DE10259677B4 - Probes for the detection of nucleic acids, in particular for the detection of pathogenic germs in foods - Google Patents

Probes for the detection of nucleic acids, in particular for the detection of pathogenic germs in foods

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Publication number
DE10259677B4
DE10259677B4 DE2002159677 DE10259677A DE10259677B4 DE 10259677 B4 DE10259677 B4 DE 10259677B4 DE 2002159677 DE2002159677 DE 2002159677 DE 10259677 A DE10259677 A DE 10259677A DE 10259677 B4 DE10259677 B4 DE 10259677B4
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sample
method according
characterized
detection
microorganisms
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DE10259677A1 (en
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Jaszczuk Wieslaw Dipl-Phys
Klauth Peter Dr Dipl-Biol
Original Assignee
Büddefeld, Jürgen, Prof. Dr.-Ing.
Jaszczuk, Wieslaw, Dipl.-Phys.
Klauth, Peter, Dr. Dipl.-Biol.
Kynast, Ulrich, Prof. Dr.
Rietz, Manfred, Prof. Dr.
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Application filed by Büddefeld, Jürgen, Prof. Dr.-Ing., Jaszczuk, Wieslaw, Dipl.-Phys., Klauth, Peter, Dr. Dipl.-Biol., Kynast, Ulrich, Prof. Dr., Rietz, Manfred, Prof. Dr. filed Critical Büddefeld, Jürgen, Prof. Dr.-Ing.
Priority to DE2002159677 priority Critical patent/DE10259677B4/en
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    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00Investigating or analysing materials by the use of optical means, i.e. using infra-red, visible or ultra-violet light
    • G01N21/62Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64Fluorescence; Phosphorescence
    • G01N21/6428Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING 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
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/68Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving nucleic acids
    • C12Q1/6813Hybridisation assays
    • C12Q1/6816Hybridisation assays characterised by the detection means
    • C12Q1/6818Hybridisation assays characterised by the detection means involving interaction of two or more labels, e.g. resonant energy transfer
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00Investigating or analysing materials by the use of optical means, i.e. using infra-red, visible or ultra-violet light
    • G01N21/62Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64Fluorescence; Phosphorescence
    • G01N21/6408Fluorescence; Phosphorescence with measurement of decay time, time resolved fluorescence
    • BPERFORMING OPERATIONS; TRANSPORTING
    • B01PHYSICAL OR CHEMICAL PROCESSES OR APPARATUS IN GENERAL
    • B01LCHEMICAL OR PHYSICAL LABORATORY APPARATUS FOR GENERAL USE
    • B01L7/00Heating or cooling apparatus; Heat insulating devices
    • B01L7/52Heating or cooling apparatus; Heat insulating devices with provision for submitting samples to a predetermined sequence of different temperatures, e.g. for treating nucleic acid samples
    • GPHYSICS
    • G01MEASURING; TESTING
    • G01NINVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N21/00Investigating or analysing materials by the use of optical means, i.e. using infra-red, visible or ultra-violet light
    • G01N21/62Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
    • G01N21/63Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
    • G01N21/64Fluorescence; Phosphorescence
    • G01N21/6428Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes"
    • G01N2021/6439Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks
    • G01N2021/6441Measuring fluorescence of fluorescent products of reactions or of fluorochrome labelled reactive substances, e.g. measuring quenching effects, using measuring "optrodes" with indicators, stains, dyes, tags, labels, marks with two or more labels

Abstract

Method for identifying and quantifying microorganisms in a sample or a food sample, comprising the following method steps:
Isolation of the microorganisms from the sample,
Transfer of the isolated microorganisms into a PCR vessel,
Amplification of the target nucleic acid by a real-time PCR as a marker for the microorganisms,
Detection of the amplified target nucleic acid by means of a probe having at least two fluorophores which form a FRET pair, the fluorophores being chosen so that a recognizable signal is emitted by the fluorophore after hybridization of the probe with the target nucleotide, that one of the Fluorescence emission of the sample environment extended life, so that the measurement signal is differentiable by a time-resolved fluorescence measurement of the autofluorescence of the sample environment,
Detecting the fluorescence emission by means of time-resolved fluorescence measurement,
- Quantification of the pathogen concentration in the original sample using a standard curve with calibration factors.

