such adsorption columns include cation exchange columns, hydrophobic columns (reverse-phase columns), and affinity columns. The adsorption column may contain one type of adsorption particle or multiple types of adsorption particles. The separation unit 11 may have one adsorption column or two or more. The separation column is not limited to those that separate components by adsorption as described here; it may also use separation particles that separate components by size exclusion, such as gel filtration columns.

[0057] The liquid flow mechanism is not particularly limited, and can be broadly adapted to those found in general liquid chromatography systems. For example, it may include a supply channel for supplying a sample or other solvent to the adsorption column, an outlet channel for discharging the liquid from the adsorption column, and a flow rate control unit provided on the supply channel or outlet channel to control the liquid flow. This flow rate control unit may include, for example, a valve or pump arranged on the supply channel or outlet channel, and a flow control unit that controls the operation of these valves or pumps. The function of this flow control unit may be performed by the pre-treatment control unit 31 described above.

[0058] In addition to the configuration described above, the liquid chromatography system may further include a switching unit to switch the connection destination of the supply channel and the discharge channel as needed. Furthermore, it may further include a sampling unit to collect the extract supplied from the discharge channel into, for example, a sample tube FC or a bottle BT.

[0059] Furthermore, the pre-processing unit 1 may also include a concentration unit 12 that concentrates the microorganisms selected as described above by filtering them using a filter or tracked membrane that does not allow the microorganisms to pass through but allows components other than the microorganisms to pass through. In this specification, concentration means increasing the concentration of microorganisms by reducing the volume of liquid to a level lower than the volume of liquid immediately before concentration. For example, if the sample is diluted before concentration, this includes concentrating the sample to a volume that is less than the volume of the diluted sample but greater than the volume of liquid before dilution.

[0060] The concentration unit 12, as shown in Figure 6, for example, comprises a bottle BT that stores a large volume of sample, a cartridge (sample tube FC) that is detachably attached to the lower end opening of the bottle BT and has a filter FC1 with fine holes formed inside that are too small to allow microorganisms to pass through, and a pump (not shown) that sucks the liquid inside the sample tube FC from the lower end. The upper end of the cartridge is inserted into the lower end opening of the bottle BT, and the liquid inside the sample tube FC is sucked out from the lower end of the sample tube FC with the pump or the like, thereby concentrating the sample on the filter FC1 of the sample tube FC. The operation of the pump and the like may be controlled by the pre-processing control unit 31 described above.

[0061] Since the filter FC1 allows components other than microorganisms to pass through, virtually only microbial cells become concentrated on the filter FC1.

[0062] The aforementioned sample tube FC, as shown in Figure 7, for example, comprises a cylindrical tube body FC2 with open ends and a filter cassette FC3 positioned in contact with the inner circumferential surface of the tube body FC2, with the aforementioned filter FC1 positioned in the middle of the tube body FC2 in a direction perpendicular to the axial direction of the tube body FC2. When this filter cassette FC3 is attached to the inner circumferential surface of the tube body FC2, a small gap S may be formed between the filter cassette FC3 and the inner circumferential surface of the tube body FC2. If a sample is supplied into the sample tube FC with this gap S formed, and part of the sample enters this gap S, accurate measurement will be hindered if the sample contains components that affect luminescence measurement.

[0063] Therefore, before supplying the sample to the sample tube FC, it is preferable to circulate a buffer solution or distilled water that does not affect ATP measurement, so that the buffer solution or distilled water can pre-fill the gap S formed between the filter cassette FC3 and the tube body FC2. To make it easier for these liquids to enter the aforementioned gap, surfactants or other agents may be added to the buffer solution or distilled water.

[0064] If the sample contains biological components other than microorganisms, such as proteins and peptides, these biological components may be aggregated. In such cases, filtering as described above may concentrate not only the microorganisms but also the biological components that have increased in size due to aggregation.

