JP2007033353A - Microbe detection system - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a microorganism detection system for detecting water-borne infectious microorganisms such as protozoa, bacteria and viruses present in water such as environmental water such as rivers and lakes and treated water of each treatment process of water and sewage.
SOLUTION: A separation / concentration unit 102 for separating and concentrating a detection target microorganism in sample water to obtain a concentrated sample, and a desalting unit for reducing the salt concentration of the concentrated sample and / or a defoaming unit for removing bubbles. Measure the fluorescence intensity of each fluorescence wavelength of the sample purification unit 104, the antibody reaction unit 106 that reacts and binds the detection target microorganism and a plurality of labeled antibodies, and the plurality of fluorescent label antibodies having different fluorescence wavelengths. The measurement unit 110 including a fluorescent scattered light measuring instrument and the sample storage unit 116 having storage means for storing the sample collected based on the measurement data obtained by the measurement unit are provided.
[Selection] Figure 1

Description

  The present invention relates to a microorganism detection system that detects water-borne infectious microorganisms such as protozoa, bacteria, and viruses present in water such as rivers and lakes and the like, and treated water of each treatment process of water and sewage.

  Since there are a wide variety of chemical substances in the environment, it is thought that environmental waters such as rivers and lakes that are raw water supply are also contaminated with various chemical substances. However, in addition to such water quality problems in the water environment, the occurrence of water-borne infectious diseases caused by protozoa such as Cryptosporidium, bacteria such as enterohemorrhagic Escherichia coli O157 and Legionella, and viruses has become a major social problem.

  In order to prevent outbreaks of these water-borne infections, it is essential to frequently monitor the causative microorganisms in the water treatment process. However, a technology for efficiently separating and concentrating microorganisms from a sample has not been established, and in order to separate and concentrate pathogenic microorganisms that are only slightly contained in the environment, there is a problem that high skill and time are required. is there.

  Cryptosporidium, Giardia, and the like are known as the causes of emerging water-borne infections. As an inspection method for these protozoa, for example, in the “Provisional Guidelines for Cryptosporidium” provided by the Ministry of Health, Labor and Welfare (amendment of the guidelines in 1998), the inspection procedures for Cryptosporidium are broadly divided into sampling, filtration concentration, separation. It consists of the steps of suspension, separation and purification, immunofluorescence staining, and microscopic observation.

In the sample collection step, 10 L for raw water such as river water and 40 L for purified water / tap water are collected in a polyethylene container having a capacity of 10 to 20 L.
In the filtration and concentration step, a hydrophilic polytetrafluoroethylene (PTFE) disc filter having a diameter of 47 mm or 90 mm and a pore size of 5 μm is used, and the sample is filtered in a pressure or suction type filter holder.

  In the exfoliation suspension step, the disc filter capturing the turbidity is put in a 50 mL centrifuge tube, and an inducer solution consisting of 0.02% sodium pyrophosphate, 0.03% EDTA-3Na, 0.01% Tween 80 is added, The turbidity containing Cryptosporidium is peeled off from the disk filter by vigorous stirring.

In the separation and purification step, Cryptosporidium is recovered from the suspension by immunomagnetic beads to which an antibody recognizing Cryptosporidium is bound.
In immunofluorescence staining and microscopic observation, Cryptosporidium is stained with a fluorescently labeled anti-cryptospodium antibody and observed with a fluorescence microscope.

  In the above-mentioned Cryptosporidium test method, the presence of Cryptosporidium can be confirmed, but it is determined whether Cryptosporidium is alive or dead, infectious to humans or not I can't do it. Therefore, in order to obtain more detailed test results, a growth activity test and an infectivity test may be performed in addition to the above-described cryptosporidium test.

  The growth activity test of Cryptosporidium includes a decapsulation test for determining the life and death from the presence or absence of sporozoites (life forms) from Cryptosporidium oocysts (sac), DAPI (4,6-diamino-2-phenylindole) and PI There is a vital staining test for determining the viability from double staining of (propidium indole).

Cryptosporidium infectivity tests include methods for evaluating infectivity to laboratory animals such as mice and methods for evaluating infectivity to cultured cells derived from human small intestine.
Notification of the Ministry of Health, Labor and Welfare “Provisional Guidelines for Cryptosporidium” (Revision of Guidelines in 1998)

  In the water purification process, in order to appropriately remove or disinfect and disinfect water-borne infectious microorganisms present in the environment and supply safe tap water, the target microorganisms are measured at a high frequency and the measurement results are processed. Need feedback.

