JP4197797B2 - Inspection method of oocyst concentration of Cryptosporidium - Google Patents

Description

[0001]
BACKGROUND OF THE INVENTION
Recently, the emergence of emerging or reviving waterborne infectious diseases caused by protozoan cryptosporidium or pathogenic E. coli O-157 has become a major social problem. In order to prevent outbreaks of these infectious diseases, it is essential to monitor the causative microorganisms in the food production process and water treatment process. In addition, when developing a new water treatment process for removing pathogenic microorganisms and supplying safe purified water, it is necessary to evaluate the presence or absence of causative microorganisms. The present invention relates to a rapid and simple method for examining microorganisms in water.
[0002]
[Prior art]
For example, there are protozoa such as Cryptosporidium and Giardia as emerging causative microorganisms of emerging aquatic infections (for example, Mitsuyoshi Hosaka (1998) “Aquatic protozoal infections—causative microorganisms and outbreaks”), water and wastewater, Vol. 40, No. 2, p. 11; Mitsumi Kaneko (1998) “Technology for detection and removal of protozoa and other pathogenic microorganisms”, water and waste water, Vol. 10, No. 4, p. 32, etc.). These protozoa exist in the form of cysts in cysts called oocysts or cysts in the environmental water. Cryptosporidium oocysts and Giardia cysts are the targets of the examination. Conventional testing methods include microscopy, immunological testing, and genetic testing.
[0003]
First, the microscopic observation method is a method in which microbial cells are stained with a dye or a fluorescent dye, and the number of microorganisms stained with the microscope or the fluorescent microscope is counted, and the cells are non-specifically stained regardless of the type of the microorganism. Therefore, it is possible to distinguish from other microorganisms (yeast, bacteria, etc.) in terms of form and size, but since skill is required to distinguish from closely related oocysts, it is generally easy to make an erroneous determination. It is said that.
Next, immunological examination methods include fluorescent antibody staining, flow cytometer analysis, fluorescence spectroscopic analysis, enzyme antibody method, Western analysis, and the like. The immunological test method can specifically detect only the target microorganism by using an antibody that specifically binds to the target microorganism or its biological component. Observation, expensive flow cytometer analysis equipment, staining procedures are complicated, and a sample containing a large amount of microorganisms is necessary. Therefore, it is not a satisfactory inspection method in terms of operability and quantitativeness.
[0004]
Furthermore, examples of genetic testing methods include Southern analysis, Northern analysis, RCR [polymerase chain reaction] analysis, RT-PCR [reverse transcription PCR] analysis, and the like. Genetic testing can detect microorganisms to be tested specifically if the gene of interest is fully considered, but requires specialized knowledge on genetic analysis, complicated testing procedures, and presence / absence of presence. Even if it can be determined, it is difficult to quantitatively evaluate the abundance.
[0005]
By the way, the fluorescence polarization method is known as a method for detecting the binding characteristics between biomolecules such as proteins and nucleic acid molecules (for example, Checovic, WJ et al., (1995) Fluorescence polarization: a new tool for cell and molecular biology, Nature, vol.375, p254). This method was first reported by Perrin in 1926 (Perrin, F. (1926) J. Phys. Rad., Vol. 1, p390) and has recently gained attention again. The measurement principle of the fluorescence polarization method is that when a solution in which fluorescent molecules are suspended is irradiated with fluorescent light having a uniform amplitude direction as incident light, the incident light is scattered by free movement of the fluorescent molecules, and reflected light in all amplitude directions is generated. However, in the state where the free movement of the fluorescent molecule is restrained by immobilization such as binding to the target molecule, the degree of polarization ( The binding state (binding amount or binding strength) between the fluorescent molecule and the target molecule can be examined using the degree of polarization) as an index. In other words, if the target molecule that can bind to the fluorescent molecule is not present in the test water, the fluorescent molecule is in a free motion state and the degree of polarization is very small, but the target molecule that binds to the fluorescent molecule is present in the test water. Since the fluorescence molecule is immobilized by the binding of the fluorescence molecule and the target molecule and the movement state is reduced, the degree of polarization increases, so the amount of the target molecule in the test water is estimated by measuring the change in the degree of polarization. it can.
[0006]
