JP2014066641A - Method for detecting escherichia coli o157 - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide Escherichia coli O157 for immunochromatography detection kit capable of using a detection method by an immuno-chromatographic method capable of specifically and quickly detecting Escherichia coli O157 from a sample to be tested of a food product and Escherichia coli O157 for immuno-chromatographic detection kit usable for the detection method in order to efficiently eliminate contamination and infection by pathogenic Escherichia coli O157.SOLUTION: A sample to be tested is treated with a surface-active agent such as polyoxyethylene (10) octylphenyl ether and benzalkonium chloride, and an antibody for pathogenic escherichia coli O157 is made to act on this treated object so as to detect pathogenic escherichia coli O157. A monoclonal antibody for pathogenic escherichia coli O157 is preferably two kinds of monoclonal antibodies that recognize different epitopes.

Description

  The present invention relates to a method for detecting E. coli O157. More specifically, the present invention relates to a detection method for E. coli O157 that is detected by allowing an antibody against E. coli O157 to act on a processed product obtained by treating a test sample with a surfactant. The present invention relates to an E. coli O157 detection kit comprising an activator.

  E. coli O157 means E. coli having the 157th antigen found as the O antigen derived from the cell wall on the cell surface. In addition, bacteria that produce verotoxin among Escherichia coli are also called enterohemorrhagic Escherichia coli, causing severe abdominal pain, watery diarrhea, bloody stool, etc. when ingesting food containing bacteria, It is known to cause complications such as hemolytic uremic syndrome (HUS). In particular, food poisoning symptoms caused by Escherichia coli O157: H7 have become a social problem in recent years, and improvement in detection accuracy of the bacteria in clinical examination is regarded as important.

  As a method for detecting verotoxin produced by Escherichia coli O157, a detection sensor for verotoxin produced by Escherichia coli O157 in which a sugar chain derivative containing galactose is immobilized through a monomolecular layer on the substrate surface (for example, a patent) Reference 1) and a method for detecting enterohemorrhagic Escherichia coli O157: H7-produced verotoxin using a support on which a calix crown derivative layer is formed (see, for example, Patent Document 2) have been reported. In addition, a monoclonal antibody that specifically recognizes the 55th asparagine on the B subunit of verotoxin (Stx) 1 produced by Escherichia coli O157: H7, and Stx1 can be specifically measured using such a monoclonal antibody. A characteristic immunoassay (see, for example, Patent Document 3) has been reported.

  A method for detecting E. coli O157 using an antigen-antibody reaction includes a transparent substrate, a metal film disposed on the transparent substrate, and an antibody against E. coli O157 disposed on the metal film by hydrophobic bonding or electrostatic bonding. A surface plasmon resonance biosensor measurement chip (see, for example, Patent Document 4), insoluble particles sensitized with an antibody that specifically binds to E. coli O157, and a test sample are brought into contact with each other. An analysis method of E. coli O157 for analyzing the degree (for example, see Patent Document 5) has been reported.

  An example of the use of a surfactant in the detection of E. coli O157 is a composition for exposing a microbial antigenic epitope, the composition comprising a general enriched medium and at least one structurally modified organic chemical. (For example, see Patent Document 6), there is a description that a surfactant solution can be used to improve the contact property with a stored antigen epitope, but the action of the composition is an internal specific antigen. It is specified that this is a modification of the metabolic pathway of the target microorganism so as to produce a defective cell wall that can be exposed, and such an effect is obtained by bringing the surfactant solution into contact with the mixture of the composition and the test sample. Has been shown to be a preferred embodiment and the use of a surfactant solution alone is not shown.

Furthermore, immunochromato strips for the detection of various E. coli O157 are commercially available, but most of the detection limit concentrations are 1.0 × 10 ⁴ CFU / mL or more, and further improvement in detection accuracy is required. Yes.

JP 2002-022745 A JP 2008-233011 A JP 2002-275200 A JP-A-11-242301 JP-A-11-133029 Special table 2002-528052 gazette

  An object of the present invention is to provide an immunochromatography method capable of specifically detecting E. coli O157 quickly and accurately, and an E. coli O157 detection kit for immunochromatography which can be used therefor.

  The present inventors have made various trials and errors in order to improve conventional methods for detecting E. coli O157 by causing an antibody against E. coli O157 to act on a test sample such as food. When the test sample is treated with a surfactant and then an antibody against E. coli O157 is allowed to act on the heated heat-treated product, the detection accuracy of E. coli O157 is higher than when no surfactant is used. I found it. The inventors continued further research and recognized different epitopes using Triton X-100, which is a non-ionic surfactant with a different category, and benzalkonium chloride, which is a cationic surfactant. We found that the detection accuracy was significantly higher when more than one species of monoclonal antibody was allowed to act, and this detection method had a low cross-reactivity and a very low false positive rate compared to the conventional product. As a result, the present invention was completed.

