DE102014010333A1 - Method for detecting at least one target analyte in a sample - Google Patents

Abstract

The invention relates to a method for detecting at least one target analyte in a sample, wherein A) a complex immobilized on a solid surface is formed comprising a) a scavenger binding molecule that specifically binds the target analyte; b) the target analyte to be detected; c) a reporter binding molecule, wherein the reporter binding molecule (aa) has a binding molecule and (bb) a double-stranded reporter DNA bound thereto, the reporter DNA in at least one of its strands i) having a first sequence with a recognition sequence for a nicking And ii) 3 'thereof has a second sequence serving as the reporter binding molecule specific sequence tag; B) the complex formed is washed; and C) adding a nicking endonuclease and a polymerase which has strand displacement activity but no 5 'to 3% exonuclease activity and isothermal amplification of the sequence tag by repeatedly recognizing the recognition sequence of the nicking endonuclease and introduce a nick into the first sequence of the reporter DNA to produce a free 3 'OH end that is 5' of the sequence tag and the nick through the polymerase with complementary nucleotides to the complementary strand, with concomitant displacement of the sequence tag the double strand is filled up; D) the displaced sequence tag is detected, thereby detecting the target analyte in the sample. Furthermore, kits and systems for carrying out the method are provided.

Description

FIELD OF THE INVENTION

The present invention relates to a method for detecting at least one target analyte in a sample based on the principle of an immunoassay. The method can be carried out automatically in a simple manner and, for example, can also be integrated into existing automated systems / devices for the detection of nucleic acids. This makes it possible to carry out nucleic acid-based (NAT) detection as well as immunoassay-based detection of at least one target analyte for a sample on an automation platform. Furthermore, a kit and a corresponding automated system for carrying out the method according to the invention are provided.

BACKGROUND OF THE INVENTION

Known methods for the detection of target analytes such as proteins, metabolites, pathogens u. Ä., Are often based on the principle of an immunoassay. The best known embodiment is the enzyme-linked immunoabsorbent assay (ELISA). In an ELISA, capture antibodies (or other suitable capture binding molecules) are immobilized on a solid surface to specifically bind a target analyte from a complex sample. The solid surface may be, for example, the interior of a reaction vessel, such as a microtiter plate. The unbound sample is washed away and subsequently a reporter antibody is added which specifically binds the target analyte (direct ELISA). Or, a detection antibody specific for the target analyte and a reporter antibody that binds the detection antibody are added (indirect ELISA). In both variants, a complex is formed which has the capture binding molecule, the target analyte and the reporter antibody. These methods are also called "sandwich" methods. In the classical ELISA, the reporter antibody is labeled with an enzyme (for example peroxidase or alkaline phosphatase) with which a detection reaction is carried out. The enzyme activity is determined and is directly proportional to the amount of bound target analyte. A variety of variations of this process are known in the art.

A further development of the classical ELISA is, for example, immuno-PCR, in which the enzyme-labeled reporter antibody has been replaced by an oligonucleotide (for example DNA) labeled antibody. The oligonucleotide serves as a marker instead of the enzyme. The oligonucleotide is amplified and detected by means of a PCR following the complex formation (see, for example Sano et al. (2000), "Immuno-PCR: Very sensitive antigen detection by means of specific antibody-DNA conjugates", Science 258 (5079): 120-122 ). Various variants of this method are known. The immuno-PCR has the disadvantage that it is prone to background effects and must be performed for the PCR recurring temperature cycles. This complicates litigation and requires specialized instruments and well-trained laboratory personnel. The complexity of the procedure also makes the integration of this method in existing automated systems more difficult or makes them more complex and therefore more expensive.

An object of the present invention was therefore to provide an alternative method for detecting at least one target analyte. It was also an object to provide a method that can be easily automated and, for example, can also be integrated into existing automated systems that can purify nucleic acids.

SUMMARY OF THE INVENTION

The present invention provides a method for detecting at least one target analyte in a sample using a reporter binding molecule labeled with a double-stranded reporter DNA. The double-stranded reporter DNA has in at least one of its strands a first sequence with a recognition sequence for a nicking endonuclease and 3 'of which has a second sequence which serves as a sequence tag specific for the reporter binding molecule. This sequence tag is amplified and detected by a specific isothermal amplification method, thereby detecting the presence (or absence) of the target analyte. The method has the advantage that it is highly specific, suitable for multiplex analysis and, due to its simple process control, can also be integrated well into existing automated systems. It is also applicable to Lab to Chip (LoC) applications and related systems. In contrast to immuno-PCR, different temperature levels are not required during the amplification, which brings considerable advantages. In particular, there is no need to melt the double-stranded DNA for amplification and to set different temperature levels. With regard to the requirements for the processing, the method according to the invention has similarities with a nucleic acid preparation and for this reason can be integrated in a simple manner into existing automated systems which are oriented for the isolation and if necessary detection of nucleic acids. This also makes an automated A system is provided which makes it possible to detect both nucleic acids and other target analytes. This allows the automated detection of multiple diagnostic targets, such as nucleic acids, proteins, metabolites and organisms by means of automated processes. The method can be used for example for the detection of microorganisms, in particular pathogens. Among other things, it provides a diagnostic system that allows you to perform a variety of different tests. Flexibility in application diversity is a significant advantage in the clinical laboratory.

• A) ein an eine feste Oberfläche immobilisierter Komplex ausgebildet wird, umfassend
• a) ein Fänger-Bindemolekül, das den Zielanalyten spezifisch bindet;
• b) den nachzuweisenden Zielanalyten;
• c) ein Reporter-Bindemolekül, wobei das Reporter-Bindemolekül (aa) ein Bindemolekül und (bb) eine daran gebundene doppelsträngige Reporter DNA aufweist, wobei die Reporter DNA in zumindest einem ihrer Stränge i) eine erste Sequenz mit einer Erkennungssequenz für eine Nicking-Endonuklease aufweist, und ii) 3' davon eine zweite Sequenz aufweist, die als für das Reporter-Bindemolekül spezifischer Sequenz-Tag dient;
• B) der gebildete Komplex gewaschen wird; und
• C) eine Nicking-Endonuklease und eine Polymerase, die eine Strang-Verdrängungs-Aktivität aber keine 5' nach 3' Exonuklease-Aktivität aufweist, zugegeben wird und eine isothermale Amplifikation des Sequenz-Tags erfolgt, indem wiederholt die Nicking-Endonuklease ihre Erkennungssequenz erkennt und einen Nick in die erste Sequenz der Reporter DNA einführt, so dass ein freies 3' OH Ende erzeugt wird, das 5' des Sequenz-Tags liegt und der Nick durch die Polymerase mit zum Gegenstrang komplementären Nukleotiden unter gleichzeitiger Verdrängung des Sequenz-Tags aus dem Doppelstrang aufgefüllt wird; und
• D) der verdrängte Sequenz-Tag detektiert wird, wodurch der Nachweis des Zielanalyten in der Probe erfolgt.

According to a first aspect, a method is provided for detecting at least one target analyte in a sample, wherein in the method according to the invention

• A) a complex immobilized on a solid surface is formed, comprising
• a) a capture binding molecule that specifically binds the target analyte;
• b) the target analyte to be detected;
• c) a reporter binding molecule, wherein the reporter binding molecule (aa) has a binding molecule and (bb) a double-stranded reporter DNA bound thereto, the reporter DNA in at least one of its strands i) having a first sequence with a recognition sequence for a nicking And ii) 3 'thereof has a second sequence serving as the reporter binding molecule specific sequence tag;
• B) the complex formed is washed; and
• C) adding a nicking endonuclease and a polymerase which has strand displacement activity but no 5 'to 3' exonuclease activity and isothermal amplification of the sequence tag by repeatedly recognizing the recognition sequence of the nicking endonuclease and introduce a nick into the first sequence of the reporter DNA to produce a free 3 'OH end that is 5' of the sequence tag and the nick through the polymerase with complementary nucleotides to the complementary strand, with concomitant displacement of the sequence tag the double strand is filled up; and
• D) the displaced sequence tag is detected, thereby detecting the target analyte in the sample.

