Microchip for acquiring information for purpose of evaluating properties or state of microorganisms, information acquisition method or evaluation system using microchip, and computer program that can be used in the information acquisition method or the evaluation system thereof

Abstract

To provide means for saving a use amount of a reagent while further improving safety, simplicity, and rapidity on the premise of the accuracy of the evaluation in an evaluation test of drug resistance and the like of a microorganism, and further capable of performing a simultaneous evaluation test of many items.SOLUTION: The inventor has found that the above problems can be solved by providing a microchip that has a reaction cell in which a detection reagent of drug resistance and the like of a microorganism is sealed or fixed in a manner that dissolves upon contact with a liquid sample, and has a function of distributing a liquid sample containing a test microorganism to the reaction cell by suction force caused by a reduced pressure state, and further by providing an efficient evaluation method and evaluation system of drug resistance and the like of a microorganism based on a signal of the detection reagent generated upon contact with the test microorganism in the microchip, as well as a computer program that can be used in the evaluation method and evaluation system.SELECTED DRAWING: Figure 11

Description

[Technical field]

[0001] The present invention relates to a detection means for microorganisms, and more specifically, to a microchip for acquiring information on the properties or state of microorganisms, such as drug resistance of the microorganisms, and an acquisition method and acquisition system for the properties or state of the microorganisms using the same, and a computer program that can be used in the acquisition method or evaluation system. [Background technology]

[0002] Evaluation of the properties or status of microorganisms such as bacteria and fungi is carried out in a wide range of fields, including for the diagnosis of infectious diseases in clinical and veterinary fields, as well as for the detection of food poisoning bacteria and hygiene indicator bacteria in the food and environmental fields. Although rapid tests using genetic and immunological methods and automated analytical equipment have become widespread in recent years, isolation and identification of microorganisms by culture methods remains the basis for evaluation of the properties or status of microorganisms through microbial testing.

[0003] For example, as one of the ways of evaluating the properties or state of microorganisms by microbiological testing, drug susceptibility testing is performed to select an antibacterial agent effective for the treatment of an infectious disease and to determine the antibacterial agent and dosage appropriate for the infected person by examining the degree of sensitivity of the bacteria to the antibacterial agent. Drug susceptibility testing is broadly divided into two methods, the diffusion method and the dilution method. The diffusion method is a qualitative test method that easily selects an antibacterial agent effective for treatment, and is represented by the disk diffusion method, in which a disk test piece impregnated with an antibacterial agent is placed on a culture medium. The dilution method is a quantitative test method for accurately measuring the MIC (Minimum Inhibitory Concentration) value. The dilution method is used when precise treatment is required, and the disk method is used in the medical field because of its economy, speed, and simplicity.

[0004] In recent years, the emergence of new antibacterial drug-resistant bacteria has led to a demand for the development of an accurate diagnostic method using highly accurate drug susceptibility testing. Among antibacterial drug-resistant bacteria, the ones that are particularly problematic are those that have extended-spectrum beta-lactamase (ESBL) and class A and B carbapenemase activity and hydrolyze a wide range of antibacterial drugs. The plate method using multi-hole plates is widely used as a method for detecting these antibacterial drug-resistant bacteria. However, the plate method requires the cultivation of resistant bacteria, so it takes at least 24 hours from the start of the test to the determination.

[0005] In contrast to the above-mentioned plate method, a test method using a microchip for the purpose of evaluating drug susceptibility has been disclosed in the patent literature.

[0006] For example, a device has been disclosed that can determine the type of enzyme produced by cells from images of each channel of cells cultured within the channels of a microchip for determining enzyme types, and from images capturing changes in cell morphology due to the effects of a reagent that combines an antimicrobial drug and an inhibitor (Patent Document 1).

[0007] Also, a technology has been disclosed in which a suspension of test bacteria is introduced into multiple flow paths of a methicillin-resistant Staphylococcus aureus (MRS) testing device, a mixture of an antibacterial agent and the suspension is cultured within the multiple flow paths, and whether the test bacteria is MRS is determined by observing the culture area of ​​the flow path (Patent Document 2). Also disclosed is a microdevice in which antibacterial agents are placed in a plurality of flow paths and an observation area is provided for detecting bacteria or fungi derived from test microorganisms using a microscope or the like (Patent Document 3).

[0008] Also disclosed is a microchip that uses a chip in which multiple types of cells are immobilized in multiple micropores on a substrate, and another chip that has multiple microchannels perpendicular to the chip (Patent Document 4). [Prior art documents] [Patent documents]

[0009] [Patent Document 1] Patent Publication No. 2021-40545 [Patent Document 2] JP 2018-113963 A [Patent Document 3] WO2013 / 038925 International Publication Pamphlet [Patent Document 4] JP 2005-46121 A [Patent Document 5] JP 2017-67595 A Summary of the Invention [Problem to be solved by the invention]

[0010] Tests for evaluating the properties of microorganisms, such as drug susceptibility tests, or tests for evaluating the status, such as microbial enumeration tests, have long been needed and are performed in a variety of ways in a variety of industries. On the other hand, there is a continuing demand for improved safety, simplicity, and speed in performing the tests, provided that the accuracy of the evaluation is maintained.

[0011] In particular, when handling pathogenic microorganisms, the risk of exposure of the tester or the test environment to the pathogenic microorganisms is a safety issue. The culture method, which has been widely used in the past, is very excellent in terms of accuracy. However, in addition to the safety issues associated with the culture operation, it takes time and effort to culture the target microorganism, and there are limitations in terms of simplicity and speed.

[0012] The drug susceptibility tests using microchips in Patent Documents 1 to 4 have problems similar to those of the culture method described above in that the effort required to introduce the sample liquid into the fine flow path of the microchip and there are concerns about liquid leakage, and because the target microorganisms need to be cultured inside the microchip, it takes a long time to obtain the test results.

[0013] The microchip test of Patent Document 5 utilizes negative pressure to rapidly introduce a sample liquid into a well in the chip, does not require the cultivation of microorganisms, and is therefore safe and simple. However, time is required for the nucleic acid amplification reaction in the introduced cell, and there remains an issue with regard to rapidity.

[0014] In addition, as a general issue, there is a demand for simultaneous evaluation of multiple items, while at the same time, there is a demand for saving on the amount of reagent used. [Means for solving the problem]

[0015] As a result of repeated studies to solve the above problems, it was found that the above problems could be solved in a remarkable way by creating something using the basic structure of the microchip of Patent Document 5 (related to the applicant's patent (Patent No. 6621636)). That is, by preferably mechanically evaluating a signal such as color development according to the properties or state of the microorganism to be evaluated by a reagent that dissolves upon contact with a liquid sample sealed or fixed in the cell of the microchip of Patent Document 5, it is possible to significantly improve safety and simplicity while ensuring the accuracy of the evaluation. Furthermore, it was found that the microorganism culture process and gene amplification process can be omitted in principle, and further, the signal obtained by the microchip of the present invention is an integrated signal of the "cell unit of the microchip", and the information of each microorganism present therein is omitted, and the processing elements for the final evaluation are simplified and abstracted, so that the speed of the evaluation can be significantly improved, and thus the present invention was completed.

[0016] The present invention comprises: (1) an invention of a microchip (hereinafter also referred to as the microchip of the present invention); (2) an invention of a method of acquiring information for evaluating the properties or state of a microorganism using the microchip of the present invention (hereinafter also referred to as the information acquisition method of the present invention); (3) an invention of a system for evaluating the properties or state of a microorganism using the microchip of the present invention (hereinafter also referred to as the evaluation system of the present invention); and (4) an invention of a computer program that can be used in the information acquisition method or evaluation system of the present invention (hereinafter also referred to as the computer program of the present invention).

[0017] (1) Microchip of the Present Invention The microchip of the present invention comprises: A microchip having a substrate for acquiring information for evaluating the properties or state of a microorganism, the microchip having a plurality of cells maintained at a reduced pressure relative to atmospheric pressure, the substrate of the microchip has a two-layer structure including a first plate-shaped part having gas impermeability and a second plate-shaped part laminated on one surface of the first plate-shaped part and having self-sealing properties; The plurality of cells are formed between the first plate-shaped portion and the second plate-shaped portion, and a reagent for detecting the properties or states of one or more types of microorganisms is sealed or fixed in some or all of the plurality of cells in a manner that dissolves upon contact with a liquid sample; The cell is a substrate of a microchip that communicates with an injection space into which a liquid sample is punctured and injected via a flow path, a microchip for acquiring information on the properties or state evaluation of microorganisms in the liquid sample from signals from one or more types of cells resulting from a contact reaction between the liquid sample introduced into each of the plurality of cells and the reagent sealed or fixed in the cell upon release of the reduced pressure state resulting from puncturing and injecting the liquid sample into the microchip; It is.

[0018] The basic structure of the microchip of the present invention is the same as that of the microchip disclosed in Patent Document 5. Specifically, it will be disclosed in the embodiment of the invention. The "substrate" of the microchip is the part that directly controls the function of the microchip of the present invention, and the microchip itself may overlap the "substrate", or the microchip of the present invention may include a housing or container for protecting the "substrate" from physical impacts and exposure to external light, separate from the "substrate" that exerts the function of the microchip. The "substrate" of the microchip and the housing or container may be inseparable or detachable.

[0019] The microchip substrate of the present invention can and preferably has a reinforcing film on the surface of the second plate-shaped part opposite to the first plate-shaped part. The reinforcing film on the second plate-shaped part can improve the function of preventing the injection needle from swaying sideways during puncture injection. Therefore, if the reinforcing film is provided, it must be provided at least at a position on the second plate-shaped part facing the injection space.

[0020] Furthermore, in the microchip substrate of the present invention, a light-shielding layer can be provided with at least a flat surface facing each of the plurality of cells removed. By providing the light-shielding layer, scattering of incident light toward the plurality of cells can be suppressed.

[0021] In the microchip of the present invention, microorganisms to be evaluated for their properties or states include, but are not limited to, bacteria, fungi, protozoa, yeasts, etc. In the case of selection as a pathogenic microorganism, bacteria and fungi, particularly bacteria, are the main targets.

[0022] The properties of microorganisms evaluated in the microchip of the present invention include drug resistance of microorganisms, classification and detection at the genetic level of microorganisms, etc., and the state of microorganisms evaluated in the same manner includes the number of microorganisms per unit volume, etc. The reduced pressure state is released by puncturing and injecting a liquid sample into the microchip of the present invention, and a signal from each of one or more types of cells due to a contact reaction between the liquid sample introduced into each of the multiple cells provided in the microchip of the present invention and the reagent sealed or fixed in the cell is detected, preferably as color development or fluorescence, to obtain information that is the basis for evaluating the properties or state of microorganisms.

[0023] The principle of signal detection for microbial drug resistance is to detect the difference in proliferation in the cells of the target microorganism according to the concentration gradient of the drug (antibacterial agent, antifungal agent, antibiotic, etc.) as a turbidity signal. In the present invention, turbidity in this case is included in the "type of color development."

