JP2001208756A - Bacterium detecting method and kit utilizing it - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a bacterium detecting method which is remarkably improved in selectivity, sensitivity, and quickness as compared with the conventional method. SOLUTION: This method by which bacteria contained in a sample is detected includes a step of providing an immunochromatograph provided with a sample migrating body having at least a sample introducing section and a bacterium catching section and an antibody which can be bonded specifically to a bacterium to be detected in a state that the antibody is already immobilized in the bacterium catching section, a step of bonding the bacterium to the antibody by introducing the sample to the sample introducing section, and a step of detecting the bacterium bonded to the antibody by bringing the bacterium into contact with a metabolite measuring reagent.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

The present invention relates to a method and a kit for detecting bacteria in a sample using an immunochromatographic device and a reagent for measuring metabolites, and more particularly, to a method for detecting a trace amount of bacteria from a sample with high sensitivity and high sensitivity. To methods and kits useful in the environmental, food and medical fields.

[0002]

2. Description of the Related Art Conventional methods for detecting bacteria include a method using an immunochromatographic device having a labeled antibody and a method for measuring adenosine triphosphate (ATP) in a solution system.
One is.

In a method for detecting bacteria using an immunochromatographic device, a sample containing the bacteria to be detected is usually introduced into an immunochromatographic device having an antibody capable of binding to the bacteria to be detected, electrophoresed, and the bacteria to be detected are sandwiched. To capture. In this method, generally, at least one type of antibody is immobilized on an immunochromatographic device, and the other antibody is carried in a state capable of migrating. A dye or an enzyme can be used as the label of the antibody. Bacteria are detected by coloring when a dye is used as the label, and by visualization by enzymatic reaction with a chromogenic substrate when an enzyme is used.

In the method of detecting bacteria by measuring ATP, a sample containing the bacteria to be detected is treated with an ATP extractant to release ATP in the bacteria and to remove AT from the bacteria.
Bacteria are detected by measuring the amount of luminescence generated by the reaction of P with luciferase and luciferin.

However, each of these conventional methods for detecting bacteria has drawbacks. The detection limit of the bacteria detection method using an immunochromatography device is limited by the affinity of an antibody that binds to the bacteria to be detected and the sensitivity of a detector that measures a label, and is about 1,000 bacteria per ml of a sample. Not satisfactory.

[0006] The detection limit of the bacteria detection method by ATP measurement is also about 1000 to 1000 bacteria per ml of sample.
Only about 0. Furthermore, in the ATP measurement method, it is not possible to specifically detect only ATP from the detection target bacteria, and it is also possible to simultaneously measure ATP from bacteria other than the detection target bacteria contained in the sample. In general, since a sample contains a plurality of types of bacteria, the obtained ATP measurement value often has an error as an index of the detection target bacteria.

[0007] In view of the above, there is a need for a method for detecting bacteria that can selectively and rapidly detect the target bacteria.

[0008]

SUMMARY OF THE INVENTION The present invention is intended to solve the above-mentioned problems, and provides a method for detecting bacteria which is significantly improved in selectivity, sensitivity and speed as compared with conventional methods. The purpose is to do.

[0009]

Means for Solving the Problems The present inventors introduce a sample into an immunochromatographic device on which an antibody capable of specifically binding to a bacterium to be detected is immobilized, and remove the sample from a sample which may contain a plurality of types of bacteria. After selectively capturing only the bacteria to be detected,
By detecting the captured bacteria using a metabolite measurement reagent, it has been found that only the target bacteria can be detected with high sensitivity and rapidity, and the present invention has been completed based on this.

[0010] The method of the present invention is a method for detecting bacteria in a sample, comprising a sample electrophoretic body having at least a sample introduction part and a bacteria capture part, and an antibody capable of specifically binding to the bacteria to be detected. Providing an immunochromatographic device comprising: a step in which the antibody is immobilized in the bacteria capturing unit; a step of introducing a sample into the sample introduction unit to bind the bacteria to the antibody; And bacteria bound to the antibody,
Detecting by contacting with a metabolite measuring reagent.

[0011] The metabolite measurement reagent can be added to at least a region containing the immobilized antibody on the immunochromatographic device.

The bacteria bound to the antibody can be transferred to a device containing the reagent for measuring metabolites.

[0013] The kit of the present invention is a kit for detecting bacteria in a sample, which includes an immunochromatography device and a device containing a metabolite measurement reagent, wherein the immunochromatography device has at least A sample electrophoresis body having a sample introduction part and a bacteria capture part, and an antibody capable of specifically binding to a bacterium to be detected are provided, and the antibody is immobilized on the bacteria capture part.

The device containing the metabolite measurement reagent may have a structure suitable for adding the metabolite measurement reagent to at least a region containing the antibody immobilized on the immunochromatographic device.

[0015] The device containing the metabolite measurement reagent may have a structure suitable for transferring bacteria bound to the antibody.

In the method and kit of the present invention, the reagent for measuring metabolites includes adenosine monophosphate (AMP), adenosine diphosphate (ADP), and adenosine triphosphate (A
TP), nicotinamide adenine dinucleotide (NA
D ⁺ ), reduced nicotinamide adenine dinucleotide (NADH), nicotinamide adenine dinucleotide phosphate (NADP ⁺ ), reduced nicotinamide adenine dinucleotide phosphate (NADPH), flavin mononucleotide (FMN), and reduced It may measure the metabolites are selected from the group consisting of flavin mononucleotide (FMNH _2).

In the method and kit of the present invention, the metabolite measuring reagent may include a metabolite-dependent enzyme, a luminescent substrate, and a metabolite extractant.

