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

Abstract

The present disclosure proposes a microorganism collecting reagent 100 (see FIG. 1) in which a surface of a magnetic particle 101 is modified with a dimethylamino group 103 serving as a microorganism binding moiety, in order to provide: a microorganism collecting reagent that can be stored for a long period of time at normal temperature and that can specifically and highly efficiently capture and concentrate microorganisms present at a low level in blood without requiring centrifugal operation; a microorganism collecting kit; and a microorganism collecting procedure using same.

Description

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

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

[0002] Infectious disease testing requires rapid pathogen detection to expedite the initiation of appropriate treatment and prevent secondary infection. Bloodstream infections, particularly those typified by sepsis, often progress to shock and worsen with even a small number of bacteria present in the bloodstream. It has been reported that in severe sepsis, the survival rate drops by 7.6% every hour if antibiotic treatment is delayed (A. Kumar et al., Crit Care Med 2006). Therefore, if a bloodstream infection is suspected, it is desirable to begin antibiotic treatment as quickly as possible, preferably within one hour. Because causative bacteria are diverse and different antibiotics are effective for each, it is advisable to test for multiple bacteria simultaneously and administer the appropriate antibiotic promptly.

[0003] However, the number of bacteria contained in the blood of patients with bloodstream infections is as low as 1 CFU (Colony forming unit) / mL in some cases, making it difficult to detect in its current state. Currently, blood samples of approximately 10 mL are collected and cultured overnight or longer to detect 10 ６ The test is conducted after the number of bacteria has been increased by more than double. Until the test results are available, broad-spectrum antibiotics or cocktails of multiple antibiotics that can cover all possible bacteria are administered. However, there is concern that administering these antibiotics may increase drug-resistant bacteria that are not affected by antibiotics. Furthermore, narrow-spectrum antibiotics that are suited to the pathogen often have a greater therapeutic effect than broad-spectrum antibiotics.

[0004] The PCR (Polymerase Chain Reaction) method can synthesize 10 genes of bacteria in one hour. ８The PCR method can amplify the DNA by about 2-fold, enabling faster detection of bacteria compared to conventional blood culture-based tests. By using the PCR method, it is possible to switch from broad-spectrum antibiotics to narrower-spectrum antibiotics at an early stage. This not only achieves a higher therapeutic effect but also suppresses the emergence of resistant bacteria, making it a promising technology for treating bloodstream infections.

[0005] However, performing PCR directly on approximately 10 mL of collected blood is not practical because the large amounts of hemoglobin and proteins present in blood inhibit PCR and require a huge amount of reagents. Therefore, it is common to concentrate and purify bacteria in the blood in advance, extract the bacterial genes, and then subject them to PCR. This concentration and purification process has traditionally been performed manually by skilled laboratory technicians. However, in hospital laboratories with limited manpower and budgets, it is desirable to obtain test results automatically with minimal human intervention, and it is also necessary to minimize the testing cost per sample. Furthermore, since a wide variety of testing equipment must be installed in a limited space, it is desirable to minimize the footprint per device.

[0006] For example, Patent Document 1 discloses a procedure for a fully automated sepsis testing device in which red blood cells, which are the most abundant in blood, are destroyed with a lysing agent, and then the target bacteria are concentrated and obtained as a pellet by centrifugation. According to Patent Document 1, the type of bacteria present in the blood of a patient with a bloodstream infection can be rapidly identified by extracting and amplifying the nucleic acid of the bacteria from the obtained bacterial pellet, and detecting the aggregation state of magnetic particles (magnetic beads) that specifically bind to the bacterial nucleic acid using magnetic resonance.

[0007] Furthermore, Patent Document 2 discloses a technology in which bacteria present in a patient's whole blood are captured using magnetic particles (magnetic beads) whose surfaces are modified with molecules having a microorganism-binding domain such as lectin, the magnetic particles are collected with a magnet, blood components are removed with Tris-buffered saline, and the bacteria are detected and quantified by ELISA. According to Patent Document 2, this technology is advantageous in terms of reducing the size of the device, since it is possible to concentrate bacteria without using a large-scale mechanism such as a centrifuge.

[0008] US Patent Application Publication No. 2017 / 0233798 JP 2014-523914 A

[0009] Sayed, SM et al. (2021) Naphthalimide-based multifunctional AIEgens Selective, fast, and wash-free fluorescence tracking and identification of Gram-positive bacteria. Analytica Chimica Acta. 1146. 41-52.

[0010] However, the testing device disclosed in Patent Document 1 requires the installation of a rotor mechanism for centrifugation, which increases the size of the device. It is also difficult to separate cells contained in blood that have a similar shape to bacteria. For example, platelets are approximately 2 μm in size, similar in size to bacteria (slightly less than 1 μm to several μm). Furthermore, there are approximately 1.5 to 4.5 billion platelets in 1 mL, far outnumbering bacteria at a concentration of 1 CFU / mL. For these reasons, it is difficult to selectively separate only bacteria using centrifugation.