[0024] In addition, there is a method in which a factor found in drug-resistant microorganisms that hinders the effectiveness of the drug against the microorganism, for example, a decomposition product of the drug by the action of a decomposition enzyme that can decompose the drug, is used as a detection signal.Specific examples include the acidometry method, in which a decrease in pH caused by the presence of an acid generated by hydrolysis of a substrate such as a β-lactam antibacterial drug by β-lactamase is detected by a color change of a pH indicator; the chromogenic method, in which a substrate that changes color when the β-lactam ring is cleaved by hydrolysis by β-lactamase is used for detection; the iodometry method, in which the substrate iodine is reduced and changes color by the acid generated by hydrolysis of a β-lactam antibacterial drug by β-lactamase; etc.

[0025] Other examples of classification and detection of microorganisms at the genetic level include the FISH (Fluorescence in situ hybridization) method. In the microchip of the present invention, for example, by encapsulating or immobilizing in the cell a nucleic acid containing a fluorescent substrate complementary to bacterial 16SrRNA or a drug resistance gene, it is possible to identify the microbial species and detect drug resistance based on hydrolysis of the β-lactam ring by β-lactamase as well as other principles.

[0026] The number of microorganisms per unit volume can be determined using as an index the color intensity or fluorescence intensity of a nuclear staining agent enclosed or fixed in the cell. In this case, the evaluation can be performed by counting the number of colored or fluorescently stained bacteria in the cell, or by detecting the fluorescence intensity of the entire cell.

[0027] A reagent for detecting the properties or state of a microorganism is sealed or fixed in the cell provided in the microchip of the present invention in a manner that dissolves when contacted with a liquid sample.

[0028] In the case of detecting drug resistance in microorganisms, at least drugs to be detected for drug resistance (antibacterial agents, antifungal agents, antibiotics, "inhibitors of enzymes that decompose or neutralize drugs and confer drug resistance on microorganisms", etc.; these drugs are also called "substrates") are preferably encapsulated or fixed in each cell with a concentration gradient.

[0029] In addition, a signal generating reagent (indicator) that generates a signal such as color or fluorescence depending on the presence of drug resistance in the microorganism is enclosed or fixed as necessary.

[0030] In addition, if necessary, encapsulation or fixation can be performed by incorporating a buffer, an inorganic salt, a surfactant, a fixing agent, a solubilizing agent, etc.

[0031] As an example of the method of encapsulation or fixation, a method in which a reagent for detecting the properties or state of the above-mentioned microorganism is dispensed into a cell before the pressure is reduced in the manufacturing process of the microchip of the present invention, and a vacuum drying process is performed can be mentioned.

[0032] (2) Information Acquisition Method of the Present Invention The information acquisition method of the present invention comprises: A method for injecting a liquid sample, which is an aqueous dilution solution containing microorganisms derived from a biological sample, into the microchip of the present invention, and acquiring visual images or image information of signals from each of a plurality of cells generated by a reaction between the liquid sample introduced into each of the cells and a reagent for detecting the properties or states of one or more microorganisms, as information for evaluating the properties or states of the microorganisms in the liquid sample; It is.

[0033] The liquid sample in the information acquisition method of the present invention is an aqueous dilution solution containing microorganisms derived from a biological sample. The type of microorganism is not particularly limited, and is the same as the above-mentioned "microorganisms to be evaluated for their properties or state in the microchip of the present invention". The liquid sample can be a colony obtained by isolating and culturing the microorganism from a living organism or the environment and adjusting its concentration. Microorganisms in a living organism that exist in the environment are also included in the microorganisms derived from the biological sample of the present invention. A sample prepared from a clinical specimen can be used as the biological sample. The clinical specimen is not particularly limited as long as it is a living organism in which the target microorganism may exist, and examples of the specimen include urine, blood, saliva, feces, mucosa, solid tissue, and biological washings. The liquid sample may be a suspension prepared by diluting to a predetermined concentration with an aqueous solvent such as physiological saline or a buffer solution, as necessary.

[0034] The means for puncturing and injecting the above-mentioned liquid sample into the microchip of the present invention is not particularly limited, but it is preferable that the means take the form of an injection chip equipped with a cylindrical reservoir for storing the liquid sample and a needle (injection needle) for injecting the liquid sample into the microchip.

[0035] The visual image or image information of the signal is preferably a visual image or image information based on coloration (including turbidity) or fluorescence of the microorganism. The visual image may be a direct visual image of the coloration or fluorescence in the cell of the microchip of the present invention, or may be imaged by a camera. The image may be imaged by an analog camera or a digital camera, but imaged by a digital camera is preferable because the image can be viewed immediately after image capture. In addition, if a digital camera is used, it is also possible to reconstruct an image visualized by computer image processing and view this. The image information is information that is a prerequisite for evaluating the properties or state of the microorganisms in the liquid sample by executing computer software.

[0036] The color or fluorescence image information representing the properties or state of the microorganisms may be image information in which a leveling process is performed by a computer using a leveling process means for leveling the variation in color or fluorescence intensity within each cell. The color or fluorescence image information may be image information based on a color difference or fluorescence intensity difference calculated by a computer for each cell between the color value or fluorescence intensity value normalized based on the reference judgment information corresponding to the properties or state of the microorganisms. Furthermore, it is preferable that the color image information is image information based on color space values ​​of a color system suitable for digitization. Examples of color space values ​​of the color system include L*a*b*, L*C*h, Hunter Lab, etc., and image information based on L*a*b* is particularly preferable. Furthermore, values ​​of color systems such as the Munsell color system and the XYZ (Yxy) color system may be used as the image information. Furthermore, conversion processing for color space such as HSV conversion and HSL conversion may be used as necessary.

[0037] The "properties or state of a microorganism" in the information acquisition method of the present invention is, as described for the microchip of the present invention, a property of a microorganism, for example, drug resistance of a microorganism, classification and detection of a microorganism at a genetic level, and a state of a microorganism, for example, the number of microorganisms per unit volume, but is not limited to these.

[0038] (3) Evaluation system of the present invention and computer program of the present invention The evaluation system of the present invention overlaps substantially with the information acquisition method of the present invention in many ways. The evaluation system is a system that evaluates desired microorganisms by executing computer software on the acquired image information, which is the subject of the information acquisition method of the present invention, in the form of "image information acquired by an image acquisition unit."

[0039] That is, the evaluation system of the present invention is: 1. A system for assessing the nature or state of a microorganism in a biological sample, comprising: (a) comprising an image acquisition unit for acquiring color (including turbidity) or fluorescence representing the properties or state of microorganisms from each cell of the microchip of the present invention as image information, and a computer; (b) a means for causing a computer to compare the image information inputted into the computer with a color value or a fluorescence intensity value normalized based on reference information for determining the properties or state of the microorganism, and for causing the computer to evaluate the properties or state of the microorganism obtained from the biological sample; The system for evaluating the properties or state of a microorganism may include a computer built in the image acquisition unit.

[0040] The image acquisition unit is preferably an imaging unit that uses an imaging device. Specifically, the imaging unit includes: (a) An imaging space is formed in which external light is blocked. (b) within the imaging space, an imaging device that acquires color or fluorescence representing the properties or state of microorganisms from each of the cells of the microchip whose position is fixed as image information is fixed in position, or an imaging device position fixing mechanism that can detachably fix the imaging device in position is provided; (c) a light source is provided in the light incidence mechanism of the imaging device in a fixed position so that light emitted from the light source is incident from a main optical path; (d) a mechanism for fixing the position of the microchip is provided so that the light receiving surface of the microchip is incident on a main optical path of the light emitted from the light source until the light receiving surface reaches a light incident mechanism of the imaging device, and the transmitted light is incident on the light incident mechanism; can be cited as a preferred embodiment. The light receiving surface of the microchip is the exposed surface of the first plate-shaped part of the microchip of the present invention, but when a light-shielding layer is provided on the microchip, the substantial light receiving surface is limited to a plane facing each of the multiple cells of the microchip. In addition, the mechanism for fixing the position of the microchip in (d) above may be provided with an optical mask mechanism that blocks light from being received on any surface other than the plane facing each of the multiple cells of the microchip, and in this case as well, the substantial light receiving surface is limited to a plane facing each of the multiple cells of the microchip.

[0041] The imaging space (a) above where external light is blocked may be formed, for example, by a dark box that blocks the entire imaging section from external light, or imaging may be performed in a darkroom.

[0042] In the computer of the evaluation system of the present invention, it is preferable to provide a means for evaluating color using a color space value of a color system suitable for numerical expression as an index. For example, this can be achieved by using color space values ​​of color systems such as L*a*b*, L*C*h, Hunter Lab, etc., particularly preferably L*a*b*, as an index related to color in the computer program disclosed herein. Furthermore, values ​​of color systems such as the Munsell color system and the XYZ (Yxy) color system can also be used as an index related to color. Furthermore, conversion processing for color spaces such as HSV conversion and HSL conversion can also be used as the above means, if necessary.

[0043] A computer may be provided with an equalization processing means for equalizing the variation in color or fluorescence intensity inside the cell for the imaging data acquired by the imaging unit. As an index of the color or fluorescence intensity, as described above, L*a*b*, L*C*h, Hunter Lab, Munsell color system, XYZ (Yxy) color system, etc., with L*a*b* being particularly preferred, may be used, and conversion processing for color space such as HSV conversion or HSL conversion may be used as necessary. A computer program for causing a computer to perform the equalization processing may, for example, include the following steps: (i) selecting an arbitrary cell from among a plurality of cells of a microchip; (ii) segmenting the cell selected in step (i) in the cell image plane and labeling each segment; (iii) calculating a color value or a fluorescence intensity for each of the divided elements labeled in step (ii) and associating the color value or the fluorescence intensity with each label; (iv) extracting a median value of the color value or the fluorescence intensity for all the labels that have been associated with the color value or the fluorescence intensity in the step (i), and specifying and storing the median value as the color value or the fluorescence intensity value selected in the step (i); (v) repeatedly performing steps (i)-(iv) on cells other than the cell selected by step (i); (vi) terminating the repetition of steps (i)-(iv) when no cells other than the cell selected in step (i) are recognized in step (v); and executing a computer program of an algorithm including:

[0044] The computer program relating to the above-mentioned leveling process is one of the computer programs defined in the present invention. Execution of the computer program can also be used as the "leveling process means" in the information acquisition method of the present invention.

[0045] In the evaluation system of the present invention, the means for having a computer evaluate the properties or state of a microorganism can be the execution of a computer program of an algorithm including the following steps (i)-(iv). As the color index described below, preferably, color space values ​​of color systems such as L*a*b*, L*C*h, Hunter Lab, Munsell color system, and XYZ (Yxy) color system, particularly preferably L*a*b*, can be used. Furthermore, values ​​of color systems such as the Munsell color system and XYZ (Yxy) color system can also be used as an index related to color. Furthermore, conversion processing for color space such as HSV conversion and HSL conversion can be used as necessary. (i) labeling each of a plurality of cells of a microchip by associating a given condition with each of the cells; (ii) further associating color values ​​or fluorescence intensities for each of the cells labeled in step (i); (iii) a step of matching each of the plurality of cells labeled with the given conditions and the color values ​​or the fluorescence intensities in steps (i) and (ii) with the color values ​​or the fluorescence intensity values ​​normalized based on the reference determination information of the predetermined property or state of the microorganism, thereby associating the properties or states of the microorganisms; (iv) for each of the plurality of cells into which the properties or state of the microorganisms have been fitted by step (iii), combining the given conditions with the evaluation of the properties or state of the microorganisms to derive an evaluation of the properties or state of the microorganisms in the liquid sample, the evaluation being weighted by factors derived from the given conditions.

