DE10259677A1 - System for detecting events, especially hybridization, in a sample, useful for identifying and quantifying microorganisms in foods, by long-lived fluorescent energy transfer with time-resolved measurement - Google Patents

Abstract

System (A) for detecting an event in a sample comprising a probe (I) with at least two fluorophores (I) that form a FRET (fluorescent resonant energy transfer) pair, is new. After the occurrence of an event, (I) emits a signal that can be differentiated from autofluorescence of the sample by time-resolved measurements. Independent claims are also included for: (1) oligonucleotide (ON), as probe for detecting a target oligonucleotide (TON) in a sample, that has a complementary detection sequence and two flanking (3', 5') self-complementary sequences that are bound to either a quencher or a fluorophore to form a FRET pair that, in the event of hybridization, generates a signal that can be differentiated as above; (2) detecting TON; and (3) device, especially for performing real-time PCR, in which the system for exciting the PCR reporter comprises a UV-LED and that for detecting the measurement signal is a photodiode.

Description

The invention relates to a probe for the detection of a molecular event, in particular for the detection hybridization of the oligonucleotide probes with a target oligonucleotide, preferably with the help 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 one Hairpin structure. They therefore have a "stem and loop structure". The loop has a nucleotide sequence - the so-called sample sequence, the is complementary to a predetermined target nucleotide sequence. The The stem is created by the hybridization of two complementary arm sequences formed, which flank the sample sequence. A fluorescent dye is at the end of one arm, while a quencher is at the end of the other arm is bound. By the tribe these two become Units in a defined spatial Closeness to each other kept that to one if possible complete Quenching the fluorescence radiation of the fluorophore through the so-called fluorescence resonance energy transfer (FRET) leads. For this it is necessary to match the FRET pair "fluorophore quencher" to each other.

If the molecular beacon hits one Target sequence, hybridize the sample sequence and the target sequence over one Area that is more stable and longer than the stem from the two arm sequences. The trunk is therefore separated into his arms and take the fluorophore and quencher a defined spatial Distance from each other. This will interrupt the FRET FRET decreases with the 6th power radius. Fluorescence emission from the fluorophore becomes detectable.

The structure and the principle of operation the molecular beacon are detailed described by Tyagi and Kramen (1996): "Molecular beacons: probes that fluoresce upon hybridization "in Nature Biotechnology 14, 303-308. They are also 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 and the choice of suitable FRET pairs is fully referred to.

As a well-known ideal fluorophore-quencher pair, that in the master formation of the MB to an almost complete deletion of the Leads to fluorescence emisson the 5 '- (2-aminoethyl) aminonaphtaline-1-sulfonic acid (EDANS) at the 5 'end and as Quencher the 4- (4'-dimethylaminophenylazobenzenic acid (DABCYL) known at the 3 'end. Likewise, the use of DABCYL in combination with fluorescein suitable at the 5 'end (G. Leone, by Schijndel H., van Gemen B., Kramen 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).

Because MBs due to the caused by FRET Quenchings in the result as long as no or hardly any fluorescence radiation emit, or spectral shift when using two fluorophores cause and thus remove the signal from the measuring range, like the Sample sequence is not hybridized with the target sequence MB in younger Proven particularly in the past as probes in detection systems, in those washing 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).

Especially in complex biological Systems such as living cells, however, have unspecific fluorescence for example more cellular Components proven to be an essential systematic source of error, which are the significance and specificity of the detection methods themselves significantly reduced with the known MB. For example, show hemoglobins or aromatic hydrocarbons when excited with UV light an unspecific fluorescence that turns out to be background radiation noticeable. The actual measurement signal is thus superimposed. This background radiation is subsequently referred to as autofluorescence the sample environment or sample environment.

The present invention lies hence the task of an improved detection system of events in a sample environment, the Evidence of the event, especially evidence of the present of a target molecule serves. The target molecule can, for example, an iligonucleotide or a peptide or Be protein (enzyme). In particular, this is intended to be a probe for detection of nucleic acids, as well as a corresponding evaluation system using this Systems are provided with which, above all, the problem of unwanted background radiation is reduced with UV excitation. Furthermore, a device for carrying out a particularly preferred embodiment of this method.

The solution to this problem according to the invention is based at its core, a probe with two fluorophores forming a FRET pair to be used, the pair being chosen so that the donor of the FRET pair a measurement signal if the event occurs emits that a across from the fluorescence emission of the sample environment has an extended lifespan. By the time difference of the lifetimes of the measurement signal and The background radiation discriminates against emissions through a time resolved Fluorescence measurement possible.

