Method for detecting bacteria in sample and system for detecting bacteria in sample

Abstract

The present invention relates to a method for detecting bacteria in a sample and a system for detecting bacteria in a sample, wherein the presence or absence of bacteria can be quickly determined using a well unit provided with a transparent nanoporous thin film without going through a blood culture process after collecting a blood sample of a subject to be tested, and sensitivity tests for various concentrations of various antibiotics can be quickly performed.

Description

Method for bacterial testing of samples and system for bacterial testing of samples

[0001] This application claims priority based on Korean patent application No. [No.] filed on October 16, 2024, and all contents disclosed in the specification and drawings of said application are incorporated into this application.

[0002] The present invention relates to a method for testing bacteria in a sample and a system for testing bacteria in a sample. More specifically, the invention relates to a method for testing bacteria in a sample and a system for testing bacteria in a sample that can rapidly determine the presence or absence of bacteria using a well unit equipped with a transparent nanoporous film without undergoing a blood culture process after collecting a blood sample from a test subject, and can also rapidly perform susceptibility testing for various concentrations of various antibiotics.

[0003] As technology advances for the convenience of humanity, many environmental pollution problems are arising in modern society. A representative example is water pollution, particularly the harmful effects on the human body caused by infectious bacteria originating from sources such as food waste and livestock barns.

[0004] Therefore, the task of diagnosing (detecting) infectious bacteria is essential for preventing secondary infections in plants, animals, and the human body.

[0005] For example, sepsis occurs due to bacterial infection; however, under current clinical methods, it takes about a day for blood cultures to diagnose sepsis after the onset of symptoms, and another day or so to perform antibiotic susceptibility testing of bacteria at various concentrations to administer appropriate antibiotics.

[0006] As such, it takes about 40 hours from the onset of sepsis symptoms to the administration of antibiotics, and for every hour of delay, the patient's survival rate decreases by about 10%. Therefore, many studies are being conducted to shorten this time.

[0007] For example, a conventional method for evaluating antibiotic susceptibility includes the steps of isolating a pure colony of a microorganism to be tested and culturing the isolated substance on a solid medium or in a liquid medium, and finally evaluating antibiotic susceptibility by performing an analysis of the biochemical and / or phenotypic characteristics of the microorganism to be tested. The above-described traditional method for evaluating antibiotic susceptibility involves, after the step of isolating the microorganism, evaluating the susceptibility of the culture in which the isolated substance was cultured using liquid medium dilution or agar diffusion analysis.

[0008] Specifically, the liquid medium dilution method involves inoculating a pure isolate of the test microorganism into a growth medium containing a specific antibiotic at a series of specified concentrations, where the minimum inhibitory concentration (MIC) or MIC-like measurement is determined. The inoculated medium is cultured for 18 to 24 hours, and visible growth is observed, measured by turbidity, pellet size, and / or the emission of color or fluorescent moiety. Additionally, the agar diffusion analysis involves placing an antibiotic-containing disc or an antibiotic gradient strip on the surface of an agar medium inoculated with the pure isolate of the test microorganism. The plate is cultured for 18 to 24 hours, and during the culture period, the antibiotic substance diffuses from the disc or strip, and the change in the effective concentration of the antibiotic as a function of the radius from the disc or strip is observed.

[0009] Currently, there are approximately 10 antibiotic susceptibility testing methods approved by the FDA. 5 Inoculation of microorganisms with CFU / mL is required. Clinical samples are generally 10 5 Because it contains much less than CFU / mL, it is difficult to directly apply FDA approval tests to clinical samples.

[0010] Typically, clinical samples are inoculated into a culture medium, and the number of microorganisms is about 10 8 They must be grown until they reach CFU / mL. Therefore, the traditional method for evaluating microbial antibiotic susceptibility described above requires 48 to 72 hours to obtain results. However, during this period, microorganisms suspected of being non-susceptible to antibiotics spread to patients and the environment, threatening the health of patients.

[0011] According to a report analyzed in 2014, it is projected that by 2050, the number of deaths caused by antibiotic-resistant bacteria will exceed the number of deaths caused by cancer. Furthermore, there is a prediction that the specific number of deaths will reach 10 million per year. As the risk of antibiotic resistance increases and the development of antibiotics to prepare for it becomes crucial, the importance of developing methods to assess antibiotic susceptibility is also growing.

[0012] Traditional antibiotic susceptibility assessment methods have the problem of taking a long time to obtain results because they require the isolation of microorganisms and the culture of the isolated samples for evaluation. Therefore, if the time required to identify infectious microorganisms and select an effective antibiotic regimen is shortened, it is possible to significantly reduce the morbidity and mortality of microorganisms, prevent the outbreak of infectious diseases, and reduce the cost of treating patients with aggressive microbial infections.

[0013] Against this technological backdrop, research on technologies capable of evaluating antibiotic susceptibility within a short period is actively underway, but the current situation is still inadequate.

[0014] Accordingly, the present invention, which aims to solve the aforementioned conventional problems, provides a method for testing bacteria in a sample and a system for testing bacteria in a sample that can rapidly determine the presence or absence of bacteria using a well unit equipped with a transparent nanoporous film without undergoing a blood culture process after collecting a blood sample from a test subject, and can rapidly perform susceptibility testing for various concentrations of various antibiotics.