[0011] Furthermore, the method described in Patent Document 2 requires the use of lectins, which are proteins. Generally, proteins are susceptible to degradation by microorganisms in the environment and to functional degradation due to oxidation. This makes it difficult to store proteins at room temperature for long periods of time. This requires the addition of preservatives such as antiseptics and antioxidants, or frozen storage. Some preservatives contain components that inhibit the activity of polymerase, an enzyme used in PCR, and therefore, the removal of these preservatives is necessary to detect bacteria using PCR.

[0012] On the other hand, the need for frozen storage increases reagent storage costs, leading to higher testing costs. Bloodstream infections occur at random times and are often treated urgently, so reagents must be prepared well in advance, which increases laboratory operating costs. If reagents could be stored at room temperature for long periods, these storage costs could be reduced.

[0013] In light of this situation, the present disclosure proposes a microbial collection technology that enables specific and highly efficient (high recovery rate) capture and concentration of microorganisms present in small amounts in blood without the need for centrifugation, and also enables long-term storage at room temperature.

[0014] In order to solve the above problems, the present disclosure proposes a microorganism collection reagent in which the surfaces of magnetic particles are modified with microorganism binding moieties using dimethylamino groups.

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

[0016] According to the technology of the present disclosure, a reagent that can be stored for a long period of time at room temperature makes it possible to efficiently capture microorganisms in blood without the need for large-scale equipment.

[0017] 1 is a diagram showing an example of the structure of a microorganism collection reagent 100 according to this embodiment;

[0023] FIG. 1 is a diagram showing reactions in a production process of the microorganism collection reagent 100;

[0024] FIG. 2 is a flowchart explaining the procedure for collecting trace amounts of bacteria from the blood of a patient with a bloodstream infection using the microorganism collection reagent 100;

[0025] FIG. 3 is a diagram showing specific steps of a bacterial collection rate evaluation experiment based on the flowchart of FIG. 3;

[0026] FIG. 4 is a diagram showing the evaluation results of the collection rate (results when water was used as the specimen solution 402 and when human whole blood was used);

[0027] FIG. 5 is a diagram showing the results of a collection rate evaluation experiment using an isotonic solution, a blood cell solution, and a plasma solution;

[0028] FIG. 6 is a diagram showing the change in bacterial collection rate corresponding to a change in plasma suspension concentration (%) (experimental results);

[0029] FIG. 7 is a diagram showing the change in bacterial collection rate corresponding to a change in sodium chloride (salt) concentration (experimental results);

[0029] FIG. 8 is a diagram showing the change in bacterial collection rate corresponding to a change in ionic strength in whole blood (experimental results);

[0029] FIG. 9 is a diagram showing the change in bacterial collection rate corresponding to a change in sodium chloride aqueous solution (salt) concentration in a blood cell suspension from an additional experiment (experimental results);

[0029] FIG. 10 is a diagram showing microscopic images of each blood cell solution concentration. Fig. 4 is a flowchart illustrating a microorganism (bacteria) collection process devised based on the discovered facts, which includes a step of adding a diluent, and is a modified example of the microorganism collection process of Fig. 3. Fig. 5 is a diagram showing an electrophoresis pattern of a product obtained by PCR after mixing a microorganism collection reagent according to this embodiment. Fig. 6 is a diagram showing magnetic particles (magnetic beads) modified with ethylpyridinium bromide. Fig. 7 is a diagram showing an example of the configuration of a microorganism testing system that automatically identifies causative bacteria of bloodstream infections and tests for drug resistance genes as necessary.

[0018] The 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 components (including element steps, etc.) are not necessarily essential unless otherwise specified or considered to be obviously essential in principle.

[0019] In order to facilitate understanding of the invention, the position, size, shape, range, etc. of each component shown in the drawings may not represent the actual position, size, shape, range, etc. Therefore, the present invention is not necessarily limited to the position, size, shape, range, etc. disclosed in the drawings.

[0020] <Structure of Microorganism Collection Reagent and Method for Producing the Reagent> (i) FIG. 1 is a diagram showing an example of the structure of a microorganism collection reagent 100 according to this embodiment. The microorganism collection reagent 100 comprises magnetic particles 101 that have been surface-modified 102 with dimethylamino groups 103. The dimethylamino groups 103 are characterized by having two methyl groups Me, which are positively charged. On the other hand, it is known that the cell walls on the surface of bacteria 104 contain negatively charged sites due to membrane proteins. This electrostatically bonds the bacteria 104 with the dimethylamino groups 103. Utilizing this effect, it is possible to capture microorganisms present in a patient's specimen. Furthermore, because the reagent does not contain proteins, it can be stored for long periods at room temperature.