  Protozoa such as Cryptosporidium, bacteria such as enterohemorrhagic Escherichia coli O157 and Legionella bacteria, virus inspection methods, particularly Cryptosporidium inspection methods, as described above, depending on the turbidity of the sample when capturing and recovering Cryptosporidium The disc filter may become clogged.

  For this reason, since the filtration rate decreases in a short time, it takes a long time for all the test steps, so that quick measurement cannot be performed and a large amount of samples cannot be processed efficiently.

In addition, since observation with a fluorescence microscope requires a high level of skill in distinguishing from other microorganisms, high-frequency measurement is desired once per hour or once a day.
Furthermore, the recovery rate of Cryptosporidium in the above-mentioned filtration concentration and exfoliation suspension is as low as several tens of percent, at most around 80%, the variation is large, and the detection error may be judged as negative. There are many points.

  Thus, in the inspection of microorganisms, the inspection accuracy is low and the operation is complicated, so that a skillful technique is required, and the burden on the worker is large. Therefore, automation of work steps and labor saving are desired.

  In the present invention, in view of the above-mentioned problems, in detecting water-borne infectious microorganisms such as Cryptosporidium from sample water, the detection target microorganisms are separated and concentrated with high efficiency from a large amount of sample water in a short time, and the detection sensitivity is increased. It is an object of the present invention to provide a microorganism detection system that can automate and save labor.

In order to achieve the above object, the present invention is a microorganism detection system for automatically detecting a detection target microorganism, wherein the detection target microorganism in a sample water is separated and concentrated to obtain a concentrated sample. And a desalting means for lowering the salt concentration of the concentrated sample and / or a defoaming means for removing bubbles, and adjusting the sample state suitable for the binding reaction between the detection target microorganism and the labeled antibody for labeling the detection target microorganism. Supply multiple types of labeled antibodies that bind specifically to the sample purification unit and the detection target microorganism and bind fluorescent substances with different fluorescence wavelengths, and react and bind the detection target microorganism to the multiple labeled antibodies. An antibody reaction unit, a measurement unit including a fluorescence scattered light measuring instrument for measuring the fluorescence intensity of each fluorescence wavelength of a detection target microorganism to which a plurality of fluorescently labeled antibodies having different fluorescence wavelengths are coupled, and a measurement data in the measurement unit. Based on data, characterized in that it comprises a sample storage unit having a storage means for storing the fractionated sample.

  The present invention is also a microorganism detection system for automatically detecting a detection target microorganism, comprising heating means for raising the temperature of the sample water to a constant temperature, and detecting the detection target microorganism in the sample water. After adsorbing to the acid-soluble carrier, a flocculant is further added and precipitated to form a concentrated sample, and a separation and concentration unit for separating and concentrating the detection target microorganism in the sample water, and the detection target microorganism A sample purifying unit for purifying a microorganism to be detected from the concentrated sample concentrated by adsorption aggregation, comprising: a desalting means for dissolving the adsorption carrier and lowering a salt concentration of the concentrated sample and / or a defoaming means for removing bubbles. A sample purification unit that adjusts to a sample state suitable for the binding reaction between the detection target microorganism and the labeled antibody that labels the detection target microorganism; and a specific purification unit that binds to the detection target microorganism and has a different fluorescence wavelength. A reaction target microorganism that supplies a plurality of types of labeled antibodies to which a fluorescent substance is bound, reacts and binds the detection target microorganism and the plurality of label antibodies, and a detection target microorganism in which a plurality of fluorescent label antibodies having different fluorescence wavelengths are combined A measurement unit including a fluorescence scattered light measuring instrument for measuring the fluorescence intensity of each fluorescence wavelength, and a sample storage unit having a storage unit for storing a sample collected based on the measurement data in the measurement unit. It is characterized by that.

  The present invention is an embodiment of the above-described microorganism detection system. In order to increase the detection accuracy of the detection target microorganism, the measurement unit circulates the sample in the measurement unit, repeatedly measures the detection target microorganism, and integrates the measurement data. Means are provided.

  In addition, in the above-described microorganism detection system according to the present invention, the sample storage unit again sends the sample collected based on the detection result of the detection target microorganism to the measurement unit, and the measurement data A means for confirming reproducibility is provided.

  Furthermore, according to the present invention, in the above-described embodiment, when the detection target microorganism is detected in the above-described microorganism detection system, the sample is determined to be stored, and the sample discharged from the measurement unit is received in the storage container. Means for storing and testing for determining whether the detected microorganism to be detected is alive and / or determining whether the detected microorganism to be detected is infectious.