Description

  • The Invention relates to a probe for detecting a molecular event, in particular for detecting the hybridization of the oligonucleotide probes with a target oligonucleotide, preferably with the aid of molecular beacons. The system according to the invention is preferably used to detect pathogenic germs in food.
  • Molecular beacons (MB) are single-stranded DNA molecules with a hairpin structure (hair pin code). You therefore have a "stem and loop structure ". The loop has a nucleotide sequence - the so-called. Sample sequence complementary to a predetermined target nucleotide sequence. Of the Stem is obtained by hybridization of two complementary arm sequences formed flanking the sample sequence. A fluorescent dye is at the end of an arm, while a quencher is at the end the other arm is tied. Through the trunk are these two Units in a defined spatial Close to each other held to one as possible complete Quenching (deleting) the fluorescence radiation of the fluorophore by the so-called fluorescence resonance energy transfer (FRET) leads. For this it is necessary to match the FRET pair "fluorophore quencher".
  • Meets the molecular beacon on a target sequence, hybridize the sample sequence and the target sequence over an area that is more stable and longer than the trunk the two arm sequences. The trunk is therefore separated into his arms and the fluorophore and the quencher take a defined spatial Distance to each other. Thus the FREI is interrupted, as the FREE with the 6th power radius decreases. Fluorescence emission of the fluorophore becomes detectable.
  • The structure and mode of operation of the molecular beacon are described in detail by Tyagi and Kramer (1996): "Molecular beacons: coarse that fluoresce upon hybridization" in Nature Biotechnology 14, 303-308. Likewise, they are the subject of US Pat. No. 5,925,517 , which is hereby incorporated by reference for disclosure purposes regarding the structure and function of the MBs as well as the selection of suitable FRET pairs.
  • When well-known ideal fluorophore quencher couple living in the tribe formation of the MB to an almost complete Clear the fluorescent emisson leads is the 5 '- (2-aminoethyl) aminonaphthalene-1-sulfonic acid (EDANS) at the 5'-end and as Quenching the 4- (4'-dimethylaminophenylazobenzoic acid (DABCYL) known at the 3'-end. Likewise, the use of DABCYL in combination with fluorescein suitable at the 5'-end (G. Leone, Schijndel H., van Gemen B., Kramer R. F. and Schön D. (1998) "Molecular Beacon Probes combined with Amplification by NASBA enable homogeneous real-time Detection of RNA "in: Nucleic Acids Research 26, 9, 2150-2155).
  • There MBs due to quenching caused by FREE in the result as long as no or hardly emit fluorescence radiation, or spectral Cause shift when using two fluorophores and thus the signal from the measuring range remove as the sample sequence does not hybridize to the target sequence MBs have become more recent Past especially proven as probes in detection systems, in which wash out excess probes not wished or possible is. This applies in particular to real-time PCR or detection of nucleic acids in living cells (Liu X, Farmerie W., Schuster S., Tan W. (2000): "Molecular beacons for DNA Biosensors with a Micrometer to Submicrometer Dimension "in: Analytical Biochemistry 283, 56-63).
  • Just in complex biological systems like living cells has become however, the non-specific fluorescence of, for example, cellular components proved as a significant systematic source of error, the meaningfulness and specificity the detection methods even with the known MB significantly reduced. For example, show hemoglobins or aromatic hydrocarbons when excited by UV light unintentionally a nonspecific fluorescence that turns out to be background radiation makes noticeable. The actual measuring signal is thus superimposed. This background radiation is also referred to below as autofluorescence the sample environment or sample milieu.
