[go: up one dir, main page]

EP1292698A2 - Procede de detection de fongicides - Google Patents

Procede de detection de fongicides

Info

Publication number
EP1292698A2
EP1292698A2 EP01943436A EP01943436A EP1292698A2 EP 1292698 A2 EP1292698 A2 EP 1292698A2 EP 01943436 A EP01943436 A EP 01943436A EP 01943436 A EP01943436 A EP 01943436A EP 1292698 A2 EP1292698 A2 EP 1292698A2
Authority
EP
European Patent Office
Prior art keywords
fungicides
detection
plate
suspension
fluorescence
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.)
Withdrawn
Application number
EP01943436A
Other languages
German (de)
English (en)
Inventor
Rolf Eymann
Heinz-Emil Hauck
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.)
Merck Patent GmbH
Original Assignee
Merck Patent GmbH
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 Merck Patent GmbH filed Critical Merck Patent GmbH
Publication of EP1292698A2 publication Critical patent/EP1292698A2/fr
Withdrawn legal-status Critical Current

Links

Classifications

    • 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

Definitions

  • the invention relates to a bioautographic method for the detection of fungicides, wherein the growth of fungi directly on a thin-layer chromatography plate (DC plate) by fluorescence quenching or
  • Fungicides are often detected by means of thin layer chromatography. After separation, the fungicides can be detected on the basis of their chemical properties, such as staining behavior, running behavior etc. or by contacting them with fungi. The latter option is generally preferred because it allows fungicides to be recognized in complex systems because of their effects.
  • agar overlay method in which an agar plate which has been inoculated with fungi is placed on a developed chromatography plate.
  • the fungicides on the chromatography plate diffuse into the agar and prevent the growth and sporylation of the fungi there.
  • the disadvantage of the known methods is the large amount of time required. In these methods, detection takes place via the formation of colored spores (e.g. Aspergilius niger forms black spores) or in special cases via the formation of colored metabolites (Rhodoturula rubra develops a reddish color after the growth phase). In both cases, however, a mostly multi-day incubation is necessary to achieve a color effect.
  • colored spores e.g. Aspergilius niger forms black spores
  • redoturula rubra develops a reddish color after the growth phase
  • the object of the present invention was therefore to develop a method for the detection of fungicides which enables rapid and sensitive bioautographic detection.
  • the present invention therefore relates to a method for the bioautographic detection of fungicides, characterized by the following method steps: a) providing a dried developed DC plate; b) placing the mushroom suspension on the TLC plate; c) incubation of the TLC plate; d) Detection of zones of inhibition by means of fluorescence quenching and / or staining with an optical brightener.
  • the mushroom suspension in step b) contains a linear heteropolysaccharide composed of partially O-acetylated glucuronic acid, glucose and rhamnose units.
  • the TLC plate is incubated in step c) for 15 to 20 hours.
  • the mushroom suspension is pre-incubated on the TLC plate in step c) before the application.
  • the fluorescence quenching in step d) is detected at 254 nm.
  • the fluorescence of an optical brightener is detected in step d) at a wavelength between 360 and 390 nm.
  • the present invention also relates to a test kit for
  • the at least one Nutrient medium, a linear heteropolysaccharide as a gelling agent and a storage-stable test strain for the preparation of the mushroom suspension contains.
  • the test kit additionally contains at least one TLC plate with a fluorescence indicator.
  • test kit additionally contains an optical brightener.
  • the method according to the invention is suitable for the general detection of fungicides, regardless of their mode of action. By selecting the type of fungus, the growth of which is detected, the detection can be oriented towards certain fungicides. To find new fungicides against a specific fungal disease, the detection method according to the invention is therefore generally carried out with the corresponding pathogen or similar fungi.
  • fungicides in e.g. Food or feed are preferred mushrooms of risk group 1, i.e. fungi harmless to health, e.g. Aspergilius niger or preferably Rhodotorula rubra, Saccharomyces boulardii or Saccharomyces cerevisiae.
  • the sample to be examined for its fungicide content is separated by thin layer chromatography.
  • a thin-layer chromatographic separation on a TLC plate that is to say the development of the TLC plate, is carried out under known conditions.
  • a developed DC plate is therefore, according to the invention, a DC plate on which at least one sample has been applied and separated by thin layer chromatography.
  • DC plates with a sorbent layer based on SiO 2 are mostly used for this.
  • other plates for example, plates coated with cellulose, polyamide or aluminum oxide.
  • silica gel-coated plates which have a diol modification or are waterproof.
