DE102007031137A1 - Real-time detection of nucleic acid targets, comprises providing a primer containing a sequence-target binding sequence to amplify nucleic acid, introducing a fluorophore and providing a probe for detection, which carries a fluorophore - Google Patents

Abstract

Real-time detection of nucleic acid targets as products of a nucleic acid-amplification reaction by a primer/probe system, comprises providing at least a primer, which contains at least one sequence-target binding sequence (TBS) to specifically amplify nucleic acid and carries modification at the 3'-end, which allows an enzymatic extension complementary to the target; introducing a fluorophore 1; and providing at least a probe for the detection, which carries a fluorophore 2 at least at the 3'-end, where one of both fluorophores is stimulated by light of suitable wavelength. Real-time detection of nucleic acid targets as products of a nucleic acid-amplification reaction by a primer/probe system, comprises providing at least a primer, which contains at least one sequence-target binding sequence (TBS) specifically to amplify nucleic acid and carries modification at the 3'-end, which allows an enzymatic extension complementary to the target; introducing a fluorophore 1; and providing at least a probe for detection, which carries a fluorophore 2 at least at the 3'-end, where at least one probe with the complementary strand is extended to the target sequence at the 3'-end primer or use of more primers hybridized with the target strand, and one of both fluorophores is stimulated by light of suitable wavelength in such a way a fluorescence resonance energy transfer takes place between fluorophore 1 and 2, which are detected by optical fluorescence method. Independent claims are included for: (1) the primer/probe system for the detection of nucleic acids, comprising: (a) at least a primer containing at least one sequence specific for the assigned nucleic acid target and exhibiting modification at the 3'-end, which allows an enzymatic extension, where optionally one of the primer is marked with a fluorophore 1; and (b) at least a probe, which at least carries a fluorophore 2 and optionally exhibits a further fluorophore 1; (2) a reagent kit for the detection of nucleic acids, comprising at least a primer/probe system and a suitable buffer and optionally further auxiliary materials; (3) a device system for the performance of real-time polymerase chain reaction (PCR) reactions, which is fully automated computer-assisted technique, comprising: thermo-cycler, which exhibits a reaction range with several temperaturable recordings for the reaction containers; optionally an immobilization system for micro-particles; a reaction range assigned lighting mechanisms (light emitting diode and/or laser) with which the stimulated light is radiationable; detector installation, which produces measured values as a function of a measured light intensity with optical installation; optionally a reference arrangement, which produces a reference value; and an evaluator, where the reaction containers contain optically transparent container bottom and/or container cap with low characteristic fluorescence, in which the fluorescence optical analysis facilitates during the thermocyclic or isothermic performance of the detection reaction; (4) a reaction container for PCR with transparent container bottom; and (5) an immuno-PCR, which conducts multiplex-immune reactions under the use of non-fluorescent beads, porous or non-porous two-dimensional or small rod-shaped carrier materials and the analyte specific amplification product of fluorescence coded microparticle.

Description

The This invention relates generally to the field of nucleic acid amplification. In particular, it concerns a "real time" procedure Detection of a nucleic acid target. Subject of the invention are also a primer / probe system and a kit for the detection of a Nucleic acid targets and their use in solution or in the array, preferably for liquid phase PCR and for Multiplex PCR.

method for the detection and quantification of nucleic acids known. Starting from an mRNA to be analyzed, first a single-stranded cDNA using a reverse transcriptase produced, with the help of PCR becomes then a double-stranded one Generated DNA amplification product. The PCR is for both qualitative as well as quantitative statements applicable. Quantification of PCR products can vary Species take place. Newer detection methods allow the in vitro Synthesis of PCR products "real time", ie homogeneous directly in the PCR reaction vessel used in each case. This so-called "real time" PCR (RT-PCR) provides a Particularly sensitive method dar. Here is in each cycle of the PCR tracked the emergence of PCR products. The measurement of amplification takes place usually in thermal cycling, which additional Means for measuring fluorescence signals during the Amplification reaction have. The amplification products are for example by fluorescence-labeled hybridization samples, the only upon binding to the target nucleic acid fluorescence signals emit or in some cases also by double-stranded DNA binding fluorescent dyes detected. A defined signal threshold is determined for all analyzed reactions and the necessary to reach the threshold cycle number Cp both for the target nucleic acid as well determines the reference nucleic acids. On the basis of for the target nucleic acid as well as the reference nucleic acid obtained Cp values can be either absolute or relative Copy numbers of the target molecule can be determined.

A recent method for detecting a specific nucleic acid molecule uses so-called "molecular beacons" (Tyagi et al. US 6,150,097A Molecular beacons are dye-labeled oligonucleotides that have a stem-loop structure. At each of the two free ends of the stem portions (the 3 'and the 5' ends), one fluorophore is coupled, one as a reporter dye and the other as a quencher dye which contributes to the fluorescence of the reporter dye deletes sufficient spatial proximity by Foerster energy transfer. The sequences of the stem portions at both ends of the molecular beacons are chosen so that when the molecular beacon folds, the stem portions hybridize exclusively to each other but not to other portions of the oligonucleotide. The distance between reporter and quencher dye is sufficiently small in the state of the hybridized stem portions, so that the fluorescent dye does not fluoresce even with appropriate excitation with light. The loop portion has a sequence that is complementary to the sequence of a target sequence. When the molecular beacons and target sequence-containing nucleic acid molecules are in solution, the loop portions and the target sequence portions may hybridize, thereby unfolding the molecular beacon resolving the hybridization of the two stem portions. This unfolding leads to an increase in the spatial distance between the reporter dye and the quencher dye and the reporter dye is excited to fluoresce. With continuous observation of fluorescence intensity, an increase in intensity can be detected as the molecular beacons detect and hybridize to the target sequences. Thus, the nucleic acid molecules can be detected quantitatively. These molecular beacons have the major disadvantage of a costly and expensive synthesis, since these probes must be labeled with both the fluorescence and the quencher dye. Quencher fluorescent dye systems are used in comparable probe systems such as Amplifluor primers, Scorpions and TaqMan probes.

One Another known system is the light cycler system. Be here the fluorescent dyes on two different oligonucleotides distributed. At the 3 'end of the first oligonucleotide is the a fluorescent dye, e.g. As fluorescein, and downstream annealing oligonucleotide has at the 5 'end another fluorescent dye, z. B. IC Red 640. Fluorescein is used by an LED as a light source stimulates and emits light via fluorescence resonance energy transfer (FRET) excites the second fluorescent molecule. That of the second dye emitted light is via a corresponding Filter detected. Excitation of the second dye can only then take place when by the addition of the two oligonucleotides to its complementary strand the two dye molecules in spatial proximity (distance 1 to 5 nucleotides) are located. The principle of this system is based on the secondary Excitation of a second fluorescent dye, the first by the Generation of the PCR product is enabled.

The systems mentioned are basically suitable for multiplex applications, but have the Disadvantage that - should different target sequences are detected side by side - the reaction approach is relatively expensive because of the increasing number of different detection probes and the increasing number of probes can affect the amplification reaction. In addition, not enough FRET pairs are known to achieve a high degree of multiplexing.

Of the The invention was therefore based on the object, methods and primer / probe systems to provide that is more cost effective and easily synthesized can be and the same or improved sensitivity exhibit. By suitable modification of the primer structures should be found a way, the expensive use each bypass specific probe oligonucleotides, wherein the Multiplex application, which for today's diagnostics always becomes more important, should not be affected.

• a) Verwendung von mindestens einem Primer, der mindestens eine für die zu amplifizierende Nukleinsäure spezifische Sequenz – im folgenden auch Targetbindungssequenz(en) (TBS) genannt – umfasst und der am 3'-Ende Modifikationen trägt, die eine enzymatische Verlängerung komplementär zum Target gestatten.
• b) Einführung eines Fluophors 1. Dabei kann dieses Fluorophor 1 in einer Ausführungsvariante direkt an einem ersten oder weiteren Primer vorliegen oder in einer Markierungsreaktion in einen Primer eingeführt werden. Alternativ kann es auch an einer Sonde vorliegen.
• c) Nach enzymatischer Verlängerung erfolgt der Einsatz von mindestens einer Sonde zum Nachweis des Targets, die mindestens am 3'-Ende ein Fluorophor 2 trägt. Dabei kann die mindestens eine Sonde mit dem Komplementärstrang des zur Targetsequenz am 3'-Ende verlängerten Primers hybridisieren oder bei Verwendung mehrerer Primer an die Targetsequenz.

According to the invention, the object is achieved by a method for the "real-time" detection of nucleic acid targets as products of a nucleic acid amplification reaction by means of a novel primer / probe system.

• a) Use of at least one primer which comprises at least one sequence which is specific for the nucleic acid to be amplified, also referred to as target binding sequence (s) (TBS), and which carries modifications at the 3 'end which permit enzymatic extension complementary to the target ,
• b) Introduction of a Fluophore 1. In one embodiment variant, this fluorophore 1 can be present directly on a first or further primer or introduced into a primer in a labeling reaction. Alternatively, it may also be present on a probe.
• c) After enzymatic extension, at least one probe is used to detect the target, which carries a fluorophore 2 at least at the 3 'end. In this case, the at least one probe can hybridize with the complementary strand of the primer extended to the target sequence at the 3 'end, or when using a plurality of primers to the target sequence.

