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WO2002048712A1 - Detection de e-coli et d'autres organismes coliformes et pathogenes dans l'eau - Google Patents

Detection de e-coli et d'autres organismes coliformes et pathogenes dans l'eau Download PDF

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Publication number
WO2002048712A1
WO2002048712A1 PCT/IN2001/000217 IN0100217W WO0248712A1 WO 2002048712 A1 WO2002048712 A1 WO 2002048712A1 IN 0100217 W IN0100217 W IN 0100217W WO 0248712 A1 WO0248712 A1 WO 0248712A1
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WO
WIPO (PCT)
Prior art keywords
buffer
water
membrane
dilution
dilution buffer
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Ceased
Application number
PCT/IN2001/000217
Other languages
English (en)
Inventor
Deepti Dileep Deobagkar
Veena Abhay Limaye
Chandrashekhar Madhav Chitale
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
India Defence Ministry of Research and Development Organization
Original Assignee
India Defence Ministry of Research and Development Organization
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by India Defence Ministry of Research and Development Organization filed Critical India Defence Ministry of Research and Development Organization
Publication of WO2002048712A1 publication Critical patent/WO2002048712A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/53—Immunoassay; Biospecific binding assay; Materials therefor
    • G01N33/569—Immunoassay; Biospecific binding assay; Materials therefor for microorganisms, e.g. protozoa, bacteria, viruses
    • G01N33/56911—Bacteria
    • G01N33/56916—Enterobacteria, e.g. shigella, salmonella, klebsiella, serratia
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
    • G01N2333/195—Assays involving biological materials from specific organisms or of a specific nature from bacteria
    • G01N2333/24—Assays involving biological materials from specific organisms or of a specific nature from bacteria from Enterobacteriaceae (F), e.g. Citrobacter, Serratia, Proteus, Providencia, Morganella, Yersinia
    • G01N2333/245—Escherichia (G)

