JP2002330799A - Method for measuring specific microorganism and apparatus for measuring specific microorganism - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method and an apparatus with which specific microorganisms can rapidly and accurately be measured by using a fluorescence in the method and apparatus for measuring the specific microorganisms included in a liquid specimen or having a possibility of being included therein. SOLUTION: This method for measuring the specific microorganisms allows capturing the specific microorganisms included in the liquid specimen or having a possibility of being included therein by a means reacting with the specimen and simultaneously and rapidly grasping the number or presence of the captured specific microorganisms. The apparatus for measuring the specific microorganism allows suppressing quenching of fluorescence with an excitation light, accurately measuring the number of the specific microorganism and preventing an overestimation with mixing of foreign substances and-keeping a high sensibility by setting a threshold value.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a specific microorganism (the meaning of a microorganism is at least a concept including bacteria and fungi), that is, a microorganism containing or possibly containing specific bacteria and fungi. The present invention relates to a method and an apparatus capable of causing a specific microorganism to emit light from a certain liquid sample and measuring it easily and quickly.

[0002]

2. Description of the Related Art Japanese Patent Laid-Open Publication No.
As described in Japanese Patent No. 0148, there is known an apparatus which detects an emission of a specific microorganism by using an antibody labeled with a fluorescent substance. Further, according to this method, the specific microorganism and all the microorganisms can be grasped in combination with the reagent that stains nonspecifically.

[0003] Specific microorganism measuring devices are often used in food factories, and it is an important element in management to confirm the presence or absence of specific microorganisms such as Escherichia coli which can cause food poisoning. In addition, with the recent introduction of HACCP, not only inspection of food itself, but also inspection of factories and work processes, for example, inspection of cooking utensils such as walls and floors, cutting boards and kitchen knives, etc. Management and environmental management have become important.

[0004]

In such a conventional method for quantifying a specific microorganism, measurement can be performed with high sensitivity because an antibody is used. However, since the antibody must be labeled with a fluorescent substance, it takes time and effort. It will be expensive. In addition, this method cannot determine whether a specific microorganism is alive or dead. In addition, since the antibody is fluorescently labeled, there are problems in the reaction accuracy, time, etc. of the antibody. In addition, when a secondary antibody is used, there are problems in operation complexity and stability, and there are problems such as temperature control, reaction time, and operation procedure.

[0005] The present invention solves such a conventional problem, and comprises a first compound that emits light from living and dead cells;
A second compound that causes dead cells to emit light at a wavelength different from the light emission; and a third compound that causes live cells to emit light at a wavelength different from the light emission.
And at least one or more of the at least one fourth compound that emits light at a wavelength different from the emission by reacting with a specific microorganism-derived substance may contain or contain a specific microorganism. When the liquid sample contains a specific microorganism by contacting it with a liquid sample having a characteristic, the specific microorganism is fluorescently stained, and then the sample contact solution is immobilized with an antibody that specifically reacts with the specific microorganism. An object of the present invention is to provide a method for quantifying a specific microorganism, characterized in that the specific microorganism stained with a fluorescent dye is measured by an optical measuring means.

SUMMARY OF THE INVENTION The present invention solves such a conventional problem, and a liquid sample containing or possibly containing a specific microorganism is immobilized with an antibody that specifically reacts with the specific microorganism. When the liquid sample contains a specific microorganism by contacting the liquid sample with the means, the specific compound is allowed to bind to the antibody immobilized on the sample reaction means, and then the first compound that causes live cells to emit light, and the dead cell is emitted by the light emitting apparatus. A second compound that emits light at a different wavelength from the third, a third compound that emits live cells at a different wavelength from the luminescence, and at least one or more types that emit light at a different wavelength from the luminescence by reacting with a specific microorganism-derived substance. Contacting one or more of the fourth compounds with the sample reaction means to fluorescently stain the specific microorganisms bound to the antibody; And to provide a particular microorganisms measuring method and measuring by optical measuring means.

It is another object of the present invention to provide a method wherein the first compound is a nucleic acid binding compound.

It is another object of the present invention to provide a method wherein the second compound is a nucleic acid binding compound.

Another object of the present invention is to provide a method for producing a compound which emits light by reacting a third compound with a substance derived from a microorganism.

Another object of the present invention is to provide a method using a microorganism-derived substance as an enzyme protein.

It is another object of the present invention to provide a method using a substance derived from a specific microorganism as an enzyme protein.

It is another object of the present invention to provide a method in which a sample reaction means can be brought into contact with a sample contact liquid while flowing the sample contact liquid.

It is another object of the present invention to provide a method in which the sample reaction means can be brought into contact with a liquid sample while flowing the sample.

Further, the sample reaction means has two electrodes disposed opposite to each other with a gap therebetween and a short-circuit portion capable of short-circuiting both electrodes so that the potential can be varied, and specifically reacts with a specific microorganism. An object is to provide a method in which an antibody is immobilized on one electrode.

The present invention solves such a conventional problem, and comprises a first compound that emits light from living and dead cells,
A second compound that causes dead cells to emit light at a wavelength different from the light emission; and a third compound that causes live cells to emit light at a wavelength different from the light emission.
And at least one or more of the at least one fourth compound that emits light at a wavelength different from the emission by reacting with a specific microorganism-derived substance may contain or contain a specific microorganism. Sample contacting means for immobilizing an antibody that specifically binds to a specific microorganism, for contacting a sample contacting solution obtained by contacting with a liquid sample having a property, and a predetermined small area of the sample reacting means. A light source that irradiates excitation light in a given wavelength range, a light receiving unit that receives light in a predetermined wavelength range that emits light by the excitation light, and light that is emitted and emitted by the light source within a predetermined period of time. The specific microorganism determining means to determine the specific microorganism when the amount of received light is within the range of the set threshold value, and to move the small constant area continuously or intermittently And moving means, and an object thereof is to provide a particular microorganisms metering device, characterized in that it comprises an integration means for integrating the quantity of a specific microorganism from signals determined to identify microorganisms from a particular microorganism judging means.

The present invention solves such a conventional problem. An object of the present invention is to provide an antibody that specifically binds to a specific microorganism for contacting a liquid sample containing or possibly containing the specific microorganism. An immobilized sample reaction means, a first compound that causes live cells to emit light, a second compound that causes dead cells to emit light at a wavelength different from the light emission, and a third compound that causes live cells to emit light at a wavelength different from the light emission A compound and a detection reagent-containing portion containing any one or more of at least one or more fourth compounds that emit at a wavelength different from the emission by reacting with a specific microorganism-derived substance; and A light source for irradiating excitation light in a predetermined wavelength range to a small predetermined area of the reaction means, a light receiving unit for receiving light in a predetermined wavelength range emitted by the excitation light, and the light source A specific microorganism judging means for receiving light emitted and emitted in a predetermined period of time and judging a specific microorganism when the amount of received light is within a set threshold range; and Provided is a specific microorganism measuring device, comprising: moving means for continuously or intermittently moving an area; and integrating means for integrating the number of specific microorganisms from a signal determined as a specific microorganism by the specific microorganism determining means. With the goal.

It is another object of the present invention to provide an apparatus in which the light receiving means is at least one photoelectric conversion element and a condenser lens for condensing light emitted by the excitation light is provided at a stage preceding the photoelectric conversion element.

It is another object of the present invention to provide a device in which a plurality of photoelectric conversion elements are linearly arranged.

It is another object of the present invention to provide an apparatus in which the sample reaction means is formed in a disk shape and the sample reaction means is movable.

It is another object of the present invention to provide an apparatus in which the area for measuring the sample reaction means is polygonal and the sample reaction means is movable.

Further, there is provided an apparatus in which a driving means for moving any one or more of a light source, a light receiving means and a specimen reaction means is provided as a moving means for moving a minute area, and the driving means is movable within a set speed range. The purpose is to do.

It is another object of the present invention to provide an apparatus provided with a reflector as a means for condensing light emitted from a light source and / or a light receiving means.

It is another object of the present invention to provide an apparatus provided with a spectroscopic section that transmits only a specific wavelength between a light source and a small area.

It is another object of the present invention to provide an apparatus in which a spectroscopic section for transmitting only a specific wavelength is provided between a very small area and a light receiving means.

It is another object of the present invention to provide an apparatus having an automatic focusing function in which a light source and / or a light receiving means focuses on a minute area in a specimen reaction means.

It is another object of the present invention to provide an apparatus having a means for keeping the distance between the light source and the specimen reaction means constant and keeping the minute area constant.

It is another object of the present invention to provide an apparatus having means for introducing light emitted from a light source into a sample reaction means using an optical fiber.

It is another object of the present invention to provide an apparatus using a lens transmitting ultraviolet light as a light source condensing means.

It is another object of the present invention to provide an apparatus having means for indicating the measurement position of the sample reaction means.

It is another object of the present invention to provide an apparatus that uses magnetism as means for indicating the measurement position of the sample reaction means.

It is another object of the present invention to provide an apparatus having means for recognizing the measurement position of the sample reaction means.

Further, it is an object of the present invention to provide an apparatus having means for moving a trajectory for recognizing a measurement position of a sample reaction means to a next recognition trajectory position while maintaining a fixed distance from the recognition trajectory position. Aim.

It is another object of the present invention to provide an apparatus in which the distance from one recognition orbit position to the next recognition orbit position is 10 to 50 μm.

