Bacteriolysis method and bacterial detection method

Abstract

Provided is a new bacteriolysis method capable of bacteriolysing multiple species of bacteria and making bacteriolysis treatment more efficient and faster. A bacteriolysis method for lysing multispecies bacterial groups in a specimen, wherein bacteriolysis of multispecies bacterial groups is made possible by a step that reacts a specimen with a surfactant and lytic enzyme in a liquid and bacteriolyses the multispecies bacterial groups present in the specimen.

Description

[Technical Field]

[0001] The present invention relates to a lysis method for lysing multiple species of bacteria in a specimen, and a bacteria detection method using the lysis method.The present invention also relates to a lysis agent and a lysis kit used in the lysis method, and a bacteria detection kit used in the bacteria detection method. [Background technology]

[0002] Various techniques for lysing bacteria in a sample have been used to detect bacteria in a sample based on their intracellular components. For example, Patent Documents 1 and 2 (JP 2017-32579 A and WO 2015 / 093544 A) disclose a technique for detecting Staphylococcus aureus by antigen-antibody reaction based on its ribosomal protein L7 / L12, and describe that the bacteria in the sample are lysed using lysostaphin as a lytic agent. Furthermore, Patent Document 3 (WO 2015 / 093545 A) discloses a technique for detecting Escherichia coli by antigen-antibody reaction based on its ribosomal protein L7 / L12, and describe that the bacteria in the sample are lysed using lysozyme as a lytic agent. [Prior art documents] [Patent documents]

[0003] [Patent Document 1] JP 2017-32579 A [Patent Document 2] International Publication No. 2015 / 093544 [Patent Document 3] International Publication No. 2015 / 093545 Summary of the Invention [Problem to be solved by the invention]

[0004] However, conventional bacteriolysis methods such as those described in Patent Documents 1 to 3 could not simultaneously lyse multiple species of bacteria. Therefore, when simultaneously detecting multiple species of bacteria, it was necessary to lyse each bacterial genus using a separate bacteriolytic agent, which made the bacteriolysis process complicated and delayed. [Means for solving the problem]

[0005] As a result of extensive research, the present inventors have discovered that by performing lysis using a lysing agent containing a surfactant and a lytic enzyme, it is possible to lyse multiple types of bacteria simultaneously, thereby making it possible to make the lysis process more efficient and rapid, and have completed the present invention.

[0006] That is, the gist of the present invention relates to, for example, the following. [1] A method for lysing a group of multiple types of bacteria including at least one or more types of Gram-positive bacteria and / or Gram-negative bacteria in a sample, the method comprising the step of treating the sample with a solution containing a surfactant and a lytic enzyme, wherein the surfactant is selected from an amine oxide and a quaternary ammonium. [2] The method according to Item 1, wherein the group of multiple species of bacteria includes both at least one species of Gram-positive bacteria and at least one species of Gram-negative bacteria. [3] The method according to item 1 or 2, wherein the surfactant is one or more amine oxides. [4] The method of claim 3, wherein the amine oxide is N,N-dimethyldodecylamine N-oxide (DDAO). [5] The method according to Item 3 or 4, wherein the concentration of the amine oxide in the solution during treatment with the specimen is 0.01 to 0.2% by mass. [6] The method according to item 1 or 2, wherein the surfactant is one or more quaternary ammonium salts. [7] The method according to item 6, wherein the quaternary ammonium salt is benzalkonium chloride. [8] The method according to Item 6 or 7, wherein the concentration of the quaternary ammonium salt in the solution during treatment with the specimen is 0.005 to 0.08% by mass. [9] The method according to any one of items 1 to 8, wherein the lytic enzyme is lysostaphin.

[10] The method according to Item 9, wherein the concentration of the lysostaphin in the solution during treatment with the sample is 0.01 μg / mL to 5.0 μg / mL.

[11] The method according to any one of items 1 to 10, wherein the Gram-positive bacteria include at least bacteria of the genus Staphylococcus.

[12] The method according to any one of items 1 to 11, wherein the solution containing a surfactant and a lytic enzyme contains any dye selected from red, blue, and green.

[13] The method according to any one of items 1 to 12, which is used to detect at least one species of bacteria in a sample by immunochromatography.

[14] A method for detecting multiple species of bacteria in a sample, comprising: Lysing multiple species of bacteria in a sample by the method according to any one of items 1 to 13; and a step of detecting the bacterial antigens released from the lysed bacteria using an antibody that undergoes an antigen-antibody reaction with the antigens; A detection method comprising:

[15] The method according to Item 14, wherein the group of multiple species of bacteria in the sample to be detected includes both at least one species of Gram-positive bacteria and at least one species of Gram-negative bacteria.

[16] The method according to Item 14 or 15, wherein the detecting step is carried out by immunochromatography.

[17] The method according to any one of items 14 to 16, wherein the bacterial antigen released by the lysing step is an L7 antigen.

[18] A lysis kit for lysing a group of multiple types of bacteria including at least one or more types of Gram-positive bacteria and / or Gram-negative bacteria in a sample, which is used in the method according to any one of Items 1 to 13, the lysis kit comprising a surfactant and a lytic enzyme.

[19] A bacteria detection kit for detecting a group of multiple species of bacteria including at least one or more species of Gram-positive bacteria and / or Gram-negative bacteria in a sample, comprising: A surfactant and a lytic enzyme used in the method according to any one of items 1 to 13; below: (a) a labeling antibody-attached member to which a labeling antibody is attached, the labeling antibody forming a first complex through an antigen-antibody reaction with an antigen derived from a target bacterium; and (b) a strip having a detection region on which a capture antibody is immobilized, which forms a second complex through an antigen-antibody reaction with the first complex; An immunochromatographic detection device including The bacteria detection kit comprising:

[20] A method for detecting the presence and / or abundance of bacteria in a specimen, comprising: (I) lysing bacteria in a sample by the method according to any one of items 1 to 13; (II) capturing and labeling antigens derived from bacteria in the sample through an antigen-antibody reaction between the sample, a capture antibody immobilized on a solid phase carrier, and a labeling antibody having a detection label; and (III) detecting bacteria in a sample based on a detection label; A method in which one of the capture antibody and the labeling antibody is one or more general-purpose antibodies that undergo antigen-antibody reactions with antigens derived from five or more genera of bacteria, including bacteria, and the other of the capture antibody and the labeling antibody is one or more specific antibodies that undergo antigen-antibody reactions with one or more genera of bacteria.

[21] The method according to Item 20, wherein two or more types of bacteria in a sample are detected using a single capture antibody immobilization site. [Effects of the Invention]

[0007] According to the present invention, by performing bacteriolysis using a surfactant and a lytic enzyme, bacteria of multiple genera (multiple bacterial species) can be lysed simultaneously, making it possible to improve the efficiency and speed of the bacteriolysis process, which in turn makes it possible to improve the efficiency and speed of detection of bacteria of multiple genera using intracellular antigens of bacteria, etc. [Brief explanation of the drawings]

[0008] [Figure 1]FIG. 1 is a cross-sectional view showing a schematic configuration of a strip-shaped detection mechanism, which is an example of the detection mechanism of a lateral flow immunochromatographic detection device. [Figure 2] Figure 2 shows the results of measuring the test line intensity when samples containing Escherichia coli (EC) or Staphylococcus aureus (SA) were detected by immunochromatography, with the final concentration of N,N-dimethyldodecylamine N-oxide (DDAO) used as a surfactant being varied. [Figure 3] Figure 3 shows the results of measuring the test line intensity when samples containing Escherichia coli (EC) or Staphylococcus aureus (SA) were detected by immunochromatography, with the final concentration of benzalkonium chloride used as a surfactant being varied. [Figure 4] Figure 4 shows the results of measuring the test line intensity when a sample containing Staphylococcus aureus (SA) was detected by immunochromatography using DDAO as a surfactant and varying the final concentration of lysostaphin used as a lytic enzyme. Figure 4(a) shows all the data, and Figure 4(b) shows an expanded view of the horizontal axis (lysostaphin final concentration) in the range of 0 to 1.0 μg / ml. [Figure 5] Figure 5 shows the results of measuring the test line intensity when a specimen containing Staphylococcus aureus (SA) was detected by immunochromatography using benzalkonium chloride as a surfactant and varying the final concentration of lysostaphin used as a lytic enzyme (Figure 5a). Figure 5b shows a graph in which the horizontal axis of Figure 5a is expanded to the range of 0 to 1.0 μg / ml. DETAILED DESCRIPTION OF THE INVENTION

[0009] The present invention will be described in detail below with reference to specific embodiments, but the present invention is not limited to the following embodiments and can be embodied in any form without departing from the spirit of the present invention.

[0010] One aspect of the present invention relates to a method for lysing a group of multiple species of bacteria. Another aspect of the present invention relates to a method for detecting a group of multiple species of bacteria in a specimen, the method comprising a step of lysing the group of multiple species of bacteria. Yet another aspect of the present invention relates to a lysis agent or lysis kit used in the lysis method, and a bacteria detection kit for detecting a group of multiple species of bacteria in a specimen.