  The microorganism detection system of the present invention separates and concentrates microorganisms to be detected from a large amount of sample water in a short time from a large amount of sample water, such as bacteria and viruses such as Cryptosporidium, such as Cryptosporidium. It is possible to react with a fluorescently labeled antibody that specifically binds to the microorganism to be detected in the concentrated sample and measure the fluorescence intensity to increase the detection sensitivity and to automate and save labor so that the water quality is safe. Can be increased.

Embodiments of a microorganism detection system according to the present invention will be described below with reference to the accompanying drawings.
First, FIG. 1 shows a general embodiment of a microorganism detection system according to the present invention.

In the present embodiment, the sample water 100 is supplied to the separation and concentration unit 102 to concentrate the detection target microorganism.
As a method for separating and concentrating the microorganism to be detected, a concentration method by filtration using a hollow fiber membrane, a membrane filter or the like, a concentration method using immunomagnetic beads to which an antibody is bound, and the like can be employed.

The separation and concentration unit 102 of the microorganism detection system according to the present invention preferably employs a method of adsorbing, aggregating, and precipitating the detection target microorganism in the sample water on an acid-soluble carrier.
Processing time can be shortened compared to conventional membrane filter filtration and immunomagnetic bead concentration methods. In addition, it is because no special skill is required, the load on the tester can be reduced and labor can be saved, and the fluctuation of the collection rate for each sample is considered to be small.
As the acid-soluble carrier, fine particles of an acid-soluble inorganic substance are generally used. Such inorganic substances include carbonates, and examples include calcium carbonate, magnesium carbonate, strontium carbonate, and barium carbonate. Calcium carbonate is most preferred because barium carbonate is toxic and magnesium carbonate and strontium carbonate are more expensive than magnesium salts to produce them.

The separation and concentration unit 102 is preferably provided with a heating means for raising the water temperature of the sample water and keeping it at a constant temperature.
The target sample water includes environmental water such as rivers and lakes. These environmental waters also vary in temperature due to climate change. For this reason, such a temperature change becomes a variation factor such as a production reaction of the acid-soluble carrier, an adsorption reaction between the detection target microorganism and the acid-soluble carrier, and a sedimentation rate of the precipitate. This may affect the recovery rate of the detection target microorganism. In particular, when the water temperature is low as in winter, there is a risk that the production reaction rate of the acid-soluble carrier decreases and the recovery rate of the detection target microorganism decreases. For this reason, it is preferable to provide the heating means described above.
As such a heating means, a heater is most commonly used.

In the separation and concentration unit 102, the microorganisms to be detected in the sample water are adsorbed on the acid-soluble carrier, and further, a flocculant is added and collected as a precipitate.
Examples of the flocculant include inorganic flocculants such as polyaluminum chloride, aluminum sulfate, iron chloride, and polyiron sulfate, and organic flocculants such as alginic acid and polyacrylic acid.

The concentrated sample (the precipitate) separated and concentrated by the separation and concentration unit 102 is sent to the sample purification unit 104.
In the case where the microorganism to be detected is adsorbed onto a carrier of fine particles of inorganic substance and separated and concentrated, a step of dissolving only the carrier is required. In this case, the sample purification unit 104 dissolves the concentrated sample (the precipitate) adsorbed by the detection target microorganism with an acid, and dissolves only the carrier.
Here, examples of the acid used include sulfamic acid and hydrochloric acid.

The sample solution obtained by dissolving the precipitate (concentrated sample) with an acid contains high-concentration salts and bubbles. These hinder the binding between the detection target microorganism and the labeled antibody that labels the detection target microorganism to be performed later. That is, there is a possibility that the detection target microorganism and the fluorescently labeled antibody are bound to each other, and the fluorescence measurement by the measuring instrument is hindered. For this reason, it is desirable to remove salts and bubbles contained in the sample solution.
Therefore, the sample purification unit 104 is configured to perform desalting to lower the salt concentration of the concentrated sample and / or to remove bubbles.

  Even when the sample water is filtered through a hollow fiber membrane or a membrane filter, the sample solution contains high-concentration salts and bubbles. For this reason, desalting and / or defoaming steps are required as well.

  Examples of the desalting and / or defoaming method include a contaminant removal filter capable of passing a detection target microorganism and capturing a contaminant larger than the detection target microorganism, a detection target microorganism, and a high-concentration salt. It is possible to employ a method of removing a solution containing a high concentration of salts in two steps using a capture filter that permeates the solution containing the solution. However, the method of desalting and / or defoaming is not limited to this, and it can also be carried out by a method using ultrafiltration or dialysis using a dialysis membrane.

