WO2002065131A1

WO2002065131A1 - Method of detecting bacterium belonging to the genus vibrio, detection reagent and antibody to be used therein

Info

Publication number: WO2002065131A1
Application number: PCT/JP2002/001064
Authority: WO
Inventors: Hiroki Tatsumi; Satoshi Fukuda; Sumio Shinoda; Shin-Ichi Miyoshi; Yoko Maehara
Original assignee: Kikkoman Corporation
Priority date: 2001-02-09
Filing date: 2002-02-08
Publication date: 2002-08-22
Also published as: JPWO2002065131A1

Abstract

A method of detecting bacterium belonging to the genus Vibrio) characterized by involving: (1) a first step of bringing an antibody, which can react with a lecithin-dependent hemolysin originating in the bacterium belonging to the genus Vibrio , into contact with a sample to induce an antigen-antibody reaction; and (2) a second step of detecting the reaction product obtained in the first step; a reagent for detecting bacterium belonging to the genus Vibrio characterized by containing an antibody which can react with a lecithin-dependent hemolysin originating in the bacterium belonging to the genus Vibrio; and an antibody which can react with a lecithin-dependent hemolysin originating in a bacterium belonging to the genus Vibrio, usable in the above detection method and detection reagent.

Description

Description Method for detecting Vibrio spp., Reagents for detection, and antibodies used therefor

The present invention relates to a method for detecting a genus Vibrio, a reagent for detecting a bacterium of the genus Vibrio, and an antibody used therefor. Background art

Vibrio parahaemolyt icus, a member of the genus Vibrio, is the causative agent of most seafood poisoning in summer in Japan. In addition to raw seafood infected with Vibrio parahaemolyticus, secondary-contaminated cooked foods and drinking water can be food-causing foods. Testing for Vibrio parahaemolyticus in foods and drinks is essential to assess safety.

The following methods were known as methods for detecting Vibrio parahaemolyticus (according to Food Sanitation Inspection Guideline Microorganisms (Japan Food Hygiene Association, 1990)).

First, samples of fish and shellfish, food and drink, etc. are cultured in saline polymyxin broth (manufactured by Nissui) at 37 ° C for 18 hours, and then spread on TCBS agar medium (manufactured by Nissui), and then incubated at 37 ° C, 18 ° C. Incubate for hours. Vibrio parahaemolyticus forms non-sucrose-degradable green-blue colonies of about 2 mm in diameter on TCBS agar medium. However, non-saccharide-degrading Vibrio bacteria often form similar colonies of the same color as Vibrio parahaemolyticus, so a primary confirmation test and possibly a secondary confirmation test were performed to distinguish them from each other. Required.

In the primary confirmation test, the properties described in parentheses are examined using the following six tests

(1) l% NaCl added ordinary agar (Oxidase)

(2) 1% NaCl added TSI agar (hydrogen sulfide, gas, glucose fermentation)

(3) SIM medium supplemented with 1% NaCl (indole, indole pyruvate, motility) (4) 1% NaCl added lysine medium (lysine decarboxylase)

(5) 1¾ NaCl added VP semi-fluid medium (Voges- Proskauer)

(6) Broth with 0, 3, 8, and 10% NaCl (Development)

If the above test does not confirm, a secondary confirmation test using the following four types of tests is further performed to identify.

(7) 〇_nitrophenyl] 3-D-galactopyranoside (0_nitrophenyl β-D-galactopyranoside, 0NPG)

(8) Orditin medium supplemented with 1% NaCl

(9) 1% NaCl added arginine medium

(10) 1% NaCl-containing medium for glycolysis test (arabinose, maltose, inosit, xylose, salicin, mannitol, mannose, sucrose)

The above confirmation test for detection of Vibrio parahaemolyticus consists of a large number of tests, which requires labor, medium costs and time. Therefore, a quick, simple, and accurate test alternative to these confirmation tests has been desired.

