Inspection device and inspection method

Abstract

[Problem] To provide an inspection device and an inspection method which are capable of quickly measuring microorganisms. [Solution] This inspection device 1 for inspecting a target microorganism X in a predetermined specimen 100 has: a fluorescent unit 11 that makes the predetermined specimen 100 fluorescent after reacting the target microorganism X with an antibody Y which causes an antigen-antibody reaction with the target microorganism X and is previously bound to a predetermined fluorescent reagent Z; and a fluorescence quantity measuring unit 12 that measures the quantity of fluorescence having a specific wavelength and emitted from the specimen 100 made fluorescent by the fluorescent unit 11.

Description

Inspection device and inspection method

[0001] The present invention relates to a testing device and a testing method, and more particularly to a testing device and a testing method for testing a target microorganism in a predetermined sample.

[0002] Conventionally, for example, a culture method has been used for qualitative and quantitative measurement of microorganisms such as viruses, bacteria, and fungi. For such a culture method, reference can be made to the technology disclosed in Patent Document 1, for example.

[0003] Japanese Patent Application Laid-Open No. 2015-198598

[0004] Incidentally, quantitative analysis of microorganisms, such as bacteria, requires a culture time of 24 to 48 hours. Quantitative analysis of fungi (mold, yeast) requires a culture time of about 2 to 7 days. Furthermore, qualitative analysis of each type requires an additional 7 days of identification culture and microscopic identification. In other words, it takes about two weeks to determine the results of qualitative and quantitative analysis of microorganisms.

[0005] The present invention has been made in view of the above circumstances, and has as its object to provide an inspection device and an inspection method that can rapidly measure microorganisms.

[0006] In order to achieve the above object, the testing device of the present invention is a testing device for testing target microorganisms in a specified specimen, and has a fluorescent unit that makes the specified specimen fluoresce after the target microorganism is reacted with an antibody that undergoes an antigen-antibody reaction with the target microorganism and that has been pre-bound to a specified fluorescent reagent, and a fluorescence amount measuring unit that measures the amount of fluorescence of a specific wavelength emitted from the specimen that has become fluorescent due to the fluorescent unit.

[0007] According to the present invention, the apparatus includes a fluorescent unit that causes the specified specimen to fluoresce after the target microorganism is reacted with an antibody that undergoes an antigen-antibody reaction with the target microorganism and that has been pre-bound to a specified fluorescent reagent, and a fluorescence amount measuring unit that measures the amount of fluorescence at a specific wavelength emitted from the specimen that has been fluoresced by the fluorescent unit. As a result, it is possible to measure the amount of fluorescence at a specific wavelength emitted from the specimen via the antigen-antibody reaction between the target microorganism and the antibody. As a result, microorganisms can be measured quickly without the need for microbial cultivation.

[0008] The fluorescent part is a fluorescent part that causes target microorganisms in the specified specimen to fluoresce when an antigen-antibody reaction occurs with an antibody that has been pre-bound to the specified fluorescent reagent, and does not cause microorganisms other than the target microorganisms to fluoresce.

[0009] The antibody is an antibody that undergoes the antigen-antibody reaction with the target microorganism, but does not undergo the antigen-antibody reaction with microorganisms other than the target microorganism.

[0010] The device has a correlation memory unit that stores data showing the correlation between the amount of fluorescence of the specific wavelength emitted from the target microorganisms and the number of the target microorganisms, and a target microorganism number calculation unit that calculates the number of the target microorganisms based on the amount of fluorescence of the specific wavelength emitted from the specified specimen measured by the fluorescence amount measurement unit, which is the amount of fluorescence of the specific wavelength emitted from the target microorganisms, and the data showing the correlation stored in the correlation memory unit, thereby making it possible to calculate the number of target microorganisms from the amount of fluorescence of the specific wavelength emitted from the target microorganisms.

[0011] The predetermined fluorescent reagent is incorporated into the antibody and is pre-bound to the antibody.

[0012] The fluorescence amount measuring unit has a light amount sensor using a photomultiplier tube and / or a semiconductor, and can measure the amount of fluorescence using the photomultiplier tube and / or the light amount sensor using the semiconductor.

