Microorganism collecting reagent, microorganism collecting kit, microorganism collecting method, and microorganism collecting device

Abstract

To provide a microorganism collecting reagent, a microorganism collecting kit, and a microorganism collecting procedure using them, which are capable of specifically and highly efficiently capturing and concentrating microorganisms present at a low level in blood without requiring centrifugal operation, and which can be stored for a long period of time at normal temperature.SOLUTION: In order to solve the above problem, the present disclosure proposes a microorganism collecting reagent 100 in which a surface of a magnetic particle 101 is modified with a microorganism binding moiety by a dimethylamino group 103.SELECTED DRAWING: Figure 1

Description

Technical Field

[0001] The present disclosure relates to a microbial collection reagent, a microbial collection kit, a microbial collection method, and a microbial collection device.

Background Art

[0002] In infectious disease testing, rapid pathogen detection is required to initiate appropriate treatment promptly and prevent secondary infections. In particular, bloodstream infections typified by sepsis can present shock symptoms and progress severely even when only a small number of bacteria are present in the bloodstream. In the case of severe sepsis, it has been reported that the survival rate decreases by 7.6% per hour when treatment with antibacterial drugs is delayed (A. Kumar et al., Crit Care Med 2006). Therefore, when a bloodstream infection is suspected, it is desirable to initiate treatment with antibacterial drugs as soon as possible, preferably within 1 hour. In this regard, since the causative bacteria are diverse and the effective antibacterial drugs differ for each, it is desirable to simultaneously test for multiple bacteria and promptly administer appropriate antibacterial drugs.

[0003] However, the bacteria contained in the blood of patients with bloodstream infections are about 1 CFU (Colony forming unit) / mL in some cases, and it is difficult to detect them as they are. Currently, about 10 mL of blood is collected and cultured overnight to increase the bacteria by 10 6 times or more before conducting the test. Until the test results are obtained, broad-spectrum antibacterial drugs or cocktails of multiple antibacterial drugs that can cover all possible bacteria are administered to address the situation. On the other hand, the administration of such antibacterial drugs is regarded as a problem because it increases drug-resistant bacteria that are not susceptible to the antibacterial drugs. Also, narrow-spectrum antibacterial drugs that are more compatible with the pathogenic bacteria often have a higher therapeutic effect than broad-spectrum antibacterial drugs.

[0004] The PCR (Polymerase Chain Reaction) method can amplify the genes of bacteria 10 times in 1 hour 8It can be amplified several times and can detect bacteria more quickly than conventional blood culture-based tests. By using the PCR method, it becomes possible to switch from broad-spectrum antibacterial drugs to antibacterial drugs with a narrower spectrum at an early stage. As a result, a higher therapeutic effect can be obtained and the emergence of resistant bacteria can be suppressed, making it a promising technology for the treatment of bloodstream infections.

[0005] However, when performing direct PCR on about 10 mL of collected blood, it is not practical because hemoglobin and proteins contained in large amounts in the blood inhibit PCR and the required amount of reagents becomes extremely large. Therefore, it is common to concentrate and purify bacteria in the blood in advance, extract the genes of the bacteria, and then subject them to a PCR reaction. Such concentration and purification have conventionally been performed manually by laboratory technicians with specialized skills. However, in a hospital laboratory with limited manpower and budget, it is desirable to obtain test results automatically without involving human hands as much as possible, and it is necessary to minimize the test cost per specimen. Furthermore, since it is necessary to arrange a variety of test devices in a limited space, it is desirable to minimize the floor area per device.

[0006] For example, Patent Document 1 discloses a procedure for destroying the most abundant red blood cells in blood with a lysing agent and then concentrating the target bacteria by centrifugation to obtain them as pellets in a fully automated sepsis testing device. According to Patent Document 1, by extracting and amplifying the nucleic acid of the bacteria from the obtained bacterial pellets and detecting the aggregation state of magnetic particles (magnetic beads) that specifically bind to the nucleic acid of the bacteria by nuclear magnetic resonance, the types of bacteria present in the blood of patients with bloodstream infections can be quickly identified.

[0007] In addition, Patent Document 2 discloses a technique in which bacteria present in a patient's whole blood are captured using magnetic particles (magnetic beads) whose surface is modified with a molecule having a microbial binding domain such as lectin, the magnetic particles are recovered with a magnet, blood components are removed with Tris-buffered saline, and the bacteria are detected and quantified by the ELISA method. According to Patent Document 2, since bacteria can be concentrated without a large-scale mechanism such as a centrifuge, it is considered advantageous in reducing the size of the apparatus.

Prior Art Documents

Patent Documents

[0008]

Patent Document 1

Patent Document 2

Non-Patent Documents

[0009]

Non-Patent Document 1

Summary of the Invention

Problems to be Solved by the Invention

[0010] However, in the inspection apparatus disclosed in Patent Document 1, it is necessary to mount a rotor mechanism for centrifugation, and there is a problem that the apparatus size increases. In addition, it is difficult to separate cells contained in blood and having a shape similar to that of bacteria. For example, platelets are about 2 μm in size, which is close to the size of bacteria (less than 1 μm to about several μm). In addition, platelets are contained at about 1.5 billion to 4.5 billion per 1 mL, which is overwhelmingly larger in number than bacteria at a concentration of 1 CFU / mL. From the above, it is difficult to selectively separate only bacteria by centrifugation.