[0046] In the computer program for deriving the above-mentioned properties or state of a microorganism, an algorithm for selecting and excluding data of a cell for which an evaluation of the properties or state of a microorganism derived for each cell in step (iii) is inconsistent with the evaluation of the properties or state of a microorganism of a large number of other cells matched with given conditions may be incorporated as a premise for step (iv).

[0047] The computer program for deriving the properties or state of the microorganisms is also one of the computer programs defined in the present invention, and is also one of the computer embodiments of the information acquisition method of the present invention. The relationship between the computer program for deriving the properties or state of the microorganisms and the computer program for the leveling process will be described later.

[0048] The "properties or state of microorganisms" in the evaluation system of the present invention are, as described for the microchip of the present invention, examples of the properties of microorganisms include drug resistance of microorganisms, classification and detection of microorganisms at the genetic level, and examples of the state of microorganisms include, but are not limited to, the number of microorganisms per unit volume. Effect of the Invention

[0049] According to the present invention, there is provided a means capable of remarkably improving safety, simplicity, and speed while ensuring the accuracy of existing evaluation methods for the properties or state of microorganisms. The present invention relates to a microchip, a method for acquiring information on the properties or state of microorganisms from the microchip, an evaluation system for the properties or state of microorganisms using the microchip, and a computer program that can be used in the evaluation system or the acquisition method.

[0050] For example, when "presence or absence or strength of resistance to a specific drug" is selected as the property of the microorganism to be evaluated in the present invention, the safety of the evaluation process with guaranteed accuracy is ensured, and miniaturization, simplification, and speed are realized. Furthermore, simultaneous evaluation of multiple items is possible, and at the same time, the efficiency of reagent use is significantly improved because the reagent is encapsulated or fixed in small cell units with good yield, and as a result, the amount of reagent used can be reduced. In this way, the present invention makes it possible to perform an evaluation process that is exactly in line with the needs of the medical field, and can contribute to the identification of drug-resistant bacterial enzyme species and the spread of appropriate drug administration based on the identification. [Brief description of the drawings]

[0051] [Figure 1] FIG. 1 is a plan view of one embodiment of a microchip of the present invention. [Diagram 2] FIG. 2 is a schematic cross-sectional view taken along line II-II shown in FIG. [Diagram 3] FIG. 2 is a schematic cross-sectional view taken along line III-III shown in FIG. [Figure 4] 2 is a plan view showing a state in which a reinforcing film is further provided over the entire surface of the embodiment shown in FIG. 1. FIG. [Diagram 5] 5 is a plan view showing a state in which the reinforcing film of FIG. 4 is applied so as to cover the injection space and the disposal space as a whole. FIG. [Figure 6] 5 is a plan view showing a state in which the reinforcing film of FIG. 4 is applied so as to cover each of an injection space and a disposal space. FIG. [Figure 7] FIG. 2 is a schematic cross-sectional view of a reinforcing film. [Figure 8] 5 is a plan view showing a state in which a light-shielding layer is further provided on the entire surface of the embodiment using the reinforcing film of FIG. 4. FIG. [Figure 9] 7 is an overall cross-sectional view of a microchip using the reinforcing film of FIG. 6 during puncture injection using a needle. [Figure 10] FIG. 10 is a partial cross-sectional view showing an enlarged view of the puncture portion in FIG. 9. [Figure 11] FIG. 1 is a schematic diagram of an evaluation system of the present invention. [Figure 12] This shows a state in which air bubbles are mixed in when a liquid sample is introduced into a reaction cell of the microchip of the present invention (a side view and a front view from the imaging viewpoint), and a division diagram using the above front view of spot division to correct this by leveling out the air bubbles. [Figure 13] FIG. 1 shows the results of a summary evaluation of the reactions of various drug-resistant bacteria to various β-lactam antibacterial agents in Example 1, in which ΔE* was calculated for each reaction cell over a reaction time of 60 minutes using the evaluation system of the present invention. [Figure 14] This shows the ROC curves of representative antibiotics for each β-lactamase. [Figure 15]FIG. 1 shows the results of a summary evaluation of the reactions of various drug-resistant bacteria to various β-lactam antibacterial agents in Example 1, in which ΔE* was calculated for each reaction cell at a reaction time of 15 minutes using the evaluation system of the present invention. [Figure 16] This figure shows the ROC curve obtained by randomly dividing the ΔE* values ​​of the test bacteria for several antibiotics into training data and validation data and performing neural network analysis. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0052] Hereinafter, an embodiment of the present invention will be described.

[0053] (1) Microchip of the Present Invention FIG. 1 is a plan view of one embodiment of the microchip of the present invention. FIG. 2 is a schematic cross-sectional view taken along line II-II in FIG. 1. FIG. 3 is a schematic cross-sectional view taken along line III-III in FIG. 1. The microchip 1 of this embodiment includes a transparent or semi-transparent chip substrate 2 as a main part. In the chip substrate 2, a reaction space 3 and a waste injection space 4 that are independent of each other are formed. In this embodiment, the term "transparent" means a state in which the other side is clearly visible because of the transparency. The term "semi-transparent" means a state in which the other side is semi-transparent and can be seen, or a state in which the other side can be seen only by light of a specific wavelength. The state in which the other side can be seen only by light of a specific wavelength means, for example, a state in which the other side can be seen only when coloring or fluorescence occurs in a cell of the microchip, when the other side is normally opaque due to a specific color (such as dark green). In addition, a state in which a color is applied to such an extent that the other side can always be seen is also included in the term "semi-transparent".

[0054] The reaction space 3 is a sealed space formed inside the chip substrate 2. The reaction space 3 is depressurized relative to atmospheric pressure, and can be, for example, 1 / 100 atmospheric pressure or less. The reaction space 3 has an injection space 3a, a plurality of reaction cells 3b, and a flow path 3c. The reaction cells 3b provided in the chip substrate 2 may be described as one representative reaction cell or as a group of a plurality of reaction cells 3b.

[0055] The injection space 3a is a space into which a liquid sample is injected by puncturing. The shape of the injection space 3a is not particularly limited, but it may be, for example, a substantially cylindrical space.

[0056] In the reaction cell 3b, a "reagent for detecting the properties or state of microorganisms" (hereinafter, abbreviated as "reagent" unless otherwise specified) 5 is sealed or fixed in a manner that dissolves upon contact with the liquid sample, in order to react with the injected liquid sample. The "sealing" includes a state in which the reagent 5 is not fixed to the inner wall surface of the reaction cell 3b in the reaction cell 3b. In contrast, the "fixing" is a state in which the reagent 5 is attached to the inner wall surface. That is, the above-mentioned "sealing" is a concept that includes attachment. The reagent 5 can be easily fixed to the inner wall surface by drying an aqueous solution of the reagent 5 before decompression in the manufacturing process of the microchip 1. As the above-mentioned drying method, freeze-drying is preferable. The reagent 5 fixed in this manner dissolves quickly upon contact with the liquid sample when the microchip 1 is used, and causes a desired reaction (including an inhibitory reaction of microbial growth) in the reaction cell 3b, and a signal corresponding to the combination of the reagent 5 and the microorganisms in the liquid sample can be emitted in the reaction cell 3b. Examples of the signal include color (including shades according to turbidity) or fluorescence. The number and arrangement of the reaction cells 3b on the chip substrate 2 are not particularly limited, but may be, for example, 5 rows in the vertical direction (up and down (short side) direction in FIG. 1) and 5 rows in the horizontal direction (left and right (long side) direction in FIG. 1), for a total of 25 cells. For such a vertical and horizontal arrangement of the reaction cells 3b, variations in elements such as the type and concentration of the reagent 5 are standardized, and reaction signals according to the variations are obtained, thereby making it possible to obtain basic information on the type or state of the microorganisms in the liquid sample. The spatial shape of the reaction cells 3b is not particularly limited, but may be, for example, a substantially cylindrical space.

[0057] The flow path 3c is a flow path that connects the injection space 3a and each reaction cell 3b. Specifically, one flow path 3c extends from the injection space 3a, and on the way from the injection space 3a to the reaction cells 3b, the flow path 3c branches into the number of reaction cells 3b, and each branched flow path 3c is connected to each reaction cell 3b. The cross-sectional shape of the flow path 3c is not particularly limited, but may be, for example, a semicircular shape.

[0058] The throw-away space 4 is a sealed space formed inside the chip substrate 2, which can be selectively provided and used in the microchip of the present invention. The throw-away space 4 is reduced in pressure relative to atmospheric pressure, and can be, for example, 1 / 100 atmospheric pressure or less. The shape of the throw-away space 4 is not particularly limited, but for example, it can be a form in which a flow path and a reaction space similar to the reaction space 3 are provided independently of the reaction space (not shown), and it can also be further simplified to be a roughly cylindrical space.

[0059] The first role of the throw-away space 4 is to prevent air from entering the reaction space 3 during the puncture injection of the liquid sample. That is, one of the best means for preventing air from entering the reaction space 3 during the puncture injection is to remove minute air particles from the injection needle in advance. Before the actual liquid sample is injected into the reaction space 3, a test puncture injection is performed into the throw-away space 4, and the air in the injection needle is literally "discarded" into the throw-away space 4, thereby preventing air from entering the reaction space 3 during the puncture injection of the actual liquid sample. Furthermore, it is possible to confirm whether the test injection has been performed or not by marking the color of the color change agent 6 sealed or fixed in the throw-away space 4 due to contact with the liquid sample. When this first role is important, it is also preferable to select a form in which the flow path or reaction cell is omitted, such as a cylindrical shape, for the throw-away space 4. Even if the throw-away space 4 has a form with a flow path or a cell, it is natural that the first role can be performed.

[0060] The second role of the throw-away space 4 is to complement the manufacturing yield of the microchip of the present invention. That is, it is assumed that the reduced pressure state in the reaction space 3 of the microchip of the present invention is not sufficiently maintained due to subtle and probabilistic malfunctions in the manufacturing process, long-term storage, or specific handling, and the suction force for the liquid sample is weak. Even if the microchip product of the present invention in such a state is used for testing, a sufficient reaction cannot be caused in the reaction cell 3b, and there is a possibility of an incorrect test, but it is not easy to identify the cause after the fact. The throw-away space 4 is designed to have the same reduced pressure quality as the reaction space 3, and by trial puncturing and injecting a liquid sample into the throw-away space 4 before puncturing and injecting the liquid sample into the reaction space 3, the reduced pressure state inside the microchip product of the present invention can be confirmed in advance by the color development of the aqueous solution (which may be a liquid sample) and the color change agent 6 sealed or fixed in the throw-away space 4. When the second role of the throw-away space 4 is emphasized, it is preferable that the throw-away space 4 is in a form in which a flow path and a reaction space similar to the reaction space 3 are provided independently of the reaction space. In this way, the dump space 4 is designed to have the same reduced pressure quality as the reaction space 3, and by performing a test puncture injection into the dump space 4 before puncturing and injecting a liquid sample into the reaction space 3, the reduced pressure state inside the microchip product of the present invention can be confirmed in advance.

