Method for detecting microorganisms

Abstract

Provided are: a method for detecting microorganisms in a sample, said method comprising (a) a step of causing microorganisms in the sample to be adsorbed on a carrier, (b) a step of recovering the carrier on which the microorganisms have been adsorbed, (c) a step of suspending the concentrated carrier in a liquid medium, (d) a step of subjecting the obtained suspension to a detection step, and (e) a step of examining presence or absence of an event derived from the microorganisms in data obtained from the detection step, wherein the carrier has, on a surface thereof, a substance that adsorbs to the microorganisms, and when the event derived from the microorganisms is recognized in step (e), it is determined that the microorganisms are present in the sample; a method for preparing a sample for use in a method for detecting microorganisms; a method for obtaining a sample containing a substance that does not bind to a carrier by separating the carrier after bringing the sample into contact with the carrier and a method for detecting microorganisms contained in the sample before treatment; and a kit for detecting microorganisms in a sample, the kit containing a carrier having, on a surface thereof, a substance that adsorbs to microorganisms.

Description

Methods for detecting microorganisms

[0001] This application claims priority to Japanese Patent Applications No. 2024-170603 and No. 2025-111623, which are incorporated herein by reference in their entirety. This disclosure relates to a method for detecting and removing microorganisms in a sample.

[0002] The presence of microorganisms in pharmaceuticals, quasi-drugs, cosmetics, and foods can adversely affect the health of patients and consumers, and in some cases, can be fatal. To date, methods for detecting microorganisms have included Gram staining, gas detection, solid-phase cytometry, and Milliflex. （登録商標） Rapid detection methods, nucleic acid amplification methods, and flow cytometry (FCM) are used. However, these methods have problems such as low detection sensitivity and long analysis times. In addition, conventional microbial detection methods have often targeted specific bacterial species (Patent Document 1, Non-Patent Document 1).

[0003] Japanese Patent Publication No. 2022-24167

[0004] The section on Dynabeads anti-E.coli O157 in the VERITAS Dynabeads catalog.

[0005] The challenge was to provide a method for detecting microorganisms that has high detection sensitivity, requires little time for analysis, and covers a wide range of target microorganisms.

[0006] The inventors diligently conducted research to solve the above problems and found that these problems could be solved by applying a carrier that adsorbs microorganisms to a sample. Furthermore, the inventors found that these microorganisms could be removed by applying a carrier that adsorbs microorganisms to a sample, thus completing the present invention.

[0007] In other words, the present disclosure provides: [Item 1] A method for detecting microorganisms in a sample, comprising the steps: (a) adsorbing microorganisms in the sample onto a carrier; (b) recovering the carrier with the adsorbed microorganisms; (c) suspending the recovered carrier with the adsorbed microorganisms in an aqueous medium or a solution that dissolves microorganisms to prepare a detection sample; (d) subjecting the detection sample to a detection step, and then (e) examining the data obtained from the detection step for the presence or absence of events of microorganism origin, wherein the carrier has a substance on its surface that adsorbs to microorganisms, and if an event of microorganism origin is observed in step (e), it is determined that microorganisms are present in the sample. [Item 2] A method for preparing a sample for use in a method for detecting microorganisms, comprising the steps: (a) adsorbing microorganisms in a sample onto a carrier; (b) recovering the carrier with the adsorbed microorganisms; and then (c) suspending the recovered carrier in an aqueous medium or a solution that dissolves microorganisms to obtain a concentrated sample. [Clause 3] A method for obtaining a sample containing a substance that does not bind to a carrier, comprising the steps: (a) contacting a sample before treatment with a carrier, and then (b) obtaining a sample after treatment separated from the carrier, wherein the carrier has a substance on its surface that is adsorbed by microorganisms. [Clause 4] The method according to Claim 3, further comprising the steps: (c) contacting the treated carrier with an aqueous medium or a solution that dissolves microorganisms to prepare a sample for detection, (d) subjecting the sample for detection to a detection step, and then (e) examining the data obtained from the detection step for the presence or absence of events of microorganism origin. [Clause 5] The method according to any one of Claims 1, 2, and 4, wherein the specific gravity of the aqueous medium is matched to the specific gravity of the carrier. [Clause 6] The method according to any one of Claims 1 to 3, wherein the carrier is magnetic beads. [Clause 7] The method according to any one of Claims 1 to 3, wherein the substance adsorbed by microorganisms is lactoferrin or a polycation. [Clause 8] The method according to any one of Claims 1 to 4, wherein the microorganism is a bacterium. [Item 9] The method according to item 1, wherein the microorganism is fluorescently stained before or after step (a), and the detection step is flow cytometry.[Clause 10] The method according to Claim 1 or 4, wherein the detection step comprises a step of amplifying nucleic acids derived from the microorganism. [Clause 11] The method according to Claim 4, wherein in step (d), the microorganism in the detection sample is fluorescently stained and subjected to a detection step which is flow cytometry. [Clause 12] The method according to Claim 1 or 4, wherein the method further comprises, if a microorganism-derived event is observed in step (e), estimating the level of microorganisms in the sample based on data obtained in the detection step. [Clause 13] A kit for removing and / or detecting microorganisms in a sample, comprising a carrier having a substance on its surface that adsorbs to microorganisms. [Clause 14] The kit according to Claim 13, further comprising an aqueous medium having a specific gravity matched to the specific gravity of the carrier. [Clause 15] The kit according to Claim 14, comprising a fluorescent stain.