[0021] Non-Patent Document 1 discloses a method for specifically staining bacteria using a dye containing a dimethylamino group. The dimethylamino group is positively charged, and electrostatically binds to negatively charged bacteria, thereby staining the bacteria. In other words, in Non-Patent Document 1, the dimethylamino group is used not for binding bacteria 104 but for staining the bacteria. In contrast, in this embodiment, magnetic particles (magnetic beads) modified with dimethylamino groups are used to realize a reagent that enables bacterial recovery without containing proteins with poor long-term stability. However, as described below (see the experimental results in Figures 5 and 6), our experiments have shown that using dimethylamino-modified magnetic particles for bacteria contained in blood can recover bacteria relatively efficiently, but the recovery rate remains at around 43%, leaving room for improvement. Furthermore, we found that plasma components may inhibit the binding of bacteria to the magnetic particles. Further improving the recovery rate is an important challenge for detecting bacteria present in small amounts in the blood of sepsis patients.

[0022] (ii) The microorganism collection reagent 100 was prepared by the following method. Figure 2 is a diagram showing the reactions in the process of preparing the microorganism collection reagent 100. As shown in Figure 2, magnetic particles 200 (SC0100, Ocean Nanotech) with a particle diameter of 100 nm and surface-modified with carboxyl groups 201 and N,N-dimethyl-1,3-propanediamine 202 (D0790, Tokyo Chemical Industry Co., Ltd.) were subjected to dehydration condensation using a kit (KSC0100-04, Ocean Nanotech) to prepare the microorganism collection 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 patient with a bloodstream infection using the microorganism collection reagent 100.

[0024] (i) Step S301 The operator (user) places blood (specimen solution 402) collected from a patient with a bloodstream infection and the microorganism collection reagent 100 into a container 400. The container 400 may be, for example, a microtube or centrifuge tube used in biological experiments, and may have a surface treated to inhibit microbial adsorption. Alternatively, the microorganism collection reagent 100 may be added directly to the blood collection tube used for blood collection. Alternatively, the microorganism collection reagent 100 may be added directly to the blood culture bottle after blood culture in the blood culture bottle.

[0025] (ii) Step S302: The operator stirs the contents of the container 400. Stirring can be performed, for example, using a thermoshaker maintained at a temperature of 25°C. The stirring time is preferably a time required for the microorganism collection reagent 100 to sufficiently bind to the majority of the bacteria in the specimen, for example, 15 minutes.

[0026] (iii) Step S303 The operator brings a magnet (for example, a magnetic stand used in biological experiments) close to the container 400, causing the aggregates of the microorganism collection reagent 100 and the microorganisms 104 dispersed in the container 400 to clump together on the inner wall of the container 400. The time required for clumping 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 combined bodies of the microorganism collecting reagent 100 and the microorganisms 104 (S309).

[0028] If it is desired to remove as many other components (for example, blood components) contained in the sample as possible, the operations from step S304 to step S307 may be performed after step S303 and before step S308.

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

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

[0031] (vii) Step S306 The operator agitates the container 400 to wash away components adhering to the combination of the microorganism collecting reagent 100 and the microorganisms 104.

[0032] (viii) Step S307 The operator again brings the magnet close to the container 400, causing the combined microorganism collection reagent 100 and microorganisms 104 to aggregate on the inner wall of the container. The operator repeats the processes of S304 to S307 until the other components have been removed to a desired level.

[0033] (ix) Steps S308 and S309 The operator discards the supernatant from the container 400 (S308), and obtains the combined bodies of the microorganism collecting 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 examples of the solution that can be used include water, saline, phosphate buffer solution (PBS), and Pluronic (registered trademark) water.

[0035] <Experiment to Evaluate Bacterial Recovery Rate> Fig. 4 is a diagram showing specific steps of an experiment to evaluate a bacterial recovery rate based on the flowchart of Fig. 3. An experiment to evaluate the recovery rate of bacteria using the microorganism collection reagent 100 was carried out according to the specific steps shown in Fig. 4.