[0011] In addition, in the method described in Patent Document 2, it is necessary to use lectin, which is a protein. Generally, proteins may be decomposed by microorganisms in the environment or their functions may deteriorate due to oxidation. For this reason, it is difficult to store proteins at room temperature for a long time. Therefore, it is necessary to add preservatives such as preservatives and antioxidants and store them frozen. Since some of the preservatives contain components that inhibit the action of polymerase, which is an enzyme for PCR, an operation for removing such preservatives is required to detect bacteria by PCR reaction.

[0012] On the other hand, when frozen storage is required, the storage cost of the reagent increases, leading to an increase in the inspection cost. In particular, since the onset timing of bloodstream infection is random and urgent, it is necessary to prepare the inspection reagent with a margin, which is a factor increasing the operation cost of the laboratory. If it can be stored at room temperature for a long time, such storage costs can be reduced.

[0013] In view of such a situation, the present disclosure proposes a microorganism collection technique that enables capture and concentration of microorganisms slightly present in blood without centrifugation operation specifically and with high efficiency (high recovery rate), and enables long-term storage at room temperature.

Means for Solving the Problems

[0014] In order to solve the above problems, the present disclosure proposes a microorganism collection reagent in which a microorganism binding part with a dimethylamino group is modified on the surface of magnetic particles.

[0015] Further features related to the present disclosure will become apparent from the description in this specification and the accompanying drawings. Also, aspects of the present disclosure are achieved and realized by elements and combinations of various elements and the aspects of the following detailed description and the appended claims. The description in this specification is merely a typical example and does not limit the scope of the claims of the present disclosure or the application examples in any sense.

Advantages of the Invention

[0016] According to the technology of the present disclosure, a reagent that can be stored for a long time at room temperature enables the efficient collection of microorganisms in blood without the need for large-scale equipment.

Brief Description of the Drawings

[0017]

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Mode for Carrying Out the Invention

[0018] Embodiments of the present invention will be described in detail with reference to the drawings. In the following embodiments, it goes without saying that the constituent elements (including element steps, etc.) are not necessarily essential unless otherwise specified or considered clearly essential in principle.

[0019] The positions, sizes, shapes, ranges, etc. of the respective constituent elements shown in the drawings may not represent the actual positions, sizes, shapes, ranges, etc. in order to facilitate understanding of the invention. For this reason, the present invention is not necessarily limited to the positions, sizes, shapes, ranges, etc. disclosed in the drawings.

[0020] <Structure of Microorganism Collection Reagent and Method for Producing the Reagent> (i) Figure 1 is a diagram showing a structural example of the microorganism collection reagent 100 according to the present embodiment. The microorganism collection reagent 100 is composed of magnetic particles 101 having a surface modification 102 with a dimethylamino group 103. The dimethylamino group 103 has two methyl groups Me, and these are characterized by being positively charged. On the other hand, it is known that there are portions on the surface of the cell wall of the bacterium 104 that are negatively charged by membrane proteins. Thereby, the bacterium 104 and the dimethylamino group 103 are electrostatically bonded. Utilizing this action, it becomes possible to collect microorganisms present in a patient's specimen. Also, since it does not contain protein, it can be stored for a long time at room temperature.

[0021] Note that Non-Patent Document 1 discloses a method of specifically staining bacteria with a dye having a dimethylamino group. The dimethylamino group is positively charged and realizes the staining of bacteria by electrostatically binding to negatively charged bacteria. That is, in Non-Patent Document 1, the dimethylamino group is used not for binding to the bacterium 104 but for staining bacteria. On the other hand, in the present embodiment, by using magnetic particles (magnetic beads) modified with a dimethylamino group, a reagent that does not contain a protein with poor long-term stability and enables the recovery of bacteria is realized. However, as will be described later (refer to the experimental results in FIGS. 5 and 6), according to our experiments, when magnetic particles modified with a dimethylamino group are used for bacteria contained in blood, the bacteria can be recovered relatively efficiently, but the recovery rate remains at about 43%, and there is room for improvement in the recovery rate. Furthermore, it has been found that the binding between bacteria and magnetic particles may be inhibited by plasma components. Further improving the recovery rate is an important issue in detecting bacteria contained only slightly in the blood of septic patients.

[0022] (ii) The microbial capture reagent 100 was prepared by the following method. FIG. 2 is a diagram showing the reaction in the production process of the microbial capture reagent 100. As shown in FIG. 2, magnetic particles 200 with a particle diameter of 100 nm (SC0100, Ocean Nanotech) surface-modified with carboxyl groups 201 and N,N-dimethyl-1,3-propanediamine 202 (D0790, Tokyo Chemical Industry) were dehydrated and condensed using a kit (KSC0100-04, Ocean Nanotech) to prepare the microbial capture reagent 100 having dimethylamino groups 103.

[0023] <Procedure for recovering microorganisms (bacteria) from a specimen> FIG. 3 is a flowchart for explaining the procedure for recovering bacteria contained in trace amounts from the blood of a bloodstream infection patient using the microbial capture reagent 100.