It is another object of the present invention to provide an apparatus having a function of recognizing a signal obtained from a microorganism contained in a sample reaction means as binarized point coordinates and measuring the number of point coordinates at which a signal is obtained. With the goal.

It is another object of the present invention to provide a device having a function of recognizing a neighboring signal as one signal among point coordinates at which a signal is obtained.

[0036]

In order to achieve the above object, a first method for measuring a specific microorganism according to the present invention comprises: a first compound for emitting live and dead cells; and a dead compound emitting light at a wavelength different from the emission. A second compound, a third compound that causes living cells to emit light at a wavelength different from the emission, and at least one or more fourth compounds that emit light at a wavelength different from the emission by reacting with a specific microorganism-derived substance. Any one in
If the liquid sample contains a specific microorganism, the specific microorganism is fluorescently stained by contacting one or more types with a liquid sample containing or possibly containing the specific microorganism. An antibody specifically reacting with the antibody is brought into contact with an immobilized specimen reaction means, and the fluorescent-stained specific microorganisms bound to the antibody are measured by an optical measurement means. Further, according to the present invention, a method for measuring a specific microorganism that can simultaneously and quickly grasp the number or presence or absence of a specific microorganism captured by a sample reaction means is obtained.

In order to achieve the above object, the second specific microorganism measuring method of the present invention comprises immobilizing an antibody specifically reacting with a specific microorganism on a liquid sample containing or possibly containing the specific microorganism. By contacting the sample reaction means,
When the liquid sample contains a specific microorganism, after binding the specific microorganism to the antibody immobilized on the sample reaction means, a first compound that emits live cells and a second compound that emits dead cells at a wavelength different from the emission. Of the second compound, a third compound that causes living cells to emit light at a wavelength different from the emission, and at least one or more fourth compounds that emit light at a wavelength different from the emission by reacting with a specific microorganism-derived substance. By contacting any one or a plurality of types with the sample reaction means, the specific microorganisms bound to the antibody are fluorescently stained,
The fluorescent-stained specific microorganism is measured by an optical measuring means. And the number or presence of the specific microorganisms captured by the sample reaction means also at the same time according to the present invention,
A method for measuring a specific microorganism that can be quickly grasped is obtained.

Further, by using the first compound as a nucleic acid-binding compound, a method for measuring a specific microorganism that can measure a living cell of a specific microorganism at a single cell level with high accuracy can be obtained.

Further, by using the second compound as a nucleic acid-binding compound, a method for measuring a specific microorganism that can measure a dead cell of a specific microorganism at a single cell level with high accuracy can be obtained.

In addition, since the third compound is a compound that emits light when it reacts with a substance derived from a microorganism, a method for measuring a specific microorganism that can measure only a specific living microorganism due to the difference in the amount of light emitted is provided. can get.

Further, by using a microorganism-derived substance as an enzyme protein, a method for measuring a specific microorganism that can determine the viability of a specific microorganism from its reactivity can be obtained.

In addition, by using a substance derived from a specific microorganism as an enzyme protein, a method for measuring a specific microorganism that can determine the viability of a specific microorganism from its reactivity can be obtained.

Further, since the sample reaction means can be brought into contact with the sample contact liquid while flowing the sample contact liquid, the efficiency of contact between the antibody immobilized on the sample reaction means and the fluorescent-stained specific microorganism can be improved. A specific microorganism weighing method is obtained.

Further, a method for measuring a specific microorganism which can improve the contact efficiency between the antibody immobilized on the sample reaction means and the specific microorganism by using the sample reaction means capable of contacting the liquid sample while flowing the liquid sample is provided. can get.

Further, the sample reaction means has two electrodes disposed opposite to each other with a gap therebetween, and a short-circuit portion capable of short-circuiting both electrodes so that the potential can be varied. Since the antibody is immobilized on one electrode, the property that the surface of the microorganism is charged to a negative charge is usually used, and the voltage is set so that the electrode on which the antibody is immobilized is on the positive side. By applying the method, a specific microorganism weighing method capable of improving the contact efficiency between the antibody and the specific microorganism can be obtained.

In order to achieve the above object, the first specific microorganism weighing device of the present invention comprises a first compound for emitting live and dead cells, a second compound for emitting dead cells at a wavelength different from the emission, Any one of a third compound that causes living cells to emit light at a wavelength different from the emission and at least one or more fourth compounds that emit light at a wavelength different from the emission by reacting with a specific microorganism-derived substance; Alternatively, for contacting a sample contact liquid obtained by contacting a liquid sample containing or possibly containing a plurality of specific microorganisms, a sample reaction means in which an antibody specifically binding to the specific microorganism is immobilized. A light source for irradiating excitation light in a predetermined wavelength range to a minute constant area of the specimen reaction unit, and a light receiving unit for receiving light in a predetermined wavelength range emitted by the excitation light. A specific microorganism determining means for receiving light emitted and emitted by the light source within a set time, and determining the specific microorganism when the received light amount is within a set threshold range; and It has moving means for moving a certain area continuously or intermittently, and integrating means for integrating the number of specific microorganisms from a signal determined as specific microorganisms by the specific microorganism determining means. According to the present invention, the number or presence or absence of the specific microorganisms captured by the sample reaction means can be simultaneously and quickly grasped, and the irradiation area and time of the excitation light can be minimized to minimize the excitation light. The quenching of the compound due to is suppressed, and furthermore, the threshold of the fluorescence intensity is provided so that the specific microorganism and the foreign substance can be distinguished, and the quantity of the specific microorganism included in a certain area can be quickly and simultaneously measured. Thus, a specific microorganism weighing device is obtained.

The second specific microorganism weighing device of the present invention specifically binds to a specific microorganism for contacting a liquid sample containing or possibly containing the specific microorganism to achieve the above object. A sample reaction means to which an antibody is immobilized, a first compound that emits live cells at a wavelength different from the emission, a second compound that emits dead cells at a wavelength different from the emission, and a second compound that emits live cells at a wavelength different from the emission. 3, a detection reagent-containing part containing one or more of the at least one or more fourth compounds that emit light at a wavelength different from the emission by reacting with a specific microorganism-derived substance, A light source for irradiating excitation light in a predetermined wavelength range on a minute constant area of the sample reaction unit, a light receiving unit for receiving light in a predetermined wavelength range emitted by the excitation light, A specific microorganism judging means for receiving light emitted and emitted by the light source within a set time, and judging the specific microorganism when the amount of received light is within a set threshold range; and It has moving means for moving the area continuously or intermittently, and integrating means for integrating the number of specific microorganisms from a signal determined as a specific microorganism by the specific microorganism determining means. In addition, according to the present invention, the number or presence or absence of the specific microorganisms captured by the sample reaction means can be simultaneously and quickly grasped, and the irradiation area and time of the excitation light can be minimized to minimize the irradiation area and time. The quenching of the compound can be suppressed, and furthermore, by setting the threshold of the fluorescence intensity, it is possible to distinguish the specific microorganism from the foreign substance, and the quantity of the specific microorganism contained in a certain area can be quickly and simultaneously measured. A specific microorganism measuring device that can be obtained is obtained.

Further, by using a photoelectric conversion element, rapid measurement becomes possible, and furthermore, by providing a condenser lens at the preceding stage, a specific microorganism measuring device capable of measuring with high sensitivity can be obtained.

In addition, by arranging the photoelectric conversion elements in a line, a specific range of detection can be performed at once, and a specific microorganism weighing device capable of speeding up weighing can be obtained.

Further, by making the sample reacting means in a disk shape, it is easy to drive the sample reacting means in the rotation direction, and it is possible to minimize the detection moving distance of both or at least one of the light source and the light receiving means. A specific microorganism measuring device that can be obtained is obtained.

Further, by making the area for measuring the sample reaction means a polygon, the driving of the sample reaction means in one direction is facilitated, and at least one of the light source, the light receiving means and the sample reaction means is moved to specify the sample reaction means. A specific microorganism measuring device capable of measuring microorganisms is obtained.

Further, by moving the minute area of the irradiation of the excitation light emitted from the light source to the specimen within the set speed range, it is possible to obtain a specific microorganism weighing apparatus capable of removing a shift in light emission, an afterimage, and an afterglow.

Further, at least one of the light emitted from the light source and the light emitted from the sample reacting means is condensed by using a reflector, so that the sample reacting means can be efficiently irradiated or received by the light receiving means. A specific microorganism weighing device that enables highly efficient detection of the microorganism is obtained.

Further, by providing a spectroscopic section between the light source and the minute area for transmitting only a specific wavelength, a specific microorganism weighing apparatus capable of irradiating a target excitation wavelength even when a light source having a wide wavelength range is used. can get.

Further, by providing a spectroscopic section for transmitting only a specific wavelength between the minute area and the light receiving means, it is possible to obtain a specific microorganism weighing apparatus capable of detecting a target fluorescence wavelength even in the case of emission in a wide wavelength range. Can be

Further, by providing a function of automatically focusing a minute area to be detected in the sample reaction means, it is possible to always detect a specific microorganism existing in a certain area, and to further improve the workability of the operator. A specific microorganism measuring device that can be obtained is obtained.

Further, by introducing excitation light from a light source using an optical fiber, a light source serving as a heat source can be installed in a separate part. A specific microorganism weighing device capable of introducing a light amount into the sample reaction means is obtained.

Further, by using an ultraviolet-transmissive lens for the light source condensing means, it is possible to obtain a specific microorganism weighing apparatus which can be introduced into the specimen reaction means without reducing the light quantity of the excitation wavelength in the ultraviolet region.