Definitions

  • F ⁇ ELD OF INVENTION This invention relates to a method far detection of E.cal ⁇ , other caiif ⁇ raas and pathogenic organisms in water far the purpose of testing the potability of water.
  • Water contaminated with sewage may have many disease causing organises such as Salmonella (causing typhoid?, vibrio ch ⁇ lerae (causing cholera), Shigella (causing dysentry) ,
  • E.c ⁇ ii (causing diarrhea ⁇ , Hepatitis ⁇ virus
  • Rota virus etc (causing gastr ⁇ enteritis ⁇ ,, parasites like E ⁇ ta oeba hist ⁇ litica (causing amoelic dysentry ⁇ , Biardia lambia (causing giardiasis) etc are presentb alongs ⁇ ith normal flora of human/animal intestine.
  • Water borne infections are mainly transmitted by consumption of untreated or contaminated water os sometimes by wjimwiing in the open lakes.
  • the potability of water is generally checked by det cting the level ⁇ f E.c ⁇ i.
  • the routine laboratory methods used are "Membrane filtration technique, and Most probable number technique" .
  • test media, systems and kits are costly, require special equipment to read the results and need trained staff.
  • Another method known in the art for detection of bacteria in water is based on laser scanning using chemscan laser scanner. This method is sensitive, rapid and accurate.
  • a rapid Direct Viable count method is also known in the art for enumeration of bacteria in mineral water. This method needs series of test media. The limitation of the above method is that it requires around 14 hours for completion of test.
  • test method Another limitation of the test method is that' it requires trained staff.
  • Another method known in the art for detection of potability ⁇ f water is based on enzymatic reaction — Ecloss test. This method is based upon enhanced cner ⁇ iluminescence as an indicator of water quality.
  • PCR polymerase chain reaction
  • RT PCR
  • peptide nucleic acid probe etc for detection of E—c ⁇ li and other bacteria in water samples.
  • the limitation of the above method is that it needs 7 to 8 hours .
  • Another limitation of the above method is that it requires fluorogenic and chemiluminogenic substrates and sophisticated instruments having U light,, —ray film developing syste ⁇ , etc.
  • Still another limitation of the above method is that it requires sterile conditions, well set laboratory as it is based upon oisicrocQlony formation with the production of the enzyme.
  • the detection of water— borne pathogens is based on amplifying specific target DNA sequence and detecting the amplified target D ⁇ #i sequence.
  • the ampli ications of DNA sequence is toy means of selected primer pairs according to a procedure known as Polymerase Chain Reaction (PCR).
  • the limitation of the above method is that it requires trained staff to do DNA isolation.
  • Another limitation of the above method is that it requires special instrumentation like PCR machine etc.
  • the primary object of the present invention is t ⁇ provide a method for detection of E—coli and other coliforms and pathogenic organisms in water.
  • Another object of the present invention is t ⁇ provide a method of testing bacteria in water which is very sensitive.
  • Still another object of the present invention is t ⁇ provide a method of testing bacteria in water which can detect bacteria in water within 3 to 6 hours.
  • Yet another object of the present invention is to provde a method of testing bacteria in water which provides a simple method for detection of bacteria in (water.
  • Further object of the present invention is t ⁇ provide a method of testing bacteria in water which can also detect viable-nonculturable bacteria in water i.e the bacteria which fail to grow on a laboratory media as sometimes bacteria enter into a dormant state as a response t ⁇ sublethal stresses such as extreme temperature, pH, etc.
  • Yet further object of the present invention is to provide a method of testing bacteria in water which can detect the previous history of the contamination and can distinguish between live and dead organism.
  • Still further object of the present invention is t ⁇ provide a method of testing bacteria in watr tsihieh can detect many other related organisms like Pseud ⁇ m ⁇ nas aerugenosa which are present in water and which make the water unsafe to use.
  • a rapid method for detection of E—c ⁇ li, other c ⁇ liforms and pathogenic organisms in water which immobilises microbes from water Cif any) on to a solid phase nitrocellulose membrane and with help of antibody generate a coloured reaction far detection of E—coli in water-
  • the method involves use of reagents namely goat anti—rabbit anitbody conjugated to horse raddish perossidase diaminobenzedene tetrahydrochloride, H 0 and Tris Buffered Saline (TBS) with
  • the other reagents used are blocking buffer, dehydrated selective nutrient medium, dilution buffer, washing buffer (TBS + 0.1% Tween 0 ⁇ besides nitrocellulose membrane.
  • the method of testing water based on the above reagents is very simple and time—efficient as it takes only 3 to & hours as compared t ⁇ conventional methods which detect viable nonculturable bacteria which fail to grow in a media.
  • the method can also detect related organisms lifee Pseud ⁇ m ⁇ nas aerugenosa which are present in water and make it unsafe for use.
  • the method does not require any sophisticated equipment such as laminar flow, ELISA reader etc but can be carried out by an unskilled person, at r ⁇ ocm te ⁇ perature.
  • the antibodies used are designed to recognise both viable and dead cells and therefore the method finds application for detection of previous history of contamination.
  • the reagents used in the method of detection as per present invention ares I. Reagent As 10X solution of blocking buffer — 2.5% casein in Tris Buffered Saline (TB) . II. Reagent B: Concentrated antibody solution — diluted in IX dilution buffer — 1:500.
  • Reagent Cs Goat anti—rabbit antibody conjugated t ⁇ horse raddish peroxidase — t ⁇ be diluted in IX dilution buffer 1:1000.
  • Reagent D Solution of diami ⁇ benzedene tetrahydrochl ⁇ ride in distilled water.
  • Reagent F vials of dehydrated selective nutrient medium.
  • Washing Buffer 50X TBS, pH7.5 + 0. t% Tween 20.
  • a drop of water sample' to be tested is. spotted on to the small pieces of nitrocellulose membrane.
  • the membrane is incubated in the diluted "Reagent A”* for 5—20 minutes with constant shaking at room temperature- The membrane is then washed in washing buffer for 2 minutes.
  • the membrane in then treated with diluted ""Reagent B” for 5— 5-15 minutes with constant shaking. It is then washed with washing buffer for 5 to 15 minutes and is incubated in the diluted "Reagent C” for about IS minutes with constant shaking at room temperature. The washing in with washing- buffer is repeated for 5—15 minutes- Two drops of "Reagent D” and ⁇ two drops of "Reagent E” are mixed in 10 ml of IX dilution buffer. The membrane is incubated in this mixture till the colour develops. This step is carried out in dark and takes about 1 t ⁇ 5 minutes. The membrane is washed in washing buffer and dried and stored.
  • TBS for IS 1 minutes with shaking.
  • the membrane was washed as above.
  • the membrane was then developed with 5mg Diamin ⁇ - benzedene tetrahydr ⁇ chloride + 30ul H O + 10 ml TBS.
  • the water sample from the State Transport stand water tank was taken. 2u of it was directly dotted on the membrane. 1ml, 10 ml and 100 ml sample was ce ⁇ trifuged and the sediment was then spatted onto the Hybond C membrane. The membrane was air—dried. Membrane was kept in 2% milk p ⁇ wder in TBS pH 7.5. The membrane was then washed with TBS pH 7.5 + Tween 20 for 2 minutes. The membrane was then kept in antibody solution diluted 1:1000 in TBS for 15 minutes and then washed with TBS + Tween 20 for 20 minutes.
  • the membrane was challenged with the diluted (1:1000) goat anti—rabbit antibody conjugated to horse raddish peroxidase for 15 minutes fallowed by washing in the same w as above.
  • the membrane was developed in dark with the solution of Diaminobenzedene tetrahydrochl ⁇ ride, H O and TBS-.
  • the reagent B was diluted to 1:500 with IX dilution buffer (imll. The membrane was kept in it for 15 minutes with constant shaking. Washing of the membrane was clone as above for 20 minutes. (4 wash, 5 minutes each). Dilution of the reagent C, 1:1000, with T . dilution buffer was done and the membrane was incubated in it for 15 minutes with constant shaking at room temperature- Washing was similar t ⁇ the above step. The next step was carried out in dark. 2 drops of reagent D and 2 drops of reagent E were mixed in 10 ml of ⁇ X dilution buffer- The membrane was washed with the washing solution.