That is, the present invention
[1] A method for detecting E. coli O157, which comprises treating a test sample with a surfactant and detecting E. coli O157 by treating the treated product with an antibody against E. coli O157. Method;
[2] The method for detecting Escherichia coli O157 according to the above [1], wherein the treated product is a heat-treated product obtained by treating a test sample with a surfactant and then heating it;
[3] The method for detecting E. coli O157 according to the above [1] or [2], wherein the antibody against E. coli O157 is a monoclonal antibody against E. coli O157;
[4] The method for detecting Escherichia coli O157 according to [3] above, wherein the monoclonal antibody against Escherichia coli O157 is a monoclonal antibody against heat-treated cells of Escherichia coli O157;
[5] The method for detecting E. coli O157 according to the above [3] or [4], wherein the monoclonal antibody against E. coli O157 is two or more kinds of monoclonal antibodies recognizing different epitopes;
[6] The method for detecting Escherichia coli O157 according to the above [5], wherein at least one of two or more kinds of monoclonal antibodies recognizing different epitopes is a monoclonal antibody labeled with a colloidal gold used for immunochromatography. ;
[7] The monoclonal antibody against E. coli O157 is a monoclonal antibody O157W-17B3 produced by a hybridoma (NITE P-1347) or a monoclonal antibody O157W-20B6 produced by a hybridoma (NITE P-1346) [3] The method for detecting E. coli O157 according to any one of [6];
[8] The method for detecting Escherichia coli O157 according to any one of the above [1] to [7], wherein the surfactant is a nonionic surfactant or a cationic surfactant;
[9] The method for detecting Escherichia coli O157 as described in [8] above, wherein the nonionic surfactant is polyoxyethylene (10) octylphenyl ether;
[10] The method for detecting Escherichia coli O157 according to the above [8], wherein the cationic surfactant is benzalkonium chloride;
[11] The detection method according to any one of [1] to [10] above, wherein E. coli O157 of 1 × 10 ³ CFU / mL or less can be detected;
[12] An E. coli O157 detection kit comprising an antibody against E. coli O157 and a surfactant;
[13] The kit for detecting E. coli O157 according to the above [12], wherein the antibody against E. coli O157 is a monoclonal antibody against E. coli O157;
[14] The kit for detecting Escherichia coli O157 according to [13] above, wherein the monoclonal antibody against Escherichia coli O157 is a monoclonal antibody against heat-treated cells of Escherichia coli O157;
[15] The kit for detecting E. coli O157 according to the above [13] or [14], wherein the monoclonal antibodies against E. coli O157 are two or more monoclonal antibodies that recognize different epitopes;
[16] The kit for detecting Escherichia coli O157 according to [15] above, wherein at least one of two or more kinds of monoclonal antibodies recognizing different epitopes is a monoclonal antibody labeled with a colloidal gold used for immunochromatography. ;
[17] The monoclonal antibody against E. coli O157 is monoclonal antibody O157W-17B3 against E. coli O157 produced by a hybridoma (NITE P-1347), or monoclonal antibody O157W-20B6 against E. coli O157 produced by a hybridoma (NITE P-1346) A kit for detecting Escherichia coli O157 according to any one of [13] to [16] above,
[18] The detection kit according to any one of the above [12] to [17], wherein the surfactant is a nonionic surfactant or a cationic surfactant;
[19] The kit for detecting Escherichia coli O157 according to the above [18], wherein the nonionic surfactant is polyoxyethylene (10) octylphenyl ether;
[20] The detection kit according to the above [18], wherein the cationic surfactant is benzalkonium chloride;
[21] A method for improving the detection accuracy of E. coli O157, comprising using a surfactant and an antibody against E. coli O157;
About.

  According to the method for detecting E. coli O157 of the present invention, pathogenic E. coli O157 in a test sample such as food can be detected effectively, and food contamination and infection by E. coli O157 can be effectively eliminated.

  As a method for detecting Escherichia coli O157 of the present invention, a test sample is treated with a surfactant, and an antibody against Escherichia coli O157 (hereinafter also referred to as “anti-E. Coli O157 antibody”) is allowed to act on the treated product. The detection method is not particularly limited, and the E. coli O157 detection kit of the present invention is not particularly limited as long as it is a kit comprising an antibody against E. coli O157 and a surfactant. Pathogenic Escherichia coli O157 can be preferably exemplified, and examples of the pathogenic Escherichia coli O157 include O157: H7 strain, O157: HNT strain and O157: H- strain.

  Examples of test samples include water, beverages, meat and processed meat products such as chicken, pork, and beef, seafood and processed fish products, dairy products such as milk and cheese, processed vegetables such as vegetables and pickles, fruits, etc. And biological samples such as animal and human urine, feces, vomit, blood, tissue, etc. In the case of a solid material, the surface of the solid material is sterilized with water or a liquid medium for sterilized Escherichia coli O157. A washed washing solution, a suspension obtained by pulverizing solids and then suspended in sterile water or a liquid medium for sterilized Escherichia coli O157, or a supernatant thereof can be used as a test sample. The sterilized water may contain a buffer that adjusts conditions such as pH of the test sample.

  Examples of the surfactant include anionic surfactants such as sodium dodecyl sulfate (SDS) and sodium deoxycholate, cationic surfactants such as benzalkonium chloride, 3-[(3- Amphoteric surfactants such as 3- (3-cholamidepropyl) dimethylammonio-1-propanesulphonate (CHAPS), polyoxyethylene (10) octylphenyl ether (trade names such as TritonX- 100 (registered trademark)), polyoxyethylene (20) sorbitan monolaurate (trade name is, for example, Tween 20 (registered trademark)), polyoxyethylene (9) octylphenyl ether (trade name is, for example, NP-40 (registered trademark) Nonionic surfactants such as trade marks)), and SDS Benzalkonium chloride, polyoxyethylene (10) octylphenyl ether, polyoxyethylene (20) sorbitan monolaurate are preferred, benzalkonium chloride and polyoxyethylene (10) octylphenyl ether are more preferred, and polyoxyethylene ( 10) Octylphenyl ether is particularly preferred.

By adding the surfactant to the test sample, E. coli O157 can be detected with higher accuracy than when no surfactant is added. For example, 1 × 10 ⁴ in the liquid test sample. Less than CFU / mL, preferably less than 5 × 10 ³ CFU / mL E. coli O157 can be detected.

  As the surfactant, the addition concentration (final concentration) when using polyoxyethylene (10) octylphenyl ether which is a nonionic surfactant is preferably 0.01 to 10 (v / v)%, 0.05 to 1.5 (v / v)% is more preferable, 0.2 to 1.0 (v / v)% is more preferable, and 0.3 to 0.7 (v / v)% is particularly preferable.

  The addition concentration (final concentration) when polyoxyethylene (20) sorbitan monolaurate which is a nonionic surfactant is used as the surfactant is preferably 0.01 to 10 (v / v)%. 0.05 to 2.0 (v / v)% is more preferable, 0.07 to 1.0 (v / v)% is more preferable, and 0.08 to 0.2 (v / v)% is particularly preferable. preferable.

  The addition concentration (final concentration) when polyoxyethylene (9) octylphenyl ether which is a nonionic surfactant is used as the surfactant is 0.005 to 0.09 (v / v)%. Preferably, 0.01 to 0.08 (v / v)% is more preferable, and 0.03 to 0.07 (v / v)% is more preferable.

  The addition concentration (final concentration) when benzalkonium chloride, which is a cationic surfactant, is used as the surfactant is preferably 0.001 to 1.0 (v / v)%, 0.005 -0.5 (v / v)% is more preferable, 0.01-0.1 (v / v)% is further more preferable, and 0.03-0.07 (v / v)% is particularly preferable.

  When CHAPS which is an amphoteric surfactant is used as the surfactant, the addition concentration (final concentration) is preferably 0.2 to 2.0 (v / v)%, and 0.3 to 1.2 ( v / v)% is more preferable.

  As the surfactant, the addition concentration (final concentration) when using an anionic surfactant sodium deoxycholate is preferably 0.02 to 0.5 (v / v)%, 0.03 -0.12 (v / v)% is more preferable.

  The addition concentration (final concentration) in the case of using SDS as an anionic surfactant as the surfactant is preferably 0.01 to 1 (v / v)%, and 0.05 to 0.5 ( v / v)% is more preferable, 0.07 to 0.2 (v / v)% is more preferable, and 0.08 to 0.12 (v / v)% is particularly preferable.