[0015] The problems solved by the present invention are not limited to those mentioned above, and other problems not mentioned will be clearly understood by those skilled in the art from the description below.

[0016] According to one aspect of the present invention for achieving the above objectives and other features of the present invention, a method for testing bacteria in a sample for testing the presence or absence of bacteria in a sample or testing bacterial antibiotic susceptibility is provided, comprising: a sample injection step of injecting a sample into a well unit; a bacteria separation step of separating bacteria from the sample in the well unit into which the sample was injected; and an analysis step of acquiring an image of the separated bacteria through an image acquisition means and analyzing the presence or absence of bacteria through the acquired image, or administering an antibiotic to the separated bacteria and then acquiring an image through the image acquisition means and analyzing antibiotic susceptibility through the acquired image; wherein the analysis step is characterized by performing an analysis based on an image obtained from the bottom of the well unit through the image acquisition means.

[0017] In one aspect of the present invention, the bacteria separation step may be performed by applying a separation force to a nanoporous thin film provided in the sample receiving space of the well unit to leave bacteria on the nanoporous thin film and discharging the remaining sample for separation.

[0018] In one aspect of the present invention, the bacteria separation step utilizes pressure or voltage as the separation force, and the nanoporous thin film used in the bacteria separation step may be formed as a transparent thin film of silicon nitride (Si3N4).

[0019] In one aspect of the present invention, the transparent nanoporous thin film of the bacteria separation step preferably has a pore diameter of 20 nm or more and a thickness of 1 μm or less.

[0020]

[0021] In one aspect of the present invention, the bacteria separation step may further include a bacteria position fixation step for creating a fixation for fixing the position of bacteria while the bacteria are present on the transparent nanoporous thin film, and a culture step for culturing the bacteria after the fixation is created.

[0022] In one aspect of the present invention, in the bacteria fixation step, the fixative may be composed of a hydrogel layer.

[0023] In one aspect of the present invention, the hydrogel layer in the bacteria fixation step may be formed using one or more of agarose, calcium alginate, and poly-N-isopropylacrylamide (poly-NIPAM).

[0024] In one aspect of the present invention, the hydrogel layer in the bacteria fixation step is

[0025] It can be formed into a laminated structure of one or more of agarose, calcium alginate, and poly-N-isopropylacrylamide (poly-NIPAM).

[0026] In one aspect of the present invention, the method further comprises a hydrogel layer removal step for removing the hydrogel layer to recover bacteria trapped in the hydrogel layer, wherein the hydrogel layer removal step is performed by injecting a monovalent cation solution into a well unit when the hydrogel layer is made of calcium alginate, and by lowering the temperature of the well unit when the hydrogel layer is made of Poly-N-isopropylacrylamide (poly-NIPAM).

[0027] In one aspect of the present invention, the method further comprises staining bacteria, wherein the staining of the bacteria may be performed using at least one of Gram staining, staining specific to a cell membrane, staining specific to a cell fluid, or staining specific to a DNA sequence.

[0028] According to another aspect of the present invention, a bacterial testing system for a sample for testing the presence or absence of bacteria in a sample or testing bacterial antibiotic susceptibility is provided, comprising: a well unit equipped with a bacterial concentration transparent filtering means configured to allow the sample to pass through while allowing bacteria to be present; a separation force application means provided in the well unit and configured to apply a separation force to the bacterial concentration transparent filtering means to separate the bacteria from the remaining sample; and an image acquisition means provided at the bottom of the well unit and configured to acquire an image of the bacteria present in the bacterial concentration transparent filtering means from the bottom.

[0029] In another aspect of the present invention, the bacteria concentration transparent filtering means is composed of a nanoporous thin film, and the separation force application means may be configured to separate bacteria and the remaining sample from the nanoporous thin film by using pressure or voltage as the separation force.

[0030] In another aspect of the present invention, the bacteria concentration transparent filtering means may be formed of transparent silicon nitride (Si3N4).

[0031] In another aspect of the present invention, the apparatus further comprises a fixation injection device for injecting a fixation for fixing the position of bacteria on the upper part of the bacteria concentration transparent filtering means, and a bacteria staining device for staining bacteria specifically, wherein the fixation used in the fixation injection device is composed of a hydrogel layer, and the bacteria specific staining may be performed with at least one of Gram staining, fluorescent staining specific to the cell membrane of bacteria, fluorescent staining specific to the cell fluid of bacteria, and fluorescent staining specific to the DNA sequence of bacteria.

[0032] The method for testing bacteria in a sample and the system for testing bacteria in a sample according to the present invention provide the following effects.

[0033] First, the present invention can determine the presence of bacteria based on whether they divide rapidly through image analysis of all bacteria contained in a sample without blood culture, and can perform bacterial susceptibility testing at various concentrations of various antibiotics in a multiple well structure, thereby enabling rapid disease diagnosis by performing the detection of bacteria in blood samples and antibiotic susceptibility testing at a low cost in a short time.