[0011] [I. Lysis method] One aspect of the present invention relates to a method for lysing a group of multiple species of bacteria in a sample (hereinafter referred to as the "lysis method of the present invention"). The lysis method of the present invention comprises a step of treating the sample in a solution containing a surfactant and a lytic enzyme to lyse the group of multiple species of bacteria present in the sample. The group of bacteria lysed by the lysis method of the present invention is then subjected to an immunological technique. The immunological technique may be any detection technique using an antigen-antibody reaction, such as immunochromatography, Western blotting, ELISA, immunoprecipitation, or immunoturbidimetry, but from the perspective of easily testing a sample, immunochromatography is particularly preferred. The group of multiple species of bacteria lysed by the lysis method of the present invention needs to be lysed so that it can be detected by an immunological technique in a subsequent step. Therefore, the lysis method of the present invention can be said to be a lysis method for detection by an immunological technique.

[0012] The step of lysing the multiple bacterial species may be carried out by any method as long as the sample is treated in a solution containing a surfactant and a lytic enzyme. For example, treatment in a solution containing a surfactant and a lytic enzyme is achieved by adding a predetermined amount of a solution containing a surfactant and a predetermined amount of a solution containing a lytic enzyme to a predetermined amount of the sample. This treatment may be referred to as a reaction. The reaction of the sample in the solution containing a surfactant and a lytic enzyme occurs when the sample, surfactant, and lytic enzyme are present in the solution, and may be mixed or stirred as needed. Here, the solution containing a surfactant and the solution containing a lytic enzyme may be provided as separate solutions, or may be provided in advance as a single solution. As another example, at least one or both of the surfactant and the lytic enzyme may be provided as a dried product and then reconstituted in any liquid for use. Such any liquid may be a liquid sample. The liquid sample may be a liquid sample itself, or, if the sample is solid, may be a suspension of the sample in a liquid such as a buffer solution. For example, a solution may be prepared by adding a dried product of at least one or both of a surfactant and a lytic enzyme directly to a liquid sample, or the dried product may be added to a liquid such as water and the resulting solution may be reacted with the sample. A solution or dried product containing a lytic enzyme and / or a surfactant may be provided in an encapsulated form. A solution may be formed by placing a dried product or capsule of a surfactant and / or a lytic enzyme in a container for the liquid sample and then adding the liquid sample. Alternatively, a dried product or capsule of a surfactant and / or a lytic enzyme may be added separately to the liquid sample. In another example, a dried product or capsule of a surfactant and a lytic enzyme may be placed in the pad of an immunochromatographic strip, and a reaction with the solution containing the surfactant and the lytic enzyme may be achieved when the liquid sample is applied to the strip. The reaction time of the sample in the solution containing the surfactant and the lytic enzyme may be any time sufficient to cause bacteriolysis, and may be, for example, 10 seconds or more, 30 seconds or more, or 1 minute or more. The upper limit of the reaction time is not particularly limited, but from the viewpoint of rapid operation, a time of 1 hour or less, 30 minutes or less, 10 minutes or less, or 5 minutes or less is preferred. The sample and the solution containing the surfactant and the lytic enzyme may be further stirred.

[0013] In the bacteriolysis method of the present invention, the plurality of bacterial species to be lysed is selected from, but not limited to, at least one or more Gram-positive bacteria and at least one or more Gram-negative bacteria. More preferably, the plurality of bacterial species includes at least one or more Gram-positive bacteria and at least one or more Gram-negative bacteria. Even more preferably, the plurality of bacterial species includes Staphylococcus bacteria, which are Gram-positive bacteria, and other bacteria.

[0014] Gram-positive and Gram-negative bacteria are bacterial classifications distinguished by Gram staining, and are known to have significant differences in the composition and structure of their cell walls. While both bacteria contain peptidoglycan, in the cell walls of Gram-positive bacteria, the peptidoglycan forms a thick layer with teichoic acid, lipoteichoic acid, and other proteins, whereas in the cell walls of Gram-negative bacteria, the peptidoglycan is localized in a relatively thin layer, surrounded by an outer layer consisting of an outer leaflet composed of lipopolysaccharides and an inner leaflet composed of phospholipids.

[0015] Due to these significant differences in cell wall composition and structure, it has been extremely difficult to simultaneously lyse Gram-positive and Gram-negative bacteria using the same lysing agent. Staphylococcus bacteria, in particular, cannot be lysed even with surfactants or enzymes capable of lysing other bacteria, posing a problem when lysing multiple bacterial species. In contrast, the lysis method of the present invention makes it possible to simultaneously lyse Gram-positive and Gram-negative bacteria, including Staphylococcus, thereby significantly improving the efficiency and speed of the lysis process.

[0016] In the present invention, Gram-positive bacteria include, but are not limited to, bacteria of the genus Staphylococcus, Bacillus, Streptococcus, Corynebacterium, Listeria, Clostridium, etc. Among these, bacteria of the genus Staphylococcus or Bacillus are preferred.

[0017] In the present invention, Gram-negative bacteria include, but are not limited to, Escherichia, Pseudomonas, Salmonella, Helicobacter, Legionella, Campylobacter, Vibrio, and Yersinia. Among these, bacteria of the genus Escherichia or Pseudomonas are preferably included.

[0018] In the bacteriolysis method of the present invention, the sample is preferably, but not limited to, a food or environmental sample. A food sample refers to any sample obtained from any food or beverage. For example, a food sample may be the food or beverage itself, or a sample obtained by diluting, crushing, swabbing, and / or suspending the food or beverage. An environmental sample refers to any sample obtained from the environment. For example, a sample obtained by swabbing the surface of a material and dispersing it in a solution may be mentioned.

[0019] In the present invention, any surfactant can be used as long as it has a bacteriolytic effect, but it is selected so as not to interfere with immunological measurement after lysis. For example, sodium dodecyl sulfate is a strong surfactant that can lyse almost all bacteria, but it destroys the structure of antibodies used in immunological techniques, making it unsuitable as a surfactant for use in the bacteriolysis method of the present invention. Examples of surfactants that do not interfere with immunological measurements after lysis, particularly immunochromatographic measurements, include, but are not limited to, amine oxides, quaternary ammonium salts, aliphatic amine salts, betaine, etc. Among these, amine oxides and quaternary ammonium salts are preferred.

[0020] Amine oxides include, but are not limited to, N,N-dimethyl-C 8-20 Examples include alkylamine N-oxides (for example, N,N-dimethyldodecylamine N-oxide, N,N-dimethylcaprylamine N-oxide), pyridine N-oxide, N-methylmorpholine N-oxide, etc. Among these, N,N-dimethyldodecylamine N-oxide, etc. are preferred.

[0021] Examples of quaternary ammonium salts include, but are not limited to, benzalkonium chloride (benzalkonium chloride), tetramethylammonium chloride, tetraethylammonium chloride, tetrapropylammonium chloride, tetrabutylammonium chloride, benzyltrimethylammonium chloride, benzyltriethylammonium chloride, hexadecyltrimethylammonium chloride, benzethonium chloride, benzalkonium chloride, cetylpyridinium chloride, etc. Among these, benzalkonium chloride, etc. is preferred.

[0022] The concentration of the surfactant in the lysis of the present invention is not limited, but is preferably, for example, 0.005% by mass or more, or 0.01% by mass or more, or 0.02% by mass or more in the liquid during reaction with the sample, and is also preferably, for example, 0.2% by mass or less, or 0.15% by mass or less, or 0.1% by mass or less.

[0023] When the surfactant is an amine oxide such as N,N-dimethyldodecylamine N-oxide, the concentration of the amine oxide in the bacteriolysis method of the present invention is not limited, but is preferably, for example, 0.01% by mass or more, 0.025% by mass or more, or 0.05% by mass or more in the solution during reaction with the specimen in order to exert the bacteriolytic effect. Furthermore, in order to maintain the activity of an enzyme used in combination, the concentration of the amine oxide in the solution during reaction with the specimen is preferably, for example, 0.2% by mass or less, 0.15% by mass or less, or 0.1% by mass or less.

[0024] When the surfactant is a quaternary ammonium salt such as benzalkonium chloride, the concentration of the quaternary ammonium salt in the bacteriolysis method of the present invention is not limited, but is preferably, for example, 0.005% by mass or more, 0.01% by mass or more, or 0.02% by mass or more in the solution during reaction with the sample in order to exert the bacteriolytic effect. Furthermore, in order to suppress aggregation of the labeling antibody, it is preferably, for example, 0.08% by mass or less, 0.075% by mass or less, or 0.065% by mass or less.

[0025] Examples of lytic enzymes include, but are not limited to, proteolytic enzymes, carbohydrate hydrolases, etc. Examples of proteolytic enzymes include lysostaphin, pepsin, glucosidase, galactosidase, achromopeptidase, etc. Examples of carbohydrate hydrolases include lysozyme, β-N-acetylglucosaminidase, etc. Among these, proteolytic enzymes are preferred, with lysostaphin being particularly preferred.