In the method and kit of the present invention, the metabolite-dependent enzyme may be luciferase, the luminescent substrate may be luciferin, and the metabolite extractant may be a surfactant.

The bacterium may belong to any of the following classes: rhodospirillum, chromatia, chlorobium, myxococcus, alkangium, cystobacter, polyangium, cytoferga, veggiatua, simonsierra, leucotrix, achromatium, peronema, spirocheta, Spirilum, Pseudomonas, Azotobacter, Rhizobium, Methylomonas, Halobacterium, Enterobacteriaceae, Vibrio, Bacteroides, Neisseria, Bayonela, Ammonia or nitrite bacteria,
Sulfur-metabolizing bacteria, iron oxide and / or manganese oxide depositing bacteria, siderocapsa, methanobacterium, aerobic or facultative anaerobic micrococcus, streptococcus, anaerobic peptococcus, bacillus, lactobacilli, coryneform bacteria, propionibacteria, actino Mrs., Mycobacterium, Frankia, Actinoplanes, Dermatofils, Nocardia, Streptomyces, Micromonospora, Rickettsia, Bartonella, Anaplasma,
Chlamydia, Mycoplasma, and Acoreplasma.

[0020]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail.

In the present invention, unless otherwise specified, a luminescence quantification method, an immunological technique, and the like, which are known in the art, can be employed. These techniques can be performed using commercially available devices, antibodies, labeling substances, and the like.

(Sample and Bacteria to be Detected) According to the method for detecting bacteria of the present invention, bacteria in a sample can be specifically detected. The sample can be any aqueous sample containing water as a solvent or dispersion medium. Examples of aqueous samples include urine, blood,
Samples of biological origin such as body fluids such as plasma, serum, saliva, milk, sweat and their fractions; samples of natural origin such as well water, ground water, tap water, fruit juice; and foodstuffs, vegetables, meat, It may be a sample in which a crushed product such as an egg is suspended in water.

Examples of bacteria to be detected include bacteria belonging to any of the following classes: Rhod
ospiriraceae, Ch
romatiaceae (Chromatiua), Chlor
obiaceae (chlorobium), Myxococc
aceae (Mixococcus), Archangiac
eae (alkangium), Cystobactera
ceae (cystobacter), Polyangiace
ae (polyangium), Cytophagaceae
(Sitefaga), Beggiatoaceae (Beguiatua), Simonsiellaceae (Simonsierra), Leucotrichaceae (Leucotrix), Achromatiaceae (Achromachium), Pelonematacee (Peronema), Spiera spiapea, Spiera and Piscesa
eudomonadaceae (Pseudomonas), A
zoobacteraceae (Azotobacter),
Rhizobiaaceae (Rhizobium), Methy
lomonadaceae (Methylomonas), Halo
bacteriaaceae (Halobacterium), En
terobacteriaceae (intestinal bacteria), Vi
brionaceae (vibrio), Bactero
idaceae (Bacteroides), Neisseri
aceae (Neisseria), Veillonellac
eae (Vayionella), ammonia or nitrifying bacteria (Organisms oxidizing amm)
onia or nitrite), sulfur metabolizing bacteria (O
rganisms metabolizing sul
fer and sulfur compound
s), bacteria that deposit iron oxide and / or manganese oxide (O
rganisms depositing irona
nd / or magnesium oxides), S
iderocapsaceae (siderocapsa), Me
anaerobic or facultative anaerobic (Aerobic acid)
r facultatively anaerobi
c), Micrococcaceae (Micrococcus), Streptococceae (Streptococcus), anaerobic (Anaerobic), Pept
ococcaceae (Peptococcus), Bacil
raceae (Bacillus), Lactobacilla
ceae (lactobacilli), coryneform bacteria (Cory)
neform group of bacteri
a), Propionibacteriaaceae (propionic acid bacteria), Actinomycetaceae
(Actinomyces), Mycobacteriacea
e (Mycobacterium), Franciaceae
(Frankia), Actinoplanaceae (Actinoplanes), Dermatophilacea
e (Dermatofilus), Nocardiaceae
(Nocardia), Streptomycetacea
e (Streptomyces), Micromonospor
aceae (micromonospora), Rickettsi
aceae (Rickettsia), Bartonellace
ae (Bartonella), Anaplasmamateae
(Anaplasma), Chlamydiaceae (Chlamydia), Mycoplasmamateae (Mycoplasma), and Acholeplasmamate
eae (achole plasma).

(Immunochromatographic Device) In the method for detecting bacteria of the present invention, an immunochromatographic device is used. The immunochromatographic device includes, as constituent elements, a sample electrophoretic body having at least a sample introduction part and a bacteria capture part, and an antibody capable of specifically binding to a detection target bacterium. As the material of the sample migrating body, any porous carrier that does not substantially exhibit nonspecific adsorption to the bacteria to be detected and allows the sample to move can be used. Examples of the porous carrier include cellulose, nitrocellulose, glass fiber filter paper, styrene resin, vinyl resin and the like.
When the affinity of the porous carrier for water is low, a surfactant may be used. The shape of the sample migration body is not particularly limited, but is preferably formed in a sheet shape. A continuous same porous carrier may be used for the sample introduction part and the bacteria capturing part of the sample migration body, or different materials may be used. The sample introduction part is preferably made of glass fiber filter paper or cellulose. The bacteria trap is preferably nitrocellulose, glass fiber filter paper. If both the sample introduction part and the bacteria trapping part are made of nitrocellulose, the flow rate of the sample becomes extremely low.Therefore, only the bacteria trapping part is made of nitrocellulose, and the sample introduction part is made of glass with a faster sample flow rate. It is preferable to use fiber filter paper or the like.

