WO1992016648A1 - Test chimioluminescent pour microorganismes - Google Patents

Test chimioluminescent pour microorganismes Download PDF

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Publication number
WO1992016648A1
WO1992016648A1 PCT/US1992/001869 US9201869W WO9216648A1 WO 1992016648 A1 WO1992016648 A1 WO 1992016648A1 US 9201869 W US9201869 W US 9201869W WO 9216648 A1 WO9216648 A1 WO 9216648A1
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Prior art keywords
chemiluminescent
microorganisms
interest
composition
light
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PCT/US1992/001869
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English (en)
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Robert Joseph Carrico
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Environmental Test Systems, Inc.
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Publication of WO1992016648A1 publication Critical patent/WO1992016648A1/fr

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    • 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
    • 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
    • C12Q1/18—Testing for antimicrobial activity of a material

Definitions

  • the field of the invention is test compositions and methods for the detection, quantitation and
  • ATP triphosphate
  • the extract is added to the
  • luciferin-luciferase reagents and if ATP is present, a burst of light is emitted within a few seconds.
  • This method measures the ATP present in cells at the time of extraction and does not take advantage of the amplifying power of the enzymes that produce ATP.
  • An additional disadvantage is that luciferase is expensive, unstable and is inhibited by a wide variety of compounds,
  • the second bioluminescence system measures NAD and NADP extracted from the cells in the sample or specimen. Following extraction for these cofactors, a dehydrogenase and its substrate, such as alcohol dehydrogenase and ethanol, are added. This reaction mixture is then added to riboflavin reductase, an aldehyde and luciferase from the marine bacterium Phntohar-t-aritrm fischeri. if the reduced cofactors are present, a burst of light is emitted within a few seconds. Again, this system does not take advantage of the amplifying power of cellular dehydrogenases. In addition it requires extra reagents and an incubation step for the extracellular reduction of the cofactors.
  • Another method for testing for bacteria reacts the specimen with a peroxide, such as sodium perborate or sodium pyrophosphate peroxide, and luminol in 0.04 to 0.12 M sodium hydroxide.
  • a peroxide such as sodium perborate or sodium pyrophosphate peroxide
  • luminol in 0.04 to 0.12 M sodium hydroxide.
  • Hematin from the cells catalyzes the oxidation of luminol by peroxide. Light emission occurs as a burst that lasts about 30 seconds. In principle the hematin catalyzes the
  • this assay is ineffective for detection of bacteria, including most anaerobic species, that do not produce hemin.
  • the specificity of the assay is
  • PMNL Polymorphonuclear leukocytes stimulated by specific substances such as opsonified zymosan emit substantial levels of light due to the production of superoxide ion by a unique NADPH oxidase present in these leukocytes.
  • the light emission can be increased 1000 fold by incubating the cells with the probes lucigenin or luminol.
  • Allen uses PMNL luminescence response to opsonified zymogen as an example to illustrate the mechanism of chemiluminescence.
  • Figure 5 he shows data related to this response as followed by use of a barbitol buffer containing calcium and magnesium ions, 0.1 g/dl glucose and albumin at pH 7.2 and zymogen opsonified with fresh autologous serum.
  • Light emission from leukocytes with or without the use of the chemiluminescent probes is specific for activation of the cells by extraneous agents such as opsonified zymosan and has not been used to detect the presence of cells in an unknown sample.
  • chemiluminescence The chemiluminescence produced withE. coli cells and lucigenin in air was less than in 100% oxygen and heat killed cells showed no increase in chemiluminescence on addition of either oxygen, paraquat or menadione.
  • Experiments were carried out on cells grown aerobically in LB medium supplemented with 0.5% glycerol, and with LB medium supplemented with 0.3% xylose.
  • Harvested cells were suspended in glycerol taxis buffer (GBT) which contained 100 ⁇ M EDTA, 10 mM potassium phosphate buffer, 1 mM magnesium sulfate, 1 mM ammonium sulfate and 10 mM glycerol (final pH 7.0).
  • GBT glycerol taxis buffer
  • lucigenin to provide a final lucigenin concentration of 40 ⁇ M.
  • Samples were placed in a luminometer and the chemiluminescent response to the addition of oxygen or air was measured. Peters et al. concluded that lucigenin may be used as a chemiluminescent probe to demonstrate continuous intracellular production of reactive oxygen metabolites in E. coli.