[0061] The third role of the throw-away space is to easily distinguish between used and unused microchip products of the present invention by visual inspection. Since most liquid samples are transparent, it cannot be denied that a used microchip product of the present invention may be mistaken for an unused product by a user who handles many microchips in a short time. To address this possibility of mix-up, when the microchip is used for the first time, the above-mentioned test puncture injection is performed into the throw-away space 4, whereby the liquid sample comes into contact with the color change agent 6, and the color change agent 6 sealed or fixed in the throw-away space 4 develops color. As a result, the used microchip product of the present invention is marked by the above-mentioned color development, making it possible to prevent subsequent mix-ups. When this third role is important, it is also preferable to select a form in which the throw-away space 4 does not include a flow channel or a reaction cell, such as a cylindrical shape. Even if the form includes a flow channel or a cell, the throw-away space 4 can fulfill the third role, as in the above-mentioned first role.

[0062] The discoloration of the color-changing agent 6 enclosed or fixed in the throw-away space 4 corresponds to changes in each element of color such as hue, brightness, saturation, etc., and to a change between colorless and colored, etc. As the color-changing agent 6, a compound or composition that changes color upon reacting with water is suitable, and examples thereof include cobalt(II) chloride, copper(II) sulfate (anhydride), the moisture / humidity indicator described in Japanese Patent No. 4537767, the color change indicator described in Japanese Patent No. 4100776, and the moisture indicator composition described in Japanese Patent No. 3298217.

[0063] The chip substrate 2 is formed in a thin, generally rectangular plate shape, but this can be changed as necessary according to requirements such as standards, and is not limited thereto. The chip substrate 2 has a double structure of a first plate-shaped portion 7 and a second plate-shaped portion 8 laminated on one surface of the first plate-shaped portion 7. The first plate-shaped portion 7 and the second plate-shaped portion 8 are bonded together in a state where they are overlapped with each other.

[0064] The first plate-shaped portion 7 is formed in a thin, approximately rectangular plate shape. A reaction space recess 7a and a dump space recess 7b are formed on the surface of the first plate-shaped portion 7 facing the second plate-shaped portion 8. The reaction space recess 7a is a recess for forming the reaction space 3. The shape of the reaction space recess 7a corresponds to the shape of each of the injection space 3a, the multiple reaction cells 3b, and the flow path 3c. The dump space recess 7b is a recess for forming the dump space 4. The shape of the dump space recess 7b corresponds to the shape of the dump space 4.

[0065] The first plate-shaped portion 7 is made of a gas-impermeable material. The material of the first plate-shaped portion 7 is not particularly limited, but examples thereof include gas-impermeable glass and synthetic resin. Examples of synthetic resin include PMMA (polymethyl methacrylate: acrylic resin), PC (polycarbonate), PS (polystyrene), PET (polyethylene terephthalate), COP, COC (cyclic polyolefin), etc.

[0066] The second plate-shaped portion 8 is formed in a thin, approximately rectangular plate shape. The second plate-shaped portion 8 covers at least the reaction space recess 7a and the dump space recess 7b. In this case, it is preferable that the second plate-shaped portion 8 covers the entire surface of the first plate-shaped portion 7. By covering the reaction space recess 7a, the second plate-shaped portion 8 forms an injection space 3a of the reaction space 3, a plurality of reaction cells 3b, and a flow path 3c between the second plate-shaped portion 8 and the first plate-shaped portion 7. In addition, by covering the dump space recess 7b, the second plate-shaped portion 8 forms the dump space 4 between the second plate-shaped portion 8 and the first plate-shaped portion 7.

[0067] The second plate-shaped portion 8 has a self-sealing property. The self-sealing property means that even if a hole is made by puncturing or the like, the hole is naturally sealed by the restoring force due to the elastic deformation of the second plate-shaped portion 8. Examples of the elastic material used for the second plate-shaped portion 8 include silicone-based elastomers, acrylic-based elastomers, urethane-based elastomers, and fluorine-based elastomers. The second plate-shaped portion further preferably has gas permeability, and PDMS (polydimethylsiloxane), which is an elastic material having self-sealing property and gas permeability, can be used.

[0068] The microchip 1 thus configured can be manufactured as follows. That is, a transparent dielectric film such as SiO2, Al2O3, TiO, etc. is coated on the surface of the first plate-shaped portion 7 that is to be joined to the second plate-shaped portion 8. The transparent dielectric film can be coated by, for example, sputtering or vacuum deposition. Next, the reagent 5 is dropped into the recess of the reaction space recess 7a corresponding to the reaction cell 3b, and the color change agent 6 is dropped into the disposal space recess 7b, and the reagent 5 in the reaction cell 3b and the color change agent 6 in the disposal space recess 7b are subjected to a drying method such as vacuum freeze drying. Next, the surface of the first plate-shaped portion 7 that is to be joined to the second plate-shaped portion 8 and the surface of the second plate-shaped portion 8 that is to be joined to the first plate-shaped portion 7 are plasma cleaned. Next, the first plate-shaped portion 7 and the second plate-shaped portion 8 are overlapped in a reduced pressure atmosphere (a state reduced in pressure relative to atmospheric pressure). As a result, the first plate-shaped portion 7 and the second plate-shaped portion 8 are joined together, and the reaction space 3 and the throw-away space 4, which are decompressed relative to atmospheric pressure, are formed between the first plate-shaped portion 7 and the second plate-shaped portion 8. The reaction space 3 and the throw-away space 4 are at substantially the same atmospheric pressure.

[0069] As a preferred embodiment of the microchip of the present invention, a reinforcing film can be further used. The reinforcing film can be used at least in the injection space 3a of the reinforcing film 10 and, if used, in a position facing the dump space 4, thereby preventing the lateral vibration of the puncture needle when the liquid sample is punctured and injected into the microchip 1 and suppressing the expansion of cracks caused by puncturing. FIG. 4 is a plan view of an embodiment in which the reinforcing film 10 is further used on the entire surface of the chip substrate 2 in the microchip 1. The reinforcing film 10 is attached to the entire surface of the surface 8a of the second plate-like portion 8. That is, the reinforcing film 10 is attached to the surface 8a of the second plate-like portion 8 at a position facing the reaction space 3 (injection space 3a, reaction cell 3b, flow path 3c) and the dump space 4, as well as at a position not facing the reaction space 3 and the dump space 4. Characters or a code L indicating identification information of the microchip 1 may be printed on the reinforcing film 10. In addition, the reinforcing film 10 does not have to be attached to the entire surface of the surface 8a of the second plate-shaped portion 8 as long as it is attached to a position facing the injection space 3a and the throw-away space 4. In FIG. 5, the reinforcing film 10 is attached to the surface 8a of the second plate-shaped portion 8 so as to integrally cover, with a rectangle, a position facing the injection space 3a and a position facing the throw-away space 4. In FIG. 6, the reinforcing film 10 is attached to the surface 8a of the second plate-shaped portion 8 so as to cover, with a circle, a position facing the injection space 3a and a position facing the throw-away space 4. In addition, the above-mentioned rectangle and circle are merely examples, and the shape is not limited to these shapes.

[0070] 7 is a schematic cross-sectional view of the reinforcing film 10. As described above, the reinforcing film 10 includes a base material 10a forming the main body of the reinforcing film 10, and an adhesive portion 10b attached to the second plate-shaped portion 8. The material and thickness of the base material 10a and the adhesive portion 10b can be, for example, the following first and second conditions. However, the material and thickness of the base material 10a and the adhesive portion 10b are not limited to the following conditions.

[0071] The first condition is that the material of the base material 10a is polyurethane or polyester (PET), and the material of the adhesive portion 10b is an acrylic adhesive or a silicone adhesive. The thickness of the base material 10a is 10-30 μm, and the thickness of the adhesive portion 10b is 20-120 μm. By making the materials and thicknesses of the base material 10a and the adhesive portion 10b satisfy the above first condition, the reinforcing film 10 has excellent elasticity.

[0072] The second condition is that the material of the base material 10a is polypropylene or polyester (PET), and the material of the adhesive portion 10b is a silicone-based adhesive. The thickness of the base material 10a is 20-100 μm, and the thickness of the adhesive portion 10b is 20-100 μm. By making the materials and thicknesses of the base material 10a and the adhesive portion 10b satisfy the second condition, a reinforcing film 10 with excellent shape retention can be obtained.

[0073] In addition, from the viewpoint of suppressing stray light when light from a light source for obtaining a desired color or fluorescent signal is directed toward the first plate-shaped portion 7, which is the light receiving surface of the microchip 1, a light-shielding layer 11 colored in a dark color such as black can be provided on the second plate-shaped portion 8 side of the reinforcement film 10, as shown in FIG. 8. It is preferable that the light-shielding layer 11 is colored in a dark color such as black only at a position on the surface of the second plate-shaped portion 8 side that does not face the reaction cell 3b. The position facing the reaction cell 3b is transparent. This allows the light irradiated to each reaction cell 3b to pass through, and stray light of the light irradiated to each reaction cell 3b can be suppressed, thereby improving detection accuracy. The lamination position at which the light-shielding layer 11 is provided is not limited as long as it does not substantially impede the effect of the present invention in the microchip 1, and it is also possible to provide the light-shielding layer 11 further outside or inside the first plate-shaped portion 7, for example.

[0074] (2) Information Acquisition Method of the Present Invention As mentioned above, the liquid sample used in the present invention is an aqueous dilute solution containing microorganisms derived from a biological sample.

[0075] That is, (a) a step of concentrating and collecting the target microorganism from the above-mentioned biological sample (typically blood or urine), or a step of collecting the target microorganism that has been cultured and grown, and (b) a step of diluting the target microorganism collected in step (a) to a predetermined concentration, thereby preparing the above-mentioned liquid sample. The concentration means in the above-mentioned (a) "concentration of the target microorganism" is not particularly limited. That is, concentration by centrifugation, concentration by filter filtration using filter paper or glass filter paper, concentration by polymer precipitation method, specifically, separation by precipitation of bacteria in the biological sample by adding a polymer, concentration of bacteria by removing water by adding a water-absorbing polymer to the biological sample, etc. can be mentioned, but are not limited to these.

[0076] (c) Next, a step of puncturing and injecting the liquid sample into the injection space 3a of the microchip 1 is performed, and a step of transferring the injected liquid sample to each reaction cell 3b through the flow path 3c by negative pressure is performed. Figures 9 and 10 are schematic cross-sectional views of the microchip 1 of Figure 6 using the reinforcing film 10, in which a cylindrical reservoir 91 storing a liquid sample and an injection chip 9 equipped with a needle N for injecting the liquid sample into the microchip are used for puncturing and injecting. Figure 9 is an overall cross-sectional view, and Figure 10 is an enlarged cross-sectional view of the puncturing part. That is, the needle N is punctured into the second plate-shaped part 8 of the chip substrate 2 toward the injection space 3a, and the tip part of the needle N is inserted into the injection space 3a. At this time, the needle N is pressed by the second plate-shaped part 8 having self-sealing properties, and it is possible to distribute the desired liquid sample to each reaction cell 3b through the flow path 3c by negative pressure even without the reinforcing film 10. However, the needle N is also pressed down by the reinforcing film 10, which suppresses deformation of the second plate-shaped portion 8 due to lateral vibration of the needle N. Moreover, the reinforcing film 10 is attached to the surface 8a of the second plate-shaped portion 8 around the puncture hole H, which also suppresses the extension of cracks from the puncture hole H. This further suppresses the inflow of air from the injection space 3a to the flow path 3c and further to the reaction cell 3b.