[0065] Therefore, for example, as shown in Figure 8, the preprocessing unit 1 may further include an aggregated component removal unit 13. This aggregated component removal unit 13 may include, for example, an aggregation promoting unit that promotes the aggregation of biological components other than microorganisms in the sample, and a removal unit that removes biological components other than microorganisms whose aggregation has been promoted by the aggregation promoting unit.

[0066] Bio-derived components such as proteins and peptides have the property of becoming more easily aggregated when the ion concentration of the solvent is reduced. Therefore, the aggregation-promoting unit 131 may add a predetermined amount of a diluent with a low ion concentration, such as pure water (preferably Water for Injection, abbreviated as WFI), to the sample. Known methods may be used for adding the diluent. Furthermore, the amount of diluent to be added may be calculated and automatically added by the pre-treatment control unit 31 described above, or the user may manually calculate the required amount of diluent and add it manually.

[0067] The removal unit separates aggregated biological components (aggregated components) from microorganisms by filtering or other means. For example, it is equipped with a filter that has a pore size that allows components larger than microorganisms to pass through without permeation, while allowing microorganisms to pass through.

[0068] The aggregated component removal unit 13 may be located upstream of the separation unit described above, and the sample that has passed through the aggregated component removal unit 13 may be supplied to the separation unit, for example, via the supply channel described above. The aggregated component removal unit 13 may consist of an aggregation promotion unit, which is a diluent adding means for adding a predetermined amount of diluent to the bottle BT provided in the concentration unit 12, and a removal unit configured by replacing the filter FC1 of the filter cassette FC3 described above with an appropriate one.

[0069] Furthermore, if the sample contains proteins or peptides as biological components other than microorganisms, it is thought that by refolding these proteins and peptides, they can pass smoothly through the filter in their original compact three-dimensional structure during filtering in the aforementioned concentration section.

[0070] Therefore, for example, as shown in Figure 8, the pre-processing unit may further include a refolding unit 14. The refolding unit 14, for example, adds a refolding agent to the sample. As the refolding agent, a wide range of substances known to be able to refold proteins and peptides can be used. Specific examples of such refolding agents include one or more selected from the group consisting of arginine, surfactants, ethylene glycol, etc.

[0071] The refolding section 14 may be provided, for example, downstream of the separation section 11, and configured to receive the sample through the discharge channel of the separation section 11. The refolding section 14 may also be equipped with an additive means for adding a predetermined amount of refolding agent to the bottle BT provided in the concentration section 12 via a refolding agent addition channel or the like.

[0072] <Pretreatment Method> The following are examples of methods and processes for selecting microorganisms to be detected from a sample using the pretreatment unit 1 configured as described above. First, a portion of the product is taken as a sample and supplied to the pretreatment unit 1. The sample supplied to the pretreatment unit 1 is first sent to the agglutination component removal unit 13, where the agglutination of components other than microorganisms (for example, biological components such as proteins and peptides) is promoted by the agglutination promotion unit, and then the agglutination components are removed by the removal unit.

[0073] Next, the sample that has passed through the filter in the removal section is supplied to the adsorption column in the separation section 11. The sample supplied to the separation section 11 is supplied to the adsorption column by the liquid flow mechanism. As the mobile phase used in this adsorption step, for example, pure water (preferably WFI) or buffer solution can be widely used, but it is preferable to use a solution with a pH of 5 to 8 or less, which is near neutral, and a mild condition with a salt concentration of about 0.01 M to 1 M.

[0074] In this separation unit 11, depending on the properties of components other than microorganisms contained in the sample, one chromatography using one type of adsorbed particle may be performed once for the pretreatment of one sample, or if components other than microorganisms cannot be separated in one step, chromatography may be performed multiple times. Alternatively, two or more chromatography methods using two or more types of adsorbed particles may be combined.