[0013] The antibody is preferably an antibody extracted by immunizing a mammal with the target microorganism or a protein antigen having the same structure as the protein structure of the target microorganism.

[0014] The antibody is preferably a monoclonal or polyclonal antibody.

[0015] The predetermined fluorescent reagent is preferably at least one of merocyanine, perylene, acridine, luciferin, pyranine, stilbene, and rhodamine.

[0016] In order to achieve the above object, the testing method of the present invention is a testing method for testing a target microorganism in a specified sample, which includes a fluorescence step of causing the specified sample to fluoresce after reacting the target microorganism with an antibody that undergoes an antigen-antibody reaction with the target microorganism and that has been pre-bound to a specified fluorescent reagent, and a fluorescence amount measurement step of measuring the amount of fluorescence of a specific wavelength emitted from the sample that has fluoresced in the fluorescence step.

[0017] According to the present invention, the method includes a fluorescence step of causing the specified specimen to fluoresce after reacting the target microorganism with an antibody that undergoes an antigen-antibody reaction with the target microorganism and that has been pre-bound to a specified fluorescent reagent, and a fluorescence measurement step of measuring the amount of fluorescence at a specific wavelength emitted from the specimen that has become fluorescent in the fluorescence step.This makes it possible to measure the amount of fluorescence at a specific wavelength emitted from the specimen via the antigen-antibody reaction between the target microorganism and the antibody.As a result, microorganisms can be measured quickly without culturing the microorganisms.

[0018] The fluorescence step is a step in which target microorganisms in the specified specimen that have undergone an antigen-antibody reaction with antibodies that have been pre-bound to the specified fluorescent reagent are made to fluoresce, while microorganisms other than the target microorganisms are not made to fluoresce.

[0019] The antibody is an antibody that undergoes the antigen-antibody reaction with the target microorganism, but does not undergo the antigen-antibody reaction with microorganisms other than the target microorganism.

[0020] The method has a correlation storage step for storing data showing the correlation between the amount of fluorescence of the specific wavelength emitted from the target microorganism and the number of the target microorganisms, and a target microorganism number calculation step for calculating the number of the target microorganisms based on the amount of fluorescence of the specific wavelength emitted from the specified specimen measured in the fluorescence amount measurement step, which is the amount of fluorescence of the specific wavelength emitted from the target microorganisms, and the data showing the correlation stored in the correlation storage step, thereby making it possible to calculate the number of target microorganisms from the amount of fluorescence of the specific wavelength emitted from the target microorganisms.

[0021] The predetermined fluorescent reagent is incorporated into the antibody and is pre-bound to the antibody.

[0022] The fluorescence amount measuring step may be a step of measuring the amount of fluorescence using a light amount sensor that uses a photomultiplier tube and / or a semiconductor.

[0023] The antibody is preferably an antibody extracted by immunizing a mammal with the target microorganism or a protein antigen having the same structure as the protein structure of the target microorganism.

[0024] The antibody is preferably a monoclonal or polyclonal antibody.

[0025] The predetermined fluorescent reagent is preferably at least one of merocyanine, perylene, acridine, luciferin, pyranine, stilbene, and rhodamine.

[0026] According to the present invention, microorganisms can be measured quickly.

[0027] 1 is a diagram showing the configuration of a testing device according to an embodiment of the present invention; FIG. 2 is a diagram showing the configuration of a testing unit of the testing device; FIG. 3 is a diagram showing the configuration of an arithmetic processing unit of the testing device; FIG. 4 is a diagram showing a sample to be tested by the testing device; FIG. 5 is a diagram showing an antibody; FIG. 6 is a diagram showing a fluorescent reagent; FIG. 7 is a diagram showing a state in which an antibody and a fluorescent reagent are bound; FIG. 8 is a diagram showing a state in which an antigen-antibody reaction has occurred between a target microorganism and an antibody; FIG. 9 is a diagram showing the configuration of a computer of the testing device; and FIG. 10 is a flowchart for explaining a testing method in the testing device.