  Note that the sample purification unit 104 adjusts the concentrated sample to a sample state suitable for the binding reaction between the detection target microorganism and the labeled antibody that labels the detection target microorganism, and thus also functions as a sample adjustment unit.

The sample purified (adjusted) by the sample purification unit 104 is sent to the antibody reaction unit 106.
In the antibody reaction unit 106, the fluorescence-labeled antibody 108 specifically bound to the detection target microorganism and bound with the fluorescent substance is bound to the detection target microorganism in the concentrated sample by an antigen-antibody reaction. At this time, when one kind of fluorescently labeled antibody is used, there is a possibility that a fluorescently labeled antibody that is non-specifically bound to a contaminant is determined as a false positive. For this reason, in the microorganism detection system according to the present invention, a plurality of antibodies having different antigen recognition sites are prepared, and the detection target microorganism is multi-labeled (stained) with a fluorescently labeled antibody 108 in which fluorescent substances having different fluorescence wavelengths are respectively bound. To do. Thereby, the detection accuracy of the detection target microorganism is improved.
The antibody itself can be obtained by a technique known to those skilled in the art. An antibody can be used regardless of its kind, such as a polyclonal antibody or a monoclonal antibody, as long as it has specificity for a specific microorganism for counting. Monoclonal antibodies are generally preferable because of their excellent specificity.
Moreover, as a pigment | dye used for the fluorescent label of an antibody, the well-known thing which shows fluorescence can be used.

Next, the concentrated sample labeled with the fluorescently labeled antibody 108 is sent to the measurement unit 110.
The measurement unit 110 includes a fluorescent scattered light measuring instrument for measuring the fluorescent scattered light intensity. In the measurement unit 110, the fluorescence scattered light intensity of the microorganism to be detected to which the fluorescent labeled antibody is bound is measured by the fluorescence scattered light measuring instrument. The measurement data is sent to the determination unit 112.

  The measuring unit 110 performs multiple labeling using a plurality of fluorescently labeled antibodies. Therefore, it is preferable that the measurement unit 110 is configured so that a plurality of fluorescence wavelengths can be measured simultaneously.

  In addition, since there are usually very few microorganisms to be detected in a sample, there is a possibility that they cannot be detected by a single measurement. Therefore, the target microorganism can be detected with higher accuracy by circulating the sample through the measurement unit and integrating the measurement data (114 in FIG. 1).

The microorganism detection system according to the embodiment of FIG. 1 includes a sample storage unit 116.
In the sample storage unit 116, the determination unit 112 determines whether to store the measured sample based on the measurement data detected by the measurement unit 110. When confirming the reproducibility of the collected sample, the stored sample is sent again to the measurement unit 110 to detect the detection target microorganism (118 in the figure).
On the other hand, when the detection target microorganism is detected, the sample discharged from the measuring unit 110 is received in a storage container and stored (120 in the figure). The stored sample can be provided to a test for determining whether or not a contaminating microorganism to be detected is alive and / or a test for determining whether it is infectious. The unnecessary sample is discarded as drainage (122 in the figure).

  Next, FIG. 2 shows Embodiment 1 (hereinafter referred to as system form 1) in which the microorganism detection system of the present invention is employed and Cryptosporidium is detected in particular.

System form 1
The separation / concentration part of the system form 1 is constructed based on a method for concentrating Cryptosporidium with calcium carbonate (G. Vesey et al. (1993) Journal of Applied Bacteriology, 75, pages 82-86).

  In this concentration method, when a calcium chloride solution and a sodium hydrogen carbonate solution are added to sample water and the pH is raised to 10 with a sodium hydroxide solution, insoluble calcium carbonate is generated. Using the calcium carbonate fine particles as a carrier, Cryptosporidium which is a microorganism to be detected in the sample water is adsorbed on the carrier, and the precipitate is collected.

In this concentration method, sulfamic acid is further added to the calcium carbonate precipitate to dissolve only calcium carbonate, and Cryptosporidium is isolated.
In this concentration method, a flocculant is added, thereby increasing the precipitation of calcium carbonate, increasing the sedimentation rate of the precipitate, and separating the supernatant and the precipitate in a short time. Examples of the flocculant include inorganic flocculants such as polyaluminum chloride, aluminum sulfate, iron chloride, and polyiron sulfate, and organic flocculants such as alginic acid and polyacrylic acid.