An antibody capable of binding specifically to the bacteria to be detected is immobilized on the bacteria capturing portion of the sample electrophoresis at a strength that does not substantially change the position due to the movement of the sample. This antibody can be any antibody that can specifically bind to the bacteria to be detected. "Able to bind specifically" means
It means that it can bind to the bacteria to be detected in the sample but cannot substantially bind to other bacteria that may be present in the same sample. The term “binding” as used herein refers to a binding by an antigen-antibody reaction, and does not include nonspecific adsorption.

An antibody capable of specifically binding to a bacterium to be detected can be immobilized on a sample electrophoresis in an appropriate amount by a method known in the art. For example, when the bacterial capturing part is nitrocellulose, the antibody can be non-specifically adsorbed by dropping a solution containing the antibody onto nitrocellulose, drying and washing. As another immobilization method, for example, the antibody may be immobilized on glass filter paper using a sugar conjugate described in Japanese Patent Application No. 8-283297.

The above antibody may be a polyclonal antibody or a monoclonal antibody, a chimeric antibody,
It may be in the form of a Fab antibody, (Fab) ₂ antibody or the like. The class of the antibody is not particularly limited, but preferably,
IgG. One skilled in the art can select an appropriate antibody according to the bacteria to be detected. Polyclonal or monoclonal antibodies that bind to certain bacteria are known in the art and are readily available. Antibodies can also be produced according to immunological methods known in the art. For example, when the bacterium to be detected is Salmonella, one or more anti-Salmonella monoclonal antibodies or anti-Salmonella polyclonal antibodies may be used as the antibody.

In the method for detecting bacteria of the present invention, when it is intended to transfer the captured bacteria to a device containing a reagent for measuring metabolites, the antibody can efficiently capture bacteria from a sample and subsequently transfer the bacteria. It has a range of binding affinities. The degree of binding affinity can be expressed using the "titer", ie, the highest dilution at which binding to the antigenic peptide is observed. Specifically, the preferred antibody titer in the present invention is 1/100 to 1
/ 10000 range.

[0029] The immunochromatographic device used in the method for detecting bacteria of the present invention may further include a water absorbing part on the sample electrophoresis body on the side opposite to the sample introducing part with respect to the bacterial capturing part. The water absorption part is a part used for quickly absorbing an excess sample, and is made of a porous substance having excellent water absorption capacity and water supply capacity. Examples of the porous substance include glass fiber filter paper and cellulose.

FIG. 1 shows an example of the configuration of an immunochromatographic device used in the method for detecting bacteria of the present invention. As shown in FIG. 1, the sample migrating body is assembled by attaching the sample introduction unit 101 on one end of the bacteria capturing unit 102 and the water absorption unit 103 on the other end. Alternatively, the sample migration unit may be assembled by sequentially arranging the sample introduction unit 101, the bacteria capturing unit 102, and the water absorption unit 103. The bacteria capturing unit includes a region 106 on which an antibody capable of specifically binding to the bacteria to be detected is immobilized. The sample migrating body is attached to the double-sided tape 104, and is held on the support 105 via the double-sided tape 104.

(Reagent for Measuring Metabolites) In the method for detecting bacteria of the present invention, a reagent for measuring metabolites for measuring metabolites such as ATP derived from captured bacteria is further used.

"Metabolite" refers to a molecule that is present in the cells of a bacterium and that is required for its biological function, which can be used to identify the presence of the bacterium in a sample. Examples of metabolites include adenosine monophosphate (also called AMP), adenosine diphosphate (also called ADP), adenosine triphosphate (also called ATP), nicotinamide adenine dinucleotide (also called NAD ⁺ ), reduced form Nicotinamide adenine dinucleotide (also called NADH), nicotinamide adenine dinucleotide phosphate (also called NADP ⁺ ), reduced nicotinamide adenine dinucleotide phosphate (NA
DPH), flavin mononucleotide (F
MN), and reduced flavin mononucleotides (also called FMNH ₂ ). AM
P, ADP, and ATP are important in the energy metabolism of organisms and are contained in all living cells. In the present invention, a particularly preferred metabolite is ATP.

The “metabolite measurement reagent” may include any combination of components that enables the extraction and measurement of metabolites from bacteria. The metabolite measurement reagent may be a luminescent reagent or a chromogenic reagent. If a luminescent reagent, it may include a metabolite-dependent enzyme, a luminescent substrate, and a metabolite extractant. An appropriate combination of the metabolite-dependent enzyme and the luminescent substrate can be selected so as to enable a desired luminescent reaction.

For example, to detect metabolites, a luminescence reaction represented by the following formula can be used: (1) When the metabolite is AMP, ADP, or ATP: AMP + substrate → ATP + enzyme reaction product Formula (1a) (Example of catalyst: pyruvate orthophosphate dikinase) ADP + substrate → ATP + enzyme reaction product Formula (1b) (Example of catalyst: pyruvate kinase) ATP + luciferin + O ₂ → product + light Formula (1c) (Example of catalyst: luciferase); (2) When the metabolite is NAD (P) H, NAD (P) ⁺ , FMN, or FMNH ₂ : NAD (P) H + FMN + H ⁺ → FMNH ₂ + NAD (P) ⁺ formula (2a) (examples of catalysts: NADH: FMN oxidoreductase or NADPH: FMN oxidoreductase) FMNH ₂ + O ₂ + R _{HO → FMN + RCOOH + H 2} O + light formula (2b) (Example of Catalyst: emission bacterial luciferase; wherein, RCHO is a long chain aliphatic aldehyde, RCOOH represents a fatty acid corresponding to the aldehyde).