[0036] A specimen solution 402 and dried Staphylococcus aureus 401 (BioBall S. aureus 30 CFU, bioMérieux Japan) were introduced into the container 400 and vortexed (by inversion for samples containing blood components) (step (i): S301). Next, 10 μL of a 20 mg / mL microorganism collection reagent 100 was added (step (ii): S302). The solution was then shaken and stirred at 25°C and 1000 rpm for 15 minutes in a thermoshaker (BSR-MS100, Thermo Fisher) to allow the microorganism collection reagent 100 to bind to the Staphylococcus aureus 403 (step (iii): S303). A relatively high concentration of beads (magnetic particles) is desirable for rapid binding of low concentrations of bacteria to the beads. The amount of beads added this time was 10 per mL of specimen 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, there will be 100 particles per side. １／３ = 4.64 beads. In other words, if the beads are arranged in a lattice pattern, the spacing between the lattices is 10 / 4.64 = 2.2 μm. Since the size of Staphylococcus aureus is approximately 1 μm, the concentration of beads is such that they are present within 1 μm of the Staphylococcus aureus. Next, after the microorganism collection reagent 100 and the Staphylococcus aureus 403 are bound, a magnetic stand 404 (Dynamag-2, Thermo Fisher) is applied to the side of the container 400 for 10 minutes, and a bound body 405 of the microorganism and the microorganism collection reagent is captured on the inner wall of the container (step (iv): S304). Next, a total of 1 mL of the supernatant was collected with a pipette 406 so as not to affect the combined microorganisms and microorganism collection reagent 405 (step (v): S308), after which the magnetic stand 404 was removed from the container 400, and 100 μL of Pluronic water was added to resuspend the collected microorganisms (step (vi)). Both the collected supernatant and the suspension containing the combined microorganisms and microorganism collection reagent 405 were cultured on a medium 407 for 18 to 24 hours, and colonies were counted for each (step (vii)). The capture rate was calculated from the ratio of the counts for both.

[0037] <Evaluation Results of Collection Efficiency from Each Experiment> (1) Figure 5 shows the evaluation results of the collection efficiency. Figure 5 shows the results when water and human whole blood were used as the specimen solution 402. For the evaluation using whole blood, purchased blood (anticoagulated with EDTA2K, Tennessee Blood Services) was used. In addition, taking into account blood sample dependency, the collection efficiency was evaluated for the blood of three different subjects.

[0038] As can be seen from the results in FIG. 5, when water was used as the specimen solution 402, a high collection rate of approximately 96% was observed, whereas when whole blood was used, the collection rate was significantly reduced to approximately 43%.

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

[0040] Step (i-1): Whole blood was centrifuged at 1500g for 15 minutes to separate it into plasma and blood cells. Step (i-2): After removing as much plasma as possible, the removed volume was filled up with isotonic solution. Step (i-3): The blood was resuspended by inversion. Step (i-4): Steps (i-1) to (i-3) were repeated three times to replace the plasma with isotonic solution. Step (i-5): Isotonic solution was added to the blood cell solution prepared in step (i-4), and the volume was filled up to the same as the original blood solution. Hereinafter, this solution will be referred to as the 100% blood cell solution.

[0041] The plasma solution was prepared as follows: Procedure (ii-1): The hematocrit value of the whole blood was measured using a blood cell counter (XN-330, Sysmex). Procedure (ii-2): The whole blood was centrifuged at 1500 g for 15 minutes to separate it into plasma and blood cells. Procedure (ii-3): After transferring only the plasma to another tube, the volume was adjusted to the volume of blood cells measured in procedure (ii-1) with an isotonic solution (hereinafter, this solution will be referred to as the 100% plasma solution).

[0042] (2) Figure 6 shows the results of a collection efficiency evaluation experiment using an isotonic solution, a blood cell solution, and a plasma solution. From the evaluation results shown in Figure 6, the inventors discovered that the collection efficiency was significantly reduced when a plasma solution was used as the specimen solution 402. Furthermore, several additional experiments not shown here suggested that biological substances, mainly proteins, contained in the plasma may inhibit the formation of the conjugate 405 between the microorganisms and the microorganism collection reagent.

[0043] (3) Therefore, in order to reduce the influence of biological substances in plasma, we attempted to improve the capture efficiency by diluting the blood. Specifically, we evaluated whether the capture efficiency could be improved by diluting a 100% plasma solution with an isotonic solution (a 154 mM sodium chloride solution) (diluting the plasma concentration from 100% to 25%). Figure 7 shows the change in bacterial capture efficiency (experimental results) corresponding to changes in plasma suspension concentration (%). As shown in Figure 7, diluting a 100% plasma solution with an isotonic solution to 25% did not result in any improvement in the capture efficiency.

[0044] (4) Next, the same experiment was conducted by diluting the solution with a 10 mM sodium chloride aqueous solution, which is lower in concentration than the isotonic solution. Figure 8 shows the experimental results of the change in bacterial capture rate corresponding to changes in sodium chloride (salt) concentration. This experiment revealed that the capture rate improved significantly to 87% when the salt concentration was diluted to 118 mM. It is possible that the lower salt concentration than that of blood denatured biological components such as proteins contained in plasma, changing the electrostatic binding properties.

[0045] (5) Based on the above results, an experiment was conducted to confirm the change in capture efficiency by serially diluting whole blood with a 10 mM sodium chloride solution. Figure 9 shows the change in bacterial capture efficiency (experimental results) corresponding to changes in the ionic strength of whole blood. Figure 9 shows that while the capture efficiency was below 50% with whole blood with an ionic strength of 154 mM, a high capture efficiency of 92% was achieved by diluting to 112.5 mM. Figure 9 also shows that the capture efficiency deteriorates and variability increases at ionic strengths around 46 mM.