[0034] Second, the present invention has the effect of enabling a direct image of a component of a specific structure where all bacteria are present using a microscope objective lens, and thereby allowing for a more rapid determination of the presence of bacteria.

[0035] Third, the present invention can be utilized for testing for the diagnosis of sepsis and the administration of appropriate antibiotics, and can also be utilized for testing the antibiotic susceptibility of tuberculosis bacteria, thereby having the effect of reducing the mortality rate of subjects through rapid diagnosis of sepsis or tuberculosis.

[0036] The effects of the present invention are not limited to those mentioned above, and other unmentioned problems will be clearly understood by those skilled in the art from the description below.

[0037] Figure 1 is a flowchart schematically illustrating the process of a method for testing bacteria in a sample according to the present invention.

[0038] FIG. 2 is a diagram illustrating an example of a process for separating a sample for bacterial testing from whole blood in a method for bacterial testing of a sample according to the present invention.

[0039] FIG. 3 is a diagram schematically illustrating a method for testing bacteria in a sample according to the present invention.

[0040] FIG. 4 is a block diagram showing the configuration of a bacterial inspection system for a sample according to the present invention.

[0041] FIG. 5 is a diagram illustrating a well unit and an image acquisition means included in a bacterial inspection system of a sample according to the present invention.

[0042] FIG. 6 is a diagram illustrating the configuration of a separation force application means and the separation process for separating bacteria by applying voltage in a bacteria inspection system of a sample according to the present invention.

[0043] Figure 7 is a photograph of bacteria concentrated on a transparent nanoporous thin film using an image acquisition means, taken using a bacteria inspection system for a sample according to the present invention.

[0044] Figure 8 is an electron microscope image taken over time by culturing bacteria in a culture medium using the bacterial inspection system of a sample according to the present invention.

[0045] Figure 9 is an electron microscope image showing the case where bacteria are fluorescently stained using the bacteria inspection system of a sample according to the present invention.

[0046] Further objects, features, and advantages of the present invention can be more clearly understood from the following detailed description and the accompanying drawings.

[0047] Before providing a detailed description of the present invention, it should be understood that the present invention is capable of various modifications and may have various embodiments, and that the examples described below and illustrated in the drawings are not intended to limit the present invention to specific embodiments, but rather include all modifications, equivalents, and substitutions that fall within the spirit and scope of the present invention.

[0048] When it is stated that one component is "connected" or "connected" to another component, it should be understood that while it may be directly connected or connected to that other component, there may also be other components in between. On the other hand, when it is stated that one component is "directly connected" or "directly connected" to another component, it should be understood that there are no other components in between.

[0049] The terms used in this specification are used merely to describe specific embodiments and are not intended to limit the invention. Singular expressions include plural expressions unless the context clearly indicates otherwise. In this specification, terms such as "comprising" or "having" are intended to indicate the existence of the features, numbers, steps, actions, components, parts, or combinations thereof described in the specification, and should be understood as not precluding the existence or addition of one or more other features, numbers, steps, actions, components, parts, or combinations thereof.

[0050] Additionally, terms such as "...part," "...unit," and "...module" described in the specification refer to a unit that processes at least one function or operation, and this may be implemented in hardware, software, or a combination of hardware and software.

[0051] Furthermore, in the description referring to the attached drawings, identical components are assigned the same reference numeral regardless of drawing symbols, and redundant descriptions thereof are omitted. In describing the present invention, if it is determined that a detailed description of related prior art could unnecessarily obscure the essence of the present invention, such detailed description is omitted.

[0052] Hereinafter, a method for testing bacteria in a sample and a system for testing bacteria in a sample according to a preferred embodiment of the present invention will be described in detail with reference to the attached drawings.

[0053] First, a method for testing bacteria in a sample according to the present invention will be described in detail with reference to FIGS. 1 to 3.

[0054] FIG. 1 is a flowchart schematically showing the process of a method for testing bacteria in a sample according to the present invention, FIG. 2 is a diagram schematically showing an example of the process of separating a sample for testing bacteria from whole blood in a method for testing bacteria in a sample according to the present invention, and FIG. 3 is a diagram schematically showing a method for testing bacteria in a sample according to the present invention.

[0055] A method for testing bacteria in a sample according to the present invention is a method for testing bacteria in a sample (e.g., a blood sample) to determine whether bacteria are present or to test bacterial antibiotic susceptibility, and as shown in FIGS. 1 to 3, it mainly comprises a sample injection step (S100), a bacteria isolation step (S200), and an analysis step (S300).

[0056] Specifically, the method for testing bacteria in a sample according to the present invention is a method for testing bacteria in a sample for testing the presence or absence of bacteria in a sample or testing the antibiotic susceptibility of bacteria, and comprises: a sample injection step (S100) of injecting a sample containing bacteria to be tested into a well member comprising a transparent nanoporous film, as shown in FIGS. 1 to 3; a bacteria separation step (S200) of separating bacteria from a sample by passing the sample through the transparent nanoporous film of the well member into which the sample was injected through the sample injection step (S100) while allowing bacteria to exist on the upper surface of the transparent nanoporous film of the well member into which the sample was injected; and an analysis step (S300) of acquiring an image of the bacteria separated through the bacteria separation step (S200) using an image acquisition means and analyzing the presence or absence of bacteria or antibiotic susceptibility through the acquired image.