  As a method of treating the test sample with a surfactant, a surfactant is added to the liquid test sample such as the cleaning solution, suspension or supernatant thereof diluted as necessary, and mixed and stirred. Method, method of washing the surface of a solid test sample with sterilized water containing a surfactant or liquid medium for sterilized Escherichia coli O157, or sterilization containing a surfactant after pulverizing the solid test sample Examples thereof include a method of suspending in water or a liquid medium for sterilized Escherichia coli O157. And the processed material which processed the test sample with surfactant, especially the processed material containing the culture medium for colon_bacillus | E._coli O157 can be made into the culture preparation prepared by culture | cultivating beforehand. The sterilized water may contain a buffer that adjusts conditions such as pH of the test sample. Moreover, it is preferable to make the processed material which processed the test sample with surfactant at 60-150 degreeC for 1 to 60 minutes, Preferably, it is set as a heat-treated material at 90-121 degreeC for 5 to 20 minutes.

  As the medium for E. coli O157, tryptosome broth (manufactured by Nissui Pharmaceutical), TA10 Broth (manufactured by Primaham), novobiocin-added mEC medium (manufactured by Merck), novobiocin-added mTSB medium (manufactured by Merck), mEC medium (Manufactured by Nissui Pharmaceutical Co., Ltd.), and the temperature suitable for the culture (enrichment) can be 32 to 50 ° C., preferably 33 to 48 ° C., more preferably 34 to 45 ° C. The culture time may be 3 to 48 hours, preferably 4 to 36 hours, more preferably 5 to 30 hours, and particularly preferably 5 to 24 hours.

Examples of the type of antibody used in the detection method or detection kit of the present invention include a monoclonal antibody and a polyclonal antibody, and the monoclonal antibody is preferable in terms of the specificity of its recognition site. The immunoglobulin class of the antibody is not particularly limited, and may be any isotype such as IgG, IgM, IgA, IgD, and IgE, but IgG is preferred. The antibody may be a whole antibody or an antibody fragment as long as E. coli O157 can be detected. Examples of such antibody fragments include Fab fragments and F (ab ′) ₂ fragments. Antibody fragments, CDRs, multifunctional antibodies, single-chain antibodies (ScFv), and the like. For example, Fab fragments can be treated with papain or the like, and F (ab ′) ₂ fragments can be treated with pepsin or the like. It can prepare by processing with.

  As a method for preparing the antibody-producing cells of the above-mentioned anti-E. Coli O157 antibody, live bacteria of E. coli O157, heated cells obtained by heating E. coli O157, or a pulverized product thereof, can be used as an immunogen as it is or with an appropriate adjuvant. A method for immunization by administration to a mammal can be exemplified, but in consideration of a treatment method when an antibody actually obtained and an antigen contained in a test sample are reacted, a heat-treated bacterium of Escherichia coli O157 It is preferable to use the body as an immunogen in terms of convenience and safety. Examples of mammals include rats, mice, and rabbits, but it is preferable to use mice for the convenience of production, and monoclonal antibodies derived from mice are preferably used. Examples of administration locations include intravenous, subcutaneous, and intraperitoneal. The interval for performing immunization is preferably from several days to several weeks, and more preferably from 1 to 4 weeks. Separation of anti-E. Coli O157 antibody-producing cells is carried out by collecting from the immunized animal 1 to 60 days, preferably 1 to 14 days after the final immunization day. Examples of antibody-producing cells include spleen cells and lymph node cells. And peripheral blood-derived cells are preferred, and spleen cells are more preferred.

  As a method for preparing the above monoclonal antibody against Escherichia coli O157 (hereinafter also referred to as “anti-E. Coli O157 monoclonal antibody”), Keller and Milstein hybridoma method (Nature 256, 495-497, 1975), trioma method, human B cell hybridoma method (Immunology Today 4, 72, 1983) and EBV-hybridoma method (MONOCLONAL ANTIBODIES AND CANCER THERAPY, pp. 77-96, Alan R. Liss, Inc., 1985) can be used. After cell fusion between the antibody-producing cells and myeloma cells, the hybridoma producing the monoclonal antibody that recognizes E. coli O157 is cultured on the medium, or administered into the abdominal cavity of the animal and allowed to grow in ascites, A hybridoma method for collecting monoclonal antibodies from the culture medium or ascites can be preferably mentioned. When heated cells are used as an immunogen, a plurality of monoclonal antibodies capable of binding to E. coli O157 heated cell antigen can be selected from the obtained monoclonal antibodies, and E. coli O157 heated cell antigen can be analyzed with high accuracy by sandwich ELISA. It is preferred to select a combination of monoclonal antibodies, for example a combination of monoclonal antibodies O157W-17B3 and O157W-20B6.

  As the myeloma cell, generally available cell lines such as mouse, rat, rabbit-derived cell and the like can be used. However, the antibody-producing cell and the myeloma cell are preferably derived from the same species, and are not fused. In this state, it is preferable that the HAT selection medium cannot survive in a HAT selection medium (including hypoxanthine, aminopterin, and thymidine) but can survive as a positive hybridoma only in a state fused with an antibody-producing cell. Examples include mouse myeloma cell lines such as -X63-Ag8-U, P3X63Ag8.653, NSI / 1-Ag4-1, NS0 / 1, and rat myeloma cell lines such as YB2 / 0.

  The cell fusion method can be performed, for example, by mixing antibody-producing cells and myeloma cells in the presence of a fusion promoter such as polyethylene glycol in a medium such as Dulbecco's modified Eagle medium (DMEM). After cell fusion is completed, the mixture is appropriately diluted with DMEM, centrifuged, and the hybridoma is selected by suspending and culturing the precipitate in a selective medium such as HAT medium. By searching for antibody-producing hybridomas and cloning by limiting dilution or the like, hybridomas producing monoclonal antibodies that recognize E. coli O157 can be obtained. As described above, antibody-producing hybridomas can be cultured in a culture medium or in vivo, and monoclonal antibodies can be collected from the culture. However, as a method for separating and purifying monoclonal antibodies from the culture or ascites, it is generally used to purify proteins. Any method can be used as long as it is used, and for example, it can be carried out by an ammonium sulfate fractionation method ordinarily used for IgG purification, chromatography using a column such as an anion exchanger or protein A or G.

