JP2004101361A - Method and method for detecting cryptosporidium - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a highly accurate Cryptosporidium detecting method capable of highly efficiently processing large quantities and to provide an apparatus for implementing the method. <P>SOLUTION: In the detection method, microorganisms in sample water are isolated through the use of a hollow fiber membrane (11), condensed, further isolated through the use of a membrane filter (14), and condensed. An oocyst outer shell of Cryptosporidium among acquired microorganisms is damaged. By performing both fluorescent emission in-situ hybridization through the use of a probe complementary to nucleic acids contained in the Cryptosporidium and immuno-staining through the use of an antibody to matter related to the Cryptosporidium to perform double-staining, a differential interference image and a fluorescent emission image of the double-stained microorganisms are acquired and analyzed to recognize the Cryptosporidium. <P>COPYRIGHT: (C)2004,JPO

Description

[0001]
TECHNICAL FIELD OF THE INVENTION
The present invention relates to a method for detecting Cryptosporidium for detecting Cryptosporidium in a sample water and an apparatus for performing the method.
[0002]
[Prior art]
At present, the detection of Cryptosporidium, a kind of chlorine-resistant pathogenic microorganism, is carried out by a test method formulated by the Ministry of Health, Labor and Welfare (former Ministry of Health and Welfare). This was a method of separating and concentrating microorganisms from 10 to 50 L of sample water using a membrane filter, purifying the microorganisms by immunomagnetic separation or density gradient centrifugation, staining with a fluorescently labeled antibody, and observing with a microscope. .
[0003]
[Problems to be solved by the invention]
However, the above test method is very time-consuming, takes a long time to process each step, and performs all the steps manually, so the detection accuracy depends on the skill of the person performing the test. Individual differences were large with respect to such factors. Also, in the microscopy work, the skill is greatly affected and the accuracy is low.
[0004]
The present invention has been made in order to solve the above-mentioned problems of the prior art, and aims to provide a method for detecting Cryptosporidium with high accuracy while enabling high-efficiency and large-scale processing, and an apparatus for performing the method. And
[0005]
[Means for Solving the Problems]
The invention according to claim 1 is
In a method for detecting Cryptosporidium for detecting Cryptosporidium in a sample water,
Separating and concentrating microorganisms in the sample water using a hollow fiber membrane, further separating and concentrating using a membrane filter,
Damaging the oocyst shell of Cryptosporidium among microorganisms separated and concentrated by the membrane filter,
Performing fluorescence in-situ hybridization using a probe complementary to the nucleic acid contained in Cryptosporidium, and performing immunostaining using an antibody against a substance related to Cryptosporidium to perform double staining,
Obtaining a differential interference image and a fluorescence emission image of the double-stained microorganism;
Analyzing the obtained differential interference image and fluorescence emission image to recognize Cryptosporidium,
Are carried out sequentially. A method for detecting Cryptosporidium.
[0006]
The invention according to claim 2 is
In a Cryptosporidium detector for detecting Cryptosporidium in sample water,
Microorganism recovery means for separating and concentrating microorganisms in the sample water using a hollow fiber membrane, further separating and concentrating using a membrane filter,
Pretreatment means for damaging the oocyst shell of Cryptosporidium among microorganisms separated and concentrated by the microorganism collection means,
Performing fluorescence in-situ hybridization using a probe complementary to the nucleic acid contained in Cryptosporidium, and staining means for performing immunostaining using an antibody against a substance related to Cryptosporidium,
Obtaining a differential interference image and a fluorescence emission image of the microorganism stained by the staining means, analyzing these images and detecting means for recognizing Cryptosporidium,
An apparatus for detecting Cryptosporidium, comprising:
[0007]
According to a third aspect of the present invention, in the apparatus for detecting cryptosporidium according to the second aspect, the pretreatment means performs at least one of heat treatment, microwave treatment, and electric treatment, and performs formalin fixation. It is characterized by the following.