Examples of application of this fluorescence polarization method (for example, Mineno, J. (1997) Protein, Nucleic Acid, Enzyme, Vol. 42, No. 1, p. 77) Analysis of interaction between protein molecules, such as measurement of reactivity between degrading enzyme and its substrate protein molecule, analysis of interaction between nucleic acid molecules such as DNA hybridization, measurement of binding between DNA and DNA-binding protein, protein and sugar chain There is an interaction analysis between different kinds of molecules such as measurement of binding. However, all of these cases are related to isolated and purified biomolecules, and no examples of application to macromolecules such as microbial cells have been reported.
[0007]
[Problems to be solved by the invention]
Conventionally known methods for testing microorganisms (especially protozoa such as Cryptosporidium) are not satisfactory in terms of ease of operation and detection accuracy, as described above. In addition, although there is an application example regarding a biomolecule isolated and purified with respect to the fluorescence polarization degree method, there is no application example to a macromolecule such as a microbial cell. Therefore, an object of the present invention is to provide a method for simply and rapidly examining the presence and concentration of microorganisms such as protozoa and pathogenic bacteria in test water based on the principle of fluorescence polarization.
[0008]
[Means for Solving the Problems]
Conventionally, with regard to the fluorescence polarization method, which was applied only to isolated and purified biomolecules, by optimizing the selection of fluorescent probes, pretreatment conditions for test water, binding solvent conditions, polarization measurement conditions, etc. Organisms such as microorganisms and cells can be detected and quantified. That is, the present invention relates to a biological component exposed on the surface of cryptosporidium oocysts or a biological component exposed on the sporozoid epidermis of cryptosporidium as a target molecule, and an anti-cryptospodium monoclonal antibody that can bind to the target molecule or fluorescence polarization to measure total fluorescence probe having a modified polyclonal antibodies FITC (fluorescein isothiocyanate) of a fluorescent material was added to the test water, determining the concentration of Cryptosporidium included from the variation of the fluorescence polarization to the subject water A method for inspecting the oocyst concentration of Cryptosporidium, characterized in that:
[0009]
DETAILED DESCRIPTION OF THE INVENTION
Prior to the description of the present invention, terms will be described first.
・ Target molecule: Probes used for testing specifically bind among proteins and sugar chains exposed on the surface of microbial cells to be tested, or biological components such as proteins, nucleic acids, carbohydrates, and lipids present in microbial cells. Means a possible biological component.
Probe: A compound that can bind to a target molecule. Specific examples include antibodies, lectins, oligonucleotides and the like.
Fluorescent probe: a probe modified with a fluorescent compound such as fluorescein, FITC [fluorescein isothiocyanate], rhodamine, Texas red, etc.
The present invention is, for protozoa cryptosporidium being particularly problematic in Sewage art are described below along the flow of the inspection procedure.
[0010]
(1) Selection of target molecule: Cryptosporidium is present in the form of oocysts encapsulating infectious worms called sporozoids in environmental water, and as target molecules, (1) living bodies exposed on the surface of oocyst skin Ingredients, {circle around (2)} biological components exposed on the sporozoid epidermis, {circle around (3)} biological components extracted from oocysts or sporozoid cell debris. In order to examine the abundance of Cryptosporidium in environmental water, (1) should be selected as a target molecule, but in order to evaluate the abundance of Cryptosporidium that retains infectivity, (2) is used as a target molecule. It only has to be selected. The biological component (3) includes nucleic acids such as DNA and RNA, proteins, carbohydrates, lipids, etc., but in order to detect Cryptosporidium in a limited way, it is necessary to consider the selection of the target molecule. For example, the base sequence of a gene characteristic of Cryptosporidium may be selected. In addition, if a gene or protein that is characteristically expressed at the time of decapsulation or infection / onset is selected as a target molecule, the abundance of Cryptosporidium that retains the ability to infect can be evaluated. Among these, considering the ease of inspection, (1) or (2) is simple.
[0011]
(2) fluorescent probes Selection of: a probe that binds specifically to a target molecule wherein the antibody and the like. Antibodies The antibody that Cryptosporidium oocysts skin and antigen are various commercially available, it is preferable in terms of ease and specificity of availability. Furthermore, in the fluorescence polarization method, the detection sensitivity may be increased when as many fluorescent probes as possible bind to the target molecule. Therefore, a polyclonal antibody is preferable to a monoclonal antibody rather than a monoclonal antibody.
[0012]