Further, by providing a means for indicating the measurement position of the sample reaction means, a specific microorganism weighing device capable of recognizing the detection position can be obtained.

Further, by providing a means for indicating the measurement position of the sample reaction means, a specific microorganism measuring apparatus capable of recognizing the detection position without being affected by the size, shape, etc. of the sample reaction means can be obtained.

Further, by providing a means for indicating the measurement position in the sample reaction means, a specific microorganism weighing device capable of recognizing the detection position according to the type of the sample reaction means can be obtained.

Further, a specific microorganism weighing device capable of simply and accurately recognizing the detection position of the sample reaction means can be obtained by using magnetic means for indicating the measurement position of the sample reaction means.

Further, by providing a means for recognizing the measurement position of the sample reaction means, it is possible to obtain a specific microorganism weighing apparatus capable of adjusting the detection target position of the sample reaction means.

In addition, since there is a means for moving the orbital position for recognizing the measurement position of the sample reaction means from the orbital position for recognizing the measurement position to the next recognizable orbital position while maintaining a constant distance, it is not necessary to detect the entire sample, so that weighing can be performed. A specific microorganism weighing device that can be speeded up is obtained.

The distance from the orbital position at which the measurement position of the sample reaction means is recognized to the next recognized orbital position is 10 to 5
By setting it to 0 μm, a specific microorganism weighing device capable of performing weighing highly correlated with the culture method can be obtained.

Further, by recognizing signals obtained from microorganisms contained in the sample reaction means as binarized point coordinates, it is possible to obtain a specific microorganism measuring device capable of easily and quickly measuring.

In addition, a specific microorganism weighing device capable of weighing highly correlated with the culture method can be obtained by recognizing, as one signal, a nearby signal among the point coordinates at which the signal was obtained.

[0068]

BEST MODE FOR CARRYING OUT THE INVENTION The first method for measuring specific microorganisms according to the present invention is a method for measuring specific microorganisms contained or possibly contained in a liquid specimen by binding them with an antibody after fluorescent staining. . A first compound that causes live cells to emit light and a second compound that causes dead cells to emit light at a wavelength different from the emission are added to a liquid sample containing or possibly containing a specific microorganism.
And a third compound that causes living cells to emit light at a wavelength different from the emission, and at least one or more fourth compounds that emit light at a wavelength different from the emission by reacting with a specific microorganism-derived substance. When one or more of them are brought into contact, the specific microorganism is fluorescently stained when the liquid specimen contains the specific microorganism. By immobilizing an antibody that specifically binds to a specific microorganism on the sample reaction means, if the sample contact solution containing the fluorescently stained specific microorganism is brought into contact with the sample reaction means, the sample reaction means is fluorescently stained. The specified microorganisms can be captured with high accuracy. Then, the fluorescent-stained specific microorganisms bound to the antibody are measured by the optical measuring means, and the number of the specific microorganisms can be easily, quickly, and accurately measured. As the optical measuring means, for example, known means using the light amount or the bright spot as an index, such a means to count specific microorganisms one by one, or from the light amount based on a calibration curve prepared in advance The total number of specific microorganisms may be counted. FIG. 11 shows an example of the concept. FIG.
As shown in the figure, the sample reaction means 51 is provided with an antibody 5 that specifically binds to a specific microorganism 61 on a substrate 52 such as a glass substrate.
3 is immobilized. Liquid sample is specific microorganism 6
In the case of containing 1, a first compound that causes the liquid specimen to emit live and dead cells, a second compound that causes dead cells to emit light at a different wavelength from the emission, and a live cell that emits light at a different wavelength from the emission By contacting any one or more of at least one or more fourth compounds that emit at a wavelength different from the emission by reacting with the third compound and a specific microorganism-derived substance, Are fluorescently stained by the compound 54. The antibody 53 binds to the fluorescent-stained specific microorganism 61 by an antigen-antibody reaction.
Is captured by Then, by measuring the fluorescent-stained specific microorganism 61 bound to the antibody 53 by an optical measuring means, it is possible to observe the luminescence at the position where the fluorescent-stained specific microorganism 61 is captured.

The second specific microorganism measurement method of the present invention is a method in which a specific microorganism contained in or possibly contained in a liquid sample is bound to an antibody and then subjected to fluorescent staining to measure the amount. By contacting a liquid sample containing or possibly containing a specific microorganism with a sample reaction means on which an antibody specifically reacting with the specific microorganism is immobilized, the specific microorganism can be removed if the liquid sample contains the specific microorganism. It binds to the antibody immobilized on the sample reaction means. Thereafter, a first compound that causes the specimen reaction means to emit live and dead cells, a second compound that causes dead cells to emit light at a different wavelength from the light emission, and a third compound that causes live cells to emit light at a different wavelength from the light emission When contacting any one or more of at least one or more of the fourth compounds that emit at a wavelength different from the emission by reacting with a specific microorganism-derived substance, the specific microorganisms bound to the antibody emit fluorescence. Since the specific microorganisms are stained, the number of the specific microorganisms can be easily, quickly, and accurately measured by optically measuring the specific microorganisms stained with the fluorescent light. As the optical measuring means, the above-mentioned means can be mentioned.

Further, since the first compound is a nucleic acid-binding compound, the staining of the nucleic acid present in the cells is used as an index, the detection can be performed at the level of one cell after the staining. This has the effect that the number of viable and dead cells of the specific microorganism can be measured with higher accuracy.

In addition, since the second compound is a nucleic acid-binding compound, the staining of the nucleic acid present in the cells is used as an index, the detection can be performed at the level of one cell after the staining. This has the effect that the number of dead cells of the specific microorganism can be measured with higher accuracy.

In addition, since the third compound is a compound that emits light when it reacts with a substance derived from a microorganism, it reacts with a metabolite of a living cell of the specific microorganism. Therefore, the viability of the specific microorganism can be determined based on the reactivity. It has the action of:

Further, the use of a microorganism-derived substance as an enzyme protein has an effect that the viability of living cells of a specific microorganism can be determined from its reactivity.

In addition, the use of a substance derived from a specific microorganism as an enzyme protein has the effect that the viability of a specific microorganism can be determined from its reactivity.

Further, since the sample reaction means can be brought into contact with the sample contact liquid while flowing the sample contact liquid, the contact efficiency between the antibody immobilized on the sample reaction means and the specific microorganism stained with fluorescence can be improved. Therefore, the fluorescent-stained specific microorganism can be more reliably captured by the sample reaction means, and the measurement can be performed with higher accuracy.

Further, since the sample reaction means can be brought into contact with the liquid sample while flowing it, the contact efficiency between the antibody immobilized on the sample reaction means and the specific microorganism can be improved. It is possible to more reliably capture the microorganisms in the sample reaction means, and it is possible to perform more accurate measurement by fluorescently staining the specific microorganism.

Further, the sample reaction means has two electrodes disposed opposite to each other with a gap therebetween and a short-circuit portion capable of short-circuiting the two electrodes so that the potential can be varied, and specifically reacts with a specific microorganism. If the antibody is immobilized on one electrode and a voltage is applied so that the electrode on which the antibody is immobilized is on the positive side, the specific microorganism whose surface is negatively charged is usually Since the particles move and gather in the direction of the positive electrode, the contact efficiency between the antibody and the specific microorganism can be improved. Therefore, the specific microorganism can be more reliably captured by the sample reaction means, and the measurement can be performed with higher accuracy. FIG. 12 shows an example of the concept. As shown in FIG. 12, the sample reaction means 51 is formed by immobilizing an antibody 53 that specifically binds to a specific microorganism 61 on a substrate 52 such as a glass substrate, and the antibodies 53 face each other with a gap therebetween. Electrodes 5 arranged in parallel
7 and 58 are fixed to one of the electrodes 57. Antibody 5
3 is fixed such that the electrode 57 on which
When the voltage is applied by the step 9, the specific microorganism 61
In the case where the specimen 4 has already been fluorescently stained, the specific microorganisms 61 which are contained in the specimen contact liquid 62 and which are usually fluorescently stained and whose surface is charged to a negative charge move toward the electrode 57 and gather. Therefore, the contact efficiency between the antibody 53 and the specific microorganism 61 that has been fluorescently stained can be improved. If the voltage is reversed so that the electrode 58 is on the positive side after the antibody 53 is bound to the fluorescent-stained specific microorganism 61, microorganisms other than the specific microorganism to be inspected may be added to the sample contact liquid 62. If so, the microorganism is
Since they are moved and gathered in the direction of 8, the separation of the specific microorganism 61 stained with fluorescent light from other microorganisms is facilitated, and high measurement accuracy is secured. The antibody 53 binds to the fluorescent-stained specific microorganism 61 by an antigen-antibody reaction, the fluorescent-stained specific microorganism 61 is captured by the sample reaction means 51, and the fluorescent-stained specific microorganism 61 bound to the antibody 53 is removed. By measuring by optical measuring means,
Light emission can be observed at the position where the specific microorganism 61 stained with fluorescent light is captured.