• A) wenigstens ein Fänger-Bindemolekül, das den Zielanalyten spezifisch bindet und
• B) wenigstens ein Reporter-Bindemolekül, wobei das Reporter-Bindemolekül (aa) ein Bindemolekül und (bb) eine daran gebundene doppelsträngige Reporter DNA aufweist, wobei die Reporter DNA in zumindest einem ihrer Stränge i) eine erste Sequenz mit einer Erkennungssequenz für eine Nicking-Endonuklease aufweist, und ii) 3' davon eine zweite Sequenz aufweist, die als für das Reporter-Bindemolekül spezifischer Sequenz-Tag dient.

According to a second aspect, there is provided a kit for detecting at least one target analyte in a sample, comprising:

• A) at least one capture binding molecule that specifically binds to the target analyte, and
• B) at least one reporter binding molecule, wherein the reporter binding molecule (aa) comprises a binding molecule and (bb) a double-stranded reporter DNA bound thereto, the reporter DNA in at least one of its strands i) having a first sequence with a recognition sequence for a nicking And ii) 3 'thereof has a second sequence serving as the reporter binding molecule specific sequence tag.

According to a third aspect, the present invention relates to the use of an automated system for detecting one or more target analytes by means of the method according to the invention.

DETAILED DESCRIPTION OF THE INVENTION

Method for detecting at least one target analyte in a sample According to a first aspect, a method for detecting at least one target analyte in a sample according to claim 1 is provided. The term "detection" includes the determination of the presence or absence of the target analyte in the sample and, in embodiments, also permits a quantitative determination of the amount of the target analyte in a sample. The individual steps of the method according to the invention are explained below.

Step A)

• a) ein Fänger-Bindemolekül, das den Zielanalyten spezifisch bindet;
• b) den nachzuweisenden Zielanalyten;
• c) ein Reporter-Bindemolekül, wobei das Reporter-Bindemolekül (aa) ein Bindemolekül und (bb) eine daran gebundene doppelsträngige Reporter DNA aufweist, wobei die Reporter DNA in zumindest einem ihrer Stränge i) eine erste Sequenz mit einer Erkennungssequenz für eine Nicking-Endonuklease aufweist, und ii) 3' davon eine zweite Sequenz aufweist, die als für das Reporter-Bindemolekül spezifischer Sequenz-Tag dient.

In step A), a complex immobilized on a solid surface is formed, comprising

• a) a capture binding molecule that specifically binds the target analyte;
• b) the target analyte to be detected;
• c) a reporter binding molecule, wherein the reporter binding molecule (aa) has a binding molecule and (bb) a double-stranded reporter DNA bound thereto, the reporter DNA in at least one of its strands i) having a first sequence with a recognition sequence for a nicking And ii) 3 'thereof has a second sequence serving as the reporter binding molecule specific sequence tag.

The formation of the complex can be carried out analogously to the immunoassay method known in the prior art. Non-limiting embodiments are explained below.

The capture binding molecule specifically recognizes and binds the target analyte. For example, an antibody can be used as catcher binding molecule. Suitable alternatives for target-analyte-specific binding molecules are known to the person skilled in the art and nonlimiting examples are also described below. According to a preferred embodiment, the scavenger binding molecule is attached to the solid surface of a support material, such as a reaction vessel, a "well" of a microtiter plate, a particle or the like and is correspondingly on this surface immobilized. The binding of the scavenger binding molecule to the surface may be covalent or non-covalent. The solid surface may, for example, be coated with the scavenger binding molecule. Corresponding embodiments for immobilizing a scavenger binding molecule (for example an antibody) on a solid surface, such as, for example, the interior of a reaction vessel (for example bottom and / or side walls) or on a particle, in particular a magnetic particle, are also known to the person skilled in the art the immunoassay method of the prior art. These can also be used in the context of the method according to the invention.

The reporter binding molecule has (aa) a binding molecule (for example, an antibody) and (bb) a double-stranded reporter DNA bound thereto. The reporter DNA has a first sequence with a recognition sequence for a nicking endonuclease in at least one of its strands. Preferably, this first sequence is in the 3 'region of the strand (see also figures). Preferably, the entire reporter DNA has only one corresponding recognition sequence. Further, the reporter DNA 3 'from the first sequence has a second sequence which serves as a specific sequence tag for the reporter binding molecule. The detection is carried out via an isothermal amplification of this sequence tag, as explained in detail below. The sequence tag is a tag sequence characterizing the reporter DNA. For example, the sequence tag may have a length of at least 12 nucleotides, for example 15 to 100 nucleotides, 18 to 50 nucleotides or 25 to 35 nucleotides. The sequence tag preferably has no dimers or hairpin forming sequence motifs. This sequence tag is specifically detected in step D). The reporter binding molecule may also have two or corresponding reporter DNAs to increase signal strength. Such an embodiment is encompassed by the term "reporter binding molecule".

The double-stranded reporter DNA can be of any length, provided the subsequent amplification is possible. Importantly, it has the first sequence and the second sequence to provide a recognition sequence for the nicking endonuclease and the sequence tag. In principle, it can also consist of these sequences. In one embodiment, the first and second sequences are in the 3 'region of the corresponding strand of the reporter DNA. Preferably, the double-stranded reporter DNA has a length of 25 to 500 bp, for example 35 to 350 bp or 50 to 250 bp. Of course, the term "double-stranded reporter DNA" also encompasses embodiments in which additionally single-stranded regions are present. Importantly, the portion of the reporter DNA that provides the first sequence (containing the recognition sequence for a nicking endonuclease) and the second sequence (sequence tag) is double-stranded. The double-stranded reporter DNA is attached to the binding molecule to provide the reporter binding molecule.

Various methods are known in the art that allow DNA to be coupled to a binding molecule, such as an antibody. Attachment of the reporter DNA to the binding molecule can be covalent or non-covalent. It should be sufficiently stable to perform the subsequent processing. Such methods are described, for example, in connection with the known immuno-PCR method and can be used to attach the reporter DNA according to the invention to the binding molecule. For example. a biotin-labeled reporter DNA can be used. The binding molecule (eg, antibody) is labeled with a biotin-binding molecule (eg, avidin or streptavidin) so that it can bind the biotin-providing reporter DNA. Furthermore, linkerbased systems can be used. Suitable methods for attaching a DNA serving as a marker, for example, in the WO2008 / 122310 described, which is referenced. Prefabricated binding molecule-DNA conjugates are preferably used as the reporter binding molecule because this significantly reduces the processing times of the assay protocol. As stated, the double-stranded DNA is preferably covalently bound to the binding molecule to provide the reporter binding molecule. For this purpose, as stated, suitable linker groups can be used. It is also possible to use reporter binding molecules that have more than one reporter DNA, and such embodiments include the term reporter binding molecule having a reporter DNA. The attachment of the reporter DNA is preferably not at the end having the sequence tag, but at the opposite end as shown in the figures. This minimizes steric hindrance.

According to a preferred embodiment, the reporter binding molecule also specifically binds the target analyte to be detected, analogously to a direct ELISA principle. However, it is also possible to additionally use another binding molecule such as, for example, another antibody which specifically binds the target analyte and a reporter binding molecule which then specifically binds this further binding molecule, analogously to an indirect ELISA principle. It is also possible to use two or more reporter binding molecules which are directed against the same target analyte (or the same further antibody, respectively) but, for example, recognize a different region of the target analyte. Appropriate Reporter binding molecules that recognize the same target analyte preferably have the same sequence tag or reporter DNA. Such embodiments are also included according to the invention.

In one embodiment, the scavenger binding molecule first binds the target analyte to be detected and, if the scavenger binding molecule is already immobilized on a solid surface, immobilizes the target analyte to be detected on the solid surface, such as the inside of a reaction vessel or the outside surface of a particle , Already at this point, one or more washing steps can be carried out to remove, for example, excess sample material. Subsequently, the corresponding reporter binding molecule can then be added to form the corresponding complex containing all three components. However, it is also possible to first add the reporter binding molecule to the sample, or the capture binding molecule and the reporter binding molecule are added at the same time. If the complex formation initially took place in solution, the immobilization of the complex on a solid surface can also take place subsequently. If the capture binding molecule is not already immobilized on a solid surface, for example, the complex formed can also be immobilized on a solid support, for example via the reporter binding molecule or other binding molecules which recognize the complex or individual components thereof and thereby immobilize a solid surface, provided they are, for example, immobilized itself. However, it is preferred that the capture analyte binding specifically to the target analyte is already bound to a solid support and thus immobilized. In the end, it is possible to make use of the known process guides from the field of ELISA and immuno-PCR.