The two different, each with a FREE donor or FREI acceptor labeled primer / probe nucleotides are designed to be after hybridization, the fluorophores 1 and 2 in a for the Free to be sufficiently close so that they are during a PCR cycle allow fluorescence of the acceptor. That is, one of the two fluorophores is lighted of suitable wavelength, so that between fluorophore 1 and 2 a FRE takes place, which detects fluorescence optics becomes.

In an embodiment of the invention, the at least a primer at the 5 'end additionally one unspecific to the target Tail up. For example, fluorophore 1 may be at the specific or non-specific part of a first primer or on another Primer present. The arrangement of the fluorophores is exchangeable, such that fluophore 1 is present on a primer and fluophore 2 on the probe can or vice versa. Alternatively, fluorophore 1 and 2 are introduced into probes. In which direction FREI takes place it does not matter. Preferably, the fluorophores are 1 and 2 covalently bound to primers and probes.

The Invention is based on a novel primer / probe design, wherein in a very particularly preferred variant, at least one primer has a partial sequence which in the 5 'region partially complementary to 3 'area is so that it forms a hairpin.

Preferably the at least one primer may have the fluorophore 1 at the 5 'end, wherein the primer is either only from a target-specific sequence exists or additionally possesses a nonspecific tail. After enzymatic extension and probe hybridization forms to detect the target, the primer at the 5 'end no hairpin or partially or completely a hairpin with itself or extended to the complementary to the target Primer sequence to give a FREE signal.

In a very preferred embodiment of the invention, the method is characterized in that it uses at least three primers. It includes the following steps:
In a first reaction, the use of a primer pair 1 and 2. The selected forward primer 1 comprises at least one target binding sequence 1 (TBS 1) and at least one target binding sequence 2 (TBS 2) and is designed so that TBS 1 can bind to the target to be amplified , Subsequently, this primer 1 is extended complementary to the target, reverse primer 2 binds to the complementary target and is also extended. Primer 1 preferably contains a TBS 1 and a TBS 2. In one variant, it contains several TBS 2, which differ slightly or completely. In this way, multiple target sequences can be detected in one reaction (eg detection of human high-risk and low-risk papillomaviruses).

In a further reaction, a primer 3 is used, which with identical or partially identical to the 5 'region of primer 1, however, does not have TBS. If appropriate, primer 3 carries the fluorophore 1 at the 5'-end. He is designed to be one Hairpin 1 completely or partially with itself or with complementary portions of the primer 1 sequence can form. The conditions are adjusted so that with primer 2 and 3 the amplification takes place. The binds at least a TBS 2 of primer 1 to one or more further target binding sections (s), which now between extended primer 1 and the complementary one Sequence of primer 2 is, creating a hairpin 2 is formed. Becomes the amplificate then with the fluorophore 2 labeled probe in The free signal formed with the fluorophore 1 can be brought in contact be detected. In a preferred embodiment carries the primer 3 fluorophore 1 and the probe with fluorophore 2 at the 3 'end completely hybridizes to the target substance. alternative the probe can be coupled with fluorophore 2 at the 3 'end via hairpin 2 and primer 1 hybridize to Targetsbstanz.

In a further embodiment of the invention, the three Primer and two probes are used, each free donor and acceptor (fluorophore 1 and 2), the used Probe 1 primer is 1-specific and probe 2 used is target-specific.

The fluorophores 1 and 2 used according to the invention are selected from fluorescent dye molecules, wherein the light which is absorbed and remitted by the first fluorophore is reabsorbed and remitted by the second fluorophore spatially close to the first fluorophore after binding of the probe (s). Preferably, various fluorophores serve as donor and acceptor so that a FREI signal can be recorded as the occurrence of donor-stimulated fluorescence of the acceptor. Alternatively, the same dye can be used as donor and acceptor and the resulting depolarization of the fluorescence can be detected. Fluorophore 1 is preferably fluorescein and fluorophore 2 LC Red 640 or LC Red 705 Examples of further preferred FREI pairs are: Fluophore 1 Fluophore 2 amino coumarin fluorescein fluorescein Rhodamine or tetramethylrhodamine fluorescein Cy3 Cy3 Cy5 Cy3 tetramethylrhodamine europium Cy5 terbium rhodamine Bodipy Bodipy dansyl fluorescein naphthalene dansyl ruthenium Cy5.

As already stated, fluorophore according to the invention 1 on a primer and Fluophore 2 on the probe or vice versa. The FREE pair may also be bound to probes. The invention used Free pairs achieve a high degree of multiplexing. It is for the inventive method numerous fluorophores for the spectral range from 350 to 750 nm, that is from ultraviolet to infrared, available.

The Amplification reaction is preferably a homogeneous liquid-phase PCR, a multiplex PCR, an asymmetric PCR, a solid phase PCR, an RT-PCR, an in situ PCR, an immuno-PCR or a nested PCR.

• a) mindestens einen Primer, der mindestens eine für das nachzuweisende Nukleinsäuretarget spezifische Sequenz umfasst und am 3'-Ende Modifikationen aufweist, die eine enzymatische Verlängerung gestatten, wobei gegebenenfalls einer der Primer mit einem Fluorophor 1 markiert ist,
• b) mindestens eine Sonde, die ein Fluorophor 2 trägt oder mehrere Sonden die jeweils Fluorophor 1 und 2 tragen.

The novel primer / probe system for the detection of nucleic acids comprises:

• a) at least one primer which comprises at least one sequence specific for the nucleic acid target to be detected and has modifications at the 3 'end which permit enzymatic extension, optionally one of the primers being labeled with a fluorophore 1,
• b) at least one probe carrying a fluorophore 2 or more probes carrying each fluorophore 1 and 2.

Preferably come for the different amplicons or polymorphisms different FREE pairs are used.

• a) einen Primer, der mindestens eine für das nachzuweisende Nukleinsäuretarget spezifische Sequenz umfasst und am 3'-Ende Modifikationen aufweist, die eine enzymatische Verlängerung gestatten, wobei dieser Primer mit dem Fluorophor 1 markiert ist,
• b) eine Sonde, die am 3'-Ende das Fluorophor 2 trägt und die für den 3'-verlängerten Komplentärstrang von a) spezifisch ist.

A preferred primer / probe system comprises:

• a) a primer which comprises at least one sequence specific to the nucleic acid target to be detected and has modifications at the 3'-end which permit enzymatic extension, this primer being labeled with the fluorophore 1,
• b) a probe carrying fluorophore 2 at the 3'-end and which is specific for the 3'-extended complement of a).

• a) drei Primer, wobei – ein Primerpaar 1 und 2 als Vorwärts- und Rückwärtsprimer fungieren und Primer 1 mindestens zwei für das nachzuweisende Nukleinsäuretarget spezifische Sequenzen (TBS 1 und 2) umfasst und beide Modifikationen aufweisen, die eine enzymatische Verlängerung gestatten, und – einen Primer 3, der mit dem 5'-Bereich von Primer 1 teilweise oder vollständig identisch ist, ebenfalls enzymatisch verlängerbar ist und Fluorophor 1 trägt, wobei er so konstruiert ist, dass er eine Haarnadel 1 vollständig oder partiell mit sich selbst oder mit komplementären Abschnitten der Primer 1-Sequenz bilden kann,
• b) eine Sonde, die am 3'-Ende Fluorophor 2 trägt und die vollständig für die Targetsequenz spezifisch ist, oder die für die Targetsequenz und Primer 1 spezifisch ist.

Another preferred primer / probe system comprises:

• a) three primers, wherein - a primer pair 1 and 2 function as forward and reverse primer and primer 1 comprises at least two sequences specific for the nucleic acid target to be detected (TBS 1 and 2) and both have modifications that allow enzymatic extension, and - a Primer 3, which is partially or completely identical to the 5 'region of primer 1, is also enzymatically extendible and carries fluorophore 1, being designed to form a hairpin 1 wholly or partially with itself or with complementary portions of the hairpin Can form primer 1 sequence,
• b) a probe which carries fluorophore 2 at the 3 'end and which is completely specific for the target sequence or which is specific for the target sequence and primer 1.

• a) drei Primer, wobei – ein Primerpaar 1 und 2 als Vorwärts- und Rückwärtsprimer fungieren und wobei Primer 1 mindestens zwei für das nachzuweisende Nukleinsäuretarget spezifische Sequenzen (TBS) umfasst und beide Modifikationen aufweisen, die eine enzymatische Verlängerung gestatten, und – Primer 3, der mit dem 5'-Bereich von Primer 1 teilweise oder vollständig identisch ist und ebenfalls enzymatisch verlängerbar ist,
• b) zwei Sonden, die jeweils Fluorophor 1 und 2 tragen und die vollständig für die Targetsequenz spezifisch sind, oder die für die Targetsequenz und Primer 1 spezifisch sind.

The invention may also be a primer / probe system comprising:

• a) three primers, wherein - a primer pair 1 and 2 function as forward and reverse primer and wherein primer 1 comprises at least two sequences specific for the nucleic acid target to be detected (TBS) and both have modifications which allow enzymatic extension, and - primer 3, which is partially or completely identical to the 5 'region of primer 1 and is also enzymatically extendible,
• b) two probes each carrying fluorophore 1 and 2 which are completely specific for the target sequence or specific for the target sequence and primer 1.