[0024] (i) Step S301 The operator (user) puts the blood (specimen solution 402) collected from a bloodstream infection patient and the microbial capture reagent 100 into a container 400. The container 400 can be, for example, a microtube or a centrifuge tube used in biological experiments, and those with a surface processed to suppress the adsorption of microorganisms can be used. Also, the microbial capture reagent 100 may be directly added to the blood collection tube used for blood collection. After performing blood culture in a blood culture bottle, the microbial capture reagent 100 may be directly added to the blood culture bottle.

[0025] (ii) Step S302 The operator stirs the contents of the container 400. Stirring can be carried out, for example, using a thermoshaker maintained at a temperature of 25 degrees. The stirring time is desirably the time until the microbial capture reagent 100 and most of the bacteria in the specimen are sufficiently bound, for example, 15 minutes.

[0026] (iii) Step S303 The operator brings a magnet (e.g., a magnetic stand used in biological experiments) close to the container 400 and aggregates the conjugate of the microbial collection reagent 100 and the microorganism 104 dispersed in the container 400 on the inner wall of the container 400. The time until aggregation depends on the properties of the blood and the shape and characteristics of the magnetic particles (magnetic beads) used, but is typically about 10 minutes.

[0027] (iv) Steps S308 and S309 The operator discards the supernatant from the container 400 (S308) and obtains the conjugate of the microbial collection reagent 100 and the microorganism 104 (S309).

[0028] If it is desired to remove other components (e.g., blood components) contained in the specimen as much as possible, after step S303, the operations from step S304 to step S307 may be performed before step S308.

[0029] (v) Step S304 The operator discards the supernatant from the container 400 following the operation of step S303.

[0030] (vi) Step S305 The operator removes the magnet from the container 400 and puts a cleaning liquid into the container 400.

[0031] (vii) Step S306 The operator stirs the container 400 to wash the components attached to the conjugate of the microbial collection reagent 100 and the microorganism 104.

[0032] (viii) Step S307 The operator brings the magnet close to the container 400 again and aggregates the conjugate of the microbial collection reagent 100 and the microorganism 104 on the inner wall of the container. The operator repeats the processes from S304 to S307 until other components are removed to a desired level.

[0033] (ix) Step S308 and Step S309 The operator discards the supernatant from the container 400 (S308) and obtains the combined microorganism capturing reagent 100 and the microorganisms 104 (S309).

[0034] The cleaning solution used in this treatment (work) is preferably one that does not affect bacteria, and for example, water, physiological saline, phosphate buffer solution (PBS), pluronic water, etc. can be used.

[0035] <Bacteria recovery rate evaluation experiment> Fig. 4 is a diagram showing specific steps of an experiment to evaluate the recovery rate of bacteria based on the flowchart of Fig. 3. An experiment to evaluate the recovery rate of bacteria using the microorganism collecting reagent 100 was carried out according to the specific steps shown in Fig. 4.

[0036] The sample solution 402 and dried Staphylococcus aureus 401 (BioBall S. aureus 30 CFU, bioMérieux Japan) were introduced into the container 400 and vortexed (inverted mixing was performed for samples containing blood components in the sample solution) (step (i): S301). Next, 10 μL of 20 mg / mL microbial collection reagent 100 was added (step (ii): S302), and the mixture was shaken and stirred at 25°C and 1000 rpm for 15 minutes in a thermoshaker (BSR-MS100, Thermo Fisher) to bind the microbial collection reagent 100 and the Staphylococcus aureus 403 (step (iii): S303). To rapidly bind the low concentration of bacteria to the beads, it is better to have a relatively high concentration of beads (magnetic particles). The amount of beads added this time was 10 per mL of sample solution 402. 13 If the volume of the sample solution 402 is converted to 10 μm×10 μm×10 μm (1 pL), the concentration is about 100 particles. Also, if the particles are uniformly distributed in a cube with a side of 10 μm, the concentration is 100 particles per side. 1 / 3=There will be 4.64 beads. That is, assuming the beads are arranged in a lattice, the spacing between the lattices is 10 / 4.64 = 2.2 μm. Since the size of Staphylococcus aureus is about 1 μm, the concentration is such that there are beads within 1 μm around Staphylococcus aureus. Subsequently, after binding the microbial collection reagent 100 and Staphylococcus aureus 403, the magnetic stand 404 (Dynamag-2, Thermo Fisher) was applied to the side of the container 400 for 10 minutes, and the conjugate 405 of the microorganism and the microbial collection reagent was collected on the inner wall of the container (step (iv): S304). Next, in order not to affect the collected conjugate 405 of the microorganism and the microbial collection reagent, the entire 1 mL supernatant was recovered with a pipette 406 (step (v): S308), then the magnetic stand 404 was removed from the container 400, and 100 μL of Pluronic water was added and resuspended (step (vi)). Both the supernatant recovered above and the suspension containing the conjugate 405 of the microorganism and the microbial collection reagent were cultured on the medium 407 for 18 to 24 hours, and each was colony counted (step (vii)). The collection rate was calculated from the ratio of the counts of both.

[0037] <Evaluation results of collection rate for each experiment> (1) Figure 5 is a diagram showing the evaluation results of the collection rate. Figure 5 shows the results when water and human whole blood were used as the sample solution 402, respectively. For the evaluation using whole blood, purchased blood (anticoagulated with EDTA2K, Tennessee blood services) was used. Also, considering the blood sample dependence, the collection rate was evaluated for the blood of three different subjects respectively.

