JP2002125695A - Method for detecting microorganism and substance derived from organism - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for specifically and efficiently concentrating low-level microorganisms and/or substances derived from organisms in samples used in the fields of therapy, clinical examination, food inspection and water examination, and to provide a method for detecting such microorganisms and/or substances derived from organisms using the above method. SOLUTION: This method for detecting microorganisms and/or substances derived from organisms is characterized by comprising the following practice: a sample that may contain the microorganisms and/or substances derived from organisms in such concentrations as to be below the detection limit for a means to be applied thereto is brought into contact with an adsorbent capable of specifically adsorbing the microorganisms and the substances derived from organisms and then the means for detecting the microorganisms and/or the substances derived from organisms is applied to the thus adsorbed microorganisms and/or the substances derived from organisms.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention
The present invention relates to a method for specifically and efficiently concentrating low-concentration microorganisms and biological substances in a sample used in the field of food inspection and water quality inspection, and a method for detecting microorganisms and biological substances using the same. More specifically, lake water,
Tap water, circulating baths, circulating water such as cooling towers, or dissolve fresh foods, dairy products, instant foods, or human or animal body fluids, blood, excreta, tissues, cells, etc.
The present invention also relates to a method for detecting a low-concentration microorganism or biological substance contained in a suspended or dispersed sample.

[0002]

2. Description of the Related Art In therapeutic, clinical, food, and water quality tests, microorganisms and biological substances in a sample can be quickly and accurately determined.
It is possible to detect with high sensitivity, treatment method, selection of treatment timing, disease, infection, diagnosis diagnosis of constitution, food contamination, determination of the presence or absence of genetic manipulation, water pollution, determination of contamination,
It is extremely important in confirming the treatment effect. Usually, these microorganisms and biological substances have been detected using techniques such as biochemical tests and immunological tests.However, if the concentration of these microorganisms or biological substances in the sample to be tested exceeds a certain level, If it is present, it can be detected, but if the presence of microorganisms or biological substances is low, the microorganisms should be cultured and the concentration increased before use for detection, or for biological substances, liquid chromatography or mass spectrometry Attempts have been made to detect gauges and the like using the latest equipment.
However, in the method of culturing microorganisms, the operation is complicated and requires a high degree of skill, and it has been conventionally difficult to obtain a result with good reproducibility even by an expert in this field. In addition, rapid diagnosis and treatment of microorganisms that require a long period of time for cultivation are not possible, which has been a great hindrance in practical use. In addition, the detection limits of biological substances that can be detected even by using the latest equipment such as high-sensitivity liquid chromatography and mass spectrometer are naturally limited, and the equipment itself is expensive and highly skilled in handling. And was not universally available. There is an increasing demand for detection of trace amounts of biological substances in recent years due to an increase in health consciousness, an increasing demand for food inspection, and an increase in awareness of environmental issues.

In recent years, the possibility of detecting biologically-derived substances having low concentrations of microorganisms and nucleic acids has been opened by genetic testing techniques such as nucleic acid amplification techniques and invader techniques typified by the polymerase chain reaction method (hereinafter, PCR method). Have been. However, detection of very low concentrations of microorganisms and biological substances having nucleic acids at very low concentrations requires special equipment and advanced technology even by the PCR method. It is.
In addition, the amount of sample that can be used is also limited by the instrument.
The problem is that detection is not possible if the concentration of the biological substance having a nucleic acid is low.

[0004]

SUMMARY OF THE INVENTION The object of the present invention is to use a conventional culture method which cannot be detected by a conventional method, or even if it is possible, requires complicated culture operations and expensive and versatile analytical instruments. An object of the present invention is to provide a method for industrially significant detection of low-concentration microorganisms and biological substances required.

[0005]

In order to solve the above-mentioned problems, the present invention provides a method in which a microorganism and / or a biological substance is contained at a concentration lower than the detection limit of the applied microorganism and biological substance detecting means. After contacting an adsorbent capable of specifically adsorbing the microorganism and the biological substance to a sample that may be adsorbed, separating the adsorbent from the sample, and then bonding the sample to the adsorbent And a method for detecting a microorganism and / or a biological substance, which comprises applying a detection means for a microorganism and / or a biological substance.

[0006]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in more detail. [Adsorbent] The adsorbent that can be used in the present invention (hereinafter, simply referred to as “target substance adsorbent”) does not indicate the raw material itself, but various materials such as organic polymers,
It is made of an inorganic polymer, an inorganic compound, a natural polymer compound, or the like, or a combination thereof, and may have various shapes as described later. The adsorbent must be water-insoluble because it can easily separate microorganisms and biological substances. It is composed of an aqueous substance serving as a sample, for example, a substance insoluble in blood, urine, aqueous protein solution and the like. Examples of the organic polymer constituting the target substance adsorbent include (co) polymers of radically polymerizable monomers such as aromatic vinyl compounds, acrylates, methacrylates, and unsaturated carboxylic acids, polyesters,
Polyolefin, polyether, polycarbonate, polyimide, polyamide, cation exchange resin, anion exchange resin, polyethylene imine, polypropylene imine,
Polylysine, glucosamine, polyallylamine and the like can be mentioned. Examples of the inorganic polymer include a hydrolyzed condensate such as an alkoxysilane and an organoalkoxysilane. Examples of the inorganic compound include silica, alumina, calcium phosphate and the like. Examples of the natural polymer include dextran, cellulose, agarose, and glucomannan. When the material is, for example, an organic polymer, there are water-soluble ones, but the target substance adsorbent must be water-insoluble, and in such a case, treatment such as crosslinking or holding on a water-insoluble substrate is required. is necessary.