  • Such plates have the advantage that they can be wetted with the aqueous mushroom suspension without there being any risk of damage or cracking of the silica gel layer.
  • a fluorescence indicator for example HPTLC-FP KG 60, WRF 254s from Merck, Darmstadt.
  • Suitable flow agents for example acetonitrile or mixtures of acetonitrile and dichloromethane and a non-polar component such as n-hexane, are known to the person skilled in the art. The person skilled in the art is also able to adapt the flow agents to the particular separation problem.
  • the TLC plate is carefully dried to remove residues of the eluent.
  • the developed DC plate is then incubated with a mushroom suspension. This can e.g. by briefly immersing the DC plate in the mushroom suspension or by spraying the mushroom suspension. In any case, care should be taken to ensure that the mushroom suspension is applied evenly.
  • the mushroom suspension can only be a certain time before
  • a nutrient medium with a form of a test strain.
  • a form of a test strain This can be a living culture of the microorganism or a storable form of the test strain, ie for example a spore preparation or preferably a lyophilisate or granulate.
  • This mixture is preferably pre-incubated for a certain period of time, usually between 2 and 5 hours. All can be used as nutrient medium for the purpose of Fungus culture known culture media are used. The use of Sabouraud broth is preferred.
  • a gelling agent is also added to the nutrient medium or the mushroom suspension.
  • These are preferably gelling linear heteropolysaccharides, which are typically of bacterial origin. Linear heteropolysaccharides comprising of O-acetylated glucuronic acid, glucose and rhamnose as Gelrite ® from Roth, Germany are particularly preferred. Such materials form stable gels in the presence of soluble salts. It has been found that this particular type of
  • a gelling agent made of linear heteropolysaccharides from partially O-acetylated glucuronic acid-glucose and rhamnose units can be processed in contrast to agar at room temperature.
  • this gelling agent can be boiled up for sterilization and sterile filtered, so that expensive autoclaving is avoided. This is not possible when using agar as a gelling agent.
  • the gelling agent composed of linear heteropolysaccharides composed of partially O-acetylated glucuronic acid-glucose and rhamnose units are added to 200 ml of nutrient medium.
  • the Do not apply the Piiz suspension according to the application as it is too stiff or does not become sufficiently firm.
  • Fungus suspensions for the method according to the invention typically contain the test strain fungus in a number of microorganisms between 10 5 and 10 8 / ml.
  • the TLC plate After compilation and typically pre-incubation of the mixture of nutrient medium, gelling agent and test strain (e.g. in the form of a spore suspension), called mushroom suspension according to the invention, the TLC plate is wetted with it.
  • the DC plate is typically incubated at 23-30 ° C. in a moist environment.
  • the duration of the incubation is typically 13 to 30 hours, preferably 15 to 24 hours. In most cases it is
  • the zones of inhibition can be detected by means of fluorescence quenching or by staining with an optical brightener on the dried TLC plate. The latter method is somewhat more sensitive.
  • DC plates with a fluorescence indicator are used for detection by means of fluorescence quenching.
  • the DC plates are irradiated at the appropriate wavelength to excite the fluorescence.
  • magnesium tungstate in the product name in general indicated by the addition F254s, is irradiated with light of the wavelength 254 nm. It was found that with this procedure the zones of inhibition are visible as glowing spots on the dark background. With this simple principle, the detection can still be carried out during the growth phase of the fungi, ie before the formation of spores or the formation of an intrinsic color. While the resulting mushroom lawn leads to complete or partial quenching of the fluorescence, the autofluorescence of the TLC plate remains clearly visible in the zones of inhibition.
  • this method is also suitable for examining the plate several times during the incubation after different incubation times and for monitoring the formation of the inhibition zones.
  • the method of detection by fluorescence quenching can only be used to a limited extent.
  • the DC plate When using optical brighteners, the DC plate is sprayed with the staining solution, which contains an optical brightener, after incubation.
  • This staining solution typically contains 0.01 to 0.1%
  • optical brightener in a basic aqueous solution, for example 0.1 M NaOH solution or 15% potassium hydroxide solution.
  • optical brighteners such as stilbene derivatives, for example disodium 4,4'-bis ⁇ (4-anilino-6-morpholino-1, 3,5-triazin-2-yl) amino ⁇ stilbene-2, are preferred as optical brighteners , 2'-disulfonate (C 40 H 38 N ⁇ 2 O 8 S 2.
  • Blankophor ® Blankophor ® (Bayer AG), Calcofluor ® White M2R (Sigma), Tinopal ® (Sigma) or Leucophor ® (Clariant, Canada) are used.
  • the optical brightener Blankophor.RTM ® BA is particularly preferably 267% of Bayer AG, Germany, are used. After a development time of 5 minutes at room temperature, the TLC plate is irradiated.