When measuring AMP, the reaction is represented by the formula (1)
a) and equation (1c). In this case, in addition to the metabolite extractant, an enzyme (eg, pyruvate orthophosphate dikinase) that catalyzes a reaction to produce ATP from AMP, a substrate for the enzyme, and an organism that depends on ATP It may include an enzyme that catalyzes a luminescent reaction (eg, luciferase) and a substrate for the enzyme. Pyruvate orthophosphate dikinase acts on AMP, phosphoenolpyruvate and pyrophosphate in the presence of magnesium ions,
It is an enzyme that reversibly catalyzes the reaction that produces TP, pyruvate, and phosphate. Thus, the metabolite measurement reagent can be, for example, a reagent containing pyruvate orthophosphate dikinase, phosphoenolpyruvate, pyrophosphate, luciferin, luciferase, and a metabolite extractant. All components of the reagents, including pyruvate orthophosphate dikinase, are readily available.

When measuring ADP, the reaction is represented by the formula (1)
b) and equation (1c). In this case, the metabolite measuring reagent is, in addition to the metabolite extractant, an enzyme that catalyzes a reaction for producing ATP from ADP and a substrate for the enzyme, and an enzyme that catalyzes an ATP-dependent bioluminescent reaction and an enzyme for that enzyme. A substrate may be included. Examples of enzymes that catalyze the reaction that produces ATP from ADP and substrates for the enzymes include: pyruvate kinase (substrate: phosphoenolpyruvate); acetate kinase (substrate: acetyl phosphate); creatine kinase (Substrate: creatine phosphate); carbamate kinase (substrate: carbamoyl phosphate); and phosphoglycerate kinase (substrate: 3-phospho-D-glycerate).

When ATP is directly measured, the reaction is represented by the formula (1)
According to c), the metabolite-dependent enzyme is preferably luciferase. Luciferase used as an ATP-dependent enzyme is any luciferase that requires ATP as a cofactor, such as Cypridina luciferase, the following beetles (Coleoptera Coleopter)
Luciferases from a) and natural or synthetic variants that retain their function: Luci
ola cruciata,
Luciola lateralis, Luciola mingrelica, Lampyris nact
iluca (Lampiris Naktilka), Photoi
nus pyralis (Photinus pyralis),
Photouris pennsylvanica (Photouris Pennsylvanica), Hotaria par
vula (Hotalia Parvula), Pyrocoel
ia miyako (pyrochoria miyako), and Pyrophorus plagiophthalam
us (Pyrophorus pragyotarumus). Luciferase may be one isolated from nature or one produced using a genetic recombination method.

When luciferase is used as a metabolite-dependent enzyme, luciferin is used as a luminescent substrate.
Luciferin that can be used in the present invention includes all of natural luciferin, luciferin derivatives, and luciferin precursors. Examples of luciferin derivatives include:
For example, the following may be mentioned: D-luciferin-
O-sulfate, D-luciferin methyl ester, D-
Luciferin-O-phosphate, D-luciferin-L-
Phenylalanine, and D-luciferin-L-Nα
-Arginine. Examples of luciferin precursors include, for example, 2-cyano-6-methoxybenzothiazole and D-cysteine.

As is well known, luciferase has a high degree of substrate specificity and generally acts only on luciferin from closely related species. One of skill in the art will readily select an appropriate luciferase and luciferin combination.

NAD (P) H, NAD as metabolites
When (P) ⁺ , FMN, or FMNH ₂ is measured, the combination of the metabolite-dependent enzyme and the luminescent substrate constituting the metabolite measurement reagent is determined based on the formulas (2a) and (2b). For example, when measuring NADH, the metabolite measuring reagent may include, in addition to the metabolite extractant, NADH: FMN oxidoreductase and luciferase as metabolite-dependent enzymes, and aldehyde as a luminescent substrate. The metabolite-dependent enzyme can also be hololuciferase (holoenzyme) (JP-A-6-153995).
See). When using phoruciferase from luminescent bacteria (having FMN as a cofactor), an enzyme that catalyzes formula (2a) is not required. Examples of luminescent bacteria that produce hololuciferase include, for example, Vibrio harveyi, Vibrio fisheri, Vibrio splendida.
dus), Vibrio cholerae (Vibrio cho)
lerae), Photobacterium phosphoreum (Photobacterium phosphore)
um), and Photobacterium leiognashi (P
photobacterium leiognathi)
Is mentioned.

The metabolite measuring reagent may also be a chromogenic reagent. The metabolite measurement reagent is a chromogenic reagent, for example, NA
In the case of a reagent for detecting DH or NADPH, a color reaction represented by the following formula can be used: NAD (P) H + O ₂ + H ⁺ → NAD (P) ⁺ + H ₂ O ₂ Formula (3a) ( Example of catalyst: NAD (P) H oxidase) H ₂ O ₂ + oxidative chromogenic substrate → color body Formula (3b) (Example of catalyst: peroxidase).

Based on equations (3a) and (3b), N
In the case of detecting ADH or NADPH, the metabolite measuring reagent includes, in addition to the metabolite extractant, an enzyme that catalyzes the reaction of the formula (3a), an oxidative chromogenic substrate (a substrate that reacts with H ₂ O ₂ to generate color) And an enzyme that catalyzes the color reaction (formula (3b)
Enzyme that catalyzes the reaction of

The enzyme catalyzing the reaction of the formula (3a) is NA
And specifically oxidize the DH or NADPH to generate H ₂ O _2, it may be any enzyme such as NAD (P) H oxidase.