If it is a probe for nucleic acid detection the "event" represents, for example, the Hybridization of the probe with the target oligonucleotide. Includes the probe of the invention however, an oligo or polypeptide portion, the event may, for example also the cleavage or the binding of the peptide to a ligand his. The "event" can therefore be general every activity represent that to a change the spatial Arrangement of the FRET pairs leads to one another, which is discriminatory Detection of the donor signal in the time-resolved fluorescence measurement enabled.

The discrimination between the fluorescence of the desired measurement signal and the non-specific background radiation is therefore only possible according to the invention by a fluorophore, which emits its signal in terms of time by at least a factor of 10 longer than the fluorescence emission of the sample environment. This prevents or significantly reduces the "background noise" that is disturbing in the currently known methods by unspecific auto-fluorescence when using known MBs. Sounds like B. the autofluorescence - ie the interfering fluorescence of the sample environment - as early as 200 ns after the end of the UV excitation in the form of a pulse and emits the molecular beacon according to the invention when hybridized to its target sequence overall over a period of 0.5 ms to 2 ms Measurement signal, the fluorescence signal which can be detected after 200 ns after the UV pulse has ended can be assigned exclusively to the emission of the probes according to the invention. A schematic representation of the detection of the autofluorescence and of the SEF fluorescence by means of time-resolved fluorescence measurement shows 1 ,

The application of the necessary pulsed UV excitation as well as the device and the method for time-resolved Fluorescence measurements correspond to those known in the prior art Method. These are, for example, from the German patent application 196 34 873 A1 known (see also R. Müller, C. Sanden, M. Sauer, M. Deimel, D.S. Ko, S. Siebert, J. Arden-Jacob, G. Deltau, N.J. Marx, K.H. Drexhage, J. Wolfrum: Time-resolved identification of single molecules in solution with a pulsed semiconductor diode lasen, Chem. Phys. Lett. 262: 716-722 (1996). Another advantage of probes according to the invention is the possibility of a much higher To apply excitation energy without causing the disturbance otherwise it was also included Autoflourescence of the sample environment increases. This used according to the invention Excitation energy (UV diode) can be, for example, 300% more than usual excitation energy used. The high quantum yield and the extremely narrow-band emission spectrum of the probes used to lead to a high emission intensity in a narrow frequency range while the UV diode is the probe can excite optimally. So that increases the specific measurement signal (Output signal) the fluorophore emission clearly compared to conventional Fluorophores. The humiliation is also advantageous the detection limit of the detection system.

In a particularly preferred embodiment become rare earth dyes (hereinafter also SEF) as fluorophores for the molecular according to the invention beacon used. These are dyes that are under others. Contain lanthanides such as europium or terbium. SEF show usually fluorescence lifetimes of about 0.5 to 2.5 ms after an excitation impulse on (Hemmilä, I. (1990) Applications of fluorescence in immunoassays, Wiley-Interscience). Are against organic dyes mostly by fluorescence lifetimes of only characterized by a few ns up to μs. The dye Cy5 only emits up to about 1 ns after the excitation a fluorescence signal (source: Amersham Bioscience).

The rare earth component may preferably be a The diketonato complex is coordinated with a chelating agent is.

Euttaphene or substituted derivatives, as chelating agents can be phenanthroline or bipyridine and their substituted descendants be used. Such functional groups are substituents suitable that are capable of coupling (with the linker). Particularly noteworthy are amine, carboxylate, isocyanate, thioisocaynate, Epoxy, thiol or hydroxy substituents.

In a preferred embodiment, the probes according to the invention have euttaphene (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 euttaphae shows 3 ,

To form an MB according to the invention for example euttaphene as a donor with Cy5 as an acceptor chromophore combined into a FRET couple become.