  • EP 0 973 036 B1 relates to a test method based on a FRET system located at the distal ends of a peptide molecule in which rare-earth fluorophores are used as donors because of their long emission times (paragraph 36). The signal detection takes place via a time-resolved fluorescence measurement, which is described as particularly advantageous for the reduction of the background radiation.
  • US 5,998,146 A is based on the same applicant and essentially corresponds in its disclosure content EP 0 973 036 B1 ,
  • LEONE et al. ( "Molecular beacon gross combined with amplification by NASBA enable homogeneous, Real-time detection of RNA ", Oxford University Press Vol. 26 1998, pages 2150-2155), generally describes the use of molecular beacons in one NASBA system.
  • TAN et al. ( "Molecular Beacons: A novel DNA probe for nucleic acid and protein studies, Journal of Chemistry, No. 6, 2000, pages 1107-1111), describes the general technological background to Molecular Beacons and others in their application in real-time PCR or RNA detection in living Cells.
  • FLUIT et al. ( "Molecular detection of antimicrobial resistance ", Clinical Microbiology Reviews, October 2001, pages 836-871), relates to the detection of bacterial resistance and discloses et al also the use of Molecular Beacons. This document therefore discloses the use of Molecular Beacons for detection of bacteria.
  • Further was a europium chelate known that used as a fluorescent marker becomes.
  • US 5,720,923 relates to a PCR machine for real-time amplification, which has a UV detector ( 30 ). This has a UV light source and photodiodes for fluorescence measurement.
  • EP 1 049 806 discloses methods for time-resolved fluorescence measurement (paragraphs 42 to 45). Furthermore, molecular beacons with a FRET system consisting of FAM as a fluorophore and methyl red as a quencher were disclosed (paragraphs 89 and 139). By contrast, examples of molecular beacons with a rare earth dye are not disclosed.
  • Of the The present invention is therefore based on the object, an improved System for detecting events in a sample environment for disposal the evidence of the event in particular the Detection of the presence of a target molecule is used. The target molecule may be, for example an oligonucleotide or else a peptide or protein (enzyme). This should in particular a probe for the detection of nucleic acids, as well a corresponding evaluation system using this system be provided, with the problem of the unwanted Background radiation is reduced by UV excitation. Furthermore is meant to carry a device a particularly preferred embodiment This method can be provided.
  • The inventive solution this The core of the task is a probe with two FRET pairs employing forming fluorophores, the pair being chosen that the Donor of FRET couple at suggestion in case of occurrence of the event a measuring signal emitted, the one opposite the fluorescence emission of the sample environment has extended life. Due to the time difference of the lifetimes of the measured signal and the background radiation becomes a discrimination of emissions through a time-resolved Fluorescence measurement possible.
  • Provided it is a probe for the nucleic acid detection For example, the "event" represents the Hybridization of the probe with the target oligonucleotide. Includes the probe of the invention however, an oligo- or polypeptide moiety, the event may be, for example also the cleavage or the binding of the peptide to a ligand be. The "event" can thus generally every activity represent a change the spatial Arrangement of FRET pairs leads to each other, which is a discriminatory Detection of the donor signal in the time-resolved fluorescence measurement allows.