The first specific microorganism weighing device of the present invention is a device for measuring a specific microorganism contained or possibly contained in a liquid sample by binding the antibody to an antibody and then binding the antibody to the antibody. The above-mentioned method has an effect that a method of measuring the amount of the specific microorganisms contained in or possibly contained in the liquid sample after fluorescent staining and binding to the antibodies can be performed with high accuracy. That is, irradiating the sample reaction means with the fluorescently stained specific microorganisms bound to the antibody with excitation light,
The number of specific microorganisms is counted based on the light emitted by the excitation light. The sample reaction means is usually about 40 mm in diameter, but since the size of the specific microorganisms contained therein is very small, 1 to 5 μm, image processing is performed using a conventional CCD camera. And the required number of pixels becomes enormous, making direct recognition difficult,
It could not be recognized without using a magnifying means of about 000 times. Therefore, the present inventors pay attention to the size of the specific microorganisms captured by the sample reaction means, and an area of 1 to 5 μm square (as large as possible if it can be measured as the brightness of the light receiving means, it is desirable that the area is as large as the photodiode. (Preferably, 30 μm square or less) is irradiated with excitation light in a predetermined wavelength range, the amount of light emitted by the excitation light is measured, and the number of specific microorganisms is weighed based on the integration. . At this time, the intensity of the emitted fluorescent light upon irradiation with the excitation light changes with time. Therefore, it is necessary to set a time within a certain range of the amount of emitted light and determine the presence or absence of the specific microorganism based on the amount of light at that time. In particular,
Impurities other than specific microorganisms do not emit fluorescence, but emit scattered light of excitation light and the like, which may affect upon receiving light, so that a case where the amount of received light shows a certain value or more is determined as a specific microorganism. . In addition, depending on the type of impurities, it is assumed that fluorescence of a specific microorganism or more may be emitted.Therefore, when the amount of light is within the range of the set light amount, the microorganism is determined to be a specific microorganism. Weigh as one specific microorganism. Then, the micro area irradiated with the excitation light is moved continuously or intermittently. For example, when comparing with the data of the conventional agar medium diffusion method, the entire area having a diameter of 40 mm is scanned, and the area is scanned. The number of specific microorganisms can be integrated and measured. Therefore,
It has the ability to prevent the occurrence of underestimation due to the quenching of the compound due to excitation light, and the occurrence of overestimation due to luminescence emitted from foreign substances. It is not necessary, and the specific microorganism can be measured in a short time.

The second specific microorganism measuring apparatus of the present invention is an apparatus for measuring a specific microorganism contained or possibly contained in a liquid sample by binding the antibody to an antibody and performing fluorescent staining. The above-mentioned method has an effect that the method of binding the specific microorganisms contained in or possibly contained in the liquid specimen to the antibody and then fluorescently staining the same to measure the amount can be performed with high accuracy. That is, irradiating the sample reaction means with the fluorescently stained specific microorganisms bound to the antibody with excitation light,
The number of specific microorganisms is counted based on the light emitted by the excitation light. The first to fourth compounds may be housed together in one detection reagent-containing part, or may be housed separately in a plurality of detection reagent-containing parts.

Further, the light receiving means is at least one photoelectric conversion element, and a condensing lens for condensing the light emitted by the excitation light is provided in front of the photoelectric conversion element, so that the fluorescence range generated by the excitation light is expanded. Since it can be introduced into the photoelectric conversion element in a state where it has been set, it has an effect that high detection sensitivity can be maintained.

Further, by arranging a plurality of photoelectric conversion elements included in the light receiving means in a linear manner, the movement in the X-axis direction (arrangement direction) becomes unnecessary, and the Y-axis direction (movement direction).
It is possible to perform the detection in a certain range by only the movement, and it is possible to perform the weighing quickly.

Further, by making the sample reaction means disk-shaped and moving the sample reaction means, detection by rotation of the sample or the light source becomes possible. As a detection method, for example,
An optical disk reading device may be used. The light source can be moved only by the radius of the sample reaction means by installing and rotating the sample reaction means. This has the effect that the detection can be automated and speeded up.

Further, by making the area for measuring the sample reaction means polygonal, and moving the sample reaction means, it becomes possible to easily detect the specific microorganisms contained in the sample reaction means. An example of the detection means is a scanner. By installing and moving the sample reaction means, this has the effect that the detection can be automated and speeded up.

As a moving means for moving a minute area, a driving means for moving one or more of a light source, a light receiving means, and a specimen reaction means is provided, and the moving means is moved within a set speed range. This has the effect of preventing the occurrence of shift, afterimage or afterglow, and preventing overestimation or underestimation.

Further, by providing a reflector as a means for collecting light emitted from the light source and / or a means for collecting light to the light receiving means, unnecessary loss of light is prevented, and sensitivity is improved by collecting light. It has the effect of being able to.

Further, by providing a spectroscopic section that transmits only a specific wavelength between the light source and the minute area, a specific excitation wavelength can be extracted even when the wavelength emitted from the excitation light source is wide. It becomes possible. Further, by providing a spectroscopic portion that transmits only a specific wavelength between the micro area and the light receiving means, it becomes possible to extract a specific fluorescence wavelength from fluorescence including various wavelengths as well as excitation light,
It has the effect that high-precision weighing becomes possible.

Further, by providing at least one of the light source and the light receiving means with an automatic focusing function for focusing on a small area in the sample reaction means, stable measurement can be performed without depending on the shape of the surface of the sample reaction means. Become. In addition, the autofocus function makes it possible to maintain a constant excitation light irradiation area, minimize the load on specific microorganisms captured and emitted by the sample reaction means, and avoid underestimation. Having.

In addition, the provision of the means for introducing the light emitted from the light source into the sample reaction means using an optical fiber enables stable excitation light to be provided even for a sample reaction means having a complicated shape. Further, the use of the optical fiber makes it possible to provide the light source unit in another part. Thereby, even when a light source that generates heat is used, the load on the sample reaction means and the specific microorganisms captured by the sample reaction means due to the heat generation can be minimized.

Further, by using a lens that transmits ultraviolet light, an ultraviolet light generating means can be used as a light source, and excitation light in the ultraviolet region can be stably provided to the sample reaction means.

Further, by providing a device indicating a measurement position in the apparatus or the sample reaction means and providing a light receiving means with a means for recognizing the measurement position, the position where at least one of the light source and the light receiving means exists can be grasped. It is possible to prevent double detection or omission of detection of the position, and to avoid overestimation or underestimation. Furthermore, the use of magnetism as the means for indicating the measurement position of the sample reaction means enables simple and accurate position recognition, and has the effect that over- or under-estimation can be avoided.

Further, when the trajectory for recognizing the measurement position of the sample reaction means is moved, it is moved to the next recognition trajectory position while maintaining a certain distance from the recognition trajectory position, thereby preventing leakage of the detection. It is possible to detect the entire reaction means. Further, by setting the distance from the position of the recognition orbit to the position of the next recognition orbit to be 10 to 50 μm, there is an effect that the correlation with the culture method, which is one of the conventional methods, can be improved. This is the minimum distance that can avoid the range of recognizing one colony by overlapping colonies in the culture method.

Also, by recognizing a signal obtained from a specific microorganism captured by the sample reaction means as binarized point coordinates and measuring the number of point coordinates at which the signal is obtained,
The specific microorganism captured by the sample reaction means can be easily and quickly measured. Further, it is possible to obtain the luminance from the binarized point coordinates and measure the activity possessed by the specific microorganism. In addition, by recognizing a nearby signal as one signal, the correlation with the culture method can be increased. Furthermore, the shape of the deposit attached to the sample reaction means can be grasped from the nearby signal, and the shape other than the specific microorganism can be removed from the measurement, which has an effect of improving the accuracy. .

Hereinafter, embodiments of the present invention will be described with reference to the drawings.

[0094]

DESCRIPTION OF THE PREFERRED EMBODIMENTS Embodiment 1 of the present invention will be described below.

The test object in the method for measuring a specific microorganism of the present invention is a liquid specimen containing or possibly containing a specific microorganism. The liquid sample may be of any type as long as it is an aqueous substance.For example, washing liquid for foods and cooking utensils, etc., after adding water to the food and grinding, centrifugation and filtration of solids and oily substances , A washing solution such as a cotton swab wiped from a cooking utensil, and an aqueous solution obtained by blowing air in a cooking chamber onto a surfactant-containing aqueous solution.

Specific microorganisms to be tested include Escherichia coli, Escherichia coli bacteria, Shigella, Cholera, Salmonella, Vibrio, Botulinum, Welsh, Staphylococci, which are pathogenic intestinal bacteria that can cause food poisoning. And Bacillus cereus, but may be bacteria that can cause infectious diseases other than food poisoning, such as Mycobacterium tuberculosis, S. pneumoniae, Pseudomonas aeruginosa, and Legionella.

Examples of the first compound that emits light from living and dead cells used for fluorescent staining of a specific microorganism include, for example, 4 ′, 6-diamidino-2-phenylindole dihydrochloride (which may be a derivative) Such compounds that non-specifically penetrate from the membrane surface of a living and dead cell and emit light when specifically bound to a nucleic acid present in the cell. Examples of the second compound that causes dead cells to emit light at a different wavelength from the above-mentioned light include, for example, non-specifically penetrating from the membrane surface of dead cells such as propidium iodide (which may be a derivative), And a compound that emits light when specifically bound to a nucleic acid present therein. Examples of the third compound that causes living cells to emit light at a wavelength different from the above-mentioned emission include, for example, 6-carboxyfluorescein diacetate,
2 ', 7' dichlorofluorescein diacetate, 6-
(N-succinimidyloxycarbonyl) -3 ′,
Penetrates into cells such as 6'-0,0'-diacetylfluorescein, dihydrodrhodamine, fluorescein diacetate, 4-azidofluorescein diacetate (these may be derivatives) and is present in living cells Enzyme proteins, for example, compounds that emit light when decomposed by esterases. Examples of the fourth compound that emits light at a wavelength different from the emission by reacting with a specific microorganism-derived substance include, for example, 4-methylumbelliferyl-
β-D-galactoside, 4-methylumbelliferyl-
β-D-glucuronide (these may be derivatives). These are decomposed by specifically reacting with β-galactosidase, an enzyme protein produced by Escherichia coli and coliform bacteria, to produce 4-methylumbelliferone. 4-Methylumbelliferone emits fluorescence when excited by ultraviolet light. The first to fourth compounds are used, for example, in a state of being dissolved in physiological saline. These may use only one kind of compound, may use two kinds of compounds, may use three kinds of compounds, or may use all kinds of compounds. May be used.