[0008] This disclosure provides a method and kit for detecting microorganisms that have high detection sensitivity, require little time for analysis, and are applicable to a wide range of microorganisms, as well as a method for preparing samples for use in the method for detecting microorganisms. This disclosure provides a method for removing microorganisms that is applicable to a wide range of microorganisms.

[0009] Figure 1 is a micrograph confirming the adsorption of fluorescently stained Staphylococcus aureus onto PEI cross-linked magnetic beads. The upper left panel shows a microscopic image of the beads. The upper right panel shows a fluorescence microscopic image of the fluorescently stained microorganisms. The lower left panel is an image overlaid with the microscopic image of the beads and the fluorescence microscopic image of the microorganisms. Figure 2 is a fluorescence histogram plot showing the results of flow cytometry performed using samples containing various amounts of microorganisms (from left to right in the upper row: 20,000 cfu / sample, 2,000 cfu / sample, 200 cfu / sample, 20 cfu / sample; from left to right in the lower row: 2 cfu / sample, 0.2 cfu / sample, 0.02 cfu / sample, 0.002 cfu / sample). The arrows indicate the presence of peaks originating from fluorescently stained bacteria. Figure 3 is an extracted section of Figure 2 for 2,000 cfu / sample, and further shows a schematic diagram of the relationship between microorganisms adsorbed on the carrier and the fluorescence peaks. Figure 4 is a micrograph showing the adsorption of Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, and Bacillus subtilis onto PEI, PLL, and LF cross-linked magnetic beads. From left to right: PEI beads, PLL beads, or LF beads. From top to bottom: Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, and Bacillus subtilis. Figure 5 shows quantitative PCR curves of microbial samples concentrated and recovered using LF beads. Top left: Staphylococcus aureus, top right: Pseudomonas aeruginosa, middle left: Escherichia coli, middle right: Bacillus subtilis, and bottom left: FEMTO PCR standard. The graph shows fluorescence histogram plots of flow cytometry results detecting fluorescently stained Staphylococcus epidermidis in the presence of PBS, human plasma, or human PBMC cells.

[0010] This disclosure provides, in one embodiment, the following method: A method for detecting microorganisms in a sample, comprising the steps: (a) adsorbing microorganisms in the sample onto a carrier; (b) recovering the carrier on which the microorganisms have been adsorbed; (c) suspending the recovered carrier on which the microorganisms have been adsorbed in an aqueous medium or a solution that dissolves microorganisms to prepare a detection sample; (d) subjecting the detection sample to a detection step, and then (e) examining the data obtained from the detection step for the presence or absence of microorganism-derived events, wherein the carrier has a substance on its surface that adsorbs to microorganisms, and if a microorganism-derived event is observed in step (e), it is determined that microorganisms are present in the sample.

[0011] Step (a) of the method disclosed herein is a step of adsorbing microorganisms in a sample onto a carrier.

[0012] The sample may be obtained from any specimen. Preferably, the sample is a liquid. If the specimen is solid or semi-solid, a liquid sample can be obtained by dissolving or suspending the specimen in an aqueous medium, or by extracting it in an aqueous medium. Specific examples of specimens include, but are not limited to, pharmaceuticals, bioproducts, food and beverages, environmental water, and bodily fluids. Sample preparation is within the scope of the skill of those skilled in the art. The method disclosed herein can be used to test for sterility of pharmaceuticals, detect microorganisms in food and beverages and the environment, and determine whether a patient is infected.

[0013] The microorganisms detected by the method of the present invention may be of any type. Examples of microorganisms include bacteria, actinomycetes, filamentous fungi, yeasts, and mycoplasmas. A wide range of microorganisms can be detected using the method of the present invention.

[0014] The detection sensitivity of the method disclosed herein is 10 －１ It is in the CFU order and extremely sensitive.

[0015] The carrier used in the method of this disclosure has a substance that adsorbs microorganisms (sometimes referred to herein as a microbial adsorbent) on its surface. The carrier may be of any material, size, or shape, as long as it can be recovered in step (b). Examples of carriers include, but are not limited to, polymer-based beads, ceramics, glass, silicone, magnetic materials, and polystyrene. In some embodiments, the carrier is spherical (beads), a column, or a column containing beads. Particularly from the viewpoint of ease of fluorescence detection, in some embodiments where fluorescence is used for detection, the carrier is preferably beads. Various types and sizes of beads, such as polymer-based beads and beads containing magnetic cores (magnetic beads), are known and commercially available. Beads with various functional groups such as amino groups, carboxyl groups, and hydroxyl groups introduced on their surface are also known and commercially available. These commercially available beads can be used in this disclosure. The beads and microbial adsorbent can be bonded by reacting the functional groups of the beads with functional groups introduced on their surface with the functional groups of the microbial adsorbent using known methods. An example of beads that can be used in this disclosure is surface-activated Dynabeads. （登録商標） Examples include, but are not limited to, magnetic beads bound with polyethyleneimine (such as those used with microbial adsorbents) or PEI Magnetic Particles from CD Bioparticles.