  • Nakano et al. discloses a method for determining superoxide dismutase in various tissues and blood cells with
  • cypridina luciferin analogs 2-methyl-6-(p- methoxyphenyl)-3,7-dihydroimidazo[1,2- ⁇ ]pyrazin-3-one and 2-methyl-6-phenyl-3,7-dihydroimidazo-[1,2- ⁇ ]pipazine-3- one (abbreviated MCLA and CLA respectively).
  • the invention provides a chemiluminescent reagent composition useful for the detection or characterization of viable microorganisms, comprising a substrate capable of metabolism by viable microorganisms of interest to produce reducing equivalents; and a chemiluminescent probe.
  • the composition may also contain a redox active agent.
  • Preferred compositions include lucigenin and the substrate; and luminol, a redox active agent and a substrate.
  • the invention also provides a method of detecting or characterizing viable microorganisms, comprising the steps of: contacting a sample suspected of contamination with microorganisms of interest with a chemiluminescent reagent composition comprising a substrate capable of being metabolized by the microorganism of interest and a chemiluminescent probe; and measuring the light.
  • the method may also be used for characterizing viable microorganisms
  • microorganisms by utilizing a substrate characteristic for the metabolism of the microorganisms of interest.
  • the method may include pretreatment of the sample to provide a concentrate containing the microorganisms of interest in which case the concentrate is contacted with the reagent composition. Pretreatment may be
  • the method produces light which appears quickly and can be measured at very low levels with commercial
  • the method provides a useful screening test for low levels of microorganisms.
  • a screening test is especially useful when a high
  • Chemiluminescent reagent compositions and a method for the detection and quantification of microorganisms are provided.
  • the method may be used to detect or characterize microorganisms including bacteria, fungi or yeast depending on the substrate used.
  • the method diverts electron flow generated by cellular metabolic processes into chemiluminescent reactions.
  • the light generated may be detected by placing the sample in a luminometer and measuring light emission.
  • the assay uses the chemical reducing activity of cell metabolism to generate chemiluminescence. Oxidationreduction reactions occur in all living cells.
  • the assay may be applied to the detection of a wide variety of microorganisms. These include prokaryotic and eukaryotic microorganisms that grow under aerobic and/or anaerobic conditions. Examples are bacteria, fungi, algae, plankton and protozoa. Organisms
  • composition to increase the light produced is a composition to increase the light produced.
  • the chemiluminescent method of the invention diverts electron flow generated by cellular metabolism into chemiluminescent reactions. Reducing equivalents occur in the form of reduced cofactors including reduced NAD, reduced nicotinamide adenine dinucleotide phosphate
  • NADP reduced flavins
  • ubiquinones reduced menaquinone
  • pyrroloquinoline quinone reduced cytochromes
  • the assay conditions should favor the activity of enzymes that reduce such cofactors because reducing equivalents are diverted from the
  • exogeneous cofactors can be included with the assay reagents to increase light production.
  • the chemiluminescent reagent compositions of this invention are generally composed of a substrate capable of metabolism by the viable microorganisms of interest to produce reducing equivalents and a chemiluminescent probe.
  • a redox active agent may also be included.
  • Other components, such as buffers and enzyme cofactors, may be included depending on the conditions optimal for metabolism in the microorganism of interest. However, it is not necessary to use reagents such as opsonified zymogen, perborate or peroxide. Substrates
  • a variety of substrates can be catabolized by
  • microorganisms and those that yield reducing equivalents are potentially useful for the present assay.
  • the substrate is chosen depending on the metabolism of the microorganism of interest and will depend on the purpose of the assay. For example a screening test designed to detect a wide variety of microorganisms should employ a substrate such as glucose which can be metabolized by many species of microorganisms. In some situations it can be advantageous to use several substrates in a cocktail in order to broaden the range of detectable
  • glucose, citrate or mixtures thereof are suitable substrates to detect the presence or absence of bacteria.
  • Some other useful substrates are glycerol, succinate, acetate, lactate, pyruvate and galactose. Others may be
  • ethanol is a suitable substrate for yeast and its use can differentiate between the presence of a bacterial contaminant such as E. coli and yeast since E. coli does not metabolize ethanol.
  • Bacteria and fungi are used to degrade waste chemicals in waste water plants.