Lecithin Dependent Hemolysin (LDH) (also known as Thermolabile Hemolysin) is a thermolabile hemotoxin with phospholipase A 2

It is a protein having (phospholipase A2) activity and lysophospho-lipase activity (Shinoda et al., J. Gen. Microbiol., 137, 2705 (1991)). Mizuguchi and Taniguchi found that the LDH gene was present in all 121 strains of Vibrio parahaemolyticus by colony hybridization and Southern blot hybridization and that 22 strains of Vibrio spp. Other than Vibrio parahaemolyticus were present. It has been reported that the LDH gene does not exist (Toshio Miwaya, Makoto Ohashi, Vibrio parahaemolyticus III, p. 131, Modern Press).

Recently, Bej et al. (J. Microbiological Methods, 36, 215 (1999)) and McCarthy et al. (Lett. Appl. Microbiol. 28, 66 (1999)) Indicates that the LDH gene was detected from Vibrio parahaemolyticus and not from Vibrio parahaemolyticus by the PCR method and DNA hybridization method, respectively. And useful.

However, the hybridization method and the PCR method using the LDH gene as a detection marker have the following disadvantages.

• The operation is complicated and time consuming to handle the DNA of microorganisms.

· Special equipment required

• May react with non-enteritis vibrio with similar DNA sequences.

An object of the present invention is to provide a method for quickly and simply and accurately detecting a Vibrio bacterium and a reagent for detection capable of solving the problems of the prior art.

The present inventors have studied the above problems and found that by detecting LDH produced by Vibrio sp. In a sample using an antibody capable of reacting with LDH derived from Vibrio sp. The present inventors have found that genus bacteria (particularly, Vibrio parahaemolyticus or Vibrio alginolyticus) can be detected, and completed the present invention. Summary of the Invention

That is, the present invention provides the following methods and products.

1. (1) a first step of bringing an antibody capable of reacting with LDH derived from a bacterium of the genus Vibrio into contact with a sample to generate an antigen-antibody reaction, and

(2) Second step for detecting the reaction product obtained in the first step

A method for detecting a bacterium of the genus Vibrio in a sample, comprising the step of:

2. The method for detecting a Vibrio bacterium according to 1 above, wherein the sample is cultured in a selective medium for Vibrio bacterium prior to the first step.

3. The detection method according to the above 1 or 2, wherein the antibody is an antibody obtained by immunizing an animal with LDH derived from a genus Vibrio as an antigen.

4. The detection method according to any one of the above 1 to 3, wherein the Vibrio bacterium is Vibrio parahaemolyt icus or Vibrio alginolyticas.

5. A reagent for detecting a Vibrio bacterium, which comprises an antibody capable of reacting with LDH derived from a Vibrio bacterium. 6. The detection reagent according to the above item 5, wherein the bacterium of the genus Vibrio is Vibrio parahaemolyticus or Vibrio alginolyticus.

7. Lecithin dependence derived from bacteria of the genus Vibrio. An antibody that can react with hemolysin. BEST MODE FOR CARRYING OUT THE INVENTION

Hereinafter, the present invention will be described in detail.

1. Detection of Vibrio spp.

The method for detecting a bacterium of the genus Vibrio of the present invention is characterized by including the following first and second steps.

In the first step, an antibody capable of reacting with LDH derived from bacteria of the genus Vibrio (hereinafter referred to as “anti-LDH antibody”) is brought into contact with a sample to generate an antigen-antibody reaction. If the sample contains Vibrio spp. That produces L dishes, the anti-LDH antibody and the LDH produced by the bacterium cause an antigen-antibody reaction.

In the second step, the reaction product obtained in the first step is detected. As described above, detection of Vibrio spp. Bacteria (particularly Vibrio parahaemolyticus or Vibrio alginolyticus) in the sample becomes possible.

In the present invention, the method of the antigen-antibody reaction (first step) and the detection of the reaction product (second step) are not particularly limited, and a known method utilizing an immune reaction can be used. Examples of the method usable in the present invention include enzyme immunoassay (EIA), radioimmunoassay (RIA), fluorescence immunoassay (FIA), electrochemical immunoassay, immunoagglutination, immunochromatography. And the like.

