WO2015093544A1 - 乳汁中のブドウ球菌を検出する方法 - Google Patents
乳汁中のブドウ球菌を検出する方法 Download PDFInfo
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- WO2015093544A1 WO2015093544A1 PCT/JP2014/083459 JP2014083459W WO2015093544A1 WO 2015093544 A1 WO2015093544 A1 WO 2015093544A1 JP 2014083459 W JP2014083459 W JP 2014083459W WO 2015093544 A1 WO2015093544 A1 WO 2015093544A1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
- G01N33/56911—Bacteria
- G01N33/56938—Staphylococcus
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/52—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from bacteria or Archaea
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
- C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
- C12Q1/14—Streptococcus; Staphylococcus
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Y—ENZYMES
- C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
- C12Y304/24—Metalloendopeptidases (3.4.24)
- C12Y304/24075—Lysostaphin (3.4.24.75)
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/543—Immunoassay; Biospecific binding assay; Materials therefor with an insoluble carrier for immobilising immunochemicals
- G01N33/54366—Apparatus specially adapted for solid-phase testing
- G01N33/54386—Analytical elements
- G01N33/54387—Immunochromatographic test strips
- G01N33/54388—Immunochromatographic test strips based on lateral flow
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
- G01N33/558—Immunoassay; Biospecific binding assay; Materials therefor using diffusion or migration of antigen or antibody
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
- G01N2333/305—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Micrococcaceae (F)
- G01N2333/31—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Micrococcaceae (F) from Staphylococcus (G)
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/914—Hydrolases (3)
- G01N2333/948—Hydrolases (3) acting on peptide bonds (3.4)
- G01N2333/95—Proteinases, i.e. endopeptidases (3.4.21-3.4.99)
- G01N2333/952—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from bacteria
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/914—Hydrolases (3)
- G01N2333/948—Hydrolases (3) acting on peptide bonds (3.4)
- G01N2333/95—Proteinases, i.e. endopeptidases (3.4.21-3.4.99)
- G01N2333/964—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue
- G01N2333/96425—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals
- G01N2333/96427—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals in general
- G01N2333/9643—Proteinases, i.e. endopeptidases (3.4.21-3.4.99) derived from animal tissue from mammals in general with EC number
- G01N2333/96486—Metalloendopeptidases (3.4.24)
Definitions
- the present invention relates to a lysis method and a detection method for detecting staphylococci that are mastitis-causing bacteria from the milk of livestock.
- Livestock milk represented by cows, sheep and goats is not aseptic and may contain some microorganisms due to disease or the environment. In particular, it is known that many microorganisms are eradicated in milk in diseases caused by infection of microorganisms in the breast. Mastitis is a typical livestock disease caused by microbial infection.
- Mastitis is inflammation of the ductal system and mammary gland tissue, and is mainly caused by microorganisms invading, colonizing and proliferating in the breast. Mastitis affects many animals, but bovine mastitis, particularly in dairy cows, is said to affect 15-40% of all dairy cows and is one of the most important diseases for dairymen.
- milk synthesis functions are inhibited, resulting in not only a decrease in milk production and, in some cases, halting lactation, but also significant economic losses such as treatment costs and milk price penalties due to reduced milk quality.
- labor burdens on dairy farmers will increase, such as individually milking mastitis-affected quarters to prevent infection.
- Mastitis is caused by infection with various microorganisms.
- staphylococci particularly Staphylococcus aureus
- Causative bacteria for intractable mastitis are known to be transmitted to other dairy cows via a milking machine or the like.
- Culture methods are widely used as a method for detecting staphylococci from milk.
- the culture method is not suitable for rapid identification of causative bacteria because it takes several days to obtain results.
- an identification method by an antigen-antibody reaction using an antibody against a component specific to a causative bacterium, particularly an immunochromatographic method can determine the result in several tens of minutes, and is widely used as a rapid and simple test method (for example Patent Document 1).
- Patent Document 2 The present inventors have also examined the immunochromatographic method as a method for detecting substances in the milk of livestock (Patent Document 2).
- Mastitis is caused by infection with various microorganisms, but the antibiotics that are effective depend on the causative bacteria.
- certain microorganisms are transmitted to the other quarter or individual, and their properties and subsequent responses differ, it is extremely important to identify causative bacteria present in milk quickly, easily, and with high sensitivity.
- infectious Staphylococcus aureus which is a cause of refractory mastitis
- the number of Staphylococcus aureus bacteria in milk is often small, and a method that can be detected quickly and with high sensitivity is required.
- the culture method widely used as a method for detecting bacteria has a problem that it takes several days to obtain a result.
- an immunoassay method based on an antigen-antibody reaction such as an immunochromatography method has an advantage that a causative bacterium can be detected quickly and easily, and early antimicrobial treatment is possible.
- the present invention solves the problem of providing a lysis method, a lysis solution and an immunochromatography apparatus for detecting whether or not the causative bacterium of mastitis is staphylococci using livestock milk. It was an issue that should be done.
- the present inventor believes that in order to detect staphylococci in milk, it is necessary to extract the target antigen in the bacterial body with high efficiency, and in order to lyse staphylococci in milk with high efficiency, It was found that the efficiency of lysis was improved by simultaneously using a plurality of surfactants, and staphylococci in milk could be detected with high sensitivity, and the present invention was completed.
- the present invention provides the following.
- a method for lysis of staphylococci comprising a step of lysing staphylococci present in milk by mixing a lytic enzyme and a lytic agent containing at least one amphoteric surfactant with milk obtained from livestock. Including.
- the lytic enzyme is lysostaphin.
- the lysis agent further comprises at least one nonionic surfactant.
- the amphoteric surfactant is any one selected from the group consisting of dimethylammoniopropane sulfonate, dodecyldimethylammoniobutylate, betaine laurate, and amidopropyl betaine.
- the method as described in any one.
- the nonionic surfactant is any selected from the group consisting of a polyoxyethylene sorbitan fatty acid ester and a polyoxyethylene alkylphenyl ether.
- the lysing agent according to [6] for use in a method for diagnosing mastitis in livestock.
- the immunochromatographic method is (1) A milk containing a specific substance is connected to the first antibody labeled with the specific substance or the first part in which the labeled specific substance is retained, downstream of the first part, and the specific A second part on which a second antibody against the substance is immobilized; and a third part connected to the first part or upstream of the second part and having a void capable of removing milk fat globules in the milk. Bringing the milk into contact with the third portion or an upstream portion thereof with respect to the test piece having; and (2) flowing the milk to the second portion or the downstream portion thereof; and The method according to [9], comprising the step of obtaining a signal detectable by the label in the downstream portion.
- the present invention also provides the following.
- a method for lysis of staphylococci in milk comprising lysing lysostaphin, an ionic surfactant, and a nonionic surfactant in milk and lysing staphylococci present in the milk
- a lysis method comprising: [2] The lysis method according to [1], wherein the ionic surfactant is an amphoteric surfactant.
- the amphoteric surfactant is dimethylammoniopropane sulfonate, dodecyldimethylammoniotilite, betaine laurate.
- nonionic surfactant is any one selected from polyoxyethylene alkylphenyl ether and polyoxyethylene sorbitan fatty acid ester in [2]
- the lysis method as described.
- the final concentration of the amphoteric surfactant is 0.03% or more and 0.2% or less, [2] or [3] The lysis method according to 1.
- the final concentration of the lysostaphin is 0.1 mg / ⁇ l or more and 200 mg / ⁇ l or less; and / or the lysing agent in the milk
- the lysis method according to [4] wherein the final concentration of the nonionic surfactant is 0.03% or more and 10% or less.
- [6] As defined in any one of [1] to [5], comprising a step of lysing staphylococci present in the milk by mixing a lysis agent in the milk, A method for detecting staphylococci in milk, further comprising a step of detecting a specific substance derived from microbial cells of staphylococci released by lysis. [7] The method according to [6], wherein the detection method is performed by an immunochromatographic method.