  • the efficiency of such plants is controlled by monitoring the level of microorganisms that oxidize specific compounds such as phenols. Therefore, such monitoring can be accomplished by means of the composition and method of this invention when phenols are used as the substrate.
  • Chemilumigenic compounds of principal interest as probes in the present invention are the same
  • aminophthalhydrazides as exemplified by luminol and dimethylbiacridylium salts such as lucigenin. Other chemiluminescent probes such as MCLA or CLA may also be used as equivalents to lucigenin. At the neutral to mildly alkaline pH used for the detection of viable microorganisms, aminophthalhydrazides undergo
  • dioxygenation by adding the elements of hydrogen peroxide to produce light, nitrogen and aminophthalates.
  • dioxygenation which forms an unstable dioxatane.
  • the dioxatane decomposes spontaneously to produce light and N-methylacridone.
  • Dimethylbiacridylium salts and aminophthalhydrazides are activated in chemiluminescent reactions by different molecular mechanisms.
  • the optimum conditions for the assay will be somewhat different when each class of compounds is used as a probe. Because it is not unusual for the assay reagents to emit some background light in the absence of cells, reaction conditions are typically optimized to obtain the highest signal/background ratio. If the level of background light is acceptable, the chemiluminescent compounds are used in excess so the maximum rate of light emission is achieved.
  • Luminol is a preferred probe. Derivatives and analogs such as isoluminol are also useful. In
  • redox active agents are not essential for chemiluminescence.
  • redox active agents such as menadione, 1-methoxyphenazine
  • methosulfate, plumbagin or phenothiazines such as Azure C and methylene blue may be used to increase
  • Azure C is a preferred redox active agent for use with lucigenin in concentrations of about 0.4 to 2.0 mM lucigenin.
  • concentrations of redox active agents that give maximum light yields depends on the agent used; however, the optimum concentrations have been found to occur in the range of 5 to 100 uM.
  • Lucigenin is reduced by cellular enzymes [Greenlee, L., et al., Biochem. 1:779-783 (1962)] and in this form can react with the low levels of superoxide ion normally produced in cells.
  • the dioxatane produced by this sequence of reactions decomposes to emit light as
  • Lucigenin concentration will usually be chosen to maximize light emission in the presence of microorganisms and minimize background light in the absence of cells. The optimal concentration for a particular situation can be determined experimentally by one skilled in the art. Light emission with lucigenin can be increased by
  • Reagent including a redox active agent that will react in its reduced form to produce superoxide ion.
  • concentrations in such an assay should be co-optimized to balance lucigenin and redox active agent concentrations versus superoxide ion production to achieve maximum light yield.
  • Experimental co-optimization of reagents is preferred because reagents become partitioned between intra- and extracellular spaces and interact at various rates with different cell enzymes.
  • redox active agents are defined as compounds which can receive electrons from the electron transport systems of the microorganisms to be detected and can transfer the
  • Redox active agents will be included in the reaction mixture at levels that maximize light emission.
  • concentration of colored redox agents might have to be limited to avoid quenching of emitted light.
  • concentrations of redox active agents will be optimized experimentally to maximize chemiluminescence. Reactions between reduced redox active agents and oxygen are reversible and equilibrium conditions will depend on the reduction potentials of the reactants in addition to factors such as pH [Afamas'ev, I.B., et al.. Arch.
  • the reagent composition may also contain other components such as buffers to control pH.
  • buffers to control pH.
  • a buffer capable of providing a pH between about pH 6 and about pH 10 is preferably included in the composition.
  • the buffer salt and pH should be optimized to minimize reagent background light while maximizing light generated
  • the physiological oxidant for aerobic microorganisms is oxygen and it cannot be removed conveniently from
  • cytochrome oxidase at the termini of electron transport chains.
  • Inhibitors can be used to intercept electron flow to oxygen.
  • electron transport to oxygen can be slowed by depriving the cells in the assay medium of factors, such as magnesium ion and phosphate, which are required for oxidative phosphorylation. Since oxidative phosphorylation and reduction of oxygen are coupled, deprivation of essential factors will slow electron flow to oxygen and cause accumulation of a larger pool of reducing equivalents for diversion to chemiluminescent reactions.
  • factors such as magnesium ion and phosphate
  • Samples may be taken from any medium of interest. Commonly such samples will be from environmental, industrial, food, veterinary, or medical sources.