  • the excitation wavelength depends on the optical brightener used. Wavelengths between 360 and 390 nm are often suitable. An excitation wavelength of 366 nm is well suited for Blankophor ® BA 267%.
  • optical brighteners show active fluorescence when irradiated in the presence of fungi, while there is little or no fluorescence when the optical brighteners interact with the fungicides or the DC plate in the area of the zones of inhibition.
  • the zones of inhibition therefore appear when irradiated as dark spots on a luminous background.
  • the incubated DC plate is first examined by means of fluorescence quenching and then treated with an optical brightener at a suitable time to further improve the detection sensitivity and examined for its fluorescence.
  • the detection method according to the invention enables the detection of fungicides to be carried out quickly, sensitively and with little effort.
  • linear heteropolysaccharide which is preferred according to the invention and consists of partially O-acetylated glucuronic acid-glucose and rhamnose units as gelling agents makes handling easier, since a filtering step can be carried out instead of autoclaving.
  • the fluidity of the mushroom suspension can easily be varied or adjusted. In this way, the interaction of the mushroom suspension with the TLC plate can be optimized.
  • the detection can be carried out at an earlier point in time by means of the method according to the invention, since already in the growth phase of the fungi by means of fluorescence quenching or by using optical brighteners the inhibitory areas can be represented with good contrast.
  • the formation of the inhibitory zones can already be tracked during the incubation and the incubation can be ended at a suitable time.
  • an additional optical brightener can be used for staining to increase detection sensitivity. Both staining methods enable the direct detection of fungal growth on TLC plates for the first time. As with other methods, there is no need to wait until the spores formed can be detected.
  • fungicide content of the sample can then be calculated from the size of the inhibition zones.
  • test kit is further provided according to the invention which can be used very easily in appropriately equipped laboratories.
  • the test kit preferably contains nutrient medium with the gelling agent preferred according to the invention, preferably in solid form for dissolving in water, a suitable test strain, usually either as lyophilisate, granules or spore preparation and preferably an optical brightener, typically in solid form or as a coloring reagent, and optional further constituents such as suitable DC plates, reference solutions, etc., which are required to carry out the method according to the invention.
  • a suitable test strain usually either as lyophilisate, granules or spore preparation and preferably an optical brightener, typically in solid form or as a coloring reagent, and optional further constituents such as suitable DC plates, reference solutions, etc., which are required to carry out the method according to the invention.
  • the test kit preferably contains a description in which the steps of the method according to the invention are explained.
  • TLC material HPTLC-FP KG 60, Diol F254s, Merck KGaA fungicides: - TBT 50 nl application (F1)
  • Test organism Rhodotorula rubra Chromatography: separation zone 50 mm, solvent acetonitrile, running time 6 min
  • test organism 200 ml of fully demineralized water, 10 g Sabouraud-2% glucose broth and 0.11 g Geirrte ® are boiled sterile. Then the test organism is called Lyophilisate or live culture added and preincubated at 35 ° C for 3 hours.
  • the developed DC plate is briefly immersed in the mushroom suspension and excess suspension is removed from the bottom of the plate.
  • the TLC plate is then incubated in a can, which is lined with moist paper, for 20 hours at room temperature.
  • the evaluation is carried out by quenching fluorescence at 254 nm.
  • the Hemmhof on the start line is a TBT degradation product in water.
  • TLC material HPTLC-FP KG 60, WRF254s, Merck KGaA
  • Fungicides - Fenpropimorph (A1)
  • Test organism Rhodotorula rubra Chromatography: separation zone 50 mm, solvent acetonitrile /
  • the developed DC plate is briefly immersed in the mushroom suspension and excess suspension is removed from the bottom of the plate.
  • the DC plate is then incubated in a can, which is lined with moist paper, for 20 hours at room temperature.
  • the evaluation is carried out by staining with an optical brightener (0.04%
  • the TLC plate is dried, wetted with the staining solution and then stored for 5 minutes at room temperature.
  • Evaluation takes place at 366 nm.
  • the zones of inhibition can be recognized as dark spots against a fluorescent background.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • Physics & Mathematics (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Analytical Chemistry (AREA)
  • Immunology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Agricultural Chemicals And Associated Chemicals (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