[0026] The concentration of the lytic enzyme in the bacteriolysis method of the present invention is not limited and may be selected appropriately depending on the type of lytic enzyme. However, from the viewpoint of exerting the bacteriolytic effect, in the case of a proteolytic enzyme such as lysostaphin, the concentration in the solution upon reaction with the sample is preferably, for example, 0.01 μg / mL or more, 0.025 μg / mL or more, or 0.05 μg / mL or more. Furthermore, from the viewpoint of preventing false positives in immunological tests, the concentration in the solution upon reaction with the sample is preferably, for example, 5.0 μg / mL or less, 3.0 μg / mL or less, or 2.5 μg / mL or less.

[0027] The bacteriolysis method of the present invention specifies that a specimen is reacted in a solution containing at least one surfactant and at least one lytic enzyme as specified in the present invention. However, the solution may further contain one or more other components as long as the intended bacteriolysis effect is not significantly impaired. Such other components include, for example, nonionic surfactants, buffers, and lysis promoters. These components may be added to promote bacteriolysis or to enable detection after bacteriolysis.

[0028] A nonionic surfactant can be added to ensure the flow of the developing solution when immunochromatography is used after lysis. The nonionic surfactant is not limited to, but any of ester ether type, ester type, and ether type can be suitably used. More specifically, polyoxyethylene alkyl ether, polyoxyethylene alkylphenyl ether, fatty acid sorbitan ester, alkyl polyglucoside, fatty acid diethanolamide, alkyl monoglyceryl ether, etc. are exemplified. Among them, Tween (登録商標) , Triton (登録商標)The concentration of the nonionic surfactant is not limited as long as it does not significantly inhibit lysis in the lysis method of the present invention, development in detection by immunochromatography after lysis, or antigen-antibody reactions in immunoassays, but is preferably 0.03% by mass or more, 0.05% by mass or more, or 0.1% by mass or more in terms of the concentration in the liquid during reaction with the sample, and is preferably 10% by mass or less, 5% by mass or less, or 3% by mass or less, from the viewpoint of not significantly inhibiting the reaction by the lytic enzyme or the antigen-antibody reaction.

[0029] The buffer solution is preferably, but not limited to, a buffer solution having a pH of 6.0 to 9.5. From the viewpoints of maintaining lytic enzyme activity during lysis, maintaining antigen-antibody reaction activity during detection after lysis, and avoiding false positives, a pH of 6.4 or higher or 6.9 or higher is preferred, and a pH of 9.0 or lower or 8.5 or lower is preferred. Examples of such buffer solutions include, but are not limited to, Tris buffer solution, MOPSO buffer solution, HEPES buffer solution, citrate buffer solution, phosphate buffer solution, acetate buffer solution, etc. Among these, Tris-HCl buffer solution, MOPSO buffer solution, etc. are preferred.

[0030] The bacteriolysis method of the present invention specifies that a specimen is reacted in a solution containing at least one surfactant and at least one lytic enzyme as specified in the present invention. For example, the solution containing a surfactant and a lytic enzyme is obtained by mixing a solution containing a surfactant with a solution containing a lytic enzyme. The solution containing a surfactant and a lytic enzyme may contain a dye to prevent accidental ingestion. The solution containing a surfactant and a lytic enzyme may be colored by adding a dye beforehand to the solution containing a surfactant and / or the solution containing a lytic enzyme, or by adding a solution containing a dye when mixing the solution containing a surfactant and the solution containing a lytic enzyme. Examples of the solution containing a dye include a buffer solution. The dye can be selected so as not to interfere with the test performed after bacteriolysis, and any dye selected from red, blue, yellow, and green can be used. For example, when an immunochromatography method using gold colloid is used in a subsequent step, yellow, blue, or green coloring is preferred because gold colloid exhibits a red color. While any dye may be used, a water-soluble dye is preferred, and food dyes are preferred to ensure safety in the event of oral ingestion.

[0031] [II. Lytic Agents] One aspect of the present invention also relates to a lytic agent for lysing a group of multiple types of bacteria, including at least one or more Gram-positive and / or Gram-negative bacteria, in a sample (hereinafter also referred to as the lytic agent of the present invention). A solution containing the lytic agent of the present invention is also referred to as a "reaction solution." The lytic agent of the present invention contains at least a surfactant and a lytic enzyme. The lytic agent of the present invention may be provided in the form of a solution or a dried product. In particular, the lytic enzyme may be padded or encapsulated and provided in a dried product or solution to maintain its activity. The surfactant and the lytic enzyme may be provided in the same form or in different forms. The lytic agent is usually provided as a single formulation containing a surfactant and a lytic enzyme. A lysis kit includes both a single formulation containing a surfactant and a lytic enzyme (i.e., the lytic agent) and a formulation in which the surfactant and the lytic enzyme are provided separately. The lytic agent of the present invention may further contain one or more other ingredients, as long as the intended lytic effect is not significantly impaired, and may also contain a dye to prevent accidental ingestion. Such other components include, for example, nonionic surfactants, buffers, and lysis promoters, which may be added to promote lysis or to enable detection after lysis. The surfactants, lytic enzymes, other components, and dyes contained in the lysis agent are those described herein.

[0032] [III. Bacterial Detection Methods] One aspect of the present invention relates to a method for detecting a group of multiple bacteria selected from at least one species of Gram-positive bacteria and at least one species of Gram-negative bacteria in a sample (hereinafter referred to as the "bacteria detection method of the present invention"). The bacteria detection method of the present invention includes the steps of lysing multiple genera of bacteria in a sample by carrying out the lysis method of the present invention and detecting bacterial antigens released from the lysed bacteria using antibodies that undergo an antigen-antibody reaction with the antigens. Therefore, the bacteria detection method of the present invention relates to an immunological technique using antibodies. The immunological technique may be any detection technique using an antigen-antibody reaction, such as immunochromatography, immunoprecipitation, immunoturbidimetry, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), or fluorescence immunoassay (FIA). From the perspective of easy sample testing, immunochromatography is particularly preferred. The multiple species of bacteria detected by the bacteria detection method of the present invention relate to some or all of the bacteria lysed by the lysis method of the present invention.

[0033] In the bacterial detection method of the present invention, the bacterial antigen is preferably an intracellular antigen. An intracellular antigen is a bacterial intracellular substance released by the bacteriolysis method of the present invention, which serves as an antigen. More specifically, from the perspective of detecting bacteria, the intracellular antigen is preferably a ribosomal protein, particularly the L7 / L12 protein. The L7 / L12 ribosomal protein is a type of ribosomal protein essential for microbial protein synthesis and is a protein commonly found in various bacteria. Furthermore, since multiple molecules of the L7 / L12 ribosomal protein exist intracellularly, detection sensitivity is high. For antibodies that undergo antigen-antibody reactions with bacterial ribosomal protein L7 / L12 and methods for producing such antibodies, see, for example, International Publication No. WO 2000 / 006603, a patent publication previously filed by the present inventors. In immunochromatography, a labeling antibody labeled for detection and a capture antibody immobilized on a strip are used. The labeling antibody and the capture antibody each bind to an intracellular antigen.

[0034] [antibody] In the present invention, an "antibody" refers to a protein that recognizes and binds to a specific antigen or substance, and is sometimes called an immunoglobulin (Ig). A typical antibody usually has two light chains and two heavy chains interconnected by disulfide bonds. There are two types of light chains, called λ chains and κ chains, and there are five types of heavy chains, called γ chains, μ chains, α chains, δ chains, and ε chains. Depending on the type of heavy chain, there are five isotypes of antibodies: IgG, IgM, IgA, IgD, and IgE.

[0035] Each heavy chain contains a heavy chain constant (CH) region and a heavy chain variable (VH) region. Each light chain contains a light chain constant (CL) region and a light chain variable (VL) region. The light chain constant (CL) region is composed of a single domain. The heavy chain constant (CH) region is composed of three domains, namely CH1, CH2, and CH3. The light chain variable (VL) region and the heavy chain variable (VH) region each contain four highly conserved regions called framework regions (FR) (FR-1, FR-2, FR-3, FR-4) and three hypervariable regions called complementarity determining regions (CDR) (CDR-1, CDR-2, CDR-3). The heavy chain constant (CH) region has three CDRs (CDR-H1, CDR-H2, CDR-H3) and four FRs (FR-H1, FR-H2, FR-H3, FR-H4), arranged from amino to carboxy terminus in the following order: FR-H1, CDR-H1, FR-H2, CDR-H2, FR-H3, CDR-H3, FR-H4. The light chain constant (CL) region has three CDRs (CDR-L1, CDR-L2, CDR-L3) and four FRs (FR-L1, FR-L2, FR-L3, FR-L4), arranged from amino to carboxy terminus in the following order: FR-L1, CDR-L1, FR-L2, CDR-L2, FR-L3, CDR-L3, FR-L4. The heavy and light chain variable regions contain binding domains that interact with antigens.