[0057] The above sample injection step (S100) is a process of injecting a sample containing bacteria to be tested into a well unit containing a transparent nanoporous thin film.

[0058] The sample injection step (S100) described above may utilize a configuration in which wells are arranged in parallel in an array form. Additionally, for sample stability, it is preferable that the well unit be equipped with a hydrophobic material on the top of the partitions between the wells. In other words, when a sample is introduced into a well, a well unit equipped with a hydrophobic material on the top of the partitions can promote a stable reaction during the reaction time due to the hydrophobicity.

[0059] In addition, in the sample injection step (S100), the transparent nanoporous thin film is provided at the bottom of the receiving space of the well unit that receives the sample, and may be made of silicon nitride (Si3N4). Preferably, the transparent nanoporous thin film has a pore diameter of 20 nm or more and a thickness of 1 μm or less.

[0060] Next, the bacteria separation step (S200) is a process of separating bacteria from the sample by passing the sample through the transparent nanoporous film of the well unit into which the sample was injected through the sample injection step (S100), while allowing bacteria to exist on the upper surface of the sample.

[0061] In the present invention, the bacteria separation step (S200) applies force to the upper and lower sides of the transparent nanoporous film, that is, causes the sample to move from the upper to the lower side of the transparent nanoporous film, while allowing bacteria to remain in a concentrated state on the upper side of the transparent nanoporous film. Here, even when the solution is discarded through the transparent nanoporous film and bacteria are present on the upper side of the transparent nanoporous film, other ions or solutions may also be present in the well unit, and some viruses, exosomes, and a very small number of blood cells may also be present.

[0062] In the present invention, the application of force between the upper and lower portions of a transparent nanoporous film is achieved by creating a pressure difference or by applying voltage, thereby positioning bacteria on the upper portion of the transparent nanoporous film and allowing the sample to be discharged through the transparent nanoporous film. Since the method of utilizing the pressure difference or the method of separating bacteria using voltage are sufficiently understood by those skilled in the art and known methods can be employed, a detailed description thereof is omitted. Here, the configuration for separating bacteria using voltage is described in the description of the bacterial inspection system for a sample according to the present invention, which is described below.

[0063] Next, the analysis step (S300) is a process of acquiring an image of the bacteria separated through the bacteria isolation step (S200) using an image acquisition means, and analyzing the presence or absence of bacteria or antibiotic susceptibility through the acquired image.

[0064] The above analysis step (S300) can acquire an image from the bottom of a transparent nanoporous thin film using a microscope or optical device equipped with an objective lens located at the bottom of the transparent nanoporous thin film, analyze the presence or absence of bacteria based on this, and analyze antibiotic susceptibility through images of the administration of various antibiotics to the bacteria.

[0065] Since the analysis of the presence or absence of bacteria and antibiotic susceptibility through the image obtained in the above analysis step (S300) may utilize known methods, a detailed description thereof is omitted.

[0066] Meanwhile, in the method for testing bacteria in a sample of the present invention, the bacteria separation step (S200) may further include a bacteria position fixing step (S210) in which, while the bacteria are concentrated and positioned on the transparent nanoporous thin film, a hydrogel is formed on the transparent nanoporous thin film (i.e., a hydrogel layer is created) to restrict the movement of the bacteria or fix the position of the bacteria.

[0067] In the above bacterial position fixation step (S210), the hydrogel layer can be formed in various ways.

[0068] In a first embodiment, the hydrogel layer may be provided with agarose, and by injecting agarose into the well unit, the position of bacteria on the upper surface of the transparent nanoporous thin film may be fixed.

[0069] In a second embodiment, the hydrogel layer may be calcium alginate.

[0070] Specifically, to crosslink calcium alginate, the process may be accomplished by injecting an alginate solution into a well unit and sequentially injecting a calcium solution or a calcium-containing solution into the well unit, or by injecting a calcium solution or a calcium-containing solution into the well unit and sequentially injecting an alginate solution into the well unit. In this case, the calcium-containing solution may be CaCl2. Alternatively, the process may be accomplished by applying an alginate solution into the well unit, removing excess alginate solution from the transparent nanoporous film using a solution removal means, and then reapplying a CaCl2 solution onto the bacteria concentrated on the transparent nanoporous film.

[0071] In a third embodiment, the hydrogel layer may be formed of Poly-N-isopropylacrylamide (poly-NIPAM).

[0072] Specifically, to crosslink Poly-N-isopropylacrylamide (poly-NIPAM), Poly-N-isopropylacrylamide (poly-NIPAM) is injected into a well unit, and heat is applied to the well unit. For example, a hydrogel layer made of Poly-N-isopropylacrylamide (poly-NIPAM) can be produced by maintaining the temperature of the well unit above a specific point.

[0073] After creating a hydrogel layer as a position fixing device for bacteria in a well unit using various methods as described above, a culture medium for culturing bacteria in the well unit can be injected (culture step (S220)). At this time, since the culture medium is injected while the position of the bacteria is fixed by the hydrogel layer, the bacteria can be cultured in a fixed position.