[0075] Of the samples supplied to the separation unit 11, those samples that eluted without being adsorbed by the adsorption particles packed in the adsorption column are recovered as a fraction containing microorganisms. At this time, it is preferable to supply WFI or a buffer solution to the adsorption column so that all of the sample that has not been adsorbed by the adsorption particles is drawn out and recovered from the adsorption column. More specifically, after supplying the sample to the adsorption column, a pressure application unit may be provided to apply pressure to the liquid in the adsorption column in order to draw out and recover all of the liquid containing microorganisms that were not adsorbed by the adsorption particles from the adsorption column. This pressure application unit applies pressure, such as pressing or pulling pressure, to the liquid in the adsorption column, thereby drawing out all of the liquid containing microorganisms that were not adsorbed by the adsorption particles from the adsorption column. The function of the pressure application unit may be performed, for example, by a pump provided in the liquid flow mechanism described above.

[0076] The sample extracted from the adsorption column is sent to the refolding unit 14, where proteins and peptides in the sample are refolded by the addition of a refolding agent. Subsequently, the sample containing microorganisms is concentrated in the concentration unit 12. In order to remove as many components other than microbial cells as possible, the concentration unit 12 may also wash the concentrate by adding distilled water or an appropriate buffer solution onto the filter FC1. This washing process may be performed by the concentration unit or by the user manually.

[0077] <Effects of this embodiment> With the microorganism detection device 100 configured in this embodiment, the amount of ATP contained in the treated liquid obtained by concentrating the microorganisms to be detected after selecting them from the sample is measured, so the amount of ATP derived from microorganisms can be measured with high accuracy. As a result, even if the sample contains biological components that contain ATP, such as proteins and peptides, as components other than microorganisms, microorganisms in the sample can be detected with high accuracy.

[0078] Since the separation unit selects microorganisms by adsorbing components other than microorganisms in the sample, it eliminates the need to separately prepare buffer solutions for eluting microorganisms from the adsorption column, compared to the method of selecting microorganisms by adsorbing them to the separation unit and then eluting them.

[0079] Since the separation unit separates microorganisms by adsorbing components other than microorganisms in the sample, compared to the method of separating microorganisms by adsorbing them to the separation unit and then eluting them, the opportunity for microorganisms to be exposed to solvents with altered pH and salt concentration is reduced, thereby suppressing the destruction of microorganisms.

[0080] Since the separation unit selects microorganisms by adsorbing components other than microorganisms in the sample, it is possible to minimize microbial loss and detect microorganisms contained in the sample with greater accuracy compared to cases where microorganisms are adsorbed into the separation unit.

[0081] Since the pre-treatment unit concentrates the microorganisms in the sample, steps such as culturing the microorganisms or amplifying ATP extracted from the microorganisms are unnecessary, allowing for even faster detection of microorganisms than before.

[0082] <Other Embodiments> The present invention is not limited to the embodiments described above. For example, the separation unit may adsorb components other than microorganisms, such as dead microorganisms or components derived from microorganisms, as opposed to live microorganisms.

[0083] For example, when producing biopharmaceuticals containing biologically derived components such as antibodies and vaccines using animal cells, extracellular vesicles secreted from animal cells may be included as components other than the microorganisms mentioned above. These extracellular vesicles contain ATP and may also be similar in size to microorganisms. Therefore, the inventors believe that they may slip through the selection process in the aforementioned pretreatment steps and contribute to background noise during ATP measurement.

[0084] Therefore, the separation unit 11 described above may further have a function to remove extracellular vesicles. Specific methods for removing extracellular vesicles using the separation unit 11 include, for example, the following: For example, an extracellular vesicle-binding factor that binds to extracellular vesicles is added to the sample and mixed, causing the extracellular vesicles and the extracellular vesicle-binding factor to bind and form a complex containing the extracellular vesicles. Next, this complex is subjected to a column packed with adsorbent particles that bind to the extracellular vesicle-binding factor, trapping the complex within the column. The sample that passes through the column is then collected to remove the extracellular vesicles.

