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WO2001061029A2 - Procede d'identification - Google Patents

Procede d'identification Download PDF

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Publication number
WO2001061029A2
WO2001061029A2 PCT/GB2001/000658 GB0100658W WO0161029A2 WO 2001061029 A2 WO2001061029 A2 WO 2001061029A2 GB 0100658 W GB0100658 W GB 0100658W WO 0161029 A2 WO0161029 A2 WO 0161029A2
Authority
WO
WIPO (PCT)
Prior art keywords
micro
organisms
concentration
electrophoretic
organism
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/GB2001/000658
Other languages
English (en)
Other versions
WO2001061029A3 (fr
Inventor
Kathleen Ann Grant
Stuart Harbron
David Ross Williams
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.)
Zetatronics Ltd
Original Assignee
Zetatronics Ltd
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
Priority claimed from GB0003554A external-priority patent/GB0003554D0/en
Priority claimed from GB0003795A external-priority patent/GB0003795D0/en
Priority claimed from GB0007771A external-priority patent/GB2348504B/en
Application filed by Zetatronics Ltd filed Critical Zetatronics Ltd
Priority to AU2001232127A priority Critical patent/AU2001232127A1/en
Publication of WO2001061029A2 publication Critical patent/WO2001061029A2/fr
Publication of WO2001061029A3 publication Critical patent/WO2001061029A3/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

Links

Classifications

    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
    • G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
    • G01N27/416—Systems
    • G01N27/447—Systems using electrophoresis
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/02—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving viable microorganisms
    • C12Q1/04—Determining presence or kind of microorganism; Use of selective media for testing antibiotics or bacteriocides; Compositions containing a chemical indicator therefor
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Q—MEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q2304/00—Chemical means of detecting microorganisms
    • C12Q2304/80—Electrochemical detection via electrodes in contact with culture medium