[0007] In the present invention, the surface of the target substance adsorbent is preferably ionic in order to increase the specific adsorption ability of microorganisms and biological substances. Here, that the surface is ionic indicates that the surface of the target substance adsorbent has at least one of an anionic charge and a cationic charge, or can be charged under specific conditions. Examples of the target substance adsorbent having an anionic surface include those in which the target substance adsorbent comprises a polymer obtained by copolymerizing a monomer having an anionic group, those obtained by anionizing a polymer, and synthetic polymers. A polymer obtained by seed-bonding or graft-polymerizing a sulfonic acid-containing monomer into a cation-exchange resin,
Examples thereof include those in which a sulfated natural polymer is supported on a water-insoluble polymer carrier, those having a polymer having an anionic group or a sulfated natural polymer on the surface of an inorganic polymer or inorganic particles, and the like. Here, examples of the anionic group include a sulfone group and a carboxyl group.

The following are examples of the above. Examples of polymers obtained by copolymerizing monomers having an anionic group: (co) polymers of carboxylic acid-containing monomers. Here, the carboxylic acid-containing monomer (hereinafter referred to as “carboxylic acid monomer”) is a polymerizable monomer having an addition-polymerizable unsaturated bond and a carboxyl group in the molecule. Specific examples include acrylic acid, methacrylic acid,
Crotonic acid, itaconic acid, fumaric acid, maleic acid and the like.

Examples of anionized (sulfonated) polymers: Sulfonable functional groups at least on their surface, eg unsaturated double bonds, aromatic groups, primary or It consists of a (co) polymer having a secondary amino group, a primary halogenated alkyl group, an aliphatic aldehyde, an aliphatic ketone, an aliphatic carboxylic acid, an aliphatic carboxylic anhydride, a hydroxyl group, etc. Some are sulfonated. Surface sulfonable (co)
Examples of the polymer include (co) polymers of sulphonable monomers such as styrene, α-methylstyrene, vinylnaphthalene, divinylbenzene, butadiene, isoprene, and vinyl alcohol, and polymerization of these monomers and other polymers. Polymerization compounds such as (co) polymers with hydrophilic monomers; polycarbonates, polyesters, polyester ethers,
Polyarylether, polyalkylene oxide, polysulfone, polyethersulfone, polyamide, polyimide, polyetherimide, polyetherketone, polyurethane, acetaldehyde condensate of aromatic compound, condensation polymerization type high molecular compound such as polyether and the like. .

[0010] Examples of the cation exchange resin include divinylbenzene cross-linked sulfonated polystyrene, and the particle size may be such that the central particle size is about 0.1 mm to 1 mm. Examples of a sulfated natural polymer supported on a water-insoluble polymer carrier: sulfated polysaccharides include heparin, dextran sulfate, cellulose sulfate, guardlan sulfate, chondroitin sulfate, heparan sulfate, dextran sulfate,
Chitin sulfate, chitosan sulfate, dermatan sulfate, amylopectin sulfate, ketalan sulfate, xylan sulfate, karonin sulfate, pectin sulfate, inulin sulfate, alginate sulfate, glycogen sulfate, polylactose sulfate, carrageenin sulfate, starch sulfate, polyglucose sulfate, laminarin sulfate, Galactan sulfate, levan sulfate, mepesulfate,
Examples include polysaccharides having antiviral properties such as fucoidan and sulfated glycyrrhizin. The sulfated polysaccharide can be supported directly on a polymer having a functional group such as an epoxy group, an amino group, an aldehyde group, a carboxyl group, a hydroxyl group, an acid chloride group, or via a coupling agent or a spacer.

Examples of the target substance adsorbent having a cationic surface include those in which the target substance adsorbent comprises a polymer obtained by copolymerizing a monomer having a cationic group, and a polymerization initiator having a cationic group. From a polymer obtained by radical polymerization using a compound having a cationic group bonded to a polymer carrier, from an anion exchange resin, or from a water-soluble polymer containing a cationic group. Supported on a polymer carrier,
Examples thereof include a polymer having a cationic group or a polymer having a cationic group-containing water-soluble polymer on the surface of an inorganic polymer or an inorganic particle.