[0085] Alternatively, the sample may be added to adsorbed particles to which an extracellular vesicle binding factor has been pre-bound, thereby binding the extracellular vesicles to the adsorbed particles and removing them. Furthermore, if the extracellular vesicle binding factor is agglutinating, such as an antibody, the extracellular vesicle binding factor and the sample may be mixed and aggregated, and then the extracellular vesicles may be precipitated and removed using a spin column or the like.

[0086] Preferably, the extracellular vesicle binding factor is capable of binding to both extracellular vesicles and adsorbed particles. For example, it is possible to use lectins that recognize sugar chains present on the surface of extracellular vesicles, antibodies that recognize and bind to surface antigens present on the surface of extracellular vesicles, or natural or synthetic structures among these proteins and peptides that have only the binding site that recognizes and binds to extracellular vesicles. These extracellular vesicle binding factors are not necessarily limited to those with high binding specificity, such as those used in affinity chromatography.

[0087] If the amount of microorganisms obtained by the pretreatment step for microbial selection is very small, making detection by subsequent ATP measurement difficult, the pretreatment step may further include a microbial culture step for culturing the target microorganisms, as shown in Figure 9, for example. Specifically, it is conceivable to add the components necessary for culturing microorganisms to the sample and culture the microorganisms for a range such as 1 hour to 24 hours, 3 hours to 20 hours, or 6 hours to 12 hours. However, since many of the microbial culture components added to cultivate microorganisms are derived from living organisms, these microbial culture components may also contain ATP that can become background noise during the ATP measurement step for detecting microorganisms.

[0088] Therefore, the microbial culture process is preferably performed on the sample before it is subjected to the separation unit 11, and more preferably on the sample before it is subjected to the agglutination component removal unit 13, so that the microbial culture components can be efficiently removed after culture.

[0089] To minimize contamination with ATP derived from microbial culture components added for culturing microorganisms, an ATP elimination solution may be added to the sample during or after microbial culture to eliminate ATP present outside the microorganisms. Then, a washing step may be performed to replace the solvent other than the microorganisms using a filter similar to the one used in the concentration step described above, before the sample is subjected to subsequent pretreatment or measurement steps.

[0090] Furthermore, in the manufacturing process of biopharmaceuticals, microbial growth inhibitors may be used to suppress the growth of microorganisms. In such cases, the sample before culturing the microorganisms may be subjected to an additional microbial growth inhibitor removal process, such as dialysis or ultrafiltration using a membrane capable of removing only the microbial growth inhibitor, adsorption removal using an ion exchange resin, or enzymatic treatment with an enzyme capable of decomposing the microbial growth inhibitor. These microbial culture and microbial growth inhibitor removal processes may be performed fully automatically according to a pre-set program, provided that the microbial culture unit 15 in the pre-processing unit has an appropriate device configuration, or the user may perform part or all of these processes separately using appropriate tools.

[0091] The microbial sorting and concentration processes performed by the pre-processing unit may be carried out using the bottles and sample tubes provided in the concentration unit, as described above, or they may be carried out in separate tanks or other containers prepared for each process.