[0077] (d) Then, the liquid sample is introduced into each reaction cell 3b, and the liquid sample comes into contact with the drug encapsulated or fixed in each reaction cell 3b, whereby the drug dissolves, and a predetermined reaction indicating the properties or state of the target microorganism contained in the liquid sample is initiated under favorable conditions by keeping the microchip 1 at a constant temperature, thereby promoting the generation of a predetermined signal.

[0078] As an example, an outline of a case where the presence or absence and the level of production of β-lactamase, which is responsible for the drug resistance of microorganisms to β-lactam antibacterial drugs (β-lactam antibacterial agents and antibiotics, and antibacterial drugs containing β-lactamase inhibitors) having a β-lactam ring, in a target microorganism is detected by the acidometry method. Naturally, the concept of β-lactam antibacterial drugs shown here can be applied to other types of antibacterial drugs as well.

[0079] The acidometry method is a method that uses the color change of a pH indicator, which shows a decrease in pH due to the -COOH produced when antibacterial agents or antibiotics that have a β-lactam ring in their molecular structure are hydrolyzed by β-lactamase, as an indicator of drug resistance to β-lactam drugs.

[0080] Hydrolysis of the β-lactam ring in β-lactam antibiotics by β-lactamase produced by drug-resistant bacteria, which serves as a detection signal for pH indicators in acidometry and other methods, proceeds as follows:

[0081] [ka]

[0082] The pH indicator is not particularly limited as long as it is capable of identifying a decrease in pH due to the decomposition of the β-lactam ring by β-lactamase through a color change, and examples of such indicators include phenol red (color change range: pH 6.8-8.4, red on the alkaline side but turns yellow on the acidic side), neutral red (color change range: pH 6.8-8.0, yellow on the alkaline side but turns red on the acidic side), bromocresol purple (color change range: pH 5.2-6.8, purple on the alkaline side but turns yellow on the acidic side), bromothymol blue (color change range: pH 6.0-7.6, blue on the alkaline side but turns yellow on the acidic side), and nitrazine yellow (color change range: pH 6.4-6.8, light blue on the alkaline side but turns yellow on the acidic side).

[0083] There is no limit to the types of β-lactam antibacterial agents that are the subject of microbial drug resistance evaluation, and they include penicillins, cephalosporins, carbapenems, monobactams, etc. Antibacterial agents containing β-lactamase inhibitors are also included.

[0084] By using the microchip of the present invention, it is possible to simultaneously evaluate drug resistance to multiple β-lactam antibacterial agents. That is, for example, when evaluating drug resistance of a target microorganism using a microchip 1 having 5×5 reaction cells 3b, even taking into account the reaction cells 3b used as controls, drug resistance to three types of β-lactam antibacterial agents can be simultaneously, safely, and quickly evaluated using one microchip 1.

[0085] For example, when three types of β-lactam antibacterial drugs are selected in advance and the microchip 1 is used to evaluate the drug resistance of the target microorganisms in the liquid sample against these β-lactam antibacterial drugs, the three types of β-lactam antibacterial drugs are separately included in the prescription, and each β-lactam antibacterial drug is prescribed with a concentration gradient. The reagents 5 (containing multiple prescriptions according to the type and prescribed concentration of the β-lactam antibacterial drug) are arranged in each reaction cell 3b so that the type and concentration gradient of the β-lactam antibacterial drug are two-dimensionally developed into a matrix, and the reagents are sealed or fixed to prepare the microchip 1. A liquid sample is introduced into each reaction cell 3b of the microchip 1 thus prepared in the above manner. In this way, the liquid sample is contacted with the reagent 5, and the reagent 5 dissolves in the water of the liquid sample, and a color reaction by the acidometry method, which is an indicator of the decomposition of the β-lactam ring by the β-lactamase produced by the target microorganism, according to the concentration of each β-lactam antibacterial drug, can be used to detect the presence or absence or the strength of drug resistance to the above three types of β-lactam antibacterial drugs in the target microorganism in the liquid sample. That is, when phenol red is used as a pH indicator, if the spot in the reaction cell 3b, which is originally red, turns yellow due to a decrease in pH caused by the action of the β-lactamase produced by the target microorganism, it can be detected that the target microorganism shows drug resistance to the β-lactam antibacterial drug indicated by the spot in terms of the concentration of the antibacterial drug. The results at each of these spots can be detected as a matrix of spots constituting the microchip 1, which have elements of the type and concentration of the antibacterial drug.

[0086] (e) The detection information shown in (d) serves as the basis for evaluating the properties or state of the target microorganism. For example, as mentioned above, the detection results of the color reaction in the spots of the target microorganism using the above-mentioned acidometry method serve as the basis for evaluating the drug resistance of the target microorganism to the three selected β-lactam antibacterial drugs.

[0087] One of the evaluation methods is to visually evaluate the color change. The color change can be preferably compared with standard information to evaluate the presence or absence or the degree of decomposition of the β-lactam antibacterial agent in each spot by β-lactamase produced by the target microorganism.

[0088] Another evaluation method is to obtain the detection result by converting the coloration into image information, that is, image information that can be processed by a computer.

[0089] The image information can be obtained by capturing an image of the reaction cell 3b that has already been colored or emitted fluorescence, preferably of the display surface (the surface opposite to the light-receiving surface) of the microchip 1. The imaging means is preferably a means capable of obtaining digital image information (such as a digital camera).

[0090] The image information may be the data obtained by imaging, or may be appropriately processed.

[0091] The above processing can be executed by a computer. Specifically, there are a leveling process by a leveling process means for leveling the variation in color or fluorescence intensity within each cell, and a process for converting the color difference or fluorescence intensity difference calculated by a computer for each cell between the color value or fluorescence intensity value normalized based on the reference judgment information corresponding to the properties or state of the microorganism, into image information. In addition, the image information by the above color is preferably image information by color space values ​​of a color system suitable for digitization. For example, color space values ​​of color systems such as L*a*b*, L*C*h, Hunter Lab, etc., are included, and L*a*b* is particularly preferable. Furthermore, it is also possible to use values ​​of color systems such as the Munsell color system and the XYZ (Yxy) color system, and a conversion process for the color space such as HSV conversion or HSL conversion can be used as necessary. These are as described as "means for solving the problem".

[0092] The image information acquired by the information acquisition method of the present invention also serves as the basis for a computer-based evaluation of the properties or state of a microorganism by the evaluation system of the present invention described below.

[0093] (3) Evaluation system of the present invention (including the computer program of the present invention) The evaluation system of the present invention is composed of (a) an image acquisition unit that acquires, as image information, color or fluorescence that indicates the properties or state of the microorganism from each of the reaction cells 3b of the microchip 1, and (b) a computer, and (c) the computer is provided with "means for making the computer compare the input image information with a color value or a fluorescence intensity value normalized based on reference determination information for the properties or state of the microorganism, and for making the computer evaluate the properties or state of the microorganism obtained from the biological sample." In addition, (d) the image acquisition unit may be equipped with a computer.

[0094] The image acquisition unit in (a) above is preferably an imaging unit that uses an imaging device, and Fig. 11 is a schematic diagram of the entire evaluation system 20 of the present invention in the form of this imaging unit. The above items (a) to (d) will be described with reference to Fig. 11.

[0095] (a) Image acquisition unit (imaging unit) 11, an evaluation system 20 includes an imaging unit 21 and a computer 22. First, the imaging unit 21 will be described.

[0096] The imaging unit 21 is largely composed of a dark box 211, which is an imaging space where external light is blocked, and an imaging mechanism 212 housed therein. The form of the dark box 211 is not limited. For example, a freely openable door (not shown) can be provided on the front surface 2111. The door can be closed to block external light when imaging, and opened to facilitate various operations when setting the microchip 1 or making various adjustments. The dark box 211 exists to block external light when imaging, and even if the dark box 211 is not present, it is sufficient if imaging is performed in, for example, a dark room or a darkened room, and the dark box 211 is not essential as a configuration of the imaging unit 21. However, the presence of the dark box 211 is preferable in the sense that imaging can be performed regardless of location. On the other hand, the imaging mechanism 212 is essential as a configuration of the imaging unit 21. 11, the imaging mechanism 212 is shown in a form in which the position fixing mechanism for imaging (position fixing mechanism 2122 for the microchip, position fixing mechanism 2124 for the imaging device) is supported at a predetermined height by the support 2121, but the present invention is not limited to this form. For example, the position fixing mechanism for imaging may be directly attached to the back surface 2112 (the surface facing the door) or the side surfaces 2113 and 2114 of the dark box 211, accompanied by a height switching means. However, in a form in which the support 2121 is provided, it is preferable to provide a function for adjusting the height of the position fixing mechanism for imaging each time while guiding the position fixing mechanism for imaging to the support 2121, thereby easily and freely adjusting the height of the position fixing mechanism for imaging for use in imaging. 11, one support pillar 2121 is provided on the back surface 2112 of the dark box 211, which is a preferred embodiment, but multiple support pillars can be provided on the back surface 2112 as necessary, and one or multiple support pillars can also be provided on the side surfaces 2113 and 2114. Furthermore, it is also possible to provide a stand-type accessory with a base (not shown) that is detachable from the dark box 211 and designed so that the support pillar 2121 stands in the height direction of the dark box 211, preferably along the back surface 2112.

[0097] A light source 213 is provided on the bottom surface 2115 of the dark box 211. The light source 213 is provided at a predetermined position where the light receiving surface of the microchip 1, which is fixed in position by a microchip position fixing mechanism 2122 described later, is on the optical path of the emitted light, for example, on the bottom surface 2115 directly below the light receiving surface of the microchip 1, or on a base (not shown) when the support 2121 is attached to the stand type.

[0098] A support 2121 rising from the bottom surface 2115 on which the light source 213 is provided is provided with a microchip position fixing mechanism 2122 and an imaging device position fixing mechanism 2124 from the lower side.

[0099] The microchip position fixing mechanism 2122 is supported by the support 2121 and fixed in position (detailed mechanism is not shown). One specific example of the embodiment is a form in which a protruding rod part protruding from the support 2121 toward the door direction is the main part, accompanied by a mechanism capable of fixing the position of the support 2121 at a desired height, and a plate holder 2123 capable of finely adjusting the position, preferably in the front (door direction) and back (support direction) directions, is provided near the tip of the protruding rod part. The plate holder 2123 is provided with a mechanism for fixing the position of the microchip 1 in a detachable state. The light receiving surface of the microchip 1 fixed in position by the plate holder 2123 can be fixed in position on the main optical path of the light emitted from the light source 213, and can be fixed in position so that the light can pass through each reaction cell 3b from the light receiving surface side. For example, the above-mentioned "light-transmitting method" includes a method of fixing the position of the microchip 1 by pressing the side of the microchip 1 to fix the position and exposing the light receiving surface of the microchip 1 toward the light source 213, a method of providing a bottom part on which the microchip 1 can be fixed and the bottom part is made of a transparent material, or a method of providing a light transmission opening at a portion of the bottom part corresponding to each reaction cell 3b. The type of the light source 213 is not limited, and can be selected according to the type of color or fluorescence detected in the microchip 1. In addition, it is preferable that the type and illuminance of the illumination can be controlled from the outside. In FIG. 11, the form in which the control in the computer 22 is performed is shown in a form in which the light source 213 and the computer main body 221 are connected by a data transfer cable 2211.