[0008]
According to a fourth aspect of the present invention, in the apparatus for detecting cryptosporidium according to the second or third aspect, the staining means performs fluorescence emission in-situ hybridization using a Cy3 fluorescently labeled probe and performs identification different from that. It is characterized by performing immunostaining using a possible labeling substance.
[0009]
According to a fifth aspect of the present invention, in the apparatus for detecting cryptosporidium according to any one of the second to fourth aspects, the detecting means detects double-stained luminescence in the sample and differentiates the fluorescence detection position. When the interference image is recognized as the target microorganism shape, a means for notifying an operator is provided.
[0010]
BEST MODE FOR CARRYING OUT THE INVENTION
Hereinafter, the present invention will be described in detail based on preferred embodiments shown in the drawings. FIG. 1 is a system diagram showing a configuration of a first embodiment of a cryptosporidium recovery unit in an apparatus for performing a cryptosporidium detection method according to the present invention. The cryptosporidium recovery unit 10A includes a hollow fiber membrane separation unit 11 having a pore size of 1 μm or less at the front stage, and a membrane filter unit 14 having a pore size of 1 μm or less at the rear stage. Among them, a sample water introduction valve V1 is provided in a sample water introduction path to the hollow fiber membrane separation unit 11, and a sample water discharge valve V2 and a suction pump 12 are provided in a drain path thereof.
[0011]
Further, a backwash pump 13 and a backwash water injection valve V3 are provided in the backwash water introduction path of the hollow fiber membrane separation unit 11, and a backwash water guide valve V4 is provided in the backwash water discharge path. ing. A membrane filter unit 14 is connected to a path on the outlet side of the backwash water induction valve V4, and a drainage valve V5 is provided in a drainage path of the membrane filter unit 14. Further, a pump 15 for injecting liquid injection and a valve V6 for injecting liquid extract are provided in a liquid injecting path of the membrane filter unit 14, and a valve V7 for discharging sample water is provided in a sample water discharging path of the membrane filter unit 14. , A sample collection unit 16 is provided.
[0012]
The operation of the Cryptosporidium recovery unit configured as described above will be described below. First, with the backwash water injection valve V3 and the backwash water induction valve V4 closed, the sample water introduction valve V1 and the sample water discharge valve V2 are opened, and the suction pump 12 is driven. Thereby, a large amount of sample water passes through the hollow fiber membrane separation unit 11 and is drained. As described above, the hollow fiber membrane separation unit 11 is used as a separation medium for the chlorine-resistant pathogenic microorganisms in the first stage, and the sample water passing through the hollow fiber membrane separation unit 11 is sucked by the suction pump 12, whereby the sample water is separated. Large-volume processing and continuous processing can be performed. For example, by setting the processing amount of the sample water to 100 L or more, the recovery amount and recovery probability of the recovery target microorganisms are improved. In this case, the upper limit of the processing amount is not particularly set, but is set according to the processing capacity (specification) of the hollow fiber membrane. In addition, the processing speed shall conform to the processing capacity (specifications) of the hollow fiber membrane.
[0013]
Next, the sample water introduction valve V1 and the sample water discharge valve V2 are closed, and the backwash water injection valve V3 and the backwash water induction valve V4 are opened. Then, the drainage valve V5 is opened while the extraction liquid injection valve V6 and the sample water discharge valve V7 provided in the connection path of the membrane filter unit 14 are closed. By driving the backwashing pump 13 in this state, backwash water is supplied to the hollow fiber membrane separation unit 11, and microorganisms including chlorine-resistant pathogenic microorganisms are guided to the membrane filter unit 14 together with the backwash water, Here, microorganisms including chlorine-resistant pathogenic microorganisms are separated from the backwash water, and the backwash water is drained through a drain valve V5. Here, the amount of backwash water of the hollow fiber membrane is much smaller than that of the sample water, and impurities of 1 μm or less are removed by the hollow fiber membrane separation unit 11. Therefore, in the separation step using the membrane filter, The load is extremely reduced as compared with the case where the sample water is directly treated, the membrane performance can be maintained for a long time, and operations such as membrane replacement are reduced. Examples of the chlorine-resistant pathogenic microorganism include Cryptosporidium, Giardia, cyclospora, microspora, and the like.