[0046] (6) In order to determine the cause of the deterioration of the capture efficiency and the increase in variability at an ionic strength of approximately 46 mM, an additional experiment was conducted. Specifically, a blood cell solution was diluted with a 100% 10 mM sodium chloride aqueous solution to change the salt concentration from 118 mM to 46 mM, and the resulting solution was used as the specimen solution 402, and a similar experiment was conducted.

[0047] Figure 10 shows the experimental results of the additional experiment, showing the change in bacterial capture rate corresponding to the change in the concentration of sodium chloride aqueous solution (salt) in the blood cell suspension. As shown in Figure 10, there was a tendency for the variation to increase as the salt concentration decreased. In particular, in the blood cell solution with a salt concentration of 46 mM, although the average capture rate was high at 90%, there were samples where the capture rate fell to 74%, suggesting that this may affect the stability of the capture rate.

[0048] (7) Therefore, to investigate the cause of the large variation, microscopic observations were performed for each concentration of blood cell solution. Figure 11 shows microscopic images of each blood cell solution concentration. As shown in Figure 11, in blood cell solutions with salt concentrations of 154 mM and 118 mM, red blood cells remained intact, but the number of red blood cells decreased from the blood cell solution with a salt concentration of 82 mM. In the blood cell solution with a salt concentration of 46 mM, most red blood cells were hemolyzed, and the remaining blood cells were found to be swollen.

[0049] The above suggests that a decrease in ionic strength may cause red blood cells to lyse, and the leaked components, such as hemoglobin (protein), may inhibit the binding of magnetic particles to bacteria. Therefore, in an additional experiment, to confirm the effect of hemoglobin on bacterial capture efficiency, a pure hemoglobin solution was used to simulate a completely hemolyzed blood cell solution with a salt concentration of 46 mM. The average capture efficiency was 61% (maximum: 72%, minimum: 50%), suggesting the possibility that the capture efficiency may be significantly reduced due to the influence of hemoglobin leaking from red blood cells.

[0050] Based on the above experimental results, it was found that when concentrating bacteria from a blood sample using the microorganism collection reagent 100, it is desirable to dilute the salt concentration in the blood with a diluent, and more specifically, the concentration should be in the range of 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 increases, which may complicate subsequent processing or increase the amount of reagent required for subsequent processing. Therefore, it is desirable to keep the dilution to a minimum, for example, 118 mM.

[0051] This configuration makes it possible to recover trace amounts of bacteria contained in whole blood with a high efficiency of over 90%. Furthermore, the use of dimethylamino groups makes it possible to produce the antibody by chemical synthesis, and it can be stored at room temperature for a long period of time.

[0052] <Procedure for Recovering Microorganisms (Bacteria) from a Specimen Including a Step of Adding Diluent> FIG. 12 is a flowchart illustrating a microorganism (bacteria) recovery process, including a step of adding a diluent, devised based on previously discovered facts, as a variation of the microorganism recovery process of FIG. 3 . Based on the flowchart of FIG. 3 , FIG. 12 adds a new step of adding a diluent. More specifically, the operator (user) adds the diluent to the container 400 along with the patient specimen containing the microorganisms 104 (step S1201). This denatures the biological material contained in the specimen. Next, the operator adds the microorganism collection reagent 100 to the container 400 (step S1202). The subsequent steps are the same as steps S302 to S309 shown in FIG. 3 .

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

[0054] This modified treatment allows for the recovery of trace amounts of bacteria contained in whole blood with a high efficiency of over 90%. Furthermore, the use of dimethylamino groups allows for chemical synthesis, enabling long-term storage at room temperature.

[0055] 13 shows the electrophoresis pattern of a product obtained by PCR after mixing a microorganism collection reagent according to this embodiment. Band 1301 indicates a band obtained by PCR amplification after mixing a blood-free solution with the microorganism collection reagent 100 to capture bacteria. Band 1302 indicates a band obtained by PCR amplification after mixing blood with the microorganism collection reagent 100 to capture bacteria.

[0056] 13, band 1303 appears in both cases. This shows that when the microorganism collection reagent 100 according to this embodiment is mixed with blood and a microorganism (bacteria) collection process is performed, PCR is not inhibited by hemoglobin in the blood. Therefore, according to this embodiment, bacteria can be efficiently collected, and by amplifying them by PCR, the bacterial genes can be detected.

[0057] <Examples of groups to be surface-modified on magnetic particles (magnetic beads)> A wide variety of causative bacteria are found in the blood of patients with bloodstream infections, including not only Staphylococcus aureus, a gram-positive bacterium, but also Escherichia coli, a gram-negative bacterium. The surface structures of gram-negative and gram-positive bacteria differ significantly, and in some cases, a sufficient capture rate may not be achieved using magnetic particles 101 having dimethylamino groups 103 alone. Therefore, magnetic particles (second microorganism capture reagent 1401) modified with a microorganism-binding moiety other than dimethylamino groups 103 may be added to the microorganism capture reagent 100.