An example of an enzyme that catalyzes the color reaction of the formula (3b) is peroxidase. in this case,
The oxidized chromogenic substrate can be a reagent comprising 3-methyl-2-benzothiazolinone-hydrazone (also called MBTH) or a derivative thereof, and a phenol or aniline derivative. Examples of phenols include phenol, 2,4-dichlorophenol, 2,4-dibromophenol, and 3,5-dichloro-2-hydroxybenzenesulfonic acid. Examples of the aniline derivative include N, N-dimethylaniline, N, N-diethylaniline, N-ethyl-N- (2-hydroxyethyl)
-M-toluidine, N-ethyl-N- (3-methylphenyl) -N'-acetylethylenediamine, N, N-dimethyl-m-anisidine, and N of aniline with alkylsulfonic acid or hydroxyalkylsulfonic acid.
Substituents are mentioned. Examples of MBTH derivatives include:
Examples include those in which substituents such as a sulfone group, an alkyl group, a halogen atom, a nitro group, and a carboxyl group are introduced into MBTH.

Examples of the metabolite extractant include a surfactant,
And an organic solvent (eg, ethanol).
Surfactants include, for example, non-ionic surfactants, cationic surfactants, and anionic surfactants. Examples of nonionic surfactants include Triton X
-100 and Triton X-200. Examples of cationic surfactants include alkyldimethylbenzylammonium chloride (benzalkonium chloride), benzethonium chloride, and cetyltrimethylammonium chloride. Examples of the anionic surfactant include sodium alkylbenzenesulfonate and sodium dodecyl sulfate. When a surfactant is used as the metabolite extractant, the metabolite measurement reagent preferably further contains a reagent (enzyme reaction inhibition inhibitor) capable of suppressing the inhibition of the enzyme reaction by the surfactant. Examples of the enzyme reaction inhibition inhibitor include α-cyclodextrin and β-cyclodextrin.
Cyclodextrin, γ-cyclodextrin, and silicone. Those skilled in the art can select an appropriate enzyme reaction inhibition inhibitor and its concentration according to the type and concentration of the surfactant.

Particularly when ATP is directly measured, the metabolite measurement reagent may further contain a metal salt. Examples of metal salts include divalent metal salts, for example, magnesium salts such as magnesium sulfate and magnesium chloride, and manganese salts such as manganese sulfate and manganese chloride. One skilled in the art can select an appropriate metal salt depending on the metabolite-dependent enzyme used.

The metabolite measuring reagent may further contain other additives as necessary, for example, an enzyme stabilizer and a buffer. Examples of enzyme stabilizers include dithiothreitol (a luciferase stabilizer), monosaccharides (eg, glucose, galactose, fructose, xylose), disaccharides (eg, sucrose, maltose), and polysaccharides. As an example of the buffer, the pH of the metabolite measurement reagent is kept within the optimal pH range of the metabolite-dependent enzyme,
Glycine-NaOH buffer, HEPES (hydroxyethylpiperazineethanesulfonic acid) buffer, and Tr
is (tris (hydroxymethyl) aminomethane) buffer. The metabolite measurement reagent may include any medium component capable of growing bacteria, but usually does not require a medium component.

The metabolite-dependent enzyme, luminescent substrate, and metabolite extractant are luciferase, luciferin,
For Triton X-200, examples of preferred concentrations of the ATP-dependent enzyme, the luminescent substrate, and the ATP extractant in the ATP measurement reagent are as follows: luciferase, 0.1 μM to 10 μM; luciferin, 0.1 μM
~ 100 μM; Triton X-200, 0.001% ~
0.5%.

[0049] The metabolite measurement reagent can be prepared in a desired form by a method known in the art. When it is intended to transfer the captured bacteria to a metabolite measurement reagent, a solid metabolite measurement reagent is preferred. If it is intended to add a metabolite measurement reagent to the immunochromatographic device,
Preferably, it is a viscous liquid or soft gel-like semi-solid metabolite measurement reagent or a solid metabolite measurement reagent having a viscosity that does not pass through the porous carrier of the immunochromatography device. The metabolite measurement reagent can be prepared in a semi-solid or solid form by including, for example, a gelling agent selected from agar, agarose, agaropectin, carrageenan, gellan gum, and the like.

For example, a liquid metabolite measuring reagent is prepared by dissolving each component of the metabolite measuring reagent in water and appropriately adjusting the pH as necessary. An appropriate pH value can be selected according to the type of metabolite-dependent enzyme. Further, for example, after preparing a liquid metabolite measurement reagent, after adding a gelling agent and heating and dissolving, dispensing into an appropriate container, cooling to an appropriate temperature, gel-like, semi-solid, or A solid metabolite measurement reagent is prepared. Metabolite-dependent enzymes
To prevent denaturation due to heat, the solution may be added at a stage where the solution in which the gelling agent is dissolved is cooling to some extent and gelling, or may be added after the solution is completely gelled.

The device containing the metabolite measurement reagent can be provided by containing the metabolite measurement reagent in a container of any shape. Examples of the shape of the container include a plate, a dish, a beaker, a flask, a tube, a cell, a vial, a bottle, and the like. The device containing the metabolite measurement reagent
It can be used to transfer bacteria immobilized on an immunochromatographic device (the device in this case is also called a “transfer device”). The transfer device has a shape with a large opening such as a plate or a dish, and the surface of the metabolite measurement reagent exposed from the opening is larger than the area of the region on the immunochromatography device where the antibody is immobilized. It is preferred to have a large area plane. The transfer device may further include an optional sheet for covering the opening to prevent drying of the metabolite measurement reagent. The sheet can be made of any material and thickness as long as it does not transmit moisture.
Examples of sheet materials include polyethylene, vinyl chloride copolymer, vinyl chloride vinyl acetate copolymer, and the like. FIG. 2 shows an example of an ATP measurement pad covered with a transparent sheet.