[0014]
Next, the pump 15 for injecting liquid injection is driven with the valve V4 for backwash water induction and the valve V5 for draining water closed and the valve V6 for injecting liquid injection opened to remove a predetermined amount of the extracting liquid into the membrane filter. It stays in the unit 14. Then, after a predetermined time has elapsed, the sample water discharge valve V7 is opened. By the subsequent processing, the concentrated sample water containing Cryptosporidium to be collected is collected in the sample collection unit 16. Here, the amount of the extraction liquid of the membrane filter unit 14 is very small compared with the backwash water of the hollow fiber membrane separation unit 11.
[0015]
As described above, by combining the first-stage treatment by the hollow fiber membrane separation unit 11 and the second-stage treatment by the membrane filter unit 14, it is possible to provide a cryptosporidium recovery unit capable of performing high-efficiency and mass-treatment.
[0016]
In the above embodiment, the extraction liquid is retained in the membrane filter unit 14 for a predetermined time. However, when the extraction liquid is injected, the sample water discharge valve V7 is kept open to respond to the injection operation of the extraction liquid. To collect concentrated sample water. In addition, by irradiating the membrane filter unit 14 with ultrasonic waves or applying electric vibration while the extracted liquid is kept in the membrane filter unit 14 for a predetermined time, the chlorine-resistant pathogen is removed from the membrane filter. Sex microorganisms can be collected in a shorter time. If necessary, repeat the above operation multiple times.
[0017]
FIG. 2 is a block diagram illustrating a configuration of a cryptosporidium detection unit in an apparatus that performs the cryptosporidium detection method. The Cryptosporidium detection unit 100 shown here mainly uses a pretreatment unit 101 that damages the oocyst shell of Cryptosporidium and a probe that is complementary to the nucleic acid contained in Cryptosporidium and emits fluorescence. A reaction section 102 for performing double staining by performing immunostaining using an antibody against a substance related to Cryptosporidium while performing situ hybridization, an immobilization section 103 for immobilizing a sample on a slide glass or the like, A differential interference image and a fluorescence emission image of the microorganism that has been superstained are obtained, and the detection unit 104 is configured to analyze the images and recognize Cryptosporidium.
[0018]
Here, the pre-processing unit 101 is a process for performing a process for making a sample easily detectable, and particularly relates to in-situ hybridization. The oocyst outer shell of Cryptosporidium is damaged by any physical or chemical method so that a DNA probe can be introduced into the shell.
[0019]
Next, in the reaction unit 102, two processes of an in-situ hybridization 102a and an immune antibody reaction 102b are executed. Among them, in the in-situ hybridization 102a, a DNA probe having a nucleotide sequence complementary to the nucleic acid contained in Cryptosporidium and having been subjected to fluorescent labeling is used. As the nucleic acid sequence, 18S rRNA universal contained in Cryptosporidium or DNA containing a specific sequence of Cryptosporidium is used. Regarding the fluorescent labeling substance, it does not matter what kind of fluorescent labeling substance is, but it should be distinguishable from the labeling substance on the immune antibody. After performing pretreatment on the sample, the sample is added together with the fluorescently labeled probe into the hybridization buffer, and hybridization is performed under certain conditions. For example, a condition of 48 ° C./1 hour is set. In the immune antibody reaction 102b, an antibody labeled with a fluorescent substance different from the fluorescent substance labeled on the DNA probe for hybridization is added to the specimen, and the specimen is allowed to stand for a certain time to cause an immune reaction. The type of the fluorescent labeling substance is not limited, but labeling is performed using a fluorescent substance different from the labeling substance for the DNA probe. Either the in-situ hybridization 102a or the immune antibody reaction 102b may be performed first, and may be performed simultaneously in some cases.