[0058] 14 shows 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 microorganism capture reagent 1401) with two or more surface modifications to the microorganism capture reagent 100, it becomes possible to stably capture a wider range of microorganisms.

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

[0060] <Microorganism Collection Kit> A microorganism collection kit can be configured by combining the microorganism collection reagent 100 and a diluent for reducing the ionic strength of the target specimen (e.g., blood). (As mentioned above, any liquid with an ionic strength lower than the salt concentration of blood (154 mM) can be used; for example, a 10 mM sodium chloride aqueous solution, water, or other liquids can be used.) The microorganism collection kit can be configured, for example, by combining a predetermined container (e.g., container 400 used for microorganism collection) containing a predetermined amount of diluent and a separate container or wrapping paper (e.g., a plastic bag) containing the microorganism collection reagent 100. The above experiment showed that a good recovery rate can be achieved by diluting the specimen by approximately 80%, so a diluent of approximately 20% by volume of water or a 10 mM sodium chloride aqueous solution relative to the specimen (e.g., blood) can be previously placed in the container included in the microorganism collection kit. The amount of sample (blood) to be processed varies depending on the application, but for example, when processing 5 mL of blood, the amount of diluent that should be placed in the container of the microorganism collection kit is 1 mL. In this way, by providing multiple types of microorganism collection kits with different diluent capacity, users can easily handle a variety of applications and the processing of various types of samples.

[0061] <Configuration Example of Microbiological Testing System> FIG. 15 is a diagram showing a configuration example of a microbiological testing system that automatically identifies the causative bacteria of bloodstream infections and tests for drug resistance genes as necessary.

[0062] The microbiological testing system according to this embodiment comprises a microbiological testing device 1500 and a computer 1517 that controls the operation of the microbiological testing device 1500. The microbiological testing device 1500 comprises a stirring mechanism 1505 that applies vibrations or the like to a blood collection container 1501 that contains patient blood 1502, bacteria (germs) 1503, and a microorganism capturing reagent 1504 to stir the contents, a magnetic particle capturing mechanism 1506, a pump 1509, a nozzle driving mechanism 1510, and a dispensing mechanism 1508 that includes 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 technician) collects blood from a patient into a blood collection container (blood collection tube) 1501. An anticoagulant (not shown) is added to the blood collection tube as needed. When the operator sets the blood collection tube into the microbiology testing device 1500, a diluent is injected from a reagent container 1511 by a nozzle 1507 of a dispensing mechanism 1508 under the control of a computer 1517 and a microcontroller 1516. A stirring mechanism 1505 mixes the patient's blood 1502 and the diluent. Next, a microorganism collection reagent 1504 is injected from a reagent container 1512 by a nozzle 1507 of the dispensing mechanism 1508. The stirring mechanism 1505 mixes the diluted patient's blood 1502 and the microorganism collection reagent 1504, promoting binding between bacteria 1503 contained in the patient's blood 1502 and the microorganism collection reagent 1504. Here, a vortex mixer, a stirrer, a shaker, or the like can be used as the stirring mechanism 1505.

[0064] After stirring for a predetermined time, under the control of the microcontroller 1516, the magnetic particle collection mechanism 1506 adsorbs the bond between the microorganism collection reagent 1504 and bacteria 1503 in the patient's blood 1502. Next, under the control of the microcontroller 1516, the nozzle 1507 of the dispensing mechanism 1508 aspirates the supernatant and then turns off the magnetic particle collection mechanism 1506. Furthermore, a cleaning solution is injected from the reagent container 1518, and the solution is again stirred by the stirring mechanism 1505, washing away residual blood components that have adhered to the bond between the microorganism collection reagent 1504 and bacteria 1503. After repeating the above washing procedure a predetermined number of times, the dispensing mechanism 1508 sucks up and discards the final cleaning solution.

[0065] Next, the microcontroller 1516 turns off the magnetic particle collection mechanism 1506, injects a diluent from the reagent container 1511, and stirs the solution using the stirring mechanism 1505 to prepare a suspension of a combination of the microorganism collection reagent 1504 and the bacteria 1503. The solution used for the suspension does not need to be a diluted solution of blood, and a dedicated suspension solution may be injected from the reagent container 1519 depending on the requirements of the subsequent nucleic acid extraction step.

[0066] Next, under the control of the microcontroller 1516, the dispensing mechanism 1508 sucks up the suspension and injects it into the nucleic acid extraction unit 1513. The nucleic acid extraction unit 1513 destroys the bacterial cell walls with a lysis reagent and extracts the bacterial nucleic acids. Furthermore, 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 carries out a gene amplification reaction. Here, the gene amplification reaction is carried out using the PCR method, the LAMP method, or another isothermal amplification technique.