  • The discrimination between the fluorescence of the desired measurement signal and the nonspecific background radiation is therefore only possible according to the invention by a fluorophore which emits its signal in temporal terms by at least a factor of 10 longer than the fluorescence emission of the sample environment. Thus, the "background noise" disturbing in the currently known methods by unspecific autofluorescence using known MBs is prevented or considerably reduced. Sounds z. B. the autofluorescence - ie the Störfluoreszenz the sample environment - after 200 ns after completion of the UV excitation in the form of a pulse and emits the molecular beacon according to the invention in hybridization to its target sequence total over a period of 0.5 ms to 2 ms Measuring signal, so that after the expiry of 200 ns after completion of the UV pulse detectable fluorescence signal can be assigned exclusively to the emission of the probes according to the invention. A schematic representation of the detection of autofluorescence and the SEF fluorescence by means of time-resolved fluorescence measurement shows 1 ,
  • The application of the pulsed UV excitation required for this, as well as the device and the methods for time-resolved fluorescence measurement, correspond to the methods known in the prior art. These are for example from the German Patent Application 196 34 873 A1 (See also R. Müller, C. Sander, M. Sauer, M. Deimel, DS Ko, S. Siebert, J. Arden-Jacob, G. Deltau, NJ Marx, KH Drexhage, J. Wolfrum: Time-resolved Phys. Lett 262, 716-722 (1996) A further advantage of the probes according to the invention is the possibility of applying a much higher excitation energy without it The interference by an otherwise also detected autofluorescence of the sample environment would increase.This excitation energy (UV diode) used according to the invention can be used, for example way 300% above the commonly used excitation energy. The high quantum efficiency and the extremely narrow emission spectrum of the probes used lead to a high emission intensity in a narrow frequency range, while the UV diode can optimally excite the probe. This also significantly increases the specific measurement signal (output signal) of the fluorophore emission compared to conventional fluorophores. Furthermore, this advantageously goes hand in hand with the lowering of the detection limit of the detection system.
  • In a particularly preferred embodiment become rare earth dyes (hereinafter also SEF) as fluorophores for the inventive molecular used beacon. These are dyes that are under other lanthanides such as europium or terbium. SEF wise usually fluorescence lifetimes of about 0.5 to 2.5 ms after an excitation impulse (Hemmilä, I. (1990) Applications of fluorescence in immunoassays, Wiley-Interscience). On the other hand are organic dyes mostly by fluorescence lifetimes of only a few ns to μs. Thus, the dye Cy5 emits only up to about 1 ns after the excitation a fluorescent signal (Source: Amersham Bioscience).
  • The Rare earth component may preferably be a diketonato complex who is coordinated with a chelating agent.
  • When Diketonat can Eutaphs or substituted derivatives, as chelating agents can phenanthroline or bipyridine and their substituted derivatives. When Substituents are those functional groups suitable for a coupling (with the linker) are capable. To name in particular Amine, carboxylate, isocyanate, Thioisocaynat-, epoxy, thiol or Hydroxy substituents.
  • In a preferred embodiment, the probes of the invention have Euttaphen (1-thenyl, 4,4,4-trifluoro-1,3-butane, 1,3-dionato) (1,10-phenanthrolinato) Eu (+ III)) as SEF on ( 2 ). This color complex is characterized by a fluorescence emission until about 1 ms after its excitation. The luminescence spectrum of Euttaphen shows 3 ,
  • to Formation of an MB according to the invention can for example, Euttaphen as a donor with Cy5 as the acceptor chromophore combined to a FRET couple become.
  • The selection of chromophores as suitable FRET partners can be accomplished by examining the fluorescence emission of a potential donor in the presence of a potential acceptor, varying the concentration of the acceptor. The absorption spectrum must overlap significantly with the emission spectrum of the donor. The interaction required for a quenching effect (especially the spatial proximity) between donor and acceptor is z. B. adjusted by concentration of potential FRET partners. If necessary, the spatial proximity can also be achieved by chemically binding a partner to a linker or spacer molecule which, when the second partner is added, can additionally bind it. If the emission of the donor decreases as the concentration of the acceptor increases, this is a clear sign of the quenching activity of the acceptor and thus of the compatibility of the dyes used as a FRET pair. Exemplary of this procedure is the representation of the decrease in the fluorescence emission of Euttaphen depending on the concentration of Cy5 in 4 ,
  • After the selection of suitable FRET pairs, these are joined together to form the probes according to the invention. This can be done, for example, by the protocol of Min Li and Paul R. Sevin (Amine Reactive Forms of a Luminescent Diethylenetriamine Pentaactic Acid Chelate of Terbium on Europium: Attachment to DNA and Energy Transfer Measurements, Bioconjugate Chem. 1997, 8, 127-132) , For this purpose, for example, the Euttaphen NH 2 is used and first isothiocynated by the treatment with CSCl 2 on its NH 2 . Subsequently, a coupling to an amino-modified linker, for example an amino-modified oligonucleotide, takes place at the thiocynate residue. For the coupling of the linker, the protocol of Li and Sevin can also be used. The linker also carries a chromophore, for example Cy5. The coupling of the dye to the linker is possible by known standard methods.