- the immunochromatographic method comprises: (1) A milk containing a specific substance is connected to the first antibody labeled with the specific substance or the first part in which the labeled specific substance is retained, downstream of the first part, and the specific A second part on which a second antibody against the substance is immobilized; and a third part connected to the first part or upstream of the second part and having a void capable of removing milk fat globules in the milk. Bringing the milk into contact with the third portion or an upstream portion thereof with respect to the test piece having; and (2) flowing the milk to the second portion or the downstream portion thereof; and The detection method according to [7], comprising a step of obtaining a signal detectable by the label in the downstream portion.
- the lysing agent according to [12] or [13], The first antibody labeled with the specific substance or the first part holding the labeled specific substance and the second antibody linked to the downstream of the first part and the second antibody against the specific substance are immobilized.
- a test piece having a second part and a third part connected upstream of the first part or the second part and having a cavity capable of removing milk fat globules in the milk.
- staphylococci in milk can be detected quickly, conveniently and with high sensitivity and on the spot.
- an appropriate treatment policy such as selection of an appropriate antibiotic and measures for preventing the spread of infection at an early stage.
- FIG. 2 is a schematic cross-sectional view of a test piece of an immunochromatography apparatus used in Example 2.
- FIG. 1 is a schematic cross-sectional view of a test piece of an immunochromatography apparatus used in Example 2.
- the present invention provides a lysing agent for lysing bacteria in milk.
- the lysing agent of the present invention includes a lytic enzyme and a specific surfactant.
- the kind of lytic enzyme in the present invention is not particularly limited, and any two or more lytic enzymes may be used in appropriate combination. Escherichia coli , a bacterium belonging to the genus Klebsiella, or Staphylococcus aureus as a mastitis-causing fungus. It is also preferable to use one or a combination of two or more enzymes.
- one or more lytic enzymes selected from lysozyme, lysostaphin, pepsin, glucosidase, galactosidase, achromopeptidase, ⁇ -N-acetylglucosaminidase and the like can be used.
- a method using lysozyme and a cell membrane lysing agent as a lytic enzyme has been proposed (Japanese Patent Laid-Open No. 63-167799).
- lysostaphin that exhibits a specific lytic action against staphylococci alone or in combination with other lytic enzymes
- a method for lysing Staphylococcus aureus with lysostaphin is disclosed in JP-A-11-28099, and those skilled in the art can easily obtain lysostaphin as a lytic enzyme.
- JP-A-11-28099 JP-A-11-28099
- Natural lysostaphin is a zinc protease produced by Staphylococcus simulans and is known to lyse these bacteria by hydrolyzing the glycylglycine bond in the glycopeptide chain of the cell wall peptidoglycan of Staphylococcus aureus or its congeners. It has been.
- lysostaphin includes, in addition to the above natural lysostaphin, addition, deletion, and / or addition to one or more amino acids in the amino acid sequence of natural lysostaphin, as long as the hydrolysis activity is not lost. Mutant lysostaphin into which mutations such as substitution have been introduced is included.
- modified lysostaphin in which other compounds such as saccharides and polyethylene glycol are bound to the natural lysostaphin or mutant lysostaphin is also included as long as the hydrolysis activity is not lost.
- lysostaphins can be obtained by the culture method or genetic engineering method described in JP-A No. 11-28099, or can be obtained by purchasing commercially available products.
- the content of the lytic enzyme in the lytic agent (when using a plurality of lytic enzymes, the content as the total amount of lytic enzymes is not particularly limited as long as an effective lysis rate is ensured for detection,
- the lower limit is that when an enzyme (or enzyme protein) with an activity of 3500 units / mg or more is used, the final concentration of the enzyme (or enzyme protein) when mixed with milk is 0.03 ⁇ g / ml or more. It is preferably 0.15 ⁇ g / ml or more, more preferably 0.3 ⁇ g / ml or more, further preferably 1.5 ⁇ g / ml or more, and particularly preferably 1 ⁇ g / ml or more.
- the upper limit of the content of the lytic enzyme can be appropriately determined from an economic viewpoint, and can be 200 ⁇ g / ml or less, preferably 100 ⁇ g / ml or less, regardless of the lower limit. More preferably, it is 75 ⁇ g / ml or less, more preferably 50 ⁇ g / ml or less, and particularly preferably 25 ⁇ g / ml or less.
- the lower limit can be such that the final concentration when mixed with milk is 0.105 units / ml or more, preferably 0.525 units / ml or more, more preferably 1.05 units / ml or more.
- the upper limit can be 700 units / ml or less, preferably 350 units / ml or less, more preferably 262.5 units / ml or less, and even more preferably 175, regardless of the lower limit.
- Units / ml or less particularly preferably 87.5 units / ml or less.
- an enzyme activity in this specification it is based on the following definition.
- 1 unit is 0.01 OD / min at 25 ° C., pH 8.0, 600 nm for a suspension using a heat-treated target bacterium (preferably Staphylococcus aureus , more preferably Staphylococcus aureus 8325-4) as a substrate.
- a heat-treated target bacterium preferably Staphylococcus aureus , more preferably Staphylococcus aureus 8325-4
- turbidity pH 8.0, 25 ° C., initial turbidity 1.0 in 1.0 mL of 0.2 mol / L Tris buffer.
- ionic surfactants Surfactants that ionize when dissolved in water are called ionic surfactants or ionic surfactants, and surfactants that do not become ions are called nonionic surfactants. Ionic surfactants are further classified into anionic (anionic) surfactants, cationic (cationic) surfactants and amphoteric surfactants.
- At least one amphoteric surfactant, at least one anionic surfactant, at least one cationic surfactant, or any combination thereof is used simultaneously with the lytic enzyme.
- at least one amphoteric surfactant or at least one nonionic surfactant or a combination thereof is used simultaneously with the lytic enzyme.
- at least one amphoteric surfactant and a nonionic surfactant are used in combination with the lytic enzyme.
- amphoteric surfactants include amino acids (alkylamino fatty acid salts), betaines (alkylbetaines), amine oxides (alkylamine oxides), and the like, but are not particularly limited. More specific examples include dimethylammoniopropane sulfonate, dodecyldimethylammoniobutylate, betaine laurate, and amidopropyl betaine.
- More specific examples include n-Tetradecyl-N, N-dimethyl-3-ammonio-1-propanesulfonate, n-Decyl-N, N-dimethyl-3-ammonio-1-propanesulfonate, n-Dodecyl-N, N-dimethyl-3-ammonio-1-propanesulfonate, n-Tetradecyl-N, N-dimethyl-3-ammonio-1-propanesulfonate, n-Hexadecyl-N, N-dimethyl-3-ammonio-1-propanesulfonate, N- Dodecyl-N, N- (dimethylammonio) butyrate.
- the content of amphoteric surfactant in the lysis agent is as long as an effective lysis rate is ensured.
- the final concentration when mixed with milk can be 0.03% or more, preferably 0.05 or more, regardless of the content of the lytic enzyme. More preferably 0.06% or more, still more preferably 0.07% or more, and particularly preferably 0.08% or more.
- the upper limit of the amphoteric surfactant content is not limited so long as it does not significantly inhibit the reaction by the lytic enzyme or the antigen-antibody reaction. In any case, it can be 1% or less, preferably 0.75. % Or less, more preferably 0.5% or less, still more preferably 0.3% or less, and particularly preferably 0.2% or less.
- any of ester ether type, ester type, and ether type can be suitably used. More specifically, polyoxyethylene alkyl ether, polyoxyethylene alkyl phenyl ether, fatty acid sorbitan ester, alkyl Polyglucosides, fatty acid diethanolamides, alkyl monoglyceryl ethers, polysorbates (those obtained by condensing a few tens of ethylene oxides with a fatty acid sorbitan ester), and the like, are not particularly limited.
- polysorbates more specifically polyoxyethylene (20) sorbitan monolaurate, polyoxyethylene (60) sorbitan monostearate, polyoxyethylene (65) sorbitan tristearate, and polyoxyethylene (20)
- examples thereof include oxyethylene (80) sorbitan monooleate and the like, and polyoxyethylene alkylphenyl ether, more specifically polyoxyethylene (10) octylphenyl ether.