Here, the cationic group includes a primary amino group, a secondary amino group, a tertiary amino group; a quaternary ammonium group, an imino group (—NH— and ＝ NH group), an amidino group, an imidino group, A hydrazino group; and a cyclic group containing a nitrogen atom such as a pyridyl group. Examples of the above include the following. Example of polymerization in which a monomer having a cationic group is copolymerized: Examples of the monomer having a cationic group include 2-dimethylaminoethyl (meth) acrylate, 2-diethylaminoethyl (meth) acrylate, and 2-dimethylaminopropyl (meth). Aminoalkyl group-containing (meth) acrylates such as acrylate and 3-dimethylaminopropyl (meth) acrylate, and quaternary salts thereof with methylene chloride, dimethyl sulfate, diethyl sulfate, and the like; 2
-(Dimethylaminoethoxy) ethyl (meth) acrylate, 2- (diethylaminoethoxy) ethyl (meth)
Acrylates, (meth) acrylates containing aminoalkoxyalkyl groups such as 3- (dimethylaminoethoxy) propyl (meth) acrylate and the like, and quaternary salts thereof with methylene chloride, dimethyl sulfate, diethyl sulfate and the like; N- (2- Dimethylaminoethyl) (meth) acrylamide, N- (2-diethylaminoethyl) (meth)
Acrylamide, N- (2-dimethylaminopropyl)
N-aminoalkyl group-containing (meth) acrylamides such as (meth) acrylamide and N- (3-dimethylaminopropyl) (meth) acrylamide, and quaternary salts thereof with methylene chloride, dimethyl sulfate, diethyl sulfate and the like. Can be Among them, 2-dimethylaminoethyl (meth) acrylate, N- (2-dimethylaminoethyl) (meth) acrylamide, and quaternary salts thereof with methylene chloride are preferable. These may be copolymerized with other polymerizable monomers. Examples of the monomer copolymerized with the cationic monomer include a crosslinkable monomer and a non-crosslinkable and nonionic monomer as shown below.

Examples of the crosslinking monomer include divinylbenzene, divinylbiphenyl, ethylene glycol di (meth) acrylate, diethylene glycol di (meth) acrylate, triethylene glycol di (meth) acrylate, tetraethylene glycol di (meth) acrylate, and propylene glycol. Di (meth) acrylate, dipropylene glycol di (meth) acrylate, tripropylene glycol di (meth) acrylate, tetrapropylene glycol di (meth) acrylate, 1,4-butanediol di (meth) acrylate,
1,6-hexanediol di (meth) acrylate, neopentyl glycol di (meth) acrylate, 2,
2'-bis [4- (meth) acryloyloxypropoxyoxyphenyl] propane, 2,2'-bis [4- (meth) acryloyloxydiethoxydiphenyl] propane, glycerin tri (meth) acrylate, trimethylolpropane tri ( Examples thereof include divinyl monomers such as meth) acrylate and pentaerythritol tetra (meth) acrylate, trivinyl monomers and tetravinyl monomers. Among them, divinylbenzene, ethylene glycol dimethacrylate and trimethylolpropane trimethacrylate are preferred. The copolymerizable non-crosslinkable and nonionic monomer is a noncrosslinkable and nonionic monomer that can be copolymerized with either a cationic monomer or a crosslinkable monomer.

As such a monomer, styrene, α
Aromatic vinyl monomers such as methyl styrene, p-methyl styrene, halogenated styrene; unsaturated nitriles such as acrylonitrile; methyl acrylate, methyl methacrylate, ethyl acrylate, ethyl methacrylate;
Acrylates such as butyl acrylate, butyl methacrylate, cyclohexyl methacrylate, 2-ethylhexyl acrylate, 2-ethylhexyl methacrylate, lauryl acrylate, lauryl methacrylate, glycidyl acrylate, glycidyl methacrylate, 2-hydroxyethyl acrylate, 2-hydroxyethyl methacrylate, and methacrylic acid Esters; diolefins such as butadiene and isoprene; carboxylic acid vinyl esters such as vinyl acetate; and vinylidene halides such as vinyl chloride and vinylidene chloride. Among them, styrene, α-methylstyrene, acrylonitrile, methyl methacrylate, butyl methacrylate, 2-hydroxyethyl acrylate, and cyclohexyl methacrylate are preferred.

Examples of a polymer obtained by polymerizing a monomer using a polymerization initiator having a cationic group: A radical polymerization initiator having a cationic group is a polymer obtained by radical polymerization using the same. It has a cationic group derived from the radical polymerization initiator at its terminal. Preferred examples of the radical polymerization initiator having a cationic group include azobis-type initiators having an amidino group, an imidino group, or a pyridium group. Those having a 10-hour half-life temperature in the range of 40 to 95 ° C. are preferable because the polymerization can be carried out under mild conditions.

A carboxyl group, a hydroxyl group, an epoxy group, an amino group, an amide group and the like are introduced into the surface of a polymer particle obtained by binding a compound having a cationic group to a polymer carrier by copolymerization or seed polymerization. By reacting these groups as a bonding portion, a compound having a cationic group can be introduced to the polymer surface. Examples of the compound having a cationic group include polyethyleneamine, polypropyleneimine, polyallylamine, polyvinylamine, polyamine compounds such as polypropyleneamine, polylysine, polyarginine, free amino acids such as polyhistidine, and monomers having the above-described cationic group. Copolymers and the like can be mentioned.