  • As already executed, enable the probes according to the invention the discrimination between probe signal and unspecific background radiation over the time-resolved Fluorescence measurement. At the same time according to the invention to the MB coupled dyes have an emission spectrum with sharp emission bands.
  • The two factors - namely the sharp emission bands of the dyes and the substantial elimination of non-specific autofluorescence even with an increase in the excitation energy - make it possible to dispense with elaborate laser and evaluation systems (eg ICCD (Intensified Coupled Charge Device)), which in the prior art Optimization of the measured signals were developed against the background of disturbing autofocus. These are located in the commercially available real-time PCR thermal cyclers in the periphery of the devices - ie except half of the actual thermoblock - and communicate with the samples via fiber optic systems that may contain filters or amplifiers.
  • Such structurally complex systems for UV excitation can now according to the invention by a commercial one UV light-emitting diode for excitation of the fluorophores and a commercially available photodiode for detecting the measuring signal be replaced. Advantageously, so that the construction of a on the implementation the method according to the invention adapted Device considerably simplified and cheaper.
  • In an advantageous embodiment are the commercial ones Diodes integrated directly into the thermoblock of a thermocycler for real-time PCR. The Integration of the diodes directly into the thermoblock allows one compact design of the thermal cycler, so that this preferably also portable or mobile.
  • The inventive probes allow u.a. the specific and meaningful real-time Detection of amplification of target nucleic acids with a low detection limit, in particular the real-time PCR in a compact and simply designed thermocycler. The counter to e.g. possibly a photomultiplier reduced sensitivity of a photodiode is due to the intense as well as time-resolved Signal of the probes according to the invention balanced. About that In addition, the probes of the invention are but also in isothermal amplification methods such as the Nucleic Acid Sequence Based Amplification (NASBA) can be used. Also, the inventive probes in the array technology Find use. This is already for the classical molecular beacons known (Liu X. et al. (2000)). This opens up for the probes according to the invention the broad field of application of the entire medical, molecular biological or biochemical analysis and diagnostics.
  • Furthermore The system of rare earth dyes as a fluorophore and a adapted to it Quencher - so the FRET couple - also in a binding to oligopeptides or proteins successfully used become. The use of the FRET pair used according to the invention is thus not limited to the coupling to nucleic acids. So it is equally possible that it is first in defined neighborhood to each other bound to a protein and in the case of a cleavage of the protein or due to a conformational in the case of binding to a ligand in spatial distance from each other lies so that the Fluorescence emission of the donor fluorophore becomes detectable. In order to are these FRET pairs proposed here also in the protein or peptide analysis can be used advantageously.
  • For example, the protocol of Li and Selvin can be used to form a protein FRET complex (Min Li and Paul R. Selvin: Amine Reactive Forms of a Luminescent Diethylenetriamine Pentaacetic Acid Chelate of Terbium and Europium: Attachment to DNA and Energy Transfer Measurements. Bioconjugate Chem., 1997, 8, 127-132). For this purpose, Euttaphen-NH 2 is again converted into its isothiocyanate form. This step can be done analogously to the coupling to DNA.