- the content of the nonionic surfactant in the lysis agent (when a plurality of nonionic surfactants are used, the content as the total amount of nonionic surfactants) is, for example, a developing solution when using an immunochromatographic method.
- the lower limit can be such that the final concentration when mixed with milk is 0.03% or more, preferably 0.05% or more. More preferably, it is 0.075% or more, more preferably 0.1% or more, and particularly preferably 0.3% or more.
- the upper limit of the content of the nonionic surfactant only needs to be a level that does not significantly inhibit the reaction by the lytic enzyme or the antigen-antibody reaction, and in any case, it can be 10% or less, preferably It is 7.5% or less, more preferably 5% or less, further preferably 3% or less, and particularly preferably 2% or less.
- any of carboxylate, sulfonate, sulfate ester, and the like can be suitably used. More specific examples include alkyl ether carboxylates, linear alkylbenzene sulfonates (LAS), ⁇ -olefin sulfonates (AOS), dialkyl sulfosuccinates, formaldehyde condensates of naphthalene sulfonates, alkyl sulfates Examples thereof include salts (AS), polyoxyethylene alkyl sulfates (AES) sulfated by adding ethylene oxide to higher alcohols, and phosphate esters such as higher alcohols and ethylene oxide adducts thereof.
- AS salts
- AES polyoxyethylene alkyl sulfates
- phosphate esters such as higher alcohols and ethylene oxide adducts thereof.
- sodium alkyl sulfates such as sodium dodecyl sulfate and sodium myristyl sulfate
- sodium N-acyl sarcosine such as sodium N-lauroyl sarcosinate, sodium N-myristoyl sarcosinate, sodium dodecylbenzene sulfonate
- hydrogen Examples thereof include sodium coconut fatty acid monoglyceride monosulfate, sodium lauryl sulfoacetate, N-acyl glutamate such as sodium N-palmitoyl glutamate, sodium N-methyl-N-acylalanine, and sodium ⁇ -olefin sulfonate.
- an amine salt type or a quaternary ammonium salt type can be suitably used as the cationic surfactant. More specific examples include distearyldimethylammonium chloride, benzalkonium chloride, hexadecyltrimethylammonium chloride. Examples thereof include bromide, hexadecyltrimethylammonium bromide, and myristyltrimethylammonium bromide.
- the lysing agent contains lysostaphin, an amphoteric surfactant, and a nonionic surfactant.
- the preferred amphoteric surfactant is selected from dimethylammoniopropane sulfonate, dodecyl dimethylammoniobutylite, betaine laurate, and amidopropyl betaine
- Preferred nonionic surfactants are polyoxyethylene alkylphenyl ethers and polyoxyethylene sorbitan fatty acid esters.
- the concentration of each component in such a lysis agent is such that the final concentration of lysostaphin is 0.1 mg / ⁇ l or more and 200 mg / ⁇ l or less in the step of mixing the lysis agent in milk and lysing the bacteria present in the milk.
- the final concentration of the amphoteric surfactant is preferably 0.03% or more and 0.2% or less, and the final concentration of the nonionic surfactant is preferably 0.03% or more and 10% or less.
- the lysis agent of the present invention can contain one or more other components in addition to the lytic enzyme and the surfactant as long as the intended effect is not significantly impaired.
- a preferable example of components other than the lytic enzyme and the surfactant is a substance having an effect of promoting lysis.
- glutaraldehyde, halogen compounds, chlorhexidine alcohols (eg, methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, 2-methyl-1-propanol, 2-methyl- 2-propanol), phenol, hydrogen peroxide, acrinol, guanidine and salts thereof, chelating agents, organic acids and salts thereof, polyhydric alcohols (eg, ethylene glycol, propylene glycol, diethylene glycol, glycerin), and 2-mercaptoethanol , Reducing agents such as, but not limited to, dithiothreitol, cystine and thiophenol.
- alcohols eg, methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, 2-butanol, 2-methyl-1-propanol, 2-methyl- 2-propanol
- phenol e.g, hydrogen peroxide, acrinol,
- the lysis agent can be used by mixing with milk.
- the mixing ratio between the milk and the lysing agent is not particularly limited as long as the final concentration of the lytic enzyme or the like is appropriately maintained and a sufficient lysis rate can be secured. If a relatively small amount of lysing agent is used for the milk, the milk is not diluted. Therefore, it can be expected that the cells can be detected with higher sensitivity. Moreover, when using a comparatively large amount of lysing agent with respect to milk, the influence by the fat globule and protein in milk is reduced, and it can be anticipated that a microbial cell can be detected in a shorter time.
- the ratio of milk in the mixture of milk and lysing agent (milk / (milk + lysing agent) x 100) is higher from the viewpoint that the detection sensitivity can be increased, and any other conditions are However, it can be, for example, 5% or more, preferably 10% or more, more preferably 20% or more, and further preferably 30% or more.
- the upper limit can be set to 100% by using a lysis agent solidified by means such as drying, whatever the other conditions.
- the ratio of milk can be 90% or less, 80% or less, 70% or less, 60% or less, or 50% or less.
- the upper limit value may be determined in consideration of easiness of mixing, stability as a solution of the lysis agent, and the like.
- milk may be simply mixed with a lysing agent.
- the temperature at the time of mixing and the treatment time for the subsequent enzyme to act are not particularly limited as long as the lytic enzyme to be used can exert its activity, and may usually be room temperature. Those skilled in the art can appropriately design the time required for the processing time after mixing in consideration of the lysis rate.
- the treatment time can be significantly shortened compared with the case where only the lytic enzyme is used.
- the treatment time in the present invention is generally from several tens of minutes to several hours, more specifically within 120 minutes, preferably within 60 minutes, and more preferably within 45 minutes.
- the treatment time can also be set to substantially 0 (milk and a lysis agent are mixed and immediately subjected to measurement). Even in such a short time, according to the present invention, a lysis rate of 90% or more is ensured, and highly sensitive detection of staphylococci can be performed.
- milk containing S. aureus at about 5 ⁇ 10 8 cells / ml is treated with the lysis agent of the present invention for about 30 minutes. It has been confirmed that a lysis rate of 100% can be achieved.
- the lysis rate is the lysis rate of the target staphylococci containing the lysate after treatment with an appropriate concentration of nonionic surfactant in advance, followed by sonication and sufficient enzyme treatment as necessary. 100%.
- the staphylococci lysed according to the present invention can be detected by various immunological methods using components derived from bacterial cells as antigens.
- immunological measurement methods include immunochromatography, aggregation reaction, enzyme immunoassay (ELISA), radioimmunoassay (RIA), or fluorescence immunoassay (FIA).
- ELISA enzyme immunoassay
- RIA radioimmunoassay
- FFA fluorescence immunoassay
- a blocking substance for preventing non-specific adsorption or a substance for preventing a cross reaction with bacteria other than the target bacteria may be used.
- Antigen-antibody reaction is performed by sandwich assay using “labeled first antibody against specific substance” retained in the first part and “second antibody against specific substance” immobilized on the second part. It can be detected by the method. Alternatively, the antigen-antibody reaction may be detected by a competition method using a labeled specific substance held in the first part and an antibody against the specific substance immobilized in the second part. However, in the present invention, a sandwich assay method in which the detection sensitivity is high and the antibody detection line appears positive is preferable.
- the immunochromatography apparatus is an apparatus for detecting a specific substance in milk by an immunochromatography method, and includes a first antibody labeled against the specific substance or a first part in which the labeled specific substance is retained. A second part linked downstream of the first part and immobilized with a second antibody against the specific substance, and linked to the first part or upstream of the second part, and in the milk A device comprising a test piece having a third portion with a void from which milk fat globules can be removed.
- the fat globule removing member (third portion) 7 is installed downstream of the sample addition member 4 and upstream of the labeled antibody-impregnated member (first portion) 1.
- the immunochromatography apparatus can be manufactured using a commercially available material by a known method.
- the material used for the first part is not particularly limited as long as it can perform immunochromatography, but is preferably a fiber matrix such as a cellulose derivative, filter paper, glass fiber, cloth, cotton, or the like.