[0067] Finally, the dispensing mechanism 1508 transfers the amplified product to a gene detection unit (not shown). The gene detection unit detects the presence or absence of the target nucleic acid by fluorescence observation, electrophoresis, gene hybridization, or the like. The microcontroller 1516 controls the operation of each functional block according to a predetermined procedure, transmits the results to an external computer 1517, and receives control commands from the external computer 1517 and controls the operation of each functional block based on those commands. Furthermore, the nucleic acid targeted by the microorganism testing device 1500 is preferably 16S ribosomal DNA when identifying bacteria, but is not limited to this. Furthermore, the effectiveness of antibiotics can be evaluated in advance by examining the region of the nucleic acid containing the drug resistance gene.

[0068] The specimens that can be targeted by the technology according to this embodiment are not limited to blood, but may also include various specimens suspected of being infected with microorganisms, such as cerebrospinal fluid, saliva, urine, lymph, nasal secretions, and swabs from affected areas. Also, cultures obtained after blood or urine cultures and their suspensions may also be included.

[0069] Summary of the Embodiments (i) This embodiment proposes a microorganism collection reagent 100 that increases the capture rate (recovery rate) of microorganisms (bacteria). The microorganism collection reagent 100 is configured by modifying the surface of magnetic particles 101 (magnetic particles: for example, 10 nm to 1 μm, preferably 100 nm or less) that are smaller than the size of the microorganisms to be captured with microorganism-binding moieties formed by dimethylamino groups 103. The methyl group _Me contained in the dimethylamino group is positively charged, and thus electrostatically binds to negatively charged microorganisms (bacteria). This enables efficient capture of microorganisms. Note that the smaller the particle size of the magnetic particles, the larger the total surface area of ​​the microorganism collection reagent can be, thereby increasing the recovery efficiency.

[0070] Furthermore, the microorganism collection reagent 100 may contain magnetic particles 101 whose surfaces are modified with microorganism binding moieties using groups other than dimethylamino groups (e.g., the ethyl group of ethylpyridinium bromide). This makes it possible to capture microorganisms that are difficult to capture using dimethylamino groups. Modification with the ethyl group of ethylpyridinium bromide makes it possible to improve the recovery rate of E. coli.

[0071] (ii) This embodiment proposes a microorganism collection kit that includes the microorganism collection reagent 100 and a diluent for reducing the ionic strength of the target specimen. A sodium chloride solution with a concentration of less than 154 mM can be used as the diluent. The diluent may be provided, for example, in advance in a container 400, and the specimen and the microorganism collection reagent 100 may be placed in the container at the actual specimen testing site.

[0072] Furthermore, the microorganism collection reagent included in the microorganism collection kit may contain, in addition to the magnetic particles modified with the above-mentioned dimethylamino groups, magnetic particles whose surfaces are further modified with microorganism binding moieties formed by groups other than dimethylamino groups (for example, the ethyl group of ethylpyridinium bromide).

[0073] (iii) This embodiment also proposes a microorganism collection method for capturing microorganisms contained in a specimen using a microorganism collection reagent in which the surface of magnetic particles is modified with microorganism-binding moieties based on dimethylamino groups. Specifically, this method includes adding a microorganism collection reagent 100, in which the surface of magnetic particles 101 is modified with microorganism-binding moieties based on dimethylamino groups, to a specimen (e.g., patient blood), binding the microorganism collection reagent to the microorganisms contained in the specimen, and recovering (collecting) the bound product of the microorganism collection reagent 100 and the microorganisms. This method allows for efficient collection of microorganisms (bacteria) using the microorganism collection reagent. A recovery rate of 43% can be achieved without adding a diluent, and the recovery rate can be further improved by adding a diluent to reduce the ionic strength of the specimen. For example, adding a diluent to reduce the ionic strength to between 82 mM and 154 mM enables a capture rate of 87% or higher.

[0074] Furthermore, when the microorganism collection reagent 100 is added to a specimen, it is preferable that one or more magnetic particles 101 are contained per 1 pL of specimen, and more preferably, about 100 magnetic particles 101 are contained per 1 pL of specimen. By containing about 100 magnetic particles 101 per 1 pL of specimen, it is possible to create a state in which at least one magnetic particle is present around the bacteria.