[0092] Furthermore, the aforementioned pretreatment process may be partially performed manually by the user, or it may be entirely performed automatically by the pretreatment unit. In particular, although the above-described embodiment described a separation unit equipped with a liquid chromatograph, some of the functions performed by each component other than the adsorption column, such as the liquid flow mechanism, may be performed manually by the user. Alternatively, instead of using an adsorption column packed with adsorbent particles, a batch method may be used in which the sample and adsorbent particles are mixed in a suitable container such as a syringe, and components other than microorganisms in the sample are adsorbed onto the resin. For example, adsorption beads can be used as the adsorbent particles. As the adsorption beads, a wide range of known beads used to adsorb bio-derived substances can be used, and there are no particular limitations on the material or shape. For example, adsorption beads commonly used for the purification of proteins and peptides, which are types of bio-derived substances, can be appropriately selected. With adsorption beads, the target bio-derived substance can be adsorbed and removed by collecting the beads after contact with the sample, so separation can be performed in a short time even if the volume of sample liquid is large. Among these, magnetic beads (beads containing paramagnetic material) that can be easily separated by an external magnetic field are preferred. Magnetic beads enable solid-liquid separation without the need for centrifugation or filter filtration, allowing for faster and more efficient adsorption and removal of biomolecules. Furthermore, using magnetic beads reduces sample movement within and between containers compared to the column method described above. Sample loss during operation is minimized, and detection targets can be obtained with high recovery rates even from minute samples. The surface of the adsorption beads can be modified using surface modifications widely used for biomolecule adsorption. For example, antibodies, avidin, streptavidin, functional groups such as carboxyl groups, amino groups, sulfone groups, or other affinity ligands may be introduced. Using such surface-modified beads allows for non-specific or specific, efficient adsorption of biomolecules simply by bringing them into contact with the bead surface, enabling simple and highly efficient adsorption operations.From the perspective of preventing contamination from adsorption beads, it is even more desirable that the adsorption beads have a material and structure that can withstand sterilization operations such as autoclaving. As an example of the operation when using the aforementioned magnetic beads in the separation unit, for example, magnetic beads can be added to a container containing a sample, mixed to adsorb biological substances onto the magnetic beads, and then a magnet can be brought close to the container to attract the magnetic beads to the container wall, and only the supernatant can be collected to separate and remove biological substances from the sample.

[0093] Furthermore, the pre-processing unit only needs to include a separation unit, and the aforementioned agglutination component removal unit, refolding unit, concentration unit, etc., may be omitted. Also, the order of each step performed in the agglutination component removal unit, separation unit, refolding unit, and concentration unit is not limited to those described above and can be rearranged. For example, the refolding step may be performed after the agglutination component removal step, followed by the adsorption step and concentration step.

[0094] Furthermore, the bacterial species contained in the sample whose ATP has been measured in the measurement unit of the above embodiment may be identified. Specifically, it is conceivable to identify the bacterial species from the DNA or RNA contained in the residual liquid after adding the luminescent reagent (sample after ATP measurement) or the residual liquid in the sample tube FC (sample before ATP measurement). More specifically, the bacterial species can be identified from the residual liquid using a DNA sequencer. In the case of RNA, the DNA sequencer can be used after synthesizing DNA by reverse transcription.

[0095] For analysis using a DNA sequencer, amplification by PCR is a possible method. Here, DNA is collected from the remaining solution, for example, using DNA collection beads, and the collected DNA is amplified by PCR. The remaining solution contains ATP extract.

[0096] ATP extracts can suitably be surfactants, mixtures of ethanol and ammonia, methanol, ethanol, trichloroacetic acid, perchloric acid, Tris buffer, etc. Examples of surfactants include sodium dodecyl sulfate, potassium lauryl sulfate, sodium monolauroyl phosphate, sodium alkylbenzenesulfonate, benzalkonium chloride, benzethonium chloride, cetylpyridinium chloride, cetyltrimethylammonium bromide, and myristyldimethylbenzylammonium chloride. Some ATP extracts inhibit the activity of enzymes that degrade DNA, and if PCR is performed on a sample after ATP measurement, the ATP extract may inactivate the PCR enzymes. Therefore, it is advisable to remove, dilute, neutralize, or mask the ATP extract before PCR.

[0097] It is possible that microorganisms present in a sample may form a biofilm. In some cases, it can be difficult to efficiently extract ATP from microorganisms protected by such biofilms. Therefore, a microbial detection device may be further equipped with a biofilm degradation unit to break down the biofilm in the sample.

[0098] Specific configurations of the biofilm degradation unit include, for example, a filtration device using a track-etched membrane, nylon mesh, etc. Other embodiments of the biofilm degradation unit include, for example, adding a biofilm-degrading enzyme to the aforementioned bottle BT to chemically degrade the biofilm. Alternatively, the biofilm may be degraded by shear stress or the like by moving the sample back and forth in a capillary tube. This biofilm degradation process may be performed by the biofilm degradation unit of the microbial detection device, or it may be performed manually by the user.