[0100] The position fixing mechanism 2124 of the imaging device is also supported by the support 2121 and fixed in position (detailed mechanism is not shown). As an example of a specific embodiment, a form in which a protruding rod part protruding from the support 2121 toward the door direction is included as a main part, with a mechanism capable of fixing the position at a desired height of the support 2121, similar to the above-mentioned microchip position fixing mechanism 2122, and an imaging device holder capable of finely adjusting the position in the front (door direction) and back (support direction) directions is provided near the tip of the protruding rod part. The imaging device holder is provided with a mechanism capable of fixing the position of the imaging device 214. The imaging device 214 may be detachable from the imaging device holder or may be fixed at all times. A representative embodiment of the imaging device 214 is a camera of the illustrated form, and it is preferably a digital camera capable of acquiring digitized image information. However, it may be in a form other than a camera, for example, a "computer device with imaging function" such as a smartphone (a form in which a computer is built into the imaging device). The imaging device 214 is fixed in position so that the light emitted from the light source 213 is incident on its light incidence mechanism (if the imaging device 214 is a camera, the camera lens) from the main optical path. By doing so, the light emitted from the light source 213 and transmitted through the microchip 1, whose position is fixed by the plate holder 2123, particularly through the reaction cells 3b group, is incident on the light incidence mechanism of the imaging device 214, and image information based on the transmitted light can be acquired.

[0101] In this way, the imaging unit 21 can obtain image information of color or fluorescence, which is an indicator of the properties or state of the microorganisms in the microchip 1, as transmitted light through the microchip 1 by light emitted from the light source 213, using the imaging device 214.

[0102] (b) Computers as components of the evaluation system As described above, the evaluation system 20 is essentially composed of the image acquisition unit 21 and the computer 22. The computer 22 is not limited in appearance or form as long as it can execute a computer program for implementing the evaluation system of the present invention by combining a storage device and an arithmetic unit in the computer main body 221. Hereinafter, such a "computer capable of executing a computer program for implementing the evaluation system of the present invention" will be referred to as an "execution computer" unless otherwise specified. The computer 22 has a monitor 222 connected to the main body 221 by a display cable 2213.

[0103] For example, as described above, the imaging device 214 may have a built-in function of an execution computer. Typically, a smartphone is provided with the functions of both the imaging device and the execution computer, and is used as the imaging device 214, allowing the smartphone to perform both imaging and the function of the execution computer. Also, a built-in form in which a microcomputer that functions as the execution computer is attached to the image acquisition unit (imaging unit 21) is also possible. Naturally, a computer terminal separate from the image acquisition unit can also be used as the execution computer.

[0104] When the imaging device 214 and the execution computer are separate, any existing or future computer input means, such as wired or wireless, can be used. In Fig. 11, the computer input means is shown in a state in which the imaging device 214 and the computer main body 221 are connected by a data transfer cable 2212. Furthermore, for example, an existing (commercially available) storage device can be connected to the imaging device 214, image information acquired by the imaging device 214 can be input to the storage device, and this can be input again to the main body 221 of the computer 22, which is the execution computer, to execute the evaluation system of the present invention. Also, a cloud system or a self-owned server device can be used as a storage means for image information or an execution computer.

[0105] (c) Evaluation step using the evaluation system of the present invention As described above, the execution computer is equipped with a computer program as "means for causing a computer to compare input image information with a color value or a fluorescence intensity value normalized based on reference judgment information for the properties or state of the microorganism, and for evaluating the properties or state of the microorganism obtained from the biological sample."

[0106] Here, the aspect of the process up to evaluation by the evaluation system of the present invention, including the execution process of the above computer program, will be described as the above "evaluation system 20 of the present invention equipped with the imaging unit 21 and the main body 221 of the computer 22 which is an execution computer." Image information captured by the imaging unit 21 is input to the execution computer main body 221 via a data transfer cable 2212.

[0107] (a) The starting state is that the microchip 1, in which the reaction for a specified color or fluorescence has been completed, is properly positioned and fixed on the plate holder 2123 provided in the imaging unit 21, and the door of the dark box 211 is closed.

[0108] (a) Next, a step of selecting and adjusting the type and illuminance of the illumination in the light source 213 is performed. The selection and adjustment is illustrated as being performed by the computer 22, which is an execution computer. Alternatively, for example, an illuminance adjuster electrically connected to the light source 213 can be used, or the selection and adjustment can be performed by an external computer. The type of light source 213 is selected depending on the type of color or fluorescence detected in the microchip 1.

[0109] (c) After the light source 213 is selected and adjusted, the image capturing device 214 performs a process of acquiring image information of the microchip 1 that is incident on the microchip 1 after passing through the microchip 1. In short, this is a process of acquiring image data of the display surface of the microchip 1.

[0110] (D) As an example of a means for evaluating the properties or state of the microorganisms by the color value or fluorescence intensity of each reaction cell 3b quantified by the execution computer 22 for the image information of the microchip 1 acquired in (C), there can be mentioned the execution of a computer program of an algorithm including the following steps (i)-(iv). At this point, the image information is in a state where it can be processed by the execution computer, that is, the image information is already stored in the storage device of the execution computer main body 221, or, in a mode not shown, the image information is stored in a storage device built into the imaging device 214 or an external storage device (such as a cloud system), and each of the stored image information is in a state where it can be processed by the execution computer. (i) labeling each of a plurality of cells of a microchip by associating a given condition with each of the cells; (ii) further associating color values ​​or fluorescence intensities for each of the cells labeled in step (i); (iii) a step of matching each of the plurality of cells labeled with the given conditions and the color values ​​or the fluorescence intensities in steps (i) and (ii) with the color values ​​or the fluorescence intensity values ​​normalized based on the reference determination information of the predetermined property or state of the microorganism, thereby associating the properties or states of the microorganisms; (iv) for each of the plurality of cells into which the properties or state of the microorganisms have been fitted by step (iii), combining the given conditions with the evaluation of the properties or state of the microorganisms to derive an evaluation of the properties or state of the microorganisms in the liquid sample, the evaluation being weighted by factors derived from the given conditions.

[0111] In the computer program for deriving the above-mentioned properties or state of a microorganism, an algorithm for selecting and excluding data of a cell for which an evaluation of the properties or state of a microorganism derived for each cell in step (iii) is inconsistent with the evaluation of the properties or state of a microorganism of a large number of other cells matched with given conditions may be incorporated as a premise for step (iv).

[0112] The "given conditions" in (i) above, in the case of evaluating drug resistance of microorganisms, include conditions for each reaction cell 3b, such as the type and concentration of a drug to be evaluated for drug resistance.

[0113] In the above (iii) "process of comparing the color value or fluorescence intensity value normalized based on the standard judgment information of the property or state of a specified microorganism with each of the plurality of cells to which the color value or fluorescence intensity is linked and labeled under given conditions, and linking the cells to the property or state of the specified microorganism", the "color value or fluorescence intensity value normalized based on the standard judgment information of the property or state of the specified microorganism" includes a negative control (NC) value.

[0114] For example, if L*a*b* values ​​are used as color space values ​​in the above-mentioned acidometry method, and the (L* value, a* value, b* value) after 5 minutes of reaction are as follows: Negative control: (L* value, a* value, b* value) = (48, 70, 50) Non-resistant strain: (L* value, a* value, b* value) = (49, 69, 48) Resistant strain: (L* value, a* value, b* value) = (70, 31, 73) The above match value is converted into the formula:

number

[0115] Here, the ΔE* threshold for each antibiotic can be determined by statistical or machine learning methods such as linear or quadratic discriminant analysis, logistic regression analysis, or support vector machines.

[0116] It is also possible to adjust color indexes such as L*a*b* values ​​and ΔE* values ​​for each antimicrobial drug as learning data and evaluate drug resistance based on this. In this case, drug resistance can be evaluated in one step based on image matrix data for all reaction cells 3b of the microchip 1, which can contribute to further accelerating the evaluation process.

[0117] (E)-1 As a prerequisite for having the computer 22 execute the process of (E) above on the image information from the microchip 1, it is preferable to execute a filtering process to provide the image information for the evaluation process. The filtering process is a conversion process (filtering) for making the image information easier to process in the evaluation process, and examples of the filtering process include setting the color gamut in the selected color space, adjusting the degree of shading according to the color gamut, smoothing / leveling process for noise removal, and processing for emphasizing local structures such as edges, points, lines, and surfaces. As preferable examples, a shading process for the acquired image information and a leveling process as described later in (E)-2 are given here.

[0118] The shading processing step executed by the execution computer includes the following algorithms (i) to (iii). (i) A pixel information extraction process step for fitting the acquired image information of the reaction cells 3b into a color gamut corresponding to a preset color space such as L*a*b*; (ii) a shading calculation process step for calculating the shading degree according to the negative (no reaction)-positive (reaction) state of the color value or fluorescence intensity observed in the reaction cell 3b from the pixel information fitted to the color gamut extracted in the above step (i); (iii) A shading process step in which a predetermined threshold value is applied to the shading calculated in the above step (ii) to binarize the shading (black and white shading).

[0119] Regarding the "fitting process into a color gamut corresponding to a color space such as L*a*b* set in advance" in the above step (i), the color of the image data of each reaction cell 3b can be quantified preferably using the color space values ​​of the color system, such as L*a*b*, L*C*h, and Hunter Lab, particularly preferably L*a*b*. It is also possible to quantify the color or fluorescence intensity using color system values ​​such as the Munsell color system and the XYZ (Yxy) color system, and a conversion process step for the color space, such as HSV conversion or HSL conversion, can be introduced into the algorithm as necessary. The quantification of these color or fluorescence intensities is performed by processing by the execution computer. Typical examples of the quantification of color or fluorescence intensity are as follows.

[0120] In the L*a*b* color space, lightness is represented by L*, and chromaticity, which indicates hue and color, is represented by a* and b*. a* indicates the red direction, -a* indicates the green direction, b* indicates the yellow direction, and -b* indicates the blue direction.

[0121] In the L*C*h color space, lightness is represented by L*, C* represents saturation, and h represents the hue angle.

[0122] In the Hunter Lab color space, the calculation formula and values ​​are different from L*a*b*, and it is expressed as HL,a,b.

[0123] In the Munsell color system, it is expressed by hue H, value V, and chroma C.

[0124] In the XYZ (Yxy) color system, reflectance is represented by Y, and chromaticity is represented by x and y.

[0125] (E)-2 Leveling process One of the preferable treatments in the present invention is "a leveling treatment for leveling out the variation in color or fluorescence intensity inside the cell."

[0126] Figure 12 shows a state in which air bubbles are mixed in when a liquid sample is introduced into a reaction cell of a microchip (a side view and a front view from the imaging viewpoint), and a division diagram using the above front view of spot division to correct this by leveling out the air bubbles.

[0127] When the liquid sample in the microchip 1 is introduced into the reaction cell 3b, there is a risk that minute air bubbles 12 may be mixed in. In that case, the wall (vertical part) in the height direction of the reaction cell 3b will have a lower light transmittance and will become dark (see the side view and front view of FIG. 12). For this reason, it is preferable to execute a computer program including an algorithm for executing a process of performing a "division process excluding the image part near the wall in the height direction" shown in the division diagram of FIG. 12 on the image information of the reaction cell 3b and excluding division elements corresponding to air bubbles from the group of division elements. This leveling process is preferable to be executed after "(i) a process of extracting pixel information by fitting the image information of the acquired reaction cell 3b group into a color gamut corresponding to a color space such as L*a*b* that has been set in advance" as a prerequisite for executing the filtering process of (E)-1 above. In the case where the filtering process is not performed, it is also possible to incorporate the data obtained by the leveling process after performing the above-mentioned "pixel information extraction process by fitting the acquired image information of the group of reaction cells 3b into a color gamut corresponding to a preset color space such as L*a*b*" into step (i) or (ii) of the algorithm shown in (e) above, and to cause a computer to execute a program including the algorithm of (e).