Antibodies against E. coli O157 such as anti-E. Coli O157 monoclonal antibody can be used as labeled antibodies. The labeling substance used for the preparation of the labeled antibody may be any labeling substance that can provide a detectable signal either alone or by reacting with another substance, such as an enzyme, a fluorescent substance, a chemiluminescent substance, or a radioactive substance. , Colloidal gold, etc. can be used, such as peroxidase, alkaline phosphatase, β-D-galactosidase, glucose oxidase, glucose-6-phosphate dehydrogenase, alcohol dehydrogenase, malate dehydrogenase, penicillinase, Catalase, apoglucose oxidase, urease, luciferase, acetylcholinesterase, etc., and fluorescent substances include fluorescein isothiocyanate, phycobiliprotein, rare earth metal chelate, dansyl chloride, tetramethylrhodamine Thiocyanate, etc., as the light-emitting substance, luminol compounds, dioxetanes, acridinium salts, and the like, a ^{3 H, 14 C, 125 I} , 131 I and the like as a radioactive material, can be respectively exemplified. When the labeling substance is an enzyme, a substrate, and if necessary, a color former, a fluorescent agent, a luminescent agent, etc. can be used for measuring the activity.

  As a method for detecting E. coli O157 by allowing an antibody against Escherichia coli O157 to act on a treated product obtained by treating the test sample with a surfactant, the treated product is allowed to act on the treated product by binding the antibody bound to an insoluble carrier. E. coli O157 is captured to form an antigen-antibody complex, and then a sandwich method in which an antibody against labeled E. coli O157 is reacted, or a labeled antibody against E. coli O157 with magnetic beads bound to the treated product is allowed to act. A magnetic bead method for detecting a labeling substance in the antigen-antibody complex by separating it by a magnetic force and an antigen-antibody complex in which an antibody against Escherichia coli O157 labeled to the treated substance is allowed to aggregate and precipitate. And then separated by centrifugation to detect the labeled substance in the antigen-antibody complex. A known immunoassay method such as a diffusion method or a radioimmunodiffusion method can be used, but a method of detecting two or more monoclonal antibodies that recognize different epitopes as the antibodies against Escherichia coli O157 on the treated product. For example, a sandwich (two antibody) method using a monoclonal antibody recognizing E. coli O157 bound to an insoluble carrier and a labeled monoclonal antibody recognizing E. coli O157 recognizing a different epitope (labeled second antibody), or different epitopes. An immunochromatography method using a monoclonal antibody in which at least one of two or more kinds of monoclonal antibodies recognizing γ is labeled with a label such as a colloidal gold used for immunochromatography is preferable.

  Examples of the insoluble carrier include polymer compounds such as polystyrene, polyethylene, polypropylene, polyester, polyacrylonitrile, fluororesin, cross-linked dextran, and polysaccharides, glass, metal, magnetic particles, and combinations thereof. In addition, the insoluble carrier can be used in various shapes such as a tray shape, a spherical shape, a fiber shape, a rod shape, a disk shape, a container shape, a cell, a microplate, a test tube, and a latex bead shape. . Furthermore, the method for immobilizing the antigen or antibody on these insoluble carriers is not particularly limited, and a physical adsorption method, a covalent bond method, an ion bond method, or the like can be used.

  As a method for detecting E. coli O157 by the immunochromatography method, an antigen-antibody complex is prepared by allowing a gold colloid-labeled antibody obtained by binding a gold colloid to an anti-E. Coli O157 antibody to act on the treated product, and the immunochromatographic strip is subjected to capillary action or the like. Presence or absence of colored lines appearing when the anti-E. Coli O157 antibody recognizing an epitope different from the gold colloid-labeled antibody fixed at a predetermined position of the immunochromatographic strip is captured by supplementing the antigen-antibody complex during the movement A method for qualitative analysis can be mentioned.

  As a method for producing the immunochromatographic strip, for example, an antibody is prepared in a PBS solution, applied linearly to a membrane, dried, and then blocked with PBS containing BSA, Tween 20 or the like at 30 to 40 ° C., then PBS An antibody-immobilized membrane that has been washed with and dried and prepared, and a glass wool sample pad for a spot of a treated product, an antibody-immobilized membrane, and an absorbent pad for absorbing a treated product, respectively, are attached in this order.

  The method for producing the gold colloid-labeled antibody to which the gold colloid is bound is not particularly limited, including a conventionally known method. For example, a gold colloid solution prepared with a potassium carbonate solution, a borate buffer solution with an anti-E. Coli O157 antibody, A method in which a solution in which a monoclonal antibody against E. coli O157 is dissolved is preferably added and reacted at room temperature for 5 minutes to 2 hours, preferably 15 to 60 minutes, followed by addition of BSA solution for reaction and centrifugation. The colloidal gold-labeled antibody is preferably applied to a glass wool conjugate pad or the like and dried to form a colloidal gold-labeled antibody carrier from the viewpoint of storage stability. A test sample can be dissolved in a treatment solution treated with a surfactant and used as a detection reagent.

  The development support is prepared by, for example, applying a buffer solution containing an antibody recognizing an epitope different from that of a colloidal gold-labeled antibody, preferably a monoclonal antibody, to a membrane in a straight line and drying, followed by blocking treatment. Can do.

  The kit for detecting E. coli O157 of the present invention is preferably a monoclonal antibody and a surfactant against E. coli O157, more preferably a monoclonal antibody and a surfactant against two or more E. coli O157 that recognize different epitopes, more preferably each. Monoclonal antibodies and nonionic surfactants or cationic surfactants against two or more E. coli O157 that recognize different epitopes, particularly preferably two or more E. coli O157 and polyoxygens that recognize different epitopes Containing ethylene (10) octylphenyl ether or benzalkonium chloride, and further, a buffer solution for dissolving the monoclonal antibody, a culture solution (medium), a preparation solution for preparing the treated product, an instruction manual, etc. Attachment of Documents and the like may be included. A more preferred embodiment of the kit for detecting E. coli O157 of the present invention includes a test strip in the immunochromatography method. In this case, it is preferable that at least one of two types of monoclonal antibodies recognizing different epitopes is a monoclonal antibody labeled with a colloidal gold used for immunochromatography.

  Preferred examples of the monoclonal antibody in the present invention include the monoclonal antibody O157W-17B3 produced by the hybridoma (NITE P-1347) and / or the monoclonal antibody O157W-20B6 produced by the hybridoma (NITE P-1346). These hybridomas are entrusted to the Patent Microorganism Depositary Center (NPMD) on June 25, 2012 (date of entrustment).

  Hereinafter, the present invention will be described more specifically by way of examples. However, the technical scope of the present invention is not limited to these examples.