Anion-exchange resin Divinylbenzene cross-linked polystyrene has a quaternary ammonium group -CH ₂ N ⁺ (CH ₃ ) ₃ (type I) or -CH ₂ N ⁺ (CH ₃ ) ₃ (C ₂ H ₄
OH) (Type II) introduced strong basic anion exchange resin, -N
Weakly basic anions into which H (CH ₃ ) ₂ , —NH (CH ₂ CH ₂ NH) _n H, and —CONH (CH ₂ ) ₃ N (CH ₃ ) ₂ are introduced. A polymer having a cationic group or a cationic group-containing water-soluble polymer on the surface of an inorganic polymer or inorganic compound. Further, a resin having a donor atom (O, N, S) called a chelate resin can also be mentioned. Examples of the chelating resin include iminodiacetic acid type, iminopropionic acid type, ethylenediamine triacetic acid type, quinoline type, aminocarboxylic acid type, amine phosphoric acid type, amidoxime type, cryptant type, polyamine type, pyridine type, picolylamine type, and thiol type , Phosphoric acid type, polyphenol type, dithiocarbamic acid type, thiourea type, isothiuronium type and dithizone type.

In the present invention, the target substance adsorbent may contain a magnetic substance such as a ferromagnetic substance and a superparamagnetic substance, which are effective for automating the inspection and the separating operation. The shape of the target substance adsorbent is not particularly limited, and may be various. For example, it may be in the form of particles, filters, fibers, sheets, tubes, plates, or the like. When the shape of the target substance adsorbent is particulate, the particle size is about 0.1 to 200 μm when used by being dispersed in a sample, and about 0.1 to 1 mm when used by filling in a column. It preferably has a particle size.

The filter is preferably a filter having an average pore diameter in the range of 0.1 to 50 μm, and examples thereof include a flat membrane, a hollow fiber membrane, a nonwoven fabric, and a woven fabric.
When the target is plasma, culture solution, or the like, the average pore size is 0.
1 μm to 10 μm is preferred. If the average pore size is 0.1 μm or less, a sufficient filtration amount cannot be obtained because the pore size becomes small. The average pore diameter is a value obtained by using a palm porometer (manufactured by Porous Materials) described in ASTM-F316, and is a value reflecting an actual transmission pore.
The amount of water passing through the filter was 10 mL / mi as a value measured at 25 ° C. ± 2 ° C. under a pressure of 0.7 kg / cm ^2.
n / m ² / mmHg or more, preferably 100 mL / min
/ M ² / mmHg or more, because the filtration pressure can be reduced. In the case of a nonwoven base material, the filaments forming the filter material may be monofilaments or multifilaments, but the average diameter (average of the major and minor diameters of the filaments observed with a scanning electron microscope) Value) is 100 μm or less, preferably 50 μm.
It is preferable that it is not more than m since the surface area of the film becomes large and the number of adsorption sites increases. Further, it may be a modified filament or a porous filament. Further, the ratio of the porosity of the filter (porosity) is 20%.
Or more, preferably 50% or more.

Examples of the target substance adsorbent having a sheet shape, a tube shape, or a plate shape include a blood collection tube, a vacuum blood collection tube, a test tube, a well plate, a microplate, a microtest tube, and an Eppendorf tube (trade name). Can be mentioned. In order to obtain the target substance adsorbent having these shapes, among the organic polymers exemplified above, thermoplastic resins such as polystyrene, polypropylene, polyethylene, polyethylene terephthalate, and norbornene-based resin can be directly molded into a desired shape. . Further, by ionizing the surface of a general-purpose blood collection tube made of glass, polystyrene, polymethyl methacrylate, or the like, a target substance adsorbent suitable for the present invention can be obtained.

[Sample] The detection method of the present invention is applied to:
This is a sample that may contain microorganisms and biological substances. Samples include, for example, various body fluids such as blood, plasma, serum, urine, and saliva, excretions such as urine and feces, tissues, cells, cell lysates, cultured cell lysates, fresh foods, processed dairy products, and instant foods. Food, lake water, tap water, circulating baths, circulating water such as cooling towers, environmental water, etc., which are dissolved, suspended and dispersed. In the present invention,
The concentration of the target substance adsorbent in the sample is a concentration range that cannot be measured by the detection means used when the sample is directly measured.

[Contact / Binding] In the present invention, the target substance adsorbent is brought into contact with the sample prepared as described above.
There are various contact methods, and there is no particular limitation. For example, when the target substance adsorbent is a particle having a particle size of less than 200 μm,
The target substance adsorbent is dispersed in the sample, and the sample is brought into contact with the target substance adsorbent by shaking. When the target substance adsorbent is a particle having a particle diameter of more than 200 μm, the column is packed and a sample is passed through the column. When the target substance adsorbent is a large one such as plastic beads having a diameter of 5 to 6 mm, one to several pieces are put into a test tube and brought into contact with the sample. When the target substance adsorbent is in the form of a filter or a fiber, the sample is filtered through the filter or the fiber so that the surface of the filter or the fiber is sufficiently in contact with the sample. When the target substance adsorbent is a test device such as a test tube or a well plate, the sample is placed in the test device and shaken. For example, when the target substance adsorbent is particles, the target substance adsorbent is added to the sample at a rate such that the surface area of the particles becomes 50 to 10,000 square cm per 1 ml of the sample.