In particular, the term "binding molecule" refers to any molecule capable of specifically binding a target substance. In the case of the scavenger binding molecule this is the target analyte. Usually, such a binding molecule binds its target substance with a higher binding affinity than it binds to other substances. Suitable embodiments are well known to those skilled in the art. Examples include, for example, antibodies, wherein the term "antibody" as used herein in addition to full length antibodies also antibody fragments such as Fab, F (ab '), Sub.2, ScFv, Fv diabodies, linear antibodies and monoclonal antibodies, polyclonal antibodies and includes single-chain antibodies. Other binding molecules can also be used, as long as they are capable of specifically binding a target analyte, for example nucleic acid ligands and lipocalins, such as, for example, anticalins. Suitable binding molecules are also in WO2008 / 122310 , Pages 14-18 and are hereby incorporated by reference. The binding molecule of the scavenger binding molecule and the reporter binding molecule may be of the same type (for example, one antibody each) or different. In one embodiment, the reporter binding molecule also specifically binds the target analyte. For example. For example, a binding molecule that binds the target analyte at a different site than the capture binding molecule can be used in the reporter binding molecule. If both specifically bind the target analyte, the binding molecules of the capture binding molecule and the reporter binding molecule are coordinated so that both simultaneously bind the target analyte and thus can form the complex. Preferably, the binding molecule is an antibody. Preferably, the binding molecule of both the capture binding molecule and the reporter binding molecule is an antibody.

The target analyte can be of a different nature. The target analyte is a molecule against which a binding molecule, such as an antibody, can be generated. The target analyte may be, for example, a protein, polypeptide, hapten, carbohydrate, lipid, cell, microorganism, bacteria, plasmodia, viruses, viroids, prions, yeasts, fungi, metabolites, pathogens or the like or derived from the above materials. In one embodiment, the target analyte is a substance generated from fungi, viruses, microorganisms, yeasts, plants, animals or humans. However, the method of the invention can also be used to detect smaller organic compounds such as drugs, metabolites, dyes or the like. The method according to the invention can ultimately detect the same target analytes as an ELISA or an immuno-PCR. The method according to the invention can therefore be used in particular in diagnostics.

The sample containing the target analyte may be, for example, a biological sample. Non-exhaustive examples are listed below. For example, the sample may be a body fluid, a cell culture medium, a tissue sample, or else a lysate, homogenate or extract, and partially or completely purified proteins or other biological molecules and mixtures thereof. The sample may be derived from, for example, a human, animal, plant or microorganism. Examples which may be mentioned are, for example, body fluids, blood, serum, plasma, leukocyte fraction, sputum, sperm, faeces, forensic samples, smears, punctates, biopsies, tissue samples, tissue separators, organs, food samples, environmental samples, plants, tumor tissue, cerebrospinal fluid, urine , Lymph and others Secretions of the respiratory system or gastrointestinal tract, tears, sputum, milk, white blood cells, myomas and the like, as well as fragments, components or derived compositions containing or suspected of containing the target analyte (s) ,

The solid surface is provided by a suitable carrier material. For this purpose, any support material can be used which is suitable for immobilizing the capture binding molecule or the complexes formed. Suitable solid surfaces are provided, for example, by wells of a plate, such as a microtiter plate, the interior of reaction vessels (eg, interior walls and / or bottom), particles, membranes, and the like. The person skilled in the art is aware of corresponding solid surfaces and carrier materials from the immunoassays used in the prior art (for example ELISA and immuno-PCR).

According to a preferred embodiment, the capture binding molecule is preferably immobilized, for example bound, to magnetic particles. This allows the processing of the target analyte bound to the capture binding molecule by means of a magnet, which has advantages in process control. Thus, for example, automated systems can be used that can process magnetic particles. Suitable systems are known to the person skilled in the art.

After step A), a corresponding complex containing the capture binding molecule, the target analyte and the reporter binding molecule is present, so that the presence of the target analyte can subsequently be detected on the basis of the reporter DNA of the bound reporter binding molecule.

Step B)

In step B), the complex formed is first washed to wash away contaminants such as unbound reporter binding molecules and any sample residues, etc. Suitable washing buffers are known to the person skilled in the art, for example from sandwich immuno-PCR methods. Preferably, two or more washing steps are performed.

Step C)

In step C) the reporter DNA is detected by isothermal amplification of the sequence tag. For this purpose, for example, the complex formed containing the scavenger binding molecule, the target analyte and the reporter binding molecule is brought into contact with a nicking endonuclease and a polymerase. For this purpose, the washed complexes can first be taken up in a suitable buffer in which the polymerase and the nicking endonuclease are active and, accordingly, the amplification can be carried out. The polymerase used has a strand displacement activity but no 5 'to 3' exonuclease activity. The isothermal amplification of the sequence tag is accomplished by the nicking endonuclease recognizing its recognition sequence and introducing a nick, and thus a single strand break, into the first sequence of the reporter DNA, thereby creating a free 3 'OH end in the strand. The nick is 5 'of the sequence tag. This nick is then filled in by the polymerase with complementary nucleotides to the complementary strand, whereby the sequence tag is displaced from the duplex. This is repeated, so that there is an amplification of the sequence tag, which is then in single-stranded form after the release (displacement) from the duplex.

The inserted nicking endonuclease recognizes a recognition sequence located in the first sequence of the reporter DNA. 3 'of this first sequence is the sequence tag. As stated, at least this portion of the reporter DNA is double-stranded. Depending on the type of nicking endonuclease, a nick is introduced into or in addition to this recognition sequence in the double strand, whereby the 5'-3 'phosphodiester bond between two nucleotides is hydrolytically cleaved. The nicking endonuclease thus acts as a phosphodiesterase, so that a single-strand break is inserted in the double strand before the sequence tag. This produces a free 3'-OH end which serves as a starting point for the polymerase. In the process according to the invention, only one strand of the double strand is cut at a time, namely the strand on which the recognition sequence for the nicking endonuclease and the sequence tag to be displaced are located. The other (complementary) strand remains intact. Endonucleases that cut not only one strand of the double strand, but both are not suitable for the process according to the invention. Suitable nicking endonucleases are known in the art, for example Nt.BstNBI, Nt.BspQI, Nb.BbvCI, Nb.BsmI, Nb.BsrDI, Nb.BtsI, Nt.AlwI, Nt.BbvCI, Nt.CviPII, Nt .BsmAI, Nb.Bpu10I and Nt.Bpu10I. Further embodiments are known to the person skilled in the art.

The introduced nick is recognized by a polymerase that has strand displacement activity but no 5 'to 3' exonuclease activity. A polymerase in the context of the present invention is an enzyme for nucleic acid replication and / or for nucleic acid repair. The polymerase fills the nick at the free 3'OH end starting with nucleotides that are complementary to the uncut counterstrand. The polymerization reaction always takes place in 5 'to 3' direction. During this process is through the Strand displacement activity of the polymerase displaces the 3 'of the Nick sequence tag from the double strand. The sequence tag is thus unhybridized as a single strand. Suitable polymerases are, for example, all polymerases which have a strand displacement activity but at the same time have no 5 'to 3' exonuclease activity. The polymerase is a DNA polymerase. Suitable examples include Vent exo, Deep Vent exo, Bst exo, the Klenow fragment of DNA polymerase I, Phi29 DNA polymerase and 9 ° Nm DNA polymerase. Other suitable DNA polymerases are also familiar to the person skilled in the art.

