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.
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
the fluorescence radiation of the fluorophore by the so-called fluorescence resonance energy transfer
For this it is necessary to match the FRET pair "fluorophore quencher".
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
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.
well-known ideal fluorophore quencher couple living in the tribe formation
of the MB to an almost complete
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).
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
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
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
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.
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
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
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
was a europium chelate known that used as a fluorescent marker
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.
The present invention is therefore based on the object, an improved
System for detecting events in a sample environment for
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.
inventive solution this
The core of the task is a probe with two FRET pairs
employing forming fluorophores, the pair being chosen
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.
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
represent a change
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.
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).
Rare earth component may preferably be a diketonato complex
who is coordinated with a chelating agent.
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
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 ,
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
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.
the probes according to the invention
the discrimination between probe signal and unspecific background radiation over the
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.
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
Device considerably simplified and cheaper.
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.
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
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.
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
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.
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
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 ,
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.
Determination of pathogens in samples such as food
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)
and the process also potentially because of the danger of propagation
pathogenic microorganisms according to the infection protection law display and
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
Germs are preferably removed by immunological methods
Specimen environment specifically isolated. It is preferred on the
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.
a concrete embodiment
25 g food suspended with 225 ml PBS buffer. The suspension
Immunomagnetic particles are added. The particles carry
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
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
to 2. Amplification of the target nucleic acids
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.
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,
However, the probes according to the invention are used.
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
a serial connection a calibration function (standard function)
for the germ
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.