In the present invention, prior to the first step, the sample is preferably cultured in a medium to increase the number of bacteria of the genus Vibrio. This makes it possible to improve the detection sensitivity of Vibrio spp. Bacteria. The medium is not particularly limited as long as it is capable of growing Vibrio spp., But it is particularly preferable to use a selective medium for Vibrio spp. By using a selective medium, it is possible to enrich Vibrio spp. In a sample. As shown in the Examples, when the detection method of the present invention is used, enteritis virus present in a sample can be obtained. Brio (Vibrio parahaemolyt icus) or Vibrio alginolyticas (Vibrio alginolyt icus) can be detected quickly, conveniently and accurately. Further, the detection method of the present invention can also be used as a method for detecting other Vibrio bacteria that can react with an anti-LDH antibody.

In the present invention, the sample is not particularly limited, but includes, for example, raw and processed fish and shellfish, food and drink, seawater, river water, dishes and cooking utensils, and the like. When the sample is in a liquid state, the sample may be filtered using a membrane filter or the like, and a sample obtained by capturing microorganisms on the filter may be used as the sample. When detecting Vibrio spp. Attached to food or cooking utensils, wipe the food or cooking utensils with a cotton swab impregnated with saline solution or buffer solution, and remove the resulting adhering substances appropriately. A sample suspended in a liquid or medium may be used as the sample. Furthermore, microbes may be extracted from food using a stomacher or the like, and the resulting extract may be used as a sample.

The “anti-LDH antibody” may be any antibody as long as it can react with LDH derived from Vibrio sp., For example, an antibody obtained by immunizing an animal with LDH derived from Vibrio sp. As an antigen is used. it can. As the anti-LDH antibody, any of a polyclonal antibody and a monoclonal antibody can be used. Furthermore, a recombinant anti-LDH antibody prepared using the anti-LDH antibody gene can also be used. Polyclonal antibodies can be obtained from the sera of sensitized animals. The sensitization can be carried out by injecting the LDH protein as an antigen into mammals, preferably, egrets, goats and cattle. Normally, the first injection is followed by a booster injection to maximize the immune response, and then sera from the sensitized animals are collected. Monoclonal antibodies can be obtained by producing hybridomas capable of producing anti-LDH antibodies by cell fusion.

LDH protein can be purified from cultures of Vibrio parahaemolyticus. The LDH gene of Vibrio parahaemolyticus is already known (Taniguclii et al., Microb. Pathog., 1, 425, (1986)). The LDH protein is produced by producing the gene in an appropriate host cell-vector system, and the LDH protein can be isolated by purifying the LDH protein. By sensitizing appropriate animals using LDH protein as an antigen, LDH antibodies are obtained. Using the gene of the anti-LDH antibody, a recombinant antibody (single chain Fv or Fab) can be produced using a microorganism such as Escherichia coli as a host.

The anti-LDH antibody may be chemically modified depending on the method of the antigen-antibody reaction (first step) and the detection of the reaction product (second step). When a method utilizing the reaction between biotin and streptavidin is employed in the first and second steps, the anti-LDH antibody is biotinylated by a known method.

Hereinafter, as a specific example of the method for detecting a bacterium of the genus Vibrio of the present invention, a method of culturing a sample in a medium, contacting an anti-LDH antibody with the sample to generate an antigen-antibody reaction, and then detecting a reaction product Will be described.

First, prepare a sample, inoculate it on a solid medium or liquid medium, and culture it under appropriate conditions. Fish, seafood and food and drink can be directly used as samples and inoculated directly into the culture medium. An extract obtained by extracting microorganisms from food or drink or the like may be used as a sample. Alternatively, microorganisms adhering to food or cooking utensils may be wiped off with a cotton swab, and then the microorganisms may be transferred to a culture medium and cultured. When a liquid sample is filtered with a membrane filter or the like and microorganisms are captured on the filter, the filter can be placed on a solid medium or the captured substance can be suspended in the liquid medium.

The medium is not particularly limited as long as it can grow Vibrio spp., But by using a selective medium for Vibrio spp., The Vibrio spp. In the sample can be concentrated. Selection media include TCBS agar medium, Vibrio agar medium, salt polymyxin broth, alkaline peptone water (Nissui, etc.), 1.5% salt-added Heart Infusion Broth (HIB) Abbreviation, manufactured by Diico). When a selective medium is used, the sample may be applied directly to the agar medium, or the sample may be cultured in a liquid medium. Further, after liquid culture, it may be applied to an agar medium. When overnight culture medium is used, the quantitative test can be performed by the most probable method.