After the nick has been recognized by the polymerase and filled in with nucleotides complementary to the uncut counterstrand (which leads to the displacement of the sequence tag), the reporter DNA is again present as a double strand that is intact in this region and again has a recognition sequence for a nicking endonuclease as well as a sequence tag. The reporter DNA thus again has the same nucleic acid sequence in this region as before the introduction of the nick by the nicking endonuclease, so that the process of introducing a nick by the nicking endonuclease, the filling of the nick and the displacement of the sequence tag by the polymerase can be repeated as often as desired (which, for example, can be controlled by the duration of the reaction), resulting in isothermal amplification of the sequence tag. The amplification is linear.

Preferably, the nicking endonuclease and the polymerase work in a similar temperature range, which is advantageous in isothermal amplification because of the ease of process control. Suitable temperature ranges for an isothermal amplification are known to those skilled in the art and will be apparent from the description herein.

The amount of isothermal amplified and released sequence tag can be modulated by several factors. The reaction time during which the isothermal amplification of the sequence tag takes place has a major influence on the amount obtained. The longer the reaction time, the more sequence tags can usually be formed and released accordingly. Another important influencing factor is the reaction temperature. As with all enzymatic reactions, the reaction takes place more effectively and thus more quickly, the more the reaction temperature coincides with the temperature optimum for the activity of the enzymes involved. Consequently, the more similar the temperature optima of the involved enzymes, ie the nicking endonuclease and the polymerase, the more efficient the isothermal amplification of the sequence tag, and the better the reaction temperature agrees with these temperature optima. The choice of the reaction temperature also plays a role in another aspect for the amplification of the sequence tag. If the reaction temperature lies below the melting temperature of the sequence tag on its complementary strand, the sequence tag can be released only after insertion of the nick by a polymerase with strand displacement activity. If the reaction temperature lies above the melting temperature of the sequence tag on its complementary strand, then this hybrid is possibly so unstable after insertion of the nick that the sequence tag can also be released without a polymerase with strand displacement activity. In this case, the sequence tag is not actively displaced, but released passively. In a preferred embodiment, the reaction temperature for isothermal amplification is selected so that the sequence tag is released by the strand displacement activity of the polymerase to ensure that the nucleus replenishes with complementary nucleotides parallel to the release of the sequence tag has been. In one embodiment, the reaction temperature in the isothermal amplification is selected so that the sequence tag is released by the strand displacement activity of the polymerase and that the reaction temperature coincides with the temperature optima of the nicking endonuclease and the polymerase. Corresponding conditions are for example in the WO 2011/020588 which describes an isothermal amplification method based on a nicking endonuclease and a polymerase.

Step D)

In step D) the detection of the displaced sequence tag takes place, whereby also the detection of the target analyte in the sample takes place. The specific detection of the amplified sequence tag in step D) thus allows the specific detection of the reporter DNA and thus allows specific detection of the presence (or absence) of the target analyte in the sample. Also, quantitative determinations are possible.

In one embodiment, the isothermal amplified sequence tag is specifically detected by means of a probe directed against the sequence tag. The probe must therefore be complementary to the liberated single-stranded sequence tag at least in a partial region in order to be able to hybridize to it. Possibilities for detecting the hybrid of sequence tag and probe are known to those skilled in the art. The probe is preferably labeled to facilitate detection. For this purpose, it can have different modifications for the purpose of labeling, for example by means of one or more fluorescent dyes, quantum dots or gold nanoparticles. Other possibilities of modification of Probes which enable detection are familiar to the person skilled in the art.

The probe is preferably an oligonucleotide, preferably a DNA probe. In a further preferred embodiment, the probe has a GC content of 40% to 60%. It may, for example, have a melting temperature of 48 ° C to 74 ° C. It may also comprise modified nucleotides, provided that it can specifically hybridize to the sequence tag. The probe may, for example, have a length of at least 12 nucleotides, preferably at least 15 nucleotides. Suitable lengths include, for example, 10 to 50 nucleotides, 15 to 40 nucleotides, 20 to 35 nucleotides and 25 to 30 nucleotides. As stated, it is sufficient if the probe hybridizes only to a subarea of the sequence tag. It can also be longer than the sequence tag.

In one embodiment, the probe is labeled with at least one fluorescent dye and detection is via measurement of the fluorescence change after the probe has bound to the released sequence tag. In a preferred embodiment, the probe belongs to the class of dual-labeled probes. These are oligonucleotides coupled to a reporter fluorophore and a quencher. The nucleotides at the 5'-end of the probe are complementary to those at the 3'-end, so that a characteristic secondary structure such as a hairpin structure can form. This principle is known, for example, molecular beacons, other examples are hydrolysis probes Taqman ^® probes, Scorpion primers and the like. Corresponding embodiments are familiar to the person skilled in the art.

In one embodiment, the detection of the sequence tag by a probe is performed by melting curve analysis. In a melting curve analysis, the double-stranded nucleic acid is melted by raising the temperature slowly and continuously. At a melting point that is specific for the hybrid of probe and sequence tag, the double strand denatures into two single-stranded molecules. By having single-stranded nucleic acids at 260 nm of absorption properties other than double-stranded, melting curve analysis at 260 nm can be performed without the need for dye labels on the probe or intercalating substances for detection in double-stranded nucleic acids. In a particularly preferred embodiment, the melting curve analysis is carried out with the aid of dyes which are specific for double-stranded DNA, for example with the aid of intercalating fluorescent dyes. At the melting temperature, the duplex formed from the probe and sequence tag denatures into two single-stranded molecules. In this case, the fluorescent dye is released, and it is registered a fluorescence decrease. Examples of double-stranded DNA intercalating fluorescent dyes SYBR ^® Green and EvaGreen ^®. The person skilled in the art is familiar with further double-stranded DNA-specific fluorescent dyes which are suitable for a corresponding melting curve analysis. In a further embodiment, the melting curve analysis is carried out with the aid of a labeled probe. Examples of such a label are fluorescent dyes or quantum dots. The skilled person is familiar with further possibilities for marking the probe. In a preferred embodiment, more than one sequence tag (and thus more than one target analyte) is detected in a multiplexing approach. According to one embodiment, correspondingly two or more, for example at least three, at least four, at least five or at least ten different target analytes are detected in a multiplexing approach. In a preferred embodiment, the nucleotide sequence of the sequence tags differs in their nucleotide sequence to the extent that in the multiplexing approach correspondingly different probes hybridize specifically to their sequence tags and thus several different sequence tags (and hence target analytes) simultaneously in a reaction vessel can be specifically detected. Cross hybridizations can be avoided.

According to one embodiment, the sequence tags of the various reporter binding molecules differ in their melting temperature with the associated complementary probe, so that in a multiplexing approach due to different melting temperatures several different sequence tags can be detected simultaneously.

The number of target analytes which can be analyzed in parallel in a multiplexing approach results, for example, from the number of different melting temperatures which can be distinguished from one another by the corresponding analyzer, combined with the number of different fluorescent dyes which are distinguished from one another at different wavelengths by the respective analyzer can. If two sequence tags have the same melting temperature when the complementary probe is bound, they can nevertheless be specifically detected together and distinguished from one another in a multiplexing approach if their respective associated probes have different fluorescent dyes which emit the fluorescence at different wavelengths that these can be detected in different fluorescence channels. Of course, this principle is also applicable if the sequence tags differ in their melting temperature. Conversely, probes that hybridize with different sequence tags can have the same fluorescent label when the melting temperatures of the hybrids of these probes differ with their associated sequence tag. These can then be detected in the same fluorescence channel, but can still be distinguished from each other by their different melting temperatures.

In principle, all devices which are capable of detecting changes in the corresponding wavelength range are suitable for detecting the sequence tag, for example by means of a suitable probe. In the case of melting curve analysis without the addition of dyes, for example, devices capable of measuring absorption differences in a wavelength range of about 260 nm are suitable. An example is a UV spectrophotometer. The skilled person is familiar with other suitable devices. If the detection of the sequence tag by means of a fluorescent dye, for example. By means of a corresponding dye-labeled probe, then all devices are suitable for the detection, which are able to measure the fluorescence of the corresponding wavelength. These include, for example, real-time PCR cyclers, their detection units or spectrophotometers. Other suitable technical devices are well known to the skilled person. Corresponding details of possible probes to be used for the detection of a released sequence tag as well as detection possibilities are also in WO 2011/020588 to which reference is accordingly made.