[0016] The microbial adsorbent can be any substance capable of adsorbing microorganisms, and may be a natural or artificial low-molecular-weight, medium-molecular-weight, or high-molecular-weight compound. A preferred microbial adsorbent is one that is positively charged under conditions in which microorganisms are negatively charged. Generally, the conditions in which microorganisms are negatively charged and the microbial adsorbent is positively charged are near-neutral conditions, with a pH of 5 to 8, preferably 6 to 8, and more preferably 6.5 to 7.5. The pH of the sample may be adjusted to the above range using a buffer. Since a wide range of microorganisms are negatively charged under near-neutral conditions, a wide range of microorganisms can be adsorbed and detected using a positively charged microbial adsorbent. Examples of microbial adsorbents include, but are not limited to, polycations. Examples of polycations include, but are not limited to, polyethyleneimine and poly-L-lysine. Polyethyleneimine and poly-L-lysine have the advantage of being inexpensive. Antibodies against microbial cell walls, such as bacterial cell wall antibodies, can also be used as microbial adsorbents. In some embodiments, the microbial adsorbent is lactoferrin.

[0017] The adsorption of microorganisms onto a carrier can be carried out by any method that brings the sample and the carrier into contact. For example, it can be done by adding the carrier to the sample and stirring as appropriate. The type and amount of carrier to be added can be appropriately determined by a person skilled in the art, taking into account the physical properties and volume of the sample, the expected amount of microorganisms present, the type of microbial adsorbent, etc. The contact conditions, such as the degree of stirring, time, temperature, pH, etc., can also be appropriately determined by a person skilled in the art. By adsorbing microorganisms onto a carrier, the recovery and concentration of microorganisms can be facilitated, and the detection sensitivity can be improved.

[0018] Step (b) of the method disclosed herein is a step of recovering the carrier on which the microorganisms obtained in step (a) have been adsorbed. The carrier can be recovered using known means such as centrifugation, gravity, or magnetism, or a combination thereof. Carriers with a high specific gravity may be recovered by sedimentation by gravity or by centrifugation. Carriers with a large size may be recovered using a filter. Magnetic beads may be recovered using magnetism. It is preferable to recover as much carrier as possible in this step to increase the concentration efficiency in the next step.

[0019] Step (c) of the method of this disclosure is a step of suspending the carrier adsorbing the microorganisms recovered in step (b) in an aqueous medium to prepare a concentrated flow cytometry sample. Alternatively, step (c) of the method of this disclosure is a step of suspending the carrier adsorbing the microorganisms recovered in step (b) in a microorganism-dissolving solution to prepare a sample for detecting microorganism-derived components. In the case of a sample with a low microbial concentration, the amount of carrier adsorbing microorganisms per unit volume becomes small, which can reduce detection sensitivity and make detection impossible. Therefore, detection sensitivity can be improved by concentrating the carrier. Even in samples with a normal microbial concentration, microorganisms can be detected more reliably by concentrating the carrier.

[0020] Microbial components include proteins, nucleic acids, lipids, and polysaccharides. Microbial components may be common to many microorganisms or to specific microorganisms. To reduce false positives and nonspecific detections in the detection process, the detected microbial components are distinct from non-microbe-derived components in the sample. In some embodiments, the detected component is nucleic acid.

[0021] The composition of the aqueous medium for suspending the carrier is not particularly limited and can be appropriately determined by those skilled in the art. The aqueous medium may be a buffer such as PBS. The aqueous medium may be an aqueous solution containing salt. The amount of aqueous medium can be appropriately changed and determined depending on the amount of carrier recovered, the structure of the flow cytometer, etc. Generally, a smaller amount of aqueous medium is preferable. The amount of aqueous medium for preparing a flow cytometry sample is usually 0.1 mL to 10 mL, but is not limited to this range. However, for concentration, the amount of aqueous medium must be less than the volume of the original sample. In the method of this disclosure, by concentrating the carrier, 10 －１ It is possible to achieve detection sensitivity on the order of CFU.