The cultivation temperature of the genus Vibrio is 15 ° C to 45 ° C, preferably about 30 ° C to 40 ° C. During the culture, the culture may be aerated or closed. If a liquid medium is used, the culture may be shaken as necessary. After the culture, an anti-LDH antibody is brought into contact with the culture to generate an antigen-antibody reaction (first step), and then the reaction product is detected (second step). Through these steps, LDH in the sample is detected, and as a result, Vibrio spp. That produces LDH is detected.

LDH may be detected from any of the whole culture, the culture supernatant, the cells, and the disrupted cells. The method of disrupting the cells may be any method as long as it does not change the antibody recognition structure of LDH, and examples thereof include ultrasonic treatment, treatment with a lytic enzyme or a lytic agent, and French press. Alternatively, the culture may be once cultured on a selective medium of Vibrio parahaemolyticus such as TCBS agar medium, and LDH may be detected directly from the colony suspected of Vibrio parahaemolyticus or after culture in the medium.

The antigen-antibody reaction and the detection of the reaction product can be performed using enzyme immunoassay (EIA), radioimmunoassay (RIA), electrochemical immunoassay, immunoagglutination, immunochromatography, and the like.

For example, when using an enzyme immunoassay, there are a sandwich method and a competitive method. In the case of the sandwich method, first, an anti-LDH antibody is bound to a carrier, then brought into contact with a sample, and then contacted with a labeled anti-LDH antibody (for example, an antibody-enzyme complex, biotinylated antibody). Finally, if necessary, a second label (for example, a biotinylated luciferase ostreptoavidin complex) is contacted. LDH can be detected by measuring the label or the second label by an enzyme reaction. In the case of the competitive method, an anti-LDH antibody is first bound to a carrier, and then a sample and a labeled LDH (eg, an LDH-enzyme complex, a biotinylated LDH) are added, and if necessary, a second labeled substance (eg, LDH can be detected by contacting the product with a biotinylated luciferase-sestrebutavidin complex) and measuring the label or the second label by an enzyme reaction.

When using the immunoagglutination method, first, an anti-LDH antibody is sensitized to latex particles, gold colloid, or the like, and then the sample is brought into contact with the sample. Thereafter, LDH force can be detected by detecting agglutination, which is a reaction product.

By the above method, Vibrio spp., Especially Vibrio parahaemolyticus or Bib Although it is possible to detect Rio alginolyticus (V. alginolyticus), a confirmation test is performed by a known method, if necessary.

Vibrio parahaemolyticus and Vibrio alginolyticus were contained in the sample.

When both (V. alginolyt icus) are present, both are detected simultaneously by the detection method of the present invention. Vibrio parahaemolyticus and Vibrio alginolyticus

If only one species (V. alginolyt icus) needs to be detected, cultivation is carried out using a selective medium in order to separate the two prior to performing the antigen-specific antibody reaction. For example, on a TCBS agar medium (Nissui), a selective medium for Vibrio spp., Vibrio parahaemolyticus, which is non-degradable by sucrose, forms a green-blue colony, whereas Vibrio arginolyticus (V alginolyt icus) is known to form yellow settlements. By using TCBS agar as the selective medium, both can be distinguished.

2. Detection reagent for Vibrio spp.

The detection reagent of the present invention is characterized by containing an anti-LDH antibody. The reagent is used in the method for detecting a bacterium of the genus Vibrio of the present invention. The reagent contains, in addition to the anti-LDH antibody, other components necessary for the detection of Vibrio spp., Or components (preservatives, pH adjusters, antioxidants, etc.) that allow each reaction step to proceed stably. May be included. The shape of the detection reagent of the present invention may be a solution or a solid. The reagent in the form of a solution is, for example, a solution in which an anti-LDH antibody is dissolved in an appropriate solvent. The solid-state reagent is, for example, one in which an anti-LDH antibody is immobilized on a solid carrier such as a well or a latex particle for immunoassay. Example

Hereinafter, the present invention will be described more specifically with reference to examples.