The sample reaction means in the method for measuring a specific microorganism of the present invention comprises a substrate such as a glass substrate on which an antibody specifically reacting with a specific microorganism to be tested is immobilized. The immobilization of the antibody on the substrate is performed by a method known per se. The outline is described below. First, in the case of a glass substrate, a long-chain alkyl group is introduced on the glass surface. This can be performed, for example, by immersing a glass substrate whose surface has been cleaned with a strong acid, an organic solvent, or the like, in a reagent composed of a 10 mM octadecyltrichlorosilane / benzene solution. Amino groups may be introduced on the glass surface. This can be performed, for example, by immersing a glass substrate whose surface has been cleaned with a strong acid, an organic solvent, or the like in a reagent composed of a 10 mM aminopropyltriethoxysilane / benzene solution. When a long-chain alkyl group is introduced into the glass surface, the surface has high hydrophobicity. However, since the antibody also has hydrophobicity, the antibody is immobilized on the glass surface by so-called hydrophobic interaction.
When an amino group is introduced to the glass surface, the antibody is immobilized on the glass surface by bonding the amino group of the antibody to the amino group of the glass surface with glutaraldehyde.
In addition, to the part where the antibody is not immobilized, the measurement accuracy such as bovine serum albumin, skimmed milk, casein, etc. is affected, so that proteins that affect the measurement accuracy do not bind. It is desirable to bind and block proteins that do not occur.

The antibody may be immobilized on the entire surface of the sample reaction means, or the immobilization region may be limited. The immobilization area can be limited by using a glass capillary having a diameter of about 10 to 100 μm for spotting the antibody solution.

A single type of antibody may be immobilized on the sample reaction means to measure a single type of specific microorganism, or a plurality of types of antibody may be immobilized to simultaneously measure a plurality of types of specific microorganisms. The fixed area may be divided and fixed, and the area may be specified.

[0101] The antibody may be a polyclonal antibody or a monoclonal antibody.
It is desirable to use a monoclonal antibody with excellent specificity. When a polyclonal antibody is used, it is preferable to use an affinity-purified antibody.
There is no particular limitation on the method for producing the antibody, a method by animal immunization using an antigen of a specific microorganism, a method by cell fusion of antibody-producing cells of a required antibody, a genetic engineering technique using a gene of the required antibody And the like.
Various types of bacterial monoclonal antibodies that can cause food poisoning are already known, and some are commercially available.

In the first method for quantifying a specific microorganism of the present invention, first, one or more of the first to fourth compounds may contain or contain a specific microorganism to be tested. Contact with a liquid sample with The contact conditions, that is, the conditions for fluorescent staining of the specific microorganisms when the liquid sample contains the specific microorganisms to be tested, can be, for example, at room temperature. Is good.

Next, a sample contact liquid obtained by bringing one or more of the first to fourth compounds into contact with a liquid sample is brought into contact with the sample reaction means. The contact condition, that is, the reaction condition for binding the antibody to the specific microorganism stained with fluorescent light may be a usual antigen-antibody reaction condition, for example, a reaction time of about 30 ° C. for several minutes.

Finally, the sample reaction means is washed with a washing solution such as distilled water or physiological saline to remove microorganisms, contaminants, unreacted first to fourth compounds, etc., which are not the object of the inspection, and then optically react. The number and presence / absence of the fluorescent-stained specific microorganism captured by the sample reaction means are measured by the measurement means.

In the second specific microorganism measurement method of the present invention, first, a liquid specimen containing or possibly containing a specific microorganism to be tested is brought into contact with a specimen reaction means. The contact conditions, that is, the reaction conditions for binding the antibody to the specific microorganism when the liquid sample contains the specific microorganism to be tested may be ordinary antigen-antibody reaction conditions, for example, at about 30 ° C. It can be something like a reaction time of several minutes.

Next, if necessary, the sample reaction means is washed with a washing solution such as distilled water or physiological saline to remove microorganisms and contaminants which are not the object of the test. One or more types are brought into contact with the sample reaction means. The contact conditions, that is, the conditions for fluorescent staining of the specific microorganism captured by the sample reaction means can be, for example, room temperature, but may be a reaction time of about 30 ° C. for several minutes.

Finally, the sample reaction means is washed with a washing solution such as distilled water or physiological saline to remove unreacted first to fourth compounds, etc., and then captured by the sample reaction means by the optical measurement means. The number and the presence or absence of the fluorescent-stained specific microorganisms are measured.

When the antibody is immobilized on the entire surface of the sample reaction means, the trajectory of the excitation light provided on the specific microorganism measuring device on the sample reaction means covers the entire sample reaction means 1 as shown in FIG. It is hoped that it is. For this purpose, the width and length of the specimen are moved and detected like the trajectory A2. In addition, as shown in FIG. 2, the trajectory for detecting the entire sample reaction means can be circular, so that the trajectory can be moved and detected like trajectory B3. The light source 4 moves on the specimen reaction means along each trajectory. FIG. 3 shows a model of the specific microorganism judging means using the fluorescence generated by the excitation light emitted from the light source 4. The fluorescence emitted from the sample reaction means correlates with the site where the fluorescence is emitted to form the specific microorganism determination means 5. The specific microorganism determining means 5 contains a mixture of scattered light generated by impurities and the like and fluorescent light generated by the same excitation light, and may not be limited to the fluorescent light emitted from the target specific microorganism. Therefore, a threshold is provided. The threshold has an upper limit 6 and a lower limit 7 of the fluorescence intensity,
The fluorescence within that range is recognized as being derived from the specific microorganism and integrated. Thereby, the fluorescence derived from impurities other than the specific microorganism is grasped, and accurate measurement is enabled. The threshold is determined by the fluorescence intensity emitted by the specific microorganism. When the specific microorganisms are close to each other, a waveform formed from the fluorescence emitted by the specific microorganisms is obtained as shown in FIG. In this case, when the adjacent specific microorganisms are detected by the agar medium diffusion method, which is one of the conventional methods, the colonies overlap with each other with the growth of the microorganisms and the expansion of the colonies, and as one colony at the time of visual confirmation. You may decide.
Therefore, in the present invention, in the case shown by the waveform 8, it was set so as to be detected as one specific microorganism. Thereby, a correlation with the agar medium diffusion method can be obtained.

The excitation light is irradiated to the sample reaction means 1 within a time range in which the fluorescence intensity does not extinguish as shown in FIG. That is, it is within the range of the threshold value 9.

[0110] As shown in FIG.
Light source 4, lens 10 as light source condensing means, light receiving unit 11
including. In order to extract a target wavelength from the excitation light emitted from the light source 4, the light is separated by the excitation light spectral filter 12.
The split excitation light passes through the prism 13 to change the optical path. The excitation light whose optical path has been changed is condensed on the surface of the sample reaction means 1 via the lens 10. Then, the fluorescence of the specific microorganism excited by the excitation light passes through the prism 13 again. At this time, the fluorescent light passes through the prism 13 as it is and reaches the light receiving unit 11. The fluorescence that has reached the light receiving unit 11 passes through the fluorescence spectral filter 14 to extract only the target fluorescence, reaches the photoelectric conversion element 15 built in the light receiving unit 11, is converted into a signal, and is recognized. Although not shown in the figure, the sample reaction means 1 or the specific microorganism weighing device includes a means for moving the sample reaction means, and receives all or a part of the fluorescence emitted from the sample reaction means 1. be able to.

The fluorescent light that has reached the photoelectric conversion element 15 is determined by the specific microorganism determining means 5 as a specific microorganism or a foreign substance, and the fluorescent light determined as the specific microorganism is integrated and its quantity is measured.

The excitation light generated from the light source 4 is
At this time, the area irradiated with the excitation light by the lens 10 is condensed to a minute constant area. In this case, the minute constant area indicates a range of about 0.2 μm to 7.0 μm on a side when set based on the size of the specific microorganism. Also, based on a comparison with the agar medium diffusion method, which is one of the most utilized microorganism detection means at present, colonies formed by a population of microorganisms cultured and grown by the agar medium diffusion method have a distance of When the colonies are close to each other, the colonies may overlap with each other. When the colonies are finally confirmed visually, there may be a case where the colonies are recognized as one colony. Therefore, the minute constant area in this case is 100 μm to 500 μm on a side based on the distance at which the colonies do not overlap with each other.
It indicates a range of about μm.

The irradiation time of the excitation light condensed by the lens 10 depends on the quenching time of the fluorescent compound and the excitation light intensity. Depending on the type of the compound, the compound may be decomposed by ultraviolet light existing in nature, and may be 2 seconds to 30 seconds.
It is desirable to irradiate the excitation light within a range of about 0 seconds.