- the material used for the second part is not particularly limited as long as it can perform immunochromatography, but is preferably nitrocellulose, mixed nitrocellulose ester, polyvinylidene fluoride, nylon or the like.
- the material used for the third part preferably has voids that can remove milk fat globules with a diameter of 1 to 10 ⁇ m contained in the milk.
- the third part needs to be arranged upstream of the second part made of a porous membrane having a pore diameter of several tens to several hundreds of nanometers, and is a position where the sample solution can first contact and pass. It is preferable that the first portion is disposed on the upstream side.
- the gap of the third part is not limited as long as milk fat globules can be separated, and the retained particle size is preferably 0.1 to 10 ⁇ m, more preferably 1 to 3.5 ⁇ m.
- the material is not particularly limited as long as it has voids in the above range, but is preferably a fiber matrix such as a cellulose derivative, filter paper, glass fiber, cloth, cotton or the like.
- Retained particle size is the particle size of milk fat globules that are retained in the third portion without allowing milk fat globules having a particle size larger than that size to pass through the void, and the average pore size of the void in the third portion
- milk fat globules having a particle size equal to or larger than 50%, preferably 60% or more, more preferably 70% or more, still more preferably 80% or more, particularly preferably 90% or more. % Or more, and most preferably 98% or more is retained in the third portion without passing through the gap.
- the percentage of milk fat globules retained can be measured by methods well known to those skilled in the art.
- the GF / B provided by GE Healthcare Biosciences is the retention particle size (the particle diameter with a retention efficiency of 98%.
- the retention particle size refers to this unless otherwise specified.
- the product catalog it is possible to confirm the above particle size by methods well known to those skilled in the art.
- the third part may use only a material having a specific holding particle size, and may be used by stepping together ones having a larger holding particle size in order to increase the separation efficiency of milk fat globules. .
- the third part is composed of two or more members having voids capable of removing milk fat globules having different particle sizes, it is a preferred aspect of the present invention, and the third part is a first part disposed downstream. It is a further preferred embodiment of the present invention when it is constituted by a member and a second member disposed upstream, and the retained particle size of the second member is larger than the retained particle size of the first member.
- the retained particle size of the first member disposed downstream is 1.0 to 2.0 ⁇ m
- the retained particle size of the second member disposed upstream is It is preferably 3.0 to 3.5 ⁇ m.
- the third part is a member having a small retained particle size and retained particles. It is preferable to configure a combination with a member having a large size.
- the first part holds the labeled first antibody against the specific substance or the labeled specific substance.
- the specific substance can be detected by a sandwich assay method.
- the specific substance labeled in the first part can be detected by the competition method.
- the sandwich assay method in which detection sensitivity is high, positive and an antibody detection line appears is preferable, the first part should retain the first antibody labeled with a specific substance. Is preferred.
- first antibody labeled with a specific substance When the first antibody labeled with a specific substance is held in the first part, two types of antibodies are used: a first antibody against the specific substance and a second antibody against the specific substance.
- the first antibody and the second antibody are antibodies that can simultaneously bind to the specific substance so that the specific substance can be detected by sandwich assay, and the first antibody recognizes the first antibody.
- the epitope of the specific substance is preferably different from the epitope of the specific substance recognized by the second antibody.
- the first antibody or specific substance held in the first portion is labeled.
- the label used in the present invention include colored particles, enzymes, radioisotopes, and the like, but it is preferable to use colored particles that can be detected visually without the need for special equipment.
- the colored particles include, but are not limited to, metal fine particles such as gold and platinum, non-metal particles, and latex particles.
- the colored particles may be any size as long as they can be transported downstream through the voids of the test piece, but a diameter of 1 nm to 10 ⁇ m is preferable. More preferably, it is 5 nm to 1 ⁇ m, and further preferably 10 nm to 100 nm.
- staphylococci in milk can be detected.
- Staphylococcus aureus refers to Staphylococcus (Staphylococcus) bacteria belonging to the genus, including Staphylococcus aureus (S. aureus), of the other Staphylococcus spp. Bacteria, and CNS (Coagulase Negative Staphylococcus ) are included.
- CNS includes Staphylococcus intermedius, Staphylococcus hyicus, Staphylococcus xylosus, Staphylococcus epidermidis (Staphylococcus epidermidis) and the like.
- staphylococci contained in milk itself containing fat globules and high-concentration proteins obtained from livestock such as cattle by using a lytic agent in which a lytic enzyme and a surfactant are appropriately mixed. can be detected.
- the specific substance to be measured in the present invention can be any substance that can be measured by an immunological method such as immunochromatography, but is preferably a bacterial component or a substance secreted by bacteria. More preferably, it is a bacterial L7 / L12 ribosomal protein. L7 / L12 ribosomal proteins have high detection sensitivity due to the presence of multiple copies in the cell.
- the antibody used in the present invention can be prepared by the method described in WO 00/06603 pamphlet.
- the bacterial ribosomal protein L7 / L12 can be prepared using the full-length protein of the bacterial ribosomal protein L7 / L12 or a partial peptide thereof as the antigen, but it is preferable to prepare the full-length protein as the antigen.
- This partial peptide or full-length protein is directly or after cross-linking with a carrier protein, and then inoculated into an animal together with an adjuvant as necessary, and an antibody (polyclonal antibody) that recognizes the L7 / L12 ribosomal protein by recovering the serum. Serum can be obtained.
- the antibody can be purified from the antiserum and used.
- the animals to be inoculated include sheep, horses, goats, rabbits, mice, rats and the like, and sheep, rabbits and the like are particularly preferable for producing polyclonal antibodies.
- the antibody it is more preferable to apply a monoclonal antibody obtained by a known method for producing a hybridoma cell. In this case, a mouse is preferable.
- the monoclonal antibody a monoclonal antibody that reacts with ribosomal protein L7 / L12 of a specific bacterium that causes mastitis and does not react with ribosomal protein L7 / L12 of a bacterium that causes mastitis other than the specific bacteria. By screening, it can be used for diagnosis of whether or not the bacterium has an infection.
- the antibody reacts with a component of a specific bacterium causing mastitis or a substance secreted by the bacterium, and does not react with a component of a bacterium causing mastitis other than the specific bacterium or a substance secreted by the bacterium.
- a monoclonal antibody an antibody that recognizes other than ribosomal protein L7 / L12 as an antigen can be used.
- the monoclonal antibody it is preferable to use a monoclonal antibody whose antigen-antibody reaction is not inhibited by contaminants other than the specific substance contained in the milk.
- milk contains a large amount of protein such as casein, which may inhibit the reaction between a specific substance and a monoclonal antibody.
- a monoclonal antibody against a specific substance is produced by a conventional method, for example, it may be preferable to appropriately select and use a monoclonal antibody in which the antigen-antibody reaction is not inhibited by casein or the like, or a monoclonal antibody that has little influence on the antigen-antibody reaction. is there.
- a monoclonal antibody that specifically reacts with an antigen After manufacturing a monoclonal antibody that specifically reacts with an antigen according to a conventional method, such a monoclonal antibody is examined whether the antigen-antibody reaction is inhibited in the presence of contaminants such as casein, It can be easily obtained by selecting a monoclonal antibody that does not substantially inhibit the reaction.
- the immunochromatography apparatus of the present invention is a combination of a container, for example, a microtube, and an additive solution, for example, an additive solution containing a lytic enzyme or a surfactant for lysing bacteria to elute the ribosomal protein L7 / L12 into the solution. It can also be provided as a kit.
- Example 1 Effect of amphoteric surfactant in the presence of lytic enzyme and nonionic surfactant in the detection of Staphylococcus aureus by enzyme immunoassay (ELISA)
- ELISA enzyme immunoassay
- Example 2 Effect of amphoteric surfactants in the presence of lytic enzymes and nonionic surfactants in the detection of Staphylococcus aureus by immunochromatographic method (1) Preparation of immunochromatographic device The immunochromatographic device was prepared as follows did. (A) Gold colloid-labeled impregnated material Mix BBInternational's gold colloid solution (particle size 60nm) 0.9mL with 0.1M potassium phosphate pH7.0, add gold antibody colloid-labeled monoclonal antibody SA-2 at 100 ⁇ g / mL at room temperature.