[0099] Furthermore, the microbial detection device of the above embodiment can also be used to measure the amount of ATP in spore-forming bacteria. In other words, the amount of ATP in spore-forming bacteria can be measured by subtracting the amount of ATP before the spore-forming bacteria germinate from the amount of ATP after the spore-forming bacteria germinate.

[0100] Specifically, by measuring ATP levels in the treated solution (concentrated solution) without adding the spore reaction solution, or, if the spore reaction solution is added, before the spore-forming bacteria germinate, the ATP level (Y [amol]) of only the bacteria in their normal state (live bacteria) before germination can be measured. Alternatively, by measuring ATP levels after adding the spore reaction solution to the sample and allowing the spore-forming bacteria to germinate, the ATP levels (X [amol]) of both spore-forming bacteria and live bacteria can be measured. Then, the ATP level of the spore-forming bacteria can be calculated from X - Y [amol]. If the X - Y value is large, it indicates that spore-forming bacteria have been generated, and the user can take measures such as cleaning with a spore-killing agent. Furthermore, it is also possible to measure the ATP level of spore-forming bacteria by killing the live bacteria in the sample using a certain method (e.g., the heat shock method), and then inducing germination of spore-forming bacteria by heating or by adding nutrients.

[0101] Furthermore, by similarly adding ATP scavenging solution to the treated solution and by extracting ATP from these solutions with an ATP extractant and then adding a luminescent reagent for measurement, it is possible to eliminate ATP derived from dead microbial cells and free ATP not derived from microorganisms in the treated solution, thereby calculating the amount of ATP derived solely from living microorganisms. In addition, by comparing the results with the case where ATP scavenging solution is not added, it is possible to estimate the ratio of dead to living microorganisms.

[0102] As mentioned above, the user may determine whether or not microorganisms are present in the sample, or the system may further include a determination unit that determines the presence or absence of microorganisms based on the amount of ATP calculated by the calculation unit.

[0103] In addition, for example, the pretreatment kit may not include the separation unit described above, but only a concentration unit. Furthermore, the concentration unit may include a concentration filter for concentrating the sample, a liquid supply unit for supplying the sample to the concentration filter, and a concentration control unit for controlling the amount and timing of the liquid supplied to the concentration filter by the liquid supply unit.

[0104] Furthermore, the concentration control unit may control the liquid supply unit so that the liquid supply unit supplies a single sample to the concentration filter in multiple portions. In addition, the liquid supply unit may, upon command from the concentration control unit, supply a portion of the same sample to the concentration filter, and after the portion of the sample has been concentrated to a predetermined volume, supply a diluent to dilute the concentrated sample to the concentration filter, and then supply another portion of the same sample to the concentration filter. Examples of the diluent include buffer solutions and aqueous solutions containing surfactants.

[0105] The liquid supply unit may, for example, include a concentrated liquid supply channel for supplying samples and diluents, or a liquid supply mechanism equipped with a pump and valves, or it may be a system in which the user repeatedly supplies predetermined amounts of samples and diluents onto a concentration filter at predetermined timings without such a channel.

[0106] The concentration unit or concentration method configured in this manner is preferable because it minimizes clogging of the concentration filter during the concentration of the sample. Furthermore, it goes without saying that the present invention is not limited to the above-described embodiments, and various modifications are possible without departing from its spirit.

[0107] The present invention will be described below with reference to examples, but the present invention is not limited to these examples. Example 1 In this Example 1, we investigated whether the pretreatment method according to the present invention can sufficiently remove ATP derived from non-microorganism components when a sample contains biological components other than microorganisms that contain ATP. First, RNase, a protein known to bind to ATP, was prepared as a biological component containing ATP.

[0108] The sample containing RNase was supplied to a cation exchange column, and the amount of RNase contained in 100 μL of the sample taken out of the cation exchange column was measured using the Bradford method.