  • Specimens from human and animal sources include urine, whole blood, plasma, serum, amniotic fluid, cerebrospinal fluid, sputum, fecal matter, throat swabs and genital swabs.
  • sample processing procedure should be adjusted for each type of sample. In general, however, the processing should not be deleterious to the metabolic functions of microbial cells. If the sample is liquid and the suspected level of microorganism contamination is below the assay sensitivity, it may be useful to
  • the cell pellet or filtered cells may then be combined with the chemiluminescent reagents for assay. Centrifugation or filtration may also be useful for separating sample components which could interfere with the
  • chemiluminescent reactions For example, a sample may contain colored matter that absorbs chemiluminescence.
  • the chemiluminescent probes and redox active agents compete with physiological oxidants for electrons passing along electron transport chains. Removal of physiological oxidants provides a larger pool of
  • the physiological oxidants can be substances like nitrate and sulfate.
  • Sample processing methods such as centrifugation and filtration can be used to remove such oxidants before the sample is combined with assay reagents. Adding the reagent composit.ion
  • concentration of all the components of the chemiluminescent test composition will be chosen to maximize light emission from the cells of interest and to minimize background light.
  • concentrations will be chosen to maximize light emission from the cells of interest and to minimize background light.
  • the reagent compositions of this invention will usually be combined with the sample a short time before the light emission is measured. Control of the reaction temperature will often be beneficial to optimize light output and reproducibility. The temperature will
  • thermophilic microorganisms typically be between about 10 and 40° C, but higher temperatures may be employed especially for the detection of thermophilic microorganisms.
  • containers may be positioned closer to a photodetector for efficient light detection. Furthermore, reagent concentrations will be more critical to assay performance than the quantity of reagents per assay. If the reagents emit background light in the absence of cells, the use of small reaction volumes will minimize this background signal.
  • the reaction mixture will contain one or more substrates.
  • the substrates will be chosen to accommodate the types of microorganisms to be detected. Substrates will commonly be added in excess. If the substrates contribute to background light, the concentration used might need to be limited to achieve the best signal to background ratio. Substrate choice and the concentration of use thereof will be well within the skill of those versed in the art of chemiluminescence measurement given the guidance in the examples.
  • the concentration of the organic compound in some situations, the concentration of the organic compound
  • chemiluminescent probe could be limited by the solubility of the compound. In virtually all situations, however, only a small portion of the probe in the reaction mixture will be consumed during the reaction and therefore the solubility limitation is usually not a problem.
  • the assay should be conducted under
  • the assay may be conducted with a cell suspension in a solution of the assay reagents.
  • a test tube containing the reaction mixture would be positioned near a
  • the swab can be saturated with a solution of assay reagents and positioned near the light detection system. If cells are collected on a porous membrane or fiberous filter, the membrane or filter may be wetted with a reagent solution and mounted near the light detection system.
  • Light emission can be measured electronically with a luminometer using photon counting if high assay
  • the light may also be measured by exposing photographic film to reaction mixtures.
  • Clouding of developed film will indicate the level of light emitted.
  • the light intensity may be compared to a calibration scale to estimate the number of
  • the light intensity can be compared to a predetermined standard to provide a yes/no answer.
  • Chemiluminescence from these assays persists for many minutes, sometimes for hours. The duration of light measurements may be adjusted to meet performance
  • luminescence can be measured for much longer periods, up to a few hours. A longer measuring period increases assay sensitivity if background light is low or decreases with time.
  • the assay may be used for the detection of both aerobic and anaerobic microorganisms and may be used for the characterization of microorganisms. For instance, if a microorganism has a distinguishing ability to
  • the electron flow can be diverted into chemiluminescent reactions and measurement of
  • the present assay may be used to evaluate the ability of a microorganism to grow and multiply in a medium containing a characteristic metabolite.
  • a selective medium containing the metabolite is inoculated with the sample and the cell population is monitored periodically by assaying aliquots of the culture medium by the chemiluminescent method.
  • the assay may also be applied to medical
  • urinary tract infections are diagnosed by the presence of a characteristic number of microorganisms per milliliter of urine. Screening tests are used to identify infected specimens.
  • the assay method may also be used in agriculture and food
  • the assay may also be used to determine antibiotic sensitivity of infective microorganisms.