Further embodiments

According to one embodiment, two or more different target analytes are detected in parallel by means of the method according to the invention. For this purpose, (at least) one specific capture binding molecule and (at least) one corresponding reporter binding molecule are used for each target analyte to be detected. As stated, the reporter binding molecule preferably also binds specifically to the target analyte (analogously to a direct ELISA), but variants are also conceivable in which at least one further binding molecule is used which mediates the binding between the target analyte and the reporter binding molecule (analogously an indirect ELISA). In order to be able to distinguish the binding and thus the presence of the various target analytes, the various reporter binding molecules used in parallel have different reporter DNAs which differ from each other at least in the region of the sequence tag to be detected later. This releases a different sequence tag for each reporter binding molecule used, allowing different bound target analytes to be distinguished within a single reaction. This principle is also explained in the figures. To detect the released different sequence tags preferably probes are used, each of which specifically bind their sequence tag. The probes can be labeled differently so that they give correspondingly different signals. Suitable embodiments which permit the parallel detection of different sequence tags by means of suitable probes have already been described above. This allows in a simple way the parallel detection of different target analytes. This is for example a particular advantage if you want to detect different target analytes, for example. Different pathogens in a sample. For example, in a fluorescence meter several fluorescences can be detected under real-time conditions simultaneously per cavity and thus reaction. Combined with a DNA melting point analysis, it would also be possible to determine 20 to 30 different target analytes per well. The ability to detect a variety of different target analytes in parallel in a sample is a particular advantage of the method of the invention.

According to one embodiment, the recognition sequence for the nicking endonuclease in the reporter DNAs of the different reporter binding molecules is identical, so that in a corresponding multiplexing approach only one kind of nicking endonuclease is needed. Also, the entire first sequence or the entire reporter DNA of the various binding molecules - except for the characteristic area of the sequence tag - be identical.

As stated, the scavenger binding molecule is preferably immobilized on a solid surface. This facilitates the processing and thus automation of the process. Preferably, the scavenger binding molecule is immobilized on magnetic particles, which facilitates the processing of the bound target analyte, as is also apparent from the following examples. The magnetic particles may have, for example, superparamagnetic, paramagnetic, ferrimagnetic or ferromagnetic properties. The magnetic particles can be processed in a simple manner by applying a magnetic field. Various processing variants are known in the prior art. For example, the magnetic particles may be collected at the bottom or side of a reaction vessel and the remaining supernatant removed. Alternatively, a magnet may be introduced into the sample to which the magnetic particles bind and thus be removed from the sample along with the magnet. Corresponding systems, which are also known from the processing of nucleic acids by means of magnetic particles, can be used here.

• – Sammeln der Proben in einem Probenbehälter, der magnetische Partikel enthält, an die ein Fänger-Bindemolekül gebunden und damit immobilisiert vorliegt. Das Fänger-Bindemolekül, bei dem es sich vorzugsweise um einen Antikörper handelt, kann beispielsweise in getrockneter Form vorliegen, wodurch es für eine Lagerung geeignet ist. Eine entsprechende Ausführungsform eignet sich insbesondere auch für einen Einsatz in einem Kit-Format.
• – Inkubieren, so dass der Zielanalyt an das Fänger-Bindemolekül bindet. Sofern getrocknete magnetische Partikel eingesetzt werden, können diese durch die Zufuhr des Probenmaterials resuspendiert werden und das Fänger-Bindemolekül kann im Rahmen der Inkubation den in der Probe vorliegenden Zielanalyten binden.
• – Abtrennen des an die magnetischen Partikel immobilisierten Zielanalyten vom Rest der Probe durch Einsatz eines Magneten. Wie ausgeführt, können die magnetischen Partikel auf verschiedenste Art und Weise bei der Prozessierung gesammelt werden, gemäß einer Ausführungsform am Boden oder Rand des Probensammelgefäßes. Optional können die eingesammelten magnetischen Partikel ein oder mehrmals gewaschen werden, um Probenreste und entsprechend etwaige Verunreinigungen zu entfernen.
• – Die gesammelten magnetischen Partikel mit dem an das Fänger-Bindemolekül gebundenen Zielanalyten werden dann mit einem Reporter-Bindemolekül in Kontakt gebracht, wodurch der Komplex aus Fänger-Bindemolekül, Zielanalyten und Reporter-Bindemolekül ausgebildet wird, der entsprechend an die magnetischen Partikel gebunden vorliegt. Wie oben ausgeführt, kann die Bindung des Reporter-Bindemoleküls an den Zielanalyten entweder direkt oder indirekt über mindestens ein weiteres Bindemolekül erfolgen, wie es auch aus den klassischen ELISA-Verfahren bekannt ist. Um das Inkontaktbringen des Reporter-Bindemoleküls mit dem an das Fänger-Bindemolekül gebundenen Zielanalyten zu erleichtern, können die zuvor eingesammelten magnetischen Partikel beispielsweise zunächst in einem kleineren Volumen einer geeigneten Lösung, beispielsweise einem Puffer, resuspendiert werden. Hierfür können aus dem ELISA und Immuno-PCR Verfahren bekannte Puffer eingesetzt werden.
• – Abtrennen der an die magnetischen Partikel immobilisierten Komplexe durch Einsatz eines Magneten und Waschen der Komplexe.
• – Zugabe der Polymerase und der Nicking-Endonuklease zur isothermalen Amplifikation des Sequenz-Tags gemäß Schritt C) des erfindungsgemäßen Verfahrens. Die entsprechenden Komponenten sowie ferner Nukleotide und ein mit den eingesetzten Enzymen kompatibler Puffer können in beliebiger Reihenfolge hinzugegeben werden. Details zu dem Amplifikationsverfahren, wodurch die Anwesenheit des Zielanalyten nachgewiesen wird, wurden bspw. bereits oben geschildert.

According to a preferred embodiment, the method is characterized by the following steps:

• Collecting the samples in a sample container containing magnetic particles to which a scavenger binding molecule is bound and thus immobilized. The scavenger binding molecule, which is preferably an antibody, may be in dried form, for example, whereby it is suitable for storage. A corresponding embodiment is particularly suitable for use in a kit format.
• - Incubate so that the target analyte binds to the capture binding molecule. If dried magnetic particles are used, they can be resuspended by supplying the sample material and the capture binding molecule can bind the target analyte present in the sample as part of the incubation.
• Separating the target analyte immobilized on the magnetic particles from the remainder of the sample by use of a magnet. As stated, the magnetic particles may be collected in a variety of ways during processing, according to one embodiment at the bottom or edge of the sample collection vessel. Optionally, the collected magnetic particles may be washed one or more times to remove sample residue and, accordingly, any contaminants.
• The collected magnetic particles with the target analyte bound to the capture binding molecule are then contacted with a reporter binding molecule, thereby forming the complex of capture binding molecule, target analyte and reporter binding molecule that is appropriately bound to the magnetic particles. As stated above, the binding of the reporter binding molecule to the target analyte can be done either directly or indirectly via at least one other binding molecule, as is known from classical ELISA methods. For example, to facilitate contacting the reporter binding molecule with the target analyte bound to the capture binding molecule, the previously collected magnetic particles may first be resuspended in a smaller volume of a suitable solution, such as a buffer. For this purpose, known from the ELISA and immuno-PCR method known buffers can be used.
• - Separating the immobilized on the magnetic particles complexes by using a magnet and washing the complexes.
• - Addition of the polymerase and the nicking endonuclease for isothermal amplification of the sequence tag according to step C) of the method according to the invention. The corresponding components as well as further nucleotides and a buffer compatible with the enzymes used can be added in any order. For example, details of the amplification method to detect the presence of the target analyte have been described above.