[0109] As a result, the amount of RNase in the sample after passing it once through the cation exchange column was approximately 6% by mass of the amount of RNase before it was supplied to the cation exchange column, indicating that RNase could be sufficiently removed. By repeatedly passing the sample through the same cation exchange column two or three times, the amount of RNase was further reduced, and by the third pass, the amount of RNase in the sample reached the detection limit. When the amount of ATP was measured at this time, the average value of the three experiments showed that the amount of RNase in the sample before it was supplied to the cation exchange column was reduced from 1.1 million amol to 13 amol.

[0110] The RNase used as a model protein in this example has the property of agglutinating. Therefore, first, pure water was added to the sample containing RNase to promote agglutination, and then the sample was filtered using a filter with a pore size of 3 μm. A filter with a pore size of 3 μm can capture the agglutinated RNase while allowing microorganisms to pass through.

[0111] Next, arginine, a refolding agent, was added to the sample that had undergone aggregation promotion and filtering (the sample after removal of aggregated components), and the sample was concentrated using a filter with a pore size of 0.4 μm. With a 0.4 μm pore size filter, microorganisms can be collected while the RNase is refolded by the refolding agent, allowing the RNase, which has a compact three-dimensional structure, to pass through and be removed from the sample at the same time as concentration.

[0112] The amount of ATP was measured in both the concentrated sample treated in this way and the untreated RNase sample. The same experiment was performed in a series of four experiments. The result showed that the average ATP content in the untreated RNase sample, which was over 5100 amol, could be reduced to approximately 1600 amol. As this result indicates, it was confirmed that the amount of ATP derived from RNase can be significantly reduced in a concentrated sample after removing the agglutinating components and adding a refolding agent.

[0113] These results suggest that ATP derived from RNase can be almost completely removed by treatment using adsorbed particles, and that RNase can be sufficiently removed by removing agglutinating components and treating with a refolding agent. As a result, even if biological components containing ATP, such as RNase, are present in the sample, it can be reasonably inferred that only ATP derived from microorganisms can be measured by the measurement unit by performing a sorting step using adsorbed particles, and more preferably by removing agglutinating components and treating with a refolding agent.

[0114] In this Example 1, RNase was used as a model and a cation exchange resin was used as the adsorbent particle. However, the method is not limited to this, and can be applied to samples containing antibodies by using, for example, a resin in which Protein A, Protein G, Protein L, etc., which have immunoglobulin binding ability, are immobilized on agarose beads. It is also possible to remove peptides with specific sequences by using an anti-peptide resin that displays antibodies against peptides with specific sequences. Furthermore, in the case of samples containing highly hydrophobic proteins or peptides, it may be possible to remove these proteins, peptides, and other biological components other than microorganisms by using a hydrophobic resin, and accurately measure only the ATP derived from microorganisms.

[0115] Example 2 In Example 2, the effect of removing extracellular vesicles from a sample was investigated. This experiment was performed in a four-part series. First, the culture supernatant from cultured animal cells was collected. 100 μL of adsorbent particles made of cation exchange resin were added to 100 μL of this culture supernatant and mixed. This mixture was injected into a syringe fitted with a filter at the tip and air-flushed to obtain the filtrate. The same procedure was repeated three more times with the obtained filtrate. In each filtrate obtained from the fourth filtration, as confirmed in Example 1, proteins, peptides, and other biological components other than microorganisms could be sufficiently removed. However, a portion of these filtrates was further treated to remove extracellular vesicles by reacting them with TIM4, which recognizes and binds to phosphatidylserine present on the surface of extracellular vesicles. Specifically, each filtrate was concentrated by centrifugation, and the phosphatidylserine (PS) in the cell vesicles contained in each filtrate was specifically bound to the Tim4 protein. Then, the cell vesicles bound to the Tim4 protein were removed using magnetic beads that had been pre-bound to the Tim4 protein. The ATP levels of the samples before and after the removal of extracellular vesicles were measured, and the results are shown in Table 1 below.

[0116] The values ​​in Table 1 represent ATP levels, and the unit is amol.