[0028]

[0023] Hereinafter, an embodiment of the present invention will be described in detail with reference to the drawings. Fig. 1 is a diagram showing the configuration of a testing device according to an embodiment of the present invention, Fig. 2 is a diagram showing the configuration of a testing unit of the testing device, Fig. 3 is a diagram showing the configuration of a processing unit of the testing device, Fig. 4 is a diagram showing a sample to be tested by the testing device, Fig. 5 is a diagram showing an antibody, Fig. 6 is a diagram showing a fluorescent reagent, Fig. 7 is a diagram showing a state in which an antibody and a fluorescent reagent are bound, Fig. 8 is a diagram showing a state in which an antigen-antibody reaction has occurred between a target microorganism and an antibody, Fig. 9 is a diagram showing the configuration of a computer of the testing device, and Fig. 10 is a flowchart for explaining a testing method using the testing device.

[0029] An inspection device 1 according to an embodiment of the present invention is capable of inspecting target microorganisms X in a predetermined specimen 100. As shown in FIG. 1, the inspection device 1 has an inspection unit 10 and a calculation processing unit 20. As shown in FIG. 2, the inspection unit 10 further has a fluorescence unit 11 and a fluorescence amount measurement unit 12. As shown in FIG. 3, the calculation processing unit 20 further has a correlation storage unit 21 and a target microorganism number calculation unit 22. The inspection device 1 can measure the fluorescence amount of the predetermined specimen 100 shown in FIG. 4 and calculate the number of target microorganisms X in the predetermined specimen 100. The target microorganisms X are target microorganisms, and are the microorganisms to be inspected by the inspection device 1.

[0030] In this embodiment, antibody Y shown in Fig. 5 and fluorescent reagent Z shown in Fig. 6 are used to measure the amount of fluorescence from a predetermined specimen 100. As shown in Fig. 7, the predetermined fluorescent reagent Z is pre-bound to antibody Y. That is, the predetermined fluorescent reagent Z is incorporated into antibody Y and pre-bound to antibody Y. Antibody Y is an antibody Y that uses target microorganism X as an antigen and undergoes an antigen-antibody reaction with target microorganism X, but does not undergo an antigen-antibody reaction with microorganisms other than target microorganism X. Antibody Y can be selected appropriately for each target microorganism X.

[0031] Here, antibody Y is preferably antibody Y extracted by immunizing a mammal with target microorganism X or a protein antigen having the same structure as the protein structure of target microorganism X. More specifically, antibody Y is preferably antibody Y extracted from the serum or chicken eggs of a mammal obtained by immunizing a mammal such as a mouse or chicken with target microorganism X or a protein antigen having the same structure as the protein structure of target microorganism X (antibody Y is preferably antibody Y extracted from the serum or chicken eggs of a mammal obtained by immunizing a mammal such as a mouse or chicken with target microorganism X, or antibody Y is preferably antibody Y extracted from the serum or chicken eggs of a mammal obtained by immunizing a mammal such as a mouse or chicken with a protein antigen having the same structure as the protein structure of target microorganism X), and antibody Y is preferably a monoclonal antibody or a polyclonal antibody. Furthermore, the predetermined fluorescent reagent Z is preferably at least one of merocyanine, perylene, acridine, luciferin, pyranine, stilbene, and rhodamine.

[0032] That is, the fluorescent unit 11 is an antibody Y that undergoes an antigen-antibody reaction with the target microorganism X, and can cause the predetermined specimen 100 to fluoresce after the target microorganism X reacts with the antibody Y that has been pre-bound to a predetermined fluorescent reagent Z. The fluorescent unit 11 can cause the predetermined specimen 100 to fluoresce by irradiating the predetermined specimen 100 with excitation light (for example, 488 nm±10 nm).

[0033] In addition, the fluorescent unit 11 is a fluorescent unit 11 that causes a target microorganism X that has undergone an antigen-antibody reaction with an antibody Y that has been pre-bound to a predetermined fluorescent reagent Z in a predetermined specimen 100 to fluoresce, and does not cause microorganisms X' other than the target microorganism X to fluoresce.