1. Preparation of Anti-E. Coli O157 Monoclonal Antibody 1-1 Materials and Methods (1) Preparation of E. coli O157 Antigen Solution E. coli O157: H7 (ATCC 43894) strain (hereinafter also referred to as “ATCC 43894 strain”) was purchased from ATCC. The ATCC 43894 strain was cultured in TA10 Broth (manufactured by Primaham), then collected, washed, dissolved in Dulbecco's PBS (-) (manufactured by Nissui Pharmaceutical), and heated by heating at 100 ° C. for 15 minutes. The treated cells were prepared as a 2.0 × 10 ⁹ CFU / mL solution and stored frozen at −20 ° C. until immunization.

(2) Immunization Ten 6-week-old BALB / c mice (manufactured by CLEA Japan) were used as test animals. For the first immunization, an antigen emulsion for initial immunization was prepared by adding an equal amount of incomplete Freund's adjuvant (Difco) to an Eppendorf tube containing 500 μl of the antigen solution and stirring with a vortex mixer. This emulsion was inoculated intraperitoneally at 150 μL per tail. Booster immunization was performed in the same manner as the initial immunization, and was performed 5 to 20 times at 2-week intervals.

(3) Measurement of antibody titer in blood Blood was collected from the tail vein of each BALB / c mouse one week after the injection of the antigen solution in the first and booster immunizations. The collected blood was allowed to stand at room temperature for 2 hours, and then centrifuged to obtain serum. Ten-fold dilutions of these sera were prepared, and the anti-E. Coli O157: H7 antibody titer in mouse blood was examined by non-competitive ELISA. As the secondary antibody, alkaline phosphatase-labeled anti-mouse IgG (H + L) antibody (manufactured by Jackson ImmunoReserch Laboratories) was used.

(4) Production of hybridoma Hybridoma was produced according to the method of Keller and Milstein (1975). That is, 100 μL of the antigen solution was injected from the tail vein into the mouse whose antibody titer was sufficiently increased. Four days after intravenous injection, the spleen was aseptically removed from the mouse. The spleen was minced, washed with RPMI 1640 (Gibco) and passed through a sterile nylon mesh (Cell Strainer, 70 μm, Becton Dickinson) to obtain a spleen cell suspension. Spleen cells were collected by centrifugation at 1,000 rpm × 10 minutes, resuspended in RPMI 1640 and counted. This spleen cell suspension and mouse myeloma cell (P3X63Ag8.653) suspension were mixed so that the number of cells became 10: 1, and centrifuged again at 1,000 rpm × 10 minutes to obtain a pellet. To this pellet, 45% polyethylene glycol having an average molecular weight of 3,350 was dropped to perform cell fusion. RPMI1640 was added to the cell solution for dilution, and then pelleted by centrifugation. To this pellet, hybridoma medium (RPMI 1640 medium containing 10% fetal bovine serum, 40 mM 2-mercaptoethanol, 100 U / mL penicillin, 100 μg / mL streptomycin), 100 μM hypoxanthine, 0.4 μM aminopterin, Add HAT selection medium containing 16 μM thymidine and dispense to a 24-well cell culture plate (Becton Dickinson) at 5 × 10 ⁶ cells / well at 37 ° C. in a 5% CO ₂ atmosphere. Cultured.

(5) Cloning by limiting dilution The culture supernatant of each well of the cell culture plate was used as the primary antibody for ELISA to examine the presence of hybridomas producing anti-E. Coli O157 antibody. The well hybridoma positive for the ATCC 43894 strain antigen by ELISA was transferred to a 96-well cell culture plate at 0.9 cells / well and cloned by limiting dilution. As feeder cells, 4-week-old BALB / c mouse thymocytes were added to each well of a 96-well cell culture plate at 5 × 10 ⁶ cells / well. RPMI1640 medium containing 10% fetal bovine serum, 40 mM 2-mercaptoethanol, 100 U / mL penicillin, 100 μg / mL streptomycin was used for the culture of the cloned hybridoma.

(6) Screening of antibodies For screening of monoclonal antibodies, the reactivity of ATCC 43894 strain heated cells was examined by non-competitive ELISA for the culture supernatant of each well. As a result, 16 types of antibody-producing hybridomas were selected.

(7) Ascites collection and monoclonal antibody purification
According to Jones et al. (1990), BALB / c mice were first injected with 0.2 mL of incomplete Freund's adjuvant intraperitoneally. One week later, 5 × 10 ⁶ cells of the selected hybridoma were inoculated per fish. After ascites retention, ascites was collected with a syringe. The collected ascites was purified by Protein G column (Amersham Pharmacia).

(8) Monoclonal Antibody Class, Subclass and Type Monoclonal antibody classes and subclasses are determined using the Monoclonal mouse immunoglobulin isotyping kit (Pharmingen), IgG1, IgG2a, IgG2b, IgG3, IgM, IgA, IgL (κ) and IgL. (Γ) was determined.

1-2 Results (1) Characteristics, Class and Subclass of Anti-E. Coli O157: H7 Monoclonal Antibody 16 types of monoclonal antibodies against E. coli O157: H7 were obtained. The results are shown in Table 1 below.

(In Table 1, + indicates positive reaction with E. coli O157 antigen.)

(2) Combination conditions of antibodies The combination of two monoclonal antibodies for detecting Escherichia coli O157: H7 is based on the detection accuracy of Escherichia coli O157: H7ATCC4394 heated cells and other cells in sandwich ELISA and the prepared immunochromatographic strip. Elected from. Table 2 below shows the results of reactivity to Escherichia coli O157 and other bacteria in the extracted immunochromato strip.

  As is clear from the results of Table 2 above, the monoclonal antibody O157W-17B3 produced by the hybridoma (NITE P-1347) and the monoclonal antibody O157W-20B6 produced by the hybridoma (NITE P-1346) were combined with high detection accuracy. Selected.

1. Detection of Escherichia coli O157 by immunochromatography (1) Preparation of colloidal gold labeled monoclonal antibody The monoclonal antibody O157W-17B3 and the monoclonal antibody O157W-20B6 were each adjusted to a concentration of 1 mg / mL with 2 mM borate buffer (pH 9.0). Prepared. 500 μL of each monoclonal antibody solution was added to 5 mL of a colloidal gold solution (Sigma Aldrich) prepared in advance with a 0.2 M potassium carbonate solution at pH 9.0, reacted at room temperature for 30 minutes, and then 625 μL of a 10% BSA solution. In addition, the mixture was further reacted for 15 minutes. Centrifugation was performed, and a 1% BSA solution was prepared so that OD ₅₂₅ = 1.0. A glass wool conjugate pad was applied to a 68μL / cm ^2, and dried.

(2) Preparation of antibody-immobilized membrane For the monoclonal antibody O157W-17B3 and the monoclonal antibody O157W-20B6, solutions were prepared so that each would be 4 mg / mL with PBS, applied linearly to a nitrocellulose membrane and dried. . Then, after blocking with PBS containing 1% BSA and 0.1% Tween 20 at 37 ° C. for 2 hours, the plate was washed with PBS and dried.