• – mindestens ein neuartiges Primer/Sondensystem,
• – geeignete Puffer und ggf. weitere Hilfsstoffe.

The invention also encompasses a reagent kit for the detection of nucleic acids

• At least one novel primer / probe system,
• - Suitable buffers and possibly other auxiliaries.

Buffers and auxiliaries are known to the person skilled in the art. They preferably comprise thermostable polymerase, dNTP mix, PCR buffer and possibly MgCl ₂ .

at the technical realization of nucleic acid assays for Routine investigations are of particular importance, the homogeneous Assay, the multiplex method and the so-called microarrays (also Called gene or biochips). In an inventive Multiplex PCR with the primer / probe system or the kit can be with the method described above several primer sets combine due to different target binding sequences (TBS) the detection of several target sequences in a reaction mixture allow. Qualitative measurements are made by following a previous defined number of amplification cycles is checked, whether the concentration of the coupled nucleic acid molecules exceeds a certain threshold. For one Quantification, this concentration is recorded after each cycle and the number of cycles to reach a certain threshold certainly. This number is a measure of the concentration the sought-after nucleic acid in a sample.

The The principle of an array experiment is all on the array gene samples simultaneously with a nucleic acid sample to hybridize. Crucial here is that by reverse Transcription of RNA from cells or tissue obtained cDNA ideally includes all genes specifically expressed there. Parallel hybridization a nucleic acid sample having a multiplicity of complementary ones Gene sampling on a DNA array results in a characteristic Hybridization pattern with corresponding hybridization intensity. This shows the decisive advantage of this technology other methods for studying gene expression. While conventional methods to study individual Restrict genes, a DNA array provides a complete Gene expression profile of the investigated cell or the investigated Tissue. Particularly interesting is the possibility given here, to study the interaction of different genes. The capture of differences in gene expression is due to the simultaneous Hybridization of normal / immortalized or fetal / adult Cells possible.

With Help of the system and method according to the invention can be used to distinguish multiple target sequences simultaneously the target sequences in the probe binding area, in the primer binding area or in the stem of the hairpin (s) preferably by melting curve analysis be differentiated.

About that In addition, for the simultaneous differentiation of several Target sequences are used several primer / probe systems, which are at least partially or completely target specific Sections and the emission wavelengths of the emitting Fluophors differ so that these can be detected by multicolor fluorescence detection can be detected differentially.

Farther can target specific for the detection of multiple target sequences Primers differ in the 3 'range and thereby completely or incompletely complementary to the target sequence in of the respective sample, thereby detecting or not detecting become.

Farther can be used for simultaneous detection of multiple target sequences different detection systems are used, wherein the target specific primer length polymorphisms to be used and may contain fluorescent labels.

About that In addition, the target sequences can be analyzed by real-time fluorescence analysis, by electrophoresis or by hybridization without amplification the target sequence, by hybridization after amplification of the Target sequence or by hybridization during amplification the target sequence are detected.

Your widespread distribution, the real-time PCR has two properties, They rarely fit together well in the lab routine: it's fast and at the same time precise. Longer than two hours hardly needs a real-time cycler, for the tiniest amounts of DNA to reproduce and quantify. The whole thing works too in high throughput. Up to 384 PCR reactions can be done simultaneously Perform devices whose cycler blocks can be equipped with appropriate microtiter plates.

Real-Time Cycler are numerous known. They allow detection from to preferably to five fluorescent dyes simultaneously for multiplex applications, have heating blocks preferably in a four-pack for 96 or 384 well microtiter plates, show fast heating / cooling rates z. B. 30 PCR cycles in 30 minutes, quantification software, Melting point analysis and much more. The invention System and method are also suitable for high-throughput laboratories, the whole mountains have to work off samples, causing up to 5000 analyzes per day with corresponding automated real-time PCR robots the latest generation are possible. If no value on Real-time quantification is placed and only interest in the endpoint PCR can pass up to 30,000 samples in just under 3 hours through appropriate PCR robots are enabled.

With The system according to the invention can be a prefabricated Real-time PCR kit can be provided, all necessary Contains ingredients. This is the single pipetting of buffer, Nuleotidmix, reporter molecules, polymerases and primer not necessary. Uses for quantification in this (classical) method of real-time PCR, the melting point determination the amplified DNA and the analysis of the fluorescence intensity threshold (CT).

The Determination of an accurate hybridization event of probes the immobilized nucleotide sequences is a crucial step for obtaining optimal results in a performance a microarray.

According to the invention the use of a primer / probe system according to the invention in a device which uses the probes as capture probes immobilized, for the simultaneous detection of target sequences during amplification of the target sequences, the randomly or systematically distributed in the reaction environment Measuring points to which the target sequences via the immobilized Capture probes hybridize, image-captured fluorescence-optically, be decoded and measured and the reaction environment thermocyclic is controllable, if necessary by means of Peltier elements, air flow, water bath, Heat radiation, induction or microwaves.

Prefers become devices for simultaneous detection of target sequences used during the amplification of the target sequences, the one optically transparent vessel bottom and / or Include vascular lids with low intrinsic fluorescence, which the fluorescence optical evaluation during the thermocyclic or allow isothermal performance of the detection reaction.

The solid support may be in any form, and may be made of various materials, particularly including various metals, glass and plastics. Preferred solid supports are nylon membranes, epoxy glass and boron fluorate glass. The advantage of using glass and Plastics may be due to the transparency of the materials, which allows for the production of microscope slides or microplates for parallel high throughput of samples and consequent cost reduction. The microarrays may be in the form of a slide or a microplate (also referred to as a microtiter plate). The microplate is a dished container with multiple (at least two) wells. Microplate-based microarrays are a multi-well microplate placed in the bottom of the microarray biochip. An example of the microplate is a well-known 96-well ELISA microtiter plate.

Farther are solid supports based on self-assembling layer systems as well for the implementation of the present invention suitable. The application can be done using automatic procedures respectively.

Around the assignment of each nucleic acid to a particular site to enable on the solid support is the Continue in different symmetrical, such as rectangular, area the same size divided to the pattern obtained in which the probes immobilized on the solid support are. The pattern allows the analysis of the results in a simplified way, as a simple and specific assignment of respective hybridization events is possible.

Prefers a device is used which in one embodiment the invention contains fluorescence-coded microparticles as measuring points, which carry the capture probes and by gravity, magnetic forces or by chemical or physical bonding to the bottom of the vessel permanently or during the duration of the measurement process are immobilized. The detection takes place inversely through the optical transparent vessel bottom or in reflected light by oil and an optically transparent lid and through the oil coating. As an alternative to oil spilling, a perforated Heated lid can be used in the thermal cycler, which takes the picture not obstructed in reflected light.

The Availability of reactive or non-reactive additives liquid plastics, eg. As thermoplastics, elastomers, Duromers, can be efficiently by wrapping or embedding in linear chain or network forming polymers Taxes. Such polymer-based microcomposites are in the form of Microcapsules with core-shell structure or of microscale matrix particles with largely homogeneous distribution of the components over the particle cross section known. The core of microcapsules can in solid, liquid or gaseous form (hollow spheres) available. For matrix particles are homogeneous and heterogeneous Known systems. According to the invention all microparticles in question, which allow immobilization. The carrier particles are preferably made of a polymer material. Preferred are the following polymeric materials, including but not limited thereto are limited to: polystyrene, polyacrylic acid, polyacrylonitrile, Polyamide, polyacrylamide, polyacrolein, polybutadiene, polycaprolactone, Polycarbonate, polyester, polyethylene, polyethylene terephthalate, polydimethylsiloxane, Polyisoprene, Polyurethane, Polyvinylacetate, Polyvinylchloride, Palyvinylpyridine, Polyvinylbenzyl chloride, polyvinyltoluene, polyvinylidene chloride, polydivinylbenzene, Polymethyl methacrylate, polylactide, polyglycolide, poly (lactide-co-glycolide), Polyanhydride, polyorthoester, polysulfone or combinations thereof. Other polymeric materials, such as carbohydrates, such as carboxymethyl cellulose, Hydroxyethylcellulose, agar, gel, a proteinaceous polymer, Polypeptides, eukaryotic and prokaryotic cells, lipids, metal, Resins, latex, rubber, silicone, such as polydimethyldiphenylsiloxane, Glass, melamine, ceramics, charcoal, kaolinite, bentonite and the like can be used in the same way. In these polymers can also be a magnet or one selected from the group magnetically responsive metal oxide are incorporated, the superparamagnetic, paramagnetic or ferromagnetic metal oxide consists. The particles may be additional functional Contain groups on the surface, such as carboxylates, Esters, alcohols, carbamides, aldehydes, amines, sulfur oxides, nitrogen oxides or halides containing a bond of analytical reactants and / or can facilitate binding of particles to particles.

method for the production of microparticles by means of reactive and non-reactive Particle formation processes are described many times. In the reactive Particle formation occurs the formation of the wall or the matrix in parallel to a polymerization, polycondensation or polyaddition process. In the non-reactive processes, film-forming polymers become direct used in a thermodynamic way for phase separation and be brought to particle formation. In encapsulation procedures solid or liquid core materials are z. B. melamine-formaldehyde resins used. In particular, melamine-formaldehyde resins are diverse and easy to use, and they can help particle formation be applied from aqueous phase. The microcapsule size may vary depending on the reaction conditions, e.g. B. Emulgatorzusatz or dispersing be adjusted. A Another possibility is the hydrothermal crosslinking of Hydroxymethyl-melamine.