Fluorescent compounds for labeling probes include F ITC [fluorescein isothiocyanate]. Modification of the fluorescent compound to the probe can be performed by a known method. Alternatively, commercially available fluorescent antibodies for other uses such as fluorescent staining can also be used.
[0013]
(3) Preparation of test water: If the Cryptosporidium oocyst concentration in the test water is expected to be lower than 10,000 / L, test using known methods such as centrifugation, filtration and collection, chemical coagulation precipitation, etc. It is desirable to concentrate Cryptosporidium in water.
When sporozoide or an extract thereof is selected as a target molecule, a known method (for example, Upton, SJ (1994) ^ A simple and reliable method of producing in vitro infection of Cryptosporidium parvum (Apicoplexa) ", FEMS Microbiol. Lett , vol.118, p45), the oocysts in the test water may be removed in advance to release the sporozoid in the oocysts.
[0015]
(4) Addition of test reagent: A fluorescent probe is added to the test water and subjected to a binding reaction. For test water, various compounds (for example, albumin, polyvinyl alcohol, polyethylene glycol, ethylenediaminetetraacetate, protease inhibitor, phosphate buffer, Tris buffer, Hepes buffer for the purpose of promoting and stabilizing the binding reaction. Etc.) may be added as necessary.
[0016]
(5) Binding reaction: Optimum temperature and time can be selected as the conditions for the binding reaction in consideration of the combination of the target molecule and the probe. Fluorescence polarization measurement may be carried out immediately after adding a test reagent. However, the fluorescence is measured after heating at room temperature to 37 ° C or cooling to ice temperature to 15 ° C and reacting for several minutes to several hours. By measuring the degree of polarization, the binding reaction can be promoted and stabilized, and optimal conditions can be selected according to the desired combination of target molecule and probe. The fluorescent probe can be added at a concentration optimized by preliminary examination. When a nucleic acid is used as a target molecule and an oligonucleotide is used as a probe, it can be annealed by heating and subsequent slow cooling.
[0017]
(6) Measurement of fluorescence polarization degree: A commercially available fluorescence polarimeter can be used for measurement of fluorescence polarization degree.
(7) Data analysis: Create a calibration curve using oocyst suspension with a known concentration as test water, and compare the measured value of fluorescence polarization with the calibration curve when the sample to be examined is test water. The oocyst concentration in the medium can be estimated.
[0018]
【Example】
A water sample containing a known amount of Cryptosporidium parvum oocyst was tested as test water and anti-Cryptosporidium antibody as a probe.
Cryptosporidium suspension was sequentially diluted with PBS buffer to prepare oocyst-diluted suspension in the range of 0 to 10,000 per mL. 0.1 mL of BSA-PBS solution [1 mg-bovine serum albumin; PBS buffer] was added to 1 mL of the diluted suspension, and FITC-labeled anti-cryptospodium monoclonal antibody (Wako Pure Chemical Industries) was further added. After adding at a final concentration of 100 pg / mL and incubating at 37 ° C. for 1 minute, the degree of fluorescence polarization was measured using a fluorescence polarimeter (BEACON ™, Pan Vera, USA).
[0019]
The results are shown in FIG. In FIG. 1, the horizontal axis represents Cryptosporidium oocyst concentration, and the vertical axis represents the degree of fluorescence polarization as a relative value with respect to the measured value of test water not containing oocyst. As is clear from this figure, the degree of fluorescence polarization increased depending on the concentration of oocysts.
From the results of this example, it is clear that quantitative evaluation according to the concentration is possible in the range where the number of Cryptosporidium oocysts is 10,000 or less.
[0020]
【The invention's effect】
According to the present invention, the amount of microorganisms in water can be quantified and evaluated extremely easily compared to the conventional method.
The present invention can not only test the abundance of a target microorganism using an antibody as a probe, but also can use a fluorescent compound capable of binding to a microorganism cell or a biological component thereof as a probe .
[Brief description of the drawings]
FIG. 1 shows an embodiment of Cryptosporidium detected using the inspection method of the present application.

Claims (1)

Cryptosporidium (Cryptosporidium) biological components exposed to oocysts envelope surface of, or, a biological component exposed to sporozoite epidermis of Cryptosporidium to a target molecule, the anti-Cryptosporidium monoclonal or polyclonal antibodies capable of binding to the target molecule it fluorescent probes modified with a fluorescent substance FITC (fluorescein isothiocyanate) was added to the test water measuring fluorescence polarization photometer, to quantify the concentration of Cryptosporidium included in the test water from the variation of the fluorescence polarization An inspection method for oocyst concentration of Cryptosporidium, which is a feature .

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