On the other hand, the metabolite measurement reagent can be added to at least the region containing the immobilized antibody on the immunochromatography device (the device in this case is referred to as “addition device”).
Also called). The addition device can be provided by accommodating each form of the metabolite measurement reagent in a container of any shape suitable for the addition. Those skilled in the art can select an appropriate device depending on the form of the metabolite measurement reagent and the like.

The material of the container for the metabolite measuring reagent is not particularly limited, and examples thereof include plastics such as polystyrene, polypropylene, polyvinyl chloride and polycarbonate; glass; and silicone resins. The volume of the reagent contained in the container can be appropriately set in consideration of the size of the immunochromatographic device, particularly the amount of the immobilized antibody, and is typically about 0.1 mL to about 1 L, preferably about 1 L.
mL to about 100 mL.

(Bacteria Detection Method and Kit) In the bacteria detection method of the present invention, first, a sample electrophoretic body having at least a sample introduction part and a bacteria capture part as described above, A sample is introduced into a sample introduction part of an immunochromatographic device containing a bindable antibody to bind bacteria to the antibody. When the sample is added to the sample introduction section of the immunochromatographic device, it migrates from the sample introduction section in a direction passing through the bacteria capturing section. The detection target bacteria that have reached the antibody immobilization region in the bacteria capturing unit are immobilized by binding to an antibody that can specifically bind to the detection target. Bacteria that do not bind to the antibody and other components in the sample migrate through the bacterial capture. The electrophoresis of the sample is preferably performed for a period of time sufficient to prevent bacteria and other components that do not bind to the antibody from substantially remaining in the bacterial capture.

Bacteria bound to the antibody are detected using a reagent for measuring metabolites. The metabolite measurement reagent can be added, for example, to at least the antibody-immobilized region of the bacterial capturing unit of the immunochromatographic device. In a preferred embodiment, the metabolite measurement reagent is added so as not to come into contact with the part other than the bacteria trapping part, particularly the water absorbing part. By adding the metabolite measurement reagent in a position-selective manner as described above, there is an advantage that measurement of metabolites from bacteria other than the detection target can be easily prevented. On the other hand, it is also possible to add a metabolite measurement reagent to the entire immunochromatographic device, in which case a semi-solid or solid metabolite measurement reagent is used to the extent that metabolites from other bacteria are not diffused, and the bacterial It is preferable that the detection (for example, the detection of luminescence) is performed only at the bacteria capturing unit. The addition of the metabolite measuring reagent to the entire immunochromatographic device has advantages such as easy operation even when the immunochromatographic device is small, and easy detection of the bacteria to be measured.

Alternatively, at least the region containing the immobilized antibody on the immunochromatographic device can be immersed in the metabolite measurement reagent in the device containing the metabolite measurement reagent. Such an immersion method is also included in an embodiment in which the metabolite measurement reagent is “added” onto the chromatographic device. in this case,
It is preferable to immerse the immunochromatographic device from which the water absorbing part has been removed.

Bacteria bound to the antibody can also be transferred to a device containing a metabolite measurement reagent. After the bacteria are immobilized, the area containing at least the antibody immobilization part of the bacterial capture part of the immunochromatographic device is brought into contact with the surface of the metabolite measurement reagent in the transfer device, and then the antibody immobilization part is The area containing the reagent and the transcription device containing the metabolite measurement reagent are separated from each other. As a result, the bacteria to be detected are peeled from the antibodies immobilized on the immunochromatographic device, and are transferred to the metabolite measurement reagent. At least the antibody-immobilized region and the surface of the metabolite measurement reagent of the bacterial capture portion of the immunochromatographic device, for example, by inverting and overlapping a transfer device containing the metabolite measurement reagent on the immunochromatographic device, Or, conversely, the immunochromatographic device can be brought into contact by inverting and overlapping the transfer device. When a transfer device is used, it is preferable that the water-absorbing section that may contain bacteria not to be detected is not brought into contact with the metabolite measurement reagent.

Next, the reaction between the metabolite measurement reagent and the metabolite can be detected by using an appropriate method or apparatus in accordance with the metabolite measurement reagent, the metabolite, the reaction to be used, and the like. When the reaction between the metabolite measurement reagent and the metabolite is a luminescence reaction or a coloring reaction, the luminescence or coloring generated by the reaction can be visually recognized, or can be simply performed using a common light detector or color detector. It can be detected and quantified.

When the metabolite measuring reagent is a luminescent reagent,
For example, a bioluminescence image analysis system device (for example, RMD
S (trade name, manufactured by Nippon Millipore Co., Ltd.) can be used to determine the number of bacteria by measuring the emission point. Alternatively, the luminescence can be measured using, for example, a luminometer using a photomultiplier tube or diode as a photodetector, or using photographic techniques such as a polaroid camera.

When the metabolite measuring reagent is a chromogenic reagent,
The reaction can be detected with the naked eye. Alternatively, the sensitivity and accuracy of the measurement can be increased by measuring the color development using a measuring device such as a densitometer.

If necessary, bacteria can be quantified from the amount of luminescence or the amount of color development. As an example of a method for quantifying bacteria in a sample, prepare a standard sample containing a known amount of bacteria to be measured in advance and create a calibration curve showing the relationship between the amount of luminescence or color development and the number of bacteria in the standard sample. And comparing the amount of luminescence or color development measured from the sample with a calibration curve. A quantification method using such a calibration curve is well known in the art.