Hereinafter, the system configuration 1 will be further described with reference to FIG. 2 while clarifying the configuration and function of each component.
In this system form 1, the sample water 1 is supplied to the concentration tank 13 by the sample water supply pump 2. A heater 39 is installed in the concentration tank. When the water temperature of the sample is low in winter, the concentration tank 13 is heated and kept at a constant temperature. While stirring the heated sample water with the stirrer 14, the calcium chloride solution 3 is subsequently supplied to the concentration tank 13 with the calcium chloride supply pump 4. Then, the sodium hydrogen carbonate solution 5 is supplied to the concentration tank 13 by the sodium hydrogen carbonate supply pump 6.

  Further, the sodium hydroxide solution 7 is supplied to the concentration tank 13 by the sodium hydroxide supply pump 8. This raises the pH to 10 and produces calcium carbonate.

  At this time, in order to enhance the effect of adsorbing fine particles of Cryptosporidium and calcium carbonate and the effect of precipitation, a coagulant 9 such as PAC (polyaluminum chloride) or sulfuric acid band is injected to form flocs and separate the precipitate more easily. You can also.

In the first system configuration, the flocculant 9 is supplied to the concentration tank 13 by the flocculant supply pump 10 to form a floc.
Thereafter, in the first system configuration, the stirrer 14 is stopped, the precipitate is naturally settled and separated from the supernatant. However, the present invention is not limited to such a configuration, and a configuration in which the precipitate adsorbed by Cryptosporidium is continuously collected using a continuous centrifuge may be employed.

In the first system configuration, the sediment deposited on the bottom of the concentration tank 13 is supplied to the sample purification unit 16 by opening the valve 15. The inside of the concentration tank 13 can be cleaned by supplying the cleaning water 11 with the cleaning water supply pump 12.
As shown in the figure, the precipitate on which Cryptosporidium is adsorbed is sent to a precipitation dissolution tank 19, where only the calcium carbonate is dissolved from the precipitate of calcium carbonate on which Cryptosporidium is adsorbed, and Cryptosporidium is concentrated. Adjust.

That is, the sulfamic acid 17 is injected into the precipitation dissolution tank 19 by the sulfamic acid supply pump 18 to dissolve calcium carbonate. At this time, hydrochloric acid or the like can be used in addition to sulfamic acid as an acid for dissolving calcium carbonate.
Since the sample solution contains salts such as calcium ions and the pH is lowered by sulfamic acid, it interferes with the binding between Cryptosporidium and the fluorescently labeled antibody that labels Cryptosporidium. Therefore, the sample solution is supplied to the desalting / defoaming tank 21 via the valve 20 before being supplied to the subsequent antibody reaction section.

  As a structure of the desalting defoaming tank 21, what was provided with the contaminant removal filter and the capture filter can be illustrated.

As the contaminant removal filter, a filter having a pore size of about 10 μm or more that can pass Cryptosporidium (diameter 5 μm) and can capture contaminants larger than Cryptosporidium is suitable.
As the capture filter, a filter having a pore size of about 1 μm or less capable of capturing Cryptosporidium and transmitting a solution containing a high concentration of salts is preferable.
A method of removing a solution containing a high concentration of salts in two stages using these filters is suitable.

However, the configuration for carrying out the desalting and defoaming is not limited to this, and other suitable examples include filtration by ultrafiltration and dialysis using a dialysis membrane.
In this system form 1, Cryptosporidium trapped in the desalting / defoaming tank 21 is washed away by supplying the washing water 22 with the pump 23, and then supplied to the antibody reaction unit 25 via the valve 24.

The antibody reaction part 25 of the system form 1 is composed of an antibody mixing tank 26 for mixing a sample containing Cryptosporidium and a Cryptosporidium-labeled fluorescent antibody, and an antibody reaction tank 27 for performing an antigen-antibody reaction.
In the antibody mixing tank 26, a fluorescently labeled antibody 28 that specifically recognizes Cryptosporidium is injected by a fluorescently labeled antibody supply pump 29 via a valve 30 and mixed with the sample.

  At this time, when one type of fluorescently labeled antibody is used, there is a possibility that a fluorescently labeled antibody that is not specifically bound to a contaminant may be determined as a false positive. Therefore, a plurality of antibodies with different antigen recognition sites are prepared, and each of them is fluorescent. In order to improve the detection accuracy of Cryptosporidium, it is preferable to multiplexly label Cryptosporidium with fluorescently labeled antibodies bound with fluorescent substances having different wavelengths.