  • In a second step is the isothiocyanate form of Euttaphen coupled with a protein / peptide labeled with Cy5. This can be done according to Takalo et al. (Harri Takalo, Veli-Matti Mukkala, Heikki Mikola, Päivi Liitti, and Ilkka Hemmila: Synthesis of Europium (III) Chelates suitable for Labeling of Bioactive Molecules, Bioconjugate Chem., 1994, 5, 278-282, see Synthesis of Chelates.pdf). For this, the protein / peptide is in the presence of the isothiocyanate form of Euttaphen in carbonate buffer (pH 9.3) at room temperature for 16 h. The purification is then via gel chromatography possible.
  • Alternatively, the coupling of Euttaphen-NH 2 via derivatives of DTA (4,6-dichloro-1,3,5-triazin-2-ylamine) according to a protocol of Karsilayan et al. (Huriye Karsilayan, Ilkka Hemmila, Harri Takalo, Airi Toivonen, Kim Petterson, Timo Lovgren, and Veli-Matti Mukkala: Influence of Coupling Method on the Luminescence Properties, Coupling Efficiency, and binding Affinity of Antibodies labeled with Europium (III) Chelates Bioconjugate Chem., 1997, 8, 71-75, see Influence of Coupling Method on the Luminescence Properties.pdf). For later industrial production by means of automatic synthesis machines, the Euttaphen NH 2 could also be used according to a protocol by Peuralahti et al. (Jari Peuralahti, Harri Hakal, Veli-Matti Mukkala, Kristiina Loman, Pertti Hurskainen, Outi Mulari, and Jari Hovinen: Introduction of Lanthanide (III) Chelates to Oligopeptides at Solid Phase, Bioconjugate Chem, 2002, 13, 870-875, see Introduction of Lanthanides (III) Chelates to Oligopeptides to Solid.pdf) to Peptides.
  • In a further alternative method, an oligopeptide may first be loaded with an isocyanate. In a second step, the condensation with an amine ligand and then the complex formation with Euttaphen. Again, the oligopeptide can be previously coupled with a dye. This procedure is shown schematically in FIG 5 ,
  • Embodiment:
  • The Probes, the method and the device according to the invention can be preferred for the detection of pathogenic germs in food and their Use quantification in the original sample.
  • to Determination of pathogens in samples such as food are diverse Known method, the predominant on the determination of the germ count or the detection of the germs capable of reproduction or based on immunological or biochemical detection reactions. In particular, the germ count determination implies a previous enrichment the pathogens, on the one hand to a long duration (up to 72 hours) of proof and the process also potentially because of the danger of propagation pathogenic microorganisms according to the infection protection law display and requires authorization.
  • With the probes according to the invention This can be a simple, inexpensive and also by less trained personnel feasible alternative proof and to quantify pathogenic germs.
  • This complex detection method comprises the following steps, which are described in detail below:
    • 1. Isolation of pathogens from the food sample
    • 1a. if necessary, lysis step
    • 2. Amplification of the target nucleic acids by a real-time PCR with delayed fluorescence measurement as a marker for the pathogens
    • 3. Quantification of the pathogen concentration in the original sample using a standard curve with calibration factors.
  • to 1. isolation of the pathogens (example Salmonella)
  • The Germs are preferably removed by immunological methods Specimen environment specifically isolated. It is preferred on the with antibodies against specific surface molecules of the to be detected Microorganisms coated paramagnetic particles recourse. For the detection of Salmonella, the so-called "Dynabeads anti-Salmonella" (Prod. be used by the company Dyna Biotech.
  • In a concrete embodiment 25 g food suspended with 225 ml PBS buffer. The suspension Immunomagnetic particles are added. The particles carry antibody against surface molecules of the to be detected Germs. The incubation takes place for Stirring for 30 minutes. Subsequently, the magnetic particles are removed via a magnet. The pellet is washed once with 20 ml of PBS buffer or TEST buffer (10 mmol / l Tris [pH 8], 150 mmol / l NaCl, 0.05% TWEEN 20).