Example 1

(1) Preparation of LDH

E. coli C600pHL591 strain in which the LDH gene of Vibrio parahaemolyticus has been cloned (Taniguchi et al., Microb. Pat og., 1, 425 (1986)) was used in にて 5 mL. The cells were cultured at 37 ° C for 8 hours with shaking, inoculated into 500 mL of HIB, and then cultured at 37 ° C for 16 hours with shaking. The cells were centrifuged at 7000Xg for 40 minutes to collect the cells. Periplasmic fractions were obtained from these cells by the osmotic shock method according to the method of Neu and Neppel (J. Biol. Chem., 240, 3685 (1965)).

To this, ammonium sulfate was added so as to obtain a 65% saturation (420 g / L), left at 4 ° C for a while, and then centrifuged at 7000Xg for 40 minutes to collect a precipitate. The resulting precipitate was dissolved in 3 mL of 0.15 M NaCl / 20 mM histidine monohydrochloride (Histidine_HCl) buffer (pH 5.5), dialyzed against the same buffer overnight, and centrifuged to remove insolubles. The supernatant was added to an anion exchanger Mono Q HR 5/5 column equilibrated with a 0.15 M NaCl / 20 mM histidine-HCl (Histidine-HC1) buffer solution (pH 5.5), and high-performance liquid chromatography was performed. That is, after the unadsorbed protein was washed with the same buffer, the protein was eluted with the same buffer containing 0.9 M NaCl, and a fraction having poma erythrocyte hemolytic activity (ie, LDH activity) was collected. The erythrocyte hemolytic activity was determined by reacting the sample with 0.5% erythrocytes in the presence of 50 ig / mL lecithin at 37 ° C for 2 hours to determine the presence or absence of hemolysis. As a control, a reaction was performed in the absence of lecithin, and the difference was determined.

(2) Preparation, purification and biotinylation of anti-LDH antibody

Completely Freund's adjuvant with LDH obtained in (1)

(Freund's complete adjuvant) to immunize the egret to prepare an anti-LDH antiserum. Subsequently, the anti-LDH antibody was purified from the anti-LDH antiserum using a MabTrap GII kit (Amersham Pharmacia Biotech) and desalted using a PD-10 column (Amersham Pharmacia Biotech). The protein concentration was measured with BCA Protein Atssay reagent (Pierce). From 1.5 mL of antiserum, 3.5 mL (1.1 mg / mL) of anti-LDH antibody (polyclonal antibody) was purified.

To 1 mL of the purified antibody, a 10-fold molar ratio of a biotinylation reagent (Sulfo-NHS-LC-Biotin, manufactured by Pierce) was added, and the mixture was left on ice for 2 hours for biotinylation. Thereafter, unreacted biotinylation reagent was removed with a PD-10 column. The biotinylated anti-LDH antibody obtained above was used as one of the detection reagents of the present invention.

(3) Preparation of sample containing bacteria Eight strains of Vibrio parahaemolyticus and 22 strains of Vibrio spp. Other than Vibrio parahaemolyticus (Tables 1 and 2) were spread on HIB agar supplemented with 1.5% sodium chloride and cultured at 37 for 18 hours. The single colony was cultured with shaking in 2 mL of HIB liquid medium containing 1.5% sodium chloride for 5 hours, 1 mL of the culture was transferred to a microtube, and a culture supernatant was obtained by centrifugation. The culture supernatant was used as a sample.

Twenty-one strains of bacteria other than the genus Vibrio (Table 3) were spread on HIB agar medium and cultured at 37 for 18 hours. The single colony was cultured with shaking in 2 mL of HIB liquid medium for 5 hours, 1 mL of the culture was transferred to a microtube, and a culture supernatant was obtained by centrifugation. The culture supernatant was used as a sample.

(4) Testing for Vibrio bacteria by enzyme-linked immunosorbent assay

Prepare a 60 πιΜ sodium carbonate buffer (ρΗ 9.6) containing anti-LDH antibody at a concentration of 10 xg / mL, and add each to a C8 MAXI BREAKAPART NUNCHI IMUNO module C8 MAXI BREAKAPART NUNC- IMMUNO MODULE, manufactured by Nunc) and left at 4 ° C for 18 hours to allow the anti-L dish antibody to adsorb to the well. The well to which the antibody was adsorbed was used as one of the reagents for detecting Vibrio bacteria of the present invention.