  The antigen-antibody reaction with many commercially available antibodies is 20 to 30 minutes at 37 ° C. and 1 hour or more at room temperature, and in order to measure more frequently, after mixing the sample and the labeled antibody, It is preferable that the mixed liquid be transferred to another container so that the next sample can be supplied.

Further, in order to measure Cryptosporidium at a high frequency, it is preferable to prepare a plurality of antibody reaction tanks 27 as shown in the figure, and supply a different antibody reaction tank for each sample by switching the valve 34.
In the first system configuration, after the first sample and the labeled antibody are mixed, the cleaning water 31 is supplied by the pump 32 via the valve 33, washed away as mixed water, and supplied to the antibody reaction tank 27.

  Next, the valve 36 is opened, and after washing the antibody reaction tank with washing water, the second sample is supplied to the antibody reaction tank, and the labeled antibody is supplied in the same manner as the first sample. Similarly, the mixed solution of the second sample and the labeled antibody is washed away with washing water and sent to the antibody reaction tank 27. At this time, by switching the valve 34, the second sample mixture is supplied to the first mixture and another antibody reaction tank.

  In order to advance the antigen-antibody reaction efficiently, the antibody reaction part 25 and / or the antibody reaction tank 27 is preferably equipped with a thermostat that keeps the sample temperature at 37 ° C. The sample that has completed the antigen-antibody reaction is supplied to the measurement unit 37 by the pump 35.

In the measurement unit 37, Cryptosporidium having a fluorescently labeled antibody bound thereto is sent to the measurement device 38 via the valve 41. Then, the fluorescence intensity of Cryptosporidium is measured with the measuring instrument 38. The measuring instrument 38 is composed of a fluorescent scattered light measuring instrument. The measuring device 38 measures the fluorescence scattered light intensity of Cryptosporidium bound with the fluorescently labeled antibody.
Next, the measurement data is sent to the determination unit 40. When multiple labeling (staining) using a plurality of fluorescently labeled antibodies is performed, it is preferable that the measuring unit be capable of simultaneously measuring a plurality of fluorescent wavelengths.
The determination unit 40 determines whether or not sample storage is necessary based on the measurement data detected by the measuring instrument 38. Furthermore, the mixing level of Cryptosporidium contained in the environmental water can be grasped based on the measurement data, and feedback control can be performed for the water purification process using this environmental water as raw water. Control of the Cryptosporidium mixing level at this time includes stopping water intake, switching the water intake, and washing the reservoir, and in the coagulation process, changes in the amount of coagulant added, addition of coagulant aid, and change in stirring strength can be mentioned. Water purification treatment for Cryptosporidium can be appropriately operated.

After the measurement by the measuring instrument 38 is completed, the sample is sent to the sample storage unit 43 by the pump 42.
When Cryptosporidium is detected in the sample storage unit 43, the sample discharged from the measurement unit 37 is separated by the separator 45, received in the storage container 44, and stored. The stored sample can be submitted to the test to determine if contaminating Cryptosporidium is alive and / or whether it is infectious. Samples that do not need to be stored are drained.
The sample stored in the storage container 44 can be provided to a test for determining whether the contaminated Cryptosporidium is alive and / or infectious. At this time, it is necessary to lower the activity of the mixed Cryptosporidium and suppress the propagation of other germs. For this purpose, it is preferable to store the sample in a refrigerated state. It is preferable to take out and store in a refrigerator at 4 ° C or the like.

System form 2
Next, FIG. 3 shows still another embodiment (hereinafter referred to as system embodiment 2) of the embodiment in which the microorganism detection system of the present invention is employed and particularly cryptosporidium is detected.

In the second system configuration, detection accuracy of fluorescently labeled cryptosporidium is further improved.
For this reason, after the sample is sent to the measuring instrument 38 by the pump 35 and measured, the sample circulation channel switching valve 46 is closed with respect to the sample storage unit 43 and the sample is temporarily stored in the reservoir 48.
Next, the valve 41 is switched, and the sample water is circulated to the measuring instrument 38 by using the pump 42 with the sample circulation channel 47 as a bypass, thereby repeatedly performing fluorescence measurement. As a result, the measurement data is integrated to improve the detection probability of the fluorescent fine particles having a low number concentration.
The system configuration No. 2 can be the same as the system configuration No. 1 in FIG. 2, and the components having the same reference numerals as those in FIG. 2 are the same as those in FIG. Has action and effect.

System form 3
Next, FIG. 4 shows still another embodiment (hereinafter, system embodiment 3) of the embodiment that employs the microorganism detection system of the present invention and detects Cryptosporidium in particular.