[0022] In some embodiments where the detection step is flow cytometry, it is preferable to suspend the carrier using an aqueous medium having a specific gravity that matches the carrier's specific gravity. This allows debris (generally low specific gravity) to float to the surface and be removed, while the carrier remains uniformly suspended without settling, preventing clogging of the flow path of the flow cytometer. Debris includes cells (in the case of biological samples) and waste derived from test equipment (such as tubes). Removing debris increases the specificity of the method disclosed herein and avoids false positives. Matching the specific gravity of the aqueous medium to the specific gravity of the carrier means making the specific gravity of the aqueous medium the same as the specific gravity of the carrier, or within a range of ±10%, preferably ±7%, and more preferably ±5% of the specific gravity of the carrier. Those skilled in the art can adjust the specific gravity of the aqueous medium using known means and methods. For example, the specific gravity of the aqueous medium can be adjusted by adjusting the salt concentration in the aqueous medium.

[0023] Step (d) of the method of this disclosure is a step of subjecting the detection sample obtained in step (c) to a detection step. The detection step may include flow cytometry, nucleic acid detection assays including a nucleic acid amplification test (NAT), and microscopic observation.

[0024] In some embodiments, the detection step is flow cytometry. Various flow cytometers are known and commercially available. The operation of a flow cytometer is within the scope of the skill of those skilled in the art. Bacterial species identification may be performed using a flow cytometer with a cell sorting function. The bacteria that have been sorted, recovered, and concentrated can be identified by, for example, rDNA sequencing.

[0025] If the detection step is flow cytometry, the microorganisms are fluorescently stained. Fluorescent staining of the microorganisms may be performed before or after step (a). If the fluorescent staining step is performed after step (a), it should be performed before step (d), preferably before step (b). Various fluorescent stains are known and commercially available. Examples of fluorescent stains include PI (Propidium iodide), DAPI (4',6-diamidino-2-phenylindole), AO (Acridine orange), and CYBR. （登録商標） Genetic staining agents such as Green (N',N'-dimethyl-N-[4-[(E)-(3-methyl-1,3-benzothiazol-2-ylidene)methyl]-1-phenylquinolin-1-ium-2-yl]-N-propylpropane-1,3-diamine) (these stain dead bacteria or total bacteria); CFDA (5(6)-Carboxyfluorescein diacetate), CFSE (5(6)-(N-Succinimidyloxycarbonyl)fluorescein Examples of active staining agents include, but are not limited to, 3',6'-diacetate, SPiDER-βGal ((2S,3R,4S,5R,6R)-2-{[3'-(Diethylamino)-5'-(fluoromethyl)-3H-spiro(isobenzofuran-1,9'-xanthen)-6'-yl]oxy}-6-(hydroxymethyl)tetrahydro-2H-pyran-3,4,5-triol), and CTC (5-Cyano-2,3-ditolyl-2H-tetrazolium chloride) (these stain viable bacteria). A person skilled in the art can appropriately select a fluorescent staining agent and fluorescently stain microorganisms.

[0026] In some embodiments, the detection step is a nucleic acid detection assay. In the methods of this disclosure, the nucleic acid detection assay may be a method for detecting nucleic acids or a quantitative method. The nucleic acid detection assay may be any method known in the art. The nucleic acid may be DNA or RNA. If it is RNA, the detection step may include a reverse transcription step. The nucleic acid detection assay may or may not include a nucleic acid amplification step. The nucleic acid detection assay may also be a method using an intercalator or probe. In some embodiments, the nucleic acid detection assay is a nucleic acid amplification test (NAT). NATs include, but are not limited to, PCR, LAMP, TMA, TRC, and NEAR methods. In some embodiments, the nucleic acid detection assay may be the Femto Bacterial DNA Quantification Kit® (ZYMO RESEARCH).

[0027] Step (e) of the method of this disclosure is to examine the presence or absence of events originating from fluorescently stained microorganisms in the data obtained from a flow cytometer, or the presence or amount of microbially derived nucleic acids detected in a nucleic acid detection assay. The data can be analyzed using known means. Such means include histogram plots, dot plots, density plots, contour plots, nucleic acid electrophoresis, and amplification plots, which are known to those skilled in the art. Events originating from fluorescently stained microorganisms appear, for example, as peaks or spikes in a histogram plot, and as dots or clusters of dots in a dot plot. In the method of this disclosure, if events originating from microorganisms are observed, it is determined that microorganisms are present in the sample. For example, in a histogram plot, the intensity of the fluorescent peaks is generally multiple (see, for example, Figure 3). Therefore, it is presumed that the number of adsorbed bacteria constitutes different peaks. For this reason, if fluorescent peaks are observed, it can be determined that microorganisms are present in the sample. In addition, if amplification bands are observed by nucleic acid electrophoresis, intercalators or probes are detected, or a decrease in the CT value in the amplification plot is observed, it is determined that microorganisms are present in the sample.

[0028] The method disclosed herein may be used to quantify microorganisms in a sample. For example, the number of microorganisms can be estimated and quantified by measuring the area undercoop (AUC) of the peaks derived from fluorescently stained microorganisms in a histogram plot. Alternatively, the number of microorganisms can be estimated and quantified based on the difference in CT values ​​between the sample and a standard sample in an amplification plot.