  • a sample for example from a throat swab, would be contacted with the chemiluminescent reagent composition and light measured.
  • a second sample would be contacted with the
  • chemiluminescent reagent composition which additionally includes an antibiotic, and light measured.
  • Environmental applications may include testing of river, lake and well waters for microbial content.
  • the chemiluminescent method of the present invention could also be used to assay for environmental contaminants such as heavy metals.
  • a microorganism whose metabolism is inhibited by the contaminant would be combined with the environmental sample along with the chemiluminescense reagents. The light emission from this mixture would be compared to light emitted by a control without the
  • the assay could also be used to determine if a toxicant is present in a sample.
  • the sample suspected of containing a toxicant would be contacted with a
  • microorganism capable of being affected by the toxicant, or a cocktail of microorganisms, and chemiluminiscent reagent composition added.
  • the light produced is then measured and compared to a control or standard curve to determine any differential in the light produced.
  • the invention has several advantages over the previous chemiluminescence and bioluminescence assays .
  • the assay reagents are relatively inexpensive.
  • the reagents are low molecular weight and
  • the cells in the sample do not have to be disrupted for the assay.
  • the enzymes involved in electron transport provide a steady flow of electrons to the chemiluminescent reactions thus providing
  • the steady-state light emission allows the assay method to be carried out by conventional pipetting techniques and so forth.
  • bioluminescence assays require rapid mixing of assay reagents with sample because the light burst occurs a few seconds later. Such rapid mixing can only be achieved with special equipment that adds to assay cost.
  • E. co l i (ATCC 23716 )
  • Bac i l lius sub ti l i s (ATCC 29056 )
  • Staphyl oc occus aureus (ATCC 25923 )
  • Sa cchrom yces ce revi s i ae (ATCC 834 )
  • a bacterium tentatively identified as E. coli was isolated from a farm yard sample.
  • Yeast were grown at 25° C in a liquid medium
  • the cell suspension was omitted from reaction mixtures and background light was measured for 2 minutes. Then the cell suspension was added and light emission was measured for another 2 minutes. In cases where background light emission changed with time, background light was measured with one solution and a fresh mixture was prepared to measure light produced with cells. Light production is expressed in arbitrary units .
  • Buffers used were 50 mM sodium phosphate, pH 6.0 and 7.0, 50 mM Tris-hydrochloride, pH 8.0 and 9.0, and 50 mM sodium carbonate, pH 10.0.
  • Other solutions were 1 M glucose, 100 mM paraquat, 100 mM juglone in acetone, 100mM plumbagin in acetone, 100 mM sodium citrate, pH 8.9, 100 mM sodium azide, 10 mM magnesium chloride.
  • lucigenin 100 mM glucose, 10 8 bacteria cells (the environmental isolate) and various buffers at pH 5 to pH 10. Significant light was produced at pH 6.0 and the intensity increased as the pH was increased. Maximum light was emitted with 50 mM Tris-hydrochloride buffer, pH 9.0. At the optimum pH, the light emitted for a 2 minute period was 19.2 units and the reagent background was 0.13 unit.
  • Magnesium chloride was included at 0.2 mM in a reaction mixture and the light production was decreased by 67%. At 0.5 mM magnesium chloride, the light was reduced by only 57%.
  • reaction mixture prepared with 50 mM sodium
  • phosphate buffer, pH 7.0 had a reagent background light production of 0.007 unit and light emission increased to 4.74 units with 107 cells.
  • Sodium azide included at 7.5 mM in a reaction mixture decreased the light production by 82%.
  • E . coli (ATCC 23716) was measured with 50 mM Tris-hydrochloride, pH 8.9, containing 0.5 mM magnesium chloride. Reaction mixtures containing 0.5 mM lucigenin, 100 mM glucose and the following levels of cells gave the indicated light production: Cells/Assay Light Emission
  • reagent background light with 0.5 mM plumbagin was 0.33 unit and 10 8 cells emitted 0.86 unit.
  • Example 2 Chemiluminescence with Baci llus subtillus Measurements with lucigenin
  • the reaction buffer was 50 mM Tris-hydrochloride buffer, pH 9.0, containing 0.5 mM magnesium chloride.