(3) Assembling the immunochromatographic strip In addition to the conjugate pad and the antibody-immobilized membrane, a glass wool sample pad for spot of a processed product treated with a surfactant and a glass wool for absorbing the processed product Separately, an absorption pad was prepared, and a sample pad, a conjugate pad, an antibody-immobilized membrane, and an absorption pad were attached in this order to obtain an immunochromatographic strip.

(4) Combination conditions of antibodies in the immunochromatography method In the above immunochromatography strip, when O157W-20B6 is a colloidal gold-labeled monoclonal antibody and O157W-17B3 is applied linearly to a nitrocellulose membrane, the detection accuracy is improved. Confirmed that it would be higher. Therefore, in the following Examples, the immunochromatographic strip of the present invention was examined by using O157W-20B6 as the colloidal gold labeled monoclonal antibody.

1. Examination of Surfactant Using Escherichia coli O157: H7 (ATCC 43894) strain as a test strain, the effect of adding various surfactants was examined. The surfactants studied were SDS (Nacalai Tesque), anionic surfactant, sodium deoxycholate (Wako Pure Chemical Industries), and benzalkonium chloride (Japanese), a cationic surfactant. Kopure Pharmaceutical Co., Ltd.), amphoteric surfactant CHAPS (Dojindo Laboratories), nonionic surfactant Triton X-100 (Sigma Aldrich), Tween 20 (MP Bio Japan), It was NP-40 (manufactured by Biovision). Tryptosoya bouillon manufactured by Nissui Pharmaceutical Co., Ltd. (casein peptone 17 g / L, soybean peptone 3 g / L, sodium chloride 5 g / L, glucose 2.5 g / L, dipotassium hydrogen phosphate 2.5 g / L) The Escherichia coli O157 (ATCC 43894) strain was used as a culture solution and added to the medium, followed by culturing at 35 ° C. for 24 hours. The number of bacteria was adjusted to 1.0 × 10 ⁴ , 1.0 × 10 ³ , 5.0 × 10 ² , and 1.0 × 10 ² CFU / mL. Each surfactant was added so that it might become the final density | concentration shown in this, and it heated in boiling water for 15 minutes, and was set as the processed material in this invention. 100 μL of the treated product is mixed with a detection reagent containing a colloidal gold-labeled monoclonal antibody, allowed to act on the colloidal gold-labeled monoclonal antibody, and then applied to the immunochromatographic strip of the present invention. Was judged. The results are shown in Table 3 below.

2. Results (1) No addition of surfactant As apparent from Table 3 above, when no surfactant is added, 1.0 ± 10 ⁴ CFU / mL, “±: thin line, positive if skilled It was confirmed that the detection limit concentration was 1.0 × 10 ⁴ CFU / mL.

(2) Addition of nonionic surfactant When the concentration of Triton X-100 is 0.5%, the number of bacteria is 1.0 × 10 ³ CFU / mL. “Positive” can be determined, and in the number of bacteria of 5.0 × 10 ² CFU / mL, “±: the line is thin, and it can be determined positive by an expert”, compared with the case where no surfactant is added As a result, it was confirmed that the detection accuracy was remarkably increased by about 20 times and the detection limit concentration was 5.0 × 10 ² CFU / mL. In addition, when the concentration of Triton X-100 is 1%, “+: clearly positive can be determined” in the number of bacteria of 1.0 × 10 ⁴ CFU / mL, and even when the concentration is 0.1% It is “+ w” in the number of bacteria of 1.0 × 10 ⁴ CFU / mL, and the detection accuracy is higher than the case where no surfactant is added, and the detection limit concentration is less than 1.0 × 10 ⁴ CFU / mL. It was confirmed that.

When the concentration of Tween 20 is 0.5 to 1.0%, it is “+ w” in the number of bacteria of 1.0 × 10 ⁴ CFU / mL, and the detection accuracy is higher than the case where no surfactant is added. It was confirmed that the detection limit concentration was less than 1.0 × 10 ⁴ CFU / mL. If Tween20 concentration is 0.1%, a "±" in the number of bacteria 1.0 × ¹⁰ 3 CFU / mL, the detection limit concentration, confirmed to be 1.0 × ¹⁰ 3 CFU / mL did. In addition, when the concentration of NP-40 is 0.05%, it is “+ w” in the number of bacteria of 1.0 × 10 ⁴ CFU / mL, and the detection accuracy is high compared with the case where no surfactant is added. It was confirmed that the detection limit concentration was less than 1.0 × 10 ⁴ CFU / mL.

(3) Addition of cationic surfactant When the concentration of benzalkonium chloride is 0.05%, it is “+ w” in the number of bacteria of 1.0 × 10 ³ CFU / mL, and 5.0 × 10 ² The number of bacteria in CFU / mL is “±”, and the detection accuracy is about 20 times higher than when no surfactant is added, and the detection limit concentration is 5.0 × 10 ² CFU / mL. confirmed. In addition, even when the concentration of benzalkonium chloride is 0.01% or 0.1%, it is “+ w” in the number of bacteria of 1.0 × 10 ⁴ CFU / mL, compared with the case where no surfactant is added. As a result, it was confirmed that the detection accuracy increased and the detection limit concentration was less than 1.0 × 10 ⁴ CFU / mL.

(4) Addition of anionic surfactant When the SDS concentration is 0.1%, it is “±” in the number of bacteria of 1.0 × 10 ³ CFU / mL, compared with the case where no surfactant is added. As a result, it was confirmed that the detection accuracy was 10 times higher and the detection limit concentration was 1.0 × 10 ³ CFU / mL. When the concentration of sodium deoxycholate is 0.05% or 0.10%, it is “+ w” in the number of bacteria of 1.0 × 10 ⁴ CFU / mL, compared with the case where no surfactant is added. As a result, the detection accuracy increased and the detection limit concentration was confirmed to be less than 1.0 × 10 ⁴ CFU / mL.

(5) Addition of amphoteric surfactant When the concentration of CHAPS is 0.5% or 1.0%, it is “+ w” in the number of bacteria of 1.0 × 10 ⁴ CFU / mL, and the surfactant is added. It was confirmed that the detection accuracy was higher than that in the case of not performing the detection, and the detection limit concentration was less than 1.0 × 10 ⁴ CFU / mL.