- gold colloid labeled antibody a colloidal gold labeled monoclonal antibody SA-2 (hereinafter referred to as “gold colloid labeled antibody”) solution. This solution was centrifuged (15000 ⁇ rpm, 5 minutes) to precipitate the colloidal gold labeled antibody, and the supernatant was removed to obtain a colloidal gold labeled antibody.
- the colloidal gold labeled antibody was suspended in 20 mm Tris-HCl buffer (pH 8.2) containing 0.25% BSA, 2.5% sucrose and 35 mm NaCl to obtain a colloidal gold labeled antibody solution.
- a 10 mm ⁇ 300 mm belt-shaped glass fiber pad was impregnated with 2 mL of the colloidal gold labeled antibody solution and dried under reduced pressure at room temperature to obtain a colloidal gold labeled antibody impregnated member 1 (first part).
- Capture site of complex of antigen and colloidal gold labeled antibody A nitrocellulose membrane having a width of 25 mm and a length of 300 mm was prepared as a membrane carrier 2 (second part) for chromatographic development of a chromatographic medium. A solution containing 1.5 mg / mL of the monoclonal antibody SA-1 was applied in a line at 1 ⁇ L / cm at a position 10 mm from the end of the chromatographic development start side in the membrane carrier 2 for chromatographic development, It was dried at 50 ° C. for 30 minutes, then immersed in 0.5% sucrose solution for 30 minutes and dried overnight at room temperature. Staphylococcus aureus ribosomal protein It was designated as capture site 3 for the complex of L7 / L12 antigen and colloidal gold-labeled antibody.
- FIG. 2 shows a cross-sectional view of the immunochromatograph device.
- 25 mm GF / DVA manufactured by GE Healthcare Biosciences
- Filter member 4 consisting of glass fiber with a thickness of 776 ⁇ m and a retention particle size of 3.5 ⁇ m
- 20 mm GF / AVA GE Healthcare Biosciences
- Filter member 7 consisting of glass fiber with a thickness of 299 ⁇ m and a retention particle size of 1.7 ⁇ m
- filter paper was prepared as the absorbing member 5.
- Test Milk milk was measured with an immunochromatograph apparatus as follows. Take 100 ⁇ l of milk with a final concentration of 1 ⁇ 10 5 (cfu / ml) Staphylococcus aureus in a microtube and prepare a lysis solution (final concentration 1% Tween20, 10 ⁇ g / ml (35 units / ml) lysostaphin, 0.1 M MOPSO pH 7.5, Zwettergent 3-12 with different concentrations, or DDMAB) (150 ⁇ l) was added and mixed for 30 minutes at room temperature. Commercial milk drinks were used for milk juice.
- the immunochromatograph device is immersed in the mixed solution from the sample addition member 4 and allowed to stand at room temperature for 30 minutes for development, and then the ribosomal protein L7 / L12 antigen and gold colloid-labeled antibody captured at the supplemental site 3
- a red-purple line that increases or decreases the capture of the complex in proportion to the capture amount was measured with an apparatus C10066 (manufactured by Hamamatsu Photonics).
- Amphoteric surfactants were added at different concentrations, and the lysis rate at each concentration was calculated with the lysis rate of the bacterial solution preliminarily treated by the same method as in Example 1 as 100%. The results are shown in Figure 3. It was revealed that the lysis rate was greatly improved depending on the concentration when any amphoteric surfactant was used.
- Example 3 Measurement of milk sample by immunochromatography The immunochromatography method was performed on milk samples from cattle with mastitis. The measurement was performed according to Example 2 (Bacterial treatment solution: final concentration 1% Tween 20, 10 ⁇ g / ml (35 units / ml) lysostaphin, 0.1 M MOPSO pH 7.5, final concentration 0.1% Zwettergent 3-12, 30 at room temperature. After the minute treatment, it was developed at room temperature for 30 minutes.) The reddish purple line that appeared was visually judged (positive +, negative-). On the other hand, in order to confirm the number of Staphylococcus aureus in milk, it quantified by the culture method.
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Abstract
Description
[1] ブドウ球菌の溶菌方法であって、家畜から得た乳汁に、溶菌酵素および少なくとも一の両性界面活性剤を含有する溶菌剤を混合して乳汁中に存在するブドウ球菌を溶菌する工程を含む、方法。
[2] 該溶菌酵素が、リゾスタフィンである、[1]に記載の方法。