[0074] As described above, by creating a hydrogel layer on a transparent nanoporous thin film to fix the position of separated bacteria on the transparent nanoporous thin film and injecting a culture medium into a well unit to culture the bacteria, the bacteria are positioned at a certain distance from the imaging means, thereby making image acquisition tasks such as auto-focusing easier.

[0075] Meanwhile, the present invention may further include a hydrogel layer removal step (S310) for removing the hydrogel layer in order to recover bacteria trapped in the hydrogel layer when the hydrogel layer is formed.

[0076] In other words, when the analysis of bacteria in the above analysis step (S300) is completed to determine the presence or absence of bacteria and / or division, etc., the hydrogel layer removal step (S310) may be further included to remove the hydrogel layer to release the fixation of the bacteria, for example, for antibiotic susceptibility testing.

[0077] The above hydrogel layer removal step (S310) can be implemented in various ways to remove the hydrogel layer created within the well unit and release the fixation of the bacteria.

[0078] In a first embodiment, when the hydrogel layer is provided with calcium alginate, the hydrogel layer can be removed by injecting a monovalent cation solution into the well unit. For example, the monovalent cation solution may be sodium carbonate.

[0079] In a second embodiment, when the hydrogel layer is made of Poly-N-isopropylacrylamide (poly-NIPAM), the hydrogel layer can be removed by lowering the temperature of the well unit.

[0080] Specifically, when the temperature of the well unit is maintained above the singularity point to crosslink Poly-N-isopropylacrylamide (poly-NIPAM), lowering the temperature of the well unit to below the singularity point can dissolve the crosslinking of Poly-N-isopropylacrylamide (poly-NIPAM).

[0081] When the hydrogel layer is removed in this manner, the immobilized state of the bacteria is released, and the bacteria become capable of being mixed with the culture medium or buffer solution. Specifically, in the process of detecting the presence or division of bacteria, the bacteria were separated from the sample by a transparent nanoporous film within the well unit and were unable to be mixed with the culture medium or buffer solution due to the hydrogel layer; however, with the removal of the hydrogel layer, the bacteria can be dispensed into various other well units in a state mixed with the culture medium or buffer solution through the open top of the well unit.

[0082] Meanwhile, in the present invention, the analysis step (S300) may include staining specific to bacteria to perform image analysis more effectively.

[0083] For example, staining specific to bacteria may include Gram staining, fluorescent staining specific to bacterial cell membranes, fluorescent staining specific to bacterial cell fluid, fluorescent staining specific to bacterial DNA sequences, etc.

[0084] The performance of specific staining for bacteria can be carried out before forming the hydrogel layer, during the process of acquiring bacterial images, or before that.

[0085] In this way, the presence or division of bacteria in a sample can be determined through bacterial analysis.

[0086] The component(s) for executing each step in the bacterial testing method of a sample of the present invention described above, and the details thereof, can be implemented by the component(s) described below in the bacterial testing system of a sample.

[0087] Next, a bacterial inspection system for a sample according to the present invention will be described in detail with reference to FIGS. 4 to 8.

[0088] FIG. 4 is a block diagram showing the configuration of a bacterial inspection system for a sample according to the present invention, and FIG. 5 is a diagram showing a well unit and an image acquisition means included in a bacterial inspection system for a sample according to the present invention. FIG. 6 is a diagram showing the configuration of a separation force application means and the separation process for separating bacteria by applying voltage in a bacterial inspection system for a sample according to the present invention, FIG. 7 is a photograph of an image of bacteria concentrated on a transparent nanoporous thin film obtained through an image acquisition means using a bacterial inspection system for a sample according to the present invention, FIG. 8 is an electron microscope photograph taken over time by culturing bacteria in a culture medium using a bacterial inspection system for a sample according to the present invention, and FIG. 9 is an electron microscope photograph showing a case where bacteria are fluorescently stained using a bacterial inspection system for a sample according to the present invention.

[0089] A bacterial testing system for a sample according to the present invention is a method for testing the presence or absence of bacteria in a sample or testing bacterial antibiotic susceptibility, and as shown in FIGS. 4 to 9, it comprises a well unit (100) equipped with a bacterial concentration transparent filtering means (110), a separation force application means (200), and an image acquisition means (300).

[0090] Specifically, the bacterial inspection system for a sample according to the present invention is a bacterial inspection system for a sample for inspecting the presence or absence of bacteria in a sample or for inspecting bacterial antibiotic susceptibility, and comprises: a well unit (100) in which a bacterial concentration transparent filtering means (110) configured to allow the sample to pass through while allowing bacteria to be present, as shown in FIGS. 4 to 9, is provided in a sample receiving space; a separation force application means (200) configured to apply a bacterial separation force to the bacterial concentration transparent filtering means (110) of the well unit (100) so that bacteria remain above the bacterial concentration transparent filtering means (110) and the sample is discharged through the bacterial concentration transparent filtering means (110); and an image acquisition means (300) provided at the bottom of the well unit (100) and configured to acquire an image of bacteria present in the bacterial concentration transparent filtering means (110) from the bottom.