[0029] In another aspect, this disclosure provides a method for preparing a sample to be used in a method for detecting microorganisms, comprising the following steps: (a) adsorbing microorganisms in the sample onto a carrier; (b) recovering the carrier on which the microorganisms have been adsorbed; and (c) suspending the recovered carrier in an aqueous medium or a solution that dissolves microorganisms to obtain a concentrated sample. Steps (a) to (c) above are as described above. Methods for detecting microorganisms include, but are not limited to, flow cytometry, observation using a fluorescence microscope, and nucleic acid detection assays including NAT. By using the above method, detection sensitivity can be increased and a wide range of microorganisms can be detected.

[0030] In another aspect, this disclosure provides a method for obtaining a sample containing a substance that does not bind to a carrier, comprising the steps of: (a) contacting a sample before treatment with a carrier, and then (b) obtaining a sample after treatment separated from the carrier, wherein the carrier has a substance on its surface that adsorbs to microorganisms. Such a method may correspond to a method for removing microorganisms from a sample. The carrier having a substance on its surface that adsorbs to microorganisms, the microorganisms adsorbed to the carrier, and the sample are as described above.

[0031] Step (a) of the method for obtaining a sample containing a substance that does not bind to a carrier is a step of adsorbing microorganisms in the sample onto a carrier. Examples of substances that do not bind to a carrier include, but are not limited to, desired molecules, compounds, cells, and compositions that are desirable to be purified. In some embodiments, the substance that does not bind to a carrier is a liquid composition. In some embodiments, the substance that does not bind to a carrier is cells.

[0032] In the method of obtaining a sample containing a substance that does not bind to the carrier in the present disclosure, step (b) is a step of obtaining a sample that does not contain the microorganism because the microorganism is adsorbed to the carrier. Step (b) is performed by removing the carrier or isolating the sample. The removal of the carrier may be performed by any method, for example, by the step of recovering the above-mentioned carrier. The isolation of the sample may be performed by any method, for example, by eluting or flowing out the treated sample, or by sucking with a pipette.

[0033] Steps (a) and (b) can be performed independently or in parallel. For example, when the carrier is beads, it can be performed by adding beads to the sample to adsorb the microorganism, and then recovering and removing the beads adsorbed with the microorganism to obtain a treated sample. For example, when the carrier is a column, by adding the sample to the column, the microorganism is adsorbed to the carrier during the process of the sample passing through the column, and a treated sample flowing out from the column can be obtained.

[0034] The method of obtaining a sample containing a substance that does not bind to the carrier of the present disclosure may further involve detecting the microorganism adsorbed to the carrier. Detecting includes the following steps: (c) contacting the treated carrier with an aqueous medium or a solution that dissolves the microorganism to prepare a detection sample; (d) subjecting the detection sample to a detection step; and then (e) examining the presence or absence of an event derived from the microorganism in the data obtained from the detection step. In addition to the matters described above, step (c) includes preparing a detection sample containing components derived from the microorganism by contacting a solution that dissolves the microorganism even when the carrier cannot be suspended, and recovering the solution after contact. Steps (d) and (e) are as described above. Examples of the method for detecting the microorganism include, but are not limited to, flow cytometry, observation using a fluorescence microscope, nucleic acid detection assays including NAT, etc. By using the above method, the detection sensitivity can be enhanced, and a wide range of microorganisms can be detected.

[0035] In another aspect, the present disclosure provides a kit for implementing the above method. That is, the present disclosure provides a kit for detecting and / or removing microorganisms in a sample, including a carrier having a microbial adsorbent. The kit of the present disclosure may further include an aqueous medium having a specific gravity adapted to the specific gravity of the carrier and / or a fluorescent staining agent. The carrier having a microbial adsorbent, the aqueous medium having a specific gravity adapted to the specific gravity of the carrier, and the fluorescent staining agent are as described above.

[0036] Using the kit of the present disclosure, for example, microorganisms in pharmaceuticals, foods, environmental water, body fluids, etc. can be detected and / or removed. Using the kit of the present disclosure, sterility tests of pharmaceuticals and their sterilization treatments, microbial tests of foods and environmental water and their sterilization treatments, investigations of the presence or absence of infections in patients, etc. can be carried out.

[0037] The terms in this specification are construed to have the meanings commonly understood in the fields of microbiology, medicine, chemistry, biochemistry, biology, pharmacy, analytical chemistry, food manufacturing, etc., unless otherwise specified.

[0038] Examples are shown below to describe the present disclosure in more detail and specifically, but the examples should not be construed as limiting the scope of the present disclosure.

[0039] (1) Preparation of polyethyleneimine-crosslinked Dynabeads （登録商標） Polyethyleneimine (PEI) was bound via the epoxy group of Dynabeads M-270 Epoxy (VERITAS) to obtain polyethyleneimine-crosslinked Dynabeads (hereinafter referred to as "beads", "PEI beads" or "PEI-crosslinked magnetic beads").

[0040] (2) Microbial detection experiment materials: For bacteria, Bioball (Staphylococcus aureus, NCTC 10788 strain, Biomérieux) was obtained and grown for use. As the bacterial fluorescent staining dye, Bacstatin-CFDA solution (Dojindo Laboratories / Kumamoto / Japan) was used.