When the sample is brought into contact with the target substance adsorbent, a polyvalent metal ion or the like may be further added as necessary. The temperature of the sample when the sample is brought into contact with the target substance adsorbent is usually 5 to 37 ° C., and the contact time is not particularly limited, but is preferably adjusted depending on the object so as to obtain a sufficiently specific adsorption efficiency. The term “specific adsorption” as used in the present invention refers to high-efficiency and high-selective adsorption from a target microorganism, a microorganism coexisting with a biological substance, a biological substance, or another substance. PCR for detection of low concentration microorganisms
Although the use of nucleic acid amplification is effective, it is not usually possible to distinguish live bacteria from dead bacteria, and even if a positive result is obtained as a result of nucleic acid amplification, for example, the effect on humans, animals, food, and the environment In the final judgment, it is inevitable to sample a part of the sample presumed to contain microorganisms and detect it after culturing, which is a major obstacle to the application of the nucleic acid amplification method. I was

In addition, when a microorganism or a biological substance in a sample is detected in a highly sensitive nucleic acid amplification method or immunochemical assay method, a false positive judgment is made such that a signal suspected of being present is generated even if it is not actually present. In many cases, this has been a major obstacle in practical terms. However, according to the present invention, after contacting an adsorbent capable of specifically adsorbing microorganisms and biological substances, the adsorbent is separated from a sample, and then the microorganisms and biological substances bound to the adsorbent are contacted. By adopting a method for detecting microorganisms and living body-derived substances, which is characterized in that the false positive is detected, the false positive rate can be significantly reduced.

[Detection of Microorganisms and Biological Substances] After the target substance adsorbent is brought into contact with the sample, the target substance adsorbent is separated from the sample. At this time, the target substance adsorbent has a particle size of 200 μm.
In the case of less than particles, they are separated, for example, by centrifugation. In addition, when the magnetic substance is contained in the target substance adsorbent, it can be easily separated from the sample by magnetism.
Microorganisms and biological substances on the target substance adsorbent separated from the sample are concentrated. Microorganisms concentrated in this way,
The biological substance is then subjected to detection. However, if necessary, the target substance adsorbent may be washed with a low-concentration buffer before being subjected to detection, or detection may be performed after separating microorganisms and biological substances from the target substance adsorbent. For example, when the microorganism or biological substance is protozoa, fungi, bacteria, rickettsia or mycoplasma, it can be separated, and when it is nucleic acid, it can be extracted. Also,
The detection can be carried out with the microorganism and the biological substance still bound to the target substance adsorbent. An appropriate method may be selected depending on the type of the microorganism, the biological substance, the material of the target substance adsorbent, and the like.

As a method for separating microorganisms and biological substances from the target substance adsorbent, there is a method in which a salt solution and a surfactant or a mixture of both are allowed to act to dissociate microorganisms and biological substances from the target substance adsorbent. . As a salt solution, a high concentration of, for example, 2 to 8 M potassium bromide, sodium bromide, sodium iodide, guanidine isothiocyanate, guanidine thiocyanate, guanidine hydrochloride,
An aqueous solution of guanidine sulfate, sodium thiocyanate, potassium thiocyanate and a salt thereof, a 1.5 to 6 M sodium chloride aqueous solution, a 0.5 to 5 mM phosphotungstic acid aqueous solution, or the like can be used. As surfactants, SDS, Triton X-100, Tween 20, 80, NP-40
and so on.

Further, nucleic acid extraction can be carried out even when microorganisms and living body-derived substances remain bound to the target substance adsorbent. In order to effectively amplify and detect a target nucleic acid, the nucleic acid typically must be isolated from cells or other residues of the sample. Various separation methods are known, including a freezing method, a treatment method with a digestive enzyme such as a protease (eg, proteinase K), a boiling method, and a method using various detergents (for example, Higuchi, filed on April 6, 1988). U.S. Patent Application No. 178,202 and European Patent Application Publication No. 07828197 issued May 22, 1991), solvent deposition methods and heating protocols. By adding a small amount of buffer solution to the separated target substance adsorbent and heating it, it is also possible to directly extract microorganisms, nucleic acids of biological substances, or directly add a commercially available nucleic acid extraction reagent It is also possible to extract nucleic acids of microorganisms and biological substances.

As described above, microorganisms and biological substances separated and extracted in the state of being concentrated on the target substance adsorbent and, in some cases, further concentrated, are detected. As the detection method, a known method can be adopted according to the type of the microorganism or the biological substance, and examples thereof include a nucleic acid amplification test and an immunochemical measurement method. Nucleic acid amplification test (NAT; nucl
The method of eic acid amplification test is not particularly limited. For example, PCR (Polymerization chain) of Roche
reaction) method, TMA (Transcript
ion Mediated amplification-hybridization protectio
n assay) method, Abbott's LCR (Ligase chain reacti
on) method, NASBA (Nucleic Acid Sequence-based Amp)
lification) (targeting RNA, characterized by constant temperature),
Eiken Chemical's LAMP method, Takara Shuzo's ICAN method, CP
T (Cyclic Probe Tech) (Target DNA, DNA
/ Amplify biotin-labeled probe using RNA chimera probe), Qβ Replicase Amplification (target RNA, isolate RNA with streptavidin particles), Inva
der Assay etc. can be used.