[0020]
Next, in the fixing section 103, the chlorine-resistant pathogenic microorganisms containing Cryptosporidium, which have been double-stained as described above, are fixed to a slide glass or a membrane filter as a fixing medium. When a slide glass is used, a solution containing a specimen is dropped and immobilized.
[0021]
Next, the detection unit 104 executes image acquisition 104a through fluorescence observation and observation with a differential interference microscope, and fluorescence image processing and shape recognition image processing 104b.
[0022]
FIG. 3 shows an example of the configuration of the apparatus when the detector 104 acquires a bright-field image and a differential interference image. This means that the excitation light source 42 whose lighting is controlled by the control means 41, a fluorescence filter system 43 including an excitation filter, a half mirror and a fluorescence filter, a differential interference filter 44, and a filter switching means 45 for switching these are imaged from the fixed plate 32. It is provided on the optical path up to the means 46. By sequentially changing or removing the filter to be applied, a fluorescence image, a bright field image, and a differential interference image can be obtained. Here, the obtained image is stored in the image recording unit 47, and the image analysis unit 48 performs image processing on the obtained image, thereby extracting the existence point of the microorganism 31.
[0023]
In this case, the excitation filter includes, for example, a B excitation filter and a G excitation filter, and first, one of the filters scans the specimen. Next, the sample is similarly scanned by the other filter, and finally, a differential interference image is obtained by the differential interference filter. As a result, the three types of images are analyzed by the image analysis means 48, and the double-stained fluorescently stained and shape-recognized portion is recognized as cryptosporidium and displayed on the image display means 49.
[0024]
Thus, according to the first embodiment described with reference to FIGS. 1 to 3, high-efficiency and mass-processing can be performed, and Cryptosporidium can be detected with high accuracy.
[0025]
In the above embodiment, the hollow fiber membrane separation unit 11 having a pore diameter of 1 μm or less is provided, but the pore diameter of the hollow fiber membrane may be 2 μm or less. That is, Cryptosporidium is generally reported to have a diameter of 3 to 5 μm. From this, it is considered that the pore diameter of the hollow fiber membrane is desirably 2 μm or less in consideration of the safety factor. In this way, by using a material having a pore size as large as possible, the permeability and long-term operability of the membrane can be ensured.Furthermore, it is possible to remove minute impurities from the target microorganism and reduce the proportion of impurities in the separated components. Can be.
[0026]
FIG. 4 is a system diagram showing the configuration of the second embodiment of the cryptosporidium recovery unit in the apparatus for performing the method for detecting cryptosporidium according to the present invention. In the figure, the same elements as those in the first embodiment shown in FIG. 1 are denoted by the same reference numerals, and description thereof will be omitted. The microorganism recovery unit 10B according to this embodiment is provided with a sample water introduction valve V0 and a sterilization processing unit 20 in a path upstream of the sample water introduction valve V1 that supplies the sample water to the hollow fiber membrane separation unit 11, Further, a dispersant introduction unit 21 is connected to a sample water introduction path between the sample water introduction valve V0 and the sterilization processing unit 20 via a dispersant injection valve V8.
[0027]
The operation of the second embodiment configured as described above will be described, particularly the parts that differ from the first embodiment in configuration. In the sample water, not only chlorine-resistant pathogenic microorganisms to be detected but also general microorganisms such as Escherichia coli and cells are present. Therefore, a sterilization treatment unit 20 using one or more of chlorine, ozone, and UV (ultraviolet) is introduced into a process preceding the hollow fiber membrane separation unit 11. Thereby, general microorganisms that are not chlorine-tolerant are killed and dissolved, and are not captured by the separation medium. On the other hand, chlorine-resistant microorganisms are not killed by the chlorine sterilization treatment. In addition, ozone or UV treatment causes a decrease in infectivity, inactivation, and the like. However, since the spores have a spore shape, there is no change in the individual shape, and they do not pass through the separation medium. As a result, the content of impurities is reduced, separation selectivity is improved, and membrane performance (including life) can be maintained.