After washing three times with Tris buffered saline (hereinafter abbreviated as “TBS”) containing 0.1% Tween 20, 300 L of 25% PROC Ace (Dainippon Pharmaceutical Co., Ltd.) was added. The mixture was added and left at 37 ° C. for 1 hour to perform blocking of the wells. After washing three times with TBS containing 0.1% Tween 20, add the sample prepared in (3) or add HIB IOOL as a control, leave it at 37 for 1 hour, and remove the solid phase antibody and LDH in the sample. The reaction was performed.

After washing three times with TBS containing 0.1% Tween 20, add 10 mL of biotinylated anti-LDH antibody (prepared with (2)) diluted 3,000-fold with 10% Block Ace and add 37 ° C. C for 1 hour, and the reaction between LDH bound to the solid phase antibody and the biotinylated anti-LDH antibody was performed. After washing three times with TBS containing 0.1% Tween 20, the biotinylated reagent kit (trade name: InteliteAB, manufactured by Kikkoman Co., Ltd.) containing a biotinylated luciferase zestrebutavidin complex solution (complex ) Add IOO L and set at 37 ° C 30 Then, the mixture was allowed to react with the biotinylated anti-LDH antibody and the complex. After washing 3 times with TBS containing Tween 20 of 0.1, cut off the cell of C8 MAXI BREAKAPART NUNC-1 (C8 MAXI BREAKAPART NUNC-1 picture NO MODULE, Nunc) and emit light. Attached to a photometric tube (Lumitube, manufactured by Kikkoman Co., Ltd.). The luminescence substrate solution of Intellite AB was added, and the luminescence was measured with a luminescence detector Lumitester-K210 (manufactured by Kikkoman). Samples showing a luminescence value more than twice that of the control HIB were judged as positive, and samples showing less than twice the amount of luminescence were judged as negative.

As a result, all Vibrio parahaemolyticus and V. alginolyticus (V. alginolyticus) were positive, while all other Vibrio and non-Vibrio bacteria were negative (Table 1). ~ 3). In other words, the enzyme immunoassay using anti-LDH antibody showed that only two species, Vibrio parahaemolyticus and V. alginolyticus, could be specifically detected from the bacteria used. .

Example 2 Detection of Vibrio spp. By latex agglutination method

Detection of Vibrio spp. Bacteria by the latex agglutination method can be performed by the following method.

First, the antibody is sensitized to the latex particles. Polybead polystyrene microspheres (diameter Ι Ι, manufactured by Polysciences, Inc.) are used as latex particles. Latex particles 2.5 Put 0.5 mL of suspension into a microphone mouth tube, add 1 mL of borate buffer (0.1 M Borate, pH 8.5), and after turbidity, centrifuge for latex Collect the particles. The procedure of resuspending this in 1 mL of borate buffer and centrifuging is repeated twice, and then resuspended in 1 mL of borate buffer. The anti-LDH antibody obtained in Example 1 (2) is added to a concentration of 25 g / mL, and left at room temperature for 16 hours under shaking at 20 rpm.

After collecting the latex particles by centrifugation, suspend them in 1 mL of borate buffer containing 1% bovine serum albumin and leave at room temperature with shaking at 20 rpm for 30 minutes. After repeating this operation twice more, the latex particles were collected by centrifugation, and 1 mL of a storage buffer (20 mM sodium phosphate, 150 mM NaCl, 1% BSA, 5% glycerol, 0.1% NaN ₃ , H7.4). The latex particles sensitized with the anti-LDH antibody obtained above are one of the detection reagents of the present invention.

The sample prepared in Example 1 (3) and the latex particles sensitized with the anti-LDH antibody described above were diluted with a diluent (phosphate buffered saline containing 0.5% bovine serum albumin and 0.1% NaN _3). ) Dilute each 10-fold with. Add 252 L of each diluted sample to each well of a V-shaped microplate (Course Yuichisha). Next, 25 diluted dilute latex particles are added dropwise thereto, and the mixture is shaken and stirred with a mixer for a microplate, and then left at room temperature for 18 hours to observe aggregation of each well.

Culture supernatants of Vibrio parahaemolyticus and Vibrio alginolyticus (V. alginolyticus) cause latex aggregation, but other culture supernatants do not. That is, by using the Latec particles sensitized with the anti-LDH antibody, Vibrio parahaemolyticus and V. alginolyticus can be efficiently detected.