[3] 溶菌剤が、さらに少なくとも一の非イオン界面活性剤を含む、[1]または[2]に記載の方法。
[4] 両性界面活性剤が、ジメチルアンモニオプロパンスルホネイト、ドデシルジメチルアンモニオブチレイト、ラウリル酸ベタイン、およびアミドプロピルベタインからなる群より選択されるいずれかである、[1]~[3]のいずれか一に記載の方法。
[5] 非イオン界面活性剤が、ポリオキシエチレンソルビタン脂肪酸エステルおよびポリオキシエチレンアルキルフェニルエーテルからなる群より選択されるいずれかである、[1]~[4]のいずれか一に記載の方法。
[6] 家畜乳汁中のブドウ球菌の溶菌のために用いる、溶菌酵素および少なくとも一の両性界面活性剤を含有する溶菌剤。
[7] 家畜の乳房炎を診断する方法において用いるための、[6]に記載の溶菌剤。
[8] [1]~[5]のいずれか一に記載の方法を含み、溶菌により放出されたブドウ球菌の菌体内由来の特定物質を検出する工程をさらに含む、家畜乳汁中のブドウ球菌の検出方法。
[9] 特定物質の検出工程が、イムノクロマトグラフ方法により実施される、[8]に記載の方法。
[10] イムノクロマトグラフ方法が、
(1)特定物質を含む乳汁を、該特定物質に対する標識された第一の抗体または標識された該特定物質が保持された第1部分と、該第1部分の下流に連結され、かつ該特定物質に対する第二の抗体が固定化された第2部分と、該第1部分または該第2部分の上流に連結され、かつ該乳汁中の乳脂肪球を除去できる空隙を有する第3部分とを有する試験片に対して、該乳汁を該第3部分またはそれより上流部に接触させる工程;および
(2)該乳汁を該第2部分またはそれより下流部まで流し、該第2部分またはそれより下流部で該標識による検出可能な信号を得る工程:を含む、[9]に記載の方法。
[11] 該第1部分に、該特定物質に対する標識された第一の抗体が保持されている、[10]に記載の方法。
[12] 第3部分がそれぞれ異なる粒子サイズの乳脂肪球を除去できる空隙を有する2種以上の部材により構成される、[10]または[11]に記載の方法。
[13] 第3部分が下流に配置された第一の部材、および上流に配置された第二の部材により構成され、第二の部材の保持粒子サイズが第一の部材の保持粒子サイズより大きい、[12]に記載の方法。
[14] [6]または[7]に記載の溶菌剤と、 該特定物質に対する標識された第一の抗体または標識された該特定物質が保持された第1部分と、該第1部分の下流に連結され、かつ該特定物質に対する第二の抗体が固定化された第2部分と、該第1部分または該第2部分の上流に連結され、かつ該乳汁中の乳脂肪球を除去できる空隙を有する第3部分とを有する試験片を含む、乳汁中の特定物質を検出するためのイムノクロマトグラフ装置とからなる、家畜乳汁中のブドウ球菌検出用キット。
[1] 乳汁中のブドウ球菌の溶菌方法であって
乳汁に、リゾスタフィン、イオン界面活性剤、および非イオン界面活性剤を含有する溶菌剤を混合して乳汁中に存在するブドウ球菌を溶菌する工程を含む、溶菌方法。
[2] 該イオン界面活性剤が両性界面活性剤である、[1]に記載の溶菌方法
[3] 該両性界面活性剤が、ジメチルアンモニオプロパンスルホネイト、ドデシルジメチルアンモニオブチライト、ラウリル酸ベタイン、およびアミドプロピルベタインから選択されるいずれかであり;ならびに/または
該非イオン界面活性剤が、ポリオキシエチレンアルキルフェニルエーテル、ポリオキシエチレンソルビタン脂肪酸エステルから選択されるいずれかである、[2]に記載の溶菌方法。
[4] 乳汁に溶菌剤を混合して乳汁中に存在するブドウ球菌を溶菌する工程において、該両性界面活性剤の終濃度が、0.03%以上0.2%以下である、[2]または[3]に記載の溶菌方法。
[5] 乳汁に溶菌剤を混合して乳汁中に存在するブドウ球菌を溶菌する工程において、該リゾスタフィンの終濃度が、0.1mg/μl以上200mg/μl以下であり;および/または
乳汁に溶菌剤を混合して乳汁中に存在するブドウ球菌を溶菌する工程において、該非イオン界面活性剤の終濃度が、0.03%以上10%以下である、[4]に記載の溶菌方法。
[6] [1]~[5]のいずれか一に定義した、乳汁に溶菌剤を混合して乳汁中に存在するブドウ球菌を溶菌する工程
を含み、
溶菌により放出されたブドウ球菌の菌体内由来の特定物質を検出する工程
をさらに含む、乳汁中のブドウ球菌の検出方法。
[7] 検出方法が、イムノクロマトグラフ方法により実施される[6]に記載の方法。
[8] 該イムノクロマトグラフ方法が、
(1)特定物質を含む乳汁を、該特定物質に対する標識された第一の抗体または標識された該特定物質が保持された第1部分と、該第1部分の下流に連結され、かつ該特定物質に対する第二の抗体が固定化された第2部分と、該第1部分または該第2部分の上流に連結され、かつ該乳汁中の乳脂肪球を除去できる空隙を有する第3部分とを有する試験片に対して、該乳汁を該第3部分またはそれより上流部に接触させる工程;および
(2)該乳汁を該第2部分またはそれより下流部まで流し、該第2部分またはそれより下流部で該標識による検出可能な信号を得る工程
を含む、[7]に記載の検出方法。
[9] 該第1部分に、該特定物質に対する標識された第一の抗体が保持されている、[8]に記載の検出方法。
[10] 第3部分がそれぞれ異なる粒子サイズの乳脂肪球を除去できる空隙を有する2種以上の部材より構成されている、[8]または[9]に記載の検出方法。
[11] 第3部分が下流に配置された第一の部材、および上流に配置された第二の部材により構成され、第二の部材の保持粒子サイズが第一の部材の保持粒子サイズより大きい、[10]に記載の検出方法。
[12] [1]~[5]のいずれか一に定義された、溶菌剤。
[13] 家畜の乳房炎を診断する方法において用いるための、[12]に記載の溶菌剤。
[14] [12]または[13]に記載の溶菌剤と、
該特定物質に対する標識された第一の抗体または標識された該特定物質が保持された第1部分と、該第1部分の下流に連結され、かつ該特定物質に対する第二の抗体が固定化された第2部分と、該第1部分または該第2部分の上流に連結され、かつ該乳汁中の乳脂肪球を除去できる空隙を有する第3部分とを有する試験片を含む、乳汁中の特定物質を検出するためのイムノクロマトグラフ装置と
を含む、乳汁中のブドウ球菌の検出用キット。
本発明における溶菌酵素の種類は特に限定されず、任意の2種以上の溶菌酵素を適宜組み合わせて用いてもよい。乳房炎の起炎菌として大腸菌(Escherichia coli)、クレブシエラ(Klebsiella)属に属する細菌、または黄色ブドウ球菌(Staphylococcus aureus)による感染が多発することから、これらの微生物に対して溶菌作用を発揮できる溶菌酵素を1種または2種以上組み合わせて用いることも好ましい。例えばリゾチーム、リゾスタフィン、ペプシン、グルコシダーゼ、ガラクトシダーゼ、アクロモペプチダーゼ、β-N-アセチルグルコサミニダーゼなどから選ばれる1種または2種以上の溶菌酵素を用いることができる。例えば、溶菌酵素としてリゾチームと細胞膜溶解剤とを用いる方法が提案されている(特開昭63-167799号公報)。
水に溶かしたときに、電離してイオンとなる界面活性剤をイオン界面活性剤またはイオン性界面活性剤といい、イオンにならない界面活性剤を非イオン(ノニオン)界面活性剤という。イオン界面活性剤はさらに、陰イオン(アニオン)界面活性剤、陽イオン(カチオン)界面活性剤および両性界面活性剤に分類される。
特に好ましい態様の一つにおいては、溶菌剤は、リゾスタフィン、両性界面活性剤、および非イオン界面活性剤を含有する。リゾスタフィン、両性界面活性剤、および非イオン界面活性剤を含有する溶菌剤において、好ましい両性界面活性剤は、ジメチルアンモニオプロパンスルホネイト、ドデシルジメチルアンモニオブチライト、ラウリル酸ベタイン、およびアミドプロピルベタインから選択されるいずれかであり;好ましい非イオン界面活性剤は、ポリオキシエチレンアルキルフェニルエーテル、ポリオキシエチレンソルビタン脂肪酸エステルである。このような溶菌剤における各成分の濃度は、乳汁に溶菌剤を混合して乳汁中に存在する該菌を溶菌する工程において、リゾスタフィンの終濃度が、0.1mg/μl以上200mg/μl以下であり、両性界面活性剤の終濃度が、0.03%以上0.2%以下であり、かつ非イオン界面活性剤の終濃度が、0.03%以上10%以下であるようにするとよい。
本発明の溶菌剤は、溶菌酵素および界面活性剤以外に、意図した効果を著しく損なわない限り、一種またはそれ以上の他の成分を含むことができる。溶菌酵素および界面活性剤以外の成分の好ましい例は、溶菌を促進する効果を有する物質である。具体的には、グルタルアルデヒド、ハロゲン化合物、クロルヘキシジン、アルコール類(例えば、メタノール、エタノール、1-プロパノール、2-プロパノール、1-ブタノール、2-ブタノール、2-メチル-1-プロパノール、2-メチル-2-プロパノール)、フェノール、過酸化水素、アクリノール、グアニジンおよびその塩、キレート剤、有機酸およびその塩、多価アルコール類(例えば、エチレングリコール、プロピレングリコール、ジエチレングリコール、グリセリン)、ならびに2-メルカプトエタノール、ジチオスレイトール、シスチンおよびチオフェノールなどの還元剤をあげることができるが、これらに限定されない。