[0036] The antibody of the present invention may be a polyclonal or monoclonal antibody, but is preferably a monoclonal antibody. Polyclonal antibodies are typically prepared from the serum of animals immunized with an antigen and are a mixture of various structurally distinct antibody molecule species. On the other hand, monoclonal antibodies refer to antibodies consisting of a single type of molecule containing a combination of a light chain variable (VL) region and a heavy chain variable (VH) region with a specific amino acid sequence. Monoclonal antibodies can be produced from clones derived from antibody-producing cells, but they can also be produced by genetic engineering using nucleic acid molecules containing gene sequences encoding the amino acids of the antibody protein. Furthermore, techniques well known to those skilled in the art include modifying the heavy and light chains, or their variable regions, CDRs, and other genetic information to improve the binding and specificity of antibodies.

[0037] The antibody of the present invention may also be an antibody fragment and / or derivative. Examples of antibody fragments include F(ab')2, Fab, and Fv. Examples of antibody derivatives include antibodies with artificially introduced amino acid mutations in the light and / or heavy chain constant regions, antibodies with modified domain configurations in the light and / or heavy chain constant regions, antibodies with two or more Fc regions per molecule, glycosylated antibodies, bispecific antibodies, antibody conjugates in which an antibody or antibody fragment is bound to a protein other than an antibody, antibody enzymes, tandem scFvs, bispecific tandem scFvs, and diabodies. Furthermore, when the above-mentioned antibody or its fragment or derivative is derived from a non-human animal, the antibody of the present invention also includes chimeric antibodies or humanized antibodies in which part or all of the sequence other than the CDRs has been replaced with the corresponding sequence of a human antibody. Unless otherwise specified, the simple term "antibody" used in the present invention also includes antibody fragments and / or derivatives.

[0038] The antibody of the present invention undergoes an antigen-antibody reaction with a certain bacterium means that the antibody specifically binds to some component of the bacterium as an antigen. The bacterial component that serves as the antigen for the antibody of the present invention is not limited. It may be a component contained in the cell wall or cell membrane, which is exposed outside the bacterial cell, or a component contained in the cytoplasm, organelles, nucleus, etc., which is not exposed outside the bacterial cell.

[0039] There are no particular limitations on the level of the antigen-antibody reaction between the antibody of the present invention and the bacteria to be detected, as long as the antigen-antibody reaction occurs to an extent that can be detected by any known detection method.

[0040] Furthermore, it is preferable that the antibodies of the present invention do not cross-react with one or more non-bacterial components that may be present in a sample. Examples of such non-bacterial components include, but are not limited to, various bioorganic compounds derived from viruses, plants, and / or animals that are not present in bacteria. Specific examples of such bioorganic compounds include proteins, sugars, glycoproteins, lipids, complex lipids, nucleic acids, etc. It is preferable that the antibodies of the present invention do not cross-react with at least one or more, usually three or more, even four or more, five or more, six or more, seven or more, eight or more, particularly nine or more, and particularly ten or more of these non-bacterial components.

[0041] The antibodies of the present invention are not limited as long as they undergo an antigen-antibody reaction with the bacteria to be detected, but it is preferable to use the following general-purpose antibodies and / or specific antibodies, and when detecting bacterial antigens in a sandwich-type manner, it is preferable to use a combination of a general-purpose antibody and a specific antibody. It is preferable that the general-purpose antibody undergoes an antigen-antibody reaction with as many genera of bacteria as possible. Specifically, the general-purpose antibody undergoes an antigen-antibody reaction with at least four genera of bacteria. Of these, it is preferable that the general-purpose antibody undergoes an antigen-antibody reaction with at least five or more genera of bacteria, and it is even more preferable that the general-purpose antibody undergoes an antigen-antibody reaction with at least six or more genera of bacteria. There are no limitations on the specific bacterial genera with which the universal antibody generates an antigen-antibody reaction, but it is preferable that the universal antibody generates an antigen-antibody reaction with at least one genera of bacteria selected from the genera Escherichia, Staphylococcus, Pseudomonas, Bacillus, Klebsiella, Serratia, Rahnella, Citrobacter, Listeria, Enterobacter, and Salmonella.

[0042] On the other hand, it is preferable that the specific antibody undergoes an antigen-antibody reaction only with bacteria of a limited genus. Specifically, the range of bacteria with which the specific antibody undergoes an antigen-antibody reaction is made to match the range of bacteria to be detected. When only a single specific antibody is used, the range of bacteria with which that specific antibody undergoes an antigen-antibody reaction is made to match the range of bacteria to be detected. On the other hand, when two or more specific antibodies are used in combination, the combined range of bacteria with which each of those specific antibodies undergoes an antigen-antibody reaction should match the range of bacteria to be detected. In particular, the latter embodiment is extremely advantageous, as it makes it possible to adjust the range of bacteria to be detected in various ways by appropriately combining multiple specific antibodies that each undergo an antigen-antibody reaction with different bacteria.

[0043] Each specific antibody of the present invention is only required to undergo an antigen-antibody reaction with bacteria of at least one genus. The specific genus of bacteria with which each specific antibody undergoes an antigen-antibody reaction is not limited, but it is preferable that the antibody undergo an antigen-antibody reaction with bacteria of at least one or more genera selected from the genera Escherichia, Staphylococcus, Pseudomonas, Bacillus, Klebsiella, Serratia, Rahnella, Citrobacter, Listeria, Enterobacter, and Salmonella.

[0044] [Labeling antibody] The labeling antibody is an antibody labeled for detection and forms a complex through an antigen-antibody reaction with an antigen derived from the target bacterium released from the lysed target bacterium. The complex thus formed is referred to as the "first complex." The type of detection label used for the labeling antibody is not particularly limited and may be selected appropriately depending on the detection method. Specific examples include metal colloids such as gold colloid, platinum colloid, and palladium colloid; non-metal colloids such as selenium colloid, alumina colloid, and silica colloid; insoluble granular substances such as colored resin particles, dye colloids, and colored liposomes; color-developing reaction-catalyzing enzymes such as alkaline phosphatase, peroxidase, and luciferase; fluorescent dyes; radioisotopes; chemiluminescent labels, bioluminescent labels, and electrochemiluminescent labels. The method for labeling the antibody is also not particularly limited. Specific examples include physical adsorption utilizing the hydrophobicity of the antibody and chemical binding utilizing the functional groups of the antibody.

[0045] [Capture antibody] The capture antibody is an antibody that forms a complex with the first complex through an antigen-antibody reaction. The complex thus formed is referred to as the second complex. When used in immunochromatography, the capture antibody is more specifically immobilized on a chromatographic development membrane carrier located in the detection area of ​​the immunochromatographic strip. The method for immobilizing the antibody on the solid phase material is not particularly limited, but specific examples include immobilization by physical adsorption utilizing the hydrophobicity of the antibody and immobilization by chemical bonding utilizing the functional groups of the antibody.

[0046] The general-purpose antibody may be a labeling antibody and the specific antibody may be a capture antibody, or the specific antibody may be a labeling antibody and the general-purpose antibody may be a capture antibody. From the viewpoint of facilitating the production of an immunochromatographic detection device, it is preferable that the general-purpose antibody is a labeling antibody and the specific antibody is a capture antibody.

[0047] The second complex is retained at the immobilized position of the capture antibody (the capture antibody immobilization site) due to an antigen-antibody reaction with the immobilized capture antibody, where the label of the labeling antibody is detected. When the label is detected at the position where the capture antibody is immobilized, the target bacterium is detected in the sample liquid.

[0048] [IV. Kit] Another aspect of the present invention relates to a lysis kit (hereinafter referred to as "the lysis kit of the present invention") for lysing multiple bacterial species selected from at least one or more Gram-positive bacteria and at least one or more Gram-negative bacteria in a sample, which is used in the lysis method of the present invention described above. The lysis kit includes a preparation containing a surfactant and a lytic enzyme (i.e., a lysis agent) and a preparation in which the surfactant and the lytic enzyme are provided separately. Such a lysis kit is used for detecting bacterial species after lysis, and more preferably relates to a lysis kit for immunochromatographic detection. The bacterial species to be lysed, the surfactant, and the lytic enzyme are as described above.

[0049] The lysis kit may be provided with the surfactant and the lytic enzyme in solution or solid form in one container or in two or more containers.

[0050] Another aspect of the present invention may relate to a detection kit for detecting a group of multiple bacteria selected from at least one or more types of bacteria, for example, at least one or more types of Gram-positive bacteria and at least one or more types of Gram-negative bacteria, for use in the aforementioned bacterial detection method of the present invention (hereinafter, appropriately referred to as the "bacterial detection kit of the present invention"). The bacterial detection kit of the present invention may include a lysis kit, or a surfactant and a lytic enzyme may be pre-disposed in an apparatus or container used in the bacterial detection kit. Such a bacterial detection kit detects a group of bacteria based on an immunological technique. The immunological technique may be any detection technique using an antigen-antibody reaction, such as immunochromatography, immunoprecipitation, immunoturbidimetry, enzyme-linked immunosorbent assay (ELISA), radioimmunoassay (RIA), or fluorescence immunoassay (FIA). However, from the perspective of simple sample testing, immunochromatography is particularly preferred. The group of multiple bacteria detected by the bacterial detection kit of the present invention relates to some or all of the bacteria lysed by the lysis method of the present invention. The surfactant and lytic enzyme used in the bacteria detection kit of the present invention are as described above.