[0041] Specific Procedure: The frozen CFDA solution was allowed to stand at room temperature for 30 minutes to thaw. Staphylococcus aureus was suspended in PBS(-) and a bacterial density dilution series was prepared (repeated 10-fold dilutions from the original solution, resulting in 8 dilution series including the original solution), and the bacterial density was measured. 2 μL of CFDA solution was added to 400 μL of each dilution series cell suspension and mixed. The mixture was incubated at 37°C for 30 minutes. 100 μL of 10% neutral buffered formalin was added to fix the bacteria. 1 μL of PEI beads was added and incubated while gently mixing. After incubation, DynaMag （登録商標） The sample was placed in a tube and allowed to stand for 2 minutes to collect the PEI beads and microbial conjugates on the tube wall, and the supernatant was removed. After removing it from the DynaMag, the sample was resuspended in a specific gravity solution (a solution whose specific gravity was matched to that of the beads) to a final volume of 500 μL. The sample was observed using a fluorescence microscope (BioZero, Keyence) (imaged at 488 nm). The results are shown in Figure 1. In the fluorescence microscope image, fluorescently stained bacteria were adsorbed onto the PEI beads, and no bacteria not bound to the PEI beads were observed. Furthermore, the sample was observed using a flow cytometer (guava easyCyteHT, Millipore, 500 μL of sample applied, detected at 488 nm). The results are shown in Figure 2. The cell suspension was seeded on an agar plate in parallel with the FCM measurement. Three days after sowing, the colony forming unit was measured, and the bacterial colony forming unit in the untreated solution was 5 x 10⁻⁶. ４The cfu / mL value was obtained. As a result, the cfu / sample values ​​for FCM were 20000, 2000, 200, 20, 20, 2, 0.2, 0.02, and 0.002 cfu / sample. In Figure 2, as indicated by the arrows, peaks due to fluorescently stained bacteria were observed when bacteria were present at a concentration of 0.2 cfu / sample or higher (the large peaks on the left end of each chart are autofluorescence from the PEI beads). This experiment demonstrated a detection sensitivity of 0.2 cfu / sample. In the above experiment, the time required from the adsorption of microorganisms onto the PEI beads to obtaining the flow cytometry results was less than one hour. It was found that microorganisms can be detected in a sample rapidly and with high sensitivity using the method disclosed herein.

[0042] As described above, the presence or absence of microorganisms can be determined from the FCM results by observing peaks other than autofluorescence, meaning it can be used as a sterility test. In addition, the microbial concentration can be estimated from the peak pattern (number and intensity of peaks) observed in the FCM results. Referring to Figure 3, which extracts the upper left figure (2000 cfu / sample) from Figure 2 and adds a schematic diagram, four peaks other than autofluorescence can be observed. Here, the fluorescence intensity (horizontal axis) of the four peaks is roughly a multiple. This is thought to reflect the number of bacteria adsorbed onto the carrier. Therefore, for example, it can be said that more fluorescent microorganisms are adsorbed onto the carrier that constitutes the peak on the right with strong fluorescence intensity. However, as the fluorescence intensity increases, that is, as the number of microorganisms bound to the carrier increases, the peak weakens. This is because the number of carriers to which two microorganisms bind is less than the number of carriers to which one microorganism binds, and this number decreases further as the number of bacteria increases. In a sterility test sample with a low bacterial concentration, the adsorption reaction between the carrier and microorganisms can be assumed to be in an ideal state according to their respective concentrations. Furthermore, the number of carriers that support more microorganisms is thought to decrease according to mathematical probability. Therefore, by applying a mathematical probability model to the obtained peak pattern, it becomes possible to estimate the microbial concentration (CFU or bacterial count) in the sample.

[0043] (1) Preparation of Lf beads and PLL beadsPreparation of lactoferrin-crosslinked Dynabeads (registered trademark): Lactoferrin (Lf) was conjugated to Dynabeads M-270 Carboxylic acid (VERITAS) to obtain lactoferrin-crosslinked Dynabeads (hereinafter referred to as "beads", "Lf beads"). Preparation of poly-L-lysine-crosslinked Dynabeads (registered trademark): Poly-L-lysine (PLL) was conjugated to Dynabeads M-270 Carboxylic acid (VERITAS) to obtain poly-L-lysine-crosslinked Dynabeads (hereinafter referred to as "beads", "PLL beads").