Fluorescence-coded microparticles are known in the art and z. In DE 699 07 630 T2 described. The dyes, when more than one dye is used to dye more than one population of particles, are selected to have substantially different emission spectra, preferably with emission maxima greater than 10 nm, more preferably with emission maxima greater than 25 nm, are separated, more preferably separated by more than 50 nm. The dyes can be selected to have emission bands that match commercially available filters or to detect multiple fluorophores with different excitation and emission bands.

According to the Invention are preferably microparticles of melamine, silica, polysulfone and polyethers, most preferably used of melamine. They have i. d. R. a diameter of 0.5-20 microns. The preferred particle size is 5-10 microns.

The preferably used microparticles are characterized by that they preferably consist of 2 layers and a core, the differ fluorescence optics and wherein at least the outer Layer temperature resistant and z. B. consists of melamine. The middle and outer layers are preferably also made of melamine. The middle layer contains particles such as As magnetic particles, with the help of a secure immobilization ensures the particle during the measurement is.

The basic element of a preferably used chip preferably consists of a glass or plastic carrier, as used in microscopy. On such a carrier, the probes (preferably on microparticles) z. B. applied by a robot. This must work with high precision and cleanliness. For each spot, billionths of a liter (usually 0.6 nl) of the oligonucleotide solution is used. After each pass, a thorough self-cleaning of the needles (rinsing, ultrasound) takes place so as not to carry off any impurities. At one end of the oligonucleotides is an amino group (NH ₂ ). This combines permanently with a chemical reaction on the carrier or on the microparticle. These so-called spots have a diameter of preferably 100 μm (0.1 mm). Due to this small amount and the small distance between the individual points (vzw 0.3 mm) have on a glass carrier up to 30,000 spots space. These serve as later docking points for the sample DNA. The unused surface of the support must finally be "blocked" by treatment with a special blocking solution, which may already be included in the final kit, which uses PCR to amplify the specific target genes, coupled with fluorescence, onto the target surface If necessary, one removes unbound DNA through several washes, and a special scanner irradiates the chips with light of a suitable wavelength, which fluoresces the fluorescence and then evaluates the resulting result: The corresponding spots are the result positive.

The Evaluation is done professionally, usually under Inclusion of reference values.

• – Thermocycler, der einen Reaktionsbereich mit mehreren temperierbaren Aufnahmen für Reaktionsgefäße aufweist,
• – Positioniereinrichtung für den Thermocycler,
• – dem Reaktionsbereich zugeordnete Beleuchtungseinrichtungen (LED und/oder Laser) mit denen Anregungslicht strahlbar ist,
• – Detektoreinrichtung, die in Abhängigkeit von einer gemessenen Lichtintensität Messwerte erzeugt, mit Optikeinrichtungen vorzugsweise für Durchlicht und Auflicht,
• – ggf. eine Referenzeinrichtung, die einen Referenzwert erzeugt,
• – Auswerteeinrichtungen.

Devices for carrying out real-time PCR reactions according to the invention on an array, which are fully computer controllable, include:

• - Thermocycler, which has a reaction area with several temperature-sensitive receptacles for reaction vessels,
• Positioning device for the thermal cycler,
• Illumination means (LED and / or laser) assigned to the reaction area, with which excitation light can be radiated,
• Detector device which generates measured values as a function of a measured light intensity, with optical devices preferably for transmitted light and reflected light,
• If appropriate, a reference device which generates a reference value,
• - Evaluation facilities.

For The use of microparticles allows the devices additionally immobilization facilities for z. B. magnetic or paramagnetic particles.

By the use of optically transparent vessel bottom and / or vascular lids with low intrinsic fluorescence, which the fluorescence optical evaluation during the thermocyclic or allow isothermal performance of the detection reaction, Measurements with reflected light and transmitted light fluorescence are possible. In incident light fluorescence commercially available Microtiter plates are used such. Nucleolink or Nucleosorb. The commercially available reaction vessels used can be coated to reduce intrinsic fluorescence become.

The Real-time proofs can be performed on the array under Use of the probe systems according to the invention but also using other probe systems such. B. Molecular Beacon, FREE probes, TaqMan, Scorpions, GenePins or Amplifluor primers are made.

Advantages:

• - Combination of real-time amplification and enables chip detection in the PCR array as a closed system an accurate determination of target sequences, where the multiplex degree only by the degree of miniaturization of array systems used is limited.
• - A primer that is universal for different Amplicon can be used already carries a fluorophore. Thereby can cost the fluorophores of the probes different amplicons are detected (Example 3).
• - The dye-labeled probes and primers must not polymorphic, since the polymorphisms are over varying Target binding sequences of unlabelled primer 1 detected can be. This makes it possible to dye-labeled Probes and primers for maximum detection sensitivity optimize and determine the specificity for the Identification of sequence differences on the optimization is not to limit labeled primer (eg, primer 1, example 3), which reduced the cost of optimizing the PCR systems.
• - Because a fluorescent dye via a primer covalently bound to the amplificate and thus only one probe can be used, higher Sensitivity can be achieved over a higher temperature range stabilizes measured values be achieved, making the system more robust than the classic two-probe system power.
• - As more target binding sequences introduced in primer 1 polymorphisms about the primer specificity to detect without, unlike the classical allele-specific PCR to include the sequence in the priming event and thus to be distorted if necessary. This is a subsequent one Sequencing or cloning of the detected source sequence easily possible.
• - The particles used leave a high number Codes to, because layer 1 (outside), the core and, where appropriate the middle layer can be coded differently. The inclusion of different layer thicknesses or core diameter as a coding parameter with the advantage overall about the same size Being able to use particles continues to increase the number of different coding options high test accuracy. The outer layer is as ring fluorescence, which is sharp against the core by the copolymerization of magnetic particles or other fluorochromes delimit. This makes it possible outer Layer and core using identical fluorochromes or but also to code using the ligand dye. In order to increase the coding possibilities for Microparticle populations considerably, so that complex Detection approaches such as complete genotyping of amplicons, Exons or genes in real-time format is possible.
• - When using fluorescently labeled capture primer or Capture probes for immobilization on the particle surface and unlabelled oligos in the liquid phase Free signals only on immobilized amplificate and not on amplificate arise in the solution, so that disturbing Background fluorescence minimized in favor of test sensitivity becomes.
• - The use of active, based on electric agents Systems for immobilizing microparticles has the advantage that the microparticles during the measuring process can be immobilized and otherwise during the PCR reaction due to thermal turbulence freely movable are. This is the usually occurring in biochips and the sensitivity limittierende mass transport of the Overcome analyte to capture probe / capture primer. intense Shaking, which is difficult to realize technically in a thermocycler is, can be omitted. Flow-dependent differences in the vessel lumen, as they are in place-coded Biochips adversely affect, lose importance.

The invention is schematically in the 1 to 3 shown. The invention also relates to a device system for the combined amplification of target sequences and detection of the amplificates on an array, in particular on a microparticle array and in the 4 and 5 explained in more detail. The fully automatic computer-controlled device is a combination of fluorescence microscope and thermocycler, but can also be designed as a fully integrated solution. The different detection and device systems accordingly 1 to 5 always work for all possible applications, such. B. for the detection of various pathogens, such. As viruses, polymorphisms, to simultaneous detection, in particular for multiplex application and / or microorray.

following the invention is based on drawings and embodiments explained in more detail, but not limited thereto shall be.

1 - Schematic representation of the use of a primer / probe system using a primer and a probe

One Primer 1 carries a covalently bonded fluorophore 1, which binds with its target binding sequence 1 at the 3 'end to the target sequence and is by a DNA polymerase under suitable conditions extended to complement the target. After denaturation the double strand can carry the probe bearing fluorophore 2 at the 3 'end at the complement to the target extended primer 1 hybridize. By irradiation of the reaction mixture with light a defined wavelength is a via fluorophore 1 mediated FREE triggered and the emission also defined wavelength correlates with that in the reaction mixture located amount of template.

2 - Schematic representation of the use of the primer / probe system using a primer and a probe for the detection of a target polymorphism

primer 1 carries a covalently attached to the 5 'end fluorophore. 1 and a target binding sequence 1 and 2, wherein the target binding sequence 2 serves for polymorphism detection. Primer 1 binds to the target binding sequence 1 of its 3 'end to the target sequence in a 1st position and becomes complementary by a DNA polymerase under appropriate conditions extended. After denaturation of the double strand forms the 5 'end of primer 1 via target binding sequence 2 a hairpin with the target sequence 2 of the gene comprising the polymorphism. In addition, the probe binds the fluorophore 2 at the 3 'end contributes to the complementary to the target elongated Immediately follow the formed hairpin. By Irradiation of the reaction mixture is with light of defined wavelength fluorophore 1 mediated FREE and an emission measured by fluorophore 2. This emission correlates with the Reaction amount of template containing the polymorphism includes. For better discrimination of target sequences with polymorphism and of target sequences without, which are only marginally differentiate in their target sequence 2, a melting curve is created. If both target sequences are contained in the reaction mixture, result two peaks in the representation of the 1st derivative of the melting curve.