Chromophores can be selected as suitable FRET partners by examining the fluorescence emission of a possible donor in the presence of a possible acceptor, the concentration of the acceptor being varied. The absorption spectrum must clearly overlap with the emission spectrum of the donor. The interaction required for a quenching effect (especially the spatial proximity) between donor and acceptor is e.g. B. set by concentration of potential FRET partners. The spatial proximity can optionally also be achieved in that one partner is chemically bound to a linker or spacer molecule which, when the second is added Partners is also able to connect this. If the emission of the donor decreases with increasing concentration of the acceptor, 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. An example of this procedure is the representation of the decrease in the fluorescence emission of euttaphae as a function of the concentration of Cy5 in 4 ,

After selecting suitable FRET pairs, these are connected to one another to form the probes according to the invention. This can be done, for example, using the protocol by Min Li and Paul R. Sevin (Amine-Reactive Forms of a Luminescent Diethylenetriaminepentaacteic Acid Chelate of Terbium an Europium: Attachment to DNA and Energy Transfer Measurements. Bioconjugate Chem. 1997, 8, 127-132) , For this purpose, the euttaphene NHZ is used, for example, and isothiocynated on its NH ₂ by treatment with CSCl ₂ . The thiocynate residue is then coupled to an amino-modified linker, for example an amino-modified oligonucleotide. The Li and Sevin protocol can also be used to link the linker. The linker also carries a chromophore, for example Cy5. The dye can be coupled to the linker using known standard methods.

As already stated, enable the probes of the invention the discrimination between probe signal and non-specific background radiation over the time-resolved Fluorescence measurement. At the same time, the show according to the MB coupled dyes have an emission spectrum with sharp emission bands.

The two factors - namely gated emission bands of the dyes and the extensive elimination the unspecific autofluorescence even when increasing the Excitation energy - enable the elimination of complex laser and evaluation systems (e.g. ICCD (Intensified Coupled Charge Device)) used in the prior art to optimize the measurement signals against the background more disturbing Autoflourescence were developed. These are in the usual ones real-time PCR thermal cyclers in the periphery of the devices - i.e. outside the actual thermoblock - and communicate with the samples about Light guide systems, in which filters or amplifiers can be accommodated are.

Such structurally complex systems for UV excitation according to the invention by now a commercial one UV light-emitting diode to excite the fluorophores and a commercially available photodiode for detection of the measurement signal be replaced. Advantageously, the construction of a on the implementation of the method according to the invention adapted Device considerably simplified and cheaper.

In an advantageous embodiment are the standard ones Diodes integrated directly into the thermal block of a thermal cycler for real-time PCR. The Integration of the diodes directly into the thermoblock enables one compact design of the thermal cycler, so that it preferably also portable or mobile.

The probes according to the invention therefore allow et al the specific and meaningful real-time detection of the Amplification of target nucleic acids with a low detection limit, especially real-time PCR in one compact and simply designed thermal cycler. The opposite e.g. a photomultiplier possibly 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 according to the invention however also in isothermal amplification methods such as the nucleic acid Sequence Based Amplification (NASBA) can be used. They can also probes according to the invention in array technology Find use. This is already for the classic molecular beacons known (Liu X. et al. (2000)). This opens up for the probes according to the invention the wide field of application of the entire medical, molecular biological or biochemical analysis and diagnostics.

In addition, the system the rare earth dyes as a fluorophore and a quencher adapted to them - that is FRET couple - also in binding to oligopeptides or proteins successfully used become. The use of the FRET pair used according to the invention is thus not limited to coupling to nucleic acids. So it is also possible that it is initially in defined neighborhood to each other is bound to a protein and in the event of a cleavage of the protein or due to a conformational in the case of binding to a ligand at a spatial distance from one another lies so that the Fluorescence emission of the donor fluorophore becomes detectable. In order to are these proposed FRET pairs also in protein or peptide analysis can be used advantageously.

For example, Li and Selvin's protocol can be used to form a protein-FRET complex (Min Li and Paul R. Selvin: Amine-Reactive Forms of a Luminescent Diethylenetriaminepentaacetic Acid Chelate of Terbium and Europium: Attachment to DNA and Energy Transfer measurements, Bioconjugate Chem., 1997, 8, 127-132). For this purpose, Euttfaphen-NH _{2 is} again converted into its isothiocyanate form. This step can be carried out analogously to the coupling to DNA.

In a second step, the isothiocyanate form of Euttfaphen is coupled with a protein / peptide that is labeled with Cy5. According to Takalo et al. (Harri Takalo, Veli-Matti Mukkala, Heikki Mikola, Päivi Liitti, and Ilkka Hemmilä: 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 incubated in the presence of the isothiocyanate form of uttfaphen in carbonate buffer (pH 9.3) at room temperature for 16 h. The purification is then possible using gel chromatography.