When recognizing the luminance after light emission, as shown in FIG. 7, the position where the fluorescent light is emitted is represented by binarization of "0" and "1". In the case of binarization, adjacent light emission is regarded as one light emission source 16. If the size of the light source and the luminance thereof are relatively different from the set size, the light source is recognized as a foreign substance 17 and is not measured as a specific microorganism by deeming it to be out of the inspection target. The distance between adjacent luminescence is based on a comparison with the agar medium diffusion method, which is one of the most widely used means for detecting microorganisms, and is formed by a population of microorganisms grown and grown by the agar medium diffusion method. If the colonies to be colonized are close to each other, the colonies may overlap with each other, and when finally confirmed visually, there may be a case where the colonies are recognized as one colony. Therefore, based on the distance at which the colonies do not overlap with each other, 100 μm on each side
About 500 μm.

When detecting light emission, if the wavelength range of the light source 4 is wide, the excitation wavelength can be adjusted and spectrally separated by the excitation light spectral filter 12. Since the excitation light spectral filter 12 can be changed according to the target to be detected, it can correspond to various fluorescent compounds. At the same time, in the case where the width of the emitted fluorescence wavelength is wide, it is possible to cope with compounds that emit various fluorescences by changing the fluorescence spectral filter 14 in accordance with the target to be detected in order to detect the target emission. .

Examples of the light source 4 include various diodes, halogen lamps, xenon lamps, cold cathode tubes, lasers, black lights, mercury lamps and the like. Of these light sources, diodes, cold-cathode tubes, black lights, etc., whose maximum excitation wavelengths are relatively limited, may be implemented without using the excitation light spectral filter 12 and the fluorescent spectral filter 14. For a halogen lamp, a mercury lamp, and the like, it may be necessary to use the excitation light spectral filter 12 and the fluorescent spectral filter 14.

The prism 13 and the lens 10 have the property of transmitting ultraviolet light as required. Quartz glass and the like have a property of transmitting ultraviolet light. This makes it possible to cope with compounds excited by ultraviolet light. The part where the sample reaction means 1 is installed has a rotating ability, and the sample reaction means 1 is installed on the upper part. The excitation light condensed by the lens 10 moves a distance corresponding to a radius from the outer peripheral portion of the sample reacting means 1 to the center or from the central portion to the outer peripheral portion. At this time, by changing the rotation speed of the specimen reaction means 1 when the position of the excitation light condensed by the lens 3 exists at the outer peripheral portion and when it exists at the central portion, the excitation light condensed by the lens 10 is changed. It is possible to prevent a shift in fluorescence, an afterimage, and an afterglow of fluorescence emitted by the excited compound when light is present at the outer peripheral portion and when light is present at the central portion.

By providing a means for recognizing the condensed position, the position of the excitation light condensed by the lens 10 is recognized so that the condensed light does not deviate from the orbit. It is set to return to.

The minute constant area for irradiating the excitation light is not limited to a polygon including a square, but may be a circle, an ellipse, or the like, as long as the specimen can be irradiated.

It is also possible to use a diffraction grating or the like as a means for dispersing excitation light or fluorescence.

Although the afterimage and the afterglow of the fluorescence are prevented by adjusting the rotation speed of the sample reaction means 1, the afterimage and the afterglow are adjusted by adjusting the moving speed of the lens 10 for irradiating the excitation light. It is also possible to prevent it.

The means for recognizing the focused position does not necessarily need to directly recognize the focused position of the excitation light, but may be any as long as it can grasp the trajectory on the sample reaction means 1.

Hereinafter, a second embodiment of the present invention will be described.

The specific microorganism measuring apparatus shown in FIG. 8 comprises a sample reaction means 1, a light source 4, a reflector 18, a lens 10,
Including 1. The excitation light emitted from the light source 4 is excited by the specific microorganisms captured by the sample reaction means 1, and the generated fluorescence is changed in the light direction by the reflection plate 18, condensed by the lens 10, and condensed by the lens 10. To detect. The fluorescent light that has reached the light receiving section 11 is determined by the specific microorganism determining means 5 as a specific microorganism or a foreign substance, and the fluorescent light determined as the specific microorganism is integrated and its quantity is measured. The light source 4 scans the sample reaction means 1. Further, the reflecting plate 18 is set in accordance with the trajectory of the light source 4.

The excitation light generated from the light source 4 is
At this time, the area irradiated with the excitation light is condensed on a minute constant area. In this case, when the minute constant area is set based on the size of the specific microorganism,
It indicates a range of about 0.2 μm to 7.0 μm on one side.
Also, based on a comparison with the agar medium diffusion method, which is one of the most utilized microorganism detection means at present, the colonies formed by a population of microorganisms cultured and grown by the agar medium diffusion method are located at the distance. Are close together,
The colonies may overlap with each other, and when finally confirmed visually, there may be a case where the colonies are recognized as one colony. Therefore, the minute constant area in this case indicates a range of about 100 μm to 500 μm on a side based on a distance at which colonies do not overlap with each other.

The irradiation time of the excitation light condensed by the lens 10 depends on the extinction time of the fluorescent compound and the excitation light intensity. Depending on the type of the compound, the compound may be decomposed by ultraviolet light existing in nature, and may be 2 seconds to 30 seconds.
It is desirable to irradiate within a range of about 0 seconds.

Examples of the light source 4 include various diodes, a halogen lamp, a mercury lamp, a xenon lamp, a cold cathode tube, a laser, a black light and the like.

The reflection plate 18 and the lens 10 each have a property of transmitting ultraviolet light as required. Quartz glass and the like have a property of transmitting ultraviolet light. This makes it possible to cope with compounds excited by ultraviolet light.

In order to reduce the intensity of the excitation light emitted from the light source 4 to the sample reaction means 1 or to prevent the external light from affecting the fluorescence, a dark field plate or the like for placing the sample reaction means 1 in a dark field environment is used. It is also possible to set up.

The light source 4 does not always move. It is also possible to move the sample reaction means 1 and detect the specific microorganism captured by the same.

The light source 4 is not always built-in. It is also possible to provide a light source section including the light source 4 outside, and to introduce excitation light into the apparatus from there using an optical fiber or the like.

It is also possible to provide a means for removing a wavelength other than the intended wavelength depending on the type of the light source. For example, an excitation light spectral filter, a diffraction grating, and the like can be given as means.

It is also possible to provide a means for removing a wavelength other than the intended wavelength depending on the type of the emitted fluorescence.
For example, a fluorescence spectroscopic filter, a diffraction grating, or the like may be used.

It is also possible to use a prism or the like instead of the reflection plate 18.

Hereinafter, a third embodiment of the present invention will be described.

As shown in FIG. 9, the sample reaction means 1 captures the handle 19 and the specific microorganisms contained in or possibly contained in the liquid sample in order to improve the handleability of the operator for the attaching / detaching operation. And a detection start line 21 for recognizing a detection start point and a sample recognition trajectory 22 for recognizing a detection trajectory.

The fluorescent-stained specific microorganism captured in the sample reaction section emits fluorescence at the position where it is captured. Further, in order to recognize the position of the excitation light focused on the sample reaction means 1, a sample recognition trajectory 22 provided in advance in the sample reaction means 1 is detected to recognize the position of the excitation light. Excitation light condensed from the lens 10 shown in FIG. 6 moves on the outer circumference with the detection start line 21 as a start point.
After reaching the detection start line 21 after the outer peripheral movement, the excitation light condensed by the lens 10 shown in FIG. 6 shifts to the innermost sample recognition trajectory 22 and recognizes the number of light emission included in the trajectory. I do. By moving the excitation light toward the center while repeating this, the number of specific microorganisms contained in the area requiring detection is detected.

The detection start line 21 may be a marking directly engraved on the sample reaction means, a marker having a reflectivity capable of recognizing the excitation light or the focused position, or the like. The same applies to the specimen recognition trajectory 22, but it is desirable to use a marker that does not affect the fluorescence.

Note that the handle does not always exist on the outer periphery.

The recognition of the detection start point is not limited to the line.

The detection trajectory may be spiral or oblique.

Note that the movement of the excitation light is not always from the outer periphery.

Hereinafter, a fourth embodiment of the present invention will be described.

FIG. 10 is a schematic view of an example of the sample reaction means in which the sample reaction means can be brought into contact with the sample contact liquid / liquid sample while flowing. Sample reaction means 1
Is composed of a flow cell having an inlet 23 for introducing a sample contact liquid / liquid sample and an outlet 24 for discharging excess sample contact liquid / liquid sample and air contained in the sample reaction means 1 in advance. The sample reaction means 1 has a structure in which at least a surface in a direction in which the light receiving unit 11 exists is a light transmitting surface 25, and the light receiving unit 11 can detect fluorescence.
The light transmitting surface 25 and the bottom surface 26 located opposite the light transmitting surface 25
It is in a parallel positional relationship and has a configuration that does not cause inclination. An antibody that specifically binds to a specific microorganism is immobilized on the bottom surface 26. Then, the light transmitting surface 25 and the bottom surface 26
And two electrodes are connected by a short-circuit portion 27 that can short-circuit both electrodes so that the potential can be varied. If a voltage is applied so that the electrode disposed on the bottom surface 26 is on the positive side, the specific microorganisms whose surface is negatively charged usually move toward the bottom surface 26 and collect, so that the antibody and the specific microorganisms are collected. The contact efficiency with the contact can be improved.