Example 3 Method of performing antigen-antibody reaction after selective culture

The enzyme immunoassay and the latex agglutination method described above can detect only Vibrio parahaemolyticus and V. alginolyticus producing LDH. Vibrio parahaemolyticus is a bacterium that causes food poisoning and requires food hygiene control, but V. alginolyticus (V. alginolyt icus) does not cause food poisoning, so it is desirable to distinguish between the two. Vibrio parahaemolyticus, which is non-sucrose-degrading on TCBS agar medium (manufactured by Nissui), which is a selective medium for Vibrio bacteria, forms a green-blue colony. alginolyt icus) is known to form yellow settlements. For this reason, it is easy to distinguish between the two. Therefore, the shape of the colonies on the TCBS agar medium of the Vibrio spp. Bacteria and the non-Vibrio spp. Bacteria used in Example 1 (4) was examined (Tables 1-3). 1. After culturing each strain for 24 hours at 37 ° C in 2 mL of HIB medium (Vibrio spp. Bacteria) or HIB medium (Non-vibrio spp. Bacteria) supplemented with 5% sodium chloride, spread on TCBS agar medium, The cells were cultured at ° C for 18 hours. The presence or absence of growth on TCBS agar medium and the color of the colonies were observed. On TCBS agar, all eight strains of Vibrio parahaemolyticus used form green-blue colonies, and all three strains of Vibrio alginolyticus (V. alginolyt icus) used form yellow colonies. (Table 1). Most Vibrio bacteria other than Vibrio parahaemolyticus and V. alginolyticus grew on TCBS agar and formed green-blue or yellow colonies (Tables 1 and 2). All non-Vibrio bacteria used did not grow or formed very small colonies on TCBS agar, and were easily distinguished from Vibrio parahaemolyticus and Vibrio alginolyticus (V. alginolyticus). It was possible (Table 3).

A single colony of Vibrio spp.grown on TCBS agar was suspended in mL of HIB liquid medium containing 1.5% sodium chloride, incubated at 37 ° C for 6 hours, and then 1 mL of the culture was transferred to a microphone mouth tube. The culture supernatant was prepared by centrifugation. When the culture supernatant was examined by the immunoassay described in Example 1 (4), Vibrio parahaemolyticus and V. alginolyticus (V. alginolyticus) were positive, and all the other strains were negative. . The selective culture of Vibrio parahaemolyticus and Vibrio alginolyticus (V. alginolyticus) can also be performed prior to the latex aggregation method described in Example 2.

After performing selective culture on TCBS agar, detection using anti-LDH antibody is performed to obtain Vibrio parahaemolyticus and Vibrio alginolyticus.

(V. alginolyt icus) respectively. Strain name EIA * TCBS ^ V. parahaemolyticus AQ3354 positive green-blue V. parahaemolyticus RIMD2210518 positive green-blue V. parahaemolyticus 93-200 positive green-blue V. parahaemolyticus 93- 347 positive green-blue tl paste V. ΓαίΙ θΐηθΐγΐ lCUS V i Q V. parahaemolyticus OP-377 positive green-blue enteritis vibrio (V. parahaemolyticus) 93-100 positive green-blue enteritis vibrio (V. parahaemolyticus) 93- 346 positive green blue Vibrio alginolyticus (V. alginolyticus)

π UτA / Λ Q 7 ΠΠ 1 Positive Yellow Ao 〖UU 1

Positive z «i Nore no Riice Js v. Aiginoiyi icus no Positive Yellow OKA87-003

V. alginolyticus

Positive yellow 0KA87-004

Vibrio hollisae (V. hollisae) CDC9041-81 negative green-blue Vibrio, hollisae (V. hollisae) CDC0075-80 negative green-blue Vibrio furnissii (V. furnissii) Q17 negative yellow picture 4119 negative yellow

* Results from the enzyme immunoassay of Example 1 (4)

«Color of colonies on TCBS agar

Table 2

Strains EIA TCBS «

16 V. metschnikovii Negative Yellow ATCC11171

17 Vibrio fluvialis (V. fluvialis) AQ0005 negative yellow

18 Vibrio * Fluvialis (V. fluvialis) AQ0012 negative Yellow

19 Vibrio anguillarum (V. anguillarum) negative yellow

IN then Μΰδώθ

l "No 7 * 1)]

20 t ri 1-^^! R · V J Funore / (V V.dllgl ΐ 1 Ί 丄 1 丄 d πrUιτlτIU Negative No growth.