Alternatively, the coupling of Euttfaphen-NH ₂ via derivatives of DTA (4,6-dichloro-1,3,5-triazin-2-ylamine) according to a protocol by Karsilayan et al. (Huriye Karsilayan, Ilkka Hemmilä, Harri Takalo, Airi Toivonen, Kim Petterson, Timo Lövgren, and Veli-Matti Mukkala: Influence of coupling Method on the Luminescence Properties, coupling Efficiency, and binding Aftinity of Antibodies labeled with Europium (III) Chelates, Bioconjugate Chem., 1997, 8, 71-75, see Influence of Coupling Method on the Luminescence Properties.pdf). For a later industrial production using automatic synthesizers, the Euttfaphen-NH _{2 could} also be based on 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 on Solid Phase, Bioconjugate Chem, 2002, 13, 870-875, see Introduction of Lanthanide (III) Chelates to Oligopeptides on Solid.pdf) to peptides.

In a further alternative method, an oligopeptide can first be equipped with an isocyanate. In a second step, the condensation takes place with an amine ligand and then the complex formation with euttaphene. Here, too, the oligopeptide can be coupled with a dye beforehand. This procedure is shown schematically in 5 ,

Embodiment:

The probes, the method and the Device according to the invention can be used for the detection of pathogenic germs in food as well as their quantification in the original sample.

For the determination of pathogens in Samples such as food are a variety of methods known the predominantly on the determination of the number of germs or the detection of the multiplicable germs or based on immunological or biochemical detection reactions. In particular, the bacterial count determination implies prior enrichment the pathogens that on the one hand have a long duration (up to 72 hours) of the proof leads and the process is also potentially due to the risk of propagation pathogenic microorganisms according to the Infection Protection Act requires approval.

With the probes according to the invention can do this simple, inexpensive and also an alternative to proof that can be carried out by less trained personnel and be provided for the quantification of pathogenic germs.

• 1. Isolierung der Pathogene aus der Lebensmittelprobe
• 1a. ggf. Lyseschritt
• 2. Amplifikation der Zielnukleinsäuren durch eine real-time-PCR mit verzögerter Fluoreszenzmessung als Marken für die Pathogene
• 3. Quantifizierung der Pathogen-Konzentration in der Ausgangsprobe über eine Standardkurve mit Kalibrierfaktoren.

This complex detection procedure comprises the following steps, which are described in detail below:

• 1. Isolation of the 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 brands for the pathogens
• 3. Quantification of the pathogen concentration in the initial sample using a standard curve with calibration factors.

to 1. Isolation of the pathogens (Example Salmonella)

The germs are preferably through immunological methods specifically isolated from the sample environment. In this case, preference is given to those to be detected using antibodies against specific surface molecules Microorganisms coated paramagnetic particles are used. To detect Salmonella, you can use the so-called "Dynabeads anti-Salmonella" (Prod. No. 710.02) can be used by Dyna Biotech.

In a specific embodiment 25 g of food suspended with 225 ml of PBS buffer. The suspension immunomagnetic particles are added. Wear the particles antibody against surface molecules of the detected Germs. Incubation is for 30 minutes with stirring. The magnetic particles are then removed using a magnet. The pellet is washed once with 20 ml PBS buffer or TEST buffer (10th mM Tris [pH 8], 150 mM NaCl, 0.05% TWEEN 20).

to 1a. Lysis of the isolated microorganisms

For Microorganisms that are not lysed by the first step of the PCR a lysis step is carried out with a lysis buffer with proteinase K (e.g. 50mM Tris-HCl (pH 8.5) 1mM EDTA, 0.1% SDS and 1 mg / ml Proteinase K). The lysis is carried out for one hour at 55 ° C, whereby is constantly shaken / stirred. After that, the DNA is over an affinity chromatography column (e.g. B. QI Aamp DNA Mini Kit from Qiagen) bound 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 if necessary, an aliquot from the lysis transferred to a PCR tube that with those for the PCR required components are assembled.

The PCR for Salmonella corresponds to the Pro tokoll by 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 here.

The PCR for Legionella pneumophila will follow the protocol of N. Wellinghausen, C. Frost, R. Marre (2001): Detection of Legionellae in Hospital Water Samples by Quantitative real time Lightcycler PCR. Appl. Environ. Microbiol. 67, 3985-3993.

Previously, a Calibration function (standard function) for the germ and for the reference germ carried out under exactly these specific PCR conditions. The principle the establishment of a calibration function and the subsequent quantification the pathogen in the initial sample corresponds to that of the average specialist known method, for example from 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 to 288).