[0091] The well unit (100) described above is a component configured such that a bacterial concentration transparent filtering means (110) is provided in a sample receiving space, configured to allow the sample to pass through while allowing bacteria to be present. The well unit may be a known type used for the analysis of various samples, but in the present invention, the bacterial concentration transparent filtering means (110) is provided inside the well.

[0092] Specifically, the well unit (100) may be configured such that the wells are arranged in parallel in an array form. Here, for sample stability, it is preferable that the well unit (100) be provided with a hydrophobic material on the top of the partitions between the wells. In other words, when a sample is introduced into the wells, the well unit (100) provided with a hydrophobic material on the top of the partitions can promote a stable reaction during the reaction time due to the hydrophobicity.

[0093] The bacteria concentration transparent filtering means (110) provided in the well unit (100) above is made of a transparent nanoporous thin film provided at the bottom of the receiving space of the well unit that receives the sample.

[0094] The above transparent nanoporous thin film can be formed from silicon nitride (Si3N4). Preferably, the above transparent nanoporous thin film has a pore diameter of 20 nm or more and a thickness of 1 μm or less.

[0095] Here, the bacterial inspection system of a sample according to the present invention may be configured to analyze the presence or absence of bacteria concentrated on the upper surface of a transparent nanoporous film or antibiotic susceptibility through an image acquisition means (300) provided on the lower surface of a transparent nanoporous film, which is a bacterial concentration transparent filtering means (110).

[0096] For example, the well unit (100) may be configured to include a well member (111) having a transparent nanoporous film, which is a bacteria concentration transparent filtering means (110), at the bottom of a receiving space; an upper support (120) having an inlet (121) and an outlet (122) formed on one side and the other side, respectively, so as to be able to inject media or remove plasma, etc.; a lower support (130) having a discharge path formed at the bottom of the well member (111) to discharge a sample discharged through the transparent nanoporous film to one side; and a transparent finishing glass (140) having a lower support (130).

[0097] In the present invention, the well unit (100) described above is not particularly limited as long as it is configured to obtain a clear image of bacteria concentrated on the upper part of a transparent nanoporous film provided inside a well member using an image acquisition means (300) provided on the lower part of a transparent nanoporous film.

[0098] Next, the separation force application means (200) is configured to apply a bacterial separation force to the bacterial concentration transparent filtering means (110) of the well unit (100) so that the bacteria remain on the upper part of the bacterial concentration transparent filtering means (110) and the sample is discharged through the bacterial concentration transparent filtering means (110).

[0099] The above separation force application means (200) may be composed of, for example, a component or device that applies force to the upper and lower sides of the transparent nanoporous film, that is, causes the sample to move from the upper to the lower side of the transparent nanoporous film while allowing bacteria to remain in a concentrated state on the upper side of the transparent nanoporous film.

[0100] In a first embodiment, the means for applying the separation force (200) may be configured to generate a pressure difference between the upper and lower parts of the transparent nanoporous film, thereby moving the sample from the upper side to the lower side of the transparent nanoporous film due to the pressure difference. Accordingly, as previously mentioned, the transparent nanoporous film allows plasma and particles smaller than the nanoporous film (e.g., blood proteins, exosomes, etc.) to escape from the sample, while bacteria remain concentrated on the transparent nanoporous film. This enables a highly sensitive test by increasing the number of bacteria using a large amount of sample, even if the blood bacterial concentration is not sufficiently high.

[0101] And in a second embodiment, the separation force application means (200) separates by applying voltage to cause bacteria to be positioned on the upper side of the transparent nanoporous film, and the sample to be discharged through the transparent nanoporous film. This method of applying voltage can be configured by providing a planar electrode on the transparent finishing glass (140) of the well unit (100).

[0102] Specifically, the separation force application means (200) for separating bacteria by applying voltage is configured such that, as shown in FIG. 6, an upper electrode (210) to which voltage is applied is provided on the upper side of a transparent nanoporous film, which is a bacteria concentration transparent filtering means (110), and a lower electrode (220) grounded is provided on the lower side of the transparent nanoporous film.

[0103] The separation force application means (200) for separating bacteria by applying such voltage is such that when a voltage is applied to the upper and lower electrodes (210, 220) while there is liquid in the upper and lower parts of the bacteria concentration transparent filtering means (110) (i.e., nanoporous thin film) (lower left figure in FIG. 6), electroosmotic flow occurs through each nanopore of the nanoporous thin film, moving the solution of the sample, and as a result, the bacteria are separated and concentrated (lower right figure in FIG. 6). At this time, it is preferable that the surface of the nanoporous thin film is not neutral but has an electric charge. To achieve this, the nanoporous thin film can be treated with O2 plasma to give the surface a negative charge, and it is also possible to apply charged particles through surface coating, etc.

[0104] The above separation force application means (200) is not specifically limited to methods that separate bacteria using the pressure difference or voltage as described above, but can separate and concentrate bacteria by applying a predetermined force to a transparent nanoporous film to form a flow from the top to the bottom of the transparent nanoporous film.