As stated, according to one embodiment, the method according to the invention serves to detect the presence or absence of two or more different target analytes in a sample. For this purpose, one set comprising (at least) one capture binding molecule specific for the target analyte and (at least) one reporter binding molecule with a characteristic sequence tag are used per target analyte to be detected. The reporter binding molecules, which each serve to detect another target analyte, thus have a double-stranded reporter DNA in which at least the sequence tag of the sequence tags of the other reporter binding molecules used in parallel in a reaction, different. This allows differentiation of the amplified sequence tags. This allows differentiation between the different reporter DNAs so that different target analytes can be specifically detected. To detect the different amplified sequence tags, corresponding probes are added, each of which can hybridize specifically to its sequence tag, but not to the sequence tags of the other reporter DNAs. Details of the samples and the possibilities of distinguishing the various probes, such as different markings and / or melting temperatures, have already been described above.

According to a preferred embodiment, magnetic particles are used which are provided with different scavenger binding molecules, each of which can bind different target analytes. As stated, the magnetic particles can be coated, for example, with the scavenger binding molecules, and other embodiments are known to those skilled in the art to immobilize scavenger binding molecules, such as antibodies, to magnetic particles. On a magnetic particle also different capture binding molecules can be applied. According to a preferred embodiment, however, different types of magnetic particles are used, wherein the magnetic particles each provided with only one type of capture binding molecule, for example. Coated, as it is also described in the embodiments. As stated, the binding molecules are preferably antibodies.

If the capture binding molecule is not already immobilized on a solid surface, then the complex of capture binding molecule and target analyte, or the final complex comprising the capture binding molecule, the target analyte and the reporter binding molecule are subsequently immobilized on a solid surface. Corresponding embodiments are known from classical ELISA methods or immuno-PCR methods and can also be used in the context of the method according to the invention.

Kit

• A) wenigstens ein Fänger-Bindemolekül, das den Zielanalyten spezifisch bindet und
• B) wenigstens ein Reporter-Bindemolekül, wobei das Reporter-Bindemolekül (aa) ein Bindemolekül und (bb) eine daran gebundene doppelsträngige Reporter DNA aufweist, wobei die Reporter DNA in zumindest einem ihrer Stränge i) eine erste Sequenz mit einer Erkennungssequenz für eine Nicking-Endonuklease aufweist, und ii) 3' davon eine zweite Sequenz aufweist, die als für das Reporter-Bindemolekül spezifischer Sequenz-Tag dient.

In a second aspect, the present invention relates to a kit for detecting at least one target analyte in a sample comprising:

• A) at least one capture binding molecule that specifically binds to the target analyte, and
• B) at least one reporter binding molecule, wherein the reporter binding molecule (aa) comprises a binding molecule and (bb) a double-stranded reporter DNA bound thereto, the reporter DNA in at least one of its strands i) having a first sequence with a recognition sequence for a nicking And ii) 3 'thereof has a second sequence serving as the reporter binding molecule specific sequence tag.

A corresponding kit is suitable for carrying out the method according to the invention according to the first aspect. Details of the capture binding molecule, the reporter binding molecule, the target analyte, the double-stranded reporter DNA, the first sequence, and the second sequence are described above in the context of the method of the invention, and we refer to the above disclosure.

In a preferred embodiment, the capture binding molecule that specifically binds a target analyte is immobilized on a solid surface of a support. Suitable embodiments are described above and include, for example, the interior (eg bottom and / or walls) of reaction vessels, microtiter plates, particles, membranes or the like. Corresponding embodiments are also familiar to the person skilled in the art from the ELISA and immuno-PCR methods. Preferably, the scavenger binding molecule is bound to particles, more preferably magnetic particles. Suitable embodiments for corresponding magnetic particles are known to the person skilled in the art.

In a preferred embodiment, the kit allows the detection of two or more different target analytes. For this purpose, the kit has, for each target analyte to be detected, a kit specific for this target analyte, comprising a capture binding molecule that specifically binds the target analyte to be detected, and a corresponding reporter binding molecule. The various reporter binding molecules used in parallel each have a double-stranded reporter DNA which, at least in the region of the sequence tag, is to be used by the reporter DNAs of other reporter binding molecules contained in the kit, which are to be used in parallel in a detection reaction differentiates. Details of this embodiment have already been described above. The differing sequence tags make it possible to differentiate between the different reporter binding molecules and thus between the different target analytes present in the sample. Preferably, the recognition sequences used for the amplification for the nicking endonuclease in the different reporter DNAs are identical, so that the same nicking endonuclease can be used. Further, such set specific to a target analyte preferably comprises at least one probe specific for the sequence tag of the corresponding reporter DNA.

In one embodiment, the kit additionally comprises a nicking endonuclease. Suitable embodiments have been described in connection with the method according to the invention and are also known to the person skilled in the art. In one embodiment, the kit further comprises a polymerase having strand displacement activity but no 5 'to 3' exonuclease activity. Suitable polymerases have been described above in connection with the method according to the invention and may be included accordingly in the kit. Preferably, the kit has a corresponding nicking endonuclease and a corresponding polymerase.

In a preferred embodiment, the kit further comprises at least one probe directed against the sequence tag. This is preferably labeled to allow easy detection and possibly differentiation between different probes, for example when the kit is used to detect two or more target analytes. In one embodiment, the probe is labeled with at least one fluorophore. Suitable fluorophores are familiar to the person skilled in the art and can be used accordingly here. If the kit has several different probes for the purpose of detecting different sequence tags, these preferably have different markers and / or different melting temperatures in hybrid with the corresponding sequence tag in order to enable differentiation.

Furthermore, the kit may also contain other consumables, such as nucleotides for the amplification reaction and / or one or more buffers, for example washing buffer, resuspension buffer and / or amplification buffer.

According to one embodiment, the kit also includes at least one sample collecting vessel magnetic particles and at least one scavenger binding molecule bound thereto, such as an antibody. Where the kit is designed to detect a target analyte, the magnetic particles are bound to a type of capture binding molecule that is specific for the target analyte. However, different capture binding molecules could also be used, as long as they are specific for the target analyte to be detected (for example, different antibodies to the same target). Again, this is discussed as an embodiment where the magnetic particles are endowed, for example coated, with one or one type of capture binding molecule, because they specifically bind a particular target analyte.

According to one embodiment, the sample collection vessel comprises two or more types of magnetic particles each having different capture binding molecules for detecting various target analytes. This provides a kit that allows you to easily detect different target analytes in a sample in parallel. Thus, according to one embodiment, a corresponding sample collection vessel has different types of magnetic particles, each type of magnetic particle being functionalized with a type of capture binding molecule. As stated, the capture molecules can be bound to the magnetic particles, for example. According to an alternative embodiment, the magnetic particles are coated with different types of capture binding molecules directed against different target analytes. Therefore, according to this embodiment, different target analytes can also bind to a magnetic particle.

• 1. Waschlösung;
• 2. Reporter-Bindemolekül, dieses liegt vorzugsweise in getrockneter und damit lagerungsfähiger Form vor;
• 3. Resuspensionslösung zur Resuspension der magnetischen Partikel; die Resuspensionslösung ist kompatibel mit den eingesetzten Bindemolekülen;
• 4. Waschlösung;
• 5. Reagenzienmischung enthaltend die Nicking-Endonuklease, die Polymerase mit Strang-Verdrängungs-Aktivität aber ohne 5' nach 3 Exonuklease-Aktivität und ein oder mehrere Detektions-Sonden, sowie optional Nukleotide; vorzugsweise liegt die Reagenzienmischung in getrockneter Form vor;
• 6. Resuspensionspuffer, dieser ist vorzugsweise kompatibel mit der Reagenzienmischung und den dort enthaltenen Enzymen, so dass dieser Puffer zur Resuspension der Reagenzienmischung genutzt werden kann, wenn diese in getrockneter Form vorliegt.

According to one embodiment, the kit comprises a cartridge which can be introduced into an automatic system on which the method according to the invention can proceed. The corresponding cassette may comprise sample containers with the following materials, which are preferably also arranged in this order in the cassette:

• 1. washing solution;
• 2. reporter binding molecule, this is preferably present in dried and thus storable form;
• 3. Resuspension solution for resuspending the magnetic particles; the resuspension solution is compatible with the binding molecules used;
• 4. washing solution;
• 5. reagent mixture containing the nicking endonuclease, the polymerase with strand displacement activity but without 5 'to 3 exonuclease activity and one or more detection probes, and optionally nucleotides; Preferably, the reagent mixture is in dried form;
• 6. Resuspension buffer, this is preferably compatible with the reagent mixture and the enzymes contained therein, so that this buffer can be used for resuspension of the reagent mixture when it is in dried form.