[0038] As can be seen from the results in Figure 5, when water was used as the sample solution 402, a high collection rate of about 96% was shown, while in the case of whole blood, it was found that the collection rate deteriorated significantly to about 43%.

[0039] Next, the collection rate was evaluated using an aqueous sodium chloride solution (hereinafter referred to as isotonic solution) with a concentration of 154 mM, which has the same ionic strength as blood, as the sample solution 402, a blood cell solution, and a plasma solution. The blood cell solution was prepared by the following procedure.

[0040] Step (i-1): Whole blood was separated into plasma and blood cells by centrifugation at 1500 g for 15 minutes. Step (i-2): After removing as much plasma as possible, it was made up to the removed volume with isotonic solution. Step (i-3): It was inverted and stirred to resuspend. Step (i-4): Plasma was replaced with isotonic solution by repeating steps (i-1) to (i-3) three times. Step (i-5): Isotonic solution was added to the blood cell solution prepared in step (i-4) and made up to the same volume as the original blood solution. Hereinafter, this solution is referred to as 100% blood cell solution.

[0041] The plasma solution was prepared as follows. Step (ii-1): The hematocrit value of whole blood was measured with a blood cell counter (XN-330, Sysmex). Step (ii-2): Whole blood was separated into plasma and blood cells by centrifugation at 1500 g for 15 minutes. Step (ii-3): Only the plasma was transferred to another tube, and the volume fraction of the blood cells measured in step (ii-1) was made up with isotonic solution (hereinafter, this solution is referred to as 100% plasma solution).

[0042] (2) Figure 6 is a diagram showing the results of a capture rate evaluation experiment using an isotonic solution, a blood cell solution, and a plasma solution. From the evaluation results shown in Figure 6, the inventors found that when a plasma solution was used as the specimen solution 402, the capture rate deteriorated significantly. Furthermore, from several additional experiments not shown here, it was found that the production of the conjugate 405 of the microorganism and the microorganism capture reagent was likely inhibited by biological substances centered on the proteins contained in the plasma.

[0043] (3) Therefore, in order to reduce the influence of biological substances in plasma, an attempt was made to improve the collection rate by diluting the blood. Specifically, a 100% plasma solution was diluted with an isotonic solution (an aqueous sodium chloride solution with a concentration of 154 mM) (diluting the plasma concentration from 100% to 25%) to evaluate whether the collection rate could be improved. Figure 7 is a diagram showing the change in the bacterial collection rate (experimental result) corresponding to the change in the plasma suspension concentration (%). As shown in Figure 7, it was found that even when the 100% plasma solution was diluted with the isotonic solution to 25%, no effect of improving the collection rate was observed.

[0044] (4) Next, the same experiment was conducted by diluting with a 10 mM aqueous sodium chloride solution having a lower concentration than the isotonic solution. Figure 8 is a diagram showing the change in the bacterial collection rate (experimental result) corresponding to the change in the sodium chloride (salt) concentration. According to this experiment, it became clear that the collection rate was significantly improved to 87% when the salt concentration was diluted to 118 mM. Since the salt concentration decreased below that of the blood, there is a possibility that biological components such as proteins contained in the plasma were denatured and the electrostatic binding properties changed.

[0045] (5) Based on the above results, an experiment was conducted to serially dilute whole blood with a 10 mM aqueous sodium chloride solution and confirm the change in the collection rate. Figure 9 is a diagram showing the change in the bacterial collection rate (experimental result) corresponding to the change in the ionic strength in whole blood. From Figure 9, it was found that the collection rate, which was less than 50% in whole blood with an ionic strength of 154 mM, was obtained at an average high collection rate of 92% by diluting to 112.5 mM. In Figure 9, it was found that the collection rate deteriorated and the variation also increased when the ionic strength was around 46 mM.

[0046] (6) In order to confirm the cause of the deterioration of the collection rate and the increase in variation when the ionic strength was around 46 mM, additional experiments were conducted. That is, a blood cell solution was diluted using a 100% 10 mM aqueous sodium chloride solution, and the diluted solution with the salt concentration ranging from 118 mM to 46 mM was used as the sample solution 402, and the same experiment was carried out.

[0047] Figure 10 is a diagram showing the change in the bacterial collection rate (experimental result) corresponding to the change in the concentration of the sodium chloride aqueous solution (salt) in the blood cell suspension by the additional experiment. As shown in Figure 10, as the salt concentration decreased, the variation tended to increase. In particular, in the blood cell solution with a salt concentration of 46 mM, although the average collection rate was as high as 90%, samples with a collection rate decreased to 74% were observed, suggesting that it might affect the collection rate stability.

[0048] (7) Therefore, in order to investigate the cause of the increasing variation, microscopic observations were performed for each concentration of the blood cell solution. Figure 11 is a diagram showing the microscopic images of each blood cell solution concentration. As shown in Figure 11, in the blood cell solutions with salt concentrations of 154 mM and 118 mM, red blood cells remained while maintaining their shape, but the number of red blood cells decreased in the blood cell solution with a salt concentration of 82 mM, and most red blood cells hemolyzed in the blood cell solution with a salt concentration of 46 mM, and swelling was confirmed in the remaining blood cells.