3 - Schematic representation of the use of the primer / probe system using three primers and a probe for real-time detection and electrophoretic detection of polymorphisms

• (A) Primer 1 comprises two target binding sequences 1 and 2 and a 5 'end which corresponds to the sequence of primer 3. Primer 1 binds to the target binding sequence 1 of its 3'-end to the target sequence of the gene to be detected and is characterized by a DNA polymerase under suitable conditions complementary extended to the target. After denaturation of the double strand binds primer 2 to the extended primer 1 and becomes complementary extended to.
• (B) The resulting duplex is denatured so that (C) Primer 3 with fluorophore 1 at the 5 'end to the complementary End of primer 1 binds and is extended.
• (D) The resulting double strand is denatured, so that in the rehybridization, a hairpin 1 between target binding sequence 2 and target sequence 2 forms. In addition, forms between the 5 'end of the primer 3 sequence and primer 1 a hairpin 2 from. The probe carrying a label with fluorophore 2 at the 3 'end, binds to the complement of the target extended Primer 3 immediately after the hairpin 1. (D1) For the selective amplification of different target sequences different primer 1 in the target binding sequence 1 and by a length marker between primer binding sequence 1 at the 3 'end and primer sequence 3 at its 5 'end of other primers 1 for other target sequences in a multiplex approach.

By Irradiation of the reaction mixture with light of defined wavelength a fluorophore 1 mediated FREI is triggered and an emission of fluorophore 2 at a defined wavelength measured. This emission correlates with that in the reaction mixture the amount of template comprising the polymorphism. For better discrimination of target sequences with polymorphism and of target sequences without, which are only marginally differentiate in their target sequence 2, a melting curve is created. If both target sequences are contained in the reaction mixture, result two peaks in the representation of the 1st derivative of the melting curve. Due to different length markers in different Primers 1 (see D1?) May additionally contain the amplicons also detected by gel electrophoresis or mass spectroscopy or be differentiated.

4a : Schematic representation of the detection of several target sequences with a combination of real-time PCR and microparticle array

When solid phase for the hybridization of amplificate different or one and the same nucleotide sequence different in their sequence, labeled with fluorophore 2 at the 3 'end and covalent via its 5' end immobilized capture probes (consisting of DNA, PTO-DNA or LNA) serve fluorescence-coded microparticles.

The microparticles used are known per se to the person skilled in the art and are characterized by the fact that they are preferably made of 2 layers and consist of a nucleus that differ fluorescence optics and where at least the outer layer temperature resistant is and z. B. consists of melamine. The middle and outer Layer preferably also consist of melamine. The middle layer Contains particles such. B. magnetic particles, with their help a safe immobilization of the particles during the measurement is guaranteed.

The invention is used, for example, in an array as follows: particles from particle population 1 (left half of the image):
The core contains z. B. 100 parts of aminocoumarin and z. B. 20 parts Nile Blue (100/20). The middle layer contains z. B. 100 parts of fluorescein and the outer layer contains z. B. 50 parts of aminocoumarin and z. B. 100 parts Nile Blue.

dye 1 in the hybridized amplificate is z. B. fluorescein as a fluorophore 1 and 2 dye at the 3 'end of the capture probe is z. B. rhodamine as Fluorophore 2, resulting in a FREE pair.

Particles from particle population 2 (right Figure half):

Of the Kern contains z. B. 100 parts of aminocoumarin and z. Eg 20 Parts Nile Blue (100/20). The middle layer contains z. B. 20 parts fluorescein and the outer layer contains z. B. 100 parts of aminocoumarin and z. B. 100 parts Nile blue.

If no amplificate hybridizes, no FREI pair between dye 1 (eg fluorescein) and dye 2 at the 3 'end of the capture probe (e.g. As rhodamine) arise. After the amplified sample with the Microparticles was brought into contact or cyclically during the amplification in the same reaction vessel the Amplificate is formed before the measurement by means of microscopic Optics to hybridize the amplification to different To measure microparticle populations, all microparticles sedimented in the beginning or by a permanent or electric stomach at the bottom of the vessel fixed.

By Fluorescence spectroscopy identifies the microparticle populations and thus determine which capture probe attached to which microparticle and whether fluorescent amplificate hybridized. To the excitation of the exemplified aminocoumarin occurs at a wavelength of 350 nm, around the first coding fluorescence the particles in the outer and in the inner To detect shift. Subsequently, z. B. fluorescein excited at a wavelength of 480 nm and at 520 nm measured to the second coding / reference fluorescence of the particles and simultaneously using a beam splitter prism (at 560 nm) to the FREI signal (here, for example, the rhodamine fluorescence) of the hybridized amplificate. By following Excitation at 680 nm and detection at 720 nm is the example called Nile Blue excited and detected to the second coding fluorescence of the core or outer layer.

The Billing of spatially resolved fluorescence signals takes place according to the following rules:

Core:

• - Determination of the quotient 1 from Aminocoumarin and Nile Blue fluorescence
• - Determination of the core diameter

Middle layer:

• - Determination of the quotient 2 from Fluorescein fluorescence and quotients 1
• Determination of the thickness of layer 2

Outer layer:

• - Determination of the quotient 3 from Aminocoumarin and Nile Blue fluorescence
• Determination of the thickness of layer 3

To These rules can be the microparticles of the present Example can be distinguished by quotient 3 both particles are measurably different.

Quotient 3

• Particle population 1: 0.5
• Particle population 2: 1

The rest Quotients and all other diameters and layer thicknesses of the particles do not differ.

The Amplificate fluorescence, e.g. B. the emission of rhodamine free signal corresponds, can be output as intensity or against a constant parameter such as the particle total diameter the z. B. detected by the fluorescein or rhodamine signal can be referenced.

Becomes the (eg rhodamine) fluorescence during each PCR cycle measured, if PCR and detection occur simultaneously, is the sample evaluated as positive if the FRET signal breaks the background intensity (Threshold Cycle). The more amplified target sequence in the sample is, the higher the signal intensity or the earlier the threshold cycle is reached. Especially the determination of the threshold cycle allows accurate multiplex quantification of target sequences too. Alternatively, for all microparticles At the end of the PCR, a melting curve is recorded (see also Example 2).

4b : Schematic representation of the detection of a polymorphism on amplificate which is immobilized on the surface of microparticles by solid-phase PCR (on a polymorphism in the factor 2 gene)

When solid phase for the immobilization of amplificate different or one and the same nucleotide sequence different in their sequence, with covalently immobilized capture primer over its 5 'end serve fluorescence-coded microparticles. The used microparticles are characterized by the fact that they are preferably made of 2 layers and a nucleus that differ fluorescence optics and wherein at least the outer layer is temperature resistant is and z. B. consists of melamine. The middle and outer layers here are also made of melamine. The middle layer contains Particles such. B. magnetic particles, with the help of a safe Immobilization of the particles ensured during the measurement is. For the sake of simplicity, here only one particle from particle population 1, wherein for the detection of multiple polymorphisms also several particle populations each with target specific Capture probes can be used.

Particle from particle population 1,

Of the Core contains 100 parts of aminocoumarin and 20 parts of Nile Blue (100/20). The middle layer contains 100 parts fluorescein and the outer layer contains 50 parts Aminocoumarin and 100 parts Nile Blue.

dye 1 in the immobilized amplificate is fluorescein and dye 2 rhodamine is at the 3 'end of the capture probe, so a FREE pair arises.

After this the sample contacted with the microparticles and thermocyclic was amplified, before the measurement by means of microscopic Optics to hybridize the amplification to different To measure microparticle populations, all microparticles in the batch sedimented or as here in the example by a permanent or electric magnet fixed to the bottom of the vessel.

Fluorescence spectroscopy identifies the microparticle populations spatially resolved and thus determines which capture primer is on which microparticle and whether fluorescence amplificate has been immobilized. For this purpose, the excitation of the aminocoumarin takes place at a wavelength of 350 nm in order to detect the first coding fluorescence of the particles in the outer and in the inner layer. Subsequently, fluorescein is excited at a wavelength of 480 nm and measured at 520 nm to the second To determine coding / reference fluorescence of the particles and simultaneously, in dehybridized probe, to detect immobilized amplicon. For the analysis of any polymorphisms, it is then spatially resolved at 480 nm excitation wavelength and at 560 nm emission wavelength to detect the FREI signal (rhodamine fluorescence) of the probe hybridized to the amplificate via a temperature step gradient. By subsequent excitation at 680 nm and detection at 720 nm, Nile blue is excited and detected to determine the second coding fluorescence of the core and the outer layer, respectively. The offsetting of the spatially resolved fluorescence signals takes place according to the following rules:

Core:

• - Determination of the quotient 1 from Aminocoumarin and Nile Blue fluorescence
• - Determination of the core diameter

Middle layer:

• - Determination of the quotient 2 from Fluorescein fluorescence and quotients 1
• Determination of the thickness of layer 2

Outer layer:

• - Determination of the quotient 3 from Aminocoumarin and Nile Blue fluorescence
• Determination of the thickness of layer 3

After these Rules may be the microparticles of the present example be distinguished by quotient 3 of both particles measurably different.

Quotient 3

• Particle population 1: 0.5
• Particle population 2: 1

The rest Quotients and all other diameters and layer thicknesses of the particles do not differ.

The Amplificate fluorescence indicating the emission of rhodamine free signal equivalent can be spent as intensity or against a constant parameter such as the total particle diameter the detected via the fluorescein or rhodamine signal can be referenced.