[0028]
Cryptosporidium, on the other hand, has a surface charge and is likely to form aggregates with other contaminants and to be present in the sample water. Therefore, the dispersant introducing unit 21 is introduced into the preceding step of the hollow fiber membrane separation unit 11, and the dispersant injection valve V8 is opened at the same time as the sampling of the test water starts, so that the dispersant such as a surfactant injected into the sample water is added. Is added to disperse the aggregate formed by the impurities and Cryptosporidium. Further, in a post-process of the dispersant introducing unit 21, a separation filter unit (not shown) having a pore size of 10 μm to 100 μm is introduced to remove impurities and microorganisms larger than the above size dispersed by the dispersant effect. You can also. Regardless of the type of the separation filter medium, a hollow fiber membrane filter, a membrane filter, a ceramic filter, a plankton net and the like can be mentioned. In addition, as a process, it is desirable that the dispersant introduction unit 21 and the separation filter unit be installed in a stage preceding the sterilization treatment unit 20.
[0029]
Thus, according to the second embodiment, general microorganisms that are not chlorine-resistant are killed and dissolved, and aggregates are dispersed to collect only microorganisms having an outer shape similar to Cryptosporidium. In addition to enabling more efficient and larger-volume processing than the embodiment described above, Cryptosporidium can be detected with high accuracy.
[0030]
By the way, in each of the above embodiments, the use of the membrane filter as the sample fixing medium has been described. However, as the material of the membrane filter, hydrophilic PTFE is preferable. In this case, after the collected sample is pretreated, it is added to a hybridization buffer together with a fluorescently labeled probe, hybridization is performed under a certain condition, and a fluorescently labeled antibody is added as it is, and an immunoreaction is also performed. After that, it is fixed by suction on the membrane filter. In this case, since the reaction is all carried out in a homogeneous system, there is an advantage that the reaction rate is higher than in a heterogeneous system.
[0031]
In addition, in performing in-situ hybridization in the above embodiment, it is necessary to perform pretreatment. For example, heat treatment, microwave treatment, or electric treatment is performed, and formalin fixation is performed. Each of these processes may be performed independently, or may be performed in combination. For example, Cryptosporidium as a specimen is immobilized in a 16% formalin solution, and subjected to microwave treatment at 500 W / 2 minutes.
[0032]
Furthermore, in the fluorescent-labeled probe used for the in-situ hybridization, the fluorescent antibody is fluorescently labeled with Cy-excited fluorescent emission Cy3, and the immune antibody is labeled with a different labeling substance, for example, FITC excited with B-excitation. Cy3 is characterized by a long excitation holding time. In the in-situ hybridization, the reaction amount is smaller than that of the immune antibody reaction, and the excitation light is weak when the same labeling is performed. In addition, in the in-situ hybridization, when the Cy3 label is compared with the FITC label, the Cy3 label is less likely to fade, and bright fluorescence is obtained for a long time.
[0033]
As a modified example of each of the above-described embodiments, an apparatus relating to a series of processes from water sampling to dyeing may be regarded as one unit, and this unit may be operated in parallel, and each unit may be operated with a temporal phase difference. It is possible. The number of units and the temporal phase difference are set appropriately. The operation cycle depends on the processing capacity and specifications of the unit. For example, three units are arranged in parallel, and the separation process using the hollow fiber membrane is performed for 40 minutes in one cycle of 120 minutes, and each is operated with a phase difference of 40 minutes. This makes it possible to obtain samples every 40 minutes instead of every 120 minutes and measurement results.
[0034]
Further, as another modified example, in-situ hybridization and double staining treatment with an immune antibody were performed, and a fluorescence image observation and a differential interference image observation with two types of excitation light were performed on the fixed sample. If double-stained luminescence is detected and the differential interference image at the fluorescence detection position is recognized as the shape of the target microorganism, the operator may be notified in some form to provide a monitoring function as well as detection. it can.