[0049] From the above, it was suggested that the decrease in ionic strength caused red blood cells to hemolyze, and the leaked components, such as hemoglobin (protein), might inhibit the binding between magnetic particles and bacteria. Therefore, as a further additional experiment, in order to confirm the effect of hemoglobin on the bacterial collection rate, an evaluation was performed using a pure hemoglobin solution simulating the completely hemolyzed blood cell solution with a salt concentration of 46 mM. As a result, the average collection rate was 61% (maximum: 72%, minimum: 50%), suggesting that the collection rate might decrease significantly due to the influence of hemoglobin leaking from red blood cells.

[0050] Based on the above experimental results, when concentrating bacteria from a blood specimen using the microbial collection reagent 100, it is desirable to dilute the salt concentration in the blood with a diluent. More specifically, it was found that the concentration range should be 82 mM or more and less than 154 mM, preferably in the range of 82 mM to 118 mM. If the dilution rate is too high, the volume of the diluted solution will increase, which may complicate the subsequent processing or increase the reagents required for the subsequent processing. Therefore, it is desirable that the dilution be minimized, for example, it can be 118 mM.

[0051] According to such a configuration, it is possible to collect trace amounts of bacteria contained in whole blood with high efficiency of 90% or more. In addition, by adopting a dimethylamino group, it can be produced by chemical synthesis and can be stored for a long time at room temperature.

[0052] <Procedure for recovering microorganisms (bacteria) from a sample, including the step of adding a diluent> Figure 12 is a flowchart for explaining a modified example of the microorganism recovery process including the step of adding a diluent, which was devised based on the facts discovered so far. In Figure 12, based on the flowchart of Figure 3, a new step of adding a diluent is added. More specifically, the operator (user) adds a diluent to the container 400 together with the patient sample containing the microorganism 104 (step S1201). This denatures the biological substances contained in the sample. Next, the operator adds the microorganism collection reagent 100 to the container 400 (S1202). The subsequent steps are the same as steps S302 to S309 shown in Figure 3.

[0053] The diluent may be a liquid with an ionic strength lower than the salt concentration of blood (154 mM). For example, not only an aqueous solution of sodium chloride at 10 mM but also water or other liquids may be used.

[0054] According to the treatment of this modified example, it is possible to collect trace amounts of bacteria contained in whole blood with high efficiency of 90% or more. In addition, by adopting a dimethylamino group, it can be produced by chemical synthesis and can be stored for a long time at room temperature.

[0055] <Amplification result by PCR> Figure 13 is a diagram showing the electrophoresis pattern of the product obtained by mixing the microorganism collection reagent according to this embodiment and performing PCR. Band 1301 indicates the band amplified by PCR after collecting bacteria by mixing the microorganism collection reagent 100 with a solution not containing blood. Band 1302 indicates the band amplified by PCR after collecting bacteria by mixing the microorganism collection reagent 100 with blood.

[0056] Referring to FIG. 13, band 1303 appears in both cases. That is, when the microbial collection reagent 100 according to the present embodiment is mixed with blood to perform microbial (bacteria) collection treatment, it can be seen that PCR is not inhibited by hemoglobin in the blood. Therefore, according to the present embodiment, bacteria can be efficiently recovered, and by amplifying it by PCR, the genes of the bacteria can be detected.

[0057] <Examples of groups for surface modification of magnetic particles (magnetic beads)> The causative bacteria contained in the blood of patients with bloodstream infections are diverse. For example, not only Staphylococcus aureus, which is a Gram-positive bacterium, but also Escherichia coli, which is a Gram-negative bacterium, may be present. The surface structures of Gram-negative bacteria and Gram-positive bacteria are very different, and in some cases, sufficient collection rates may not be obtained only with magnetic particles 101 having a dimethylamino group 103. Therefore, magnetic particles (second microbial collection reagent 1401) modified with a microbial binding part other than the dimethylamino group 103 may be added to the microbial collection reagent 100.

[0058] FIG. 14 is a diagram showing magnetic particles modified with ethylpyridinium bromide. The ethyl group 1402 of ethylpyridinium bromide is known to bind well to Escherichia coli. By adding magnetic particles (second microbial collection reagent 1401) subjected to two or more surface modifications to the microbial collection reagent 100, it becomes possible to stably collect a wider range of microorganisms.

[0059] According to such a configuration, it becomes possible to recover various types of trace bacteria contained in whole blood with a high efficiency of 90% or more.

[0060] <Microbial collection kit> The above-described microorganism collection reagent 100 and a diluent for reducing the ionic strength of a target specimen (such as blood) (as described above, any liquid with an ionic strength lower than the salt concentration of blood (154 mM) is acceptable. For example, not only an aqueous solution of 10 mM sodium chloride, but also water or other liquids may be used) can be set to form a microorganism collection kit. The microorganism collection kit can be configured, for example, by combining a predetermined container (such as container 400 used for microorganism collection) that pre-stores a predetermined amount of diluent, and a container or medicine wrapping paper (such as a plastic bag) that stores the microorganism collection reagent 100 separately from the container, as a set. From the above experiments, it was found that a good recovery rate can be obtained if the specimen is diluted to about 80%. Therefore, for a target specimen (such as blood), about 20% by volume of water or an aqueous solution of 10 mM sodium chloride as a diluent can be pre-stored in the container included in the above microorganism collection kit. The amount of the target specimen (blood) varies depending on the application. For example, when processing 5 mL of blood, the amount of diluent to be stored in the container of the microorganism collection kit is 1 mL. In this way, by providing multiple types of microorganism collection kits with different storage volumes of diluent, users can easily cope with various applications and the processing of various types of specimens.