  • to 1a. Lysis of the isolated microorganisms
  • For microorganisms, which are not lysed by the first step of the PCR is done a lysis step with a lysis buffer with proteinase K (eg 50 mmol / l Tris-HCl (pH 8.5) 1 mmol / l EDTA, 0.1% SDS and 1 mg / ml proteinase K). The lysis takes place for one Hour at 55 ° C, being constantly shaken / stirred. After that, the DNA is over an affinity chromatography column (e.g. QIagen DNA Mini Kit from Qiagen) and eluted. An aliquot (<50 μl) is used for the PCR used.
  • to 2. Amplification of the target nucleic acids
  • The paramagnetic particles including the bound microorganisms - or possibly an aliquot from the lysis - be transferred to a PCR tube, the with the for the PCR required components is populated.
  • The PCR for Salmonella conforms to the protocol of W. Chen, G. Martinez, A. Mulchandani (2000): Molecular Beacon: A Real Time Polymerase Chain Reaction Assay for Detecting Salmonella, Analytical Biochemistry, 280, 166-172. However, the probes according to the invention are used.
  • The PCR for Legionella pneumophila is used according to the protocol of N. Wellinghausen, C. Frost, R. Marre (2001): Detection of Legionellae at Hospital Water Samples by Quantitative real time Lightcycler PCR. Appl. Environ. Microbiol. 67, 3985-3993 carried out.
  • before was over a serial connection a calibration function (standard function) for the germ also for the reference germ under exactly these specific PCR conditions. The principle the establishment of a calibration function and the following quantification the pathogen in the original sample corresponds to the average expert known methods, for example, Fortin N Y, Mulchandani A. and Chen W. ("Use of Real-time Polymerase Chain Reaction and Molecular Beacons for the Detection of Escherichia coli O157: H7 (2001) in: Analytical Biochemistry 289, 281-288).
  • In the PCR method probes according to the invention are used which have the following structure:
    5 ' SEF-CGCTATACTGACACTGAGCGACGAAAGCGTAGCG_Cy5 3 '
  • Euttaphen was used as the SEF. The linker used was a C6 amino linker with the in 6 shown structure.
  • Thermocycler with thermoblock
  • The thermocycler 1 ( 7 ) has a pattern generator 2 with an exit 3 on, for triggering and synchronization of the pulse signals. For the generator, a programmable timer stone, a commercially available microcontroller board or a PC with I / O card, including the corresponding additional function, can be used depending on requirements and configuration level. In addition, there is a control module clocked via the trigger signal 4 for the UV diode and a synchronized sense amplifier with sample / hold circuit 5 provided for the photodiode. The analog clocked output signal 6 can be stored digitally for further evaluation with commercially available analog / digital converters and further processed.
  • The thermoblock 8th (please refer 8th ) may consist of a cuboid aluminum heating block for receiving the sample vessel and the sensor components for the real-time detection. For this he includes Peltier elements 9 with coolers 10 and fans 11 , A UV LED diode 12 is below the sample vessel 13 in a hole 14 accommodated. On the side next to the sample vessel is a commercially available photodiode 15 arranged.

Claims (16)

  1. Method of identification and quantification of microorganisms in a sample or food sample, comprising the following method steps: - Isolation of microorganisms from the sample, - transfer of the isolated microorganisms in a PCR tube, - Amplification of the target nucleic acid by a real-time PCR as a marker for the microorganisms, - Proof the amplified target nucleic acid by means of a probe with at least two fluorophores, which is a FRET pair form, wherein the fluorophores are selected so that only after Hybridization of the probe with the target nucleotide is a recognizable Signal emitted by the fluorophore is one opposite the Fluorescence emission of the sample environment has extended life, so that the measurement signal through a time-resolved Fluorescence measurement differentiable from the autofluorescence of the sample environment is - To capture the fluorescence emission by means of time-resolved fluorescence measurement, - Quantification the pathogen concentration in the original sample using a standard curve Calibration factors.
