WO2002065131A1 - Procédé de détection d'une bactérie du genre vibrion, réactif de détection et anticorps à cet effet - Google Patents

Procédé de détection d'une bactérie du genre vibrion, réactif de détection et anticorps à cet effet Download PDF

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Publication number
WO2002065131A1
WO2002065131A1 PCT/JP2002/001064 JP0201064W WO02065131A1 WO 2002065131 A1 WO2002065131 A1 WO 2002065131A1 JP 0201064 W JP0201064 W JP 0201064W WO 02065131 A1 WO02065131 A1 WO 02065131A1
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vibrio
antibody
ldh
sample
detecting
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PCT/JP2002/001064
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English (en)
Japanese (ja)
Inventor
Hiroki Tatsumi
Satoshi Fukuda
Sumio Shinoda
Shin-Ichi Miyoshi
Yoko Maehara
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Kikkoman Corporation
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Priority to JP2002564598A priority Critical patent/JPWO2002065131A1/ja
Publication of WO2002065131A1 publication Critical patent/WO2002065131A1/fr

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911—Bacteria

Definitions

  • 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.
  • Vibrio parahaemolyt icus a member of the genus Vibrio, is the causative agent of most seafood poisoning in summer in Japan.
  • 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.
  • 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.
  • 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.
  • LDH Lecithin Dependent Hemolysin
  • the hybridization method and the PCR method using the LDH gene as a detection marker have the following disadvantages.
  • 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
  • the present invention provides the following methods and products.
  • a method for detecting a bacterium of the genus Vibrio in a sample comprising the step of:
  • Vibrio bacterium is Vibrio parahaemolyt icus or Vibrio alginolyticas.
  • 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.
  • the method for detecting a bacterium of the genus Vibrio of the present invention is characterized by including the following first and second steps.
  • 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.
  • the reaction product obtained in the first step is detected.
  • detection of Vibrio spp. Bacteria (particularly Vibrio parahaemolyticus or Vibrio alginolyticus) in the sample becomes possible.
  • 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.
  • the method usable in the present invention include enzyme immunoassay (EIA), radioimmunoassay (RIA), fluorescence immunoassay (FIA), electrochemical immunoassay, immunoagglutination, immunochromatography. And the like.
  • the sample prior to the first step, 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • a recombinant antibody single chain Fv or Fab
  • 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).
  • first step the antigen-antibody reaction
  • second step the detection of the reaction product
  • the anti-LDH antibody is biotinylated by a known method.
  • 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.
  • 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.
  • 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).
  • a selective medium 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.
  • the culture may be aerated or closed. If a liquid medium is used, the culture may be shaken as necessary.
  • 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).
  • first step an anti-LDH antibody is brought into contact with the culture to generate an antigen-antibody reaction
  • second step the reaction product 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.
  • 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.
  • EIA enzyme immunoassay
  • RIA radioimmunoassay
  • electrochemical immunoassay immunoagglutination
  • immunochromatography and the like.
  • an enzyme immunoassay there are a sandwich method and a competitive method.
  • 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.
  • a labeled anti-LDH antibody for example, an antibody-enzyme complex, biotinylated antibody
  • a second label for example, a biotinylated luciferase ostreptoavidin complex
  • 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.
  • LDH labeled LDH
  • a second labeled substance eg, LDH can be detected by contacting the product with a biotinylated luciferase-sestrebutavidin complex
  • 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.
  • Vibrio spp. Especially Vibrio parahaemolyticus or Bib
  • Rio alginolyticus V. alginolyticus
  • Vibrio parahaemolyticus and Vibrio alginolyticus were contained in the sample.
  • V. alginolyt icus 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.
  • 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.
  • 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.
  • 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 ⁇ ⁇ 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)).
  • 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.
  • Histidine_HCl histidine monohydrochloride
  • 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.
  • Histidine-HC1 histidine-HC1 buffer solution
  • 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.
  • 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.
  • TBS Tris buffered saline
  • PROC Ace Dainippon Pharmaceutical Co., Ltd.
  • 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.
  • 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.
  • C8 MAXI BREAKAPART NUNC-1 C8 MAXI BREAKAPART NUNC-1 picture NO MODULE, Nunc
  • Bacteria by the latex agglutination method can be performed by the following method.
  • the antibody is sensitized to the latex particles.
  • Polybead polystyrene microspheres (diameter ⁇ ⁇ , 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.
  • the latex particles sensitized with the anti-LDH antibody obtained above are one of the detection reagents of the present invention.
  • Example 1 (3) 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.
  • a diluent phosphate buffered saline containing 0.5% bovine serum albumin and 0.1% NaN 3
  • V. alginolyticus 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.
  • 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.
  • 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.
  • Vibrio parahaemolyticus and V. alginolyticus were positive, and all the other strains were negative. .
  • the selective culture of Vibrio parahaemolyticus and Vibrio alginolyticus can also be performed prior to the latex aggregation method described in Example 2.
  • 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. ⁇ ⁇ 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)
  • 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
  • Vibrio fluvialis V. fluvialis
  • Vibrio spectacularus V. splendidus
  • 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.
  • the present invention provides a reagent for detecting Vibrio bacteria, which comprises an antibody capable of reacting with LDH derived from Vibrio bacteria.
  • Vibrio parahaemolyticus and Vibrio 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.
  • the present invention is useful in fields such as food hygiene, medical treatment, and scientific research on bacteria of the genus Vibrio.