[0061] <Configuration example of microorganism inspection system> FIG. 15 is a diagram showing a configuration example of a microorganism inspection system that automatically identifies the causative bacteria of bloodstream infections and inspects drug resistance genes as needed.

[0062] The microorganism inspection system according to this embodiment includes a microorganism inspection device 1500 and a computer 1517 that controls the operation of the microorganism inspection device 1500. The microorganism inspection device 1500 includes a stirring mechanism 1505 that applies vibrations or the like to a blood collection container 1501 containing patient blood 1502, bacteria (germs) 1503, and a microorganism collection reagent 1504 to stir the contents, a magnetic particle collection mechanism 1506, a dispensing mechanism 1508 including a pump 1509, a nozzle drive mechanism 1510, and reagent containers 1511 - 1512 and 1518 - 1519, a nucleic acid extraction unit 1513, a nucleic acid amplification unit 1514, a nucleic acid detection unit 1515, and a microcontroller 1516.

[0063] First, an operator (a medical institution's laboratory technician) collects a patient's blood into a blood collection container (blood collection tube) 1501. An anticoagulant (not shown) is added to the blood collection tube as necessary. When the blood collection tube completed by the operator is set in the microorganism inspection device 1500, a diluent is injected from the reagent container 1511 by the nozzle 1507 of the dispensing mechanism 1508 under the control of the computer 1517 and the microcontroller 1516. The stirring mechanism 1505 stirs the patient blood 1502 and the diluent. Subsequently, the microorganism collection reagent 1504 is injected from the reagent container 1512 by the nozzle 1507 of the dispensing mechanism 1508. The stirring mechanism 1505 stirs the diluted patient blood 1502 and the microorganism collection reagent 1504 to promote the binding of the bacteria 1503 contained in the patient blood 1502 and the microorganism collection reagent 1504. Here, as the stirring mechanism 1505, a vortex mixer, a stirrer, a shaker, etc. are applicable.

[0064] After stirring for a predetermined time, under the control of the microcontroller 1516, the magnetic particle collection mechanism 1506 adsorbs the conjugate of the microbial collection reagent 1504 and the bacteria 1503 in the patient blood 1502. Next, the nozzle 1507 of the dispensing mechanism 1508 sucks out the supernatant and then turns off the magnetic particle collection mechanism 1506 under the control of the microcontroller 1516. Further, a cleaning solution is injected from the reagent container 1518, and the solution is stirred again by the stirring mechanism 1505 to wash away the residue of the blood components attached to the conjugate of the microbial collection reagent 1504 and the bacteria 1503. After repeating the above cleaning procedure a predetermined number of times, the dispensing mechanism 1508 sucks up and discards the last cleaning solution.

[0065] Next, the microcontroller 1516 turns off the magnetic particle collection mechanism 1506, injects a diluent from the reagent container 1511, and uses the stirring mechanism 1505 to stir the solution to prepare a suspension of the conjugate of the microbial collection reagent 1504 and the bacteria 1503. The solution used for suspension does not necessarily have to be a blood diluent, and a dedicated suspension solution may be injected from the reagent container 1519 according to the requirements of the subsequent nucleic acid extraction step.

[0066] Subsequently, under the control of the microcontroller 1516, the dispensing mechanism 1508 sucks up the suspension and injects the suspension into the nucleic acid extraction unit 1513. The nucleic acid extraction unit 1513 destroys the cell wall of the bacteria with a lysing reagent and extracts the nucleic acid of the bacteria. Further, under the control of the microcontroller 1516, the dispensing mechanism 1508 transfers the nucleic acid extract to the nucleic acid amplification unit 1514. The nucleic acid amplification unit 1514 performs a gene amplification reaction. Here, the gene amplification reaction is performed using the PCR method, or the LAMP method or other isothermal amplification techniques.

[0067] Finally, the dispensing mechanism 1508 transfers the amplified product to a gene detection unit (not shown). The gene detection unit detects whether or not the target nucleic acid is present by fluorescence observation, electrophoresis, gene hybridization, or the like. The microcontroller 1516 controls the operations of the respective functional blocks according to a predetermined procedure, transmits the results to an external computer 1517, receives control commands from the external computer 1517, and controls the operations of the respective functional blocks based thereon. Also, the nucleic acid targeted by the microorganism inspection apparatus 1500 is preferably 16s ribosomal DNA when identifying bacteria, but is not limited thereto. Furthermore, it is also possible to preliminarily evaluate the effectiveness of an antibacterial agent by looking at the region of the drug resistance gene among the nucleic acids.