本発明においては、溶菌剤は、乳汁に混合して用いることができる。乳汁と溶菌剤との混合比は、溶菌酵素等の終濃度が適切に維持され、十分な溶菌率が確保できる限り、特に限定されない。乳汁に対して比較的少量の溶菌剤を用いる場合は、乳汁が希釈されない。したがって、より高い感度で菌体を検出できると期待できる。また乳汁に対して比較的大量の溶菌剤を用いる場合は、乳汁中の脂肪球やタンパク質による影響が減じられ、より短時間で菌体を検出できると期待できる。乳汁と溶菌剤との混合物における乳汁の割合(乳汁/(乳汁+溶菌剤)×100)は、高いほど検出感度を上げることができるとの観点からは、他の条件がいずれの場合であっても、例えば5%以上とすることができ、10%以上とすることが好ましく、20%以上とすることがより好ましく、30%以上とすることがさらに好ましい。上限値は、他の条件がいずれの場合であっても、乾燥等の手段によって固形化した溶菌剤を用いることにより、乳汁の割合を100%とすることができる。また、他の条件がいずれの場合であっても、乳汁の割合は90%以下とすることができ、80%以下、70%以下、60%以下、または50%以下とすることができる。上限値は、混合の容易さ、溶菌剤の溶液としての安定性等を考慮して決定してもよい。
本発明により溶菌されたブドウ球菌は、菌体由来の成分を抗原として、各種の免疫学的方法により検出することができる。免疫学的測定方法としては、例えば、イムノクロマトグラフ法、凝集反応法、酵素免疫測定法(ELISA法)、放射能免疫測定法(RIA法)、または蛍光免疫測定法(FIA法)などをあげることができるが、これらに限定されることはない。免疫学的方法の実施に際し、非特異吸着を防ぐためのブロッキングのための物質や、対象菌以外の菌との交差反応を防ぐための物質を用いてもよい。
抗原抗体反応は、第1部分に保持されている「特定物質に対する標識された第一の抗体」と、第2部分に固定化された「特定物質に対する第二の抗体」を用いて、サンドイッチアッセイ法により検出することができる。あるいは抗原抗体反応は、第1部分に保持されている標識された特定物質と、第2部分に固定化された特定物質に対する抗体を用いて競合法にて検出してもよい。但し、本発明においては、検出感度が高く、陽性で抗体検出ラインが出現するサンドイッチアッセイ法が好ましい。
本発明により、乳汁中のブドウ球菌を検出できる。本発明で「ブドウ球菌」というときは、特に記載した場合を除き、スタフィロコッカス(Staphylococcus)属に属する細菌をいい、これにはStaphylococcus aureus(黄色ブドウ球菌)、それ以外のスタフィロコッカス属の細菌、およびCNS(Coagulase Negative Staphylococcus、コアグラーゼ陰性ブドウ球菌)が含まれる。CNSには、Staphylococcus intermedius、Staphylococcus hyicus、Staphylococcus xylosus、Staphylococcus epidermidis(表皮ブドウ球菌)等が含まれる。本発明においては、溶菌酵素と界面活性剤が適切に配合された溶菌剤を用いることにより、牛等の家畜から得られた、脂肪球と高濃度のタンパク質を含有する乳汁そのものに含まれるブドウ球菌を検出することができる。
本発明に用いる抗体は、国際公開第00/06603号パンフレットに記載の方法で作製することができる。細菌リボゾームタンパク質L7/L12を抗原とする場合は、細菌リボゾームタンパク質L7/L12の全長タンパク質あるいはその部分ペプチドを抗原として用いて作製することができるが、全長タンパク質を抗原として作製することが好ましい。この部分ペプチドあるいは全長タンパク質をそのまま、またはキャリアタンパク質と架橋した後必要に応じてアジュバントとともに動物へ接種せしめ、その血清を回収することでL7/L12リボソームタンパク質を認識する抗体(ポリクローナル抗体)を含む抗血清を得ることができる。また抗血清より抗体を精製して使用することもできる。接種する動物としてはヒツジ、ウマ、ヤギ、ウサギ、マウス、ラット等であり、特にポリクローナル抗体作製にはヒツジ、ウサギなどが好ましい。また、抗体としてはハイブリドーマ細胞を作製する公知の方法により取得したモノクローナル抗体を適用することがより好ましいが、この場合はマウスが好ましい。当該モノクローナル抗体として、乳房炎の原因となる特定の細菌のリボゾームタンパク質L7/L12と反応し、前記特定の細菌以外の乳房炎の原因となる細菌のリボゾームタンパク質L7/L12とは反応しないモノクローナル抗体をスクリーニングすることにより、当該細菌による感染症にかかっているかどうかの診断に役立てることが可能となる。
本発明においては、上述した試験片をそのまま使用してイムノクロマトグラフ装置としてもよいし、ケースに収納して、イムノクロマトグラフ装置としてもよい。前者の場合は、試料となる乳汁の量が多い場合に、容器に入った試料に直接試験片の一端を浸して使用することが好ましい。また、後者の場合は、試料となる乳汁の量が少ない場合に、ピペット等で所定量を計量して試験片に滴下して使用することが好ましい。後者の場合のケースの形状は、試験片を収納できればいかなる形状でもよい。ケースの材料はいかなる材料でもよく、ポリプロピレンやポリカーボネートなどが好ましいものとしてあげられる。
(a)リボゾームタンパク質L7/L12に対するモノクローナル抗体の作製
金コロイド標識する抗体には、Staphylococcus aureus(黄色ブドウ球菌)リボソームタンパク質L7/L12モノクローナル抗体を使用した。国際公開WO00/06603号パンフレットの実施例5に記載の方法に準じて、黄色ブドウ球菌Staphylococcus aureusのL7/L12リボゾームタンパク質を取得し、同タンパク質を用いてモノクローナル抗体を作製した。当該モノクローナル抗体は、上記L7/L12リボゾームタンパク質の別のサイトに同時に結合しうる2種(SA-1およびSA-2)の組合せを選択した。
10μg/mlのモノクローナル抗体SA-1とPBS溶液100μlを96穴ELISAプレート(Nunc社Maxsorp ELISAプレート)に分注し4℃で一晩吸着させた。上澄み除去後、1%牛血清アルブミン溶液(PBS中)200μlを添加し、室温で1時間反応させてブロッキングした。上澄み除去後、洗浄液(0.02%Tween20、PBS)で数回洗浄した。市販の牛乳に懸濁した黄色ブドウ球菌(約5×108cells/ml)を表1に記載の溶液で室温にて30分処理後、同牛乳にて100倍に希釈したものを100μl添加し、室温にて1時間反応させた。さらに上澄み除去後、パーオキシダーゼ標識したモノクローナル抗体SA-2を0.02% Tween20、PBSにて最終濃度1μg/mlになるように希釈してそれぞれ100μl添加し、室温にて1時間反応させた。上澄み除去後さらに洗浄液で数回洗浄したのち、TMB溶液(KPL社製)を100μlずつ加え室温10分間反応させた後1 mol/lの塩酸を100μl添加して反応を停止したのち450nmの吸光度を測定した。
(1)イムノクロマトグラフ装置の作製
イムノクロマトグラフ装置は以下のように作製した。
(a)金コロイド標識含浸部材 BBInternational社製金コロイド溶液(粒径60nm)0.9mLに0.1M リン酸カリウム pH7.0を混合し、金コロイド標識するモノクローナル抗体SA-2を100μg/mL加え室温で10分間静置し、この抗体を金コロイド粒子表面に結合させた後、金コロイド溶液における最終濃度が1%となるようにウシ血清アルブミン(BSA)10%水溶液を加え、この金コロイド粒子の残余の表面をBSAでブロッキングして、金コロイド標識したモノクローナル抗体SA-2(以下、「金コロイド標識抗体」と記す)溶液を調製した。この溶液を遠心分離(15000×rpm、5分間)して金コロイド標識抗体を沈殿せしめ、上清液を除いて金コロイド標識抗体を得た。この金コロイド標識抗体を0.25%BSA、2.5%スクロース、35mm NaClを含有する20mmトリス塩酸緩衝液(pH8.2)に懸濁して金コロイド標識抗体溶液を得た。10mm×300mmの帯状のグラスファイバーパットに、金コロイド標識抗体溶液2mLを含浸せしめ、これを室温で減圧乾燥させて金コロイド標識抗体含浸部材1(第1部分)とした。
幅25mm、長さ300mmのニトロセルロース膜をクロマトグラフ媒体のクロマト展開用膜担体2(第2部分)として用意した。 モノクローナル抗体SA-1 1.5mg/mLが含有されてなる溶液を、このクロマト展開用膜担体2におけるクロマト展開開始点側の末端から10mmの位置に1μL/cmでライン状に塗布して、これを50℃で30分間乾燥し、その後、0.5%スクロース溶液に30分浸し、一晩室温で乾燥させた。Staphylococcus aureusリボソームタンパク質 L7/L12抗原と金コロイド標識抗体との複合体の捕捉部位3とした。
マイクロチューブに終濃度1×105(cfu/ml)の黄色ブドウ球菌を添加した乳汁を100μl分取し、溶菌処理液(終濃度1% Tween20、10μg/ml(35 units/ml) リゾスタフィン、0.1M MOPSO pH7.5、濃度の異なるZwettergent 3-12、またはDDMAB)150μlを加えて混合し室温で30分間処理した。牛乳汁は市販の飲用品を用いた。同混合溶液に上記イムノクロマトグラフ装置を試料添加用部材4から浸漬して、室温で30分静置して展開後、上記補足部位3で補足されたリボソームタンパク質L7/L12抗原と金コロイド標識抗体との複合体の捕捉の有無を捕捉量に比例して増減する赤紫色のラインを装置C10066(浜松ホトニクス製)にて測定した。両性界面活性剤を濃度を変えて添加し、実施例1と同様の方法にてあらかじめ溶菌処理した菌液の溶菌率を100%として各濃度における溶菌率を算出した。結果を図3に示す。いずれの両性界面活性剤を用いた場合においても濃度に依存して溶菌率が大きく向上していることが明らかとなった。