[0075] (iv) This embodiment also proposes a microorganism collection device (a device including some of the functions (microorganism collection function) of the microorganism testing 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-mentioned microorganism collection method. Specifically, the microorganism collecting device includes an agitation mechanism 1505 that agitates the specimen (e.g., blood) and microorganism collecting reagent 100 contained in a blood collection container 1501 to promote binding between bacteria 1503 contained in the specimen and magnetic particles (microorganism collecting reagent 1504), a magnetic particle collecting mechanism 1506 that collects the bound bacteria 1503 and magnetic particles (microorganism collecting reagent 1504), a dispensing mechanism 1508 that aspirates the supernatant liquid from the container and injects a cleaning solution and a diluent into the blood collection container 1501, and a microcontroller (controller) 1516 that controls the operation of the agitation mechanism 1505, the magnetic particle collecting mechanism 1506, and the dispensing mechanism 1508. Here, the microcontroller 1516 injects the diluent into the blood collection container 1501 to reduce the ionic strength of the specimen (blood) in the blood collection container 1501. Specifically, the microcontroller 1516 injects a diluent into the blood collection container 1501 to reduce the ionic strength to between 82 mM and 154 mM. For example, injecting a diluent of water or a 10 mM sodium chloride solution, approximately 20% of the volume of the sample, into the blood collection container 1501 can reduce the ionic strength to between 82 mM and 154 mM. After dilution, the microcontroller 1516 controls the dispensing mechanism 1508 to aspirate the supernatant and wash the conjugate with a washing solution. Finally, the washing solution is aspirated. This configuration allows the user to automatically obtain a conjugate of bacteria 1503 and a microorganism collection reagent 1504 contained in the sample with simple operations. The microorganism collection method disclosed in this technology causes minimal damage to bacteria, and the obtained conjugate can be appropriately suspended and cultured directly on an agar medium. This allows the bacterial count to be quantified by colony counting. In addition to culture, the method can also be used for other conventional microorganism tests, such as staining and observation under an optical microscope.

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

[0077] 100, 1504 Microorganism collection reagent 101, 200 Magnetic particle 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 product obtained by PCR after mixing with microorganism collection reagent 1302 Electrophoresis pattern of product obtained by PCR after mixing with microorganism collection reagent and blood 1303 Target band (75 bp) 1401 Second microorganism collection reagent 1402 Ethyl group of ethylpyridinium bromide 1500 Microorganism testing device 1501 Blood collection container 1502 Patient's blood 1503 Bacteria 1505 Stirring mechanism 1506 Magnetic particle collection mechanism 1507 Nozzle 1508 Dispensing mechanism 1509 Pump 1510 Nozzle driving mechanism 1511, 1512, 1518, 1519 Reagent container 1513 Nucleic acid extraction unit 1514 Nucleic acid amplification unit 1515 Nucleic acid detection unit 1516 Microcontroller 1517 Computer

Claims

1. A microbial collection reagent in which a surface of magnetic particles is modified with a microbial binding part by a dimethylamino group.

2. The microbial collection reagent according to claim 1, 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. The microbial collection reagent according to claim 1, further comprising magnetic particles whose surface is modified with a microbial binding part by a group other than the dimethylamino group.

4. The microbial collection reagent according to claim 3, wherein the group other than the dimethylamino group is an ethyl group of ethylpyridinium bromide.

5. A microbial collection kit comprising the microbial collection reagent according to claim 1 and a diluent for reducing the ionic strength of a sample to be targeted.

6. The microbial collection kit according to claim 5, wherein the diluent is an aqueous sodium chloride solution having a concentration of less than 154 mM.

7. The microbial collection kit according to claim 5, wherein the microbial collection reagent further comprises magnetic particles whose surface is modified with a microbial binding part by a group other than the dimethylamino group.

8. The microbial collection kit according to claim 7, wherein the group other than the dimethylamino group is an ethyl group of ethylpyridinium bromide.

9. A microbial collection method for collecting microorganisms contained in a sample using the microbial collection reagent according to claim 1, comprising adding the microbial collection reagent to the sample, binding the microbial collection reagent to the microorganisms contained in the sample, and recovering the conjugate of the microbial collection reagent and the microorganisms.

10. The microbial collection method according to claim 9, further comprising reducing the ionic strength of the sample by adding a predetermined diluent.

11. The microbial collection method according to claim 10, wherein the sample is blood, and when reducing the ionic strength, the ionic strength is reduced to 82 mM or more and less than 154 mM.

12. The microbial collection method according to claim 9, wherein the microbial collection reagent is added so that one or more of the magnetic particles are contained per 1 pL of the sample.

13. A microorganism collection device that collects microorganisms contained in a specimen using a microorganism collection reagent containing magnetic particles, comprising: a stirring mechanism that is housed in a container and stirs the specimen and the microorganism collection reagent to promote the binding of bacteria contained in the specimen to the magnetic particles; a magnetic particle collection mechanism that collects the conjugate of the bacteria and the magnetic particles; a dispensing mechanism that sucks out the supernatant in the container and injects a washing liquid and a dilution liquid into the container; and a control unit that controls the operations of the stirring mechanism, the magnetic particle collection mechanism, and the dispensing mechanism, wherein the control unit controls the dispensing mechanism to inject the dilution liquid into the container, thereby reducing the ionic strength of the specimen in the container and performing suction of the supernatant and washing of the conjugate with the washing liquid.

14. The microorganism collection device according to claim 13, wherein 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.

15. The microorganism collection device according to claim 14, wherein the control unit injects 20% of the dilution liquid of the amount of the specimen into the container.

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