Definitions

  • the present invention relates to a method and apparatus for the detection of the presence of specific micro-organisms in a fluid. It is particularly applicable, but in no way limited, to identifying microbial pathogens.
  • said fluid sample is obtained from a human or animal body and said method provides an indication of the cause of an infection in said human or animal body.
  • an apparatus for carrying out the methods described herein comprising:-
  • the present invention is a method for determining a characteristic fingerprint for a micro-organism.
  • the micro-organism may be a bacterium, a fungus, a virus or it may be an individual animal cell, for example a blood cell, or a plant cell, for example an alga.
  • the present invention is also a method for detecting the presence of a micro- organism in a sample.
  • the micro-organism may be any micro-organism that is susceptible to an enzyme composition.
  • the micro-organism is preferably a bacterium.
  • the sample may also be obtained from a food material by homogenising the food to cause any micro-organism present to be transferred from the food to the homogenising solution.
  • the sample may be obtained from the atmosphere by drawing air through a liquid to cause any micro- organisms present in the atmosphere to be transferred to the liquid.
  • the sample containing the micro-organism Prior to measurement, the sample containing the micro-organism may be contacted with nutrients and incubated at a suitable temperature to cause the micro-organisms in the sample to grow and divide.
  • the nutrients added and the temperature used are chosen such that only one or only one group of micro- organisms are caused to grow and divide.
  • Enzymes that may be used in this invention include but are not limited to: lysozyme, mutanolysin, lyticase, chitinase, mucopeptide amidohydrolase, N-acetylglucosamine deacetylase, ⁇ (1-6) and ⁇ (1-3) glycanases, proteases, and mannase.
  • Preferred enzymes are lysozyme and mutanolysin.
  • An example of the use of lysozyme is given in Example 12 and an example of mutanolysin is given in Example 13.
  • the present invention further provides a method for determining a characteristic fingerprint for a micro-organism.
  • the fingerprint comprises zeta potential values or electrophoretic mobility values obtained in the presence of an enzyme composition and under one or more different conditions.
  • composition of the solution is chosen such that it contains a predetermined concentration of a surface-active agent.
  • a surface-active agent may be a non-ionic detergent, an anionic detergent, a cationic detergent or a zwitterionic detergent. Examples of surface-active agents include the following:
  • Alkyltrimethylammonium bromides : BIGCHAP ⁇ N,N-bis(3-D-Glucon- Dodecyltrimethylammonium bromide amidopropyl)-cholamide ⁇ Hexadecyltrimethylammonium bromide Decanoyl-N-methylglucamide Tetradecyltrimethylammonium bromide n-Decyl- ⁇ -D-glucopyranoside Benzalkonium chloride n-Decyl- ⁇ -D-glucopyranoside Benzethonium chloride n-Decyl- ⁇ -D-maltopyranoside Benzyldimethyldodecylammonium bromide Deoxy-BIGCHAP ⁇ N,N-bis(3-Glucon- Benzyldimethylhexadecylammonium bromide amido-propyl)-deoxycholamide ⁇ Benzyltrimethylammonium methoxide n-Dodecyl-
  • the composition of the solution is chosen such that it contains a predetermined concentration of an organic solvent.
  • the solvent may be a water-miscible solvent such as an alcohol, acetonitriie, DMSO, or THF.
  • the solvent may be a water-immiscible solvent, such as toluene, ethyl acetate, or ether used at a concentration at or below its maximum solubility in water so that a single aqueous phase is obtained.
  • organic solvents can be used in this method and the examples given above are by way of illustration only.
  • composition of the solution is chosen such that it contains a predetermined concentration of a heavy metal salt.
  • Suitable heavy metal salts are salts of copper, mercury, lead, calcium, chromate and the like.
  • composition of the solution is chosen such that it contains a predetermined concentration of a chelating agent.
  • the chelating agents may be ethylene diamine, DTPA (diethylenetriaminepenta-acetic acid), EDTA (ethylenediamine tetra-acetic acid), EGTA (ethylene glycol-bis( ⁇ -aminoethyl ether)
  • N,N,N',N'-tetra-acetic acid Dimercaprol (2,3-dimercapto-1-propanol), HEDTA (N- hydroxyethylethylenediaminetriacetic acid), citric acid, gluconates, or NTA (nitrilotri- acetic acid).
  • composition of the solution is chosen such that it contains a predetermined concentration of a polyol or polyether.
  • Suitable polyethers include polyethylene glycol and polyproylene glycol.
  • Polyols include monosaccharides, disacchahdes, polysaccharides and carbohydrates in general.
  • the sample containing the micro-organism is mixed with two or more different solutions and the electrophoretic mobility of the micro-organism in each of the different solutions is measured.
  • the electrophoretic mobility data obtained under these defined but differing conditions provide a fingerprint characteristic of the micro-organism.
  • the electrophoretic mobility data may be transformed to zeta potential data.
  • the sample containing the micro-organism is mixed with a solution of predetermined composition to form a first mixture and the electrophoretic mobility of the micro-organism in the first mixture is measured. At least one more different solution is mixed with this first mixture to form second and subsequent mixtures, and the electrophoretic mobility of the micro-organism in each of these subsequent mixtures is measured.