[0034] As shown in Figure 8, in a specific specimen 100, target microorganism X, which has undergone an antigen-antibody reaction with antibody Y previously bound to a specific fluorescent reagent Z, can be made to fluoresce by applying the specific fluorescent reagent Z. The fluorescent unit 11 is configured to have a cell holder filled with specimen 100 inside a casing that blocks 100% of external light, and excitation light can be irradiated from the sides (first to fourth sides) of the cell holder.

[0035] The fluorescence amount measuring unit 12 can measure the amount of fluorescence at a specific wavelength (e.g., 512 nm±5 nm) emitted from the specimen 100 that has been fluoresced by the fluorescence unit 11. The fluorescence amount measuring unit 12 has a light amount sensor using a photomultiplier tube and / or a semiconductor, and can measure the amount of fluorescence using the light amount sensor using a photomultiplier tube and / or a semiconductor. The amount of fluorescence measured by the fluorescence amount measuring unit 12 can be displayed digitally.

[0036] The correlation storage unit 21 can store data showing the correlation between the amount of fluorescence of a specific wavelength emitted from the target microorganism X and the number of the target microorganisms X. The data showing the correlation includes data correlating the amount of fluorescence of a specific wavelength emitted from the target microorganism X with the number of the target microorganisms X, mathematical data of the amount of fluorescence of a specific wavelength emitted from the target microorganism X and the number of the fluorescent target microorganisms X, etc.

[0037] The target microorganism count calculation unit 22 can calculate the number of target microorganisms X based on the amount of fluorescence of a specific wavelength emitted from a specified specimen 100 measured by the fluorescence amount measurement unit 12, which is the amount of fluorescence of a specific wavelength emitted from the target microorganism X, and data indicating the correlation stored in the correlation memory unit 21.

[0038] The amount of fluorescence measured by the fluorescence amount measuring unit 12 and the number of target microorganisms X calculated by the target microorganism number calculating unit 22 can be displayed digitally.

[0039] The inspection device 1 has a general configuration as a computer. That is, as shown in Fig. 9, the inspection device 1 has a central processing unit (CPU, GPU, DSP) 1B, a storage device (ROM, RAM, hard disk, cache memory) 1C, an input device (keyboard, touch panel, mouse) 1D, a display device (liquid crystal display) 1E, etc., which are connected to each other via a bus 1A, and these interact with each other to function as an arithmetic processing unit 20. The storage device 1C functions as a computer-readable storage medium.

[0040] The inspection method using the inspection device 1 configured as above will be described with reference to the flowchart in Figure 10. In the following, the explanation will be given on the assumption that the correlation storage unit 21 has performed the correlation storage step and stored in advance data indicating the correlation between the amount of fluorescence of a specific wavelength emitted from the target microorganism X and the number of the target microorganism X.

[0041] First, in step S10, a predetermined specimen 100 is prepared. That is, an antibody Y shown in Fig. 5 and a fluorescent reagent Z shown in Fig. 6 are prepared, and the predetermined fluorescent reagent Z and the antibody Y are preliminarily bound together as shown in Fig. 7. The predetermined fluorescent reagent Z is incorporated into the antibody Y and preliminarily bound to the antibody Y.

[0042] Next, in step S20, the fluorescent unit 11 irradiates excitation light onto the specified specimen 100 after reacting the target microorganism X with antibody Y, which is an antibody Y that undergoes an antigen-antibody reaction with the target microorganism X and has been pre-bound to a specified fluorescent reagent Z, causing the specified specimen 100 to fluoresce (fluorescence step).

[0043] Next, in step S30, the fluorescence amount measuring unit 12 measures the amount of fluorescence of a specific wavelength emitted from the specimen 100 that has been fluoresced by the fluorescent unit 11 (fluorescence amount measuring step).

[0044] Next, in step S40, the target microorganism count calculation unit 22 calculates the number of target microorganisms X based on the amount of fluorescence of a specific wavelength emitted from a specified specimen 100 measured by the fluorescence amount measurement unit 12, which is the amount of fluorescence of a specific wavelength emitted from the target microorganism X, and the data indicating the correlation stored in the correlation memory unit 21 (target microorganism count calculation step).