[0068] Note that the sample targeted by the technique according to the present embodiment is not limited to blood, and may be various samples suspected of being infected with microorganisms, such as cerebrospinal fluid, saliva, urine, lymph fluid, nasal discharge, and exudate from the affected area. Also, it may be a culture or a suspension thereof after blood culture or urine culture.

[0069] <Summary of the Embodiment> (i) This embodiment proposes a microorganism collection reagent 100 that enhances the collection rate (recovery rate) of microorganisms (bacteria). The microorganism collection reagent 100 is configured by modifying the surface of magnetic particles (magnetic particles: for example, 10 nm or more and 1 μm or less, preferably 100 nm or less) that are smaller than the size of the microorganism to be collected with a microorganism binding portion formed by dimethylamino groups 103. Since the methyl groups _Me contained in the dimethylamino groups are positively charged, they electrostatically bind to negatively charged microorganisms (bacteria). As a result, microorganisms can be efficiently collected. Note that the smaller the particle size of the magnetic particles, the larger the total surface area of the microorganism collection reagent can be, so the recovery efficiency can be increased.

[0070] In addition, the microorganism collection reagent 100 may contain magnetic particles 101 whose surface is modified with a microorganism-binding portion formed by a group other than a dimethylamino group (for example, the ethyl group of ethylpyridinium bromide). This makes it possible to collect microorganisms that are difficult to collect by the dimethylamino group. By modifying the ethyl group of ethylpyridinium bromide, it becomes possible to improve the recovery rate of Escherichia coli.

[0071] (ii) This embodiment proposes a microorganism collection kit in which the above microorganism collection reagent 100 and a diluent for reducing the ionic strength of a target sample are set. As the diluent, an aqueous sodium chloride solution with a concentration of less than 154 mM can be used. The diluent may be, for example, stored in a container 400 in advance and provided, and at the actual site of sample inspection, the sample and the above microorganism collection reagent 100 may be put into it for use.

[0072] In addition, in the microorganism collection reagent included in the microorganism collection kit, in addition to the magnetic particles modified with the above dimethylamino group, magnetic particles whose surface is further modified with a microorganism-binding portion formed by a group other than the dimethylamino group (for example, the ethyl group of ethylpyridinium bromide) may be included.

[0073] (iii) This embodiment also proposes a microorganism collection method for collecting microorganisms contained in a specimen using a microorganism collection reagent in which a microorganism binding part formed by a dimethylamino group is modified on the surface of magnetic particles. Specifically, this method includes adding a microorganism collection reagent 100, in which a microorganism binding part formed by a dimethylamino group is modified on the surface of magnetic particles 101, to a specimen (for example, patient blood), binding the microorganism collection reagent to the microorganisms contained in the specimen, and recovering (collecting) the conjugate of the microorganism collection reagent 100 and the microorganisms. By doing so, microorganisms (bacteria) can be efficiently recovered using the microorganism collection reagent. A recovery rate of 43% can be obtained without adding a diluent, and the collection rate (recovery rate) can be further improved by adding a diluent to reduce the ionic strength of the specimen. For example, when the ionic strength is reduced to 82 mM or more and less than 154 mM by adding a diluent, the collection rate can be made 87% or more.

[0074] When adding the microorganism collection reagent 100 to the specimen, it is preferable that one or more magnetic particles 101 are contained per 1 pL of the specimen, and more preferably, about 100 magnetic particles 101 are contained per 1 pL of the specimen. By having about 100 magnetic particles 101 per 1 pL of the specimen, a state where at least one magnetic particle exists around the bacteria can be created.

[0075] (iv) This embodiment also proposes a microorganism collection device (a device including a part of the functions (microorganism collection function) of the microorganism inspection device 1500) that collects microorganisms contained in a specimen using a microorganism collection reagent containing magnetic particles 101. The microorganism collection device has a device configuration that realizes the above microorganism collection method. Specifically, the microorganism collection device includes a stirrer mechanism 1505 that promotes the binding between bacteria 1503 contained in the specimen and magnetic particles (microorganism collection reagent 1504) by stirring the specimen (for example, blood) and the microorganism collection reagent 100 contained in the blood collection container 1501, a magnetic particle collection mechanism 1506 that collects the conjugate of bacteria 1503 and magnetic particles (microorganism collection reagent 1504), a dispensing mechanism 1508 that sucks out the supernatant liquid in the container and injects a washing liquid and a diluent into the blood collection container 1501, and a microcontroller (control unit) 1516 that controls the operations of the stirrer mechanism 1505, the magnetic particle collection mechanism 1506, and the dispensing mechanism 1508. Here, the microcontroller 1516 reduces the ionic strength of the specimen (blood) in the blood collection container 1501 by injecting a diluent into the blood collection container 1501. Specifically, the microcontroller 1516 injects a diluent into the blood collection container 1501 so as to reduce the ionic strength to 82 mM or more and less than 154 mM. For example, when water or a 10 mM sodium chloride aqueous solution with a volume of about 20% of the amount of the specimen is injected into the blood collection container 1501 as the diluent, the ionic strength can be reduced to 82 mM or more and less than 154 mM. After dilution, the microcontroller 1516 controls the dispensing mechanism 1508 to suck out the supernatant liquid and wash the conjugate with the washing liquid. Finally, the washing liquid is sucked out. According to such a configuration, the user can automatically obtain the conjugate of bacteria 1503 and the microorganism collection reagent 1504 in the specimen with a simple operation. The microorganism collection method disclosed in this technology causes less damage to bacteria, and the obtained conjugate can be appropriately suspended and cultured directly on an agar medium. Thereby, it is possible to quantify the number of bacteria by colony counting. In addition to culturing, it can also be appropriately used for other conventional microorganism inspections such as staining and observation with an optical microscope.