[0051] The bacteria detection kit of the present invention comprises the above-mentioned capture antibody and labeling antibody. The capture antibody is usually provided in an appropriate form depending on the type of solid phase carrier (such as a container including a porous membrane, a container including a flow path, or a plate capable of holding a solution). The labeling antibody is usually provided in the form of an aqueous reagent containing the labeling antibody in an aqueous medium or a dry reagent in which the labeling antibody is dried.

[0052] In addition to the above-mentioned capture antibody and labeling antibody, the bacteria detection kit of the present invention includes one or more reagents, a detection device or its components, and / or instructions describing the procedures for carrying out the method of the present invention, which are necessary for carrying out the method of the present invention using these antibodies. The types of reagents, the contents of the instructions, and other components included in the kit of the present invention may be determined appropriately depending on the specific type of immunoassay method.

[0053] When the bacteria detection kit of the present invention includes a detection device or its components, the device constituted by such a kit is a device equipped with the components necessary to carry out the method of the present invention using the labeling antibody and / or capture antibody of the present invention (hereinafter referred to as the "device of the present invention"). The specific components of the device of the present invention can be appropriately adjusted depending on the type of immunoassay, which is a specific embodiment of the method of the present invention. As mentioned above, examples of immunoassays include, but are not limited to, various known immunoassays such as ELISA (enzyme-linked immunosorbent assay) using an antibody-supported microtiter plate; latex particle agglutination assay using antibody-supported latex particles (e.g., polystyrene latex particles); immunochromatography using an antibody-supported membrane; and sandwich assay using a labeling antibody labeled with colored particles, color-developing particles, enzymes, or fluorophores, and a capture antibody immobilized on a solid phase carrier such as magnetic particles. The device of the present invention is equipped with the components necessary to carry out such various immunoassays. Two or more types of bacteria in a sample can also be detected using a single capture antibody immobilization site. In this case, one or more types of capture antibodies are immobilized on a single capture antibody immobilization site. By using a single capture antibody immobilization (linking) site, two or more types of bacteria in a sample can be detected simultaneously (total amount of bacteria can be detected).

[0054] Specific examples of immunochromatographic detection devices include lateral flow and flow-through devices. The lateral flow method involves developing a target analyte and a labeling antibody in parallel across a membrane containing a detection region with a capture antibody immobilized on its surface, thereby detecting the target substance captured in the detection region of the membrane. Lateral flow kits are broadly classified into dipstick and cassette types. Dipstick kits develop the target solution by immersing the immersion region (which may be a sample pad) of the detection device in the sample solution, whereas cassette kits develop the target solution by adding the sample to the sample addition member (sample pad) (3) of the detection device. The flow-through method involves passing a target analyte and a labeling antibody vertically through a membrane with a capture antibody immobilized on its surface, thereby detecting the target substance captured on the membrane surface. The method of the present invention can be applied to both lateral flow and flow-through devices.

[0055] Both lateral flow-type devices and flow-through-type immunochromatographic detection devices are publicly known, and procedures other than those described in this disclosure can be appropriately designed by a person skilled in the art based on common technical knowledge. Below, the schematic configuration of the detection mechanism of a lateral flow-type immunochromatographic detection device is described with reference to the drawings, but this is merely one example of the schematic configuration of the detection procedure, and the configuration of the lateral flow-type immunochromatographic detection device is not limited in any way to the embodiment illustrated in the drawings.

[0056] FIG. 1 is a cross-sectional view showing the schematic configuration of a strip-shaped detection mechanism, which is an example of the detection mechanism of a lateral flow immunochromatographic detection device. The immunochromatographic detection device (10) of FIG. 1 is configured on a substrate (5) with a strip-shaped labeling antibody-attached member (conjugate pad) (2) (to which a labeling antibody is attached) and a specimen-adding member (sample pad) (3) arranged at one end (upstream of specimen flow B) of the strip lengthwise on a chromatographic development membrane carrier (1), and an absorption member (absorption pad) (4) arranged at the other end (downstream of specimen flow B). The labeling antibody attached to the labeling antibody-attached member (conjugate pad) (2) may be eluted into the specimen solution. A capture antibody-immobilized site (6) to which a capture antibody is immobilized is arranged at the center of the strip lengthwise on the chromatographic development membrane carrier (1), and, if necessary, a control reagent-immobilized site (7) to which a control reagent is immobilized is also arranged. The control reagent is a reagent that does not bind to the analyte but binds to the labeling antibody. Furthermore, a bacteriolytic agent may be placed on the strip so that it can be dissolved into the sample solution.

[0057] During use, when specimen A is applied to the specimen application member (sample pad) (3), it passes through the labeling antibody attachment member (conjugate pad) (2) and flows through the chromatographic development membrane carrier (1) in the direction of specimen flow B. During this process, the analyte in the specimen (in this invention, the bacterial antigens L7 / L12) binds to the labeling antibody, forming a first analyte-labeling antibody complex. When specimen A passes through the capture antibody immobilization site (6), the analyte in the specimen binds to the capture antibody, forming a second capture antibody-analyte-labeling antibody complex. Furthermore, when specimen A passes through the control reagent immobilization site (7), the labeling antibody that has not bound to the analyte binds to the control reagent, thereby confirming the completion of the test (i.e., specimen A has passed through the capture antibody immobilization site (6)). Here, the presence or absence or amount of the analyte can be detected by detecting the label of the labeling antibody in the second complex of capture antibody-analyte-labeling antibody present at the capture antibody immobilization site (6) by known means. If necessary, the label of the labeling antibody may be sensitized by known techniques to facilitate detection.

[0058] The labeling antibody attachment member (conjugate pad) (2), the specimen addition member (sample pad) (3), and / or the control reagent immobilization site (7) can be optionally omitted. If this system does not have the labeling antibody attachment member (conjugate pad) (2), a test similar to the above can be performed by applying specimen A and the labeling antibody, either premixed or separately, simultaneously or sequentially to one end of the chromatographic development membrane carrier (1).

[0059] Furthermore, even if the capture antibody and labeling antibody are interchanged, a detection kit capable of similar detection can be constructed.

[0060] Other aspects of the present invention are listed in items 22 to 34 below.

[22] A method for detecting the presence and / or abundance of bacteria in a specimen, comprising: (I) treating a specimen with a solution containing a surfactant and a lytic enzyme; (II) capturing and labeling antigens derived from bacteria in the sample through an antigen-antibody reaction between the sample, a capture antibody immobilized on a solid phase carrier, and a labeling antibody having a detection label; and (III) detecting bacteria in a sample based on a detection label; A method in which one of the capture antibody and the labeling antibody is one or more general-purpose antibodies that undergo antigen-antibody reactions with antigens derived from five or more genera of bacteria, including bacteria, and the other of the capture antibody and the labeling antibody is one or more specific antibodies that undergo antigen-antibody reactions with one or more genera of bacteria. According to this embodiment, even if multiple types of bacteria are present in a sample, they can be lysed simultaneously, making it possible to make the lysis process more efficient and rapid, and as a result, making detection more efficient and rapid.

[23] The method according to Item 22, wherein the bacteria include both at least one species of Gram-positive bacteria and at least one species of Gram-negative bacteria.

[24] The method of claim 22 or 23, wherein the surfactant is one or more amine oxides.

[25] The method of claim 24, wherein the amine oxide is N,N-dimethyldodecylamine N-oxide (DDAO).

[26] 26. The method according to item 24 or 25, wherein the concentration of the amine oxide in the solution during treatment with the specimen is 0.01 to 0.2% by mass.

[27] The method of any one of items 22 or 23, wherein the surfactant is one or more quaternary ammonium salts.

[28] The method of claim 27, wherein the quaternary ammonium salt is benzalkonium chloride.

[29] The method according to Item 27 or 28, wherein the concentration of the quaternary ammonium salt in the solution during treatment with the specimen is 0.005 to 0.08% by mass.

[30] 30. The method according to any one of items 22 to 29, wherein the lytic enzyme is lysostaphin.

[31] The method according to Item 30, wherein the concentration of the lysostaphin in the solution during treatment with the sample is 0.01 μg / mL to 5.0 μg / mL. According to the aspects of items 23 to 31, by treating with a solution containing a specific surfactant and an enzyme adjusted to a specific concentration, a more reliable bacteriolysis treatment can be achieved, which not only makes the bacteriolysis treatment more efficient and rapid, but also enables more reliable detection.

[32] The method according to any one of Items 22 to 31, wherein the Gram-positive bacteria include at least bacteria of the genus Staphylococcus.

[33] The method according to any one of Items 22 to 32, wherein the solution containing a surfactant and a lytic enzyme contains any dye selected from red, blue, and green.