[0045] As described above, the testing device 1 of the present invention includes a fluorescent unit 11 that causes a specific specimen 100 to fluoresce after the target microorganism X reacts with antibody Y, the antibody Y being pre-bound to a specific fluorescent reagent Z, and the target microorganism X, and a fluorescence measurement unit 12 that measures the amount of fluorescence at a specific wavelength emitted from the specimen 100 that has been fluoresced by the fluorescent unit 11. This makes it possible to measure the amount of fluorescence at a specific wavelength emitted from the specimen 100 via the antigen-antibody reaction between the target microorganism X and antibody Y. As a result, microorganisms can be measured quickly without culturing the microorganisms. (Note that, as a means of qualitative and quantitative analysis using an antigen-antibody reaction, the ELISA method, which uses two antibodies to determine color, is generally used. However, this method takes about five hours, is very difficult to operate, requires a large measuring device, and has high running and initial costs. While this method is feasible in medical settings, it is difficult to operate in the food industry, where rapid analysis is most needed. Therefore, it is believed that the present invention will be widely used.)

[0046] In addition, the device has a correlation memory unit 21 that stores data showing the correlation between the amount of fluorescence of a specific wavelength emitted from the target microorganism X and the number of target microorganisms X, and a target microorganism number calculation unit 22 that calculates the number of target microorganisms X based on the amount of fluorescence of a specific wavelength emitted from a specified specimen 100 measured by a fluorescence amount measurement unit 12, which is the amount of fluorescence of a specific wavelength emitted from the target microorganism X, and the data showing the correlation stored in the correlation memory unit 21, so that the number of target microorganisms X can be calculated from the amount of fluorescence of a specific wavelength emitted from the target microorganisms X.

[0047] Furthermore, the testing method of the present invention includes a fluorescence step in which a predetermined specimen 100 is made to fluoresce after the target microorganism X is reacted with antibody Y, which undergoes an antigen-antibody reaction with target microorganism X and has been pre-bound to a predetermined fluorescent reagent Z, and a fluorescence amount measurement step in which the amount of fluorescence of a specific wavelength emitted from the specimen 100 that has become fluorescent in the fluorescence step is measured, thereby making it possible to measure the amount of fluorescence of a specific wavelength emitted from the specimen 100 via the antigen-antibody reaction between the target microorganism X and antibody Y. As a result, microorganisms can be measured quickly without culturing the microorganisms.

[0048] Furthermore, the method has a correlation storage step for storing data showing the correlation between the amount of fluorescence of a specific wavelength emitted from the target microorganism X and the number of target microorganisms X, and a target microorganism number calculation step for calculating the number of target microorganisms X based on the amount of fluorescence of a specific wavelength emitted from a specified specimen 100 measured in the fluorescence amount measurement step, which is the amount of fluorescence of a specific wavelength emitted from the target microorganism X, and the data showing the correlation stored in the correlation storage step, thereby making it possible to calculate the number of target microorganisms X from the amount of fluorescence of a specific wavelength emitted from the target microorganisms X.

[0049] X: Target microorganism X': Microorganism other than target microorganism X Y: Antibody 1: Testing device 1A: Bus 1B: Central processing unit 1C: Storage device 1D: Input device 1E: Display device 10: Testing unit 11: Fluorescence unit 12: Fluorescence amount measuring unit 20: Arithmetic processing unit 21: Correlation storage unit 22: Target microorganism number calculation unit 100: Specimen

Claims

1. A testing device for testing a target microorganism in a specified specimen, the testing device having a fluorescent unit that makes the specified specimen fluoresce after the target microorganism is reacted with an antibody that undergoes an antigen-antibody reaction with the target microorganism and that has been pre-bound to a specified fluorescent reagent, and a fluorescence amount measuring unit that measures the amount of fluorescence of a specific wavelength emitted from the specimen that has become fluorescent due to the fluorescent unit.