NCMB829

Vibrio splendidus (V. splendidus)

21 Negative yellow stroke 4716

22 Vibrio cholera (V. cholerae) non-01 NCTC4716 Negative Yellow ώϋ ti nori -i ref v. Ciioierae no u 丄 ΐώ ^ 115¾ 佥 1 soil 臾 / 1 C glue -J \ V. 1¾ 1 Tsuchi 1 ± i

OP;

Norichi ^! , 8 hu (V τ 1η11ΐ 丄 τ 11τι1ί rises i 1i1c'no i Π W9Π U all '1' aw? F¾ t ± i

26 V. mimicus 48Pt negative Green-blue

27 Vibrio vulnificus (V. vulnificus) negative green-blue strain B

28 Vibrio parnificus (V. vulnificus) negative green-blue strain C

29 Vibrio 'Harveyi' OKA82-557 negative No growth

30 V. damsel a ATCC33539 negative greenish blue

* Results from the enzyme immunoassay of Example 1 (4)

«Color of colonies on TCBS agar Table 3

Table 3 (continued)

Industrial applicability

According to the present invention, by detecting an LDH produced by a Vibrio bacterium in a sample using an antibody capable of reacting with LDH derived from a Vibrio bacterium, a Vibrio bacterium (particularly, Vibrio parahaemolyticus or Vibrio spp. alginolyt icus) was detected. In addition, the present invention provides a reagent for detecting Vibrio bacteria, which comprises an antibody capable of reacting with LDH derived from Vibrio bacteria.

According to the conventional method based on the Food Hygiene Inspection Guidelines, when identifying Vibrio parahaemolyticus and suspicious colonies on the selective agar medium, one day for the primary confirmation test (six types) and one day for the secondary confirmation test (four types) A total of 2 days (10 tests) were required.

In contrast, in the enzyme immunoassays described in Examples 1 and 3, Vibrio parahaemolyticus and Vibrio alginolyticus (V. alginolyticus) can be identified within a single test day from a colony on a selective agar medium. It is. Also, in the latex agglutination method described in Examples 2 and 3, it is possible to identify from the colonies on the selective agar medium the next day by one type of test. It has been shown that the use of the method of the present invention makes the operation simpler than the conventional method and shortens the inspection time by one to two days.

INDUSTRIAL APPLICABILITY The present invention is useful in fields such as food hygiene, medical treatment, and scientific research on bacteria of the genus Vibrio.

Claims

The scope of the claims

1. (1) contacting an antibody capable of reacting with lecithin-dependent hemolysin derived from a bacterium of the genus Vibrio with a sample to generate an antigen-antibody reaction, and

(2) a second step of detecting a reaction product obtained in the first step,

A method for detecting a bacterium of the genus Vibrio in a sample, comprising:

2. The method for detecting Vibrio bacteria according to claim 1, wherein the sample is cultured in a selective medium for Vibrio bacteria prior to the first step.

3. The detection method according to claim 1, wherein the antibody is an antibody obtained by immunizing an animal with lecithin-dependent hemolysin derived from a bacterium of the genus Vibrio as an antigen.

4. The detection method according to claim 2, wherein the antibody is an antibody obtained by immunizing an animal with lecithin-dependent hemolysin derived from a bacterium of the genus Vibrio as an antigen.

5. The detection method according to any one of claims 1 to 4, wherein the Vibrio bacterium is Vibrio parahaemolyticus or Vibrio alginolyticus.

6. A reagent for detecting Vibrio bacteria, which comprises an antibody capable of reacting with lecithin-dependent hemolysin derived from Vibrio bacteria.

7. The detection reagent according to claim 6, wherein the Vibrio bacterium is Vibrio parahaemolyticus or Vibrio arginolitis.

8. An antibody capable of reacting with lecithin-dependent haemolysin from Vibrio spp.