  2. Method according to claim 1, characterized by a Enrichment of microorganisms via a microorganism specific ligands coated carrier.
  3. Method according to claim 2, characterized by Use one with antibodies coated microparticle for the enrichment of microorganisms.
  4. Method according to one of claims 1 to 3, characterized in that followed by a lysis step to isolate the pathogens.
  5. Method according to one or more of claims 1 to 4, characterized in that a probe is used in at the difference between the lifetime of the signal and those the sample environment is at least a factor of 10.
  6. Method according to one or more of claims 1 to 5, characterized in that the used Probe as a fluorophore comprises a rare earth dye.
  7. Method according to Claim 6, characterized that the Rare earth dye a diketonate complex or derivatives thereof containing a chelating agent or derivatives thereof.
  8. Method according to claim 7, characterized in that that the chelated binder is selected is from the group of phenanthroline or bipyridine as well as their Derivatives.
  9. Method according to claim 8, characterized in that that the Having derivative substituents with the following functional groups: Amine, carboxylate, isocyanate, Thioisocaynat-, epoxy, thiol or Hydroxy groups.
  10. Method according to one of the preceding claims 1 to 9, characterized in that the FRET pair formed by Euttaphen and Cy5.
  11. Method according to one or more of claims 1 to 10, characterized in that as a probe for detecting the target oligonucleotide in the sample environment, an oligonucleotide is used, which is complementary to the target oligonucleotide detection sequence and two flanking the sample sequence at the 3'- and 5'-end comprises mutually complementary oligonucleotide sequence segments connected to either a quencher or a fluorophore to form a common FRET pair, with the FRET pair selected in that, in the case of hybridization of the detection sequence to the target sequence, a signal is emitted from the fluorophore that is differentiable from the autofluorescence of the sample environment by a time-resolved fluorescence measurement.
  12. Method according to one or more of claims 1 to 11, characterized in that a device for carrying out a Real-time PCR with a heating block, means for cooling, means for heating, means to stimulate PCR reporters as well as means to capture the the PCR reporters emitted measuring signals is used, wherein as a means of stimulating the reporter a UV LED and as a means to Acquisition of the measuring signals a photodiode can be used.
  13. Method according to claim 12, characterized in that that the UV LED and the photodiode are integrated into the heating block.
  14. Method according to one or more of claims 1 to 13, marked by a NASBA.
  15. Oligonucleotide as a probe for the detection of a Zieloligonukleotids in a sample environment comprising one to the target oligonucleotide complementary Detection sequence and two the sample sequence at the 3 'and 5' end flanking, mutually complementary oligonucleotide sequence sections, the with either a quencher or a fluorophore to form a common FRET pairs, the FRET pair of Euttaphen and Cy5 is formed so that in the Case of hybridization of the detection sequence with the target sequence a signal is emitted from the fluorophore by a time-resolved fluorescence measurement of autofluorescence of the sample environment is differentiable.
  16. Use of an oligonucleotide according to claim 15 in a method according to one or more of claims 1 to 14th
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DE102008033871A1 (en) 2008-06-26 2009-12-31 InBio Prof.Jürgen Büddefeld Dr.Peter Klauth Prof.Manfred Rietz GbR (vertretungsberechtigter Gesellschafter: Dr.Peter Klauth, 41189 Mönchengladbach) Use of two different fluorophores for configuring fluorescence resonance energy transfer pair, where the first fluorophore acts as donor fluorophore and the second fluorophore acts as acceptor fluorophore
DE202009008015U1 (en) 2009-04-07 2010-10-07 InBio Prof.Jürgen Büddefeld Dr.Peter Klauth Prof.Manfred Rietz GbR (vertretungsberechtigter Gesellschafter: Dr.Peter Klauth, 41189 Mönchengladbach) Modification of phyllosilicates for luminescence activation
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