Automated system and use

According to a third aspect, the present invention relates to the use of an automated system for detecting one or more target analytes by means of the method according to the first aspect of the present invention. Details have already been explained and reference is made to the corresponding disclosure.

The inventive method is easy to automate due to the simple procedure. Corresponding automated processing permits a CLIA (clinical laboratory improvement amendment) "Waiver" or a "Medium Complexity" classification, which is an essential prerequisite for the operation of diagnostic systems, for example in the USA.

According to one embodiment, the automated system has at least one unit for sample processing, for example in order to be able to carry out the pipetting steps required for carrying out the method according to the invention. As stated, the scavenger binding molecule is preferably bound to magnetic particles to permit processing by means of a magnetic field. Therefore, according to one embodiment, the device has at least one magnet which allows the magnetic particles to be processed.

Furthermore, the automated system has at least one heating module, for example in order to enable the temperature setting for the isothermal amplification and possibly a melting curve analysis.

Furthermore, the automated system has a detection module. The detection module is tuned to the type of proof. Preferably, as indicated, labeled (eg fluorescently labeled) probes are used for the detection. The detection module allows the detection of the released amplified sequence tag or allows the detection of the inserted labeled probe. Suitable detection modules are described above in connection with the method according to the invention. For example. The system can have an integrated Real Time PCR Cycler as a detection module.

The design of the associated consumables can be designed in such a way that the user only has to insert his sample into the device in addition to the consumables. The sample can be processed automatically until the resulting diagnostic result. This is especially true when magnetic particles are used with one or more scavenger binding molecules immobilized thereon, as described above. Corresponding consumables are described in connection with the kit according to the invention and can be presented in the usual for such automated systems cartridges containing the consumables.

Furthermore, the automated system preferably has at least one unit for purifying at least one nucleic acid from the sample. The method according to the invention is designed such that it can be carried out on simply structured devices / systems, so that methods based on nucleic acid (NAT) and immunoassay-based methods can be executed simultaneously or with one system.

According to one embodiment, the automated system has a unit for carrying out the method according to the invention according to the first aspect and at least one unit for purifying at least one nucleic acid from a sample. If magnetic particles are used for the processing according to the invention in the process, it is advantageous to use magnetic particles, such as magnetic silica particles or the like, also for the isolation of the nucleic acids.

The method according to the invention makes it possible by its simplicity to integrate detection based on an immunoassay in an automated system which can also perform nucleic acid-based detection (NAT). This is a particular advantage because an automated system can be used in terms of process flows for multiple diagnostic targets, such as nucleic acids, proteins, metabolites, organisms, especially pathogenic organisms. The steps required by the method according to the invention are very similar to a nucleic acid preparation or the detection of a nucleic acid with respect to the requirements of the device. By similar processing, the implementation of both types of assays (nucleic acid purification (and optionally subsequent detection) and parallel detection of other targets (target analytes), especially targets that are not nucleic acids) is greatly facilitated. The advantage of such an automated system is that it provides a diagnostic platform that allows for a variety of different tests. This flexibility is a significant advantage.

Terms used in the singular, such as "a" scavenger binding molecule, "a" reporter DNA, "a" buffer, and the like, are intended to encompass "at least one," "at least one," and the like also the majority and vice versa, unless expressly provided otherwise or results from the context.

It is preferred to select and combine preferred embodiments as described herein, and the specific subject matter resulting from such combination of preferred embodiments is also within the scope of the present disclosure.

The invention is not limited by the example methods and embodiments described herein. Numeric ranges include the digits that define the range. The headings used herein are not a limitation of the various aspects or embodiments of the invention which are to be regarded as overall disclosure.

EMBODIMENTS AND FIGURE DESCRIPTION

The examples are for illustration only and do not limit the invention.

1 explains the principle of the invention using an embodiment in which different target analytes (shown two, but it can also be several) are detected by the method according to the invention. FIG. 1) shows a reaction vessel (for example a "well" of a microtiter plate) which is functionalized with two different capture binding molecules, here a first capture antibody a) which specifically binds the target analyte shown as a triangle in the following pictures and one second capture antibody b) which specifically binds the target analyte shown in the following pictures as a rhombus. In Figure 2), to this plate / well coated with the antibodies specific for the various target analytes is added a solution (for example, a sample or a prepared sample) containing the desired target analytes (or to test whether it contains the target analytes ). There is an incubation, so that the target analytes contained can bind to the respective capture binding molecule and thereby also be immobilized on the surface. Optionally, one or more washing steps can already be carried out at this point in order to wash away unbound sample material or impurities. In 3) the target analytes bound to the capture binding molecules are contacted with a reporter binding molecule, here again an antibody. Of the Reporter antibody a) binds specifically to the target analyte shown as a triangle, the reporter antibody b) binds specifically to the target analyte shown as a rhombus. The reporter antibodies have, in addition to the antibody (binding molecule), a double-stranded reporter DNA bound to it. As stated, this can also be double-stranded only in a partial region, for example the end not bound to the binding molecule. According to this embodiment, the double-stranded reporter DNA has a first sequence in the 3 'region of the upper strand (not separately marked in the figure), which provides a recognition sequence for a nicking endonuclease (this recognition sequence is in the reporter DNA a) and b) equal) as well as 3 'thereof a specific for the respective reporter DNA sequence tag, which is characterized in the case of the reporter antibody a) as a dashed line and in the case of the reporter antibody b) as a gray line. The dashed line of the sequence tag a) serves only to distinguish it from the sequence tag b), in both cases the reporter DNA is double-stranded in this region and hybridizes correspondingly normally to the opposite strand. Details are also in 2 explained in more detail. At least the sequence tags of the different reporter DNAs of the reporter binding molecules a) and b) differ in their sequence to allow discrimination between the reporter binding molecules and thus the target analyte. Binding of the reporter binding molecule to the target analyte produces a complex containing the capture binding molecule, the target analyte, and the reporter binding molecule. In 4) the reagents required for the linear amplification of the sequence tag ("Amp Mix") are added, in particular a nicking endonuclease recognizing the recognition sequence of the reporter DNA, a polymerase which has a strand displacement activity but not 5 'to 3'. Having exonuclease activity, as well as nucleotides. As a result, a partial segment (sequence tag) of the double-stranded reporter DNA is linearly amplified. The amplification takes place isothermally, which is a particular advantage of the method according to the invention. The linear amplified sequence tags of the reporter binding molecules a) and b) are shown in FIG. 5). These are detected, for example by means of detection probes specific for the respective sequence tag, which hybridize to the released and thus single-stranded sequence tag. The detection probes are preferably labeled. The probes may be added together with the amplification reagents, or separately. The probes are preferably labeled differently and / or differ in terms of their melting temperature in hybrid with the sequence tag, so that a distinction between the different sequence tags and thus Zielanalyten is possible. Corresponding probes are known to the person skilled in the art. Preferably, the probe is a fluorescence-labeled oligonucleotide probe which is reverse-complementary to the sequence tag or a partial region thereof. As a result, a plurality of fluorescences can be detected simultaneously under real-time conditions per cavity in a fluorescence meter (see FIG. 6)). By a DNA melting point analysis, it is also possible to determine 20 to 30 target analytes in parallel per cavity and thus reaction in parallel. Details on this are, for example, also in WO2008 / 122310 disclosed. The method according to the invention therefore makes it possible to carry out a multiplex detection within a single cavity or reaction and therefore within a single sample. For this reason, several diagnostic test results can be determined simultaneously with one and the same biological sample. The method is simple in terms of process management and thus less error-prone and can waive different temperature cycles o. Ä., As they are required in an immuno-PCR.