[34] The method according to any one of Items 22 to 33, which is used to detect at least one species of bacteria in a sample by immunochromatography. [Example]

[0061] The present invention will be described in more detail below with reference to examples. However, these examples are merely examples shown for the convenience of explanation, and the present invention is not limited to these examples in any sense.

[0062] Example 1: Preparation of immunochromatographic test strip for detecting multiple species of bacteria including at least one species of Gram-positive bacteria and / or Gram-negative bacteria -Generating universal antibodies Pseudomonas aeruginosa was used as the immunogen. Antibodies against P. aeruginosa ribosomal protein L7 / L12 were produced according to the method described in International Publication No. 2000 / 06603. Specifically, Escherichia coli transformed with an expression vector incorporating DNA encoding the entire amino acid sequence of P. aeruginosa ribosomal protein L7 / L12 were cultured in LB medium or similar medium, and the fusion protein was purified using an affinity column using the tag sequence derived from the expression vector. Using this full-length P. aeruginosa L7 / L12 protein as an immunogen, mice were immunized four times using standard hybridoma isolation methods, with the immunogen concentration adjusted to 0.4 mg / mL in PBS and an equal volume of Freund's adjuvant added. The immunogen was then immunized with an immunogen dose of 50 μg per immunization. After confirming an increase in serum antibody titer by blood sampling, mouse spleen cells were isolated. The isolated mouse spleen cells were fused with myeloma cells to obtain various hybridomas. The obtained hybridomas were cultured in HAT medium, and the antibodies in the culture supernatant were used for screening. Screening was performed by ELISA, and hybridomas producing antibodies simultaneously reactive with bacterial lysates of at least four bacterial species: Escherichia coli (EC), Staphylococcus aureus (SA), Pseudomonas aeruginosa (PA), and Bacillus subtilis (BS) were selected. Following standard methods for monoclonal antibody production, the selected hybridomas were cultured in TIL Media I medium supplemented with 10% fetal bovine serum (FBS) and administered intraperitoneally to mice, and the ascites fluid was collected. The collected ascites fluid was centrifuged to separate floating matter and red blood cells, and then filtered through a 0.45 μm filter. The resulting filtrate was passed through a Protein G column to adsorb the antibody, and the resulting general-purpose antibody was purified from the mouse ascites.

[0063] Preparation of specific antibodies I Similarly, various hybridomas were obtained using the same procedure as for obtaining general antibodies, except that Haemophilus influenzae (HI) was used as the immunogen and the ribosomal protein L7 / L12 of H. influenzae was used as the immunogen. Hybridomas producing antibodies reactive with bacterial lysates of at least two bacterial species, Escherichia coli (EC) and Pseudomonas aeruginosa (PA), were then selected. Specific antibody I was then produced using the same procedure as for obtaining general antibodies.

[0064] Preparation of specific antibodies II Similarly, various hybridomas were obtained using the same procedure as for obtaining generic antibodies, except that Staphylococcus aureus (SA) was used as the immunogen and the ribosomal protein L7 / L12 of Staphylococcus aureus was used as the immunogen. Hybridomas that produced antibodies reactive with bacterial lysates of at least two species of bacteria, Staphylococcus aureus (SA) and Bacillus subtilis (BS), were then selected. Specific antibody II was produced by the procedure.

[0065] - Preparation of immunochromatographic test strips The obtained general-purpose antibody was used as a labeling antibody, and specific antibodies I and II were used as capture antibodies to prepare the following immunochromatographic test strip.

[0066] - Preparation of membrane carrier for immunochromatography Specific antibodies I and II were mixed in 10 mM sodium phosphate buffer solution at 1.5 mg / mL each, and a solution containing 3% (v / v) trehalose was prepared. The resulting solution was applied to a commercially available nitrocellulose membrane cut to a width of 2.5 cm and a length of 15 cm. 2 The solution was applied in a volume of 1 μL per sample to form a single line, and then dried to prepare a membrane carrier for immunochromatographic development.

[0067] Preparation of labeling antibodies using gold colloid as a label and antibody-attached materials using gold colloid as a label: A commercially available colloidal gold solution (particle size 60 nm) was mixed with 1 / 10 the amount of a general-purpose antibody to prepare a solution with an antibody concentration of 0.1 mg / mL. This solution was left to stand at room temperature for 30 minutes to allow the antibody to bind to the surface of the colloidal gold particles. A BSA solution was then added to the colloidal gold solution to a final concentration of 0.1% for blocking, preparing a labeling antibody solution using colloidal gold as a label. This antibody solution was impregnated into a commercially available glass fiber sheet and then dried to prepare a labeling antibody-attached member using colloidal gold as a label.

[0068] Assembling the immunochromatographic test strip: In addition to the immunochromatographic development membrane carrier and the labeling antibody-immobilized member using gold colloid as a label prepared by the procedure described above, cotton cloth was prepared as the sample addition member and filter paper as the absorption member. These members were then attached to a commercially available polyethylene substrate and cut to a width of 5 mm to prepare an immunochromatographic test strip with the same configuration as in Figure 1 (the control reagent immobilization site 7 is omitted).

[0069] Example 2: Evaluation of lysis and detection performance with various reaction solution (lysing agent) compositions Preparation of bacterial solution for evaluation: Escherichia coli (EC) and Pseudomonas aeruginosa (PA) were selected as Gram-negative bacteria, and Staphylococcus aureus (SA) and Bacillus subtilis (BS) were selected as Gram-positive bacteria, and each was prepared with physiological saline to a concentration of 1e6 cfu / ml. The solution without bacteria was also prepared with physiological saline.

[0070] Preparation of various reaction solutions: Using a 0.1 M Tris-HCl (pH 7.4) buffer solution containing 0.2% Tween 20 for immunochromatographic development as a mother solution, various reaction solutions were prepared under the conditions shown in Table 1. The concentrations of the various surfactants were 0.1% (final concentration after mixing with the bacterial solution for evaluation (sample) was 0.05%), and the concentrations of the various bacteriolytic enzymes were 2.0 μg / ml (final concentration after mixing with the bacterial solution for evaluation (sample) was 1.0 μg / ml).

[0071] [Table 1]

[0072] Immunochromatographic detection performance evaluation: The above bacterial solution for evaluation and the above various reaction solutions were mixed at a 1:1 ratio, and the immunochromatographic test strip prepared in Example 1 was inserted to evaluate bacterial lysis and detection performance based on antigen-antibody reaction. The evaluation was performed by visually judging the degree of red color caused by the gold colloid in the test line area. The results are shown in Table 2. Visually positive was represented as +, weakly visually positive as ±, and visually negative as -. [Table 2]

[0073] Under conditions without surfactants (2-1), only weakly positive signals for PA and BS were observed. This is thought to be due to slight bacteriolysis caused by Tween 20 added for immunochromatographic development. On the other hand, under conditions with various surfactants (2-2 to 2-11), all results were visually negative without bacteria, but with bacteria, the results varied depending on the bacterial species. In particular, EC and SA were difficult to lyse with most surfactants, but DDAO (condition 2-2) or benzalkonium chloride (condition 2-3) resulted in a positive result for EC and a weak positive result for SA, allowing bacteriolysis and immunochromatographic detection.

[0074] Next, to enhance the SA signal, a lytic enzyme (lysostaphin or lysozyme) was added to the reaction mixture containing DDAO or benzalkonium chloride (conditions 2-12 to 2-15). Under conditions with lysostaphin (2-12, 2-13), a positive SA signal was observed. Furthermore, the absence of bacteria resulted in a negative result, while bacteria other than SA also showed a positive result. Under conditions with lysozyme (2-14, 2-15), there was no change compared to the absence of lysozyme (2-2, 2-3), and the SA signal remained weakly positive.

[0075] The above results demonstrate that by using DDAO or benzalkonium chloride as a surfactant and lysostaphin as a lytic enzyme, multiple bacterial species, including at least one Gram-positive and / or Gram-negative bacteria, can be simultaneously lysed and detected by immunochromatography.

[0076] Example 3: Concentration study of DDAO or benzalkonium chloride Preparation of bacterial solution (sample) for evaluation: Escherichia coli (EC) was selected as the gram-negative bacterium, and Staphylococcus aureus (SA) was selected as the gram-positive bacterium, and each was prepared in saline at a concentration of 1e5 cfu / ml. A solution without bacteria was also prepared in saline.

[0077] Preparation of various reaction solutions: A mother solution was prepared by adding 2.0 μg / ml of lysostaphin enzyme (final concentration after mixing with the evaluation bacterial solution (sample) of 1.0 μg / ml) and 0.2% by mass of Tween 20 for immunochromatographic development to 0.1 M Tris-HCl (pH 7.4) buffer solution. Various reaction solutions were prepared using this mother solution containing 0 to 0.5% by mass of DDAO (final concentration after mixing with the evaluation bacterial solution (sample) of 0 to 0.25% by mass) or 0 to 0.5% by mass of benzalkonium chloride (final concentration after mixing with the evaluation bacterial solution (sample) of 0 to 0.25% by mass).