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Abstract

La présente invention concerne un procédé de détection d'une bactérie du genre vibrion caractérisé en ce qu'il implique, d'une part (1) une première opération par laquelle on prend un anticorps capable de réagir avec une hémolysine à dépendance lécithinique provenant de la bactérie du genre vibrion, et on le met en contact avec un échantillon de façon à induire une réaction antigène-anticorps, et d'autre part (2) une seconde opération par laquelle on détecte le produit de réaction obtenu par la première opération. L'invention concerne également un réactif permettant de détecter une bactérie du genre vibrion caractérisé en ce qu'il contient un anticorps capable de réagir avec une hémolysine à dépendance lécithinique provenant de la bactérie du genre vibrion. L'invention concerne enfin un anticorps capable de réagir avec une hémolysine à dépendance lécithinique provenant de la bactérie du genre vibrion, convenant pour le procédé de détection et le réactif de détection de l'invention.
PCT/JP2002/001064 2001-02-09 2002-02-08 Procédé de détection d'une bactérie du genre vibrion, réactif de détection et anticorps à cet effet WO2002065131A1 (fr)

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2006017554A (ja) * 2004-06-30 2006-01-19 Daikin Ind Ltd 磁気ビーズ及び微生物検出方法
JP2013085503A (ja) * 2011-10-17 2013-05-13 Osaka Prefecture Univ 腸炎ビブリオの検出方法
CN106950368A (zh) * 2017-03-11 2017-07-14 中国海洋大学 一种病原性溶藻弧菌的检测试纸
CN110734491A (zh) * 2019-11-21 2020-01-31 上海交通大学医学院 检测副溶血性弧菌的试剂盒及检测方法
CN113121682A (zh) * 2019-12-30 2021-07-16 中国科学院上海营养与健康研究所 特异性识别o3:k6型副溶血性弧菌的单克隆抗体及其应用

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
BEJ ET AL.: "Detection of total and hemolysin-producing vibrio parahaemolyticus", JOURNAL OF MICROBIOLOGICAL METHODS, vol. 36, pages 215 - 225, XP002909008 *
MCCARTHY ET AL.: "Evaluation of alkaline phosphatase- and digoxigenin-labelled probes for detection of the thermolabile hemolysis gene of vibrio parahaemolyticus", LETTERS IN APPLIED MICROBIOLOGY, vol. 28, 1999, pages 66 - 70, XP002909007 *
SHINODA ET AL.: "Purification and characterization of a lecithin-dependent haemolysin", JOURNAL OF GENERAL MICROBIOLOGY, vol. 137, 1991, pages 2705 - 2711, XP002909006 *

Cited By (8)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
JP2006017554A (ja) * 2004-06-30 2006-01-19 Daikin Ind Ltd 磁気ビーズ及び微生物検出方法
JP2013085503A (ja) * 2011-10-17 2013-05-13 Osaka Prefecture Univ 腸炎ビブリオの検出方法
CN106950368A (zh) * 2017-03-11 2017-07-14 中国海洋大学 一种病原性溶藻弧菌的检测试纸
CN106950368B (zh) * 2017-03-11 2019-02-15 中国海洋大学 一种病原性溶藻弧菌的检测试纸
CN110734491A (zh) * 2019-11-21 2020-01-31 上海交通大学医学院 检测副溶血性弧菌的试剂盒及检测方法
CN110734491B (zh) * 2019-11-21 2022-03-25 上海交通大学医学院 检测副溶血性弧菌的试剂盒及检测方法
CN113121682A (zh) * 2019-12-30 2021-07-16 中国科学院上海营养与健康研究所 特异性识别o3:k6型副溶血性弧菌的单克隆抗体及其应用
CN113121682B (zh) * 2019-12-30 2022-08-26 中国科学院上海营养与健康研究所 特异性识别o3:k6型副溶血性弧菌的单克隆抗体及其应用

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