Examples of the immunochemical assay include enzyme immunoreaction (EIA), chemiluminescence (CLTA), and fluorescence (FIA). Examples of the microorganism and the biological substance that can be detected by the microorganism and the biological substance detection method of the present invention include protozoa, fungi, bacteria, rickettsas, mycoplasmas, nucleic acids, antigens, antibodies, and proteins. Protozoa include amoeba, trichomonas, malaria pathogens, toxoplasma, chrysomonas, euglena and the like. Examples of fungi include ascomycetes such as Aspergillus, blue mold and Aspergillus, and fungi represented by incomplete fungi such as Candida and Cryptococcus. Bacteria include Bacillus subtilis, Diphtheria, Botulinum, Haemophilus influenzae, Neisseria gonorrhoeae, Staphylococcus, Methicillin-resistant Staphylococcus aureus, Streptococcus, Escherichia coli, Methanobacterium,
Cholera, Pseudomonas aeruginosa, Legionella and the like.
Ricketts include trachoma and parrot pathogens,
Q fever pathogen, typhus fever pathogen and the like. Examples of the mycoplasma include mycoplasma pneumonia and urea mycoplasma. As nucleic acids, DNA, RN
A, mRNA, tRNA and the like. Antigens and antibodies include ferritin, ASO, T ₃ , T ₄ , α-fetoprotein, HBs antigen, CEA (carcinoembryonic antigen), HC
V surface antigen, HIV surface antigen, anti-Treponemal pallidum, anti-carbiolipin antigen, anti-HBC antibody, anti-HIV
All substances measured by the immune reaction, such as antibodies, are included. Examples of the protein include α-lipoprotein and β-lipoprotein.

[0030]

EXAMPLES The present invention will be described below in detail with reference to examples, but the present invention is not limited to these examples.
In the following description, “parts” means “parts by weight” unless otherwise specified.

Reference Example 1 (1) Acetone is added to oil-based ferrofluid ferricolloid HC50 (trade name, manufactured by Taiho Kogyo KK) to precipitate particles, which are then dried to obtain a lipophilic treatment. Ferrite-based superparamagnetic material (particle size:
0.01 μm). Then, the superparamagnetic material 4 obtained above
To 0 parts, 90 parts of cyclohexyl methacrylate, 10 parts of chloride of trimethylaminoethyl methacrylate and 3 parts of benzoyl peroxide (polymerization initiator) are added, and the system is mixed and stirred to uniformly disperse the superparamagnetic material, thereby obtaining a monomer composition. Was prepared. On the other hand, 10 parts of polyvinyl alcohol, 0.05 part of sodium lauryl sulfate and polyethylene oxide nonyl phenyl ether 0.1 part.
One part was dissolved in 1000 parts of water to prepare an aqueous monomer composition. The above monomer composition was added to the aqueous monomer composition thus prepared, and the mixture was preliminarily stirred with a homogenizer, and then subjected to a dispersion treatment with an ultrasonic disperser, whereby oil droplets (oil phase) having an average particle diameter of 1 μm were formed in the aqueous medium. (Oil droplet dispersion) was prepared. Next, the obtained suspension was charged into a two-neck flask with a stirrer having a capacity of 2 liters,
The temperature of this system was raised to 75 ° C., and the monomers in the oil droplets were polymerized (suspension polymerization) for 5 hours while stirring under a nitrogen atmosphere to produce magnetic polymer particles.
The obtained particles were photographed with an optical microscope, the diameter of 200 particles was measured, and the average was determined. The result was 1.2 μm.

(2) The magnetic polymer particles obtained in the above (1) are dispersed in a 5 mM aqueous sodium hydroxide solution,
For 12 hours to obtain carboxy-modified magnetic polymer particles. (3) 1 g of the obtained carboxy-modified magnetic polymer particles were suspended in 20 mL of 10 mM MES buffer solution (pH 6.0), and a 30% aqueous solution of polyethyleneimine (Wako Pure Chemical Industries, Ltd., number average molecular weight 70,000), 33 μL, Water-soluble carbodiimide reagent EDC · hydrochloride (1-ethyl-3
0.05 g of (3-dimethylaminopropyl) carbodiimide hydrochloride) was added and reacted at room temperature for 2 hours to obtain an adsorbent.

Example 1 The aqueous suspension of the polyethyleneimine-immobilized adsorbent obtained in Reference Example 1 was purified and adjusted to a solid concentration of 5% with physiological saline. 1000 ml of sample water from which solid foreign substances had been removed in advance using cellulose filter paper was transferred from a cooling tower water in which the presence of Legionella bacterium collected from the cooling tower was estimated to a plastic container capable of being sealed. To this sample water, 20 ml of a 5% aqueous solution of polyethyleneimine immobilized adsorbent was added, and the mixture was added at room temperature for 2 hours.
The mixture was stirred at a rotation speed of 10 times per minute for 5 minutes to perform the adsorption treatment on the adsorbent.