[0105] Next, the image acquisition means (300) is a component configured to be provided at the bottom of the well unit (100) and to acquire an image of bacteria present in the bacteria concentration transparent filtering means (110) from the bottom.

[0106] The above image acquisition means (300) may be composed of a microscope or optical device equipped with an objective lens.

[0107] Meanwhile, the bacterial inspection system of the sample of the present invention may further include a fixation injection device (400) for injecting a fixation to restrict the movement of bacteria or fix the position of bacteria when bacteria are present on the upper surface of a transparent nanoporous thin film.

[0108] The above-mentioned fixture injection device (400) is not particularly limited as long as it is a device capable of injecting a fixture into the sample receiving space of the well unit (100).

[0109] In the present invention, the fixation injected through the fixation injection device (400) may be composed of hydrogel.

[0110] The above hydrogel may be composed of agarose, calcium alginate, and poly-N-isopropylacrylamide (poly-NIPAM).

[0111] In the case where the above hydrogel is composed of calcium alginate, to crosslink the calcium alginate, an alginate solution may be injected into the well unit (100) and a calcium solution or a solution containing calcium may be injected sequentially into the well unit (100), or a calcium solution or a solution containing calcium may be injected into the well unit (100) and an alginate solution may be injected sequentially into the well unit (100). At this time, the solution containing calcium may be CaCl2. Alternatively, the process may be carried out by applying an alginate solution into the well unit (100), removing the excess alginate solution from the transparent nanoporous film, and then applying a CaCl2 solution again onto the bacteria concentrated on the transparent nanoporous film.

[0112] When the above hydrogel is formed of Poly-N-isopropylacrylamide (poly-NIPAM), Poly-N-isopropylacrylamide (poly-NIPAM) is injected into a well unit to crosslink the Poly-N-isopropylacrylamide (poly-NIPAM), and heat is applied to the well unit. For example, by maintaining the temperature of the well unit above a specific point, it can be formed into a hydrogel layer of Poly-N-isopropylacrylamide (poly-NIPAM).

[0113] After creating a fixation in the well unit (100) formed by various methods as described above, a culture medium (not shown) for culturing bacteria can be injected into the well unit (100). At this time, since the culture medium is injected while the position of the bacteria is fixed by the hydrogel layer, the bacteria can be cultured at the fixed position.

[0114] As described above, by creating a hydrogel on a transparent nanoporous film to fix the position of separated bacteria on the transparent nanoporous film and injecting a culture medium into a well unit (100) to culture the bacteria, the bacteria are positioned at a certain distance from the image acquisition means (300), thereby making image acquisition tasks such as auto-focusing easier.

[0115] Meanwhile, the bacteria inspection system of the present invention may further include a bacteria staining device (500) for staining bacteria specifically in order to more effectively analyze the image obtained by the image acquisition means (300).

[0116] The above bacterial staining device (500) is not specifically limited to any device capable of performing staining specific to bacteria.

[0117] In the present invention, the bacteria-specific staining by the bacteria staining device (500) may be Gram staining, fluorescent staining specific to the cell membrane of bacteria, fluorescent staining specific to the cell fluid of bacteria, or fluorescent staining specific to the DNA sequence of bacteria.

[0118] Specific staining of bacteria by the above-mentioned bacteria staining device (500) can be performed during the process of acquiring a bacteria image or before forming a hydrogel layer.

[0119] In this way, the presence or division of bacteria in a sample can be determined through bacterial analysis.

[0120] According to the method and system for testing bacteria in a sample of the present invention as described above, the present invention has the advantage of being able to determine the presence or absence of bacteria from whether bacteria divide rapidly through image analysis of all bacteria contained in a sample without blood culture, and to perform bacterial susceptibility testing at various concentrations of various antibiotics in a plurality of well structures, thereby enabling rapid disease diagnosis by performing testing for the presence or absence of bacteria in a blood sample and testing for bacterial antibiotic susceptibility in a short time at low cost.

[0121] Secondly, the present invention allows for the direct imaging of a specific structural component where all bacteria are present using a microscope objective lens, thereby enabling more rapid determination of the presence of bacteria. It can be utilized for testing for the diagnosis of sepsis and the administration of appropriate antibiotics, as well as for testing the antibiotic susceptibility of tuberculosis bacteria, thus offering the advantage of reducing patient mortality through rapid diagnosis of sepsis or tuberculosis.

[0122] The embodiments described in this specification and the accompanying drawings are merely illustrative of a part of the technical concept included in the present invention. Accordingly, since the embodiments disclosed in this specification are intended to explain, not limit, the technical concept of the present invention, it is obvious that the scope of the technical concept of the present invention is not limited by these embodiments. All variations and specific embodiments that can be easily deduced by a person skilled in the art within the scope of the technical concept included in the specification and drawings of the present invention should be interpreted as being included within the scope of the rights of the present invention.