乳房炎の牛からの乳汁サンプルを対象としてイムノクロマトグラフ法を実施した。測定は実施例2に準じて行い(溶菌処理液:終濃度1% Tween20、10μg/ml(35 units/ml) リゾスタフィン、0.1M MOPSO pH7.5、終濃度0.1% Zwettergent 3-12。室温で30分処理の後、室温で30分間展開。)、出現した赤紫色のラインを目視により判定した(陽性+、陰性-)。一方、乳汁中の黄色ブドウ球菌の数を確認するために、培養法にて定量を行った。トリプチケースソイII5%ヒツジ血液寒天培地(日本ベクトンデッキンソン社製)に乳汁を100μl播種後、37℃で24時間培養し、出現したコロニーを計数した。培養法によって算出した黄色ブドウ球菌の数およびイムノクロマト法による判定結果を表2に示す。Zettergent 3-12を添加することによって103 (cfu/ml)オーダーの場合も検出することが可能となった。
Claims (14)
- 乳汁中のブドウ球菌の溶菌方法であって
乳汁に、リゾスタフィン、イオン界面活性剤、および非イオン界面活性剤を含有する溶菌剤を混合して乳汁中に存在するブドウ球菌を溶菌する工程を含む、溶菌方法。 - 該イオン界面活性剤が両性界面活性剤である、請求項1に記載の溶菌方法
- 該両性界面活性剤が、ジメチルアンモニオプロパンスルホネイト、ドデシルジメチルアンモニオブチライト、ラウリル酸ベタイン、およびアミドプロピルベタインから選択されるいずれかであり;ならびに/または
該非イオン界面活性剤が、ポリオキシエチレンアルキルフェニルエーテル、ポリオキシエチレンソルビタン脂肪酸エステルから選択されるいずれかである、請求項2に記載の溶菌方法。 - 乳汁に溶菌剤を混合して乳汁中に存在するブドウ球菌を溶菌する工程において、該両性界面活性剤の終濃度が、0.03%以上0.2%以下である、請求項2または3に記載の溶菌方法。
- 乳汁に溶菌剤を混合して乳汁中に存在するブドウ球菌を溶菌する工程において、該リゾスタフィンの終濃度が、0.1mg/μl以上200mg/μl以下であり;および/または
乳汁に溶菌剤を混合して乳汁中に存在するブドウ球菌を溶菌する工程において、該非イオン界面活性剤の終濃度が、0.03%以上10%以下である、請求項4に記載の溶菌方法。 - 請求項1~5のいずれか1項に定義した、乳汁に溶菌剤を混合して乳汁中に存在するブドウ球菌を溶菌する工程
を含み、
溶菌により放出されたブドウ球菌の菌体内由来の特定物質を検出する工程
をさらに含む、乳汁中のブドウ球菌の検出方法。 - 検出方法が、イムノクロマトグラフ方法により実施される請求項6に記載の方法。
- 該イムノクロマトグラフ方法が、
(1)特定物質を含む乳汁を、該特定物質に対する標識された第一の抗体または標識された該特定物質が保持された第1部分と、該第1部分の下流に連結され、かつ該特定物質に対する第二の抗体が固定化された第2部分と、該第1部分または該第2部分の上流に連結され、かつ該乳汁中の乳脂肪球を除去できる空隙を有する第3部分とを有する試験片に対して、該乳汁を該第3部分またはそれより上流部に接触させる工程;および
(2)該乳汁を該第2部分またはそれより下流部まで流し、該第2部分またはそれより下流部で該標識による検出可能な信号を得る工程
を含む、請求項7に記載の検出方法。 - 該第1部分に、該特定物質に対する標識された第一の抗体が保持されている、請求項8に記載の検出方法。
- 第3部分がそれぞれ異なる粒子サイズの乳脂肪球を除去できる空隙を有する2種以上の部材より構成されている、請求項8または9に記載の検出方法。
- 第3部分が下流に配置された第一の部材、および上流に配置された第二の部材により構成され、第二の部材の保持粒子サイズが第一の部材の保持粒子サイズより大きい、請求項10に記載の検出方法。
- 請求項1~5のいずれか1項に定義された、溶菌剤。
- 家畜の乳房炎を診断する方法において用いるための、請求項12に記載の溶菌剤。
- 請求項12または13に記載の溶菌剤と、
該特定物質に対する標識された第一の抗体または標識された該特定物質が保持された第1部分と、該第1部分の下流に連結され、かつ該特定物質に対する第二の抗体が固定化された第2部分と、該第1部分または該第2部分の上流に連結され、かつ該乳汁中の乳脂肪球を除去できる空隙を有する第3部分とを有する試験片を含む、乳汁中の特定物質を検出するためのイムノクロマトグラフ装置と
を含む、乳汁中のブドウ球菌の検出用キット。
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ES14871368T ES2699886T3 (es) | 2013-12-18 | 2014-12-17 | Procedimiento para la detección de estafilococos contenidos en la leche |
DK14871368.8T DK3085770T3 (en) | 2013-12-18 | 2014-12-17 | Method for detecting staphylococci in milk |
US15/105,440 US10151751B2 (en) | 2013-12-18 | 2014-12-17 | Method for detecting Staphylococcus contained in milk |
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US16/163,847 US10732179B2 (en) | 2013-12-18 | 2018-10-18 | Method for detecting Staphylococcus contained in milk |
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US16/163,847 Division US10732179B2 (en) | 2013-12-18 | 2018-10-18 | Method for detecting Staphylococcus contained in milk |
US16/184,384 Division US10527616B2 (en) | 2013-12-18 | 2018-11-08 | Method for detecting Staphylococcus contained in milk |
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JP (1) | JP6314156B2 (ja) |
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ES (1) | ES2699886T3 (ja) |
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JP2018151185A (ja) * | 2017-03-10 | 2018-09-27 | 東洋紡株式会社 | イムノクロマト試験片 |
WO2021085651A1 (ja) * | 2019-11-01 | 2021-05-06 | 旭化成株式会社 | 検体中の黄色ブドウ球菌を検出するための抗体、並びに斯かる抗体を用いて黄色ブドウ球菌を検出するための方法、試薬、及びキット |
JPWO2020111223A1 (ja) * | 2018-11-30 | 2021-09-30 | 旭化成株式会社 | 乳房炎の原因菌の検出方法 |
WO2023095845A1 (ja) | 2021-11-24 | 2023-06-01 | 旭化成株式会社 | 溶菌方法、及び細菌検出方法 |
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US20180128828A1 (en) * | 2016-11-04 | 2018-05-10 | James W. Stave | Direct detection of microorganisms in patient samples by immunoassay |
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JP2018151185A (ja) * | 2017-03-10 | 2018-09-27 | 東洋紡株式会社 | イムノクロマト試験片 |
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WO2023095845A1 (ja) | 2021-11-24 | 2023-06-01 | 旭化成株式会社 | 溶菌方法、及び細菌検出方法 |
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EP3085770A4 (en) | 2017-08-16 |
ES2699886T3 (es) | 2019-02-13 |
US10151751B2 (en) | 2018-12-11 |
JP6314156B2 (ja) | 2018-04-18 |
US20160313327A1 (en) | 2016-10-27 |
DK3085770T3 (en) | 2019-01-07 |
EP3085770A1 (en) | 2016-10-26 |
US10732179B2 (en) | 2020-08-04 |
US20190049446A1 (en) | 2019-02-14 |
US10527616B2 (en) | 2020-01-07 |
EP3085770B1 (en) | 2018-11-07 |
US20190079089A1 (en) | 2019-03-14 |
JPWO2015093544A1 (ja) | 2017-03-23 |
NZ721267A (en) | 2017-04-28 |
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