After the adsorption treatment of Reference Example 1, the plastic container was
The adsorbent was allowed to settle and separated at the bottom of the plastic container.
The supernatant was removed by suction with a suction tube, 10 ml of physiological saline was added to the obtained adsorbent, quantitatively transferred to a 20 ml test tube, and set on a magnetic separation stand. The particles of the cell adsorbent and the supernatant were separated by magnetic operation, the supernatant was discarded, and 3 ml of Tris-HCl buffer solution (pH 7.4) was added to obtain a cell adsorbent particle dispersion. Separate the supernatant and volume 20ml
To a centrifuge tube. The same operation was further repeated twice, and the obtained cell suspension was centrifuged at 30,000 rpm for 30 minutes, and the supernatant was removed. Then, 200 u of Tris-HCl buffer (pH 7.4) was added.
1 cell suspension. DNA Extrac from this cell suspension
Nucleic acids were extracted using tor Kit (manufactured by Wako Pure Chemical Industries, Ltd.) according to the attached protocol. Nested-P
By the CR method, the DNA was amplified by the amplification process of 35 times of 1st-PCR and 25 times of 2nd-PCR. The resulting amplification products of the 1st-PCR and 2nd-PCR were subjected to electrophoresis using 3% agarose, the DNA was visualized by ethidium bromide staining, and photographed on a Polaroid (registered trademark) photograph. Obtained purpose DN
The amount of A was determined from the fluorescence intensity of the band as-, +, ++, ++.
+, +++++.

EXAMPLE 2 1000 ml of the same cooling tower water used in Example 1
After filtration and removal of coarse foreign substances by a cellulose filter in the same manner, bacteria were collected by suction filtration with a membrane filter (manufactured by Nippon Millipore) having a pore size of 0.22 μm made of polytetraethylene. After suction filtration, transfer the membrane filter to a sealable 20 ml test tube containing 5 ml of a buffer solution containing a nonionic surfactant, shake with a vortex mixer for 5 minutes, and separate presumed bacterial cells from the membrane filter. , Suspended. The suspension supernatant was transferred to a 20 ml centrifuge tube, and the same operation was repeated again with 5 ml of the buffer solution to obtain 10 ml of a cell suspension. Thereafter, a Tris-HCl buffer (pH 7.4) 20
After obtaining 0 ul of the cell suspension, the cell concentration was determined in five steps of-, +, ++, +++, and +++ by PCR measurement.

Example 3 American Tissue Culture Collection ATC
C No. Advance with 33152 1.5x10 ^2/1
1000 ml of the Legionella bacterium model suspension adjusted to a cell concentration of 00 ml was adsorbed and concentrated in the same manner as in Example 1, and the cell concentration was determined to be −, +, ++, ++ by PCR measurement.
Judgment was made by five-stage display of +, +++++. Example 4 Legionella bacterium model suspension 1000 m similar to Example 3
1 was subjected to PCR measurement in the same manner as in Example 2. Example 5 200 u of Tris-HCl buffer solution (pH 7.4) obtained in Example 2
1 100 ul of the cell suspension was applied to a BCYEα agar medium (manufactured by Eiken Chemical Co., Ltd.) using an application rod, and cultured at 37 ° C. for 5 days in an incubator. After the culture, the formation of bacterial colonies was observed on the surface of the plate medium, and the morphology, the swelling state of the colonies, and the color suggested the presence of Legionella bacteria. The calculated bacterial cell concentration from the number of colonies was 1.8.
x10 was ² / 100ml.

The concentration of bacterial cells in the specimen determined in Examples 1 to 4 will be summarized. Example 1: ++ Example 2: ++++ Example 3: ++ Example 4: ++ These results indicate that the determination of the concentration of Legionella by PCR measurement using the adsorbent according to the present invention was performed using a membrane filter (Japan). Compared to the case of PCR measurement after suction filtration and collection of bacteria by Millipore Corporation, it strongly suggests that it gives a judgment result closer to the culture method, and it is effective for rapid control of the water quality of the cooling tower. I understand. In general, the filtration method collects live bacteria and dead bacteria without distinction.
In the determination by the R method, the measurement result tends to be higher than the actual presence of viable bacteria. In comparison, the adsorbent according to the present invention specifically adsorbs and concentrates only viable bacteria. Presumed.

Reference Example 3 (1) Similar to Reference Example 1, an oil-based ferrofluid ferricolloid
A ferromagnetic superparamagnetic material (particle size: 0.01 μm) having a lipophilic surface was obtained from HC50 (trade name, manufactured by Taiho Kogyo KK). Then, 95 parts of styrene, 5 parts of methyl methacrylate and 3.5 parts of benzoyl peroxide (polymerization initiator) are added to 75 parts of the superparamagnetic substance obtained above, and the superparamagnetic substance is uniformly mixed and stirred by mixing the system. It was dispersed to prepare a monomer composition. on the other hand,
An aqueous monomer composition was prepared by dissolving 6 parts of polyvinyl alcohol, 0.23 part of sodium lauryl sulfate and 0.5 part of polyethylene oxide nonylphenyl ether in 1300 parts of water. The above monomer composition was added to the aqueous monomer composition thus prepared, and the mixture was preliminarily stirred with a homogenizer, and then subjected to dispersion treatment with an ultrasonic disperser, whereby oil droplets (oil phase) having an average particle diameter of 2.6 μm were formed. A suspension (oil droplet dispersion) dispersed in an aqueous medium was prepared. next,
The resulting suspension was charged into a 3 liter three-necked flask equipped with a stirrer, the system was heated to 75 ° C, and the monomers in the oil droplets were polymerized (suspended) for 9 hours while stirring under a nitrogen atmosphere. Polymerization) to produce magnetic polymer particles. The obtained particles were photographed with an optical microscope, the diameter of 200 particles was measured, and the average was determined. The result was 2.5 μm.