L'invention concerne un procédé de détection bioautographique de fongicides, selon lequel on fait incuber une suspension de champignons sur une plaquette de chromatographie sur film mince développée, et l'on détecte les zones d'inhibition se créant, par extinction de fluorescence ou à l'aide d'un azurant optique.
EP01943436A 2000-06-21 2001-05-23 Procede de detection de fongicides Withdrawn EP1292698A2 (fr)

Applications Claiming Priority (3)

Application Number Priority Date Filing Date Title
DE10029512 2000-06-21
DE10029512 2000-06-21
PCT/EP2001/005915 WO2001098529A2 (fr) 2000-06-21 2001-05-23 Procede de detection de fongicides

Publications (1)

Publication Number Publication Date
EP1292698A2 true EP1292698A2 (fr) 2003-03-19

Family

ID=7645836

Family Applications (1)

Application Number Title Priority Date Filing Date
EP01943436A Withdrawn EP1292698A2 (fr) 2000-06-21 2001-05-23 Procede de detection de fongicides

Country Status (6)

Country Link
US (1) US20030129684A1 (fr)
EP (1) EP1292698A2 (fr)
JP (1) JP2004500893A (fr)
AU (1) AU2001266015A1 (fr)
DE (1) DE10123987A1 (fr)
WO (1) WO2001098529A2 (fr)

Families Citing this family (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP2919004A1 (fr) * 2014-03-14 2015-09-16 Merck Patent GmbH Essais biologiques sur des plaques TLC/HPTLC modifiées
RU2568410C1 (ru) * 2014-10-08 2015-11-20 Федеральное государственное бюджетное научное учреждение "Северо-Кавказский зональный научно-исследовательский институт садоводства и виноградарства" Способ количественного определения фунгицида артафит 10%, врк в растительном материале

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5882882A (en) * 1995-04-12 1999-03-16 Biolog, Inc. Gel matrix with redox purple for testing and characterizing microorganisms

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See references of WO0198529A3 *