The kit of the present invention comprises the above-mentioned immunochromatographic device and a device containing a metabolite measuring reagent.
Each component of the kit of the present invention can be usually provided by being sealed with a package to avoid contamination with various bacteria, and packed with appropriate instructions.

[0063]

The present invention will be described in more detail with reference to the following examples. The present invention is not limited to the following examples.

<Examples> Specific examples of Salmonella detection using the bacteria detection method of the present invention will be described below.

(Immobilization of Salmonella antibody) Approximately 5 cm ×
About 2 cm from the short side end of about 0.9 cm of nitrocellulose (manufactured by Millipore), 10 μL of a phosphate buffered saline (PBS) solution containing 1 mg / mL of an anti-Salmonella polyclonal antibody was added to the length of nitrocellulose. An anti-Salmonella polyclonal antibody was immobilized by dripping in a line shape having a width of about 1 mm from end to end in a direction perpendicular to the side, and then drying in a light-shielded state at 40 ° C. for 20 minutes. The titer of the used anti-Salmonella polyclonal antibody against Salmonella was 1/2000. The obtained immobilized nitrocellulose was infiltrated into a 0.1 M Tris solution (pH 8.2) containing 1% skim milk, and blocked by shaking at 30 ° C. for 30 minutes using a shaker. After blocking, the nitrocellulose was washed with 0.1 M Tris solution (pH 8.2) at 30 ° C.
After washing three times in 0 minutes, 40 minutes in an incubator.
Dry at 4 ° C. for 4 hours. The antibody-immobilized nitrocellulose after drying was used as a porous carrier for the bacteria capturing part.

(Construction of Immunochromatography Device) As a porous carrier of the sample introduction part 101 and the water absorption part 103, glass fiber pieces of about 2 cm × about 0.9 cm were used.

About 7 cm × about 0.9 cm double-sided tape 10
4 was stretched on the surface of a support 105 of a polyvinyl plate of about 7 cm × about 0.9 cm. On the other side of the double-sided tape 104,
The above-mentioned nitrocellulose (bacterial capturing part 102) immobilized with the anti-Salmonella antibody was adhered, and then the sample introduction part 101 and the water absorbing part 1 were attached to both ends of the nitrocellulose, respectively.
03 in the same manner as the arrangement shown in FIG. 1 except that a part of the sample introduction part 101 and a part of the bacteria capturing part 102, and a part of the water absorbing part 103 and a part of the bacteria capturing part 102 are overlapped. The sample electrophoresis body 100 was formed by bonding, and an immunochromatography device was produced. The sample introduction part is on the upstream side, and the water absorption part is on the downstream side.

(Electrophoresis of Salmonella dispersion) 200 μl of Salmonella dispersion (1, 5, 10, 100 or 1)
000 cells / ml) was added to the sample introduction unit 101 of the immunochromatographic device prepared above, and left for 5 minutes.
During this time, the added dispersion liquid migrated from the sample introduction unit 101 to the water absorption unit 103 through the antibody immobilization unit 106 in the bacteria capturing unit 102.

(ATP Measurement) A Salmonella dispersion was used as a sample.
The immunochromatographic device 5 minutes after addition to the introduction section (Fig. 3
(A)) shows an ATP measurement pad shown in FIG.
Peel off the transparent sheet of the transfer device
Thus, the surface of the ATP measurement reagent overlaps with the region containing the bacteria trapping part.
(FIGS. 3B to 3C). This AT
The P measurement pad is a 2 cm polystyrene made 5 mm deep.
1.5 × 10 on a 2 × 2 cm plate ^-7M Lucifera
(ATP-dependent enzyme), 1.5 × 10^-6Lucifer of M
Phosphorus (luminescent substrate) and 0.1% Triton X-2
Gelation to ATP measurement reagent containing 00 (ATP extractant)
Add about 10% agarose as an agent and dissolve by heating
After filling, the mixture was cooled.

After maintaining the above mounting state at 37 ° C. for 10 minutes, the ATP measurement pad was peeled off from the immunochromatography device. Two minutes later, the amount of luminescence from the measurement pad was measured using an optical detector (K-210, manufactured by Kikkoman) (FIG. 3 (d)), and Salmonella was detected. As a result, detection was possible even when the concentration of the Salmonella dispersion was 5 bacteria / ml (FIG. 4). Thus, according to the method of the present invention, bacteria can be selectively detected with very high sensitivity and quickly.

[0071]

According to the method for detecting bacteria of the present invention, the bacteria to be detected can be selectively detected with high sensitivity and speed with high reliability. According to the present invention, a high-performance bacteria detection kit useful in the fields of environmental measurement, food management measurement, and medical diagnosis is provided.

[Brief description of the drawings]

FIG. 1 is a configuration diagram of an immunochromatographic device used in the present invention.

FIG. 2 is a schematic view showing an ATP measurement pad used in an example.

FIG. 3 is a schematic diagram showing an operation process of detecting bacteria by the method of the present invention. (A) shows an immunochromatographic device to which a sample containing bacteria has been added. (B) shows that the immunochromatographic device after electrophoresis of the sample containing bacteria has A
The operation | movement which attaches a TP measurement pad is shown. (C) shows at least the antibody-immobilized region of the immunochromatographic device and AT
Fig. 3 shows a state where an ATP measurement reagent contained in a P measurement pad is brought into contact with the reagent. (D) shows the measurement of the amount of luminescence due to the bioluminescence reaction from the ATP measurement pad peeled off from the immunochromatographic device.

FIG. 4 is a graph showing the detection sensitivity of Salmonella according to an example of the present invention.