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  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Immunology (AREA)
  • Engineering & Computer Science (AREA)
  • Hematology (AREA)
  • Chemical & Material Sciences (AREA)
  • Urology & Nephrology (AREA)
  • Biomedical Technology (AREA)
  • Molecular Biology (AREA)
  • Microbiology (AREA)
  • Physics & Mathematics (AREA)
  • Biotechnology (AREA)
  • Virology (AREA)
  • Tropical Medicine & Parasitology (AREA)
  • Food Science & Technology (AREA)
  • Medicinal Chemistry (AREA)
  • Cell Biology (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • General Physics & Mathematics (AREA)
  • Pathology (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

L'invention concerne un procédé permettant de détecter E-coli, et d'autres organismes coliformes et pathogènes dans l'eau. Ce procédé comprend l'utilisation des réactifs suivants : une solution à 10 % de tampon de blocage comprenant 2,5 % de caséine dans un tampon TBS (Tris Buffered Saline), une solution concentrée d'anticorps dilués dans un tampon de dilution 1X, des anticorps de chèvre anti-lapin conjugués avec de la peroxydase de raifort diluée dans un tampon de dilution 1X, un révélateur, un tampon de dilution, un tampon de lavage, de petits morceaux de membrane de nitrocellulose et un milieu nutritif sélectif déshydraté.
PCT/IN2001/000217 2000-12-13 2001-12-11 Detection de e-coli et d'autres organismes coliformes et pathogenes dans l'eau Ceased WO2002048712A1 (fr)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
IN1151DE2000 2000-12-13
IN1151/DEL/2000 2000-12-13

Publications (1)

Publication Number Publication Date
WO2002048712A1 true WO2002048712A1 (fr) 2002-06-20

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Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/IN2001/000217 Ceased WO2002048712A1 (fr) 2000-12-13 2001-12-11 Detection de e-coli et d'autres organismes coliformes et pathogenes dans l'eau

Country Status (1)

Country Link
WO (1) WO2002048712A1 (fr)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005036168A3 (fr) * 2003-10-10 2005-09-15 Univ Cincinnati Procede de detection de materiaux biologiques dans un liquide

Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1981002790A1 (fr) * 1980-03-18 1981-10-01 Ciba Geigy Ag Nouveau support solide pour proteines a des fins analytiques
WO1989001162A1 (fr) * 1987-07-28 1989-02-09 Biotechnology Australia Pty. Ltd. Procedes de detection
GB2234587A (en) * 1989-08-02 1991-02-06 Chisso Corp ELISA kit for detecting bacteria comprising polyclonal antibodies
JPH05322897A (ja) * 1992-05-21 1993-12-07 Mitsubishi Yuka B C L:Kk フィルターを用いる細菌の酵素免疫測定法
US5415997A (en) * 1987-07-28 1995-05-16 Biotechnology Australia Pty Limited Method for detecting low levels of microorganisms
EP0982590A1 (fr) * 1998-07-01 2000-03-01 Nitto Denko Corporation Conjugué marqué et méthode de détection utilisant ce conjugué

Patent Citations (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1981002790A1 (fr) * 1980-03-18 1981-10-01 Ciba Geigy Ag Nouveau support solide pour proteines a des fins analytiques
WO1989001162A1 (fr) * 1987-07-28 1989-02-09 Biotechnology Australia Pty. Ltd. Procedes de detection
US5415997A (en) * 1987-07-28 1995-05-16 Biotechnology Australia Pty Limited Method for detecting low levels of microorganisms
GB2234587A (en) * 1989-08-02 1991-02-06 Chisso Corp ELISA kit for detecting bacteria comprising polyclonal antibodies
JPH05322897A (ja) * 1992-05-21 1993-12-07 Mitsubishi Yuka B C L:Kk フィルターを用いる細菌の酵素免疫測定法
EP0982590A1 (fr) * 1998-07-01 2000-03-01 Nitto Denko Corporation Conjugué marqué et méthode de détection utilisant ce conjugué

Non-Patent Citations (6)

* Cited by examiner, † Cited by third party
Title
ACTA BIOQUIM. CLIN. LATINOAM., vol. 26, no. 3, 1992, pages 295 - 298 *
B.E. RICE ET AL.: "Development of a rapid and specific colony-lift immunoassay for detection and enumeration of campylobacter jejuni, C. coli and C. lari", CLINICAL AND DIAGNOSTIC LABORATORY IMMUNOLOGY, November 1996 (1996-11-01), pages 669 - 677 *
Computer-translation of JP 05 322 897 A, retrieved from internet *
DATABASE CAPLUS [online] M.E. SANZ ET AL.: "Development of a Dot-Blot assay for the detection of adhesives in enterotoxigenic escherichia coli", accession no. STN Database accession no. 1994:72784 *
DATABASE WPI Week 199402, Derwent World Patents Index; AN 1994-013257 *
PATENT ABSTRACTS OF JAPAN *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2005036168A3 (fr) * 2003-10-10 2005-09-15 Univ Cincinnati Procede de detection de materiaux biologiques dans un liquide

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