2. The testing device according to claim 1, wherein the fluorescent unit is a fluorescent unit that causes target microorganisms in the specified specimen that have undergone an antigen-antibody reaction with antibodies that have been pre-bound to the specified fluorescent reagent to fluoresce, and does not cause microorganisms other than the target microorganisms to fluoresce.

3. The testing device according to claim 1, wherein the antibody undergoes the antigen-antibody reaction with the target microorganism, but does not undergo the antigen-antibody reaction with microorganisms other than the target microorganism.

4. An inspection device as described in claim 2, further comprising: a correlation memory unit that stores data indicating the correlation between the amount of fluorescence of the specific wavelength emitted from the target microorganisms and the number of the target microorganisms; and a target microorganism number calculation unit that calculates the number of the target microorganisms based on the amount of fluorescence of the specific wavelength emitted from the specified specimen measured by the fluorescence amount measurement unit, which is the amount of fluorescence of the specific wavelength emitted from the target microorganisms, and the data indicating the correlation stored in the correlation memory unit.

5. The testing device according to claim 1, wherein the predetermined fluorescent reagent is incorporated into the antibody and is pre-bound to the antibody.

6. An inspection device according to claim 1, wherein the fluorescence amount measuring unit has a light amount sensor using a photomultiplier tube and / or a semiconductor, and measures the amount of fluorescence using the light amount sensor using the photomultiplier tube and / or the semiconductor.

7. The testing device according to claim 1, wherein the antibody is an antibody extracted by immunizing a mammal with the target microorganism or a protein antigen having the same structure as the protein structure of the target microorganism.

8. The testing device according to claim 1, wherein the antibody is a monoclonal antibody or a polyclonal antibody.

9. The inspection device according to claim 1, wherein the predetermined fluorescent reagent is at least one of merocyanine, perylene, acridine, luciferin, pyranine, stilbene, and rhodamine.

10. A testing method for testing a target microorganism in a specified specimen, the testing method comprising a fluorescence step of causing the specified specimen to fluoresce after reacting the target microorganism with an antibody that undergoes an antigen-antibody reaction with the target microorganism and that has been pre-bound to a specified fluorescent reagent, and a fluorescence amount measurement step of measuring the amount of fluorescence of a specific wavelength emitted from the specimen that has fluoresced in the fluorescence step.

11. The testing method described in claim 10, wherein the fluorescence step is a step of causing target microorganisms in the specified specimen that have undergone an antigen-antibody reaction with antibodies that have been pre-bound to the specified fluorescent reagent to fluoresce, while preventing microorganisms other than the target microorganisms from fluorescing.

12. The testing method according to claim 10, wherein the antibody undergoes the antigen-antibody reaction with the target microorganism, but does not undergo the antigen-antibody reaction with microorganisms other than the target microorganism.

13. The testing method according to claim 10, further comprising: a correlation storage step for storing data showing the correlation between the amount of fluorescence of the specific wavelength emitted from the target microorganism and the number of the target microorganisms; and a target microorganism number calculation step for calculating the number of the target microorganisms based on the amount of fluorescence of the specific wavelength emitted from the specified specimen measured in the fluorescence amount measurement step, which is the amount of fluorescence of the specific wavelength emitted from the target microorganisms, and the data showing the correlation stored in the correlation storage step.

14. The method of claim 10, wherein the predetermined fluorescent reagent is incorporated into the antibody and is bound to the antibody in advance.

15. The inspection method according to claim 10, wherein the fluorescence amount measuring step includes a light amount sensor using a photomultiplier tube and / or a semiconductor, and measures the fluorescence amount using the light amount sensor using the photomultiplier tube and / or the semiconductor.

16. The testing method according to claim 10, characterized in that the antibody is an antibody extracted by immunizing a mammal with the target microorganism or a protein antigen having the same structure as the protein structure of the target microorganism.

17. The testing method according to claim 10, wherein the antibody is a monoclonal antibody or a polyclonal antibody.

18. The inspection method according to claim 10, wherein the predetermined fluorescent reagent is at least one of merocyanine, perylene, acridine, luciferin, pyranine, stilbene, and rhodamine.

Images