[Explanation of symbols]

 DESCRIPTION OF SYMBOLS 101 Sample introduction part 102 Bacterial capture part 103 Water absorption part 104 Double-sided tape 105 Support 106 Antibody immobilization area

Claims (14)

[Claims] 1. A method for detecting bacteria in a sample, comprising: a sample migrating body having at least a sample introduction part and a bacteria capturing part; and an antibody comprising an antibody capable of specifically binding to the bacteria to be detected. Providing a chromatographic device, wherein the antibody is immobilized on the bacterial capture;
Introducing a sample into the sample introduction part to bind the bacterium to the antibody; and detecting the bacterium bound to the antibody by contacting the antibody with a metabolite measurement reagent. 2. The method according to claim 1, wherein the metabolite measurement reagent is added to at least a region containing the antibody immobilized on the immunochromatographic device. 3. The method of claim 1, wherein the bacteria bound to the antibody are transferred to a device containing the metabolite measurement reagent. 4. The method according to claim 1, wherein the metabolite measuring reagent is adenosine monosaccharide.
Acid (AMP), adenosine diphosphate (ADP),
Nosin triphosphate (ATP), nicotinamide adenine di
Nucleotide (NAD⁺), Reduced nicotinamide
Nindinucleotide (NADH), nicotinamide ade
Nindinucleotide phosphate (NADP ⁺), Reduction type Nico
Tinamide adenine dinucleotide phosphate (NADP
H), flavin mononucleotide (FMN), and
Flavin mononucleotide (FMNH)_TwoConsisting of
2. The method according to claim 1, wherein a metabolite selected from the group can be measured.
the method of. 5. The metabolite measurement reagent according to claim 1, wherein the metabolite measurement reagent includes a metabolite-dependent enzyme, a luminescent substrate, and a metabolite extractant.
The method described in. 6. The method according to claim 5, wherein said metabolite-dependent enzyme is luciferase, said luminescent substrate is luciferin, and said metabolite extractant is a surfactant. 7. The method according to claim 1, wherein the bacterium belongs to any of the following classes: rhodospirillum, chromatia, chlorobium, myxococcus, alkangium, cystobacter, polyangium, cytofaga, veggiatua, simimonsiera, Leukotrix,
Achromium, peronema, spirochetes, spirils, pseudomonas, azotobacter, rhizobium, methylomonas, halobacterium, enterobacteria, vibrio, bacteroides, neisseria, bayonera, ammonia or nitrite bacteria, sulfur metabolizing bacteria, iron metabolizing bacteria, iron oxide and / or manganese oxide Deposited bacteria, siderocapsa, methanobacterium, aerobic or facultative anaerobic micrococcus, streptococcus, anaerobic peptococcus, bacillus, lactobacilli, coryneform bacteria, propionibacteria, actinomyces, mycobacterium, franchia, actinoplanes , Dermatophylls, Nocardia, Streptomyces, Micromonospora, Rickettsia, Bartonella, Anaplasma, Chlamydia, Mycoplasma, and Acole Plasma. 8. A kit for detecting bacteria in a sample, the kit comprising an immunochromatographic device and a device containing a metabolite measuring reagent, wherein the immunochromatographic device has at least a sample introduction part and A kit comprising: a sample electrophoretic body having a bacteria capturing portion; and an antibody capable of specifically binding to a bacteria to be detected, wherein the antibody is immobilized on the bacterial capturing portion. 9. A device containing the metabolite measurement reagent,
The kit according to claim 8, wherein the kit has a structure suitable for adding the reagent for measuring metabolites to at least a region including the antibody immobilized on the immunochromatographic device. 10. The kit according to claim 8, wherein the device containing the metabolite measurement reagent has a structure suitable for transferring bacteria bound to the antibody. 11. The metabolite measuring reagent is adenosine monophosphate (AMP), adenosine diphosphate (ADP), adenosine triphosphate (ATP), nicotinamide adenine dinucleotide (NAD ⁺ ), reduced nicotinamide Adenine dinucleotide (NADH), nicotinamide adenine dinucleotide phosphate (NADP ⁺ ), reduced nicotinamide adenine dinucleotide phosphate (NADP ⁺ )
H), flavin mononucleotide (FMN), and reduced flavin mononucleotide (may measure metabolites selected from the group consisting of FMNH _2), kit of claim 8. 12. The metabolite measurement reagent according to claim 8, comprising a metabolite-dependent enzyme, a luminescent substrate, and a metabolite extractant.
The kit according to 1. 13. The kit according to claim 12, wherein said metabolite-dependent enzyme is luciferase, said luminescent substrate is luciferin, and said metabolite extractant is a surfactant. 14. The kit according to claim 8, wherein said bacterium belongs to any of the following classes: rhodospirillum, chromatia, chlorobium, myxococcus, alkangium, cystobacter, polyangium, cytofaga, veggiatua, simimonsiera, Leukotrix,
Achromium, peronema, spirochetes, spirils, pseudomonas, azotobacter, rhizobium, methylomonas, halobacterium, enterobacteria, vibrio, bacteroides, neisseria, bayonera, ammonia or nitrite bacteria, sulfur metabolizing bacteria, iron metabolizing bacteria, iron oxide and / or manganese oxide Deposited bacteria, siderocapsa, methanobacterium, aerobic or facultative anaerobic micrococcus, streptococcus, anaerobic peptococcus, bacillus, lactobacilli, coryneform bacteria, propionibacteria, actinomyces, mycobacterium, franchia, actinoplanes , Dermatophylls, Nocardia, Streptomyces, Micromonospora, Rickettsia, Bartonella, Anaplasma, Chlamydia, Mycoplasma, and Acole Plasma.