[0078] Immunochromatographic detection performance evaluation: The bacterial solution for evaluation and the various reaction solutions described above were mixed at a 1:1 ratio, and the immunochromatographic test strip prepared in Example 1 was inserted to evaluate bacterial lysis and detection performance through antigen-antibody reaction. The evaluation was performed by photographing the redness of the gold colloid in the test line area with a camera and then quantifying it through image processing (the G value, which indicates red absorption among RGB, was used; this is shown as "test line intensity" in the graphs described below). The higher the test line intensity, the higher the redness, and a test line intensity of 10 or higher is considered visually positive.

[0079] Figure 2 shows the relationship between DDAO concentration and test line intensity. No poor immunochromatographic development was observed at any concentration of DDAO added. The results of the evaluation of Escherichia coli (EC) showed that the addition of DDAO promoted bacteriolysis compared to the absence of DDAO, resulting in a higher test line intensity due to the antigen-antibody reaction. The results of the evaluation of Staphylococcus aureus (SA) showed that the addition of DDAO promoted bacteriolysis at all concentrations compared to the absence of DDAO, resulting in a higher test line intensity due to the antigen-antibody reaction. However, the behavior varied depending on the final DDAO concentration. When the final DDAO concentration was in the range of 0 to 0.05% by mass, the test line intensity increased, then remained nearly constant between 0.05 and 0.1% by mass, and decreased above 0.1% by mass (resulting in a higher test line intensity than without DDAO). When the final DDAO concentration is up to 0.1% by mass, a synergistic effect of lysostaphin enzyme and DDAO is exerted on the lysis of Staphylococcus aureus. However, when the final DDAO concentration exceeds 0.1% by mass, the activity of lysostaphin enzyme is inhibited, which is thought to result in a decrease in the lysis efficiency and the intensity of the test line.

[0080] Next, the relationship between the final concentration of benzalkonium chloride and test line intensity is shown in Figure 3. When the final concentration of benzalkonium chloride was between 0 and 0.05% by mass, no poor liquid development was observed in immunochromatography. However, when the final concentration exceeded 0.05% by mass, poor development was observed in some parts of the liquid flow (the gold colloid-labeled antibody was not developed on the nitrocellulose membrane and remained in the sample application area). When the final concentration exceeded 0.075% by mass, the tendency for poor liquid development became more pronounced. The evaluation results for Escherichia coli (EC) showed that the lysis efficiency and test line intensity due to the antigen-antibody reaction were higher at final benzalkonium chloride concentrations up to 0.075% by mass compared to when no benzalkonium chloride was present. When the final concentration exceeded 0.075% by mass, the test line intensity decreased due to the aforementioned poor liquid flow development. In the evaluation of Staphylococcus aureus (SA), the addition of benzalkonium chloride promoted bacteriolysis compared with the absence of benzalkonium chloride, resulting in a higher test line intensity due to the antigen-antibody reaction. However, this behavior varied depending on the final benzalkonium chloride concentration. Test line intensity increased when the final benzalkonium chloride concentration was between 0 and 0.065% by mass, but decreased when the final benzalkonium chloride concentration exceeded 0.07% by mass. Up to a final benzalkonium chloride concentration of 0.065% by mass, a synergistic effect of lysostaphin enzyme and benzalkonium chloride was exerted on the lysis of Staphylococcus aureus. However, a final benzalkonium chloride concentration exceeding 0.07% by mass is thought to cause the aforementioned poor liquid flow and inhibition of lysostaphin enzyme activity, resulting in a decrease in bacteriolysis efficiency and test line intensity. The above results demonstrate that adding DDAO as a surfactant at a final concentration of preferably 0.2% by mass, more preferably up to 0.1% by mass, in combination with lysostaphin enzyme efficiently lyses bacteria of multiple genera (Escherichia coli and Staphylococcus aureus) and detects them by immunochromatography. Furthermore, it was shown that adding benzalkonium chloride as a surfactant, preferably at a final concentration of 0.08% by mass, more preferably up to 0.065% by mass, in combination with lysostaphin enzyme, efficiently lyses bacteria of multiple genera (Escherichia coli and Staphylococcus aureus) and detects them by immunochromatography.

[0081] Example 4: Lysostaphin concentration study Preparation of bacterial solution (sample) for evaluation: To examine the effect of lysostaphin, Staphylococcus aureus (SA) was prepared in saline at a concentration of 1e6 cfu / ml, and saline was used as the solution without bacteria.

[0082] Preparation of various reaction solutions: A mother solution was prepared by adding 0.15% by mass (final concentration after mixing with the evaluation bacterial solution (sample) of 0.075% by mass) of DDAO or 0.05% by mass (final concentration after mixing with the evaluation bacterial solution (sample) of 0.025% by mass) of benzalkonium chloride, and 0.2% by mass of Tween 20 for immunochromatographic development to 0.1 M Tris-HCl (pH 7.4) buffer solution. Various reaction solutions containing 0 to 50 μg / ml of lysostaphin enzyme (final concentration after mixing with the evaluation bacterial solution (sample) of 0 to 25 μg / ml) were prepared.

[0083] Immunochromatographic detection performance evaluation: The bacterial solution for evaluation and the above-mentioned various reaction solutions were mixed in a 1:1 ratio, and the immunochromatographic test strip prepared in Example 1 was inserted to evaluate the detection performance (test line intensity) based on bacterial lysis and antigen-antibody reaction.

[0084] Figure 4 shows the relationship between the final lysostaphin concentration and test line intensity when DDAO was used as a surfactant. Figure 4(a) shows all data, and Figure 4(b) shows an enlarged view of the horizontal axis (final lysostaphin concentration). The addition of lysostaphin tended to promote the lysis of Staphylococcus aureus (SA) compared to the absence of lysostaphin. When the final lysostaphin concentration exceeded 0.01 μg / ml, sufficient lysis (catalytic enzyme activity) was observed, resulting in a high test line intensity due to the antigen-antibody reaction. On the other hand, when the final lysostaphin concentration exceeded 5.0 μg / ml, a test line (false positive due to lysostaphin) was observed (test line intensity ≥ 10) even in the absence of bacteria. Therefore, it was confirmed that a final lysostaphin concentration in the range of 0.01 to 5.0 μg / ml is optimal.

[0085] Figure 5 shows the relationship between the final lysostaphin concentration and test line intensity when benzalkonium chloride was used as a surfactant. Figure 5(a) shows all data, and Figure 5(b) shows an enlarged view of the horizontal axis (lysostaphin final concentration). The addition of lysostaphin tended to promote the lysis of Staphylococcus aureus (SA) compared to the absence of lysostaphin. When the final lysostaphin concentration exceeded 0.01 μg / ml, sufficient lysis (catalytic action of the enzyme) was observed, resulting in a high test line intensity due to the antigen-antibody reaction. On the other hand, when the final lysostaphin concentration exceeded 5.0 μg / ml, a test line (false positive likely due to lysostaphin) was observed (test line intensity ≥ 10) even in the absence of bacteria. Therefore, it was confirmed that a final lysostaphin concentration in the range of 0.01 μg / ml to 5.0 μg / ml is optimal.

[0086] The above results demonstrated that the use of lysostaphin as a lytic enzyme at a final concentration ranging from 0.01 μg / ml to 5.0 μg / ml in combination with the surfactants DDAO or benzalkonium chloride efficiently lyses Staphylococcus aureus and detects it by immunochromatography.

[0087] Example 5: Confirmation of bacteriolysis and detection performance near boundary conditions Preparation of various reaction solutions: To confirm the preferred range of combinations of the above-mentioned surfactant DDAO or benzalkonium chloride and the lysostaphin enzyme, a reaction solution was prepared under the conditions shown in Table 3 using a mother solution prepared by adding 0.2% by mass of Tween 20 to 0.1 M Tris-HCl (pH 7.4) buffer for immunochromatographic development. [Table 3]

[0088] Preparation of bacterial solution (sample) for evaluation: Escherichia coli (EC) and Pseudomonas aeruginosa (PA) were selected as Gram-negative bacteria, and Staphylococcus aureus (SA) and Bacillus subtilis (BS) were selected as Gram-positive bacteria, and each was prepared in saline at a concentration of 1e6 cfu / ml. Furthermore, as a representative bacterial mixture, a solution containing both Escherichia coli (EC) and Staphylococcus aureus (SA) at a concentration of 1e6 cfu / ml was prepared in saline. A solution without bacteria was also prepared using saline.

[0089] Immunochromatographic detection performance evaluation: The bacterial solution for evaluation and the various reaction solutions described above were mixed at a 1:1 ratio, and the immunochromatographic test strip prepared in Example 1 was inserted to visually evaluate the detection performance based on bacterial lysis and antigen-antibody reaction. The results are shown in Table 4. Under all conditions, the results were visually negative when there were no bacteria, and visually positive when there were Escherichia coli (EC), Pseudomonas aeruginosa (PA), Staphylococcus aureus (SA), Bacillus subtilis (BS), and a mixture of Escherichia coli (EC) and Staphylococcus aureus (SA). [Table 4] These results demonstrate that the use of surfactants DDAO or benzalkonium chloride and lysostaphin enzyme at appropriate concentrations enables efficient lysis and immunochromatographic detection of bacteria from multiple genera.