Although the detection of Nukleiäurepolymorphismen also accordingly 4a can be carried out, it is preferable to proceed as follows. After sufficient amplificate has been immobilized on the microparticles by the PCR, a melting curve is run, ie, in the critical temperature range between 50 ° C and 70 ° C, measurements are carried out in the step gradient with a distance between steps of 2 ° C. Alternatively, it is sufficient to carry out a two-point measurement in the region of the melting curve peak of each amplicon (probe area or stem area of the hairpin) or else to detect the signals via the step gradient in smaller steps or continuously. The signals acquired for a microparticle population can be averaged or individually analyzed and displayed.

He follows the melting point determination for a polymorphism on a particle at two points, then the absolute values of the signals are compared with each other or a quotient two signals are formed. If a signal is detected, means this is the presence of the corresponding polymorphism in the sample.

The Signal arises when sufficient amplificate over the capture primer immobilized on the particle surface the probe is hybridized to the amplificate and the 5 'end of the Target specific section of the catch primer under training a hairpin with the nucleotide position (s) of the polymorphism hybridized. Mismatches in the trunk of the hairpin can over the melting point analysis are detected. While the amplicon shown in the diagram without mismatch a hairpin forms, occurs with variation of the stem sequence of the hairpin in the polymorphic Nucleotide at this point on a mismatch, so that in the same temperature, the hairpin does not form.

By Combination of several particle populations in a detection reaction, may have multiple amplificates of different or the same Gene fragments are examined simultaneously for polymorphisms.

5 : Schematic representation of a device system (A) and a reaction environment (B) for the combined amplification of target sequences and detection of the amplicons at the microparticle array

• (A) The fully automatic computer-controlled device is a combination of fluorescence microscope and thermocycler based on its performance parameters. On a tripod 6 with motorized (z-drive), the optical components are at least consisting of a long-distance optics 5 equipped with a 20 mm objective and elements for beam guidance and image composition for fluorescence detection. The excitation takes place via at least three different LEDs 3 or laser 4 in the spectral ranges 350-400 nm; 450-500 nm; 550-600 nm. The specific wavelengths of the excitation light depend on the fluorescent dyes used and, if required, are matched by suitable filters 2 fine-tuned. The light emitted by the fluorescent dyes is emitted through appropriate emission filters 1 separated from the excitation light and recorded by a sensitized CCD camera 3 , The stage has an x / y drive with an accuracy of less than 0.1 mm and accommodates the thermocycler with sample holder (here for 96 reaction vessels with 0.2 ml capacity). Among the reaction vessels, which have a planar bottom, is a magnet 11 (Permanent agent or an electromagnet) positioned so that paramagnetic microparticles are well immobilized during the measurement process. An automatic computer control includes the PCR program and thus the temperature profile of the PCR reaction, the filter changer, the CCD camera, the image acquisition and image processing, the xx / y / z drive and the control of the excitation light, so that during the PCR reaction as well as the output of the amplificate fluorescence for the different particle populations can be guaranteed.
• (B) As reaction vessels z. B. Nucleosorb 8-bar 12 used, which are closed with a glass cover glass.
• (C) The PCR master mix is used together with the sample DNA 16 pipetted into the reaction vessel and covered with oil. Thereafter, the reaction vessel is closed with a coverslip and the PCR / measurement process started. As the PCR cycles proceed, fluorescence measurements are preferably at the annealing stage under isothermal conditions. Alternatively, a melting curve is recorded at the end of the PCR. The microparticles are preferably permanently immobilized during the PCR by covalent coupling, sedimentation or magnetic attraction. When electromagnets are used, the particles can be immobilized or free-floating during the PCR and are re-immobilized for each measurement.

1

emission filter

2

filter for excitation light

3

CCD camera

4

LED or laser for stimulation

5

Long-distance optics

6

tripod

7

z drive

8th

thermocycler

9

x / y-drive

10

crop mark

11

magnet

12

Nukleosorb 8 bars

13

Reaction vessel receiving in the thermoblock

14

cover

15

Oil overlay

16

Master Mix + Sample DNA

example 1

Detection of herpes simplex virus in a sample A volume of 5 μl of smears from lip blisters of purified sample DNA is added to 15 μl of master mix of the following composition: Primer 1 400nM Primer 2 300 nM probe 100nM in Taq DNA polymerase, nucleotides, magnesium chloride, Tris / HCl buffer and Tween 20.

The reaction batch is pipetted into a LightCycler capillary and then transferred to the LightCycler and thermocyclically amplified. The procedure is schematic in 1 shown. Primer 1 carries a covalently bound fluorescein at the 4th nucleotide from the 3 'end, binds with its target binding sequence 1 at the 3' end to the target sequence of the herpes simplex virus genome and becomes complementary by a DNA polymerase under appropriate conditions extended to the target. After denaturation of the double strand, the probe bearing a rhodamine label at the 3 'end can hybridize to the primer extended to complement the target. By irradiation of the reaction mixture with light of a wavelength of 480 nm, a fluorescein-mediated FREI is triggered and the emission at 560 nm correlates with the amount of template present in the reaction mixture.

To Completion of the amplification is for all samples, which have a sufficiently high measurement signal, a melting curve recorded.

sequences

• Primer 1: 5'-CAT CAC CGA CCC GGA GAG GGA C-3 '
• G at position 20 (shaded gray) enters covalently FITC molecule.
• Primer 2: 5'-GGG CCA GGC GCT TGT TGG TGT A-3 '
• Probe: 5'-GGTGAGGACAAAGTCCTGGAT-3'-Rhodamine

Example 2:

Detection of a factor 2 polymorphism

A volume of 5 μl of patient blood purified sample DNA is added to 15 μl of master mix of the following composition: Primer 1 (FITC-modified) 300 nM Primer 2 300 nM probe 100nM in Taq DNA polymerase, nucleotides, magnesium chloride, Tris / HCl buffer and Tween 20.

The reaction batch is pipetted into a LightCycler capillary and then transferred to the LightCycler and thermocyclically amplified. The procedure is schematic in 2 shown. Primer 1 carries a covalently bound fluorescein at the 5 'end and a target binding sequence 2, binds with the target binding sequence 1 of its 3' end to the target sequence of the human factor 2 gene and is extended by a DNA polymerase under suitable conditions complementary to the target , After denaturation of the double strand, the 5 'end of primer 1 forms a hairpin with the target sequence two comprising the polymorphism. In addition, the probe, which carries a rhodamine label at the 3 'end, binds to the complementary to the target-extended primer immediately following the hairpin. Irradiation of the reaction mixture with 480 nm wavelength light initiates a fluorescein-mediated FREI and measures emission of rhodamine at 560 nm. This emission correlates with the amount of template in the reaction mixture which comprises the polymorphism. To better distinguish target sequences with polymorphism and target sequences without, which differ only slightly in their target sequence 2, a melting curve is created. If both target sequences are contained in the reaction mixture, two peaks result in the representation of the first derivative of the melting curve.

To Completion of the amplification is for all samples, which have a sufficiently high measurement signal, a melting curve recorded.

alternative is the distinction of the formed amplicons due to the Length marker in primer 1b electrophoretically possible.

sequences

• Primer 1 (FITC-modified): FITC-5'-ggctcgctccaataaa agtgactctc a-3 '
• Primer 2: 5'-ccaggtggtggattcttaagtc-3 '
• Probe: 5'-gctgcccatgaatagcactgggagcatt-3'-rhodamine

Example 3:

Simultaneous detection of herpes simplex DNS and internal standard in a sample

A volume of 5 μl of patient blood purified sample DNA is added to 15 μl of master mix of the following composition: Primer 1a 10nM Primer 1b 10nM Primer 2 300 nM Primer 3 (FITC-modified) 300 nM Probe 1 100nM Probe 2 100nM Internal Stan dard 100 fm in Taq DNA polymerase, nucleotides, magnesium chloride, Tris / HCl buffer and Tween 20.

The reaction batch is pipetted into a LightCycler capillary and then transferred to the LightCycler and thermocyclically amplified. The procedure is schematic in 3 shown. Upon completion of the amplification, a melting curve is recorded for all samples having a sufficiently high measurement signal to detect amplicon of the internal standard and, for HSV positive samples, HSV amplicon.

sequences

• Primer 1a: 5'-actgcacgctcacacggacg gtgcagtaagtcctgg CAT CAC CGA CCC GGA GAG GGA C-3 '
• Primer 1b: 5'-actgcacgctcacacggacgttgttgttgt tgttgttgtt gttgttgttg ttgttgttgt gcgtgcagt gcggtccac
• CATCACCGAC CCGGACAGCC CA-3 '
• Primer 2: 5'-GGG CCA GGC GCT TGT TGG TGT A-3 '
• Primer 3: FITC-5'-actgcacgctcacacggacg-3 '
• Probe 1: 5'-tcagttcggcggtgaggaca-3'-rhodamine
• Internal standard: 5'-CATCACCGAC CCGGACAGCC CACCAGCGTG GACCGCTGT CCT CAC CGC CGA ACT GA GGCCAG GCGGTGAAGG GCAATCAGCT GTTGCCCGTC TCGCTGGTGA AAAGAAAAAC CACCCTGGCG CCCAATACGC AAACCGCCTA CACCAACAAG CGCCTGGCCC-3 '

Example 4:

Combination of probe system for real-time PCR with a microparticle array for the simultaneous detection of different target sequences of a factor 2 and a factor 5 polymorphism. The procedure is schematic in 4 shown.