2. Triton X-100
Ten different strains of E. coli O157 were used as test strains, and the detection accuracy was examined using Triton X-100. Ten types of test strains were cultured at 35 ° C. for 24 hours using the above tryptosome broth, then 1.0 × 10 ² , 1.0 × 10 ³ , 1.0 × 10 ⁴ , 1.0 × 10 ⁵ and 1.0 × 10 ⁶ CFU / mL. Thereafter, Triton X-100 was added to 1 mL of the prepared solution so as to have a final concentration of 0.5%, and heated in boiling water for 15 minutes to obtain a treated product. Using the immunochromatographic strip of the present invention, the effect of adding each concentration of Triton X-100 was evaluated. Further, the same evaluation was performed for the case where Triton X-100 was not added (no addition of Triton X-100). The results are shown in Table 4 below.

As is clear from Table 4 above, the detection accuracy of the E. coli O157: H7 ATCC 43894 strain, RIMD05091896 strain, RIMD05091924 strain, RIMD05091971 strain, RIMD0500991 strain, RIMD05090929 strain, and RIMD05091980 strain was almost 10 times higher. The detection accuracy of E. coli O157: H7 strain RIMD05091974 was almost 100 times higher. In the E. coli O157: H7 RIMD05091944 strain and the E. coli O157: HNT RIMD05091894 strain, “±” became “+ w” at 1.0 × 10 ⁴ CFU / mL. Therefore, no. 1-No. In any of the 10 strains of E. coli O157: H7, the detection accuracy was significantly higher than when no Triton X-100 was added, and the detection limit concentration was less than 10 ⁴ CFU / mL except for the RIMD05091980 strain. It was confirmed that there was.

1. Company A Strip Using an O157 test strip (Company A strip) sold by Company A, the effect of adding Triton X-100 was examined. The extraction sample was prepared in the same manner as in Example 3. According to the attached instruction, 150 μL of the extraction sample was dispensed into a test tube, the strip was added to the test tube, and the determination was made visually after 15 minutes. As in Example 3, 10 types of strains having different strain numbers of E. coli O157 were used as test strains, and the effect of adding Triton X-100 at each concentration was evaluated. Further, the same evaluation was performed for the case where Triton X-100 was not added. The results are shown in Table 5 below.

As is clear from Table 5 above, the detection accuracy of E. coli O157: H7 strain ATCC 43894, RIMD05091896, RIMD0509191, RIMD05091944, and RIMD05091980 was almost 10 times higher. In the E. coli O157: H7 RIMD05091971 and RIMD05091974 strains, “±” becomes “+ w” in the number of bacteria of 1.0 × 10 ⁴ CFU / mL, and in the RIMD05090929 strain, 1.0 × 10 ⁴ CFU / mL In the number of bacteria, “+ w” became “+”. The detection accuracy of the RIMD05091924 strain and E. coli O157: HNT in the RIMD05091894 strain did not change. Therefore, even when A company strip is used, 1-No. In 10 strains of 10 types of Escherichia coli O157: H7, it was confirmed that the detection accuracy was generally higher when Triton X-100 was added compared to the case where Triton X-100 was not added.

2. Company B Strip Using a test strip for O157 (Company B strip) sold by Company B, the effect of adding Triton X-100 was examined. The extraction sample was prepared in the same manner as in Example 3. According to the attached instruction, 1 mL of the extraction sample was dispensed into a test tube, the strip was added to the test tube, and the judgment was made visually after 10 minutes. As in Example 3, 10 types of strains having different strain numbers of E. coli O157 were used as test strains, and the effects of adding Triton X-100 at each concentration were evaluated. Moreover, it evaluated similarly about the case where TritonX-100 is not added. The results are shown in Table 6 below.

As is apparent from Table 6 above, the detection accuracy of E. coli O157: H7 strain ATCC 43894, RIMD05091924, RIMD0509171, RIMD05091891, and RIMD05090929 was almost 10 times higher. In the RMD05091974 strain of E. coli O157: H7, “±” became “+ w” at 1.0 × 10 ³ CFU / mL. In the E. coli O157: H7 RIMD05091980 strain and the E. coli O157: HNT RIMD05091894 strain, “±” became “+ w” at 1.0 × 10 ⁴ CFU / mL. The detection accuracy in the RIMD05091896 strain and the RIMD05091944 strain did not change. Therefore, when Company B strip is used, 1-No. In 10 strains of 10 types of Escherichia coli O157: H7, it was confirmed that the detection accuracy was generally higher when Triton X-100 was added compared to the case where Triton X-100 was not added.

1. Examination of culture medium Using Escherichia coli O157: H7 (ATCC 43894) strain as a test strain, TA10 Broth (tryptose 10.0 g / L, meat extract 5.0 g / L, yeast extract 5.0 g / L, sodium extract 5 manufactured by Primaham 0.0 g / L, glucose 0.5 g / L, disodium hydrogen phosphate 7.0 g / L, potassium dihydrogen phosphate 15.0 g / L, salts 0.95 g / L), mEC medium supplemented with Merck novobiocin The effect of adding Triton X-100 or benzalkonium chloride was examined using three types of culture solutions of mTSB medium supplemented with Merck novobiocin. After culturing the ATCC 43894 strain at 35 ° C. for 24 hours, the number of bacteria was 1.0 × 10 ⁶ , 1.0 × 10 ⁵ , 1.0 × 10 ⁴ , 5.0 × 10 ³ , 1.0 × 10 ^3. , 5.0 × 10 ² CFU / mL. Thereafter, Triton X-100 is added to 1 mL of the prepared solution to a final concentration of 0.5% or benzalkonium chloride to a final concentration of 0.05%, and heated in boiling water for 15 minutes to be treated. It was. After 100 μl of the treated product was reacted with the detection reagent (gold colloid-labeled monoclonal antibody), the immunochromato strip of the present invention was added, and the determination was made visually 15 minutes later. The results are shown in Table 7 below.

As apparent from Table 7 above, when no surfactant was used, the detection limit concentrations when cultured in the above three types of culture solutions were all 1.0 × 10 ⁴ CFU / mL. When Triton X-100 was added to the culture solution to a final concentration of 0.5%, the detection limit concentration was 1.0 × 10 ³ CFU / mL in any of the above three media. When benzalkonium chloride is added to the culture solution to a final concentration of 0.05%, the detection limit concentration in TA10 Broth is 1.0 × 10 ³ CFU / mL, and novobiocin-added mEC medium and novobiocin-added mTSB medium The detection limit concentrations in both were 5.0 × 10 ³ CFU / mL. Therefore, although there is some variation depending on the type of culture medium, the detection accuracy will be significantly higher when 0.5% Triton X-100 or 0.05% benzalkonium chloride is added to the culture medium. Was confirmed.