2 shows the basic structure of the double-stranded reporter DNA and explains how the sequence tag is linearly amplified by means of the method according to the invention. As shown in Figure 1), the double-stranded reporter DNA according to the embodiment shown has a first sequence (white) with a recognition sequence (see arrow) for a nicking endonuclease in the 3 'region of the strand shown above, and 3' thereof a second sequence (checkered), which serves as specific for the respective reporter binding molecule sequence tag. The corresponding sections on the opposite strand are also marked differently in the illustration, but they are less relevant for the explanation of the principle according to the invention. Upon addition of a nicking endonuclease (e.g., Nt.BstNBI), it recognizes its recognition sequence in the first sequence and, in the embodiment shown, cuts in the recognition sequence (see Figure 2)). This introduces a "nick" into the strand which creates a free 3 'OH end within the strand, 5' of the sequence tag. The strand area 3 'of the nick is subsequently released as a sequence tag. The opposing strand remains intact. The polymerase also added (for example, Vent exo ^- ), which has no 5 'to 3' exonuclease activity, prolongs the 3 'OH end and displaces the sequence tag from the opposite strand due to its strand displacement activity, thereby reducing the sequence Tag is released in single-stranded form (see Figure 3)). The released sequence tag is in 2 marked with an arrow. At the same time, the sequence tag is replicated by the polymerase (the complementary strand serves as a template), so that again an intact, double-stranded reporter DNA molecule is present (see FIG. 3)). This reaction (cutting, filling, displacement and thus release of the sequence tag as a single strand) is repeated several times, so that the sequence tag is linearly amplified. Of the liberated sequence tag is detected, preferably by using a labeled probe. This hybridizes to the liberated single-stranded sequence tag and thus allows detection (see 4)). By different labeling of probes specific for different sequence tags and / or by using probes with different melting temperatures, different target analytes can be detected in parallel. For example, as in 4) the 2 Illustrated a Taq Man Probe (Taq Man Probe) to be used.

In 3 a preferred embodiment of the present invention is shown. Here, instead of immobilizing the capture binding molecules (antibodies) to the surface of a reaction vessel (eg, its bottom and / or interior walls), an embodiment is shown in which the capture antibodies (a) and b) specific for the various target analytes are attached magnetic particles are bound. This can be done in the form of a coating. A sample collection vessel is used for liquid sample materials (for example, blood, plasma or serum) comprising the magnetic particles with the preferably dried capture antibodies (see FIG. 1)). According to the embodiment shown, two different types of magnetic particles are employed, type a) having a capture binding molecule (antibody) specific for a target analyte (shown as a triangle), and type b) the magnetic particles having a capture binding molecule (Antibody) which specifically binds a second target analyte (shown as a rhombus). These different types of magnetic particles can coexist in a sample collection vessel. However, it is also possible to choose an embodiment in which two or more scavenger binding molecules specific for different target analytes are present on the same particles. In 2), the liquid sample is taken up into the sample collection vessel, whereby the dried magnetic particles are resuspended. As part of the incubation, the target analytes can bind to their specific capture binding molecules. By magnetic separation and removal of the supernatant, the target analytes bound to the magnetic particles via the capture binding molecules can be enriched, as shown in FIG. 3). One or more wash steps can be performed at this point to remove sample residue. The magnetic particles with the target analytes bound thereto are then preferably resuspended in a smaller volume of a suitable buffer. In 4) the reporter binding molecules are then added. According to the embodiment shown, these bind their respective target analytes specifically and thus directly, but indirect methods are also possible. By binding the reporter binding molecules to the corresponding target analytes, a complex is formed which has the capture molecule, the target analyte and the reporter binding molecule. The complexes are immobilized on the magnetic particles via the capture binding molecules. The complexes thus formed are separated by application of a magnetic field and preferably washed one or more times (see FIG. 5)). In 6) the detection of the bound target analyte is carried out by isothermal amplification of the sequence tag of the reporter DNA. Details of this detection method have already been explained above.

QUOTES INCLUDE IN THE DESCRIPTION

This list of the documents listed by the applicant has been generated automatically and is included solely for the better information of the reader. The list is not part of the German patent or utility model application. The DPMA assumes no liability for any errors or omissions.

Cited patent literature

• WO 2008/122310 [0015, 0018, 0069]
• WO 2011/020588 [0030, 0038]

Cited non-patent literature

• Sano et al. (2000), "Immuno-PCR: Very sensitive antigen detection by means of specific antibody-DNA conjugates", Science 258 (5079): 120-122 [0003]

Claims (13)

Method for detecting at least one target analyte in a sample, wherein A) a complex immobilized on a solid surface is formed, comprising a) a capture binding molecule that specifically binds the target analyte; b) the target analyte to be detected; c) a reporter binding molecule, wherein the reporter binding molecule (aa) has a binding molecule and (bb) a double-stranded reporter DNA bound thereto, the reporter DNA in at least one of its strands i) having a first sequence with a recognition sequence for a nicking And ii) 3 'thereof has a second sequence serving as the reporter binding molecule specific sequence tag; B) the complex formed is washed; and C) adding a nicking endonuclease and a polymerase which has strand displacement activity but no 5 'to 3' exonuclease activity and isothermal amplification of the sequence tag by repeatedly recognizing the recognition sequence of the nicking endonuclease and introduce a nick into the first sequence of the reporter DNA to produce a free 3 'OH end that is 5' of the sequence tag and the nick through the polymerase with complementary nucleotides to the complementary strand, with concomitant displacement of the sequence tag the double strand is filled up; and D) the displaced sequence tag is detected, thereby detecting the target analyte in the sample. The method of claim 1, wherein two or more different target analytes are detected in parallel, wherein for each target analyte to be detected in each case a specific capture binding molecule and a reporter binding molecule with different sequence tag are used. The method of claim 1 or 2, wherein the scavenger binding molecule is immobilized on a solid surface, wherein preferably the scavenger binding molecule is immobilized on magnetic particles. Method according to one or more of claims 1 to 3, characterized in that the amplified sequence tag is detected by means of a directed against the sequence tag probe. Method according to one or more of claims 1 to 4, characterized by the following steps: Collecting the sample in a sample container containing magnetic particles to which the scavenger binding molecule is bound; - incubate so that the target analyte binds to the capture binding molecule; Separating the target analyte immobilized on the magnetic particles from the remainder of the sample by use of a magnet; - contacting with a reporter binding molecule, thereby forming the complex of capture binding molecule, target analyte and reporter binding molecule immobilized on the magnetic particles; Separating the complex immobilized on the magnetic particles by using a magnet and washing the complex; - Addition of the polymerase, the nicking endonuclease and the sequence tag specific probe and isothermal amplification of the sequence tag according to step C). Kit for detecting at least one target analyte in a sample comprising: A) at least one capture binding molecule that specifically binds to the target analyte, and B) at least one reporter binding molecule, wherein the reporter binding molecule (aa) comprises a binding molecule and (bb) a double-stranded reporter DNA bound thereto, the reporter DNA in at least one of its strands i) having a first sequence with a recognition sequence for a nicking And ii) 3 'thereof has a second sequence serving as the reporter binding molecule specific sequence tag. Kit according to claim 6, characterized in that the capture binding molecule is immobilized on a solid surface, preferably bound to magnetic particles. Kit according to claim 6 or 7, for detection of two or more different target analytes, the kit for each target analyte to be detected each having a specific capture binding molecule and a corresponding reporter binding molecule and the different reporter binding molecules each having a double-stranded reporter DNA with different sequence Day. Kit according to one or more of claims 6 to 8, characterized in that it comprises one or more of the following components: - a nicking endonuclease; A polymerase which has strand displacement activity but no 5 'to 3' exonuclease activity; and / or a probe directed against the sequence tag. Kit according to one or more of claims 6 to 9, characterized in that it comprises a sample collecting vessel with magnetic particles and at least one capture scavenger molecule bound thereto, wherein preferably the Sample collection vessel has two or more types of magnetic particles, each having different capture binding molecules for detecting various target analytes. Use of an automated system for detecting one or more target analytes by means of the method according to one or more of claims 1 to 5. Use according to claim 11, characterized in that the automated system comprises at least one unit for carrying out the method according to one or more of claims 1 to 11 and further at least one unit for purifying at least one nucleic acid from a sample. Use according to claim 11 or 12, characterized in that the automated system comprises a detection module, preferably a real-time cycler module.

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