[0044] (2) Microbial detection experiment using PEI beads, PLL beads and LF beads Specific procedure: Cultured Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, and Bacillus subtilis were suspended in PBS (-) to obtain a bacterial suspension (900 μL). 100 μL of 10% neutral buffered formalin was added to immobilize the bacteria. 1 μL of PEI beads, PLL beads or LF beads was added to the bacterial suspension (1000 μL) to which 10% neutral buffered formalin had been added, and the mixture was incubated while gently mixing. After incubation, it was set on DynabMag and allowed to stand for 2 minutes, and each bead-microorganism conjugate was collected on the tube wall and the supernatant was removed. It was removed from DynabMag and resuspended in a specific gravity solution (solution with a specific gravity adjusted to that of the beads) to make the final sample volume 500 μL. 5 μL of DAPI was added to each sample to stain the nucleoid. The samples were observed with a fluorescence microscope (BioZero, Keyence) (imaging at 488 nm). The results are shown in Fig. 4. In the fluorescence microscope image, fluorescently stained bacteria were adsorbed to the beads, and no bacteria that were not bound to the beads were observed. From the above experiment, it was shown that in addition to the polycationic PEI beads and PLL beads, LF beads can also concentrate and recover bacteria. （登録商標）

[0045] Specific procedure for microbial concentration experiment using LF beads: Cultured Staphylococcus aureus, Pseudomonas aeruginosa, Escherichia coli, and Bacillus subtilis were suspended in PBS(-). 2 μL of LF beads were added to the bacterial suspension (2 mL) and incubated while gently mixing. After incubation, DynaMag （登録商標） The samples were placed in a tank and allowed to stand for 2 minutes. The LF beads and microbial conjugates were collected on the tube wall, the supernatant was removed, and the beads were recovered. Microorganisms adsorbed onto the LF beads were dissolved and neutralized using the Kaneka Simple DNA Extraction Kit version 2 (registered trademark) (Kaneka), and 1 μL was quantitatively measured using the Femto Bacterial DNA Quantification Kit (registered trademark) (Zymo Research) (LF-beads). As a control, the same bacterial suspension without added beads was dissolved and neutralized using the Kaneka Simple DNA Extraction Kit version 2 (registered trademark) (Kaneka), and 1 μL was quantitatively measured using the Femto Bacterial DNA Quantification Kit (registered trademark) (Zymo Research) (Control). The measurement results are shown in Figure 5. From the above experiment, quantitative PCR curves obtained using the Femto Bacterial DNA Quantification Kit (registered trademark) (Zymo Research) showed that for all bacterial species, the PCR curves obtained from samples using LF beads were shifted to the left compared to those of the control, indicating that LF beads enrich the microorganisms.

[0046] This embodiment demonstrates that microorganisms in a sample can be detected using a nucleic acid amplification-based assay by the method of this disclosure. Furthermore, the results of quantitative PCR in this embodiment demonstrate that microorganisms in a sample can be enriched by using a carrier that adsorbs microorganisms.

[0047] Microbial detection test material in biological samples: Bacteria were obtained from Bioball (Staphylococcus epidermidis, strain NCTC6513, Biomerieux), grown, and used. Bacstatin-CFDA solution (Dojin Chemical Laboratories Co., Ltd. / Kumamoto / Japan) was used as the bacterial fluorescent staining dye.

[0048] Specific procedure: The frozen CFDA solution was allowed to stand at room temperature for 30 minutes to thaw. Staphylococcus epidermidis was placed in PBS(-), human plasma, and human peripheral blood-derived mononuclear cell (hPBMC) suspension (PBS(-), with 1.87 x 10⁻¹ human peripheral blood-derived mononuclear cell cells in the suspension. ６ The bacterial density was measured after suspending the cells / sample (58% viable) in a suspension solution. 2 μL of CFDA solution was added to 400 μL of cell suspension and mixed. The mixture was incubated at 37°C for 30 minutes. 100 μL of 10% neutral buffered formalin was added to fix the bacteria. 1 μL of Lf beads was added and incubated while gently mixing. After incubation, the sample was placed in a DynaMag® and allowed to stand for 2 minutes to collect the beads and microbial conjugates on the tube wall, and the supernatant was removed. The sample was removed from the DynaMag and resuspended in a specific gravity solution (a solution whose specific gravity was matched to the specific gravity of the beads) to a final volume of 500 μL. The sample was observed using a flow cytometer (guava easyCyteHT, Millipore, 500 μL of sample applied, detected at 488 nm). The results are shown in Figure 6. During FCM measurement, the suspension was seeded onto an agar plate. Three days after seeding, the colony forming unit was measured. Although 111 cfu of bacteria were added to the sample, the actual number of cells (bacteria) detected in human plasma was 40 cfu / sample, and in the human peripheral blood mononuclear cell suspension it was 16 cfu / sample. This is presumed to be due to the bactericidal and bacteriostatic effect of the plasma or cells. In the above experiment, the time required from the adsorption of microorganisms onto the beads to obtaining the flow cytometry results was less than one hour. It was found that by using the method disclosed herein, microorganisms can be detected in samples rapidly and with high sensitivity from a variety of specimens.