[0117] These results indicate that removing extracellular vesicles in addition to removing proteins and peptides using cation exchange columns can further reduce the background noise when measuring ATP derived from microorganisms.

[0118] According to the present invention, even if the product itself, which is the subject of investigation to determine whether or not microorganisms are present, contains ATP-containing biological components, microorganisms contained in the sample can be detected with high accuracy.

[0119] 100...Microbial detection device 1...Pre-processing unit 11...Separation unit 12...Concentration unit 2...Measurement unit

Claims

1. A sample pretreatment kit for sorting microorganisms from a sample in order to measure ATP derived from microorganisms, comprising a separation unit containing separation particles for separating microorganisms from at least a portion of non-microorganism components contained in the sample.

2. The pretreatment kit for microbial detection according to claim 1, wherein the separated particles adsorb the target component by one or more actions selected from the group consisting of cation exchange, hydrophobic interaction, and specific interaction.

3. The pretreatment kit for microbial detection according to claim 1 or 2, wherein the separation unit further comprises an adsorption column containing the separated particles and a pressure application unit for applying pressure to the sample in the adsorption column, and the pressure application unit discharges all of the liquid containing microorganisms from the adsorption column.

4. A pretreatment kit for microbial detection according to any one of claims 1 to 3, further comprising a removal unit for removing aggregated components contained in the sample.

5. A pretreatment kit for microbial detection according to any one of claims 1 to 4, further comprising a concentration unit for concentrating a sample, wherein the concentration unit concentrates a sample to which a refolding agent for refolding components other than microorganisms contained in the sample has been added by filtering.

6. A pretreatment kit for microbial detection according to any one of claims 1 to 5, further comprising a concentration unit for concentrating a sample by filtering, wherein the concentration unit comprises a concentration filter for concentrating the sample and a liquid supply unit for supplying the sample to the concentration filter, the liquid supply unit supplies one sample to the concentration filter in multiple portions, a portion of the same sample is supplied to the concentration filter and after the portion of the sample is concentrated to a predetermined liquid volume, a diluent for diluting the concentrated sample is supplied to the concentration filter, and then another portion of the same sample is supplied to the concentration filter.

7. The microorganism detection pretreatment kit according to any one of claims 1 to 6, wherein the component other than the microorganism is one or more selected from the group consisting of proteins, peptides, lipids, polysaccharides, nucleic acids, organelles and metabolites.

8. A microorganism detection system comprising a microorganism pretreatment kit according to any one of claims 1 to 7, and a measuring unit for measuring the amount of ATP derived from microorganisms selected by the microorganism pretreatment kit.

9. A method for detecting microorganisms, comprising separating microorganisms and at least a portion of non-microorganism components contained in a sample using separation particles to select microorganisms, and measuring the amount of ATP derived from the selected microorganisms.

10. The method for detecting microorganisms according to claim 9, comprising removing components other than microorganisms using one or more liquid chromatography methods selected from the group consisting of cation exchange chromatography, hydrophobic chromatography, and affinity chromatography.

11. The method for detecting microorganisms according to claim 10, further comprising a pressure application step of applying pressure to the liquid in the separation column used in the liquid chromatography, thereby draining all of the liquid containing microorganisms from the separation column.

12. A method for detecting microorganisms according to any one of claims 9 to 11, further comprising the step of removing components other than the microorganisms by filtering after adding a diluent to the sample.

13. A method for detecting microorganisms according to any one of claims 9 to 12, further comprising the step of removing components other than the microorganisms by filtering after adding a refolding agent to the sample.

14. A method for detecting microorganisms according to any one of claims 9 to 13, further comprising the steps of supplying a portion of the same sample to a concentration filter so that the portion of the sample is concentrated to a predetermined liquid volume, supplying a diluent to dilute the concentrated sample to the concentration filter, and then supplying another portion of the same sample to the concentration filter.

15. A method for detecting microorganisms according to any one of claims 9 to 13, further comprising a culture step of culturing microorganisms in the sample.

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