Probes according to the invention which have the following structure are used in the PCR method:
5 'SEF-CGCTATACTGACACTGAGCGACGAAAGCGTAGCG Cy5 3'

Euttaphene was used as SEF. A C6 amino linker with the following structure was used as the linker 6 ,

thermocycler with thermoblock

The thermal cycler 1 ( 7 ) has a pattern generator 2 with an exit 3 on for triggering and synchronization of the pulse signals. A programmable timer block, a commercially available microcontroller board or a PC with an I / O card, including a corresponding additional function, can be used for the generator, depending on requirements and expansion level. Next to it 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 digitally saved and further processed for further evaluation using commercially available analog / digital converters.

The thermoblock 8th (please refer 8th ) can consist of a cuboidal aluminum heating block for holding the sample vessel and the sensor components for real-time detection. It also 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. A commercially available photodiode is located to the side of the sample vessel 15 arranged.

Claims (23)

System for the detection of an event in a sample environment comprising a probe with at least two fluorophores which form a FRET pair, characterized in that the fluorophores are chosen such that a signal is emitted by the fluorophore only after the occurrence of the event, a time-resolved fluorescence measurement can be differentiated from the autofluorescence of the sample environment. System according to claim 1, characterized in that the arrangement the fluorophores can be changed relative to one another by the event is. System according to claim 1 or 2, characterized in that that the Difference between the life of the signal and that of the Sample environment is at least a factor of 10. System according to one of claims 1 to 3, characterized in that that the Probe comprising a rare earth dye with a fluorophore. System according to claim 4, characterized in that the rare earth dye a diketonate complex or derivatives thereof comprising a chelating agent or derivatives thereof coordinated. System according to claim 5, characterized in that the chelating agent selected is from the group of phenanthroline or bipyridine and their derivatives. System according to claim 6, characterized in that derivatives Have substituents with the following functional groups: amine, Carboxylate, isocyanate, thioisocaynate, epoxy, thiol or hydroxy groups. System according to one of the preceding claims, characterized characterized that the FRET pair of euttaphene and Cy5 is formed. System according to one of claims 1 to 8 by an oligonucleotide as a probe. System according to claim 9, characterized in that the Probe one to a target in the essentially complementary Oliogonucleotide section includes. System according to claim 9, characterized in that the signal detectable after hybridization of the oligonucleotide section with the target molecule becomes. System according to one of claims 1 to 8, characterized in that the probe is an oligo is peptide. Oligonucleotide as a probe for the detection of a target oligonucleotide in a sample environment comprising one to the target oligonucleotide complementary detection sequence and two the sample sequence on 3'- and 5 'end flanking, complementary to each other Oligonucleotide sequence sections with either a quencher or a fluorophore to form a common FRET pair are characterized in that the FRET pair is selected so that in In case of hybridization of the detection sequence with the target sequence Signal from the fluorophore is emitted by a time-resolved fluorescence measurement can be differentiated from the autofluorescence of the sample environment. Method for the detection of target oligonucleotides in a sample environment include the following steps: - incubate a target nucleic acid with a system according to one of claims 1 to 12 or with one The oligonucleotide according to claim 13 - Irradiation of the incubation batch - To capture the fluorescence emission by means of time-resolved fluorescence measurement. A method according to claim 14, countersigned by a real-time PCR for the amplification of the target nucleic acids. A method according to claim 15, characterized in that either the target nucleic acid and the oligonucleotide is bound as a probe on a support. A method according to claim 16 characterized by a NASBA. Use of the system according to one of claims 1 to 12 or the oligonucleotide according to claim 13 for identification and quantification of microorganisms, in particular microorganisms and food. Use of the system according to claim 18, characterized by enrichment of the microorganisms via ligands specific to microorganisms coated carrier. Use according to claim 19, characterized by antibody coated microparticles. Use according to one of claims 18 to 20, characterized by specifying a calibration actuator to quantify the Microorganisms. Device in particular for carrying out a real-time PCR with a heating block, means for cooling, means for heating, means for stimulating PCR reporters and means for recording the of measurement signals sent to the PCR reporters, characterized in that that the Means for stimulating the reporters by means of a UV LED and the means for Acquisition of the measurement signals can be represented by a photodiode. Device according to claim 22, characterized in that the UV LED and the photodiode are integrated in the heating block.