[0123] [Explanation of the symbol]

[0124] S100: Sample injection step

[0125] S200: Bacteria isolation step

[0126] S210: Bacteria position fixation step

[0127] S220: Culture stage

[0128] S300: Analysis phase

[0129] S310: Hydrogel layer removal step

[0130] 100: Well unit

[0131] 110: Bacteria Concentration Transparent Filtering Means

[0132] 111: Well absence

[0133] 120: Upper support

[0134] 121: Inlet

[0135] 122: Outlet

[0136] 130: Lower support

[0137] 140: Clear finish glass

[0138] 200: Means for applying separation force

[0139] 210: Upper electrode

[0140] 220: Lower electrode

[0141] 300: Means of image acquisition

[0142] 400: Fixture injection device

[0143] 500: Bacteria staining device

Claims

1. A method for testing bacteria in a sample to determine the presence or absence of bacteria in the sample or to test bacterial antibiotic susceptibility, Sample injection step of injecting a sample into a well unit; A bacterial separation step performed to separate bacteria from the sample in a well unit into which the sample is injected; and The method includes an analysis step in which an image is acquired through an image acquisition means for the isolated bacteria and the presence or absence of the bacteria is analyzed through the acquired image, or antibiotic susceptibility is analyzed through an image acquired through the image acquisition means after administering an antibiotic to the isolated bacteria. The above analysis step is characterized by being performed to analyze based on an image obtained from the lower part of the well unit through the image acquisition means. Method for testing bacteria in a sample.

2. In Paragraph 1, The above bacteria isolation step The method is characterized by applying a separation force to a nanoporous thin film provided in the sample receiving space of the well unit to leave bacteria on the nanoporous thin film and discharging the remaining sample for separation. Method for testing bacteria in a sample.

3. In Paragraph 2, The above bacterial separation step utilizes pressure or voltage as the separation force, and The nanoporous thin film used in the above bacteria separation step is characterized by being formed as a transparent thin film of silicon nitride (Si3N4). Method for testing bacteria in a sample.

4. In Paragraph 3, The transparent nanoporous thin film of the above bacteria separation step is Characterized by a pore diameter of 20 nm or more and a thin film thickness of 1 μm or less Method for testing bacteria in a sample.

5. In Paragraph 2, The above bacteria isolation step The method is characterized by further including a bacterial position fixing step for creating a fixation for fixing the position of bacteria while bacteria are present on the transparent nanoporous thin film, and a culture step for culturing bacteria after the fixation is created. Method for testing bacteria in a sample.

6. In Paragraph 5, The fixative in the above bacteria fixation step is characterized by being composed of a hydrogel layer. Method for testing bacteria in a sample.

7. In Paragraph 6, In the above bacteria immobilization step, the hydrogel layer Characterized by being formed using one or more of agarose, calcium alginate, and poly-N-isopropylacrylamide (poly-NIPAM). Method for testing bacteria in a sample.

8. In Paragraph 7, In the above bacteria immobilization step, the hydrogel layer Characterized by being formed of a laminated structure of one or more of agarose, calcium alginate, and poly-N-isopropylacrylamide (poly-NIPAM). Method for testing bacteria in a sample.

9. In Paragraph 7, The method further includes a hydrogel layer removal step of removing the hydrogel layer to recover bacteria trapped in the hydrogel layer, and The above hydrogel layer removal step is characterized by injecting a monovalent cation solution into a well unit when the hydrogel layer is composed of calcium alginate, and lowering the temperature of the well unit when the hydrogel layer is composed of Poly-N-isopropylacrylamide (poly-NIPAM). Method for testing bacteria in a sample.

10. In Paragraph 1, It further includes staining bacteria, The staining of the bacteria is characterized by being composed of at least one of Gram staining, staining specific to the cell membrane, staining specific to the cytoplasm, or staining specific to the DNA sequence. Method for testing bacteria in a sample.

11. A bacterial testing system for a sample to test for the presence or absence of bacteria in the sample or to test bacterial antibiotic susceptibility, A well unit equipped with a bacterial concentration transparent filtering means configured to allow a sample to pass through while allowing bacteria to be present; A separation force application means provided in the well unit and configured to apply a separation force to the bacteria concentration transparent filtering means to separate bacteria from the remaining sample; and Characterized by including an image acquisition means provided at the bottom of the well unit and configured to acquire an image of bacteria present in the bacteria concentration transparent filtering means from the bottom. Sample bacterial testing system.

12. In Paragraph 11, The above-mentioned bacteria concentration transparent filtering means is formed of a nanoporous thin film, and The above separation force application means is characterized by being configured to separate bacteria and the remaining sample from a nanoporous thin film by utilizing pressure or voltage as the separation force. Sample bacterial testing system.

13. In Paragraph 12, The above-mentioned bacteria concentration transparent filtering means is Characterized by being formed as a transparent thin film of silicon nitride (Si3N4). Sample bacterial testing system.

14. In Paragraph 10, The above-mentioned bacteria concentration transparent filtering means further includes a fixation injection device for injecting a fixation material to fix the position of bacteria, or a bacteria staining device for staining bacteria specifically, and The fixture used in the above fixture injection device is composed of a hydrogel layer, and The specific staining of the bacteria is characterized by comprising at least one of Gram staining, fluorescent staining specific to the bacterial cell membrane, fluorescent staining specific to the bacterial cell fluid, and fluorescent staining specific to the bacterial DNA sequence. Sample bacterial testing system.

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