This system form 3 is provided with another form for determining whether to store the measured sample based on the measurement data detected by the measuring instrument 38.
In the third system configuration, the valve 45 operates under the control of the determination unit 40 based on the measurement data, and the samples are sequentially stored in the storage container 44 provided in the autosampler 49. The autosampler 49 sequentially samples samples into a plurality of storage containers 44 while rotating in the direction of arrow A in the figure.

  When confirming the reproducibility of the collected sample, the target sample is fed by the storage sample supply pump 50 to the reservoir 48 of the measuring unit 37 by the pump 50 and circulated to the measuring instrument 38 as described above.

  On the other hand, when Cryptosporidium is detected, the sample discharged from the measuring unit 37 is received in the storage container 44 and stored. The stored sample can be provided to a test for determining whether a contaminating microorganism to be detected is alive and / or infectious. At this time, the sample storage unit 43 is preferably stored refrigerated at 4 ° C. or the like in order to prevent germs and the like from growing in the sample.

  The system configuration No. 3 can have the same configuration as the system configuration No. 2 in FIG. 3, and the components having the same reference numerals in FIG. 4 are the same as those in FIG. Has action and effect.

System form 4
Next, FIG. 5 shows still another embodiment (hereinafter referred to as system embodiment 4) of the embodiment that employs the microorganism detection system of the present invention and detects Cryptosporidium in particular.

In this system configuration No. 4, a hollow fiber membrane is used for the separation and concentration section.
In this system form 4, sample water 1 is supplied to the external pressure type hollow fiber membrane 51 that captures Cryptosporidium via the valve 60 by the sample water supply pump 2, and a predetermined amount of the sample water 1 is filtered and filtrated. Is discharged.
Cryptosporidium and other particles are trapped on the hollow fiber membrane 51.

  Next, the surfactant 53 is supplied to the hollow fiber membrane container 52 via the valve 61 by the surfactant supply pump 54. The surfactant 53 functions as a turbid dispersant that facilitates peeling of Cryptosporidium trapped on the hollow fiber membrane 51. As the surfactant 53, for example, 1% PET (0.02% sodium pyrophosphate, 0.03% EDTA-3Na, 0.01% Tween 80) or the like can be used.

  Next, the backwash air 57 is supplied via the valve 65 by the air pump 58 to pressurize the hollow fiber membrane 51. At the same time, the ultrasonic wave generated by the ultrasonic generator 63 is irradiated from the ultrasonic transducer 59 to the surface of the hollow fiber membrane 51. At this time, Cryptosporidium on the hollow fiber membrane 51 is peeled from the hollow fiber membrane 51 by the pressure of backwashing the hollow fiber membrane 51 and the physical action of ultrasonic waves. The concentrated sample solution containing Cryptosporidium remaining in the hollow fiber membrane container 52 is supplied to the sample purification unit 16 via the valve 15.

  On the other hand, the cleaning water 55 is supplied to the hollow fiber membrane container 52 via the valve 62 by the cleaning water supply pump 56, and the cleaning liquid rinsed inside the hollow fiber membrane 51 and the container 52 is also sent to the sample purification unit 16.

  As described above, when a hollow fiber membrane or a membrane filter is used for the separation and concentration unit, an operation for dissolving the inorganic substance is not required. Therefore, the configuration of the sample purification unit 16 may be only the desalting and defoaming tank 21. .

  The system configuration No. 4 can be the same as the system configuration Nos. 1 and 2 in FIGS. 2 and 3, and the components having the same reference numerals as those in FIGS. Has the same actions and effects as in FIGS. Note that the system form 4 is different from the system form 2 in that the measuring unit 37 does not include the reservoir 48.