  • a reaction mixture containing 0.5 mM lucigenin and 100 mM glucose gave reagent background light of 0.11 unit. Then 6.7 ⁇ 10 7 cells were added and the light emission was
  • reaction mixtures containing 0.5 mM lucigenin, 100 mM glucose and 4.4 ⁇ 10 7 cells were prepared with various buffers from pH 6.0 to pH 10.0. The most intense light emission occurred at pH 10. The reagent background light was 6.0 units. With 4.4 ⁇ 10 7 cells per milliliter at pH 10, the following levels of light were measured for successive two minute integration periods:
  • Sodium citrate was also found to be an effective substrate for obtaining light emission with lucigenin.
  • 100 mM sodium citrate, pH 8.9 was added in place of glucose, to the reaction mixtures described above.
  • Two minute integrated light emission for this mixture as well as the emission from endogeneous substrate (no added substrate) were as follows:
  • This work employed 1.0 mL reaction mixtures made up with 100 ⁇ L of 10 mM luminol (in 50 mM sodium carbonate buffer, pH 10.0), 100 ⁇ L 1 M glucose, 5 ⁇ L of a redox active agent, 745 ⁇ L of 50 mM sodium carbonate buffer, pH 10.0 and 100 ⁇ L of cell suspension containing 4.4 ⁇ 10 8 cells per milliliter.
  • a mixture containing 5 ⁇ L of 100 mM plumbagin but no cells produced reagent background light of 0.93 units. Addition of cells increased the light production to 3.94 units. Plumbagin and luminol were both found to be essential for light production with this reaction mixture.
  • a reaction mixture containing 5 ⁇ L of 100 mM juglone produced reagent background light of 0.65 units. When cells were included, the light yield increased to 1.74 units.
  • Reaction mixtures were prepared with 100 ⁇ L of 5 mM lucigenin, 100 ⁇ L 1 M glucose, 100 ⁇ L of the cell
  • reaction mixture was prepared with 50 ⁇ L ethanol in place of the glucose. The following light emission was measured for successive two minute integration periods. The light emitted due to endogeneous substrates is also presented for comparison.
  • Reaction mixtures were prepared with 100 ⁇ L 10 mM luminol (in 50 mM sodium carbonate buffer, pH 10), 100 ⁇ L 1 M glucose, 10 ⁇ L 100 mM paraquat, 100 ⁇ L of cell suspension and 690 ⁇ L 50 mM sodium carbonate, pH 10.0. Light emission from this reaction and one without the cell suspension (reagent control) was integrated during successive two minute intervals:
  • juglone Five microliters of 100 mM juglone was included in a reaction mixture in place of paraquat. Light emission in the absence of cells was 1.05 units and in the presence of cells was 2.27 units. Thus, juglone also functioned as a redox active agent in this system.
  • Example 5 Luminescence generated with E. coli, lucigenin and various redox active agents.
  • Example__6 Chemiluminescence in the presence of sodium cyanide as a metabolic inhibitor.
  • Various levels of sodium cyanide were included in reactions composed of 50 mM Bis Tris Propane buffer, pH 10.0, 2.5 mM glucose, 0.01 mM methylene blue, 0.2 mM lucigenin and 2.2 ⁇ 107 E. coli cells/reaction.
  • Light emission was measured as in Example 5.
  • Light emitted as a function of sodium cyanide concentration was as follows:
  • Example 7 Azure C as redox active agent.
  • a 200 ⁇ L reaction mixture containing 250 mM Bis Tris Propane buffer, pH 10.0, 0.5 mM lucigenin, 2.5 mM glucose and 80 ⁇ M Azure C was prepared at ambient temperature and light emission was integrated for one minute with the BioOrbit luminometer. The integrated background light was 117 units and the emission increased to 3850 units when about 5 ⁇ 10 5 E. coli cells (barn yard isolate) were added to the mixture.
  • Another reaction mixture was prepared without Azure C and light emission without cells was 103 units and with cells was 352 units.

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Abstract

L'invention concerne un procédé et une composition d'un réactif pour la détection ou la caractérisation des microorganismes par émission de lumière. La composition comprend un substrat capable de métabolisme par des microorganismes viables d'intérêt et une sonde chimioluminescente, et peut également comprendre un agent actif rédox. Le procédé peut être utilisé pour détecter ou caractériser des bactéries, des champignons ou une levure dans divers échantillons provenant de l'environnement, l'industrie, les produits alimentaires, des sources vétérinaires ou médicales. La composition et le procédé de détection sont particulièrement utiles pour détecter la bactériurie.