(2) 100 g of the magnetic particles obtained above
r. 1 part of SDS (Emal 10N) as an emulsifier, 1 part of nonionic emulsifier Emulgen 910, 50 parts of styrene
, 30 parts of TBAS-Q (2-methyl-3-sulfopropylacrylamide) and 1 part of azoisobutyronitrile were mixed and stirred. Thereafter, polymerization was carried out at 70 ° C. for 6 hours, and the obtained particles were measured for 200 particles under an optical microscope.
Sulfonated magnetic particles having an average particle size of 2.8 μm were obtained.

Example 6 Escherichia coli HB101 strain was inoculated on a culture plate and incubated at 37 ° C.
After culturing for 0 hours, the cells were re-inoculated into a liquid medium, cultured, finally diluted with a culture solution, and optically 1 × 1 using a wavelength of 620 nm.
An E. coli suspension of 0E3 E. coli / ml was obtained and used as a standard suspension. This standard suspension was placed in a 5 ml test tube, diluted sequentially with physiological saline, and 1 × 10 ² , 5 × 10, 1 × 10,
5, 1 / ml suspension was prepared. In this suspension,
Concentration 20mg / m prepared from five human fecal samples
One hundred ul of the stool suspension was added to each, to form an E. coli suspension line.

Example 7 Reference Example 2 was applied to each of the E. coli suspension trains prepared in Example 6.
5 mg of the sulfonated particles synthesized in
50 ul of an aqueous mM zinc chloride solution was added. A 5 ml test tube was set on a shaker and shaken at room temperature for 20 minutes to bring Escherichia coli into contact with the sulfonated magnetic particles and adsorb them. The magnetized magnetic particles of the adsorbed magnetic particles are separated by a magnet, washed with a Tris-HCl buffer (pH 7.4), and washed with a Tris-HCl buffer 100.
ul suspension was obtained, and thereafter, the presence of Escherichia coli was determined by PCR in the same manner as in Example 1, and those which were clearly stained by ethidium bromide staining were determined to be significant presence of Escherichia coli. (Total 25 times).

Example 8 45,000 r from the E. coli suspension train prepared in Example 6
After ultracentrifugation at pm for 30 minutes and removing the supernatant with a pipette, Tris-HCl buffer (pH 7.4) was used in the same procedure as in Example 7.
100 ul of suspension was obtained. Thereafter, a PCR reaction was performed in the same procedure as in the actual example, and the presence or absence of Escherichia coli was determined.

Table 1 shows the numbers in Examples 7 and 8 in which the presence of Escherichia coli was clearly determined in five samples at each concentration.

[Table 1] Compared with Example 8, when the sulfonated magnetic particles according to the present invention were used to specifically adsorb and concentrate Escherichia coli, even if the bacteria were present at an extremely low concentration, they were detected as very positive with high sensitivity. It turns out that it is possible. On the other hand, when the PCR reaction is performed after concentration by centrifugation, which has been widely used in the past, the interference factor presumed to be due to the contaminants has an effect, and the determination ratio of the presence in low-concentration E. coli samples decreases However, it was strongly suggested that false negative judgments could be made in the evaluation of actual samples.

[0044]

According to the method for detecting microorganisms and biological substances of the present invention, microorganisms and biological substances in a sample are specifically adsorbed, concentrated and separated with high efficiency prior to detection, and are contained in the sample. It is possible to efficiently and selectively detect microorganisms and biological substances even when the amount of the existing microorganisms and biological substances is extremely small.

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Claims (7)

[Claims] 1. A method in which a microorganism and / or a biological substance is specifically contained in a sample which may be contained at a concentration lower than a detection limit of a detection means for detecting the microorganism and / or the biological substance. A method for detecting a microorganism and a biological substance, which comprises contacting an adsorbent capable of being adsorbed on the adsorbent, and then applying a detecting means for the microorganism and / or the biological substance bound to the adsorbent. 2. The sample according to claim 1, wherein the sample is a body fluid, blood, excrement, tissue, cell, food, environmental water, circulating water, or a solution, suspension or dispersion thereof. A method for detecting microorganisms and biological substances. 3. The method according to claim 1, wherein the microorganism and the biological substance are protozoa,
The method for detecting a microorganism and a biological substance according to claim 1 or 2, wherein the method is a fungus, bacteria, rickettcher, mycoplasma, nucleic acid, antigen, antibody or protein. 4. An adsorbent capable of specifically adsorbing a microorganism and a biological substance is in the form of particles, a filter,
The method for detecting a microorganism and a biological substance according to claim 1, 2 or 3, wherein the method is in the form of a fiber, a sheet, a tube, or a plate. 5. The adsorbent capable of specifically adsorbing a microorganism and a biological substance is made of an organic polymer, an inorganic polymer, an inorganic compound or a natural polymer. 2. The method for detecting a microorganism or biological substance according to claim 1. 6. The method for detecting a microorganism and a biological substance according to any one of claims 1 to 5, wherein the means for detecting the microorganism and the biological substance is a nucleic acid amplification method or an immunochemical assay. 7. An adsorbent used for the detection method according to claim 1.