Also Published As

Publication number Publication date
US20030129684A1 (en) 2003-07-10
DE10123987A1 (de) 2002-01-03
WO2001098529A2 (fr) 2001-12-27
WO2001098529A3 (fr) 2002-04-18
JP2004500893A (ja) 2004-01-15
AU2001266015A1 (en) 2002-01-02

Similar Documents

Publication Publication Date Title
DE69330818T2 (de) Fluoreszenzfarbstoff, Pyryliumsalze oder ähnhliche Salze enthaltend, Nachweisverfahren von Nukleinsäure durch Verwendung desselben
DE102009033368B4 (de) Massenspektrometrische Sepsisdiagnose
DE69624593T2 (de) Verfahren zum nachweis von bakterien
DE102007058516B4 (de) Identifizierung von Erregern in Körperflüssigkeiten
DE69531007T2 (de) Medium zum nachweis von enterococci in einer probe
DE69506394T2 (de) Verfahren zum kalibrieren chemischer tests
DE102014000646B4 (de) Massenspektrometrische Resistenzbestimmung durch Metabolismus-Messung
WO1993024652A1 (fr) Procede de preparation et d'hybridation de sondes specifiques
EP2801825B1 (fr) Détermination par spectrométrie de masse des résistances de microbes
DE19814715A1 (de) Gelatinemembranfilter, Verfahren zu ihrer Herstellung und ihre Verwendung
DE1767748A1 (de) Mittel zur Bestimmung der weiblichen Fruchtbarkeitsperiode
WO2015024576A1 (fr) Iode à liaison polymère utilisée comme agent d'extinction pour des mesures de fluorescence
DE102020105123B3 (de) Verfahren zum spektrometrischen Charakterisieren von Mikroorganismen
DE2728077A1 (de) Verfahren zum faerben von mikroorganismen
DE19602345A1 (de) Verfahren zur Verbesserung des Wachstums und des kolorimetrischen Nachweises von Bakterien, Hefen, Pilzen oder Kokken
EP1292698A2 (fr) Procede de detection de fongicides
DE2953720C2 (de) Verfahren zum Nachweisen von Bakterien in flüssigen Proben
DE102004046024A1 (de) Antibiotikum und Verfahren zur Herstellung
WO2006005727A1 (fr) Photostabilisation de colorants fluorescents
DE202024102518U1 (de) Ein Extraktionssystem zur Extraktion von Bikaverin aus Fusarium oxysporum
DE19933078A1 (de) Verfahren und Kit zur Bestimmung von DNA-Doppel-/Einzelstrangbrüchen und Vorrichtung zum kontrollierten Filtrieren von insbesondere DNA-Moleküle und/oder andere Moleküle/Molekülkomplexe enthaltenden flüssigen Medien mit Well-Filterplatten
DE2462631C2 (de) Superoxiddismutase, Verfahren zu deren Herstellung und deren Verwendung
DE2738041A1 (de) Antimikrobale wirkstoffe und verfahren zu ihrer herstellung
EP0115547B1 (fr) Procédé pour la détermination quantitative de la mutagénicité des agents chimiques utilisant des phages dans un test in vitro
DE1642501C (de) Neue biocide Zusammensetzungen

Legal Events

Date Code Title Description
PUAI Public reference made under article 153(3) epc to a published international application that has entered the european phase

Free format text: ORIGINAL CODE: 0009012

17P Request for examination filed

Effective date: 20021023

AK Designated contracting states

Kind code of ref document: A2

Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LI LU MC NL PT SE TR

Designated state(s): AT BE CH CY DE DK ES FI FR GB GR IE IT LI LU MC NL PT SE TR

AX Request for extension of the european patent

Extension state: AL LT LV MK RO SI

STAA Information on the status of an ep patent application or granted ep patent

Free format text: STATUS: THE APPLICATION IS DEEMED TO BE WITHDRAWN

18D Application deemed to be withdrawn

Effective date: 20041130