[0076] (v) Note that the technology of the present disclosure is not limited to the above embodiments and includes various modifications. The above embodiments and examples have been described in detail to explain the technology of the present disclosure clearly, and are not necessarily limited to those having all the configurations described. Also, it is possible to replace a part of the configuration of one example with the configuration of another example, and it is also possible to add the configuration of another example to the configuration of one example. Further, it is possible to add, delete, or replace other configurations for a part of the configuration of each example.

Explanation of Signs

[0077] 100, 1504 Microorganism collection reagent 101, 200 Magnetic particles 102 Surface modification 103 Dimethylamino group 104 Microorganism (bacteria) 201 Carboxyl group 202 N, N-Dimethyl-1, 3-propanediamine 400 Container 401 Dried Staphylococcus aureus 402 Specimen solution 403 Staphylococcus aureus 404 Magnetic stand 405 Conjugate of microorganism and microorganism collection reagent 406 Pipette 407 Culture medium 1301 Electrophoresis pattern of the product obtained by mixing microorganism collection reagents and performing PCR 1302 Electrophoresis pattern of the product obtained by mixing microorganism collection reagent and blood and performing PCR 1303 Target band (75bp) 1401 Second microorganism collection reagent 1402 Ethyl group of ethylpyridinium bromide 1500 Microorganism inspection device 1501 Blood collection container 1502 Patient blood 1503 Bacteria 1505 Stirring mechanism 1506 Magnetic particle collection mechanism 1507 Nozzle 1508 Dispensing mechanism 1509 Pump 1510 Nozzle drive mechanism 1511, 1512, 1518, 1519 Reagent containers 1513 Nucleic acid extraction unit 1514 Nucleic acid amplification unit 1515 Nucleic acid detection unit 1516 Microcontroller 1517 Computer

Claims

1. A microorganism collection reagent in which a microorganism binding part with a dimethylamino group is modified on the surface of magnetic particles.

2. In Claim 1, The microorganism collection reagent, wherein the particle size of the magnetic particles is 10 nm or more and smaller than the size of the microorganism to be collected.

3. In Claim 1, The microorganism collection reagent, further comprising magnetic particles in which a microorganism binding part with a group other than the dimethylamino group is modified on the surface.

4. In Claim 3, The microorganism collection reagent, wherein the group other than the dimethylamino group is an ethyl group of ethylpyridinium bromide.

5. A microorganism collection kit comprising the microorganism collection reagent according to Claim 1 and a diluent for reducing the ionic strength of the specimen to be tested.

6. In Claim 5, The microorganism collection kit, wherein the diluent is an aqueous sodium chloride solution having a concentration of less than 154 mM.

7. In Claim 5, The microorganism collection kit, wherein the microorganism collection reagent further comprises magnetic particles in which a microorganism binding part with a group other than the dimethylamino group is modified on the surface.

8. In Claim 7, The microorganism collection kit, wherein the group other than the dimethylamino group is an ethyl group of ethylpyridinium bromide.

9. A microorganism collection method for collecting microorganisms contained in a specimen using the microorganism collection reagent according to Claim 1, comprising: adding the microorganism collection reagent to the specimen; binding the microorganism collection reagent to the microorganisms contained in the specimen; and recovering the conjugate of the microorganism collection reagent and the microorganisms.

10. In Claim 9, further comprising reducing the ionic strength of the specimen by adding a predetermined diluent.

11. In Claim 10, the specimen is blood, and when reducing the ionic strength, the ionic strength is reduced to 82 mM or more and less than 154 mM.

12. In Claim 9, The microorganism collection method, wherein the microorganism collection reagent is added so that one or more magnetic particles are contained per 1 pL of the specimen.

13. A microorganism collection apparatus for collecting microorganisms contained in a specimen using a microorganism collection reagent containing magnetic particles, comprising: a stirrer mechanism housed in a container for promoting the binding between the bacteria contained in the specimen and the magnetic particles by stirring the specimen and the microorganism collection reagent. A magnetic particle collection mechanism for collecting the conjugate of the bacterium and the magnetic particles, A dispensing mechanism for sucking out the supernatant liquid in the container and injecting a cleaning liquid and a dilution liquid into the container, A control unit for controlling the operations of the stirring mechanism, the magnetic particle collection mechanism, and the dispensing mechanism, The control unit controls the dispensing mechanism and injects the dilution liquid into the container to reduce the ionic strength of the sample in the container, and sucks out the supernatant liquid and cleans the conjugate with the cleaning liquid. A microorganism collection device.

14. In claim 13, The control unit injects the dilution liquid into the container so as to reduce the ionic strength to 82 mM or more and less than 154 mM. A microorganism collection device.

15. In claim 14, The control unit injects the dilution liquid of 20% of the amount of the sample into the container. A microorganism collection device.

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