A volume of 5 μl of patient blood purified sample DNA is added to 15 μl master mix of the following composition (oligonucleotide sequences for factor 5 and factor 2 see below): Primer 1 (liquid phase) 300 nM Primer 1 (FITC-labeled capture primer for coating the microparticles by EDC coupling: 100nM Primer 2 (liquid phase) 300 nM Probe (rhodamine-labeled) 100nM in Taq DNA polymerase, nucleotides, magnesium chloride, Tris / HCl buffer and Tween 20.

The reaction mixture is pipetted in a nucleosorb (black) -8er bar and then in the thermocycler of the measuring station ( 5 ), covered with oil, closed with a glass cover glass, thermocyclically amplified and detected after the amplification by melting curve analysis (see 4 and 5 ).

Microparticles:

It will be the particles with the properties 4a coated accordingly with capture primer for specific for the amplicon sequences of factor 5 and factor 2 gene by means of EDC coupling.

sequences

Factor 2:

• Primer 1 (FITC-modified, for solid-phase immobilization): Aminohexyl-5'-ttgttgttgttgttgggctcgctccaataaa agtgactctc a-3 '
• Gray background: polymorphism
• Bold italics: FITC marking

• Primer 1 (liquid phase): ggctcgctccaataaaagtgactctca-3 '
• Gray background: polymorphism

• Primer 2 (liquid phase): 5'-ccaggtggtggattcttaagtc-3 '
• Probe: 5'-gctgcccatgaatagcactgggagcatt-3'-rhodamine

Factor 5:

• Primer 1 (FITC-modified, for solid-phase immobilization): Aminohexyl-5'-ttgttgttgttgaggaccggagatccctggacaggc-3 '
• Gray background: polymorphism
• Bold italics: FITC marking

• Primer 1 (liquid phase): Aggaccggagatccctggacaggc 5'-3 '
• Gray background: polymorphism

• Primer 2 (liquid phase): 5'-cttgaaggaaa tgccccatta tttag-3 '
• Probe: 5'-ctgaaaggtt acttcaagga caaaatacct gtat-3'-rhodamine

Legend to the pictures

sequences

• Primer 1: 5'-CAT CAC CGA CCC GGA GAG GGA C-3 '
• G at position 20 (shaded gray) enters covalently FITC molecule.
• Primer 2: 5'-GGG CCA GGC GCT TGT TGG TGT A-3 '
• Probe: 5'-GGTGAGGACAAAGTCCTGGAT-3'-Rhodamine

Example 2:

Detection of a factor 2 polymorphism

A volume of 5 μl of patient blood purified sample DNA is added to 15 μl of master mix of the following composition: Primer 1 (FITC-modified) 300 nM Primer 2 300 nM probe 100nM in Taq DNA polymerase, nucleotides, magnesium chloride, Tris / HCl buffer and Tween 20.

The reaction batch is pipetted into a LightCycler capillary and then transferred to the LightCycler and thermocyclically amplified. The procedure is schematic in 2 shown. Primer 1 carries a covalently bound fluorescein at the 5 'end and a target binding sequence 2, binds with the target binding sequence 1 of its 3' end to the target sequence of the human factor 2 gene and is extended by a DNA polymerase under suitable conditions complementary to the target , After denaturation of the double strand, the 5 'end of primer 1 forms a hairpin with the target sequence two comprising the polymorphism. In addition, the probe, which carries a rhodamine label at the 3 'end, binds to the complementary to the target-extended primer immediately following the hairpin. Irradiation of the reaction mixture with 480 nm wavelength light initiates a fluorescein-mediated FREI and measures emission of rhodamine at 560 nm. This emission correlates with the amount of template in the reaction mixture which comprises the polymorphism. To better distinguish target sequences with polymorphism and target sequences without, which differ only slightly in their target sequence 2, a melting curve is created. If both target sequences are contained in the reaction mixture, two peaks result in the representation of the first derivative of the melting curve.

To Completion of the amplification is for all samples, which have a sufficiently high measurement signal, a melting curve recorded.

alternative is the distinction of the formed amplicons due to the Length marker in primer 1b electrophoretically possible.

sequences

• Primer 1 (FITC-modified): FITC-5'-ggctcgctccaataaa agtgactctc a-3 '
• Primer 2: 5'-ccaggtggtggattcttaagtc-3 '
• Probe: 5'-gctgcccatgaatagcactgggagcatt-3'-rhodamine

Example 3:

Simultaneous detection of herpes simplex DNS and internal standard in a sample

A volume of 5 μl of patient blood purified sample DNA is added to 15 μl of master mix of the following composition: Primer 1a 10nM Primer 1b 10nM Primer 2 300 nM Primer 3 (FITC-modified) 300 nM Probe 1 100nM Probe 2 100nM Internal Stan dard 100 fm in Taq DNA polymerase, nucleotides, magnesium chloride, Tris / HCl buffer and Tween 20.

The reaction batch is pipetted into a LightCycler capillary and then transferred to the LightCycler and thermocyclically amplified. The procedure is schematic in 3 shown. Upon completion of the amplification, a melting curve is recorded for all samples having a sufficiently high measurement signal to detect amplicon of the internal standard and, for HSV positive samples, HSV amplicon.

sequences

• Primer 1a: 5'-actgcacgctcacacggacg gtgcagtaagtcctgg CAT CAC CGA CCC GGA GAG GGA C-3 '
• Primer 1b: 5'-actgcacgctcacacggacgttgttgttgt tgttgttgtt gttgttgttg ttgttgttgt gcgtgcagt gcggtccac CATCACCGAC CCGGACAGCC CA-3 '
• Primer 2: 5'-GGG CCA GGC GCT TGT TGG TGT A-3 '

• Primer 3: FITC-5'-actgcacgctcacacggacg-3 '
• Probe 1: 5'-tcagttcggcggtgaggaca-3'-rhodamine
• Internal standard: 5'-CATCACCGAC CCGGACAGCC CACCAGCGTG GACCGCTGT CCT CAC CGC CGA ACT GA GGCCAG GCGGTGAAGG GCAATCAGCT GTTGCCCGTC TCGCTGGTGA AAAGAAAAAC CACCCTGGCG CCCAATACGC AAACCGCCTA CACCAACAAG CGCCTGGCCC-3 '

Example 4:

Combination of probe system for real-time PCR with a microparticle array for the simultaneous detection of different target sequences of a factor 2 and a factor 5 polymorphism. The procedure is schematic in 4 shown.

A volume of 5 μl of patient blood purified sample DNA is added to 15 μl master mix of the following composition (oligonucleotide sequences for factor 5 and factor 2 see below): Primer 1 (liquid phase) 300 nM Primer 1 (FITC-labeled capture primer for coating the microparticles by EDC coupling: 100nM Primer 2 (liquid phase) 300 nM Probe (rhodamine-labeled) 100nM in Taq DNA polymerase, nucleotides, magnesium chloride, Tris / HCl buffer and Tween 20.

The reaction mixture is pipetted in a nucleosorb (black) -8er bar and then into the thermocycler of the measuring station ( 5 ), covered with oil, closed with a glass cover glass, thermocyclically amplified and detected after the amplification by melting curve analysis (see 4 and 5 ).

Microparticles:

It will be the particles with the properties 4a coated accordingly with capture primer for specific for the amplicon sequences of factor 5 and factor 2 gene by means of EDC coupling.

sequences

Factor 2:

• Primer 1 (FITC-modified, for solid-phase immobilization): Aminohexyl-5'-ttgttgttgttgttgggctcgctccaataaa agtgactctc a-3 '
• Gray background: polymorphism
• Bold italics: FITC marking

• Primer 1 (liquid phase): ggctcgctccaataaaagtgactctca-3 '
• Gray background: polymorphism

• Primer 2 (liquid phase): 5'-ccaggtggtggattcttaagtc-3 '
• Probe: 5'-gctgcccatgaatagcactgggagcatt-3'-rhodamine

Factor 5:

• Primer 1 (FITC-modified, for solid-phase immobilization): Aminohexyl-5'-ttgttgttgttgaggaccggagatccctggacaggc-3 '
• Gray background: polymorphism
• Bold italics: FITC marking

• Primer 1 (liquid phase): 5'-aggaccggagatccctggacaggc-3 ''
• Gray background: polymorphism

• Primer 2 (liquid phase): 5'-cttgaaggaaa tgccccatta tttag-3 '
• Probe: 5'-ctgaaaggtt acttcaagga caaaatacct gtat-3'-rhodamine

SEQUENCE LISTING

QUOTES INCLUDE IN THE DESCRIPTION

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Cited patent literature

• - US 6150097 A [0003]
• - DE 69907630 T2 [0044]

Claims (32)