When the specific microorganism has already been fluorescently stained, a washing solution such as distilled water or physiological saline is supplied into the flow cell from the inlet 23 to supply microorganisms, contaminants, and unreacted first to non-reacted substances which are not to be inspected. After removing Compound 4 and the like from the inside, excitation light is irradiated, and the number of specific microorganisms is counted based on light emitted by the excitation light.

If the specific microorganism has not been stained with fluorescent light, a washing solution such as distilled water or physiological saline is supplied into the flow cell from the inlet 23 as necessary to remove microorganisms and contaminants not intended for the inspection from the inside. After that, one or more of the first to fourth compounds are supplied from the inlet 23, and the specific microorganism is fluorescently stained. Then, a cleaning liquid such as distilled water or physiological saline is supplied into the flow cell from the input port 23 to remove unreacted first to fourth compounds and the like from the inside, and then irradiated with excitation light and emitted by the excitation light. The number of specific microorganisms is counted based on the light emitted.

According to the above configuration, when the specific microorganism is introduced into the sample reaction means 1, the gap between the light transmitting surface 25 and the bottom surface 26 opposite to the light transmitting surface 25 is maintained at a constant thickness. The microorganisms can be spread over a certain area without overlapping each other. As a result, it is possible to prevent underestimation due to overlapping, and the light receiving unit 11
Accurate weighing is possible.

By providing a filter in front of the inlet 23 for removing foreign substances, the sensitivity for detecting a specific microorganism can be improved.

It is to be noted that interference can be prevented by configuring the light transmitting surface 25 so as not to affect excitation light such as ultraviolet rays.

By setting the distance between the light transmitting surface 25 and the bottom surface 26 located opposite to the light transmitting surface 25 in the range of 0.5 to 15 μm, it is possible to prevent the specific microorganisms from overlapping and to perform accurate discrimination. Become.

[0151]

As is clear from the above embodiments, according to the present invention, the specific microorganisms contained in or possibly contained in the liquid sample are captured by the sample reaction means, and the number of the specific microorganisms captured is determined. Alternatively, it is possible to provide a specific microorganism measuring method capable of simultaneously and quickly grasping the presence / absence. This method has the advantages of being able to accurately measure viable and dead cells and dead cells, so that the status of environmental pollution by a specific microorganism can be grasped in real time, and that measures for preventing pollution can be taken promptly.

Further, since the first compound is a nucleic acid-binding compound, even if a liquid specimen contains metabolites or contaminants of cells, viable and dead cells can be measured accurately and in a short time. And a method for measuring a specific microorganism capable of performing the method.

In addition, since the second compound is a nucleic acid-binding compound, dead cells can be measured accurately and in a short time even when cell metabolites or contaminants are present in the liquid sample. And a method for measuring a specific microorganism capable of performing the method.

Further, by using the third compound as a compound which emits light by reacting with a substance derived from a microorganism, it is possible to provide a method for measuring a specific microorganism which can confirm the presence or absence of a specific microorganism living. Further, by using a microorganism-derived substance as an enzyme protein, it is possible to provide a method for measuring a specific microorganism which can confirm the presence or absence of a specific microorganism living with higher sensitivity.

[0155] Further, by using a substance derived from a specific microorganism as an enzyme protein, the presence or absence of the specific microorganism can be easily confirmed, and a method for measuring a specific microorganism that can monitor the specific microorganism can be provided. In addition, it is possible to provide a method for measuring a specific microorganism, which eliminates the need for pretreatment and the like, and can further shorten the inspection time.

In addition, since the sample reaction means can be brought into contact with a sample contact liquid or a liquid sample while flowing the same, the efficiency of contact between the antibody immobilized on the sample reaction means and the specific microorganism can be improved. A method for measuring microorganisms can be provided.

Further, the sample reaction means has two electrodes disposed opposite to each other with a gap therebetween and a short-circuit portion capable of short-circuiting both electrodes so that the potential can be varied, and specifically reacts with a specific microorganism. Since the antibody is immobilized on one electrode, the property that the surface of the microorganism is charged to a negative charge is usually used, and the voltage is set so that the electrode on which the antibody is immobilized is on the positive side. By applying the method, it is possible to provide a specific microorganism measurement method capable of improving the contact efficiency between the antibody and the specific microorganism.

According to the present invention, it is possible to provide a specific microorganism measuring apparatus for measuring a specific microorganism contained in or possibly contained in a liquid sample. This device suppresses the quenching of fluorescence by the excitation light, and enables the number of specific microorganisms to be accurately measured.
This prevents overestimation due to foreign matter contamination and maintains high sensitivity.

Further, high detection sensitivity can be obtained by using at least one photoelectric conversion element as the light receiving means and providing a condenser lens for condensing light emitted by the excitation light in front of the photoelectric conversion element. Further, by arranging the photoelectric conversion elements in a linear manner, it is possible to detect a specific microorganism present on the sample reaction means by scanning in one direction, which has the effect of speeding up the measurement of the specific microorganism. A specific microorganism measuring device can be provided.

Further, by making the sample reacting means in a disk shape, the driving of the sample reacting means in the rotating direction is facilitated, whereby the detection speed can be improved. Further, the detection moving distance of at least one of the light source and the light receiving means can be minimized. Along with this, it is possible to provide a specific microorganism weighing device having an effect that the detection time can be shortened and the specific microorganism weighing device can be simplified.

Further, by moving the minute area of the irradiation of the excitation light emitted from the light source to the sample reaction means within the set speed range, it is possible to prevent a shift in light emission, an afterimage, and an afterglow. It is possible to provide a specific microorganism weighing device having the effect of preventing the microorganism.

Further, by using a reflector to condense at least one of the light emitted from the light source and the light emitted from the sample reaction means, the sample reaction means can be irradiated or received with high efficiency. This makes it possible to provide a specific microorganism weighing device having an effect that the detection can be performed with high efficiency and the device configuration can be simplified.

Further, by providing a spectroscopic section capable of dispersing the wavelength of the light source and a spectroscopic section capable of dispersing the fluorescence wavelength, it is possible to irradiate the target wavelength and detect the emitted target fluorescence wavelength, thereby providing a highly sensitive target. A specific microorganism weighing device capable of detecting a microorganism can be provided.

Also, the surface of the sample reaction means is automatically recognized, and the detection position is recognized by focusing the excitation light.
It is possible to detect the entire sample reaction means, and it is possible to provide a specific microorganism weighing device capable of preventing over- or under-estimation of a sample by preventing double detection or detection leakage accompanying the detection.

In addition, by introducing excitation light from a light source using an optical fiber, a light source serving as a heat source can be installed in a separate part, whereby a specific microorganism or the like captured by the sample reaction means can be removed. It is possible to prevent the influence of heat and to avoid an increase in error. Further, even when the sample reaction means has a complicated shape, it is possible to provide a specific microorganism weighing device having an effect that constant light can be always introduced into the sample reaction means and more accurate measurement can be performed.

Further, by using an ultraviolet light transmitting lens for the light source condensing means, the light can be introduced into the sample reaction means without reducing the amount of light having a wavelength in the ultraviolet region, and can be detected based on the wavelength in the ultraviolet region. It is possible to provide a specific microorganism measuring device having an effect of performing measurement based on a compound.

Further, by providing the device or the sample reaction means with a means for indicating the measurement position, the detection position can be recognized, thereby preventing erroneous detection or adjusting the detection speed. Further, it is possible to provide a specific microorganism measuring apparatus having an effect that simple and accurate detection can be performed by using magnetism as a means for indicating a measurement position of the sample reaction means.

[0168] Further, since there is provided a means for moving the trajectory position at which the measurement position of the sample reaction means is recognized from the position at which the measurement is recognized to the next recognition trajectory position at a constant distance, it is not necessary to detect the entire sample reaction means. Speeding up is possible. In addition, erroneous recognition of microorganisms located between the orbits can be prevented, and more accurate measurement can be performed. Furthermore, the distance between each orbit is 1
By setting the thickness to 0 to 50 μm, it is possible to provide a specific microorganism weighing device having an effect that a correlation with the conventional method can be obtained.

In addition, by recognizing the fluorescence obtained from the sample reaction means as binary point coordinates of “0” and “1”, specific microorganisms can be easily and quickly weighed. Those that were recognized as foreign substances were removed as non-targets, and among those that were also recognized as specific microorganisms as their positional relationship, those that were recognized as neighboring specific microorganisms when compared with the conventional agar medium diffusion method By recognizing one specific microorganism, it is possible to provide a specific microorganism measuring device having an effect of having a correlation with the conventional method.

[Brief description of the drawings]

FIG. 1 is a configuration diagram of an orbit A on a sample reaction unit of excitation light according to a first embodiment of the present invention.

FIG. 2 is a configuration diagram of a trajectory B of the excitation light on the specimen reaction means of the first embodiment.

FIG. 3 is a diagram showing a waveform model according to the first embodiment;

FIG. 4 is a diagram showing a waveform model when a specific microorganism is present adjacent to the first embodiment.

FIG. 5 is a diagram showing a fluorescence quenching model of Example 1;

FIG. 6 is a view showing a specific microorganism weighing device of the first embodiment.

FIG. 7 is a diagram showing a binarization model according to the first embodiment;

FIG. 8 is a view showing a specific microorganism measuring device according to a second embodiment of the present invention.

FIG. 9 is a view showing a sample reaction means according to a third embodiment of the present invention.