  • the electrophoretic mobility data obtained under these defined but differing conditions provide a fingerprint characteristic of the micro-organism.
  • the electrophoretic mobility data may be transformed to zeta potential data.
  • a sample containing one or more unknown micro-organisms is obtained. Aliquots of the sample are then mixed with two or more different solutions and the electrophoretic mobility of the micro-organism or micro-organisms in each of the different solutions is measured.
  • the electrophoretic mobility data may be transformed to zeta potential data. The electrophoretic mobility and/or zeta potential data obtained under these defined but differing conditions is compared to the fingerprints of known microorganisms to determine the identity of the one or more micro-organisms present in the sample.
  • the database will provide further information about the micro-organism or micro-organisms present. For example in a clinical or veterinary application, the database may provide therapeutic information relating to the treatment of any disease associated with the presence of the micro-organism.
  • the sample containing one or more unknown microorganisms is mixed with a solution of predetermined composition to form a first mixture and the velocity, displacement, zeta potential or electrophoretic mobility measured. At least one more different solution is mixed with this first mixture to form second and subsequent mixtures, and the velocity, displacement, zeta potential or electrophoretic mobility of the micro-organism in each of these subsequent mixtures is measured.
  • the electrophoretic mobility data may be transformed to zeta potential data. The velocity, displacement, zeta potential or electrophoretic mobility data obtained under these defined but differing conditions is compared to the fingerprints to determine the identity of the one or more micro-organisms present in the sample.
  • the database will provide further information about the micro-organism or microorganisms present. For example in a clinical or veterinary application, the database may provide therapeutic information relating to the treatment of any disease associated with the presence of the micro-organism.
  • the sample containing the micro-organism may be derived from a number of sources.
  • known micro-organisms are cultured to provide the sample.
  • the sample may be from any source for which the identity of any micro-organisms present in the sample is required.
  • the sample may be a sample obtained and removed from a human or animal subject: the sample may be of urine or blood, or it may be derived from swabbing the throat, or from faeces.
  • the sample may also be obtained from a water supply.
  • the sample may be a food material, or it may be obtained from a food material by swabbing the surface of the food and transferring the swab to a liquid to cause any microorganism present to be transferred from the surface of the food to the solution.
  • the sample may also be obtained from a food material by homogenising the food to cause any micro-organism present to be transferred from the food to the homogenising solution.
  • the sample may be obtained from the atmosphere by drawing air through a liquid to cause any micro-organisms present in the atmosphere to be transferred to the liquid. Prior to measurement, the sample containing the micro-organism may be contacted with nutrients and incubated at a suitable temperature to cause the micro-organisms in the sample to grow and divide.
  • Buffers chosen for the measurement of zeta potentials of the initial test bacterial species (all buffers used at 5mM)
  • the bacterial strains used in this study were Escherichia coli W3110, Bacillus cereus, Enterococcus faecalis and Pseudomonas aeruginosa. Cultures were grown in nutrient broth at 37° C. with shaking until the optical density at 600 nm was 0.3.
  • the bacterial strains used in this study were Escherichia coli W3110, Bacillus cereus, Enterococcus faecalis and Pseudomonas aeruginosa. Cultures were grown in nutrient broth at 37° C. with shaking until the optical density at 600 nm was 0.3. An aliquot of each culture (100 ⁇ l) was added to 10 ml of 5 mM MES buffer, pH 6.0, containing 5% sucrose and either 0, 0.005, 0.05 or 0.5 mM CTAB. The buffer solution was filtered through a 0.2 ⁇ m filter prior to use to remove small particles that may interfere with subsequent electrophoretic measurements. Electrophoretic mobilities and the derived zeta potentials were obtained by analysing the solutions in a Malvern Zetasizer 2000. The results are shown in Figure 3. Example 4 - Effect of Stains
  • Electrophoretic mobilities and the derived zeta potentials were obtained by analysing the solutions using a Malvern Zetasizer 2000. The results are shown in
  • the bacterial stains used in this study were Escherichia coli W3110, Bacillus cereus ATCC 11778, Enterococcus faecalis NCTC 12697, Pseudomonas aeruginosa NCTC 6750, Staphylococcus saprophyticus NCTC 7292, Proteus mirabilis NCTC 10374. Cultures were grown in nutrient broth at 37°C, with shaking, until the optical density at 600 nm was 0.3.
  • the bacterial stains used in this study were Escherichia coli W31 10, Bacillus cereus ATCC 1 1778, Enterococcus faecalis NCTC 12697, Pseudomonas aeruginosa NCTC 6750, Staphylococcus saprophyticus NCTC 7292, Proteus mirabilis NCTC 10374. Cultures were grown in nutrient broth at 37°C, with shaking, until the optical density at 600 nm was 0.3.