Method for "real-time" detection of Nucleic acid targets as products of a nucleic acid amplification reaction by means of a primer / probe system comprising the steps: a) Use of at least one primer containing at least one for the nucleic acid-specific sequence to be amplified - target binding sequence (s) (TBS) - includes and carries at the 3'-end modifications, which complements one enzymatic extension allow to the target, b) Introduction of a fluorophore 1, c) for the detection use of at least one probe, the at least at the 3 'end carries a fluorophore 2, wherein the at least one probe with the complementary strand of the Target sequence at the 3'-end extended primer or at Using multiple primers can hybridize to the target strand, in which one of the two fluorophores by light of suitable wavelength is excited, so that a FREI takes place between fluorophore 1 and 2, which is detected by fluorescence optics. Method according to claim 1, characterized in that that the at least one primer at the 5 'end additionally one to the target unspecific tail. Method according to claim 1 or 2, characterized that the fluorophore 1 is attached to one primer or to another Probe is located or introduced. Method according to one of claims 1 to 3, characterized in that the fluorophores 1 and 2 are covalent bound to primers and probes. Method according to one of claims 1 to 4, characterized in that the at least one primer a sequence specific for the target sequence to be amplified (TBS), in the 5 'region partially complementary to the 3' region so that it forms a hairpin. Method according to one of claims 1 to 5, characterized in that the fluorophore 1 to the specific or unspecific part of a first primer or on another Primer present. Method according to one of claims 1 to 6, characterized in that the at least one primer is the fluorophore at the 5 'end, wherein for the detection of the target, the primer on 5'-end partially or completely a hairpin with it itself or the complementary to the target extended Forms primer sequence. Method according to one of claims 1 to 7, characterized in that at least three primers used and that includes the following steps: - in a first reaction use of a primer pair 1 and 2, wherein the forward primer 1 at least one target binding sequence 1 (TBS 1) and and at least one target binding sequence 2 (TBS 2) comprises, so that TBS 1 binds to the target to be amplified, then primer 1 complementary to the target is extended, reverse primer 2 on the complementary target binds and also prolongs becomes, In a further reaction between primers 3 which is identical or partial to the 5 'region of primer 1 is identical and optionally the fluorophore 1 at the 5'-end carries, and primer 2 the amplification takes place, wherein Primer 3 is designed to complete a hairpin 1 or partially with itself or with complementary ones Sections of the primer 1 sequence forms, and wherein the at least a TBS 2 to at least one other target binding section binds (s) between extended primer 1 and the complementary sequence of primer 2 is (s), whereby a hairpin 2 is created, - then one with the fluorophore 2 labeled probe is contacted with the amplificate and that the FREI signal formed with the fluorophore 1 is detected. Method according to claim 8, characterized in that that primer 3 carries fluorophore 1 and the probe with fluorophore 2 at the 3 'end completely hybridized to the Targetsbstanz. Method according to claim 8, characterized in that that primer 3 carries fluorophore 1 and the probe with fluorophore 2 at the 3 'end via hairpin 2 and primer 1 to the target substance hybridized. Method according to claim 8, characterized in that that two probes are used, each containing fluorophore 1 and 2, where probe 1 is primer 1 specific and probe 2 is target specific is. Method according to one of claims 1 to 11, characterized in that the Fluophore 1 and 2 are selected from fluorescent dye molecules, wherein the light which is absorbed and remitted by the first fluorophore is reabsorbed and remitted by the second second fluorophore spatially close to the first fluorophore after binding of the probe (s) to the first fluorophore. Process according to claim 12, characterized in that the following fluorophores are used: Fluorophore 1 Fluorophore 2 fluorescein LC Red 640 or LC Red 705 amino coumarin fluorescein fluorescein Rhodamine or tetramethylrhodamine fluorescein Cy3 Cy3 Cy5 Cy3 tetramethylrhodamine europium Cy5 terbium rhodamine Bodipy Bodipy dansyl fluorescein naphthalene dansyl ruthenium Cy5. Method according to one of claims 1 to 13, characterized in that the amplification reaction a homogeneous liquid-phase PCR, a multiplex PCR, an asymmetric PCR, a solid-phase PCR, an RT-PCR, an in situ PCR, an immuno-PCR or a nested PCR. Method according to one of claims 1 to 14, characterized in that for the simultaneous distinction of several target sequences, the target sequences in the probe binding area, in the primer binding region, in the stem of the hairpin (s) by melting curve analysis or by using multiple fluorophores or length markers be differentiated. Primer / probe system for the detection of nucleic acids full: a) at least one primer containing at least one specific for the nucleic acid target to be detected Sequence comprises and at the 3 'end has modifications that a allow enzymatic extension, optionally one of the primers is labeled with a fluorophore 1, b) at least a probe, at least one carrying a fluorophore 2 and optionally another, the fluorophore 1 has. The primer / probe system of claim 16 comprising: a) a primer containing at least one for the Nucleic acid target specific sequence and at the 3 'end Has modifications that allow enzymatic extension allow this primer to be labeled with the fluorophore 1, b) a probe carrying the fluorophore 2 at the 3 'end and the for the 3'-extended complement strand of a) is specific. The primer / probe system of claim 16 comprising: a) three primers, where A primer pair 1 and 2 as forward and reverse primers, and wherein primers 1 at least two for the nucleic acid target to be detected comprises specific sequences and both have modifications, which allow an enzymatic extension, and - primer 3, which is partial or complete with the 5 'region of primer 1 is identical, is also enzymatically extendable and Fluorophore 1 carries, being designed so that it a hairpin 1 completely or partially with itself, with complementary sections of the primer 1 sequence or can form complementary sections of the target, b) a probe which carries fluorophore 2 at the 3 'end and which is complete is specific for the target sequence, or for the target sequence and primer 1 is specific. A primer / probe system according to claim 16 comprising: a) three primers, wherein - a primer pair 1 and 2 function as forward and reverse primer and wherein primer 1 comprises at least two sequences specific for the nucleic acid target to be detected and both have modifications permitting enzymatic extension, and Primer 3, which is partially or completely identical to the 5 'region of primer 1, is also enzymatically extendable, b) two probes, each carrying fluorophore 1 and 2, which are completely specific for the target sequence, or which are specific for the target sequence Target sequence and primer 1 are specific. Reagent kit for the detection of nucleic acids full a) at least one primer / probe system according to a of claims 16 to 19, b) suitable buffers and, if appropriate other auxiliaries. Use of a primer / probe system after a of claims 16 to 19 or a reagent kit according to claim 20 for the simultaneous differentiation of several target sequences, wherein several primer / probe systems are used which are at least partially or completely in the target sequence specific Sections and the emission wavelengths of the emitting Fluophors differ so that these can be detected by multicolor fluorescence detection can be detected differentially. Use of a primer / probe system after a of claims 16 to 19 or a reagent kit according to claim 20 for the detection of multiple target sequences, wherein the target-specific Primers differ in the 3 'range and thereby completely or incompletely complementary to the target sequence in the respective sample and thereby detected or not detected become. Use of a primer / probe system after a of claims 16 to 19 or a reagent kit according to claim 20 for the simultaneous detection of multiple target sequences under Use of different detection systems, wherein the used target-specific primer length polymorphisms and may contain fluorescent labels. Use of a primer / probe system after a of claims 16 to 19 or a reagent kit according to claim 20, where the target sequences are determined by real-time fluorescence analysis, by electrophoresis or by hybridization without amplification the target sequence, by hybridization after amplification of the Target sequence or by hybridization during amplification the target sequence are detected. Use of a primer / probe system after a of claims 16 to 19 or a reagent kit according to claim 20, in a microarray, which uses the probes as capture probes or a subset of a primer immobilized as capture primer comprises. Device system for implementation Real-time PCR reactions that can be controlled fully automatically by computer is, comprising: - Thermocycler, which has a reaction area with several temperature-sensitive receptacles for reaction vessels, - possibly an immobilization system for microparticles, - the Reaction area associated lighting devices (LED and / or Laser) with which excitation light can be radiated, - detector device, depending on a measured light intensity Generated measured values, with optical devices, - possibly. a reference device that generates a reference value, - evaluation facilities, in which Reaction vessels are used which have an optical transparent vessel bottom and / or tube lid with low intrinsic fluorescence, which are the fluorescence optical Evaluation during the thermocyclic or isothermal Enable the detection reaction. Device system for the simultaneous detection of Target sequences during the amplification of the target sequences according to claim 26, characterized in that the reaction vessels represent a microarray which immobilizes the probes as capture probes distributed randomly or systematically in the reaction environment Measuring points to which the target sequences on the immobilized capture probes hybridize, imaging fluorescence optics be detected, decoded and measured and the reaction environment thermocyclic is controllable, preferably by means of Peltier elements, air flow, Water bath, heat radiation or microwaves. Device system according to claim 26 or 27, characterized in that the reaction vessels comprise measuring points, with microparticles, preferably fluorescence-coded, being used as measuring points, carry the capture probes and are immobilized by gravity, magnetic forces or by chemical or physical bonding to the vessel bottom permanently or for the duration of the measurement process. Device system according to one of the claims 26 to 28, characterized in that the vessel bottom is transparent or non-transparent and the vessel lid transparent, with the measurement through the transparent vessel bottom from below or from above and through the non-transparent vessel bottom from above. Reaction vessel for PCR with transparent Vessel bottom. Use of a device system according to a of claims 26 to 30 for fluorescence optical evaluation during thermocyclic or isothermal conduct the detection reaction by incident light and transmitted light fluorescence. Immuno-PCR in the multiplex immune reaction under Use non-fluorescent beads, porous or non-porous flat or rod-shaped carrier materials takes place and in a second reaction step the analyte-specific Amplification products of fluorescence-coded microparticles accordingly Claims 1-27 be demonstrated.