It is a conceptual diagram which shows the outline | summary of the microorganisms detection system concerning this invention. BRIEF DESCRIPTION OF THE DRAWINGS It is a conceptual diagram explaining Embodiment 1 about the microorganisms detection system which concerns on this invention. It is a conceptual diagram explaining Embodiment 2 about the microorganisms detection system which concerns on this invention. It is a conceptual diagram explaining Embodiment 3 about the microorganisms detection system which concerns on this invention. It is a conceptual diagram explaining Embodiment 4 about the microorganisms detection system which concerns on this invention.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Sample water 2 Sample water supply pump 3 Calcium chloride solution 4 Calcium chloride supply pump 5 Sodium hydrogen carbonate solution 6 Sodium hydrogen carbonate supply pump 7 Sodium hydroxide solution 8 Sodium hydroxide supply pump 9 Flocculant 10 Flocculant supply pump 11 Washing water DESCRIPTION OF SYMBOLS 12 Washing water supply pump 13 Concentration tank 14 Stirrer 15 Valve 16 Sample refinement | purification part 17 Sulfamic acid solution 18 Sulfamic acid supply pump 19 Precipitation dissolution tank 21 Desalination defoaming tank 22 Washing water 23 Washing water supply pump 24 Valve 25 Antibody reaction part 26 Antibody Mixing Tank 27 Antibody Reaction Tank 28 Fluorescent Labeled Antibody 29 Fluorescent Labeled Antibody Supply Pump 31 Wash Water 32 Wash Water Supply Pump 37 Measuring Unit 38 Measuring Instrument 39 Heater 40 Judging Unit 41 Valve 42 Pump 43 Sample Storage Unit 44 Sample Storage Container 45 Flow Road Replacement valve 46 Sample circulation channel switching valve 47 Sample circulation channel 48 Reservoir 49 Autosampler 50 Storage sample supply pump 51 Hollow fiber membrane 52 Hollow fiber membrane container 53 Surfactant 54 Surfactant supply pump 55 Washing water 56 Washing water supply Pump 57 Backwash air 58 Air pump 59 Ultrasonic vibrator 63 Ultrasonic generator

Claims (5)

A microorganism detection system for automatically detecting microorganisms to be detected,
A separation and concentration unit for separating and concentrating the target microorganisms in the sample water to obtain a concentrated sample;
A sample purification unit comprising a desalting means for reducing the salt concentration of the concentrated sample and / or a defoaming means for removing bubbles, and adjusting the sample state suitable for the binding reaction between the detection target microorganism and the labeled antibody that labels the detection target microorganism When,
An antibody reaction unit that specifically binds to a detection target microorganism and supplies a plurality of types of labeled antibodies that are combined with fluorescent substances having different fluorescence wavelengths, and reacts and binds the detection target microorganism and the plurality of labeled antibodies;
A measurement unit comprising a fluorescence scattered light measuring instrument for measuring the fluorescence intensity of each fluorescence wavelength of a detection target microorganism to which a plurality of fluorescently labeled antibodies having different fluorescence wavelengths are bound;
A microorganism detection system comprising: a sample storage unit having storage means for storing a sample collected based on measurement data obtained by the measurement unit. A microorganism detection system for automatically detecting microorganisms to be detected,
A heating means is provided to raise the temperature of the sample water to a constant temperature. After the microorganisms to be detected in the sample water are adsorbed to the acid-soluble carrier, a flocculant is added and precipitated to form a concentrated sample. And a separation and concentration unit for separating and concentrating the detection target microorganism in the sample water,
A sample purification unit for purifying a detection target microorganism from a concentrated sample concentrated with the detection target microorganism adsorbed and agglomerated, comprising a desalting means and / or a foam for dissolving the adsorption carrier and reducing the salt concentration of the concentrated sample. A sample purifying unit that includes a defoaming means for removing, and adjusts the sample state suitable for the binding reaction between the detection target microorganism and the labeled antibody that labels the detection target microorganism;
An antibody reaction unit that specifically binds to a detection target microorganism and supplies a plurality of types of labeled antibodies that are combined with fluorescent substances having different fluorescence wavelengths, and reacts and binds the detection target microorganism and the plurality of labeled antibodies;
A measurement unit comprising a fluorescence scattered light measuring instrument for measuring the fluorescence intensity of each fluorescence wavelength of a detection target microorganism to which a plurality of fluorescently labeled antibodies having different fluorescence wavelengths are bound;
A microorganism detection system comprising: a sample storage unit having storage means for storing a sample collected based on measurement data obtained by the measurement unit. The microorganism detection system according to claim 1 or 2, further comprising means for circulating the sample in the measurement unit, repeatedly measuring the detection target microorganism, and integrating the measurement data in order to increase the detection accuracy of the detection target microorganism. A featured microorganism detection system. 3. The microorganism detection system according to claim 1 or 2, further comprising means for re-feeding the sample collected by the sample storage unit based on the detection result of the detection target microorganism to the measurement unit and confirming reproducibility of the measurement data. A microorganism detection system characterized by that. In the microorganism detection system according to any one of claims 1 to 4, when a detection target microorganism is detected, determination of storage of the sample is performed, and the sample discharged from the measurement unit is received in a storage container, stored, and detected. A microorganism detection system comprising means for performing a test for determining whether a detection target microorganism is alive and / or determining whether the detected detection target microorganism is infectious.

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