[0090] Example 6: pH study of reaction solution Preparation of various reaction solutions: A mother solution was prepared by adding 0.15% by mass (final concentration after mixing with the evaluation bacterial solution (sample) of 0.075% by mass) DDAO or 0.05% by mass (final concentration after mixing with the evaluation bacterial solution (sample) of 0.025% by mass) benzalkonium chloride, 5.0 μg / ml of lysostaphin enzyme (final concentration after mixing with the evaluation bacterial solution (sample) of 2.5 μg / ml), and 0.2% by mass of Tween 20 for immunochromatographic development to 0.1 M Tris-HCl (or MOPSO) buffer, and the pH was adjusted to prepare reaction solutions with different pH values ​​(6.0 to 9.5).

[0091] Preparation of bacterial solution (sample) for evaluation: Escherichia coli (EC) and Pseudomonas aeruginosa (PA) were selected as Gram-negative bacteria, and Staphylococcus aureus (SA) and Bacillus subtilis (BS) were selected as Gram-positive bacteria, and each was prepared with physiological saline to a concentration of 1e6 cfu / ml. The solution without bacteria was also prepared with physiological saline.

[0092] Immunochromatographic detection performance evaluation: The bacterial solution for evaluation and the various reaction solutions described above were mixed at a 1:1 ratio, and the immunochromatographic test strip prepared in Example 1 was inserted. The detection performance based on bacterial lysis and antigen-antibody reaction was visually evaluated. The results are shown in Table 5. At pH 6.0 to 6.2, the test line for Staphylococcus aureus (SA) was negative, and at pH 9.5, the result was weakly positive. This pH is thought to be due to a decrease in the enzymatic activity of lysostaphin. At other pH values, the absence of bacteria resulted in a visually negative result, while Escherichia coli (EC), Pseudomonas aeruginosa (PA), Staphylococcus aureus (SA), and Bacillus subtilis (BS) were visually positive. [Table 5] The above results demonstrated that by using a reaction solution with a pH in the range of 6.4 to 9.0, lysis and immunochromatographic detection of multiple bacterial species, including at least one species of Gram-positive bacteria and / or Gram-negative bacteria, can be efficiently performed.

[0093] Example 7: Study of coloration of reaction solution ·Selection of coloring agents We investigated the coloring of reaction solutions as a means of preventing accidental ingestion in food processing facilities. We obtained commercially available water-soluble dyes (26 types, including Blue No. 1, Yellow No. 4, and Green No. 3) and measured their absorption spectra. We selected four dyes (Blue No. 1, Yellow No. 4, Green No. 3, and a mixture of Blue No. 1 and Yellow No. 4) whose absorption did not interfere with the gold colloid (red) used for labeling.

[0094] Preparation of color reaction solution A mother liquid was prepared by adding 0.15% by mass (final concentration after mixing with the evaluation bacterial solution (sample) of 0.075% by mass) of DDAO or 0.05% by mass (final concentration after mixing with the evaluation bacterial solution (sample) of 0.025% by mass) of benzalkonium chloride, 5.0 μg / ml of lysostaphin enzyme (final concentration after mixing with the evaluation bacterial solution (sample) of 2.5 μg / ml), and 0.2% by mass of Tween 20 for immunochromatographic development to 0.1 M Tris-HCl (pH 7.4) buffer solution, and the colorants were added under the conditions shown in Table 6. [Table 6]

[0095] Preparation of bacterial solution (sample) for evaluation: Escherichia coli (EC) and Pseudomonas aeruginosa (PA) were selected as Gram-negative bacteria, and Staphylococcus aureus (SA) and Bacillus subtilis (BS) were selected as Gram-positive bacteria, and each was prepared with physiological saline to a concentration of 1e6 cfu / ml. The solution without bacteria was also prepared with physiological saline. Immunochromatographic detection performance evaluation: The bacterial solution for evaluation and the various reaction solutions described above were mixed at a 1:1 ratio, and the immunochromatographic test strip prepared in Example 1 was inserted, and the detection performance based on bacterial lysis and antigen-antibody reaction was visually evaluated. The results are shown in Table 7. When various colorings were applied, the results were negative when there were no bacteria and positive when there were bacteria, just as in the case without coloring. [Table 7] The above results demonstrated that even when the reaction solution was colored to prevent accidental ingestion, lysis and immunochromatographic detection of multiple bacterial groups, including at least one species of Gram-positive and / or Gram-negative bacteria, was efficiently performed. [Industrial Applicability]

[0096] The present invention can be widely applied to fields such as food and medical fields, including bacterial lysis and bacterial detection, and is of great utility value. [Explanation of symbols]

[0097] 10 Immunochromatography detection device 1. Chromatographic development membrane carrier 2. Labeling antibody attachment member (conjugate pad) 3. Sample pad 4 Absorption material (absorption pad) 5 Base material 6 Capture antibody immobilization site 7. Control reagent fixing site Sample A B. Sample flow

Claims

1. A method for lysing a group of multiple types of bacteria including at least one or more types of Gram-positive bacteria and Gram-negative bacteria in a specimen, the method comprising the step of treating the specimen with a solution of pH 6.4 to 9.0 containing a surfactant and a lytic enzyme, wherein the surfactant is selected from N,N-dimethyldodecylamine N-oxide (DDAO) having a concentration of 0.01 to 0.1% by mass in the solution upon treatment with the specimen, and benzalkonium chloride having a concentration of 0.005 to 0.07% by mass in the solution upon treatment with the specimen, and the lytic enzyme is lysostaphin having a concentration of 0.01 μg / mL to 5.0 μg / mL in the solution upon treatment with the specimen.

2. The method of claim 1 , wherein the group of multiple species of bacteria includes at least one or more species of Gram-positive bacteria and at least one or more species of Gram-negative bacteria.

3. 2. The method of claim 1, wherein the Gram-positive bacteria include at least bacteria of the genus Staphylococcus.

4. The method of claim 1 , wherein the solution containing the surfactant and the lytic enzyme contains a dye selected from the group consisting of red, blue, and green.

5. The method according to claim 1, which is used to detect at least one type of bacteria in a specimen by immunochromatography.

6. A method for detecting a group of multiple species of bacteria in a sample, comprising: A step of lysing a group of multiple types of bacteria in a specimen by the method of claim 1; a step of detecting the bacterial antigens released from the lysed bacteria using an antibody that undergoes an antigen-antibody reaction with the antigens; A detection method comprising:

7. The method according to claim 6, wherein the group of multiple species of bacteria in the sample to be detected includes both at least one species of Gram-positive bacteria and at least one species of Gram-negative bacteria.

8. The method of claim 6 , wherein the detecting step is carried out by immunochromatography.

9. The method of claim 6, wherein the bacterial antigen released by the lysis step is the L7 antigen.

10. A bacteriolysis kit for use in the method of claim 1, for lysing a group of multiple types of bacteria including at least one or more types of Gram-positive bacteria and Gram-negative bacteria in a specimen, the bacteriolysis kit comprising a surfactant selected from N,N-dimethyldodecylamine N-oxide (DDAO) and benzalkonium chloride, and lysostaphin as a lytic enzyme.

11. A bacteria detection kit for detecting a group of multiple types of bacteria including at least one or more types of Gram-positive bacteria and Gram-negative bacteria in a sample, comprising: The method according to claim 1, wherein the surfactant is selected from N,N-dimethyldodecylamine N-oxide (DDAO) and benzalkonium chloride, and the lysostaphin is used as a lytic enzyme. below: (a) a labeling antibody-attached member to which a labeling antibody is attached, the labeling antibody forming a first complex through an antigen-antibody reaction with an antigen derived from a target bacterium; and (b) a strip having a detection region on which a capture antibody is immobilized, which forms a second complex through an antigen-antibody reaction with the first complex; An immunochromatographic detection device including The bacteria detection kit comprising:

12. 1. A method for detecting the presence and / or abundance of bacteria in a sample, comprising: (I) lysing bacteria in a specimen by the method of claim 1; (II) capturing and labeling antigens derived from bacteria in the specimen through an antigen-antibody reaction between the specimen, a capture antibody immobilized on a solid phase carrier, and a labeling antibody having a detection label; and (III) detecting bacteria in a specimen based on a detection label; A method in which one of the capture antibody and the labeling antibody is one or more general-purpose antibodies that undergo antigen-antibody reactions with antigens derived from five or more genera of bacteria, including bacteria, and the other of the capture antibody and the labeling antibody is one or more specific antibodies that undergo antigen-antibody reactions with one or more genera of bacteria.

13. The method according to claim 12, wherein two or more types of bacteria in a sample are detected using a single capture antibody immobilization site.

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