[0049] Specific procedure for removing microorganisms from the sample: Staphylococcus epidermidis is cultured in LB medium, and the McFarland turbidity standard solution number is 5, with an estimated viable cell count concentration of 15 x 10⁻¹⁰. ８ The cfu / mL was obtained. The bacterial culture solution was serially diluted with physiological saline to obtain a concentration of 15 x 10 at each dilution stage. ８ , 15x10 ７ , 15x10 ６, 15x10 ５ , 15x10 ４ , 15x10 ３ , 15x10 ２ A bacterial suspension of cfu / mL was obtained. Human peripheral blood mononuclear cells were suspended in saline solution and 1.1 x 10⁶ cells were added. ５ Human peripheral blood mononuclear cell saline suspensions were prepared at a cell / mL concentration. To 1800 μL of the human peripheral blood mononuclear cell saline suspension, serially diluted bacterial suspensions (7 series) or 200 μL of physiological saline as a control were added. Each cell-bacterial mixture was dispensed into two 1.5 mL tubes, one of which contained PEI cross-linked magnetic beads, and the other served as a control without the beads. After stirring at room temperature for 1 hour, all tubes were placed in a magnetic bead collection stand. 100 μL of the supernatant was collected from each tube, taking care not to touch the PEI cross-linked magnetic beads. Three plates of each sample were spread onto agar plates (10 cm² dish), and the colony forming unit was measured after 24 hours. The results are shown in Table 1 below.

[0050] After adding PEI-crosslinked magnetic beads to a sample containing microorganisms and human peripheral blood-derived mononuclear cells, the carrier and sample were separated to obtain a treated sample with reduced microbial counts or sterile material, containing human peripheral blood-derived mononuclear cells that do not bind to the carrier. Therefore, it is demonstrated that the method of this disclosure makes it possible to remove microorganisms from a sample, or to obtain a treated sample containing a carrier-non-binding substance with reduced microbial counts or sterile material.

[0051] This disclosure can be used to detect and / or remove various microorganisms present in pharmaceuticals, bio-products, quasi-drugs, cosmetics, food and beverages, environmental water, bodily fluids, etc. This disclosure is particularly useful for sterility testing and sterilization treatment of bio-products such as cell therapies and antibody drugs used in regenerative medicine, microbial testing and sterilization treatment of food and beverages and environmental water, and investigation of infection in patients. Therefore, this disclosure is applicable in the pharmaceutical, bio-product, quasi-drug, cosmetics, food, and environmental industries.

Claims

1. A method for detecting microorganisms in a sample, comprising the following steps: (a) adsorbing microorganisms in the sample onto a carrier; (b) recovering the carrier on which the microorganisms have been adsorbed; (c) suspending the recovered carrier on which the microorganisms have been adsorbed in an aqueous medium or a solution that dissolves microorganisms to prepare a detection sample; (d) subjecting the detection sample to a detection step; and (e) examining the presence or absence of microorganism-derived events in the data obtained from the detection step, wherein the carrier has a substance on its surface that adsorbs to microorganisms, and if a microorganism-derived event is observed in step (e), it is determined that microorganisms are present in the sample.

2. A method for preparing a sample for use in a method for detecting microorganisms, comprising the following steps: (a) adsorbing microorganisms in the sample onto a carrier; (b) recovering the carrier on which the microorganisms have been adsorbed; and (c) suspending the recovered carrier in an aqueous medium or a solution that dissolves microorganisms to obtain a concentrated sample.

3. A method for obtaining a sample containing a substance that does not bind to a carrier, comprising the steps of: (a) bringing a sample before treatment into contact with a carrier, and then (b) obtaining a sample after treatment separated from the carrier, wherein the carrier has a substance on its surface that is adsorbed by microorganisms.

4. The method according to claim 3, further comprising the steps: (c) preparing a detection sample by contacting the treated carrier with an aqueous medium or a solution that dissolves microorganisms; (d) subjecting the detection sample to a detection step, and then (e) examining the presence or absence of microorganism-derived events in the data obtained from the detection step.

5. The method according to any one of claims 1, 2, and 4, wherein the specific gravity of the aqueous medium is matched to the specific gravity of the carrier.

6. The method according to any one of claims 1 to 3, wherein the carrier is a magnetic bead.

7. The method according to any one of claims 1 to 3, wherein the substance adsorbed to the microorganism is lactoferrin or a polycation.

8. The method according to any one of claims 1 to 4, wherein the microorganism is a bacterium.

9. The method according to claim 1, wherein the microorganism is fluorescently stained before or after step (a), and the detection step is flow cytometry.

10. The method according to claim 1 or 4, wherein the detection step includes a step of amplifying nucleic acids derived from the microorganism.

11. The method according to claim 4, wherein in step (d), the microorganisms in the detection sample are fluorescently stained and subjected to a detection step which is flow cytometry.

12. The method according to claim 1 or 4, further comprising estimating the level of microorganisms in a sample based on data obtained in the detection step if a microorganism-derived event is observed in step (e).

13. A kit for removing and / or detecting microorganisms in a sample, comprising a carrier having a substance on its surface that adsorbs to microorganisms.

14. The kit according to claim 13, further comprising an aqueous medium having a specific gravity matched to the specific gravity of the carrier.

15. The kit according to claim 14, comprising a fluorescent staining agent.

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