[0035]
【The invention's effect】
As is clear from the above description, according to the present invention, the microorganisms in the sample water are each separated and concentrated by using the hollow fiber membrane first, and then using the membrane filter, and complementing the nucleic acid contained in Cryptosporidium. Fluorescence in-situ hybridization using a suitable probe, immunostaining using an antibody against a substance related to Cryptosporidium, double staining, and analyzing the differential interference image and the fluorescence emission image Since Cryptosporidium is recognized, it is possible to provide a Cryptosporidium detection method and a detection apparatus with high accuracy and high throughput, and with high accuracy.
[Brief description of the drawings]
FIG. 1 is a system diagram showing a configuration of a first embodiment of a cryptosporidium recovery unit in an apparatus for performing a cryptosporidium detection method according to the present invention.
FIG. 2 is a block diagram showing a configuration of a cryptosporidium detection unit in an apparatus for performing the cryptosporidium detection method according to the present invention.
FIG. 3 is a configuration example of a device in a case where a bright field image and a differential interference image are acquired by a detection unit illustrated in FIG. 2;
FIG. 4 is a system diagram showing a configuration of a second embodiment of a cryptosporidium recovery unit in an apparatus for performing the cryptosporidium detection method according to the present invention.
[Explanation of symbols]
10A, 10B Microorganism recovery unit 11 Hollow fiber membrane separation unit 12 Suction pump 13 Backwash pump 14 Membrane filter unit 15 Pump for injecting liquid injection 16 Sample recovery unit 20 Sterilization treatment unit 21 Dispersant introduction unit 31 Microorganism 32 Immobilization plate 41 Control means 42 Excitation light source 43 Fluorescence filter system 44 Differential interference filter 45 Filter switching means 46 Imaging means 47 Image recording means 48 Image analysis means 49 Image display means 100 Cryptosporidium detection unit 101 Preprocessing unit 102 Reaction unit 103 Fixing unit 104 Detection unit

Claims (5)

In a method for detecting Cryptosporidium for detecting Cryptosporidium in a sample water,
Separating and concentrating microorganisms in the sample water using a hollow fiber membrane, further separating and concentrating using a membrane filter,
Damaging the oocyst shell of Cryptosporidium among the microorganisms separated and concentrated by the membrane filter,
Performing fluorescence in-situ hybridization using a probe complementary to the nucleic acid contained in Cryptosporidium, and performing immunostaining using an antibody against a substance related to Cryptosporidium to perform double staining,
Obtaining a differential interference image and a fluorescence emission image of the double-stained microorganism;
Analyzing the obtained differential interference image and fluorescence emission image to recognize Cryptosporidium,
Are carried out sequentially. A method for detecting Cryptosporidium. In a Cryptosporidium detector for detecting Cryptosporidium in sample water,
Microorganism recovery means for separating and concentrating microorganisms in the sample water using a hollow fiber membrane, further separating and concentrating using a membrane filter,
Pretreatment means that damages the oocyst shell of Cryptosporidium among the microorganisms separated and concentrated by the microorganism collection means,
Performing fluorescence in-situ hybridization using a probe complementary to the nucleic acid contained in Cryptosporidium, and staining means for performing immunostaining using an antibody against a substance related to Cryptosporidium,
Obtaining a differential interference image and a fluorescence emission image of the microorganism stained by the staining means, a detection means for analyzing these images to recognize Cryptosporidium,
An apparatus for detecting Cryptosporidium, comprising: The cryptosporidium detection apparatus according to claim 2, wherein the pretreatment means performs at least one of heat treatment, microwave treatment, and electrical treatment, and performs formalin fixation. The said staining means performs fluorescence emission in-situ hybridization using a fluorescent labeling probe, and performs immunostaining using a different identifiable labeling substance. Cryptosporidium detection device. The detection means comprises means for notifying an operator when double-stained luminescence is detected in the sample and the differential interference image at the fluorescence detection position is recognized as the target microorganism shape. Item 5. The apparatus for detecting Cryptosporidium according to any one of Items 2 to 4.

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