PCT/US1992/001869 1991-03-18 1992-03-09 Test chimioluminescent pour microorganismes WO1992016648A1 (fr)

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Cited By (8)

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WO1995014105A2 (fr) * 1993-11-18 1995-05-26 Aquaculture Diagnostics Limited Antibiogramme
WO2002055729A1 (fr) * 2001-01-09 2002-07-18 Ursula Schies Procede et dispositif de detection de micro-organismes sur des surfaces
WO2004027084A1 (fr) * 2002-09-20 2004-04-01 Queen's University At Kingston Detection de molecules biologiques par division differentielle de substrats et de produits d'enzyme
WO2004047614A2 (fr) * 2002-11-26 2004-06-10 Expressive Constructs, Inc. Procedes, biocapteurs et necessaires de detection et d'identification de champignons
WO2010129532A3 (fr) * 2009-05-05 2011-01-06 Trustees Of Boston University Procédé et dispositif de détection rapide de la résistance ou de la sensibilité d'une bactérie à un antibiotique
US9017963B2 (en) 2002-01-31 2015-04-28 Woundchek Laboratories (Us), Inc. Method for detecting microorganisms
US9097672B2 (en) 2010-06-18 2015-08-04 Queens's University At Kingston Method and system for detecting biological molecules in samples
US9562253B1 (en) 2012-11-09 2017-02-07 Point Of Care Diagnostics, Llc Distinguishing between a bacterial and non-bacterial infection at the point of care

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WO1995014105A3 (fr) * 1993-11-18 1995-06-08 Aquaculture Diagnostics Limite Antibiogramme
WO1995014105A2 (fr) * 1993-11-18 1995-05-26 Aquaculture Diagnostics Limited Antibiogramme
WO2002055729A1 (fr) * 2001-01-09 2002-07-18 Ursula Schies Procede et dispositif de detection de micro-organismes sur des surfaces
DE10100581B4 (de) * 2001-01-09 2007-11-15 Ursula Schies Verfahren und Vorrichtung zum Nachweis von Mikroorganismen auf Oberflächen
US9017963B2 (en) 2002-01-31 2015-04-28 Woundchek Laboratories (Us), Inc. Method for detecting microorganisms
US8377686B2 (en) 2002-09-20 2013-02-19 R. Stephen Brown Detection of biological molecules by differential partitioning of enzyme substrates and products
WO2004027084A1 (fr) * 2002-09-20 2004-04-01 Queen's University At Kingston Detection de molecules biologiques par division differentielle de substrats et de produits d'enzyme
JP2005538728A (ja) * 2002-09-20 2005-12-22 クィーンズ ユニバーシティー アット キングストン 酵素基質および酵素生成物の分配差による生体分子の検出
US7402426B2 (en) 2002-09-20 2008-07-22 Queen's University At Kingston Detection of biological molecules by differential partitioning of enzyme substrates and products
US8632966B2 (en) 2002-09-20 2014-01-21 Queen's University At Kingston Detection of biological molecules by differential partitioning of enzyme substrates and products
WO2004047614A2 (fr) * 2002-11-26 2004-06-10 Expressive Constructs, Inc. Procedes, biocapteurs et necessaires de detection et d'identification de champignons
WO2004047614A3 (fr) * 2002-11-26 2005-02-10 Expressive Constructs Inc Procedes, biocapteurs et necessaires de detection et d'identification de champignons
WO2010129532A3 (fr) * 2009-05-05 2011-01-06 Trustees Of Boston University Procédé et dispositif de détection rapide de la résistance ou de la sensibilité d'une bactérie à un antibiotique
US8785148B2 (en) 2009-05-05 2014-07-22 Fraunhofer, Usa, Inc. Method and device for rapid detection of bacterial antibiotic resistance/susceptibility
US9611501B2 (en) 2009-05-05 2017-04-04 Trustees Of Boston University Method and device for rapid detection of bacterial antibiotic resistance/susceptibility
US9097672B2 (en) 2010-06-18 2015-08-04 Queens's University At Kingston Method and system for detecting biological molecules in samples
US9562253B1 (en) 2012-11-09 2017-02-07 Point Of Care Diagnostics, Llc Distinguishing between a bacterial and non-bacterial infection at the point of care

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