FIG. 10 is a view showing a sample reaction means according to a fourth embodiment of the present invention.

FIG. 11 is a conceptual diagram showing an example of the theory of an immunoassay method.

FIG. 12 is a conceptual diagram showing an example of a sample reaction unit using electrodes.

[Explanation of symbols]

 DESCRIPTION OF SYMBOLS 1 Sample reaction means 2 Orbit A 3 Orbit B 4 Light source 5 Specific microorganism judgment means 6 Upper limit 7 Lower limit 8 Waveform 9 Threshold 10 Lens 11 Light receiving part 12 Excitation light spectral filter 13 Prism 14 Fluorescence spectral filter 15 Photoelectric conversion element 16 Light emitting source REFERENCE SIGNS LIST 17 Foreign matter 18 Reflector 19 Handle 20 Sample reaction part 21 Detection start line 22 Sample recognition trajectory 23 Input port 24 Discharge port 25 Light transmission surface 26 Bottom 27 Short circuit part 51 Sample reaction means 52 Substrate 53 Antibody 54 Compound 57 Electrode 58 Electrode 59 Short-circuit part 61 Specific microorganisms 62 Sample contact liquid

──────────────────────────────────────────────────の Continued on the front page (51) Int.Cl. ⁷ Identification symbol FI Theme coat ゛ (Reference) G01N 33/48 G01N 33/48 P 33/569 33/569 A B F term (Reference) 2G043 AA03 BA16 BA17 CA04 DA01 DA02 DA05 GA06 GA07 GA08 GB19 HA01 HA02 HA05 HA15 JA02 JA04 KA03 KA05 KA09 LA01 NA05 2G045 AA28 CB21 FA37 FB03 FB12 GC15 2G057 AA04 AB03 AB04 AC01 BA05 BB02 4B029 AA07 BB01 CC01 FA01 4B06QQA Q0101

Claims (31)

[Claims] 1. A first compound that causes live and dead cells to emit light,
A second compound that causes dead cells to emit light at a wavelength different from the light emission; and a third compound that causes live cells to emit light at a wavelength different from the light emission.
And at least one or more of the at least one fourth compound that emits light at a wavelength different from the emission by reacting with a specific microorganism-derived substance may contain or contain a specific microorganism. When the liquid sample contains a specific microorganism by contacting it with a liquid sample having a characteristic, the specific microorganism is fluorescently stained, and then the sample contact liquid is immobilized with an antibody that specifically reacts with the specific microorganism. And measuring the fluorescent-stained specific microorganisms bound to the antibody by an optical measuring means. 2. A liquid sample containing a specific microorganism is contacted with a sample reaction means on which an antibody specifically reacting with the specific microorganism is immobilized, whereby the liquid sample contains the specific microorganism. In this case, after binding the specific microorganism to the antibody immobilized on the sample reaction means, a first compound that emits live cells and a second compound that emits dead cells at a wavelength different from the emission, A third compound that emits light at a wavelength different from the emission, and any one or more of at least one or more fourth compounds that emit light at a wavelength different from the emission by reacting with a specific microorganism-derived substance Is contacted with the sample reaction means to fluorescence-stain the specific microorganisms bound to the antibody, and the fluorescence-stained specific microorganisms are measured by an optical measurement means. Biometric method. 3. The method for quantifying a specific microorganism according to claim 1, wherein the first compound is a nucleic acid-binding compound. 4. The method according to claim 1, wherein the second compound is a nucleic acid binding compound. 5. The method for quantifying a specific microorganism according to claim 1, wherein the third compound is a compound that emits light when it reacts with a substance derived from a microorganism. 6. The method for measuring a specific microorganism according to claim 5, wherein the microorganism-derived substance is an enzyme protein. 7. The method for measuring a specific microorganism according to claim 1, wherein the substance derived from the specific microorganism is an enzyme protein. 8. The method for measuring a specific microorganism according to claim 1, wherein the sample reaction means can be brought into contact with the sample contact liquid while flowing the sample contact liquid. 9. The method for measuring a specific microorganism according to claim 2, wherein the sample reaction means can be brought into contact with the liquid sample while flowing the liquid sample. 10. A sample reaction means having two electrodes disposed opposite to each other with a gap therebetween and a short-circuit portion capable of short-circuiting both electrodes so that the potential can be varied, and specifically reacts to a specific microorganism. The method for measuring a specific microorganism according to any one of claims 1 to 9, wherein the antibody is immobilized on one of the electrodes. 11. A first compound that causes live cells to emit light, a second compound that causes dead cells to emit light at a wavelength different from the light emission, and a third compound that causes live cells to emit light at a wavelength different from the light emission. A liquid that contains or may contain any one or more of the at least one fourth compound that emits at a wavelength different from the emission by reacting with the specific microorganism-derived substance. A sample reaction unit for immobilizing an antibody that specifically binds to a specific microorganism, for contacting a sample contact solution obtained by contacting with a sample, and a wavelength range predetermined in a minute constant area of the sample reaction unit. A light source that irradiates the excitation light with light, a light receiving unit that receives light in a predetermined wavelength range that emits light by the excitation light, and a predetermined time period in which the light emitted and emitted by the light source is set. A specific microorganism determining means for determining a specific microorganism when the amount of received light is within a range of a set threshold value; a moving means for continuously or intermittently moving the small constant area; An apparatus for measuring a specific microorganism, comprising an integrating means for integrating the quantity of a specific microorganism from a signal determined as a specific microorganism by the microorganism determining means. 12. A sample reaction means for immobilizing an antibody which specifically binds to a specific microorganism, for contacting a liquid sample containing or possibly containing a specific microorganism, and a first means for emitting live and dead cells. And a second compound that causes dead cells to emit light at a wavelength different from the light emission, a third compound that causes living cells to emit light at a wavelength different from the light emission, and the light emission by reacting with a specific microorganism-derived substance. A detection reagent-containing portion accommodating any one or more of at least one or more kinds of fourth compounds emitting at different wavelengths, and a predetermined wavelength range in a minute constant area of the sample reaction means. A light source for irradiating excitation light, light receiving means for receiving light in a predetermined wavelength range emitted by the excitation light, and light emitted and emitted by the light source within a predetermined period of time. A specific microorganism determining means for receiving light, and determining that the received light quantity is within a range of a set threshold, as a specific microorganism; a moving means for continuously or intermittently moving the minute constant area; An apparatus for measuring a specific microorganism, comprising an integrating means for integrating the number of the specific microorganism from a signal determined as a specific microorganism by the means. 13. The specific microorganism weighing device according to claim 11, wherein the light receiving means is at least one photoelectric conversion element, and a condenser lens for condensing light emitted by the excitation light is provided in front of the photoelectric conversion element. . 14. The specific microorganism weighing device according to claim 13, wherein a plurality of photoelectric conversion elements are arranged linearly. 15. The specific microorganism weighing device according to claim 11, wherein the sample reaction means has a disk shape, and the sample reaction means is movable. 16. The specific microorganism measuring apparatus according to claim 11, wherein the area for measuring the sample reaction means is a polygon, and the sample reaction means is movable. 17. A driving means for moving one or more of a light source, a light receiving means, and a specimen reaction means as a moving means for moving a minute area, and moving within a set speed range. 17. The device for measuring a specific microorganism according to any one of claims 16 to 16. 18. The specific microorganism weighing device according to claim 11, wherein a reflection plate is provided as a light collecting means for collecting light emitted from the light source and / or a light collecting means for the light receiving means. 19. The specific microorganism weighing device according to claim 11, further comprising a spectroscopic unit that transmits only a specific wavelength between the light source and the minute area. 20. The specific microorganism weighing device according to claim 11, further comprising a spectroscopic unit that transmits only a specific wavelength between the minute area and the light receiving unit. 21. The specific microorganism measuring apparatus according to claim 11, wherein the light source and / or the light receiving means has an automatic focusing function for focusing on a minute area in the specimen reaction means. 22. The specific microorganism weighing apparatus according to claim 11, further comprising means for fixing the distance between the light source and the sample reaction means and for stabilizing the minute area. 23. The specific microorganism weighing device according to claim 11, further comprising means for introducing light emitted from the light source to the sample reaction means using an optical fiber. 24. The specific microorganism weighing apparatus according to claim 11, wherein a lens transmitting ultraviolet light is used as a light source condensing means. 25. The specific microorganism weighing device according to claim 11, further comprising means for indicating a measurement position of the sample reaction means. 26. The specific microorganism weighing apparatus according to claim 25, wherein magnetism is used as a means for indicating a measurement position of the sample reaction means. 27. The specific microorganism weighing device according to claim 11, further comprising means for recognizing a measurement position of the sample reaction means. 28. The method according to claim 27, further comprising, when moving a trajectory for recognizing the measurement position of the sample reaction means, moving to a next recognition trajectory position while maintaining a certain distance from the recognition trajectory position. Microorganism measuring device. 29. The specific microorganism measuring apparatus according to claim 28, wherein the distance from the position of the recognition orbit to the position of the next recognition orbit is 10 to 50 μm. 30. The apparatus according to claim 11, further comprising a function of recognizing a signal obtained from a microorganism contained in the sample reaction means as binarized point coordinates and measuring the number of point coordinates at which the signal is obtained.
30. The specific microorganism weighing device according to any one of claims to 29. 31. The specific microorganism measuring apparatus according to claim 30, having a function of recognizing a nearby signal as one signal among the point coordinates at which the signal is obtained.