[0128] Specifically, the leveling process includes the following steps: (i) selecting an arbitrary cell from among a plurality of cells of a microchip; (ii) segmenting the cell selected in step (i) in the cell image plane and labeling each segment; (iii) calculating a color value or a fluorescence intensity for each of the divided elements labeled in step (ii) and associating the color value or the fluorescence intensity with each label; (iv) extracting a median value of the color value or the fluorescence intensity for all the labels that have been associated with the color value or the fluorescence intensity in the step (i), and specifying and storing the median value as the color value or the fluorescence intensity value selected in the step (i); (v) repeatedly performing steps (i)-(iv) on cells other than the cell selected by step (i); (vi) terminating the repetition of steps (i)-(iv) when no cells other than the cell selected in step (i) are recognized in step (v); It is possible to introduce a computer program execution process of an algorithm including the above.

[0129] A representative embodiment of the division in the above step (ii) is "9 divisions" as shown in the division diagram of FIG. 12, but is not limited thereto. In particular, the number of divisions can be changed, and whether or not the vicinity of the wall in the height direction is included in the division elements can be changed as appropriate. In addition, the "median" shown in step (iv) may be the average value (arithmetic mean, geometric mean, harmonic mean) of the calculated color values ​​or fluorescence intensities of each division element, or the color value or fluorescence intensity of the division element that is in the middle (the fifth in the case of 9 divisions) in the order of the color values ​​or fluorescence intensities of each division element may be the median value of the color values ​​or fluorescence intensities of the cell that includes that division element. In addition, it does not necessarily have to be the "median" and can be changed as appropriate, for example, to the "maximum value", "the intermediate value between the median value and the maximum value", etc. In addition, it is preferable that the above-mentioned ΔE* value is used as an index for the above-mentioned color value or fluorescence intensity. EXAMPLES

[0130] Examples of the present invention are shown below, but the present invention is not limited to these examples.

[0131] [Example 1] Evaluation of β-lactam antibacterial resistance using the microchip of the present invention A test for evaluating drug resistance of "bacterial strains known to be resistant to β-lactam antibacterial agents" was carried out using the microchip of the present invention in an embodiment in which a reinforcing film was provided over the entire surface as shown in FIG.

[0132] The detection principle was the acidometry method, and the evaluation based on the color change by the acidometry method was performed by the ΔE* value collated with the L*a*b* value. The ΔE* threshold was evaluated by setting an arbitrary threshold value for each combination of antibiotic and β-lactamase, and by using the results of neural network analysis of the ΔE* values ​​of all antibiotic substrates.

[0133] <Test species> Escherichia coli or Klebsiella pneumoniae Among the strains, those that had been confirmed to have the following properties by broth microdilution and PCR were evaluated for their resistance to β-lactam antibiotics.

[0134] (a) Strains confirmed to produce β-lactamase: 20 strains (b) β-lactam antibiotic-susceptible strains (Non-AMR. AMR is an abbreviation for Antimicrobial Resistance): 20 strains (c) ESBL (Extended-spectrum beta-lactamase)-producing strains: 20 strains (d) Class A CPE (Carbapenemase-producing Enterobacteriaceae (Hereinafter referred to as carbapenemase-producing strains): 10 strains (e) Class B CPE (carbapenemase-producing strains): 8 strains

[0135] <Reagent composition and final concentration> A reagent having the following composition was prepared and was fixed to the inner wall of a given reaction cell.

[0136] (a) Substrate (antibacterial agent): 0.5 mg / mL each CEZ (cefazoline), CTX (cefotaxime), CFS (cefsulodin), CTRX (ceftreaxone), CPDX (cefpodoxime), AZT (aztreonam), CMZ (cefmetazole), IPM (imipenem) (b) β-lactamase inhibitors (used in combination with antibacterial agents): 0.2 mg / mL each TAZ (tazobactam) and AVI (avibactam) were used as combination drugs CEZ / TAZ and CAZ / AVI. (c) Buffer: CAPS 3mM (d) pH indicator: phenol red (PR) 0.6 mM (e) Salt: NaCl 100 mM (f) Surfactant: Tween 20 0.5% (g) Fixing agent: n-butyl alcohol (h) Solubilizer: n-Octyl-β-D-Thioglucoside 6mM (i) pH=8.2

[0137] <Adhesion of reagents> About 5 μL of each of the fixing reagents with the above formulation was injected into the predetermined reaction cell of the first plate-shaped part (made of polycarbonate (PC)) of the microchip, and then dried under reduced pressure to fix the reagents. Thereafter, a second plate-shaped part (made of polydimethylsiloxane (PDMS)) was attached to the reaction cell side surface of the first plate-shaped part under vacuum, and a reinforcing film (polypropylene (PP) sheet) was attached to the opposing side of the first plate-shaped part under atmospheric pressure. In this way, the microchip of the present invention was produced in which the inside was in a reduced pressure state compared to atmospheric pressure and the predetermined reagent was fixed in the predetermined reaction cell in a state in which it dissolves when it comes into contact with the liquid sample introduced, and the following tests were performed.

[0138] <Drug susceptibility testing using acidometry> Colonies obtained by the above-mentioned isolation and culture of various bacteria were picked up, and a liquid sample prepared in a picking tool with physiological saline to a certain concentration according to McFarland turbidity was placed in a reservoir, and the liquid sample was injected from the puncture injection port on the microchip prepared above with an inoculation needle of an injection chip (see FIG. 9). After injection, the liquid sample moved to each reaction cell through a flow path due to the negative pressure generated in the microchip. The reaction cells were left to stand at room temperature for 60 minutes, and the presence or absence and the degree of coloring of the reaction cells were confirmed. The presence or absence and the degree of coloring were evaluated using the evaluation system 20 shown in FIG. 11, with the L*a*b* values ​​calculated by an execution computer from the image data of the microchip obtained by a digital camera, and further the color difference (ΔE*) was calculated from the lightness L* and chromaticity a*b* based on the calculated value and the negative control value, and the reaction of each drug-resistant bacteria to various β-lactam antibacterial drugs was evaluated using the ΔE* value as an index. The results are shown in FIG. 13. As shown in Figure 13, various antibiotics reacted with various β-lactamases, and ΔE* values ​​were detected. ROC curves were used to determine positive results by setting arbitrary thresholds for the ΔE* values ​​for each β-lactamase. ROC curves for representative antibiotic substrates are shown in Figure 14. The thresholds for the ΔE* values ​​in this study were set by determining the appropriate cutoff value for the ΔE* value using the Youden index from the ROC curve, as shown in Table 1.

[0139] [Table 1]

[0140] However, the reactivity of β-lactamase with antibiotic substrates is actually complicated, and there are combinations with low ΔE* values ​​after 15 minutes of short reaction time, such as the reaction values ​​of Cefsulodin and Ceftazime against ESBL and class A CPE in FIG. 15. Therefore, it is desirable to further improve the accuracy by using information on multiple antibiotics to distinguish them. In the neural network analysis used in this embodiment, the ΔE* values ​​of the antibiotics shown in FIG. 15 were randomly divided into teacher data and validation data, and then an optimal regression model was created using the teacher data with a statistical software or the like. Next, using this created model, the predicted values ​​for various β-lactamase types were calculated for each strain from the ΔE* values ​​of various antibiotics. Next, an ROC curve was obtained from the predicted values ​​for various β-lactamase types for each of the teacher data and validation data that did not contribute to the model creation. The result is the ROC curve in FIG. 16. In addition, "non-resistant" in Figure 16 stands for Non-AMR (strains susceptible to β-lactam antibiotics), and "CP" stands for CPE. The results of differentiation based on the appropriate cutoff value to determine the resistance type are shown in Table 2.

[0141] [Table 2]

[0142] As the results show, it is possible to appropriately differentiate various β-lactamases by multivariate analysis of the reaction values ​​of multiple antibiotics. [Explanation of symbols]

[0143] 1: Microchip 2: Chip substrate 3: Reaction space 3a: Injection space 3b: Reaction cell 3c: Flow path 4: Abandoned space 5: Reagents 6: Discoloring agent 7: First plate-shaped part 7a: Reaction space recess 7b: Recessed portion for waste space 8: Second plate-shaped part 8a: Surface 9: Injection tip 91: Reservoir N: Needle 10: Reinforcement film 10a: Base material 10b:Adhesive part 11: Light blocking layer 12: Air bubbles H:Puncture hole L: Letters and symbols 20: Rating System 21: Imaging unit 211: Dark box 2111:Front 2112: Back 2113, 2114: Side 2115: Bottom 212: Imaging mechanism 2121: Post 2122: Microchip position fixing mechanism 2123: Plate holder 2124: Imaging device position fixing mechanism 213: Light source 214: Imaging device 22: Computer 221: Computer main unit 2211, 2212: Data transfer cable 2213: Display cable 222: Monitor

Claims

1. A microchip having a substrate for acquiring information for evaluating the properties or state of a microorganism, the microchip having a plurality of cells maintained in a reduced pressure state relative to atmospheric pressure, the substrate of the microchip has a two-layer structure including a first plate-shaped part having gas impermeability and a second plate-shaped part laminated on one surface of the first plate-shaped part and having self-sealing properties; The plurality of cells are formed between the first plate-shaped portion and the second plate-shaped portion, and a reagent for detecting the properties or states of one or more types of microorganisms is sealed or fixed in some or all of the plurality of cells in a manner that dissolves upon contact with a liquid sample; The cell is a substrate of a microchip, which communicates with an injection space into which a liquid sample is punctured and injected via a flow path, A microchip for acquiring information on the properties or state evaluation of microorganisms in the liquid sample from signals from each of one or more types of cells resulting from a contact reaction between the liquid sample introduced into each of the multiple cells and the reagent sealed or fixed in the cells, when the reduced pressure state is released following puncture injection of the liquid sample into the microchip.

2. 2. The microchip according to claim 1, wherein a reinforcing film is provided on a surface of the second plate-shaped portion of the substrate opposite the first plate-shaped portion, at least at a position facing the injection space of the second plate-shaped portion.

3. 2. The microchip according to claim 1, wherein a light-shielding layer is provided on the substrate with at least a flat surface facing each of the plurality of cells removed.

4. 4. The microchip according to claim 1, wherein the property of the microorganism to be evaluated in the microchip is drug resistance of the microorganism.

5. 5. The microchip according to claim 4, wherein a reagent for detecting drug resistance of a microorganism, which is a property of the microorganism, contains a drug to be detected as the drug resistance of the microorganism.

6. 6. The microchip according to claim 5, wherein the reagent for detecting drug resistance of a microorganism, which is a property of the microorganism, further contains a signal generating reagent that indicates resistance to a drug to be detected, the drug resistance of the microorganism.

7. 7. The microchip according to claim 6, wherein the signal of the signal generating reagent which indicates resistance to a drug for which drug resistance of a microorganism is to be detected is color or fluorescence.