1. Examination of cross-reactivity Cross-reactivity tests were conducted using 60 types of bacteria other than E. coli O157 as test strains. Using the tryptosome broth as a medium, each test bacterium was cultured at 35 ° C. for 24 hours, and then prepared so that the number of bacteria was 1.0 × 10 ⁸ CFU / mL. Thereafter, Triton X-100 was added to 1 mL of the prepared solution to a final concentration of 0.5%, and heated with boiling water for 15 minutes to obtain an extracted sample. 100 μL of the extracted sample was reacted with a detection reagent containing a colloidal gold-labeled monoclonal antibody, and then applied to the immunochromatographic strip (test strip) of the present invention. After 15 minutes, the determination was made visually. The results are shown in Table 8 below.
Further, the A company strip and the B company strip were also visually determined in accordance with the attached instructions in the same manner as in Example 4. The results are shown in Table 8 below.

  As apparent from Table 8 above, in each of the 60 types of test bacteria, the immunochromatographic strip and the Company A strip of the present invention were No. No. 4 Citrobacter freundii NBRC16624 strain and Positive for 57 Salmonella Urbana ATCC9261 strains. Citrobacter Freundi and Salmonella Urbana have been reported to have a common O antigen with E. coli O157 [(Nishiuchi et al. 2000. Structure and serologic properties of O-specific polysaccharide from Citrobacter freundii possessing cross-reactivity with Escherichia coli O157: H7. FEMS Immunol. Med. Microbiol. 28, 163-170) and (Koichi Minami et al. 2000. Salmonella Urbana (O30) with the common antigen of enterohemorrhagic Escherichia coli O157 and severe neurological sequelae A case of salmonellosis left behind. Pediatric infection immunity 12 (1), 19-22)], consistent with the results here. Therefore, it was confirmed that the immunochromatographic strip of the present invention and the Company A strip did not have cross-reactivity with antigens other than the common O antigen with E. coli O157: H7 even when Triton X-100 was added.

  Further, the strip of Company B, when at least Triton X-100 is added, has the above-mentioned Citrobacter Freundi NBRC16624 strain and No. In addition to 57 Salmonella Urbana ATCC9261 strains, Citrobacter rodentium NBRC105723 strain, Cronobacter sakazaki BRC102416 strain, Edward Sierra Tarda NBRC105688 strain, Erwinia carotobola IFO3380 strain, Escherichia Brattae NBRC111725 strain E. coli RIMD050092014 and ATCC33780, Escherichia coli O111: H8RIMD05092026, Escherichia coli O26: H46RIMD0509624, Escherichia Fagussonii NBRC102419, Escherichia Hermannii ATCC33650, ATCC33651 Denshia alkaline tumefaciens NBRC105687 strain, both Salmonella Paratyphi B ATCC51962 strain and the like showed cross-reaction, E. coli O157: inability specifically detect H7 O antigen was confirmed.

  The detection method of Escherichia coli O157 of the present invention has a very high detection accuracy and low cross-reactivity compared to the case where a surfactant is not used by treating a test sample with a surfactant. It was confirmed that the false positive rate was very small compared to the conventional product. Therefore, the present invention is useful in preventing contamination and infection by pathogenic E. coli O157, and can be used particularly in the food industry.

Claims (21)

  A method for detecting Escherichia coli O157, which comprises treating a test sample with a surfactant and reacting an antibody against Escherichia coli O157 with the treated product to detect Escherichia coli O157.   The method for detecting Escherichia coli O157 according to claim 1, wherein the treated product is a heat-treated product obtained by treating a test sample with a surfactant and then heating.   The method for detecting E. coli O157 according to claim 1 or 2, wherein the antibody against E. coli O157 is a monoclonal antibody against E. coli O157.   The method for detecting E. coli O157 according to claim 3, wherein the monoclonal antibody against E. coli O157 is a monoclonal antibody against heat-treated cells of E. coli O157.   The method for detecting Escherichia coli O157 according to claim 3 or 4, wherein the monoclonal antibodies against Escherichia coli O157 are two or more kinds of monoclonal antibodies that recognize different epitopes.   6. The method for detecting Escherichia coli O157 according to claim 5, wherein at least one of two or more kinds of monoclonal antibodies recognizing different epitopes is a monoclonal antibody labeled with a colloidal gold used for immunochromatography.   The monoclonal antibody against E. coli O157 is a monoclonal antibody O157W-17B3 produced by a hybridoma (NITE P-1347) or a monoclonal antibody O157W-20B6 produced by a hybridoma (NITE P-1346). 6. The method for detecting E. coli O157 according to any one of 6 above.   The method for detecting Escherichia coli O157 according to any one of claims 1 to 7, wherein the surfactant is a nonionic surfactant or a cationic surfactant.   The method for detecting Escherichia coli O157 according to claim 8, wherein the nonionic surfactant is polyoxyethylene (10) octylphenyl ether.   The method for detecting Escherichia coli O157 according to claim 8, wherein the cationic surfactant is benzalkonium chloride. The detection method according to claim 1, wherein E. coli O157 of 1 × 10 ³ CFU / mL or less can be detected.   An E. coli O157 detection kit comprising an antibody against E. coli O157 and a surfactant.   The kit for detecting E. coli O157 according to claim 12, wherein the antibody against E. coli O157 is a monoclonal antibody against E. coli O157.   The kit for detecting E. coli O157 according to claim 13, wherein the monoclonal antibody against E. coli O157 is a monoclonal antibody against heat-treated cells of E. coli O157.   15. The kit for detecting E. coli O157 according to claim 13 or 14, wherein the monoclonal antibody against E. coli O157 is two or more kinds of monoclonal antibodies recognizing different epitopes.   The kit for detecting Escherichia coli O157 according to claim 15, wherein at least one of the two or more kinds of monoclonal antibodies recognizing different epitopes is a monoclonal antibody labeled with a colloidal gold used for immunochromatography.   The monoclonal antibody against E. coli O157 is a monoclonal antibody O157W-17B3 against E. coli O157 produced by a hybridoma (NITE P-1347) or a monoclonal antibody O157W-20B6 against E. coli O157 produced by a hybridoma (NITE P-1346) The kit for detecting Escherichia coli O157 according to any one of claims 13 to 16.   The detection kit according to any one of claims 12 to 17, wherein the surfactant is a nonionic surfactant or a cationic surfactant.   The kit for detecting Escherichia coli O157 according to claim 18, wherein the nonionic surfactant is polyoxyethylene (10) octylphenyl ether.   The detection kit according to claim 18, wherein the cationic surfactant is benzalkonium chloride.   A method for improving the detection accuracy of E. coli O157, comprising using a surfactant and an antibody against E. coli O157 in combination.