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  • Life Sciences & Earth Sciences (AREA)
  • Chemical & Material Sciences (AREA)
  • Health & Medical Sciences (AREA)
  • Organic Chemistry (AREA)
  • Molecular Biology (AREA)
  • Physics & Mathematics (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Analytical Chemistry (AREA)
  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
  • Wood Science & Technology (AREA)
  • Immunology (AREA)
  • Zoology (AREA)
  • Engineering & Computer Science (AREA)
  • Pathology (AREA)
  • Biophysics (AREA)
  • Toxicology (AREA)
  • Biotechnology (AREA)
  • Microbiology (AREA)
  • General Physics & Mathematics (AREA)
  • Electrochemistry (AREA)
  • Chemical Kinetics & Catalysis (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • Genetics & Genomics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)

Abstract

La présente invention concerne un procédé d'identification d'au moins un micro-organisme dans un échantillon fluide. Ce procédé comporte plusieurs opérations. (i) Mise en culture éventuelle de l'échantillon pour augmenter si nécessaire ne nombre de micro-organismes jusqu'à un niveau convenu. (ii) Application d'un champ électrique sur une partie du fluide considéré. (iii) Mesure de la vitesse, du déplacement, du potentiel zêta ou de la mobilité électrophorétique de tous les micro-organismes présents après application du champ électrique. (iv) Nouvelle mesure de la vitesse, du déplacement, du potentiel zêta ou de la mobilité électrophorétique de l'échantillon fluide après incubation en présence d'un peptide bioactif. (v) Comparaison des vitesses, déplacements, potentiels zêta ou mobilités électrophorétiques mesurés avec des tables de vitesses, déplacements, potentiels zêta ou mobilités électrophorétiques de micro-organismes connus dans des conditions expérimentales sensiblement identiques de façon à déterminer ceux des micro-organismes qui sont encore éventuellement présents.
PCT/GB2001/000658 2000-02-17 2001-02-16 Procede d'identification Ceased WO2001061029A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU2001232127A AU2001232127A1 (en) 2000-02-17 2001-02-16 Identification method

Applications Claiming Priority (6)

Application Number Priority Date Filing Date Title
GB0003554.3 2000-02-17
GB0003554A GB0003554D0 (en) 2000-02-17 2000-02-17 Identification method
GB0003795A GB0003795D0 (en) 2000-02-19 2000-02-19 Identification method
GB0003795.2 2000-02-19
GB0007771A GB2348504B (en) 1999-03-30 2000-03-30 Improved method for detecting micro-organisms
GB0007771.9 2000-03-30

Publications (2)

Publication Number Publication Date
WO2001061029A2 true WO2001061029A2 (fr) 2001-08-23
WO2001061029A3 WO2001061029A3 (fr) 2002-04-04

Family

ID=27255536

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/GB2001/000658 Ceased WO2001061029A2 (fr) 2000-02-17 2001-02-16 Procede d'identification

Country Status (3)

Country Link
AU (1) AU2001232127A1 (fr)
GB (1) GB2363842A (fr)
WO (1) WO2001061029A2 (fr)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012076431A1 (fr) * 2010-12-06 2012-06-14 Syngenta Limited Détecteur de pathogènes
CN106589135A (zh) * 2016-11-25 2017-04-26 东北农业大学 一种靶向抗菌肽及其制备方法和应用

Family Cites Families (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0720658A1 (fr) * 1993-09-23 1996-07-10 E.I. Du Pont De Nemours And Company Procede electrophoretique d'isolation et de separation de microorganismes
US5994067A (en) * 1995-11-14 1999-11-30 The United States Of America As Represented By The Secretary Of The Army Method and kit for rapid detection of toxins and bacteria
GB2333981B (en) * 1998-02-10 2000-05-03 Brookdale Tool Co Ltd A power tool attachment
EP1068527A2 (fr) * 1998-03-31 2001-01-17 Zetatronics Limited, University of Hertfordshire Procede rapide de detection de micro-organismes et d'evaluation de l'activite antimicrobienne

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2012076431A1 (fr) * 2010-12-06 2012-06-14 Syngenta Limited Détecteur de pathogènes
CN106589135A (zh) * 2016-11-25 2017-04-26 东北农业大学 一种靶向抗菌肽及其制备方法和应用

Also Published As

Publication number Publication date
AU2001232127A1 (en) 2001-08-27
GB2363842A (en) 2002-01-09
GB0103921D0 (en) 2001-04-04
WO2001061029A3 (fr) 2002-04-04

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