8. 8. The microchip according to claim 7, wherein the drug resistance of the microorganism to be detected in the microchip is drug resistance due to a decomposition enzyme of a predetermined drug.

9. 9. The microchip according to claim 8, wherein the decomposition enzyme of the predetermined drug is β-lactamase.

10. A method for obtaining a visual image or image information of a signal from each of a plurality of cells generated by a reaction between the liquid sample introduced into each of the plurality of cells and a reagent for detecting the properties or state of one or more types of microorganisms, as information for evaluating the properties or state of the microorganisms in the liquid sample.

11. The method according to claim 10, wherein the visual or image information of the signal from each cell is a visual or image information based on color or fluorescence that represents the nature or state of the microorganism.

12. The method according to claim 11, wherein the color or fluorescence image information representing the properties or state of the microorganism is image information that has been subjected to equalization processing by a computer using an equalization processing means for equalizing variations in color or fluorescence intensity within each cell.

13. The method according to claim 11 , wherein the color image information is image information based on color space values ​​of a color system.

14. The method according to claim 13, wherein the image information in the color space values ​​of the color system is image information in the L*a*b* system.

15. The method according to claim 13, wherein in the method, the color or fluorescence image information representing the properties or state of the microorganism is image information based on a color difference or fluorescence intensity difference calculated by a computer for each cell between a color value or a fluorescence intensity value normalized based on reference determination information corresponding to the properties or state of the microorganism.

16. A method for injecting an aqueous dilution solution containing microorganisms derived from a biological sample into the microchip described in claim 4 as a liquid sample, and acquiring visual images or image information of signals from each of a plurality of cells generated by a reaction between the liquid sample introduced into each of the cells and a reagent for detecting drug resistance of one or more types of microorganisms, as information for evaluating the drug resistance of the microorganisms in the liquid sample.

17. 1. A system for assessing the nature or state of a microorganism in a biological sample, comprising: (1) An image acquisition unit that acquires color or fluorescence representing the properties or state of microorganisms from each cell of the microchip according to any one of claims 1 to 3 as image information, and a computer, (2) a means for making the computer compare the image information input to the computer with a color value or a fluorescence intensity value normalized based on reference determination information for the properties or state of the microorganism, and for making the computer evaluate the properties or state of the microorganism obtained from the biological sample; A system for evaluating the properties or state of a microorganism, comprising:

18. The evaluation system according to claim 17 , wherein the image acquisition unit includes a built-in computer.

19. The evaluation system according to claim 17 , wherein the image acquisition section that acquires the color or fluorescence as image information is an imaging section that uses an imaging device.

20. In the evaluation system, the imaging unit (1) An imaging space is formed in which external light is blocked, (2) Within the imaging space, an imaging device is fixed in position, which acquires color or fluorescence representing the properties or state of microorganisms from each of the cells of the microchip whose position is fixed as image information, or a position fixing mechanism for the imaging device is provided, which can detachably fix the imaging device in position; (3) A light source is provided in the light incidence mechanism of the imaging device in a position-fixed state so that emitted light is incident from a main optical path, (4) A mechanism for fixing the position of a microchip is provided so that the light emitted from the light source hits a light receiving surface of the microchip, which is fixed in position, on a main optical path until the light reaches a light incidence mechanism of the imaging device, and the transmitted light is made incident on the light incidence mechanism; 20. The evaluation system of claim 19.

21. The evaluation system according to claim 20, wherein (4) the mechanism for fixing the microchip in position is provided with an optical mask mechanism for blocking light emitted from the light source from being received on the light receiving surface of the fixed microchip except for the planes facing each of the multiple cells of the microchip.

22. 20. The evaluation system according to claim 19, further comprising a means for evaluating the color using a color space value of a color system as an index in the computer of the evaluation system.

23. 23. The evaluation system according to claim 22, wherein in the computer of the evaluation system, the color space values ​​of the color system are color space values ​​according to L*a*b*.

24. 20. The evaluation system according to claim 19, further comprising an equalization processing means for equalizing variations in color or fluorescence intensity inside the cell for the image data, in the computer of the evaluation system.

25. The leveling processing means in the evaluation system includes the following steps: (1) A step of selecting an arbitrary cell from among a plurality of cells of a microchip; (2) segmenting the image information of the cell selected in step (1) in the cell image plane and labeling each segment; (3) calculating the color value or the fluorescence intensity for each of the divided elements labeled in the step (2) and associating the color value or the fluorescence intensity with each label; (4) extracting a median value of the color value or the fluorescence intensity for all the labels that have been associated with the color value or the fluorescence intensity in the step (3), and specifying and storing the median value as the color value or the fluorescence intensity value selected in the step (1); (5) repeatedly executing steps (1)-(4) for cells other than the cell selected by step (1); (6) terminating the repetition of steps (1)-(4) when no cells other than the cell selected in step (1) are recognized in step (5); 25. The evaluation system of claim 24, wherein the evaluation system is a computer program execution of an algorithm comprising:

26. The means for evaluating the properties or state of a microorganism in the evaluation system includes the following steps: (1) A step of labeling each of a plurality of cells of a microchip by associating the cells with given conditions; (2) further associating color values ​​or fluorescence intensities for each of the cells labeled in step (1); (3) a step of matching the color value or the fluorescence intensity value normalized based on the reference determination information of the property or state of a predetermined microorganism with each of the plurality of cells that have been linked and labeled with the given conditions and the color value or the fluorescence intensity by the steps (1) and (2), and linking the cells by applying the property or state of the microorganism; (4) for each of the plurality of cells into which the properties or states of the microorganisms have been assigned by step (3), combining the given conditions with the evaluation of the properties or states of the microorganisms to derive an evaluation of the properties or states of the microorganisms in the liquid sample, the evaluation being weighted by the factors derived from the given conditions; 20. The evaluation system of claim 19, wherein the evaluation system is a computer program execution of an algorithm comprising:

27. 27. The evaluation system according to claim 26, wherein an algorithm for selecting and excluding data of a cell from which an evaluation of the microbial property or state derived for each cell by step (3) is inconsistent with the evaluation of the microbial property or state of a large number of other cells matched with given conditions is incorporated as a premise for step (4).

28. In the evaluation system: (1) The microchip used is the microchip according to claim 4, (2) The property of the microorganism being evaluated is the drug resistance of the microorganism, (3) The color or fluorescence indicating the drug resistance of the above microorganism is a color or fluorescence observed due to a reaction between a drug resistance substance produced by the microorganism and the corresponding drug; 20. The evaluation system of claim 19.

29. An imaging unit for use in the evaluation system according to claim 19, (1) An imaging device that acquires color or fluorescence representing the properties or state of microorganisms from each of the cells of the microchip that is fixed in position as image information is fixed in position, or an imaging device position fixing mechanism that can detachably fix the imaging device in position is provided; (2) A light source is provided in the light incidence mechanism of the imaging device in a position-fixed state so that the emitted light is incident from a main optical path, (3) A mechanism for fixing the position of the microchip is provided so that the light emitted from the light source hits the light receiving surface of the microchip on a main optical path until the light reaches the light input mechanism of the imaging device, and the transmitted light is input to the light input mechanism; Imaging unit.

30. The imaging unit of claim 29, wherein (3) the mechanism for fixing the position of the microchip is provided with an optical mask mechanism for blocking light emitted from the light source from being received on the light receiving surface of the fixed position microchip except for the planes facing each of the multiple cells of the microchip.

31. An imaging unit for use in the evaluation system according to claim 19, (1) An imaging space that is shielded from external light is provided, (2) Within the imaging space, an imaging device is fixed in position, which acquires color or fluorescence representing the properties or state of microorganisms from each of the cells of the microchip whose position is fixed as image information, or a position fixing mechanism for the imaging device is provided, which can detachably fix the imaging device in position; (3) A light source is provided in the light incidence mechanism of the imaging device in a position-fixed state so that emitted light is incident from a main optical path, (4) A mechanism for fixing the position of the microchip is provided so that the light emitted from the light source hits the light receiving surface of the microchip on a main optical path until the light reaches the light input mechanism of the imaging device, and the transmitted light is input to the light input mechanism; Imaging unit.

32. The imaging unit of claim 31, wherein (4) the mechanism for fixing the position of the microchip is provided with an optical mask mechanism for blocking reception of light emitted from the light source on the light receiving surface of the fixed position microchip except for the planes facing each of the multiple cells of the microchip.

33. A computer program for injecting an aqueous dilution solution containing microorganisms derived from a biological sample as a liquid sample into the microchip according to any one of claims 1 to 3, and leveling color or fluorescent image information representing the properties or states of the microorganisms from each of a plurality of cells generated by a reaction between the liquid sample introduced into each of the plurality of cells and a reagent for detecting the properties or states of one or more types of microorganisms, the computer program comprising an algorithm of the following steps: (1) A step of selecting an arbitrary cell from among a plurality of cells of a microchip; (2) segmenting the image information of the cell selected in step (1) in the cell image plane and labeling each segment; (3) calculating the color value or the fluorescence intensity for each of the divided elements labeled in the step (2) and associating the color value or the fluorescence intensity with each label; (4) extracting a median value of the color value or the fluorescence intensity for all the labels that have been associated with the color value or the fluorescence intensity in the step (3), and specifying and storing the median value as the color value or the fluorescence intensity value selected in the step (1); (5) repeatedly executing steps (1)-(4) for cells other than the cell selected by step (1); (6) Terminating the repetition of steps (1)-(4) when no cells other than the cell selected in step (1) are recognized in step (5).

34. In the computer program: (1) The property of the microorganism is the drug resistance of the microorganism. (2) The color or fluorescence indicating the drug resistance of the above microorganism is a color or fluorescence observed due to a reaction between a drug resistance substance produced by the microorganism and the corresponding drug; 34. A computer program according to claim 33.

35. A computer program for evaluating the properties or state of microorganisms based on color or fluorescent image information representing the properties or state of the microorganisms from each of a plurality of cells produced by a reaction between a liquid sample introduced into each of a plurality of cells and a reagent for detecting the properties or state of one or more types of microorganisms, the computer program comprising an algorithm for the following steps: (1) labeling each of the plurality of cells of the microchip by associating it with a given condition; (2) further associating color values ​​or fluorescence intensities for each of the cells labeled in step (1); (3) a step of matching the color value or the fluorescence intensity value normalized based on the reference determination information of the property or state of the microorganism with each of the plurality of cells that have been linked and labeled with the given conditions and the color value or the fluorescence intensity in the steps (1) and (2), and linking the cells by applying an evaluation of the property or state of the microorganism; (4) For each of the multiple cells to which the evaluation of the microbial property or state has been assigned by step (3), a step of combining the given conditions with the evaluation of the microbial property or state to derive an evaluation of the microbial property or state in the liquid sample, the evaluation being weighted by the factor derived from the given conditions.

36. In the computer program: (1) The property of the microorganism is the drug resistance of the microorganism. (2) The color or fluorescence indicating the drug resistance of the above microorganism is a color or fluorescence observed due to a reaction between a drug resistance substance produced by the microorganism and the corresponding drug; 36. A computer program according to claim 35.

37. The computer program according to claim 35, wherein the computer program includes, as a prerequisite for step (4), a step of selecting and excluding data of a cell from which an evaluation of the microbial properties or states derived for each cell by step (3) is inconsistent with the evaluation of the microbial properties or states of a number of other cells matched with given conditions.

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